a microbial oasis in the hypersaline atacama subsurface...

A Microbial Oasis in the Hypersaline AtacamaSubsurface Discovered by a Life Detector Chip:

Implications for the Search for Life on Mars

Victor Parro,1 Graciela de Diego-Castilla,1 Mercedes Moreno-Paz,1 Yolanda Blanco,1

Patricia Cruz-Gil,1 Jose A. Rodrıguez-Manfredi,2 David Fernandez-Remolar,3 Felipe Gomez,3

Manuel J. Gomez,1 Luis A. Rivas,1 Cecilia Demergasso,4,5 Alex Echeverrıa,4 Viviana N. Urtuvia,4

Marta Ruiz-Bermejo,1 Miriam Garcıa-Villadangos,1 Marina Postigo,1 Monica Sanchez-Roman,3

Guillermo Chong-Dıaz,5,6 and Javier Gomez-Elvira2

Abstract

The Atacama Desert has long been considered a good Mars analogue for testing instrumentation for planetaryexploration, 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, andseveral cores and powder samples from up to 5 m deep were analyzed in situ with LDChip300 (a Life DetectorChip containing 300 antibodies). Here, we show the discovery of a hypersaline subsurface microbial habitatassociated 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 than0.5 g of ground core sample. The results were supported by oligonucleotide microarray hybridization in the fieldand finally confirmed by molecular phylogenetic analysis and direct visualization of microbial cells bound tohalite crystals in the laboratory. Geochemical analyses revealed a habitat with abundant hygroscopic salts likehalite (up to 260 g kg - 1) and perchlorate (41.13 lg g - 1 maximum), which allow deliquescence events at lowrelative humidity. Thin liquid water films would permit microbes to proliferate by using detected organic acidslike acetate (19.14 lg g - 1) or formate (76.06 lg g - 1) as electron donors, and sulfate (15875 lg g - 1), nitrate(13490 lg g - 1), or perchlorate as acceptors. Our results correlate with the discovery of similar hygroscopic saltsand possible deliquescence processes on Mars, and open new search strategies for subsurface martian biota. Theperformance demonstrated by our LDChip300 validates this technology for planetary exploration, particularlyfor the search for life on Mars. Key Words: Atacama Desert—Life detection—Biosensor—Biopolymers—In situmeasurement. Astrobiology 11, 969–996.

1. Introduction

The space science community agrees on the need to ex-plore the martian subsurface for evidence of intact organic

molecules (Kminek and Bada, 2006; Shkrob et al., 2010). Infact, ESA’s ExoMars mission aims to search for life or its re-mains by analyzing samples taken from a drill hole of at least2 m in depth (http://www.esa.int/SPECIALS/ExoMars/SEM10VLPQ5F_0.html ). Dartnell et al. (2007) suggested, aftera series of simulation experiments on the effect of solar radi-ation on biological material (bacteria), that a minimum depthof 7.5 m would be needed to find viable cryopreserved cells.

Different robotic missions have shown that Mars is a saltyplanet with a volcanic basement (Murchie et al., 2009).Chlorides and bromides precipitated as cementing salts ineolian deposits (McLennan et al., 2005) or likely as sedi-mentary deposits that infilled shallow basins (Osterloo et al.,2008) due to a strong oversaturation of solutions that leachedto the Mars surface under high evaporative rates. Chloride-bearing salts are excellent matrices for the preservation ofbiological remains (Fish et al., 2002; Stan-Lotter et al., 2006),and their hygroscopic properties can produce deliquescenceevents under low relative humidity (Davila et al., 2008). Infact, the high content of perchlorate (ClO�4 ) at the Phoenix

1–3Departments of 1Molecular Evolution, 2Instrumentation, and 3Planetology and Habitability, Centro de Astrobiologıa (INTA-CSIC),Madrid, Spain.

4Centro de Biotecnologıa, 5Centro de Investigacion Cientıfica y Tecnologica para la Minerıa, and 6Department of Geological Sciences,Universidad Catolica del Norte, Antofagasta, Chile.

ASTROBIOLOGYVolume 11, Number 10, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/ast.2011.0654

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lander site on Mars (Hecht et al., 2009) might promote theformation of stable liquid saline water on present-day Mars(Zorzano et al., 2009).

The Atacama Desert is one of the most accurate terrestrialanalogues for martian environments because it combinesthe formation of two key inorganic compounds: chloridesand perchlorates. Similar to events on Mars, harsh aridconditions promoted an extreme oversaturation of theground and surface water solutions, which resulted innearly exclusive precipitation of halite, the end member inevaporation from brines, with no other mineral phase(Chong-Dıaz et al., 1999). The bounding regions of the SalarGrande (Cordillera de la Costa, region de Atacama, Chile)are characterized by saline subsoils associated with nitratedeposits that contain chlorides, sulfates, chlorates, chro-mates, iodates, and perchlorates. These salts are older anddifferent from those of the more central region of the SalarGrande (Chong-Dıaz et al., 1999).

Though the Atacama Desert has been studied for manyyears as a Mars analogue (Cameron, 1969; Cabrol et al., 2001;Glavin et al., 2004; Shafaat et al., 2005; Skelley et al., 2005),very few studies have focused on the microbiology or mo-lecular biomarker content of the subsoil. Lester et al. (2007)described the microbiology from the surface to 15 cm deep,and Bobst et al. (2001) reported a 100 m drilling in the Salarde Atacama (23�S, 68�W) only for paleoclimatic studies.Gramain et al. (2011) described the archaeal community,identified via nested PCR and cultivation, that occupied apure halite core up to 15 m deep from the Salar Grande.However, most of the microbiological and life-detectionstudies on the Atacama Desert have focused mainly on thesurface or very close to it (Navarro-Gonzalez et al., 2003;Lester et al., 2007; Weinstein et al., 2008). Cabrol et al. (2007)used a fluorescence detection system on board a rover (Zoe)to detect fluorescent signals from biological material (e.g.,chlorophyll) along different transects. Piatek et al. (2007) useda combined orbital image analysis and a field rover withinstrumentation aimed to examine the mineralogy, geomor-phology, and chlorophyll potential of field sites on the sur-face. We did not find in the literature any systematic studythat involved the search for life or traces of it or the micro-biology of the Atacama subsurface below a few centimeters.Further, there have been no studies conducted in whichtechniques based on immunological biosensors were used.

Here, we present the results of the July 2009 astrobiologicalfield campaign, AtacaMars2009, in which we tested a newin situ life-detection instrument designed for planetary ex-ploration and study of the geomicrobiology of the Atacamasubsurface at the west side of the Salar Grande. The key in-strument was Signs Of LIfe Detector V3.0 (SOLID3) and itsLDChip300 biosensor (a Life Detector Chip with 300 anti-bodies), which was designed to detect a broad range of mo-lecular biomarkers with different specificities and sensitivitiesat low parts per billion levels for proteins and peptides or 104

to 105 cells per milliliter (Parro et al., 2005, 2008a, 2011a; Rivaset al., 2008; Parro, 2010). The LDChip300 biosensor containsantibodies against universal biomolecules like DNA, nucleo-tides, amino acids, and peptidoglycan, and includes anti-bodies that target general groups of bacteria (e.g., Gram-positive cells or Bacillus spp. spores) and bacterial strains fromdifferent phylogenetic groups and different extreme environ-ments (it contains a relatively high redundancy of antibodies

against environmental extracts and bacterial strains from theacidic sulfur- and iron-rich environment of Rıo Tinto in Spain).LDChip300 also contains some antibodies that target relevantmetabolic pathways.

LDChip300 detected bacteria, archaea, and molecularbiomarkers, all of which were especially abundant around2 m deep. This suggests the presence of a microbial oasis inthe Atacama subsurface. The results obtained in the fieldwere exhaustively checked and validated in the laboratorywith conventional techniques, which confirmed the presenceof a new subsurface hypersaline environment. The mineral-ogical composition together with the extremely low liquidwater availability suggests important analogies with somemartian environments. Our LDChip may open a new era forlife-detection systems in planetary exploration.

2. Materials and Methods

2.1. Logistics, base camp, and tasks

The campaign was carried out such that it was completedby July 31, 2009, wintertime in the region. A base camp foreight researchers and two assistants was set up at about 1 kmdistance from the drilling site (GPS coordinates: 20�58¢7.30†S,70�2¢2.95†W) (Fig. 1A). The camp included a tent that wasused as a field laboratory with space for three people andsome equipment (a microcentrifuge, a water bath, a scanner,a handheld sonicator, tubes, and other materials and reac-tants). Drilling was usually done by two people, with theassistance of two more during the more complicated opera-tions (removing the cores, changing the corers, etc). Threepeople were stationed permanently in the field laboratoryprocessing and analyzing samples, one of whom performedgeophysical studies and assisted in the lab.

2.2. Drilling and sample collection

The apron at the base of a mountain of volcanic originnearby the Salar Grande was chosen as a Mars analogue forthe drilling and life-detection campaign (Fig. 1A). We mea-sured temperature extremes of - 3�C and 39�C at the drillingsite during the campaign. The drilling was done with aCARDI EN 400, a 4.08–horse power gas-powered motor, and85 mm diameter commercial off-the-shelf diamond-impreg-nated coring drill bits and rods. The drilling operations weredifficult due to the limited power of the apparatus. Tominimize cross contamination and assist drilling, com-pressed air was injected inside the corer to help transportchips up and out of the hole. No filtering devices were usedto avoid potential contamination from the compressed air. Ifany contamination were to come from the compressed air,we would have detected it nearly uniformly in all coresamples, which we did not. Rather, we obtained clearlydifferentiated patterns along the entire hole. Samples rangingfrom 500 g to 3 kg of this material were collected with avacuum sampling system (a commercial aspirator with anew and sterile bag) and were labeled and stored for lateranalysis (Table 1). Samples were collected in plastic bags orwrapped in aluminum foil, and were then stored at roomtemperature (in the shade) inside closed boxes for as long as2 h. The samples were then carried out to the field laboratoryto be analyzed or further stored until transport to the labo-ratory in the Centro de Astrobiologıa, Madrid, Spain.

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2.3. Raman spectroscopy in the field

Intact cores were immediately photographed, analyzedwith Raman spectroscopy, and then wrapped in aluminumfoil to be transported to the laboratory in Spain. Ramanspectra were taken in the field with a portable AvantesAvaSpec-2048TEC Raman spectrometer with 10 cm - 1 spec-tral resolution, a 50 mW, 532 nm diode laser, and a fiber opticprobe custom-made by InPhotonics according to these pa-rameters. The Raman excitation source, made by PD-LD, is a‘‘Volume Bragg Grating’’–stabilized module, which consistsof a stabilized laser diode and the associated electronics. This

module provides a high-stability output around 532 nm,with a typical spectral line width of 0.08 nm.

2.4. Antibody production, purification, and labeling

The antibodies used in this research (see Table 2 in Ap-pendix) were the result of several years of work aimed at thedetection of molecular biomarkers for planetary exploration.At the time the present study began we had a collection of300 antibodies, which constituted the so-called im-nunosensor LDChip300. The criteria for antibody productionand selection were explained previously by Parro et al.

FIG. 1. AtacaMars2009 drilling campaign. (A) The drilling site in one apron located at the base of a mountain to the west ofthe Salar Grande (Atacama Desert, Chile). (B) Geological profile of the core with the depth scale and a photograph of thecollected core pieces (left), showing the white halite veins. Color images available online at www.liebertonline.com/ast

LIFE-DETECTION EXPERIMENT IN ATACAMA SUBSURFACE 971

(2005), Parnell et al. (2007), Parro et al. (2008a), Rivas et al.(2008), and Parro et al. (2011a). Because we are developingan instrument for use on Mars, our choice of antibodies isguided by the types of molecular biomarkers found in or-ganisms living on Earth under similar, Mars-like conditions.Consequently, LDChip300 contains antibodies raisedagainst whole microbial cells (archaea and bacteria), extra-

cellular polymers, environmental extracts from terrestrialanalogues for Mars or Europa, proteins and peptides fromwell-conserved anaerobic metabolic pathways (nitrogenfixation, sulfate reduction, nitrate reduction, etc.), exopo-lysaccharides (EPS), and universal molecular biomarkerslike DNA, amino acids, and other biomolecules (Table 2).All the antibodies were purified by Protein A affinity pu-rification and fluorescently labeled as reported previouslyby Parro et al. (2005) and Rivas et al. (2008).

2.5. Design and construction of antibody microarrays

Antibody microarrays were constructed as described byParro et al. (2005) and Rivas et al. (2008) with some modifi-cations, as follows: (i) we used a commercial protein printingbuffer 2 · (Whatman, Schleicher & Schuell, Sandford, ME,USA) and 0.02% Tween 20 as a spotting solution; (ii)printing was done in a duplicate pattern on epoxy-activatedglass slides (Arrayit Corp., Sunnyvale, CA, USA) with aMicroGrid II TAS arrayer (Biorobotics, Genomic Solutions,UK). Two microarray designs were performed: one for amanual procedure for the systematic analysis of all thesamples with standard microscope slides (see Section 2.6.1)and another to test the automatic procedure with the SOLID3instrument (Section 2.6.2). For the manual procedure, up tonine identical micoarrays (9 LDChip300) containing a du-plicate spot pattern for the 300 antibodies were spotted on amicroscope slide so that each LDChip300 fits with one of thenine flow cells in a MultiArray Analysis Module (MAAM)device (see Section 3.1; Parro, 2010).

To test SOLID3, the microarray was also designed andconstructed with a MicroGrid II TAS arrayer (BioRobotics,Digilab, Holliston, MA, USA). Five LDChip300 replicas wereprinted in five parallel microarrays so that they fit into thefive different flow cells on the SOLID3 Sample Analysis Unit(Parro et al., 2011a). Antibodies were printed at 1 mg mL - 1 inprotein printing buffer (Whatman, Schleicher & Schuell,Sandford, ME, USA) on a specially designed glass slide (di-mensions 75 · 27 · 0.15 mm) activated with Superepoxychemistry (custom made by Arrayit Corporation, CA, USA).

In all cases, after printing, the antibody microarrays werekept at room temperature until used. We verified that prin-ted antibodies were stable in printing buffer for at least oneyear at room temperature and could be transported to thefield with no significant loss of activity (data not shown).SOLID3 was run and operated as described previously(Parro et al., 2011a).

2.6. Sandwich microarray immunoassay (SMI)procedures

To analyze solid samples by SMI, the potential molecu-lar biomarkers present in the sample have to be extractedinto a liquid solution or suspension and then incubatedin the presence of the capturing antibodies previously im-mobilized on the microarray. After a second incubationwith fluorescent tracer antibodies, the positive antigen-an-tibody reactions are read by fluorescence emission either byCCD device or a scanner. In this campaign, we analyzed thesamples by a manual procedure or automatically by usingthe SOLID3 instrument. Because SOLID3 is designed forsingle use for planetary exploration and it needs a cleanroom to load a new microarray chip, only five assays can be

Table 1. List of the Samples Collected

during the Drilling Campaign

Sample ID Depth (cm) Comments

0 0–5 Chips and powder5 5–10 Chips and powder

10 10–11 Chips and powder20 20–25 Chips and powder25 25–30 Chips and powder30 30–33 Chips and powder35 35–38 Chips and powder40 39–40 Chips and powder45 40–45 Chips and powder60 60–68 Chips and powder (hard crust)68 68–70 Stones90 90–95 Chips and powder (hard crust)

100 100–104 Chips and powder105 105–108 Chips and powder110 110–112 Chips and powder115 115–118 Chips and powder120 120–122 Chips and powder122 122–125 Small core125 125–130 Small core130 130–131 Chips and powder131 131–134 Small core135 135–138 Small core140 140–142 Chips and powder142 142–144 Stones145 145–150 Small core180 180–185 Chips and powder185 185–200 Chips and powder200 200–205 Chips and powder216 216–220 Chips and powder241 241–245 Chips and powder250 250–255 Chips and powder286 286–290 Chips and powder296 296–305 Chips and powder305 305–310 Chips and powder330 330–335 Chips and powder340 340–345 Chips and powder350 350–355 Chips and powder375 375–380 Chips and powder384 384–388 Chips and powder389 389–300 Chips and powder391 391–395 Chips and powder398 398–401 Powder (white)404 404–408 Chips and powder408 408–412 Chips and powder431 431–433 Chips and powder (gray)436 436–440 Chips and powder447 447–450 Chips and powder455 455–560 Chips and powder460 460–465 Chips and powder (hard crust)470 470–475 Chips and powder489 489–499 Chips and powder (gray-brown)507 507–508 Chips and powder (gray crust)508 508–511 Chips and powder

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done. Therefore, the samples were assayed by manualprocedure with use of the small and portable MAAM devicefor nine simultaneous analyses (see Section 3.1).

2.6.1. Manual SMI using the MAAM. Immediately beforeuse, the nine microarrays containing printed slides weretreated and washed with a BSA-containing solution to blockall free binding sites on the slides and to remove the excess ofnon-covalently-bound antibodies. For that purpose, theslides were inverted and dropped onto a 0.5 M Tris-HCl pH9, 5% (w/v) bovine serum albumin (BSA) solution for 2 minand then immersed into 0.5 M Tris-HCl pH 8, 2% BSA so-lution (TBSB, Tris buffered saline–BSA), with gentle agitationfor 1 h. After drying with a short centrifugation, slides wereready for immediate use to analyze samples by a sandwichimmunoassay. The samples to be analyzed were processedas follows: (i) up to 0.5 g of dust or ground core samples weresonicated in 1 mL of TBSTRR buffer (0.4 M Tris-HCl pH 8, 0.3M NaCl, 0.1% Tween 20) by using a manual sonicator for3 · 1 min cycles (Dr. Hielscher 50W DRH-UP50H sonicator,Hielscher Ultrasonics, Berlin, Germany); (ii) the sonicatedsamples were filtered through 20-micron nylon filters to re-move sand and coarse material. Then 50 lL of the filtrate wasinjected into one of the nine flow cells of the MAAM deviceto put it in contact with LDChip300, where it was left toincubate at ambient temperature (20–30�C) for 1 h withmixing by pipetting every 15 min. To attain more homoge-neous conditions between different microarray incubations,we avoided extreme temperatures ( - 1�C to 5�C during thenight or up to 35–39�C during the day) by incubating duringmiddle morning or late afternoon, when the ambient tem-perature in the field laboratory was in the range of 15–30�C.Then the microarrays were washed out by passing 3–5 mL ofthe same buffer through the flow cells and evacuating thelast volume by pipetting air. The positive antibody-antigenreactions were revealed by incubation with 50 lL of a cock-tail containing 300 fluorescent antibodies at a final concen-tration of approximately 100 lg mL - 1 for the whole mixtureand an average around 0.4 lg mL - 1 of each antibody. After1 h incubation at ambient temperature, the microarrays werewashed out again to remove the excess fluorescent cocktail;the slide was separated from the MAAM, rinsed by dippinginto a 50 mL falcon tube filled with distilled water, and fi-nally dried by a short centrifugation in a small and portablemicrocentrifuge designed for slides (Arrayit Corp.). Finally,the slides were analyzed in the field laboratory by illumi-nating them with 635 nm light and imaging their fluorescentemission at 650 nm with a GenePix 4100A scanner. The im-ages were quantitatively analyzed in the field by usingGenePix Pro software (Genomic Solutions) installed on alaptop computer. A blank was always run in parallel withonly buffer as an ‘‘antigenic’’ sample and then revealed withthe same fluorescent antibody or antibody cocktail as the realsamples. The final fluorescence intensity (F) for each anti-body spot was calculated with the equation F = (Fsample -Fblank - 3Favcontrol spots), where F is the fluorescent intensity at635 nm minus the local background as quantified by thesoftware (GenePix Pro) and where Fsample is the total fluo-rescence signal of the sample, Fblank the total fluorescencesignal of the blank, and Favcontrol spots the average fluorescentsignal of the control spots. Said control spots are located indifferent parts of the microarray and consist of BSA, only

buffer, and others corresponding to the pre-immune antisera(more than 50). Because, theoretically, they should not ex-hibit any fluorescent signal, we use them as a baseline forfluorescence. We always subtract 2–3 times the Favcontrol spots

as a stringency cutoff to minimize false positives. Those spotshaving obvious defects (missing or very tiny spots, an arti-fact due to a bad wash or dust in the array) or those dupli-cated spots whose standard deviation was 0.2 times higherthan the mean were not considered for quantification.

2.6.2. Automatic SMI in SOLID3. SOLID3 is the thirdprototype of the SOLID (Signs Of Life Detector) instrumentconcept devoted to the detection of molecular biomarkers inplanetary exploration (see Section 3.1). It is an analytical in-strument, based on antibody microarray technology, that candetect a broad range of molecular-sized compounds, fromthe amino acid size level to whole cells, with sensitivities at1–2 ppb (ng mL- 1) for biomolecules and 104 to 105 cells mL - 1

(Fernandez-Calvo et al., 2006; Rivas et al., 2008; Parro et al.,2011a). SOLID3 is currently in Technology Readiness Level(TRL) 5–6, although some of the internal elements are in theTRL 9 stage (Parro et al., 2011a). The instrument accepts bothsolid (dust or ground rock or ice) and liquid samples. Thelimit of sample volume ranges from 0.5–1 cm3 to 2.5 cm3

(mL) at final processing volume (including the extractionbuffer supplied by the instrument). SOLID3 is able to per-form both sandwich and competitive immunoassays andconsists of two separate functional units: a Sample Pre-paration Unit (SPU) for 10 different extractions by ultra-sonication, and a Sample Analysis Unit (SAU) for fluorescentimmunoassays. The SAU consists of five different flow cells,with an antibody microarray LDChip300 in each one. It isalso equipped with an exclusive optical package and a CCDdevice for fluorescence detection.

After loading up to 0.5 g of dust or ground core sampleinto one of the homogenizing chambers of SOLID3’s SPU,the instrument was set to run autonomously as described inParro et al. (2011a). After being started, SOLID3 adds 2 mL ofextraction-incubation buffer, TBSTRR, to the homogenizingchamber. A sonicator horn pushes a ring membrane forward,hermetically closing the chamber, and advances to the son-ication position. After four cycles of sonication, the linearactuator pushes the sample forward through a filtering sys-tem (15 lm pore size). The filtrate is injected into the SAU,floods one of the flow cells, and contacts one of theLDChip300 antibody microarrays. An internal recirculationcircuit allows the sample to be re-circulated for up to 1 h inorder to enhance the reaction kinetics between antigens andspotted antibodies. After the incubation time, the remainingsample is discarded into the waste deposit, and the micro-array cell is washed out with the incubation buffer to removethe nonbound sample. Then the buffer is injected into theauxiliary chambers that are pre-loaded with a cocktail of 300lyophilized fluorescent antibodies. The buffer dissolves theantibodies and floods the microarray chamber, where it is re-circulated for up to 1 h. An additional wash removes theexcess of fluorescent antibodies and leaves the microarrayready for fluorescent detection by excitation with a laserbeam via total internal reflection through the glass support,which acts as a waveguide. The fluorescent signal is capturedby a highly sensitive CCD detector and stored as a FlexibleImage Transport System (i.e.,*.fits) image file that can be


processed and analyzed by conventional microarray soft-ware as described in Section 2.6.1. One of the microarrays isincubated with only the buffer (blank) and revealed withfluorescent antibodies. The blank image is used as a baselineto calculate spot intensities in the tested sample, as describedin Section 2.6.1.

2.7. Environmental DNA extractionand PCR amplification

Total DNA was extracted from 2 g of sample (powder orground cores) by using the commercial DNA EZNA soilDNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) withsome modification: after cell lysis, the extract was partiallydesalinated by using Amicon 30 filters (Millipore) followingthe manufacturer’s protocol. We used the obtained DNA forsimultaneous fluorescent labeling and PCR amplification byusing the bacterial universal primers for 16S rRNA gene 16SFas forward (positions 8–27 of Escherichia coli 16S rRNA, 5¢-AGAGTTTAGTCATGGCTCA) and 16SR as reverse (posi-tions 1057–1074, 5¢-CACGAGCTGACGACAGCCG). Thereactants were previously set up in a gelified mixture (Bio-tools S.A, Madrid, Spain) containing the buffer, the nucleo-tide precursors, the fluorescent nucleotide Cy3-dUTP, theprimers, and the enzyme as follows: 10 · buffer (5.0 lL),50 mM MgCl2 (3.0 lL), 10 mM dACTPs (0.5 lL), 1 mM dTTP(6.25 lL), 2 nm/lL Cy3-dUTP (5.0 lL), forward primer10 mM 16SF (1.0 lL), reverse primer 10 mM 16SR (1.0 lL),Taq-platinum 5U/lL (Invitrogen) (1.0 lL), and distilled H20(27 lL). The gelification process was carried out by BiotoolsS.A. (Madrid, Spain). The samples prepared in this way canbe stored for long periods at ambient temperature. Ad-ditionally, we prepared PCR mixtures without fluorescentnucleotides: 10 · Buffer (5.0 lL), MgCl2 50 mM (1.5 lL),dNTPs 10 mM (1.0 lL), primer 16SF 10 mM (1.0 lL), primer16SR 10 mM (1.0 lL), Taq-platinum 5U/ul (1.0 lL), and dH20(40 lL). Gelified samples were reconstituted by adding dis-tilled nuclease-free water and the template DNA. For simul-taneous labeling and PCR amplification, the field thermocyclerwas programmed as follows: 95�C, 5 min; 10 · (95�C 20 s, 50�C30 s, 68�C 60 s); 25 · (95�C 20 s, 48�C 30 s, 68�C 60 s + 5 s percycle); 68�C 10 min; 4�C.

2.8. Oligonucleotide microarray hybridizationin the field laboratory

Oligonucleotide microarrays containing specific probes forprokaryotic microorganisms, like our previously reportedprokaryotic acidophile microarray (PAM), were printed andtreated as described by Garrido et al. (2008). This microarraywas specially developed for the detection of prokaryoticacidophiles in several field campaigns to the extremely acidicenvironment of Rıo Tinto (Huelva, Spain), but it also con-tains specific oligonucleotide probes for the 16S and 23SrRNA genes from other universal groups: Alpha-, Beta-,Gamma-, and Deltaproteobacteria, as well as Firmicutes (lowGC content), high-GC-content Gram-positive bacteria, Ar-chaea, and so on. Fluorescently labeled 16S rRNA geneamplicons were checked by agarose gel electrophoresis thenpurified with Qiagen PCR purification kit columns (Qiagen,CA, USA) and set to hybridize in HibIt hybridization buffer(Arrayit Corp.) at 50�C for 12 h in a water bath. They werethen washed and scanned for fluorescence as previously

described (Garrido et al., 2008). Because the probes for Beta-and Gammaproteobacteria correspond to sequences from the23S rRNA gene and we used only the amplicon from 16SrRNA gene for fluorescent labeling, we could not detectthese types of bacteria in hybridization with the oligonucle-otide microarray.

2.9. Determination of total protein and sugarcontent in the field laboratory

Total protein and sugar content in the samples was de-termined in the field laboratory as follows: 1 g of sample(powder) was subjected to 3 · 1 min ultrasonication cycles in2 mL of distilled water with 1–2 min stops by using ahandheld sonicator (Dr. Hielscher 50W DRH-UP50H soni-cator, Hielscher Ultrasonics, Berlin, Germany). Samples werecentrifuged at 2000g to sediment the mineral particles, andthe supernatants were directly assayed for protein concen-tration by using the bicinchoninic acid protein assay reagent(Pierce, Rockford, IL, USA) (Smith et al., 1985) and sugarcontent as described by Dubois et al. (1956), respectively. ANanoDrop (NanoDrop Int.) instrument was used for spec-trophotometric measurements.

2.10. Agarose gel electrophoresis

For DNA fractionation and visualization a pre-stainedagarose gel and a portable FlashGel System (Cambrex, EastRutherford, NJ, USA) were used and run following themanufacturer’s recommendations. A personal digital camera(Panasonic) was used to take pictures of the gel.

2.11. Phylogenetic analysis by cloning and sequencingof the bacterial 16S rRNA gene

The extracted environmental DNA was used as templatefor PCR amplification of the bacterial 16S rRNA gene withthe primer pair 16SF-16SR. The product from this reactionwas used as template for a second PCR amplification withprimers 63F (positions 43–63, 5¢CAGGCCTAACACATGCAAGTC) and 907R (positions 906–926, 5¢-CCGTCAATTCMTTTGAGTTT). The amplicon was cloned into vector pTOPO-TA (Invitrogen) and sequenced from both ends in an ABI3710 automatic sequencer (PE Biosystems, General Electrics).Sequences were assembled with the package Phred-Phrap-Consed, with use of a Perl wrapper that automaticallyclustered and processed the chromatograms correspondingto individual clones (Ewing et al., 1998; Gordon, 2004).Putative chimeras were discarded after screening the as-sembled clone sequences with Bellerophon (Huber et al.,2004). The remaining clone sequences were assigned to tax-onomical categories with use of the Ribosomal DatabaseProject (RDP) web server (Cole et al., 2009). Complementarytaxonomical information was obtained by comparing theclone sequences against the NCBI NR database with BLAST(Altschul et al., 1997). The alignments generated by RDPwere used as input for Mothur application (Schloss et al.,2009) to calculate distance matrices ignoring terminal gaps,cluster clone sequences into operative taxonomical units(OTUs), generate rarefaction curves, identify OTUs sharedacross samples, and calculate the Ace and Chao1 estimatorsfor species richness and the Simpson Index (D) for commu-nity diversity.

974 PARRO ET AL.

2.12. Phylogenetic analysis by cloning and sequencingof the archaeal 16S rRNA gene

For archaeal 16S rRNA gene amplification, two rounds ofPCR amplification were performed with the universal ar-chaeal primer pair 21F-958R (Delong, 1992) for the firstround and the specific pair for the domain ArchaeaARC344F-ARC915R (Stahl and Amann, 1991) for the secondround. The thermocycler was programmed as follows: 94�C,7 min; 30 · (94�C, 1 min; 56�C, 1 min); 72�C, 7 min; 4�C forthe first round, and 94�C, 4 min; 10 · (94�C, 1 min; 68�C - 1�Cper cycle, 1 min; 72�C, 1.5 min); 20 · (94�C, 1 min; 60�C, 1min; 72�C , 1.5 min); 72�C, 30 min; 4�C, for the second round.We used the Go-Taq Green master mix and the GoTaqpolymerase (Promega Corp., WI, USA) in a 50 lL final re-action volume.

2.13. DAPI staining

To visualize cells with optical microscopy, the microor-ganisms from the ground samples were separated from theinorganic and humic fractions with physical dispersiontechniques, such as low- and high-speed centrifugation. Two5 g aliquots of the samples analyzed by DAPI were re-suspended in 50 mL of extraction buffer (1.5 M NaCl, 0.15%Tween 20, 10 mM Tris pH8, and 100 mM EDTA), then incu-bated for 2 h at room temperature in a roller and subse-quently centrifuged at 2000g for 2 min. The pellet, whichconsisted mainly of minerals and coarse material, was dis-carded, and the supernatant was centrifuged at 10,000g for10 min. The resulting new pellet was washed in 300 lL of theextraction buffer and centrifuged at 15000g for 5 min. Thesupernatant was discarded, and a few cells were collectedwith a sterile loop-spreader from the upper section of the wetpellet, spread on a microscope slide, left to dry, and incu-bated with 5 lL of 1 lg mL - 1 DAPI-blue stain solution for1 min. The slide was rinsed with water and then with 80%ethanol to remove the non-specific-bound DAPI stains. Cellswere visualized in an Olympus Bx40 microscope at · 100augmentations. Images were captured with an OlympusDP70 camera. Additionally, small pieces of core sampleswere subjected to a quick stain with DAPI in an Eppendorftube, washed with buffer, and observed directly under themicroscope. Under these conditions, we visualized cells withflagellar movement.

2.14. Biochemical extractions

Extracts were prepared from 20 to 40 g of different sam-ples with GuHCl buffer (4 M guanidine-hydrochloride, 0.5 MEDTA, 0.5 M Tris pH 7.4) following the procedure describedby Tuross and Stathoplos (1993). The final extracts were re-suspended in sterile distilled water, dialyzed ( > 1200 Dacutoff) against water, and finally lyophilized. These extractswere used for the identification of amino acids by gaschromatography–mass spectrometry (see Section 2.15).

2.15. Determination of amino acids

For the identification of amino acids, the samples werefirst hydrolyzed with 6 M HCl at 110�C for 24 h and thenfreeze-dried to remove water, HCl, and any volatile organics.The hydrolyzed samples were analyzed, in a way similar tothat described by Ruiz-Bermejo et al. (2007), by high-pressure

liquid chromatography after derivatization with phenyl-isothiocyanate (Thermo-Fischer) with use of the followingconditions: Solvent A: 50 mM NH4OAc buffer, pH 6.5; Sol-vent B: 100 mM NH4OAc-CH3CN (50:50), pH 6.5; 0 min 0% B(100% A), 45 min 70% B (30% A), 46 min 70% B (30% A),48 min 100% B (0% A). The flow was 2 mL min - 1, the columnwas thermostated at 52�C, and the chromatogram registeredat 254 nm. High-pressure liquid chromatography analyseswere carried out on a Surveyor (ThermoFinnigan, ScientificInstruments Services, Ringoes, NJ, USA) with a photodiodearray detector with a Kromasil 100 C18 5 lm 25 · 0.46 col-umn. The identifications of the amino acids were verified bycomparing the retention times and UV absorbance spectrawith external standards, purchased from Sigma-Aldrich andFluka (Sigma-Aldrich, St. Louis, MO, USA).

2.16. Determination of total organic carbon

Up to 2 g of sample were dried by incubating at 110�C for24 h. Total organic carbon was estimated by the weight ofsample lost after a second incubation at 550�C for 3 h.

2.17. Testing the deliquescence propertiesof the drilled samples

To test the capacity of the collected samples to absorb anddeliquesce atmospheric water vapor, we proceeded as fol-lows: 2 g of samples was exhaustively dried at 110�C for 24 h(until no further weight loss was measurable); then theywere stored in identical 100 mL glass beakers covered byaluminum foil (ajar) in a cold room at 4�C and below 75%relative humidity. Over the next 20 days, the samples wereweighted daily. The water-weight gain was plotted as thepercentage of the initial dried weight. Deliquescence, definedas the process by which a substance absorbs moisture fromthe atmosphere until it dissolves in the absorbed water andforms a solution, was clearly visible to the naked eye. Someof the samples even showed liquid water accumulating at thebottom of the beaker after only 5 days.

2.18. Ion chromatography analysis

Samples (10 g) were sonicated (3 · 1 min cycles) in 20 mLof water, and the mineral particles were removed by cen-trifugation. The supernatants were collected and loaded intoa Metrohm 861 Advanced compact ion chromatographer IC(Metrohm AG, Herisau, Switzerland) undiluted or at dilu-tion values of either 50% or 20%. For all the anions, exceptperchlorate, the column Metrosep A supp 7-250 was used with3.6 mM sodium carbonate (NaCO3) as eluent. For perchloratemeasurements, we used a Metrosep A supp 4-250 column with1.9 mM Na2CO3, 1.9 mM NaHCO3, 25% (w/v) acetonitrile aseluent. The pH of the water solutions was measured withan inoLab pH meter (WTW, GmbH & Co. KG, Weilheim,Germany) after 24 h of solution stabilization.

2.19. Scanning electron microscopy (SEM)

Scanning electron micrographs were made with gold-coated pieces of core samples by using a JEOL JSM-5600 LVinstrument ( JEOL, Tokyo, Japan). To examine the micro-morphology and mineralogy, it was operated at accelerationvoltages of 10–20 kV and 2–3 kV. The phase compositionswere measured by using energy-dispersive X-ray (INCA


X-Sight, Oxford), with acceleration voltages of 10 kV forminerals and 2 kV for microorganisms.

3. Results

3.1. LDChip300 detected in situ microbial biomarkersin the Atacama subsurface

A 1-week duration drilling campaign was carried out inthe apron at the base of a mountain to the west of SalarGrande (Materials and Methods; Fig. 1). We drilled a 5.2 mdeep hole (reported here) and another one of 75 cm to theeast of Salar Grande (not shown). More than 40 samples thatcontained the chips and came up and out of the hole werecollected. Additionally, several intact core pieces from 160 to310 cm deep were also recovered (Fig. 1B). This 1.5 m of solidcore corresponded to several hard layers of solid material.The rest of the core mostly corresponded to soft or weaklycemented material, except approximately 1 m that we lost atthe bottom of the hole (from approx. 4 to 5.20 m) when wefinished the drilling operations. The loss of this core was dueto a failure in the clamping system in the corer tip. Thegeological profile of the whole core showed thin gypsumcrusts at the upper parts followed by fine-grained halite-cemented sand layers with and without pebbles (Fig. 1B).The recovered strongly compacted cores contained halite-cemented breccia and halite veins. These samples were an-alyzed with a field-portable Raman spectrometer renderingspectra with peaks identified as halite and anhydrite (CaSO4)crystals (Fig. 2).

Forty powder- and chip-containing samples recoveredfrom the core (from 0 to - 5 m) were analyzed in the field byfluorescent sandwich antibody microarray immunoassaywith LDChip300 (Figs. 3 and 4; see Materials and Methods).The fluorescent signal of each of the 300 antibodies (Fig. 3)was quantified and plotted, which generated an im-munogram from each sample (Fig. 4). Positive reactions wereidentified with several antibodies in several samples. Espe-cially significant were those taken at depths around 2 m(samples 185, 200, and 216), where positive reactions corre-

sponded to antibodies raised against environmental bio-chemical extracts, microbial extracts, and biologicalcompounds like DNA, EPS, or lipoteichoic acid polymersfrom Gram-positive bacteria, and others (Fig. 3 and Table 3).Among the microorganisms, we detected Gram-negativeAlphaproteobacteria (weak signal from an Acetobacteraceae)and Gammaproteobacteria (Acidithiobacillus, Halothiobacillus,and Pseudomonadaceae), Gram-positive Actinobacteria andFirmicutes (Bacillales), Bacteroidetes, and the archaeon Ha-lorubrum spp. The LDChip results confirm that complexpolymers (EPS and lipoteichoic acids) were present in theanalyzed samples, which, in turn, constitutes direct evidenceof viable life or the remains of recently viable life withabundances especially high at *2 m. A strong fluorescentsignal was obtained with an anti-steroid antibody in most ofthe samples below 2 m. We hypothesize that organomineralcomplexes bearing structurally similar compounds producedeither by bacteria or fungi may have been responsible forsuch a high signal. A likely candidate was ergosterol, a tri-terpene lipid that forms part of the fungal cell membranesand functions like cholesterol in animal cells (Weete et al.,2010). To verify the authenticity of the antigen-antibody re-actions, some samples were heated over a flame in sterilealuminum foil for 10 min in the field laboratory and thenanalyzed with LDChip300. Most (at least 90%) of the fluo-rescent signals disappeared from the immunograms, whichindicates that the signals detected in the biochip were indeeda consequence of a sandwich immunoassay against organic(most probably biological) complex polymers.

3.2. Biochemical and molecular analysesin the field supported the LDChip300 results

We successfully extracted DNA from two out of eightsamples assayed in the field: one from the surface (sample 0)and the other from a depth of 2 m (sample 200). The last onealso had the strongest molecular biomarker signals obtainedwith LDChip300 (Figs. 3 and 4). The failure in obtainingDNA from the other samples may have been due to a lowernumber of cells. The obtained DNA was checked

FIG. 2. Raman spectroscopy identified anhydrite and halite in the field. (A) Detailed image of one of the cores used forRaman analysis. (B) A characteristic Raman spectrum showing halite (NaCl) and the other abundant mineral, anhydrite(CaSO4). Color images available online at www.liebertonline.com/ast

976 PARRO ET AL.

FIG. 3. LDChip300 detected microbes and polymeric biological remains at 2 m deep under the Atacama surface. (A) Apicture showing the nine flow cells for nine simultaneous LDChip300 assays in the MAAM device (see Section 2.6.1). (B) Apicture of the SOLID3 instrument in the field, showing its two functional units: the Sample Preparation Unit (SPU) and theSample Analysis Unit (SAU) (See Section 2.6.2). (C) LDChip300 image obtained with sample 200 (top) indicating some of thespots with the highest fluorescent signals. The cartoon shows how capturing antibodies (Y forms) bind biological polymers(rhombi) and how they are sandwiched by fluorescent (F) tracer antibodies. The image obtained with the negative control(bottom) was used as a baseline (blank) to calculate spot intensities in the test samples. Long yellow rectangles encompass afluorescent spot gradient. (D) Immunogram showing the fluorescence intensity of the positive spots. The peak numbers,antibody name, and immunogen can be seen in Table 3 (see also Table 2). Circles and squares indicate the main antibodyclusters: (I) antibodies against whole extracts from sediments, biofilms, and water from Rıo Tinto rich in Gammaproteo-bacteria; (II) Gammaproteobacteria (Halothiobacillus spp., Acidithiobacillus spp.); (III) Desulfosporosinus, Pseudomonas,Salinibacter; (IV) different peptides and proteins; (V) cell wall polymeric components like lipoteichoic acids, lipidA, orpeptidoglycan; (VI) nitroaromatic compounds and nucleotide derivatives. Color images available online at www.liebertonline.com/ast


spectrophotometrically and by using PCR amplification ofthe 16S rRNA gene with bacterial and archaeal universalprimers (See Sections 2.11 and 2.12, Materials and Methods).Additionally, the PCR amplicon from sample 200 wasfluorescently labeled and used to hybridize to an oligonu-cleotide microarray designed for the analysis of prokaryoticdiversity (Garrido et al., 2008). The results further confirmthe presence of bacteria, mainly from the Gram-positive low-GC-content Firmicutes group, as well as from the high-GC-content Gram-positive bacteria (Fig. 5 and Table 3). Totalprotein and sugar contents were also determined in the fieldlaboratory, showing a peak in the samples around 2 m deep(Fig. 6), which is in agreement with LDChip300 and DNA

microarray analyses, although sugars still remain high to thebottom of the hole. All these data support and validate the insitu LDChip300 results and are consistent with the presenceof a subsurface microbial habitat.

3.3. Geochemical analyses revealed a hypersalinehabitat in the Atacama subsurface

The analysis of all powder- and chip-containing samplescollected during drilling by ion chromatography showed aspecific geochemical pattern at around 2 m deep (Fig. 6),which is in agreement with the results obtained in the field.A slight decrease in the pH of water solutions is coincidentwith a high increase of salt content (NaCl) and conductivity,both decreasing just when nitrates, perchlorates, and formatecontents increase and remain steady down to the bottom ofthe borehole (5.2 m). In contrast, sulfate content is slightlyhigher in the first 2–3 m. A peak of total protein and sugarprofiles is consistent with the salinity (halite), conductivity,the maximal deliquescence capacity, and LDChip300 results,while the pH of the water solution used for ion chromatog-raphy analysis (Section 2.18) showed a small acidification.This subsurface habitat is characterized by the presence ofhigh amounts of nitrates, sulfates, and perchlorates, all ofwhich are capable of working as electron acceptors for bac-terial respiration. The presence of small organic acids is alsohighly remarkable, with propionate and acetate dominatingin the upper part, while formate increases from 2 m deep(Fig. 6). All of them can be used as electron donors to supportbacterial nitrate, sulfate, and perchlorate reduction. Ad-ditionally, amino acids were also detected by gas chroma-tography–mass spectrometry in polymeric material extractedwith guanidine-hydrochloride buffer (see Section 2.14, Ma-terials and Methods) from different depths (Table 4). Theseamino acids may come either from extracellular or intracel-lular fractions because we cannot rule out the cell lysis withthe extraction method used.

3.4. Microscopy and molecular phylogeny confirmed thepresence of bacteria and archaea in the Atacamasubsurface

Further analyses in the laboratory were performed to ob-tain new information and to validate the results obtainedin the field. Core samples were assayed for microscopicevidence of cells either by SEM or optical microscopy.The scanning electron microscope images (Fig. 7) show thepresence of cellular and biofilm morphologies together withmineralized cellular moulds excavated in the halite, whichhas a high signal for organic carbon. Fluorescent DNA-specific staining (DAPI) and microscopy analysis revealedthe presence of cells with different morphologies: coccoid-,rod-, and comma-shaped, and vibrioid (Fig. 8A). After ad-dition of water to the sample, some of these cells showedflagellar movement under the microscope, with and withouta quick DAPI staining (see Section 2.13). These cells weremetabolically active probably due to the water captured bythe core during transport and storage in a cold room.

Phylogenetic analyses of the bacterial 16S rRNA gene (Fig.8B) indicated that the most abundant clones found at 2 mdeep were closely related to the Gammaproteobacteria class(most of them Vibrio spp. and some Pseudomonas spp.). Ad-ditionally, many clones corresponded to the phylum

FIG. 4. LDChip300 immunoprofiling identified a newhabitat in the Atacama subsurface. Ordered immunogramsobtained after analyzing samples from different depths withLDChip300. The arrow indicates sample 200, collected at 2 mdeep. Note that the most abundant and intense fluorescentsignals are in and around this sample (see Fig. 3 and Table 3for details). See also text for explanation.

978 PARRO ET AL.

Table 3. LDChip300-Detected Microbial Compounds at 2 m Deep

LDChip300

Phylum/group No. AntibodyImmunogen

(See also Table 2) PAM (16 S) 16S rRNA seq.

Gamma-proteobacteria

1 IA1C1 3.2 damp water cellular fraction(Gamma, Nitrospira)

Gammaproteobacteria

2 IA1S1 3.2 damp water supernatant Vibrionaceae3 IA2S1 Green filaments EPS (Eukaryotes,

Gamma .)Xanthomonadaceae

4 IC1C1 3.2 beach cells (Nitrospira, Gamma)5 IC6C1 3.1 beach under-crusts cells6 ID1C1 Jarosite Nac. Cells (Gamma, Nitrospira,

others)7 ID1C3 Jarosite Nac. Sonicated Cells after

EDTA8 IVE3C1 Acidithiobacillus ferrooxidans intact cells9 IVE6C1 A. caldus

10 IVE7C1 Halothiobacillus neapolitanus12 IVF2C1 Shewanella gelidimarina (marine broth

15�C)13 IVF2S2 S. gelidimarina (Alteromonadaceae)21 IVI1C1 Pseudomonas putida

(Pseudomonadaceae)Pseudomonadaceae

23 IVI6C3 Azotobacter vinelandii(Pseudomonadaceae)

Alphaproteobacteria 14 IVG1C1 Acidocella aminolytica(Acetobacteraceae)

15 IVG2C1 Acidiphilium spp.

Actinobacteria 11 IVE8C1 Acidimicrobium ferrooxidans HGC_G + Actinobacteria

Firmicutes 20 IVI19C1 Desulfosporosinus meridiei (Clostridiales.G + SRB)

LGCa_G + Firmicutes

28 Lmo Listeria monocitogenes (Bacillales,Listeriaceae)

LGCb_G + Bacillaceae

26 G + Bact Gram + LTA (crossreacts Bacillales) LGCc_G + Paenibacillaceae19 IVH1C1 Bacillus subtilis spores (Bacillaceae) Lactobacillaceae43 LTA LTA, L. monocitogenes (Bacillales,

Listeriaceae)Leuconostaceae

Acidobacteria 16 IVG3C1 Acidobacterium capsulatum(Acidobacteria)

Deinococcus 17 IVG4C1 Thermus scotoductus (Deinococci-Thermales)

18 IVG4C2 T. scotoductus

Bacteroidetes 22 IVI21S1 Salinibacter ruber PR1 (Bacteroidetes)Euryarchaeota 24 IVJ8C1 Halorubrum spp. (Halobacteriaceae) Halorubrum spp.

Epsilonproteobac. 27 Hpyl Helicobacter pylori (Gram-negativeepsilon div.)

Nucleic acidsand derivatives

29 dsDNA(hu) dsDNA (Human plasma)

46 cGMP(bio) cGMP-2¢-succi-BSA49 cGMP(ups) cGMP-BSA50 GMP_N GMP-BSA51 cAMP_sh cAMP-BSA52 Thiamine Thiamine-BSA

Cell wall 41 EPS_SP EPS Santa Pola (Alicante, Spain)42 Lectin Lectin potato specific for NAG-NAM

oligomers44 LipidA LipidA (monoclonal Ab, clon 43)25 Cortisol Cortisol-17-BSA47 Digoxin Digoxin (monoclonal Ab, mouse)

Xenobiotics 45 Atrazine Atrazine-BSA48 DNP Dinitrophenol-BSA

(continued)


Firmicutes (most of them Bacillus spp.), and others from thephylum Actinobacteria (Microbacterium spp.). These resultsare again consistent with those obtained with LDChip300(Table 3), where the antibodies show positive reactions thatcan be grouped into Gammaproteobacteria (Shewanella,Pseudomonas, strains or natural extracts enriched in thesetypes of microorganisms like Acidithiobacillus, Halothioba-cillus, and environmental extracts from Rıo Tinto), the Gram-positive Firmicutes, and Actinobacteria. Additionally, weobtained several sequences closely related to the archaealgenus Halorubrum. Again, this result is in agreement withLDChip and corroborated the first report about archaea inthe Atacama subsoil.

4. Discussion

4.1. LDChip300 detected microbes and molecularbiomarkers in the Atacama subsurface

We previously described the performance of former ver-sions of LDChip (Parro et al., 2008b; 2011a, 2011b; Rivas et al.,2008). Herein, we demonstrate the unprecedented capacity ofan immunosensor chip for the detection of microbes andmolecular biomarkers in the subsurface of one of the bestMars analogues on Earth: the Atacama Desert (Fig. 3).Samples were immediately analyzed in the field after theircollection from the core. Thirty samples can be assayed si-multaneously with LDChip300 and the results obtained after3 h, which allowed us to perform nearly real-time monitor-ing. It was highly relevant that several positive reactionsobtained around 2 m deep corresponded to antibodiesagainst Gram-positive bacterial antigens from the groupFirmicutes (Table 3). This is the case for IVI19C1, raisedagainst Desulfosporosinus meridiei (Clostridiales, Gram-

positive sulfur-reducing bacteria); Lmo, for Listeria mono-citogenes (Bacillales, Listeriaceae); G + Bact, Gram-positivelipoteichoic acids (cross reacts with Bacillales); IVH1C1, forBacillus subtilis spores (Bacillaceae); or LTA, for LTA from theGram-positive bacterium Listeria monocitogenes (Bacillales,Listeriaceae). The presence of bacteria from the Firmicutesgroup was demonstrated in the field by DNA extractionfrom the same sample, PCR amplification and fluorescentlabeling of the bacterial 16S rRNA gene, and hybridizationwith a phylogenetic oligonucleotide microarray (Fig. 5).

Some of the antibodies raised against the Rıo Tinto acidicenvironment or acidophilic strains showed significant fluo-rescent signals, which indicates the presence of similar mo-lecular biomarkers or microorganisms, or both. This is thecase for peaks 1–7 in Fig. 3 (Table 3), which correspond toextracellular polymeric material or cellular fractions obtainedfrom water, biofilms (filaments), sulfur-rich samples (drycrusts, sediments, jarosite and iron-sulfate precipitates), andpeaks 8 and 9 from iron-sulfur bacteria of Acidithiobacillusgenus. The presence of molecular biomarkers and bacteriacharacteristic of acidic environments might be related to thefact that Atacama soils may contain strong acids in accu-mulated dust particles, as reported by Quinn et al. (2005).They concluded that extremely low pH resulting from acidaccumulation combined with limited water availability andhigh oxidation potential may result in acid-mediated reac-tions at the soil surface during low-moisture transient wet-ting events (i.e., thin films of water). These micro-nichesmight support the growth of sulfur-oxidizing bacteria likeAcidithiobacillus spp. and Hallothiobacillus spp., iron-reducingbacteria like Acidocella spp. and Acidiphilium spp., which maytolerate up to pH 5 (Lu et al., 2010), or iron-oxidizing bacterialike Acidimicrobium spp.

Table 3. (Continued)

LDChip300

Phylum/group No. AntibodyImmunogen

(See also Table 2) PAM (16 S) 16S rRNA seq.

Peptides 30 ApsA Adenylylsulfate reductase peptide(Desulfovibrio desulfuricans)

31 ASB_prot Bacterial ATPsynthase F1 subunitAlpha (prot)

32 Beta-Glu Beta-glucanase (E. coli)33 CcdA CcdA peptide (Cytochrome

biogenesys. Geobacter)34 DhnA DhnA (Dehydrin) peptide (Nostoc)35 Hsp70 Chaperon Hsp70 (M. tuberculosis)36 ModA Molibdate transport peptide

(Leptospirillum ferrooxidans)37 NifH2 NifH nitrogenase peptide

(L. ferrooxidans)38 NorB NorB protein (Nitrobacter hamburgensis)39 Pfu_fer Ferritin (Pyrococcus furiosus) protein40 Stv Streptavidin

List of the antibodies showing positive reaction in LDChip300 with sample 200 and comparison to the results obtained witholigonucleotide microarray (PAM) in the field, and by cloning and sequencing of the 16S rRNA gene in the laboratory.

Column No. indicates the number of each of the peaks of the immunogram in Fig. 3. The column PAM (16S) indicates the group of bacteriadetected after fluorescent hybridization of bacterial 16S rRNA gene in the field: HGC_G + , high-GC-content Gram-positive bacteria;LGCa,b,c_G + , low-GC-content groups a, b, and c of Gram-positive Firmicutes (see Fig. 5). Alpha, Beta, Gamma: division of proteobacteria.LTA, lipoteichoic acids. EPS, Exopolysaccharides. dsDNA, double-stranded DNA. cGMP and cAMP, cyclic guanine and adeninemonophosphate nucleotides, respectively.

980 PARRO ET AL.

The relatively high fluorescent signal obtained with anti-bodies IVI21S1, IVJ8C1, EPS_SP raised against Salinibacterruber, Halorubrum spp., and exopolymeric substances fromthe Santa Pola solar saltern (Alicante, Spain), respectively,indicates the presence of archaea at 2 m deep. This fact wasconfirmed by molecular phylogenetic studies in the labora-tory by amplification and sequencing the archaeal 16S rRNAgene. Archaea have been reported in superficial samplesfrom the Atacama (Lizama et al., 2001; Demergasso et al.,2004), but until very recently they were not detected in thesubsurface (Gramain et al., 2011). Our work further confirmsand expands the presence of these prokaryotes in the Ata-cama soil subsurface.

Sugars and proteins followed a parallel pattern up to near3 m, where proteins drop dramatically but sugars continuewith relatively high levels. The peak in protein content couldbe associated with a more active microbial population, as aconsequence of higher water availability due, in turn, to the

presence of materials with higher deliquescence capacity.The microbial activity would produce acids that might ex-plain the small drop in the pH of water solutions in thesesamples. By contrast, the permanent sugar level below 3 mmight be due to the accumulation of extracellular polysac-charides (EPS) produced as a response to lower water ac-tivity, as it has been reported in some bacteria (e.g., Robersonand Firestone, 1992) or in soil environments (Foster, 1981).The strong correlation between protein content, halite, smallacidification, and maximal deliquescence capacity (Fig. 6)with LDChip results, together with microscopic and molec-ular phylogenetic analyses, indicates the presence of an ac-tual or recently colonized habitat, a true microbial oasis.

4.2. A hygroscopic mineral-driven subsurfacehypersaline habitat: implications for Mars

We described a new hypersaline subsurface habitat wherethe presence of hygroscopic minerals (halite, perchlorate, andanhydrite) may be responsible for water retention. Samplescontained 10–41 ppm (mg Kg - 1) of perchlorate and a peakof NaCl of 260 g Kg - 1 at 2 m depth, high enough to promotedeliquescence. In fact, these samples showed the higherdeliquescence capacity (Figs. 6 and 9) by accumulatingnearly 60% of the initial weight of liquid water in 20 days(e.g., sample 200). After 5 days, no significant differenceswere observed; but later, a clear increase in the capacity ofwater absorption and deliquescence was recorded for sample200 and those in close proximity to it, coincident with thehighest halite content. Moreover, deliquescence increaseswhen organic matter (ca. 1.9% in sample 200) is removed(Fig. 10). These results are consistent with those of Davilaet al. (2008), which demonstrated that water vapor condensesand deliquesces on the halite surface and pores at low rela-tive humidity (75%). The ability of halite and perchloratesalts to absorb water and form a liquid solution (deliquesce)is highly relevant for the search for life on Mars. Zorzanoet al. (2009) demonstrated that small amounts of sodiumperchlorate spontaneously absorb moisture and melt into aliquid solution under martian atmospheric conditions. Inaddition, Davila et al. (2010) reported the deliquescence ca-pacity of hygroscopic salts and their hypothetical capacity tosupport microbial growth or metabolic activity under sim-ulated martian conditions.

Our AtacaMars field campaign demonstrates that sub-surface hygroscopic salt environments may be true oasis formicrobial life and further supports the possibility of a sub-surface martian habitat based on the deliquescence capacityof similar hygroscopic salts. We hypothesize an ecosystemfueled by reduced compounds from hydrothermal activity(H2, SH2, acetate, formate, etc.). Potential martian microor-ganisms could use perchlorates, nitrates, or sulfates as elec-tron acceptors as shown in the Atacama subsurface (Fig. 11).Strong gradients of salts can be formed, and microorganismswould use flagella to move in search of nutrients. Whenwater is scarce, microorganisms produce abundant exopo-lymeric material that helps them to avoid desiccation andattaches them to mineral surfaces, where they form smallbiofilms or cell clusters. After the death of the microorgan-isms, the polymeric remains (EPS, proteins, nucleic acids,etc.) or other metabolites are retained by electrostatic chargesof mineral and salt crystals. These interactions with minerals,

FIG. 5. Detection of bacteria by DNA hybridization insample 200. Microarray image showed positive fluorescentspots (top figure) corresponding to universal eubacterial probes(UNI1392, EU338, Y2R), high-GC-content Gram-positivebacteria (Actinomycetes group, probe HGC236), the low-GC-content Gram-positives Firmicutes including the Lactobacillussubgroup (LGC354a), the Bacillus subgroup (LGC354b), andthe Streptococcus subgroup (LGC354c); Deltaproteobacteria(DELTA495a); and Archaea (1Af). This last probe correspondsto a highly degenerated oligonucleotide considered specific forArchaea. Because the fluorescent probe was amplified withprimers for Eubacteria, we did not expect positive results with1Af unless it came from similar sequences from unknownbacteria. (Bottom figure) Plot showing the quantification offluorescent signals from nonsaturated images. Color imagesavailable online at www.liebertonline.com/ast


in addition to high salt concentrations, hinder enzymaticdegradation. Similar niches could be present in the martiansubsurface, where small organic acids can be supplied fromthe partial photodestruction of complex meteoritic organicmatter. Halite and perchlorate salts have two main advan-tages to sustain a hypothetical subsurface martian ecosys-tem: they trap and form liquid water and simultaneouslylower its freezing point. Additionally, high salt concentra-tions inhibit enzymes like DNAses, which allows betterpreservation of biological polymers.

4.3. LDChip for life detection on Mars

Our LDChip is suited to the search for microbial life inplanetary exploration, particularly for Mars. We hypothesizethat microorganisms living in similar habitats under similarphysicochemical parameters share similar molecular mech-anisms to deal with such conditions. Those mechanisms willproduce similar biomolecules that constitute a good set ofmolecular biomarkers. We suggest two strategies for targetselection: (i) a direct approach, in which well-known bio-molecules or their diagenetic products from those listed byParnell et al. (2007) and Parro et al. (2008a) are used as

FIG. 6. Biogeochemical analyses of the samples collected along the whole core. Sample names and depths, the assayed com-pound or parameter, the units, and the scale are indicated at the top of the figure. Arrows point to sample 200. The horizontal linesdelimit the samples with the higher halite and protein concentration, the highest deliquescence capacity, and a small decrease inpH, defining a geological unit for a subsurface habitat. (Deliq) Deliquescence capacity of the samples after 20 days at 4�C and 75%relative humidity, expressed as the percentage (% dw) of adsorbed water with respect to the initial dry weight (2 g per sample),showing a maximum of 58% at sample 200 (See Figs. 9 and 10).

Table 4. Amino Acids Detected in Polymeric

Material ( > 1200 Da) Extracted by Guanidine

Chloride Buffer

Sample Amino acids

0 Gly, Ala, + 3 unidentified amines5 Glu, Ser, Gly, Ala, + 3 unidentified amines

60 Asp (traces), Glu (traces), Ser, Gly, Ala,unidentified amines

140 Asp (traces), Glu (traces), Ser, Gly, Ala,5 unidentified amines

200 Asp, Glu, Ser, Gly, Ala, unidentified amines286 Asp (traces), Glu (traces), Ser, Ala, Pro, Val,

2 unidentified amines305 Asp (traces), Glu (traces), Ser, Gly, Ala, 2

unidentified amines398 Asp, Glu, Ser, Gly, Ala, 2 unidentified amines431 Asp (traces), Glu (traces), Ser, Gly, Thr, Ala460 ND507 Asp, Glu, Ser, Gly, Ala, 3 unidentified amines

ND, Not detected.

982 PARRO ET AL.

antigens to produce antibodies; and (ii) a shotgun strategywhere whole or fractionated biochemical extracts from ter-restrial Mars analogues are used as targets for antibodyproduction (Parro et al., 2008a, 2011a; Rivas et al., 2008; Parroand Munoz-Caro 2010). We have produced polyclonal anti-bodies against biochemical extracts from environments thatare acidic and iron-sulfur-rich, hydrothermal, permanentlyfrozen, hypersaline, and so on (Table 2). Samples were takenfrom water, sediments, mineral deposits (sulfate precipitates,jarosite, hematite, etc.), rocks, and subsurface cores duringdrilling campaigns. Simultaneously, we can produce anti-bodies against different kinds of universal macromoleculeslike EPS, anionic polymers, cell wall components, nucleicacids, nucleotides and derivatives, phylogenetically con-served proteins, and so on. There are commercial antibodiesagainst single- or double-stranded DNA, all recognizingstructures and features of terrestrial DNA. In the case of an

eventual martian DNA made up of slightly different nucle-otides, it probably would not be recognized with antibodiesagainst terrestrial counterparts, except in those cases wherethe antibodies were recognizing common structures. It ispossible to make antibodies against modified nucleotides,but it is unknown which modifications to target. Alternativenucleobases (e.g., xantine, aminated, or methylated deriva-tives of the four known nucleotides) can be used as targets.However, considering that some nucleobases (e.g., adenine)can be obtained abiotically (Yuasa et al., 1984), the detectionof these types of compounds by itself is not a biomarker,unless they were forming part of a polymer like DNA or asecondary structure like a double or triple helix. Universalmodified nucleotides that are widely used for life as signal-transducers, for example, cyclic adenosine monophosphate(cAMP) and cyclic guanosine monophosphate (cGMP) (Go-melsky, 2011), are good targets for antibody production.

FIG. 7. Microbial forms under the Atacama surface bound to halite crystals. Scanning electron microscope images andenergy dispersive X-ray (EDX) spectra showed halite crystals with incipiently mineralized microbial cells. (A) Halite crystalwith elongated morphology and rough surface showing nanoparticles and mineralized microbial cell-like structures (arrows)on its surface. EDX spectrum of the crystal (inset) indicates that it is composed of Na and Cl (halite). (B) Close-up of twomicrobial cells from (A) attached to the halite crystal surface (arrows). (C) Halite crystal showing empty voids whichcorrespond to mineralized moulds of degraded microbial cells, most probably bacteria (arrows), some of them with clearvibriod form. Note that EDX spectrum displays Na, Cl, C, O, and N. The four latter elements come, most probably, fromorganic compounds of microbial cells. The pie chart shows the percentage of the main elements. (D) Close-up of twomicrobial cells attached to the halite crystal surface (arrow) and the EDX spectrum of one of them. The EDX spectracorrespond to the green squares. Color images available online at www.liebertonline.com/ast


There are commercial antibodies against these compounds(Table 2), and we have also produced our own stock. In fact,LDChip300 showed strong signals for antibodies againstthese compounds (see peaks 46, 49, and 50, all against cGMP,in Fig. 3 and Table 3), which indicates that some microor-ganisms might be producing and even secreting cGMP or itsderivatives. In fact, it has been recently reported that theAlphaproteobacterium Rhodospirillum centenum secretes rel-

atively high amounts of cGMP to trigger the formation ofmetabolically dormant cells, called cysts, which are highlyresistant to desiccation (Berleman and Bauer, 2004; Mardenet al., 2011). High salt concentration inhibits degrading en-zymes, among them the nucleases, which may explain why,in this hypersaline environment, DNA and nucleotidederivatives are well-preserved. Additionally, the binding tomineral surfaces may be an additional factor of stabilization

FIG. 8. Microbial cells and phylogenetic analysis of bacterial DNA from 2 m deep. (A) Sample 200 was stained with DNA-specific stain (DAPI). Rod- and comma-shaped or vibrioid forms (arrows) can be detected. Bar, 5 lm. (B) Phylogenetic treeobtained with the 16S rRNA gene sequences from sample 200 (see text for explanations). Sequences obtained from sample 200were named as 4E1..Color images available online at www.liebertonline.com/ast

984 PARRO ET AL.

and aggregation (Romanowski et al., 1993). Whether thepositive signals obtained with anti-cGMP antibodies are in-deed due to the presence of cGMP is yet to be determined.Positive signals from a SMI indicate that we are detecting atleast a dimer, an oligomeric or polymeric compound, orother types of aggregate, because at least two antigen-bind-ing sites are necessary; one to bind to the capturing antibodyand another to bind to the fluorescent tracer.

LDChip300 contains antibodies against other universalcompounds like protoporphyrin IX, lipidA, peptidoglycan,amino acids, vitamin K12, some antibiotics, and antibodiesagainst proteins and peptides from relevant microbial metab-olisms: nitrogen fixation; sulfate, nitrate, and perchlorate re-duction; iron oxi-reduction; and so on. (Table 2). Theoretically,it is possible to produce antibodies against any type of terres-trial biomolecule or even molecules that could be consideredan extraterrestrial substitute. But, given that we know onlyterrestrial life, we must focus on making antibodies for that.

5. Conclusion

We have discovered a new hypersaline subsurface habitatin the Atacama. The presence of highly hygroscopic saltdeposits associated with the microorganisms indicates thatthis habitat constitutes a true microbial oasis. Hygroscopicsalts promote the deliquescence of water on the mineralsurfaces and form thin liquid films that can maintain mi-crobial growth. Our LDChip300 detected microbes andmolecular biomarkers in this hypersaline subsurface envi-ronment. The relatively high number of antibodies against arational and structured panel of target molecular biomarkersmakes LDChip a suitable tool for the search for life forplanetary exploration. Halite- and perchlorate-rich subsur-face environments on Mars should be considered targets forfuture life-detection missions. The known destructive effectson organic matter by thermal oxidation promoted by per-chlorates (Navarro-Gonzalez et al., 2010) are not a constraintfor our LDChip-SOLID system (Parro et al., 2011a). The useof a SOLID-like Life Detector Chip and instrumentation aspart of the payload of future missions could be an importantcontribution to the search for life on Mars.

FIG. 9. Deliquescence capacity of the samples at different depths. (Top) Photographs showing the aspect of the samples after20 days of deliquescence assay (see Section 2.17). The accumulation of liquid water is clearly visible. (Middle) The water retainedby each of the samples was estimated as the percentage of the initial dry weight along the time course: 1, 5, 7, 9, 18, and 20 days.(Bottom) Liquid water recovered from some of the samples. Color images available online at www.liebertonline.com/ast

FIG. 10. The presence of ca. 2% organic matter in thesamples diminishes the deliquescence capacity to about 50%.Two aliquots of 2 g of sample 200 were dried, their organicmatter kept ( + OM) or baked at 550�C to destroy it (-OM),and finally subjected to the deliquescence capacity assay for20 days (see Materials and Methods). The adsorbed waterwas measured as the percentage of the initial dry weight(gray) and as the total liquid water recovered (black). Totalorganic carbon in sample 200 was 1.98 – 0.15%.


Acknowledgments

This work has been funded by the Spanish Ministerio deCiencia e Innovacion (MICINN) grant No. AYA2008_04013. Wethank Patricio Raul Arias for his assistance in the field work,Companıa Minera Cordillera for their help during the fieldcampaign, and Drs. Josefa Anton and Fernando Santos (Uni-versidad de Alicante, Spain) for providing halophilic strains.

Abbreviations

BSA, bovine serum albumin; cAMP, cyclic adenosinemonophosphate; EPS, exopolysaccharides; cGMP, cyclic gua-nosine monophosphate; LDChip, Life Detector Chip; MAAM,MultiArray Analysis Module; OTUs, operative taxonomicunits; PAM, prokaryotic acidophile microarray; RDP, the Ri-bosomal Database Project; SAU, Sample Analysis Unit; SEM,scanning electron microscopy; SMI, sandwich microarrayimmunoassay; SPU, Sample Preparation Unit; SOLID, SignsOf LIfe Detector.

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Address correspondence to:Vıctor Parro

Department of Molecular EvolutionCentro de Astrobiologıa (INTA-CSIC)

Carretera de Ajalvir km 4Torrejon de Ardoz, 28850

MadridSpain

E-mail: [email protected]

Submitted 23 March 2011Accepted 1 September 2011

(Table 2 in Appendix follows/)

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nS

amp

le/C

ult

ure

con

dit

ion

sIm

mu

nog

en/F

ract

ion

Hos

tS

up

p.

Ref

eren

ces

1IA

1C1

Rıo

Tin

to(3

.2w

ater

dam

)W

ater

Cel

lula

rfr

acti

on

Rb

Riv

aset

al.,

2008

2IA

1S1

Rıo

Tin

to(3

.2w

ater

dam

)W

ater

Su

per

nat

ant

Rb

Riv

aset

al.,

2008

3IA

1S2

Rıo

Tin

to(3

.2w

ater

dam

)W

ater

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

4IA

1C2

Rıo

Tin

to(3

.2w

ater

dam

)W

ater

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

085

IA1S

100

Rıo

Tin

to(3

.2w

ater

dam

)W

ater

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

6IA

1C3

Rıo

Tin

to(3

.2w

ater

dam

)W

ater

ED

TA

was

hed

cell

s(s

on

icat

ed)

Rb

Riv

aset

al.,

2008

7IA

2C1

Rıo

Tin

to(3

.1st

ream

)G

reen

fila

men

tsC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

088

IA2S

1R

ıoT

into

(3.1

stre

am)

Gre

enfi

lam

ents

Su

per

nat

ant

Rb

Riv

aset

al.,

2008

9IA

3C1

Rıo

Tin

to(3

.1st

ream

)B

lack

fila

men

tsC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0810

IA3S

1R

ıoT

into

(3.1

stre

am)

Bla

ckfi

lam

ents

Su

per

nat

ant

Rb

Riv

aset

al.,

2008

11IC

1C1

Rıo

Tin

to(P

lay

a3.

2)S

edim

ents

Cel

lula

rfr

acti

on

Rb

Riv

aset

al.,

2008

12IC

1S1

Rıo

Tin

to(P

lay

a3.

2)S

edim

ents

Su

per

nat

ant

Rb

Riv

aset

al.,

2008

13IC

1S2

Rıo

Tin

to(P

lay

a3.

2)S

edim

ents

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

14IC

2C1

Rıo

Tin

to(3

.1w

ater

dam

)D

ark

red

sed

imen

tsC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0815

IC2S

2R

ıoT

into

(3.1

wat

erd

am)

Dar

kre

dse

dim

ents

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

16IC

2C3

Rıo

Tin

to(3

.1w

ater

dam

)D

ark

red

sed

imen

tsE

DT

Aw

ash

edce

lls

(so

nic

ated

)R

bR

ivas

etal

.,20

0817

IC3C

1R

ıoT

into

(Mai

nsp

rin

g)

Bro

wn

fila

men

tsC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0818

IC3S

2R

ıoT

into

(Mai

nsp

rin

g)

Bro

wn

fila

men

tsS

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0819

IC3C

3R

ıoT

into

(Mai

nsp

rin

g)

Bro

wn

fila

men

tsE

DT

Aw

ash

edce

lls

(so

nic

ated

)R

bR

ivas

etal

.,20

0820

IC4C

1R

ı oT

into

(3.2

wat

erd

am)

Bro

wn

fila

men

tsC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0821

IC4S

2R

ıoT

into

(3.2

wat

erd

am)

Bro

wn

fila

men

tsS

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0822

IC5C

1R

ıoT

into

(Pla

ya

3.1)

Red

cru

sts

Cel

lula

rfr

acti

on

Rb

Riv

aset

al.,

2008

23IC

5S1

Rıo

Tin

to(P

lay

a3.

1)R

edcr

ust

sS

up

ern

atan

tR

bR

ivas

etal

.,20

0824

IC6C

1R

ıoT

into

(Pla

ya

3.1)

Red

sed

imen

t1–

2cm

un

der

cru

stC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0825

IC6S

1R

ıoT

into

(Pla

ya

3.1)

Red

sed

imen

t1–

2cm

un

der

cru

stS

up

ern

atan

tR

bR

ivas

etal

.,20

0826

IC7C

1R

ıoT

into

(3.2

wat

erd

am)

Dri

edw

all

sed

imen

tsC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0827

IC8C

1R

ıoT

into

(3.1

stre

am’s

ban

ks)

Gre

en-o

ran

ge

sed

imen

tsC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0828

IC8S

1R

ıoT

into

(3.1

stre

am’s

ban

ks)

Gre

en-o

ran

ge

sed

imen

tsS

up

ern

atan

tR

bR

ivas

etal

.,20

0829

IC9C

1R

ıoT

into

(3.1

min

e’s

ruin

s)R

ed-g

rey

sulf

ate-

rich

pre

cip

itat

esC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0830

A13

8R

ıoT

into

(‘‘N

acim

ien

to’’)

Yel

low

mat

s(c

entr

alst

ream

)W

ho

leG

u/

HC

lex

trac

tio

nR

bR

ivas

etal

.,20

0831

A14

0R

ıoT

into

(3.0

stre

am)

Pin

ksu

per

fici

alla

yer

Cel

lula

rfr

acti

on

Rb

Riv

aset

al.,

2008

32A

141

Rıo

Tin

to(3

.0st

ream

)P

ink

sup

erfi

cial

lay

erS

up

ern

atan

tR

bR

ivas

etal

.,20

0833

A14

3R

ıoT

into

(3.2

wat

erd

am)

Wal

lse

dim

ents

.L

ith

ified

ov

erg

row

thW

ho

leG

u/

HC

lex

trac

tio

nR

bR

ivas

etal

.,20

0834

A15

2R

ıoT

into

(3.0

stre

am)

Pin

ksu

per

fici

alla

yer

Wh

ole

Gu

/H

Cl

extr

acti

on

Rb

Riv

aset

al.,

2008

35ID

1C1

Rıo

Tin

to(M

ain

spri

ng

)Ir

on

sulf

ate–

rich

pre

cip

itat

esC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0836

ID1S

1R

ıoT

into

(Mai

nsp

rin

g)

Iro

nsu

lfat

e–ri

chp

reci

pit

ates

Su

per

nat

ant

Rb

Riv

aset

al.,

2008

37ID

1S2

Rıo

Tin

to(M

ain

spri

ng

)Ir

on

sulf

ate–

rich

pre

cip

itat

esS

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0838

ID1C

3R

ıoT

into

(Mai

nsp

rin

g)

Iro

nsu

lfat

e–ri

chp

reci

pit

ates

ED

TA

was

hed

cell

s(s

on

icat

ed)

Rb

Riv

aset

al.,

2008

39ID

2S2

Pen

ad

eH

ierr

o(1

48m

dee

p)

4-59

csa

mp

le(M

AR

TE

pro

ject

)S

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0840

ID3S

2P

ena

de

Hie

rro

(96

md

eep

)4-

39c

sam

ple

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

41ID

4S2

Pen

ad

eH

ierr

o(1

54m

dee

p)

4-61

asa

mp

le(M

AR

TE

pro

ject

)S

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0842

ID5S

2P

ena

de

Hie

rro

(141

md

eep

)4-

56c

sam

ple

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

(con

tin

ued

)

989

Ta

bl

e2.

(Co

nt

in

ue

d)

Ab

No

Ab

nam

eS

ourc

e/S

trai

nS

amp

le/C

ult

ure

con

dit

ion

sIm

mu

nog

en/F

ract

ion

Hos

tS

up

p.

Ref

eren

ces

43ID

7S2

Pen

ad

eH

ierr

o(8

4–97

md

eep

)8-

42b

+43c

+45a

bc+

46b

c(M

AR

TE

pro

ject

)S

up

ern

atan

tfr

om

ED

TA

/g

uan

idin

ium

Rb

Riv

aset

al.,

2008

44ID

10S

2P

ena

de

Hie

rro

(119

–127

md

eep

)8-

54a+

54c+

56c

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

A/

gu

anid

iniu

mR

bR

ivas

etal

.,20

08

45ID

11S

2P

ena

de

Hie

rro

(138

md

eep

)8-

60b

+60c

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

A/

gu

anid

iniu

mR

bR

ivas

etal

.,20

08

46ID

12S

2P

ena

de

Hie

rro

(147

–152

md

eep

)8-

63b

+64b

+64c

+65b

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

A/

gu

anid

iniu

mR

bR

ivas

etal

.,20

08

47ID

13S

2P

ena

de

Hie

rro

(2–3

md

eep

)8-

2a(M

AR

TE

pro

ject

)S

up

ern

atan

tfr

om

ED

TA

/g

uan

idin

ium

Rb

Riv

aset

al.,

2008

48ID

18S

2P

ena

de

Hie

rro

(93

md

eep

)8-

45b

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

A/

gu

anid

iniu

mR

bR

ivas

etal

.,20

08

49ID

14S

2P

ena

de

Hie

rro

(105

md

eep

)8-

49b

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

A/

gu

anid

iniu

mR

bR

ivas

etal

.,20

08

50ID

15S

2P

ena

de

Hie

rro

(119

md

eep

)8-

54a

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

A/

gu

anid

iniu

mR

bR

ivas

etal

.,20

08

51ID

16S

2P

ena

de

Hie

rro

(152

md

eep

)8-

65b

(MA

RT

Ep

roje

ct)

Su

per

nat

ant

fro

mE

DT

A/

gu

anid

iniu

mR

bR

ivas

etal

.,20

08

52ID

17C

1R

ıoT

into

(3.2

wat

erd

am)

Yel

low

Fe-

S–r

ich

salt

pre

cip

itat

esC

ellu

lar

frac

tio

nR

bR

ivas

etal

.,20

0853

ID17

S1

Rıo

Tin

to(3

.2w

ater

dam

)Y

ello

wF

e-S

–ric

hsa

ltp

reci

pit

ates

Su

per

nat

ant

Rb

Riv

aset

al.,

2008

54A

hy

dY

ello

wst

on

eN

atio

nal

Par

kH

yd

rog

enob

acte

rac

idop

hil

us

(Bac

teri

a,A

qu

ifica

e)W

ho

lece

lls

Rb

Sch

wei

tzer

etal

.,20

05

55A

cyan

Yel

low

sto

ne

Nat

ion

alP

ark

Cy

anid

ium

spp

.(E

uk

ary

ote

,R

ho

do

ph

yta

)W

ho

lece

lls

Rb

Sch

wei

tzer

etal

.,20

05

56A

185

Aci

dip

hil

ium

spp

.B

atch

cult

ure

Wh

ole

cell

s(i

nta

ct+

son

icat

ed)

Rb

Par

roet

al.,

2005

57A

139

Lep

tosp

iril

lum

ferr

oox

idan

sB

atch

(N2

fix

ing

)S

on

icat

edce

lls

Rb

Par

roet

al.,

2005

58A

186

L.

ferr

oox

idan

sB

atch

cult

ure

Wh

ole

cell

s(i

nta

ct+

son

icat

ed)

Rb

Par

roet

al.,

2005

59IV

E1C

1L

.p

her

rifi

lum

(LP

H2)

Fer

men

tor

Wh

ole

cell

sR

bR

ivas

etal

.,20

0860

IVE

1S1

L.

ph

erri

filu

m(L

PH

2)F

erm

ento

rC

ult

ure

sup

ern

atan

tR

bR

ivas

etal

.,20

0861

IVE

1C2

L.

ph

erri

filu

m(L

PH

2)F

erm

ento

rIn

solu

ble

cell

pel

let

fro

mS

100

Rb

Riv

aset

al.,

2008

62IV

E1S

100

L.

ph

erri

filu

m(L

PH

2)F

erm

ento

rS

olu

ble

cell

ula

rfr

acti

on

S10

0R

bR

ivas

etal

.,20

0863

IVE

1BF

L.

ph

erri

filu

m(L

PH

2)F

erm

ento

rB

iofi

lmR

bR

ivas

etal

.,20

0864

IVE

2C1

L.

ph

erri

filu

msp

p.

Bat

ch+

Fe2

+W

ho

lece

lls

Rb

Riv

aset

al.,

2008

65IV

E2S

1L

.p

her

rifi

lum

spp

.B

atch

+F

e2+

Cu

ltu

resu

per

nat

ant

Rb

Riv

aset

al.,

2008

66IV

E2S

100

L.

ph

erri

filu

msp

p.

Bat

ch+

Fe2

+S

olu

ble

cell

ula

rfr

acti

on

S10

0R

bR

ivas

etal

.,20

0867

A18

2A

cid

ith

ioba

cill

us

ferr

oox

idan

sB

atch

+F

e2+

Wh

ole

cell

sR

bP

arro

etal

.,20

0568

A18

3A

t.fe

rroo

xid

ans

Bat

ch+

Fe2

+W

ho

lece

lls

(so

nic

ated

)R

bP

arro

etal

.,20

0569

IVE

3C1

At.

ferr

oox

idan

sB

atch

+F

e2+

Wh

ole

cell

sR

bR

ivas

etal

.,20

0870

IVE

3S1

At.

ferr

oox

idan

sB

atch

+F

e2+

Cu

ltu

resu

per

nat

ant

Rb

Riv

aset

al.,

2008

71IV

E3C

2A

t.fe

rroo

xid

ans

Bat

ch+

Fe2

+In

solu

ble

cell

pel

let

fro

mS

100

Rb

Riv

aset

al.,

2008

72IV

E3S

100

At.

ferr

oox

idan

sB

atch

+F

e2+

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

73A

184

At.

thio

oxid

ans

Bat

ch+

SW

ho

lece

lls

(in

tact

+so

nic

ated

)R

bP

arro

etal

.,20

0574

IVE

4C1

At.

thio

oxid

ans

Bat

ch+

SW

ho

lece

lls

Rb

Riv

aset

al.,

2008

75IV

E4C

2A

t.th

ioox

idan

sB

atch

+S

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

0876

IVE

4S10

0A

t.th

ioox

idan

sB

atch

+S

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

(con

tin

ued

)

990

Ta

bl

e2.

(Co

nt

in

ue

d)

Ab

No

Ab

nam

eS

ourc

e/S

trai

nS

amp

le/C

ult

ure

con

dit

ion

sIm

mu

nog

en/F

ract

ion

Hos

tS

up

p.

Ref

eren

ces

77IV

E5C

1A

t.al

bert

ensi

sB

atch

+S

Wh

ole

cell

sR

bR

ivas

etal

.,20

0878

IVE

5C2

At.

albe

rten

sis

Bat

ch+

SIn

solu

ble

cell

pel

let

fro

mS

100

Rb

Riv

aset

al.,

2008

79IV

E5S

100

At.

albe

rten

sis

Bat

ch+

SS

olu

ble

cell

ula

rfr

acti

on

S10

0R

bR

ivas

etal

.,20

0880

IVE

6C1

At.

cald

us

Bat

ch+

SW

ho

lece

lls

Rb

Riv

aset

al.,

2008

81IV

E6S

2A

t.ca

ldu

sB

atch

+S

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

82IV

E6C

2A

t.ca

ldu

sB

atch

+S

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

0883

IVE

6S10

0A

t.ca

ldu

sB

atch

+S

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

84IV

E7C

1H

alot

hio

baci

llu

sn

eap

olit

anu

sB

atch

+S

Wh

ole

cell

sR

bR

ivas

etal

.,20

1185

IVE

8C1

Aci

dim

icro

biu

mfe

rroo

xid

ans

Bio

mas

sfr

om

DS

MN

�103

31W

ho

lece

lls

Rb

Th

isw

ork

86IV

E8S

2A

cid

imic

robi

um

ferr

oox

idan

sB

iom

ass

fro

mD

SM

N�1

0331

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Th

isw

ork

87IV

F1S

1S

hew

anel

lag

elid

imar

ina

Bat

ch(M

arin

eb

roth

15�C

)C

ult

ure

sup

ern

atan

tR

bR

ivas

etal

.,20

0888

IVF

2C1

S.

gel

idim

arin

aB

atch

(Mar

ine

bro

th4�

C)

Wh

ole

cell

sR

bR

ivas

etal

.,20

0889

IVF

2S2a

S.

gel

idim

arin

aB

atch

(Mar

ine

bro

th4�

C)

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

90IV

F2S

2bS

.g

elid

imar

ina

Bat

ch(M

arin

eb

roth

4�C

)S

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0891

IVF

2C2

S.

gel

idim

arin

aB

atch

(Mar

ine

bro

th4�

C)

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

0892

IVF

2S10

0S

.g

elid

imar

ina

Bat

ch(M

arin

eb

roth

4�C

)S

olu

ble

cell

ula

rfr

acti

on

S10

0R

bR

ivas

etal

.,20

0893

IVF

3C2

Psy

chro

serp

ens

burt

onen

sis

Bat

ch(M

arin

eb

roth

15�C

)In

solu

ble

cell

pel

let

fro

mS

100

Rb

Riv

aset

al.,

2008

94IV

F4C

1P

.bu

rton

ensi

sB

atch

(Mar

ine

bro

th4�

C)

Wh

ole

cell

sR

bR

ivas

etal

.,20

0895

IVF

4S1

P.

burt

onen

sis

Bat

ch(M

arin

eb

roth

4�C

)C

ult

ure

sup

ern

atan

tR

bR

ivas

etal

.,20

0896

IVF

4S2a

P.

burt

onen

sis

Bat

ch(M

arin

eb

roth

4�C

)S

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0897

IVF

4S2b

P.

burt

onen

sis

Bat

ch(M

arin

eb

roth

4�C

)S

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0898

IVF

4S10

0P

.bu

rton

ensi

sB

atch

(Mar

ine

bro

th4�

C)

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

99IV

F5C

1P

sych

roba

cter

frig

idic

ola

Bat

ch(H

arp

o’s

med

ium

15�C

)W

ho

lece

lls

Rb

Riv

aset

al.,

2008

100

IVF

5S1

Ps.

frig

idic

ola

Bat

ch(H

arp

o’s

med

ium

15�C

)C

ult

ure

sup

ern

atan

tR

bR

ivas

etal

.,20

0810

1IV

F5C

2P

s.fr

igid

icol

aB

atch

(Har

po

’sm

ediu

m15

�C)

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

0810

2IV

F5S

100

Ps.

frig

idic

ola

Bat

ch(H

arp

o’s

med

ium

15�C

)S

olu

ble

cell

ula

rfr

acti

on

S10

0R

bR

ivas

etal

.,20

0810

3IV

F6C

1C

ryob

acte

riu

mp

sych

rop

hil

um

Bat

ch(T

SA

)W

ho

lece

lls

Rb

Riv

aset

al.,

2008

104

IVF

6S1

C.

psy

chro

ph

ilu

mB

atch

(TS

A)

Cu

ltu

resu

per

nat

ant

Rb

Riv

aset

al.,

2008

105

IVF

6S2

C.

psy

chro

ph

ilu

mB

atch

(TS

A)

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

106

IVF

6C2

C.

psy

chro

ph

ilu

mB

atch

(TS

A)

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

0810

7IV

F6S

100

C.

psy

chro

ph

ilu

mB

atch

(TS

A)

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

108

IVF

7C1

Col

wel

lia

psy

chre

ryth

raea

Bat

hcu

ltu

re(M

arin

eb

roth

)W

ho

lece

lls

Rb

Riv

aset

al.,

2011

109

IVF

7S1

C.

psy

chre

ryth

raea

Bat

hcu

ltu

re(M

arin

eb

roth

)C

ult

ure

sup

ern

atan

tR

bT

his

wo

rk11

0IV

F7S

2C

.p

sych

rery

thra

eaB

ath

cult

ure

(Mar

ine

bro

th)

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Th

isw

ork

111

IVG

1C1

Aci

doc

ella

amin

oly

tica

DS

M11

237

Bat

ch(D

SM

Zm

ediu

mN

�269

)W

ho

lece

lls

Rb

Riv

aset

al.,

2011

112

IVG

2C_1

85A

cid

iph

iliu

msp

p.

Bat

ch(D

SM

Zm

ediu

mN

�269

)W

ho

lece

lls

Rb

Th

isw

ork

113

IVG

2C1

Aci

dip

hil

ium

sp.

Bat

ch(D

SM

Zm

ediu

mN

�269

)W

ho

lece

lls

Rb

Riv

aset

al.,

2011

114

IVG

3C1

Aci

dob

acte

riu

mca

psu

latu

mD

SM

1124

4B

atch

(DS

MZ

med

ium

N�2

69)

Wh

ole

cell

sR

bR

ivas

etal

.,20

11

115

IVG

4C1

Th

erm

us

scot

odu

ctu

sB

atch

(TY

G)

Wh

ole

cell

sR

bR

ivas

etal

.,20

1111

6IV

G4C

2T

.sc

otod

uct

us

Bat

ch(T

YG

)In

solu

ble

cell

pel

let

fro

mS

100

Rb

Riv

aset

al.,

2011

117

IVG

5C1

Su

lfob

acil

lus

acid

oph

ilu

sB

iom

ass

fro

mD

SM

No

1033

2W

ho

lece

lls

Rb

Riv

aset

al.,

2011

118

IVG

6C1

T.

term

oph

ilu

sB

atch

(TY

G)

Wh

ole

cell

sR

bT

his

wo

rk11

9IV

H1C

1B

acil

lus

subt

ilis

(sp

ore

s)B

atch

(Sch

aeff

erm

ediu

m)

Wh

ole

spo

res

Rb

Fer

nan

dez

-Cal

vo

etal

.,20

0612

0IV

I1C

1P

seu

dom

onas

pu

tid

aB

atch

(LB

)W

ho

lece

lls

Rb

Riv

aset

al.,

2008

(con

tin

ued

)

991

Ta

bl

e2.

(Co

nt

in

ue

d)

Ab

No

Ab

nam

eS

ourc

e/S

trai

nS

amp

le/C

ult

ure

con

dit

ion

sIm

mu

nog

en/F

ract

ion

Hos

tS

up

p.

Ref

eren

ces

121

IVI1

C2

P.

pu

tid

aB

atch

(LB

)In

solu

ble

cell

pel

let

fro

mS

100

Rb

Riv

aset

al.,

2008

122

IVI1

S10

0P

.p

uti

da

Bat

ch(L

B)

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

123

IVI1

RB

P.

pu

tid

aB

atch

(LB

)R

ibo

som

efr

acti

on

Rb

Riv

aset

al.,

2008

124

IVI2

C1

Bac

illu

ssp

p.

(en

vir

on

.is

ol.

)*B

atch

(LB

)W

ho

lece

lls

Rb

Riv

aset

al.,

2008

125

IVI2

S2

Bac

illu

ssp

p.

(en

vir

on

.is

ol.

)*B

atch

(LB

)S

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0812

6IV

I2C

2B

acil

lus

spp

.(e

nv

iro

n.

iso

l.)*

Bat

ch(L

B)

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

0812

7IV

I2S

100

Bac

illu

ssp

p.

(en

vir

on

.is

ol.

)*B

atch

(LB

)S

olu

ble

cell

ula

rfr

acti

on

S10

0R

bR

ivas

etal

.,20

0812

8IV

I3C

1S

hew

anel

laon

eid

ensi

sB

atch

(LB

)W

ho

lece

lls

Rb

Riv

aset

al.,

2008

129

IVI3

S2

S.

onei

den

sis

Bat

ch(L

B)

Su

per

nat

ant

fro

mE

DT

Aw

ash

Rb

Riv

aset

al.,

2008

130

IVI3

C2

S.

onei

den

sis

Bat

ch(L

B)

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

0813

1IV

I3S

100

S.

onei

den

sis

Bat

ch(L

B)

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

132

IVI4

C1

Bu

rkh

old

eria

fun

gor

um

Bat

ch(L

B)

Wh

ole

cell

sR

bR

ivas

etal

.,20

0813

3IV

I4S

2B

.fu

ng

oru

mB

atch

(LB

)S

up

ern

atan

tfr

om

ED

TA

was

hR

bR

ivas

etal

.,20

0813

4IV

I4C

2B

.fu

ng

oru

mB

atch

(LB

)In

solu

ble

cell

pel

let

fro

mS

100

Rb

Riv

aset

al.,

2008

135

IVI4

S10

0B

.fu

ng

oru

mB

atch

(LB

)S

olu

ble

cell

ula

rfr

acti

on

S10

0R

bR

ivas

etal

.,20

0813

6IV

I5C

1S

.on

eid

ensi

sA

nae

rob

ic(f

um

arat

e)W

ho

lece

lls

Rb

Riv

aset

al.,

2008

137

IVI5

S1

S.

onei

den

sis

An

aero

bic

(fu

mar

ate)

Cu

ltu

resu

per

nat

ant

Rb

Riv

aset

al.,

2008

138

IVI5

C2

S.

onei

den

sis

An

aero

bic

(fu

mar

ate)

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

0813

9IV

I5S

100

S.

onei

den

sis

An

aero

bic

(fu

mar

ate)

So

lub

lece

llu

lar

frac

tio

nS

100

Rb

Riv

aset

al.,

2008

140

IVI6

C3

Azo

toba

cter

vin

elan

dii

Bat

chcu

ltu

re(L

B)

ED

TA

was

hed

cell

s(s

on

icat

ed)

Rb

Riv

aset

al.,

2008

141

IVI7

C1

B.

subt

ilis

168

Bat

chcu

ltu

re(L

B)

veg

etat

ive

cell

sW

ho

lece

lls

Rb

Riv

aset

al.,

2008

142

IVI8

C1

B.

subt

ilis

3610

Bio

film

Wh

ole

cell

sR

bR

ivas

etal

.,20

0814

3IV

I8S

1B

.su

btil

is36

10B

iofi

lmC

ult

ure

sup

ern

atan

tR

bR

ivas

etal

.,20

0814

4IV

I9C

1D

ein

ococ

cus

rad

iod

ura

ns

Bio

mas

sfr

om

DS

MZ

No

2053

9W

ho

lece

lls

Rb

Riv

aset

al.,

2008

145

IVI1

0C1

Des

ulf

ovib

rio

vu

lgar

is(v

ulg

aris

)B

iom

ass

fro

mD

SM

ZN

o64

4W

ho

lece

lls

Rb

Riv

aset

al.,

2008

146

IVI1

1C1

Geo

bact

ersu

lfu

rred

uce

ns

Bio

mas

sfr

om

DS

MZ

No

1212

7W

ho

lece

lls

Rb

Riv

aset

al.,

2008

147

IVI1

2C1

Geo

bact

erm

etal

lire

du

cen

sB

iom

ass

fro

mD

SM

ZN

o72

10W

ho

lece

lls

Rb

Riv

aset

al.,

2008

148

IVI1

3C1

Th

erm

otog

am

arit

ima

Bio

mas

sfr

om

DS

MZ

No

3109

Wh

ole

cell

sR

bR

ivas

etal

.,20

0814

9IV

I14C

1V

erru

com

icro

biu

msp

inos

um

Bio

mas

sfr

om

DS

MZ

No

4136

Wh

ole

cell

sR

bR

ivas

etal

.,20

0815

0IV

I15C

1M

eth

ylo

mic

robi

um

cap

sula

tum

Bio

mas

sfr

om

DS

MZ

No

6130

Wh

ole

cell

sR

bR

ivas

etal

.,20

0815

1IV

I16C

1P

lan

ctom

yce

sli

mn

oph

ilu

sB

iom

ass

fro

mD

SM

ZN

o37

76W

ho

lece

lls

Rb

Riv

aset

al.,

2008

152

IVI1

7C1

Hy

dro

gen

obac

ter

ther

mop

hil

us

Bio

mas

sfr

om

DS

MZ

No

6534

Wh

ole

cell

sR

bR

ivas

etal

.,20

0815

3IV

I19C

1D

esu

lfos

por

osin

us

mer

idie

iB

iom

ass

fro

mD

SM

No

1325

7W

ho

lece

lls

Rb

Riv

aset

al.,

2011

154

IVI2

0C1

Sal

inib

acte

rru

ber

M8

Bat

ch(S

W25

%m

arin

esa

lt)

Wh

ole

cell

sR

bR

ivas

etal

.,20

1115

5IV

I21C

1S

.ru

ber

PR

1B

atch

(SW

25%

mar

ine

salt

)W

ho

lece

lls

Rb

Riv

aset

al.,

2011

156

IVI2

1C2

S.

rube

rP

R1

Bat

ch(S

W25

%m

arin

esa

lt)

Inso

lub

lece

llp

elle

tfr

om

S10

0R

bR

ivas

etal

.,20

1115

7IV

I21S

1S

.ru

ber

PR

1B

atch

(SW

25%

mar

ine

salt

)C

ult

ure

sup

ern

atan

tR

bT

his

wo

rk15

8IV

J1C

1H

alof

erax

med

iter

ran

eiB

atch

(SW

25%

mar

ine

salt

)W

ho

lece

lls

Rb

Riv

aset

al.,

2008

159

IVJ2

C1

Met

han

ococ

coid

esbu

rton

iiB

iom

ass

fro

mD

SM

ZN

o62

42W

ho

lece

lls

Rb

Riv

aset

al.,

2008

160

IVJ3

C1

Th

erm

opla

sma

acid

oph

ilu

mB

iom

ass

fro

mD

SM

ZN

o17

28W

ho

lece

lls

Rb

Riv

aset

al.,

2008

161

IVJ4

C1

Met

han

obac

teri

um

form

icic

um

Bio

mas

sfr

om

DS

MZ

No

1535

Wh

ole

cell

sR

bR

ivas

etal

.,20

0816

2IV

J5C

1M

eth

anos

arci

na

maz

eii

Bio

mas

sfr

om

DS

MZ

No

3647

Wh

ole

cell

sR

bR

ivas

etal

.,20

0816

3IV

J6C

1P

yro

cocc

us

furi

osu

sB

iom

ass

fro

mD

SM

No

3638

Wh

ole

cell

sR

bR

ivas

etal

.,20

1116

4IV

J8C

1H

alor

ubr

um

sp.

Bat

ch(S

W25

%m

arin

esa

lt)

Wh

ole

cell

sR

bR

ivas

etal

.,20

1116

5IV

J9C

1H

alob

acte

riu

msp

.B

atch

(SW

25%

mar

ine

salt

)W

ho

lece

lls

Rb

Riv

aset

al.,

2011

(con

tin

ued

)

992

Ta

bl

e2.

(Co

nt

in

ue

d)

Ab

No

Ab

nam

eS

ourc

e/S

trai

nS

amp

le/C

ult

ure

con

dit

ion

sIm

mu

nog

en/F

ract

ion

Hos

tS

up

p.

Ref

eren

ces

166

A-E

coli

1E

sch

eric

hia

coli

E.

coli

cult

ure

All

sero

typ

esR

bU

SB

iolo

gic

al(E

3500

-26)

167

A-H

py

lori

Hel

icob

acte

rp

ylo

riB

atch

cult

ure

(AT

CC

4350

4)W

ho

lece

lls

Rb

BIO

DE

SIG

N(B

6566

0R)

168

A-L

mo

no

L.

mon

ocy

tog

enes

Bat

chcu

ltu

re(A

TC

C43

251)

Wh

ole

cell

sR

bB

IOD

ES

IGN

(B65

420R

)16

9A

-My

cob

My

coba

cter

ium

gen

us

Bat

chcu

ltu

reM

.tu

berc

ulo

sis

Gen

us-

spec

ific

anti

gen

sex

trac

tR

bB

IOD

ES

IGN

(B47

827R

)17

0A

-Pae

rP

.ae

rug

inos

aB

atch

cult

ure

(P.

aeru

gin

osa)

Ou

ter

mem

bra

ne

pro

tein

extr

act

Gp

igB

IOD

ES

IGN

(B47

578G

)17

1A

-Sal

mS

alm

onel

lasp

.(O

+H

Ag

s)B

atch

cult

ure

Mix

S.

ente

riti

dis

,ty

ph

ym

.,h

eid

elb

urg

Rb

BIO

DE

SIG

N(B

6570

1R)

172

A-G

+Ag

Gra

m-p

osi

tiv

ean

tig

enC

ult

ure

Gra

m-p

osi

tiv

ean

tig

enM

(Mab

)B

IOD

ES

IGN

(C11

333M

)17

3A

-G+B

acG

ram

-po

siti

ve

Bac

teri

aC

ult

ure

Lip

ote

ich

oic

acid

Gra

m-p

osi

tiv

esM

(Mab

)B

IOD

ES

IGN

(C65

380M

)17

4A

-PG

Pep

tid

og

lyca

nC

ult

ure

(Str

epto

cocc

us

mu

tan

s)In

solu

ble

PG

extr

act

ob

tain

edb

yT

CA

M(M

ab)

CH

EM

.IN

TE

R.

(MA

B99

7)

175

A-l

ipid

AL

ipid

A(E

.co

liO

157)

Cu

ltu

reW

ho

lece

lls

pre

po

fli

pid

AG

oat

BIO

DE

SIG

N(B

6569

2G)

176

A-L

TA

Lip

ote

ich

oic

acid

Cu

ltu

reN

ativ

eS

trep

toco

ccu

sp

yro

gen

esR

bB

IOD

ES

IGN

(B30

103R

)17

7A

-NA

GN

-Ace

tylg

luco

sam

ine

NA

Go

np

rote

ins

NA

G-K

LH

con

jug

ated

M(M

ab)

BIO

DE

SIG

N(H

6710

8M)

178

A-D

NA

1ss

,d

sDN

AB

ov

ine

ss,

dsD

NA

Cal

fth

ym

us

DN

AM

(Mab

)U

SB

iolo

gic

al(D

3878

-05)

179

A-D

NA

2D

NA

102

(hu

man

)d

sDN

AA

uto

imm

un

eH

uS

chw

eitz

eret

al.,

2005

180

A-d

sDN

A(h

u)

dsD

NA

(hu

man

pla

sma)

dsD

NA

Au

toim

mu

ne

Hu

BIO

DE

SIG

N(K

0711

2H)

181

A-R

NA

pII

RN

Ap

oly

mer

ase

IIE

uk

ary

oti

cR

NA

po

lW

hea

tg

erm

RN

Ap

ol

II(p

uri

fied

)M

(Mab

)B

IOD

ES

IGN

(M55

701M

)18

2A

-Gro

EL

Gro

EL

(E.

coli

)R

eco

mb

inan

tG

roE

L(p

uri

fied

)R

bS

igm

a-A

ldri

ch(G

6532

)18

3A

-Hsp

70H

SP

-70

Bat

chcu

ltu

re(M

.tu

berc

ulo

sis)

HS

P-7

0(p

uri

fied

pro

tein

)M

(Mab

)B

IOD

ES

IGN

(H86

313M

)18

4A

-Las

nas

eL

-Asp

arag

inas

e(E

.co

li)

Bat

chcu

ltu

reL

-Asp

arag

inas

e(p

uri

fied

)R

bB

IOD

ES

IGN

(K59

171R

)18

5A

-BG

alb

eta

dG

alac

tosi

das

e(E

.co

li)

Bat

chcu

ltu

reB

eta

dG

alac

tosi

das

e(p

uri

fied

)R

bB

IOD

ES

IGN

(K03

450R

)18

6A

-Glu

Ox

idG

luco

seo

xid

ase

Bat

chcu

ltu

re(A

sper

gil

lus

nig

er)

Glu

cose

ox

idas

e(p

uri

fied

)R

bB

IOD

ES

IGN

(K59

137R

)18

7A

-GS

TG

luta

thio

ne-

S-t

ran

sfer

ase

Rec

om

bin

ant

(S.

jap

onic

um

)G

ST

(pu

rifi

ed)

Rb

Sig

ma-

Ald

rich

(G77

81)

188

A-S

tvS

trep

tav

idin

Cu

ltu

re(S

trep

tom

yce

sav

idin

ii)

Str

epta

vid

in(p

uri

fied

)R

bS

igm

a-A

ldri

ch(S

6390

)18

9A

-Th

ioT

hio

red

ox

in(E

.co

li)

Rec

om

bin

ant

Th

iore

do

xin

(pu

rifi

ed)

Rb

Sig

ma-

Ald

rich

(T08

03)

190

A-B

glu

B.

subt

ilis

bet

a-g

luca

nas

eR

eco

mb

inan

t(B

.su

btil

is)

Bet

a-g

luca

nas

eS

DS

-PA

GE

pu

rifi

edR

bR

ivas

etal

.,20

08

191

A-C

snB

.su

btil

isch

ito

san

ase

Rec

om

bin

ant

(B.

subt

ilis

)C

snS

DS

-PA

GE

pu

rifi

edR

bR

ivas

etal

.,20

0819

2A

-Hfe

rH

um

anfe

rrit

inF

rom

hu

man

liv

erF

erri

tin

(pu

rifi

ed)

M(M

ab)

BIO

DE

SIG

N(H

5371

5)19

3A

1484

NIF

H3

pep

tid

ese

q.

AK

GIL

KY

AN

SG

GV

RL

KL

Hco

nju

gat

eR

bF

ern

a nd

ez-C

alv

oet

al.,

2006

194

A14

85N

IFD

2p

epti

de

seq

.Y

RS

MN

YIS

RH

ME

EK

YK

LH

con

jug

ate

Rb

Fer

nan

dez

-Cal

vo

etal

.,20

0619

5A

1487

HS

CA

2p

epti

de

seq

.T

FT

IDA

NG

ILD

VR

AL

KL

Hco

nju

gat

eR

bF

ern

and

ez-C

alv

oet

al.,

2006

196

A14

90F

DX

2p

epti

de

seq

.H

VII

TE

GF

DN

LS

PM

EK

LH

con

jug

ate

Rb

Fer

nan

dez

-Cal

vo

etal

.,20

0619

7A

1492

GL

NB

2p

epti

de

seq

.V

ET

AL

RIR

TG

ET

GD

AK

LH

con

jug

ate

Rb

Fer

nan

dez

-Cal

vo

etal

.,20

0619

8A

1494

MO

DA

2p

epti

de

seq

.M

LA

PL

HK

KIV

YA

NT

LK

LH

con

jug

ate

Rb

Fer

nan

dez

-Cal

vo

etal

.,20

0619

9A

1496

NIF

S2

pep

tid

ese

q.

LP

VN

KE

GR

VE

IET

LK

KL

Hco

nju

gat

eR

bF

ern

and

ez-C

alv

oet

al.,

2006

200

Lec

tin

Po

Lec

tin

po

tato

pro

tein

(no

tA

b)

SD

S-P

AG

Ep

uri

fied

pro

tein

No

tap

pli

cab

le—

CA

LB

IOC

HE

M(4

3178

8)20

1A

-cA

MP

1C

ycl

icA

MP

Cy

clic

AM

P3¢

-5¢-c

AM

P-2

¢-BS

Aco

nju

gat

edR

bS

igm

a-G

eno

sys

(A06

70)

202

A-c

AM

P2

Cy

clic

AM

PH

um

ancA

MP

Su

ccin

yla

ted

cAM

P-B

SA

con

ju-

gat

edR

bU

SB

iolo

gic

al(C

8450

-05)

203

A-c

AM

P3

Cy

clic

AM

PC

ycl

icA

MP

Su

ccin

yla

ted

cAM

P-B

SA

con

ju-

gat

edS

hee

pB

IOD

ES

IGN

(Q55

101S

)

204

A-A

spA

spar

tate

Pu

rifi

edA

spA

sp-K

LH

con

jug

ated

Rb

Sig

ma-

Ald

rich

(A96

84)

205

A-G

luG

luta

mat

eP

uri

fied

Glu

Glu

-KL

Hco

nju

gat

edR

bS

igm

a-A

ldri

ch(G

6642

)

(con

tin

ued

)

993

Ta

bl

e2.

(Co

nt

in

ue

d)

Ab

No

Ab

nam

eS

ourc

e/S

trai

nS

amp

le/C

ult

ure

con

dit

ion

sIm

mu

nog

en/F

ract

ion

Hos

tS

up

p.

Ref

eren

ces

206

A-T

rpT

ryp

top

han

Try

pto

ph

anT

rp-g

luta

rald

ehy

de-

Po

ly-l

ysi

ne

Rb

US

Bio

log

ical

(T90

13-0

5)20

7A

-po

lyH

isP

oly

His

tid

ine

Po

ly-H

isH

is-T

agg

edfu

sio

np

rote

inM

(Mab

)S

igm

a-A

ldri

ch(H

1029

)20

8A

-Co

rtis

ol

Co

rtis

ol

Co

rtis

ol

Co

rtis

ol-

21-h

emis

ucc

.-T

hy

rog

lob

uli

nR

bS

igm

a-A

ldri

ch(C

8409

)

209

A-E

stra

d1

Est

rad

iol

17-b

eta-

Est

rad

iol

17-b

eta-

Est

rad

iol-

BS

AM

(Mab

)B

IOD

ES

IGN

(E86

022M

)21

0A

-Est

rad

2E

stra

dio

l17

-bet

a-E

stra

dio

lE

stra

dio

l-17

-bet

a-6-

BS

Aco

nju

-g

ated

M(M

ab)

BIO

DE

SIG

N(H

3110

0M)

211

A-P

enP

enic

illi

nP

enic

llin

and

der

ivat

ives

Pen

icil

lin

-BS

Aco

nju

gat

edM

(Mab

)B

IOD

ES

IGN

(G13

011M

)21

2A

-Tc

Tet

racy

clin

eT

etra

cycl

ine

Tet

racy

clin

e-B

SA

Rb

US

Bio

log

ical

(T29

65-0

5)21

3A

-Vit

B12

Vit

amin

B12

Cy

ano

cob

alam

ine

Vit

amin

B12

-BS

Aco

nju

gat

edR

bU

SB

iolo

gic

al(C

8400

-10)

214

A-A

traz

ine

Atr

azin

eA

traz

ine

Atr

azin

e-B

gG

Rb

BIO

DE

SIG

N(K

8210

2R)

215

A-H

BV

Ag

Hep

atit

isB

vir

us

surf

ace

Ag

Hep

atit

isB

vir

us

surf

ace

anti

gen

Hig

hly

pu

rifi

edH

epat

itis

Ban

ti-

gen

Go

atA

BC

AM

(ab

9216

)

216

A-S

urf

acti

n_N

Su

rfac

tin

Sig

ma

S35

23S

urf

acti

n-A

min

e-B

SA

Rb

Th

isw

ork

217

A-A

SB

_113

62A

rch

aeog

lobu

sfu

lgid

us

AT

Psy

nth

ase,

sub

un

itB

(Arc

hae

a)P

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk21

8A

-AS

F1_

1135

5T

her

mot

oga

mar

itim

aA

TP

syn

thas

eF

1,su

bu

nit

Alp

ha

(Bac

teri

a)P

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk

219

A-B

aFE

RB

acte

rio

ferr

itin

Bac

teri

ofe

rrit

inP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bR

ivas

etal

.,20

11**

220

A-C

rReT

s_97

7T

.sc

otod

uct

us

Ch

rom

ate

red

uct

ase

(mem

bra

ne)

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Riv

aset

al.,

2011

221

A-C

spA

-113

57P

.p

uti

da

Co

ldsh

ock

pro

tein

AP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk22

2A

-Dsr

A-1

1365

Dsr

A(A

rch

aeog

lobu

sfu

lgid

us)

Dis

sim

ilat

ory

sulfi

tere

du

ctas

eP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk22

3A

-Dsr

B_1

1368

Dsr

B(A

rch

aeog

lobu

sfu

lgid

us)

Dis

sim

ilat

ory

sulfi

tere

du

ctas

eP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk22

4A

-EF

G_1

1359

Th

erm

otog

am

arit

ima

Elo

ng

atio

nfa

cto

rG

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

225

A-F

dh

FF

DH

F(E

.co

liK

12)

Sel

eno

po

lyp

epti

de

sub

.F

orm

ate

DeH

ase

HP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk

226

A-F

eReT

s_98

3Ir

on

red

uct

ase

(T.

scot

odu

ctu

s)Ir

on

red

uct

ase

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

227

A-H

tpG

Htp

G(N

osto

cP

CC

7310

2)H

tpG

ho

mo

log

ou

sto

HS

P90

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

228

A-H

up

LH

up

L(A

t.fe

rroo

xid

ans)

Ni-

Fe

mem

bra

ne

hy

dro

gen

ase

Lch

ain

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

229

A-H

up

SH

up

S(L

.fe

rroo

xid

ans)

Ni-

Fe

mem

bra

ne

hy

dro

gen

ase

Sch

ain

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

230

A-H

yfG

Hy

fG(E

.co

li)

Hy

dro

gen

ase

4,co

mp

on

ent

BP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk23

1A

-Mcr

B_1

1378

Mcr

B(M

eth

anoc

occo

ides

burt

onii

)M

eth

yl

Co

Mre

du

ctas

eI,

Bsu

bu

nit

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

232

A-N

AD

H_1

1363

Nu

oF

(P.

pu

tid

a)N

AD

Hd

ehy

dro

gen

ase

I(q

uin

on

e)P

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk23

3A

-Nif

D_1

1466

Nif

D(G

.m

etal

lire

du

cen

s)N

ifD

pro

tein

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

234

A-N

irS

_113

69N

irS

(P.

aeru

gin

osa)

Nit

rite

red

uct

ase

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

235

A-N

OR

1_11

375

NO

R1

(N.

ham

burg

ensi

s)N

itri

teo

xid

ore

du

ctas

eB

eta

sub

un

itP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk23

6A

-NR

A-1

1912

NR

A(G

.m

etal

lire

du

cen

s)N

itra

tere

du

ctas

esu

bu

nit

Alp

ha

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

237

A-O

mp

AO

mp

A(E

.co

li)

Om

pA

mem

bra

ne

pro

tein

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

238

A-O

pp

AO

pp

A(B

acil

lus

subt

ilis

)O

lig

op

epti

de

bin

din

gp

rote

insu

bA

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Th

isw

ork

239

A-P

fuD

PS

DP

S(P

yro

cocc

us

furi

osu

s)F

e-b

ind

ing

and

sto

rag

ep

rote

inD

pS

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Riv

aset

al.,

2011

**24

0A

-Pfu

FE

RF

erri

tin

(P.

furi

osu

s)F

erri

tin

Fe-

bin

din

gan

dst

ora

ge

pro

tein

Pu

rifi

edre

com

bin

ant

po

lip

epti

de

Rb

Riv

aset

al.,

2011

**24

1A

-Rb

cL_R

B11

374

Rb

cL(A

t.fe

rroo

xid

ans)

Ru

bis

cola

rge

sub

un

itP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk24

2A

-Ru

stic

yan

inR

ust

icy

anin

(At.

fero

oxid

ans)

Ru

stic

yan

inP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bT

his

wo

rk24

3A

-Sso

DP

SD

PS

(Su

lfol

obu

sso

lfat

aric

us)

Fe-

bin

din

gan

dst

ora

ge

pro

tein

Dp

SP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bR

ivas

etal

.,20

11**

244

A-A

BC

tran

sA

BC

tran

spo

rter

(T.

scot

odu

ctu

s)A

BC

-tra

nsp

ort

erp

rote

inP

uri

fied

reco

mb

inan

tp

oli

pep

tid

eR

bR

ivas

etal

.,20

1124

5A

-Ap

sA-1

1754

Ap

sA(D

esu

lfov

ibri

od

esu

lfu

rica

ns)

HM

ML

RE

MR

EG

RG

PIY

CC

on

jug

ate

Rb

Th

isw

ork

(con

tin

ued

)

994

Ta

bl

e2.

(Co

nt

in

ue

d)

Ab

No

Ab

nam

eS

ourc

e/S

trai

nS

amp

le/C

ult

ure

con

dit

ion

sIm

mu

nog

en/F

ract

ion

Hos

tS

up

p.

Ref

eren

ces

246

A-B

fRB

FR

(D.

des

ulf

uri

can

s)C

AE

NF

AE

RIK

EL

FF

EP

Co

nju

gat

eR

bT

his

wo

rk24

7A

-Ccd

AC

cdA

(Geo

bact

ersu

lfu

rred

uce

ns)

CL

LG

EK

RL

QV

HR

KP

AG

YC

on

jug

ate

Rb

Th

isw

ork

248

A-c

yd

A-1

1758

Cy

dA

(S.

onei

den

sis)

YIL

KK

RD

LP

FA

RR

SF

AC

Co

nju

gat

eR

bT

his

wo

rk24

9A

-Dh

nA

1D

hn

A(N

osto

cP

CC

7310

2)L

RN

NA

FK

QD

KD

YH

LA

CC

on

jug

ate

Rb

Th

isw

ork

250

A-D

hn

A2

Dh

nA

(Nos

toc

PC

C73

102)

SG

RK

AF

QR

PF

EE

GV

KL

CC

on

jug

ate

Rb

Th

isw

ork

251

A-F

tsZ

Fts

ZP

seu

dom

onas

pu

tid

aC

PF

EG

RK

RM

QIA

DE

GIR

Co

nju

gat

eR

bT

his

wo

rk25

2A

-GG

DE

F_1

1760

Sen

sory

bo

x/

GG

DE

FD

om

ain

IDL

DE

FK

RIN

(DT

)FG

HK

EG

DK

VC

Co

nju

gat

eR

bT

his

wo

rk25

3A

-Gro

elG

roE

L(G

.m

etal

lire

du

cen

s)E

TE

MK

EK

KA

RV

ED

AL

CC

on

jug

ate

Rb

Th

isw

ork

254

A-I

CD

H_1

1755

ICD

H-N

AD

(C.

hy

dro

gen

ofor

-m

ans)

VL

(ES

)IK

KN

KV

AL

KG

PC

Co

nju

gat

eR

bT

his

wo

rk

255

A-I

siA

1Is

iA,

PS

II(N

osto

cP

CC

7310

2)E

ISR

YK

PE

IPM

GE

QG

CC

on

jug

ate

Rb

Th

isw

ork

256

A-I

siA

2Is

iA,

PS

II(N

osto

cP

CC

7310

2)F

HF

EW

ND

PK

LG

LIL

CC

on

jug

ate

Rb

Th

isw

ork

257

A-K

tran

s_11

750

K+

-tra

nsp

ort

er(G

.m

etal

lire

du

-ce

ns)

LA

MS

LG

RK

GE

GG

TIV

CC

on

jug

ate

Rb

Th

isw

ork

258

A-N

ifD

1_12

288

Nif

D1

(Bu

rkh

old

eria

xen

ovor

ans)

VF

DK

AD

PD

KR

PD

FV

SC

Co

nju

gat

eR

bT

his

wo

rk25

9A

-Nif

D3_

1228

9N

ifD

3(B

.x

enov

oran

s)V

FG

GD

KK

LD

KII

DE

ICC

on

jug

ate

Rb

Th

isw

ork

260

A-N

ifH

1_12

291

Nif

H1

(B.

xen

ovor

ans)

CN

ER

QT

DK

EL

EL

AE

AL

Co

nju

gat

eR

bT

his

wo

rk26

1A

-Nif

H2_

1229

3N

ifH

2(B

.x

enov

oran

s)E

FA

PE

SK

QA

EE

YR

QL

CC

on

jug

ate

Rb

Th

isw

ork

262

A-O

mcS

Om

cS(G

.su

lfu

rred

uce

ns)

CH

DP

HG

KY

RR

FV

DG

SI

Co

nju

gat

eR

bT

his

wo

rk26

3A

-Ph

aC1

Ph

aC(P

seu

dom

onas

pu

tid

a)C

SL

AP

DS

DD

RR

FN

DP

AC

on

jug

ate

Rb

Th

isw

ork

264

A-P

haC

2P

haC

(P.

pu

tid

a)C

LG

ER

AG

AL

KK

AP

TR

LC

on

jug

ate

Rb

Th

isw

ork

265

A-P

hcA

1P

hcA

(Nos

toc

PC

C73

103)

NT

EL

QS

AR

GR

YE

RA

AC

Co

nju

gat

eR

bT

his

wo

rk26

6A

-Ph

cA2

Ph

CA

(Nos

toc

PC

C73

104)

TP

GP

QF

AA

DS

RG

KS

KC

Co

nju

gat

eR

bT

his

wo

rk26

7A

-Pu

fM1

Pu

fM(R

hod

osp

iril

lum

rubr

um

)S

RL

GG

DR

EV

EQ

ITD

RC

Co

nju

gat

eR

bT

his

wo

rk26

8A

-Pu

fM2

Pu

fM(R

hod

osp

iril

lum

rubr

um

)S

RL

GG

DR

EV

EQ

ITD

RC

Co

nju

gat

eR

bT

his

wo

rk26

9A

-RR

OR

ub

red

ox

in(D

.des

ulf

uri

can

sG

20)

CH

TQ

DE

TM

KA

LE

IKK

DV

Co

nju

gat

eR

bT

his

wo

rk27

0A

-So

dA

So

dA

(G.

sulf

urr

edu

cen

s)C

ML

DY

GL

KR

PD

YIE

AF

Co

nju

gat

eR

bT

his

wo

rk27

1A

-So

dF

So

dF

(G.

sulf

urr

edu

cen

s)C

AR

IDK

DF

GS

FD

KF

KE

EC

on

jug

ate

Rb

Th

isw

ork

272

A-W

shR

Wat

erst

ress

pro

tein

(P.

pu

tid

aF

1)C

VR

FT

DE

GK

LD

LP

KG

TC

on

jug

ate

Rb

Th

isw

ork

273

A-A

eKaC

_SA

EK

AC

pep

tid

eA

EK

AC

-sy

nth

etic

Co

nju

gat

eR

bT

his

wo

rk27

4A

-EP

S_S

PE

xo

po

lysa

cch

arid

esS

ola

rsa

lter

n(S

anta

Po

la,

Ali

can

te,

Sp

ain

)E

PS

extr

act

Rb

Th

isw

ork

275

A-L

PS

_NL

PS

(Pse

ud

omon

assp

.)L

PS

-BS

AR

bT

his

wo

rk27

6A

-Lip

idA

_43

Lip

idA

Lip

idA

Mab

Hy

cult

bio

tech

.(H

M20

46)

277

A-A

MP

_NA

MP

AM

P-O

H-B

SA

Rb

Th

isw

ork

278

A-c

AM

P_N

cAM

PcA

MP

-OH

-BS

AR

bT

his

wo

rk27

9A

-cA

MP

_sh

cAM

PS

ucc

iny

late

dcA

MP

-BS

AR

bA

bca

m(a

b83

2)28

0A

-cG

MP

(bio

)cG

MP

2¢O

-su

ccin

yl

gu

ano

sin

e-3¢

,5¢

cGM

PR

bM

BL

,In

c.(J

M-3

568-

100)

281

A-c

GM

P_N

cGM

PcG

MP

-OH

-BS

AR

bT

his

wo

rk28

2A

-Co

A_S

Co

enzy

me

AC

oA

-SH

-BS

AR

bT

his

wo

rk28

3A

-Dig

ox

inD

igo

xin

Dig

ox

in-K

LH

Mab

SIG

MA

(D81

56)

285

A-d

init

rop

h.

Din

itro

ph

eno

lD

init

rop

hen

ol-

BS

AR

bS

IGM

A(D

9656

)28

6A

-cG

MP

(up

s)cG

MP

2¢-M

on

osu

ccin

yl

cycl

icG

MP

-KL

HR

bU

PS

TA

TE

287

A-G

MP

_NG

MP

GM

P-O

H-B

SA

Rb

Th

isw

ork

(con

tin

ued

)

995

Ta

bl

e2.

(Co

nt

in

ue

d)

Ab

No

Ab

nam

eS

ourc

e/S

trai

nS

amp

le/C

ult

ure

con

dit

ion

sIm

mu

nog

en/F

ract

ion

Hos

tS

up

p.

Ref

eren

ces

288

A-P

roto

IX_N

Pro

top

orp

hy

rin

IXP

roto

ph

orp

hy

rin

IX-O

H-B

SA

Rb

Th

isw

ork

289

A-T

hia

min

eT

hia

min

eT

hia

min

e-B

SA

Rb

US

Bio

log

ical

(T50

04)

290

A-A

rgA

rgin

ine

Arg

inin

eA

sym

met

ric

NG

-NG

-dim

eth

yl

arg

inin

eR

bS

anta

Cru

zB

iote

ch.s

c-57

626

292

A-A

la_N

Ala

Ala

-NH

2-B

SA

Rb

Th

isw

ork

294

A-a

a_N

Am

ino

acid

sC

ys-

SH

-BS

AR

bT

his

wo

rk29

5A

-Gly

_NG

lyG

ly-N

H2-

BS

AR

bT

his

wo

rk29

6A

-His

_NH

isH

is-N

H2-

BS

AR

bT

his

wo

rk29

8A

-Ph

e_N

Ph

eP

he-

NH

2-B

SA

Rb

Th

isw

ork

299

A-T

yr_

NT

yr

Ty

r-N

H2-

BS

AR

bT

his

wo

rk30

0A

-Val

_NV

alV

al-N

H2-

BS

AR

bT

his

wo

rk30

1P

-139

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Riv

aset

al.,

2008

302

P-1

496

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Riv

aset

al.,

2008

303

P-I

A1S

2P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0830

4P

-IA

1C1

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Riv

aset

al.,

2008

305

p-I

A2S

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bT

his

wo

rk30

6p

-IA

3C1

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Th

isw

ork

307

P-I

C1S

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0830

8P

-IC

1C1

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Riv

aset

al.,

2008

309

p-I

C4C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bT

his

wo

rk31

0p

-IC

7C1

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Th

isw

ork

311

P-I

C8C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0831

2p

-IC

9C1

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Th

isw

ork

313

P-I

D4S

2P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0831

4P

-IV

E1C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0831

5P

-IV

E3C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0831

6P

-IV

E4C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0831

7P

-IV

E5C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0831

8P

-IV

E6C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0831

9P

-IV

F2C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0832

0p

-IV

G2C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bT

his

wo

rk32

1p

-IV

G4C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bT

his

wo

rk32

2P

-IV

I2C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0832

3P

-IV

I8C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bR

ivas

etal

.,20

0832

4p

-IV

I10C

1P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bT

his

wo

rk32

5p

-IV

I120

C1

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Th

isw

ork

326

p-A

BC

tran

sP

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bT

his

wo

rk32

7p

-CrR

eTs-

977

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Th

isw

ork

328

p-F

eReT

s-98

3P

re-I

mm

un

ese

rum

IgG

frac

tio

n(p

rote

inA

pu

rifi

ed)

Pre

-Im

mu

ne

seru

mR

bT

his

wo

rk32

9p

-FeR

eTs-

984

Pre

-Im

mu

ne

seru

mIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rum

Rb

Th

isw

ork

330

p-p

oo

lP

re-I

mm

un

ese

rap

oo

lIg

Gfr

acti

on

(pro

tein

Ap

uri

fied

)P

re-I

mm

un

ese

rap

oo

lR

bR

ivas

etal

.,20

08

C1,

cell

ula

rfr

acti

on

;C

2,in

solu

ble

cell

pel

let

fro

mS

100;

C3,

ED

TA

was

hed

cell

s;S

1,cu

ltu

resu

per

nat

ant;

S2,

sup

ern

atan

tfr

om

ED

TA

was

h.

Rb

,p

oly

clo

nal

anti

bo

die

sfr

om

rab

bit

;M

(Mab

),m

on

ocl

on

alA

bs

fro

mm

ou

se;

Mab

,m

on

ocl

on

alan

tib

od

y;

Hu

,h

um

an;

Gp

ig,

Gu

inea

pig

.*E

nv

iro

nm

enta

lis

ola

ted

fro

mR

ıoT

into

(kin

dly

pro

vid

edb

yF

elip

eG

om

ez-G

om

ez).

**A

nti

gen

s(p

uri

fied

pro

tein

s)w

ere

kin

dly

pro

vid

edb

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a microbial oasis in the hypersaline atacama subsurface...

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