Underground Habitats in the Rio Tinto Basin: A Model for Subsurface Life Habitats on Mars

ASTROBIOLOGYVolume 8, Number 5, 2008© Mary Ann Liebert, Inc.DOI: 10.1089/ast.2006.0104

Special Paper

Underground Habitats in the Río Tinto Basin: A Model for Subsurface Life Habitats on Mars

David C. Fernández-Remolar,1 Olga Prieto-Ballesteros,1 Nuria Rodríguez,1

Felipe Gómez,1 Ricardo Amils,1,2 Javier Gómez-Elvira,1 and Carol R. Stoker3

Abstract

A search for evidence of cryptic life in the subsurface region of a fractured Paleozoic volcanosedimentary de-posit near the source waters of the Río Tinto River (Iberian pyrite belt, southwest Spain) was carried out byMars Astrobiology Research and Technology Experiment (MARTE) project investigators in 2003 and 2004. Thisconventional deep-drilling experiment is referred to as the MARTE ground truth drilling project. Boreholeswere drilled at three sites, and samples from extracted cores were analyzed with light microscopy, scanningelectron microscopy–energy dispersive spectroscopy (SEM-EDS), X-ray diffraction (XRD), and Fourier trans-form infrared spectroscopy. Core leachates were analyzed with ion chromatography, and borehole fluids wereanalyzed with ion and gas chromatography. Key variables of the groundwater system (e.g., pO2, pH, and salin-ity) exhibit huge ranges probably due to surficial oxygenation of overall reducing waters, physical mixing ofwaters, and biologically mediated water-rock interactions. Mineral distribution is mainly driven by the pH ofsubsurface solutions, which range from highly acidic to neutral. Borehole fluids contain dissolved gases suchas CO2, CH4, and H2. SEM-EDS analyses of core samples revealed evidence of microbes attacking pyrite. TheRío Tinto alteration mechanisms may be similar to subsurface weathering of the martian crust and provide in-sights into the possible (bio)geochemical cycles that may have accompanied underground habitats in extensiveearly Mars volcanic regions and associated sulfide ores. Key Words: Río Tinto—Subsurface habitats—Mars ana-logue. Astrobiology 8, 1023–1047.

1023

Introduction

THE DISCOVERY OF SULFATE DEPOSITS ON MARS (Gendrin etal., 2005; Squyres et al., 2004, 2006) and the predomi-

nance of iron minerals on that planet support the idea thatiron and sulfur geochemistry prevailed as essential pro-cesses that may have played a role in modifying surfaceand subsurface martian environments. Moreover, giventhat Mars has lost its atmosphere, which would have pro-vided greenhouse warming and an ultraviolet shield(Fanale et al., 1992; Squyres and Kasting, 1994; Brain andJakosky, 1998), the most obvious region to search for po-tential habitats on Mars is the subsurface (McKay, 2001).Aquifers of acidic brines would have provided the ingre-dients needed to support a feasible metabolic pathway for

organisms in martian aquifers in the past or, plausibly, inthe current epoch; putative acidic cryptobiospheres can besustained by iron and sulfur chemolithotrophy. The studyof terrestrial analogues with Mars-like geochemistry is es-sential preparation for those involved in the search for ev-idence of possible microbial life on Mars. To this end, theMars Astrobiology Research and Technology Experiment(MARTE) (Stoker et al., 2004) focused on the exploration ofsubsurface deposits at Río Tinto, Spain. Drilling, asepticsampling, and associated analysis was performed to searchfor life in a sulfide deposit and the surrounding sulfide-free rocks located at Peña de Hierro, an abandoned minesite located in the catchment area of the Río Tinto River,Iberian pyrite belt, southwest Spain (Fig. 1) (Fernández-Remolar et al., 2004; Stoker et al., 2004).

1Centro de Astrobiología (INTA-CSIC), Torrejón de Ardoz, Madrid, Spain.2Centro de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.3NASA Ames Research Center, Moffet Field, California.

FERNÁNDEZ-REMOLAR ET AL.1024

FIG. 1. Geological, stratigraphical, and hydrogeological features of the Peña de Hierro area. (A) Geological map showingthe location of Boreholes 1, 4, and 8, the geometry of normal and thrust faults, and the lithological units in the area. (B)General cartography of the Río Tinto fluvial basin. (C) Lithology and phreatic level of Boreholes 1, 4, and 8.

Artesian springs that outcrop along faults at the southernend of Peña de Hierro source the Río Tinto (Fig. 1). The riverhas an average pH of 2.3 along its 100 km length but reachesa maximum pH of 4 in the river estuary when it contacts theAtlantic Ocean (Fernández-Remolar et al., 2003). The artesiansprings host microbial communities that are comprised of arange of acidophilic iron- and sulfur-oxidizing microorgan-isms, some of which are facultative anaerobes (González-To-ril et al., 2003; Amils et al., 2007). The MARTE project inves-tigated the hypothesis that a subsurface microbial ecosystem(referred to here as a “bioreactor”), dependent on the anaer-obic oxidation of iron and sulfur minerals, might be livingin the Peña de Hierro subsurface aquifers. To test this hy-pothesis, rock and groundwater fluids were sampled fromdifferent depths in a sulfide deposit and from rocks of shalecompositions located down gradient (in a groundwater flowsense) from the sulfides. The sampling points were selectedto determine whether the microbial populations of the com-munities change as a function of systematic changes in thefluid geochemistry (i.e., subsurface aquifer fluids prior tocontact with sulfides, fluids in contact with sulfides, fluidsin shales down gradient from the sulfides, and post-surface-emergence river fluids).

We characterized the Peña de Hierro subsurface on the ba-sis of core mineralogy and petrography, leached ions fromcore samples, and groundwater fluids. In this way, we couldestimate the environmental parameters and hypothesizeabout the types of biogeochemical processes that could re-sult from interactions between the microbial communitiesand the underground water-rock matrix.

In 2003 and 2004, deep drilling and coring were carriedout by way of a commercial coring rig at Peña de Hierro, atthree locations designated Boreholes 1, 4, and 8 (Fig. 1). To

study spatial changes in microbial and hydrogeochemicalprocesses, approximately 165 m of the pyrite stockwork wascored from Boreholes 4 and 8, and 59 m of dark shale wascored from Borehole 1. The drill cores were selected to allowus to sample the hydrothermally emplaced sulfide depositsand the volcanosedimentary rock that hosts the sulfide ores.

Geological and Hydrogeological Settings at the RíoTinto Headwaters

The Peña de Hierro area (Fig. 1) lies on the north flank ofthe Río Tinto Anticline, which comprises a thick vol-canosedimentary succession composed of, in order of de-creasing age, basaltic lavas, rhyolitic materials, fine ashes andtuffites, and green/purple shales and dark shales. Hy-drothermal activity has left a record of massive sulfide lensesor stockwork veins (Fig. 2) that include pyrite and quartz,which occur at the upper part of the Iberian pyrite belt vol-canic sequence (Leistel et al., 1998).

The Peña de Hierro stratigraphy is inverted as a conse-quence of tectonism associated with the Hercynian orogeny,which produced an inverted anticline that propagates alonga 110°N thrusting front (Figs. 1 and 2). This compressivestructure is intersected by NNE-SSW normal fractures thatdirect the flow and emergence of acidic streams that encir-cle Peña de Hierro (Fig. 2). The inverted stratigraphy is over-lain by a Tertiary age gossan unit that originated from thein situ weathering of the sulfide complex (Figs. 1 and 2).

The complete sedimentary record of the Peña de Hierro isthe result of 3 different events that started in the Late De-vonian to the early Carboniferous oceans and ended in Ter-tiary to Quaternary times when the Paleozoic deposits werestrongly weathered. At the end of the Devonian (around 360

SUBSURFACE HABITATS IN THE RIO TINTO MARS ANALOGUE 1025

FIG. 2. Photograph of the Peña de Hierro open pit outcrops showing the relationship between geometry of faults (F1 andF2) and gossan deposits (Gs) that evidence fluid movement from surface to aquifers during the Tertiary; the At (gossanizedacidic tuff) and St (stockwork with iron oxides) lithologies are the same as those represented in the vertical log of Borehole4, Fig. 1C.

Ma), the first Varsican extensional event divided a homoge-nous Devonian shallow platform that formed on the South-Portuguese Iberian Terrain and partitioned it through nor-mal faulting into several tectonic blocks (Colmenero et al.,2002). Regional extension led to the development of conti-nental and marine volcanosedimentary systems that were as-sociated with the accumulation of a thick volcanoclasticrecord comprised of several volcanosedimentary sequences(Leistel et al., 1998), which consisted of basalts, andesites, andrhyolites. In the Peña de Hierro and Río Tinto areas, thispetrological record corresponds to a second felsic volcanicepisode (second ryolithic sequence VA2 as shown in Leistelet al., 1998) that produced Tournaisian (�355 Ma) depositscomposed mainly of pyroclastic and rhyolitic deposits cross-cut by peperitic sills (Boulter, 1993; Colmenero et al., 2002).The submarine magmatic activity induced convection withinfractured, brecciated, porous rocks of the ocean floor. As aresult, the volcanosedimentary sequences experienced in-tense hydrothermal alteration (i.e., chloritic/sericite haloes)and extensive iron sulfide deposits, which appear as stock-work pyrite-quartz networks that were emplaced within thesedimentary structures.

During the Upper Visean (�334 Ma), the convergence be-tween the South Iberian Zone and the autochthonous Ossa-Morena Zone reworked the volcanosedimentary materials,which produced a diachronous, highly folded sequence, theCulm Group, in the form of dark greywackes and shales thatcovered the Carboniferous volcanosedimentary record. Theconvergent plate movement culminated in an extensive col-lision that deformed the sedimentary record, which had orig-inated under the extensional early Carboniferous episode.The main impact of the regional tectonic activity was the de-velopment of a fold and thrust belt that propagates south-ward along the Paleozoic sequence (Quesada, 1998). In thePeña de Hierro area, the fold and thrust belt has inverted theentire Paleozoic sequence along a thrusting front that runsalong layers of post-volcanic dark purple shales. The pres-ence of in situ gossan deposits on Peña de Hierro supportsthe hypothesis that acidic weathering of the sulfide ore bodyoccurred during the Tertiary period [65–2 Ma, Moreno et al.(2003)]. Glauconitic marine sands that cover the gossan onthe Las Cruzes ore body (Seville) were dated as 6.7 millionyears old (Moreno et al., 2003), so the gossanized alterationhorizon associated with the sulfide ore bodies should pre-date the Tortonian (Upper Miocene 11.6–7.3 Ma). Moreover,the age of an old Río Tinto ferruginous aqueous sedimen-tary terrace at El Alto de la Mesa (Fernández-Remolar et al.,2005) indicates that an acidic river environment has been inexistence since the Gelasian (Upper Pliocene). Human activ-ity during the Bronze Age interfered with the natural sys-tem and, since around 3800 BP, has exposed sulfide materi-als to the atmosphere (Nocete et al., 2005). Mining activitycontinued throughout the Tartessian domain (3200 BP), andthe Phoenicians, Carthaginians, and Romans (2800–1800 BP)sustained an intense period of mining (Pérez Macías, 1996;Carrasco Martiañez, 2000). During the Visigothic and Ara-bian kingdoms (1500–500 BP), mining of the Iberian pyriticore bodies was reduced to a few locations or a simple har-vest of sulfur and iron from the Río Tinto sediments. Min-ing activity was somewhat reactivated again when the Span-ish Crown (ca. 1600–1700) acquired the gold and silver minesin the region. During the early 1800s, however, the United

Kingdom began an era of extensive mineral extraction thatled to the creation of open-pit mines. This type of mining ac-tivity significantly impacted the landscape of the area bychanging the relief and exposing millions of tons of ore bodyto the atmosphere. As a consequence, the weathering rate ofsulfides increases during the rainy seasons when the acidicsurface solutions mix with the subsurface artesian fluids thatsupply the river headwaters (Fernández-Remolar et al., 2003).

Interestingly, the presence of several in situ gossan levels,along with three iron-rich sedimentary terraces that wereoriginated by the river (Fernández-Remolar et al., 2005), sug-gests that several past warm and wet climatic periods alsoresulted in escalated weathering of the sulfide ores. By con-sidering the geochronological data mentioned above, re-searchers have concluded that these climatic episodes wouldhave started during the late Miocene (�6.5 Ma) and contin-ued in the late Pliocene (2 Ma). The existence of intermedi-ate-age and young ferric terraces imply that at least twoepisodes of weathering occurred during the Quaternary (Fer-nández-Remolar et al., 2005).

While the Río Tinto is an interesting geochemical analogueto some areas of Mars, the geotectonic environment repre-sented at Río Tinto is obviously not replicated anywhere onMars. Other differences include surface temperatures thatwere never as high on Mars as in the Río Tinto Basin, andthe supply of oxygen to the martian crust might never haveattained oxidation rates as high as those of the Río TintoBasin. In spite of these differences, the initiation of hy-drothermal processes on Mars (due, perhaps, to volcano-iceinteractions, impact-related hydrothermal activity, or veryearly plate tectonic-type activity) followed by oxidativeaqueous activity may have produced rocks roughly similarin geochemistry to those present at Río Tinto, even if the tec-tonic histories of the sites are very different. There is a greatdeal to be learned at Río Tinto, much of which may apply tothe study of Mars’ unique history. This is especially true withregard to the study of some geochemical processes mediatedby seasonal redox cycles for sulfur, iron, and carbon.

Techniques and Methods

To explore and describe the underground habitats of theRío Tinto basement demands a multidisciplinary approachthat involves both geological and microbiological methodsin the field and in the laboratory as well. First, geologicaland hydrogeological surface surveys and mapping were car-ried out to determine the spatial distribution of the geolog-ical and hydrogeological units associated with the putativeacidic bioreactor. Later, fast-turnoff transient electromag-netic geophysical sounding (Jernsletten, 2005) was appliedto localize the subsurface aquifer that sources acidic head-waters of the Río Tinto and to help plan the drilling for Bore-holes 1, 4, and 8. The well locations were selected to studymicrobial and hydrogeochemical processes and monitor spa-tial changes from the putative bioreactor zone in the sulfidedeposit to the downstream sulfide-free locations where thereactions would, hypothetically, be completed.

In September and October of 2003, Boreholes 1 and 4 weredrilled on Peña de Hierro (Fig. 1), and in September 2004 anadditional core was obtained from Borehole 8, which is closeto Borehole 4. Coring was performed with a Boart-Longyear(Salt Lake City, UT) HQ wireline system that produced 60


mm diameter cores within a plastic liner. Since the acquisi-tion of sterile samples was imperative for analysis of the sub-surface microbiology, a chemical tracer (NaBr) was incorpo-rated into the drilling fluid to identify possible samplecontamination produced during drilling. Cores were re-trieved in 1 meter sections, which were encased in plasticliners. These liners were flushed with N2 gas at the borehole,sealed, and then transported to a field laboratory within onehour. The laboratory was located at Museo Geominero in thetown of Ríotinto, which is approximately 6 km from the bore-hole.

In the laboratory, the cores were placed in an anaerobicchamber filled with nitrogen gas. Reducing conditions weremaintained in the chamber by inflow of hydrogen. Powderedsamples were extracted from each 1-meter section of core byfirst cutting open the liner, then breaking open the core witha pneumatic bladed rock cutter, and grinding powderedsamples out of the center of the fresh core face with a ster-ilized bit placed in a rotary mill. All these procedures wereperformed inside the anaerobic chamber. Sample sets for bi-ological, geochemical, and geological analysis were extractedfrom each location in the core segment that showed evidenceof alteration. If no alteration was seen, the samples were ex-tracted from the approximate middle of the core section. Allequipment touching the rock cores was cleaned and steril-ized prior to use on each sample.

Immediately after drilling, wells were completed by in-stalling PVC casings set in clean gravel packing. Perforatedscreens were inserted in the casing to sample groundwatersin areas of interest, which were selected according to pre-liminary field results (Figs. 3, 4, and 5). The screened inter-vals were isolated by bentonite plugs in the gravel pack andinflatable packers or baffles inside the casing. Multi-level dif-fusion samplers (MLDS) were installed in the screened in-tervals and used to measure dissolved constituents at 0.5 mintervals. The MLDS consisted of PVC rods on which werestrung (1) PVC baffles to isolate the sampling intervals, (2)heat-sealed, gas-filled segments of polyethylene tubing to actas gas diffusion cells, and (3) water-filled polyethylene mi-crocentrifuge tubes capped with porous nylon membranesto act as liquid diffusion cells. The MLDS were left in placefor several months to allow borehole fluids to equilibratewith fluids from the surrounding formation.

Geochemical and gas analysis was performed, as follows:rock leachates were produced by adding 5 ml sterile anoxicwater to 0.5 g powdered core subsample and allowing themto stand for 1 hour in an anoxic chamber before filtrationthrough pre-rinsed nylon 0.2 �m filters. Water samples fromthe MLDS for metal analysis were filtered through 0.2 �mfilters then adjusted to a 0.5 N HCl solution. Anion concen-trations were determined on another filtered aliquot by ionchromatography by way of a Dionex (Sunnyvale, CA) 4010isystem with an AS14A column, bicarbonate-buffered eluent,and suppressed-conductivity detection. Transition metalconcentrations were determined by ion chromatography byway of a CS5A column, a pyridine-2-6-dicarboxylic acid elu-ent, and post-column reaction with 4-(2-pyridylazo) resorci-nol followed by colorimetric detection at 530 nm. Dissolvedgases in borehole fluids were sampled by allowing them toequilibrate across a submerged sealed polyethylene tube forseveral months. Tubes were removed to the field laboratoryand analyzed within 1 hour by gas chromatography by a

Carle gas chromatograph with a molecular sieve column andthermal conductivity detector, with use of purified N2 as acarrier gas.

Mineralogical and geochemical analyses of rock sampleswere performed by X-ray diffraction (XRD), reflective andtransmissive light microscopy, and variable pressure scan-ning electron microscopy coupled to an electron dispersivespectrograph. Tables 1–3 show the procedures performed oneach sample analyzed. X-ray diffraction for the characteri-zation of mineralogy was performed on crushed rock sam-ples with a Seifert XRD 3003 TT diffractometer system operating in a diffraction range between 5° and 60°. Mi-crostructural and microcompositional analysis was accom-plished by coating rock samples with gold by way of aSC7620 Thermo VG Scientific sputter-coating device, thenanalyzing them with a JEOL JSM-5600V scanning electronmicroscope coupled to an Oxford INCA X-sight EDAX En-ergy Dispersive X-ray Microanalysis Probe.

Samples for biological analysis were prepared asepticallyunder anaerobic conditions. Culture-independent detectionof microorganisms was done by epifluorescent microscopyafter staining the samples with 4�,6-diamino-2-phenylindoledihydrochloride (DAPI) and by fluorescent in situ hy-bridization (FISH) with specific probes (FISH and CARD-FISH) (see González-Toril et al., 2006). Chemolithotrophic en-richment cultures were performed in minimal Mackintoshmedia with the addition of ferrous iron (20 gL�1) or sterilerock samples to provide electron donors (González-Toril etal., 2006). Anaerobic enrichments for denitrifying microor-ganisms were performed as described by Stevens andMcKinley (1995) with the addition of 10 gL�1 Na2S2O3 and1 gL�1 KNO3. Anaerobic enrichments for sulfate reducerswere performed as described by González-Toril et al. (2006)and for anaerobic methanogen enrichments as described bySanz et al. (1997).

Spatial integration of acquired data by depth was essen-tial to characterizing the aquifer environmental conditionsand, hence, the underground habitats of the putative Peñade Hierro bioreactor zone (Figs. 3–5).

Peña de Hierro Subsurface Data from Core Sampling:Stratigraphy, Mineralogy, and Geobiology

Shale lithologies were characteristic of the samples col-lected from Borehole 1, whereas Boreholes 4 and 8 (compareFig. 3 to Figs. 4 and 5) contained gossan and pyrite stock-work. Situated to the south of the major thrust fault (Fig. 1),Borehole 1 was drilled through 59 m of a monotonous lithol-ogy dominated by dark greywacke shales, on which threedifferent horizons with distinctive degrees of alteration aresuperimposed: a weathered shale horizon (�7.5 to �13 m),a non-altered horizon (�13 to �40 m), and an altered hori-zon (below �40 m). Above the shales, 7.5 m of mine tailingsoccur. Below �40 m, the shale is dissected by quartz veinsand heavily altered to a dark clayey matrix with a mineral-ogy similar to that of the upper non-altered horizon but withincreasing sericite and chlorite content and secondary sul-fides that appear as new phases. Some veins that cross cutthe shale are carbonate-bearing, a reflection of the chemistryof fluids accompanying their emplacement during faulting.

Drilling operations at Borehole 4 recovered 166.35 metersof cores from which six different horizons can be recognized


(Fig. 4). From the surface at �11 m to around �89 m, fivedifferent alteration zones could be identified as distinct hori-zons above the water table: (i) an acid volcanosedimentarytuff rich in iron oxides, (ii) an in situ gossan with a matrix ofhematite and goethite (Figs. 6A and 7), (iii) a highly alteredpyrite stockwork weathered to iron oxides and sulfates (Fig.8B, 8G), (iv) a low alteration stockwork (Figs. 6B and 8H)with occasional traces of sulfates and iron oxides (Fig. 8A),and (v) a cherty chlorite–rich stockwork that shows local ev-

idence of alteration or secondary mineral generation as cryp-tocrystalline oxides and carbonates (Fig. 9C).

Evidence of acidic alteration such as pyrite and silicate dis-solution along with an increasing pH (Fig. 4) occurs in lo-calized places in the stockwork. Evidence of dissolutionabove –89 m includes traces of hydrothermal minerals (i.e.,barite) coated by iron oxides (Fig. 8A). Between –89 m and–159 m, the pyrite stockwork contains cracks and pores filledwith sulfates and oxyhydroxides (Fig. 8F), which have a max-


FIG. 3. Log of Borehole 1 showing lithology, pH of rock leachates, sulfate and bromide concentration in rock leachate(bromide) and fluids (bromide in solution), as well as ion and gas concentration in underground water (ions and gas con-centration in ppm). Sodium bromide was used as a chemical tracer included in the drilling fluid, and the bromide con-centration from the rock leachate was used to calculate the contamination factor.


FIG. 4. Log of Borehole 4 showing lithology, pH of rock leachates, sulfate and bromide concentration in rock leachate(bromide) and fluids (bromide in solution), as well as ion and gas concentration in underground water (ions and gas con-centration in ppm). Sodium-bromide was used as a chemical tracer included in the drilling fluid, and the bromide con-centration from the rock leachate was used to calculate the contamination factor.


FIG. 5. Log of Borehole 8 showing lithology, pH of rock leachates, sulfate and bromide concentration in rock leachate(bromide) and fluids (bromide in solution), as well as ion and gas concentration in underground water (ions and gas con-centration in ppm). Sodium bromide was used as a chemical tracer included in the drilling fluid, and the bromide con-centration from the rock leachate was used to calculate the contamination factor.

imal occurrence between �90 and �155 m. Carbonates oc-curred as vein fillings and isolated minerals (Fig. 6B) below�107 m. At �159 m and below, a chloritized tuff with dis-seminated pyrite and carbonate filled veins was found with

no evidence of any additional alteration (Fig. 6B), which sug-gests low to very low acidic weathering at this depth.

Alteration stratigraphy of Borehole 8 is somewhat differ-ent from the site of Borehole 4 (Figs. 1 and 5). The upper 31

m of Borehole 8 consists of a weathered tuff-gossan complexthat covers around 12.5 m of pyrite stockwork, which is com-prised of iron oxides. However, the unaltered stockwork ex-tends from around �43 to �151 m and alternates with twoslightly oxidized horizons with iron oxides starting at �66m and �103.5 m (Fig. 5). From �151 m to �166 m, the pyritestockwork shows millimeter- to centimeter-size fracturesfilled by sulfates and iron oxides.

Scanning electron microscopy and energy dispersive spec-troscopy analyses provide direct evidence of geomicrobio-logical interactions over time in the pyrite aquifer (Fig. 8).Two different lines of evidence relate the present environ-mental conditions to subsurface geobiological processeswithin the alteration horizons: the first is the secondary min-eralogy, which was correlated between the cores and thewalls of boreholes; the second is the association of distinctmicrobial communities with different mineral substrates.

Evidence of recent acidic water flows can be observed inthe gossan horizon (Fig. 7A, 7B), at around �20 m and be-low, where 2–4 mm long and 20 �m diameter fungal fila-ments adopt a consistent orientation within the fractures(Fig. 7B). The fungi are coated by ferric minerals, and they

are found in fractures that were filled first with quartz andthen iron oxides (Fig. 7C, 7D). XRD analysis indicated thatthe associated ferric mineral assemblage is dominated bygoethite (Figs. 6 and 7; Table 2), which is compatible withmoderate- to low-acidity waters. Moreover, SEM analysisshowed that intracrystalline porosity left by acid-leached sul-fides (Fig. 7B, 7D) is used by fungi and other heterotrophicmicroorganisms as microhabitats in the volcanosedimentarybasement.

Evidence of bacteria residing in micrometer-sized frac-tures in pyrite was found with the use of SEM-EDS analysis(Fig. 8B–8E) of samples taken from various depths of coresections along Borehole 4. Table 2 shows the location of sam-ples that were analyzed using this technique, which includedintact hyrothermal materials, weakly altered pyritic stock-work (Fig. 8H), highly weathered stockwork with secondaryiron oxides (Fig. 8A) and sulfates (Fig. 8G), and deep chlo-ritized tuffs (Figs. 9C) with moderate weathering and sec-ondary iron oxides and carbonates. Only a few mineral sur-faces were found to be covered by microstructures ofpossible microbial origin. Interestingly, pyrite surfaces (Figs.8C–8F and 9A, 9B) and, to a minor extent, quartz and chlo-


TABLE 1. DATASET FOR BOREHOLE 1

Core number Start depth End depth(core length) (m) (m) XRD SEM-EDS Ion chromatography* Gas chromatography FISH

1-0 0 �6.21-1 (1.10) �6.2 �7.41-2 (1.47) �7.4 �8.51-3 (1.38) �8.5 �10.151-4 (1.04) �10.15 �12.45 11-5 (0.40) �12.45 �14.65 11-6 (1.43) �14.65 �16.2 11-7 (1.57) �16.2 �17.7 X s, 1 X1-8 (1.56) �17.7 �19.15 s, 1 X1-9 (1.52) �19.15 �20.6 s, 1 X1-10 (1.73) �20.6 �22.11-11 (1.57) �22.1 �23.551-12 (1.41) �23.55 �24.95 X1-13 (1.46) �24.95 �26.45 s1-14 (1.00) �26.45 �27.91-15 (1.33) �27.9 �28.85 s1-16 (1.44) �28.85 �30.35 s1-17 (1.58) �30.35 �31.85 s1-18 (1.46) �31.85 �33.25 s1-19 (1.44) �33.25 �34.65 s1-20 (0.77) �34.65 �36.15 s1-21 (3.02) �36.15 �39.9 s1-22 (2.53) �39.9 �42.9 s1-23 (2.95) �42.9 �45.451-24 (2.34) �45.45 �47.75 X s1-25 (1.54) �47.75 �49.45 s, 1 X1-26 (1.00) �49.45 �51.55 s, 1 X1-27 (0.65) �51.55 �52.65 X s, 1 X1-28 (0.80) �52.65 �53.5 s, 1 X1-29 (1.14) �53.5 �55.5 s, 1 X1-30 (1.06) �55.5 �56.15 s, 1 X1-31 (1.12) �56.15 �57.7 s, 1 X1-32 (0.89) �57.7 �58.2 s, 1 X1-33 (0.59) �58.2 �59 s, 1 X

*s, leachate from solid sample; 1, subsurface solutions.



4-0 0 2.84-1 (1.70) �2.8 �5.2 s X4-2 (0.50) �5.2 �7.74-3 (1.37) �7.7 �9.5 s X4-4 (0.28) �9.5 �10.754-5 (1.03) �10.75 �12.35 X s X4-6 (0.46) �12.35 �13.35 X4-7 (0.55) �13.35 �14.154-8 (1.18) �14.15 �16.4 X X s X4-9 (0.40) �16.4 �18.95 X X X4-10 (0.55) �18.95 �21.45 X s X4-11 (0.28) �22.45 �22.754-12 (1.24) �22.75 �25.75 X s X4-13 (0.28) �25.75 �274-14 (1.95) �27 �28.75 s X4-15 (2.13) �28.75 �30.9 s X4-16 (3.02) �30.9 �33.95 s X4-17 (2.98) �33.95 �36.95 X X s X4-18 (3.04) �36.95 �39.95 X s X4-19 (3.04) �39.95 �43 X s X4-20 (3.02) �43 �46 s X4-21 (1.15) �46 �47.2 s X4-22 (2.65) �47.2 �49.75 s X4-23 (3.00) �49.75 �52.75 s X4-24 (3.05) �52.75 �55.75 X s X4-25 (3.05) �55.75 �58.75 X s X4-26 (3.05) �58.75 �61.75 s X4-27 (3.05) �61.75 �64.75 s X4-28 (2.85) �64.75 �67.6 X s X4-29 (3.05) �67.6 �70.6 s X4-30 (3.05) �70.6 �73.65 s X4-31 (2.95) �73.65 �76.6 X s X4-32 (3.09) �76.6 �79.55 X X s X4-33 (3.00) �79.55 �82.55 s X4-34 (3.00) �82.55 �85.55 s X4-35 (3.00) �85.55 �88.55 X s, 1 X X4-36 (1.80) �88.55 �90.35 X s, 1 X X4-37 (–) �90.35 �90.354-38 (3.05) �90.35 �90.4 s X4-39 (2.95) �90.4 �96.35 X s, 1 X X4-40 (3.00) �96.35 �99.35 s, 1 X X4-41 (3.05) �99.35 �102.3 X s, 1 X X4-42 (3.05) �102.3 �105.35 s, 1 X X4-43 (2.95) �105.35 �108.35 s, 1 X X4-44 (2.95) �108.35 �111.3 s X4-45 (3.00) �111.3 �1114.25 X s X4-46 (1.50) �114.25 �117.25 X s X4-47 (1.50) �117.25 �118.75 X4-48 (3.00) �118.75 �121.75 s X4-49 (3.00) �121.75 �124.75 X s X4-50 (2.60) �124.75 �127.354-51 (2.95) �127.35 �130.3 X s X4-52 (1.60) �130.3 �131.9 X X4-53 (1.53) �131.9 �133.54-54 (2.90) �133.5 �136.4 X X s, 1 X X4-55 (3.00) �136.4 �139.4 X s, 1 X X4-56 (3.00) �139.4 �142.4 X s, 1 X X4-57 (2.95) �142.4 �145.35 X s, 1 X X4-58 (2.95) �145.35 �148.3 X s, 1 X X4-59 (2.95) �148.3 �151.25 X s, 1 X X4-60 (1.80) �151.25 �153.15 X s X4-61 (1.90) �153.15 �155.05 s X4-62 (2.70) �155.05 �157.75 X X s4-63 (2.80) �157.75 �160.55 s4-64 (2.90) �160.55 �163.454-65 (2.90) �163.45 �166.35 s X

*S, leachate from solid sample; 1, subsurface solutions.



8-1 (1.00) 0 �4.28-2 (1.00) �4.2 �6.38-3 (0.85) �6.3 �7.258-4 (–) �7.25 �9.358-5 (0.45) �9.35 �10 s8-6 (0.35) �10 �10.558-7 (–) �10.55 �11.18-8 (–) �11.1 �12.258-9 (1.10) �12.25 �13.358-10 (–) �13.35 �13.68-11 (0.90) �13.6 �14.8 s8-12 (1.00) �14.8 �16.78-13 (2.10) �16.7 �18.88-14 (–) �18.8 �22.78-15 (0.80) �21.7 �22.58-16 (3.00) �22.25 �25.5 s8-17 (0.65) �25.5 �26.38-18 (0.90) �26.3 �27.258-19 (0.80) �27.5 �28.35 s8-20 (2.15) �28.35 �30.58-21 (1.80) �30.5 �32.8 s8-22 (1.45) �32.8 �34.25 s8-23 (2.85) �34.25 �37.1 X s8-24 (2.95) �37.1 �40.05 s8-25 (3.00) �40.05 �43.05 s8-26 (1.75) �43.05 �45.95 s8-27 (1.55) �45.95 �47.58-28 (1.20) �47.5 �48.7 s8-29 (2.95) �48.7 �51.56 s8-30 (2.95) �51.56 �54.68-31 (2.90) �54.6 �57.5 s8-32 (3.000) �57.5 �60.58-33 (3.00) �60.5 �63.5 s8-34 (2.70) �63.5 �66.28-35 (0.01) �66.2 �66.38-36 (1.70) �66.3 �68.18-37 (2.90) �68.1 �718-38 (3.00) �71 �74 s8-39 (3.00) �74 �77 s8-40 (2.95) �77 �80 s8-41 (2.95) �80 �82.95 s8-42 (3.00) �82.95 �85.958-43 (2.90) �85.95 �88.85 s8-44 (2.90) �88.85 �91.75 s8-45 (2.95) �91.75 �94.7 s8-46 (2.95) �94.7 �97.65 s8-47 (2.95) �97.65 �100.6 s8-48 (3.00) �100.6 �103.6 s, 1 X8-49 (3.00) �103.6 �106.6 s, 1 X8-50 (3.00) �106.6 �109.6 s, 1 X8-51 (2.95) �109.6 �112.55 s, 1 X8-52 (3.00) �112.55 �115.55 s, 1 X8-53 (3.00) �115.55 �118.55 s, 1 X8-54 (2.95) �118.55 �121.5 s, 1 X8-55 (2.95) �121.5 �124.45 s8-56 (2.95) �124.45 �127.35 s8-57 (2.95) �127.35 �130.3 s8-58 (3.00) �130.3 �133.3 s8-59 (2.95) �133.3 �136.25 s8-60 (2.95) �136.25 �139.2 s8-61 (2.95) �139.2 �142.15 s, 1 X8-62 (2.95) �142.15 �145.1 s, 1 X8-63 (2.95) �145.1 �148.05 s, 1 X

(continued)



TABLE 3. DATASET FOR BOREHOLE 8 (CONT’D)


8-64 (3.00) �148.05 �151.05 X s, 1 X8-65 (3.00) �151.05 �154.05 s, 1 X8-66 (2.05) �154.05 �157 X s, 1 X8-67 (3.00) �157 �160 s, 1 X8-68 (3.00) �160 �163 X s, 1 X8-69 (3.00) �163 �166 s, 1 X

*S, leachate from solid sample; 1, subsurface solutions.

FIG. 6. XRD spectra of pyrite stockwork core samples. (A) Cores 4-8a (�13.5 m), 4-9a (�14.5 m), and 4-17a (�17.5 m) and(B) Cores 4-32b (�75 m), 4-36b (�87 m), 4-45a (�112 m), 4-52b (�131 m), 4-54a �134 m), 4-58c (�147 m), and 4-62a (�152)obtained from Borehole 4; sample depths have been provided for locating the cores in the section. Samples recovered fromthe top of the Peña de Hierro gossan (A) have been analyzed showing hematite (He) as the main mineral composition, andgoethite (Go) as a minor one. The secondary minerals detected at the top of Borehole 4 are mainly hematite (He) and goethite(Go) and quartz (Qz), which remain as a relic of the stockwork weathering under acidic conditions. In (B) primary rock-forming minerals in the sulfide ore are quartz (Qz), pyrite (Py), barite (Ba), feldspars (Fs), chlorite (Cl), and mica (Mc).Siderite (Sd), under microbial and inorganic weathering, can act as ionic sources for the acidic waters feeding Río Tinto.Weathering by-products such as iron oxides and sulfates are found in the microscope images shown in Figs. 7, 8, and 10.


FIG. 7. Core sample 4-12a obtained at around �21 m in Borehole 4 showing a quartz crack (A and B) filled with a mix-ture of iron oxyhydroxides and sulfates (schwertmannite) that preserve fungal filaments (C). Quartz porosity observed in(B) may result from the weathering under microbial attack of pyrite disseminated in a quartz matrix of hydrothermal ori-gin. The resulting porosity is now being used by microbial heterotrophs (fungi) as microhabitats (D).

rite surfaces (Figs. 8C and 9C) showed evidence of possiblemicrobial activity.

In shallower regions, where the pyrite deposits are altered,spheroidal carbonaceous structures, grouped and in somecases forming clusters, were found associated with quartzdeposits and chlorites (Figs. 8B and 9A, 9B). Below �110 m,structures enriched in carbon occur in the sulfide-rich mate-rials. At �115 m, carbonaceous networks that are clusters offilaments occur inside pyrite cracks (Fig. 8C). At �162 m,chains of encapsulated 2.5-micron-diameter carbonaceousparticles occur in fractures within highly altered pyritestockwork (Fig. 8D). In some areas, aggregates of carbona-ceous spherical structures are associated with pits on thepyrite surface (Fig. 8E). Some micron-sized sinuous marksend in pits, which is consistent with (but not proof of) mi-crobial attack on the ore.

Near the bottom of Borehole 4 (at �151.5 m) platelike cir-cular structures of likely carbonate composition are found(Fig. 9C), which could be the result of carbonate microbialmediation (Van Lith et al., 2003; Sanchez-Roman et al., 2008).These are somewhat similar to chains of encapsulated car-bonaceous spheroids found at 162 m in Borehole 8 (Fig. 8D).

Analysis of fluorescence of subsurface samples preparedby FISH also provides evidence of geomicrobial activity un-derground. Figure 10 shows an example from Borehole 8where cocci-like morphologies were detected at �40.5 m byway of the eubcy3 fluorofore (Fig. 10).

Peña de Hierro Downhole Aqueous Geochemistry

Preliminary hydrogeochemical analyses of Borehole 4 pro-vided a dynamic view of the water chemistry. The pH of un-derground waters averaged 3.5, which is in agreement withoxic waters enriched in ferric iron as observed in the river.Although the sulfate concentrations for these fluids rarelyexceed 100 ppm, they are close to 200 ppm in some samplesat interval depths between �91 and �93 m. Ferric and fer-rous ion concentrations were analyzed in the subsurface flu-ids to determine whether iron oxidation or reduction was fa-vored (i.e., when the ferric to ferrous iron concentrationexceeds 1.5, the precipitation of ferric iron compounds is fa-vored). The highest ferric concentration (164.3 ppm, pH �3.7) was found at �93.5 m, and it remained high (140 ppm,pH � 4.0) at �104 m. Ferrous ion reached a maximum con-centration of 345 ppm (pH � 4.1) at �107 m below surface.

In Borehole 8, chemical analyses of core leachates showed

high sulfate concentrations below �150 m, where some lev-els with anion enrichments higher than 10,000 ppm werefound (Fig. 5). Borehole fluid samples of underground wa-ter taken at around �100 m had a pH of 5.5 and showed ev-idence of sulfate and iron reduction in the form of black ironsulfides accompanied by H2S.

Above �110 m, sulfate concentration of borehole fluids ishigher than 458 ppm (pH � 4.0), reaches a maximum of 1400ppm (pH � 4.4 at �115.5 m), and finally decreases to 171ppm (pH � 4.4) at �158 m depth. In this borehole, ferrousiron is 5.5 times higher than ferric iron, which indicates netiron reduction in the aquifer. However, although there is adecrease in the total iron concentration down hole (Fig. 5),the ferrous iron decreases with depth much faster than fer-ric iron, which remains as a major cation in the fluid.

Underground water samples taken from Borehole 1 (Fig.3) had a pH of 6.5 above �18 m, which is comparable to thecore leachate pH of cores recovered below �30 m. Comparedto the fluid from the leachates extracted from Boreholes 4and 8, those taken from core samples from Borehole 1showed more enrichment in sulfates, averaging 193 ppm(pH � 7.0). At this site, high sulfate enrichments in rockleachates were detected below –50 m, with the highest sul-fate concentration (1,342 ppm) found at –55.8 m. The increasein pH from 6.5 to 9.9 at depths greater than �30 m (Fig. 3)strongly supports an absence of acidic chemistry at the rock-water interface; but bicarbonate, silica, or sulfide producersmight buffer the underground solutions on mineral sub-stratums. Water samples collected from MLDS emplaced inBorehole 1 also showed sulfate enrichment in all the sam-ples. The average sulfate concentration was slightly greaterthan 225 ppm (average pH � 7) and reached a maximum of709 ppm (pH � 7.2) at �57.5 m. In Borehole 1, the averageferric and ferrous concentrations, (81.7 and 56 ppm, respec-tively) were lower than in Borehole 4.

While the maximum ferric concentration (323.3 ppm,pH � 6.0) in Borehole 1 was found at –10.5 m, the ferric toferrous ratio below this depth was maintained throughoutthe water column at between 1.5 and 0.65, which suggestsnet production of reduced iron compounds. The presence ofblack deposits on the MLDS equipment was evidence of ironsulfide precipitation in the water column and, hence, the re-lease of H2S into the aquifer fluid.

Four gases—hydrogen, methane, carbon dioxide, and oxy-gen—were detected in the diffusion cells from the MLDSplaced inside the boreholes. In Borehole 1, the most abun-


FIG. 8. SEM images of various core samples with possible geobiological features that show signatures of possible micro-bial attack, source materials, and by-products of weathering that allow characterization of underground processes and habi-tats. (A) Sample 4-32a obtained at �72 m from the pyritic stockwork horizon has barite (Ba) crystals that show clear evi-dence of dissolution under acidic conditions followed by an oxyhydroxide precipitation (Oxyh) that suggests a pH increasein the aquifer. (B) Sample 4-18a obtained just below �35 m shows spheres of average size 2.5 microns located in a quartzvein. The inset EDS spectrum shows high counts per second for carbon. (C) Sample 4-46b at �115 m shows a carbonaceousnetwork attached to a pyrite deposit, which may be a microbial structure. (D) Sample 8-68c at �162 m shows chains ofsmall encapsulated 2.5-micron-sized carbonaceous particles. (E) Sample 4-54b at �134 m shows aggregates of possible mi-crobial cocci on pyrite (Py) that are associated with micron-sized pits (white arrows) connected to sinuous traces possiblycaused by microbial attack on pyrite. (F) Core sample 4-55c at �139 m depth shows iron oxide spheroids (Ox) with fibrousmicrostructure coating the pyrite (Py). Atomic percentage obtained through EDS analyses suggests there are ferrousiron–bearing oxides. (G) Sample 4-17a at �32 m depth shows iron-bearing sulfates (Fe-S) (probably jarosite) associatedwith nonfoliated phyllosilicates filling a crack. (H) Sample 4-24a at �51 m shows pristine primary mineralogy of hy-drothermal origin with pyrite (Py) and chlorite (Cl).

dant gases were carbon dioxide and methane, with an aver-age of 10,796 and 6,790 ppm, respectively, whereas oxygenand hydrogen showed lower average concentrations, 1,422and 8.6 ppm, respectively.

Interestingly, methane concentration in Borehole 1reached a maximum value at �19 m. Oxygen concentrationcorrelated inversely with that of methane to �19 m, then de-creased and became undetectable below �20 m. In Borehole4, carbon dioxide was the predominant gas, with an averageconcentration of around 7,794 ppm, and oxygen appeared ata much lower average concentration, 406 ppm. In this bore-hole, hydrogen and methane in the diffusion cells are minorcompounds that averaged 94 and 17.7 ppm, respectively. InBorehole 8, methane was found in discontinuous levels from�142.5 m to �158 m (Fig. 5). Below this, all levels showedmethane at increasing concentration from 15 to 34 ppm. Bycontrast, detectable hydrogen concentrations were foundcontinuously beginning with the shallowest sample at �101

m all the way to the aquifer bottom, where it occurred at aconcentration that ranged between 8 and 17 ppm (Fig. 5).

Evidence of sulfide precipitation suggests net productionof H2S through sulfate reduction in all three boreholes. Al-though H2S concentrations have not been measured, thegreater presence of black precipitates in Boreholes 1 and 8suggests a lower oxygen fugacity than in Borehole 4. On theother hand, the detection of sulfur oxidizers (Amils et al.,2007) in deep aquifer regions, where H2S should be producedand net reduction of the ferric cation is favored, suggests thestability of the ferrous sulfates associated with the sulfides.

As mentioned above, NaBr was used in drilling fluid soas to introduce it into the groundwater as a contaminationtracer. Underground water fluxes can be roughly estimatedby the bromide concentration in fluids sampled more than1 year post drilling (Figs. 3–5). In Borehole 1, the bromideconcentration correlates positively with the abundance ofmethane and hydrogen (Fig. 3) but is inversely related to


FIG. 9. SEM-EDS analyses of samples 8-23c (A, B) recovered from �37 m depth and 4-60a (C) recovered from �151.5 mdepth show elemental variations related to the mineral composition and microbial activity. (A) An SEM image showing apyrite surface with traces of alteration in the form of small cracks and voids and a stippled pattern (right side of image).The pyrite surface is partially covered by clusters of carbon-rich spheroidal structures. The EDS analysis shows peaks forS and Fe (A1, A2), and an intense peak for Si in one area (A3) indicates a quartz vein. (B) Magnification of (A) showing thecluster of spheroids that EDS analysis confirms are enriched in carbon (B1) compared to the pyrite surface (B2) where car-bon is not detected. (C) SEM image showing 40-micron-diameter circular plates whose elemental composition is charac-terized by C, P, Ca, and Fe (C1) whereas the composition of the spheroid-free mineral surface (C2) shows peaks for Si, Al,and Mg, consistent with a Mg rich chlorite, but lower concentration in C and Fe.

FIG. 10. Image is an epifluorescence photomicrograph obtained from sample 8-25b at -40.5 m prepared after staining thesamples with 4�,6-diamino-2-phenylindole dihydrochloride (DAPI) and by fluorescent in situ hybridization with specificprobes (FISH and CARD-FISH) to detect bacteria using the fluorofore eubcy3. The fluorescent dots are bacterial colonies.

oxygen, which suggests high influx of oxygenated waterabove �18 m. Borehole 4 water samples between �85 and�100 m were low in bromide, whereas those from below�100 m had a higher concentration of bromide. The water-table depth was 85 m. The bromide distribution is also some-what inversely correlated to the methane occurrence. The gasand bromide profiles suggest oxygenated water flow at thetop of the water table that could decrease hydrogen ormethane concentrations in the fluids. Below �130 m, the in-flow of oxygenated water is much slower, which correlateswith an increase in the concentration of methane. In Bore-holes 4 and 8, methane and hydrogen concentrations are typ-ically inversely correlated, implying methanogenesis. InBorehole 8, the bromide concentration only increases slightlydownhole to a maximum at around �153 m (Fig. 5), whichsuggests a slower underground flow and, therefore, a lesspermeable substrate.

Discussion: a Model for the Río Tinto Underground Habitats

The combination of mineral and geobiological information(Figs. 6–9) with the microbiological analysis (Fig. 10) sup-ports the hypothesis that the structures observed in SEM(Figs. 8 and 9) are microbial in origin. Differences in the mi-crobial habits at different levels and changes in the geo-chemistry of subsurface fluids support a range of environ-mental conditions in the Peña de Hierro subsurface. Thefilament clumps found in the sample 4-46b at �115 m (Fig.8C) occur in a region of the aquifer where subsurface flowis apparently higher than below �130 m. Such differences inthe underground water movement are essential physico-chemical properties of environments that affect the micro-bial communities. The association between these microbial-like structures and pyrite minerals strongly suggests there isa dependence of microbes on this mineralogy, which wouldbe consistent with iron- or sulfur-based chemolithotrophy.However, fungal-sized elements (Fig. 7C, 7D) are abundantin shallow regions; this suggests that heterotrophic eukary-otes dominate in the higher levels of the vadose zone abovethe water table.

The above SEM and optical microscopy observations areconsistent with a study (R. Amils et al., in preparation) of mi-crobial communities, which has detected iron and sulfur ox-idizers and methanogens in several horizons of the pyriticstockwork and deeper regions of the aquifer.

We present next a model of possible microbial habitats,both aerobic and anaerobic, that we have inferred from theobservations described above. Boreholes 4 and 8, in terms ofthe mineralogical, structural, hydrogeological, and geo-chemical variables, provide insight into the habitats in theiron sulfide–rich brines. Moreover, Borehole 1 has providedinsights into the aquifer in the Carboniferous shale, whichstores, rather than produces, iron- and sulfur-rich brines thatare produced upstream in the sulfide aquifers of Peña de Hierro. The geochemistry and hydrogeology of this Car-boniferous shale aquifer suggest strong anaerobic condi-tions. As sulfur and iron production are only possible in thepyrite, the occurrence of sulfides that were observed in thecores of Borehole 1 can be explained by a two-step processthat consists of the weathering of primary sulfide to sulfate

and a secondary reduction of sulfate to sulfide under re-ducing conditions.

For Boreholes 4 and 8, at least four different habitats arepossible (Fig. 11). The topmost habitat, which occurs fromthe surface to around –30 m, corresponds to a vadose envi-ronment, which receives seasonal rainwater that has weath-ered and removed nearly all the sulfide minerals. Het-erotrophs are expected to dominate the subsurface microbialcommunities that occur in the zone where ferric iron is mo-bilized via rainwater recharge during the wet season. Thepresence of fungal filaments entombed in ferrous iron likelyrepresents the terminal site of precipitation of the dissolvediron in the vadose zone during the dry seasons, where fila-ments are coated by mobilization and precipitation of dis-solved ferric iron (Figs. 7, 8B) that is supplied from waterflowing laterally or vertically during the wet season. Meta-bolic reactions are represented by Reaction 1:

(CH2O)n � nO2 � nCO2 � nH2O (1)

which corresponds to the surface and subsurface organicdegradation by fungal and bacterial communities. In the va-dose zone, diagenesis leads to hematite formation from oxy-hydroxides by simple mineral dehydration (Fernández-Re-molar et al., 2004) as shown in Reaction 2:

2FeOOH � Fe2O3 � H2O (2)

The next habitat identified (�30 to �43 m) is an oxygenicand vadose zone where sulfide minerals persist that couldmaintain a community of chemolithotrophic microbesthrough oxidation of sulfur and iron. This, in turn, wouldgenerate ferric sulfates and oxides as by-products of iron sul-fide weathering (Fig. 8A, 8B, 8G). The presence of iron andsulfur oxidizers in this region (R. Amils et al., in preparation)suggests that this process is comprised of three main reac-tions mediated by microbes, which are shown in Fig. 11 andutilize Reactions 3–5:

FeS2 � H2O � 3/2O2 � 2SO4� � Fe2� � 2H� (3)

Fe2� � Fe3� � e� (4)

FeS2 � 14Fe3� � 8H2O � 15Fe2� � 2SO4� � 16H� (5)

The third habitat occupies the saturated zone beneath thewater table where iron and sulfide oxidation occurs by mi-crobial mediation (Reactions 3 and 4). It produces not onlysulfate and ferric iron but also protons that acidify the aquiferfluids, all of which results in the generation of ferric-sulfatephases. The resulting acidic solutions attack the host rock,which includes silicates and carbonates. Silica and carbondioxide are released, as is summarized in Reaction 6:

CO3(Fe, Ca, Mg) � H� � (Fe, Ca, Mg)2� � HCO3� (6)

We hypothesize that high demand for oxygen due to mi-crobial respiration on the surface and in oxygenated areas ofthe aquifer would induce anoxia in the deeper regions. Thisoxygen uptake would support the existence of an anoxicaquifer habitat that could sustain microbial life through theanoxygenic alteration of sulfides (Fig. 11) and explain thepresence of sulfate under oxygen depletion (see Reaction 5).Obviously, this habitat could host anaerobic areas in whichoxygen is completely absent. In this case, the alterationwould be induced by the ferric iron that was previously ox-



FIG. 11. Proposed underground habitats inferred for Borehole 4 on the basis of lithology, mineralogy, geochemistry, andSEM-EDS analysis. The distribution of sulfate and oxide mineralogy suggests geochemical processes related to undergroundconditions such as acidity, oxygen content, and redox conditions. Mineralogy from the top to the bottom includes an oxi-dized iron mineralogy (goethite and hematite), an oxidized iron and sulfur horizon (ferric sulfates), a sulfur-oxidizing levelpossibly mediated by microbial reduction of iron (ferrous sulfates), and a reducing iron mineralogy (secondary pyrite). Ionand gas hydrochemistry, combined with the microbial data, support iron and sulfur redox processes under anaerobiosis.Presence of methane and detection of methanogens strongly support the dominance of anaerobic environments that favoriron and sulfur reduction at the bottom of the aquifer.

idized either in shallower areas or in deeper areas exposedto oxygenated waters (possibly introduced through frac-tures). Such mechanisms could produce sulfates, ferrousions, and hydronium ions after the ferric consumption andsulfide oxidation (Schippers and Sand, 1999). Such a processhas been reported in Río Tinto surface water masses as fer-ric reduction with sulfide as electron donor, and the simplereduction of iron by microbial respiration as well (Malki etal., 2006). The acidification of the fluid can be partiallybuffered by silicate weathering but also by carbonate weath-ering that introduces carbon dioxide into the deep anaero-bic habitat. Heterotrophic metabolism could also be a sourceof carbon dioxide; organic matter degradation with ferriciron as the terminal electron acceptor is another way of gen-erating carbon dioxide and reducing iron (Bridge and John-son, 1998).

The higher hydrogen influx (i.e., decrease in pH) in thewater column between –91 and –105 m (Fig. 11) suggests hy-drogen generation through inorganic pathways (Drobner etal., 1990; Rickard et al., 1997), though microbial sources ofhydrogen should not be discarded, given that bacteria canproduce H2 at much higher rates than abiotic processes. Indeeper areas, however, methane occurs at a higher concen-tration than hydrogen, which could be a consequence of hy-drogen consumption at deeper aquifer regions or the in-creased production of methane through methanogenesis,which has been detected at the surface (Rodríguez et al., 2004)and in the subsurface (R. Amils et al., in preparation). Giventhat carbon dioxide, present as dissolved inorganic carbon,is also needed for methanogenesis, we infer that pyrite bioleaching would favor methanogenesis. Methanogens, ifpresent, would produce methane, as represented by the netReaction 7:

CO2 � 4H2 � CH4 � 2H2O (7)

The possibility of microbial sulfate reduction in Borehole4 adds another possible mechanism for recycling organicmatter in the anaerobic habitat, which can be represented bythe net Reaction 8:

SO4� � 5H2 � H2S � 4H2O (8)

The detection of secondary iron sulfides in subsurface flu-ids, which was performed with use of Chemin 4 (Sarrazin etal., 2007), supports the idea that sulfate-reducing bacteria cangrow and produce H2S, which is known to produce pyriteand release hydrogen after reacting with ferrous iron (Drob-ner et al., 1990; Rickard et al., 1997). This is also an effectivemechanism for producing hydrogen in the environment asshown in Reaction 9 (see also Fig. 11):

2H2S � Fe2� � FeS2 � 2H2 (9)

Iron may also be oxidized by oxygen-mediated microbialrespiration that might occur under microaerophilic condi-tions in the aquifer. The mineral expression of all these pro-cesses operating in the microaerophilic region of the aquiferwould be ferrous mineralogies bearing sulfate or sulfidegroups (Fig. 11).

The more localized oxidation found in Borehole 8 samplessuggests that the prevailing conditions there are anaerobic.In this site, therefore, there may be little or no oxygenicaquifer. Pyrite weathering, which occurs in the highly frac-tured area below �150 m, would be the main process that

causes hydrogen and sulfur recycling through the pyrizita-tion process (Reaction 9) (Drobner et al., 1990). Ferric ironsources may originate in the Borehole 4 area and be trans-ported through transverse faults to the anoxic aquifer locatedin Borehole 8. However, the existence of an altered stock-work with oxide and sulfates, beneath gossan deposits, sug-gests that ferric iron is produced above by rainfall input ofoxygen. The detection of microbe-like structures in Borehole8 by SEM-EDS (Fig. 8D) suggests that the geochemical pro-cesses driving the pyrite weathering under aerobic andanaerobic conditions may be produced microbiologically.

The hydrogeological, geochemical, and mineralogical dataobtained in Borehole 1 also suggest the existence of an anaer-obic aquifer in which reducing gases such as H2S andmethane are produced and hydrogen is exhausted (Figs. 3and 12). Given the alteration degree of the host rock, thisaquifer must be located below �48 m, where the fluids areable to alter the rock matrix to phyllosillicates. Sulfate andferric ions are sourced and transported from the pyrite orebody through faults. Anaerobic, pH-neutral conditions exist(Figs. 3 and 12) at this depth. The fluids probably originate


FIG. 12. Underground habitats inferred for Borehole 1.This anaerobic habitat has neutral pH (water sampled at �18m gave a pH of around 7) and is dominated by methano-genesis in the deepest horizon and aerobic sulfide oxidationat the top.

from the Peña de Hierro acidic aquifer. It is likely that lat-eral recharge from neutral aquifers, microbial sulfur-reduc-ing activity, and mineral neutralization increases the pH ofthese fluids. The thrust fault seems to affect the deeper ar-eas of both Boreholes 1 and 8. This and other reverse andnormal faults may provide a pathway to transport ions downgradient from the source area at Peña de Hierro to the greenshale aquifer.

Potential Mars Underground Habitats

Recent discoveries of surface mineral deposits on Marshave provided new insights into the environments that formpossible habitats on the planet. Oxides, sulfate, and phyl-losilicate mineralogies (Kargel, 2004b; Kargel and Marion,2004; Squyres et al., 2004; Poulet et al., 2005; Bibring et al.,2006; Wang et al., 2006) are interpreted as weathering andsedimentation by-products of the martian crust materials un-der acidic to neutral conditions. Interestingly, these materi-als are associated with weathered and non-weathered sili-cate materials that originated during volcanogenic andclimatic events that predate the dry and cold conditions char-acteristic of modern Mars (Kargel, 2004a). Although it is notclear what processes are involved in producing such miner-

alogies, iron sulfates and oxides suggest that acidic and ox-idizing conditions must be supported by surficial oxidizingcompounds sourced in the martian atmosphere (Fig. 12).Kargel (2004a) presented a broad overview of martian hy-drogeology, which included some aqueous hydrochemistryaspects, and he pointed out that the periods of aqueous ac-tivity that have affected rock compositions are also the likelyperiods when martian geomorphologic character was estab-lished. In both cases, geologically significant periods of time,but not eons of time, were involved in imprinting the sur-face and crust with its ubiquitous indicators of an active wetand warm history. Moreover, weathering in acidic solutionswithout the necessity of major climate change may be thecase, given the possible existence of acid brines on Mars thatare liquid at current environmental conditions (Kargel andMarion, 2004).

A Preliminary Model of Biogeochemical Cycles onMars Based on the Río Tinto Subsurface Regions

A model that represents the types of biogeochemical cy-cles that may have operated on Mars by analogy to the pro-cesses we observed in the Río Tinto deposits is shown in Fig.13. On Mars, oxidants and sulfate would be supplied from


FIG. 13. This figure illustrates the main features of a model for the biogeochemical/weathering history of Mars based onthe Río Tinto analogue. See the text for a detailed explanation of the weathering stages. It shows the dominant reservoirsof atmosphere, surface and subsurface rock, and possibly shallow surface water, along with the chemical reactions and in-puts in each environment.

the atmosphere, and sulfur-bearing gases, carbon dioxide,and water would be sourced from volcanism. Under thehigher UV radiation on Mars, sulfur-bearing acids and oxi-dants would be produced and provided to the planet’s sur-face in the form of an acidic-oxidizing rain. These com-pounds could eventually interact with the martian volcanicbasement and act as strong weathering agents of the surfaceand subsurface. During the neutralization of the atmosphericsolutions, acidic leaching could provide huge quantities ofsilica and phyllosilicates to the martian sedimentary basins.Along these lines, Benison and LaClair (2003) and Crowleyet al. (2007) discussed the similarities in the origin of mart-ian and terrestrial redbeds.

Normal faults that affected the martian crust may haveacted as efficient pathways for the transportation of acidicfluids to and through the subsurface regions. In the shal-lower regions of the martian basement, the influx of acidicsurface solutions likely reacted with subsurface volcanic ma-terials in such a way that silicate-bearing minerals wouldhave been weathered, and reduced elements such as ironwould have been oxidized. All other cations, such as calciumand magnesium, would have been released to the weather-ing solutions. Solutions enriched in ferric and sulfate ionscould eventually have emerged on the surface as acidicsprings from shallow subsurface fluids. Those fluids thatreached deeper regions would be expected to precipitate fer-rous and sulfidic compounds after oxidant depletion. Sucha situation exists under the current hydrogeochemicalregime at Río Tinto.

If subsurface pyrite deposits were formed as a result ofvolcanic activity on Mars, they would have provided energysources for subsurface microbial communities. Under thesecircumstances, pyrite could be weathered abiotically underoxidizing or biotically under reducing conditions, so the al-teration pattern would follow the same type of mineral andgeochemical distribution observed in the Río Tinto subsur-face. Such pyrite deposits exposed to abiotic weatheringwould likely provide extensive subsurface deposits of sec-ondary sulfides (Burns, 1988).

Surface waters of ancient martian basins would have re-ceived chemicals and sediments sourced from acidic leach-ing of the martian crust. In this sense, sulfur and iron redoxprocesses would have occurred in much the same way asthey did recently at Río Tinto, with oxidized compoundsforming near the surface and reduced species forming at thebottom of the water masses. Though these compoundswould not have occurred to any significant degree in thepresence of such surface oxidizing agents as atmospheric ox-idants and UV radiation, iron oxides could have remainedas relicts of aqueous-driven processes as a result of the dia-genetic evolution from ferric sulfates.

An early Mars with a thicker atmosphere that was sus-tained by a high gas release due to volcanism would alsohave higher production rates of oxidants than would be thecase in modern times. Under this scenario, the atmosphericproduction of oxidants and acids would have provided theoxidizing and acidifying potential to drive the weatheringprocesses that are presently observed in the Río Tinto sub-surface. However, the acidic, sulfur-rich oxidizing leachatesthat alter the Peña de Hierro subsurface are derived fromweathering of the pyrite ores when neutral rainwater per-colated through the subsurface environment via fractures

(Fernández-Remolar et al., 2003). Large sulfide depositscould have formed via hydrothermal processes in associa-tion with early Mars volcanism (Burns and Fisher, 1990a,1990b) and, when weathered under the eventual acidicweathering processes (Chevrier and Mathé, 2007), couldhave produced sulfate-bearing precipitates on the martiansurface (Burns, 1987, 1988). An atmospheric origin for theacidic and oxidizing weathering solutions could explain theextensive weathering observed by the different Mars explo-ration probes. The model shown in Fig. 13 presents this op-tion as a main source of the redox processes on Mars. How-ever, sulfide sources may have been involved as additionalagents of weathering in specific areas of unusually high sul-fide concentration (Fig. 13).

According to our model (Fig. 13), two main processeswould be expected if external sulfuric and oxidizing solu-tions were introduced into the martian silicate crust: neu-tralization of acidic solutions and oxidant exhaustion afteruptake through redox consumption. The external incomingacidic solutions should react with the martian silicate crustin several ways and release silica and cations, as was dis-cussed by McLennan (2003). In some cases, the acidic attackon feldspars and phyllosilicates of hydrothermal originmight produce weathering clays that could be transportedduring humid periods to sedimentary basins. Reduction un-der oxidant exhaustion would be a two-stage reaction: a pri-mary oxidation between the martian subsurface mineralogyand aqueous solutions, which would favor production of ox-idized ions, and a secondary process involving the reductionof oxidized compounds in deeper regions where atmo-spheric oxidants had been consumed. Here, ferric cationsand sulfate would replace the oxidants derived from theatmosphere to provide the oxidizing potential in deeper re-gions. Obviously, the oxidation of ferrous iron-bearing com-pounds would be a source for ferric sulfate production at theshallower regions of the martian crust. Under the supposedstrict anoxic conditions of the martian subsurface, the finaldestination of iron and sulfur would be secondary sulfidedeposits (Burns, 1987). These would fill the undergroundcracks that are natural conduits for underground fluids (seeFig. 13). Moreover, weathering under low concentrations ofoxidants would produce hydrogen through serpentinization(Schulte et al., 2006), which would promote the productionof methane if biological or geochemical conditions were fa-vorable.

In this model scenario, the distribution of mineralogieswould be analogous to the Río Tinto subsurface. As in thePeña de Hierro subsurface, the mineral compounds mightbe partitioned into different weathering areas that rangefrom the upper regions, where ferric deposits would domi-nate, to the deepest, where sulfides would appear followinga redox gradient that resulted from oxidant depletion (Fig.13). As in Río Tinto, ferrous sulfates would appear in a hori-zon below the ferric materials where ferric iron is reduced.Therefore, the main difference between the martian subsur-face and Río Tinto is the origin of the acidic and oxidizingfluids, but not the processes and by-products. However, laterexhumation of secondary pyrite, as well as hydrothermal sul-fide deposits, would produce a weathering fluid when ex-posed to an oxidizing atmosphere. Interestingly, Borehole 1does not contain any reservoir for the production of sulfurand iron under weathering but receives these substances


from the sulfide sources upstream in the Peña de Hierroaquifer. As indicated before, conditions in this undergroundhabitat are dominated by strong anoxic conditions with theaccumulation of sulfides and the presence of carbonates,which maintain the pH at around 7. Analogous to what wasobserved in Río Tinto Borehole 1, the presence of atmo-spheric CO2 on Mars would favor the formation of a car-bonate/bicarbonate buffer, which would play a role in thewater chemistry in the deepest regions of the martianaquifers.

Conclusions

Although more work is warranted to identify the natureof biogeochemical processes at Río Tinto, a remarkable hy-pothesis that has emerged from our data integration is that,under anoxic conditions, the reduction of ferric to ferrousiron through pyrite alteration initiates a process that pro-duces hydrogen as a final by-product, which is then avail-able for methanogenesis and sulfate reduction. Another in-teresting point is that the acidification of subsurface fluidsdue to pyrite weathering favors the release of carbon diox-ide from carbonates into solution, which is then available formicrobial activity. Given that CO2 is the most stable phaseof inorganic carbon under acidic conditions, it should be ac-cessible for microbial growth. Iron oxidation can also be ex-pected when oxygen is introduced into the system. As thismolecule is rapidly taken up by microbes, the oxygen oc-currence at deep areas of Borehole 4 remains an unresolvedpuzzle.

The detection of ferric sulfates such as jarosite in ancientMars sediments (Squyres et al., 2004) suggests the possibil-ity that underground habitats existed on Mars that were sup-ported by analogous processes to those observed in RíoTinto, where habitats are supported by iron and sulfur re-dox chemistry. The occurrence of oxygen at microaerophilicconcentrations and the ferric and sulfate ions produced atthe surface could potentially be used by underground mi-crobes to generate methane and hydrogen sulfide gases.Given that some acidic solutions could still exist in modernmartian aquifers and ferric iron has a strong buffer effect,these underground habitats on Mars could be active in thepresent. If this is the case, microbial hydrogen production,pyritization, or serpentinization (Oze and Sharma, 2005),along with oxidant production through photochemical path-ways (Krasnopolsky, 2003), would provide the hydrogen in-flux needed to sustain the different metabolic pathways ofmicrobial communities in subsurface anoxic habitats on Marsin much the same way as these processes operate in theanaerobic underground habitat of the Río Tinto. Moreover,as the production of oxidizing and acidic solutions is prob-ably external to the martian aquifers, the production of neu-tral solutions enriched in sulfides and carbonates is expected,as was observed in the Río Tinto Borehole 1 aquifer. Al-though the reduction and neutralization is assisted by pos-sible microbial processes in the Río Tinto, in early deep mar-tian aquifers, neutral waters may have formed as well aftermineral neutralization and oxidant exhaustion. Moreover, asthe presence of microbes on early Mars cannot be ruled out,such processes, from oxidizing and acidifying aquifers to re-ducing and neutral aquifers, could have been acceleratedthrough biological catalysis. Considering the Río Tinto sub-

surface as a model, we propose as a working hypothesis thatMars may have hosted, and may host in the present, two dis-tinct subsurface aqueous environments and potential habi-tats—one that is acidic and oxidizing, another neutral andreducing—that are juxtaposed; and gradients and dynamicinterfaces between the domains are logical consequences. Assuch, two different underground microbial communitiescould exist with these metabolic pathway requirements and,therefore, coexist in intermediate environments between thetwo habitats.

The occurrence of vigorous iron-based microbial ecologi-cal communities and evidence of extensive lithological andmineralogical alteration caused by these communities inplaces such as Río Tinto and Iron Mountain, California (Druschel et al., 2004), offer a new perspective on the possi-ble origins of banded iron formations and redbeds on Earthand on layered iron oxide–rich deposits on Mars. Our con-clusions support prior speculation about a possible role ofiron-metabolizing microbial life on Mars (Catling, 2000).However, it should be acknowledged that current environ-mental conditions on Mars are extremely unlike those atthese proposed terrestrial analog sites. While evidence con-tinues to suggest warmer, wetter conditions in Mars’ past,the possibility of simultaneous cryogenic, hypersaline, andacidic conditions must be considered (Marion et al., 2003;Kargel, 2004a, 2004b). How might life have originated andevolved in such multiply extreme conditions? Iron-basedmetabolic pathways may provide one possible means of extracting biochemical energy even under such extreme con-ditions. Río Tinto does not represent all these extreme con-ditions, but it is possible that the microbial adaptive schemeshypothesized for Río Tinto conditions (e.g., ion pumping toallow sulfuric acid and heavy metal tolerance) may be partlyshared by halophilic and psychrophilic microorganisms. En-vironmental tolerances and optimum growth conditions ofRío Tinto’s flora should be determined experimentally.

The weathering process in a silicate-rich crust (Fig. 13) byway of microbial mediation, if it has ever existed on Mars,would be produced through the iron and sulfur redox cou-ples used by microbial recycling. In this case, the microbialmediation could have produced intermediate sulfate-rich so-lutions via the reduction of ferric iron when oxidizing thesulfide that originates in anaerobic conditions. Thus, thepresence of these intermediate sulfate compounds could bea tracer of microbial activity in a world ruled by the geo-chemical cycles of sulfur and iron. The proposed chemicalsystem, which ranges from neutral to acidic, has been mod-eled thermodynamically at moderate to cryogenic tempera-tures (Marion et al., 2006), though some thermodynamic pa-rameters are still unavailable for a few chemical species ofinterest. Nevertheless, mineral assemblages like those atMeridiani Planum have been reproduced with that model.Interestingly, the detection of methane (Formisano et al.,2004) on Mars and salt hydrates and minerals such as jarositecould be linked to acidic brine chemistry comparable to whatis hypothesized as one possible life-bearing acidic oceanhabitat on Europa (Kargel et al., 2000, 2004b).

Acknowledgments

This study was supported by the projects NRA-02-OSS-01ASTEP “Mars Astrobiology Research and Technology Ex-


periment (MARTE)” a Mars analog drilling project to searchfor subsurface life at Río Tinto, ESP 2003 03692 “Sonda parala exploración remota del subsuelo de Marte” and ESP 2006-09487 “Estudio de los procesos de oxidación superficiales ysubterráneos en las Fuentes ácidas del Río Tinto: construc-ción de modelos geoquímicos para la interpretación de am-bientes primitivos en Marte.” Authors appreciate the strongsupport provided by the MARTE Science and TechnologyTeams, the CAB scientific and technical personnel, and theCAB director Professor Juan Pérez-Mercader. We also ap-preciate the analytical support provided by the Chemin teamlead at Río Tinto, Philippe Sarrazin, which was most usefulin the field. Finally, we sincerely appreciate the suggestionsgiven by two anonymous reviewers and the excellent edito-rial support provided by Sherry Cady, which greatly im-proved the manuscript.

Abbreviations

DAPI, 4�,6-diamino-2-phenylindole dihydrochloride;FISH, fluorescent in situ hybridization; MARTE, Mars As-trobiology Research and Technology Experiment; MLDS,multi-level diffusion samplers; SEM-EDS, scanning electronmicroscopy–energy dispersive spectroscopy; XRD, X-ray dif-fraction.

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Address reprint requests to:David C. Fernández-Remolar

Centro de Astrobiología, INTA-CSICCtra Ajalvir km. 4

28850 Torrejón de ArdozSpain

E-mail: [email protected]


Underground Habitats in the Rio Tinto Basin: A Model for Subsurface Life Habitats on Mars

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