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Physicochemical Conditions and Microbial DiversityAssociated with the Evaporite Deposits in the Lagunade la Piedra (Salar de Atacama, Chile)Nunzia Stivaletta a , Roberto Barbieri a , Federica Cevenini a & Purificación López-García ba Dipartimento di Scienze della Terra e Geologico-Ambientali, Università di Bologna, ViaZamboni 67, 40126, Bologna, Italyb Unité d’Ecologie, Systématique et Evolution, CNRS UMR 8079, Bâtiment 360, 91405, OrsayCedex, FrancePublished online: 07 Jan 2011.

To cite this article: Nunzia Stivaletta , Roberto Barbieri , Federica Cevenini & Purificacin Lpez-Garca (2011) PhysicochemicalConditions and Microbial Diversity Associated with the Evaporite Deposits in the Laguna de la Piedra (Salar de Atacama,Chile), Geomicrobiology Journal, 28:1, 83-95, DOI: 10.1080/01490451003653102

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Geomicrobiology Journal, 28:83–95, 2011Copyright © Taylor & Francis Group, LLCISSN: 0149-0451 print / 1521-0529 onlineDOI: 10.1080/01490451003653102

Physicochemical Conditions and Microbial DiversityAssociated with the Evaporite Deposits in the Laguna de laPiedra (Salar de Atacama, Chile)

Nunzia Stivaletta,1 Roberto Barbieri,1 Federica Cevenini,1and Purificacion Lopez-Garcıa2

1Dipartimento di Scienze della Terra e Geologico-Ambientali, Universita di Bologna, Via Zamboni 67,40126 Bologna, Italy2Unite d’Ecologie, Systematique et Evolution, CNRS UMR 8079, Batiment 360, 91405 Orsay Cedex,France

The Salar de Atacama, located in Northern Chile, is a widesalt flat that is characterized by several salt lakes, which are lo-cally called lagunas. The Laguna de la Piedra is one of the saltlake systems that is located in the northernmost sector of the Salarde Atacama. The present paper examines some physicochemicalproperties of the Laguna de la Piedra as well as the microbial di-versity of the evaporite deposits. Under extreme desiccation andambient UV flux, the evaporite deposits can create favorable en-dolithic microniches for the development of microorganisms. Inthe Laguna de la Piedra these deposits host a variety of halophilicmicroorganisms, which were investigated by using an optical andenvironmental scanning electron microscope (ESEM) as well asmolecular diversity studies based on the small subunit ribosomal(SSU) rRNA of Bacteria, Archaea and Eucarya. We detected a sin-gle phylotype of halophilic archaea and a oxytrichid ciliate. Withinthe bacteria, a variety of Cyanobacteria, Bacteroidetes, Alpha-,Beta- and Deltaproteobacteria, as well as members of the candi-date division TM6, were identified.

Keywords evaporite, endoliths, Salar de Atacama, halophiles, sulfatereducing bacteria

INTRODUCTIONThe study of the interactions between the physical settings

and biota in extreme environments enables in-depth analysisand a more comprehensive evaluation of complex ecosystemswherein certain conditions may be observed approaching thephysical limits to life on earth. The occurrence of microbial lifethat is associated with these particular environments opens upnew perspectives regarding how communities adapt and thrivein otherwise rather hostile environments. Some of these environ-

Received 21 October 2009; accepted 21 January 2010.Address correspondence to N. Stivaletta, Dipartimento di Scienze

della Terra e Geologico-Ambientali, Universita di Bologna, Via Zam-boni 67, 40126 Bologna, Italy. E-mail: nunzia.stivaletta@unibo.it

ments, and their associated parameter values – such as high UVradiation, extreme temperatures, and high variability of salinityin the oceans – could also depict the environmental conditionswhere early life arose (Kasting and Ackerman 1986; Nisbet andSleep 2001).

This, together with the fact that microorganisms were theonly inhabitants of earth for at least two billion years after lifefirst appeared, makes the study of extreme ecosystems rathervaluable for advancing those hypotheses regarding early life onearth. In addition, extreme terrestrial environments, such as theAtacama Desert, show some analogies to Martian environments(McKay et al. 1992; Navarro-Gonzales et al. 2003). Therefore,studies on their microbial communities may have implicationsfor the search of life and its putative traces beyond the earth.

Hypersaline EnvironmentsSalinity is among the most significant factors shaping the

microbial community structure (Lozupone and Knight 2007).Hypersaline environments that are characterized by salt con-centrations approaching saturation are extreme (Rothschildet al. 1994; Litchfield et al. 1998). Halophiles that thrive inhypersaline conditions include the taxa of the three domains oflife, Archaea, Bacteria and Eucarya. At the highest salt concen-trations (i.e., close to the point of halite precipitation), however,extreme halophilic archaea (haloarchaea belonging to the or-der Halobacteriales) predominate (Rodriguez-Valera et al. 1981;Oren 1994). Some bacteria of the genus Salinibacter, belong-ing to the phylum Bacteroidetes, co-occur in some hypersalineponds with haloarchaea (Anton et al. 2002) and have also beendetected in salt lakes from the Atacama Desert (Demergassoet al. 2008).

Molecular studies have revealed that a number of otherbacterial groups, such as many Cytophaga, Flexibacter and Bac-teroides (CFB group, Bacteroidetes) and Proteobacteria, are themain bacterial inhabitants of certain hypersaline environments(Demergasso et al. 2004). Microorganisms inhabiting salty soils

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of hot and cold deserts are subjected to osmotic stress due tothe high salt concentrations that are derived from accumulatedsodium, calcium, magnesium, chloride, sulfate and nitrate. Inaddition, in desert areas, environmental conditions that are dom-inated by dryness, temperature excursions, and high UV radia-tion levels force microorganisms to find refuge a few millimetersbelow the rock surface. Such an endolithic lifestyle enablessufficient amounts of nutrients, moisture, and protection forsurvival.

In spite of the recent work carried out on the microbialcharacterization of evaporitic deposits, including those of theAtacama Desert (Spear et al. 2003; Maier et al. 2004; Sørensenet al. 2005; Drees et al. 2006; Warren-Rhodes et al. 2006;Wierzchos et al. 2006; Sahl et al. 2008; Oren et al. 2009;Stivaletta et al. 2010), molecular diversity studies remainpatchy and efforts to couple local physicochemical data at themicroscale and the type of local microbial communities are stillscarce. Studies on microbial diversity in the evaporitic basinsin northern Chile usually concern salt lake waters (Zuniga et al.

1991; Lizama et al. 2000; Demergasso 2004, 2008). In only a fewcases the microbial communities of the salt lakes sediments havebeen investigated (Demergasso 2003; Dorador 2008, 2009).

Extensive evaporite deposits generally occur in saline saltlakes in arid environments. The crystalline nature of these de-posits can facilitate relatively deep light penetration (1–2 cm)into single clear crystals and create niches for photosynthetichalophilic communities (Oren et al. 1995; Douglas and Yang2002; Hughes and Lawley 2003; Dong et al. 2007; Stivalettaand Barbieri 2009). An endolithic lifestyle should therefore bepreferential in evaporite environments where the precipitatingsalts may have advantageous physical properties, such as lightcolor and hygroscopy.

The Chilean central Andes contain a large number of closedbasins that are occupied by salares, which represent a com-bination of evaporite crusts and saline lakes or playas (locallycalled lagunas) (Stoerz and Ericksen 1974; Risacher et al. 2003).One of these basins is the Salar de Atacama (northern Chile,Figure 1A–B), which is a wide system of salt lakes that includes

FIG. 1. (A) Map of northern Chile showing the main morphostructural units (after Risacher et al. 2003). (B) Location of the Laguna Cejas in the Salar deAtacama. The Laguna de la Piedra is located in the northern portion of the Laguna Cejas (square). (C) Satellite map of the Laguna de la Piedra consisting of fourdistinct hypersaline ponds. Figure available in color online.

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the Laguna de la Piedra (Figure 1C), which is a small complexof salt lakes that are the subject of the present study.

Various combinations of high solar and UV radiation, nega-tive water balance, day to night variation in temperature, highsalinity conditions, altitude, and the occurrence of hydrothermalactivity in turn make the northern Chile salt lakes conditions ex-treme.

The present study aims at characterizing the physicochemicalcharacteristics of the salt lakes of the Laguna de la Piedra as wellas their microbial diversity and relationships with the associatedevaporite deposits.

METHODS

Sampling and Physicochemical AnalysisWater and sediment samples were collected in December

2007. In situ measurements with a portable pH meter providedthe pH values and water temperature. The water samples werecollected from ponds 1 and 4 (Figure 1C), and subsequentlyplaced in 100 ml plastic bottles. Anion (Cl, SO4, Br, NO3, F)analysis was performed in the laboratory via a DIONEX ICS-90ion chromatography system. For cation (Na, Mg, K, Ca) anal-ysis, the water samples were previously filtered in situ at 0.5micron and acidified with HNO3 and subsequently analyzed inthe laboratory with an AAS (Atomic Absorption Spectrometer)Thermo S series. Moreover, the alkalinity (HCO–

3 ) was deter-mined by the acidimetric titration. In accordance with their highsalinity, the water samples required a dilution of up to 1:50,000prior to analysis.

The sediment samples were collected 1) on the southern sideof the pond 1 (Figure 1C) (samples labeled LP1), from thin,surficial salt crusts that precipitated in the loose sand next tothe shoreline, and 2) in the gypsum and halite thick precipi-tates in the pond 4 (samples labeled LP4). One sample location(LP1) was, therefore, placed in an emerged area, whereas theother is placed in submerged materials. The samples were thenplaced in plastic boxes and kept under ambient light and temper-ature. The mineral composition of the sediments was obtainedwith a Philips PW 1710 X-ray diffractometer (XRD) by usingCuKα radiation, 40 kV and 30 mA power supply. XRD patternswere collected from 3◦ to 65◦ (2θ ). The elemental compositionswere determined with an OXFORD-SATW light elements X-rayspectroscope (EDX).

Optical and Environmental Scanning Electron Microscopy(ESEM)

Within a few days after sampling, small aliquots (2 ml) ofwater samples were centrifuged at 5000 rpm for 20 minutes inorder to concentrate the cells. The obtained pellets were thenplaced on a microscope slide, sealed with a cover slip and ob-served with a transmitted light microscope. Freshly cut surfaceswere investigated with a FEI Quanta 200 environmental scan-ning electron microscope (ESEM) with a simultaneous GSEDand GBSD detector. The ESEM observations were performed at

the following conditions: working distance 9–12 mm, excitationenergy 20–25 KV.

DNA Extraction, PCR Amplification, Cloning andSequencing

We extracted DNA from a sample that was collected at thesampling site LP4. A small LP4 evaporite fragment was groundon a sterile agatha mortar and approximately 200 µl of thepowder that was generated was used for nucleic acid purifi-cation. Nucleic acids were extracted by using the PowerSoilDNA isolation kit (MoBio Laboratories) following the manu-facturer’s recommendations. DNA was resuspended in 50 µlof sterile 10 mM Tris/HCl, pH 8.5, and conserved at −20◦C.Bacterial SSU rRNA genes were amplified by a polymerasechain reaction (PCR) by using the bacterial-specific primer 63F(5′-CAGGCCTAACACATGCAAGTC) and the prokaryotic re-verse primer 1492R (5′-GGTTACCTTGTTACGACTT). To am-plify the archaeal SSU rRNA genes, two combinations of theprimers A-21F (5′-TTCCGGTTGATCCTGCCGGA), ANMEF(5′-GGCTCAGTAACACGTGGA) with the prokaryotic 1492Rprimer were used, both yielding amplicons of the expected size.

Eukaryotic SSU rRNA genes were amplified by using theprimers 82F (5′-GAAACTGCG AATGGCTC) and 1498R (5′-CACCTACGGAAACCTTGTTA). PCR reactions were per-formed under the following conditions: 30 cycles (denaturationat 94◦C for 15 s, annealing at 50◦C or 55◦C for 30 s, extensionat 72◦C for 2 min) preceded by 2 min denaturation at 94◦C,and followed by 7 min extension at 72◦C. SSU rDNA clonelibraries were constructed by using the Topo TA Cloning sys-tem (Invitrogen) following the manufacturer’s instructions. Wegenerated one bacterial, two archaeal and one eukaryotic SSUrRNA gene libraries, in which 48 to 96 clones were screenedfor each library. From the clones that had inserts of the expectedsize, we obtained 34, 28 and 12 long partial (800–1000 bp) SSUrRNA gene sequences for the bacteria, archaea and eukaryotes,respectively (Cogenics, France).

Phylogenetic AnalysisThe sequences obtained from the LP4 sample were compared

to those in GenBank by BLAST (Altschul et al. 1997). Weretrieved the closest hits in order to include them in an alignmentthat also contained the sequences from the closest cultivatedmembers and a few representative sequences of the major taxafound. The sequences were aligned by using MUSCLE (Edgar2004) in which the multiple alignment was then manually editedby using the program ED from the MUST package (Philippe1993).

Neighbor-joining (NJ) trees were constructed for thedifferent prokaryotic taxa in order to choose the representativesubsets of the sequences for further phylogenetic analyses.Gaps and ambiguously aligned positions were excluded fromour analysis, resulting in 655, 1000 and 1068 positions for, re-spectively, the set of bacteria, archaea and eukaryotes presented

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here. Phylogenetic trees were then reconstructed by using thesedatasets by maximum likelihood (ML) using TREEFINDER(Jobb et al. 2004), in turn applying a general time reversiblemodel of sequence evolution (GTR), and taking the among-siterate variation into account by using a four-category discreteapproximation of a � distribution. ML bootstrap proportionswere inferred using 1000 replicates.

Phylogenetic trees were viewed by using the programFIGTREE (http://tree.bio.ed.ac.uk/software/figtree/). Our se-quences were deposited in GenBank, with accession numbersGU363349-GU363384

DESCRIPTION OF THE STUDY AREAIn northern Chile several distinct morphostructural units were

identified (Risacher et al. 2003; Figure 1A), which display aregular North-South alignment. From West to East they are:

i) the Coastal Cordillera: a mountain chain (mean altitude1500 m a.s.l.) that is located close to the Pacific coast andconsisting of marine sediments and volcanic andesitic rocksof Mesozoic age;

ii) the Central Depression (altitude of 800 to 1400 m a.s.l.),where the Atacama hyperarid conditions occur, is charac-terized by detrital and lacustrine sediment of middle Tertiaryto Holocene ages. This region is considered as the driest andoldest desert on earth (Clarke 2006);

iii) the Precordillera, which is made up of Paleozoic and Meso-zoic sedimentary, metamorphic, and igneous rocks. TheCordillera de Domeyko, at an elevation of 3500–4000 ma.s.l. is the highest part of the Precordillera and it alsocontains continental evaporite formations on its easternfringe;

iv) the Pre-Andean Depression: this large intramontane basin(2500 m a.s.l.) contains the Salar de Atacama, which isthe largest evaporitic basin of Chile (ca. 3000 Km2), filledwith Tertiary and Holocene clastic and evaporite sedimentsthat are of continental origin. Anticlinal folds and domes ofgypsum and salty rocks border the Salar de Atacama on itswestern side (Cordillera de la Sal);

v) the Western Cordillera (Chilean Altiplano): this elevated(4000 m) plateau comprises rhyolitic ignimbrites and issurrounded by numerous volcanoes reaching 6500 m a.s.l.,which often delineate interior drainage basins incorporatingsaline lakes and salt flats.

The arid and semi-arid climate regime was established inthe region in the Middle Miocene (Houston and Hartley 2003;Dunai et al. 2005; Hartley et al. 2005) as a result of the uniquephysiographic characteristics that block the eastern and westernaccess to humid air. The extreme aridity of the Atacama Desertalso depends on the cold oceanic Humboldt current and thePacific anticyclone which prevent the humid air from reachingnorthern Chile (Parrish and Curtis 1982).The Coastal Cordilleraand the Central Depression, which also include the Atacama

Desert, represent the most arid regions with annual rainfalls ofeven less than 1 mm (Clarke 2006).

The environmental conditions of the salares that are locatedin high altitude areas (that also include the Salar de Atacama)are characterized by (1) high solar radiation, compared to thesea level values (Cabrera et al. 1994), due to the less dense airat high altitude, and the consequent decrease of the filtration ofthe solar radiation, (2) strong daily temperature cycles, and (3)an evaporation level that exceeds precipitation. Between 75%and 90% of the precipitation inputs in the Western Cordillera islost by natural evapotranspiration and the remaining part is lostby runoff (Salazar 1997). Moreover, recent volcanism events innorthern Chile led to the presence of some heat flow in the waterof many salares, in turn creating a range of water temperatureswithin the same lake.

Laguna de la Piedra (Salar de Atacama)The Laguna de la Piedra is the northern portion of the Laguna

Cejas, which is one of the salt lake systems that are located inthe northernmost sector of the Salar de Atacama (Figures 1B–C). The Salar de Atacama, which is the largest salt flat in Chile,is primarily characterized by permanently dry and hard crusts(Figure 2) that are produced by the repeated corrosion and re-deposition of the salt crusts during previous floods (Stoerz andEricksen 1974). Differently, soft saline surfaces and salty soils,which are related to the fluctuation of the near-surface ground-water levels, characterize the lagunas (Figure 3A). The Lagunade la Piedra comprises two high salinity main ponds and twofurther smaller ponds that are aligned in a north-south direction(Figure 1C) and occupy a variable flooded area depending onthe season.

Whereas these ponds stand out distinctly and are separatedfrom one another during the summer, the drowning of the in-termediate zones during winter, because of the rising of thegroundwater level, allows for communication among the threelargest ponds, as observed on September 2008. The laguna issurrounded by large strips of species-poor halophilic vegetation.Salt precipitates abundantly in the area (Figure 3A) because ofthe high evaporation rate and the sodium chloride-rich waters(Table 1). Whereas thick salt crusts (Figures 3B-C-D-E) pre-cipitate in the ponds of the southern part of the Laguna de laPiedra (ponds 2, 3 and 4, Figure 1C), salt precipitates as surficialpatinae and thin and soft crusts in the larger and less saline pond1 (Figure 3A, 4A) as a result of the evaporation of the inter-stitial water. Beneath the thin salt crusts and sediment surfaceof the pond 1 colored, millimetric horizons are visible (Figures4B–C). The typical chromatic succession includes a pale pinkhorizon at the base of the thin surficial salt crust, which is fol-lowed by green and purple horizons in the sediment just beneaththe crusts or patinae (Figure 4C). These chromatic triplets arelocally repeated down sediment. One of the sampling sites (siteLP1) comes from these thin surficial crusts.

Ponds 4 and 3 are characterized by thick halite crusts, whichproduce mamelon-like structures that are particularly well

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FIG. 2. (A) Panoramic view of the Salar de Atacama showing a high level of roughness of the surface. Figure available in color online.

developed in pond 4 (Figures 3 C–D), where they are approx. 1–1.5 m width. Because of the limited depth of pond 4 (approx. 1mwater depth at the sampling time) some of the mamelons appearon the surface, where the salt desiccation leads to the formationof white salt patinae (Figure 3E). By sectioning the salt crustsof the mamelons, 0.5 to 1 cm thick green horizons are visiblea few mm beneath the surface (Figures 4D–E) and are imme-diately followed by purple horizons of comparable thickness.Both horizons appear continuous and parallel to the surface.Such a colored stratification suggests the development of mi-crobial communities in evaporite deposits just below the surface(endoevaporites). The green horizons are capped by a few mmthick, beige-colored salt crust; a similar coloration, likely due –on the surface – to dusty components, also affects the underlyingsalt. A mamelon of pond 4 was the second sampling site (siteLP4).

RESULTS AND DISCUSSIONWater Chemistry. A total dissolved solute (TDS) of 143

(pond 1) and 169 g/l (pond 4), with an average pH and tempera-ture of 7.5 and 25◦C, respectively, were measured from the watersamples. Table 1 summarizes the most significant cation and an-ion components in the waters of the two saline ponds. The cationanalysis of the water determined the concentration of the ele-ments (mg/l) as follows: Na+ >Mg2+ >K+ >Ca2+. The anionanalysis determined the concentration of the elements (mg/l) asfollows: Cl– >SO2−

4 >HCO–3 >Br− >NO−

3 >F–. Both pondsfollow the same trend of element concentrations and, in partic-ular, Na and Cl reached high values with a maximum of 61130and 78502 mg/l, respectively in pond 4. The waters have a Na-Cl(SO4) composition as a consequence of the weathering of vol-canic rocks and Neogene evaporite deposits and sulfur oxidationin the crater (Riascher et al. 2003 and references therein).

TABLE 1Cations and anions of the hypersaline pool (1 and 4) waters of the Laguna de la Piedra expressed in mg/l

Sample Ca2+. Mg2+ Na+ K+ HCO−3 Cl− SO2−

4 NO−3 Br− F− TDS

LP1W 988 2415 48235 2550 149 70435 18806 1,4 27,9 0,00 143579LP4W 1248 2669 61130 2546 857 78502 22099 2,0 36,6 0,00 169051

Total dissolved solute (TDS) is expressed in mg/l.

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FIG. 3. Laguna de la Piedra (December 2007). (A) The main hypersaline pool (1) characterized by salt precipitated as thin and soft crusts. (B) The hypersalinepools characterized by thick salt crusts. (C-E) The hypersaline pool (4) showing the mamelon-like structures (approx. 1–1.5 m width), with patinae on the surfacedue to the salt desiccation (E). Figure available in color online.

Characteristics of Endoevaporite Deposits. In desert areas,the nature of the rocks is relevant for endolithic colonizationsince microbial survival is strongly affected by the interactionwith the chemical and physical characteristics of the microenvi-ronment. X-ray diffraction revealed that the sediment collectedfrom the two sampling sites (LP1 and LP4) were dominatedby halite (NaCl) and sulfates, including gypsum (CaSO4·2H2O)and glauberite (Na2Ca(SO4)2).

The nature of the colored horizons was investigated by op-tical and environmental scanning electron microscopy (ESEM)(Figures 5–6). Green horizons from both of the sampled sites

revealed the presence of subspherical (although frequentlycompressed), smooth-surfaced and green colored cells ofapprox. 5 µm in diameter (Figures 5A, B, 6A–C). The cellsare arranged in clusters (Figures 5A and B) and enveloped in athick slime. These cell clusters are interpreted as dense coloniesof cyanobacterial cocci with an abundant extra polymericsubstance (EPS), that surrounds and binds the single cell andthe colonies as a whole (Figures 5B, 6A–C). Similarly tocyanobacteria, which are among the most versatile of all livingorganisms (Krumbein et al. 2004), most bacteria are able toproduce EPS polysaccharides, either as wall polysaccharides or

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MICROBIAL DIVERSITY IN SALT LAKE EVAPORITES 89

FIG. 4. Microbial colonies in the evaporite sediments of the Laguna de la Piedra. (A–C) LP1 sampling site (23◦ 3′26.01′′S and 68◦13′1.51′′O, pool 1): thin saltcrusts and sand showing colored horizons just beneath the surface (B, C). (D, E) Thick salt (halite and gypsum) crusts form mamelon-like structures in the pond4 (LP4 sampling site: 23◦ 3′25.40′′S and 68◦13′5.26′′O). Just beneath the surface these crusts exhibit colored horizons similar to those of the previous samplingsite. Figure available in color online.

as extracellular excretions into the surrounding environments,for retaining water and nutrient resources as well as preventingwater loss from single cells (Potts 1994).

Because of bacterial lysis, the cell membrane can be dis-rupted and the cell compressed and deformed, as is likely thecase for the cell colonies that are described from the greenhorizons (Figures 6A–D) as a result of the stressed conditionsproduced by the sampling and consequent removal from theirnatural environment. A further explanation for cell deformationis their adaptation to a spore-forming stage, which occurs inorder to maintain survival under adverse conditions, such asdehydration.

The purple layers revealed a dense accumulation of cells ofapprox. 2 µm in diameter (Figures 5C–D), which likely belongto Chromatium-like, anoxygenic purple photosynthetic bacteria(Alphaproteobacteria). In both sampling sites the green and pur-ple horizons showed a random distribution of siliceous frustules

of pennate diatoms (Figure 5E). Isolated cells with a subspher-ical shape occur within the microcavities that are produced bythe dissolution of halite crystals (Figure 6D). Moreover, fibrousaggregates and tubular mineralogies (Figures 6E–F) of gypsumand glauberite occur in the samples, in turn showing the precip-itation of halite cubic crystals on their surface (Figure 6F).

The abundance of EPS substances such as those associatedwith the cyanobacterial colonies of the green horizons, increasesthe preservation potential of microbes because it exerts a pro-tective effect. Moreover, slimelike morphologies have been de-tected in the fossil evaporites (sulfates) of a sabkha environmentin the Sahara Desert (Barbieri et al. 2006), where their preser-vation has been assigned to early gypsification.

Evaporite minerals such as halite can facilitate the coloniza-tion of microorganisms because of the hygroscopic properties(Davila et al. 2008). The condensed water vapor into aqueoussolutions on the crystal surface and/or within the pore space

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FIG. 5. Transmitted light micrographs of the colored horizons from the LP4 sampling site. (A–B) Green horizon: cells of coccoid cyanobacteria (orderChroococcales) embedded in thick EPS (the jelly envelope that encapsulates cells). (C-D) Cells of purple bacteria belonging to the family Chromatiaceae family.(E) Diatoms belonging to the order Pennales. Scale bars: 10 micron. Figure available in color online.

between the crystals and the halite dissolution creates mi-croniches that are favorable for the development of microbiallife. Colonization by microbes is therefore selective and seemsto be largely dependent on the mineral composition of the par-ticles that comprise a given surface. EDX analyses (Figure 6G)revealed that the EPS of the embedded clusters and individ-ual cells are characterized by a robust carbon peak, along withamounts of calcium (Ca), sulfur (S), sodium (Na) and chlorine(Cl).

Microbial Diversity. In both of the sampled sites, we ob-served the same characteristic coupling between the green andpurple horizons. In order to explore the composition of the en-dolithic microbial community, a molecular survey of the bacte-rial, archaeal and eukaryotic SSU rRNA genes that were presentwas carried out in the submerged sampled site (LP4). We suc-ceeded in amplifying the genes from a variety of bacterial lin-eages, as well as archaea and eukaryotes.

In the case of archaea, all of the clones that were analyzedfrom two independent gene libraries constructed with differ-ent primer combinations corresponded to the same phylotype(Figure 7), in which a halophilic archaeon was closely related tothe members of the genus Haladaptatus (Roh et al. 2009). Thisfinding is in agreement with the salty nature of the environment

that was investigated. Interestingly, the members of this genushave also been isolated from sulfur springs (Savage et al. 2007),which is relevant in the light of the sulfate-reducing bacteria thatare associated with this sample (see later).

Concerning eukaryotes, all the clones from the SSU rRNAgene library belonged to an oxytrichid ciliate (Figure 8), whichare often found in hypersaline lakes (Esteban and Finlay 2003;Finlay et al. 2006). Whereas diatom frustules have been detectedfrom both of the sampled sites by using optical and ESEMmicroscopy, diatom genes were entirely missing at the LP4 site.This may be due to several factors:

i) the natural heterogeneity of these substrates, with differentdistribution of microorganisms depending on the micro-environmental conditions, and to the fact that DNA wasextracted from a relatively small sample volume;

ii) the limited number of eukaryotic clones that was analyzed,which might have prevented the detection of putative diatomgenes;

iii) finally, the last factor may be related to PCR amplificationbias. Because ciliates have two nuclei, they multiply thenumber of genes in their macronucleus and, by initiallycontributing many more gene copies per cell than other

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FIG. 6. ESEM micrographs of the colored horizons from the LP4 sampling site. (A-C) Collapsed cells embedded in extracellular substances among cubic halitecrystals. (D) Cells inside a partially dissolved halite crystal. (E-F) Gypsum crystals in fibrous aggregates (E) and tubular morphologies. (G) Representative EDXspectrum of cells and crystals.

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FIG. 7. ML phylogenetic tree showing the position of archaea in the microbial community of LP4 sampling site. Numbers at nodes are bootstrap values; onlythose >50% are provided.

eukaryote to a sample, they may bias the PCR reactiontowards ciliate genes.

Concerning the diversity of bacteria, remarkable proportionsof cyanobacteria have been detected, together with Proteobacte-

ria and CFB (Bacteroidetes) (Figure 9). All of the cyanobacterialSSU rRNA genes that were detected were grouped together andbranched very close to members of the Chroococcales genusEuhalothece (Figure 9), which is a genus of halophilic cyanobac-teria (Garcia-Pichel et al. 1998). These cyanobacteria have also

FIG. 8. ML phylogenetic tree showing the position of the oxytrichid ciliate detected in the LP4 sampling site. Numbers at nodes are bootstrap values; only those>50% are provided.

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FIG. 9. ML phylogenetic tree showing the position of representative bacterial sequences in the community of the LP4 sampling site. Numbers at nodes arebootstrap values; only those >50% are provided. Unc., uncultured.

been found to be associated with living stromatolites in thehypersaline Shark Bay (Goh et al. 2009).

A variety of clones belonging to the Bacteroidetes, particu-larly related to the Cytophagales were also present. The mem-bers of this group are most often associated with cyanobacteria.Since they are able to degrade long organic molecules, it is likelythat they live upon the exopolymeric substances produced by thecyanobacteria. The members of the versatile Proteobacteria, inparticular of the Alpha, Gamma and Beta subdivisions, havealso been detected. The representatives closely related to theChromatiaceae, which were expected based on microscopy ob-servations, were not identified.

This absence may be partially explained by the fact that a rel-atively low number of clones was explored and, consequently,we did not reach a saturation plateau for the bacterial diver-sity, in turn indicating that the diversity of bacteria is not yetfully described. Other phylotypes within the Alpha- and Be-

taproteobacteria were, however, identified, as well as a group ofDeltaproteobacteria sequences, whose closest relative in Gen-Bank comes from evaporitic crusts in Guerrero Negro (Sahlet al. 2008).

Deltaproteobacteria, which are typical sulfate reducers, mustbe adapted to halophilic life in these particular environmentssince both conditions, the presence of large amounts of sul-fate (gypsum) and salt (halite), are present at the macro- andmicroscale (Figure 6).

The largest proportion of bacterial clones that was analyzedsurprisingly belonged to the candidate division TM6 (Figure 9).The presence of these organisms may partially be explained bythe local reducing conditions that are related to sulfur cycling.The members of this candidate division, so far lacking cultivatedmembers, have been found in soil, anaerobic digesters and sul-fidic anoxic ponds (Briee et al. 2007), in turn suggesting thatthey may be anaerobic. They have also been found in a recent

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study in all of the microbial mats from the hypersaline lagoonof Shark Bay in a recent study which also indicates that theymay be halotolerant (Allen et al. 2009).

The organisms inhabiting the salty (sulfate + chloride) de-posits of the Laguna de la Piedra indicate a dual environment:on the one hand, these organisms are typically associated withhypersaline evaporitic systems, containing haloarchaea as wellas cyanobacteria and their often-associated CFB companionsand on the other hand organisms that, being necessarily halo-tolerant or halophilic, thrive under anaerobic conditions, suchas sulfate-reducing bacteria and, likely, the ones belonging tothe candidate division TM6. These anaerobic microorganismsare most likely occurring in the innermost part of the evaporiticcrusts.

CONCLUDING REMARKSIn the present work, we report diverse endolithic communi-

ties in evaporite deposits. Sulfate and chloride minerals appearto be the optimal refuge for such endolithic communities that arelocated in the extreme conditions of the Atacama Desert. Thecombination of the negative water balance, i.e., desiccation, andthe high UV radiation, in turn induced the colonization of thesubsurface rather than the growth on the surface of halophilicmicroorganisms.

The results of the present investigation showed that theendoliths in the Laguna de la Piedra encompass halophiliccyanobacteria, which occupy a few mm thickness of the translu-cent minerals located just below the surface in order to ensurethe necessary quantity of solar energy for photosynthesis. More-over, the availability of large sulfate amounts in the evaporitesediments in turn supports the activity of sulfate reducing bac-teria, such as Deltaproteobacteria.

ACKNOWLEDGMENTSThis work was financially supported by Progetto Strategico

Atacama of the University of Bologna. The authors acknowl-edge two anonymous reviewers for useful suggestions. Thanksto Bruno Capaccioni and Piero Trentini for chemical analysis ofthe water and Massimo Tonelli for technical assistance duringthe ESEM analysis.

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