Hydrometallurgy 94 (2008) 155–161

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Hydrometallurgy

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Evaluation of Leptospirillum spp. in the Río Tinto, a model of interestto biohydrometallurgy

Antonio García-Moyano a, Elena González-Toril b,⁎, Mercedes Moreno-Paz b, Víctor Parro b, Ricardo Amils a,b

a Centro de Biología Molecular (UAM-CSIC), Cantoblanco, Madrid 28049, Spainb Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid 28850, Spain

⁎ Corresponding author. Tel.: +34 915 20 19 36; fax: +E-mail addresses: amoyano@cbm.uam.es (A. García-

glezte@inta.es (E. González-Toril), morenopm@inta.es (Mparrogv@inta.es (V. Parro), ramils@cbm.uam.es (R. Amil

0304-386X/$ – see front matter © 2008 Elsevier B.V. Adoi:10.1016/j.hydromet.2008.05.046

A B S T R A C T

A R T I C L E I N F O

Available online 3 June 2008

Keywords:

Members of the Leptospiriprokaryotes in the Río Tintowell as in biohydrometallur

LeptospirillumRío TintoBiohydrometallurgyMolecular ecologyNitrogen fixationIron oxidationFISHCARD-FISH

gical operations. The main objective of this work was to better understand andcontrol industrial biohydrometallurgical processes by studying the role of chemolithoautotrophic bacteria ofthis genus in the Tinto ecosystem.Different strains of Leptospirillum were isolated from the Tinto basin and physiologically and geneticallycharacterized by Pulsed Field Gel Electrophoresis (PFGE) and DNAmicroarrays. Certain metabolic capabilities,such as iron oxidation, pyrite leaching and nitrogen fixation, were determined for each strain.Complementary molecular ecology techniques (fluorescence in situ hybridization (FISH and CARD-FISH)and 16S rRNA gene cloning) were used to study the microbial diversity and the distribution of leptospirilli

llum genus have recently been found to be not only the most representative, a natural extreme acidic habitat, but also in other acidic environments (AMD), as

along the iron gradient of the Tinto ecosystem.© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Highly acidic environments are relatively scarce worldwide andare generally associated with mining activities (Ehrlich, 2002;Rawlings, 2002; Van Geen et al., 1997). The natural oxidation anddissolution of sulfidic minerals when exposed to oxygen and waterresults in acid production and the process can be greatly enhanced bymicrobial metabolism (Ehrlich, 2002; Nordstrom and Alpers, 1999;Nordstrom and Southam,1997). Furthermore, low pH facilitates metalsolubilization, so as a result acidic water tends to be highly metal-liferrous (Johnson and Hallberg, 2003).

Río Tinto is one of the largest (92 km) extreme acidic environmentsin the world. The river rises in the heart of the Iberian Pyritic Belt, oneof the richest metal sulfide ore deposits on Earth, which has beenmined for centuries (VanGeen et al.,1997). It exhibits a rather constantacidic pH (mean value 2.3), and a high concentration of heavy metals(González-Toril et al., 2003; López-Archilla et al., 2001). Since thesulfidic ores present in the area are dominated by pyrite (FeS2), ferricion is the predominant oxidant in the river and its bufferingcharacteristics make it responsible for the constant pH of theecosystem (González-Toril et al., 2003). These extreme conditions arethe product of themetabolic activity of chemolithotrophic prokaryotic

34 915 20 10 74.Moyano), etoril@cbm.uam.es,. Moreno-Paz),

s).

ll rights reserved.

microorganisms, including iron- and sulfur-oxidizing bacteria that arefound in high numbers in its waters. Most of thesemicroorganisms areautotrophic, thus in addition to promoting the extreme conditions ofthe habitat they are also primary producers (González-Toril et al.,2003; López-Archilla et al., 2001).

Conventional microbial ecology studies showed that Leptospirillumis present in the Tinto ecosystem (García-Moyano et al., 2007;González-Toril et al., 2003; López-Archilla et al., 2001). Members ofthis genus are frequently found in metal-rich acidic environmentsassociated to metal sulfide leaching such as in biohydrometallurgyoperations or acid mine drainage (AMD) systems (Johnson andHallberg, 2003; González-Toril et al., 2003; Tyson et al., 2005). Despitethe fact that their peculiar energy transduction systemmakes reducediron their sole source of energy, they were relegated to a minor role inmetal sulfide bioleaching, with bioenergetic considerations focusingmost of the attention on the sulfur-oxidizing microorganisms (mainlyAcidithiobacillus spp.) (Rawlings et al., 1999).

It was only when the importance of ferric iron in the oxidation ofmetal sulfides was demonstrated (Sand et al., 2001) and culture-independent techniques in the study of AMD environments andbiohydrometallurgical operations were introduced that the realimportance of the members of this genus was established (Gonzá-lez-Toril et al., 2003). The physiology of Leptospirillum appearshomogeneous, contrary to other acidophilic chemolithotrotrophs,such as members of the genus Acidithiobacillus, which possessesheterogeneous metabolisms (Karavaiko et al., 2003). The members ofthe genus Leptospirillum can be grouped into three species on the

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basis of 16S rRNA gene phylogeny: L. ferrooxidans, L. ferriphilum, andthe recently described “L. ferrodiazothrophum” with only onerepresentative (Coram and Rawlings, 2002; Hippe, 2000; Tyson etal., 2005). The three species are known to be strict chemolithotrophiciron-oxidizers (Coram and Rawlings, 2002; Hippe, 2000; Tyson et al.,2005). Moreover, they are autotrophic, able to fix carbon dioxide, andmembers of at least two species are also able to fix nitrogen (Holden,1991; Norris et al., 1995; Parro and Moreno-Paz, 2003; Tyson et al.,2005). These characteristics make this group of bacteria one of themost important microorganisms in biohydrometallurgical operations(Rawlings et al., 1999). Furthermore, as a consequence of theirhomogeneous phylogeny, their identification and quantificationalong the iron gradient course of the Río Tinto can be easilyaccomplished, facilitating our understanding of the part they play inthe iron cycle in a natural environment and its extrapolation tobiohydrometallurgical operations.

Themain objective of this workwas to evaluate the role of differentspecies of Leptospirillum in the iron cycle operating in Río Tinto, amodel of interest in biohydrometallurgy (González-Toril et al., 2003).A combination of both classical microbiological methodologies(isolation and physiological characterization) and culture-indepen-dent techniques (based on the sequence of 16S rRNA gene) have beenapplied to study the ecology of this genus in the Tinto ecosystem.

2. Materials and methods

2.1. Area description and sampling

Samples were collected on May 2005 from eight stations locatedalong the Río Tinto course (Table 1 and García-Moyano et al., 2007).Water and sediment samples for DNA extraction and FISH analysiswere collected and processed as has been described elsewhere(González-Toril et al., 2006). Data on water temperature, conductivity(conductimeter Orion 122, ORíon research, USA), pH and redoxpotential (pHmeter Crison 506 pH/Eh), and oxygen concentration(Orion 810 oxymeter) were obtained in duplicate in each station.Chemical analyses of dissolved heavy metals were carried out asdescribed previously (González-Toril et al., 2006). Total iron contentand its oxidation state were determined by the 2-2'-bipyridylcolorimetric method (Easton, 1972).

2.2. Isolation and growth of leptospirillum species

Samples from Río Tinto water were inoculated in Mackintoshmedium (Mackintosh, 1978). Isolation of members of the genus Lep-

Table 1Location of the Río Tinto sample sites and physico-chemical characteristics of the watersamples

Station GPS T(°C)

pH Eh(mV)

Conductivity(mS/cm)

Fe2+

(ppm)Fe3+

(ppm)

Iz–Iz 37°43'15"N6°33'03"W

21 2.0 465 30.5 2450 6050

UMA 37°43'13" N6°33'23"W

25 2.6 360 33.9 4321 1935

NUR 37°43'22"N6°33'25"W

18 2.4 513 15.4 828 3920

ANG 37°43'15"N6°33'10"W

23 2.2 483 27.5 3358 2612

AG 37°43'29"N6°33'36"W

14 2.6 413 5.2 378 1450

3.2 37°43'20"N6°33'48"W

23 2.5 580 6.63 260 1350

SLS 37°40'05"N6°32'54"W

23 2.7 387 6.32 993 598

LPC 37°25'25"N6°36'36"W

24 3.2 519 2.5 bdl1 197

1Below detection limit (b0.5 ppm).

tospirillum was performed by serial dilutions, as described previously(González-Toril et al., 2006). Fluorescent in situ hybridization (FISH)with the specific probes for the genus was used to confirm the identityof the isolates as well as to detect contaminant microorganisms,frequently co-isolated. A complete list of the specific probes used forFISH are described in section 4.9. Leptospirillum isolates were grown in250-mL flasks containing 100 mL of Mackintosh medium. Inoculawere standardized at 2% (v/v) and cultures were incubated at 30 °Cunder constant shaking at 100 rpm. Physiological experiments wereperformed in triplicate. Moreover, the isolates were also grown inflasks containing Mackintosh medium without the nitrogen source(ammonium sulfate). In this case, to ensure nitrogen-free growthconditions, cells grown in ammonium sulfate were extensivelywashed by centrifugation with nitrogen-free Mackintosh media toremove any traces of ammonium. Growth curves were monitoredafter five cycles of growth in nitrogen-free conditions. For leachingexperiments, the iron sulfate was substituted by pyrite with anequivalent concentration of ferrous iron, together with 1000 ppm offerric sulfate. Parameters such as pH, redox potential and conductivitywere followed over time. Iron speciation was determined by acolorimetric method (Easton, 1972) and cell growth was estimatedby DAPI staining.

2.3. DNA extraction, 16S rRNA gene amplification, clone library andsequencing

Environmental samples were processed as previously described(González-Toril et al., 2006). Grown cultures were also harvested byfiltering and the filter used for extracting DNA as described inGonzález-Toril et al. (2006). Amplification of 16S rRNA gene wascarried out with the primer set 8F/1492R (Lane, 1991) as describedpreviously (González-Toril et al., 2006). Amplified 16S rRNA geneproducts (N1400 bp)were purified byGeneCleanTurbo Column (Q-BioGene Inc. CA, USA), cloned using the Topo Ta Cloning Kit (Invitrogen,CA, USA) as previously described (García-Moyano et al., 2007). Insertswere sequenced using a Big-Dye sequencing kit (Applied Biosystem)following the manufacturer's instructions.

2.4. Intact DNA preparation and pulsed field gel electrophoresis (PFGE)

Intact DNA was obtained by enzymatic treatment of the cellsenclosed in agarose plugs as described (Smith and Condemine, 1990)with somemodifications (Marín et al., 1997). Plugs were washed twicein TE buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 8) prior to enzymaticdigestion. Ten units of restriction enzyme SpeI (New England BioLabs)were added to the solution containing the agarose-plug and incubatedfor 4 h. PFGE was carried out in a Contour-clamped HomogeneousElectric Field type (CHEF) BioPulsator (BioRad), at 14 °C in 1% agarosegels in 0.5x TBE buffer (100mMTris, 100mMboric acid, 0.2 mM EDTA,pH 8). Fragment sizewas determined by comparison to CHEF DNA SizeStandards 8–49 kb (BIO-RAD) or chromosomes from Saccharomycescerevisiae YP80 and Schizosaccharomyces pombe 972 h-(BioRad).

2.5. Analysis of the nitrogen metabolism

Direct amplification of nifH gene coding for the nitrogenase wasperformed with the primers described elsewhere (Zehr and McRey-nolds, 1989). Genomic DNA from L. ferrooxidans strain 3.2 andL. ferriphilum strains nto, ye, rt and rt2 was isolated by using FastDNASpin Kit (Bio 101). DNA was digested with HindIII and EcoRI enzymes(New England BioLabs) for 3 h at 37 °C. Fragments were separated in a1% agarose gel and transferred to a nylon Hybond-N+ membrane(Amersham) in alkaline buffer as described (Amils et al., 1998). Afragment of the nifH gene from L. ferrooxidans 3.2 was used as a probe(Parro and Moreno-Paz, 2003). Radio-labelling was performed with2.5 mCi/mL of [γ32-P]-dCTP and 150 µg/mL of DNA by using the

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Megaprime DNA Labelling Systems (Amersham). The product waspurified by passage through a QIAquick PCR column (Qiagen). Theprotocols of pre-hybridization, hybridization and washing previouslydescribed (Amils et al., 1998) were employed. Detection was achievedby X-ray autoradiography in AGFA Curix film at different exposuretimes at −70 °C. Films were developed in Kodak equipment.

2.6. RNA extraction, amplification and cDNA labeling

Cells were recovered by filtration and the RNA was extractedfollowing the RNA extraction kit for soil Fast-RNA (Bio101), its yielddetermined spectrophotometrically (Nanodrop ND-1000, NanoDroptechnologies), and its quality checked by the Bioanalyzer 2100 (Agilenttechnologies). Total RNA was amplified through a method based onthe T7 RNA polymerase linear amplification as described (Moreno-Pazand Parro, 2006), but using the reagents and components of theMessageAmp II aRNA kit (Ambion). Amplified antisense RNA (2 μg)was labeled with the CyScribe first-strand cDNA labeling kit(Amersham Biosciences) with Cy5 or Cy3-dUTP following thesuppliers recommendations.

2.7. Microarray construction, hybridization and analysis

Shotgun DNA microarrays were constructed from a polymerasechain reaction-amplified gene library from L. ferrooxidans 3.2, printedwith the MicroGrid II TAS arrayer (BioRobotics, Genomic solutions),treated and hybridized as previously described (Moreno-Paz andParro, 2006; Parro and Moreno-Paz, 2003). Scanning, data analysis,sequencing and sequence analysis were performed as described(Moreno-Paz and Parro, 2006).

2.8. Phylogenetic analysis

16S rRNA gene sequences were assembled by using GeneDocsoftware (Nicholas and Nicholas, 1997) and the complete sequenceimported and aligned later on the ARB software package (http://www.arb-home.de) (Ludwig et al., 2004). The rRNA alignments werecorrected manually and alignment uncertainties were eliminated inthe phylogenetic analysis. Phylogenetic trees were generated usingparsimony, neighbour-joining, and maximum-likelihood analyseswith a subset of 160 nearly full-length sequences (N1400 bp). Filtersexcluding highly variable positions were used. In all cases, general treetopology and clusters were stable. Consensus trees were generated.

2.9. FISH, CARD-FISH and confocal laser scanning microscopy

Hybridization and DAPI or SYBR Green staining were carried out aspreviously described (Amann et al., 1990). Cy-3, Cy-5, FITC-labelledprobes for FISH were provided by Bonsai Technology (Barcelona,Spain). HRP-labelled probes and fluorochromes AlexaFluor488 andAlexaFluor534 were purchased from Molecular Probes (Invitrogen).Sequences of the specific probes employed in this study included theNitrospira group probe Ntr712 (5'-CGC CTT CGC CAC CGG CCT TCC-3')used in conjunctionwith the competitor Ntr712c (5'-CGC CTT CGC CACCGG TGT TCC-3') (Daims et al., 2001) and Lep634 (5'-AGT CTC CCA GTCTCC TTG -3') specific for “L. ferrodiazotrophum”, and the specificLep636 (5'-CCA GCC TGC CAG TCT CTT-3') and Lep154 (5'-TTG CCC CCCCTT TCG GAG-3') probes designed for L. ferrooxidans and L. ferriphilumrespectively (González-Toril et al., 2003).

Sediment samples were hybridized using the Catalyzed ReporterDeposition (CARD) method as has been described elsewhere(Pernthaler et al., 2002). Hybridization temperature was 46 °C andformamide concentration 35%, for each probe. An Axioskop micro-scope (Zeiss, Germany) equippedwith the proper filter set was used tovisualize the FISH hybridizations. Cell counting was carried out asdescribed (Kepner and Pratt, 1994). Biofilm samples were analyzed in

a LSM510 scanning confocal microscope (Zeiss, Oberkochen, Ger-many) equipped with an Ar ion laser (458 to 514 nm) and two He/Nelasers (543 and 633 nm). All images were recorded with aplanapochromat 63x (1.4; oil immersion) objective. Image processingwas performed with the LSM510 software package (version 1.6).

3. Results

3.1. Physico-chemical properties of selected sampling sites

A complete analysis of the physico-chemical characteristics of thedifferent sampling sites is given in Table 1. The selected stations arelocated at three different points along the river, two in the origin(origin 1 and origin 2) and one in the middle course (García-Moyanoet al., 2007). Waters from origin 1 (NUR, UMA, ANG and Iz–Iz stations)are characterized by their extreme pH and metal concentration,especially iron. Some of them spring directly from Peña de Hierromine site before joining other tributaries. Samples from origin 2 (AGand station 3.2), on the other hand, contain less ferric iron in solution.Both bodies of water meet together to form the Río Tinto. The maincourse of the river (SLS, LPC) is characterized by the gradual dilution ofmetals produced by the neutral tributaries. Both, origin 1 and 2correspond to the headwaters, and are characterized by highconcentration of metals in solution, especially iron (Table 1). Themiddle course is characterized by the dilution effect produced byneutral tributaries, which causes metal concentrations to decreasethus creating a gradient along the course of this part of the river. Thelower area is under the influence of the Atlantic Ocean and was notincluded in this study.

3.2. Isolation and phylogeny of strains

Nine strains belonging to the genus Leptospirillumwere isolated fromRío Tinto waters. As revealed by FISH and restriction analysis of the16S rRNA gene,most of the isolates belonged to the L. ferriphilum species(eight). Only one was identified as L. ferrooxidans. Four L ferriphilumstrains were selected for further comparative analysis.

3.3. Growth in mineral media

Each of the nine isolates exhibited the ability to oxidize iron.Growth phases could be correlated with variations in the physico-chemical parameters. During exponential growth, the concentrationof ferric iron increased rapidly, modifying the pH, redox potential andconductivity of the solution. Conductivity diminished dramaticallywhile the redox potential rose and pH also increased slightly. In thestationary phase, ferrous iron was exhausted, growth ceased and theparameters tended to stabilize. No differences between L. ferriphilumand L. ferrooxidans isolates were detected, with the exception ofL. ferriphilum strains which showed shorter generation times (15.5±0.5 h) than L. ferrooxidans 3.2 (20±0.6 h).

When growing on pyrite in the presence of ferric iron, thegeneration times were longer for both species. During the first 24 h,ferric iron and redox potential dropped noticeably. Consequently,ferrous iron increased and the conductivity rose faintly. Later, cellulardensity rose exponentially, the redox potential recovered andconductivity increased. The stationary phase was characterized bythe stabilization of the physico-chemical parameters.

3.4. Genomic variability

PFGE profiles for all the analyzed strains were quite different, evenamong isolates belonging to the same species, based on their 16S rRNAgene sequence (Fig. 1). No extra chromosomal elements were detectedin any of the isolates when intact DNA was analyzed by PFGE. Theestimated genome size ranged between 2Mb for isolates L. ferriphilum

Fig. 1. PFGE profiles. A, profiles for intact DNA from different Río Tinto Leptospirillum isolates digested with SpeI. B, enzymatic digestion for L. ferriphilum strain nto. CHEF DNA Sizestandards 8–48 kb was used for size determination.

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ye and nto and 3 Mb for isolate L. ferriphilum rt. L. ferrooxidans 3.2exhibited an intermediate genome size value of 2.5 Mb.

3.5. Nitrogen metabolism in l. ferriphilum

All the L. ferriphilum isolates were able to grow in Mackintoshmedium supplemented with 10 mM KNO3 as well as in a mediumdeprived of any nitrogen source after an extensive cell wash to remove

Fig. 2. Search for of nifH gene in Leptospirillum spp. The presence of nifH gene was analyzed iATCC23270 (Act) andAcidiphilium cryptum JF5 (Acd)were used as controls. Figure A shows thenifH gene from L. ferrooxidans 3.2.

any traces of exogenous nitrogen in the media and the dilution of theendogenous organic nitrogen by five growth cycles. In both, nitrogenlimiting and non-limiting conditions, generation times were compar-able, around 15 h. However, in the nitrogen deprived cultures, growthstopped after 140 h, although iron oxidation continued. The culture inthe nitrogen-limitingmedium reached 4.5×107 107 cells mL−1. Despitethese results, attempts to identify the genes involved in the nitrogenfixation were fruitless. Direct amplification with the specific primers

n two strains of L. ferriphilum (rt and ye), L. ferrooxidans 3.2, Aciditiobacillus ferrooxidansdigestedDNA for the different strains. Figure B shows the hybridizationwith 32P- labelled

Table 2Comparative nitrogen fixation gene expression for L. ferriphilum strain ye andL. ferrooxidans strain 3.2 grown in the presence and the absence of a nitrogen source

L. ferriphilum ye L. ferrooxidans 3.2

Operon or gen Product or function +NH4+ −NH4

+ +NH4+ −NH4

+

nifHDKENX Mo–Fe nitrogenase − − − +glnB PII regulator − + − +nifA2 NifA regulator − − − +amtB Ammonium

transporter− + − +

nifS-nifU-hesB-hscB-hscA

chaperones/Fe–Sclusters

− − − +

Hybridization signals for total RNA were detected with the L. ferrooxidans DNA array(Parro andMoreno-Paz, 2003) in the conditions described in theMaterials andmethodssection. +, positive hybridization signal, −, no hybridization signal detected.

Fig. 4. Representative fluorescence in situ hybridization. Laser scanner confocal image ofa biofilm from AG station hybridized with a specific probe for L. ferrooxidans (LEP636).Sample was stained with SYBR Green and the probe was Cy3 labeled. L. ferrooxidanscells are seen in red between nonspecifically labeled green cells.

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for the nitrogenase structural gene was not achieved. Southern-blothybridization with a fragment of the same gene only gave a positivesignal with the DNA extracted from L. ferrooxidans 3.2 (Fig. 2).Furthermore, the hybridization using a DNA array specifically designedfor L. ferrooxidans 3.2 confirmed the absence of a homologous genecoding for the nitrogenase in different Río Tinto L. ferriphilum isolates.However, positive hybridization signals were obtained for other genes,also expressed in L. ferrooxidans 3.2 under nitrogen starvationconditions, like glnB and amtB (Table 2).

Fig. 3. Consensus phylogenetic tree for the genus Leptospirillum. The phylogenetic tree was obtained using the 16S rRNA genes from isolated strains (3.2 and RT) and environmentalRío Tinto samples (in bold characters).

Table 3Occurrence of Leptospirillum spp. in Río Tinto samples analyzed by fluorescence in situhybridization using specific probe

Station L. ferrooxidans L. ferriphilum “L. ferrodiazotrophum”

Iz-Iz ++ Bdl BdlUMA ++ Bdl +NUR ++ Bdl +ANG ++ Bdl +AG ++ Bdl Bdl3.2 ++ Bdl BdlSLS ++ Bdl BdlLPC + Bdl Bdl

(++), high hybridization signal; (+), low hybridization signal; bdl, below detection limit(b0.1%.).

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3.6. Phylogeny and species distribution

From more than 550 clones retrieved from Río Tinto samples, 160gave a 16S rRNA gene sequence identity≥97% with any of the Leptos-pirillum species when analyzed by BLAST. All of themwere used to builda phylogenetic tree. The consensus tree is represented in Fig. 3 andclearly shows the three known species L. ferrooxidans, L. ferriphilum and“L. ferrodiazotrophum”. Most of the clones, 156, were related to thespecies L. ferrooxidans. Only four clones clustered with any of the othertwo species. 16S rRNA gene sequences from cultured cells were alsoincluded in the phylogenetic analysis.

3.7. FISH and CARD-FISH identification and quantification

Members of the different Leptospirillum species were detected byfluorescence in situ hybridization (FISH and CARD-FISH) in samplesfrom water, biofilms and sediments along the course of the river(Fig. 4). L. ferrooxidans is the best represented species of this genus inthe Río Tinto basin, while the other two species appeared in lownumbers or were absent (Table 3). Cells that hybridized with thespecific probe for “L. ferrodiazotrophum” accounted for less than 4% ofthe total cells detected by FISH. Moreover, this species was onlydetected in the sampling stations from origin 1, all of whichcorrespond to springs from a mine site. Hybridization values withthe specific probe for L. ferriphilum were below the limit of thedetection method in all the samples.

4. Discussion

4.1. Isolation and physiological characterization of leptospirilli fromRío Tinto

Nine strains of Leptospirillum were isolated using Mackintoshmedia from samples collected at different stations along the Río Tinto.All of them were physiologically and genotypically characterized.Analysis of their 16S rRNA gene sequence allowed their taxonomic andphylogenetic identification. Most of the isolates (80%) corresponded tomembers of the L. ferriphilum species. All the members of this genusare strict iron oxidizers. All of the isolates were able to grow in mediasupplemented with either soluble ferrous iron or pyrite. Their growthpatterns correlated with the physico-chemical evolution of the media.No difference could be detected at the physiological level betweenL. ferriphilum and L. ferrooxidans isolates.

4.2. Nitrogen fixation

Although the first reports on the presence of genes responsible fornitrogen fixation in leptospirilli were done by Holden (1991) andNorris and collaborators (Norris et al., 1995), it is only recently thatthe complete nif operon has been characterized in L. ferrooxidans(Parro and Moreno-Paz, 2003). Another nif operon highly homologous

with the L. ferrooxidans has been also identified in the genome of“L. ferrodiazotrophum” (Tyson et al., 2005), while no genes related tonitrogen fixation have been detected up to now for L. ferriphilum(Tyson et al., 2005). Given the important genomic differences amongL ferriphilum strains detected in the Tinto basin, we considered acomparative study of nitrogen fixation among members of thedifferent leptospirilli species to be of interest.

All Río Tinto L. ferriphilum isolates were able to develop in anitrogen-free medium after five growth cycles in nitrogen-free media.Although the growth rate was limited, we attempted to detect theputative nifH gene that could explain this important property. Despitethe use of different approaches (direct amplification, southern-blotand DNA array hybridizations), none gave a positive result. Thereforewe have to conclude that the L. ferriphilum isolates from Río Tinto arenot able to fix atmospheric nitrogen by a mechanism similar to that ofthe other two species of Leptospirillum. It has been suggested thatammonium ions absorbed from the atmosphere could explain thegrowth of acidophilic chemolithotrophs in free-nitrogen media(Holden 1991). Another possibility is that growth in the absence ofan external source of nitrogen could be due to an efficientmaintenance metabolism, capable of scavenging nitrogen from themedium or the inoculum. In any case it is clear from these results thatL. ferriphilum is dependent on dissolved inorganic nitrogen sources orthe presence in the system of other diazotrophs for an efficientgrowth. This dependence could explain the specific distribution ofLeptospirillum spp. found in Río Tinto.

4.3. Ecology of leptospirillum in Río Tinto

Eight different stations along the river course were selected tostudy the specific distribution of leptospirilli, not only in the watercolumn, but also in biofilms and in the anaerobic sediments of theriver. 16S rRNA gene amplification and cloning from the differentsampling stations retrieved more than 160 sequences related to Lep-tospirillum. These sequences were used to build a phylogenetic tree forthe genus (Fig. 3). Most of the clones clustered together within thespecies L. ferrooxidans. Only four clones related to any of the other twospecies, “L. ferrodiazotrophum” or L. ferriphilum, were retrieved.

Results from the 16S rRNA gene library were confirmed with insitu hybridization techniques using the specific probes designedfor each of the species (González-Toril et al., 2003). As shown inTable 3 L. ferrooxidans is the best represented species of the genusLeptospirillum in the Tinto basin. Of the other two species, only“L. ferrodiazotrophum” was detected at less than 4%, and only in themost extreme acidic stations. Although L. ferriphilum isolates wereobtained in greater amounts than L. ferrooxidans using Mackintoshmedia, they were not detected by hybridization with their specificprobe, which agrees with the cloning results mentioned above. Theseresults indicate that the isolation of L. ferriphilum over the other Lep-tospirillum species is an artifact introduced by the selective enrich-ment character of the media used for isolation and is not related to thereal proportion of these microorganisms in the natural system. Asimilar situation was reported years ago for Acidithiobacillus ferroox-idans, which draws attention to the risks of interpreting microbialecology results using only isolation experiments.

The physiological differences among Leptospirillum speciescould explain the distribution found in Río Tinto. Both L. ferriphilumand “L. ferrodiazotrophum” appear together in the communities ofsome AMD systems characterized for extreme acidic conditions, e.g.Iron Mountain (Edwards et al., 1999). In fact, cells belonging to“L. ferrodiazotrophum” that were detected in this study appeared onlyin places where the physico-chemical conditions are most extreme,such as those encountered at origin 1. Although nothing is yet knownabout the physiology of “L. ferrodiazotrophum”, it is well establishedthat L. ferriphilum has optimal pH values lower than L. ferrooxidans(Coram and Rawlings, 2002). Moreover, L. ferriphilum dominates most

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of the biohydrometallurgical operations in reactors, where thephysico-chemical conditions, mainly pH, are controlled in order tooptimize the processes (Rohwerder et al., 2003). Although tempera-ture has been suggested as a possible differential growth factoramong leptospirilli species (Sand et al., 1992), we think that this factorcan not explain the results observed in the Tinto basin, in whichdifferential temperatures of more than 10 °C between sampling sitesdoes not translate into any measurable change in diversity. Asmentioned, another possible explanation could be related to thedifferential dependency on nitrogen sources. While both L. ferroox-idans and “L. ferrodiazotrophum” are able to fix atmospheric nitrogen,L. ferriphilum seems to be dependent on the presence of soluble ni-trogen compounds.

Although our understanding of the microbial ecology of ironoxidation in natural systems is still in its infancy, it is clear from theseresults that a careful monitoring of the populations of leptospirillispecies might help to understand their influence on the efficiency ofbioleaching operations and eventually lead to the determination ofthe most suitable one for a given bioleaching operation. Recent datashowed that L ferrooxidans instead of L. ferriphilum is the leptospirillidominating several bioleaching reactors (Rawlings, personal commu-nication and Amils et al., in preparation), suggesting that we do not yetcontrol the role of these critical microorganisms in biohydrometal-lurgical processes. The situation is worse if we consider the complexmicrobial ecology of a heap leaching operation where control is muchmore difficult along the column of mineral due to the nature of thesubstrate and the microenvironments that can be established.

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