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Hydrometallurgy
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t

Interfacial insights of pyrite colonized by Acidithiobacillus thiooxidans cells under acidic conditions
R.H. Lara a, D. Valdez-Prez b, A.G. Rodrguez c, H.R. Navarro-Contreras c, R. Cruz a, J.V. Garca-Meza a,
a b c

Institute of Metallurgy, Engineering Faculty, UASLP, Sierra Leona 550, Lomas 2, 78210, San Luis Potos, SLP, Mxico Institute of Physics, UASLP, Salvador Nava 110, 78210, San Luis Potos, SLP, Mxico Coordination for the Innovation and the Application of Science and Technology, UASLP, Sierra Leona 550, Lomas 2, 78210, San Luis Potos, SLP, Mxico

a r t i c l e

i n f o

a b s t r a c t
Studies of interfacial processes involving leaching bacteria and sulde minerals (MS) are necessary to understand and improve the bioleaching processes of mining industries. Interfacial studies of the role of extracellular polymeric substances (EPSs) during cell attachment to MS have been restricted mainly to iron-oxidizing bacteria, neglecting interfacial mechanisms associated with the biooxidation of reduced sulfur compounds (e.g., elemental sulfur, S0); nevertheless the reduced sulfur compounds may affect MS weathering or dissolution. The importance of sulfur-oxidizing microorganisms is evidenced by the fact that S0 was generally added to promote the growth of Acidithiobacillus thiooxidans in some bioleaching processes. Preliminary research coupled either epiuorescence and atomic force microscopy (AFM) or Raman spectroscopy and AFM to analyze biolms of A. ferrooxidans on pyrite or the surface features of pyrite in biotic and abiotic experiments. Here, we applied the combination of AFM, Raman and principles of epiuorescence for the study of biolms of the A. thiooxidans on previously oxidized pyrite. Our results showed that A. thiooxidans forms a monolayered biolm, wherein the contact resulted in a strong adhesion force (467 pN) between the cells and the altered surface, perhaps due to an irreversible binding mechanism. The observations strongly suggested an intimate contact stage during the dynamic interfacial mechanisms of S0 biooxidation on the pyrite surface. During this process, an overproduction of EPS was recorded (ca. 100%), indicating that EPS plays a key role during microorganism/surface interactions. 2010 Elsevier B.V. All rights reserved.

Article history: Received 22 September 2009 Received in revised form 15 February 2010 Accepted 15 February 2010 Available online 24 February 2010 Keywords: Acidithiobacillus thiooxidans Biolms Pyrite Raman spectroscopy Atomic force microscopy Epiuorescence

1. Introduction The sulfur-oxidizing microorganisms (SOM: archaea or bacteria) play a major role in the cycling of sulfur in the biosphere. The ability of SOM has been applied in the mining industry to the bioleaching of metals from metal suldes (MSs) to remove pyritic sulfur from coal and to the desulfurization of wastewater (Gourdon and Funtowicz, 1998). SOM such as Acidithiobacillus thiooxidans and A. caldus are not able to oxidize acid-insoluble MS such as pyrite alone but grow on the sulfur released after the iron has been oxidized by iron-oxidizing microorganisms (IOMs) (Rawlings et al., 1999; Dopson and Lindstrm, 1999). In the bioleaching of base and precious metals from MS, the main role of Fe(II) oxidizers (A. ferrooxidans and Leptospirillum ferrooxidans) is the regeneration of the oxidizing agent, the Fe(III) ions, while the SOM (e.g. A. thiooxidans and A. caldus) contribute to the overall process by the oxidation of the intermediary (reduced) sulfur compounds to sulfuric acid (Sasaki et al., 1998).

Corresponding author. Geomicrobiology Area, Institute of Metallurgy, Engineering Faculty, UASLP, Sierra Leona 550, Lomas 2, 78210, San Luis Potos, SLP, Mxico. Tel./fax: + 52 444 825 4326. E-mail address: jvgm@uaslp.mx (J.V. Garca-Meza). 0304-386X/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2010.02.014

According to Schippers and Sand (1999), the degradation of an acidinsoluble MS arises via the thiosulfate mechanism by means of electron extraction, by the indirect attack of hydrated Fe(III) ions from the crystal lattice. The main reduced sulfur obtained is the thiosulfate, which via a series of reactions subsequently yields sulfate and protons (via tetrathionate and other polythionates) or elemental sulfur (S0). The S0 is a by-product produced in signicant amounts (1020% S0) during the chemical or electrochemical oxidation of pyrite at pH b 2, moderate temperature and pressure, and in the absence of SOM (Hamilton and Woods, 1981; Mycroft et al., 1990; Schippers and Sand, 1999; Rohwerder et al., 2003). S0 is also produced by the biological activity of IOMs. Because S0 may be inert under acidic conditions (Rohwerder and Sand, 2007), the produced S0 may accumulate and form a layer on the MS surface that may act as a barrier against the diffusion of oxygen or Fe(III) ions, affecting the reactivity and delaying the complete oxidation of MS (Mustin et al., 1993; Dopson and Lindstrm, 1999; Fowler and Crundwell, 1999; Bevilaqua et al., 2002; Nava et al., 2008). Additionally, A. thiooxidans causes bioacidication, providing protons to maintain Fe (III) is its oxidized state (Schippers and Sand, 1999); in fact, S0 was generally added to promote the growth of A. thiooxidans in some bioleaching process (Liu et al., 2008). It is well known that A. ferrooxidans may contribute both sulfurand iron-oxidizing capabilities to the bioleaching process. However, A.

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ferrooxidans is not the dominant species in commercial bioleaching tanks due to the solution conditions (e.g., pH and temperature), but the interfacial chemistry of the MS, e.g., at high redox potential, results in more extensive pyrite oxidation by L. ferrooxidans than by A. ferrooxidans. Thus, at a more positive potential (N 800 mV, typically found in bioleaching tanks due to Fe(III) accumulation) L. ferrooxidans outcompetes A. ferrooxidans and favors the proliferation of SOM-like A. caldus and A. thiooxidans because more reduced S-compounds may be potentially recoverable by them at the pyrite surface (Rawlings et al., 1999). All the former showed the essential role of SOM during the commercial bioleaching process. It had been demonstrated that the attachment of cells as biolms to MS surfaces enhances bioleaching of MS (Pogliani and Donati, 1999; Gehrke et al., 1998; Sand et al., 2001; Rodrguez et al., 2003; Sharma et al., 2003), and that diverse species such as A. ferrooxidans and A. thiooxidans differ in their capability of attachment (Porro et al., 1997). Worldwide studies have demonstrated that most of microbial communities spend a large part of their life cycle within a biolm, including aquatic (saturated) environments (Stoodley et al., 2002). Biolms are highly organized systems of microorganisms, embedded in a selfproduced, gelatinous and highly hydrated matrix, composed of extracellular polymeric substances (EPSs), ions, gases, colloidal and particulate compounds, and open water channels. The EPS matrix represents 5090% of the biolm, and its secretion is an essential characteristic of the biolm development, rst and foremost for the initial attachment of the cells to the surface (Neu et al., 2003) e.g., mineral particles. Since 1995, Sand, Gehrke and collaborators have highlighted the importance of the biolm matrix as a reactive space wherein the electrochemical mechanisms/surface reaction takes place, specically the initial attachment of EPS-complexed Fe(III) ions to the pyrite surface by electrostatic interactions and their acceleration of the dissolution rate of this MS (Kinzler et al., 2003). Furthermore, the amount of EPS-bound Fe(III) ions varies with the metabolic activity of the Fe oxidizers like A. ferrooxidans (Sand and Gehrke, 2006), and the amount of Fe(III) in the close vicinity of the MS surface plays a key role in the oxidative activity of the attached cells of A. ferrooxidans and L. ferrooxidans (Rohwerder et al., 2003). However, the researchers suggested that SOM completely lack complexed Fe(III) or other positive charged groups in their EPS, which could be indicative that mechanisms other than electrostatic forces are important during cell attachment. Despite the vital role of SOM biolms during the bioleaching process, their attachment to the MS surface has been scarcely studied (e.g., Dopson and Lindstrm, 1999; Lizama et al., 2003; Liu et al., 2003) and therefore, the interfacial processes are poorly understood. Recently, Mangold et al. (2008a,b) coupled atomic force microscopy (AFM) and epiuorescence microscopy to analyze the biolms of A. ferrooxidans on pyrite. Those studies demonstrated that such a combination is a powerful tool for the investigations of biolms on opaque materials and ultimately increase knowledge of the biological interfacial process. AFM was previously applied to study the morphology of pyrite surfaces after interaction with A. thiooxidans (Liu et al., 2003), and Edwards et al. (1999) combined Raman spectroscopy and AFM to obtain information on the bulk surface-speciation and the surface features of pyrite, respectively, in biotic and abiotic experiments. We applied the combination of AFM, Raman and principles of epiuorescence to the study of biolms of the SOM A. thiooxidans on previously oxidized pyrite. In our study, AFM was additionally used to quantify adhesion forces associated with the bacteriummineral interaction. The adhesion force implicit in biolms formation is useful in understanding the interfacial characteristics of the bacteriummineral system, and it has been extensively utilized for the study of non-leaching bacteria (e.g., Vadillo-Rodrguez et al., 2004; Lower et al., 2005; Touhami et al., 2006; Tsang et al., 2006; Atabek and Camesano, 2007; Busscher et al., 2008).

Instead of using epiuorescence microscopy, we examined the stained biolms using a confocal laser scanning microscope (CLSM). Raman spectroscopy was subsequently used for a precise identication of S0 or polysulde (S2 n ) species on the pyrite surface. Because A. thiooxidans is not able to oxidize pyrite or other acid-insoluble MS, the study using this set of tools was perform after the production of S0/S2 n on pyrite surface via chemical (with Fe(III) and H2O2) and electrochemical oxidation. Abiotic oxidation permits the overproduction of both S0 and S2 n directly from the pyrite. The aims of this work using AFM, CLSM and Raman spectroscopy are twofold: rst, to propose a methodology based on microscopic and spectroscopic methods to obtain information on the surface-speciation and the surface characteristics of pyrite colonized by A. thiooxidans biolms, and second, to evaluate the role of bacterial EPS during contact mechanisms between this SOM and oxidized pyrite under acidic conditions. This is the rst work of a series of studies on the interfacial characterization of SM in the presence of A. thiooxidans for acidinsoluble and acid-soluble MS, in order to elucidate the role of SOMs in the biochemical process of MS dissolution. 2. Materials and methods 2.1. Mineral samples Pyrite samples were obtained from Zacatecas, Mexico. Mineralogical analysis of samples indicated a pyrite purity of 99.5% wt, with minor amounts of chalcopyrite (CuFeS2, 0.3% wt) and sphalerite (ZnS, 0.2% wt). X-ray diffraction patterns and scanning electron microscopy coupled to energy dispersive X-ray spectroscopy (SEM-EDS) analyses were also carried out in pristine (unoxidized) pyrite samples. These analyses conrmed mineral identity and the presence of inclusions of chalcopyrite and sphalerite ( 0.5% wt). Crystal samples were selected for the construction of massive pyrite electrodes (MPEs): mineral sections (1.2 to 1.5 cm2) were mounted in epoxy resin with a silver epoxy electrical contact on the backside, and the MPE surface was polished until a mirror-like surface was obtained. 2.2. Potentiostatic MPE oxidation
Because S0/S2 n species are required to sustain the metabolic activity of the SOM, A. thiooxidans, the pyrite was chemically oxidized with Fe (III) and H2O2 as well as electrochemically oxidized. Electrooxidation was preferred because it was less time-consuming and resulted in more S0/S2 n production than chemical oxidation. Further, the electrochemical oxidation of MPE resulted in a well-controlled formation of S0/S2 n , which completely covered the surface of the MPE, allowing a better comparison between the assays (see Section 3). Preliminary voltammetric analysis of MPE was carried out to establish the potential values where reduced sulfur compounds were electrochemically formed. This analysis was carried out in acidied ATCC-125 (American Type Culture Collection) medium at pH= 2, using an autolab PGSTAT 30 coupled to a PC and a classic Pyrex glass three electrode cell. Hence, the working electrode was the MPE, the counter electrode was a graphite rod (AlfaAesar, 99.9995% purity), and the reference electrode was a saturated sulfate electrode (SSE, 0.615 V vs. SHE, the standard hydrogen electrode). The potentials tested were from 0.5 to 1.11 V vs. SHE. According to this setup, the formation of reduced sulfur compounds on MPE is favored at potentials between 0.91 and 1.11 V vs. SHE (3600 s). The reduced sulfur compounds may be generated at different potentials during electrochemical studies according to the type and amount of the impurities in the iron sulde or the defects and disorder in the lattices, among others (Hamilton and Woods, 1981; Gerischer, 1990; Kelshall et al., 1999; Cruz et al., 2001). Accordingly, the formation of S0 and/or S2 n species on electrooxidized MPE surfaces was conrmed by Raman spectroscopy after each electrooxidation.

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In order to validate that the formation of S0 species on electrooxidized surfaces evolves toward crystalline structures during weathering of pyrite in acidic conditions, an additional experiment using pristine pyrite grains was performed (106150 m, Taylor series). This experiment consisted of chemical oxidation using Erlenmeyer asks containing 5 g of pristine pyrite grains, 75 mL of acidied ATCC-125 growth medium, 2000 mg/L of Fe(III), and a daily addition of 1 mL of H2O2 during 15 days under orbital shaking (pH = 2, 2830 C). The formation of crystalline S0 species was conrmed by Raman spectroscopy. Similar experiments were carried out using non-electrooxidized MPE under acidic conditions with and without A. thiooxidans to compare the development of S0/S2 n species and the Raman response with those obtained after MPE electrooxidation. 2.3. Bacterial strain and cultivation Acidithiobacillus thiooxidans strain ATCC-8085 was used in this study. Acidithiobacillus thiooxidans were cultivated aerobically at 28 30 C in 50 mL of media ATCC-125, which contained per liter of distilled water: 10 g of S0 (Baker), 3 g of KH2PO4, 0.4 g of (NH4)2SO4, 0.5 g of MgSO47H2O, 0.25 g of CaCl22H2O, and 0.01 g of FeSO47H2O. The media was dispensed into 250 mL Erlenmeyer asks and were sterilized by autoclaving at 121 C for 15 min. However, S0 was sterilized separately using 2 h of UV irradiation with intermittent shaking. Final pH of medium was adjusted to 2 with H2SO4. The exponential growth phase was reached in 810 days with an average biomass of between 107 and 108 cells/mL. A planktonic (suspended) inoculum was used for the biotic experiments. 2.4. Biolm formation
Electrooxidized MPE (see Section 2.2) was used as the S0 and/or S2 n source for A. thiooxidans. For its sterilization, the electrooxidized MPE was exposed to UV irradiation for 24 h; afterward, the MPE was placed in a ask with 100 mL of the ATCC-125 (pH = 2) and 108 cells of A. thiooxidans. The culture was incubated aerobically at 2830 C and 150 rpm for 5 days. Similar contact times have been used for A. ferrooxidans or A. thiooxidans biolms formation on pyrite under various conditions (Mangold et al., 2008a, b; Harneit et al., 2006). This biotic assay was done in triplicate. An abiotic (uninoculated) control was also carried out in triplicate in order to compare chemical and biological oxidation of S0. After 5 days, the MPE of biotic and abiotic control assays were collected, dried with a direct current of nitrogen, and preserved in a desiccator under inert conditions until their analysis.

specic surface, and 10% of the obtained curves were randomly analyzed. The Si3N4 cantilever showed a free resonance frequency between 90 and 115 kHz and a constant between 1.08 and 2.03 N/m during the collection of these curves. In the absence of external electric elds, the interaction forces measured by AFM force-separation curves are mainly van der Waals interactions, short-range repulsive interactions, adhesion and capillary forces (Garcia and Prez, 2002). For a monolayer attached to a substratum, there are two main models currently used to described the adhesion forces that are determined by cantilever tip-surface and surface-monolayer attach forces (Garcia and Prez, 2002; Curry and Kim, 2004): FAdh = 4R FAdh = 3R 1 2

2.5. Surface analysis of massive pyrite electrodes (MPEs) Before the assays, AFM observation and analysis were carried out for pristine (un-electrooxidized) and electrooxidized surfaces (see Section 2.2). After the bioassay, the same analyses were done for biotic (electrooxidized MPE with exposure to A. thiooxidans) and abiotic control (electrooxidized MPE without A. thiooxidans cells). The AFM analysis of MPE surfaces was performed with a Nanoscope Multimode IIIa digital instrument. Narrow and wide regions were visualized to obtain topographic images by tapping mode (scan rate between 0.5 and 1 Hz). The silicon cantilever showed a free resonance frequency between 275 and 325 kHz and a constant between 31.18 and 44.536 N/m during these experiments. The non-electrooxidized MPE exposed to the culture media without microorganisms (during 5 days and under the same conditions) was also analyzed by AFM. Force-separation curves were acquired using contact mode in 1 m2 areas. The force-separation curve is a representation of the extending and retracting curves associated with the interaction forces between the cantilever tip and substratum, which allows quantication of the adhesion forces mediating bacteria attachment to MS (Kendall and Lower, 2004). At least 300 curves were taken from each

FAdh is the adhesion force, R is the radii of cantilever tip, and is the surface energy for the JohnsonKendallRoberts (JKR) and the DerjaguinMullerToporov (DMT) models (Johnson et al., 1971; Derjaguin et al., 1975). The JKR model is commonly used for the assessment of high adhesion forces and large tip radii. In contrast, the DMT model is used for the assessment of low adhesion forces and small tip radii (Garcia and Prez, 2002). Indeed, the JKR model only takes into account the adhesive interactions inside the contact zone and neglects interactions between the surfaces outside the contact zone (Curry and Kim, 2004). Accordingly, the DMT model seems the most appropriate to evaluate the interactions between the biolm and the mineral surface. Although it is desirable to characterize cell mineral adhesion forces using colloidal tips (see Kendall and Lower, 2004), the cellmineral adhesion forces were measured in this study with a triangular tip cantilever. Finally, roughness (Ra, nm) and root mean square (Rq, nm) of MPE surfaces were also evaluated in biotic and abiotic assays to generate a complete description of MPE surface. After AFM analysis, the same MPE exposed to A. thiooxidans (biotic surfaces) were analyzed by CLSM (Leica DMI4000B with a 63 immersion objective). The biolms were previously stained during 30 min in the dark, using sequentially applied lectin Canavalia ensiformis (Con-A) and uorochrome Acridine Orange (AO), respectively, for the epiuorescence of EPS compounds (-mannose, -glucose) and genetic material (RNA and DNA). Subsequently, HEPES (2%, pH = 7.4) and acetic acid (5% in ethanol) solutions were used for washing and dissolving the stain, respectively. Raman spectroscopy analysis was carried out in pristine MPE, in electrooxidized MPE, in MPE electrooxidized after the biotic and the abiotic assays, and in the non-electrooxidized MPE exposed to the culture media without microorganisms. For spectroscopic analysis, a triple monochromator Raman Jobin Yvon T64000 spectrometer equipped with an optical microscope (Olympus BH2-UMA) was used. The detector was a charged coupled device (CCD) cooled by liquid nitrogen. Pyrite surfaces were excited by an Ar+ laser beam at 514 nm (Stabilite 2017, Spectra Physics) with a power of 2 mW, and focused with a diameter of about 0.8 m on the sample. Collection time was 60 s in each analysis. Raman performance was validated using a Si wafer by assuming a single sharp peak or a wavenumber of 521 cm 1. Raman backscattering showed a signal/noise ratio greater than 100 for Si analysis, ensuring a good Raman performance during mineral surface analysis. The vibrational range was 100750 cm 1 as species show their main active modes within this interval the S0/S2 n (e.g., Sasaki et al., 1998; Toniazzo et al., 1999). At least 10 Raman spectra were collected from each specic surface. 3. Results Raman analysis was carried out to evaluate the status of sulfur compounds on MPE surfaces, e.g., pristine, electrooxidized (before the

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assays), and electrooxidized after the biotic and abiotic assays. Raman spectra collected from pristine MPE surfaces is presented in Fig. 1a. Typical peaks at 343 (v2) and 381 cm 1 (v1) conrm the mineral identity (Sasaki et al., 1998). Meanwhile, the Raman spectra of the electrooxidized MPE before the assays (Fig. 1c) showed additional peaks at 155 (v2), 222 (v3) and 465470 cm 1 (v1), which is a clear species on these surfaces signature of the formation of S0/S2 n (Mycroft et al., 1990; Sasaki et al., 1998; Toniazzo et al., 1999). The obtained Raman spectra of the MPE surface after the abiotic assay (Fig. 1d) showed similar peaks to those observed from electrooxidized surfaces (Fig. 1c), but the v1 was sharper in the electrooxidized MPE species after abiotic assay than before the assay, indicating that S2 n 0 were completely oxidized to S after 5 days. The Raman spectra of the MPE surface after 5 days of exposure to A. thiooxidans showed low intensity, noisy peaks, similar to those obtained in the pristine MPE surfaces (Fig. 1e and a, respectively), indicating S0 species were totally consumed on electrooxidized MPE surfaces after 5 days of biotic contact (Fig. 1e). Raman spectra from the non-electrooxidized MPE after 5 days in culture media without A. thiooxidans also showed low intensity, noisy peaks associated with those of pristine samples (Fig. 1f). AFM images from non-electrooxidized MPE after 5 days in culture media with A. thiooxidans were not clearly dened (Fig. 2l). This fact and the impossibility of tracking the status of sulfur compounds on mineral surfaces rendered it difcult to perform a deeper analysis of interfacial processes by AFM or Raman spectroscopy. Finally, the Raman spectra collected from oxidized mineral grain samples (Fig. 1b) were fairly similar to those obtained on abiotic control surfaces (Fig. 1d), thus conrming the formation, stability, and progressive crystallization of S0 during pyrite weathering under the tested acidic conditions. It is important to note that the Raman backscattering obtained from electrooxidized MPE was lower than that observed in the Si wafer, which showed a signal-to-noise ratio greater than 100 (Fig. 1g), whereas the ratio obtained from MPE surfaces was around 6. This difference is indicative of factors affecting Raman backscattering from MPE surfaces. Such factors could be associated with the semiconducting properties of pyrite, changes in crystallographic plane orientation of resulting species in biotic surfaces (e.g., polycrystalline structures), and disorder in mineral lattices composing such species, among others. In spite of these factors, Raman peaks collected from MPE surfaces were discernable, and therefore, the Raman analysis carried out here is valid. After the potentiostatic electrooxidation of MPE, a contrasting surface transformation can be observed by AFM: the formation of widespread nano-scale size structures of altered mineral (Fig. 2b). However, the surface of the electrooxidized MPE after the abiotic assay showed bigger structures (Fig. 2c) than those observed in the electrooxidized MPE before the assays (Fig. 2b). These results indicated a further surface alteration after 5 days of immersion in the acidic medium under abiotic conditions. The AFM analysis of the electrooxidized MPE after the biotic assay showed the presence of attached cells of A. thiooxidans (Fig. 2d to i). Biolms comprised a monolayer of bacterial cells with a typical size between 1 and 2 m (Fig. 2e to i). Attached cells were easily discernable, and the details associated with the underlying substratum were clearly distinguishable (Fig. 2g). In fact, attached cells seem to be imbedded in the resulting nano-scale size structures (Fig. 2e to i). From previous results, we conclude that such nano-scale size structures observed on electrooxidized surfaces by AFM (Fig. 2b) are mainly S0/S2 n species, in agreement with Raman spectroscopy data (Fig. 1c). In a similar way, the increase in height of the nanoscale size structures in abiotic MPE (Fig. 2c) and non-electrooxidized MPE (exposed 5 days to the media; Fig. 2j and k) can be associated with the progressive accumulation of S0 well-crystallized species, as the

Raman spectroscopy (Fig. 1d) and AFM (Fig. 2c) indicated. Fig. 2j and k show evidence of scant pyrite oxidation, evidenced by limited formation of nano-scale structures (ca. 10 nm height; Table 2). The adhesion forces of the attached cells were quantied as described in Section 2.5. Several force measurements were acquired on each specic MPE surface. Examples of force-separation curves obtained in this study are presented in Fig. 3. Adhesion forces in pristine and electrooxidized MPEs (before the assay) were evaluated in order to monitor the evolution of surface characteristics. The forces associated with the interactions between MPE surfaces and A. thiooxidans cells forming biolms were evaluated by comparing force data obtained for biotic and abiotic assays. The adhesion force of pristine MPE was omitted due to its low magnitude, which was below the experimental standard error of the value of the forces of the electrooxidized surface. This last surface exhibited an adhesion force of 1483 pN. It is worthwhile to note the important repulsive interactions observed during the nearing of the cantilever tip to the surface (see the extending curve in Fig. 3b). Afterward, an attractive interaction (see the retracting curve in Fig. 3b) is observed (Kendall and Lower, 2004). The global interaction may result from the combination of repulsive forces between the cantilever tip, the electronic 0 species on characteristics of S2 n , and the attractive force of S electrooxidized surfaces before the assays. Force data of the MPE surfaces from abiotic assays were 4400 pN (Table 1). These results indicated that the formation and accumulation of S0 with a higher crystallization degree enhance an attractive force (Fig. 3c). Finally, the measured adhesion forces of the MPE surface from the biotic (4867 pN) and abiotic assays (4400 pN) (Table 1, Fig. 3c and d) indicated an adhesion force of 467 pN for A. thiooxidans. This value indicates an attractive interaction associated with the adhesion of A. thiooxidans cells to the MPE and is the same order of magnitude reported for non-leaching bacteria forces under various conditions (Vadillo-Rodrguez et al., 2004; Lower et al., 2005). Table 2 shows the Ra, Rq on electrooxidized MPE before and after the biotic and abiotic assays. In the MPE from the biotic assay, Ra, Rq were calculated directly on biolms and adjacent-surrounding biolm areas (not on top of attached cells). It is well known that Ra, Rq are parameters that can be indicative of a coarse or uneven surface and in this way reect changes in the surface topography as a result of external perturbations. In this study, we obtained Ra, Rq information from the same AFM images presented in Fig. 2 but using respective 2D images with several areas (Table 2). The Ra average from electrooxidized surfaces was 20 nm. The Ra, Rq of the abiotic system were the highest ( 67 nm and 84 nm, respectively), conrming a further alteration of the electrooxidized MPE after 5 days under acidic conditions (Fig. 2c) that seems to result in an increase of the nanoscale size structures. However, comparing the Ra, Rq values of the MPE surfaces from the biotic (adjacent-surrounding biolms areas) and abiotic systems, resulted in a minor Ra, Rq of the MPE from the biotic system ( 36 nm). Thus, the depletion of the nano-scale size structures of S0 observed by AFM (Fig. 2d to i) and Raman spectroscopy (Fig. 1e) in MPE surfaces after 5 days of exposure to A. thiooxidans is clearly associated with biotic oxidation of S0. This last is in agreement with Raman spectra obtained from non-electrooxidized MPE and after 5 days in culture media without A. thiooxidans (Fig. 1f), species can be attributed where the absence of detectable S0/S2 n exclusively to the small extent of chemical alteration of the MPE. Fig. 4 shows CLSM images from planktonic cells (Fig. 4a to c) and biolms on MPE surfaces from the biotic assay (Fig. 4d to i). Fig. 4 makes clear the differences between the planktonic and attached cells of A. thiooxidans. The relative intensity of EPS epiuorescence in the observed stained cells (at least 50 cells) associated with biolms is

Fig. 1. Raman spectra collected from different MPE surfaces: (a) pristine (non-electrooxidized) MPE; (b) chemically oxidized grain mineral particles; (c) electrooxidized MPE before the assays; (d) electrooxidized MPE after abiotic assay; (e) electrooxidized MPE after biotic assay; (f) non-electrooxidized MPE and after 5 days in culture media without A. thiooxidans, and (g) Si wafer backscattering. 20 A. Collection time of 60 s. = 514 nm.

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Fig. 3. Force-separation (extending and retracting) curves of MPE surfaces in air using contact mode: (a) pristine (non-electrooxidized) MPE; (b) electrooxidized MPE before the assays; (c) electrooxidized MPE after abiotic assay, and (d) electrooxidized MPE after biotic assay. The area of the obtained curves was 1 m2. Scan rate of 0.51 Hz.

Table 1 Interaction forces calculated from each specic MPE surface (n = 30). Area of collection was 1 m2 (scan rate of 0.51 Hz). Contact mode. MPE surface Pristine Electrooxidized (1.11 V/SHE) Abiotic assays Biotic assays/biolms: collected on top of attached cells Biotic assays/biolms: surrounding attached cells Non-electrooxidized and after 5 days in culture media Adhesion force (pN) 24 2 1483 180 4400 200 4867 115 28 2 36 3

Table 2 Root mean square (Rq) and roughness (Ra) values collected from different MPE surface areas. MPE surface Electrooxidized (1.11 V/SHE) Area (m m) 55 2.5 2.5 11 0.25 0.25 55 11 55 11 55 11 55 11 Number of areas 5 5 35 19 50 25 35 15 25 45 30 40 RMS (Rq) nm 24.35 2.18 24.64 1.49 22.85 2.15 22.04 2.96 83.77 9.78 80.54 4.12 73.41 9.69 52.67 6.51 45.13 3.58 37.41 4.83 7.55 2.41 2.10 0.92 Roughness (Ra) nm 19.41 1.78 19.63 1.06 18.25 1.66 17.58 2.42 66.97 8.32 62.56 3.25 55.12 5.79 41.71 6.21 36.08 3.01 30.27 4.72 5.66 2.02 1.48 0.74

Abiotic assay Biotic assays (collected on top of attached cells) Biotic assays (surrounding attached cells) Non-electrooxidized MPE, after 5 days in culture media

68.42 15.28, whereas that from planktonic cells is 33.60 5.7. The average number of cells comprising biolms was 27 3 in most of the cases (Fig. 4g). 4. Discussion and conclusions Acidithiobacillus thiooxidans forms monolayered biolms strongly attached to the electrooxidized MPE surface, suggesting an intimate physical contact between the bacteria and the pyrite. The contact resulted in a strong adhesion force (467 pN) between the cells and the altered surface. Assuming that each cell of A. thiooxidans has a volume of 0.135 m3 (from a length, height and width of 1.5, 0.3 and 0.3 m, respectively) and a cell density of 1 g/cm3 (suspended or planktonic cell), the cell force was 1.13 10 3 pN, equivalent to the weight of a single cell. It is important to notice that such a strong adhesion force was recorded after the sulfur biooxidation by A. thiooxidans (Fig. 1e),

suggesting a molecularly mediated, irreversible binding mechanism to MPE (see below). After the attachment of microorganisms, the Ra, Rq of the MPE surface adjacent-surrounding (not on attached cells) biolms was lower (67 nm) than of the abiotic assay (84 nm) (Table 2), due to the decrease of the nano-scale size structures formed in the electrooxidized MPE after the assays (Fig. 2d to l). Such structures are principally S0/S2 n , as the Raman analysis indicated (Fig. 1c), and decreased after 5 days in the biotic assay (Fig. 1e). This depletion of the nano-scale size structures of S0 observed by AFM and Raman spectroscopy in biotic

Fig. 2. Three dimension images of AFM from MPE surfaces: (a) pristine (non-electrooxidized) MPE; (b) electrooxidized MPE before the assays; (c) electrooxidized MPE after abiotic assay; (d to i) electrooxidized MPE after biotic assay; (j and k) non-electrooxidized MPE and after 5 days in culture media without A. thiooxidans, and (l) non-electrooxidized MPE and after 5 days in culture media with A. thiooxidans. Images were acquired in air using tapping mode and a scan rate of 0.51 Hz. Height of elements is shown in the gure.

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42 R.H. Lara et al. / Hydrometallurgy 103 (2010) 3544

Fig. 4. CLSM images of A. thiooxidans cells: (a to c) planktonic (suspended) cells; (d to i) after 5 days of cells attachment in biolms on electrooxidized MPE. Epiuorescence of exopolysaccharides are in green, whereas genetic material is in red. An overlap of both epiuorescence images is presented in the low-left side of images (a to g). Image h is a zoom of the overlap showed in (g); image (i) showed the epiuorescence of exopolysaccharides in biolms.

assays showed the feasibility of the oxidation of S0 by A. thiooxidans and also suggested a dynamic interfacial mechanism. The surfaces of the abiotic MPE showed the presence of well-crystallized S0 after 5 days under acidic conditions. The formation of S0 clusters of variable height (expressed as Ra, Rq) could be a consequence of the heterogeneous corrosion of electrooxidized surfaces due to a random distribution of defect points and impurities, among others (composing mineral lattices). Consequently, the observed topography in the resulting surface after the biooxidation of S0 species would expose a pyrite structure with variable corrosion patterns. Since in our experiment, the Fe(II)/Fe(III) ratio was the same in both cultures with A. thiooxidans and in the abiotic control (0.01 g of iron; see Section 2.3), the aforementioned results indicate that the differences are

clearly a consequence of the oxidation of the S0 generated on the electrooxidized MPE by A. thiooxidans, which formed a monolayered biolm attached to the MPE surface, resulting in a strong biolm/ mineral interaction. According to Fowler and Crundwell (1999), A. ferrooxidans removes sulfur by a direct contact mechanism, and the bacteria directly oxidizes the reduced sulfur compounds due to its sulfur oxidation capacity. Direct contact of A. thiooxidans with the pyrite surface was previously suggested by Takakuwa et al. (1977), Konishi et al. (1995) and Liu et al. (2003). Recently, Florian et al. (2009) and Echeverra and Demergasso (2009) reported that A. thiooxidans attach to pyrite in pure and mixed cultures. According to Rohwerder and Sand (2007), the concept of a biolm matrix as a reaction space implies that EPS chains are in direct contact

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R.H. Lara et al. / Hydrometallurgy 103 (2010) 3544 43

with the MS surface and that oxidative attack is performed by EPScomplexed Fe(III) through a contact mechanism. However, Harneit et al. (2006) showed that the attached cells of A. thiooxidans and A. ferrooxidans grown in S produced up to 85 times more EPS than planktonic cells but that the amount of Fe ions in their EPS was undetectable. The ndings of Rohwerder and Sand support our results, which also indicate that EPS plays a key role in the attachment of A. thiooxidans cells of the pyrite surface because the microorganisms secreted more EPS ( 100%) than did planktonic cells. Such overproduction of EPS is directly associated with the increase of epiuorescence in attached cells (Rapposch et al., 2000), reecting considerable EPS thickness (Fig. 4e). The second observation of Harneit et al. (2006), the amount of Fe ions in their EPS was undetectable, suggests that the attachment of A. thiooxidans to the pyrite does not require EPS-complexed Fe(III) ions but relies on other, different mechanisms in addition to electrostatic forces (Rohwerder et al., 2003). Like us, several authors considered the attachment of Acidithiobacilli to minerals by means of EPS, although recent observations of A. ferrooxidans cells indicate an irreversible binding to pyrite through molecularly mediated binding between specic adhesins (EPS and outer membrane proteins) and the substrate surface (Chi et al., 2007; Chandraprabha et al., 2010) and that A. ferrooxidans can colonize a solid surface by pili-mediated sliding, twitching motility, and adherence, resulting in a strong pili substrate bond (Li et al., 2010). Hence, the strong adhesion force recorded after 5 days (when the S0 was completely consumed; Fig. 1e), as well as the elevated production and secretion of EPS by the attached A. thiooxidans after 5 days (Fig. 4), suggested a further role of EPS in deepening adhesion, e.g., trapping and concentrating nutrients and ions, and in ensuring a suitable microenvironment. We also suggest that A. thiooxidans attaches directly to electrooxidized MPE and that the S0 attachment does not require EPS-complexed Fe(III) ions because the biooxidation of the elemental S0 to sulfate occurs via enzymatic catalysis, e.g., the sulfur-dioxygenase or sulfur oxygenase (catalyzing the oxidation of S0 to sulte, HSO 3 ) and a sulte: 2 acceptor oxidoreductase or dehydrogenase (HSO 3 to SO4 ) (Kelly, 1985; Schippers and Sand, 1999; Rother et al., 2001; Rohwerder and Sands, 2003; Sand and Gehrke, 2006; Rohwerder and Sands, 2007). The CLSM, AFM and Raman data and the obtained adhesion force values strongly suggest an strong bond during the dynamic interface mechanisms of S0 biooxidation on pyrite surface. In this study, we offer experimental evidence that indicates S0 is stable during pyrite weathering in acidic conditions (abiotic assays). These reduces sulfur compounds are only oxidized by SOM (biotic assays), as was previously indicated by Rohwerder et al. (2003). We found that the electrooxidation of MPE allows a better development of the insoluble sulfur species, dened by Ra, Rq during AFM analysis and identied by Raman spectroscopy. This approach facilitated the observation and the comparison between the MPE in the biotic and the abiotic trials. Finally, the major biolm was established on the electrooxidized MPE after biotic assays rather than after the MPE was chemically altered, a nding that was facilitated by the AFM and CLSM analysis and the correlation among the results as a whole. Therefore, electrooxidized MPE may be useful for further investigation of the evolution of insoluble reduced sulfur compounds and biolm formation during different stages of MS colonization, studies that are necessary to understand the complete interfacial processes between pyrite and oxidizing microorganisms. Acknowledgments Financial support for this work comes from the Mexican Council of Science and Technology (CONACyT) (project no. 05-49321). The authors thank Dr. Amauri Pozos and Dr. Jaime Ruiz-Garca for access to the CLSM (Basics Sciences Laboratory) and the AFM (Colloids and Interfaces Laboratory) equipment at UASLP, respectively. The authors

also thank Erasmo Mata-Martinez for mineral section preparation, Francisco Galindo-Murillo for MPE preparation and Keila Nery Alvarado-Estrada for CLSM analyses and technical assistance. Special thanks to Dr. Miguel Angel Vidal-Borbolla for his comments on the manuscript.

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