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Institute of Metallurgy. 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. elemental sulfur.V. Lara a. San Luis Potosí. 78210. S0). All rights reserved. indicating that EPS plays a key role during microorganism/surface interactions. A. 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. R. Schippers and Sand. San Luis Potosí. García-Meza).. in fact. Engineering Faculty. 2003). S0 was generally added to promote the growth of A. 1990.g. 1999. (J. moderate temperature and pressure. D. affecting the reactivity and delaying the complete oxidation of MS (Mustin et al. 1981..R.014 According to Schippers and Sand (1999). the Fe(III) ions. 1999). thiooxidans forms a monolayered biofilm. During this process. A. wherein the contact resulted in a strong adhesion force (467 pN) between the cells and the altered surface. UASLP. San Luis Potosí./fax: + 52 444 825 4326.. Introduction The sulfur-oxidizing microorganisms (SOM: archaea or bacteria) play a major role in the cycling of sulfur in the biosphere. 0304-386X/$ – see front matter © 2010 Elsevier B. 78210. 1999.G. 1999. ferrooxidans may contribute both sulfurand iron-oxidizing capabilities to the bioleaching process. we applied the combination of AFM. México.2010. Preliminary research coupled either epifluorescence and atomic force microscopy (AFM) or Raman spectroscopy and AFM to analyze biofilms of A.V. However. perhaps due to an irreversible binding mechanism.. A.. .1016/j. Cruz a. Valdez-Pérez b. 2007).V. and in the absence of SOM (Hamilton and Woods. Bevilaqua et al. e l s ev i e r. The ability of SOM has been applied in the mining industry to the bioleaching of metals from metal sulfides (MSs) to remove pyritic sulfur from coal and to the desulfurization of wastewater (Gourdon and Funtowicz.H. thiooxidans and A. 2008). SLP. ferrooxidans and Leptospirillum ferrooxidans) is the regeneration of the oxidizing agent. nevertheless the reduced sulfur compounds may affect MS weathering or dissolution. 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. Rodríguez c. 100%). Tel. 78210. while the SOM (e.. 1998). Dopson and Lindström. A. Lomas 2°. thiooxidans causes bioacidification. Here. J. It is well known that A. México Institute of Physics. 78210. ferrooxidans on pyrite or the surface features of pyrite in biotic and abiotic experiments. H. Geomicrobiology Area. Interfacial studies of the role of extracellular polymeric substances (EPSs) during cell attachment to MS have been restricted mainly to iron-oxidizing bacteria. Fowler and Crundwell. 2008). which via a series of reactions subsequently yields sulfate and protons (via tetrathionate and other polythionates) or elemental sulfur (S0). Because S0 may be inert under acidic conditions (Rohwerder and Sand. by the indirect attack of hydrated Fe(III) ions from the crystal lattice.V.Author's personal copy Hydrometallurgy 103 (2010) 35–44 Contents lists available at ScienceDirect Hydrometallurgy j o u r n a l h o m e p a g e : w w w. 2002. Dopson and Lindström. México 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 sulfide minerals (MS) are necessary to understand and improve the bioleaching processes of mining industries. UASLP. SOM such as Acidithiobacillus thiooxidans and A. 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 Biofilms Pyrite Raman spectroscopy Atomic force microscopy Epifluorescence 1. Mycroft et al. San Luis Potosí. providing protons to maintain Fe (III) is its oxidized state (Schippers and Sand. S0 is also produced by the biological activity of IOMs. an overproduction of EPS was recorded (ca. The observations strongly suggested an intimate contact stage during the dynamic interfacial mechanisms of S0 biooxidation on the pyrite surface. Nava et al. doi:10.02. García-Meza a. Lomas 2°. México Coordination for the Innovation and the Application of Science and Technology. © 2010 Elsevier B. ⁎ Corresponding author. Rohwerder et al.⁎ a b c Institute of Metallurgy. Engineering Faculty. thiooxidans in some bioleaching process (Liu et al.. thiooxidans on previously oxidized pyrite. E-mail address: jvgm@uaslp.g. the main role of Fe(II) oxidizers (A. All rights reserved. Sierra Leona 550. UASLP. Sierra Leona 550. The main reduced sulfur obtained is the thiosulfate. The S0 is a by-product produced in significant amounts (10–20% S0) during the chemical or electrochemical oxidation of pyrite at pH b 2. Navarro-Contreras c. Salvador Nava 110. 1993. Raman and principles of epifluorescence for the study of biofilms of the A. UASLP. Our results showed that A. 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.. caldus) contribute to the overall process by the oxidation of the intermediary (reduced) sulfur compounds to sulfuric acid (Sasaki et al.. In the bioleaching of base and precious metals from MS. Sierra Leona 550. Additionally. 1999). SLP. neglecting interfacial mechanisms associated with the biooxidation of reduced sulfur compounds (e. the degradation of an acidinsoluble MS arises via the thiosulfate mechanism by means of electron extraction. Lomas 2°. SLP. 1998).hydromet.

SHE. in biotic and abiotic experiments. Abiotic oxidation permits the overproduction of − both S0 and S2 n directly from the pyrite..2 to 1. Recently. pH and temperature). the standard hydrogen electrode). 0. at high redox potential. the counter electrode was a graphite rod (AlfaAesar. Sand. Accordingly. ferrooxidans (Rohwerder et al.91 and 1. results in more extensive pyrite oxidation by L. Potentiostatic MPE oxidation − Because S0/S2 n species are required to sustain the metabolic activity of the SOM. We applied the combination of AFM. ferrooxidans and L. (2008a. 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 biofilms. AFM was previously applied to study the morphology of pyrite surfaces after interaction with A. and its secretion is an essential characteristic of the biofilm development. and the MPE surface was polished until a mirror-like surface was obtained. SHE. Vadillo-Rodríguez et al. Rodríguez et al. allowing a better comparison between the assays (see Section 3). 1999. at a more positive potential (N 800 mV. Lara et al. typically found in bioleaching tanks due to Fe(III) accumulation) L. This analysis was carried out in acidified ATCC-125 (American Type Culture Collection) medium at pH= 2. ferrooxidans and favors the proliferation of SOM-like A. the researchers suggested that SOM completely lack complexed Fe(III) or other positive charged groups in their EPS. It had been demonstrated that the attachment of cells as biofilms to MS surfaces enhances bioleaching of MS (Pogliani and Donati. their attachment to the MS surface has been scarcely studied (e. ions. 2003).11 V vs. Hence. Despite the vital role of SOM biofilms during the bioleaching process. 2003). These analyses confirmed mineral identity and the presence of inclusions of chalcopyrite and sphalerite (∼ 0. All the former showed the essential role of SOM during the commercial bioleaching process. mineral particles. Mexico.. 1999. colloidal and particulate compounds. However. The EPS matrix represents 50–90% of the biofilm. the electrochemical − oxidation of MPE resulted in a well-controlled formation of S0/S2 n . / Hydrometallurgy 103 (2010) 35–44 ferrooxidans is not the dominant species in commercial bioleaching tanks due to the solution conditions (e. Because A. CLSM and Raman spectroscopy are twofold: first. thiooxidans because more reduced S-compounds may be potentially recoverable by them at the pyrite surface (Rawlings et al. the working electrode was the MPE. Since 1995. composed of extracellular polymeric substances (EPSs). 1999. The aims of this work using AFM. 2006. 2. the formation of reduced sulfur compounds on MPE is favored at potentials between 0. and the reference electrode was a saturated sulfate electrode (SSE. 2003. 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. 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 sulfide or the defects and disorder in the lattices.H. Mineral samples Pyrite samples were obtained from Zacatecas.5 cm2) were mounted in epoxy resin with a silver epoxy electrical contact on the backside. and Edwards et al. 1981. ferrooxidans on pyrite. Busscher et al. (1999) combined Raman spectroscopy and AFM to obtain information on the bulk surface-speciation and the surface features of pyrite. first and foremost for the initial attachment of the cells to the surface (Neu et al. Raman spectroscopy was subsequently used for a precise identification − of S0 or polysulfide (S2 n ) species on the pyrite surface. the interfacial processes are poorly understood. In our study. Sand et al... thiooxidans differ in their capability of attachment (Porro et al. and second. the amount of EPS-bound Fe(III) ions varies with the metabolic activity of the Fe oxidizers like A. 1997). 1990.. Gehrke and collaborators have highlighted the importance of the biofilm matrix as a reactive space wherein the electrochemical mechanisms/surface reaction takes place. Further. 2005. 1998. Thus. and open water channels. According to this setup. Tsang et al..11 V vs. gelatinous and highly hydrated matrix.. to evaluate the role of bacterial EPS during contact mechanisms between this SOM and oxidized pyrite under acidic conditions. . Sharma et al.5% wt).. e. A... The potentials tested were from 0. Materials and methods 2.. Gerischer.g.2...g. Biofilms are highly organized systems of microorganisms. 2003). gases. and that diverse species such as A. Gehrke et al. specifically 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. thiooxidans is not able to oxidize pyrite or other acid-insoluble MS.9995% purity). Cruz et al..5 to 1. Kelshall et al. 2004.. 2. the − formation of S0 and/or S2 n species on electrooxidized MPE surfaces was confirmed by Raman spectroscopy after each electrooxidation.Author's personal copy 36 R.. 2006). ferrooxidans outcompetes A. AFM was additionally used to quantify adhesion forces associated with the bacterium–mineral interaction. Preliminary voltammetric analysis of MPE was carried out to establish the potential values where reduced sulfur compounds were electrochemically formed. 2003) and therefore. we examined the stained biofilms using a confocal laser scanning microscope (CLSM). 0.. This is the first work of a series of studies on the interfacial characterization of SM in the presence of A.g. 2003) e. which completely covered the surface of the MPE.b) coupled atomic force microscopy (AFM) and epifluorescence microscopy to analyze the biofilms of A.. 2002).3% wt) and sphalerite (ZnS. SHE (3600 s). 99. Liu et al.. Instead of using epifluorescence microscopy.. ferrooxidans than by A. Crystal samples were selected for the construction of massive pyrite electrodes (MPEs): mineral sections (1. 1999). and it has been extensively utilized for the study of non-leaching bacteria (e. among others (Hamilton and Woods. Mangold et al. using an autolab PGSTAT 30 coupled to a PC and a classic Pyrex® glass three electrode cell. 2003..615 V vs. ferrooxidans. 2001). Lizama et al. with minor amounts of chalcopyrite (CuFeS2. The adhesion force implicit in biofilms formation is useful in understanding the interfacial characteristics of the bacterium–mineral system. embedded in a selfproduced. thiooxidans on previously oxidized pyrite. which could be indicative that mechanisms other than electrostatic forces are important during cell attachment. 0. Atabek and Camesano.. Mineralogical analysis of samples indicated a pyrite purity of 99. Dopson and Lindström. 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. but the interfacial chemistry of the MS. 2008). thiooxidans (Liu et al. including aquatic (saturated) environments (Stoodley et al. 2007.g. the pyrite was chemically oxidized with Fe (III) and H2O2 as well as electrochemically oxidized. Raman and principles of epifluorescence to the study of biofilms of the SOM A. respectively. Touhami et al. thiooxidans. ferrooxidans and A.5% wt. Lower et al. 2006.1. Those studies demonstrated that such a combination is a powerful tool for the investigations of biofilms on opaque materials and ultimately increase knowledge of the biological interfacial process. 2003). Electrooxidation was preferred because it was less time-consuming and resulted in more − S0/S2 n production than chemical oxidation..g.. Furthermore. 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. thiooxidans for acidinsoluble and acid-soluble MS. ferrooxidans (Sand and Gehrke.. in order to elucidate the role of SOMs in the biochemical process of MS dissolution. Worldwide studies have demonstrated that most of microbial communities spend a large part of their life cycle within a biofilm.2% wt). caldus and A. 2001.

Accordingly. Results Raman analysis was carried out to evaluate the status of sulfur compounds on MPE surfaces. thiooxidans to compare − the development of S0/S2 n species and the Raman response with those obtained after MPE electrooxidation..Author's personal copy R. for the epifluorescence of EPS compounds (α-mannose.01 g of FeSO4·7H2O. 0. Sasaki et al.4) and acetic acid (5% in ethanol) solutions were used for washing and dissolving the stain. For its sterilization. Similar experiments were carried out using non-electrooxidized MPE under acidic conditions with and without A. Although it is desirable to characterize cell– mineral adhesion forces using colloidal tips (see Kendall and Lower. the MPE was placed in a flask with 100 mL of the ATCC-125 (pH = 2) and ∼ 108 cells of A. Derjaguin et al. 28–30 °C).08 and 2. The silicon cantilever showed a free resonance frequency between 275 and 325 kHz and a constant between 31.H. the same MPE exposed to A. Lara et al. In contrast. 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 Pérez.5 g of MgSO4·7H2O. At least 10 Raman spectra were collected from each specific surface. The non-electrooxidized MPE exposed to the culture media without microorganisms (during 5 days and under the same conditions) was also analyzed by AFM. 2. / Hydrometallurgy 103 (2010) 35–44 37 In order to validate that the formation of S0 species on electrooxidized surfaces evolves toward crystalline structures during weathering of pyrite in acidic conditions. Subsequently.536 N/m during these experiments.3. A planktonic (suspended) inoculum was used for the biotic experiments. and 0. Finally. The biofilms were previously stained during 30 min in the dark. AFM observation and analysis were carried out for pristine (un-electrooxidized) and electrooxidized surfaces (see Section 2. pristine. 1999). 75 mL of acidified ATCC-125 growth medium. and in the non-electrooxidized MPE exposed to the culture media without microorganisms. After the bioassay. specific surface.g. Raman performance was validated using a Si wafer by assuming a single sharp peak or a wavenumber of 521 cm− 1.5.03 N/m during the collection of these curves. The JKR model is commonly used for the assessment of high adhesion forces and large tip radii. the DMT model seems the most appropriate to evaluate the interactions between the biofilm and the mineral surface. 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. thiooxidans (biotic surfaces) were analyzed by CLSM (Leica DMI4000B with a 63× immersion objective). short-range repulsive interactions. roughness (Ra. the interaction forces measured by AFM force-separation curves are mainly van der Waals interactions. Final pH of medium was adjusted to 2 with H2SO4. Toniazzo et al. Taylor series). The exponential growth phase was reached in 8–10 days with an average biomass of between 107 and 108 cells/mL. respectively. 2002. 1998. respectively.18 and 44. Indeed. Collection time was 60 s in each analysis. 2002).8 μm on the sample. in electrooxidized MPE. Bacterial strain and cultivation Acidithiobacillus thiooxidans strain ATCC-8085 was used in this study. α-glucose) and genetic material (RNA and DNA). e. thiooxidans. which allows quantification of the adhesion forces mediating bacteria attachment to MS (Kendall and Lower.4. Surface analysis of massive pyrite electrodes (MPEs) Before the assays. For a monolayer attached to a substratum. The media was dispensed into 250 mL Erlenmeyer flasks and were sterilized by autoclaving at 121 °C for 15 min. and γ is the surface energy for the Johnson–Kendall–Roberts (JKR) and the Derjaguin–Muller–Toporov (DMT) models (Johnson et al. Spectra Physics) with a power of 2 mW. the MPE of biotic and abiotic control assays were collected. adhesion and capillary forces (Garcia and Pérez. 0. An “abiotic” (uninoculated) control was also carried out in triplicate in order to compare chemical and biological oxidation of S0. Curry and Kim. 2004). the cell–mineral adhesion forces were measured in this study with a triangular tip cantilever. Pyrite surfaces were excited by an Ar+ laser beam at 514 nm (Stabilite 2017. R is the radii of cantilever tip.4 g of (NH4)2SO4.25 g of CaCl2·2H2O. the DMT model is used for the assessment of low adhesion forces and small tip radii (Garcia and Pérez. and a daily addition of 1 mL of H2O2 during 15 days under orbital shaking (pH = 2. and preserved in a desiccator under inert conditions until their analysis. nm) of MPE surfaces were also evaluated in biotic and abiotic assays to generate a complete description of MPE surface. the electrooxidized MPE was exposed to UV irradiation for 24 h. 0. After AFM analysis. The formation of crystalline S0 species was confirmed by Raman spectroscopy. thiooxidans) and abiotic control (electrooxidized MPE without A. Force-separation curves were acquired using contact mode in 1 μm2 areas.. Raman backscattering showed a signal/noise ratio greater than 100 for Si analysis.. Raman spectroscopy analysis was carried out in pristine MPE. After 5 days.2).. 3. the same analyses were done for biotic (electrooxidized MPE with exposure to A. The detector was a charged coupled device (CCD) cooled by liquid nitrogen. and focused with a diameter of about 0. This experiment consisted of chemical oxidation using Erlenmeyer flasks containing 5 g of pristine pyrite grains. thiooxidans cells). in MPE electrooxidized after the biotic and the abiotic assays. 2. 1971. Narrow and wide regions were visualized to obtain topographic images by tapping mode (scan rate between 0. pH = 7. 2008a. Similar contact times have been used for A. 3 g of KH2PO4. ferrooxidans or A. using sequentially applied lectin Canavalia ensiformis (Con-A) and fluorochrome Acridine Orange (AO). 2004): FAdh = 4πRγ FAdh = 3πRγ ð1Þ ð2Þ 2. For spectroscopic analysis. In the absence of external electric fields. Biofilm formation − Electrooxidized MPE (see Section 2. The vibrational range was 100–750 cm− 1 as − species show their main active modes within this interval the S0/S2 n (e. afterward.5 and 1 Hz). HEPES (2%. The AFM analysis of MPE surfaces was performed with a Nanoscope Multimode IIIa digital instrument. The culture was incubated aerobically at 28–30 °C and 150 rpm for 5 days. The Si3N4 cantilever showed a free resonance frequency between 90 and 115 kHz and a constant between 1. 2004). which contained per liter of distilled water: 10 g of S0 (Baker).. However. thiooxidans. The force-separation curve is a representation of the extending and retracting curves associated with the interaction forces between the cantilever tip and substratum. 2000 mg/L of Fe(III).2) was used as the S0 and/or S2 n source for A. an additional experiment using pristine pyrite grains was performed (106–150 μm. Harneit et al. and 10% of the obtained curves were randomly analyzed. 2006). This biotic assay was done in triplicate. dried with a direct current of nitrogen. ensuring a good Raman performance during mineral surface analysis.. Acidithiobacillus thiooxidans were cultivated aerobically at 28– 30 °C in 50 mL of media ATCC-125. b. 2002). S0 was sterilized separately using 2 h of UV irradiation with intermittent shaking.. nm) and root mean square (Rq. a triple monochromator Raman Jobin Yvon T64000 spectrometer equipped with an optical microscope (Olympus BH2-UMA) was used.g. electrooxidized (before the . At least 300 curves were taken from each FAdh is the adhesion force. 1975). thiooxidans biofilms formation on pyrite under various conditions (Mangold et al.. 2004).

Lara et al.H. / Hydrometallurgy 103 (2010) 35–44 .Author's personal copy 38 R.

and progressive crystallization of S0 during pyrite weathering under the tested acidic conditions. Table 2 shows the Ra. Finally. 1g). 4d to i). 2g). the surface of the electrooxidized MPE after the abiotic assay showed bigger structures (Fig. the measured adhesion forces of the MPE surface from the biotic (4867 pN) and abiotic assays (4400 pN) (Table 1. 1e). we conclude that such nano-scale size structures observed on electrooxidized surfaces by AFM − (Fig. The AFM analysis of the electrooxidized MPE after the biotic assay showed the presence of attached cells of A. In a similar way. In this study. 1d) and AFM (Fig. These results indicated a further surface alteration after 5 days of immersion in the acidic medium under abiotic conditions. Raman spectra collected from pristine MPE surfaces is presented in Fig. 2c) indicated. thiooxidans were not clearly defined (Fig. However. thiooxidans showed low intensity. 2j and k) can be associated with the progressive accumulation of S0 well-crystallized species.. which is a clear − species on these surfaces signature of the formation of S0/S2 n (Mycroft et al. This difference is indicative of factors affecting Raman backscattering from MPE surfaces. From previous results. Adhesion forces in pristine and electrooxidized MPEs (before the assay) were evaluated in order to monitor the evolution of surface characteristics. similar to those obtained in the pristine MPE surfaces (Fig. 2e to i). the increase in height of the nanoscale size structures in abiotic MPE (Fig. 2l). thiooxidans cells to the MPE and is the same order of magnitude reported for non-leaching bacteria forces under various conditions (Vadillo-Rodríguez et al. comparing the Ra. the depletion of the nano-scale size structures of S0 observed by AFM (Fig. the Raman spectra of the electrooxidized MPE before the assays (Fig. The global interaction may result from the combination of repulsive forces between the cantilever tip. 1c). The adhesion forces of the attached cells were quantified as described in Section 2.Author's personal copy R. respectively). 1a. The relative intensity of EPS epifluorescence in the observed stained cells (at least 50 cells) associated with biofilms is Fig. 2j and k show evidence of scant pyrite oxidation. The Ra. (f) non-electrooxidized MPE and after 5 days in culture media without A. Biofilms comprised a monolayer of bacterial cells with a typical size between 1 and 2 μm (Fig. 1c). 2d to i) and Raman spectroscopy (Fig. 1990. 1998. (d) electrooxidized MPE after abiotic assay. the Raman spectra collected from oxidized mineral grain samples (Fig. thiooxidans (Fig. It is well known that Ra. In the MPE from the biotic assay. 222 (v3) and 465–470 cm− 1 (v1). Fig. Raman spectra from the non-electrooxidized MPE after 5 days in culture media without A. 1e) in MPE surfaces after 5 days of exposure to A. thiooxidans. Rq are parameters that can be indicative of a coarse or uneven surface and in this way reflect changes in the surface topography as a result of external perturbations.H. Lara et al. 10 nm height. confirming a further alteration of the electrooxidized MPE after 5 days under acidic conditions (Fig. among others. 1999). 1e and a. 2005). Rq information from the same AFM images presented in Fig. 20 A. Attached cells were easily discernable. Thus. Examples of force-separation curves obtained in this study are presented in Fig. Raman peaks collected from MPE surfaces were discernable. Such factors could be associated with the semiconducting properties of pyrite. Meanwhile. 1f). whereas the ratio obtained from MPE surfaces was around 6. 2 but using respective 2D images with several areas (Table 2). Force data of the MPE surfaces from abiotic assays were ∼ 4400 pN (Table 1). thiooxidans cells forming biofilms were evaluated by comparing force data obtained for biotic and abiotic assays. Several force measurements were acquired on each specific MPE surface. changes in crystallographic plane orientation of resulting species in biotic surfaces (e. as the Raman spectroscopy (Fig. 2c) and non-electrooxidized MPE (exposed 5 days to the media. 3b) is observed (Kendall and Lower. These results indicated that the formation and accumulation of S0 with a higher crystallization degree enhance an attractive force (Fig. stability. thiooxidans also showed low intensity.. we obtained Ra.. thiooxidans is clearly associated with biotic oxidation of S0.. The Raman spectra of the MPE surface after 5 days of exposure to A. (b) chemically oxidized grain mineral particles. The Ra average from electrooxidized surfaces was ∼ 20 nm. 2b). resulted in a minor Ra. Afterward. noisy peaks. evidenced by limited formation of nano-scale structures (ca. but the v1 was sharper in the electrooxidized MPE − species after abiotic assay than before the assay. Sasaki et al. Typical peaks at 343 (v2) and 381 cm− 1 (v1) confirm the mineral identity (Sasaki et al. respectively). 2e to i). The obtained Raman spectra of the MPE surface after the abiotic assay (Fig. 3b). Collection time of 60 s. and (g) Si wafer backscattering. 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. (e) electrooxidized MPE after biotic assay. and the attractive force of S electrooxidized surfaces before the assays. Table 2). Fig. The forces associated with the interactions between MPE surfaces and A.g. 4a to c) and biofilms on MPE surfaces from the biotic assay (Fig. attached cells seem to be imbedded in the resulting nano-scale size structures (Fig. 1d). Toniazzo et al. 3c and d) indicated an adhesion force of 467 pN for A. Fig. 2c) that seems to result in an increase of the nanoscale size structures. Rq on electrooxidized MPE before and after the biotic and abiotic assays. 2004). indicating S0 species were totally consumed on electrooxidized MPE surfaces after 5 days of biotic contact (Fig. This last surface exhibited an adhesion force of 1483 pN. 1c) showed additional peaks at 155 (v2). and the details associated with the underlying substratum were clearly distinguishable (Fig. / Hydrometallurgy 103 (2010) 35–44 39 assays). This fact and the impossibility of tracking the status of sulfur compounds on mineral surfaces rendered it difficult to perform a deeper analysis of interfacial processes by AFM or Raman spectroscopy.. an attractive interaction (see the retracting curve in Fig. Fig. λ = 514 nm. Rq of the MPE from the biotic system (∼ 36 nm).. 2b) are mainly S0/S2 n species. polycrystalline structures). a contrasting surface transformation can be observed by AFM: the formation of widespread nano-scale size structures of altered mineral (Fig. 2004. 1b) were fairly similar to those obtained on abiotic control surfaces (Fig. However. After the potentiostatic electrooxidation of MPE. The adhesion force of pristine MPE was omitted due to its low magnitude. thiooxidans.. 1f). indicating that S2 n 0 were completely oxidized to S after 5 days. and disorder in mineral lattices composing such species. 1. the Raman analysis carried out here is valid. Rq of the abiotic system were the highest (∼ 67 nm and 84 nm. This value indicates an attractive interaction associated with the adhesion of A. 1998). It is important to note that the Raman backscattering obtained from electrooxidized MPE was lower than that observed in the Si wafer. 3. AFM images from non-electrooxidized MPE after 5 days in culture media with A. the electronic − 0 species on characteristics of S2 n . . noisy peaks associated with those of pristine samples (Fig. Rq values of the MPE surfaces from the biotic (adjacent-surrounding biofilms areas) and abiotic systems. and electrooxidized after the biotic and abiotic assays. Finally. 3c). which was below the experimental standard error of the value of the forces of the electrooxidized surface. thiooxidans. Ra.5. Raman spectra collected from different MPE surfaces: (a) pristine (non-electrooxidized) MPE. 4 shows CLSM images from planktonic cells (Fig. Fig. Rq were calculated directly on biofilms and adjacent-surrounding biofilm areas (not on top of attached cells). in agreement with Raman spectroscopy data (Fig. − species can be attributed where the absence of detectable S0/S2 n exclusively to the small extent of chemical alteration of the MPE. 2d to i). thiooxidans (Fig. thus confirming the formation. This last is in agreement with Raman spectra obtained from non-electrooxidized MPE and after 5 days in culture media without A. 4 makes clear the differences between the planktonic and attached cells of A. Lower et al. 1d) showed similar peaks to those observed from electrooxidized surfaces (Fig. (c) electrooxidized MPE before the assays. which showed a signal-to-noise ratio greater than 100 (Fig. 2b). and therefore. In fact. In spite of these factors. 2c) than those observed in the electrooxidized MPE before the assays (Fig.

Author's personal copy 40 R. Lara et al. / Hydrometallurgy 103 (2010) 35–44 .H.

69 52. 1e). thiooxidans (Fig. Contact mode.01 30.78 80.5–1 Hz. respectively) and a cell density of 1 g/cm3 (suspended or planktonic cell). suggesting a molecularly mediated.12 73. height and width of ∼ 1. Images were acquired in air using tapping mode and a scan rate of 0.32 62.66 ± 2.5–1 Hz.15 22. (b) electrooxidized MPE before the assays.79 41. (d to i) electrooxidized MPE after biotic assay.92 Roughness (Ra) nm 19. 2d to l).25 ± 1.10 ± 0. 4g). (c) electrooxidized MPE after abiotic assay.18 24.41 2. (c) electrooxidized MPE after abiotic assay.71 ± 6.35 ± 2.H. After the attachment of microorganisms.5–1 Hz). equivalent to the weight of a single cell. Rq of the MPE surface adjacent-surrounding (not on attached cells) biofilms was lower (67 nm) than of the abiotic assay (84 nm) (Table 2).97 ± 8.27 ± 4. suggesting an intimate physical contact between the bacteria and the pyrite. MPE surface Electrooxidized (1.3 and 0. the Ra.83 7.Author's personal copy R.72 5.56 ± 3.11 V/SHE) Abiotic assays Biotic assays/biofilms: collected on top of attached cells Biotic assays/biofilms: 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. 1c).08 ± 3. Lara et al.5 1×1 0. The area of the obtained curves was 1 μm2.135 μm3 (from a length.85 ± 2.78 19. This depletion of the nano-scale size structures of S0 observed by AFM and Raman spectroscopy in biotic Fig. 1e). MPE surface Pristine Electrooxidized (1. Scan rate of 0.66 17.06 18. and decreased after 5 days in the biotic assay (Fig.25 × 0.28.64 ± 1. Height of elements is shown in the figure.58 37.60 ± 5.51 45.41 ± 1.58 ± 2.11 V/SHE) Area (μm × μm) 5×5 2. thiooxidans.77 ± 9. the cell force was ∼ 1.13 × 10− 3 pN.74 Abiotic assay Biotic assays (collected on top of attached cells) Biotic assays (surrounding attached cells) Non-electrooxidized MPE.54 ± 4.25 55.5.42 66.41 ± 9. thiooxidans. and (d) electrooxidized MPE after biotic assay. (j and k) non-electrooxidized MPE and after 5 days in culture media without A.96 83.25 5×5 1×1 5×5 1×1 5×5 1×1 5×5 1×1 Number of areas 5 5 35 19 50 25 35 15 25 45 30 40 RMS (Rq) nm 24. (b) electrooxidized MPE before the assays. due to the decrease of the nano-scale size structures formed in the electrooxidized MPE after the assays (Fig.42 ± 15.13 ± 3.21 36.3 μm. irreversible binding mechanism to MPE (see below). and (l) non-electrooxidized MPE and after 5 days in culture media with A. Discussion and conclusions Acidithiobacillus thiooxidans forms monolayered biofilms strongly attached to the electrooxidized MPE surface. 2.67 ± 6. Area of collection was 1 μm2 (scan rate of 0. Such structures are principally − S0/S2 n . thiooxidans has a volume of 0. The average number of cells comprising biofilms was 27 ± 3 in most of the cases (Fig. 4. Table 1 Interaction forces calculated from each specific MPE surface (n = 30). whereas that from planktonic cells is 33.63 ± 1. . Three dimension images of AFM from MPE surfaces: (a) pristine (non-electrooxidized) MPE.48 ± 0.41 ± 4. after 5 days in culture media 68.12 ± 5. 0. Assuming that each cell of A.7.5 × 2.55 ± 2. The contact resulted in a strong adhesion force (467 pN) between the cells and the altered surface. It is important to notice that such a strong adhesion force was recorded after the sulfur biooxidation by A. 3. Force-separation (extending and retracting) curves of MPE surfaces in air using contact mode: (a) pristine (non-electrooxidized) MPE. / Hydrometallurgy 103 (2010) 35–44 41 Fig.49 22. as the Raman analysis indicated (Fig.02 1.04 ± 2.

Rq) could be a consequence of the heterogeneous corrosion of electrooxidized surfaces due to a random distribution of defect points and impurities. A. thiooxidans. and the bacteria directly oxidizes the reduced sulfur compounds due to its sulfur oxidation capacity. the observed topography in the resulting surface after the biooxidation of S0 species would expose a pyrite structure with variable corrosion patterns. which formed a monolayered biofilm attached to the MPE surface. Since in our experiment. 4. The surfaces of the abiotic MPE showed the presence of well-crystallized S0 after 5 days under acidic conditions. ferrooxidans removes sulfur by a direct contact mechanism. resulting in a strong biofilm/ mineral interaction. image (i) showed the epifluorescence of exopolysaccharides in biofilms. see Section 2. (1995) and Liu et al. Epifluorescence of exopolysaccharides are in green. the concept of a biofilm matrix as a reaction space implies that EPS chains are in direct contact . Florian et al. The formation of S0 clusters of variable height (expressed as Ra. the aforementioned results indicate that the differences are clearly a consequence of the oxidation of the S0 generated on the electrooxidized MPE by A.3). Direct contact of A. assays showed the feasibility of the oxidation of S0 by A. According to Rohwerder and Sand (2007). / Hydrometallurgy 103 (2010) 35–44 Fig. According to Fowler and Crundwell (1999). thiooxidans and also suggested a dynamic interfacial mechanism.01 g of iron. Konishi et al. thiooxidans with the pyrite surface was previously suggested by Takakuwa et al. the Fe(II)/Fe(III) ratio was the same in both cultures with A. Consequently. whereas genetic material is in red. Recently.Author's personal copy 42 R. (d to i) after 5 days of cells attachment in biofilms on electrooxidized MPE. Lara et al. CLSM images of A. An overlap of both epifluorescence images is presented in the low-left side of images (a to g). thiooxidans cells: (a to c) planktonic (suspended) cells. thiooxidans and in the abiotic control (0. thiooxidans attach to pyrite in pure and mixed cultures. (2009) and Echeverría and Demergasso (2009) reported that A.H. Image h is a zoom of the overlap showed in (g). (2003). (1977). among others (composing mineral lattices).

4). J. Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans. Special thanks to Dr. The findings of Rohwerder and Sand support our results. Rohwerder and Sands. Colloid Interface Sci. Effect of contact deformations on adhesion of particles. 1985. as was previously indicated by Rohwerder et al. Electrochim.. W. Noël... 189 (23).Q. Huang.. 2743–2747.A.F. 1999. thiooxidans to the pyrite does not require EPS-complexed Fe(III) ions but relies on other.P. T. 17–24.J. Leng. M.M. 65 (12). J.. M. Hydrometallurgy 83. 144. A. O. Jaime Ruiz-García for access to the CLSM (Basics Sciences Laboratory) and the AFM (Colloids and Interfaces Laboratory) equipment at UASLP. e. S. 1998. 155–179. 245–254.Y. Hence. Sand and Gehrke. 62.. Pace. 1990.F. Yin... Miguel Angel Vidal-Borbolla for his comments on the manuscript. Leite.. Such overproduction of EPS is directly associated with the increase of epifluorescence in attached cells (Rapposch et al. B. Schippers and Sand. J. Adv. 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