Adhesion Studies of Acidithiobacillus ferrooxidans on Different Surfaces by Atomic Force Microscopy D. Bevilaqua, O. Garca, C. Fugivara, A. Benedetti State University, Sao Paulo, Brazil Diz-Prez, F. Sanz University of Barcelona, Spain oswaldo@iq.unesp.br

ABSTRACT The adhesion of Acidithiobacillus ferrooxidans on different surfaces was evaluated by atomic force microscopy (AFM). Chalcopyrite (CuFeS2) and bornite (Cu5FeS4), true substrates for the bacterium and mica and graphite (non-energy substrates), were incubated in the presence of At. ferrooxidans in a mineral salts solution at pH 1.8. Before the incubation the cells were centrifuged to strip exopolymeric substances (EPS), which is believed to be essential for its attachment on mineral surfaces. EPS was not necessary for the initial attachment on both hydrophobic sulfides but its excretion was time- and substrate-dependent. Bacterial attachment was not detected on mica, probably due the hydrophilic nature of this mineral and the low ionic strength of the medium. On the other hand, the adhesion on graphite, also a hydrophobic material, was very rapid, which could be attributed mainly to the van der Walls interactions. Key words: chalcopyrite, bornite, bacteria, HOPGraphite, mica, metal sulfide

INTRODUCTION Acidithiobacillus ferrooxidans, formerly Thiobacillus ferrooxidans (Kelly, 2000) is an acidophilic chemolithoautotrophic bacterium capable to utilize either ferrous iron (Fe2+) or reduced sulphur compounds, including mineral sulfides, as the sole energy sources for its growth. Due to its capacity to oxidize metal sulfides, this bacterium is one of the most important microorganisms utilized in industrial operations of metal leaching, such as copper, uranium and gold. During the last decade, direct and indirect mechanisms of oxidation of metal sulfides by this microorganism have been proposed and discussed (Boon, 2001; Crundwell, 2001; Sand et al., 2001; Tributsch, 2001). Independently of which reaction mechanism (or both) is correct; the previous step of bacterium attachment to the metal sulfide surfaces has been detected and showed by different authors using several techniques (Escobar et al. 1997; Porro et al. 1997; Sampson et al. 2000; Bengrine et al. 2001). Even though the adhesion of At. ferrooxidans on mineral surfaces has been demonstrated in some cases to be tenacious and often irreversible (Karan et al. 1996; Srihari et al. 1991), still remains unclear which forces drive and how exactly the bacterium-surface interaction occurs. A priori, this interaction will depend on the physical-chemical properties of the surfaces and cell envelope, mediated by the ionic strength of the grown medium. Several publications have been devoted to explain the role of cell adhesion (van Loosdrecht et al. 1987; Devasia et al. 1993; Curutchet & Donati 2000; Sampson et al. 2000). These authors have shown that sulphur grown cells of At. ferrooxidans present more hydrofobicity than those grown in ferrous iron medium, and in turn, this condition could be associated with more or less observed adhesion. More recently, other studies suggested that proteins (Ohmura et al. 1996; Somasundaran et al. 1998; Sharma et al. 2001) and exopolymeric substances (EPS) present in the cell envelope (Kinzler et al. 2001; Gehrke et al. 1998; Escobar et al. 1997) might play a very important role in the initial stages of adhesion. Blake et al. (2001), for example, indicated that the specific and high affinity adhesion of At. ferrooxidans to pyrite could be mediated by the protein aporusticyanin located on the outer surface of the bacterium. Chalcopyrite (CuFeS2) and bornite (Cu5FeS4) are oxidized by At. ferrooxidans at rather different rates, being the former very slow as compared to the second (Bevilaqua, 1999). Both copper sulfides have been not yet analyzed regarding to its At. ferrooxidans adhesion pattern nor if this pattern can explain the different reaction rates. Dealing with the direct interaction between the cell envelope and the surface, in the present contribution, the lipopolysaccharide-containing EPS was carefully removed from the cells in order to verify if these compounds are a prerequisite for bacterial attachment to these substrates. Indeed for sake of comparison and in order to investigate the nature of the bacterial-substrate interaction, the adhesion of At. ferrooxidans on two inert surfaces has been also studied, taken into account the different surface properties, hydrophilicity in the case of monocrystalline cleaved mica (Kopta and Salmeron, 2000; Xiao et al. 1995) and hydrophobicity in the case of graphite (HOPG) (Guntherodt et al. 1992; Kogan et al. 2001). Atomic force microscopy (AFM) was used to follow cell adhesion as one of the most effective techniques for biomaterial imaging due to the optimal high resolution attained either ex situ and also in situ, in vivo, single bacterium cell on a well defined surface (Telegdi et al. 1998 and Fang et al. 2000). Only few works deal with AFM to analyze cell attachment and most of them were performed operating in a contact mode (Telegdi et al. 1998; Fang et al. 2000; Kinzler et al. 2001), which results in possible damage or deformation of the cell itself. Tapping mode AFM was used in our case.

MATERIALS AND METHODS Bacterial strain and growth conditions: Acidithiobacillus ferrooxidans strain LR was used in this work. (Garcia Jr, 1991). The culture was grown in a mineral salts medium (Tuovinen & Kelly 1973), at pH 1.8 using ferrous sulfate as the energy source. The cells for attachment experiments were obtained after growth for 48 hours in a shaker (150 rpm and 30C) by successive washing and centrifugation (5000g) to eliminate residual ferric ion from the medium. To strip EPS from the cells, the washed suspension was finally centrifuged at 12000g for 25 min (Pogliani & Donati 1999; Gehrke et al. 1998; Sand et al.

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1995). The EPS free cells were then washed in water purified to 18 M twice and suspended in 10 mL of the mineral salts solution of medium T&K (Tuovinen & Kelly 1973), without ferrous iron in water purified to 18 M. The cell suspension was standardized by the modified Lowry protein determination method (Hartree 1972). Mineral samples: Research-grade chalcopyrite (CuFeS2) and bornite (Cu5FeS4) used in this study were obtained from Wards Natural Science Establishment (Rochester, NY). Samples were cut in pieces of approximately 1 cm2 using a diamond saw. One face was hand polished through four sizes of silicon carbide paper with a final polish using alumina suspension of 0.3 m particle size. Imaging of biological samples with AFM requires a near atomically flat surface in order to discriminate topographical features. Initial values of roughness for chalcopyrite and bornite surfaces were typically around 4 nm and 5 nm, respectively. To eliminate impurities the samples were rinsed with acetone, ethanol and water purified to 18 M (15 minutes each one in an ultrasonic bath), and then were blown dry with pure Argon and kept in desiccators before using. Monocrystalline mica (Green Mica) was obtained from Assheville-Shoonmaker (USA) and graphite (highly oriented pyrolitic graphite HOPG from Advanced Ceramics Corporation (Lakewood, USA). Before adhesion experiments both materials were cleaved in clean atmosphere. Adhesion experiments: All substrates were incubated individually in 1 mL of iron-free mineral salts solution of T&K medium in water purified to 18 M containing a cell suspension (~ 5 x 1010 cells). The samples were taken out from the solution after different times of incubation, rinsed thoroughly with water purified to 18 M to remove any remaining unattached bacteria and dried in purified Argon. A Nanoscope III Extended Multimode Atomic Force Microscope (Digital Instruments, Santa Barbara, CA, USA) operating in a tapping-mode in air was used to image cells. The Digital Nanoscope software (version 4.42r8) was used to analyze the topographic images.

RESULTS AND DISCUSSION Figure 1 depicts a set of images corresponding to At. ferrooxidans-LR cells attached on chalcopyrite at different incubation times. Cell attachment is clearly observed for incubation time shorter than 3 days (not shown in figure 1), but the EPS in this copper sulfide mineral was detected as small chains surrounding the bacteria (see image 1A). Images 1A, 1B and 1C show that the cell adhesion on chalcopyrite takes place in some surface sites at early incubation times and then the surface cell density remains almost time constant. After 14 days incubation time, massive EPS secretion becomes evident far from the cell this massive secretion was followed by the appearance of small grains all over the surface, which were size increasing with time (see images 1B and 1C). The bacteria dimensions were as expected (Jensen & Webb 1995), 1.5-2 m length, 0.5-0.8 m width and 0.5 m height. Figure 1D corresponds to a chalcopyrite surface exposed for 28 days to the same working media without the presence of the cell. This surface was used as the blank mineral surface, observing no corrosion damage produced by the low pH medium. In Figure 2 the set of images shows the evolution of cell adhesion on bornite surface during experimental time course. Apparently there are no great differences regarding cell density on bornite surface compared to chalcopyrite at early incubation stages. However, after 10 days the attached cell on bornite increases significantly as can be observed in image 2B. Unlike in chalcopyrite surface, the EPS formation was not evident for the bornite case (Figures 2A and 2B). In addition, some diplobacilli could be seen in bornite surface (see image 2B), which was not detected in chalcopyrite experiments. This cell morphology indicates that cellular division took place, which would be associated with active metabolism of the bacteria on bornite surface.

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Figure 1. AFM images of chalcopyrite surface after different immersion times in the cell suspension: (A) 3 days, (B) 14 days and (C) 28 days. Left images are large scale (20x20) m2 area and 400 nm of Z range. Right images are real zoom of the corresponding left one, (5x5) m2 area and 400 nm Z range, except for the image A, (1.5x1.5) m2 and 100 nm Z range. (20x20) m2 image (D) shows the blank mineral surface after 28 days immersion in the suspension media without the presence of the bacteria (400 nm Z range).

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Figure 2. AFM images of bornite surface after different immersion times in the cell suspension: (A) 3 days and (B) 10 days. Left images are large scale (20x20) m2 and right images correspond to a real zoom of (5x5) m2. All images Z scaled to 400 nm. Image (C) shows the blank mineral surface after 28 days immersion in the suspension media without the presence of the bacteria (400 nm Z range). Figure 3 shows the attachment of bacterial cells to the inert HOPGraphite surface also for different incubation times. The cell attachment was also observed at short incubation times (for instance 30 min of incubation), increasing appreciably with time (see image 3C). The strong cell adhesion

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observed was not followed by the production of EPS after 4 days (see image 3C) on the HOPG surface, since even after 10 days of incubation there was no evidence of its formation on the images (images not shown in figure 3). HOPG offers a well defined, atomically flat surface, very convenient for cell size measurements as shown in the image profile 3B. These results denote that At. ferrooxidans-LR did not require the formation a priori of EPS molecules as a link for the surface attachment. Finally, freshly cleaved monocrystalline mica surface was also incubated in the presence of bacteria and imaged at different times. However, in this case no evidence of cell attachment was observed even after 15 days of incubation, indicating that no specific interaction took place as opposed to graphite (Figure 4). Hence, the role of the bacteria attachment can be better explained from the point of view of hydrophobic and hydrophilic van der Walls interactions between the mineral surface and the outermost part of the cell membrane. Since mica substrate due to the hydroxyl ending groups offers a hydrophilic surface, it generally appears covered by at least a monolayer of strongly absorbed water (Kopta and Salmeron, 2000; Xiao et al. 1995). Therefore, the displacement of adsorbed water to permit closer approach of the two surfaces (cell and surface substrate) is energetically unfavorable (Fletcher 1996). Then, the cells most probably were suspended over the adsorbed water layer on the mica surface and subsequently removed during the washing process.

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Figure 3. AFM images of a HOPG surface after different immersion times in the cell suspension: (A) and (C) correspond to (15x15) m2 images at 1 hour and 4 days of exposition time respectively. (B) and (D) are zoomed images of (3x3) and (2x2) m2 respectively. (B) bacteria profile from image (B) 400 nm Z range for all images.

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Figure 4. AFM images of a cleaved mica surface after 15 days of immersion time in the cell suspension. As stated by Fletcher (1996), there are many examples of bacteria attaching preferentially to hydrophobic surfaces. The hydrophobic interactions act either as the primary mechanism of adhesion or facilitate a close approach to allow further adhesion interactions. The strong attachment of bacteria on the cleaved atomically flat HOPG surface (Guntherodt et al. 1992; Kogan et al. 2001) reported here indicates that hydrophobic forces act as the primary mechanism of adhesion and play a key role in the bacterial attachment. Therefore, the bacterial adhesion is mainly derived by van der Waals interactions between the cell envelope and the carbon graphite structure. As both mineral substrates, chalcopyrite and bornite, offer also a hydrophobic surfaces to the cell, we conclude that attractive van der Waals interactions are the main forces promoting cell adhesion on both mineral surfaces. The results with an inert substrate as graphite also indicate that the formation of EPS was not a prerequisite for bacterial attachment in the conditions used in our experiments, but also it is not a prerequisite in the case of sulfide mineral surfaces as was demonstrated at low incubation times. After some days of incubation the formed EPS remains around the cell and may increase the contact interaction area and consequently the general adhesion of the cell.

ACKNOWLEDGMENTS Support for this work was received from the Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq) Brazil and Fundao Andes, Chile. The authors also thank to Scientific-technical Services of the University of Barcelona for the technical assistance.

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Adhesion Studies of Acidithiobacillus ferrooxidans on Different Surfaces by Atomic Force MicroscopyABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGMENTSREFERENCES