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Adhesion of Acidithiobacillus ferrooxidans to mineral surfaces
International Journal of Mineral Processing 94 (2010) 135–139
Contents lists available at ScienceDirect
International Journal of Mineral Processing 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 / i j m i n p r o
Adhesion of Acidithiobacillus ferrooxidans to mineral surfaces Preston Devasia a, K.A. Natarajan b,⁎ a b
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India Department of Materials Engineering, Indian Institute of Science, Bangalore 560 012, India
a r t i c l e
i n f o
Article history: Received 28 May 2009 Received in revised form 18 January 2010 Accepted 7 February 2010 Available online 12 February 2010 Keywords: Acidithiobacillus ferrooxidans Bioleaching Direct mechanism Adhesion
a b s t r a c t Direct contact mechanism in bioleaching implies prior mineral adhesion of Acidithiobacillus ferrooxidans and subsequent enzymatic attack. Prior bacterial adaptation to sulfide mineral substrates influences bacterial ferrous ion oxidation rates. It is highly beneficial to understand major biooxidation mechanisms with reference to solution- and mineral-grown cells in order to optimize bioleaching reactions. For A. ferrooxidans grown in the presence of solid substrates such as sulfur, pyrite and chalcopyrite, bacterial adhesion is required for its enzymatic machinery to come into close contact for mineral dissolution. But when grown in solution substrate such as ferrous ions and thiosulfate, such an adhesion machinery is not required for substrate utilization. Proteinaceous compounds were observed on the surface of sulfur-grown cells. Such an induction of relatively hydrophobic proteins and down regulation of exposed polysaccharides leads to changes in cell surface chemistry. Sulfur-grown and pyrite- and chalcopyrite-grown bacterial cells were found to be more efficient in the bioleaching of chalcopyrite than those grown in the presence of ferrous ions and thiosulfate. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Acidithiobacillus ferrooxidans is the most widely used microorganism in the bioleaching of several sulfide minerals. Its ability to oxidize ferrous ions and reduced valence sulfur compounds to ferric ions and sulfate results in the solubilisation of metals from their sulfides. The role of acidic ferric sulfate in the solubilisation of such sulfide minerals is generally referred to as the indirect leaching mechanism (Natarajan, 1998). On the other hand, bacterial adhesion to the mineral surfaces is a prerequisite in the direct enzymatic attack of the mineral. Relative roles of direct and indirect bioleaching mechanisms in the presence of A. ferrooxidans have been reported earlier (Crundwell, 2001). However, specific involvement of A. ferrooxidans in the direct mineral attack and bacterial attachment mechanisms have not yet been clearly understood. In this paper, probable adhesion mechanisms of A. ferrooxidans to sulfur and chalcopyrite are examined in relation to the bioleaching of chalcopyrite. Bacterial cell surface hydrophobicity and changes in isoelectric points are examined in relation to modifications in surface chemistry, when A. ferrooxidans are grown in the presence of solid and
⁎ Corresponding author. Tel.: + 91 80 23600120; fax: + 91 80 23600472. E-mail address: [email protected] (K.A. Natarajan). 0301-7516/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.minpro.2010.02.003
solution substrates. Induction of the bacterial adhesion machinery and its relevance in bioleaching mechanisms are demonstrated. 2. Materials and methods 2.1. Bacterial strain used and growth conditions A. ferrooxidans MAL 4-1 used in this study was isolated from Malanjkhand copper mines India (Devasia, 1995). It was maintained on 9 K medium (Silverman and Lundgren, 1959). Ferrous iron-grown cells were obtained by growing A. ferrooxidans in 9 K medium for 2 days, on a rotary shaker (240 rpm) at 30 °C. The culture was filtered through Whatman No. 1 filter paper to remove precipitates. The filtrate was centrifuged at 15,000 rpm for 15 min using a SS34 rotor (Sorvall RC5B). The pellet was washed and resuspended in distilled water adjusted to pH 2.0 with sulfuric acid. Thiosulfate-grown cells were obtained by culturing A. ferrooxidans in 9 K− medium (without ferrous sulfate) supplemented with 10 g of sodium thiosulfate per liter. The pH of the medium was 4.5. Sulfur-grown cells were obtained by growth on 9 K− mineral salt medium containing 10 g sulfur powder per 100 ml. The medium was adjusted to pH 2.3, inoculated and incubated at 30 °C on a shaker (240 rpm) for 10 days. For growth of A. ferrooxidans on thiosulfate and sulfur it was found necessary to supplement the medium with trace amounts of iron (10 mg of ferric chloride per liter). The culture was filtered through Whatman No. 1 and harvested by centrifugation. Pyrite-grown and chalcopyritegrown cells were obtained from 9 K− medium supplemented with 4 g
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of the mineral per 100 ml of the solution. The cells were harvested as described above. 2.2. Minerals Hand-picked pure samples of pyrite and chalcopyrite were obtained from Wards, New York (USA) and analytical grade sulfur particles from Sigma Aldrich Chemicals. Through mineralogical, X-ray and chemical analysis, the purity of pyrite and chalcopyrite was determined to be 99.8%. Sulfur powder was 99.98% pure. 2.3. Hydrophobicity measurements The hydrophobicity of cells in suspensions was measured by liquid– liquid partition in aqueous and organic phase in the same manner as described previously (Rosenberg et al., 1980) using n-octane. 2.4. Ruthenium red adsorption Cell suspensions (109 cells/ml) were incubated with 1 ml of ruthenium red (100 mg/l) as described by Figueroa and Silverstein (1989). The suspension was incubated with agitation on a shaker (240 rpm) for 1 h. The cells were pelleted in a microfuge at 1800 ×g and the ruthenium red adsorption to cells was measured by determining the reduction in absorbance at 535 nm of the supernatant. 2.5. Substrate utilization Four sets of three flasks were prepared, each containing 100 ml of 9 K medium (Silverman and Lundgren, 1959). The first set was prepared with no sulfur powder (control), second set with 0.01 g, the third set with 0.1 g and the fourth with 1 g of sulfur powder. The flasks were inoculated with 5× 109 cells and the ferrous iron in solution was determined colorimetrically using 1, 10-orthophenanthroline (Vogel, 1951). 2.6. Leaching Leaching was carried out in 500 ml Erlenmeyer flasks. To 100 ml of 9 K− medium 5 g of the mineral was added and the pH was adjusted to 2.3. The pH was monitored frequently using a Systronics make pH meter and maintained at 2.3 using either sulfuric acid or sodium hydroxide. It was inoculated with 5 × 109 cells of A. ferrooxidans MAL 4-1. The flasks were incubated at 30 °C on a rotary shaker at 240 rpm. 2.7. Chemical analysis Dissolved copper concentrations were determined by atomic absorption spectrometry. Ferrous iron was determined by 1, 10orthophenanthroline and total iron after reduction of ferric iron by hydroxylamine hydrochloride (Vogel, 1951). Total protein associated with chalcopyrite concentrate during leaching was estimated as reported by Murthy and Natarajan (1992).
culture by filtration when added to fresh medium were found to settle to the bottom of the flask. The sulfur separated from the culture of A. ferrooxidans was mixed with m-xylene, a nonpolar reagent (Rosenberg et al., 1980). The mixture was vortexed for 1 min and incubated at room temperature for 15 min. The procedure was repeated twice. 9 K− medium was then added and the mixture was allowed to stand. The sulfur was then observed to be in the organic phase which partitioned above the aqueous phase. Based on the above results a preliminary model to explain adhesion of A. ferrooxidans on sulfur particles is proposed. The sulfur powder which is initially hydrophobic spreads on the surface of the aqueous medium despite having a specific gravity of 2.1 (Sander et al., 1984). During incubation with A. ferrooxidans it becomes hydrophilic and settles to the bottom of the container, after bacterial adhesion. From thermodynamic considerations, the extent of adhesion will be determined by the surface properties of all the three phases, i.e., the bacteria, liquid media and sulfur. If the effect of electric charges as well as specific biochemical interactions (receptor–ligand) is not considered (Absolom et al., 1983), the change in free energy function could be expressed as: adh
ΔG
= γBS –γBL −γSL
where ΔGadh is the free energy of adhesion, γBS is the bacterium–sulfur interfacial tension, γBL is the bacterium–liquid interfacial tension and γSL is the sulfur–liquid interfacial tension. Since adhesion will be favoured when ΔGadh is minimized, the value of γBS should be minimized or γBL and/or γSL increased. In the above system a hydrophobic secretion is postulated to be produced by A. ferrooxidans, which brings down the interfacial tension between bacterial cells and the surface of sulfur particles (γBS) to enhance contact and adhesion. All the results described support the above model of adhesion. There is no significant modification brought about in the surface tension of the aqueous medium as the fresh sulfur powder floated on the culture filtrate. The adhesion of bacteria makes the sulfur particles hydrophilic due to the exposed surface of bacteria away from the sulfur surface. The bacteria are held to the surface because of a hydrophobic secretion by A. ferrooxidans, which lowers the γBS thus favouring adhesion. After interaction with m-xylene the sulfur particles become hydrophobic as evidenced by their partitioning into the xylene phase which moves above the aqueous phase. Thus a hydrophobic secretion by A. ferrooxidans is involved in bringing about adhesion, and the secretion can be solubilised by m-xylene. Since m-xylene is a nonpolar reagent, hydrophobic substances can easily be solubilised. To check whether any surface modification occurs on A. ferrooxidans by any hydrophobic secretion, two methods, namely hydrophobic interaction chromatography and phase partitioning, were utilized to investigate its surface (Devasia et al., 1993). The results of phase partitioning with n-octane are shown in Fig. 1. With 0.1 to 0.3 ml of n-octane,
3. Results and discussion 3.1. Surface chemistry Fresh sulfur when added to 9 K− medium floats on the surface of the medium. However in flasks inoculated with A. ferrooxidans and grown for 10 days, when kept at rest, the sulfur was found to settle to the bottom of the flask. This phenomenon of settling of sulfur was not observed in control flasks which were not inoculated with A. ferrooxidans. The culture wherein sulfur particles were found to settle was passed through a Whatman No. 1 filter paper. To an aliquot of the filtrate, a fresh sample of sulfur powder was added. The sulfur was found to remain on the surface of the filtrate. However, sulfur particles recovered from the
Fig. 1. Hydrophobicity of Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate, sulfur, pyrite and chalcopyrite.
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approximately 20% of cells grown on ferrous iron or thiosulfate got partitioned into n-octane. A second phase of increased partitioning (up to 40%) were noticed at 0.4 and 0.5 ml of n-octane. In contrast, sulfur and mineral-grown cells showed higher partitioning (up to 35%) even at 0.1 ml of n-octane. Further increase in n-octane volumes resulted in increased partitioning, reaching a value of 45% at 0.5 ml of n-octane. Thus, ferrous iron and thiosulfate-grown cells showed markedly different patterns of partitioning in n-octane as compared to cells grown on sulfur, pyrite and chalcopyrite. It should be noted that the population of cells obtained by growth on sulfur, pyrite and chalcopyrite may not be homogeneous. In sulfur oxidation, partially oxidized species of sulfur appear in solution that freely suspended A. ferrooxidans oxidize (Shrihari et al., 1993). Similarly in some phases of pyrite and chalcopyrite oxidation, ferrous iron released into solution that freely suspended A. ferrooxidans may oxidize (Devasia et al., 1996). The mineral-grown cells were more hydrophobic than the ferrous iron or thiosulfate-grown cells. The greater hydrophobicity of the mineral-grown cells may help in their adhesion to mineral surfaces. The observations of Arredondo et al. (1994) suggested that it would be worthwhile to check for any change in the composition of the exposed polysaccharides in mineral-grown cells compared to solution-grown cells. Ruthenium red adsorption tests demonstrated that ruthenium red, which binds specifically to polysaccharides, exhibited higher binding to solution-grown cells. Solution-grown cells removed about 15% of the ruthenium red from solution whereas mineral-grown cells adsorbed only up to 3% of the dye. The above observation suggests a down regulation of the exposed polysaccharides in mineral-grown cells. This also allows the exposure of the hydrophobic proteins as expected from the other results discussed earlier. It can be concluded that the preferential exposure of polysaccharide moieties is favoured when the cells are utilizing solution substrates. This changes to an exposure of hydrophobic regions of proteins when grown on minerals where adhesion is required for growth. The surface chemistry of A. ferrooxidans grown on minerals is different from that when it is grown on ferrous iron or thiosulfate (Devasia et al., 1993; Devasia et al., 1996). The cells grown on minerals have a relatively higher hydrophobicity than solution-grown cells as measured by phase partitioning and hydrophobic interaction chromatography. The electrophoretic mobilities of mineral-grown and solution-grown cells are distinctly different as demonstrated through free flow electrophoresis. It has further been demonstrated that the isoelectric point (IEP) of minerals shifts on their interaction with A. ferrooxidans. For example, IEP of bacterial cells grown in the presence of ferrous iron and thiosulfate shifted from an initial value of pH 2 to values closer to pH 4 when grown in the presence of sulfur, pyrite and chalcopyrite. Such a significant positive shift in the IEP of bacterial cells was due to the presence of –NH3 groups from proteinaceous secretion as proved by FTIR surface analysis. With antibodies raised against sulfurgrown cells it was possible to examine for any difference in surface components. Enzyme Linked ImmunoSorbent Assay (ELISA) of the cells grown in different conditions revealed that all the mineral-grown cells share common epitopes which are not present in ferrous iron grown or thiosulfate-grown cells. Functional groups characteristic of proteins were found on the surface of sulfur-grown cells. To check whether a proteinaceous substance was indeed responsible for the differences in surface chemistry, the sulfur-grown cells were treated with proteinase K. When the isoelectric point of these cells was measured, it was found that it had shifted to near that of ferrous iron-grown and thiosulfategrown cells (Devasia et al., 1993; Devasia, 1995; Devasia et al., 1996) indicating the removal of originally present surface proteins. Ruthenium red, which specifically binds to exposed polysaccharides, was also found to have less binding to sulfur-grown cells compared to solution-grown cells suggesting that there is a down regulation of exposed polysaccharides on sulfur-grown cells with the synthesis of the proteinaceous substance.
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Fig. 2. Utilization of ferrous ions by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur in the absence of added sulfur.
3.2. Substrate utilization The growth curves showing utilization of ferrous iron by A. ferrooxidans grown under different conditions are shown in Fig. 2. Cells grown on ferrous iron utilize ferrous iron without any lag phase and complete oxidation is seen by 48 h. Cells grown on thiosulfate and sulfur however showed a significantly low level of ferrous oxidation up to 72 h after which the rate increases sharply. The oxidation of ferrous iron is completed by 120 h. This very low level of early oxidation was observed for thiosulfate and sulfur-grown cells because the ferrous oxidation machinery is an inducible one, whereas the sulfur pathway is constitutive (Kulpa et al., 1986a, b). When the above experiment was carried out in the presence of 0.01 g of sulfur per 100 ml of 9 K medium, some changes are observed (Fig. 3). While the profile of ferrous iron oxidation by ferrous irongrown and thiosulfate-grown cells remains almost the same, the sulfur-grown cells take longer time to complete the ferrous oxidation. This shows that the added sulfur is preferred by the cells previously grown on sulfur. When the quantity of added sulfur was further increased to 0.1 g, the oxidation of ferrous iron by sulfur-grown cells is further delayed (Fig. 4). When sufficiently more quantity of sulfur was added a distinct difference in the pattern of all the three sets of cells was observed (Fig. 5). The rate of ferrous utilization even by ferrous iron-grown cells is reduced. In thiosulfate-grown cells, a dramatic decrease in ferrous iron oxidation was observed. The levels were comparable to those observed in sulfurgrown cells. These results are very interesting. It had earlier been discussed that the ferrous iron oxidation machinery is an inducible one.
Fig. 3. Utilization of ferrous by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur in the presence of 0.01 g of sulfur.
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Fig. 4. Utilization of ferrous by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur in the presence of 0.1 g of sulfur.
Fig. 6. Bioleaching of iron from chalcopyrite by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur.
Since thiosulfate-grown cells do not possess both these mechanisms (ferrous iron oxidation and adhesion machinery) to start with, the addition of sufficiently large amounts of sulfur can change the priority of induction of machinery to that for adhesion. The presence of 1 g of sulfur compared to 0.1 g or lower amounts of sulfur provides sufficient surface for the 5 × 109 cells in suspension to interact with for induction of the adhesion machinery. The induction of the machinery for adhesion suggests physiological changes in the bacteria in response to a mineral. The induction of adhesion and hence the utilization of sulfur is preferred over the induction of ferrous oxidation in thiosulfate-grown cells where both machineries (adhesion and ferrous iron oxidation) are absent to start with. It is clear that the induction of adhesion is preferred over the induction of ferrous iron oxidation. These results suggest that more of the population of cells found in nature and under leaching environments may be adhering to the mineral surfaces.
Biodissolution of iron from chalcopyrite (particle size 425–500 μm) brought about by A. ferrooxidans grown on sulfur, and thiosulfate and ferrous iron as a function of leaching period is illustrated in Fig. 6. Sulfurgrown cells release iron from chalcopyrite with no appreciable lag phase which is prominently evident in the case of soluble substrate-grown cells. Both thiosulfate and ferrous iron-grown cells show a lag period of 12 days during which there is no solubilisation of iron. After this period there is a sharp increase in solubilisation. However the solubilisation of iron by sulfur-grown cells is much higher than those observed with solution-grown cells during the period of this study. In Fig. 7 one can see
the concomitant solubilisation of copper from chalcopyrite (particle size 425–500 μm). The trend as observed for iron release by A. ferrooxidans is also observed with respect to copper solubilisation. The pattern of cell adhesion as revealed by the protein content associated with the chalcopyrite supports the role of adhesion in leaching (Fig. 8). The increase in copper and iron solubilisation after a period of time by solutiongrown cells can also be attributed to higher surface protein (bacterial adhesion) associated with the mineral. The lag period observed in the case of solution-grown cells is supposed to be required for the synthesis of the proteinaceous cell surface appendage for firm adhesion and subsequent leaching. The interfacial proteinaceous cell surface appendage is synthesized when the cells are grown on solid substrates such as sulfur and sulfide minerals. This enables bacterial adhesion leading to higher leaching rates. The proteinaceous cell surface appendage is not produced when the cells are grown in solution substrates such as ferrous iron or thiosulfate. The total protein measured on the chalcopyrite is different and is an indicator of the population of cells adhering to the mineral. The more cells (or total protein) the higher the growth of the organism and hence leaching. The fact that thiosulfate-grown cells show leaching comparable only to ferrous iron-grown cells indicates that the adhesion of cells to mineral is the first and rate limiting step in the leaching of minerals by A. ferrooxidans in the direct mechanism, primarily because adhesion is an induced process. The evidence provided above clearly indicates the role of adhesion in leaching. To quantify the effect of adhesion it will be necessary to obtain mutants in adhesion which can be tested for leaching. It is well known in microbiology that mutants (a population of cells which lacks specific function) can be used to delineate the role
Fig. 5. Utilization of ferrous by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur in the presence of 1 g of sulfur.
Fig. 7. Bioleaching of copper from chalcopyrite by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur.
3.3. Role of growth conditions and adhesion in the leaching of chalcopyrite
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not possess the ferrous iron utilization machinery. The use of these cells in leaching clearly demonstrated that the adhesion of the cells to the mineral plays a major role in hastening the process of dissolution. The induction of the adhesion machinery in sulfur-grown cells results in further extension of the lag phase observed in thiosulfate and ferrous iron-grown cells. The fact that the thiosulfate-grown cells behaved in a manner which mostly resembled the ferrous iron-grown cells points to the fact that adhesion is indeed the first and rate limiting step in the leaching of metals from their minerals. In future studies the generation of mutants in adhesion will allow a clearer delineation of the role of adhesion in leaching.
References Fig. 8. Proteins associated with chalcopyrite during bioleaching by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur.
of the bacteria in carrying out specific functions. If the mutants, which lack specific function, can be rescued by introducing the specific gene, the role of the gene in carrying out that function can be confirmed and studied. It is also clear that the direct mechanism plays a larger role in leaching than the indirect mechanism as sulfur-grown cells are shown to leach better than ferrous iron-grown cells. While the direct mechanism may be more important, the indirect mechanism also plays a role (Devasia et al., 1996). However both mechanisms may come into play during longer periods of leaching. The importance of adhesion in leaching is demonstrated in this work. 4. Conclusions This study provides a perspective on the process of adhesion of A. ferrooxidans to mineral surfaces. It was recognized during this study that A. ferrooxidans can be cultured in solution substrate or on solid substrate for the purpose of comparison of the process of adhesion. The solution substrates include ferrous iron and thiosulfate. The solid substrates used in this study include minerals like sulfur, pyrite and chalcopyrite. In mineral-grown conditions, since the substrate is insoluble, the adhesion of bacteria is required for its enzymatic machinery to come into close contact for the dissolution of the mineral. But when grown with solution substrate, the adhesion machinery is not required for substrate utilization. These differing growth conditions thus provided a means of looking for differences in A. ferrooxidans when adhesion is required and when it is not required. Learning about these differences would be an important step in understanding the mechanism of adhesion of A. ferrooxidans to mineral surfaces. Machinery for adhesion can be induced by growth on sulfur. Such machinery is not present in cells grown on thiosulfate and ferrous iron. Further the thiosulfate-grown cells like the sulfur-grown cells do
Absolom, D.R., Lamberti, F.V., Policova, Z., Zingg, W., van Oss, C.J., Neumann, A.W., 1983. Surface thermodynamics of bacterial adhesion. Applied and Environmental Microbiology 46, 90–97. Arredondo, R., García, A., Jerez, C.A., 1994. Partial removal of lipopolysaccharide from Thiobacillus ferrooxidans affects its adhesion to solids. Applied and Environmental Microbiology 60, 2846–2851. Crundwell, P.K., 2001. How do bacteria interact with minerals? In: Ciminella, V.S.T., Gracia Jr, O. (Eds.), Biohydrometallurgy: Fundamentals, Technology and Sustainable Development. Elsevier, B.V. Amsterdam, pp. 149–157. Devasia, P., 1995. Adhesion of Thiobacillus ferrooxidans to mineral surfaces. Doctoral Thesis, Indian Institute of Science, Bangalore, India. Devasia, P., Natarajan, K.A., Sathyanarayana, D.N., Rao, G.R., 1993. Surface chemistry of Thiobacillus ferrooxidans relevant to adhesion on mineral surfaces. Applied and Environmental Microbiology 59, 4051–4055. Devasia, P., Natarajan, K.A., Rao, G.R., 1996. Role of bacterial growth conditions and adhesion in the bioleaching of chalcopyrite by Thiobacillus ferrooxidans. Minerals and Metallurgical Processing 13, 82–86. Figueroa, L.A., Silverstein, J.A., 1989. Ruthenium red adsorption method for measurement of extracellular polysaccharides in sludge flocs. Biotechnology and Bioengineering 33, 941–947. Kulpa, C.F., Roskey, M.T., Mjoli, N., 1986a. Construction of chromosomal gene banks of Thiobacillus ferrooxidans and induction of the iron oxidation pathway. Biotechnology and Applied Biochemistry 8, 330–341. Kulpa, C.F., Mjoli, N., Roskey, M.T., 1986b. Comparison of iron and sulfur oxidation in Thiobacillus ferrooxidans: inhibition of iron oxidation by growth on sulfur. In: Ehrlich, H.L., Holmes, D.S. (Eds.), Biotechnology and Bioengineering Symposium. John Wiley, New York, p. 289. Murthy, K.S.N., Natarajan, K.A., 1992. The role of surface attachment of Thiobacillus ferrooxidans on the biooxidation of pyrite. Minerals and Metallurgical Processing 9, 20–24. Natarajan, K.A., 1998. Microbes, Minerals and Environment. Geological Survey of India, Bangalore. Rosenberg, M., Gutnick, D., Rosenberg, E., 1980. Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiology Letters 9, 29–33. Sander, U.H.F., Fisher, H., Rothe, U., Kola, R., 1984. Sulfur, Sulfur Dioxide and Sulfuric Acid. Verlag Chemie International Inc. Shrihari, Bhavaraju S.R., Modak, J.M., Kumar, R., Gandhi, K.S., 1993. Dissolution of sulfur particles by Thiobacillus ferrooxidans: substrate for unattached cells. Biotechnology and Bioengineering 41, 612–616. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans. I. An improved medium and a harvesting procedure for securing high cell yields. Journal of Bacteriology 77, 642–647. Vogel, A.I., 1951. Textbook of Quantitative Inorganic Analysis, 2nd edition. English Language Book Society, London.
Contents lists available at ScienceDirect
International Journal of Mineral Processing 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 / i j m i n p r o
Adhesion of Acidithiobacillus ferrooxidans to mineral surfaces Preston Devasia a, K.A. Natarajan b,⁎ a b
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India Department of Materials Engineering, Indian Institute of Science, Bangalore 560 012, India
a r t i c l e
i n f o
Article history: Received 28 May 2009 Received in revised form 18 January 2010 Accepted 7 February 2010 Available online 12 February 2010 Keywords: Acidithiobacillus ferrooxidans Bioleaching Direct mechanism Adhesion
a b s t r a c t Direct contact mechanism in bioleaching implies prior mineral adhesion of Acidithiobacillus ferrooxidans and subsequent enzymatic attack. Prior bacterial adaptation to sulfide mineral substrates influences bacterial ferrous ion oxidation rates. It is highly beneficial to understand major biooxidation mechanisms with reference to solution- and mineral-grown cells in order to optimize bioleaching reactions. For A. ferrooxidans grown in the presence of solid substrates such as sulfur, pyrite and chalcopyrite, bacterial adhesion is required for its enzymatic machinery to come into close contact for mineral dissolution. But when grown in solution substrate such as ferrous ions and thiosulfate, such an adhesion machinery is not required for substrate utilization. Proteinaceous compounds were observed on the surface of sulfur-grown cells. Such an induction of relatively hydrophobic proteins and down regulation of exposed polysaccharides leads to changes in cell surface chemistry. Sulfur-grown and pyrite- and chalcopyrite-grown bacterial cells were found to be more efficient in the bioleaching of chalcopyrite than those grown in the presence of ferrous ions and thiosulfate. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Acidithiobacillus ferrooxidans is the most widely used microorganism in the bioleaching of several sulfide minerals. Its ability to oxidize ferrous ions and reduced valence sulfur compounds to ferric ions and sulfate results in the solubilisation of metals from their sulfides. The role of acidic ferric sulfate in the solubilisation of such sulfide minerals is generally referred to as the indirect leaching mechanism (Natarajan, 1998). On the other hand, bacterial adhesion to the mineral surfaces is a prerequisite in the direct enzymatic attack of the mineral. Relative roles of direct and indirect bioleaching mechanisms in the presence of A. ferrooxidans have been reported earlier (Crundwell, 2001). However, specific involvement of A. ferrooxidans in the direct mineral attack and bacterial attachment mechanisms have not yet been clearly understood. In this paper, probable adhesion mechanisms of A. ferrooxidans to sulfur and chalcopyrite are examined in relation to the bioleaching of chalcopyrite. Bacterial cell surface hydrophobicity and changes in isoelectric points are examined in relation to modifications in surface chemistry, when A. ferrooxidans are grown in the presence of solid and
⁎ Corresponding author. Tel.: + 91 80 23600120; fax: + 91 80 23600472. E-mail address: [email protected] (K.A. Natarajan). 0301-7516/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.minpro.2010.02.003
solution substrates. Induction of the bacterial adhesion machinery and its relevance in bioleaching mechanisms are demonstrated. 2. Materials and methods 2.1. Bacterial strain used and growth conditions A. ferrooxidans MAL 4-1 used in this study was isolated from Malanjkhand copper mines India (Devasia, 1995). It was maintained on 9 K medium (Silverman and Lundgren, 1959). Ferrous iron-grown cells were obtained by growing A. ferrooxidans in 9 K medium for 2 days, on a rotary shaker (240 rpm) at 30 °C. The culture was filtered through Whatman No. 1 filter paper to remove precipitates. The filtrate was centrifuged at 15,000 rpm for 15 min using a SS34 rotor (Sorvall RC5B). The pellet was washed and resuspended in distilled water adjusted to pH 2.0 with sulfuric acid. Thiosulfate-grown cells were obtained by culturing A. ferrooxidans in 9 K− medium (without ferrous sulfate) supplemented with 10 g of sodium thiosulfate per liter. The pH of the medium was 4.5. Sulfur-grown cells were obtained by growth on 9 K− mineral salt medium containing 10 g sulfur powder per 100 ml. The medium was adjusted to pH 2.3, inoculated and incubated at 30 °C on a shaker (240 rpm) for 10 days. For growth of A. ferrooxidans on thiosulfate and sulfur it was found necessary to supplement the medium with trace amounts of iron (10 mg of ferric chloride per liter). The culture was filtered through Whatman No. 1 and harvested by centrifugation. Pyrite-grown and chalcopyritegrown cells were obtained from 9 K− medium supplemented with 4 g
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of the mineral per 100 ml of the solution. The cells were harvested as described above. 2.2. Minerals Hand-picked pure samples of pyrite and chalcopyrite were obtained from Wards, New York (USA) and analytical grade sulfur particles from Sigma Aldrich Chemicals. Through mineralogical, X-ray and chemical analysis, the purity of pyrite and chalcopyrite was determined to be 99.8%. Sulfur powder was 99.98% pure. 2.3. Hydrophobicity measurements The hydrophobicity of cells in suspensions was measured by liquid– liquid partition in aqueous and organic phase in the same manner as described previously (Rosenberg et al., 1980) using n-octane. 2.4. Ruthenium red adsorption Cell suspensions (109 cells/ml) were incubated with 1 ml of ruthenium red (100 mg/l) as described by Figueroa and Silverstein (1989). The suspension was incubated with agitation on a shaker (240 rpm) for 1 h. The cells were pelleted in a microfuge at 1800 ×g and the ruthenium red adsorption to cells was measured by determining the reduction in absorbance at 535 nm of the supernatant. 2.5. Substrate utilization Four sets of three flasks were prepared, each containing 100 ml of 9 K medium (Silverman and Lundgren, 1959). The first set was prepared with no sulfur powder (control), second set with 0.01 g, the third set with 0.1 g and the fourth with 1 g of sulfur powder. The flasks were inoculated with 5× 109 cells and the ferrous iron in solution was determined colorimetrically using 1, 10-orthophenanthroline (Vogel, 1951). 2.6. Leaching Leaching was carried out in 500 ml Erlenmeyer flasks. To 100 ml of 9 K− medium 5 g of the mineral was added and the pH was adjusted to 2.3. The pH was monitored frequently using a Systronics make pH meter and maintained at 2.3 using either sulfuric acid or sodium hydroxide. It was inoculated with 5 × 109 cells of A. ferrooxidans MAL 4-1. The flasks were incubated at 30 °C on a rotary shaker at 240 rpm. 2.7. Chemical analysis Dissolved copper concentrations were determined by atomic absorption spectrometry. Ferrous iron was determined by 1, 10orthophenanthroline and total iron after reduction of ferric iron by hydroxylamine hydrochloride (Vogel, 1951). Total protein associated with chalcopyrite concentrate during leaching was estimated as reported by Murthy and Natarajan (1992).
culture by filtration when added to fresh medium were found to settle to the bottom of the flask. The sulfur separated from the culture of A. ferrooxidans was mixed with m-xylene, a nonpolar reagent (Rosenberg et al., 1980). The mixture was vortexed for 1 min and incubated at room temperature for 15 min. The procedure was repeated twice. 9 K− medium was then added and the mixture was allowed to stand. The sulfur was then observed to be in the organic phase which partitioned above the aqueous phase. Based on the above results a preliminary model to explain adhesion of A. ferrooxidans on sulfur particles is proposed. The sulfur powder which is initially hydrophobic spreads on the surface of the aqueous medium despite having a specific gravity of 2.1 (Sander et al., 1984). During incubation with A. ferrooxidans it becomes hydrophilic and settles to the bottom of the container, after bacterial adhesion. From thermodynamic considerations, the extent of adhesion will be determined by the surface properties of all the three phases, i.e., the bacteria, liquid media and sulfur. If the effect of electric charges as well as specific biochemical interactions (receptor–ligand) is not considered (Absolom et al., 1983), the change in free energy function could be expressed as: adh
ΔG
= γBS –γBL −γSL
where ΔGadh is the free energy of adhesion, γBS is the bacterium–sulfur interfacial tension, γBL is the bacterium–liquid interfacial tension and γSL is the sulfur–liquid interfacial tension. Since adhesion will be favoured when ΔGadh is minimized, the value of γBS should be minimized or γBL and/or γSL increased. In the above system a hydrophobic secretion is postulated to be produced by A. ferrooxidans, which brings down the interfacial tension between bacterial cells and the surface of sulfur particles (γBS) to enhance contact and adhesion. All the results described support the above model of adhesion. There is no significant modification brought about in the surface tension of the aqueous medium as the fresh sulfur powder floated on the culture filtrate. The adhesion of bacteria makes the sulfur particles hydrophilic due to the exposed surface of bacteria away from the sulfur surface. The bacteria are held to the surface because of a hydrophobic secretion by A. ferrooxidans, which lowers the γBS thus favouring adhesion. After interaction with m-xylene the sulfur particles become hydrophobic as evidenced by their partitioning into the xylene phase which moves above the aqueous phase. Thus a hydrophobic secretion by A. ferrooxidans is involved in bringing about adhesion, and the secretion can be solubilised by m-xylene. Since m-xylene is a nonpolar reagent, hydrophobic substances can easily be solubilised. To check whether any surface modification occurs on A. ferrooxidans by any hydrophobic secretion, two methods, namely hydrophobic interaction chromatography and phase partitioning, were utilized to investigate its surface (Devasia et al., 1993). The results of phase partitioning with n-octane are shown in Fig. 1. With 0.1 to 0.3 ml of n-octane,
3. Results and discussion 3.1. Surface chemistry Fresh sulfur when added to 9 K− medium floats on the surface of the medium. However in flasks inoculated with A. ferrooxidans and grown for 10 days, when kept at rest, the sulfur was found to settle to the bottom of the flask. This phenomenon of settling of sulfur was not observed in control flasks which were not inoculated with A. ferrooxidans. The culture wherein sulfur particles were found to settle was passed through a Whatman No. 1 filter paper. To an aliquot of the filtrate, a fresh sample of sulfur powder was added. The sulfur was found to remain on the surface of the filtrate. However, sulfur particles recovered from the
Fig. 1. Hydrophobicity of Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate, sulfur, pyrite and chalcopyrite.
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approximately 20% of cells grown on ferrous iron or thiosulfate got partitioned into n-octane. A second phase of increased partitioning (up to 40%) were noticed at 0.4 and 0.5 ml of n-octane. In contrast, sulfur and mineral-grown cells showed higher partitioning (up to 35%) even at 0.1 ml of n-octane. Further increase in n-octane volumes resulted in increased partitioning, reaching a value of 45% at 0.5 ml of n-octane. Thus, ferrous iron and thiosulfate-grown cells showed markedly different patterns of partitioning in n-octane as compared to cells grown on sulfur, pyrite and chalcopyrite. It should be noted that the population of cells obtained by growth on sulfur, pyrite and chalcopyrite may not be homogeneous. In sulfur oxidation, partially oxidized species of sulfur appear in solution that freely suspended A. ferrooxidans oxidize (Shrihari et al., 1993). Similarly in some phases of pyrite and chalcopyrite oxidation, ferrous iron released into solution that freely suspended A. ferrooxidans may oxidize (Devasia et al., 1996). The mineral-grown cells were more hydrophobic than the ferrous iron or thiosulfate-grown cells. The greater hydrophobicity of the mineral-grown cells may help in their adhesion to mineral surfaces. The observations of Arredondo et al. (1994) suggested that it would be worthwhile to check for any change in the composition of the exposed polysaccharides in mineral-grown cells compared to solution-grown cells. Ruthenium red adsorption tests demonstrated that ruthenium red, which binds specifically to polysaccharides, exhibited higher binding to solution-grown cells. Solution-grown cells removed about 15% of the ruthenium red from solution whereas mineral-grown cells adsorbed only up to 3% of the dye. The above observation suggests a down regulation of the exposed polysaccharides in mineral-grown cells. This also allows the exposure of the hydrophobic proteins as expected from the other results discussed earlier. It can be concluded that the preferential exposure of polysaccharide moieties is favoured when the cells are utilizing solution substrates. This changes to an exposure of hydrophobic regions of proteins when grown on minerals where adhesion is required for growth. The surface chemistry of A. ferrooxidans grown on minerals is different from that when it is grown on ferrous iron or thiosulfate (Devasia et al., 1993; Devasia et al., 1996). The cells grown on minerals have a relatively higher hydrophobicity than solution-grown cells as measured by phase partitioning and hydrophobic interaction chromatography. The electrophoretic mobilities of mineral-grown and solution-grown cells are distinctly different as demonstrated through free flow electrophoresis. It has further been demonstrated that the isoelectric point (IEP) of minerals shifts on their interaction with A. ferrooxidans. For example, IEP of bacterial cells grown in the presence of ferrous iron and thiosulfate shifted from an initial value of pH 2 to values closer to pH 4 when grown in the presence of sulfur, pyrite and chalcopyrite. Such a significant positive shift in the IEP of bacterial cells was due to the presence of –NH3 groups from proteinaceous secretion as proved by FTIR surface analysis. With antibodies raised against sulfurgrown cells it was possible to examine for any difference in surface components. Enzyme Linked ImmunoSorbent Assay (ELISA) of the cells grown in different conditions revealed that all the mineral-grown cells share common epitopes which are not present in ferrous iron grown or thiosulfate-grown cells. Functional groups characteristic of proteins were found on the surface of sulfur-grown cells. To check whether a proteinaceous substance was indeed responsible for the differences in surface chemistry, the sulfur-grown cells were treated with proteinase K. When the isoelectric point of these cells was measured, it was found that it had shifted to near that of ferrous iron-grown and thiosulfategrown cells (Devasia et al., 1993; Devasia, 1995; Devasia et al., 1996) indicating the removal of originally present surface proteins. Ruthenium red, which specifically binds to exposed polysaccharides, was also found to have less binding to sulfur-grown cells compared to solution-grown cells suggesting that there is a down regulation of exposed polysaccharides on sulfur-grown cells with the synthesis of the proteinaceous substance.
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Fig. 2. Utilization of ferrous ions by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur in the absence of added sulfur.
3.2. Substrate utilization The growth curves showing utilization of ferrous iron by A. ferrooxidans grown under different conditions are shown in Fig. 2. Cells grown on ferrous iron utilize ferrous iron without any lag phase and complete oxidation is seen by 48 h. Cells grown on thiosulfate and sulfur however showed a significantly low level of ferrous oxidation up to 72 h after which the rate increases sharply. The oxidation of ferrous iron is completed by 120 h. This very low level of early oxidation was observed for thiosulfate and sulfur-grown cells because the ferrous oxidation machinery is an inducible one, whereas the sulfur pathway is constitutive (Kulpa et al., 1986a, b). When the above experiment was carried out in the presence of 0.01 g of sulfur per 100 ml of 9 K medium, some changes are observed (Fig. 3). While the profile of ferrous iron oxidation by ferrous irongrown and thiosulfate-grown cells remains almost the same, the sulfur-grown cells take longer time to complete the ferrous oxidation. This shows that the added sulfur is preferred by the cells previously grown on sulfur. When the quantity of added sulfur was further increased to 0.1 g, the oxidation of ferrous iron by sulfur-grown cells is further delayed (Fig. 4). When sufficiently more quantity of sulfur was added a distinct difference in the pattern of all the three sets of cells was observed (Fig. 5). The rate of ferrous utilization even by ferrous iron-grown cells is reduced. In thiosulfate-grown cells, a dramatic decrease in ferrous iron oxidation was observed. The levels were comparable to those observed in sulfurgrown cells. These results are very interesting. It had earlier been discussed that the ferrous iron oxidation machinery is an inducible one.
Fig. 3. Utilization of ferrous by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur in the presence of 0.01 g of sulfur.
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Fig. 4. Utilization of ferrous by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur in the presence of 0.1 g of sulfur.
Fig. 6. Bioleaching of iron from chalcopyrite by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur.
Since thiosulfate-grown cells do not possess both these mechanisms (ferrous iron oxidation and adhesion machinery) to start with, the addition of sufficiently large amounts of sulfur can change the priority of induction of machinery to that for adhesion. The presence of 1 g of sulfur compared to 0.1 g or lower amounts of sulfur provides sufficient surface for the 5 × 109 cells in suspension to interact with for induction of the adhesion machinery. The induction of the machinery for adhesion suggests physiological changes in the bacteria in response to a mineral. The induction of adhesion and hence the utilization of sulfur is preferred over the induction of ferrous oxidation in thiosulfate-grown cells where both machineries (adhesion and ferrous iron oxidation) are absent to start with. It is clear that the induction of adhesion is preferred over the induction of ferrous iron oxidation. These results suggest that more of the population of cells found in nature and under leaching environments may be adhering to the mineral surfaces.
Biodissolution of iron from chalcopyrite (particle size 425–500 μm) brought about by A. ferrooxidans grown on sulfur, and thiosulfate and ferrous iron as a function of leaching period is illustrated in Fig. 6. Sulfurgrown cells release iron from chalcopyrite with no appreciable lag phase which is prominently evident in the case of soluble substrate-grown cells. Both thiosulfate and ferrous iron-grown cells show a lag period of 12 days during which there is no solubilisation of iron. After this period there is a sharp increase in solubilisation. However the solubilisation of iron by sulfur-grown cells is much higher than those observed with solution-grown cells during the period of this study. In Fig. 7 one can see
the concomitant solubilisation of copper from chalcopyrite (particle size 425–500 μm). The trend as observed for iron release by A. ferrooxidans is also observed with respect to copper solubilisation. The pattern of cell adhesion as revealed by the protein content associated with the chalcopyrite supports the role of adhesion in leaching (Fig. 8). The increase in copper and iron solubilisation after a period of time by solutiongrown cells can also be attributed to higher surface protein (bacterial adhesion) associated with the mineral. The lag period observed in the case of solution-grown cells is supposed to be required for the synthesis of the proteinaceous cell surface appendage for firm adhesion and subsequent leaching. The interfacial proteinaceous cell surface appendage is synthesized when the cells are grown on solid substrates such as sulfur and sulfide minerals. This enables bacterial adhesion leading to higher leaching rates. The proteinaceous cell surface appendage is not produced when the cells are grown in solution substrates such as ferrous iron or thiosulfate. The total protein measured on the chalcopyrite is different and is an indicator of the population of cells adhering to the mineral. The more cells (or total protein) the higher the growth of the organism and hence leaching. The fact that thiosulfate-grown cells show leaching comparable only to ferrous iron-grown cells indicates that the adhesion of cells to mineral is the first and rate limiting step in the leaching of minerals by A. ferrooxidans in the direct mechanism, primarily because adhesion is an induced process. The evidence provided above clearly indicates the role of adhesion in leaching. To quantify the effect of adhesion it will be necessary to obtain mutants in adhesion which can be tested for leaching. It is well known in microbiology that mutants (a population of cells which lacks specific function) can be used to delineate the role
Fig. 5. Utilization of ferrous by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur in the presence of 1 g of sulfur.
Fig. 7. Bioleaching of copper from chalcopyrite by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur.
3.3. Role of growth conditions and adhesion in the leaching of chalcopyrite
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not possess the ferrous iron utilization machinery. The use of these cells in leaching clearly demonstrated that the adhesion of the cells to the mineral plays a major role in hastening the process of dissolution. The induction of the adhesion machinery in sulfur-grown cells results in further extension of the lag phase observed in thiosulfate and ferrous iron-grown cells. The fact that the thiosulfate-grown cells behaved in a manner which mostly resembled the ferrous iron-grown cells points to the fact that adhesion is indeed the first and rate limiting step in the leaching of metals from their minerals. In future studies the generation of mutants in adhesion will allow a clearer delineation of the role of adhesion in leaching.
References Fig. 8. Proteins associated with chalcopyrite during bioleaching by Acidithiobacillus ferrooxidans grown on ferrous ion, thiosulfate and sulfur.
of the bacteria in carrying out specific functions. If the mutants, which lack specific function, can be rescued by introducing the specific gene, the role of the gene in carrying out that function can be confirmed and studied. It is also clear that the direct mechanism plays a larger role in leaching than the indirect mechanism as sulfur-grown cells are shown to leach better than ferrous iron-grown cells. While the direct mechanism may be more important, the indirect mechanism also plays a role (Devasia et al., 1996). However both mechanisms may come into play during longer periods of leaching. The importance of adhesion in leaching is demonstrated in this work. 4. Conclusions This study provides a perspective on the process of adhesion of A. ferrooxidans to mineral surfaces. It was recognized during this study that A. ferrooxidans can be cultured in solution substrate or on solid substrate for the purpose of comparison of the process of adhesion. The solution substrates include ferrous iron and thiosulfate. The solid substrates used in this study include minerals like sulfur, pyrite and chalcopyrite. In mineral-grown conditions, since the substrate is insoluble, the adhesion of bacteria is required for its enzymatic machinery to come into close contact for the dissolution of the mineral. But when grown with solution substrate, the adhesion machinery is not required for substrate utilization. These differing growth conditions thus provided a means of looking for differences in A. ferrooxidans when adhesion is required and when it is not required. Learning about these differences would be an important step in understanding the mechanism of adhesion of A. ferrooxidans to mineral surfaces. Machinery for adhesion can be induced by growth on sulfur. Such machinery is not present in cells grown on thiosulfate and ferrous iron. Further the thiosulfate-grown cells like the sulfur-grown cells do
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