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Biochemistry of sulfur extraction in bio-corrosion of pyrite by Thiobacillus ferrooxidans
597
B i o c h e m i s t r y o f sulfur extraction in bio-corrosion o f pyrite b y
Thiobacillus f errooxidans J.A. Rojas-Chapana and H. Tributsch* Hahn-Meitner Institut, Department Solare Energetik, Glienicker Strasse 100, 14109 Berlin, Germany
By adding a small amount of the aminoacid cysteine in acidic solution exposed to pyrite, the duration of the lag phase in the growth of Thiobacillusferrooxidans is minimized and the leaching rate of this sulfide is increased three times compared to the normal process without this biochemical additive. In the presence of cysteine, pyrite can be oxidized in absence of oxygen or bacteria with a leaching rate comparable with that attained by bacteria under normal leaching conditions. It seems that the sulfhydril group of cysteine participates in a binding process with pyrite. Free-SH groups from the pyrite surface would be the counterpart for the formation of the corresponding disulfide. This thiol-disulfide reaction means that cysteine is consumed by the pyrite surface with the subsequent release of iron-sulfur species. This result is accounted for by the fact that pyrite without bacteria can be completely oxidized. Bacteria would take advantage of this abiotic corrosion process by uptake and oxidation of the released species which are continuously supplied to the thiobacilli-biotope. The reactivity of this thiol biochemical compound with the pyrite surface, which improves the bacterial corrosion rate, suggested the investigation of other mono-thiol molecules with the aim to mask the SH pyritic groups. Tert-butyl mercaptan [(CH3)3C-SH: TBM] can be applied to the pyrite surface to stop the bacterial corrosion. Permanent disulfide bonds between pyrite and the blocking SH-agent are the cause for this protection.
1. INTRODUCTION Enhancement of bacterial activity at pyrite surfaces has frequently been investigated with the aim to improve the recovery of metals from sulfide ores. Strategies to exploit the leaching reactions performed by sulfide oxidizing-bacteria consider the oxidation of sulfur to sulfate and the oxidation of ferrous to ferric iron. Attempts to improve the metal recovery include also the modification of area of exposed pyrite by means of surface active agents or bio-macromolecules. These procedures must be done in a economical and environmentally safe manner.
* corresponding author
598 Recent studies on this matter have shown that the recovery of precious metals such as gold from sulfide ores by using bacterial leaching techniques is economical when carbohydrates, proteins and other substances of biological origin are added to the tank reactors. Thus, it has been reported that for selective Cu recovery from chalcopyrite ores the leaching media for bacteria should include suitable nutrient supplement of potato and corn (1). It has been shown, for example, that dissolution of metallic ions from alloys is increased by the addition of aminoacids and/or albumina (2). Concerning this matter, several experimental observations associated with the metabolism of T. ferrooxidans such as intermediary sulfur-colloid formation (3), sulfide oxidation in absence of ferric ions (4), SHpolarographic waves (5) suggest that thiol-groups are involved in sulfide leaching by T. ferrooxidans and that bacteria might cause mineral dissolution by other means than purely ferrous/ferric electrochemical attack. In order to elucidate such a possibility, several experiments were performed with thiolbiochemical compounds, especially among aminoacids. These experiments were based on modifying pyrite surfaces (100 nm thick) with thiol groups, on adding thiol compounds to bacterial suspensions and on studying the effect of thiol compounds on the dissolution of pyrite in sterile culture solutions. Similar experiments have been performed to identify thiols for protection of the pyrite surface against corrosion.
2. MATERIAL AND METHODS
2.1. Organism Strain R2 of Thiobacillusferrooxidans was cultured in Tuovinen-medium (6). When the culture reached the exponential phase of growth (about 4 days of cultivation), it was centrifuged at 3000 X g for 10 min, and the bacterial sediment was washed 3 times with the same medium without ferrous ions (buffer). 2.2. Preparation of synthetic pyrite layers Synthetic pyrite films were prepared by metal organic chemical vapour deposition (MOCVD) technique (7) and proved to be suitable substrates for bioleaching by T. ferrooxidans, on which quantitative time lapse measurements could be performed with a high resolution video microscope technique. 2.3. Cultivation on synthetic pyrite layers The bacterial suspension was adjusted to 50.000 cells ~t1-1 (cells count were determined microscopically with a Neubauer counting chamber) in Tuovinen buffer and inoculated into prepared culture chambers containing pyrite layers as energy substrate. A detailed description for the construction of the culture chamber and the inoculation conditions was previously published (8). 2.4. Pyrite layers wetted with bacterial suspension including aminoacids Washed cells (adjusted to ca. 50.000 cells ~t1-1) were resuspended in Tuovinen-buffer solutions, pH 1.6 containing 10-2 - 10-5 M aminoacid, and reinoculated in culture chambers containing pyrite as energy source.
599
2.5. Treatment of pyrite with tert-butyl mercaptan (TBM) Exposure of the pyrite surface to a S-alkylmercapto substrate such as tert-butyl mercaptan [(CH3)3C-SH] was carried out by wetting the layers (concentrations ranging from 10 to 100 mg/1) with this agent in an isolated chamber on account of the toxicity and unpleasant odor of this compound. After ca. 1 hour exposure to the thiol-TBM, the pyrite layers were rinsed with plenty of water, dried and fitted in culture chambers.
2.6. Measurement of oxidizing activity by determination of the degree of corrosion For time dependent corrosion-measurements with pyrite layers it was neccesary to integrate the pyrite surface for determination of corroded and untouched areas. For this purpose the software Photoshop from Microsoft was used.
2.7. Transmission electron microscopy (TEM) The preparations were observed and recorded using a Philips CM 12 transmission electron microscope at an accelerating voltage of 120 kV and a resolution of 2 A~.
3. RESULTS When synthetic pyrite layers were prepared with the MOCVD-reactor in presence of different aminoacids and exposed to Tuovinen-buffer solution a significantly increased sulfide dissolution was observed for cysteine, homocysteine, and methionine. Only those aminoacids which contain SH groups or sulfur were able, when attached to the pyrite surface, to trigger a progressive dissolution in absence of bacteria (9). In order to further study this phenomenon, cysteine was added to bacterial suspensions in contact with pyrite layers.
3.1. Cysteine (Cys) induced leaching of pyrite Figure 1 shows the relationship between bacterial corrosion of pyrite and the amount of aminoacid added to the culture system. The corrosive properties of cysteine, as seen in the experiments with aminoacid-modified layers (9), are evident also here. Addition of bacteria increased corrosion approximately by a factor 2.5. These results confirm that soluble cysteine can react with the pyrite surface and dissolve it. This surface chemical process appears to aid bacterial leaching of pyrite. As showed in figure 1, a concentration of ca. 10 -5 M of cysteine was sufficient to generate a significant increase of corrosion. Similar effects were observed up to concentrations of 10-4 M. However, at high concentrations of cysteine (>>10 -3 M) a deletereous effect on the bacterial viability was found, which may be attributed to the organic nature of this compound. Furthermore, the increase of cysteine concentration slowly leads to the reoxidation of this aminoacid and the appearance of cystine clusters which implies a suppression of its corrosive properties. Methionine, when added to the culture medium was incapable of corroding pyrite which points out that a demethylation is necessary to expose the reactive SH group of this aminoacid (formation ofhomocysteine).
600 120
100
80
~
bact. corrosion without cys.
60
FeS2/Cys 1e-2 M + bact. "~
40
0
9 I,,,,4
0
20
----II--
FeS2/Cys le-3 M + bact.
------O-----
FeS2/Cys le-4 M + bact.
--
FeS2/Cys 1e-5 M + bact.
0
cj
0 -20
I 0
.
,
.
10
, 20
.
, 30
Time (days) Figure 1. Effect of the concentration of cysteine on the bacterial corrosion of pyrite.
Also visual observations of culture chambers (with a synthetic pyrite layer) (see Fig. 2) show that cysteine alone acts as a corrosion trigger and bacteria act synergistically to enhace the cysteine-mediated primary attack.
Figure 2. Chambers incubated in darkness at 28 ~ during a week. A) pyrite/buffer without corrosion-signs; B) pyrite/bacterial suspension. Corrosion 25 %; C) cys-pyrite. Corrosion 50 %; I)) cys-pyrite/bacteria. Corrosion 100%.
601 On account of the observed corrosion of pyrite by SH-aminoacids and the experimental evidence that polipeptide containing -SH residues (cysteine) can react spontaneously with iron disulphides and form bonds similar to those present in biological Fe-S centers (10), the possibility that selected thiol-compounds might also inhibit the corrosion process of pyrite must be considered. 3.2. Tert-butyl disulfid (TBDS) as a chemical factor responsible for pyrite passivity Synthetic pyrite layers prepared with tert-butyl disulfide (TBDS) as sulfur precursor (11) have shown a complete resistance against bacterial attack and a corrosive action of sulfide-oxidizing bacteria (Thiobacilli) was not detected. Thus, Thiobacilli inoculated on these layers not only encounter a barrier that masks the energy contained in the pyritic substrate, but it was also found that the modified pyrite surface has a negative effect on bacteria viability. The expected formation of a protecting film on pyrite layers by TBDS is believed to be due to a cleavage of this compound into two identical sulfides [(CH3)3-C-SH] tert-butyl mercaptan (TBM), which bind to terminal pyrite sulfur atoms. TBM as cleaving product of the symmetric disulfide TBDS was investigated for its possible ability to reduce the corrosion of pyrite layers. Pyrite layers treated with this thiol agent revealed that below a critical concentration of this inhibitor bacteria can resist and weakly corrode the pyrite surface. This concentration was found to be approximately 10 mg/1. Above this concentration, TBM becomes adsorbed on the pyrite surface and neutralizes bacterial activity. On the other hand, regions of the same pyrite layer not exposed to the action of TBM showed clearly visible corrosion (see Fi~. 3).
Figure 3. Region of a pyrite layer (100 nm thick) examined by TEM. On the left untreated pyrite with typical corrosion pattern. On the fight TBM-pretreated pyrite region without corrosion.
602 4. DISCUSSION Under natural bacterial leaching conditions of pyrite (moderate concentrations of Fe 3+ and moderately positive redox potential) the following cysteine mediated sulfur recovery process can be imagined (see Fig. 4).
Figure 4. Two cysteine molecules react with pyrite, made up of interfacial {Fe++, S} and bulk FeS2 (1). The interaction disrupts the structure while leading to the formation of an ironcysteine complex, a cysteine-pyrite complex (bonded via a sulfur bridge) and an interfacial SH group (2). The cysteine-pyrite complex reorganizes and leads to the liberation of an ironsulfur-cysteine complex. This complex is the supposed chemical energy carrier (3) for T. Ferrooxidans with a cyclic turnover of cysteine and Fe +++ (4). Cysteine is secreted by T. ferrooxidans and interacts with the FeS2 surface via the bacterial capsule (exopolysaccharides) (3-12). This interaction disrupts the pyrite surface so that iron-sulfur species can be extracted. Subsequently a sulfur colloid-forming reaction takes place. These sulfur colloids serve as temporary energy reservoirs until sulfur species are transportated into the interior of the cell (3). During the natural leaching of pyrite by T. ferrooxidans cysteine or a cysteine containing biological molecule (glutathion) may play the role of trigger for the leaching process and of carrier for the chemical energy. As shown in figure 4, cysteine interacts with both a reactive sulfur produced as surface states or via anodic oxidation of pyrite and iron species that are being generated by waterinduced interfacial corrosion (see Fig. 5).
/
OCC
~
CHNH 2
Fe
HO~
~S
F e ~ SCH2CH/
OH2 (A)
NH2
~COOH (B)
Figure 5. Ferrous ions complexed with cysteine of which (A) ist most stable in acid solution while (B) predominates at a high pH.
603 Pyrite material typically shows lattice dislocations, stoichiometry deviations and deviations from crystalline order at the interface. They generate surface states, which expose dangling bonds for an interfacial chemistry, which determines cysteine adsorption and reaction. Concerning the pathway of abiotic cysteine/homocysteine-degradation of pyrite after the formation of an asymmetric disulfide cys-S-S-pyrite, we have only poor knowledge at present. Once this disulfide is formed, the next step must involve cleavage of pyrite bonds to yield an iron-sulfur-cysteine complex for transport away from the surface. Additional dangling bonds are expected to be generated during the release of the complex. A subsequent step would be the readsorption of cysteine to the formed surface states to continue the biochemical corrosion. According to figure 4, the formation and release of an iron-sulfur-cysteine complex may hint at an adaptive metabolic pathway acquired by T. ferrooxidans for pyrite disolution. This might explain, why the coupling of bacteria to the system cysteine-pyrite results in higher corrosion rates and increased biomass formation. Following this model, the iron-sulfur species extraxted from pyrite and bound to cysteine represents an energy entity which can be transported towards the cell wall by means of cysteine as a cartier. Within the extracellular polymeric layer (at the level of the cell wall) the dissociation of the iron-sulfur complex of cysteine occurs with the formation of colloidal sulfur particles and the respective transport of the sulfur and iron species into the electron transport chain of T. ferrooxidans. Thus it seems that the corrosion of pyrite in solution of thiol-aminoacids reflects a mechanism evolved by Thiobacilli with the aim to obtain energy from insoluble sulfides sources, which cannot be disintegrated by electrons extraction with ferric ions. The effect of low concentrations of thiol-aminoacids, such as produced during the autolysis of bacterial cells or through the release of a specific metabolites such as cysteine or glutathione, should therefore be to increase the rate of solubilisation of metal-sulfides. Figure 6 describes schematically the interaction between bacteria and pyrite. Bacteria condition the pyrite surface with a biofilm (exopolysacharides) rich in thiol biochemical compounds, for example, cysteine. The modified surface favors the chemotaxis and trigger a biochemical degradation of this sulfide.
Figure 6. Schematic diagram of the system pyrite-cysteine-T, ferrooxidans cells
604 The thiol chemistry involved in this leaching process also opens opportunities for a suppression of the leaching process. Thus, a possible mechanism for an interaction between pyrite and TBDS seems to be analogous to that described for cysteine i.e., the formation of disulfide bridged pyrite-S-S-C(CH3)3 would be a prerequisite for pyrite protection. Theoretically free SH-groups from pyrite and their counterpart from TBM, could convert into the corresponding disulfide forms. A simple adsorption model is proposed to explain the observed inhibition of bacterial corrosion in the presence of TBM (see Fig. 7).
Figure 7. The presence of TBM on the pyrite surface attached via a S-S bridge apparently stabilizes and masks the interface. The pyritic thiol-groups become screened and unaccesibles for both cellular and exogeneous -SH groups.
TBM, with its sulfur group interacts with a dangling sulfur bond in the pyrite interface to form a-S-S- bridged interfacial complex. Unlike in the cysteine reaction, this disulfide is sufficiently stable to shield pyrite from bacterial chemotaxis and attack.
5. OUTLOOK The outlined properties of cysteine as a possible sulfur carrier and as a promoting agent in pyrite leaching by T. ferrooxidans suggest a possible application in biohydrometallurgical operations. Where bacteria do not anymore become active due to lack of oxygen access, cysteine may still extract sulfur and transport it to aerated zones. However it has also to be considered that cysteine forms complexes with Fe 2+/3+, whereby the redox potential of this couple is significantly altered. This may interfere with the energy harvesting process of iron oxidizing bacteria. In conclusion, in an environment cysteine/pyrite, T. ferrooxidans finds ideal conditions for leaching and for its viability. From our laboratory-experiments it can be deduced that the oxidation of solid sulfides can be considerably accelerated in presence of biochemical thiol-compounds. It is suggested that using a pyrite feedstock and a bacterial leaching technology improved with the application of thiol-aminoacids, a high leaching rate may be attained economically and compatible with the environment. Thus, for successful leaching the addition of cysteine/homocysteine to dumps which have ceased for not apparent reason may represent an inexpensive, alternative effort of metal-recovering.
605 REFERENCES
1. P. A. Rusin and J.E. Sharp, Enhancement of bioleaching systems for sulfide ores using nutrient additives, US Patent No 94-194676 (1994). 2. Y. Sano and S. Takeda, Shika Igaku, 55 (2) (1992) 125. 3. J.A. Rojas-Chapana, M. Giersig and H. Tributsch. Fuel, 75 (1996) 923. 4. J.A Rojas-Chapana, P. Friebe, R. Murillo and H. Tributsch (in preparation). 5. H. Tributsch and J.C. Bennett, J. Chem. Tech. Biotechnol., 31(1981) 627. 6. O. Tuovinen and D. Kelly, Arch. Mikrobiol., 88 (1973) 285. 7. A. Ennaoui, S. Fiechter, and H. Tributsch, Solar Energy Materials and Solar Cells. 29 (1993) 289. 8. H. Tributsch, J. A. Rojas-Chapana and C.C. B~a'tels, Corrosion., 54 (1998) 216. 9. H. Tributsch and J.A. Rojas-Chapana, Verfahren zur mikrobiellen Laugung von Sulfiderzen, german patent application No P54598DE-Zie (1998). 10. G. Berthon (Ed.), Handbook of metal-ligand interactions in biological fluids, Marcel Dekker Inc, New York, 1995. 11. H. Tributsch and J. A. Rojas-Chapana, Verwendung von Di-tert-Butylsulfid (TBDS) und/oder tert-Butylmercaptan (TBM) als Korrosionsinhibitoren der mikrobiellen Korrosion von Metallen, german patent application No P58098DEZie (1998). 12. T.Gehrke, J.Telegdi, D.Thierry and W.Sand, Appl. Environ. Microbiol., 64 (1998) 2743. 13. P. C. Yocelyn (ed.), Biochemestry of the SH group, Academic Press Inc, London, 1972.
B i o c h e m i s t r y o f sulfur extraction in bio-corrosion o f pyrite b y
Thiobacillus f errooxidans J.A. Rojas-Chapana and H. Tributsch* Hahn-Meitner Institut, Department Solare Energetik, Glienicker Strasse 100, 14109 Berlin, Germany
By adding a small amount of the aminoacid cysteine in acidic solution exposed to pyrite, the duration of the lag phase in the growth of Thiobacillusferrooxidans is minimized and the leaching rate of this sulfide is increased three times compared to the normal process without this biochemical additive. In the presence of cysteine, pyrite can be oxidized in absence of oxygen or bacteria with a leaching rate comparable with that attained by bacteria under normal leaching conditions. It seems that the sulfhydril group of cysteine participates in a binding process with pyrite. Free-SH groups from the pyrite surface would be the counterpart for the formation of the corresponding disulfide. This thiol-disulfide reaction means that cysteine is consumed by the pyrite surface with the subsequent release of iron-sulfur species. This result is accounted for by the fact that pyrite without bacteria can be completely oxidized. Bacteria would take advantage of this abiotic corrosion process by uptake and oxidation of the released species which are continuously supplied to the thiobacilli-biotope. The reactivity of this thiol biochemical compound with the pyrite surface, which improves the bacterial corrosion rate, suggested the investigation of other mono-thiol molecules with the aim to mask the SH pyritic groups. Tert-butyl mercaptan [(CH3)3C-SH: TBM] can be applied to the pyrite surface to stop the bacterial corrosion. Permanent disulfide bonds between pyrite and the blocking SH-agent are the cause for this protection.
1. INTRODUCTION Enhancement of bacterial activity at pyrite surfaces has frequently been investigated with the aim to improve the recovery of metals from sulfide ores. Strategies to exploit the leaching reactions performed by sulfide oxidizing-bacteria consider the oxidation of sulfur to sulfate and the oxidation of ferrous to ferric iron. Attempts to improve the metal recovery include also the modification of area of exposed pyrite by means of surface active agents or bio-macromolecules. These procedures must be done in a economical and environmentally safe manner.
* corresponding author
598 Recent studies on this matter have shown that the recovery of precious metals such as gold from sulfide ores by using bacterial leaching techniques is economical when carbohydrates, proteins and other substances of biological origin are added to the tank reactors. Thus, it has been reported that for selective Cu recovery from chalcopyrite ores the leaching media for bacteria should include suitable nutrient supplement of potato and corn (1). It has been shown, for example, that dissolution of metallic ions from alloys is increased by the addition of aminoacids and/or albumina (2). Concerning this matter, several experimental observations associated with the metabolism of T. ferrooxidans such as intermediary sulfur-colloid formation (3), sulfide oxidation in absence of ferric ions (4), SHpolarographic waves (5) suggest that thiol-groups are involved in sulfide leaching by T. ferrooxidans and that bacteria might cause mineral dissolution by other means than purely ferrous/ferric electrochemical attack. In order to elucidate such a possibility, several experiments were performed with thiolbiochemical compounds, especially among aminoacids. These experiments were based on modifying pyrite surfaces (100 nm thick) with thiol groups, on adding thiol compounds to bacterial suspensions and on studying the effect of thiol compounds on the dissolution of pyrite in sterile culture solutions. Similar experiments have been performed to identify thiols for protection of the pyrite surface against corrosion.
2. MATERIAL AND METHODS
2.1. Organism Strain R2 of Thiobacillusferrooxidans was cultured in Tuovinen-medium (6). When the culture reached the exponential phase of growth (about 4 days of cultivation), it was centrifuged at 3000 X g for 10 min, and the bacterial sediment was washed 3 times with the same medium without ferrous ions (buffer). 2.2. Preparation of synthetic pyrite layers Synthetic pyrite films were prepared by metal organic chemical vapour deposition (MOCVD) technique (7) and proved to be suitable substrates for bioleaching by T. ferrooxidans, on which quantitative time lapse measurements could be performed with a high resolution video microscope technique. 2.3. Cultivation on synthetic pyrite layers The bacterial suspension was adjusted to 50.000 cells ~t1-1 (cells count were determined microscopically with a Neubauer counting chamber) in Tuovinen buffer and inoculated into prepared culture chambers containing pyrite layers as energy substrate. A detailed description for the construction of the culture chamber and the inoculation conditions was previously published (8). 2.4. Pyrite layers wetted with bacterial suspension including aminoacids Washed cells (adjusted to ca. 50.000 cells ~t1-1) were resuspended in Tuovinen-buffer solutions, pH 1.6 containing 10-2 - 10-5 M aminoacid, and reinoculated in culture chambers containing pyrite as energy source.
599
2.5. Treatment of pyrite with tert-butyl mercaptan (TBM) Exposure of the pyrite surface to a S-alkylmercapto substrate such as tert-butyl mercaptan [(CH3)3C-SH] was carried out by wetting the layers (concentrations ranging from 10 to 100 mg/1) with this agent in an isolated chamber on account of the toxicity and unpleasant odor of this compound. After ca. 1 hour exposure to the thiol-TBM, the pyrite layers were rinsed with plenty of water, dried and fitted in culture chambers.
2.6. Measurement of oxidizing activity by determination of the degree of corrosion For time dependent corrosion-measurements with pyrite layers it was neccesary to integrate the pyrite surface for determination of corroded and untouched areas. For this purpose the software Photoshop from Microsoft was used.
2.7. Transmission electron microscopy (TEM) The preparations were observed and recorded using a Philips CM 12 transmission electron microscope at an accelerating voltage of 120 kV and a resolution of 2 A~.
3. RESULTS When synthetic pyrite layers were prepared with the MOCVD-reactor in presence of different aminoacids and exposed to Tuovinen-buffer solution a significantly increased sulfide dissolution was observed for cysteine, homocysteine, and methionine. Only those aminoacids which contain SH groups or sulfur were able, when attached to the pyrite surface, to trigger a progressive dissolution in absence of bacteria (9). In order to further study this phenomenon, cysteine was added to bacterial suspensions in contact with pyrite layers.
3.1. Cysteine (Cys) induced leaching of pyrite Figure 1 shows the relationship between bacterial corrosion of pyrite and the amount of aminoacid added to the culture system. The corrosive properties of cysteine, as seen in the experiments with aminoacid-modified layers (9), are evident also here. Addition of bacteria increased corrosion approximately by a factor 2.5. These results confirm that soluble cysteine can react with the pyrite surface and dissolve it. This surface chemical process appears to aid bacterial leaching of pyrite. As showed in figure 1, a concentration of ca. 10 -5 M of cysteine was sufficient to generate a significant increase of corrosion. Similar effects were observed up to concentrations of 10-4 M. However, at high concentrations of cysteine (>>10 -3 M) a deletereous effect on the bacterial viability was found, which may be attributed to the organic nature of this compound. Furthermore, the increase of cysteine concentration slowly leads to the reoxidation of this aminoacid and the appearance of cystine clusters which implies a suppression of its corrosive properties. Methionine, when added to the culture medium was incapable of corroding pyrite which points out that a demethylation is necessary to expose the reactive SH group of this aminoacid (formation ofhomocysteine).
600 120
100
80
~
bact. corrosion without cys.
60
FeS2/Cys 1e-2 M + bact. "~
40
0
9 I,,,,4
0
20
----II--
FeS2/Cys le-3 M + bact.
------O-----
FeS2/Cys le-4 M + bact.
--
FeS2/Cys 1e-5 M + bact.
0
cj
0 -20
I 0
.
,
.
10
, 20
.
, 30
Time (days) Figure 1. Effect of the concentration of cysteine on the bacterial corrosion of pyrite.
Also visual observations of culture chambers (with a synthetic pyrite layer) (see Fig. 2) show that cysteine alone acts as a corrosion trigger and bacteria act synergistically to enhace the cysteine-mediated primary attack.
Figure 2. Chambers incubated in darkness at 28 ~ during a week. A) pyrite/buffer without corrosion-signs; B) pyrite/bacterial suspension. Corrosion 25 %; C) cys-pyrite. Corrosion 50 %; I)) cys-pyrite/bacteria. Corrosion 100%.
601 On account of the observed corrosion of pyrite by SH-aminoacids and the experimental evidence that polipeptide containing -SH residues (cysteine) can react spontaneously with iron disulphides and form bonds similar to those present in biological Fe-S centers (10), the possibility that selected thiol-compounds might also inhibit the corrosion process of pyrite must be considered. 3.2. Tert-butyl disulfid (TBDS) as a chemical factor responsible for pyrite passivity Synthetic pyrite layers prepared with tert-butyl disulfide (TBDS) as sulfur precursor (11) have shown a complete resistance against bacterial attack and a corrosive action of sulfide-oxidizing bacteria (Thiobacilli) was not detected. Thus, Thiobacilli inoculated on these layers not only encounter a barrier that masks the energy contained in the pyritic substrate, but it was also found that the modified pyrite surface has a negative effect on bacteria viability. The expected formation of a protecting film on pyrite layers by TBDS is believed to be due to a cleavage of this compound into two identical sulfides [(CH3)3-C-SH] tert-butyl mercaptan (TBM), which bind to terminal pyrite sulfur atoms. TBM as cleaving product of the symmetric disulfide TBDS was investigated for its possible ability to reduce the corrosion of pyrite layers. Pyrite layers treated with this thiol agent revealed that below a critical concentration of this inhibitor bacteria can resist and weakly corrode the pyrite surface. This concentration was found to be approximately 10 mg/1. Above this concentration, TBM becomes adsorbed on the pyrite surface and neutralizes bacterial activity. On the other hand, regions of the same pyrite layer not exposed to the action of TBM showed clearly visible corrosion (see Fi~. 3).
Figure 3. Region of a pyrite layer (100 nm thick) examined by TEM. On the left untreated pyrite with typical corrosion pattern. On the fight TBM-pretreated pyrite region without corrosion.
602 4. DISCUSSION Under natural bacterial leaching conditions of pyrite (moderate concentrations of Fe 3+ and moderately positive redox potential) the following cysteine mediated sulfur recovery process can be imagined (see Fig. 4).
Figure 4. Two cysteine molecules react with pyrite, made up of interfacial {Fe++, S} and bulk FeS2 (1). The interaction disrupts the structure while leading to the formation of an ironcysteine complex, a cysteine-pyrite complex (bonded via a sulfur bridge) and an interfacial SH group (2). The cysteine-pyrite complex reorganizes and leads to the liberation of an ironsulfur-cysteine complex. This complex is the supposed chemical energy carrier (3) for T. Ferrooxidans with a cyclic turnover of cysteine and Fe +++ (4). Cysteine is secreted by T. ferrooxidans and interacts with the FeS2 surface via the bacterial capsule (exopolysaccharides) (3-12). This interaction disrupts the pyrite surface so that iron-sulfur species can be extracted. Subsequently a sulfur colloid-forming reaction takes place. These sulfur colloids serve as temporary energy reservoirs until sulfur species are transportated into the interior of the cell (3). During the natural leaching of pyrite by T. ferrooxidans cysteine or a cysteine containing biological molecule (glutathion) may play the role of trigger for the leaching process and of carrier for the chemical energy. As shown in figure 4, cysteine interacts with both a reactive sulfur produced as surface states or via anodic oxidation of pyrite and iron species that are being generated by waterinduced interfacial corrosion (see Fig. 5).
/
OCC
~
CHNH 2
Fe
HO~
~S
F e ~ SCH2CH/
OH2 (A)
NH2
~COOH (B)
Figure 5. Ferrous ions complexed with cysteine of which (A) ist most stable in acid solution while (B) predominates at a high pH.
603 Pyrite material typically shows lattice dislocations, stoichiometry deviations and deviations from crystalline order at the interface. They generate surface states, which expose dangling bonds for an interfacial chemistry, which determines cysteine adsorption and reaction. Concerning the pathway of abiotic cysteine/homocysteine-degradation of pyrite after the formation of an asymmetric disulfide cys-S-S-pyrite, we have only poor knowledge at present. Once this disulfide is formed, the next step must involve cleavage of pyrite bonds to yield an iron-sulfur-cysteine complex for transport away from the surface. Additional dangling bonds are expected to be generated during the release of the complex. A subsequent step would be the readsorption of cysteine to the formed surface states to continue the biochemical corrosion. According to figure 4, the formation and release of an iron-sulfur-cysteine complex may hint at an adaptive metabolic pathway acquired by T. ferrooxidans for pyrite disolution. This might explain, why the coupling of bacteria to the system cysteine-pyrite results in higher corrosion rates and increased biomass formation. Following this model, the iron-sulfur species extraxted from pyrite and bound to cysteine represents an energy entity which can be transported towards the cell wall by means of cysteine as a cartier. Within the extracellular polymeric layer (at the level of the cell wall) the dissociation of the iron-sulfur complex of cysteine occurs with the formation of colloidal sulfur particles and the respective transport of the sulfur and iron species into the electron transport chain of T. ferrooxidans. Thus it seems that the corrosion of pyrite in solution of thiol-aminoacids reflects a mechanism evolved by Thiobacilli with the aim to obtain energy from insoluble sulfides sources, which cannot be disintegrated by electrons extraction with ferric ions. The effect of low concentrations of thiol-aminoacids, such as produced during the autolysis of bacterial cells or through the release of a specific metabolites such as cysteine or glutathione, should therefore be to increase the rate of solubilisation of metal-sulfides. Figure 6 describes schematically the interaction between bacteria and pyrite. Bacteria condition the pyrite surface with a biofilm (exopolysacharides) rich in thiol biochemical compounds, for example, cysteine. The modified surface favors the chemotaxis and trigger a biochemical degradation of this sulfide.
Figure 6. Schematic diagram of the system pyrite-cysteine-T, ferrooxidans cells
604 The thiol chemistry involved in this leaching process also opens opportunities for a suppression of the leaching process. Thus, a possible mechanism for an interaction between pyrite and TBDS seems to be analogous to that described for cysteine i.e., the formation of disulfide bridged pyrite-S-S-C(CH3)3 would be a prerequisite for pyrite protection. Theoretically free SH-groups from pyrite and their counterpart from TBM, could convert into the corresponding disulfide forms. A simple adsorption model is proposed to explain the observed inhibition of bacterial corrosion in the presence of TBM (see Fig. 7).
Figure 7. The presence of TBM on the pyrite surface attached via a S-S bridge apparently stabilizes and masks the interface. The pyritic thiol-groups become screened and unaccesibles for both cellular and exogeneous -SH groups.
TBM, with its sulfur group interacts with a dangling sulfur bond in the pyrite interface to form a-S-S- bridged interfacial complex. Unlike in the cysteine reaction, this disulfide is sufficiently stable to shield pyrite from bacterial chemotaxis and attack.
5. OUTLOOK The outlined properties of cysteine as a possible sulfur carrier and as a promoting agent in pyrite leaching by T. ferrooxidans suggest a possible application in biohydrometallurgical operations. Where bacteria do not anymore become active due to lack of oxygen access, cysteine may still extract sulfur and transport it to aerated zones. However it has also to be considered that cysteine forms complexes with Fe 2+/3+, whereby the redox potential of this couple is significantly altered. This may interfere with the energy harvesting process of iron oxidizing bacteria. In conclusion, in an environment cysteine/pyrite, T. ferrooxidans finds ideal conditions for leaching and for its viability. From our laboratory-experiments it can be deduced that the oxidation of solid sulfides can be considerably accelerated in presence of biochemical thiol-compounds. It is suggested that using a pyrite feedstock and a bacterial leaching technology improved with the application of thiol-aminoacids, a high leaching rate may be attained economically and compatible with the environment. Thus, for successful leaching the addition of cysteine/homocysteine to dumps which have ceased for not apparent reason may represent an inexpensive, alternative effort of metal-recovering.
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