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Bacterial leaching, the biooxidation of metal sulfides to soluble metal sulfates and sulfuric acid, is effected by specialized bacteria. Three species of mesoacidophilic, chemolithotrophic bacteria are mainly involved: Thiobacillus ferrooxidans, Thiobacillus thiooxidans, and Leptospirillum ferrooxidans. T. ferrooxidans oxidizes reduced sulfur compounds to sulfate and iron(II) to iron(III) ions. T. thiooxidans is able to oxidize only reduced sulfur compounds, whereas L. ferrooxidans can oxidize only iron(II) ions (1, 4, 13, 15).

Recently, we described for pyrite (FeS₂) degradation an iron(III) ion-mediated leaching mechanism via thiosulfate and polythionates (18). In this thiosulfate mechanism, the Fe-S₂ bond is cleaved, after the S₂ group has been oxidized by iron(III) hexahydrate ions to a thiosulfate group. Hydrolysis yields thiosulfate and an iron(II) ion. Thiosulfate is consecutively oxidized via tetrathionate, disulfane-monosulfonic acid, and trithionate to sulfate. Elemental sulfur occurs as a by-product only. The function of T. ferrooxidans and L. ferrooxidans is to supply the oxidizing iron(III) ions.

Although FeS₂ is the most abundant metal sulfide on earth, it is not the most valuable one. Consequently, experiments were performed in order to evaluate whether the thiosulfate mechanism is also valid for other metal sulfides. A survey of mineralogical data on metal sulfides indicated that the structure of pyrite is almost unique (2, 28). For further tests, the differently structured metal sulfides sphalerite (ZnS), chalcopyrite (CuFeS₂), galena (PbS), hauerite (MnS₂), orpiment (As₂S₃), realgar (As₄S₄), and molybdenite (MoS₂) were selected. For these, the formation of sulfur compounds by iron(III) ion-mediated chemical oxidation was analyzed. For the experiments, the metal sulfides were crushed, pulverized, and heat sterilized under N₂. Fifty milliliters of a sterile 10 mM FeCl₃ solution at pH 1.9 was added to 1 g of each metal sulfide powder in shake flasks. The suspension was sampled for analysis of reduced sulfur compounds (high-pressure liquid chromatography-diode array detection), sulfate (ion chromatography), metal ions (atomic absorption spectroscopy), iron(III)-iron(II) ions (photometry), and pH (electrode) as previously described (18). Experiments were done in triplicate. The results are shown in Table 1.

The oxidation products in the case of FeS₂ and MoS₂ consisted of up to 90% sulfate and about 1 to 2% polythionates. Because the valence bands of FeS₂ and MoS₂ are derived only from the metal orbitals, the valence bands do not contribute to the chemical bond between the metal and the sulfur moiety in the crystal (2, 25). Consequently, these metal sulfides are degradable only by an oxidizing attack, e.g., by iron(III) ions. None of these compounds is soluble in acid (proton attack) (21, 25, 26). Furthermore, both metal sulfides consist of pairs of sulfur atoms. These properties hold for WS₂ as well (25, 26). Consequently, FeS₂, MoS₂, and WS₂ are oxidized by the same, indirect thiosulfate mechanism. In leaching experiments with L. ferrooxidans and FeS₂ the same sulfur compounds resulted (18). Thus, the same dissolution mechanism is active in bioleaching.

In contrast, all other metal sulfides mentioned above have valence bands, to which metal and sulfur orbitals contribute (2, 25, 26). Consequently, they are acid soluble. The experiments with these metal sulfides yielded elemental sulfur in amounts of more than 90%. Even MnS₂, a disulfide like FeS₂, but easily acid soluble, yielded mainly elemental sulfur. To explain the differences in end products, another mechanism for dissolution was sought.

Experiments with T. thiooxidans on the bioleaching of ZnS demonstrated, in agreement with published data (5, 11), that a pure culture is able to dissolve ZnS and produce zinc(II) ions and sulfate at an almost constant pH (Fig. 1). It is known that ZnS is soluble in acid. Chemical experiments demonstrated the dissolution of this compound, too; however, elemental sulfur was formed (because sulfur-oxidizing bacteria were not present). Consequently, the pH started to increase and the dissolution of ZnS concomitantly decreased. To explain the mechanism of dissolution, the formation of intermediary polysulfides according to the work of Steudel (22) has to be considered. The mechanism becomes obvious from the following equations. The dissolution of a metal sulfide (MS) is started by proton attack (equation 1) and a consecutive oxidation of H₂S by Fe(III) ions (equation 2).

(1)

(2)

Due to the capability of Fe(III) ions to break metal sulfide bonds more effectively than protons (25, 26), the H₂S^·+ radical may preferentially be formed in one reaction without intermediately occurring H₂S (equation 3).

(3)

Polysulfide formation starts with dissociation of the strong acid H₂S^·+ to a HS^· radical (equation 4).

(4)

Two of the HS^· radicals may react to a disulfide (equation 5).

(5)

The disulfide may react again with Fe(III) ions (equation 6) or with another HS^· radical (equation 7).

(6)

(7)

Tetrasulfide occurs by dimerization of two HS₂^· radicals (analogous to equation 5), or trisulfide occurs by reaction of a HS₂^· with a HS^· radical. Further chain elongation to polysulfides proceeds by analogous reactions.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Leaching of ZnS by T. thiooxidans R20 (16). The strain had been adapted to grow on ZnS, before the experiment was started by addition of 109 cells to 1 g of ZnS (pulverized) in 50 ml of salt solution in shake flasks at 28°C in the dark. Tt, assays with T. thiooxidans; c., sterile control assays. Data are means of three parallel assays. Standard deviations were less than 15%. The experiment was reproduced twice.

In acidic solution, polysulfides decompose to rings of elemental sulfur, mainly S₈ rings (>99%) (equation 8).

(8)

Formation of thiosulfate (polythionates and sulfate) occurs by side reactions (equations 9 and 10).

(9)

(10)

This oxidation mechanism does not necessarily require the presence of Fe(III) ions. An electron transfer from a semiconductive metal sulfide to an O₂ molecule is also possible. The O₂ molecule is reduced via a superoxide radical and a peroxide molecule to water (27). However, Fe(III) ions, usually present in acidic leach biotopes, are much more efficient in extracting electrons from a metal sulfide lattice than is O₂ (25, 26).

The series of reactions 1 to 8 inherently explains the formation of elemental sulfur as the main sulfur compound oxidation product of acid-hydrolyzable metal sulfides. Thiosulfate, and consequently polythionates and sulfate, may also arise by side reactions (equations 9 and 10) (22).

According to this polysulfide mechanism, elemental sulfur is formed. Since sulfur is reasonably stable under experimental and usually also environmental conditions, only in the case of the presence of sulfur-oxidizing bacteria can degradation occur (yielding the protons needed for a further dissolution of ZnS). This mechanism allows for the first time the unequivocal explanation of the ability of T. thiooxidans to leach some metal sulfides, i.e., the ones which are susceptible to hydrolysis by proton attack. Polysulfides were detected on the surface of oxidized chalcopyrite by Hackl et al. (8), confirming this mechanism. The degradation of metal sulfides via polysulfides is summarized in Fig. 2. As a consequence, two indirect oxidation mechanisms for metal sulfides exist.

View larger version (6K): [in this window] [in a new window]   FIG. 2.   Scheme for metal sulfide (MS) oxidation via polysulfides.

One mechanism is exclusively based on the oxidative attack of iron(III) ions on the acid-insoluble metal sulfides FeS₂, MoS₂, and WS₂. Here, the main sulfur intermediate is thiosulfate. The second mechanism allows for a dissolution by an attack of iron(III) ions and/or by protons. In this case, the main sulfur intermediate is polysulfide (and consequently elemental sulfur). The two mechanisms may be simplified by the following equations:

Thiosulfate mechanism (FeS₂, MoS₂, and WS₂) Polysulfide mechanism (e.g., ZnS, CuFeS₂, or PbS) Consequently, bioleaching of metal sulfides means that the bacterial function is to generate sulfuric acid biologically to supply protons for hydrolysis attack and/or to keep the iron ions in an oxidized state [as iron(III) ions] for an oxidative attack (Fig. 3).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Bioleaching proceeds by two different indirect mechanisms via thiosulfate or via polysulfides and sulfur and is based on the properties of metal sulfides (MS). Dashed lines indicate occurrence of intermediate sulfur compounds.

Our conclusions are in agreement with data from the literature. In the absence of iron ions, T. ferrooxidans can solubilize acid-hydrolyzable, synthetic, iron-free sulfides like ZnS, CdS, NiS, CoS, CuS, and Cu₂S (14, 24-26) but not the insoluble sulfides FeS₂ (6), MoS₂, and WS₂ (25, 26). In the case of the former compounds, the leaching rates correlate with their solubility products (24, 26). This finding proves that the solubility product, besides pH, is decisive for the leachability of a metal sulfide in the case of a proton attack. We consequently conclude that in the absence of iron ions T. ferrooxidans acts like T. thiooxidans by acid production (sulfur oxidation). This conclusion is in agreement with the recent finding that the solubilization of Cu²⁺ from a copper ore is determined by the sulfur-oxidizing activity of T. ferrooxidans (23). The addition of iron ions to cultures of T. ferrooxidans growing with hydrolyzable, synthetic, iron-free sulfides generally enhanced leaching rates (25, 26). Because of the additional oxidative iron(III) ion attack, dissolution rates are much higher than those with T. ferrooxidans lacking iron ions or T. thiooxidans alone (5).

Thus, in summary bioleaching is effected by two indirect leaching mechanisms: via thiosulfate or via polysulfides and sulfur. Both mechanisms combine characteristics of the previously differentiated direct and indirect leaching mechanisms. Direct leaching means an attack on the crystal lattice of a metal sulfide through enzymatic oxidation by attached cells (4). This work shows that the mineralogy is also a contributing factor to the degradation pathway. The knowledge of these two mechanisms has implications for biotechnology and environmental problems connected to bioleaching. Sulfur compound-metabolizing enzymes are involved in metal sulfide oxidation. Their regulation or inhibition might influence the balance of sulfur compounds (9). For example, changing the balance from sulfur to sulfate would increase dissolution rates in bioleaching plants for gold recovery (12). Furthermore, cyanide consumption would be reduced (7). On the other hand, the formation of environmentally harmful acid rock drainage (17, 19, 20) might be reduced, if the oxidation could be stopped at the stage of elemental sulfur.