INTRODUCTION

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Thiobacillus ferrooxidans is a gram-negative acidophilic chemolithoautotroph, using CO₂ as a carbon source and obtaining its energy for growth from the oxidation of ferrous iron, sulfur, and reduced sulfur compounds (26). T. ferrooxidans was initially isolated from acidic copper-leaching waters and believed to be the dominant bacterium responsible for metal sulfide solubilization (18). Iron oxidation in response to pH (1, 28), organic acids (40), anions (2, 17), and cations (21, 38) has been extensively studied. Sulfur oxidation has received substantially less attention, with limited references to certain anions (26).

Thiobacilli have considerable economic importance in the treatment of acid mine drainage (10, 22) and desulfurization of waste gases (SO₂ and H₂S) (10, 12, 29). The use of bacteria in the mining industry is a growing field of interest (4, 25). Levels of tolerance of key metals by T. ferrooxidans growth on Fe²⁺ are as follows: Cd²⁺, 0.75 M; Ni²⁺, 1 M; Zn²⁺, 1 M; Cu²⁺, 0.6 M; Co²⁺, 0.15 M; Cr³⁺, 0.075 M; Pb²⁺, 1 mM; Hg⁺, 0.1 mM; Hg²⁺, 10 µM; and Ag⁺, 1 µM (21). The naturally occurring counterion sulfate is not inhibitory at 0.14 M (26, 39), and the concentration may reach as high as 1.25 M during bacterial leaching of sulfide minerals (7).

Metal extraction from mineral ore by T. ferrooxidans is achieved through two reactions: the oxidation of ferrous to ferric iron (2Fe²⁺ + 1/2 O₂ + 2H⁺  2Fe³⁺ + H₂O) and that of sulfide/sulfur to sulfuric acid (H₂S + 2O₂  H₂SO₄ or S⁰ + 1 1/2O₂ + H₂O  H₂SO₄). Uranium solubilization from uraninite, for example, requires only iron oxidation (UO₂ + 2Fe³⁺  2Fe²⁺ + UO₂²⁺, 2Fe²⁺ + 1/2 O₂ + 2H⁺  2Fe³⁺ + H₂O), while zinc solubilization from sphalerite necessitates sulfide oxidation (ZnS + 2O₂  ZnSO₄). Metal extraction becomes complicated when ores contain mineral combinations (19, 27). In a pyrite-sphalerite mixture T. ferrooxidans will oxidize both sulfide (ZnS + 2O₂  ZnSO₄) and iron plus sulfide [4FeS₂ + 15O₂ + 2H₂O  2Fe₂(SO₄)₃ + 2H₂SO₄], creating difficulty in further zinc recovery from the leachate. Low concentrations of ferric sulfate are beneficial in the indirect leaching of mineral ores. Higher concentrations, however, result in the production of jarosite, a ferric iron precipitate which can cover the ore surface, preventing further leaching from occurring. Higher jarosite levels also produce an additional disposal problem.

We propose to show that the iron and sulfur oxidation activities of T. ferrooxidans can be differentially controlled through the use of specific anions and inhibitors. Under certain conditions iron oxidation can be blocked with little to no effect on sulfur oxidation and vice versa. Through this type of manipulation we hoped to achieve specific metal extraction from an ore sample, with the absence or at least reduction of contaminating metals.

	MATERIALS AND METHODS

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Media. T. ferrooxidans strain SM-4 (20) was grown in modified 9K medium (M9K): 0.4 g of (NH₄)₂SO₄, 0.1 g of K₂HPO₄, 0.4 g of MgSO₄ · 7H₂O, and 33.3 g of FeSO₄ · 7H₂O per liter, adjusted to pH 2.3 with H₂SO₄. Cells used for sulfur oxidation were grown in Starkey no. 1 medium (33) after adaptation on sulfur (37): 0.3 g of (NH₄)₂SO₄, 3.5 g of KH₂PO₄, 0.5 g of MgSO₄ · 7H₂O, 0.25 g of CaCl₂, and 18 mg of FeSO₄ · 7H₂O per liter, adjusted to pH 2.3 with H₂SO₄. Powdered sulfur (10 g of BDH sulfur per liter) was spread evenly over the surface after inoculation. Thiobacillus thiooxidans strain SM-6 grown on sulfur was used for most of the growth experiments on sulfur, since sulfur-adapted T. ferrooxidans was not available. The results of key experiments, however, were later confirmed with sulfur-adapted T. ferrooxidans strain SM-4.

Culture procedures. Iron-grown cells were cultured in M9K using a 10% inoculum. The flasks were incubated at 25°C and placed on a rotary shaker at 150 rpm for 48 h. The culture was passed through Whatman no. 1 filter paper to remove the majority of the precipitated ferric iron. The supernatant was centrifuged at 8,000 × g for 10 min. The cell pellet was resuspended in 0.1 M -alanine sulfate buffer (pH 2.3) and centrifuged at 1,000 × g for 5 min to allow further ferric iron sedimentation. The supernatant was transferred to a secondary tube and centrifuged at 10,000 × g for 10 min. The cells were centrifuged a fourth time, generating a final suspension of 50 mg of cells (wet weight) per ml in the same buffer. The protein concentration was determined using bovine serum albumin as the standard (37).

Determination of iron and sulfur oxidation using cell suspensions. The rates of iron and sulfur oxidation were measured using a Gilson oxygraph equipped with a Clark oxygen electrode at 25°C. The reaction vessel contained 10 µl of cell suspension (sulfur- or iron-grown cells), 0.1 ml of sulfur suspension (32 g of BDH S⁰ in 100 ml, plus 500 ppm of Tween 80) (for sulfur oxidation) or 0.5 µmol of FeSO₄ · 7H₂O (for iron oxidation), and various concentrations of potassium salts of anions, sucrose, or -alanine buffer (all at pH 3.0 unless otherwise stated) to make a total volume of 1.2 ml. The effect of azide and cyanide was studied in 0.1 M -alanine sulfate at pH 3. For sulfur oxidation by iron-grown cells, however, 100 µl of the cell suspension was required for accurate rate determinations. All tests whose results are shown in Fig. 1 were performed at pH 3 rather than pH 2.3 (growth pH) because of the lower sulfur oxidation activity at pH 2.3. Duplicate experiments using the same batch of cells were impossible to carry out for all of the conditions tested due to the instability of activity after more than 2 days of storage. Results, however, were reproducible with other batches of cells, although absolute activities varied from 10 to 20%. Standard deviations in the activity determinations fell within 10% of the stated values except for sulfur oxidation by iron-grown cells, where the values could deviate by as much as 20%. It should be noted that all of the experiments testing iron and sulfur oxidation were repeated with T. ferrooxidans ATCC 19859, with similar results.

Determination of iron and sulfur oxidation and carbon dioxide fixation using growing cell cultures. The rates of iron and sulfur oxidation in growing cell cultures were measured using a Micro-oxymax respirometer (Columbus Instruments) at Cominco Research Limited, Trail, British Columbia, Canada. The reaction vessel contained a 5% inoculum, 12 mmol of FeSO₄ · 7H₂O (for iron oxidation) or 1 g of BDH sulfur sprinkled on the surface plus 18 mg of FeSO₄ · 7H₂O per liter (for sulfur oxidation), various concentrations of anionic salts or inhibitors, and M9K at pH 2.3, making a total volume of 100 ml. The reaction was stirred with a magnetic stirrer, and both O₂ consumption (oxidation) and CO₂ consumption (autotrophic growth) were measured at 26°C.

Shake flask leaching of metals. Flotation tailings, provided by Cominco Research Limited, contained 3.3% Zn as sphalerite and 5.5% Fe as pyrite (FeS₂) and were in the form of a finely ground powder. Five grams of tailings was placed in a 250-ml Erlenmeyer flask with 100 ml of M9K at pH 2.3 with or without additional potassium phosphate. The flask was inoculated with 5 ml of T. ferrooxidans SM-4 (grown on FeSO₄) and left stationary at 25°C for 24 h, followed by shaking on rotary shaker at 180 rpm for the remainder of the experiment. Five-milliliter samples were taken at time zero, 2 and 14 days, filtered through Whatman no. 1 filter paper, and analyzed for dissolved Fe and Zn content by atomic absorption spectrophotometry. The extent of metal leaching was calculated as percent extraction from the total metal content of the tailings.

	RESULTS AND DISCUSSION

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Effect of anions on iron and sulfur oxidation. Cell suspensions were initially used to determine the effects of anions and selective inhibitors on the iron and sulfur oxidation activities of T. ferrooxidans. The reaction in each case was carried out at pH 3, an acidic pH comparable to natural growing conditions. The specific activities of iron (Fig. 1A) and sulfur (Fig. 1B and C) oxidation in buffer concentrations ranging from 0.01 to 0.5 M were determined. Enzymes required for iron and sulfur oxidation are differentially expressed depending on the bacterial growth substrate (14, 23, 37). Cells grown on ferrous iron showed only low levels of sulfur-oxidizing activities (Fig. 1B) compared to sulfur-grown cells (Fig. 1C). Although not shown in Fig. 1, iron oxidation experiments were also carried out at pHs 2.3 and 1.8, and they showed a stronger inhibition by the potassium salts. Sucrose was not inhibitory at all pH values, while -alanine was less inhibitory than the potassium salts at lower pH values.

View larger version (52K): [in this window] [in a new window]   FIG. 1.   Effects of concentrations of various anions, sucrose, and -alanine on the oxidation of ferrous iron and sulfur in iron-grown T. ferrooxidans strain SM-4 and of sulfur in sulfur-grown T. ferrooxidans strain SM-4. Please note the different scales on the y axes.

Effects of azide and cyanide on iron and sulfur oxidation. The sulfur and iron oxidation pathways and their interactions are surrounded by a great deal of controversy. Sugio et al. suggest that ferric iron reduction is coupled to sulfur oxidation under both aerobic and anaerobic conditions (34, 35). An alternate theory proposed by Corbett and Ingledew suggests that the iron and sulfur oxidation pathways are two separate entities but that ferric iron can replace oxygen as a terminal electron acceptor under anaerobic conditions (5). Iron- and sulfur-dependent oxygen uptake occurs via two separate oxidases (24). Azide at low concentrations is a specific inhibitor of the terminal oxidase of ferrous iron oxidation, but not that of sulfur oxidation (24). The results in Table 1 agree with the concept of two terminal oxidases being differentially inhibited by azide. Inhibition of iron oxidation by 50% required only 0.7 µM azide, while that of sulfur oxidation required 19 to 32 µM azide. Cyanide is also a specific inhibitor of the terminal oxidase. It behaved in a manner similar to that of azide, preferentially inhibiting iron oxidation. As shown in Table 1, only 3.2 µM cyanide was required to cut the iron oxidation in half, while 77 to 323 µM was necessary to produce a similar effect on sulfur oxidation.

Effects of anions and inhibitors on cell growth. The second part of this paper deals with the use of the above-mentioned anions and inhibitors on growing cell cultures. Cells growing on single substrates were monitored in a Micro-oxymax respirometer. Iron oxidation and sulfur oxidation were measured in terms of cumulative oxygen consumption. Since we did not have sulfur-grown T. ferrooxidans SM-4 available for these growth experiments, we used sulfur-grown T. thiooxidans SM-6 instead for experiments on sulfur. Important findings were later confirmed with sulfur-adapted T. ferrooxidans. Figure 2 shows the effects of increasing concentrations of phosphate on iron and sulfur oxidation by growing cells. The control in each case contained a 5% inoculum along with ferrous sulfate or precipitated sulfur as the substrate. Cumulative oxygen consumption was plotted as a function of time for each of the reaction vessels. Iron oxidation (Fig. 2A) was decreased by half in the presence of 50 mM phosphate. Sulfur oxidation (Fig. 2B), on the other hand, increased to twice the control level in the presence of 50 mM phosphate. Similar results were obtained with 50 mM chloride, which also inhibited iron oxidation and stimulated sulfur oxidation, although slightly less extensively (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Effect of phosphate concentration on oxygen consumption. (A) Growth of T. ferrooxidans on Fe2+; (B) growth of T. thiooxidans on S0.

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Effect of phosphate concentration on carbon dioxide consumption. (A) Growth of T. ferrooxidans on Fe2+; (B) growth of T. thiooxidans on S0.

Effect of anions on metal leaching. Leaching of Fe and Zn from pyrite and sphalerite by T. ferrooxidans was differentially affected by phosphate concentrations, as shown in Fig. 4. Phosphate at or above 25 mM inhibited the solubilization of Fe completely while allowing the zinc solubilization to proceed with only some rate reduction. The effect at 75 and 100 mM was essentially the same as that at 50 mM. Although not shown in Fig. 4, KCl at 50 mM inhibited Fe solubilization by 84 and 35% after 2 and 14 days, respectively, and did not inhibit Zn solubilization at all. KNO₃ also inhibited the leaching of Fe more strongly than that of Zn, inhibiting Fe solubilization by 86% and Zn solubilization by only 16% at 50 mM after 14 days. The effect of NaN₃ and NaCN at 0.1 to 5 µM was not apparent after 14 days because of their volatility at pH 2.3 as HN₃ and HCN escaping from the flasks through the cotton plugs. After 2 days, however, 1 µM NaN₃ and 5 µM NaCN inhibited Fe solubilization by 65% without significantly affecting Zn solubilization (14 and 25% inhibition for NaCN and NaN₃, respectively). These preliminary leaching experiments support the concept of differential leaching of Fe and Zn by inhibiting iron oxidation but not sulfur oxidation, thus favoring the solubilization of Zn from ZnS over the solubilization of Fe from FeS₂. Detailed studies (L. Harahuc, H. M. Lizama, and I. Suzuki, submitted for publication; I. Suzuki and L. Harahuc, June 1998, Canadian Patent Office) of selective solubilization of metals from sulfide ores by this method support its potential application in bacterial leaching.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Effect of phosphate concentration on the extraction of Fe and Zn from a mixture of pyrite and sphalerite.