Microbiological Leaching of Metallic Sulfides

Microbiological Leaching of Metallic Sulfides W. E. RAZZELL1 AND P. C. TRUSSELL British Columibia Resear ch Council, Vancouver, British Colunbia, Canada ABSTRACT RAZZELL, W. E. (British Columbia Research Council, Vancouver, British Columbia, Canada) AND P. C. TRUS- SELL. Microbiological leaching of metallic sulfides. Appl. Microbiol. 11:105-110. 1963.-The percentage of chalcopyrite leached in percolators by Thiobacillus ferrooxidans was dependent on the surface area of the ore but not on the amount. Typical examples of ore leaching, which demonstrate the role of the bacteria, are presented. In stationary fermentations, changes in KH2PO4 concentration above or below 0.1 %O decreased copper leaching as did reduction in the MgSO4 7H20 and increase in the (NH4)2SO4 concentration. Bacterial leaching of chalcopyrite was more effective than nonbiological leaching with ferric sulfate; ferric sulfate appeared to retard biological leaching, but this effect was likely caused by formation of an insoluble copper-iron complex. Ferrous sulfate and sodium chloride singly accentuated both bacterial and nonbiological leaching of chalcocite but jointly depressed bacterial action. Sodium chloride appeared to block bacterial iron oxidation without interfering with sulfide oxidation. Bacterial leaching of millerite, bornite, and chalcocite was greatest at ph 2.5. The economics of leaching a number of British Columbia ore bodies was discussed. The ability of acidophilic bacteria to accelerate the solution of copper from metallic sulfides in industrial copper-leaching operations has been well documented (Bryner et al., 1954; Bryner and Anderson, 1957). Bryner and Jameson (1958) reported the isolation of discrete strains of bacteria from operations in Utah and Mexico which were capable of oxidizing pyrite, chalcopyrite, and, to some extent, molybdenite. However, their data reveal a higher rate of solution of iron than of copper from chalcopyrite, which is not consistent with the formula for the substrate (CuFeS2) given in previous data (Bryner et al., 1954) or with our results recorded below. We recently discussed the properties of similar bacteria found in acidic British Columbia mine waters (Razzell and Trussell, 1963) and report here on a number of aspects of the microbiological leaching process which have not been mentioned previously. Most of these studies have been performed with chalcopyrite, since this mineral is 1 Present address: Syntex Institute for Molecular Biology, Palo Alto, Calif. Received for publication 7 September 1962 most difficult to leach biologically and is chemically more stable under acid conditions than other metallic sulfides. MATERIALS AND METHODS Inorganic salts solution. Throughout most of this work the solution of Silverman -and Lundgren (1959) for growing cells on iron was employed. Exceptions are noted. M1inerals. All metallic sulfides were obtained from Ward's Natural Science Establishment, Inc., Rochester, N.Y. With the exception of millerite, which, in addition to nickel (31.5 %) and sulfur (24 %), contained I I % Fe and 7.1 % Cu, analyses corresponded to within a few percentages of the theoretical values. The minerals were crushed, ground, and graded by passing through standard sieves. Percolators and stationary cultures. Percolators similar to those described by Bryner et al. (1954) were loaded with a mixture of 200 g of acid-washed sand, 2.5 to 10 g of mineral, and 250 ml of solution, or with coarsely crushed ore and 100 ml of solution. Sterile air saturated with water was used to circulate the solutions. Stationary cultures, consisting of 50 ml of salts solution and 0.5 g of mineral, were set up in 300-ml screw-capped bottles. These were incubated in a flat position to provide the maximal exposure of the liquid to air and of mineral to liquid. Inoculum. Apart from preliminary experiments with Thiobacillus ferrooxidans, isolated as previously described (Razzell and Trussell, 1963) and grown on silicic acid- FeSO4 plates, most experiments were performed with T. ferrooxidans grown in liquid FeSO4 cultures (Silverman and Lundgren, 1959) at an initial ph of 1.6 (Razzell and Trussell, 1963), washed, and resuspended with dilute inorganic salts solution at ph 2.5. Growth of cells at ph 1.6 yields suspensions free of iron salts. Preliminary tests on chalcopyrite with T. thiooxidans and Candida sp. isolated from natural leaching operations, with or without T. ferrooxidans, showed that they did not contribute to, or detract from, leaching. - RESULTS Effect of size and quantity of ore particles on rate of biological leaching. In agreement with previous studies (Temple and Delchamps, 1953; Bryner and Anderson, 1957), copper was released more rapidly from fine particles than from coarse ones (Table 1). About three times as 105

106 RAZZELL AND TRUSSELL APPL. MICROBIOL. much copper was leached frorn particles of less than 32 mesh in 7 days as from particles of 70 to 140 mesh in 58 days. Although industrial leaching usually would not be conducted on finely ground ore, finely ground material was found advantageous in accelerating laboratory experiments. The percentage of copper leached was independent of the quanitity of mineral in the percolator over the range of 2 to 10 g (Table 2). In 44 days, 12 to 14 %c of the copper in the chalcopyrite was released irrespective of the amount of chalcopyrite present. For routine experiments, 10 g of mineral of - 325 mesh were most commonly used in percolators and 0.5 g was used in stationary cultures. Microbiological leaching of chalcopyrite ores by percolation. The leachability of ore samples was determined by placing 300 g of ore (-25 +40 mesh, that is, 0.25 to 0.37 in. diam) in a percolator with 50 ml of inorganic salts solution and 50 ml of water and constantly circulating the solution. About 1,400 ppm of Cu were released in 20 days from high pyrite ore compared with 100 to 200 ppm in the sterile control (Table 3). In the presence of high pyrite, the ph tended to drop, probably to the point which depressed copper leaching. On the other hand, the low ph kept the iron in solution and high iron concenitrations resulted. All iron in the inoculated percolator was in the ferric state. Controls to show the extent of chemical leaching were important for future work with ores containing both metallic sulfides and copper oxides, since oxides are readily TABLE 1. Effect of particle size of chalcopyrite on rate of leaching in percolators Copper concn (ppm) Time 70-140 140-200 -200-325 +105)* (-105 +74) (-74) (-44) days 7-29 160 180 13-64 414 420 21 44 114 476 680 33-141 540 1,180 46 51 - - 58 66 - - * Mesh size of screens (particle diamn in,u). TABI,E 2. Effect of qutantity of chalcopyrite on rate and extent of leaching in percolators Time Copper conon (ppm) Chalcopyrite (g) 2 4 6 8 10 days 20 152 462 615 885 680 28 350 624 1,012 1,206 1,000 36 414 634-1,402 1,640 44 423 841 1,530 1,860 Per cent Cu released 14 14-12 13 soluble under the acid conditions created by, and necessary for, bacterial action. Table 4 shows the results obtained with a coarse chalcopyrite ore low in pyrite which required periodic additions of acid to maintain the ph at 2.5 and to promote bacterial leaching. Here, the soluble iron concentration, particularly at ph levels above 3, was low. Effect of inorganic ions on biological leaching of chalco- TABLE 3. Day no. 0 5 20 31 34 56 66 84 85 88 91 98 Comparison of spontaneous and microbiological leaching of coarse chalcopyrite ore rich in pyrite Time between draining* days 3 22 10 28 1 4 7 7 Cu+t ppm 65 165 107 250 Without cells, plus Hg++ Fe-++ Total ph Cu++ Fe ppm ppnm Ppm <10 <10 2.5 108 <10 <10 2.6 900 <50 190 3.3 1,390 35 280 3.4 1,350 540 1,225 210 800 96 148 195 l 350 With cells Fe+++ ppm 10 10 3,200 6,200 3,250 7,500 2,150 8,400 840 1,880 3,700 3,800 ph 2.5 3.2t 2.5 2.3 1.9 1.8 1.8 1.7 2.2t 2.1 1.9 1.9 * Percolator containing cells drained and refilled with halfstrength salts solution (ph 2.5 on dayrs 31, 56, 84, and 91). t Adjusted to ph 2.5. TABLE 4. Mlicrobiological leaching of coarse chalcopyrite ore low in pyrite Day no. Time between Cu++ Fe+++ p1h draining* da3s ppm ppmt 10 3.7t 20 3.4t 29 1,240 5 3.7 30 1 425 0 2.5 34 4 840 10 3.2t 40 10 1,900 610 2.9 41 1 380 127 2.5 47 6 600 130 3.lt 72 31 1,400 30 3.7t 74 33 1,400 360 2.7 89 15 430 10 3.5t 99 25 1,900 600 3.0 100 1 530 280 2.9 121 22 1,580 10 3.6 131 10 300 0 3.6 149 28 500 10 3.2 160 39 520 10 3.7 171 50 640 10 3.6t 181 10 800 640 2.6 188 17 1,100 1,200 2.6 * Percolator drained and refilled with half-strength salt solution (ph 2.5 on days 29, 30, 40, 41, 74, 99, and 171; on day 121, refilled with salt solution, ph 3.6). t Adjusted to ph 2.5.

VOL. 11, 1963 LEACHING OF METALLIC SULFIDES 107 pyrite. Percolators are suitable for studies with coarse ores, but stationary fermentations are more efficient and reliable for leaching finely ground minerals. The stationary fermentations require less mineral and produce more rapid and complete leaching of copper than do percolators (Razzell and Trussell, 1963). Attempts to conduct leaching in shaken flasks have been consistently unsuccessful. It is not certain whether this was because the bacteria could not maintain contact with the mineral surfaces, or because they were killed by the grinding action of the mineral particles. The effect of varying the concentrations of the components of the inorganic salts solution on chalcopyrite leaching in stationary culture is shown in Table 5. Since ammonia from the atmosphere is taken up readily by solutions at ph 2.5, an ammonium-ion requirement is difficult to demonstrate and was not attempted. In Table 5, "soluble" and "total" Cu++ and Fe+++ are indicated; the former indicates the concentration of these ions in solution, the latter, the concentration in solution after treating a stirred sample, containing liquid and suspended solids, with 6 N HCl. Apparently some copper and iron, after release from chalcopyrite, recombine into a fraction which is insoluble in weak H2SO4 but soluble in HCI (Razzell and Trussell, 1963). Under the conditions of the HCl treatment, the chalcopyrite itself is not affected. Low phosphate decreased the rate of copper and iron release from chalcopyrite; high phosphate either inhibited the release or precipitated the ions as insoluble phosphates. Reducing the MgSO4-7H20 concentration from 0.1 to 0.01 % and increasing the (NH4)2SO4 from 0.1 to 0.5 7 depressed copper leaching. Effect of iron sulfate and sodium chloride on bacterial leaching of chalcopyrite and chalcocite. Table 6 illustrates the effect of ferric sulfate and bacteria, separately and together, on leaching chalcopyrite in stationary cultures. The same results have been obtained using ferrous sulfate additions in place of ferric sulfate. In the absence of cells, most of the soluble iron was found in the reduced state, the concentration of Cu increased with the quantity of added Fe, and only a portion of the added (and released) iron remained in solution. On the other hand, in the presence of cells, most of the iron was found in the oxidized form, and the amount of Cu released appeared to drop TABLE 5. Effect of concentration of inorganic ions on microbiological leaching of chalcopyrite (225 days) in stationary culture No. (NH4)2 KH,PO4 MgSO4 CaC12 NaCl SO4 7H20 Soluble Total Cull Fe+++ Cull Fe+++ % 70^% % % ppm ppm ppm ppm 1 0.1 0.005 0.1 0.01 0.1 936 90 1,310 1,020 2 0.1 0.1 0.1 0.01 0.1 1,250 886 2,740 2,120 3 0.1 0.1 0.01 0.01 0.1 990 886 2,150 1,920 4 0.5 0.1 0.01 0.01 0.1 900 547 1,620 1,440 5 0.1 0.5 0.01 0.01 0.1 490 <30 600 510 as the iron additions increased. As in the absence of cells, the percentage of soluble Fe dropped as the added iron increased, particularly between 57 and 90 days. Bacteria are more effective than the ferric ion for leaching chalcopyrite, and iron, when present with bacteria, tends to suppress the yield of soluble copper (Table 6). The reason for this is probably the same as mentioned above; much of the copper which is released from the chalcopyrite is bound by iron into an insoluble form. The amounts of iron (all Fe+++) in solution at 90 days, when the copper levels are 1,000 to 1,600 ppm, should be 1,000 to 1,600 ppm plus the amounts added; this is clearly not the case. Unfortunately, this explanation was not appreciated at the time of these experiments and determination of "total" copper and iron by hydrochloric acid treatment was not done. Table 7 shows the effect of ferrous sulfate, sodium chloride, and bacteria on the leaching of chalcocite (Cu2S). Although this mineral has the advantage of leaching rapidly under the influence of bacteria, it is much less stable to acid than chalcopyrite. In the absence of cells, both ferrous iron and sodium chloride, alone and in combination, increased the amount of soluble copper by three TABLE 6. Effect of ferric sullfate and bacteria on leaching chalcopyrite in stationary cutlture Addition Amt at 23 dayst Amt at 57 days Amt at 90 days Fe... Cells* Cut+ Fe++ Fe... Cu"+ Fe++ Fe+++ Cut+ Fe++ Fe++t ppmn ppm ppmt pptm pp,n ppm ppm ppm ppm ppnm _ - 93 0 10 220 0 11 290 - - 500-440 350 50 640 226 170 740 420 100 1,000-430 670 50 740 472 283 800 920 120 2,000-6001,315 125 1,050 750 680 1,100 630 220 _ --+ 1,000 0 245 1,350 0 530 1,560-600 500 + 660 60 700 825 0 752 1,300 0 480 1,000 + 480 0 1,080 800 0 1,21011,600 0 600 2,000 + 520 0 1,560 700 0 1,60011,050 0 700 * To bottles containing no added cells, 100 ppmi of Hg(NO3)2 were added. t All determinations were for soluble copper and iron contents. TABLE 7. Effect of ferrous sulfate, sodiunm chloride and cells on the leaching of copper from chalcocite (CU2S) Addition Amt at 3 days Amt at 6 days Fe"+ NaCl Cells Cu++ Fe++ Fe... Cu+' Fe++ Fe+++ ppm % ppm ppm ppm ppm ppm ppm 0 0-360 480 1,000 0-700 975 25 1,260 845 115 0 1.2-1,200 1,500 1,000 1.2-1,100 950 40 1,400 580 260 0 0 + 760 1,360 1,000 0 + 1,400 200 300 2,900 0 200 0 1.2 + 1,120 2,850 1,000 1.2 + 1,120 900 45 1,750 600 240

108 RAZZELL AND TRUSSELL APPL. MICROBIOL. times over that in the control flasks at 6 days. Bacteria alone increased the soluble copper concentration about three times and, in the presence of ferrous iron and sodium chloride separately, increased the soluble copper about six times. Thus, the soluble copper concentration at the end of 6 days was of the order of 3,000 ppm. Much of the added iron was converted into an insoluble form in the presence of bacteria. Copper, liberated from the Cu2S, probably combined with the Fe+++ produced by the bacteria, and an insoluble Fe-Cu precipitate formed, just as in chalcopyrite leaching. Preliminary tests with sodium chloride had shown that chalcopyrite leaching will occur under conditions which block iron oxidation (Razzell and Trussell, 1963). This effect of sodium chloride seems to apply also to chalcocite leaching here, in which case the iron remains in solution as Fe++. In addition, both FeSO4 and NaCl exert some nonspecific salting effect, which increases the rate of spontaneous leaching, and neither alone interferes with bacterial leaching. Effect of acidity on leaching of copper and nickel. The optimal ph for the leaching of Cu from chalcopyrite has been found to be near 2.3 (Razzell and Trussell, 1963). The effect of ph on the bacterial leaching of millerite (impure), bornite, and chalcocite is shown in Table 8. In all cases, leaching, both spontaneous and biological, was more rapid at ph 2.5 than at 4.0. Although bacterial leaching of Ni from millerite was greater than spontaneous leaching at 5 and 12 days, no difference was found after 19 days. With bornite and chalcocite, bacterial leaching was far more rapid than spontaneous leaching. For practical purposes, bacterial leaching of most sulfides with the particular organism used here is best done at a low ph. The leaching of copper from chalcopyrite ores from natural deposits in British Columbia is shown in Table 9. During leaching of these ores, the ph was held at 2.5 and the quantity of H2SO4 used is expressed as per cent by weight of the ore. The relationship between the percentage of acid required, copper in the ore, and the profit realizable when copper sells for 32 cents per lb is shown in Fig. 1. All samples in Table 9 were economical for leaching on the basis of acid requirement. Rocher Deboule had the highest rate of bacterial and spontaneous leaching of all ore samples. Britannia, which leached well biologically, showed low spontaneous leaching. East Jersey and Sunro were about equivalent to Britannia TABLE 8. Effect of ph on the microbiological leaching of copper and nickel from minerals* Amt at 5 days Amt at 12 days Amt at 19 days Mineral ph Bacteria - Cu++ Ni'+ Cu++ Ni++ Fe+++ Cu++ NiP+ Fe+++ Millerite 2.5-55 476 155 770 260 260 1,310 400 2.5 + 95 616 240 1,270 420 300 1,440 520 4.0 - <10 153 <10 270 <20 6 403 4.0 + <10 231 <10 432 < 20 106 576 Bornite 2.5-355 1,740 20 3,200 130 2.5 + 860 2,400 220 5,400 525 4.0-17 140 <20 180 4.0 + 45 210 <20 410 Chalcocite 2.5-660 2,300 2,550 2.5 + 960 3,000 3,400 4.0-75 205 205 4.0 + 108 220 260 * All minerals ground to -325 mesh. Acidity adjusted daily to indicated ph. Millerite was contaminated with chalcopyrite Cu:Ni = 1:23. All recovered iron was in ferric state. Results expressed in ppm. Ore TABLE 9. Bacterial leaching of chalcopyrite ores of different alkalinities CU++ (ppm) Fe!'! (ppm) Per cent H2SO4* 45 100 137 230 230 230 137 230 230 days days days days dayst dayst days days dayst Rocher Deboule 0.30 490 2,700 3,700 4,800 4,800 2,400 355 670 1,000 Britannia 0.18 190 283 735 1,000 1,400 37 447 658 750 East Jersey 2.90 110 890 960 1,000 1,150 425 22 0 425 Sunro 0.37 130 380 677 920 920 155 220 274 1,260 Chalcopyrite 0.0 410 940 1,120 1,610 2,010 402 580 1,710 * Per cent by weight of H2SO4 to ore, to maintain ph 2.5 during leaching. t Soluble Cu and Fe after treatment with 4 N HCl. t Sterile control.

VOL. 11l,1963 LEACHING OF METALLIC SULFIDES 109 50 20 within 10 days after an initial 30-day leaching of some soluble copper oxides. The most critical factor in leaching, which governs the t \\\\\\ success %COPPER IN ORE and economic usefulness of the process, is the % COPPER IN OE \alkalinity of the gangue about the mineral particles, since bacterial action will not proceed appreciably above ph 3. \\ N \ \ \ Furthermore, alkaline gangues favor surface precipitation of iron, which carries down some copper from solution \ 7 and may further slow leaching by presenting a physical barrier preventing access of bacteria to the mineral surface. Even an ore high in pyrite may thus be rendered difficult to leach unless ground extremely fine and would probably 05 \ \ \ f be uneconomical if coarse. Measures designed to overcome the loss of copper through precipitation must be balanced by a consideration of their effects on the bacteria. Although.4 oxidation of metallic sulfides is independent of iron oxidai--.- --s- - - tion and, therefore, will proceed in 1% chloride, a concentration (1.5 N) of hydrochloric acid sufficient to dissolve 0 2 03 the iron-copper precipitates will kill the bacteria outright because the ph is too low (less than zero). However, a good rinse after such acid treatment would dilute the acid, and fresh bacteria could be introduced. For example, I I I I I I dumps of leaching waste could be treated in discrete sec- 2 3 4 5 6 tions at monthly intervals, and the effluent from an un- PERCENT ACID REQUIRED treated section (adjusted to ph 2 with H2SO4 if necessary) FIG. 1. Relation among copper in ore, amount of acid required, could be used as a source of bacteria for a treated, rinsed andi profit. Profit, as per cent of the market value of copper recovered section. by conventional procedures of precipitation on detinned scrap iron, Results with Rocher Deboule ore, which contains chala function of (i) the alkalinity of the rock being leached and (ii) copyrite as the principal mineral, suggest that an accelera- as ( the copper content of the rock. Assumed copper price = S2 cents per lb. tion of leaching may result from as yet undefined components, since this ore leached more rapidly than pure in leaching rate, but seemed to have less copper bound chalcopyrite. The effects of salts on chalcocite leaching witth iron into an insoluble form after initial leaching. might be taken to suggest that Rocher Deboule ore has a Allthough Britannia, East Jersey, and Sunro were of rela- salt mixture which accelerates its leach rate; on the other tively low Cu content, their leaching rates correspond hand, the samples we obtained may not have been repre- well with that of a sample of chalcopyrite which had sentative of the chalcopyrite ore body, since no mineral- veiry ess,entially no gangue contamination. ogical examination of these particular pieces was made. Other ores, too high in alkalinity to be leached economi- DIscUSSION cally for copper alone even when bacterial sulfur oxidation serves as a source rhe most useful general procedures for examining fac- of acid, may be leached if nickel sulfides are present, tor s afectng eacing pper t copris although no procedure is yet available which sttioarywill permit the separate recovery of copper and nickel culitures, containing 0.5 g of mineral of less than 325 mesh. 050 l ofinogani sats slutin iocultedwith150 5 l i aw150 from the leach liquor without a loss of acidity. in Mg It should be noted that we have found these, or similar, of cell N in cells grown on FeSO4 at ph 1.6, or by means itosholdi be ted ta eah f si milar, percolators containing about 250 g of ore in 0.25-in,leron-oxidizing bacteria i each of six natural commercial of cutbes, and incubated at 37 C. Favorable conditions yield leach operations (Razzell and Trussell, 1963), and we are results in less than 4 weeks. confident that all such operations contain the bacteria; Percolators may be scaled up or down with little effect it is their natural habitat and they are very successful in it. on the final percentage of copper recovered from the ore, LITERATURE CITED provided the particle size is constant. For example, we obtained good results with a percolator (5 in. diam, 30 in. high), using 600 ml of a 1:1 mixture of inorganic salts and water and 4 kg of chalcopyrite ore (-1 to +0.5 in.); large volumes of leach solution were available for tests BRYNER, L. C., AND R. ANDERSON. 1957. Microorganisms in leaching sulfide minerals. Ind. Eng. Chem. 49:1721-1724. BRYNER, L. C., J. V. BECK, D. B. DAVIS, AND D. G. WILSON. 1954. Microorganisms in leaching sulfide minerals. Ind. Eng. Chem. 46:2587-2592.

110 RAZZELL AND TRUSSELL APPL. MICROBIOL. BRYNER, L. C., AND A. K. JAMESON. 1958. Microorganisms in leaching sulfide minerals. Appl. Microbiol. 6:281-287. RAZZELL, W. E., AND P. C. TRUSSELL. 1963. Isolation and properties of an iron-oxidizing Thiobacillus. J. Bacteriol. 85: 595-603. SILVERMAN, M. P., AND D. G. LUNDGREN. 1959. Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans. I. An improved medium and a harvesting procedure for securing high cell yields. J. Bacteriol. 77:642-647. TEMPLE, K. L., AND E. W. DELCHAMPS. 1953. Autotrophic bacteria and the formation of acid in bituminous coal mines. Appl. Microbiol. 1:225-258.