APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/98/$04.0010

Apr. 1998, p. 1237–1241 Vol. 64, No. 4

Copyright © 1998, American Society for Microbiology

Development of a Laboratory-Scale Leaching Plant for MetalExtraction from Fly Ash by Thiobacillus Strains

CHRISTOPH BROMBACHER,1 REINHARD BACHOFEN,1 AND HELMUT BRANDL2*

Institute of Plant Biology, Department of Microbiology, University of Zurich, CH-8008 Zurich,1 andInstitute of Environmental Sciences, University of Zurich, CH-8057 Zurich,2 Switzerland

Received 23 September 1997/Accepted 5 January 1998

Semicontinuous biohydrometallurgical processing of fly ash from municipal waste incineration was per-formed in a laboratory-scale leaching plant (LSLP) by using a mixed culture of Thiobacillus thiooxidans andThiobacillus ferrooxidans. The LSLP consisted of three serially connected reaction vessels, reservoirs for a flyash suspension and a bacterial stock culture, and a vacuum filter unit. The LSLP was operated with an ashconcentration of 50 g liter21, and the mean residence time was 6 days (2 days in each reaction vessel). Theleaching efficiencies (expressed as percentages of the amounts applied) obtained for the economically mostinteresting metal, Zn, were up to 81%, and the leaching efficiencies for Al were up to 52%. Highly toxic Cd wascompletely solubilized (100%), and the leaching efficiencies for Cu, Ni, and Cr were 89, 64, and 12%, respec-tively. The role of T. ferrooxidans in metal mobilization was examined in a series of shake flask experiments.The release of copper present in the fly ash as chalcocite (Cu2S) or cuprite (Cu2O) was dependent on themetabolic activity of T. ferrooxidans, whereas other metals, such as Al, Cd, Cr, Ni, and Zn, were solubilized bybiotically formed sulfuric acid. Chemical leaching with 5 N H2SO4 resulted in significantly increased solubi-lization only for Zn. The LSLP developed in this study is a promising first step toward a pilot plant with a highcapacity to detoxify fly ash for reuse for construction purposes and economical recovery of valuable metals.

Biohydrometallurgy, an interdisciplinary field involvinggeomicrobiology, microbial ecology, microbial biochemistry,and hydrometallurgy (23), is a novel promising technology forrecovering valuable metals from industrial waste materials(e.g., bottom and fly ash, galvanic sludge, and filter dust) andfor detoxifying these materials for environmentally safe depo-sition. Biohydrometallurgical processing of solid waste allowsrecycling of metals, similar to natural biogeochemical metalcycles, and diminishes the demand for resources, such as ores,energy, and landfill space. Fly ash from municipal waste incin-eration (MWI) is a concentrate containing a wide variety oftoxic heavy metals (e.g., Cd, Cr, Cu, and Ni). The zinc concen-trations in fly ash (3%, wt/wt) can be similar to the concentra-tions in ores subjected to conventional mining (16), whichmakes MWI fly ash a suitable candidate for economical zincrecovery. The low acute and chronic toxicity of fly or bottomash for a variety of microorganisms (8) and the low mutageniceffect (17) of this material have been demonstrated previously.However, the deposition of materials containing heavy metalsresults in a severe risk that spontaneous metal leaching mayoccur due to natural weathering processes and uncontrolledbacterial activities (18, 21, 23). Agenda 21 adopted at the 1992Earth Summit in Rio de Janeiro, Brazil, established that thereis a strong requirement to support sustainable development,including ecological treatment of wastes and safe disposal ofwastes. Biological metal leaching of fly ash is a step in thisdirection.

Biohydrometallurgy is a technology that is cleaner and con-sumes less energy than technologies used in the pyro- andhydrometallurgical industries. The latter technologies are well-established, and many of them are patented, whereas patentsfor biohydrometallurgical processing of industrial wastes are

rarely published (7). The first effort to develop biohydromet-allurgical treatment of industrial waste was made 20 years ago,and greater efforts are necessary now. This is an importantsubject of research and should result in a wide range of inves-tigations and applications in the future. However, the previousdata on biohydrometallurgical treatment of fly ash or otherindustrial waste obtained with bacteria or fungi included resi-dence times for leaching of up to 50 days (4–6, 11, 25). Most ofthese experiments were performed on a small scale with lowamounts of heavy-metal-containing material.

In this paper, a semicontinuous laboratory-scale leachingplant (LSLP) is described; this LSLP achieved high leachingefficiencies, which resulted in an elevated load of elements inthe effluent. Treatment times were found to be less than treat-ment times obtained with batch extraction procedures. A mix-ture of Thiobacillus thiooxidans producing sulfuric acid andThiobacillus ferrooxidans oxidizing reduced metal compounds(19) was used to perform the leaching experiments. The resultswere compared to chemical (abiotic) leaching efficiencies. Inaddition, we investigated whether T. ferrooxidans was neededfor leaching of fly ash. Metals can be biotically released fromfly ash by mechanisms such as direct enzymatic reduction,indirect action resulting from extracellular metabolic products,or acid formation (nonenzymatic dissolution), as previouslyshown in an anaerobic system (10). It was possible to differ-entiate among these release mechanisms in an oxic acidic flyash-Thiobacillus system.

MATERIALS AND METHODS

Bacterial strains, medium, and culture conditions. T. thiooxidans DSM622and T. ferrooxidans DSM2391 were cultivated in a medium containing (per liter)0.1 g of K2HPO4, 0.25 g of MgSO4 z 7H2O, 2.0 g of (NH4)2SO4, 0.1 g of KCl, and8.0 g of FeSO4 z 7H2O. Elemental sulfur (1%, wt/vol) was added, and the pH wasadjusted with sulfuric acid to 2.5 to 2.7. The mixed culture was grown undernonsterile conditions either in 250-ml baffled Erlenmeyer flasks on a rotaryshaker (140 rpm) or in an aerated and stirred 1,000-ml beaker. Growth wasmonitored by monitoring the pH (with a Hamilton single-pore electrode), thecell counts (with a Neubauer counting chamber), and the Fe(II) concentration(12).

* Corresponding author. Mailing address: Institute of Environmen-tal Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057Zurich, Switzerland. Phone: 41 1 635 61 25. Fax: 41 1 635 57 11. E-mail:HBRANDL@UWINST.UNIZH.CH.

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Samples and sample preparation. A 500-kg portion of fly ash retained byelectric filters at the MWI plant in Hinwil, Switzerland, was collected by workersat Sulzer Chemtech (Winterthur, Switzerland) at different times on one day andwas homogenized in a cement mixer to obtain representative homogeneoussamples. The ash was washed with water to remove water-soluble compoundsand dried on a vacuum filter. For experiments the ash was ground and dried at80°C for 48 h. The concentrations of selected elements are listed in Table 1. Thevalue for loss of combustion at 950°C represents the inorganic carbon content.

Semicontinuous LSLP. The LSLP consisted of three serially connected reac-tion vessels (designated RV-A, RV-B, and RV-C), each having a volume of 1dm3 (Fig. 1). Pulp from the fly ash storage solution (100 g liter21) and thebacterial stock culture (109 cells/ml) were mixed in equal amounts and fed every12 h semicontinuously into the first reaction vessel (RV-A) with a peristalticpump at an overall dilution rate of 0.021 h21 (0.5 day21). RV-A and RV-B wereconnected by an overflow connector. The pulp was pumped from RV-B to RV-Cwith a peristaltic pump at the same rate to flush settled fly ash particles. This

resulted in a pulp concentration in the LSLP of 50 g liter21 and a mean residencetime of 6 days (2 days in each reaction vessel). The bacterial stock culture and thethree reaction vessels were aerated at a rate of about 2 volumes of air per volumeof reactor per min. After RV-C, the pulp was transported by gravity flow into a2-liter vacuum glass filter unit, and the particle-free, metal-rich solution wascollected in a 5-liter collecting vessel.

Determination of mobilization mechanisms. Forty milliliters of an 8% (wt/vol)fly ash suspension (acidified with sulfuric acid to pH 5.4) was diluted with 40 mlof a Thiobacillus culture (109 cells/ml) after different treatments (see below) andincubated for 8 days on a rotary shaker (150 rpm) at room temperature (23 to24°C). All samples were incubated in triplicate. Several mechanisms of metalmobilization were distinguished, as described below.

The direct enzymatic effect on the release of metals was determined by dilutingthe ash suspension with bacterial stock cultures (pH 1.1). The cells were in directcontact with the fly ash. Growth of T. ferrooxidans might have been stimulated byincreased energy available from oxidation of reduced solid particles.

Leaching with cell-free spent medium revealed that indirect solubilization byextracellular metabolic products occurred. The stock culture was centrifuged at23,700 3 g, and the supernatant was filtered through a 0.22-mm-pore-size Teflonfilter to obtain cell-free spent medium. The cell-free spent medium was checkedfor viable cells by incubating a 5-ml sample in 80 ml of fresh medium.

Cell-free spent medium (see above) was autoclaved (12 min, 121°C) to obtaina sterile leaching solution without enzymatic activities to evaluate the leachingability of the acid formed. The solution was checked for precipitates and forchanges in redox state after the heat treatment.

Forty milliliters of fresh uninoculated medium was added to the fly ash sus-pension and used as a control.

Elements such as Cd or Zn might have been chemically mobilized duringpreparation of the ash suspension due to the acidification to pH 5.4.

Chemical leaching with sulfuric acid. Eighty milliliters of the fly ash suspen-sion was leached at a concentration of 5% (wt/vol) with a maximum of 11 ml of5 N sulfuric acid (final pH, pH 2) at the following pH values: at an initial pH of10, at pH 8 (corresponding to the carbonate buffer pH), at pH 4 (correspondingto the potassium-aluminum buffer pH) (2), and at pH 2 (a possible end point ofa leaching experiment). The concentrations of the solubilized metals and the acidconsumption were measured. The pH at each value was controlled with a pH-Stat (Metrom Impulsomat model 614). Fly ash was suspended in distilled waterand stirred at a constant pH for 24 h. A new suspension was prepared for eachpH step.

Analytical procedures. Metal analyses were performed by using inductivelycoupled plasma atomic absorption spectroscopy (ICP-AES; Spectro AnalyticalInstruments, Kleve, Germany) and standard addition methods at the followingwavelengths: Al, 396.2 nm; Cd, 228.8 nm; Cr, 267.7 nm; Cu, 324.8 nm; Fe, 261.2nm; Mn, 294.9 nm; Ni, 352.5 nm; and Zn, 206.2 nm. Prior to the inductivelycoupled plasma analysis, the samples were centrifuged at 23,700 3 g for 15 min,acidified with 5 drops of concentrated HNO3 per 30 ml of aqueous solution,passed through a glass fiber filter (Whatman type GF/C) to guarantee particle-free suspensions, and stored at 4°C.

RESULTS AND DISCUSSION

LSLP. A semicontinuous three-stage leaching plant for ex-traction of heavy metals from MWI fly ash was developed.Biohydrometallurgical processing of fly ash from MWI poses,especially at high pulp densities, severe problems due to thehigh content of toxic metals in the fly ash and the saline andstrongly alkaline environment. It is necessary to obtain reducedtreatment times for high pulp concentrations without addi-tional acidification; this is important for reducing the capitaland maintenance costs of a pilot plant.

RV-A of the LSLP (Fig. 1) was filled with 500 ml of abacterial culture (pH 1.5) and 250 ml of a fly ash suspension(10%, wt/vol) for the first adaptation phase. After 36 h, an-other 250-ml aliquot of the suspension was added to obtain thefinal 5% (wt/vol) solution. When the pH in RV-A droppedbelow 2, the LSLP was started. After 48 h (corresponding tofour discontinuous feeding cycles) the solution was pumped toRV-C. After 48 h, the microorganisms produced sufficientamounts of sulfuric acid in each reaction vessel to maintainsteady-state conditions despite the alkaline pH of the fly ashpulp (pH 9 during the experiment). A distinct pH cascadeoccurred from one reaction vessel to the following reactionvessel. The pH fluctuated during the steady state depending onthe fly ash present; in RV-A the pH fluctuated between 3.7 and4, in RV-B the pH fluctuated between 2.7 and 3.2, and in RV-C

FIG. 1. Schematic diagram of the LSLP. A, B, and C, serially connectedRV-A, RV-B, and RV-C, respectively; 1, fly ash reservoir; 2, bacterial stockculture; 3, peristaltic pump; 4, filter unit; 5, collecting vessel; 6, to vacuum pump;solid lines, liquid flow; dashed lines, air flow.

TABLE 1. Concentrations of selected elements in fly ash from theMWI plant in Hinwil, Switzerlanda

Element Detectionmethod(s)b

Concn in flyash (g kg21)

SE(%)

Al A, B, C 70 5.5Ca A, B, C 132 5.5Cd B 0.49 15.0Cl B 4.7 15.0Cr A, B 0.7 7.9Cu B 1.1 15.0F B 8.0 15.0Fe A, B, C 28 5.5Hg B ,0.01 15.0K A, B, C 12 5.5Mg A, B, C 14 5.5Mn A, B, C 0.77 5.5Na A, B, C 11 5.5Ni A, B 0.14 7.9Pb B, C 8.9 11.1S B, D 30 7.9Si A, B 100 7.9Sn B 9.3 15.0Ti A, B 12 7.9V B 0.13 15.0Zn B, C 31 7.9Loss of combustion (1 h, 950°C) 126 NDc

a Data reproduced from reference 27 with permission. The measured concen-trations allowed us to determine approximate leaching efficiencies.

b A, X ray fluorescence with a glass specimen (standard error, 65%); B, X rayfluorescence with a compacted powder specimen (standard error, 615%); C,inductively coupled plasma atomic absorption spectroscopy (standard error,65%); D, infrared detection after oxidation at 2,000°C (standard error, 65%).

c ND, not determined.

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the pH fluctuated between 1.2 and 1.5. Before the bacterialstock was added to RV-A, growth parameters [pH, Fe(II)concentration, cell number] were monitored (Fig. 2). The me-dium was fully replenished with fresh medium every 72 h (cor-responding to an overall dilution rate of 0.01 liter h21) to avoidaging of the bacterial stock culture.

Samples used for metal analysis were removed after 168 hfrom each reaction vessel, from the collection vessel, and (ascontrols) from the fly ash storage vessel and the bacterial stockculture. For all elements, the concentrations of soluble metalsincreased continuously with increasing mean residence time inthe LSLP (Fig. 3). In RV-C the following amounts of metalswere solubilized (per kilogram of fly ash): Al, 37 g; Zn, 25 g;Fe, 3.1 g; Cu, 0.98 g; Mn, 0.53 g; Cd, 0.49 g; Ni, 0.09 g; and Cr,0.08 g. Ferrous iron added to the bacterial stock culture as anelectron source for T. ferrooxidans precipitated in the first tworeaction vessels (RV-A and RV-B) in high amounts, resultingin a decrease in the soluble iron level. At higher pH values ironeither precipitated as hydroxide or became adsorbed on fly ash

particles. In addition, ferric iron coprecipitates with other met-als (e.g., As, Cd, Cr, Cu, Pb, and Zn) (10). In RV-A, up to 45%of the iron added to the medium precipitated; in RV-B about32% of the iron added precipitated. Only at the very low pH inRV-C (pH 1 to 1.3) was 10% net iron leaching observed.Leaching of Pb with sulfur-oxidizing bacteria like members ofthe genus Thiobacillus is not very effective, because of the lowsolubility of PbSO4 in aqueous solutions (15, 20). Sulfate waspresent in the medium at high levels. Ferrous sulfate wasadded as an energy source for T. ferrooxidans, and sulfate wasproduced by T. thiooxidans as a metabolic product of sulfuroxidation. Therefore, mobilized Pb immediately precipitatedas PbSO4 and remained with the leached fly ash in the glassfilter unit. To verify this result, ChemEQL (a computer pro-gram used to calculate thermodynamic equilibrium concentra-tions and precipitation conditions) was used. Precipitation wascalculated for the following conditions: (i) the maximum ex-pected Pb concentration, 445 ppm (2.15 mM); (ii) the mini-mum expected sulfate concentration, 1,380 ppm (14.4 mM)(only the sulfate from the medium was considered; the sulfateformed due to bacterial oxidation of S° was not taken intoaccount); and (iii) the maximum Cl2 and F2 concentrations(Cl2 and F2 form soluble Pb complexes), 235 ppm (6.63 mM)and 400 ppm (21 mM), respectively. Concentrations were cho-sen by using the method of Vonmont (27). As shown in Table2, only 1.2 to 2.6% of the Pb stayed in solution under veryacidic conditions. At pH values between 1 and 4, the solubilitywas ,1%. Although Pb concentrations close to 10 g kg21 canbe found in fly ash (Table 1), the leaching efficiencies (ex-pressed as percentages of the amounts applied) were very low(usually ,5%).

Determination of mobilization mechanisms. X ray fluores-cence analyses carried out by workers at Amt fur Gewasser-schutz und Wasserbau des Kantons Zurich in 1991 (1) indi-cated that reduced copper species (chalcocite [Cu2S] andcuprite [Cu2O]) were present in MWI fly ash, whereas zinc andother metals were present in their fully oxidized forms. Thus,copper release from fly ash should be directly affected andenhanced by T. ferrooxidans, whereas Zn, Al, Cd, Cr, and Niare released primarily due to the acidic environment. Thesedifferent mobilization mechanisms could be distinguished by aseries of batch experiments.

In batch cultures with inoculated medium the pH decreasedwithin 8 days from 3.6 to 1.6 as a result of biotically formedsulfuric acid. In freshly filtered cell-free spent medium and

FIG. 2. Monitoring of growth and activity of a mixed culture of T. thiooxidansand T. ferrooxidans (maximum growth rate, 0.055 h21; doubling time, 12.7 h).The experiment was performed in an aerated 2-liter beaker before the culturewas used as a stock culture in the LSLP. During the operation of the LSLP thepH remained between 1.0 and 1.3, and the cell count increased slightly to 4 3 109

cells per ml. Symbols: ‚, pH; {, Fe(II) concentration; E, cell count.

FIG. 3. Amounts of solubilized metals obtained from MWI fly ash (50 gliter21), expressed as percentages of the amounts present in a LSLP with threeserially connected reaction vessels (RV-A, RV-B, RV-C). The mean reactiontime in each vessel was 2 days. Negative values indicate metal precipitation. p,RV-A; , RV-B; h, RV-C.

TABLE 2. Dissolution equilibrium for Pb from MWI fly ash asdetermined by ChemEQL at high sulfate concentrations (50 g offly ash liter21; maximum total Pb concentration, 445 mg liter21;minimum H2SO4 concentration, 1,380 mg liter21; maximum Cl2

concentration, 235 mg liter21; maximum F2 concentration,400 mg liter21)a

pHAmt of dissolved total Pb

ppm %

0.5 11.51 2.591 5.22 1.171.5 3.23 0.732 2.63 0.592.5 2.50 0.563 2.57 0.583.5 2.76 0.624 2.93 0.66

a The minimum and maximum concentrations were calculated according todata in Table 1.

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autoclaved sterile spent medium the pH remained constant at3.6; in uninoculated medium the pH remained constant at 5,and the pH remained constant at 5.4 in assays in which distilledwater was used instead of medium. Acidification of the fly ashpulp (chemical mobilization) led to significant extraction yieldsfor Cd, Ni, and Zn (Fig. 4), which could be slightly increased byusing uninoculated sterile medium as the lixiviant (leachingsolution) due to the sulfuric acid present in the culture me-dium. The level of Al dissolution was low, whereas the Cr andCu concentrations in both experiments were below the detec-tion limits.

By comparing the amounts of leached copper in filteredcell-free spent medium (0.89 g kg of fly ash21; standard devi-ation [sx] 5 0.03 g kg21; n 5 3) and autoclaved sterile spentmedium (0.70 g kg21; sx 5 0.04 g kg21; n 5 3), it was concludedthat in contrast to other elements significant amounts of cop-per (as determined by a paired t test [one sided], P 5 0.02)were mobilized by metabolic products of T. ferrooxidans.Leaching with cell-free spent medium, which indicated that asolubilizing mechanism involving extracellular components waspresent, was significantly more effective than leaching withautoclaved spent medium, in which excreted enzymes wereinactivated. It is known that several components involved inthe electron transport chain in the genus Thiobacillus (rusti-cyanin, cytochromes, iron-sulfur proteins) are located in theperiplasmic space (3, 24) and might, therefore, also be presentin cell-free spent medium and catalyze oxidation of reducedmetal compounds. It is possible that heat treatment (autoclav-ing) of spent medium leads to aggregation of nonenzymaticcompounds or changes in the redox state. The two solutionswere checked for precipitates (DA660 5 0.003) and for theirredox potentials (Eh) (the values for cell-free spent mediumand autoclaved spent medium were 830 and 810 mV, respec-tively). The results show that aggregation and altered redoxconditions due to autoclaving did not occur.

In contrast, for all of the other elements examined (Al, Cd,Cr, Ni, and Zn), the difference between filtered cell-free spentmedium and autoclaved cell-free spent medium was not sig-nificant. Therefore, it was concluded that the mobilization ofthese metals from fly ash was caused only by the acidic envi-ronment.

The maximal extraction yields for all elements were ob-tained with samples incubated with both T. thiooxidans and T.

ferrooxidans. The data indicate that there was efficient releaseof most heavy metals from fly ash due to biotically formedsulfuric acid (the pH decreased within 8 days from 3.6 to 1.6).Copper solubilization increased significantly (1.10 g kg of flyash21; sx 5 0.05 g kg21; n 5 3; paired t test [one sided]; P 50.002), as well did mobilization of Al (n 5 3; P 5 0.003), Cd(n 5 3; P 5 0.03), Cr (n 5 3; P 5 0.0005), and Zn (n 5 3; P 50.03) compared to samples incubated with cell-free spent me-dium.

Chemical leaching with sulfuric acid. The value for loss ofcombustion at 950°C, of 12.6% (Table 1), indicates that con-siderable amounts of inorganic carbon occurred as carbonatesin the fly ash. These carbonates, along with other constituents,caused the pH of the fly ash suspension to be more than 10.Most of the metals were mobilized due to sulfuric acid, the acidnecessary to neutralize and acidify MWI fly ash was deter-mined, and the chemical (abiotic) leaching efficiencies of sul-furic acid at different pH values were assessed.

The dissolution of metals in fly ash in alkaline environments(pH 8 and 10) was low. Less than 0.03 g of Al or Zn and lessthan 0.01 g of Cd, Cr, Cu, Fe, or Ni were solubilized per kg offly ash. To lower the pH of the fly ash suspension from .10 to4, 175 ml of H2SO4 (95 to 97% pure) per kg of fly ash had tobe added. This resulted in release of 15 g of Al kg of fly ash21

(corresponding to 22% of the total amount present), 20 g of Znkg21 (75%), 1.44 g of Fe kg21 (5%), 0.49 g of Cu kg21 (53%),0.38 g of Cd kg21 (76%), 0.02 g of Ni kg21 (14%), and 0.01 gof Cr kg21 (2%). Large amounts of Al (45 g kg21; 64%) andZn (32 g kg21; 100%) were solubilized at pH 2, along with 7.4 gof Fe kg21 (26%), 0.96 g of Cu kg21 (88%), 0.44 g of Cd kg21

(89%), 0.03 g of Ni kg21 (22%), and 0.08 g of Cr kg21 (11%),when 311 ml of H2SO4 (95 to 97% pure) was added. Theleaching efficiencies were comparable to those of the LSLP.

Conclusions. The ability of microorganisms to leach andmobilize metals from solid materials is based on the followingthree mechanisms: (i) redox reactions, (ii) formation of inor-ganic acids, and (iii) excretion of complexing agents (e.g., or-ganic acids). T. ferrooxidans mobilizes metals from solids byredox reactions. Electron transfer from minerals to microor-ganisms either occurs directly in the case of physical contactbetween organisms and solids or is based on the biotic oxida-tion of Fe12 to Fe13 when ferric iron catalyzes metal solubi-lization as an oxidizing agent (9, 14). For solubilization of thereduced copper compounds (chalcocite [Cu2S] and cuprite[Cu2O]) present in the fly ash, these release mechanisms areimportant. The Ni leaching efficiency is also considerably in-creased by biological activities compared with chemical leach-ing with sulfuric acid due to the presence of Fe(III) in theleaching solution. The results obtained for bacterial leaching ofheavy metals from anaerobically digested sludge also con-firmed that the solubilization rates of metals obtained withmixed cultures of T. thiooxidans and T. ferrooxidans werehigher than the solubilization rates obtained with single cul-tures (26).

The three-stage LSLP described here is a promising firststep toward the establishment of a semicontinuous or contin-uous bioleaching leaching plant. The lab work showed thepracticability of biotic fly ash leaching despite the presence ofsaline and strongly alkaline material. When acidophilic au-totrophic Thiobacillus strains are used, no aseptic LSLP set-upis required. The possibility of contaminants which interferewith the Thiobacillus strains is minimal, due to the very acidicenvironment and the absence of organic compounds as carbonsources. Such conditions should reduce the capital and main-tenance costs of a pilot plant. A scaled-up version of the LSLP,a pilot plant with three or more reaction vessels for biotic

FIG. 4. Amounts of solubilized metals obtained from MWI fly ash (40 gliter21), expressed as percentages of the amounts present with different lixiviantswithin 8 days. All samples were incubated in triplicate. The release of metals dueto acidification of the fly ash pulp was defined as chemical mobilization. h,inoculated medium; , filtered cell-free spent medium; , autoclaved sterilespent medium; p, uninoculated medium; o, chemical mobilization.

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leaching of industrial waste, seems to be technically feasible.Large-scale reactor leaching of this type has been used previ-ously, especially for gold recovery (13, 22). A large-scale bi-oleaching plant should allow us to detoxify fly ash for reuse inconstruction, while valuable metals, especially zinc, should beeconomically recovered for recycling in metal-manufacturingindustries.

ACKNOWLEDGMENTS

The statistical assistance of C. Luchsinger (Institute of AppliedMathematics, University of Zurich) is acknowledged.

Financial support was provided by the Swiss National Science Foun-dation within the Priority Program Environment.

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