Environ. Sci. Technol. 1992. 26.340-346
WI, 1990; Chapter 6, pp 111-160. Chiou, C. T.; Lee, J.-F.; Boyd, S.A. Enuiron. Sci. Technol. 1990, 24, 1164. Gschwend, P. M.; Wu, S.-C. Enuiron. Sci. Technol. 1985, 19, 90. Boyd, S.A.; Mikesell, M. D.; Lee, J.-F. In Reactions and Movement of Organic Chemicals in Soils; Sawhney, B. L., Brown, K., Eds.; Special Publication 22; Soil Science Society of America: Madison, WI, 1989; Chapter 8, pp 209-228. Madhun, Y. A.; Freed, V. H.; Young, J. L.; Fang, S. C. Soil Sci. SOC.Am. J . 1986, 50, 1467. Chin, Y.-P.; Weber, W. J., Jr. Enuiron. Sci. Technol. 1989, 23, 978. Smith, J. A.; Chiou, C. T.; Kammer, J. A.; Kile, D. E. Enuiron. Sci. Technol. 1990,24, 676. Ong, S.K.; Lion, L. W. Water Res. 1991,25, 29. Brusseau, M. L.; Jessup, R. E.; Rao, P. S.C. Enuiron. Sci. Technol. 1991, 25, 134. Hamaker, J. W.; Thompson, J. M. In Organic Chemicals in the Soil Environment; Goring, C. A. I., Hamaker, J. W., Eds.; Marcel Dekker: New York, 1972; Vol. I, Chapter 2; pp 49-143. Kenaga, E. E.; Goring, C. A. I. In Aquatic Toxicology;Eaton, J. C., Parrish, P. R., Hendricks, A. C., Eds.; American Society for Testing and Materials: Philadelphia, PA, 1980; p p 78-115. Mingelgrin, U.; Gerstl, Z. J. Enuiron. Qual. 1983, 12, 1. Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kile, D. E. Enuiron. Sci. Technol. 1986, 20, 502. Chiou, C. T.; Kile, D. E.; Brinton, T. I.; Malcolm, R. L.;
Leenheer, J. A.; MacCarthy, P. Enuiron. Sci. Technol. 1987, 21, 1231. Garbarini, D. R.; Lion, L. W. Enuiron. Sci. Technol. 1986, 20, 1263. Huffman, E. W. D.; Stuber, H. A. In Humic Substances in Soil, Sediment, and Water, Aiken, G. R., McKnight, D. M.; Wershaw, R. L., MacCarthy, P., Eds.; Wiley: New York, 1985; pp 433-457. Mackay, D.; Shiu, W. Y. J . Phys. Chem. Ref. Data 1981, 10, 1175. Chiou, C. T.; Kile, D. E.; Malcolm, R. L. Enuiron. Sci. Technol. 1988, 22, 98. Steelink, C. In Humic Substances in Soil, Sediment, and Water, Aiken, G. R., McKnight, D. M., Wershaw, R. L., MacCarthy, P., Eds.; Wiley: New York, 1985; pp 457-476. Schnitzer, M. In Soil Organic Matter; Schnitzer, M., Khan, S. U., Eds.; Elsevier, New York, 1978; pp 1-58. Boyd, S.A.; Jin, X.; Lee, J.-F. J. Enuiron. Qual. 1990,19, 734. Gauthier, T. D.; Seitz, W. R.; Grant, C. L. Enuiron. Sci. Technol. 1987, 21, 243. Karickhoff, S.W.; Brown, D. S.; Scott, T. A. Water Res. 1979, 13, 241. Fujita, T.; Iwasa, J.; Hansch, C. J. Am. Chem. SOC.1964, 86, 5175.
Received for review May 13,1991. Accepted September 11,1991. The use of trade and product names in this article is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey.
Solubilization of Zinc Oxide from Filter Dust with Penicillium simplicissimum: Bioreactor Leaching and Stoichiometry Wolfgang Burgstaller,
* p t
Hermann Strasser,+ Hans Wobking,t and Franz Schlnnert
Institute of Microbiology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria, and Montanwerke Brixlegg, 6230 Brixlegg, Austria
Filter dusts from metal-processing industries contain valuable metals which should be recycled. Some disadvantages of conventional recycling methods can be avoided by applying biohydrometallurgical methods. Fungi are suitable for such processes because they are fairly resistant to heavy metals. Furthermore, leaching activity occurs in a pH range from 2 to 7. This is important because filter dusts generally raise the pH of the medium. Leaching of zinc from zinc oxide contained in filter dust was possible in a bioreactor (stirred tank reactor) especiallyconstructed for this purpose. Zinc solutions of 0.41 M were obtained after 9 days of leaching. The zinc concentration was doubled compared to leaching experiments in shake-flask cultures. The solubilization of zinc oxide was caused by the action of protons originating from citric acid produced by the fungus. A further increase in solubilized zinc seemed difficult to achieve, because zinc and citric acid precipitated at the high zinc and citric acid concentrations present at the end of the leaching period. The lack of a cheap growth substrate for the fungus appeared to be the greatest handicap toward an application on an industrial scale.
Introduction Large amounts of filter dusts develop during pyrometallurgical processes of winning metals. These mixtures +University of Innsbruck. Montanwerke Brixlegg.
*
340
Environ. Sci. Technol., Vol. 26,No. 2, 1992
of metal oxides are of great economic value (1). For example, the filter dust used in this work contained 50% zinc, 20% lead, and 6% tin, on average. Pyrometallurgical as well as hydrometallurgical recycling shows some disadvantages (1): (i) recovery rates are low and it is difficult to win the metals in a pure form; (ii) costs of chemicals and of precautions against environmental pollution are high; (iii) an economic recycling process is only guaranteed if a high turnover is possible. The use of biohydrometallurgical recycling methods is intended to avoid some of these disadvantages. Important characteristics of leaching processes with microorganisms are as follows: (i) the pollution of the environment is negligible; (ii) a selective extraction of metals is possible in certain cases; (iii) the costs of chemicals are low when autotrophic bacteria can be used for leaching processes, or when organic wastes can be used as growth substrates for heterotrophic microorganisms; (iv) energy requirements are low. The leaching of zinc from filter dust of a copper smelting plant turned out to be possible with the fungus Penicillium simplicissimum (2). The filter dust consisted of a mixture of metal oxides, half of which being zinc oxide. About 80-90% of the total zinc was solubilized after 6-10 days of cultivation in a shake-flask culture (25 g/L filter dust; 13 g/L zinc in solution at the end of the leaching period). Leaching was caused by the action of protons originating from citric acid excreted by the fungus (3). Zinc and, to a minor extent, copper and lead were solubilized whereas tin was not solubilized at all (3). In the medium used, the fungus produced considerable amounts of citric acid only
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when filter dust was present in the culture broth and was adsorbed to the mycelial pellets (4). For 1mol of zinc oxide present in the medium the fungus produced 0.67 mol of citric acid. The need for an organic carbon source was regarded as the major disadvantage in comparison to leaching processes with the autotrophic bacterium Thiobacillus spp. However, the use of Thiobacillus thiooxidans had its limits, because the presence of filter dust in the medium resulted in an alcalinization of the culture broth. The ability of T. thiooxidans to produce sulfuric acid is inhibited at pH values higher than 5 (5). When filter dust was leached with T. thiooxidans, dust could only be added in small portions [maximum 1% (w/v) per addition] and at long intervals. Consequently, the leaching periods for 25 g of filter dust/L lasted for more than 3 weeks. Numerous investigations are concerned with the leaching of various materials with heterotrophic microorganisms in shake-flask cultures (3). Yet leaching in a bioreactor is rarely dealt with in literature. Leaching experiments with P. simplicissimum in shake-flask cultures showed that a sufficient supply of oxygen and carbon source was essential for an optimal zinc yield. Therefore, we attempted to construct a bioreactor that was both suitable and as simple as possible in order to exceed the limits observed with leaching in shake-flask cultures. Subsequently, the influence of various parameters (stirrer rate, aeration rate, addition of carbon and nitrogen source, addition of filter dust, inoculation density, preparation of the inoculum) on the leaching of filter dust in the 10-L bioreactor was investigated. In view of the economy of the process, molasses was used as the sole source of carbon and nitrogen. Additionally, by treatment of filter dust or zinc oxide with different acids (organic and inorganic), the stoichiometry of the extraction of zinc from zinc oxide was examined, and biotic and abiotic leaching processes were compared. Materials and Methods
Inoculum for the Bioreactor. A 100-mL aliquot of separately sterilized medium was added to sterile 500-mL flasks containing 2.5 g of filter dust (mixture of metal oxides: 50-60% Zn, 21% Pb, 6% Sn, 1% Cu, 0.3% As, 0.2% C1; particle size 10-50 Fm). Medium 1consisted of the following (grams per liter): molasses 250; KH2P040.8; pH 7.0 with HC1. Medium 2 contained the following (grams per liter): sucrose 150 or glucose 87; NH4N032; KH2P040.8; MgSO4-7H200.4; pH not adjusted. The flasks were inoculated with a spore suspension of P. simplicissimum [isolated from heavy-metal-contaminated soil (4)] at a concentration of 2 X lo6 spores/mL of medium and incubated for 6 days (30 OC, 250 rpm). Four different types of inocula were tested the standard inoculum (as described), a standard inoculum treated with a mechanical homogenizer in order to break up the pellets, a suspension of spores produced in surface culture (4),and a suspension of spores produced in submerged culture (6). Construction of the Bioreactor. A bioreactor was constructed which was stirred from the top, because the fine particles of the filter dust would have damaged the bearings mounted at the bottom. The bioreactor consisted of a prefabricated 10-L glass vessel provided with a stainless steel top plate with a central stirrer bearing and five openings (sampling port, air inlet and outlet, temperature sensor, and dosage port). Four steel baffles were mounted in the vessel. The stirrer shaft with two flabblade turbines was driven by a stirrer (IKA RW 20; maximum speed 2000 rpm). An external infrared heater controlled by a temperature sensor was used for maintaining the temperature. Cooling was provided by a cold water tube wound around the vessel. An aluminum construction
Table I. Moles of Consumed Protons per Moles of Zinc Solubilized from Filter Dust or Zinc Oxide in Biotic ( P . simplicissimum ) and Abiotic Leaching Experiments
available protons, mM
acid citric acid
malic acid" hydrochloric acid iron(II1) chloride citric acid"
solubilized zinc, mM
mol of protons/mol of zinc
Abiotic Leaching 92.10" 47.17 172.17" 85.58 242.46" 109.74 1050.00" 556.OOb 200.00" 98.65 2.63 1.27 159.00 75.84 289.10 133.12 379.30 174.37
1.95 2.01 2.21 1.89 2.03 2.07 2.10 2.17 2.18
Biotic Leaching 448.80" 209.60' 120.42" 61.71bfd 288.00" 139.43e 954.00" 470.06
2.14 1.95 2.07 2.03
" If all carboxyl groups are fully dissociated. Zinc oxide was used instead of filter dust. "After 13 days of incubation (shakeflask culture). dAfter 4 days of incubation (shake-flask culture). the fourth day of a bioreactor run. fOn the ninth day of a bioreactor run. guaranteed sufficient stability even when high stirring rates were used. For sterilization the top part of the aluminum construction with the affixed stirrer was removed. After the filter dust (25 g/L) was added to the vessel, the top plate mounted with temperature sensor, sparger, and sampling device was clamped to the vessel and the whole unit was sterilized in an autoclave for 30 min. Culture Conditions for Bioreactor Runs. Medium 1and medium 2 were used for leaching experiments (the inoculum was grown in the respective medium). The carbon source needed for 9 L of medium was dissolved in 4 X 2 L of deionized water; the rest of the components were dissolved in 500 mL of deionized water. The sterile medium was poured into the bioreactor via a sterile funnel at an aeration rate of 100 L/h in order to avoid sedimentation of the dust into the sparger. Immediately after the bioreactor was filled with the medium, inoculation was carried out with 100 mL of pellets. The temperature was 30 OC. When the leaching rate of a standard run was at its maximum, cooling of the bioreactor became necessary. Aeration rate and stirring rate as well as time and amount of addition of filter dust and carbon source were varied according to the course of the respective fermentation. Nitrogen source (25 mM) was added at days 3 and 5. The loss of liquid due to evaporation was balanced daily by addition of deionized water before a sample was taken. Samples (50 mL; volumetric flask!) were taken daily. During sampling, the culture broth was stirred in order to guarantee representative distribution of the solids (dust and biomass). An antifoam agent (silicone) was added when necessary. Abiotic Leaching of Filter Dust and Zinc Oxide. Suspensions of filter dust (25 g/L) or zinc oxide (17 g/L) were leached with various amounts of citric acid and hydrochloric acid as well as with malic acid, and iron(II1) chloride (Table I). Solubilized zinc was determined after 24 h of contact. The ratio of added protons and solubilized zinc was then calculated. The buffering capacity of citric acid from leaching cultures was compared to that of an equimolar solution of citric acid. The pH of the equimolar citric acid solution was adjusted with NaOH to that of the culture filtrate. A Environ. Sci. Technol., Vol. 26, No. 2, 1992 341
Table 11. Operating Parameters of a Bioreactor Run under Standard Conditions
o ~w 8 Zinc ~
3 0.7
w Citric acid
working volume, L rpma
9 300-1000 0.6-2.0
VVm*
0
2
4
6
8
1
0
1
2
TIME (d) Figure 1. Citric acid production and zinc extraction from a filter dust (50% zinc oxide) by P. simplicissimum in a 10-L bioreactor with glucose as carbon source (for fermentation parameters, see Table 11). Filter dust was added on days 4 (1.5%), 5 (0.5%), 6 (1.0%), and 7 (1.o %).
defined amount of protons (HC1) was then added to both solutions. The amount of buffered protons was calculated from the difference in the pH values before and after addition of HC1. From this, conclusions about the degree of dissociation could be drawn. The interrelation between the concentrations of zinc and citric acid and the precipitation of zinc citrate was also investigated. A solution of zinc oxide and citric acid (concentrations comparable to the ones measured in bioreactor experiments) was shaken for 72 h. Then zinc and citric acid in solution were determined, and the amount of zinc and citric acid which had precipitated was calculated. In order to evaluate the solubility product of Zn3Cit2, a defined amount of Zn3Citzwas solubilized at different pH values (1,3, 4, 5, 6, 7, 8). Zinc and citric acid which has gone into solution was subsequently determined. Analytical Methods. All samples were filtrated (0.2 pm) in order to guarantee a particle-free solution for analysis. With samples from the bioreactor, the solids (consisting of filter dust and biomass) were dried after filtration (0.2 pm) at 80 OC for 24 h and then ashed at 500 "C for 4 h. The biomass was calculated from the difference in weight. pH was measured in all filtrates. Solubilized zinc was determined by atomic absorption spectrophotometry (2). In order to calculate zinc yields, it was necessary to determine the zinc content of the leached filter dust (zinc yields could not be calculated from the zinc found in solution, because the high biomass production led to a concentration of the solutes). For that purpose the ashed solids from a sample were digested in a covered Teflon cup in a steel pressure bomb (0.1 g of leached filter dust, 6 mL of 65% HNO,, 4 h at 180 "C). Sugar consumption (sucrose, glucose, fructose) and the production of organic acids (mainly citric acid) were determined by HPLC (2). Organic acids were also quantified by isotachophoresis (2). Consumed sugar, produced citric acid, solubilized zinc, the pH value, and the concentration of biomass were quantified daily for samples taken from the bioreactor in order to evaluate the effect of any variation in the operation parameters.
Results Standard Leaching Experiments in the Bioreactor. Figure 1 shows the solubilization of zinc from filter dust during a representative leaching experiment in the bioreactor. A survey of operational parameters is given in Table 11. The most striking features of this leaching 342
Environ. Sci. Technol.. Voi. 26, No. 2, 1992
temperature, "C filter dust (day 0), % carbon source (day 0), mM PH (day 0 ) inoculum inoculation density pH (at the end of the run) filter dust (at end of run), % addition of carbon source addition of "*NOB addition of filter dust leaching period, days
30 2.5 (w/v)
438 6.5-7.5 pellets from the preculture 100 mL of pellets 4-5 5-8 (w/v) 438 mM minus the concn measd in the culture broth days 3 and 5 (25 mM each) days 4, 6, 8, and 10 (more often when necessary) 9-14
uRotations per minute. *Liter of air per liter of medium and minute. 150.0 125.0 0
zN
100.0
n
Z Q
50.0 25.0 0.0
0
2
4
6
8
1 0 1 2
TIME (d)
Flgure 2. Biomass formation, sugar consumption, and zinc balance during the leaching of a fiiter dust (50% zinc oxide) in a 1O-L bioreactor by P. simplicissimum (for fermentation parameters, see Table 11. Data are from the same run as in Figure 1: (A) biomass (g of dry wt/L), (B) solubilized zinc (g/L), (C) sugar consumption (mol/L.day), (D) zinc yield (in percent of total zinc added as filter dust), (E) zinc measured in solution plus zinc determined in the leached residue (in percent of total zinc added as fiiter dust).
process were the continuous, stoichiometrically connected increases in citric acid and zinc concentrations. Furthermore, a high glucose consumption (0.19 mollday, on average; Figure 2) as well as biomass production (up to 100 g of dry weight/L; Figure 2) was observed. Data concerning the zinc balance are also given in Figure 2. The content of solubilized zinc in the culture broth (0.425 M) was doubled compared to the highest values reached in shake-flask cultures [0.220 M (291. The highest yield reached was 66.7% solubilized zinc at a pulp density of 45 g/L (Figure 2). At the beginning of the investigation, precautions were taken to guarantee sterility during the handling of the bioreactor. Later on, sterility turned out to be unnecessary for this leaching process. Microscopic examination revealed that no other organisms grew under these conditions. Deionized water was replaced by tap water without difficulties. Between 25 and 33 "C, no significant alteration of the rate of solubilization of zinc was observed. An attempt to accelerate the leaching process by increasing the temperature was not successful. The fungus was not able to grow at 40 "C. Several leaching experiments in the bioreador run under standard conditions failed to show growth of the fungus.
o.201 I
I
”
v
z N
&-A
0.10
YD
n
4
pH
Sum of odded filter dust 85 g/1
0.7
i
0.0 -.
0
1
2
3
4
TIME (d)
5
TIME (d) Flgure 3. Influence of initial nitrogen concentration and inoculation density on the solubilization of zinc from a filter dust (50% zinc oxide) by P . simplicissimum in a IO-L bioreactor until the fourth day of ferinoculated with 100 mL of pellets; (B) mentation: (A) 25 mM “,NO3, 12.5 mM “,NO, inoculated with 100 mL of pellets; (C)25 mM “,NO3, inoculated with 200 mL of pellets; (D) 50 mM “,NO3, inadded on day oculated with 200 mL of pellets. (A, D) 25 mM “,NO3 3; (B, C) no ”,NO3 added on day 3.
Possible reasons for this phenomenon will be discussed later. Carbon Source. Three different carbon sources were used. Sucrose was readily split by P. simplicissimum into glucose and fructose. Both sugars were consumed by P. simplicissimum. The hghest concentration of zinc reached with sucrose as carbon source was 0.61 M. In order to simplify the calculation of sugar balances, sucrose was replaced by glucose. The maximum of solubilized zinc with glucose as a carbon source was 0.46 M. Sucrose and glucose seemed equally suited for this leaching process. With molasses as carbon source, the zinc concentration in the culture broth never exceeded 0.26 M. The consumption of sugar per day varied between 0.04 and 0.34 M (0.19 mol of sugar/L-day), on average; Figure 2). Therefore sugar had to be added almost every day during the leaching period. It was essential that the carbon source was never totally exhausted. If this happened, the pH value in the culture broth began to rise and subsequently zinc and citric acid simultaneously precipitated. Nitrogen Source. An increase in the initial concentration of ammonium nitrate slightly stimulated the zinc extraction rate until the third day (Figure 3). The addition of ammonium nitrate on the third day was essential to maintain the ability of the fungus to excrete citric acid. Inoculum and Inoculation Density. Only inoculation of the bioreactor with the standard inoculum guaranteed growth of the fungus in the bioreactor. When spores were used as inoculum, the combination of stirring and the presence of filter dust inhibited the germination of the spores. It was not important whether the standard inoculum was 2,4, or 6 days old. When the inoculation density of the standard inoculum was doubled, the zinc extraction rate was 50% higher on day 3 (Figure 3). This increased zinc extraction rate leveled off until the end of the run. Filter Dust Addition and Adsorption. Each addition of filter dust increased the pH value of the culture broth, or at least briefly interrupted the acidification by the fungus. Therefore additions of filter dust were made in portions not exceeding 15 g/L. Furthermore, filter dust was added every second day only beginning with day 4. Starting the fermentation with 5% (w/v) fiiter dust instead of 2.5% (w/v) resulted in total inhibition of growth. The adsorption of the filter dust onto the mycelium was es-
Flgure 4. Influence of aeration on citric acid production and zinc extraction from a fllter dust (50% zinc oxide) by P . simplicissimum In a IO-L bioreactor with sucrose as carbon source (for fermentation parameters, see Table I). Filter dust was added on days 5 (1.5 %), 7 (lS%), 9 (1.5%), and 10 (1.5%).
sential for citric acid production. Whether or not a run leached a considerable amount of zinc could be judged from a “phenotypic”characteristic: high yields of zinc were always linked with complete adsorption of the filter dust. Because of this adsorption, the mycelial pellets took on a dark grey coloring and sedimented within minutes when the stirrer and the aeration were turned off. Fermentations with poor yields were characterized by a whitish coloring due to precipitated zinc citrate and a sedimentation of the biomass after hours only. Oxygen Supply. The oxygen supply was an important parameter concerning the production of citric acid by P. simplicissimum. In shake-flask cultures, the leaching period was shortened from 13 to 6 days when baffled flasks were used. In the bioreactor, low stirring and aeration rates allowed biomass formation but no citric acid was produced. The upper limit for these two parameters was judged empirically according to foam production. Interruption of the oxygen supply stopped citric acid production immediately. After the air was turned on, citric acid production started again (Figure 4). Stoichiometry of the Solubilization of Zinc Oxide. The anion and the protons of an organic acid are able to leach metals (“acidolysis”and “complexolysis”). Therefore, both mechanisms were tested for solubilizing zinc oxide by citric acid. With 150 mM citric acid and 25 g/L filter dust (which contained 220 mM zinc oxide), 100% of the zinc could be found in solution after 24 h of contact (end pH 4.3). With 150 mM potassium citrate, only 17.9% of the zinc was solubilized (end pH 9.2). Therefore, further investigations centered on the action of protons. Abiotic leaching experiments with organic and inorganic acids (Table I) confirmed the following equation for the solubilization of zinc oxide: ZnO
+ 2H+ = Zn2++ H 2 0
The amount of zinc oxide solubilized by the different acids corresponded to the maximum amount of protons available from the acids (Table I). On the basis of a proton/zinc ratio of 21 for the solubilization of zinc oxide, it was proved that the citric acid produced by the fungus was responsible for nearly all of the solubilized zinc oxide. The differences between calculated zinc concentrations and the ones measured in culture broths were within 20%. This was true for shake-flask cultures as well as bioreactor runs. From the amount of solubilized zinc oxide in leaching experiments and the fact that zinc oxide absorbs protons Environ. Sci. Technol., Vol. 26, No. 2, 1992 343
Table 111. Increased Buffering Capacity of Citric Acid Which Was in Contact with Filter Dust"
parameter
A
concn,mM PH H+ added as HC1, mM pH after addition of protons H+ buffered by citric acid, mM
citric acidb B A
B
120.00 147.37 147.37 3.89' 3.73 4.06c 4.05 208.33 208.33 403.23 403.23 1.13 2.52 0.70 1.46 115.67d 205.22d 153.82d 362.44d 120.00
" Values are the mean of three experiments; standard deviation 5 pm) from the atmosphere is roughly determined by the sedimentation rate of a given particle (which in turn is a function of particle size, its reactivity characteristics, charge, etc.) and its content of pollutants (12). In Holarctic regions such as Sweden, the composition and amounts of atmospheric fallout vary with latitude (B), owing to differences in climate and distance to the sources.
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0 1992 American Chemical Society
