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(27) Barlin, G. B.; Pausacker, K. H.; Riggs, N. V. J. Chem. SOC. 1954, 3122, part 111. (28) Rawat, B. S.; Agrawal, M. C. Indian J . Chem. 1978,17A, 299. (29) Nigh, W. G. In Oxidation in Organic Chemistry; Trahanovsky, W. S., Ed.; Academic Press: New York, 1973; Vol. 5-B, pp 51-53. (30) Wheeler, 0. H.; Gonzalez, D. Tetrahedron 1964,20, 189. (31) Sharma,L. R.; Manchanda, A. K.; Singh, G.; Verma, R. S. Electrochem. Acta 1982, 27, 223. (32) Weber, E. J.; Wolfe, N. L. Environ. Toxicol. Chem. 1987, 6 , 911. (33) Scow, K. M.; Simkins, S.; Alexander, M. Appl. Environ. Microbiol. 1986, 51, 1028. (34) You, I. S.; Bartha, R. J. Agric. Food Chem. 1982,30,274. (35) Hammett, L. P. Physical Organic Chemistry, 2nd ed; McGraw-Hill Book Co.: New York, 1970. (36) Bollag, J.-M.; Minard, R. D.; Liu, S.-Y. Enuiron. Sci. Technol. 1983, 17, 80-83. (37) Sillen, L. G.; Martell, A. E. Stability Constants for Metal-Ion Complexes;The Chemical Society: London, 1971; Supplement No. 1. (38) Smith, R. M.; Martell, A. E. Critical Stability Constants; Plenum Press: New York, 1975; Vol. 2.
Received for review December 28, 1988. Revised manuscript received August 20,1989. Accepted November 27,1989. This work was supported by the U S . Department of Energy, Grant DE-FG22-86PC90524; Richard P. Noceti, Technical Project Officer,provided useful comments.
Anaerobic Microbial Remobilization of Toxic Metals Coprecipitated with Iron Oxide Aroklasamy J. Francls" and Cleveland J. Dodge Department of Applied Science, Brookhaven National Laboratory, Upton, New York 11973
An anaerobic N2-fixingClostridium sp. solubilized Cd, Cr, Ni, Pb, and Zn coprecipitated with goethite (a-FeOOH) by (i) direct action due to enzymatic reduction of ferric iron and the release of metals associated with iron and (ii) indirect action due to metabolic products. The extent of dissolution depended upon the nature of the association of the metals with goethite. Substantial amounts of Cd and Zn, which were closely associated with iron, were released due to direct action. Nickel was solubilized by direct and indirect actions, while a small amount of Cr was solubilized only by direct action. The nature of association of P b in the coprecipitate was not affected by the presence of other cations and it was solubilized by indirect action. In the presence of bacteria, the concentration of soluble P b decreased due to biosorption. These results show that there could be significant remobilization of toxic metals coprecipitated with iron oxides in wastes, contaminated soils, and sediments due to microbial iron reduction.
Introduction Iron oxides scavenge transition and heavy metals in soils, sediments, and energy wastes (1-8). These oxides are a major sink for metals in the terrestrial and aquatic environments and play an important role in regulating their availability. Sorption and coprecipitation are the predominant processes by which most of the metals are retained by iron oxide. Sorption is a process by which metals are bound to the surface of an existing solid by adsorption and surface precipitation (9),whereas coprecipitation is 00 13-936X/90/0924-0373$02.50/0
the simultaneous precipitation of a chemical element with other elements and includes mixed-solid formation, adsorption, and inclusion ( 1 0 , I I ) . Although coprecipitation has not been as well studied as adsorption, it appears to remove trace metals from solution more efficiently (12-14). Toxic metals such as As, Cd, Co, Cr, Cu, Hg, Ni, Pb, Se, U, and Zn from fossil- and nuclear-fuel cycle waste streams, geothermal fluids, and electroplating wastes are currently removed by coprecipitation with ferric iron or are under consideration for such treatment (3,15-17). The removal of toxic metals from waste streams by coprecipitation with iron seems to be a very efficient method and economically feasible, but problems remain with the disposal of the solids generated in the process and with the ultimate fate of the coprecipitated metals in the environment. Significant dissolution of metals from the coprecipitate can be brought about by chemical and microbiological action. In general, solubility of iron oxide depends upon the degree of crystallinity. Amorphous iron oxides are orders of magnitude more soluble than goethite or hematite ( I ) , while amorphous synthetic goethite is 2-100 times more soluble than a well-crystallized goethite (18). Microorganisms play a significant role in the dissolution of amorphous and crystalline forms of iron oxides by direct action or by indirect action. Direct action involves enzymatic reductive dissolution of iron from higher oxidation state to lower oxidation state and indirect action is due to the production of metabolites (19-23). However, we have little information on microbially mediated disso-
0 1990 American Chemical Sc)ciety
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lution and remobilization of metals coprecipitated with iron. In this paper, we report for the first time the anaerobic microbial remobilization of toxic metals Cd, Cr, Ni, Pb, and Zn coprecipitated with goethite.
Materials and Methods Culture Conditions. A N2-fixing Clostridium sp. (ATCC 53464) isolated from coal-cleaning residue was grown in a medium containing the following: glucose, 5.0 g; NH4Cl, 0.5 g; glycerol phosphate, 0.3 g; MgS04.7H20, 0.2 g; FeS04.7H20,2.8 mg; CaC12.2H20,0.5 g; peptone, 0.1 g; yeast extract, 0.1 g; deionized water, 1000 mL; pH, 6.8 f 0.1. The medium was first prereduced by boiling and purging with N2 gas for 15 min to remove the dissolved oxygen. It was then cooled under a N2 atmosphere in an anaerobic glovebox and 40-mL quantities were dispensed in 60-mL serum bottles. The serum bottles were closed with butyl rubber stoppers, sealed with aluminum caps, and autoclaved. The medium was inoculated with 0.2 mL of an early logarithmic growth phase of the culture (optical density at 600 nm, 0.41) and incubated at 24 f 1 OC. Growth of the bacteria was measured at 600 nm with a Bausch and Lomb Spectronic-20 spectrophotometer. Preparation of Iron Oxide (Goethite) Metal Coprecipitate. Iron oxide coprecipitate was synthesized as described by Atkinson et al. (24) with some modifications. Stock solutions were prepared containing 0.25 M of each of the following metals in 100 mL of deionized water: Cd(N03)2.4H20(Alfa Products, 95+ % ), Cr(N03)3.9H20 (Alfa Products, 98.5%); Ni(N03)2.6H20(Alfa Products, 97+ %); Pb(N03)2(Mallinckrodt, analytical reagent grade); Zn(N03).xH20 (Alfa Products, 95+%). Each stock solution was acidified with 0.05 mL of Ultrex nitric acid, and 5 mL of each was added to a 1.5-L Pyrex flask. Fe(NO3),-9H20,50 g (Mallinckrodt, 98.5%) and 800 mL of deionized water were then added, and the pH was adjusted to 12 by adding 200 mL of 2.5 N KOH. A reddish brown precipitate formed immediately. The precipitate was aged for 24 h at 60 "C and then washed several times with deionized water until the nitrate levels in the supernatant were less than 5 mg/L. The washed precipitate was dried in an oven at 60 "C for 18 h, pulverized to a fine powder in an agate mortar, and stored in a desiccator. The structure of the iron oxide was confirmed by X-ray diffraction with a Phillips XRG 3100 Model analyzer. To determine the behavior of Pb in the absence of other cations, we prepared a separate goethite coprecipitate with only P b (NO3), and ferric iron, using the same procedure described. Chemical Characterization of Metal Coprecipitates. Total Metal Content. A 50-mg aliquot of the coprecipitate was dissolved in 40 mL of 50% HCl in duplicate. The solution was filtered through a 0.22-pm Millipore filter, and the metals were analyzed by atomic absorption spectrophotometry. Exchangeable Cations. To determine the exchangeable fraction of metals, 40 mL of a 1M MgC1, solution was added to 50 mg of the coprecipitate in duplicate. The solution was shaken continuously for 2 h on a wrist-action shaker. The samples were then filtered through a 0.22-pm Millex filter, acidified with 0.20 mL of Ultrex HN03, and analyzed for each metal. Dissolution Profile of the Metals. A 40-mL aliquot of 50% HC1 was added to 50 mg of the mixed-metal-goethite coprecipitate. Six sets of duplicate samples were incubated and each sample was mixed by agitation at periodic intervals. At 0, 2,4,6,8, and 10 h a set of samples was filtered through a 0.22-pm Millex filter and analyzed for each metal. 374
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Dissolution of Metals from Goethite Coprecipitate. We determined whether the metals were released by direct action due to enzymatic reductive dissolution or by indirect action (nonenzymatic) due to production of organic acid metabolites in a series of experiments as described elsewhere (20) with modifications. A 50-mg sample of the mixed-metal- or lead-goethite coprecipitate was incubated in 40 mL of medium in 60-mL serum bottles fitted with butyl rubber stoppers under N2 atmosphere for 40 h at 24 "C. All samples were incubated in triplicate. The treatments consisted .of the following: (i) Dissolution in Sterile Medium (Control). To determine the chemical dissolution of the coprecipitate in the uninoculated bacterial growth medium (control), we added the prereduced medium to acid-washed bottles containing the coprecipitate. The samples were then sealed with butyl rubber stoppers, autoclaved, and incubated for 40 h. (ii) Dissolution in the Presence of Bacteria. Dissolution of the coprecipitate in the presence of bacteria was determined by inoculating the autoclaved medium containing the coprecipitate with 0.2 mL of a 24-h-old culture. This treatment allowed the bacterial cells to come in direct contact with the precipitate. At the end of incubation, the total gas production and pH were determined. (iii) Dissolution by Cell-Free Spent Medium. To determine whether metals were released from the coprecipitate by extracellular components produced by the bacterium, a cell-free spent medium was prepared. Cells were grown in culture medium in the absence of coprecipitate. After 40 h of incubation (optical density at 600 nm, 0.61), the cells were separated from the culture medium by centrifugation at 13600g for 30 min and filtration through a 0.22-pm Durapore filter in a 1.5-L Teflon-coated pressure filtration device (Millipore Co.) inside the anaerobic glovebox. The spent medium was divided into two equal aliquots. The first aliquot was immediately tested for its ability to solubilize metals from the coprecipitate, while the second aliquot was used to test for nonenzymatic dissolution as described below. (iv) Nonenzymatic Dissolution of Metals. The second aliquot of filtered spent medium was transferred to serum bottles, sealed with butyl rubber stoppers, and autoclaved to inactivate the enzymes. This heat-treated spent medium was filtered again through the pressure filtration device with a 0.22-pm Durapore filter to remove any denatured cellular material. The pH of the autoclaved and filtered spent medium was measured, and the dissolution of metals from the coprecipitate was determined as described before. The filtered spent medium and the heat-treated spent medium were checked for cells by direct microscopic examination and for viable cells by incubating an aliquot in a fresh growth medium. (v) Dissolution of Metals by Synthetic Medium. To determine the effect of acid metabolites and pH of the medium on the dissolution of metals from the coprecipitate, a synthetic medium was prepared by adding metabolic acids to prereduced growth medium containing only inorganic salts (NH4C1,0.50 g; MgS04.7H,0, 0.20 g; and CaC1,.2H20, 0.5 g/L). The metabolic acids, acetic (3.44 mM), butyric (7.90 mM), and lactic (2.62 mM), were added in the same proportions as found in an inoculated culture medium of Clostridium sp. (25). The final pH of the synthetic medium was 3.1. A t the end of incubation, the samples were filtered through a 0.22-km Millex filter and the filtrate was acid-
Table-I. Metal Content of Goethite Coprecipitate metal
mmol added
Fe Cd Cr Ni Pb Zn
125 1.25 1.25 1.25 1.25 1.25
Fe Pb
125 1.25
mmol in coprecipO
% metal
incorp
Mixed Metals 78.4
98.0 f 0.00
0.62 f 0.01 0.83 f 0.00 0.62 f 0.01 0.67 0.01 0.86 f 0.06
66.6 50.0 53.8 69.2
Lead Only 100 f 2 0.77 f 0.02
80.0 61.5
50.0
*
f l standard error of the mean.
Table 11. Exchangeable Cations in Goethite Coprecipitate metal
mmol/g of dry wt
%
Mixed Metals Fe Cd Cr Ni Pb Zn Fe Pb
