SUMMARY

Pseudomonas MGF-48, a gram-negative, motile, oxidase negative, catalase positive, yellow pigmented bacterium, isolated from electroplating effluent, was found to accumulate heavy metals, especially uranium. Uptake of uranium was high, rapid and the amount increased in direct proportion to concentration, e.g., from 50 to 200 mg/l uranium. The largest amount of uranium uptake was 174 mg per gram dry weight bacterial biomass, observed to occur in stationary phase, when incubation was at 25C. Uptake was determined by flow injection analysis. Maximum uranium accumulation was obtained at pH 6.5, with 86% of the uranium being removed within 5 min of incubation. Release of uranium bound to the cells was accomplished by addition of sodium carbonate and EDTA solution (0.1 M). The solution was reusable, serving as a biosorbent. Cells immobilized in polyacrylamide gel yielded 90% uranium removal. Pseudomonas MGF-48 showed excellent efficiency in biosorbing uranium both when immobilized and as free cells. The results of this study indicates that the bacterium was capable of accumulating several metals. Accumulation of uranium was higher than other metals. We concluded that Pseudomonas MGF-48 shows excellent potential for bioremediation of uranium-polluted aqueous effluents.

Many industries, including mining and electroplating, discharge aqueous effluents containing relatively high levels of heavy metals, e.g. uranium, cadmium, mercury, and copper. Untreated effluent from these manufacturing processes have an adverse impact on the environment (6,8,11,13,16,17). A specific problem associated with heavy metals in the environment is accumulation in the food chain and persistence in the environment.

Physical and chemical methods have been designed to remove metal ions from effluents, but, in general, these methods are commercially impractical, either because of high operating cost or difficulty in treating the solid wastes generated (6,18).

Bioremediation of industrial wastes containing heavy metals has been demonstrated by several biotechnology companies employing bioaccumulation (5,6). Biosorption, bioprecipitation, and uptake by purified biopolymers derived from microbial cells provide alternative and/or additive processes for conventional physical and chemical methods (18). Intact microbial cells, live or dead, and their products can be highly efficient bioaccumulators of both soluble and particulate forms of metals (3,12,18). The cell surfaces of all microorganisms are negatively charged owing to the presence of various anionic structures. This gives bacteria the ability to bind metal cations. Various microbial species, mainly Pseudomonas, have been shown to be relatively efficient in bioaccumulation of uranium, copper, lead, and other metal ions from polluted effluents, both as immobilized cells and in the mobilized state (5). We report here the bioaccumulation of several metals by a bacterium, identified as a Pseudomonas sp., strain MGF-48, isolated from the effluent of a metal melting factory in the south of Tehran.

MATERIALS AND METHODS

Isolation procedures. Approximately 50 water, soil, and sludge samples were collected from the effluents of metal melting and electroplating factories in the south of Tehran, Iran. Samples were diluted 10-10,000 fold in sterile distilled water and plated on Trypticase Soy Agar (Difco) plates containing 1 mM Cd (Cd(NO₃)₂4H₂0), 1 mM Cu (CuSO₄5H₂0), 1 mM Zn (ZnSO₄7H₂O), or 1 mM Pb (Pb(NO₃)₂). Plates were incubated at 30C for 72 hr and colonies were randomly picked, isolated, and purified. In this preliminary screening, colonies showing resistance to Cd, Cu, Zn and Pb were selected for further study.

Bacterial cell growth conditions. The bacterium, MGF-48 was grown in 500 ml flasks containing a glucose and mineral salts medium (glucose, 10 g; NH₄Cl, 2.67 g; Na₂HPO₄, 5.35 g; distilled water, 1000 ml) amended with 6 ml mineral salts solution (CaCl₂2H₂0, 0.1 g; MgSO₄7H₂O, 10 g; MnSO₄7H₂O, 0.07 g; FeSO₄7H₂O, 0.4 g; distilled water, 1000 ml). The glucose was autoclaved separately as a solution (1 %), with the pH adjusted to 7.0 before autoclaving. Flasks were incubated at 30C for 72 h, with shaking (200 rpm).

Heavy metals uptake experiments. Bacterial cells were harvested by centrifugation at 9000 x g for 20 min at 4C and washed twice with distilled water. The cells were suspended in deionized water to a final concentration of 2.5 mg dry weight/ml. Ca.10 ml of the susension was added to 40 ml of selected concentrations of uranyl nitrate (UO₂(NO₃)₂6H₂O). The suspensions were incubated at room temperature on a shaker with 100 rpm for 1 hr then centrifuged at 9000 x g for 20 minutes. The bacterial cells were heated to 105C overnight after which the dry weight was measured. The harvested cells, 4 mg dry weight, were mixed with 0.5 ml concentrated nitric acid and incubated in a waterbath (100C) for 1 h, after which the mixture was cooled to 25C, the volume brought to 5 ml with distilled water, and the concentration of uranium measured by flow injection (model FIA-1 Autoanalyzer, Beijing, China).

The identical protocol was followed using lead (Pb(NO₃)₂), cadmium (Cd(NO₃)₂4H₂0), copper (CuSO₄5H₂0), nickel (NiCl₂6H₂0), zinc (ZnSO₄7H₂0), silver (Ag(NO₃), cobalt (CoCl₂6H₂0), and chromium (K₂Cr₂O₇) at concentrations of 50 - 100 mg/l. Metal uptake was measured by atomic absorption spectrophotometry (Perkin-Elmer, Norwalk, CT, USA, Model No. 300 S). Results were expressed as mean of experiments done in triplicate and the data compared with those measuring loss of uranium from uninoculated controls.

Recovery of uranium from cells. To measure uranium bioaccumulation, i.e., the amount of uranium taken up by MGF-48 cells, the cells were washed with distilled water for 15 min and mixed with 0.1 M sodium citrate, EDTA, Na₂CO₃, or nitric acid. The concentration of uranium released was measured and cells were reused, serving as a biosorbent.

Immobilization of bacterial cells. The bacterial isolate MGF-48 was grown in glucose mineral medium, using 100 ml in 500 ml flasks and incubating at 37C, with shaking at 200 rpm. After 36 hours incubation at 37C, cells were harvested by centrifugation at 9000 x g for 10 min at 4C, and washed three times with MES buffer (2-[N-Morpholino]ethanesulfonic acid). Ca. 50 mg (dry weight) of freshly harvested bacterial cells were suspended in 9 ml MES buffer and mixed with 6 ml of 20% polyacrylamide stock solution (18.2 g acrylamide and 1.8 g N,N-methylene-bis-acrylamide dissolved in 50 ml distilled water, and diluted with distilled water to a final volume of l00 ml) and l00 µl potassium persulphate (10%). Ca. 10 µl of N,N,N',N' tetra methyl ethylenediamine (TEMED) was added to the mixture, with polymerization at 25C for 1 h. The gel was ground into small pieces (20 mesh), washed thoroughly with MES buffer, and suspended in MES buffer for heavy metal ion uptake measurement.

In batch ion uptake experiments, immobilized bacterial cells (ca. 50 mg dry weight in 15 ml of gel) were suspended in 20 ml MES buffer amended with 100 mg/l of uranium, cadmium, or copper, and packed into a column (1.5 cm x 15 cm). After exposure for 60 min, the buffer was drained off and the concentration of heavy metal ion in the buffer determined by atomic absorption spectrophotometry. Experiments were carried out in triplicate and controls (gels without bacteria) were included. Removal efficiency of the immobilized bacterial cells is defined as the percentage of added heavy metal ion removed, including the amount absorbed by the bacterial cells.

RESULTS

Isolation of heavy metal resistant bacteria. A total of 50 bacterial strains resistant to heavy metals were isolated from samples of metal melting and electroplating effluent. Some of the strains were resistant to all four metals tested in this study (Cu, Pb, Cd, Zn). Only one strain was capable of accumulating uranium, when transferred to GMS broth containing uranium nitrate. Preliminary identification indicated that the bacteria was a Gram-negative, motile, yellow pigmented, oxidase negative, catalase positive, aerobic rod shaped bacterium. Following the criteria of Bergey's Manual of Determinative Bacteriology (2) and employing the Biolog system (microplate Pl 001) for Gram negative bacteria (Biolog, Inc., Hayward, CA, U.S.A.), the isolate was identified as a Pseudomonas sp., strain MGF-48. Biochemical characteristics of the strain are listed in Table 1.

Effect of growth stage and uranium concentration on uranium accumulation. When bacterial suspensions were prepared from 24, 48, and 72 hr cultures incubated at 30C with shaking (200 rpm) and uranium uptake measured, employing a 50 ml uranium nitrate solution (50 mg/1), the amount of uranium accumulated was 163.5, 155.5 and 162.5 mg per gram dry weight of cells, respectively. No significant difference relative to culture time was evident. Thus, in subsequent experiments, three day cultures were used. Uranium uptake by the cells was measured after incubation in media amended with concentrations of uranium ranging from 50 mg/l to 500 mg/l. It was found that the amount of uranium taken up by the cells increased with increase in concentration of uranium from 50 - 200 mg/l (Figure 1). No increase in uptake of uranium was observed at concentrations greater than 200mg/l. The highest concentration of uranium taken up by Pseudomonas MGF-48 was 174 mg/g dry weight bacterial biomass, in the stationary phase of growth and incubation at 25C. Uptake was determined using flow injection at 541 nm.

Effects of pH on uranium accumulation. As shown in Figure 2, the initial pH of the solution had a significant effect on uranium accumulation. At pH 6.5, maximum accumulation of uranium was obtained (86% within the first 5 min) and at higher and lower pH, the amount of uranium accumulation was less.

Recovery of uranium. As shown in Figure 3, uranium was released from cells by addition of sodium carbonate, sodium citrate, EDTA, and nitric acid. Because of the formation of a very stable uranyl-carbonate-complex, U-EDTA, in order to recover the accumulated uranium, the cells required washing 3 times with EDTA and sodium carbonate. The amount of uranium recovered in the first wash was 95% and after washing twice, no further increase in recovery was observed. However, when the cells were washed a third time, recovery increased to 94.4%. Recovery was significantly less when the cells were washed with sodium citrate and nitric acid (77.9 and 78.1 respectively), compared to EDTA and sodium carbonate.

Metal accumulation. Pseudomonas MGF-48 was capable of accumulating several metals (Pb, Cd, Cu, Ag, Ni), with the results, U > Pb > Cd > Cu (Figure 4). Affinity for Cr and Co was much lower and the amount of biosorption of Pb, Cd, Cu, Ag, and Ni was 57.5, 54, 26, 18 and 6 mg/g dry weight bacterial biomass, respectively (initial concentration of 100 mg/1). Bacterial cells grown in a sulfate limiting medium did not show significantly increased copper removal (20.5 mg/g dry weight bacterial biomass at 50 µg/ml and 27.5 mg/g at 100 µg/ml).

Heavy metal uptake by immobilized cells. The efficiency of removal of uranium, cadmium, and copper by immobilized Pseudomonas MGF-48 cells is shown in Table 2. Uranium was absorbed efficiently (more than 90%) and could be recovered by elution with 0.1 M Na₂CO₃. No significant changes were observed in removal efficiency of immobilized cells, compared to free cells. Maximum removal was obtained at a flow rate of 44 ml/h at 25C.

DISCUSSION

It is well recognized that microorganisms have a high affinity for metals and can accumulate both heavy and toxic metals by a variety of mechanisms (1 8,19,21). Microorganisms highly effective in sequestering heavy metals include bacteria, fungi, algae, and actinomycetes (23,25). These have been used to remove metals from polluted industrial and domestic effluent on a large scale. Wong et al. (23) isolated Pseudomonas putida II-11, from electroplating effluent, showing that it accumulated Cu(II), up to 6.5% dry weight, from a Cu(II) containing solution. Other investigators have demonstrated the capabilities of several bacteria in removing uranium, Cd, Pb, and other toxic metals from polluted effluents (1,5,10,14,18).

In this study, an isolate, Pseudomonas MGF-48, was found to be highly efficient in accumulating uranium, up to 174 mg/g dry weight bacterial biomass, when grown in GMS broth. The maximum accumulation of uranium was achieved when the bacterium was in stationary phase of growth at pH 6.5 and incubated at 25C. Uptake was relatively rapid under these conditions (86% of uranium removed within 5 min) (Figure 4).

In addition to uranium accumulation, Pseudomonas MGF-48 was capable of taking up Pb, Cd, and Cu when incubated in concentrations of 50-100 mg/l. Uranium uptake was much greater than observed for the other metals examined in this study. Furthermore, Pseudomonas MGF-48 exhibited a specificity for uranium, as well as accumulating Pb, Cd, and Cu. Specificity for a given metal ion by a bacterial species has been reported by other investigators (3,10,12,15). For example, Philips et al. (14) and Lovely et al. (4) showed uranium reduction by Desulfovibrio desulfuricans. Simons et al.(17) used Saccharomyces and Candida species to remove Ag and Cu from effluents. The investigations reported here were undertaken to isolate a bacterial strain capable of efficient removal of heavy metals from polluted effluents, e.g., uranium, cadmium, mercury, lead and copper.

Several principal sites of uranium complex formation in biological systems have been proposed, including accumulation in the cell wall, carbohydrate or protein polyphosphate-uranium complexation, complexing with the carboxyl group of the peptidoglycans in the cell wall, or entering into cells via an energy-dependent mechanism (18). Macaskie (7) and Strandberg et al. (20) provide an excellent overview of these alternatives giving detailed description of mechanisms involved in metalmicrobe interactions. Pseudomonas MGF-48 appears to accumulate uranium in the cell wall and along the external cell surfaces, as well as internally. As shown in Figure 5, electron dense areas in the cell envelope and such areas inside the cells are evident. Uptake was rapid, i.e., occurring within the first 5 min of incubation. Most of the uranium absorbed was removed when the cells were washed with Na₂CO₃ and EDTA. Cultures incubated for 21 days exhibited some uranium removal. These findings suggest that uranium uptake involves both surface phenomena and diffusion,the latter most likely a result of increased membrane permeability. Since surface adsorption (metabolism independent biosorption) is frequently reversible without causing damage to the biomass and impairment in subsequent use, it offers an efficient, inexpensive, and feasible method for removal of metals from aqueous systems (18).

Macaskie and Dean (6), Malone (8), Marques et al. (9), and Wong (24) demonstrated bioaccumulation for a wide range of heavy metals, including some in the uranium family. Uranium and other metals such as cadmium, lead and silver, were removed activiely by incorporating pre-grown cells in polyacrylamide gels. In the studies reported here, more than 90% of the uranium was removed by cells immobilized in polyacrylamide gel. In conclusion, Pseudomonas MGF-48 demonstrates high efficiency of uranium bioaccumulation and therefore, is an excellent candidate for use in bioreactors to remove uranium from polluted aqueous effluents.

Safety aspects of the use of the organism for bioremediation are being evaluated. Biochemical tests, based on the use of BIOLOG, indicate that the organism is probably not Pseudomonas aeruginosa; however, the species has not been determined. Molecular tests (i.e., 16s RNA/DNA) to determine the species are being conducted. If the organism is determined to be a pathogen, steps to permit biological control (i.e., insertion of a suicide gene) or transferring the genetic properties to a non-pathogen species will be considered.

ACKNOWLEDGEMENTS

We thank Drs. Meysami and Movassaghi at the Atomic Energy Organization of Iran for assistance with the atomic absorption and flow injection, and Dr. Khodashenas at the Reza Institute, Tehran, Iran for preparing the electron micrographs.

This work was supported in part by a grant from the University of Tehran, Tehran, Iran, and the Office of Naval Research contract No. N00014-95-1 to the University of Maryland Biotechnology Institute, University of Maryland System.

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Table 1. Selected taxonomic characteristics of Pseudomonas MGF-48, a bioaccumulator of uranium.

Characteristic	Result
Growth on MacConky agar	+
Catalase	+
Oxidase	-
H2S production (TSI)	-
Gluconate oxidation	-
Indol	-
Nitrate reduction	+
Arginine decarboxylase	-
Ornithine	-
Lysine	-
Phenylalanine	-
Glucose	+
Galactose	-
Mannose	+
Xylose	+
Lactose	-
Sucrose	-
Maltose	+
Saliein	-
Dnase	-
Esculin hydrolysis	+
Gelatin hydrolysis	-
Simmons citrate	+
Insulin	-
Trehalose	+
Arabinose	+
Glycerol	+
Mannitol	-
Ramnose	-
Sorbitol	-
Christensen urea	-

Table 2. Efficiency of metal removal by immobilized cells of Pseudomonas MGF-48.

Metal*	Initial conc. (mg/l)	Final conc. (mg/l)	Efficiency
Cadmium	100	72	28
Copper	100	79	21
Uranium	100	<10	>90

*Cadmium, Cd(NO₃)₂4H₂O; copper, CuSO₄5H₂O; uranium, UO₂(NO₃)₂6H₂O.