Appl Microbiol Biotechnol (1991) 35:406-410 0175759891001525

Applied Microbiology Biotechnology © Springer-Verlag 1991

Uranium accumulation by Pseudomonas sp. EPS-5028 Ana M. Marquis, Xavier Roca, M. Dolores Simon-Pujol, M. Carmen Fuste, and Francisco Congregado Laboratorio de Microbiologia, Facultad de Farmacia, Nrcleo Universitario de Pedralbes Universidad de Barcelona, 08028 Barcelona, Spain Received 19 December 1990/Accepted 12 February 1991

Summary. Pseudomonas sp. EPS-5028 was examined for the ability to accumulate uranium from solutions. The uptake o f uranium by this microorganism is very rapid and is affected by p H but not by temperature, metabolic inhibitors, culture time and the presence of various cations and anions. The a m o u n t of u r a n i u m absorbed by the cells increased as the uranium concentration of the solution increased up to 55 mg u r a n i u m / g cell dry weight. Electron microscopy indicated that uranium accumulated intracellularly as needle-like fibrils. U r a n i u m could be removed chemically f r o m the cells, which could then be reused as a biosorbent.

Potentially, any microorganism or cell fraction that exposes negatively charged groups on its surface, should have an affinity for metal cations (Ehrlich 1986). Metal adsorption to negatively charged groups is rapid, reversible and occurs whether or not a carbon or energy source is present in the m e d i u m (Norberg and Persson 1984). W o r k with a variety of organisms has clearly shown that bacteria excreting exopolysaccharides provide significantly enhanced metal immobilization (Scott and Palmer 1988). In this work we report on the uptake o f uranium by Pseudomonas sp. EPS-5028, an exopolysaccharide-producing microorganism, for the first step in recovering uranium from aqueous systems.

Introduction Materials and methods Aqueous effluents emanating from the mining industry and different factories contain dissolved heavy metals (e.g. uranium, cadmium, mercury). I f these discharges are emitted without treatment, they m a y have an adverse i m p a c t on the environment (Scott et al. 1986). Conventional methods for removing dissolved heavy metals include chemical precipitation, chemical oxidation and reduction, ion exchange and filtration (Norberg and Persson 1984). Such processes m a y be ineffective or extremely expensive when the initial heavy metal concentrations are not very high (Shumate et al. 1978). The uptake and accumulation of heavy metals by microbial biomass is receiving increasing attention in a biotechnological context since microbe-based technologies m a y provide an alternative or adjunct to conventional techniques of metal r e m o v a l / r e c o v e r y f r o m polluted effluents and waste-waters. Intact microbial cells, living or dead, and derived microbial products can be highly efficient bioaccumulators of both soluble and particulate forms of metals, especially from dilute external concentrations ( G a d d and de R o m e 1988).

Offprint requests to: A. M. Marqu6s

Organism. Pseudomonas sp. strain EPS-5028, used throughout the study, was isolated from soil (Congregado et al. 1985) and maintained on Trypticase Soy Agar (BBL, Cockeyville, Md, USA) with a weekly transfer to fresh medium. Uranium uptake experiments. Cells were cultured at 30°C in glucose mineral salts medium (GMS), containing (per litre distilled water): glucose, 10 g; NH4C1, 2.67 g; Na2HPO4, 5.35 g; and 6 ml of a mineral salt solution containing (per litre distilled water) CaC12.2H20, 0.1 g; MgSOa.7H20, 10 g; MnSOa.7H20, 0.075 g; FeSO4-7H20, 0.4 g. Glucose was autoclaved separately. The pH was adjusted to 7.0 using 0.1 M NaOH before sterilization. After 48 h, unless noted otherwise, bacteria were harvested by centrifugation at 9000g for 20 min, washed three times with deionized distilled water and resuspended in aqueous uranyl nitrate solution to give a final cell density of 2.5 mgml -~ dry weight. Uranyl nitrate hexahydrate (Merck, Darmstadt, FRG) solutions (40 ml) were prepared with deionized distilled water so that the addition of 10 ml cell suspension provided the appropriate dilution to give the desired initial uranium concentration. Unless otherwise stated, the suspension was shaken for 24 h at 30°C and, at desired intervals, samples were removed for supernatant uranium determination. The amount of metal ion in the residual solution was determined with inductively coupled plasma spectroscopy (ICP) (Jobin Yvon-JY-38 spectrometer; Paris). Results were expressed as the means of analysis from three replicate flasks, compared with the loss of uranium from uninoculated controis.

407 When the bacteria were treated chemically with HgCi2 (1% solution), AgNO3 (10% solution) or formaldehyde (10% solution), the washed cells were exposed to the chemical agent at room temperature for 10 min and then washed three times with distilled water before contact with uranyl nitrate. To study the effect of metal bolic inhibitors, cells wet exposed to uranium in the presence of 2,4-dinitrophenol (5 x 10 -3 M) or sodium azide (10 -3 M). To test the effect of heat, bacterial suspensions were heated for 10 rain in boiling water, collected by centrifugation,' and used for the experiments.

Release of uranium from EPS-5028 cells. The EPS-5028 cells that had taken up uranium for 15 rain were washed with distilled water and resuspended one, two or three times, with stirring at room temperature, in 0.1 M sodium citrate solution, 0.1 M EDTA solution, 0.1 M Na2CO3 solution, 0.1 M potassium oxalate solution or 0.1 M nitric acid. The uranium content of the biomass was determined following acid digestion~ The dry weights of bacterial biomass were determined. Approximately 4 mg dry weight of cells was digested at 100° C in a water bath for 1 h with 0.5 ml concentrated HNO3. The digests were cooled, made up to5 ml with distilled deionized water and the uranium concentration determined by ICP. Controis were included in aH experiments. In those instances where the cells were exposed to uranium after chemical treatment, they were rewashed three times with deionized distilled water before recontact with uranyl nitrate. Ion effects on the uptake of uranium. The effect of various cations on the uptake of uranium by Pseudomonas sp. EPS-5028 cells was investigated. Cells (dry weight 104.5 rag) were suspended in 50 ml of a solution containing 10-4 M uranium and 10 -4 M of one metal ion. Cadmium, chromium, silver, zinc, arsenic, lead and mercury ions were studied and were added as CdC12, KzCrO4, AgNO3, ZnC12, NazHAsO4.7H20, Pb(NO3)2 and HgClz, respectively. The effect of various anions: HCO~-, SO 2-, SzO33-, HzPO~and HPO42- (applied as sodium salts) on the uptake of uranium was also studied. Precultured Pseudomonas sp. EPS-5028 (dry weight 114.2 mg) was suspended in 50 ml of a solution containing 10 -3 M of one anion and 2.10 -4 M uranium at pH 4.0. The suspensions were stirred continuously for 24 h at 30 ° C. After 5 rain, 1 h and 24 h the cells were collected by centrifugation. The amount of uranium taken up by the cells was estimated from the metal ion contents in the residual solution by ICP. Electron microscopy. The location of metals on the bacterial cells was visualized by transmission electron microscopy. Uranium-exposed cells were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4-7.6) for 2 h at 4 ° C. The specimens were centrifuged (8000 g, 5 min) and washed twice in 0.1 M phosphate buffer. The cells were stained with 1% osmium tetraoxide in phosphate buffer for 1 h at 4 ° C. Cell dehydration was done in a graded series of acetone solutions and the cells were embedded in Spurr resin. Ultrathin sections were cut with a diamond knife on a Reichert Ultracut microtome (Wien, Austria) and mounted on coated 200-mesh copper grids. These were then examined in a Hitachi (Tokyo, Japan) H-800 (H-8010 scanning system) transmission electron microscope at 100 kV. Some sections were stained with 2% uranyl acetate for 30 min.

w o u l d affect t h e a s s o c i a t i o n o f u r a n i u m w i t h b a c t e r i a l biomass. Since p h y s i o l o g i c a l state c a n affect u r a n i u m u p t a k e b y cells, t h e effect o f t h e age o f t h e c u l t u r e o n t h e u p t a k e o f u r a n i u m was s t u d i e d . U p t a k e e x p e r i m e n t s w e r e c a r r i e d o u t b y s u s p e n d i n g 48-, 72- a n d 9 6 - h - o l d cells in 5 0 m l u r a n i u m s o l u t i o n ( 5 0 l x g / m l ) f o r 1 h. T h e amounts of metal removed did not change substantially w i t h age o f t h e culture. A f t e r 1 h, t h e q u a n t i t i e s o f u r a n i u m t a k e n u p b y cells were 23.3, 18.7 a n d 20.5 m g U / g d r y cells r e s p e c t i v e l y . S u b s e q u e n t u r a n i u m a c c u m u l a t i o n e x p e r i m e n t s w e r e p e r f o r m e d w i t h cells f r o m 2 - d a y o l d cultures: U r a n i u m a c c u m u l a t i o n b y cells o f P s e u d o m o n a s sp. EPS-5028 was s t u d i e d o v e r t h e r a n g e 5 - 5 0 0 Ixg U / m l . T h e a m o u n t o f m e t a l t a k e n u p b y n o n - g r o w i n g cells inc r e a s e d r a p i d l y d u r i n g t h e first 5 m i n a n d t h e n inc r e a s e d slightly w i t h time. As t h e m i c r o b i a l b i o m a s s u s e d in t h e s e e x p e r i m e n t s was c u l t u r e d in t h e a b s e n c e o f u r a n i u m a n d was t h o r o u g h l y w a s h e d b e f o r e e x p o sure to u r a n i u m , t h e u r a n i u m u p t a k e m e a s u r e d was c o n s i d e r e d a p r o p e r t y o f the cells a n d n o t a s s o c i a t e d w i t h cell growth. This r a p i d u p t a k e i s s i m i l a r to t h e effect o b s e r v e d b y S t r a n d b e r g et al (1981) w h e n u s i n g P. aeruginosa cells. S i m i l a r o b s e r v a t i o n s w e r e m a d e b y Friss a n d M y e r s - K e i t h (1986) in a s t u d y o f u r a n i u m acc u m u l a t i o n b y Streptomyces longwoodensis. H o w e v e r , o t h e r i n v e s t i g a t o r s h a v e r e p o r t e d s l o w e r rates o f u p t a k e as well as a m o r e c o m p l e x t i m e c o u r s e ( b i p h a s i c ) t h a n t h a t o b s e r v e d in this s t u d y ( S a k a g u c h i et al. 1981; F a i l l a et al. 1976). T h e a m o u n t o f u r a n i u m t a k e n u p b y cells i n c r e a s e d a l m o s t l i n e a r l y w i t h i n c r e a s e in t h e c o n c e n t r a t i o n o f u r a n i u m u p to 100 ~tg/ml a n d r e a c h e d the m a x i m u m at 200 lxg/ml ( F i g . l ) . T h e m a x i m u m a m o u n t o f u r a n i u m t a k e n u p b y cells was 55 m g U / g d r y weight. As s h o w n in Fig. 1, t h e h i g h e s t u r a n i u m a c c u m u l a t i o n r a t i o (98%)

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Results and discussion It has l o n g b e e n k n o w n t h a t m i c r o o r g a n i s m s a c c u m u late m e t a l s . H o w e v e r , in t h e p a s t two d e c a d e s m i c r o o r g a n i s m s h a v e b e e n i n c r e a s i n g l y s t u d i e d for t h e p u r p o s e of removing metals from waste or process solutions for t r e a t m e n t a n d r e s o u r c e recovery. O u r initial c o n c e r n has been focused on the identification of factors that

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500

Uranium in solution ( p g / m l )

Fig. 1. Effect of extracellular uranium concentration on the uptake of uranium by Pseudomonas sp. EPS-5028 after 1 h contact

408

was found at the lowest metal concentration studied. This ratio decreased as the uranium concentration in solution increased. When the initial concentration of uranium was low (Fig. 1) a linear relationship was observed (r = 0.99) between the reciprocal of the amounts of uranium absorbed per unit of weight of biomass and the inverse of the amount of residual uranium concentration in solution at equilibrium (plots not shown). These results indicate that the uptake of uranium by Pseudomonas sp. EPS-5028 obeys the following Langmuir isotherm: Qe=Q°bC/(l+bC), where b is a constant related to the energy or net enthalpy of adsorption, QO is the number of moles of solute adsorbed per unit weight of adsorbent in forming a complete monolayer on the surface, Qe is the amount of uranium adsorbed per unit weight of biomass, and C is the u r a n ium concentration remaining in solution at equilibrium (Gadd et al. 1988). As shown in Fig. 2, the initial solution pH had a significant effect on metal uptake. The lowest rate of uranium uptake was at pH 1.0. Despite differences in the rate of uranium uptake, the total capacity for metal accumulation was very high. At pH 3.0 maximum uranium accumulation was obtained (92% of uranium removed in 5 min). The initial and total uptake of uranium decreased as the pH in increased from 3.0 to 11.0. No effort was made to control the solution pH during the experiments. The pH was gradually modified during 24 h to a value between 6.0 and 7.0 as uranium was adsorbed or complexed by the cells. Initial pHs of 1.0 and 3.0 were not modified by cells during the process. Strandberg et al. (1981) have observed that as uranium is taken up by cells, the pH increases from 4.0 to 5.5-6.0, indicating a release of free hydroxyl ions. They suggest that UO22+ could be the form of the bound metal and, in fact, UO 2+ complexes readily with a variety of anions. We observed the same modification of initial solution pH by cells in the absence of uranium. Thus, the release of free hydroxyl ions is not associated with the presence of uranyl ions in the medium. We associated the pH increase with cellular viability. When high uranium concentrations (>200 ~tg U/ml) were used or the cells were exposed to uranium in the presence of metabolic inhibitors or the cells were pretreated with heavy metals, no cells could be cultured, and the initial pH was not modified during the process. The amount of uranium removed by EPS-5028 cells scarcely varied over a temperature range of 20-50 ° C. After 1 h cell contact with 50 txg U/ml, 23.1 mg U / g dry weight was taken up at 20 ° C, 23.2 mg U / g dry weight at 30 ° C, 23.4 mg U/g dry weight at 40 ° C, and 23.3 mg U/g dry weight at 50 ° C. These results show that the uptake of uranium is temperature-independent and is therefore presumably not directly mediated by any metabolic process. To confirm this, cells were exposed to uranium in the presence of the metabolic inhibitors 2,4dinitrophenol (5 x 10 -3 M) or sodium azide (10 -3 M). As shown in Fig. 3, the uptake of uranium was not affected by treatment with these metabolic inhibitors. The uptake of uranium by EPS-5028 cells is therefore very rapid, not affected by temperature and obeys the Lang-

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Fig. 2. Effect of initial pH on uranium uptake by EPS-5028 cells: O, pH 1.0; A, pH 3.0; n, pH 5.0; *, pH 7.0; ©, pH 9.0; • , pH 11.0

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Fig. 3. Effect of metabolic inhibitors on the uptake of uranium. Uptake experiments were carried out by suspending cells in 50 ml uranium solution at pH 4.2 and 30° C for 1 h. Control, living cells; Ag, AgNO3 (10% solution); Hg, HgC12 (1% solution); DNP, 2,4dinitrophenol (5 × 10 -3 M); A.S., sodium azide (10 -3 M); Q, heatkilled cells; F, formaldehyde (10% solution)

muir isotherm, which suggests that is depends on physicochemical adsorption at the cell surface and not metabolic activity. Bacteria were pretreated for 10 min with continuous stirring at room temperature with HgCla (1% solution), AgNO3 (10% solution) and formaldehyde (10% solu-

409 tion). These pretreatments were lethal for the species (no cells could be cultured from the treated cell preparations). The cellular Hg and Ag concentrations, before and after exposure to uranium, were not determined. As shown in Fig. 3 with mercury and formaldehyde pretreatments, there was a small difference in the uptake. Both treatments could modify some structures involved in uranium uptake. To determine whether EPS-5028 cells take up more uranium after heat treatment they were boiled in a water bath for 10 min (no cells could be cultured after this treatment). When the initial uranium concentration was 50 ktg/ml, the amount of uranium absorbed during 1 h by living (control) and heat-killed cells was very similar. Almost all uranium in solution in the medium was absorbed. With higher uranium concentrations (200500 ~tg/ml) after 1 h contact living cells took up 50 mg U/g cell dry weight and heat-killed cells took up 78 mg U / g cell dry weight. Thus, the uranium accumulation capacity of EPS-5028 cells is increased markedly by heat treatment at high concentrations of uranium. There were differences in the relative efficiency of desorption between the agents used. As Fig. 4 shows, all five treatments removed the bound uranium but in different quantities: Na2CO3 and EDTA were more effective, removing 92.5 and 89%, respectively, of the bound uranium with the first wash. The amount of uranium released was slightly increased with the second and the third washes (98% with Na2CO3 and 95.2% with EDTA). Potassium oxalate (0.1 M), sodium citrate (0.1 M) and nitric acid (0.1 M) removed 80, 74.4 and 41.4%, respectively, of the bound uranium. To determine whether surface binding sites were altered by these treatments, the treated cells were washed and reexposed to uranium. Ammonium-carbonate-treated cells (95.2% of bound uranium removed) did not modify the metal uptake. The effect of various cations on the uptake of uranium by EPS-5028 cells was studied. In the presence of 10-aM of uranium and 10-aM of cadmium, chromium, silver, zinc, arsenic, lead or mercury the uptake of uranium was 5.1-5.3 x 10 -5 mol U/g dry wt. Only after

24 h metal contact in the presence of arsenic was the uptake of uranium by the cells lower (4.4 x 10-5 mol/g dry weight). In the control experiment, without the presence of additional cations, the uptake was 5.15.2 x 10-5 mol U / g dry weight. The presence of anions (10 - 3 M carbonate, sulphate, thiosulphate, hydrogenmonophosphate or dihydrogenphosphate) did not modify the uptake of uranium by EPS-5028. Control cells took up 8.5 × 10 -5 mol U / g dry wt after 5 min and 1 h contact and 8.7 × 10-5 mol U / g dry wt after 24 h. The amount of uranium taken up by the cells in the presence of these anions was the same (8.4-8.7 x 10 -5 mol U/g dry weight). To verify the location of the accumulated uranium, cultures were examined by electron microscopy. Being electron opaque, uranium appeared as areas of darkening when samples were viewed. Bacteria in the absence and in the presence of 50 and 500 lxg U/ml in the medium were examined. A thin section of a uranium-free EPS-5028 cell is shown in Fig. 5a. Significant electron20 ¸

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Fig. 4. Release of uranium from EPS-5028 cells that had been in contact with uranium for 15 min: *, Na2CO3; O, EDTA; zX, HNO3; O, potassium oxalate; rq, sodium citrate

Fig. 5 a, b. Electron micrographs of EPS-5028 resting cells, a Cells in the absence of uranium ( x 50000). b Cell showing intracellular accumulation of uranium after 1 h exposure to 50 ktg U/ml ( x 50000)

410 dense material was present in cells that had contact with uranium. Electron microscopic examinations showed that uranium accumulated intracellularly as needle-like fibrils (Fig. 5b). At higher uranium concentrations and with longer contact times the number of cells with electron-dense particles increased. Although many bacteria possessed large needle-like fibrils, others appeared unloaded. This p h e n o m e n o n has been observed previously by Strandberg et al. (1981), who showed that only 32% o f S. cerevisiae and 44% of P. aeruginosa cells had uranium deposits. G o d d a r d and Bull (1989) also described uneven distribution within a population of bacteria containing silver deposits. Two possibilities were suggested: (a) only a proportion of the bacterial cells is responsible for metal accumulation; (b) metal deposits are removed by attrition, a well-known p h e n o m e n o n in bioreactors. Mullen et al. (1989) also described that the binding o f La was not uniform, particularly in P. aeruginosa samples. There may be physiological reasons why individual cells within a culture do or do not bind metals, although these mechanisms have not been explored. Brierley et al. (1989) consider that intracellular accumulation can be an energy-dependent function requiring active respiration by the microbial cell. Active uptake usually requires a specific transport system. Toxic metals rarely breach an energized plasma membrane unless they pass through specialized transporters to be detoxified within the cell or rapidly p u m p e d out again (Mullen et al. 1989). However, there is no direct evidence for the presence of a uranium transporter in Thiobacillusferrooxidans or in any other microorganism studied (DiSpirito et al. 1983). We do not know how uranium enters the cell so rapidly (metabolism has been discounted). Once inside the cell, uranium appears to be localized. There is circumstantial evidence that the uranium may be b o u n d in the cytoplasmic fraction of Pseudomonas sp. EPS-5028. Treating biomasS with Na2CO3 removed the uranium from the cells, without an apparent effect on the cell surface (there were no changes in subsequent metal uptake) and without loss of viability (cells could be cultured from the treated cell preparation). On the basis of these results, further studies will be undertaken to devise a practical approach to recover uranium from aqueous systems by microorganisms that accumulate uranium in large amounts.

Acknowledgements. We thank Servei d'Espectrosc6pia i Microsc6pia de la Universitat de Barcelona for technical assistance. This work was supported by grant PA86-0299 from the Comisi6n Interministerial de Ciencia y Tecnologia.

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