Elimination of Bicarbonate Intederence in the Binding of U(Vl) in Mill-Waters to Freeze-dried Chlorella vulgaris

Benjamin Greene, Michael T. Henzl. J. Michael Hosea, and Dennis W. Darnall Department of Chemistry, Box 3C, New Mexico State University, Las Cruces, New Mexico 88003

Accepted for publication August 15, 7985

Freeze-dried preparations of Chlorella vulgaris will ac- cumulate U(VI) from alkaline, bicarbonate-containing waters collected from uranium mill process streams, pro- vided that the pH is pre-adjusted to between 4.0 and 6.0. Bicarbonate ion complexes the uranyl ion in these waters and seriously interferes with the binding of U(VI) to the algal cells at pH values above 6.0. No binding of U(VI) to the algae occurred at the natural pH of 8.0 when Chlorella vulgaris was suspended in untreated mill-waters con- taining up to 2.5 x 10-4M U(VI). However, when the pH of these waters was lowered from 8.0 to near 5.0, with nitric acid, nearly quantitative binding of U(VI) to the alga was achieved. Binding is rapid and largely unaffected by ions including Na', CI-, NOT, -OAc, and SO:-. Our re- sults indicate that provided steps are taken to eliminate bicarbonate interference, such as adjustment of the pH to near 5.0, dried algal biomass could prove useful for the removal and recovery of U(VI) from high carbonate- containing waters.

The accumulation of U(V1) from aqueous solutions by microorganisms has been the subject of a number of Live cultures, dried preparations, and cell fragments of various microorganisms (algae, yeast, or bacteria) including Chlorella regularis,6.8 Rhizopus arrhizus,1-3,9,10 Streptomyces viridochromogenes,' Penicillium d i g i t a t ~ m , ~ ? ~ . ' ' , 1 2 Zoogloea ramigera,' and Chlorella vulgar i~'~ all exhibit high binding capacities for U(V1) under controlled conditions. Different mech- anisms for the accumulation of U(V1) by microorga- nisms have been proposed, but there is general agree- ment that the binding involves complexation of uranyl ion by ligands in or on the cell surface.

While the exact nature of the binding sites probably varies with the cell wall composition of the ~ r g a n i s m , ' ~ there seem to be similar ion-exchange principles in- volved with any microorganism to which U(V1) binding has been reported. For example, some workers have observed that algal-bound U(V1) can be stripped from the cells by treatment with sodium carbonate solu- tions.8,'2,'6 This is a consequence of the fact that car-

bonate forms exceptionally stable complexes with U(V1) (log p3 = 16.2)." Thus, the presence of carbonate or bicarbonate presents a major restriction in the indus- trial use of microorganisms for accumulation of U(V1) from aqueous solutions.

A method for the removal of U(V1) from aqueous solutions which utilizes the ion-exchange like prop- erties of biomass might prove to be an attractive al- ternative to the use of commercial ion-exchange resins. However, few reports have demonstrated biosorption of U(V1) from authentic uranium-containing waters. Nakajima and co-workers8 reported high yields in the binding of U(V1) to Chlorella regularis and Strepto- myces viridochromogenes from seawater media. Brier- ley and Brierley14 obtained poor binding of U(V1) to Spyrogyra or Chama in uranium mill wastewaters. Our experiments with Chlorella vulgaris and uranium mill waters that contained bicarbonate indicated that no binding to the alga occurred at the natural pH of the waters, near pH 8. Herein, we describe experiments that we performed to determine under what conditions the binding of U(V1) to Chlorella vulgaris in uranium mill waters could be improved.

EXPERIMENTAL

Materials

Chlorella vulgaris was cultivated outdoors in stain- less-steel or polyurethane tanks. The growth medium contained KH2P04 (0.3 g/L), MgS04-7H20 (0.3 g/L), KN03 (2.0 g/L), and urea (0.6 g/L) in tap water. The pH was maintained near 7.0 with carbon dioxide. The cells were harvested by centrifugation, dialyzed against deionized-distilled water (to remove salts), and lyo- philized. A composite stock was made by combining several batches and sieving with a 40-mesh screen. The

Biotechnology and Bioengineering, Vol. XXVIII, Pp. 764-767 (1986) 0 1986 J o h n Wiley & Sons, Inc. CCC 0006-3592/86/050764-04$04.00

biomass was stored in a sealed container in a freezer ( - 20°C).

Stock solutions of 1 .OOOmM U(V1) were prepared by dissolving UO2(C2H3O2),-2H2O in deionized-dis- tilled water or in 0.05M sodium acetate (pH 5). Solu- tions of lower U(V1) concentrations were prepared daily by dilution of the stock solutions. All solutions were stored in acid-washed plastic containers.

Several water samples were obtained from uranium ore process streams at United Nuclear Homestake Mining Corporation (near Grants, NM). The compo- sition of the water samples is described in Table 1. All water samples were stored in plastic containers at 4°C.

Methods

All studies were performed using freshly washed al- gae. Washing was performed by suspending the dried algae in a O.05M sodium acetate buffer at either pH 2.0 or 8.0, agitating, centrifuging, and decanting the supernatant. This procedure was repeated twice to in- sure complete removal of extracellular material that might interact with U(V1) in solution. Experiments were performed by adding a uranium solution to the washed algal pellet and agitating for a designated time (usually 0.25-2 h). The reaction mixture, or an aliquot thereof, was centrifuged and the supernatant was decanted into plastic test tubes. Control experiments showed that no loss of U(V1) from the solutions occurred as a result of precipitation by extracellular materials or as a result of pH adjustments.

Instrumentation

Uranium concentrations in the supernatant solutions were determined using a Spectrametrics V direct- current argon plasma emission spectrophotometer (DCP). All instrumental parameters were opti- mized, including argon pressure and flow rates, slit widths, and viewing zone at the 424.2 nm wavelength.

Table I. area of New Mexico.

Composition of mill waters collected in the Ambrosia Lake

Uranium (VI) Bicarbonate Water sample PH (M) (M)

A (mill water) 8.0 1.3 x 10-4 0.180 B (mill water) 8.0 7.8 x 10-5 0.017 C (mill water) 8.0 4.2 X 0.01 1 D (mill water) 8.0 2.5 x 10-4 0.039

The pH, uranium (VI) concentrations, and bicarbonate concen- trations of mill waters collected from the United Nuclear Homestake Mining Corporation near Grants, NM, are shown. Samples were collected in plastic containers and stored in the laboratory at 4°C. The pH was measured with a glass electrode, uranium (VI) deter- mined by DCP spectrophotometry, and bicarbonate was determined by titration. The samples were also known to contain ca. 0.03M sulfate.

5 t \

a0 01 a2 Salt C o n c e n t r a t i o n , M

Figure 1. Effect of different salts on binding of U(V1) to Chlorella vulgaris from sodium acetate solutions at pH 8. Washed Chiorella vulgaris was suspended for 2 h at I .5 mdmL in solutions that con- tained 0.05M sodium acetate at pH 8, 0.lmM U(V1) acetate, and a different salt concentration. The salt solutions used were (0) sodium acetate, (0) sodium bicarbonate, (0) sodium chloride, (B) sodium hydrogen phosphate, (A) sodium nitrate, and (A) sodium sulfate.

Emission signals were usually integrated for 10 s , and 5 replicate measurements were obtained for each anal- ysis and recorded by a Texas Instruments Silent 700 Recorder.

RESULTS AND DISCUSSION

The Effect of Salts on U(VI) Binding to Chlorella vulgaris

The binding of U(V1) to Chlorella vulgaris was ex- amined in the presence of different salts at pH 8.0. Sodium salts of acetate, sulfate, chloride, and nitrate, at concentrations as high as 0.2M, did not interfere with the binding (Fig. 1 ) . However, bicarbonate and, to a lesser extent, phosphate strongly inhibited the binding under the same conditions. When the U(V1) concentration was 0.1 mM, levels of bicarbonate above ImM reduced the binding of U(V1) to the algae by greater than 95%. At a concentration of 0.2M, phos- phate reduced the binding of U(V1) to the algae by ca. 70%.

n 1.0- z = 0 0.81

0 U 0.21

z u 1 2 3 4 5 6 7 8 9 1 0

LL

PH

Figure 2. The effect of pH on the binding of U(V1) to Chlorella vuigaris. Chlorella vulgaris, 1.5 mg/mL, was reacted for 2 h with 0.lmM U(V1) acetate in (0) 0.05M sodium acetate or (B) 0.5M sodium bicarbonate at different pH. Adjustments in pH were made with nitric acid or sodium hydroxide.

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The Effect of pH on U(W) Binding to Chlorella vulgaris

Bicarbonate interference is pH dependent, as illus- trated in Figure 2. Shown for comparison is the pH profile for U(V1) binding to Chlorellu vulgaris from sodium acetate solutions. In both solutions, the de- creased binding at low pH values probably reflects protonation of weakly basic coordinating groups on the algal surface. In the bicarbonate solutions, the weak- ened interaction between the algae and U(V1) is prob- ably due to the formation of stable U(V1)-carbonate complexes (log p2 = 16.3)." Acetate is a much weaker ligand than carbonate for coordination to U(V1) (log p2 = 4.4),20 which suggests why strong binding of U(V1) persists to higher pH values in acetate solutions than in carbonate solutions. At pH values much above 8.0 in the acetate solutions, the formation of stable U(V1)- hydroxide complexes probably results in weakened in- teraction of U(V1) with coordinating groups on the algae.

These observations have enormous practical signif- icance. They show that algal biomass will be incapable of removing U(V1) from carbonate-containing waters unless the pH of the water is decreased to ca. 5. This behavior is depicted in Figure 3, which demonstrates the removal of U(V1) from authentic mill water samples as a function of pH. Uranium is removed to a very limited extent by the algae near pH 8, the natural pH of the mill waters. However, as the pH is decreased by addition of concentrated nitric acid, binding of U(V1)

+ - . I m

+ I

a3 CJ C 0

I

c 5.0 1

; 1 .A I 7

0.0- 30 60 90 120 = o Time, minutes

Figure 4. Kinetics of U(VI) binding to Chiorella vulgaris in mill water. Washed Chlorella vulgaris was suspended at 1.5 mg/mL in mill water (sample B, Table I), which was acidified to pH 5.0 with nitric acid. The mixture was stirred rapidly and aliquots were with- drawn at the times indicated, centrifuged (6 min at 3600 rpm) and decanted. The dotted line is an extrapolation of the graph to time zero.

to the algae occurs in a much higher yield. Near pH 5 , recoveries of U(V1) from the mill waters averaged better than 90% at an algal concentration of 5 mg/mL.

Kinetics in Binding of U(VI) to Chlorella vulgaris in pH-adjusted Mill Water

The U(V1) was rapidly adsorbed by Chlorellu vul- guris from mill water samples that were adjusted to pH 5 . Figure 4 shows that more than 90% of the U(V1) was removed from a typical mill water sample within 10 min at pH 5.0, employing an algae concentration of 1.5 mg/mL. The sample contained 0.12mM U(V1) and 0.18M bicarbonate. These results are in agreement with other reports on the kinetics of binding of U(V1) to microorganisms.8

u) '0 7 15-01

I- I

10.ot c

7

X 3Q01i i v-1 3

0.0 5 6 7 8

PH Figure 3. Effect of pH on binding of U(V1) to Chlorelln vulgaris from mill-water and bicarbonate solutions. Algal biomass was sus- pended for 2 h at 5.0 mg/mL and different pH in mill water samples that contained U(VI), and in O.05M sodium bicarbonate that con- tained (0) 0.lmM U(V1) acetate. The U(V1) concentrations and the bicarbonate concentrations in the mill water samples from the United Nuclear Homestake Mining Corporation (near Grants, NM) were (0) 0.13mM U (VI) and 0.18M bicarbonate, (0) 0.78 mM U(V1) and 0.17 M bicarbonate, (W) and 0.042mM U(V1) and O.OIIM bicarbo- nate. The initial pH of the mill water samples was 8.0 for all.

c 0 - 20-oll 2 10.0 0 c 0 0 3 OD

0 5 10 mg/rn l A lgae

Figure 5. Binding of U(V1) in mill water to Chlorella vulgaris as a function of algae concentration. Washed Chlorelln vulgaris was sus- pended for 2 h in U(V1)-containing mill water samples A (0) and D (0) (described in Table I), which were acidified to pH 5.0 with nitric acid.

766 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 28, MAY 1986

The Effect of Algal Mass on U(VI) Binding in pH-Adjusted Mill Water

Increasing amounts of Chlorella vulgaris were added to mill water samples that were adjusted to pH 5.0. The results are presented in Figure 5. An algal con- centration of ca. 2.5 mg/mL was sufficient to reduce the U(V1) concentrations by greater than 90%. Other experiments (shown in Fig. 3) indicated that no binding of U(V1) occurred under similar conditions when the pH of the mill-waters was maintained at the natural pH of 8.0.

We have also successfully employed a contact sys- tem which incorporates polyacrylamide-immobilized Chiorelfa vulgaris for the removal of U(V1) from pH adjusted mill waters. Our results with the binding and the stripping (with bicarbonate solutions) are in agree- ment with the results of Nakajima and co-workers,8 who used immobilized Chlorella regularis and Strep- tomyces viridochrornogenes for the removal of U(V1) from aqueous solutions.

CONCLUSIONS

The binding of U(V1) to Chlorella vulgaris was greatly inhibited by bicarbonate at pH values above 6.0. In fact, other workers have exploited this phenomenon by using bicarbonate to strip bound U(V1) from bio- mass derived from several different microorganisms. Since significant concentrations of bicarbonate are often present with U(V1) in waters from uranium mining and milling operations, the use of Chlorella vulgaris, and probably other microorganisms as well, to adsorb U(V1) is ineffective when the natural pH of the waters is near 8. However, when the pH of the waters is adjusted to near 5 .O, the interference of bicarbonate complexation of U(V1) is virtually eliminated. This is probably be- cause at pH values lower than ca. 6.3 (the pK, value for carbonic acid),*] the interfering ligands will be protonated.

Algal biomass was very effective in removing U(V1) from mill waters having high bicarbonate concentra- tions, provided that the samples were adjusted to pH 5.0. Initial U(V1) concentrations ranging from ca. 4.2 x 10-SM to 2.5 x lOP4M were rapidly reduced by 90-95% with a single algae treatment at 5 mg/mL. The high binding capacity of the algal biomass com-

bined with the estimated low cost of biomass produc- tion makes dried Chlorella vulgaris an attractive method for recovery of U(V1) under these conditions.

The authors gratefully acknowledge financial support from the New Mexico Water Resources Research Institute. Tech- nical assistance by Jerold Kacsir, Adriana Uranga, and Cathy Hernandez, and Bob Gholson, who provided the water sam- ples, is also gratefully appreciated.

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