Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 91-93, 2005

Accepted
Sorption of Eu(III) on Pseudomonas fluorescens in the Presence of Citric AcidYoshinori Suzuki,*, a, b Takuya Nankawa,b Takahiro Yoshida,b Takuo Ozaki,b Toshihiko Ohnuki,bArokiasamy J. Francis,c Satoru Tsushima,a Youichi Enokida,d and Ichiro YamamotoaaDepartment of Materials, Physics, and Energy Engineering, Graduate School of Engineering,
Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8603, JapanbAdvanced Science Research Center, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-1195, Japanc
Environmental Sciences Department, Brookhaven National Laboratory, Upton, New York 11973, USAdEcotopia Science Institute, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8603, Japan
Received: November 15, 2004; In Final Form: April 21, 2005
We studied the sorption of Eu(III) on Pseudomonas fluorescens in the absence and presence of citric acid by abatch method. The cells were placed in a solution containing 2 µM of Eu(III) and 0, 100, or 1000 µM of citricacid at pH 3 – 9 for 5 hours. In the absence of citric acid, almost 100% of Eu(III) was sorbed on P. fluorescens atpHs below 7; above 7, sorption decreased with an increase in pH. The time course of Eu(III) sorption on P. fluorescens showed that a fraction of it was desorbed into the solution at alkaline pHs, suggesting that the bacterium
may release some exudates. With citric acid present, we found that at higher concentrations there was lower sorption of Eu(III), reflecting the formation of Eu(III)-citrate complexes competes with the Eu(III)-cell-surfacecomplexes. This decrease in Eu(III) sorption was significant in alkaline pHs. These findings suggest that citricacid which is ubiquitously found in the environment enhances migration of trivalent actinides in the alkaline environment.

1. Introduction
Naturally occurring organic substances and microorganisms
in the environment interact with heavy metals, including
actinides.1−3 Elucidation of their interactions is important inestimating the mobility of actinides in the environment, and inusing microorganisms to remediate contaminated soils
containing actinides.4, 5 Many investigations have assessed theeffect of the naturally-occurring organic acids, fulvic acid andhumic acid on the sorption of metal ions on inorganic
substances, such as metal oxides and clay minerals.6−8 Theyeither increase or decrease the metal-ion sorption depending on
the pH of the soil solution and properties of the inorganic
substances. For example, fulvic acid increases Yb(III) sorption on alumina in acidic solutions and decreases it in alkaline
solutions.9 The hydrated Yb(III) ion did not significantly sorb
on alumina at acidic pH while it showed a high affinity with itat alkaline pH. The uptake of Yb(III) in acidic solutions wasenhanced due to the sorption of the fulvic acid complexed with
Yb(III) on alumina; in alkaline solutions it decreased due tothe formation of Yb(III)-fulvate complexes that have a low
affinity for alumina. Citric acid, which is a low molecularorganic substance ubiquitously found in the environment,10
also affects the sorption of metal ions on inorganic substances.
Redden et al.11 reported that citric acid enhances the sorptionof U(VI) on goethite, but reduces its sorption on kaolinite atacidic pH. These findings demonstrate that the presence oforganic substances affects the mobility of metal ions.
Sorption behavior of metal ions on microorganisms has
been investigated widely. Several experiments have suggested
that the predominant functional groups on cell surfaces
involved in the sorption of metal cations are carboxyl andphosphate functional groups. 12, 13 However, the effects oforganic acids on metal-ion sorption on microorganisms still are
not clearly understood. Yoshida et al.14 reported the negative*Corresponding author. E-mail: h042306d@mbox.nagoya-u.ac.jp.
FAX: +81-52-789-3785.

effect of a siderophore, desferrioxamine B (DFO), on the sorption of Fe(III), Eu(III), and Hf(IV) on Pseudomonas fluorescens, the degree of which correlated well with the stabilityof the DFO complexes.
In this study, we investigated Eu(III) sorption on P. fluorescens and the effect of citric acid on the sorption by a batch
method. Pseudomonas fluorescens is a ubiquitous soilbacterium. Europium(III) is a good analogue for trivalentactinides, such as Am(III) and Cm(III), which are problematicelements in high-level radioactive waste. We previously
reported that P. fluorescens did not degrade the 2:2 Eu(III)citrate complex at pH 7 when the cell concentration was quite
low.15 In the present study, we examined the pH dependence(3 – 9) of Eu(III) sorption on P. fluorescens at high cell concentrations in the absence and presence of citric acid to assess theeffect of biosorption on the migration behavior of Eu(III) andEu(III)-citrate complexes.
2. Experimental
2.1. Culture. Pseudomonas fluorescens (ATCC 55241)
isolated from the low-level radioactive waste disposal site atWest Valley, NY, USA16 was used. It was aerobically grownin 250 mL of nutrient medium containing the following ingredients per liter: beef extract, 3 g; polypeptone, 5 g; and, NaCl,
5 g. The culture was incubated at 30 ± 1 ˚C on a rotary shaker
at 100 rpm in the dark. Cells were harvested during the earlystationary phase by centrifugation at 10,000 g for 5 min, washedthree times with a 0.1 M NaCl solution, and resuspended in thesame solution.
2.2. Eu(III) sorption on P. fluorescens. One milliliter ofthe cell suspension and 50 mL of a solution containing 2 µM
EuCl 3 , 0.1 M NaCl, 5 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES), and 0 – 1 mM citric acid
were mixed in a 100-mL Erlenmeyer flask. The cell concentration was 167 ± 9 mgdry weight dm−3. The initial pH of the solution was adjusted at 3 – 9 with HCl or NaOH. During thefive-hour exposure of the cells to Eu(III), the solution was© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

92Suzuki

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005
40

Dissolved fraction of Eu(III) /%Dissolved fraction of Eu(III) /%100806040Citric acid
0 µM
10 µM

200234567893020

100102pHagitated by a stirrer. Then, an aliquot of the solution was withdrawn. The optical density at 600 nm (OD600) of the aliquot
was measured as an indicator of cell concentration. The withdrawn aliquot was filtered through a 0.20-µm membrane filter.
The concentration of Eu(III) in the filtrate was analyzed byinductively-coupled plasma-atomic emission spectroscopy(Shimadzu ICPS-7000, Shimadzu Corporation, Kyoto, Japan).
Blank tests, i.e. without cells, were conducted by the sameprocedures.
3. Results and Discussion
3.1. Effect of citric acid on solubility of Eu(III). Figure 1
shows the pH dependence of the dissolved fraction of Eu(III)in the absence and presence of citric acid without bacterial cells.
The pH values were measured after 5 hours of agitation. In theabsence of citric acid, approximately 90% of Eu(III) wasdissolved in solution at pH 3. The percentage decreased slightly
with an increase of pH in the acidic pH region, and had risen toapproximately 75% at pH 7. The dissolved fraction decreased
dramatically with a rise in pH from 7 to 9 when it was almost0%. It was reported that the decline in the dissolved Eu(III)fraction with an increase of pH is due to the formation of Eu(III)precipitates, such as EuOHCO3 or Eu(OH)3.17, 18
In the presence of 10 µM citric acid, at acidic pHs, thedissolved Eu(III) fraction was almost comparable to that in thesolution without citric acid, but above pH 6, the dissolved fraction rose. At alkaline pHs, the formation of stable Eu(III)citrate complexes prevented the formation of insoluble Eu(III)species. These results signify that solubility of Eu(III)increases in the presence of citric acid at neutral and alkalinepHs.
3.2. Eu(III) sorption on P. fluorescens in the absence ofcitric acid. Open circles in Figure 2 represent the pH dependence of the dissolved fraction of Eu(III) in the absence and presence of citric acid at a cell concentration of 167 ± 9 mgdry weightdm−3. Europium(III) was not detected in solution below pH 7,
showing that almost 100% of dissolved Eu(III) was sorbed onP. fluorescens at these pHs. The sorption fell slightly with anincrease of pH above pH 7. The sorption of Eu(III) on minerals
has been widely investigated.19, 20 Goethite and boehmite did
not greatly sorb Eu(III) below pH 6, though significant sorption
was observed above it.21 On the other hand, P. fluorescens
showed a substantially higher affinity with Eu(III) at acidic pH
under the present experimental conditions. It was reported thattransport of P. fluorescens was highly retarded in the porous
medium, such as alluvium sand, quartz sand, or silica beads.22−24
These facts indicate that P. fluorescens may play an important345pH

6789Figure 2. The pH dependence of the dissolved fraction of Eu(III) in
the presence of 167 ± 9 mg dry weight L−1 cells. The solutions contained 2µM Eu(III), 0.1 M NaCl, 5 mM HEPES, and 0, 100 or 1000 µM citricacid. The aqueous phase was separated with a 0.20-µm membranefilter.

10Dissolved fraction of Eu(III) /%Figure 1. The pH dependence of the dissolved fraction of Eu(III) in
the absence of cells. The solutions contained 2 µM Eu(III), 0.1 M
NaCl, and 5 mM HEPES with or without 10 µM citric acid.

Citric acid0 µM
10 µM
1000 µM

Initial pHpH 3.0pH 9.7864200123456Time /hFigure 3. The time dependence of the dissolved fraction of Eu(III) inthe presence of 158 mg dry weight L−1 cells at pH 3.0 and 9.7. The solutions contained 2 µM Eu(III), 0.1 M NaCl and 5 mM HEPES. The pHof solutions changed from 3.0 and 9.7, to 3.1 and 8.4, respectively,during 5 hours of agitation.

role in regulating the migration of Eu(III) in an acidic environment, wherein those minerals show less association with Eu(III).
Haas et al.12 reported that U(VI) sorption on Shewanella
putrefaciens decreases at alkaline pH due to formation ofUO2(CO3)34− and UO2(OH)3−. These species are repulsed by
the cell surface that is negatively charged at alkaline pH. In
this study, the decrease in Eu(III) sorption is not attributable tothe formation of anionic complexes because concentrations ofdissolved anionic hydrolytic and carbonate Eu(III) species atpH 7–9 were calculated to be less than 0.02 µM,25 which corresponds to < 1% of the total Eu(III) concentration present insolution.
Figure 3 shows the time dependence of the dissolved fraction of Eu(III) in the presence of 158 mgdry weight dm−3 cells at
pHs 3.0 and 9.7. In the solution whose initial pH was 3.0(final pH, 3.1), 94% of Eu(III) was sorbed on P. fluorescens
within 5 min, wherein the amounts of sorbed Eu(III) increasedwith time. After 5 hours contact, almost 100% was sorbed on
the cells. In the solution whose initial pH was 9.7 (final pH,8.4), 97% of Eu(III) was sorbed on P. fluorescens within 5min; thereafter, the amount declined with time, and after 5hours contact, the percentage of Eu(III) sorbed was 92%. The
OD600 value of the solution before and after the 5 hours contact

Sorption of Eu(III) on Pseudomonas fluorescens

were 0.455 and 0.450, respectively, showing the cell concentration in the solution was almost constant during the contact.
Ozaki et al.26 observed that the desorption of Eu(III) originallysorbed on Chlorella vulgaris occurred via exudates from thecells. In the present study, the decreasing trend in Eu(III) sorption with time at alkaline pH suggested that P. fluorescens
excreted some organic substance which desorbed Eu(III) on
the cell surface by complexation with Eu(III).
3.3. Eu(III) sorption on P. fluorescens in the presence ofcitric acid. Figure 2 shows the pH dependence of the dissolved
fraction of Eu(III) in the presence of citric acid at a cell concentration of 167 ± 9 mgdry weight dm−3. With 100 µM citric acid,Eu(III) was almost completely sorbed on the cells below pH 7;
Eu(III) sorption decreased by 2 – 8% compared to that in theabsence of citric acid above pH 7. In the presence of 1000 µM
citric acid, Eu(III) was completely sorbed on the cells at pH 3,
and the sorption of Eu(III) decreased with a rise in pH. Thisdecrease in Eu(III) sorption on P. fluorescens with an increaseof citric-acid concentration suggests that citric acid competes
for Eu(III) with functional groups on the cell’s surface. The fallin Eu(III) sorption on the cells was significant at alkaline pH,
suggesting that stability of Eu(III)-citrate complexes increases
with a rise in pH, and this increasing tendency is greater thanthat of Eu(III)-cell-surface complexes. Thus, citric acid apparently reduces the sorption of trivalent actinides on microorganisms, especially at alkaline pHs, by forming stable complexes
with them.
4. Conclusions
We investigated Eu(III) sorption on P. fluorescens and theeffect of citric acid on the sorption by a batch method. The cellwalls of P. fluorescens displayed a high affinity for Eu(III) inacidic pHs, indicating P. fluorescens retained in geomedia may
retard the migration of trivalent actinides in the acidic environment. In alkaline pHs, sorption of Eu(III) on P. fluorescens wasinhibited by exudates released from the cells. In the presence ofcitric acid, citric acid competes for Eu(III) with functionalgroups on the cell’s surface, consequently, sorption of Eu(III)decreased in alkaline pHs. These findings suggest that the bothreactions, the biosorption and the complex formation withorganic acids, are important for estimating the behavior of trivalent actinides in the environments.
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