Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 77-80, 2005

Accepted
Adsorption of Th(IV) and Pu(IV) on the Surface of Pseudomonas fluorescens and Bacillus
subtilis in the Presence of Desferrioxamine Siderophore
Takahiro Yoshida,*, †, a Takuo Ozaki,a Toshihiko Ohnuki,a and Arokiasamy J. Francisb
aAdvanced Science Research Center, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-1195, Japan
Environmental Sciences Department, Brookhaven National Laboratory, Upto, New York 11973

b

Received: November 15, 2004; In Final Form: April 21, 2005
Adsorption of Th(IV) and Pu(IV) on a Gram-negative bacterium Pseudomonas fluorescens and a Gram-positivebacterium Bacillus subtilis in the presence of siderophore desferrioxamine B (DFO) was studied. Thorium(IV)and Pu(IV) were dissociated from DFO during adsorption on the cells. Thorium(IV) adsorption on bacterial cells
in the presence of DFO was larger than that of Pu(IV) because of the smaller stability of the Th(IV)-DFO complex
than that of the Pu(IV)-DFO complex. On the other hand, adsorption of Pu(IV) was larger than that of Fe(III),
wherein the stability of the Pu(IV)- and Fe(III)-DFO complex is comparable. P. fluorescens showed a higher
affinity for Th(IV) and Pu(IV) than B. subtilis, though potentiometric titration of bacterial cells indicated that
surfaces of P. fluorescens and B. subtilis cells showed similar proton binding properties.

1. Introduction

2. Experimenl

Migration behavior of actinides in the environment is of great
concern in assessing the safety of radionuclide waste disposal.
Chelating reagents, derived from biological activity and industrial drainage, have potential to enhance solubility of actinides
through complexation.1, 2 Siderophores are natural chelating
reagents, which are produced by microorganisms. They form
strong complexes with otherwise insoluble Fe(III) to supply
bioavailable Fe as a siderophore complex.3, 4 Siderophores also
form strong complexes with tetravalent actinides and enhancetheir solubility.5−7
Microorganisms adsorb metals complexed with organic
ligands8−10 or degrade metal-organic ligands complexes.11 The
Fe(III)-siderophore complex is not adsorbed on the cell surfacesof microorganisms, such as Pseudomonas fluorescens.10 On theother hand, some microorganisms, such as Escherichia coli and
Bacillus subtilis take up the Fe(III)-siderophore complexes via
their transport systems in the cell membrane.12, 13 John et al.
revealed that the Pu(IV)-siderophore complex was recognized
by Fe-siderophore uptake systems of Microbacterium flavescens.14
However, interactions of actinides-siderophore complexes withthe cell surfaces of microorganisms are not fully known.
In this study, we examined adsorption behavior of Th(IV) andPu(IV) on microorganisms in the presence of a siderophore to
elucidate the influence of microorganisms on the mobility oftetravalent actinides complexed with a siderophore. A Gramnegative bacterium Pseudomonas fluorescens and a Gram-positive bacterium Bacillus subtilis, both of which are ubiquitous in
the environment, were used in this study. We conducted batch
experiments for the adsorption of Th(IV) and Pu(IV) on bacteria
in the presence of a linear trihydroxamate siderophore, desferrioxamine B (DFO). Bacterial cell surfaces were characterized
by means of potentiometric titration in order to estimate thebinding property of the cell surfaces of these two bacteria.

2.1. Reagents. Desferrioxamine B was purchased from
Sigma Co. Ltd. and used without further purification. A Pu(IV)stock solution was prepared by purification using anion exchange
resin (Dowex 1-X8) to remove Am. The concentration of Pu(IV)in the stock solution was analyzed by UV-visible spectroscopy.
Plutonium was handled in a glovebox. Thorium(IV) nitrate waspurchased from Yokosawa Chem. Co. Ltd. and used without
further purification.
2.2. Culture. Pseudomonas fluorescens (ATCC 55241) andBacillus subtilis (IAM 1069) were grown in rich media containing the following ingredients: beef extract, 3 g L−1; polypeptone, 5 g L−1; and NaCl, 5 g L−1. They were incubated for 2days at 30 ˚C on a rotary shaker at 100 rpm. The cells were
harvested at the stationary phase by centrifugation at 10000 g
for 5 minutes and washed twice with a 0.1 M NaCl solution.
2.3. Adsorption experiments. The adsorption of Th(IV)and Pu(IV) in the presence of DFO by P. fluorescens and B.
subtilis was measured at pH 3–9 by batch methods. Bacterial
cells were contacted with 20 µM of the 1:1 Th(IV)-DFO or
Pu(IV)-DFO complexes in 1.5 mL of 0.1 M NaCl solution
containing 1 mM tris(hydroxylmethyl)aminomethane (Tris) incentrifuge tubes composed of polypropylene. Biomass used in
the experiments was 0.48 – 0.89 g L−1 on a dry weight basis.
After adjusting the pH of the suspensions by HCl or NaOH,
the suspensions were left to stand at room temperature.
Amounts of Th(IV) and Pu(IV) adsorbed on bacterial cells
were measured after 3 hours of contact: our preliminary
kinetics experiments showed that the adsorption of theseelements reached equilibrium within 3 hours. After separating
the cells by centrifugation at 5200 g for 10 minutes, concentrations of Th and Pu in the supernatant were analyzed. Thorium
was analyzed by inductive coupled plasma mass spectroscopy(ICP-MS) (Hewlett Packard, HP4500) and Pu was analyzed byliquid scintillation counting.
Adsorption of DFO bound with Th(IV) on bacterial cells was
also examined by the following procedures. P. fluorescens
cells or B. subtilis cells were contacted with 20 µM of the 1:1
Th(IV)-DFO complex. After separating the cells by centrifugation, DFO remaining in the supernatant was analyzed by highpressure liquid chromatography-electrospray ionization-mass
spectrometry (HPLC-ESI-MS) as Fe(III)(HDFO)+ after allowing*Corresponding author. E-mail: yoshida-takahiro@aist.go.jp.
FAX: +81-29-861-3643.
†Present address: Research Center for Deep Geological Environments
National Institute of Advanced Industrial Science and Technology
Higashi 1-1-1, Tsukuba 305-8567, Japan© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

Yoshida

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

DFO to complex with Fe(III) by adding 0.5 mL of 100 µM
Fe(III)(NO 3 ) 3 solution to 0.5 mL of the supernatant. The
concentration of the Fe(III)-DFO complex was determined by
HPLC (WATERS, alliance 2695) using an Xterra C 1 8
column (WATERS) with the mobile phase of a mixture of
0.2% (w/v) formic acid and 7.5% (v/v) methanol. For ESI-MS
detection, the single-ion recording (SIR) mode was used forFe(III)(HDFO)+ ion at m/z = 614.3 by using mass spectrometer
ZQ2000 (WATERS). Adsorption of 20 µM Pu(IV) on P. fluorescens cells in the presence of an excess DFO concentration(200 and 2000 µM) was also done.
2.4. Potentiometric titration. The surface of P. fluorescens and B. subtilis cells was characterized with regard to
the site density of the cell surface by means of potentiometrictitration. Bacterial cell suspensions containing 3.0 gdry weight L−1
P. fluorescens cells or 3.3 gdry weight L−1 B. subtilis cells in 50
mL of a 0.1 M NaCl solution were titrated by stepwise addition of 0.01 mL of 0.1 M HCl or carbonate-free 0.1 M NaOH
at 3 minutes intervals in an Ar atmosphere at room temperature.
Proton dissociation from the cell surface can be describedby the following eq 1
HL0 = H+ + L−,(1)

Pu, P. fluorescens
Th, P. fluorescens
Pu, B. subtilisTh, B. subtilis

60

50
Eu(III)40
Γ (µM g -1 )

78

30

20

10
Fe(III)
0
2

3

4

5

6

where w is the dry weight of the bacterial cells per liter (g L−1);
Ca and Cb are concentrations (M L−1) of the added acid andbase, respectively; and [H+] and [OH−] are the concentrationsof H+ and OH− (M L−1) calculated from the pH measured. The
concentration of proton binding sites (LT) on the cell surface
can be described by the following mass action eq 3
Ka = [L− ][H+]/[HL0 ],(3)

where Ka is the acidity constant of proton binding sites. The
concentrations of proton binding sites and their pKa values
were calculated using the computer program FITEQL 4.0.15
We adopted a three-site model, which has been widely used forcharacterization of the bacterial cell surface.16, 17
2.5. Speciation of Fe(III) and Th(IV) in DFO system.
Speciation of Fe(III) and Th(IV) in DFO ligand was calculated
at pH 3 – 9. Protonation and deprotonation constants of the
Fe(III)- and Th(IV)-DFO complexes were used from literatures.18, 19 Hydrolysis of Fe(III) and Th(IV) is quite strong inaqueous solution. Metal hydroxide species Th(H −1 DFO),
hydroxylated species of the Th(IV)-DFO complex, was considered in the calculation. However, hydrolysis of Fe(III)-DFO
complex was not considered in the calculation because we did
not found any thermodynamic data of the hydrolysis of thecomplex.
3. Results
Figure 1 shows adsorption density of Th(IV) and Pu(IV) on
bacterial cells in the presence of DFO. Adsorption of Pu(IV) on
P. fluorescens cells increased from 3 to 19 µM g−1 with a decreaseof pH from 7.3 to 3.0 while adsorption of Pu(IV) on B. subtilis
was smaller than 3 µM g−1 at about pH 3 – 8. Adsorption ofTh(IV) on P. fluorescens cells and B. subtilis cells was larger
than that of Pu(IV) on each species. It increased with a decreaseΓ (µM g -1)

(2)

9

20
20 µM DFO
200 µM DFO
2000 µM DFO

15= (Ca − Cb + [OH−] − [H+])/w,

8Figure 1. Adsorption density of Th(IV) and Pu(IV) on P. fluorescens
and B. subtilis in the presence of DFO. Initial concentrations ofFe(III), Eu(III), Th(IV), Pu(IV), and DFO were 20 µM. Data for
Fe(III) and Eu(III) are plotted based on the reference 10.

where L − is the amount of proton binding sites on the cellsurface. The net surface charge excess expression for a given
amount of cells is equal to the difference between the total
base added and the equilibrium H+ and OH− ion concentration
at any given point of the titration curve calculated by eq 2,
Charge excess (M gdry weight−1)

7

pH

10

5

0

2

3

4

5

6

7

8

9

pH

Figure 2. Adsorption density of Pu(IV) on P. fluorescens cells at
various DFO concentrations.

in pH.
We also measured percent adsorption of Th(IV) and DFO in
20 µM of the 1:1 Th(IV)-DFO complex solution on 1.7 gL−1 P.
fluorescens cells or 1.6 gL − 1 B. subtilis cells at pH 5.5,
showing that P. fluorescens adsorbed approximately 97% ofTh(IV) and 2% of DFO, while B. subtilis adsorbed approximately 91% of Th(IV) and 7% of DFO.
Figure 2 shows adsorption of Pu(IV) on P. fluorescens cells
at 20, 200, and 2000 µM DFO. Adsorption density of Pu(IV)on P. fluorescens cells shows a decreasing tendency with anincrease of the DFO concentration from 20 to 2000 µM.
Figure 3 shows charge excess plots of cell surfaces andcalculated fitting curves. The point of zero charge (pzc) of P.
fluorescens and B. subtilis surfaces is 7.2 and 7.5, respectively.
The calculated concentrations of proton binding sites and pKavalues are listed in Table 1 Calculated weighted sum of squares
divided by degrees of freedom (WSOS/DF) values, which canbe used as an indicator of the reliability of fitting of potentiometric titration curve drawn by FITEQL, for P. fluorescens
and B. subtilis were 1.0 and 0.7, respectively. Fitting procedures with a WSOS/DF smaller than 20 can be considered toJ. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

Adsorption of Th(IV) and Pu(IV) on the Surface
80

Charge excess (µM g -1)

4a). The predominant Th(IV)-DFO complex species at pH 3 – 7
is Th(IV)(HDFO)2+ (Figure 4b). Above pH 7.8, more than 50%of the Th(IV)-DFO complex is present as Th(IV)(DFO)+. Five
percent of the Th(IV)-DFO complex is present as metal hydroxide species, Th(H −1DFO), at pH 9. At acidic pH, Th(IV)(HDFO)2+
is protonated to Th(IV)(H2DFO)3+.

P. fluorescens (exp)
P. fluorescens (calc)
B. subtilis (exp)
B. subtilis (calc)

60

79

40

4. Discussion
20

0

-20

-40

3

4

5

6

7
pH

8

9

10

11

Figure 3. Surface charge excesses of P. fluorescens and B. subtilis
cells. Solid and dotted lines represent best-fitting curves based on thethree-site model.

TABLE 1: Summary of Concentrations (LT) and pKa Values
for the Proton Binding Sites on the Surfaces of P. fluorescens
and B. subtilis Cells
P. fluorescens

functional groups
−1

pKa
4.8
6.8
10.5

LT (µmol g )
61
30
81

carboxyl
phosphateamine

LT (µmol g−1)
56
28
75

carboxyl
phosphateamine

B. subtilis
pKa
4.6
7.1
10.4

be reliable.15 pKa values showed that these species had acidic(4.6 – 4.8), neutral (6.8–7.1), and basic (10.4–10.5) functionalgroups. Concentrations of these proton binding sites on these
two species were almost comparable to each other.
Figure 4 shows calculated speciation of the Fe(III)- andTh(IV)-DFO complex at pH 3–9. The predominant species of
the Fe(III)-DFO complex at pH 3 – 8 is Fe(III)(HDFO)+ andfraction of protonated Fe(III)(H2DFO)2+ is negligible (Figure

Thorium(IV) was adsorbed on P. fluorescens and B. subtilis,
while DFO was not adsorbed on them, indicating that theTh(IV)-DFO complex dissociated during the adsorption ofTh(IV) on the cells. This finding is in agreement with theresult of the Eu(III) adsorption on P. fluorescens cells in thepresence of DFO: the Eu(III)-DFO complex dissociated andonly Eu(III) adsorbed on P. fluorescens cells.10 We did notanalyze the DFO concentration in the Pu(IV)-DFO complex
solution because of analytical constrains. The decreasing
tendency of Pu(IV) adsorption on P. fluorescens cells with anincrease of DFO concentration (Figure 2) suggests that thesurface of P. fluorescens cells competes with DFO for Pu(IV).
This finding indicates that the affinity of hydrated Pu(IV) withbacterial cells is higher than that of the Pu(IV)-DFO complex,
suggesting the dissociation of the Pu(IV)-DFO complex during
the Pu(IV) adsorption.
In Figure 1, adsorption of Fe(III) and Eu(III) on P. fluorescens cells in the presence of DFO is shown. The lines areillustrated based on published data.10 Adsorption of Fe(III) on
P. fluorescens is significantly smaller than that of Th(IV) andPu(IV). Europium(III) adsorption is higher than those ofTh(IV) and Pu(IV). Stability constants of the metal-DFO
complexes decrease in the order of Pu(IV) (log K = 30.8)5 >Fe(III) (log K = 30.6)18 > Th(IV) (log K = 26.6)19 > Eu(III) (log
K = 15).6 Adsorption density of Eu(III), Th(IV), and Pu(IV)on P. fluorescens cells decreased in the order Eu(III) > Th(IV)> Pu(IV), which corresponds to the increasing order of thestability constant of the DFO complexes. Adsorption of
hydrated Eu(III) on P. fluorescens cells does not change significantly at pH 3–8,20, 21 indicating that affinity of P. fluorescenscell surfaces with metal ions is not changed significantly atthese pHs. These facts indicate that pH dependence of adsorption density of metal ions on cells is dominantly controlled by
the stability of the metal-DFO complexes. Chemical speciations of metal-DFO complexes are protonated or deprotonateddepending on the solution pH, leading to a different adsorption
density of metals on the cells. Three hydroxamate groups in
DFO bind Eu(III) above pH 8.1.10 Below pH 8.1, the Eu(III)DFO complex successively dissociates with decreasing pH andEu(III) exists as a hydrated ion at pH < 4, resulting in the

(a)

(b)
1

1

Th(DFO) +Th(HDFO) 2+

Fe(HDFO) +
0.8

0.6

0.6

Fi

Fi

0.8

0.4

0.4

0.2

0.2

Fe(H 2 DFO) 2+

0
3

4

5

Th(H 2 DFO) 3+

0
6

7

8

9

3

4

5

pH

Figure 4. Chemical species of (a) the Fe(III)-DFO and (b) the Th(IV)-DFO complexes as a function of pH.

6

pH

7

8

9

Th(H -1 DFO)

80

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

Yoshida10

ical speciation and structure of cell surfaces. Microorganisms in
the environment have the potential to affect the movement of
tetravalent actinides-DFO complexes by adsorption on theircells.

higher adsorption of Eu(III) on cells at acidic pH.
The Th(IV)-DFO complex is successively protonated or
deprotonated with a change of pH. In Th(IV)(HDFO)2+, Th(IV)is coordinated with three hydroxamate groups of DFO whereinamine group is protonated. An increase of pH above 7 resultsin the deprotonation of amine and the change of Th(HDFO)2+ toTh(IV)(DFO)+. On the other hand, Th(IV)(HDFO)2+ is protonated to Th(IV)(H2DFO)3+ with decreasing pH below 4 (Figure4b), leading to the dissociation of bonds between the hydroxamate group and Th(IV). Stability of the Th(IV)-DFO complex
decreases in the order of Th(IV)(DFO)+ > Th(IV)(HDFO)2+ >Th(H2DFO)3+, resulting in higher adsorption of Th(IV) at lower
pH. On the other hand, chemical speciation of the Fe(III)-DFO
complex does not significantly change at pH 3 – 9, resulting in
the constantly low adsorption density within the pH range
examined.
Adsorption of Pu(IV) on P. fluorescens cells was greaterthan that of Fe(III) (Figure 1), though stability constants of the
Fe(III)- and Pu(IV)-DFO complexes are comparable. Ferriciron in a cyclic trihydroxamate siderophore, desferrioxamine E
is fully encapsulated, being coordinated with 6 oxygen atoms
from 3 hydroxamate groups.22 On the other hand, Pu(IV) in a
desferrioxamine E is coordinated with 9 oxygen atoms from 6
oxygen atoms in 3 hydroxamate groups on one side of Pu(IV)and 3 oxygen atoms from 3 water molecules from the otherside23 because of its larger ionic radius.24 These facts suggestthat structure of DFO complex may affect adsorption of metals
on bacterial cells. Functional groups on the cell surface would
easily access Pu(IV) in DFO from the side coordinated withwater molecules than Fe(III) in DFO. Similarly, Th(IV) withthe large ionic radius24 would be coordinated to DFO from its
one side, allowing functional groups of the cell surface easily
to access Th(IV) from the other side, resulting in the subsequent Th(IV) adsorption.
pKa values and concentrations of proton binding sites arealmost identical in P. fluorescens and B. subtilis cells (Table1). Lipopolysaccharide in the outer cell membrane of Gramnegative bacteria contains carboxyl groups and phosphategroups.25 Peptidoglycan, a proposed structure of surfaces ofGram-positive bacteria, also contains carboxyl and phosphategroups in their structure.25 We assigned acidic and neutral
functional groups of bacterial cell surfaces to carboxyl andphosphate groups, respectively (Table 1). The basic group on
bacterial cell surfaces is assigned to amine functional group,
found as a component on bacterial cell surfaces.25 Although
the structure and chemical composition of the bacterial cellsurfaces are not identical in the Gram-negative and the Grampositive bacteria, P. fluorescens cells and B. subtilis cells show
similar proton binding properties to each other. It is also
reported that a wide range of bacterial species, such as Gramnegative bacteria, E. coli and P. aeruginosa, and Gram-positive bacteria, B. subtilis and Streptococcus faecalis, show
comparable pKa of surface functional groups and their concentrations.26 However, P. fluorescens cells had a higher affinity
with Th(IV) and Pu(IV) than B. subtilis cells (Figure 1), indicating that the affinity of bacterial cell surfaces with tetravalentactinides is not completely correlated with their affinities with
proton.
P. fluorescens and B. subtilis adsorbed Th(IV) and Pu(IV) in
the presence of DFO from solution. Thorium(IV) and Pu(IV)are adsorbed on bacterial cells as a dissociated ion. DFO
forms strong complexes with tetravalent actinides, whose
stability constants are comparable to that of the Fe(III)-DFO
complex. However, adsorption behavior of tetravalentactinides on cells in the presence of DFO is controlled by not
only the stability constant of the DFO complexes but also chem-

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