Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 73-76, 2005

Accepted
Associations of Eu(III) with Gram-Negative Bacteria, Alcaligenes faecalis, Shewanella
putrefaciens, and Paracoccus denitrificans
Takuo Ozaki,*, a Takaumi Kimura,b Toshihiko Ohnuki,a and Arokiasamy J. FranciscaAdvanced Science Research Center, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-1195, Japan
Department of Materials Science, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-1195, Japanc
Environmental Sciences Department, Brookhaven National Laboratory, Upton, New York 11973, USAb

Received: November 15, 2004; In Final Form: April 21, 2005
We studied the association of Eu(III) with Gram-negative bacteria, Alcaligenes faecalis, Shewanella putrefaciens,
and Paracoccus denitrificans by a batch method and time-resolved laser-induced fluorescence spectroscopy(TRLFS). The kinetics study showed that the Eu(III) adsorption on the bacteria rapidly proceeded. The Eu(III)adsorption on A. faecalis and P. denitrificans at pHs 3, 4, and 5, and that on S. putrefaciens at pHs 4 and 5 reacheda maximum within 5 minutes after contact. For P. denitrificans, the percent adsorption of Eu(III) decreased after
the maximum percent adsorption was attained, which suggests the existence of exudates with an affinity with
Eu(III). TRLFS showed that the coordination of Eu(III) on these bacteria is multidentate through an inner-spherical process. The ligand field of Eu(III) on P. denitrificans was as strong as the ones observed for halophilic
microorganisms, while that of A. faecalis and S. putrefaciens was the typical one observed for non-halophilic
microorganisms. The coordination environment of Eu(III) on the bacteria differed from each other, though they
are categorized as Gram-negative bacteria with the similar cell wall components.

1. Introduction
Migration behavior of radioactive nuclides in the environment is greatly affected by bacteria.1, 2 Gram-negative bacteria
are ubiquitously found in the environment. Francis et al.
showed that a Gram-negative bacterium Pseudomonas fluorescens selectively affects the environmental behavior of bidentate citric-acid metal complexes through their degradation while
it shows no association with tridentate or binuclear complexes
because they are recalcitrant to its degradation.3 In this study,
we used three Gram-negative bacterial species, Alcaligenes
faecalis, Shewanella putrefaciens, and Paracoccus denitrificans
to study the association of Gram-negative bacteria with Eu(III).
A. faecalis widely prevails in soil, waters, and even in digestiveorgans of mammals. This bacterial species is famous for theability to oxidate As(III)O2− to As(V)O43−, wherein the protein
involved in the reaction has been shown to contain Mo.4, 5 S.
putrefaciens is outstanding for its ability to grow on a wide
variety of compounds as a terminal electron acceptor, such as
O2, NO3−, NO2−, Mn(IV)-oxides, Fe(III)-oxides, and U(VI).6, 7
P. denitrificans grows both aerobically and anaerobically, and it
anaerobically reduces a fixed nitrogen oxide. 8, 9 However,reduction of metal oxides, such as Mn(IV)-oxides and Fe(III)oxides is not reported.
Associations of trivalent f-elements (lanthanides and actinides)with bacteria, especially coordination characteristics of theseelements on them have not clearly been understood. Mainfunctional groups on the cell surface of bacteria are carboxyl
and phosphate groups.10−12 Despite the relatively small variety
in the kinds of functional groups, a wide variety of coordination environments are observed for Eu(III) on Gram-positive,
Gram-negative, and halophilic bacteria,13 which suggests thesteric effects derived from the polymeric structure on theadsorption characteristics as well as the contribution fromminor functional groups to its adsorption on the cell surface. It
is important to precisely assess the association of f-elements*Corresponding author. E-mail: tozaki@popsvr.tokai.jaeri.go.jp.
FAX: +81-29-282-5927.

with bacteria in that these elements may be leaked out of radioactive contaminants. In this study, we used Eu(III) because of its
informative fluorescence properties. Europium(III) is a goodanalogue of Am(III) and Cm(III), and a study on the association of Eu(III) with bacteria is of great help in assessing that ofAm(III) and Cm(III), whose long half-lives and high-energy αparticle emission can pose high risks to human health.
Time-resolved laser-induced fluorescence spectroscopy is a
powerful tool to characterize the coordination environment ofEu(III) with regard to the number of water molecules in the
inner-sphere of Eu(III) and the strength of its ligand field bothin the inner- and outer-sphere.14 Previously, we used this technique and successfully characterized the association of Eu(III)with biopolymers (cellulose, chitin, and chitosan) and a unicellular alga Chlorella vulgaris.15, 16
In the present study, we examined the adsorption behaviorof Eu(III) on Gram-negative bacteria A. faecalis, S. putrefaciens, and P. denitrificans by a batch method. Using timeresolved laser-induced fluorescence spectroscopy (TRLFS),
we examined the coordination environment of Eu(III) adsorbedon them.
2. Experimental
2.1. Preparation of the microorganisms. One cubic centimeter of cell suspensions at the exponential growth phase from
Alcaligenes faecalis, Shewanella putrefaciens, and Paracoccus
denitrificans were transferred to 250 cm3 of culture media. Thesebacteria were grown aerobically at 30 ˚C in a nutrient medium at
pH 6.8 containing the following components: polypepton, 5 g
dm−3; beef extract, 3 g dm−3; and NaCl, 5 g dm−3. The cultures
were incubated on a rotary shaker at 100 rpm.
2.2. Measurement of adsorption behavior of Eu(III) on
the bacteria. We used radioactive 152Eu(III) in this study. The
adsorption kinetics and distribution ratios (log Kd) of Eu(III)was studied by a batch method at pHs 3, 4, and 5. At thesepHs, the hydrolysis of Eu(III) can be neglected. Bacterial cells
were harvested at the late exponential phase by centrifugationfor 10 minutes at 10000 g. The cell pellets were washed four

© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

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

times with a 0.5% NaCl solution and resuspended in it. For
the adsorption kinetics study, 0.75 cm3 of cell suspension wasadded to a 15-cm3 Eu(III) solution containing 0.5% NaCl and5.25 cm3 of the suspension was withdrawn at 5, 10, and 15
minutes after contact. Subsequently, the cells were filteredthrough a 0.20-µm membrane filter (ADVANTEC MFS, Inc.,DISMIC-25). To measure the log Kd, 0.25 cm3 of bacterial
suspension was added to 5-cm3 solutions of Eu(III). After 10
minutes of contact, the cells were filtered through a 0.20-µm
membrane filter (ADVANTEC MFS, Inc., DISMIC-25). The
pH of the contact solution was maintained at 3.00 ± 0.05, 4.00
± 0.05, or 5.00 ± 0.05, using NaOH or HClO4 during the contact.
The concentration of Eu(III) in the contact solution was approximately 1⋅10−6 mol dm−3. Europium(III) in the supernatant was
analyzed by γ-spectroscopy (Packard Co., COBRAII). The
distribution ratio Kd (cm3 g−1) was calculated according to theeq A:
Kd = (C0 – C) V / C W,

(A)

where C0 is the initial concentration of Eu(III) in the aqueous
phase; C, the equilibrium concentration of the metal ion in the
aqueous phase; V, the volume of the aqueous phase (cm3); and,W, the dry-weight of the bacterial cells (g). The dry-weight ofthe bacterial cells were 0.004 to 0.009 g.
2.3. TRLFS. The coordination environment of Eu(III)adsorbed on bacteria was studied by TRLFS. Samples wereprepared by adding bacterial cells to a solution of 1⋅10−3 mol
dm−3 Eu(III) at pHs approximately 4–6 containing 0.5% NaCl.
The cells were kept standing for approximately 2 hours after
contact. Aliquots of cell suspension were centrifuged to settle
the cells, which then were exposed to light of a wavelength of
394 nm from a XeCl excimer laser pumped dye laser (Lambda
Physik, COMPex 201) pumping PBBO (Lambda Physik) in a
1,4-dioxane solution in a dye laser head (Lambda Physik,
SCANmate 2).17
To measure the luminescence lifetime of Eu(III), the emission light was collected into a monochromator (Oriel, 77257)
using an optical fiber and detected by a photomultiplier tube
(Hamamatsu, R3896). The signal was fed into a digitizing
oscilloscope (Hewlett Packard, 54519A) connected to apersonal computer through a GP-IB interface. The luminescence decay curves were fitted by a single component exponential function. The number of water molecules, NH2O, of
Eu(III) adsorbed on bacterial cells was estimated according to
the eq B:
NH2O = 1.05⋅10−3 (1 / τobs) − 0.44,(B)

where τobs (s) is the luminescence lifetime.18
To obtain the emission spectra of Eu(III), the emission light

RE/M = I (5D0 → 7F2) / I (5D0 → 7F1),

75

pH 5
pH 4
pH 3

50

25

3. Results
3.1. Kinetics of Eu(III) adsorption on the bacteria. Figures
1a-c show the adsorption kinetics of Eu(III) at pHs 3, 4, and
5 for Alcaligenes faecalis, Shewanella putrefaciens, andParacoccus denitrificans. Non-specific adsorption of Eu(III)on the membrane filter and the wall of the contact vessels was
not observed at these pHs.
Figure 1a shows the adsorption kinetics of Eu(III) for A.
faecalis. At all pHs, the equilibrium was attained within 5
minutes. The percent adsorption increased with an increase in
pH. Similarly, the adsorption of Eu(III) by S. putrefaciens
increased with a rise in pH (Figure 1b). The Eu(III) adsorption
reached equilibrium within 5 minutes at pHs 4 and 5, while
kinetics at pH 3 slowed and Eu(III) adsorption did not reachequilibrium up to 15 minutes. For the adsorption of Eu(III) on
P. denitrificans, the equilibrium at pH 3 was attained within 5
minutes, and afterward the percent slightly decreased (Figure1c). At pHs 4 and 5, the adsorption of Eu(III) reached amaximum within 5 minutes. The percent adsorption at 15
minutes was higher at higher pHs.
3.2. Distribution ratio of Eu(III) for the bacteria. Figures
2a-c show the logarithmic distribution coefficient (log Kd) ofEu(III) for the bacterial cells at 10 minutes after contact at pHs
3, 4, and 5. The values are the average of 3 replicates. Figure
2a shows log Kd of Eu(III) for A. faecalis. A. faecalis exhibiteda higher log Kd for Eu(III) at higher pHs. log Kd at pHs 3, 4,
and 5 was approximately 2.4, 3.6, and 4.6, respectively. S.
putrefaciens also showed an increasing tendency in log Kd witha rise in pH (Figure 2b). log Kd at pHs 3, 4, and 5 was 2.3, 3.8,
and 5.1, respectively. Figure 2c shows that log Kd for P. denitrificans was higher than those for A. faecalis and S. putrefaciens. log Kd at pHs 3, 4, and 5 was approximately 3.6, 4.6, and5.8, respectively.
3.3. Coordination environment of Eu(III) on the bacteria.
Figure 3 shows the Coordination-Environment diagram (CEdiagram), on which ∆NH2O and RE/M for Eu(III) adsorbed on the
100

75

pH 5
pH 4
pH 3

50

25

0

0
5

10

Time / min

15

75

pH 5
pH 4
pH 3

50

25

(c)

(b)

(a)
0

(C)

where I (5D0 → 7F2) and I (5D0 → 7F1) are calculated from thepeak areas at 614 nm (5D0 → 7F2) and 592 nm (5D0 → 7F1),
respectively.

100

Percent adsorption / %Percent adsorption / %100

was focused on a polychromator (ISA Jogin-Yvon, HR-320)
using an optical fiber and detected by a gated multichannel
diode array (Princeton Instruments, Inc., DIDA-512). The
spectrometer was connected to Spectrometric Multichannel
Analyzer system (Tokyo Instruments, Inc., SMA) installed on
the personal computer. The relative peak intensity ratio (RE/M)
is defined by the ratio (C)Percent adsorption / %

74

20

0
0

5

10

Time / min

15

20

0

5

10

15

20

Time / minFigure 1. Time dependence in the percent adsorption of Eu(III) on (a) Alcaligenes faecalis, (b) Shewanella putrefaciens, and (c) Paracoccus denitrificans.

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

Associations of Eu(III) with Gram-Negative Bacteria
7

6

6

5

log K dlog K d

5

(b)

4
3

4
3

4
3

2

2

2

1

1

1

0

0
3

4

5

0
3

pH

(c)

5

log K d6

4. Discussion

7

7

(a)

4

3

5

pH

4

5

pH

Figure 2. Logarithmic distribution coefficient (log Kd) of Eu(III)determined for (a) Alcaligenes faecalis, (b) Shewanella putrefaciens,and (c) Paracoccus denitrificans.

9

6
4
3

∆N H2O

6

2

8
1

7
10

5
9

3

12

11

hydrated Eu(III) ion

0

0

1

2

3

75

4

R E/M
Figure 3. CE diagram obtained for the bacterial species listed above.
The large closed circle represents the result obtained for hydrated
Eu(III) ion with no interaction other than water molecules. pHs are as
follows: (1) 4.62, (2) 5.29, (3) 5.62, and (4) 5.73 for Alcaligenes
faecalis, (5) 4.46, (6) 4.86, (7) 5.34, and (8) 5.67 for Shewanella
putrefaciens, (9) 4.26, (10) 4.68, (11) 5.26, and (12) 5.68 forParacoccus denitrificans.

three bacterial species were plotted. On the CE diagram,∆NH 2O indicates the number of water molecules in the innersphere of Eu(III), calculated according to the equation ∆NH2O =9 − NH 2O. In this equation, we assumed that the number ofwater molecules in the inner-sphere of hydrated Eu(III) ion is 9
based on previous research.19 ∆NH2O represents the number ofcoordination sites occupied in the inner-sphere of Eu(III) byligands other than water molecules. A. faecalis showed a∆NH 2O of 4.60 at pH 5.3 to 5.61 at pH 5.7. RE/M was between
1.50 and 1.54. No clear correlation among pH, ∆NH2O and RE/M
was observed. The ∆NH2O in a 0.5% NaCl solution was almost
0 and the RE/M in the solution was not affected by the presenceof NaCl.
S. putrefaciens showed a large ∆NH 2O. The largest ∆NH 2O
was 6.33 at pH 5.3, and the smallest one was 5.51 at pH 4.5.
The RE/M for Eu(III) on S. putrefaciens ranged from 2.07 to
2.37. No clear pH dependence was observed. P. denitrificans
showed a small ∆NH 2O, which ranged from 3.99 to 4.15. On
the other hand, RE/M observed for P. denitrificans was larger
than those on the other two bacterial species: the maximum
was 3.13 at pH 5.7 and the smallest was 2.73 at pH 4.3.

Takahashi et al. showed that the hydrolyzed Eu(III) species
exhibits an extremely large τobs−1.20 In this study, τobs−1 smaller
than those corresponding to NH20 = 9 was found, showing that
the Eu(III) species on the cell surface was not precipitated.
The Eu(III) concentration used in the adsorption experiments
was lower than that used in the TRLFS experiments. These
facts signify that the accumulation of Eu(III) in the bacterial
cells is not due to its precipitation on the cell surface.
Accumulation of metals in bacteria usually involves two steps.
The first step is the metal adsorption on the bacterial cell surface
without involving biological activity. The second step, which
generally proceeds very slowly, is their absorption into thecytoplasm of the bacterial cells.21, 22 Our observations suggestthat the association observed between Eu(III) and A. faecalis
can be attributed to the first step. Previously, we reported a
rapid adsorption of trivalent f-elements on microorganisms.13, 23
Similarly, the Eu(III) adsorption on S. putrefaciens at pHs 4 and
5 and that on P. denitrificans at pHs 3 – 5 rapidly reached amaximum but the slow increase in the adsorption as the second
step was not observed. The adsorption of Eu(III) on P. dinitrificans at pH 3 showed a maximum at 5 minutes after contact
and then the percent adsorption decreased. It is suggested that
P. dinitrificans exudes some substance with an affinity with
Eu(III) at the pH. Chlorella vulgaris was shown to excretesome exudates which desorb Eu(III) and Cm(III) from its cellsurface.16
The adsorption of cations on bacterial cells through the exchangeof H+ on the functional groups is commonly observed.24, 25 If theadsorption of Eu(III) on cell surfaces is through the exchangewith 3 H+ in functional groups on the cell surface, the slope ofthe log Kd versus pH plots is predicted to be 3 in the pH range
where the hydrolysis of Eu(III) does not occur. However, allbacterial species showed a smaller slope in the log Kd versus
pH plots than the predicted value: A. faecalis, S. putrefaciens,
and P. denitrificans showed a slope of approximately 1.1, 1.4,
and 1.1, respectively. This cannot be explained by the mechanisms mentioned above. The presence of exudates cannot
clearly explain this, either. The adsorption of Eu(III) on
biopolymers, cellulose, chitin, and chitosan produced a slopeof the log Kd versus pH plots of approximately 1 at acidic pHs,
wherein the coordination of ether oxygen was suggested.26 In
the adsorption of Eu(III) on these three bacterial species,
involvement of functional groups such as the ether oxygenmight be present. P. fluorescens showed a negative slope in
the log Kd versus pH plots between pH 3 – 5.13 The main cellwall components of Gram-negative bacteria are lipopolysaccharide and peptidoglycan, where the possible adsorption sites
for Eu(III) are carboxyl groups and phosphate groups.27 Thesefindings indicate that the structure of coordination sites on P.
fluorescens and that on the three other bacterial species used inthis study is not identical to each other, though all four species
are categorized as Gram-negative bacteria based on the characteristics of the cell wall structure.
TRLFS shows high selectivity and sensitivity for trivalent felements. This technique is applicable to the adsorbed specieson the surface of a solid phase in the presence of a waterphase. Ligands in the inner-sphere and the ones in the outersphere of an atom produce its coordination environment, withthe characteristic ligand field surrounding the central atom.
TRLFS can predict the coordination environment of Eu(III)including both the inner- and outer-sphere, owing to its luminescence properties.14 The fluorescence lifetime of excited
Eu(III) is related to the number of water molecules in the
inner-sphere (NH 2O), which is calculated by the eq B.28 The
relative intensity of 5D0 → 7F2 (electric dipole) and 5D0 → 7F1(magnetic dipole) emissions (RE/M) is related to the strength ofligand field of Eu(III), based on which we can characterize the

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Ozaki

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005
29

coordination environment for both the inner- and outer-sphere.
Unknown coordination environments of Eu(III) can be characterized based on the location of a RE/M-∆NH2O (= 9 − NH2O) plotof the Eu(III) on the CE diagram.14 We previously showed thatthe ligand field of Eu(III) (RE/M) on halophilic microorganisms,
such as Halomonas sp. and Halobacterium salinarum is stronger
than that on the non-halophilic microorganisms, such as C.
vulgaris and B. subtilis.13 In this study, RE/M increased in theincreasing order of A. faecalis, S. putrifaciens, and P. denitrificans. Note that RE/M for P. denitrificans was larger than that for
non-halophilic microorganisms and comparable to that observedfor Eu(III) adsorbed on halophilic microorganisms.13 Thissuggests that the structure of the coordination site for Eu(III)on P. denitrificans is similar to that of halophilic Gram-negative bacteria, such as Halomonas sp., implying its adaptability
to saline conditions.
Coordination sites of trivalent f-elements on a Gram-negative bacterium, Pseudomonas aeruginosa are reported to becarboxyl groups and phosphate groups. 30 One molecule ofthese groups can provide at most bidentate coordination with
Eu(III). However, ∆N H 2O on the bacteria in this study waslarger than 3. Especially, S. puterfaciens showed ∆NH2O of aslarge as 6. These facts signify that the adsorption of Eu(III) on
the bacteria is through inner-spherical coordination, wherein
more than one functional groups are involved. Reportedly,∆NH2O on a Gram-negative bacterium P. fluorescens was larger
than that on a Gram-positive bacterium B. subtilis.13 ∆NH 2O
obtained for A. faecalis and S. putrefaciens is almost equal tothat for P. fluorescens, while that for P. denitrificans is comparable to that of B. subtilis. These findings demonstrate that thecoordination environment of Eu(III) differs even in Gramnegative bacteria, though only a few kinds of functional groups
are involved in the coordination. Further investigation would
be required to achieve the overall understanding of the association mechanisms between f-elements and bacteria.
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