Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 107-110, 2005

AcceptedCs Accumulation Behavior by Pseudomonas fluorescens
Atsushi Nakao,*, a, b Takahiro Yoshida,b Takuo Ozaki,b Toshihiko Ohnuki,b Shinya Funakawa,a andTakashi Kosakia
aGraduate school of agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Advanced Science Research Center, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-1195, Japanb

Received: November 15, 2004; In Final Form: April 21, 2005
Cs accumulation by Pseudomonas fluorescens was examined under conditions of growth and resting to elucidate
the interaction between Cs and bacteria. In the growth condition, P. fluorescens was cultured in citric acid media
containing 50 µM CsCl and 0, 0.1, 1.0, and 10 mM K for 64 h at 30 ˚C. Adsorption and desorption behaviors ofCs were examined by the contact of the cells with 50 µM CsCl solution under the resting condition. Cs-accumulated P. fluorescens was exposed to a 1 M CH3COONH4 solution to examine the reversibility of Cs accumulation.
In the growth condition, P. fluorescens did not accumulate Cs irrespective of the presence of K. In the restingcondition, the cells quickly adsorbed approximately 5 µmol Cs/gcell dry-weight and subsequently released approximately 90% of the adsorbed Cs with 1 M CH3COONH4. The amount of Cs adsorption by cells of P. fluorescens
varied with changing pH and ionic strength of the solution. These results indicate that Cs accumulation by P.
fluorescens occurs mainly by reversible adsorption on the cell walls, but not by intracellular accumulation undernutrient conditions.

1. Introduction
Radiocesium (137Cs) released by nuclear weapons tests andnuclear power plant accidents tends to remain in surface soils
for a long period.1, 2 Cesium-137 in surface soils may be transferred to plants grown in the contaminated soil. 3, 4 Thus, themigration behavior of 137Cs within soils should be elucidated
for predicting 137Cs contamination in plants.
Bacteria may have an important role in regulating thetransfer of 137Cs from soil to plants, because they have theability to accumulate metal cations on their cell surfaces andinside their cells.5, 6 It has been reported that K concentrations
in solution affect the accumulation of Cs by bacteria, and that
K transport systems largely determine the Cs-uptake ability ofbacteria.7, 8 Some bacteria, such as Rhodococcus erythropolis
and Escherichia coli, are known to have a specific K transportsystem (Kup) through which Cs is transported into cells. R.
erythropolis has been shown to accumulate less Cs from solutions with higher K concentrations.8 Pseudomonas fluorescens,ubiquitous in the terrestrial environment, accumulates negligible amounts of Cs compared to that by these bacteria. 7However, the effect of K on Cs accumulation by P. fluorescens
is still unknown.
Bacterial cell surfaces have functional groups, including
carboxylic, phosphate, amine, and hydroxyl groups. Thesesurface functional groups are deprotonated with increasing pH
and thus provide the bacteria with a net negative surface
charge.10 Bacterial cell walls have a high affinity for various
metal cations through coordination with these functionalgroups. 5 P. fluorescens is known to have these functionalgroups on its cell walls. However, little is known about theinteraction between Cs and its cell walls.
The purpose of this study was to investigate the effect of K
on Cs accumulation by P. fluorescens, and to elucidate theassociation of Cs with P. fluorescens. The association of Cs
with growing cells was investigated in media with 0 – 10 mM
K. Adsorption and desorption experiments were conducted*Corresponding author. E-mail: na_4_ka@popx.tokai.jaeri.go.jp.
FAX: +81-29-282-5927.

using resting cells at pHs 3.0 – 9.0 and 1.0 – 100 mM NaCl at
pH 6.0, and the time course of Cs adsorption and desorption
was measured.
2. Materials and Methods
Associations of Cs with growing cells. We used P. fluorescens (ATCC 55241) that was isolated from a low-level
radionuclides waste disposal site at West Valley, New York.11
P. fluorescens was pre-cultured at 30 ˚C for 24 h in 50 mL ofculture media containing 1 mM KCl, 10 mM citric acid, 0.1 M
NaCl, 6.7 mM (NH4)2SO4, 10 mM Na2HPO4, and 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). ThepH of the medium was adjusted to 6.0 before autoclaving at120 ˚C for 20 min.
The pre-cultured cell suspensions were inoculated into 50
mL culture media containing 0, 0.1, 1.0 or 10 mM KCl, 50µM
CsCl, 10 mM citric acid, 0.1 M NaCl, 6.7 mM (NH4)2SO4, 10mM Na2HPO4, and 10 mM HEPES; then, these cultures wereallowed to stand at 30 ˚C. Five milliliters of the cell suspension
was withdrawn at 18, 30, 42, and 64 h after inoculation. Cell
growth was determined on the basis of the optical density of
the culture medium measured at 600 nm (OD). The concentration of citric acid was measured by high-pressure liquid chromatography-mass spectroscopy (HPLC-MS) (Alliance 2695,Waters Corporation, Milford, MA, USA). The Cs concentration was measured by a model AA6200 atomic absorption
spectrophotometer (AAS) (Shimadzu Co., Kyoto, Japan).
Cs adsorption on and desorption from resting cells.
Pseudomonas fluorescens cells grown in a beef-extract solution(3 g L−1 beef extract, 5 g L−1 polypepton, and 5 g L−1 NaCl)
were used for the adsorption and desorption experiment. Cells
were harvested by centrifugation at 4000 rpm for 10 min. Thecell pellet was washed three times with a 10 mM NaCl solution, and cells were resuspended in a 10 mM NaCl solution toobtain a 20 mg mL−1 cell suspension on a dry weight basis.
In adsorption experiments, 20 mg of the cell pellet wascombined with 10 mL of a 50 µM CsCl solution containing 10mM NaCl. In order to examine the time course of the Cs
adsorption, cells were maintained with Cs at pH 7.0 for 0.5, 3,© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

Nakao108 J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

3. Results and Discussions
Growth of P. fluorescens and its association with Cs.
Figures 1a and 1b show the time courses of the OD and thecitric acid concentration, respectively, in the media containing
different Cs concentrations after the inoculation of P. fluorescens. In the absence of K, the OD did not increase and theconcentration of citric acid slightly decreased from 20 mM to17 mM over 64 h. These results indicate that P. fluorescens
cells did not grow in the absence of K.
In the medium containing 0.1 mM KCl, the OD increased
gradually to 0.28 and the citric acid concentration decreased to0.23 mM for 64 h. In the media containing 1.0 and 10 mM
KCl, the OD increased to 0.58 and 0.60, respectively, for 42 h,when the citric acid concentration decreased to below thedetection limit, and then gradually decreased through 64 h.
These results indicate that the cell growth was influenced by K
in the medium. The growth of Rhodopseudomonas capsulata
cells is enhanced by 0.1 and 1.0 mM Cs even in the absence ofK. R. capsulata has a specific K transporter (i.e. Kup), whichshows low selectivity between K and Cs.12 However, in this(a)0.6
0.5
0.40 mM K

0.30.1 mM K
1 mM K

0.20 mM K
0.1 mM K
1.0 mM K
10 mM K

403020100-10020

0.1
020

40

60

4060

80

Time/h080

100Time/hCitric acid concentration/mM

5010 mM K

Figure 2. Time-course of the accumulation of Cs by P. fluorescens
during the growth of the cells, which was calculated from the reduction of Cs concentration in the growth media. The sampling sets werein triplicates.

2510.0(b)20150 mM K

100.1 mM K
1 mM K

510 mM K

00204060

80

100

Cs accumulation/µmol g -1

Optical density at 600 nm

0.7study, the presence of Cs without K did not result in the growthof P. fluorescens cells.
Figure 2 shows the time-course of the accumulation of Cs
by P. fluorescens, which was calculated from the reduction ofCs concentration in medium solutions. In the media withdifferent K concentration, Cs accumulation was not shown andthere was little effect of the difference of K on the Cs accumulation by P. fluorescens during the growth period. It has beenreported that Cs is transported into bacterial cells in the same
way as K to some degree, because of their similar chemical
properties.12, 13 Tomioka et al.7 showed that Cs was not accumulated by P. fluorescens in the medium containing K as aconstituent of an yeast extract medium. In the present study,
we revealed that Cs was not accumulated by P. fluorescens in
the culture media containing 0 – 10 mM K. This suggested thatP. fluorescens selectively transported K into the cells against
Cs and that Cs did not function in place of K for the growth ofP. fluorescens.
Adsorption and desorption of Cs under resting conditions.
The time course of the adsorption of Cs on P. fluorescens atresting conditions (Figure 3) showed that the amount adsorbed
reached approximately 5.0 µmol Cs/gcell dry-weight at 30 min, andthen remained constant up to 24 h. The pHs of the solutions
were between 6.6 and 7.0 during the experiment. Note that theamount of cell pellet in contact with Cs solutions was approximately 10 times higher than the maximum amount obtained in
the growth experiment. The rapid adsorption of metals on
bacterial cells has been widely reported.14, 15
The amount of Cs accumulated increased from 1.8 to 4.2µmol Cs/gcell dry-weight with a rise in the pH of the solution from
3.5 to 7.5 (Figure 4). The final pHs were 3.5, 6.7, and 7.5,changing variously from their starting values of 3.0, 7.0, and9.0, respectively. The P. fluorescens cells have carboxyl andphosphate functional groups on their surfaces.16 These funcCs accumulation/µmol g-1

12, and 24 h. In order to examine the effect of pH and ionicstrength on the Cs adsorption by P. fluorescens, cells were also
maintained with Cs at pHs 3.0, 7.0, and 9.0 and in 1, 10, and100 mM NaCl for 12 h. After the controlled contact, supernatants were separated from the cells by centrifugation at 4000rpm for 10 min. The Cs concentration in the supernatant wasmeasured by AAS.
The cells maintained with the Cs solution containing 10 mM
NaCl at pH 7.0 for 12 h were used for desorption experiments
after washing the cells once with distilled water at pH 6.8 andcollecting them by centrifugation at 4000 rpm for 10 min. The
collected cells were resuspended in a 1 M CH3COONH4 solution for 3, 12, 24, and 36 h. The suspensions were centrifuged
at 4000 rpm for 10 min. The Cs concentrations in the supernatants were measured by AAS.

8.0

6.0
4.0

2.0

Time/hFigure 1. Time-course of the (a) OD and (b) citric acid concentrations (mM) in medium solutions containing Cs of 50 µM and K of 0,0.1, 1.0, or 10 mM after the inoculation of P. fluorescens. The bars
show the standard deviation for triplicate measurements of individual
K concentrations.

0.0010203040Time/hFigure 3. Time-course of Cs concentration adsorbed on the cells of P.
fluorescens after exposure to a 50 µM CsCl solution of 10 mL.

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005 109

Cs Accumulation Behavior by Pseudomonas fluorescens
5.0

Cs accumulation/µmol g -1

Cs accumulation/µmol g -1

6.0
5.0
4.0
3.0
2.0
1.04.0

3.0
2.0

1.0
0.0

0.0
3.0

5.0

7.00

9.0

pH
Figure 4. Effect of pH on the accumulation of Cs by P. fluorescens
cells. The final pHs are shown in the figure.

tional groups deprotonate with increasing pH and consequently
associate with cations.10 The pH dependence in the Cs adsorption showed that the adsorption of Cs occurred through theexchange with H+ on functional groups of the cell surfaces ofP. fluorescens.
Figure 5 shows Cs accumulation by P. fluorescens cells at 1,10, and 100 mM NaCl at pH 6.9 ± 0.1. The amount of the Cs
accumulated was 4.5, 3.0, and 1.9 µmol Cs/gcell dry-weight at 1.0,
10, and 100 mM NaCl, respectively. The decrease in theamount of Cs accumulation with increasing Na concentration
results from competition in the adsorption of Cs and Na by thefunctional groups of the cell surfaces. Intracellular Cs accumulation through Na transport channels is unlikely to occur
because no Cs was accumulated under the growing condition(Figure 2).
The time course of the desorption of Cs from the Cs accumulated P. fluorescens at pH 7.0 for 12 h (Figure 6) showedthat the adsorbed Cs was almost completely desorbed within
24 h by 1 M CH3COONH4. A 1 M CH3COONH4 solution istypically used as an extract solution to estimate the amount of
the reversible cations in soils.17 Thus, Cs is reversibly adsorbedon the functional groups of the cell surfaces of P. fluorescens.
The Cs desorption rate was slower than the Cs adsorption rate.
Ohnuki and Kozai 18 reported that the desorption and theadsorption rates were almost equivalent in the case of smectite.
P. fluorescens has peptidoglycan layers on its cell walls, which
consist of a cross-linked mesh structure.19 The results suggestthat adsorption of Cs on the cell surfaces of P. fluorescens
differs from that on the reversible site in smectite.
Oughton et al.20 reported that approximately 10% of radiocesium was extracted with 1 M CH3COONH4 from contaminated soils collected at 3 places in the 30 km exclusion zone
surrounding the Chernobyl reactors. Even with a heated andconsequently highly reactive acid solution (pH 2.0, 80 ˚C),
more than 60% of the radiocesium could not be extracted. 20
The Cs fixation in soils can be explained as an irreversible
adsorption of Cs on micaceous clays through the formation of
inner-sphere complexes.21, 22 On the other hand, it was reported
that Cs fixation on clay minerals was reduced by adding humic
substances because of a decrease in the average affinity of thecation adsorption sites of Cs.23 It is well known that reversible
cations in the adsorption phase are available to plants. Our
results suggest that Cs adsorption on the bacterial cell surfacesof P. fluorescens plays a less important role in restricting themigration of Cs than micaceous clays but might enhance theplant availability of Cs, because Cs was adsorbed on the bacterial cells reversibly.
No investigation has been conducted on preferential adsorption of Cs between micaceous clays and P. fluorescens. P.
fluorescens releases strong chelate reagents (i.e. siderophore),2420

40

60

80

100

Concentration of NaCl/mM
Figure 5. Effect of the NaCl concentration on the accumulation of Csby P. fluorescens cells.

100Cs desorbed/%

1.07550

250010203040Time/hFigure 6. Time-course of the Cs concentration desorbed from cells ofP. fluorescens by a 1 M CH3COONH4 solution. The Cs fraction wasdefined to be 100% when all the adsorbed Cs was desorbed.

which may enhance Cs desorption from micaceous clays by
dissolving the inner-sphere structure complexes. Even thoughP. fluorescens accumulated only a small amount of Cs, further
investigation should be performed to elucidate the effects ofbacteria on the migration behavior of Cs in soils.
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