Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 95-98, 2005

Accepted
Effect of Eu(III) on the Degradation of Malic Acid by Pseudomonas fluorescens and theSubsequent Production of Pyruvic Acid
Takuya Nankawa,*, a Yoshinori Suzuki,b Takuo Ozaki,a Toshihiko Ohnuki,aand Arokiasamy J. FranciscaAdvanced Science Research Center, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-1195, Japan
Department of Materials, Physics and Energy Engineering, Graduate School of Engineering, Nagoya University,
Furo-cho, Chikusa, Nagoya 464-8603, 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 effect of Europium, Eu(III), on the degradation of malic acid by the bacterium Pseudomonas fluorescens. Its breakdown depended upon the ratio of Eu(III)- to malic acid-concentrations; the higher the ratio, theless malic acid was degraded. The resulting chemical species of Eu(III), determined by calculations using thermodynamic data, indicated that predominant ones were Eu(Mal)2− and EuMal+; free Eu(III) constituted less than 1%of the total Eu(III). The degradation of malic acid was independent of Eu(Mal)2− concentrations. Even thoughmore than 80% of malic acid was present as free malic acid, its degradation was retarded with an increase in theratio of Eu(III) to malic acid. According to the speciation calculation, the degradation of malic acid was hinderedby the presence of EuMal+, as well as by free Eu(III). We also assessed the amount of pyruvic acid generated,which is the major degradation product of malic acid complexed with Eu(III).

1. Introduction
Lanthanide elements are widely and increasingly used insuperconductors, magnets, fluorescent materials, glass additives,and MRI contrast reagents. 1−4 Accordingly, environmental
pollution might well increase as a result of their expanding
industrial and medical use. Several authors have noted the toxicity of gadolinium(III) and lutetium(III) to Escherichia coli and
Vibrio fischer.5−7 In previous investigations of the degradationof malic acid we found that the presence of this acid masked the
toxicity of Eu(III).8 In addition, at higher concentrations ofEu(III), the breakdown of malic acid was retarded. These results
suggest that the toxicity of lanthanides depends on the particularchemical species present. However, we did not then identified
the chemical species of Eu(III) and malic acid.8
The degradation of malic acid is followed by the appearanceof at least two kinds of organic acid metabolites.8 One of them,
pyruvic acid, remained in the medium forming complexes with
Eu(III) after all the malic acid had disappeared.8 If pyruvic acidmasks the toxicity of Eu(III), then the concentration of pyruvic
acid should be nearly the same as, or higher than, the concentration of Eu(III). However, we did not measure its concentration in our previous work.8
In this study, we determined the concentrations of the chemical species using the computer codes MEDUSA and HYDRA;9
we discuss the toxicity of Eu(III) in terms of its chemical
species. We also assessed the amount of pyruvic acid generated to identify the type of pyruvate-Eu(III) complexes formedin the medium.
2. Experimental
2.1. Culture
Pseudomonas fluorescens (ATCC 55241)8, 10 was grown in amineral-salts solution containing the following: NaCl, 5.6 mg
dm −3 ; (NH 4 ) 2 SO 4 , 88 mg dm −3 ; KCl, 75 mg dm −3 ; β-glyc*Corresponding author. E-mail: nankawa@popsvr.tokai.jaeri.go.jp.
FAX: +81-29-282-5927.

erophosphoric acid disodium salt, 22 mg dm −3 ; 2-[4-(2hydrixyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES),
4.76 mg dm−3; and trace elements (MgSO4·7H2O, 7.0 mg dm−3;
CaCl 2 ·2H 2 O, 2.8 mg dm − 3 ; MnSO 4 ·5H 2 O, 1.2 mg dm − 3 ;CuSO 4 ·5H 2 O, 0.15 mg dm −3 ; CoCl 2 ·2H 2 O, 0.15 mg dm −3 ;FeSO4·7H2O, 1.5 mg dm−3; Na2MoO4·2H2O, 0.090 mg dm−3;
and ZnSO4·7H2O, 0.10 mg dm−3). Malic acid and Eu(III)Cl3
solutions were sterilized separately by passing them through a
0.20-µm pore-sized Millipore HA filter (ADVANTEC MFS,
Inc., DISMIC-25).
2.2. Biodegradation experiments. Mineral-salt media
containing 1 mM malic acid and 0, 0.05, or 0.1 mM Eu(III)Cl3
were used to study biodegradation. The pH of the medium
was adjusted to 6.6. The molar ratios of Eu(III) to malic acid
were 0:1, 0.05:1, or 0.1:1 in media containing 0, 0.05, or 0.1 mM
Eu(III), respectively.8
P. fluorescens cells were grown in the mineral-salts medium
containing 1 mM malic acid. Cell growth was determined bymeasuring the optical density (OD) at 600 nm. Cell suspensions (0.5 cm3) were withdrawn from the medium at the late
exponential growth phase (OD of 0.02) and added to 49.5 cm3of the media contained in 100 cm 3 Erlenmeyer flasks. The
samples were incubated at 30 ˚C in the dark while being shaken
at 100 rpm. Aliquots were withdrawn from the flasks every 24
h up to 264 h to measure pH, the concentrations of organic
materials, and Eu(III). The latter value was obtained after first
filtering the media through a 0.20-µm pore-size Millipore HA
filter (ADVANTEC MFS, Inc., DISMIC-25), and then acidifying 0.5 cm3 of the filtered solution with 2 cm3 of 0.2 M HNO3.
2.3. Analytical methods. The concentration of malic acidwas measured by running the filtered solutions through a highpressure liquid chromatography system (Waters, Alliance2695) equipped with an organic-acid column (Waters, P/N
023694) with the mobile phase of 0.2 w/v% formic acid. The
flow rate was 0.7 cm3 min−1. The column’s temperature was
60 ˚C. The elute was subjected to ESI-Mass spectroscopy(Waters, ZQ 2000) operated in the negative ion mode for m/z =50 – 500 under the following conditions: capillary voltage,–3.07 V; cone voltage, –23 V; desolvation temperature, 150© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

96

Nankawa

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005
3−1

˚C; sheath gas, N2; flow rate, 360 dm h . It also was analyzed
using a Photodiode Array Detector (Waters, Alliance 2996) todetect UV absorption of the organic acid coexisting with malic
acid. The concentration of malic acid was estimated from the
ESI-Mass spectra detected at m/z = 133.1, and that of the coexisting acid from the absorption intensity detected by PhotodiodeArray Detector. The Eu(III) concentration was determined byinductively-coupled plasma atomic emission spectroscopy (ICPAES) (Shimadzu, ICP-7000).
2.4. Determination of Speciation. We employed the computer
programs HYDRA and MEDUSA to determine the speciationof Eu(III) and malic-acid complexes based on their reported
stability constants.9 The acidity (pK) of malic acid (Mal) andmalic acid mono-anion are 3.24 and 4.67, respectively.11 The
stability constant (log K) of the 1:1 molar ratio Eu(III)-malic
acid complex (EuMal+) is 4.85, and that of the 1:2 molar ratio
complex (Eu(Mal)2−) is 8.11.12
3. Results and Discussion
3.1. Degradation of malic acid by Pseudomonas fluorescens. Figures 1a and 1b illustrate the degradation of malic(a)Percentage of malic acidremaining in the medium

100
80
60
40
20
0
0

150

100

50

200

250

200

250

(b)

6.9

pH

Time (h)

6.7

6.5

0

150

100

50

Time (h)Figure 1. Biodegradation of malic acid by P. fluorescens in the presence of Eu(III) with an Eu(III) to malic acid molar ratio of ▲ 0.05:10,● 0.1:10, ■ 0.2:10, △ 0.05:1, ○ 0.1:1, and no Eu(III) (* 1 mM malic
acid, × 10 mM malic acid); (a) Malic-acid concentration vs contact
time and (b) pH vs contact time.

acid in media containing 1 mM malic acid and 0, 0.05, and 0.1
mM Eu(III), expressed as the Eu:malic acid molar ratio 0:1,
0.05:1, and 0.1:1 medium, respectively. We also include ourpreviously reported data (those in 0:10, 0.05:10, and 0.1:10
medium).8 In 0:1 and 0:10 media, the concentrations of malic
acid decreased with time (Figure 1a). Malic acid was notdegraded in 0.1:1 and 0.05:1 media during the test period (264h). The acid was broken down in 0.2:10, 0.1:10, and 0.05:10
media. Its degradation occurred as fast in the 0.05:10 medium
as in the 0:10 medium. The rate of degradation of malic aciddecreased with the increasing ratio of Eu(III) to malic acid.
The initial pH of the medium was 6.55 – 6.62 (Figure 1b),
but it gradually increased with time to almost 6.9 in thosemedia where malic acid was degraded. In all media, theconcentration of Eu(III) did not change whatever its initialconcentration.
Although the concentration of Eu(III) in the 0.2:10 medium(0.2 mM) is higher than that in the 0.05:1 medium (0.05 mM),
malic acid was broken down in the former but not the latter.
These results suggest that toxicity of Eu(III) does not dependon its concentration; hence, we calculated its speciation so that
we could discuss the toxicity of Eu(III) in terms of individual
chemical species.
3.2. Speciation of Eu(III). Table 1 summarises the initialconcentrations of Eu(III) species in solution in each medium.
In the 0.1:1 and 0.05:1 ratio media where malic acid was notdegraded, more than 80% of it was present as the free acid,indicating that Eu(III) retarded the degradation of free malic
acid. In the 0.05:1 medium at pH 6.61 (the initial pH), approximately 61% of Eu(III) (0.031 mM) formed Eu(Mal)2−, andapproximately 37% (0.019 mM) formed EuMal+. In addition,
the concentration of free Eu(III) was 3.0 × 10−4 mM and that ofmonohydrated Eu(III) was 1.5 × 10−5 mM. The concentrationsof EuMal+, free Eu(III), and monohydrated Eu(III) decreased
with the decreasing ratio of Eu(III) to malic acid. In contrast,
the amount of Eu(Mal)2− fell from 0.059 to 0.031 mM as theratio of Eu(III) to malic acid dropped from 0.1:1 to 0.05:1; itthen increased to 0.19 mM at the ratio of 0.2:10, while theconcentration of malic acid rose from 1.0 to 10 mM. Because
malic acid was broken down in the 0.2:10 medium but not in
0.05:1 medium, this result suggests that Eu(Mal)2− does nothinder the degradation of malic acid by P. fluorescens.
The chemical species of Eu(III) in solutions at pH 4.0 – 7.0(Figures 2a and 2b) showed that predominant ones, Eu(Mal)2−and EuMal+, remained unchanged during cell growth, eventhough the pH in the medium rose from 6.5 to 6.9.
Fuma et al. reported that the toxicity of Gd(III) inhibited thegrowth of E. coli in media containing more than 0.3 mM,
although growth was observed in those media containing lessthan 0.1 mM Gd(III).5 Weltje and colleagues studied the effectof Lu(III) on the metabolic rate of the Gram-negative Vibrio
fischeri in the presence of organic acids, such as EDTA, NTA,
citrate, oxalate, and malate.7 They found that metabolic rate

TABLE 1: Speciation of Eu(III) in Media Containing Various Ratios of Eu(III) to Malic Acid
Eu(III) to Malic
acid ratio
in the medium

Mal

Eu(III)

Eu(Mal)2−

EuMal+Eu3+

EuOH2+

Mal2−

(mM)

(mM)

(mM)

(mM)

(mM)

(mM)

(mM)

0:1
0 : 100.1 : 10.05 : 1
0.2 : 100.1 : 100.05 : 10

1
10
1.0
1.0
10
10
10

0
0
0.1
0.05
0.2
0.1
0.05

0
0
0.059
0.031
0.19
0.095
0.047

0
0
0.039
0.019
0.011
0.00540.002600
6.8 × 10−4
3.0 × 10−4
1.6 × 10−5
7.8 × 10−6
3.8 × 10−600
3.9 × 10−5
1.5 × 10−5
7.2 × 10−7
3.5 × 10−7
1.8 × 10−7

0.98
9.8
0.83
0.89
9.5
9.7
9.8

Percentage of malic
acid remaining in themedium at 72 after
inoculation (%)
0
0
100
100
88
65
15

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

Effect of Eu(III) on the Degradation of Malic Acid

(a)

(b)

0

0

97

-

Eu(Mal)2

-1

-1

-

-2

Log concentration ofEu(III) species (mM)

Log concentration ofEu(III) species (mM)

Eu(Mal)2
EuMal+

-3

Eu3+

-4
-5

-2
-3
-4

4.5

5.0

5.5

6.0

EuOH 2+
-6

7

6.5

Eu3+

-5

EuOH2+
Eu(OH)2+-6
4.0

EuMal+

4.0

4.5

5.5

5.0

6.0

7

6.5

pH

1.0

8

0.8

6

0.6

4

0.4

2

0.2
0.0

0
50

100

150

200

8

0.8

6

0.6

4

0.4

2

0.2

0

250

0

50

100

10

1.0

8

0.8

6

0.6

4

0.4

2

0.2
0.0
50

100

150

200

250

Time (h)Malic acid concentration (mM)

1.2

(b)

Pyruvic acid concentration (mM)

Malic acid concentration (mM)Time (h)

0

150

200

250

0.0

Time (h)

12

0

1.0

1.2

12

(d)

10

1.0

8

0.8

6

0.6

4

0.4

2

0.2

0

0.0
0

50

100

150

200

Pyruvic acid concentration (mM)

0

1.2

(c)

10

Pyruvic acid concentration (mM)

(a)

10

12

Malic acid concentration (mM)

1.2

12

Pyruvic acid concentration (mM)

Malic acid concentration (mM)

pH
Figure 2. Calculations of the speciation of Eu(III)-malate complexes in media containing (a) 0.05 mM Eu(III) and 1 mM malic acid (the 0.05:1
medium), and (b) 0.2 mM Eu(III) and 10 mM malic acid (the 0.2:10 medium). The concentration of each species is expressed on a logarithmic
scale.

250

Time (h)Figure 3. Time course of changes in the concentration of ○ malic acid and ● pyruvic acid in the medium after exposure to P. fluorescens; (a)without Eu(III), and with Eu(III) with a malic acid to Eu(III) molar ratio of (b) 0.05:10, (c) 0.1:10, and, (d) 0.2:10.

abruptly decreased with increasing free Lu3+ concentration from10−3 to 10−2 mM and fell almost to zero when the concentration
was above 10−2 mM. In our study, a concentration of freeEu(III) in the 0.05:1 medium of 3.0 × 10−7 mM retarded themetabolism of malic acid by P. fluorescens; this value is muchless than those reported for E. coli and V. fischer.
Trivalent lanthanide ions bind to the surface of humanerythrocytes by electrostatic interaction, forming pores on themembrane.13 Further, this effect is caused by positively charged
species, especially free Eu3+. From our study, we suggest that
EuMal+ may affect the degradation of malic acid because theconcentration of free Eu(III) is very low.
According to Table 1, lower levels of Eu(III) generated
lower concentrations of EuMal+, Eu3+, and EuOH2+ with thesame ratio of malic acid to Eu(III). On the other hand, fromcomparing media containing the same amount of Eu(III), we
see that if the malic-acid concentration increases ten-fold theconcentration of EuMal+ decreases 1/8 fold, and those of Eu3+and EuOH2+ drop 1/100 fold because two atoms of malic acidcan coordinate to one Eu(III).
3.3. Pyruvic acid concentration in the malic-acid medium.
In our previous work, we found that two types of organic
metabolites were produced following the degradation of 10mM malic acid or in medium containing 0.1:10 and 0.05:10

Eu:malic acid.8 These organic metabolites were eluted from
LC at 8.85 and 12.03 min. To characterize the organic acids
produced by P. fluorescens during the inoculation period, we
measured the elution time and ESI-Mass spectra of standard
acid solutions and compared them to those of these organicacid metabolites. The standards used were organic acids in the
TCA cycle; fumaric-, maleic-, succinic-, oxaloacetic-, citric-,
isocitric-, glyceric-, glyoxilic-, and pyruvic acid. Because the
ESI-Mass spectrum and elution time of pyruvic acid is similar
to that of the organic acid eluted at 8.85 min, the most probable designation for it is pyruvic acid. Pyruvic acid is an intracellular metabolite of L-malic acid that reacts with the cellularenzyme malate dehydrogenase; the pyruvic acid so generated
is excreted into the medium.14
The m/zs of the other unidentified organic acid detected at
12.03 min were 115 and 161. We could not plausibly assign
them to any organic acid in the TCA cycle. We are pursuingtheir identification.
The pyruvic acid generated remained in solution in themedium and formed complexes with Eu(III). Figures 3a-d
show the concentrations of malic acid and pyruvic acid. In themedium containing only 10 mM malic acid, the concentrationof pyruvic acid reached its maximum (0.08 mM) at about 48 h
and fell to zero at 72 h after inoculation. This finding demon-

98

Nankawa

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

strated that P. fluorescens metabolized pyruvic acid.
In the 0.05:10 medium (Figure 3b), pyruvic-acid concentration was 0.60 mM at 144 h after inoculation and decreased toapproximately 0.2 mM at 264 h. In the 0.1:10 medium (Figure3c), its concentration was 0.58 mM at 192 h after inoculation,and had reached to 0.15 mM at 264 h. In the 0.2:10 medium(Figure 3d), the amount of pyruvic acid still was increasing at
264 h because malic acid still was present in the solution.
For media with ratios 0.05:10 and 0.1:10 Eu(III) to malic
acid, the maximum concentrations of pyruvic acid were 0.60
and 0.58 mM, respectively, compared with 0.08 mM in thesolution without Eu(III). These results suggest that theamounts of pyruvic acid produced are not directly correlatedwith the concentration of Eu(III). The concentrations ofpyruvic acid remaining in these media after malic acid wascompletely degraded was higher than that of Eu(III), suggestingthat they were enough to mask the toxicity of Eu(III), andfurther, that the pyruvic acid generated was metabolicallydegraded by P. fluorescens.
Appanna et al. studied the biodegradation of trivalent cationcitrate complexes by P. fluorescens.15, 16 They showed that
Al(III)-citrate was metabolized intracellularly, while organic
acids, such as oxalic acid and glyoxlic acid, were excreted into
the medium. With Ga(III)-citrate, its β-hydroxyaspartate derivative was released into the solution, while Ga(III) predominatedin the solution after citric acid was broken down.16
The stability constant of the 1:1, 1:2, and 1:3 Eu-pyruvic
acid complexes are 1.88, 3.3, and 3.8, respectively.17 Although
the stability constants of the 1:1 and 1:2 Eu(III)-malic acidcomplex are greater than those of their respective pyruvic
acids, more malic acid was degraded in the presence of Eu(III).
Acknowledgements. The present study was partially
supported by a Grant-in-Aid of the Ministry of Education,
Culture, Sports, Science and Technology and by a Grant-in-Aid
for Scientific Research from Japan Society for the Promotion of
Science to T.O.

References(1) R. A. Fishier, J. E. Gordon, and N. E. Phillips, Annu. Rev.
Phys. Chem. 47, 283 (1996).
(2) J. M. D. Coey and H. J. Sun, J. Magn. Mater. 87, 251 (1990).
(3) K. Binnemans, P. Lenaerts, K. Driesen, and C. GörllerWalrand, J. Mater. Chem. 14, 191 (2000).
(4) P. Carvan, J. J. Ellison, T. J. McMurry, and R. B. Lauffer,Chem. Rev. 99, 2293 (1999).
(5) S. Fuma, H. Takeda, K. Miyamoto, K. Yanagisawa, Y.
Inoue, N. Ishii, K. Sugai, C. Ishii, and Z. Kawabata, Bull.
Environ. Contam. Toxicol. 66, 231 (2001).
(6) E. J. Plummer and L. E. Macaskie, Bull. Environ. Contam.
Toxicol. 44, 173 (1990).
(7) L. Weleje, L. R. C. W. Verhoof, W. Verweij, and T. Hamers,
Environ. Sci. Technol. 38, 6597 (2004).
(8) T. Nankawa, Y. Suzuki, T. Ozaki, T. Ohnuki, and A. J.
Francis, J. Alloys Comp. accepted.
(9) I. Puigdomenech, MEDUSA and HYDRA software forchemical equilibrium calculations, version 19, January
1999, Royal Institute of Technology (KTH), Stochholm,
Sweden.
(10) A. J. Francis, C. J. Dodge, and J. B. Gillow, Nature 356,
140 (1992).
(11) T. Moeller, D. F. Martin, L. C. Thompson, R. Ferrús, G.
R. Feistel, and W. J. Randall, Chem. Rev. 65, 1 (1965).
(12) S. A. Ali, N. A. Dobrynina, L. I. Martynenko, and V. G.
Gontar, Russ. J. Inorg. Chem. 25, 1637 (1980).
(13) K. Wang, R. Li, Y. Cheng, and B. Zhu, Coord. Chem.
Rev. 190-192, 297 (1999).
(14) W. Kniche and F. Radler, Eur. J. Biochem. 123, 547 (1982).
(15) V. D. Appanna and R. D. Hamel, Recent Res. Dev. Microbiol.
3, 615 (1999).
(16) V. D. Appanna, R. D. Hamel, and R. Lévasseur, Cur.
Microbiol. 47, 47 (2003).
(17) G. R. Choppin and R. E. Cannon, Inorg. Chem. 19, 1889(1980).