Journal of Radioanalytical and Nuclear Chemistry, Vol. 266, No. 2 (2005) 199204 02365731/USD 20.00 AkadØmiai Kiad, Budapest ' 2005 AkadØmiai Kiad, Budapest Springer, Dordrecht Biodegradation of Eu(III)-citrate complexes by Pseudomonas fluorescens Y. Suzuki, 1,2 * T. Nankawa, 2 T. Yoshida, 2 T. Ozaki, 2 T. Ohnuki, 2 A. J. Francis, 3 S. Tsushima, 1 Y. Enokida, 4 I. Yamamoto 1 1 Department of Materials, Physics, and Energy Engineering, Graduate School of Engineering, Nagoya University, Furocho, Chikusa-ku, Nagoya, 464-8603, Japan 2 Advanced Science Research Center, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-1195, Japan 3 Environmental Sciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA 4 Ecotopia Science Institute, Nagoya University, Furocho, Chikusa-ku, Nagoya, 464-8603, Japan (Received October 4, 2004) We have investigated the structure of Eu(III)-citrate complexes in aqueous solution and their degradability by Pseudomonas fluorescens. Analysis of 1 : 1, 1 : 2, and 1 : 5 Eu(III) : citrate solutions at pH 7 by electrospray ionization mass spectrometry (ESI-MS) showed that the 2 : 2 Eu(III)-citrate complex is the predominant complex species at a low citrate/Eu(III) ratio, while at a high ratio, a 1 : 2 Eu(III)-citrate complex is formed preferably. Studies on the biodegradation of Eu(III)-citrate complex by P. fluorescens have shown that a 2 : 2 Eu(III)-citrate complex is resistent to degradation while a 1 : 2 complex transforms to a 2 : 2 complex with the degradation of excess citric acid. Introduction Trivalent actinides [An(III)], such as Am(III) and Cm(III) present in high-level radioactive waste (HLW) are problematic because they emit high-energy - particles and have long half-lives. It is essential to study the mobility of An(III) in the environment in order to evaluate the safety of the geological disposal of HLW. At neutral pH, the mobility of An(III) is low because of their low solubility and high affinity with inorganic particles. 1,2 On the other hand, in the presence of chelating substances, An(III) show high solubility and low affinity with these particles because they form soluble complexes. 3 Citric acid is one of the naturally-occurring chelating substances, 4 and it forms stable and soluble complexes with An(III). 5 Citric acid is readily metabolized both aerobically and anaerobically as a carbon source by a variety of microorganisms. 6,7 It is known that the soil microorganism Pseudomonas fluorescens degrades citrate complexes such as Fe(III)-, and Zn(II)-, and Ni(II)-citrate complexes. 8,9 The mobility of An(III)- citrate complexes may be linked to their biodegradability. It has been proposed that the biodegradability of metal citrate complexes by P. fluorescens is related to their structure. 8,9 Mononuclear bidentate complexes are readily degraded while mononuclear tridentate, binuclear, and polynuclear complexes are resistent. Neither the biodegradability of An(III)-citrate complexes by P. fluorescens nor their structure in aqueous solution is fully understood. Since Eu(III) is a good analogue of An(III), it is helpful to study the environmental behavior of Eu(III)- citric complexes to estimate that of An(III)-citrate * E-mail: h042306d@mbox.nagoya-u.ac.jp complexes. In the present study, we investigated the stoichiometric structure of Eu(III)-citrate complexes by electrospray ionization mass spectrometry (ESI-MS), and studied their biodegradability by P. fluorescens using a batch method. ESI-MS, which is often employed for the determination of the stoichiometries of metal- ligand complexes, 10,11 was used to obtain the stoichiometries of Eu(III)-citrate complexes at various Eu(III)/citrate ratios at neutral pH. The stoichiometries of the U(VI)-citrate complexes obtained from ESI-MS analysis showed good agreement with those found in titrimetric and spectrophotometric studies. 12 Experimental ESI-MS analysis of Eu(III)-citrate complexes Eu(III)-citrate complexes were investigated at pH 7 by ESI-MS. Solutions of EuCl 3 and citric acid were prepared by dissolving EuCl 3 •6H 2 O (Wako Pure Chemical Industries, Ltd., Osaka, Japan, 99.9%) and citric acid (Wako, 98.0%) in water purified with a Milli- Q system (Millipore Corporation, Bedford, MA, USA). Samples were prepared by mixing an appropriate amount of these solutions with acetonitrile, giving solutions containing 0.50 mM EuCl 3 and 0.50, 1.0, or 2.5 mM citric acid in 20% (v/v) acetonitrile/H 2 O. Twenty percent (v/v) acetonitrile/H 2 O was used to ease solvent evaporation in the ion source. The pH of the samples was adjusted to 7.0 with NaOH. ESI-MS measurements were performed using high-pressure liquid chromatography-mass spectroscopy (HPLC-MS) (Alliance 2695, Waters Corporation, Milford, MA, USA). Samples were directly introduced with a flow rate of 10 l . min 1 .