Biodegradation of metal-citrate complexes by Pseudomonas fluorescens depends on the nature of the complex formed between the metal and citric acid. Bidentate Fe(III)-, Ni-, and Zn-citrate complexes were readily biodegraded, but the tridentate Cd-and Cu-citrate, and U-citrate complexes were not. The biodegradation of Ni-and Zn-citrate commenced after an initial lag period; the former showed only partial (70%) degradation, whereas the latter was completely degraded. Uptake studies with 14 C-labeled citric acid and metal-citrate complexes showed that cells grown in medium containing citric acid transported free citric acid at the rate of 28 nmol min ؊1 and Fe(III)-citrate at the rate of 12.6 nmol min ؊1 but not Cd-, Cu-, Ni-, U-, and Zn-citrate complexes. However, cells grown in medium containing Ni-or Zn-citrate transported both Ni-and Zn-citrate, suggesting the involvement of a common, inducible transport factor. Cell extracts degraded Fe(III)-, Ni-, U-, and Zn-citrate complexes in the following order: citric acid ‫؍‬ Fe(III)-citrate > Ni-citrate ‫؍‬ Zn-citrate > U-citrate. The cell extract did not degrade Cd-or Cu-citrate complexes. These results show that the biodegradation of the U-citrate complex was limited by the lack of transport inside the cell and that the tridentate Cd-and Cu-citrate complexes were neither transported inside the cell nor metabolized by the bacterium.Citric acid is a multidentate ligand and forms stable complexes with various metal ions (19). The rate and extent of biodegradation of several metal-citrate complexes by microorganisms vary. For example, Pseudomonas pseudoalcaligenes degraded Mg-citrate at a much lower rate than Ca-, Fe(III)-, and Al(III)-citrate (29). Studies with a Klebsiella sp. showed that citric acid and Mg-citrate were readily degraded, whereas Cd-, Cu-, and Zn-citrate were resistant to biodegradation (9). Both these studies also showed that metal toxicity was not responsible for the lack of or the lower rate of degradation of certain metal-citrate complexes but gave no other explanation. Biodegradation studies with Pseudomonas fluorescens showed that bidentate complexes of Fe(III)-, Ni-, and Zn-citrate were readily biodegraded, whereas complexes that involve the hydroxyl group of citric acid, the tridentate Cd-and Cu-citrate complexes, and U-citrate complex were not biodegraded (17). No relationship between biodegradability and stability of the complexes was observed. The tridentate Fe(II)-citrate complex, although recalcitrant, was readily biodegraded after oxidation and hydrolysis to the bidentate Fe(III)-citrate form, denoting a structure-function relationship in the metabolism of the complex (16).Microorganisms have selective transport systems for the uptake of metals of known biological function (11,23,28). Heavy metals are transported into cells by preexisting transport systems and are subsequently either chemically neutralized, sequestered, or removed from the cells by rapid efflux (6,35). Metals affect the transport of citric acid in bacteria. For example, the...

Biodegradation of 1:1 nickel:citric acid by Pseudomonas fluorescens proceeded after a lag (∼17 h) at the rate of 11 ( 1 µmol h -1 , with only partial mineralization of the complex. The incomplete degradation of the complex was not attributed to changes in its structure, but was due to the toxicity of the Ni released. Addition of 1:1 Ni:citric acid inhibited glucose metabolism by the bacterium. The toxicity of the released Ni was evident only when it attained a threshold concentration of >0.3 mM in the culture medium. Speciation calculations showed that Ni released after metabolism of the complex was present as Ni 2+ ion and nickel carbonate. Addition of iron as a ferric hydroxide or 1:1 Fe:citric acid to 1:1 Ni:citric acid resulted in the complete metabolism of the Ni-citrate complex, with concurrent removal of the released Ni from solution by coprecipitation with iron.

Clostridium sphenoides, which uses citric acid as its sole carbon source, metabolized equimolar Fe(III)-citrate with the degradation of citric acid and the reduction of Fe(III) to Fe(II), but not the U(VI)-citrate complex. However, in the presence of excess citric acid or added glucose it was reduced to U(IV)-citrate. In contrast, Clostridium sp" which ferments glucose but not citrate, reduced Fe(III)-citrate to Fe(II)-citrate and U(VI)-citrate to U(IV)-citrate only when supplied with glucose, These results show that complexed uranium is readily accessible as an electron acceptor despite the bacterium's inability to metabolize the organic ligand complexed to the actinide. These results also show that the metabolism of the metal-citrate complex depends upon the type of complex formed between the metal and citric acid. Fe(III) forms a bidentate complex with citric acid and was metabolized, whereas U forms a binuclear complex with citric acid and was recalcitrant.

The hlgh-grade uranium deposit at Cigar Lake, Canada, is being investi-MATERIALS AND METHODS Water Sample Water samples were collected from plezometers located throughout the deposit down to 450 m below the surface (Figure 2). Samples were collected by J. Cramer, AECL-Research, Plnawa, Canada, using a down-hole sampling rig '