Microbial Transformations of Actinides: Implications on Radioactive
Waste Disposal and Environmental Remedaition

                                     Arokiasamy J. FRANCIS
                                                        Environmental Sciences Department
                                                        Brookhaven National Laboratory, Upton,

 The presence of the actinides Th, U, Np, Pu, and Am in transuranic (TRU) and mixed wastes is a major concern because of their potential for migration from the waste repositories and long-term contamination of the environment. The actinides in TRU and mixed wastes may be present in various forms, such as elemental, oxide, coprecipitates, inorganic, and organic complexes, and as naturally occurring minerals depending on the process and waste stream. The actinides exist in various oxidation states: Th (III, IV); U (III, IV, V, VI); Np (III, IV, V, VI, VII); Pu (III, IV, V, VI, VII); and Am (III, IV, V, VI, VII).

 In addition to the radionuclides the TRU waste consists a variety of organic materials (cellulose, plastic, rubber, chelating agents) and inorganic compounds (nitrate and sulfate).

 Significant microbial activity is expected in the waste because of the presence of organic compounds and nitrate, which serve as carbon and nitrogen sources and in the absence of oxygen the microbes use nitrate and sulfate as alternate electron acceptors.
  Biodegradation of the TRU waste can result in gas generation and pressurization of containment areas, and waste volume reduction and subsidence in the repository. Although the physical, chemical, and geochemical processes affecting dissolution, precipitation, and mobilization of actinides have been investigated, we have only limited information on the effects of microbial processes.

 Microorganisms have been detected in TRU wastes, Pu-contaminated soils, low-level radioactive wastes, backfill materials, natural analog sites, and waste-repository sites slated for high-level wastes. Microbial activity could affect the chemical nature of the actinides by altering the speciation, solubility and sorption properties and thus could increase or decrease the concentrations of actinides in solution. Actinides may be present initially as soluble or insoluble forms and, after disposal, may be converted from one to the other by microorganisms. Under appropriate conditions, actinides can be solubilized or precipitated by direct (enzymatic) or indirect (nonenzymatic) actions of microorganisms.
  These include (i) oxidation-reduction reactions, (ii) changes in pH and Eh, (iii) chelation, or the production of specific sequestering agents, (iv) biosorption and bioaccumulation by biomass and biopolymers, (v) bioprecipitation reactions leading to the formation of stable minerals, and (vi) biotransformation of actinides complexed with organic and inorganic ligands. Free-living bacteria suspended in the groundwater fall within the colloidal size range and may have strong radionuclide sorbing capacity, giving them the potential to transport radionuclides in the subsurface. Microbial activities are influenced by electron donors and acceptors and the extent of dissolution and precipitation could be significant, particularly under anaerobic conditions.
  In anaerobic environments, actinides can be reduced enzymatically from a higher oxidation state to a lower one, which affects their solubility and bioavailability. For example, reduction of U6+ to U4+ decreases its solubility. Key microbial processes involved in the mobilization or immobilization of selected actinides of interest is summarized in Table 1. Among the actinides, biotransformation of uranium has been extensively studied, whereas we have only limited understanding of the microbial transformations of other actinides such as Th, Np, Pu, and Am present in TRU and mixed wastes [1, 2].

 Chelating agents are present in TRU and mixed wastes because they are widely used for decontaminating nuclear reactors and equipment, in cleanup operations, and in separating radionuclides.
  Many organic compounds form stable complexes with actinides, and increase their solubilization and leaching. Plutonium forms very strong complexes with a variety of organic ligands. Naturally occurring organic complexing agents, such as humic and fulvic acids, and likewise microbially produced complexing agents, such as citrate, and siderophores, as well as synthetic chelating agents and the products or intermediates from waste degradation may be an important source of agents affecting the solubility and mobility of actinides.
  Biotransformation of actinide-organic complexes should result in the degradation of the organic ligand and precipitation of the actinide [3].

Remediation of Radionuclide Contaminated Soils and Wastes, and Materials.
 Fundamental understanding of the mechanisms of microbiological transformations of various chemical forms of uranium present in wastes and contaminated soils and water has led to the development of novel bioremediaition processes. One process uses anaerobic bacteria to stabilize the radionuclides and toxic metals from the waste, with a concurrent reduction in volume due to the dissolution and removal of nontoxic elements from the waste matrix. In an another process, uranium and other toxic metals are removed from contaminated soils and wastes by extracting with the chelating agent citric acid. Uranium is recovered from the citric acid extract after biodegradation/photodegradation in a concentrated form as UO3×2H2O for recycling or appropriate disposal [4, 5].

Stabilization of Uranium by Reductive Precipitation by Anaerobic Bacteria.
  Immobilization of uranium is brought about by bioreduction and bioprecipitation reactions. Uranium is reduced by a wide variety of facultative and strict anaerobic bacteria under anoxic conditions in the presence of suitable electron donors.

 Speciation of uranium in microbial cultures by x-ray absorption near edge spectroscopy (XANES) and X-ray photoelectron spectroscopy (XPS) showed that soluble U(VI) was reduced to insoluble U(IV) by the anaerobic bacterium, Clostridium sp [Fig 1]. Treatment of uranium- and toxic-metal contaminated sediment and sludge with the anaerobic bacterium Clostridium sp. removed a large fraction of soluble non-toxic metals such as Ca, K, Mg, Mn2+, Na, and Fe2+, enriched and stabilized Cd, Cr, Cu, Ni, Pb, U and Zn, and reduced the overall volume and mass [Figs. 2 and 3]. In this novel approach to treating wastes, the unique metabolic capabilities of the dual-action anaerobic bacteria were exploited to solubilize and/or precipitate radionuclides and toxic metals directly by enzymatic action and indirectly by the production of organic acid metabolites.  
The non-hazardous materials in the solid phase were solubilized and removed from the waste, thereby reducing its volume. The remobilized radionuclides and toxic metals are stabilized by precipitation reactions and redistributed with stable mineral phases of the waste.

 Consequently, the potential exists for he use of anaerobic bacteria to concentrate, contain and stabilize U in contaminated groundwaters and in waste with concurrent reduction in waste volume. Reactive barrier technology is based on the activities of these anaerobic bacteria. However, the long-term stability of bacterially immobilized U in the natural environment is poorly understood.


Removal and Recovery of uranium from contaminated soils and wastes.
  Citric acid, a naturally occurring compound, is a multidentate ligand, which forms stable complexes with various metal ions.
  It forms different types of complexes with transition metals and actinides including formation of a bidentate, tridentate, binuclear, or polynuclear complex species. Biodegradation of metal citrate complexes is dependent upon the type of complex formed between the metal and citric acid; bidentate complexes are readily biodegraded whereas the tridentate complexes are recalcitrant [3]. Pseudomonas fluorescens metabolized the bidentate complexes whereas complexes involving the hydroxyl group of citric acid, and the binuclear U-citrate complex are not [Fig. 4].
  The presence of the free hydroxyl group of citric acid is the key determinant in effecting biodegradation of the metal complex. The lack of degradation was not due to their toxicity but was limited by the transport and/or metabolism of the complex by the bacteria. No relationship was observed between biodegradability and stability of the complexes.

 For decontamination, uranium must be removed and recovered from the contaminated site, so that the site is restored. Various soil washing techniques have been developed including physical methods, such as wet-screening, attrition scrubbing, or chemical methods consisting of treating with organic and inorganic acids, salts, bases, and chelating agents. For example, nitric acid, hydrochloric acid, phosphoric acid, sulfuric acid, sodium carbonate, ammonium carbonate, sodium hydroxide, oxalic acid, citric acid, EDTA, and DTPA have been used to extract radionuclide and toxic metals. Many of the inorganic chemicals used are corrosive, which irreparably damages the soil. Furthermore, all chemical extraction methods generate secondary waste streams which create further problems of hazardous waste disposal.

 Among the several organic complexing agents used in extracting metals, citric acid appears to be the most preferred because it is a naturally occurring organic complexing agent. It is environmentally friendly, exhibits relatively consistent removal efficiency, and is cost-effective. Citric acid extract is subjected to biodegradation, followed by photodegradation [Fig.5]. Several metal citrate complexes are readily biodegraded, and the metals are recovered in a concentrated form, along with the bacterial biomass. Uranium forms a binuclear complex with citric acid and is recalcitrant.
  The supernatant containing this complex is separated, and exposed to light; it rapidly degrades with the precipitation of uranium. Uranium is recovered as UO3×2H2O in a concentrated form for recycling, or for disposal [Fig. 5]. This treatment process, unlike others, does not generate additional hazardous wastes for disposal and causes little damage to the soil which is then be returned to normal use.

 This process has significant potential for commercialization because (i) it can be applied to a variety of materials and waste forms; (ii) mixed waste is separated into radioactive and hazardous waste; (iii) uranium is separated from the toxic metals and recovered for recycling or disposal; (iv) it does not generate secondary waste streams; (v) it causes little damage to soil; and (vi) environmentally and economically important metals are removed in a concentrated form. The use of combined chemical, photochemical, and microbiological treatments of contaminated materials will be more efficient than present methods and result in considerable savings in clean up and disposal costs.

 Microorganisms can alter the stability and mobility of actinides in radioactive wastes and in the natural environment. Such microbial transformations of uranium have been extensively studied. The direct implication of microorganisms in precipitating actinides is important because of the potential application in bioremediating contaminated sites, in pre-treating radioactive wastes, and in processes critical to nuclear-waste repositories. Although a wide variety of microorganisms are present in radioactive wastes and natural radioactive mineral deposits, the extent to which they regulate the mobility of the actinides is not fully understood. Furthermore, the effects of microbial activities on TRU and mixed waste and their potential for treatment of certain waste forms to stabilize the actinides and reduce the volume of the waste have not been fully exploited.

 Fundamental understanding of the mechanisms of microbial transformations of different chemical forms of actinides under various environmental and microbial process conditions such as aerobic, anaerobic (denitrifying, fermentative, and sulfate reducing) and repository relevant conditions will be useful in predicting the long-term performance of waste repositories and in developing novel strategies for waste management and remediation of contaminated sites.

Acknowledgment. This research was sponsored by the Environmental Remediation Sciences Division, Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy under contract No. DE-ACO2-76CH00016.

1. Francis, A.J. 2001. Microbial transformations of plutonium and implications for its mobility. In“Plutonium in the Environment” A. Kudo, (Ed) Elsevier Science Ltd., Co., UK. Pp 201-219.
2. Francis, A.J., C.J. Dodge, F. Lu, G. Halada, and C.R. Clayton. 1994. XPS and XANES studies of uranium reduction by Clostridium sp. Environ. Sci. Technol. 28:636-639.
3. Francis, A.J., C.J. Dodge, J.B. Gillow. 1992. Biodegradation of metal citrate complexes and its
implications for toxic metal mobility. Nature, 356:140-142.
4. Francis, A.J. and C.J. Dodge 1998. Remediation of soils and wastes contaminated with uranium and toxic metals. Environ. Sci. Technol.32: 3993-3998.
5. Francis, A.J. 1999. Bioremediation of radionuclide and toxic metal contaminated soils and wastes. In
Bioremediation of Contaminated Soils, pp 239-271. Agronomy Monograph No. 37. ASA, CSSA, SSSA, Madison, WI.