Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 17-20, 2005

Accepted
Biotransformation of Radioactive Waste: Microbial Reduction of Actinides and Fission
Products
Jonathan R. Lloyd,*, a Joanna C. Renshaw,a, b Ian May,b Francis R. Livens,c Ian T. Burke,cRobert J. G. Mortimerc,c and Katherine Morrisca

The Williamson Research Centre for Molecular Environmental Studies and The School of Earth,Atmospheric and Environmental Sciences, The University of Manchester, Manchester M13 9PL, UKb
Centre of Radiochemistry Research and The School of Chemistry, The University of Manchester,Manchester M13 9PL, UK
cSchool of Earth and Environment, University of Leeds, Leeds, LS2 9JT, UK
Received: November 15, 2004; In Final Form: November 15, 2004
The microbial reduction of radionuclides has attracted recent interest as these transformations can play crucial
roles in controlling the mobility of key redox active actinides and fission products in a range of environments and,
if harnessed, may offer the basis of biotechnological processes for the remediation of radioactive waste. This
review focuses on recent research on the reduction of radionuclides including U(VI), Np(V), Pu(IV), and Tc(VII).
Rapid advances over the last decade have resulted in a detailed understanding of some of these transformations at
a molecular level. Where known, the mechanisms of metal reduction are discussed, alongside the environmental
impact of such transformations and possible biotechnological applications that could utilise these activities.

1. Introduction
The release of radionuclides from nuclear sites and theirsubsequent mobility in the environment is a subject of intense
public concern. Natural sources of radioactivity include U(present in Earth’s crust at concentration of 1.8 ppm), Th, Ra
isotopes, and radon, while significant quantities of natural andartificial/manmade radionuclides were also released as aconsequence of nuclear weapons testing in the 1950s and1960s, and via accidental release e.g. from Chernobyl in 1986.1
The major burden of anthropogenic environmental radioactivity, however, is from the controlled discharge of process
effluents produced by industrial activities allied to the generation of nuclear power. The inventory of radionuclides generated during the last 60 years of operating fission reactors islong and includes 237Np, Pu isotopes, Am, 3H, 14C, 85Kr, 90Sr,
99
Tc, 129I, 137Cs, several of which (notably key actinides, 99Tc
and 129I) are redox active. Wastes containing some or all ofthese radionuclides are produced at the many steps in the
nuclear fuel cycle, and vary considerably from low level, highvolume radioactive effluents produced during uranium mining
to the intensely radioactive plant, fuel and liquid wastes
produced from reactor operation and fuel reprocessing. All
wastes pose a potential threat to the environment and require(1) treatment prior to release, and (2) a much deeper understanding of the biological and chemical factors controlling themobility of radionuclides in the environment should they bedispersed by accident or as part of a controlled/monitored
release e.g. in effluents. The aim of this review is to describe
what is known about the interactions of anaerobic metalreducing with key redox-active radionuclides, and whereappropriate to discuss how such reductive biotransformations
can impact on the mobility of radionuclides in the environment. In addition, as there is intense interest in harnessingthese natural processes for in situ and ex situ remediation ofradioactive waste, the biotechnological applications ofradionuclide-microbe interactions are also discussed. Indeed,*Corresponding author. E-mail: jon.lloyd@manchester.ac.uk.
FAX: +44-161-275-3947.

research programmes aimed at remediating large areas of land
contaminated by radionuclides in the US, at so called “superfund sites”, have resulted in significant advances in the understanding of the mechanisms of metal and radionuclide reductionin the subsurface (for examples see www.lbl.gov/NABIR/).
2. Reduction of Fe(III)Microbial metal reduction has been studied intensively overthe last two decades, and the best studied examples of dissimilatory metal-reducing prokaryotes are the wide range ofArchaea and Bacteria that are able to conserve energy throughthe reduction of Fe(III) (ferric iron) to Fe(II) (ferrous iron).
The environmental relevance of Fe(III) reduction have been
well documented.2, 3 Indeed, geochemical and microbiologicalevidence suggest that the reduction of Fe(III) may have been
an early form of respiration on Earth,4 and is a candidate forthe basis of life on other planets.5 On modern Earth, Fe(III)can be the dominant electron acceptor for microbial respirationin many subsurface environments.6 As such, Fe(III)-reducing
communities can be responsible for the majority of the organic
matter oxidized in such environments. Recent studies haveshown that a range of important xenobiotics that contaminate
aquifers can also be degraded under anaerobic conditions by
Fe(III)-reducing microorganisms.7 In addition to playing animportant role in the degradation of organic material, Fe(III)reducing microorganisms can influence the mineralogy of sediments through the reductive dissolution of insoluble Fe(III)oxides. These processes can result in the release of potentially
toxic levels of Fe(II), and also trace metals and radionuclides
that were bound by the Fe(III) minerals. Depending on thechemistry of the water, a range of reduced minerals can also be
formed including magnetite (Fe 3 O 4 ), siderite (FeCO 3 ) andvivianite (Fe3(PO4)2 · 8H2O), resulting in a change in structureof the sediments. Finally, Fe(III)-reducing microorganisms
can also impact directly on the fate of other high valence contaminant metals and radionuclides through direct enzymatic
reduction, and also via indirect reduction catalysed by biogenic
Fe(II). The bioreduction of U(VI) and other actinides, in addition to fission products such as Tc(VII) by Fe(III)-reducing© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

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Lloyd

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

microorganisms will be discussed in detail in this review andcan result in immobilization of these potentially mobile
radionuclides in sediments.8

unable to reduce U(VI). Evidence was presented to suggestthat the mechanism of U(VI) reduction was distinct from thoseof Fe(III) and Mn(IV) reduction, but may share components of
the nitrite reducing pathway.17

3. Reduction of actinides and fission products
The metabolic diversity of Fe(III)-reducing bacteria is large,
and a wide range of other redox active metals can be reducedin lieu of Fe(III), including other more toxic transition metals
such as Cr(VI), Hg(II), Co(III), Pd(II), Au(III), Ag(I), Mo(VI),and V(V), metalloids such as As(V) and Se(VI), and radionuclides including U(VI), Np(V), and Tc(VII).6 Indeed, because
many radionuclides of concern are both redox active and lesssoluble when reduced, bioreduction offers much promise forcontrolling the solubility and mobility of target radionuclides
in contaminated sediments e.g. the reduction of U(VI) (theuranyl ion; UO22+) to U(IV) (uraninite; UO2)9 or the reductionof the fission product Tc(VII) (the pertechnetate ion; TcO4−) toTc(IV) (TcO2).10
4. Reduction of U(VI)The first demonstration of dissimilatory U(VI) reduction
was by Lovley and coworkers9 who reported that the Fe(III)reducing bacteria Geobacter metallireducens (previously
designated strain GS-15) and Shewanella oneidensis (formerly
Alteromonas putrefaciens and then Shewanella putrefaciens)
can conserve energy for anaerobic growth via the reduction of
U(VI). It should be noted, however, that the ability to reduce
U(VI) enzymatically is not restricted to Fe(III)-reducing
bacteria. Other organisms including a Clostridium sp.11 andthe sulfate-reducing bacteria Desulfovibrio desulfuricans12 andD. vulgaris13 also reduce uranium, but are unable to conserve
energy for growth via this transformation. To date, D. vulgaris
remains the only organism in which the enzyme system
responsible for U(VI) reduction has been characterised indetail. Purified tetraheme cytochrome c3 was shown to function as a U(VI) reductase in vitro, in combination with hydrogenase, its physiological electron donor. 13 In vivo studies
using a cytochrome c3 mutant of the close relative D. desulfuricans strain G20 confirmed a role for cytochrome c 3 inhydrogen-dependent U(VI) reduction, but suggested additional
pathways from organic electron donors to U(VI), that bypassed
the cytochrome.14
More recent studies have identified a homologous cytochrome(PpcA), a triheme periplasmic cytochrome c7 of the Fe(III)reducing bacterium Geobacter sulfurreducens that may also playa role in U(VI) reduction.15 The protein was able to reduce
U(VI) in vitro, while a ppcA deletion mutant supplied withacetate as an electron donor had lower activity against U(VI).
Additional (if indirect) evidence linking the activity of thisperiplasmic protein with U(VI) reduction in vitro included theprecipitation of the reduced product U(IV) in the periplasm,
and the lack of impact of protease treatment of whole cells on
the ability to reduce U(VI).15 This final result is important, as
it implies that U(VI) and Fe(III) may be reduced by different
mechanisms in G. sulfurreducens. A significant portion of
U(VI) would seem to be reduced in the periplasm, while thereduction of insoluble Fe(III) oxides was inhibited dramatically by protease treatment, presumably due to removal of
surface bound cytochromes required for reduction of the extracellular electron acceptor. More recent studies from our laboratory have identified U(V) as a key unstable intermediate in
the reduction of uranium by G. sulfurreducens, and as thisorganism is unable to reduce stable Np(V), it would seem thatthe specificity for hexavalent actinides is extremely high in
Geobacter species.16 The mechanism of U(VI) reduction by a
Shewanella putrefaciens strain has also been investigated.17 A
novel screening method was used to identify mutants that were

5. Reduction of Np(V) and Pu(IV)Although 238U remains the priority pollutant in most medium
and low level radioactive wastes, other actinides including230
Th, 237Np, 241Pu, and 241Am can also be present.1 Th(IV) andAm(III) are stable across most Eh values encountered in radionuclide-contaminated waters but the potentials for Pu(V)/Pu(IV)and Np(V)/Np(IV), in common with that of U(VI)/U(IV) are
more electropositive than the standard redox potential of ferrihydrite/Fe2+ (approximately 0 V).3 Thus, Fe(III)-reducing bacteria
have the metabolic potential to reduce these radionuclides enzymatically, or via Fe(II) produced from the reduction of Fe(III)oxides. This is significant because the tetravalent actinides areamenable to bioremediation due to their high ligand-complexing
abilities,18 and are also immobilized in sediments containing
active biomass.19 Thus, although it is possible for Fe(III)-reducing
bacteria to reduce and precipitate actinides in one step, e.g. thereduction of soluble U(VI) to insoluble U(IV) (see above),some transformations do not result in direct formation of an
insoluble mineral phase but in the formation of a cation moreamenable to bioprecipitation. This is illustrated when considering highly soluble Np(V) (NpO2+), which was reduced tosoluble Np (IV) by Shewanella putrefaciens, with the Np(IV)removed as an insoluble phosphate biomineral by a phosphateliberating Citrobacter sp.20 However, not all Fe(III)-reducing
bacteria are able to reduce Np(V), as recent work in our laboratory has shown that cells of Geobacter sulfurreducens areunable to reduce this actinide, suggesting a surprising degreeof specificity for hexavalent actinides such as U(VI).16 Thus,
bioremediation strategies that use Geobacter species to treat
mixed actinide wastes must be designed and monitored withcare. On this note, some studies have suggested that the reduction of Pu(IV) to (III) can be achieved by Fe(III)-reducing
bacteria, although the Pu(III) was reported to reoxidize spontaneously.21 Although this may lead to solublization of sediment-bound Pu(IV), it will yield a trivalent actinide that is alsoamenable to bioremediation using a range of microbially
produced ligands.18 The biochemical basis of these transformations remain uncharacterised.
6. Reduction of Tc(VII)The fission product technetium is another long-lived
radionuclide that is present in nuclear waste and has attracted
considerable recent interest. This is due to a combination of its
mobility as the soluble pertechnetate ion (Tc(VII); TcO4−),
bioavailability as an analog of sulfate and long half-life (2.13 ×
105 years).22 Like Np(V), Tc(VII) has weak ligand complexing
capabilities and is difficult to remove from solution using
conventional “chemical” approaches. Several reduced formsof the radionuclide are insoluble, however, and metal-reducing
microorganisms can reduce Tc(VII) and precipitate theradionuclide as a low valence oxide.
Although microbial metabolism was known to decrease thesolubility of Tc from earlier studies,23, 24 Lloyd and Macaskie
were the first to unequivocally demonstrate direct enzymatic
reduction of Tc(VII) by microorganisms.25 In this study, anovel phosphorimager technique was used to confirm reductionof the radionuclide by Shewanella putrefaciens and Geobacter
metallireducens, with similar activities subsequently detected inlaboratory cultures of Rhodobacter sphaeroides, Paracoccus
denitrificans, some Pseudomonads,26 Escherichia coli,27 and arange of sulfate-reducing bacteria.28−30 Other workers haveused this technique to show that Thiobacillus ferrooxidans andJ. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

Biotransformation of Radioactive Waste
31

T. thiooxidans and the hyperthermophile Pyrobaculum
islandicum 32 are also able to reduce Tc(VII). It should be
stressed that Tc(VII) reduction has not been shown to support
growth in any of these studies, and seems to be a fortuitousbiochemical side reaction in the organisms studied to date.
Finally, X-ray absorption spectroscopy studies have recently
identified insoluble Tc(IV) as the final oxidation state produced when Tc(VII) is reduced enzymatically by Geobacter
sulfurreducens,10 E. coli (Lloyd and Sole unpublished), andShewanella putrefaciens.33 Recent studies have also shown that
Tc(VII) can be reduced by indirect microbial processes via, forexample, biogenic sulphide,29 Fe(II) 10 or U(IV). 26 Tc(VII)
reduction and precipitation by biogenic Fe(II) is particularly
efficient, and may offer a potentially useful mechanism for the
remediation of Tc-contaminated sediments containing active
concentrations of Fe(III)-reducing bacteria.10
The biochemical basis of Tc(VII) reduction has been beststudied in Escherichia coli. Initial studies demonstrated thatanaerobic, but not aerobic, cultures of E. coli reduced Tc(VII)with the reduced radionuclide precipitated within the cell. 27
Results obtained from studies conducted with wild type cells
and 34 defined mutants defective in the synthesis of regulatory
or electron transfer proteins were used to construct a model forTc(VII) reduction by E. coli. The central tenet of this model isthat the hydrogenase 3 component of FHL catalyzes thetransfer of electrons from dihydrogen to Tc(VII). According
to this model, the formate dehydrogenase component (FdhH) isrequired only if formate, or a precursor, is supplied as an electron donor for Tc(VII) reduction in place of hydrogen. Thismodel has been validated by the observations that a mutant
unable to synthesize hydrogenase 3, was unable to reduce
Tc(VII) when either hydrogen or formate was supplied as anelectron donor.27
The identification of hydrogenase 3 of FHL as the Tc(VII)
reductase of E. coli opened up the way for a program to screen
for organisms with naturally enhanced activities against Tc(VII).
Several organisms documented to have naturally high activities
of FHL or uptake hydrogenase were tested, resulting in the identification of several strains of sulfate-reducing bacteria that wereable to couple the oxidation of formate or hydrogen to Tc(VII)
reduction.28 Rates of reduction in some strains were approximately 64 fold greater than those recorded in anaerobic culturesof E. coli.34 Desulfovibrio desulfurican30 and related strains21
were also able to utilise formate as an efficient electron donor
for Tc(VII) reduction. This is consistent with the existence of arudimentary FHL complex (consisting of a formate dehydrogenase coupled to a hydrogenase via a cytochrome) located in the
periplasm of these strains.35 Accordingly, the site of reduced
Tc precipitation was identified as the periplasm in D. desulfuricans,30 and more recent studies have confirmed a role for a
periplasmic Ni-Fe hydrogenase in Tc(VII) reduction by a relative in the δ subclass of the Proteobacteria, the sulfate-reducing
bacterium Desulfovibrio fructosovorans.36 Subsequent studies
on the development of a bioprocess to treat Tc(VII)- contaminated water have focused on the use of immobilized cells of
sulfate-reducing bacteria such as D. desulfuricans which arerobust and capable of treating low concentrations of Tc(VII)against a high background of contaminating nitrate ions, which
is often noted in nuclear waste.30, 34 Finally, recent work fromour laboratories have suggested that reduced, insoluble Tc(IV)phases are surprisingly resistant to remobilization under oxidizing
conditions, e.g. in the presence of high nitrate concentrations.37
Thus, long-term immobilization of Tc can be achieved in contaminated sediments by in situ remediation using indigenous
Fe(III)-reducing bacteria.
7. Future directions
Although the environmental relevance of metal reduction

19

processes has only become apparent recently, rapid advancesin the understanding of these important biotransformations havebeen made. However, we still have much to learn about the
precise mechanisms involved, and the full impact of such reactions on a range of biogeochemical cycles. Given the availabilityof genomic sequences for key metal-reducing microorganisms,
new post-genomic and proteomic approaches and the possibilityof combining these tools with advanced techniques from otherbranches of science and technology (e.g. isotopic, spectroscopic
and computational tools) rapid advances in these areas are
predicted.
Acknowledgements. The author thanks the UK NERC andthe Natural and Accelerated Bioremediation Research(NABIR) program of the U.S. Department of Energy for financial support.
References(1) J. R. Lloyd and J. C. Renshaw, Met. Ions Biol. Syst. 23,
205 (2005).
(2) D. R. Lovley, Microbiol. Rev. 55, 259 (1991).
(3) B. Thamdrup, Adv. Microbiol. Ecol. 16, 41 (2000).
(4) M. Vargas, K. Kashefi, E. L. Blunt-Harris, and D. R.
Lovley, Nature 395, 65 (1998).
(5) K. H. Nealson and B. L. Cox, Curr. Opin. Microbiol. 5,296 (2002).
(6) J. R. Lloyd, FEMS Microbiol. Rev. 27, 411 (2003).
(7) J. R. Lloyd, R. T. Anderson, and L. E. Macaskie, Bioremediation, eds. R. Atlas and J. Philp, Washington DC: ASM
Press. 293-317 (2005).
(8) J. R. Lloyd, D. R. Lovley, Cur. Opin. Biotechnol. 12, 248(2001).
(9) D. R. Lovley, E. J. P. Phillips, Y. A. Gorby, and E. Landa,
Nature 350, 413 (1991).
(10) J. R. Lloyd, V. A. Sole, C. V. Van Praagh, and D. R.
Lovley, Appl. Environ. Microbiol. 66, 3743 (2000).
(11) A. J. Francis, J. Alloys Compd. 213/214, 226 (1994).
(12) D. Lovley and E. J. Phillips, Appl. Environ. Microbiol. 58,
850 (1992).
(13) D. R. Lovley and E. J. P. Phillips, Appl. Environ. Microbiol.
60, 726 (1994).
(14) R. B. Payne, D. A. Gentry, B. J. Rapp-Giles, L. Casalot,
and J. D. Wall, Appl. Environ. Microbiol. 68, 3129 (2002).
(15) J. R. Lloyd, C. Leang, A. L. Hodges Myerson, S. Ciufo, S.
J. Sandler, B. Methe, and D. R. Lovley, Biochem. J. 369,
153 (2003).
(16) J. C. Renshaw, L. J. C. Butchins, F. R. Livens, I. May, J.
M. Charnock, and J. R. Lloyd, Environ. Sci. Technol. (inpress).
(17) R. Wade Jr. and T. J. DiChristina, FEMS Microbiol. Lett.
184, 143 (2000).
(18) J. R. Lloyd and L. E. Macaskie, Environmental MicrobeMetal Interactions, ed. D. R. Lovely, Washington DC: ASM
Press. 277−327 (2000).
(19) V. F. Peretrukhin, N. N. Khizhniak, N. N. Lyalikova, andK. E. German, Radiochem. 38, 440 (1996).
(20) J. R. Lloyd, P. Yong, and L. E. Macaskie, Environ. Sci.
Technol. 34, 1297 (2000).
(21) P. A. Rusin, L. Quintana, J. R. Brainard, B. A. Strietelmeier,C. D. Tait, S. A. Ekberg, P. D. Palmer, T. W. Newton, and
D. L. Clark, Environ. Sci. Technol. 28, 1686 (1994).
(22) R. E. Wildung, K. M. McFadden, and T. R. J. Garland,
Environ. Qual. 8, 156 (1979).
(23) J. Henrot, Health Phys. 57, 239 (1989).
(24) L. Pignolet, K. Fonsny, F. Capot, and Z. Moureau, HealthPhys. 57, 791 (1989).
(25) J. R. Lloyd and L. E. Macaskie, Appl. Environ. Microbiol.
62, 578 (1996).

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J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005(26) J. R. Lloyd, J. Chesnes, S.Glasauer, D. J. Bunker, F. R.
Livens, and D. R. Lovley, Geomicrobiol. J. 19, 103 (2002).
(27) J. R. Lloyd, J. A. Cole, and L. E. Macaskie, J. Bacteriol.
179, 2014 (1997).
(28) J. R. Lloyd, A. Mabbett, D. R.Williams, and L. E. Macaskie,
Hydrometallurgy 59, 327 (2001).
(29) J. R. Lloyd, H.-F. Nolting, V. A. Solé, K. Bosecker, andL. E. Macaskie, Geomicrobiol. J. 15, 43 (1998).
(30) J. R. Lloyd, J. Ridley, T. Khizniak, N. N. Lyalikova, and L.
E. Macaskie, Appl. Environ. Microbiol. 65, 2691 (1999).
(31) N. N. Lyalikova and T. V. Khizhnyak, Microbiol. 65, 468(1996).
(32) K. Kashefi and K. Lovley, Appl. Environ. Microbiol. 66,

Lloyd1050 (2000).
(33) R. E. Wildung, Y. A. Gorby, K. M. Krupka, N. J. Hess, S.
W. Li, A. E. Plymale, J. P. McKinley, and J. K. Fredrickson,
Appl. Environ. Microbiol. 66, 2451 (2000).
(34) J. R. Lloyd, G. H. Thomas, J. A. Finlay, J. A. Cole, and L.
E. Macaskie, Biotechnol. Bioeng. 66, 123 (1999).
(35) H. D. Peck, Sulfate-Reducing Bacteria: Contempary Perspectives, eds. J. M. Odom and R. Singleton. New York: SpringerVerlag (1993).
(36) G. De Luca, P. Philip, Z. Dermoun, M. Rousset, and A.
Vermeglio, Appl. Environ. Microbiol. 67, 4583 (2001).
(37) I. T. Burke, J. R. Lloyd, F. R. Livens, C. Boothman, R. J.
G. Mortimer, and K. Morris (submitted).