J. Chem. T echnol. Biotechnol.

1998, 71, 15È26

Bioaccumulation of Lanthanum, Uranium and

Thorium, and Use of a Model System to Develop
a Method for the Biologically-Mediated Removal
of Plutonium from Solution
Ping Yong & Lynne E. Macaskie*
School of Biological Sciences, The University of Birmingham, Birmingham B15 2TT, UK
(Received 7 March 1997 ; revised version received 17 June 1997 ; accepted 3 July 1997)

Abstract : Removal of La3`, UO2` and Th4` from aqueous solution by a Citro-
2
bacter sp. was dependent on phosphatase-mediated phosphate release and the
residence time in a plug-Ñow reactor (PFR) containing polyacrylamide gel-
immobilized cells. In a stirred tank reactor (STR) lanthanum phosphate accumu-
lated on the biomass rapidly, in preference to uranium or thorium phosphates.
Thorium removal was not a†ected by the presence of uranium but was promoted
in the presence of lanthanum. Analysis of the accumulated polycrystalline
material by X-ray powder di†raction (XRD) analysis and proton induced X-ray
emission (PIXE) suggested the formation of a mixed crystal of lanthanum and
thorium phosphate. La3`, UO2` and Th4` are analogues of the corresponding
species of Pu3`, PuO2` and 2Pu4`. The La/U/Th model system was used to
2 problems in the bioremediation of wastes containing
identify some potential
plutonium and to develop a method for the biologically-mediated removal of
plutonium from solution, in a test solution of 239Pu “spikedÏ with a 241Pu tracer.
( 1998 SCI.

J. Chem. T echnol. Biotechnol. 71, 15È26 (1998)

Key words : bioremediation ; Citrobacter ; lanthanum ; uranium ; thorium ; plu-

tonium

INTRODUCTION a†ected by the composition of the challenge solution,

Heavy metal uptake by microorganisms has recently
received a great deal of attention for its potential in the
remediation of metal-bearing aqueous wastes.1 Micro-
organisms are capable of concentrating and retaining
metallic elements from aqueous solution because they
o†er abundant metal-binding functional groups, e.g.,
carboxyl, hydroxyl, sulphydryl and phosphate.1h4 The
mechanism of heavy metal uptake varies between di†er-
ent organisms and metallic species,1 and uptake is also
* To whom correspondence should be addressed.
Contract grant sponsor : British Nuclear Fuels.
15
( 1998 SCI. J. Chem. T echnol. Biotechnol. 0268-2575/98/$17.50.
the pH, and the presence of co-ions and complexing
ligands.1,5h7 More than one heavy metal ion usually
exists in wastewaters.8,9 Metal uptake by biomass may
be a†ected by other cations, or by anions coexisting in
solution with the “targetÏ metals, and the pattern of
metal removal from a metal mixture may be di†erent
from that obtained from a single metal solution. Galun
et al.5 observed that approximately 0É45 mmol dm~3 of
ferric ion inhibited 50% of the uranium uptake by Peni-
cillium digitatum at an initial uranyl concentration of
0É42 mmol dm~3, while Brady et al.9 noted that more
than 99% of Zn, Cr(VI) and Cu was removed from elec-
troplating wastewater by non-viable yeast biomass
Printed in Great Britain
16
under conditions where the removal of Cr(VI) was only
50È60% from tannery wastewater. Carvalho et al.10
reported that Cd, Cu and Zn uptake by the alga
Ascophyllum nodosum from a mixed solution was com-
petitive for available binding sites on the cell surface.
Simple competition might be anticipated between diva-
lent metal species.11
Biosorptive techniques for metal removal are well
established ; where this is the major mechanism, non-
living biomass can, in some cases, remove heavy metals
more efficiently than living cells,12 for example, dead
biomass of Saccharomyces cerevisiae removed 40%
more uranium or zinc than corresponding live cul-
tures.13 Metal bioprecipitation and biocrystallization is
a newly-developed approach which overcomes satura-
tion constraints via the input of biochemical activity,
which generates precipitant ligands continuously to
form extensive cell-bound biomineral deposits. For
example, metal can be removed via H S production by
2
sulphate-reducing bacteria to form metal sulphide pre-
cipitates.2 Sulphate-reducing bacteria are metal resist-
ant because the H S intercepts the incoming metal ;
2
precipitation withholds the metals from the cells.
Another example of biocrystallization is metal removal
as the hydroxide and carbonate precipitates by Alcali-
genes eutrophus.3 This organism harbours two plasmids
(pMOL30 and pMOL28) which control heavy metal
resistance, the mechanism of which is an efflux of metal
cations and precipitation of the metals under the local-
ized alkaline conditions at the cell surface.14 Both of
these examples rely on the metabolic activity of live
biomass. Other components or constraints of the waste-
water may be toxic to the living cells, e.g. extremes of
pH and high salt concentration.
Metal uptake by resting cells of a Citrobacter sp. is an
enzymatically-mediated inorganic metal phosphate pre-
cipitation process based on the continual liberation of
inorganic phosphate from a supplied phos-
phomonoester substrate to form metal phosphate bio-
mineral on the cell surface. The phosphate ligand is
generated via phosphatase activity in growth-decoupled
and immobilized cells, with no requirement for cofac-
tors or metabolic activity ; hence the exposure to heavy
metals and other components of the waste is decoupled
from cell metabolism and growth. Metal uptake can be
in excess of 900% the cellular dry weight.15 In this
process the phosphate ligand is generated at a high con-
centration locally, in juxtaposition to the extracellular
polymeric matrix, which provides foci for the onset of
metal precipitation and “tethersÏ the precipitate to the
cells.16 Compared with this, the addition of inorganic
phosphate directly to a very dilute metal solution may
require large quantities of the ligand for precipitation ;
furthermore the precipitate may form as a colloidal sus-
pension in need of subsequent settling-out or further
downstream processing. At high phosphatase activities
the rate-limiting step is not the production of inorganic
P. Y ong, L . E. Macaskie
phosphate but chemical constraints17,18 which may be
metal-speciÐc. This system is also a†ected by various
physicochemical factors, e.g. pH, and the presence of
co-ions and complexing ligands.19h23
A variety of metals can be removed from solution by
this method, e.g. Cd2`, Cu2`, Pb2`, Sr2`,
La3` 7,15,24,25 uranium4,19 and the transuranic ele-
ments americium and plutonium.18 The latter, together
with neptunium (which is not removed by this
method18), comprises a troublesome component of
wastes from nuclear fuel reprocessing.26 Am3` was
readily removed from solution in a similar way to La3`
but the removal of plutonium was poor.18 For routine
studies the use of transuranic elements is problematic
due to the long-lived radiotoxicity of these elements.
Their complex solution chemistry, attributable to multi-
ple coexisting valence states and extensive hydrolysis
behaviour27,28 makes rigorous study difficult. In addi-
tion, solid state analysis requires appreciable amounts
of material.
The present study was initiated to evaluate the scope
for promotion of plutonium removal by the formation
of mixed metal phosphate crystals in association with
more benign elements. Due to the well-established
properties of hydrogen uranyl phosphate as a “hostÏ
lattice for intercalation of other metal species,29h32 and
the large excess of uranyl ion in typical nuclear fuel
cycle wastes,26 uranyl ion, UO2`, was routinely used. A
2
model system for preliminary tests used Th(IV) and
La(III) as “surrogateÏ elements to predict the behaviour
of Pu(IV) and Am(III). The latter is formed via radioac-
tive decay of 241Pu.26 This b-emitting isotope is present
in nuclear wastes alongside the long-lived a-emitter
239Pu.26 For the purpose of this study the valence
forms of actinides (An) likely to be present in solution
are An(III), An(IV) and An(VI). The hexavalent species
Pu(VI) (PuO2`) would be removed alongside U(VI)
2
(UO2`), probably intercalated within the lattice of
2
HUO PO 4H O30 to form a hybrid crystal. Penta-
2 4 2
valent Pu(V) does not persist in most solutions but dis-
proportionates readily according to :33,34
2PuO` ] 4H` ] Pu4` ] PuO2` ] 2H O
2 2 2
i.e. 2Pu(V) ] Pu(IV) ] Pu(VI)
Since of all the oxidation states of plutonium Pu4`
forms the strongest complexes, complexing agents sta-
bilize tetravalent plutonium against disproportionation.
Additionally, due to a strong tendency to Pu(IV) poly-
merization in low H` concentration the part of the
Pu(IV) primarily disproportionated into Pu(III) and
Pu(VI) is transformed back into tetravalent plu-
tonium.34 For this study, Pu was assumed to be as
Pu(IV).
Most studies on actinide elements have been done in
acidic solution due to their extensive hydrolysis at pH
Biologically mediated thorium and plutonium removal
values above 3É0.27,28 In contrast, the optimal pH for
phosphatase activity is 5É0È7É5 ;22,35 metal removal by
Citrobacter is more efficient at neutral pH but chelating
ligand (e.g. citrate) is required to minimize metal hydro-
lysis.22,23 This is not dissimilar to “actualÏ wastes which
may contain tributyl phosphate,26 and EDTA or,
indeed, citrate28 as complexing ligands.
The present study utilized a model system comprising
the valence-stable species La3`, Th4` and UO2` pre-
2
sented to biomass of Citrobacter sp. as the metalÈcitrate
complexes. The purpose of this investigation was to
evaluate the scope for the promotion of removal of the
“difficultÏ metal species Th(IV)20,23 prior to further
application of this technique to the removal of Pu(IV).
Previous studies had shown removal of 50% of the
input plutonium at an input concentration
60 nmol dm~3 18 but for bioprocess use greater effi-
ciency would be required. We report on the application
of this technique for the removal of Th(IV) with greatly
improved efficiency via the formation of mixed crystals,
and show also that this approach can be applied to the
removal of Pu(IV) in preliminary tests.
2 MATERIALS AND METHODS
All experiments were done using at least two di†erent
batches of cells. Determinations were made in triplicate
and statistical variations were \3%. Data from repre-
sentative batches are shown. Reproducibility was within
^5% throughout.
2.1 Growth and immobilization of cells
The Citrobacter sp.20,22,36 was cultured in minimal
medium, which contained (g dm~3) : Tris bu†er, 12É0 ;
(NH )SO , 0É96 ; glycerol 2-phosphate (G2P, 5É5 H O ;
4 4 2
disodium salt ; BDH Ltd), 0É67 ; KCl, 0É62 ;
MgSO . 7H O, 0É063 ; FeSO . 7H O, 0É000 32 ; glyc-
4 2 4 2
erol, 2É0. The pH was adjusted to 7É0 with 2 mol dm~3
HCl. Cultures were grown in 3 dm3 batches (30¡C) with
forced aeration, inoculated using 50 cm3 of overnight
culture from cells previously maintained by two daily
subcultures in the minimal medium, and harvested in
stationary phase (24 h, OD \ D 1É20). The cultures
600
were harvested by centrifugation, washed in isotonic
saline (8É5 g dm~3 NaCl) twice and resuspended in
saline (1/20 of the original volume) for experiments
using free cells.
For preparation of immobilized cells, 5 g wet weight
of cells were immobilized in a polyacrylamide gel (PAG)
and shredded as described previously.15,20 The shred-
ded gel was divided into Ðve equal parts by weight,
washed with isotonic saline three times, and packed into
Ðve replicate columns (height : diameter ratio of 3É5 : 1 ;
total vol. \ approx. 30 cm3).
17
2.2 Determination of phosphatase speciÐc activity
Phosphatase speciÐc activity was determined by the
release of p-nitrophenol (PNP) from p-nitrophenyl
phosphate (PNPP) as described previously.20,21,36 One
unit is deÐned as nmol of PNP liberated per min per
mg bacterial protein, with OD of the cells converted
600
to bacterial protein using a conversion factor, with bac-
terial protein determined by the method of Lowry
(0É276 mg protein cm~3 at OD \ 1 ; path
600
length \ 1 cm).36
2.3 Metal uptake by free cells in a stirred tank reactor
(STR)
The concentrated cell suspension was diluted (to 0É15È
0É3 mg cm~3 dry weight as noted in individual
experiments) into 20 cm3 of 20 mmol dm~3 MOPS (3-
[N-morpholino]-propanesulphonic acid adjusted to pH
7 with 0É2 mol dm~3 NaOH) bu†er, 5 mmol dm~3
G2P, various concentrations (1 mmol dm~3 or as
described in individual experiments) of metal nitrate
(uranyl, lanthanum or thorium, individually or mixed)
and citrate bu†er (pH 7) as appropriate with or without
0É1 mol dm3 ammonium acetate (NH Ac : adjusted with
4
0É1 mol dm3 HCl to pH 7) at 30¡C. Timed samples
were removed and centrifuged (12 000 rpm, 3 min), and
the supernatants were stored in an ice bath before
assay.
2.4 Metal uptake by PAG-immobilized cells in a plug
Ñow reactor (PFR)
The columns of PAG-immobilized cells were challenged
with an upÑow of composition MOPS (20 mmol dm~3,
pH 7) and citrate bu†er (0É5 mmol dm~3, pH 7), metal
nitrate (0É3 mmol dm~3 of lanthanum, uranyl and
thorium, individually or mixed at an equal concentra-
tion of 0É3 mmol dm~3 for each), G2P (5 mmol dm~3),
with 0É1 mol dm~3 ammonium acetate (pH 7) at 30¡C.
Metal removal by the bioreactor was determined by
assay of the residual metal in the column outÑow and
comparison with the metal content of the inÑow solu-
tion. Column challenge solution was prepared freshly
from 100-fold concentrated stock solutions (stored at
4¡C).
2.5 Assay of metal ions
2.5.1 Assay for a single metal in solution
Solutions for metal assay, 0É03 cm3 of the calibrant
(standard solutions of uranyl nitrate ; thorium nitrate ;
lanthanum nitrate, BDH Ltd), the supernatant (metal
concentration up to 1É2 mmol dm~3) or column inÑow,
or outÑow solution were transferred into a test tube
18 P. Y ong, L . E. Macaskie

containing 1É97 cm~3 of appropriate solution (that dorf tubes at room temperature. Plutonium stock
giving maximum sensitivity) for each metal : solutions (239Pu (a-emitter) ; in 2 mol dm~3 HNO and
3
3 mol dm~3 HCl (solution I) for thorium ; 241Pu (b-emitter) ; in 1 mol dm~3 HNO ) were from
3
0É1 mol dm~3 HCl (solution II) for uranium ; AEA Technology (Harwell Laboratory, UK). Citrobac-
0É1 mol dm~3 acetate/HCl bu†er (pH 3É3, solution III) ter cell suspensions in isotonic saline (8É5 g dm~3 of
for lanthanum, respectively.37 Metals were visualized by NaCl) were diluted (to a Ðnal concentration of 0É15È
the addition of 0É1 cm3 of 0É15% (wt/vol.) arsenazo III 0É3 mg cm~3 dry weight) in a solution containing (Ðnal
(BDH Ltd) with estimation of the blue complex at concentration) approx. 0É4 kmol dm~3 Pu (0É05 cm3 of
652 nm (Phamacia LKB-Novaspec.II each stock Pu solution) ; the Ðnal concentrations of
spectrophotometer).37 239Pu (bulk isotope, a-emitter, half-life : 2É4 ] 104
years)33 and 241Pu (tracer, b-emitter, half-life : 13É5
2.5.2 Assay for metals in a mixed metals solution years)33 were approx. 0É391 and 0É009 kmol dm~3,
Samples of a mixed solution of thorium, uranium and respectively) in 100 mmol dm~3 MOPS/NaOH bu†er
lanthanum were assayed using a simultaneous spectro- (pH 7), 5 mmol dm~3 G2P with or without (as
photometric method37,38 as follows. The solution of appropriate) citrate bu†er (2 mmol dm~3, pH 7),
mixed metal was transferred into three test tubes 100 mmol dm~3 ammonium acetate and lanthanum
(0É03 cm3 sample for each) containing 1É97 cm3 of either nitrate (0É1 mmol dm~3). The mixtures were neutralized
solution I, solution II or solution III, as appropriate. To by the addition of 1 mol dm~3 NaOH and the Ðnal pH
each test tube was added 0É1 cm3 arsenazo III was checked before addition of the cells. The Ðnal con-
(0É15% wt/vol.) and the absorbances were measured at centration of nitrate was approx. 300 mmol dm~3
660 nm for solutions I and III, and 652 nm for solution which is similar to that found in some wastes.26
II (Phamacia LKB-Novaspec.II spectrophotometer). Samples (50 mm3) were withdrawn at timed intervals,
The concentrations of Th, U and La (C , C and C ) the cells removed by centrifugation and 10 mm3 of the
Th U La
in the mixture were calculated from :37 supernatant assayed for b-activity by spotting onto
Ðlter paper in triplicate and counting using a “phosphor-
(A /b [ A /b )/(c /b [ c /b ) ImagerÏ technique, as described previously.39
1 1 2 2 1 1 2 2
[ (A /b [ A /b )/(c /b [ c /b )
C \ 2 2 3 3 2 2 3 3 (1)
Th (a /b [ a /b )/(c /b [ c /b ) 2.7 Solid state analyses
1 1 2 2 1 1 2 2
[ (a /b [ a /b )/(c /b [ c /b )
2 2 3 3 2 2 3 3
2.7.1 Crystal structure identiÐcation, and elemental
(A /a [ A /a )/(c /a [ c /a ) analysis of the accumulated metal precipitates
1 1 2 2 1 1 2 2
[ (A /a [ A /a )/(c /a [ c /a ) Crystal structure identiÐcation and elemental analysis
C \ 2 2 3 3 2 2 3 3 (2)
U (b /a [ b /a )/(c /a [ c /a ) of the accumulated metal precipitates were done for the
1 1 2 2 1 1 2 2 samples of mixed metal phosphate precipitated by free
[ (b /a [ b /a )/(c /a [ c /a )
2 2 3 3 2 2 3 3 cells. The cells (0É15È0É30 mg cm~3 dry cells) were chal-
(A /a [ A /a )/(b /a [ b /a ) lenged in suspension (100 cm3) with a mixture of
1 1 2 2 1 1 2 2 MOPS bu†er (pH 7, 20 mmol dm~3), citrate bu†er (pH
[ (A /a [ A /a )/(b /a [ b /a )
C \ 2 2 3 3 2 2 3 3 (3) 7, 2 mmol dm~3), glycerol 2-phosphate
La (c /a [ c /a )/(b /a [ b /a ) (10 mmol dm~3), ammonium acetate (100 mmol dm~3)
1 1 2 2 1 1 2 2
[ (c /a [ c /a )/(b /a [ b /a ) and the metal ions mixed as appropriate (1 mmol dm~3
2 2 3 3 2 2 3 3
for each metal) overnight at 30¡C. The metal-loaded
where a, b and c are the molar absorption coefficients of cells were centrifuged, washed with water (three times)
the thorium, uranium and lanthanum arsenazo III com- and then acetone, and dried at room temperature.
plexes, and were obtained by a least-squares Ðt of the Crystal structure identiÐcation and elemental analyses
data on the absorbances and the concentrations of three were done as described below using the same sample
pure standard metal ion solutions under the conditions preparations for each.
appropriate to each metal, A is the absorbance of the
metal mixture being determined experimentally, and the 2.7.2 X-ray powder di†raction (XRD)
subscripts 1, 2 and 3 refer to determinations in solution The crystal structure of the precipitates was identiÐed
I (at 660 nm), II (at 652 nm) and III (at 660 nm), respec- by XRD. The samples were ground to a powder, and
tively. The validity of the method was demonstrated were laid on a single silicon crystal plate with XRD
previously.37 spectra obtained using a high precision powder di†rac-
tometer (School of Physics, The University of
2.6 Plutonium uptake by cell suspensions Birmingham) for data collection. Exposure times were
up to 16 h to monochromatic Cu Ka1 radiation pro-
Plutonium uptake experiments were done in a scaled- duced using an incident-beam cured-crystal germanium
down (0É5 cm3 Ðnal volume) system in 1É5 cm3 Eppen- monochromater with asymmetric focusing at 25¡C. The
Biologically mediated thorium and plutonium removal
scale error of 2h was 0É007¡ and the specimen surface
displacement was 0É0305 mm which was checked by a
standard reference material (Ag). The powder di†raction
patterns were recorded from 5 to 60¡ (2h) with a step
length of 0É05¡ (2h).
2.7.3 Proton induced X-ray emission (PIXE)
The ground sample was wet with acetone, then placed
on a thin pioloform Ðlm on an aluminium target, and
dried at room temperature. Quantitative elemental
analyses were done by proton induced X-ray emission
using the Oxford Scanning Proton Microprobe40h42
(Department of Nuclear Physics, University of Oxford).
Elemental maps were obtained of approx.
2 mm ] 2 mm specimen areas held within the proton
beam. Matrix major element composition and thick-
ness, which are needed to calculate PIXE corrections,
were determined by simultaneously-determined Ruther-
ford Back Scattering (RBS) spectra. The intrinsic accu-
racy of PIXE using the RBS correction was
demonstrated by comparison of data obtained by PIXE
with that determined by other methods.43
3 RESULTS AND DISCUSSION
3.1 Metal removal from mixed metal solution
Metal uptake by Citrobacter is a microprecipitation of
metal phosphate, localized on the cell surface. There are
two component reactions of this biocrystallization
process :
Phosphatase
Glycerol 2 [ phosphate ÈÈÈÈÕ Glycerol
(enzyme)
] Phosphate (1)
Ksp
Phosphate ] Metal ion ÈÈÈÕ Metal phosphate
precipitate (2)
where K is the solubility product of the metal phos-
sp
phate (3É7 ] 10~23, 2É0 ] 10~11 and 1É0 ] 10~20 for
LaPO , HUO PO and Th(HPO ) , respectively) ;44
4 2 4 42
here K is, more correctly, the conditional solubility
sp
product, and is a constant for a given metal and “carrierÏ
ionic matrix. The free metal ion concentration in the
challenge solution (metal available for the formation of
metal phosphate) is governed by the nature of the
coexisting complexing ligand(s) and their concentration.
In the case of citrate :
K1
Citrate ] Metal ion AB CitrateÈMetal (3)
where K is the stability constant of the metalÈcitrate
1
complex (Log K : 6É97, 8É5 and 13É0 for La3`, UO2`
1 2
19
and Th4`, respectively).20,44 The metal phosphate solu-
bility product (K ) and metalÈcitrate complex stability
sp
constant (K ) are metal-dependent. The three metals
1
would compete for phosphate and citrate in the chal-
lenge solution ; thus, metal removal from a mixture may
be di†erent from its removal from a single metal solu-
tion. Although it would be predicted that Th(HPO )
42
should be removed from solution in preference to
HUO PO on the basis of the solubility product alone
2 4
the reverse was observed,23 and probably reÑects the
relative strengths of the metalÈcitrate complexes
(above), and hence the low concentration of free Th4`
ion. Citrate was routinely incorporated to suppress the
formation of hydroxylated species45 and also as a “sur-
rogateÏ for complexing ligands which may exist in real
industrial waste (see Introduction). For promotion of
metal removal, ammonium ion was routinely incorpor-
ated. NH UO PO . nH O and Th(NH PO ) . nH O
4 2 4 2 4 42 2
are more insoluble than the corresponding hydrogen-
salts and routine use of NH` accelerated metal removal
4
and reduced the Ñow residence time necessary in a
PFR.22,23
3.2 Metal uptake by free cells in a STR
Initial tests in an STR showed the di†erent metal accu-
mulation behaviour of the three test metals. Lanthanum
precipitation was completed rapidly (within 60 min)
either alone or in combination with the other metals
(not shown) ; a similar result was seen also with
uranium, where uptake was maximal within 2 h (Fig.
1(A)). Removal of thorium from 1 mmol dm~3 citrate,
undetectable within 7 h, was improved in the presence
of lanthanum, and similarly in the three-metal mixture
(Fig. 1(B)). This was attributable to the coexisting lanth-
anum, since thorium removal was not promoted from
citrate supplemented with uranyl ion alone (Fig. 1(B)).
Participation of nucleation sites was possible, i.e. lanth-
anum phosphate precipitate initially coated on the cell
surface could act as a focus (“seedÏ) which accelerated
the crystallization progress for thorium precipitation.
Alternatively a mixed crystal could be formed, pro-
moting the incorporation of thorium. These possibilities
were evaluated using X-ray powder di†raction analysis
of the precipitated material (Fig. 2).
3.3 Solid state analysis of the precipitated material
For solid state analysis the suspensions were incubated
overnight. In contrast to the data of Fig. 1(B) thorium
removal, and co-removal of uranium and thorium was
seen after the extended incubation (up to 90% Th
removal). No interpretable XRD pattern was obtained
for precipitate obtained from thorium-supplemented
solution alone (Fig. 2(A)), in accordance with the lack of
crystallinity concluded from previous studies.23 The
20
Fig. 1. Metal uptake from mixed metals solution by free cells
in an STR. (A) Uptake of uranium. The concentrated cells
were diluted with a solution (20 cm3) containing
20 mmol dm~3 MOPS bu†er (pH 7), 0É1 mol dm~3 NH Ac
(pH 7), 5 mmol dm~3 G2P, 1 mmol dm~3 citrate (pH 7) with 4
0É15 mmol dm~3 uranyl nitrate only + ; 0É15 mmol dm~3
uranyl and 0É15 mmol dm~3 lanthanum nitrate | ;
0É15 mmol dm~3 uranyl and 0É15 mmol dm~3 thorium
nitrate L ; 0É15 mmol dm~3 uranyl, 0É15 mmol dm~3 lantha-
num and 0É15 mmol dm~3 thorium nitrate ). (B) Uptake of
thorium. The concentrated cells were diluted with a solution
(20 cm3) containing 20 mmol dm~3 MOPS bu†er (pH 7),
0É1 mol dm~3 NH Ac (pH 7), 1 mmol dm~3 citrate (pH 7),
4 with 0É15 mmol dm~3 thorium nitrate
5 mmol dm~3 G2P
only + ; 0É15 mmol dm~3 thorium and 0É15 mmol dm~3
uranyl nitrate | ; 0É15 mmol dm~3 thorium and
0É15 mmol dm~3 lanthanum nitrate L ; 0É15 mmol dm~3
thorium, 0É15 mmol dm~3 uranyl and 0É15 mmol dm~3
lanthanum nitrate ). Data are from a representative batch of
cells. The phosphatase speciÐc activity was 568 units at
harvest, and the cell density was 0É226 mg cm~3 dry weight.
X-ray powder di†raction patterns for the precipitate
obtained from solutions supplemented with lanthanum
alone or co-supplemented with thorium were very
similar (Fig. 2(B, C)), but two extra peaks were detected
in the thorium-supplemented sample (Fig. (2C)). These
were clearly visible as a non-deÐned peak area in the
sample of Th(NH PO ) (Fig. 2(A)) but not LaPO
4 42 4
(Fig. 2(B)). Formation of a mixed Th(NH PO ) /LaPO
4 42 4
species was suggested. There was no di†erence in the
XRD pattern obtained for the precipitate whether
P. Y ong, L . E. Macaskie
Fig. 2. X-ray powder di†raction analysis of samples obtained
from uranium-unsupplemented solution. Samples of precipi-
tate obtained from solutions supplemented with thorium
nitrate (A), or lanthanum nitrate alone (B), or as a mixture (C)
were examined as described in the text. Areas of interest are in
bold.
obtained from uranium-challenged samples alone22 or
from cell suspensions co-supplemented with lanthanum
and/or thorium or all three metals (not shown), and the
XRD patterns were identical (not shown). This was
possibly because the X-ray di†raction pattern of the
uranium phosphate crystal was sufficiently strong to
mask peaks attributable to LaPO or a mixed La/Th
4
crystal (cf. Fig. 2(B C)). More detailed interpretation of
the spectra was difficult in the absence of reference data
on the mixed crystals, but it was concluded that the
presence of lanthanum promoted formation of a mixed
crystal with thorium, in accordance with the data of
Fig. 1(B). The area of interest highlighted in Fig. 2(A)
clearly corresponds to the new peaks visible in the pre-
cipitate of LaPO /Th(NH PO ) . nH O (Fig. 2(C)). It is
4 4 42 2
likely that the thorium phosphate in the presence of
lanthanum loses its amorphous character and acquires
a more crystalline structure. The XRD data were rein-
forced using quantitative elemental analysis by PIXE.
This shows (Table 1) that the expected metals were
present in the precipitate obtained from the two-metal
mixtures following overnight incubation, with approx-
imately 10È20% more La than U and 10% more U
than Th. The high degree of error (or sample
heterogeneity) precluded conclusive interpretation of
the La/Th mixed crystal. There was a higher ratio of La
to both U and Th in the samples from the three-metal
Biologically mediated thorium and plutonium removal 21

TABLE 1
Concentrationsa of Metals in the Precipitates from Mixed Metal Solutions as Determined by PIXE

(a) Precipitate from L a and T h mixed solution (b) Precipitate from L a and U mixed solution

Sample T ha L aa T h/Lab Sample Ua L aa U/Lab

1 7É51 7É36 1É02 1 5É39 6É75 0É80

2 15É7 18É5 0É85 2 11É3 16É7 0É68
3 4É03 6É26 0É64 3 10É2 12É2 0É84
4 16É6 13É4 1É24 4 15É6 16É8 0É93
5 11É5 16É5 0É70 5 5É39 6É75 0É80
Mean ^ SEM (5) : 0É89 ^ 0É24 Mean ^ SEM (5) : 0É81 ^ 0É09

Sample Ua L aa U/Lab

(c) T h and U mixed solution

Sample T ha Ua T h/Ub

1 4É58 5É03 0É91

2 1É86 2É20 0É85
3 5É31 5É05 0É83
4 4É45 6É39 0É88
5 7É46 8É26 0É90
Means ^ SEM (5) : 0É87 ^ 0É03
(d) Precipitate from U, T h and L a mixed solution

Sample L aa Ua T ha U/L ab T h/L ab T h/Ub L a/U/T hb

1 14É2 10É7 9É26 0É75 0É65 0É87 1É33 : 1 : 0 : 0É86

2 7É78 6É09 6É17 0É78 0É79 1É01 1É27 : 1É0 : 1É01
3 17É5 10É4 11É5 0É59 0É66 1É11 1É68 : 1É0 : 1É11
4 17É5 11É3 9É85 0É65 0É56 0É87 1É55 : 1É0 : 0É87
5 7É19 4É40 4É57 0É61 0É64 1É04 1É63 : 1É0 : 1É04
0É68 0É66 0É98 1É49 : 1É0 : 0É98
Means (^SEM) (5) : (^0É08) (^0É08) (^0É11) (^0É18) : 0 : (^0É11)

a Concentration units : mol dm~3 in the precipitates obtained from cell suspensions incubated in the presence of the
metals for 18 h. The samples were prepared as described in Section 2.
b Molar ratio.

mixture but no evidence could be obtained from the
XRD pattern for the presence of La UO PO alone.46
1@3 2 4
No standard di†raction data are available for
lanthanumÈthorium phosphate.46 It is likely that there
was sufficient peak overlap in XRD pattern of samples
obtained from the uranium-supplemented solution to
preclude crystal structure determination by XRD alone.
However, the high sensitivity and intrinsic accuracy of
the PIXE technique showed conclusively the mixed
nature of the precipitate. The molar ratio of La : Th : U
was approx. 1É5 : 1 : 1 (Table 1(d)). The ratio of metal
removal as seen in the solid sample was promoted to
1 : 1 for Th/U in the presence of La3` from a ratio of
0É87 : 1 for Th/U without lanthanum (Table 1(c, d)). It is
likely that the Ðnal precipitate was a mixture of LaPO
4
per se and a co-crystal of La/Th and U phosphates but
the exact nature of the mixture could not be deter-
mined, against a “backgroundÏ of NH UO PO .
4 2 4
The results of the preliminary tests (Fig. 1, Table 1)
and the solid-state studies clearly suggested facilitation
of thorium removal by its incorporation into a mixed
crystal, although a longer incubation time was required
to visualize the e†ect of uranium, in accordance with
the longer timescale required for the removal of
uranium as compared with lanthanum.22,23 This could
restrict application in a Ñow-through process where
short residence times are desirable. Accordingly, tests
were done in a Ñow-through system.
22
3.4 Metal uptake by PAG-immobilized cells in a PFR
In a PFR, the efficiency of metal removal for the three
metals at a given Ñow rate (e.g. 1 cm3 min~1, shown in
Fig. 3) was in the order of La [ U [ Th whether from a
single or mixed metal solution. At this Ñow rate the per-
centage removal of La, U and Th was 80%, 60% and
17% respectively from the single metal challenge, and
50%, 34% and 17%, respectively, from the metal
mixture (Fig. 3). Removal of lanthanum and uranium
was proportionally less from the mixture in each case
(50/80 \ 0É62 ; 34/60 \ 0É57), suggesting competition for
available phosphate. This is consistent with a previous
study which demonstrated a requirement for an excess
of phosphate in order to achieve efficient metal precipi-
tation in vitro.17 From a single metal solution the effi-
ciency of metal removal was in accordance with the
metal phosphate insolubility in the challenge solution :
Fig. 3. Removal of metals from aqueous solution by PAG-
immobilized cells in a PFR. The reactor was challenged with
solutions containing 20 mmol dm~3 MOPS (pH 7),
0É5 mmol dm~3 citrate (pH 7), 0É1 mol dm~3 NH Ac (pH 7)
4
and 5 mmol dm~3 G2P with : L 0É3 mmol dm~3 metal
nitrate only, or … three metal nitrates in a mixture at an
equal concentration of 0É3 mmol dm~3 for each (total metal
concentration was 0É9 mmol dm~3). (A) : Lanthanum
removal ; (B) : uranium removal ; (C) : thorium removal. The
phosphatase speciÐc activity of this representative batch was
568 units at harvest.
P. Y ong, L . E. Macaskie
high for lanthanum, lower for uranium and very low for
thorium in the presence of citrate.17 The removal of
thorium was very poor as compared with the other
metals in the PFR either in a mixture or in a single
metal challenge solution. The time taken for deposition
of metal phosphate was previously identiÐed as a criti-
cal factor inÑuencing bioreactor efficiency.17,18 In the
case of thorium, dissociation of the thoriumÈcitrate
complex was probably the limiting factor (possibly not
occurring within the Ñow residence time), and it was
concluded that use of a PFR for the removal of thorium
from solution would not be a realistic bioprocess
option, except at very long residence times which would
be inappropriate for an industrial Ñow-through process.
The potential for use of a batch contactor (STR) for the
removal of thorium was therefore evaluated further.
3.5 Further development of a system to remove the
tetravalent actinides thorium and plutonium
3.5.1 Removal of thorium in an ST R : further studies
Uranium, thorium and the transuranic elements are
chemically and radiologically toxic, even at a very low
concentration. It would be meaningless to describe the
efficiency of a bioprocess using “% removalÏ as a cri-
terion if the residual concentration of metal is still
above the permissible limit of discharge. There will be a
Ðnal equilibrium concentration for residual metal in the
presence of biomass regardless of the uptake mecha-
nism ; this equilibrium, and the time for its attainment,
will be a†ected by various physiochemical factors and
will also vary according to the microorganism and
metallic species.9,47,48 In a PFR the equilibrium is not
reached at short residence time, this is especially so for
thorium (see above).
As described previously, metal uptake by Citrobacter
is a biocrystallization system in which there are reac-
tions (1) and (2), and it can be a†ected markedly also by
reaction (3), i.e. the residual metal ion after precipitation
is dependent on the solubility product of the metal
phosphate, the rate of phosphate release, and the activ-
ity of free metal ion in the challenge solution, which is,
in turn, dependent on the presence of complexing
ligands and the time required for dissociation of the
metalÈligand complex. In accordance with the solubility
products of metal phosphates (see earlier), it was shown
that the priority of metal precipitation and the efficiency
of metal removal, were in the order lanthanum,
uranium and thorium under the same conditions.23 The
rate of phosphate release was limited by the phospha-
tase speciÐc activity.20 Although this could be manipu-
lated physiologically during pre-growth of the cells35 it
was concluded not to be the rate-limiting factor in the
case of thorium.18 The ionic matrix and pH of the chal-
lenge solution control the efficiency of metal removal
from a solution of a given phosphate concentration
Biologically mediated thorium and plutonium removal 23

(solution pH a†ects the solubility product of metal

phosphate),22,23 while the incorporation of ammonium
ion accelerates uranium and thorium uptake by chang-
ing the form of metal phosphate to one which has a
lower solubility product.22,23 Citrate is necessary to
prevent metal hydrolysis (see earlier), and, as a metal
complexing ligand, citrate sequesters metals, and pro-
tects the phosphatase from uranium toxicity.22
However, at the same time citrate reduces the efficiency
of metal removal because the concentration of free
metal ion (particularly Th) is decreased in aqueous solu-
tion.23 A comparative study of uranium and thorium
toxicity showed that thorium had little detrimental
e†ect on phosphatase activity,23 probably due to the
strength of the thoriumÈcitrate complex (see earlier) and
low concentration of free thorium. The e†ect of citrate
was investigated further using an STR (Fig. 4). This
conÐrmed that citrate a†ected the residual concentra-
tion of thorium (Fig. 4(A)), but had negligible e†ect on
that of uranium (Fig. 4(B)) in the presence of ammon-
ium acetate. It was shown (Fig. 4(A)) that no thorium
was removed from a challenge solution of
0É3 mmol dm~3 in the presence of 1 mmol dm~3
citrate, in accordance with a previous study.23 However,
up to 95% of the thorium was removed from the same
solution by reducing the citrate concentration to Fig. 4. The uptake of metals from single metal solutions by
0É3 mmol dm~3. At a lower concentration free cells in an STR. The cells were resuspended (20 cm3) in
(0É1 mmol dm~3) thorium removal was only 70% from 20 mmol dm~3 MOPS bu†er (pH 7), 5 mmol dm~3 G2P
with 0É3 mmol dm~3 metal nitrate supplemented with
0É3 mmol dm~3 citrate, but 95% removal was restored 1 mmol dm~3 citrate (pH 7) and 0É1 mol dm~3 NH Ac (+) ;
by adjusting the ratio of citrate : metal to 1 : 1. The rate or 0É3 mmol dm~3 citrate and 0É1 mol dm~3 NH 4Ac (…).
4
of thorium removal was reduced in the ammonium- Tests were done also using 0É1 mmol dm~3 metal with
unsupplemented solution (Fig. 4(A)), but 60% of the 0É3 mmol dm~3 citrate and 0É1 mol dm~3 NH Ac (L) ;
4
thorium was removed after 7 h. It is concluded that 0É1 mmol dm~3 metal with 0É1 mmol dm~3 citrate and
0É1 mol dm~3 NH Ac (>) ; 0É1 mmol dm~3 with
citrate plays a major role in thorium uptake by Citro- 0É1 mmol dm~3 citrate4 and without NH Ac ()).(A) : thorium ;
bacter. This is in accordance with the value of the (B) : uranium. The cell density was 0É226 4mg cm~3 dry weight,
thoriumÈcitrate stability constant (see earlier) and is a and the phosphatase speciÐc activity of this representative
major factor which limits the residual concentration of batch was 568 units at harvest.
thorium.
From the above it is concluded that the optimal con-
dition to achieve the highest efficiency of thorium system highlight the recalcitrance to removal of the
removal in solution of pH 7 is in the presence of tetravalent actinides, attributable to the complex solu-
ammonium ion and complexing ligand (citrate) at a tion chemistry of these elements. While Th(IV) is stable,
concentration as low as possible in accordance with plutonium can co-exist in all four oxidation states (III,
that required to prevent metal hydrolysis and maintain IV, V and VI) in the same solution.34 Each species has a
metal solubility. This should be balanced against the di†erent pattern of hydrolysis and complexation behav-
need for protection of the enzyme from uranyl toxicity iour which, in a solution containing other metals, will
in a mixed solution containing an excess of UO2`. In inÑuence the amenability of the metal to removal by
2
the case of Th4` a simple batch contactor would be physico-chemical or biotechnological methods. In most
preferable to a Ñow-through system ; much of the Th4` situations, and under the present conditions (pH 5È7,
was removed within 2È3 h, together with UO2` and aerobic) the plutonium would be present mainly as
2
La3`. A batch contacter format was adopted for testing Pu(IV) ; this is the most stable condition but one which
the application of the system to the removal of plu- requires stabilization against hydrolysis and poly-
tonium. merization by formation of the citrate complex. The
tests using Th(IV) (above) show that the presence of
3.5.2 Application of the system to the removal of citrate, although essential for metal stability, inhibits
plutonium from solution metal removal.
The above experiments with thorium in the model Preliminary tests to evaluate the removal of plu-
24 P. Y ong, L . E. Macaskie

tonium from solution were done in a carrier solution 4 CONCLUSIONS

containing 2 mM citrate or 100 mmol dm~3 NH Ac
4 It was shown that the bioremediation of solutions con-
with or without 0É1 mmol dm~3 La3`. Here the plu-
tonium concentration was 0É4 kmol dm~3. No removal
of plutonium was seen within 9 h in the presence of
2 mmol dm~3 citrate (Table 2). Citrate binds very
strongly to Pu4` (log K \ 15É54 and log K \ 30É0 for
1 2
the plutoniumÈcitrate complex) ;34 in turn, this retards
the formation of plutonium phosphate (K \ 2
sp
] 10~28 for Pu(HPO ) . nH O44). Previous studies
42 2 i
have shown that the presence of ammonium acetate
could accelerate the removal of UO` and Th4`.22,23
2 accelerates and facilitates removal of the “difficultÏ tetra-
There was a signiÐcant removal of plutonium from
solution when 100 mmol dm~3 NH Ac was used in lieu
4 the crystallization behaviour of the metal phosphate
of 2 mmol dm~3 citrate (Table 2). The present study
has shown that thorium removal is accelerated in the
presence of La3` (Fig. 1). Accordingly, incorporation of
La3` into the plutonium-challenge solution (with
100 mmol dm~3 NH Ac without citrate) greatly facili-
4
tated the removal of plutonium ; up to 100% of the plu-
tonium disappeared from the challenge solution within
1 h (Table 2). Similarly to the thorium removal, there
was probably an La/Pu mixed phosphate crystal
formed on the cell surface but the radiohazard of plu-
tonium precluded the use of sufficient metal to obtain a
solid in sufficient quantity for conÐrmation of this. No
loss of plutonium occurred in the same solution without
cells (Table 2), while acetate (100 mmol dm~3 ; log
K \ 2É02 and log K \ 3É34 for Pu4`)34 and nitrate
1 2
(300 mmol dm~3 ; log K \ 0É54)44 kept the Pu4`
1
mobile and would have protected against hydrolysis.
taining tetravalent actinides (Th4` and Pu4`) is more
difficult than for hexavalent (UO2`) and trivalent
2
(Am3` 18 and La3`) ions. However, plutonium removal
was achieved from a solution of only 0É4 kmol dm~3
Pu regardless of a background of approx.
300 mmol dm~3 NO~ which is a competitive inhibitor
3
of phosphatase activity (K for NO~
3
15É9 mmol dm~3).49 With La3` present, the formation
of mixed crystals of actinide/lanthanum phosphates
valent species ; other studies have shown that it may be
rather than metal phosphate insolubility per se that
governs the efficiency of metal removal.18
ACKNOWLEDGEMENTS
The authors wish to acknowledge, with thanks, the
Ðnancial support of British Nuclear Fuels plc (to P.Y.)
and valuable discussions with Dr H. Eccles of that
Company. They also wish to thank Dr G. Grime
(Scanning Proton Microprobe Unit, Department of
Nuclear Physics, University of Oxford) for his help in
the PIXE analysis, Dr J. I. Langford and R. PÑaumer
(School of Physics & Space Research, University of
Birmingham) for assistance with XRD, and Dr. J. R.
Lloyd for his assistance with Pu assay using the “phos-
phorImagerÏ technique.

TABLE 2
The Removal of Plutonium by Citrobacter in Cell Suspensionsa

Residual concentration of Pu (% of initial concentration)

T ime (h) : 0 1 6 9

100 mmol dm~3 NH Ac and

4
0É1 mmol dm~3 La3` without cells 100 100 100 100
2 mmol dm~3 citrate BI 100 100 100 100
with cells BII 100 100 100 100
100 mmol dm~3 NH Ac BI 100 64É2 36É0 23É4
4
with cells BII 100 88É1 44É9 39É3
100 mmol dm~3 NH Ac and BI 100 0 0 0
4
0É1 mmol dm~3 La3` with cells BII 100 0 0 0

a The experiment was done as described in Section 2. All samples were with 100 mmol dm~3
MOPS/NaOH bu†er (pH 7), 5 mmol dm~3 G2P and 0É4 kmol dm~3 plutonium nitrate. The cell
densities in the suspensions corresponded to 0É428 and 0É483 mg dry wt cm~3, and the phosphatase
speciÐc activities at harvest were 486É3 and 194É5 for two batches of cells (BI and BII), respectively.
The plutonium solution was a mixture of 239Pu(a) and 241Pu(b). Alpha activity is not detected by
the phosphorImager method using clingÐlm-wrapped samples.39 239Pu gave no phosphorImager
signal per se. The decay product of 241Pu, 241Am is an a,c-emitter (0É06 MeV c). Controls using
238U (c-emission of 0É048 MeV) at 1 mmol dm~3 applied concentration (cf 241Pu was \ 0É01 lM)
gave a negligible phosphorImager signal.
Biologically mediated thorium and plutonium removal
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