Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 85-86, 2005

Accepted
Microbially Mediated Removal of Np(V) by Desulfovibrio desulfuricans– Implication of Microbial Immobilization at the Radioactive Waste Repository –Toru Nagaoka*
Biotechnology Sector, Environmental Science Research Laboratory, Central Research Institute of Electric Power
Industry (CRIEPI), 1646 Abiko, Chiba 270-1194, JAPANReceived: November 15, 2004; In Final Form: April 21, 2005
We investigated the capacity of the sulfate-reducing bacterium Desulfovibrio desulfuricans to facilitate theremoval of Np(V) from aqueous solution. For comparison, we also examined the removal of Np(V) by bentonite
under aerobic and anaerobic conditions with sodium sulfide. Np(V) removal (probably after reduction to Np(IV))
was most rapid in the presence of cells, whereas there was little or no removal by bentonite under aerobic conditions. In the presence of cells, Np in suspension declined by 95% or more within 10 h, suggesting that D. desulfuricans enzymatically reduces mobile Np(V) to immobile Np(IV) and eventually to stable Np, though chemical
reduction by cell metabolite H2S may contribute. This finding highlights the importance of understanding microbial effects when considering the migration of radioactive elements from waste repositories.

1. Introduction
When considering the safe disposal of radioactive waste
containing various transuranic nuclear fuel cycle elements,
neptunium (Np) is particularly problematic because its longlived isotope, Np-237 (half-life, 2.1 × 106 years), is predominant at historical sites.1 Moreover, in its most stable form,
NpO2+ is soluble and highly mobile in the environment.
Generally, the reducing form of transuranic elements isimmobile in the natural environment, whereas the oxidizing
form is more mobile. Therefore, one approach to achieving
the removal of transuranic elements from aqueous solution
may be to utilize metal-reducing microorganisms to reduce theelements to insoluble and immobile forms. For instance, Lovley
et al. demonstrated that uranium (U) can be enzymaticallyreduced by a dissimilatory Fe(III)-reducing microorganism to
an insoluble form, and that this can serve as a mechanism forimmobilizing U in sediment and forming U deposits.2 In thatregard, it was recently reported that technetium(VII), a fission
product of U-235, is enzymatically reduced to a lower valenceby the microorganisms Shewanella putrefaciens,3 Geobacter
mitallireducens4 and Clostridium sp.,5 and that Np(V) is reduced
cooperatively by the microorganisms Citrobacter sp. and S.
putrefaciens.6 Thus, certain microorganisms are able to reduceradionuclides enzymatically (direct microbial reaction) and/or
chemically by their metabolites (indirect microbial reaction)
into insoluble, immobile forms in the environment. Our research
has focused on the stabilization and immobilization of Np(V) via
microbial removal for safe disposal in subsurface repositories.
2. Materials and Methods
Microorganism. We used the sulfate-reducing bacterium
Desulfovibrio desulfuricans in this experiment. D. desulfuricans(ATCC 7747) was cultured on modified ATCC 1249 medium
containing (g/L in distilled deionized water) CaCl2, 0.1; Na2SO4,0.8; NH4Cl, 1.0; KH2PO4, 0.5; MgSO4·7H2O, 2.0; Yeast extract,1.0; and sodium lactate, added to a final concentration of 60
mM from 60 wt% syrup solution. The pH was adjusted to 7.2.
To avoid precipitating iron sulfide during reactions, no ferrous*Corresponding author. E-mail: nagaoka@criepi.denken.or.jp.
FAX: +81-4-7183-3347.

sulfate was added to the medium. After boiling the medium toremove O2 and then cooling it under O2-free N2 gas through aheated copper column, it was pipetted into 10-mL serum
bottles that were then aluminum-capped using butyl rubber
stoppers prior to sterilization. After autoclaving, filter-sterilized sodium ascorbate and sodium thioglycolate were added tothe vials to obtain the reducing conditions necessary for cellgrowth. The cells were inoculated into each vial under an N2atmosphere and cultured at 35 ˚C in an incubator.
Np solution. Np-237 solution in HNO 3 (2 mol/L) was
obtained from AEA Tech. PLC, Harwell, UK. For use in theexperiments, the original Np stock solution was diluted to aconcentration of 5.5 × 10−4 mol/L with HNO3 (2 mol/L). The
oxidation states of Np(V) were confirmed by solvent extraction using PMBA (1-phenyl-3-methyl-4-benzoyl-pyrazol-5one) as the extractant.7
Bentonite. Bentonite mined in Japan was used in the experiment. X-ray diffraction analysis and X-ray photospectroscopy
showed that the bentonite used was comprised mainly of montmorillonite and quartz, with various minor components,
including pyrite, calcite and beidellite.8
Np removal by bentonite under aerobic conditions over awide range of pHs. To assess the effect of pH on Np removal
by bentonite under aerobic conditions, 5 mL of 0.5 g/L
bentonite suspension, with 0.01 M NaNO 3 as a backgroundelectrolyte, were added to 10-mL glass serum vials, after which
Np solution was added to a final 1.0 × 10−6 mol/L, and the pH
was adjusted with HNO3 and NaOH. The vials were then stoppered with cotton to allow equilibration with atmospheric CO2and shaken at 35 ˚C. After 24 h, pH and Eh (vs. NHE) werethen measured using a pH-ORP meter (TRX-90Si, Toko Co.,Japan). The suspensions were then filtered through a 0.22 µmfilter (Millipore), and the filtrate was subjected to Gamma-spectrometry (GL2020S, Canberra Inc., USA) to determine the Np
concentration, which was measured as the spectrum peak area
counted at 92.29 keV.9 The amount of Np adsorbed (i.e., solidphase) was calculated as the differences between the known
total amount of Np added and the measured dissolved concentration.
Np removal by bentonite under anaerobic conditions with
Na2S. For comparison, Np removal by bentonite mediated bychemical reduction with Na2S was also assessed, along withremoval under aerobic conditions. The reactions were carried© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

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

3. Results and Discussion
Effect of pH on Np removal by bentonite under aerobicconditions. The effect of pH on Np(V) removal by bentonite
under aerobic conditions is shown in Figure 1. At pHs ranging
from 1 to 10, there was no effect on Np adsorption whileadsorption was enhanced at pH > 10. This result is in good
agreement with previous researches.10 With Np in aqueous
solution under aerobic conditions, NpO2+ is the most stable andpredominant species over a wide pH range;11 indeed, it waspresent over nearly the entire pH range in the present experiment, which explains why pH did not affect adsorption at mostof the pHs tested. In sum, this result shows that there is little
or no adsorption of Np by bentonite under aerobic conditions.
It would therefore seem inappropriate to use bentonite alone as
an adsorbent to prevent Np(V) from migrating out of subsurface repositories into the environment even though it is a very
effective adsorbent for other radionuclides.
Microbial and chemical removal of Np(V). Microbial
removal of Np(V) from bentonite suspension was investigatedas shown in Figure 2. For comparison, we also assessed Np
removal mediated by chemical reaction under anaerobic condition with Na2S (without cells), as well as under aerobic conditions. The pH and Eh (vs. NHE) conditions for this series
were nearly constant throughout: under an aerobic condition,
pH = 7.2 ± 0.2 and Eh = 333 ± 22 mV; with 0.01 mol/L Na2S,
pH = 7.2 ± 0.1 and Eh = –152 ± 20 mV; and with cells, pH =7.8 ± 0.2 and Eh = –179 ± 14 mV. The removal of Np wasachieved in the presence of D. desulfurican and under anaerobic
conditions with H2S, whereas there was little or no removal
under aerobic conditions. In the presence of cells, Np removal
was most rapid, and the level of Np in the suspension wasreduced by 95% or more within about 10 h, suggesting that D.
desulfuricans reduces soluble Np(V) to insoluble Np(IV) enzymatically, although chemical reduction by H2S produced by thecells cannot be ruled out. In addition, the reducing reagents(i.e. sodium ascorbate and thioglycolate) in the medium could
affect the Np reduction as reported previously.5 Moreover, theformation of low-solubility products of tetravalent actinide
phosphate12 should be investigated, as the medium contained
phosphate for cell growth. Nonetheless, our findings suggestthat sulfate reducing bacterium D. desulfuricans could remove
Np from aqueous solution through direct and/or indirect microbial reactions and affect migration of Np in the subsurface
environment.

100

Np absorption, %out in a bentonite suspension (0.5 g/L) also containing 0.01
mol/L NaNO3 in 10-mL serum vials under an N2 atmosphere.
After boiling and cooling the suspension under O2-free N2 gas,
the suspension was dispensed into vials, and then the vials
were aluminum-capped with butyl rubber stoppers, and Na2S
solution was added to a final concentration of 0.01 mol/L. One
hour later, the Np solution was added to the vials, at the indicated times, and pH and Eh (vs. NHE) were measured using apH-ORP meter in a glove bag filled with O 2 -free N 2 , after
which the suspensions were filtered through a 0.22 µm filter(Millipore), and the filtrate was analyzed as described above.
Np removal by microbial reactions. To assess the effectof D. desulfuricans on Np removal from bentonite suspension,
microbial Np removal experiments were carried out in 10-mL
glass serum vials containing 0.1 g/L bentonite suspended in 5
mL of cell cultivation medium. After sterilizing the vials, thecells were added and shaken in an incubator at 35 ˚C for 5 days.
At that point, Np solution was added to the vials to a final
concentration of 1 × 10 −6 mol/L, and the vials were again
shaken in the incubator under the same conditions used for cellcultivation. At the indicated times the pH, Eh, and Np concentration were analyzed as described above.

Nagaoka

80604020002468101214pHFigure 1. Adsorption of Np(V) on bentonite over a wide range of pHs
under aerobic conditions.

100Np in solution, %8680
6040200020406080

100Time, hr
Figure 2. Microbial mediated removal of Np(V). Removal of Np bybentonite under aerobic conditions (●), with 0.01 mol/L Na2S (○) andwith cells (□). Conditions: bentonite, 0.5 g/L; NaNO 3, 0.01 mol/L;
Np, 1 × 10−6 mol/L.

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