Environ. Sci. Technol. 1994, 28, 1686-1690
Solubilization of Plutonium Hydrous Oxide by Iron-Reducing Bacteria Patrlcla A. Rusin,'#t Leticla Quintana,?James R. Brainard,'g* Betty A. Strietelmeier,* C. Drew Tait,l Scott A. Ekberg,l Phillip D. Palmer,* Thomas W. Newton,* and David L. Clark* MBX Systems, Inc., 325 South Euclid, Suite 123, Tucson, Arizona 85719,and CST-3 and CST-14, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
The removal of plutonium from soils is challenging because of its strong sorption to soils and limited solubility. Microbial reduction of metals is known to affect the speciation and solubility of sparingly soluble metals in the environment, notably iron and manganese. The similarity in reduction potential for a-FeOOH(s) and hydrous PuO2(s) suggests that iron-reducing bacteria may also reduce and solubilizeplutonium. Bacillus strains were used to demonstrate that iron-reducing bacteria mediate the solubilization of hydrous PuOz(s) under anaerobic conditions. Up to -90% of the PuOz was biosolubilized in the presence of nitrilotriacetic acid (NTA) within 6-7 days. Biosolubilization occurred to a lesser extent (-40% ) in the absence of NTA. Little Pu02 solubilization occurred in sterile culture media or in the presence of a non-ironreducing Escherichia coli. These observations suggest a potentially attractive, environmentally benign strategy for the remediation of Pu-contaminated soils.
Introduction Several genera of bacteria have been shown to reduce and solubilize Mn(1V) and Fe(II1) (1-6). These metals often serve as alternate electron acceptors in the absence of oxygen for anaerobic respiration or as an electron sink for reoxidation of reducing equivalents. These and other microbially mediated metal reductions play an important role in the biogeochemical cycles of metals (7). Like iron and manganese, plutonium is a redox active metal. The aqueous redox chemistry of plutonium is more complex than that of iron or manganese with oxidation states from I11 to VI known to exist in aqueous solutions. The aqueous chemistry of plutonium, especially under environmentally relevant conditions, is further complicated by the strong tendency for the aqueous cations to hydrolyze, to form complexes with a wide variety of anions, and to precipitate and adsorb strongly to surfaces. Although the form(s) of plutonium that are present in the environment are still the object of active investigation, oxides and hydroxides of Pu(1V)appear to be predominant (8-10). As with Fe(II1) and Mn(IV), the mobility and bioavailability of Pu(1V) in the environment is severely limited because of the low solubility of the oxides and hydroxides. The determination of accurate values for the solubility products of plutonium(1V) oxyhydroxides is difficult, and there are wide variations and uncertainties in the reported solubility products; recent determinations have given estimates of 10-56.8(8) and 10-57.8(9). The solubility of plutonium(II1) hydroxide is much greater, KBp= 10-22.6(11). Consequently, the reduction of plutonium(1V) oxyhydroxides to Pu(II1) is expected to greatly increase the solubility of plutonium in the environment. ~~
~~
+ MBX Systems. t
CST-3, Los Alamos National Laboratory. CST-14, Los Alamos National Laboratory.
1686
Envlron. Scl. Technol., Vol. 28, No. 9, 1994
Table 1. Reduction Potentials for Metal Oxyhydroxidesa
reaction UOz2++ 2e- = uoz(S) l/2&MnO2(s) + 2H+ + e- = '/zMn2+ + H2O yMnOOH(s) + 3H+ + e- = Mn2+ + 2H20 CrO42- 5H+ + 3e- = Cr(OH)&) Fe(OH)&) + 3H+ + e- = Fez+ + 3H20 a-FeOOH(s) + 3H+ + e- = Fez+ + 2H20 PuOu(H20)(s) + 4H+ + e- = Pu3++ (y + 2)H20
+
EH'
E'
ref
1.15
1.29
0.94 0.64
11,18 19
1.50
0.61
19
1.24 1.01
0.43 -0.13
19 20
0.67
-0.22
19
-0.63'
-0.27
21
EH' is the standard reduction potential. E' is the reduction potential at pH 7, Mnf(aq) = lo4 M. * This reduction potential is reported for pH 7.
Some strains of bacteria mediate the reduction of a remarkable variety of electron acceptors, usually under anaerobic conditions. For example, Shewanella putrefaciens can reduce nitrate, nitrite, Mn(IV), Cr(VI), and trimethylamine N-oxide (6,12-14); Geobacter metallireductans can reduce Mn(IV), Fe(III), and U(V1) (14, 15); while Bacillus circulans MBX 1 and Bacillus polymyxa MBX 2 reduce Mn(1V) and Fe(II1) (16). The electrochemical potential for the reduction of plutonium(1V) oxyhydroxide to Pu(II1) is similar to the potentials for iron and other metals known to be microbially reduced. The standard reduction potentials and reduction potentials under dilute conditions (pH 7, Mn+(aq)= 10-6 M) for several metal oxyhydroxides are compared in Table 1.The similarity in reduction potentials for hydrous PuOz and a-FeOOH suggest that those microbes capable of reducing goethite (and other electron acceptors) might also reduce plutonium(1V) oxyhydroxides under similar conditions and, thus, reductively solubilize plutonium. Microbial metal reduction has been utilized for the solubilization and extraction of metals from refractory ores (16, 17). Bioreduction has also been used for the precipitation of metals such as chromium (22)and uranium (14) from contaminated water. The use of microbially mediated redox changes in metals for waste treatment and environmental remediation is attractive because the process can be carried out under environmentally benign conditions, does not produce hazardous secondary wastes, and is potentially applicable for the treatment of a variety of metals and matrices. In addition, the potential for microbially mediated metal migration from contaminated sites is an important factor in assessingrisk and in choosing remediation options. This paper describes the solubilization of hydrous Pu02(5) by Fe(II1)-reducing strains of B. polymyxa and B. circulans. To our knowledge, this is the first report of bacterially mediated solubilization of plutonium oxides. 0013-936X/94/0928-1686$04.50/0
0 1994 American Chemical Society
Materials and Methods
Hydrous Plutonium Oxide. The solid plutonium substrate used in these experiments was prepared by hydrolysis and precipitation of Pu(1V) in aqueous base as described previously (23). Briefly, a stock solution containing 239Pu(IV) in nitric acid was purified by ionexchange chromatography and checked for purity by visible spectroscopy. The hydrous oxide precipitate was formed by adding a 0.6-mL aliquot of the Pu(1V) stock solution [0.0005 mol of Pu(IV)] to 100 mL of 0.15 M "4OH. A dark green precipitate of hydrous Pu02 formed immediately. The precipitate was washed 3-4 times with 100 mL of distilled water until the wash pH was neutral. The moist precipitate was aged at room temperature for 18-24 months prior to these experiments. The hydrous Pu02 was not sterilized in these experiments. However, sterile media controls were run with each experiment as a check for bacterial contamination of the media and hydrous oxide. These controls confirmed that no significant bacterial growth occurred in the absence of an inoculum, suggesting that the hydrous oxide was sterile. Culture Media, Plutonium solubilization experiments were performed in either tryptic soy broth (TSB, Difco) or mineral salts medium (MSM). The composition of the MSM on a per liter basis was as follows: NaHC03,2.5 g; CaCly2H20,O.l g; KC1,O.l g; NH&1,1.5g; NaH2POqH20, 0.6 g; NaC1,O.l g; MgC12-6H20,O.l g; MgSOq7H20,O.l g; MnCly4H20,0.005 g; glucose, 3.6 g; pH 7.0. Most of the plutonium solubilization experiments reported here were performed with media amended with 50 mM nitrilotriacetic acid (NTA, Sigma). In some experiments, the NTA was omitted. Bacterial Cultures. All Bacillus strains used in these experiments are facultative anaerobes that were isolated from soil, sediment, or ore samples. The Bacilli were identified according to Bergey's Manual of Systematic Bacteriology (24). The Escherichia coli was E. coli K12 derivative no, QC771(GC4468). Bacillus circulans strains SD 1and MBX 1, B. polymyxa strains MBX 2 and MBX 10, Bacillus isolate MBX 25, and E . coli K12 were grown anaerobically for 24 h to a target optical density of A600 = 0.1 (cm-l) in either MSM or TSB prior to the solubilization experiments. Cell-free spent medium in MSM (SMSM) was produced by an anaerobic culture of B. circulans SD 1for 4 days at 25 "C, centrifugation of the culture at 1500g for 10 min, and filtration of the supernatant (0.22 pm, Gelman). Plutonium Solubilization Experiments. Microbial solubilization of PuO2 was determined by placing a 20- or 10-pL aliquot of a hydrous PuO2 suspension in screw-cap centrifuge tubes with 50 mL of bacterial culture. Minimal headspace was maintained to facilitate the rapid establishment of anaerobiosis. In one case, cell-free spent medium from a culture of isolated SD 1 was used. The experiments were conducted using TSB or MSM containing 1.3-1.6 or 0.4-0.6 pmol of hydrous Pu02, respectively. Tests were performed in duplicate. The cultures and appropriate controls were incubated at room temperature (21-25 "C) on a rotator at 15 rpm. At intervals, bacteria in solution were enumerated by the spread plate technique, and the cultures were centrifuged for 30 min at 1500g to separate the solubilized plutonium from the remaining solid hydrous Pu02 and the bacterial cells. Duplicate aliquots of the supernatant were analyzed for soluble
plutonium by liquid scintillation counting (LSC). In some experiments, the centrifuged bacteria and solid Pu02 were resuspended in fresh culture media and incubated for an additional period on the rotator. Except for the photoacoustic spectroscopy experiments described below, all of the manipulations were carried out under an air atmosphere. At the end of all experiments, the residual hydrous Pu02 solids were completely dissolved in 10 mL of 2 M HN03/0.5% HF for 24 h and analyzed by LSC to obtain a mass balance. Results were analyzed using one-way analysis of variance (ANOVA). Photoacoustic Spectroscopy. Photoacoustic spectroscopy (PAS) was used to obtain absorbance spectra of the solubilized Pu species formed in the bacterial culture supernatants. All PAS experiments were carried out with B. circulans SD 1 in MSM containing NTA. Bacterial solubilization experiments were conducted as described above, except that centrifugation of the cultures after 4 days was performed for 30 min at 7000g and that centrifugation and transfer of the culture supernatant to PAS cells was done under an argon atmosphere. Aliquots of the PAS supernatant were analyzed by LSC to verify that similar quantities of Pu were solubilized. The pulsed photoacoustic instrument used has been described in detail previously (25). The large spectral range expected for electronic transitions of Pu(III), Pu(IV), and Pu(1V) colloid precluded obtaining the entire PAS spectrum with a single dye. Consequently, the PAS spectra of culture and control supernatants were obtained over a period of several days following the solubilization experiment. The following dyes were used to record the indicated signal ranges: coumarin 460, 450-475 nm; coumarin 480, 470-495 nm; coumarin 500, 495-535 nm; coumarin 540,530-575 nm; rhodamine 610,575-601 nm; and rhodamine 640,600-620 nm. Monitoring Stability of Pu(II1)-NTA. In order to investigate the stability of Pu(II1)-NTA under neutral aqueous conditions, we prepared a sample of 242Pu(III)NTA and monitored its redox state by UV-vis spectroscopy. The sample was prepared by sparging 3 mL of 1M NTA (pH = 6.76) with argon and adding 0.1 mL of 0.23 M 242Pu(III)stock solution to make a Pu(II1) final concentration of 0.0076 M Pu(II1). The pH of the Pu(111)-NTA solution was readjusted to pH = 6.66 and transferred to an argon-purged spectrometer cell; UV-vis spectra were recorded on a Perkin Elmer Lamda 9 spectrophotometer.
Results and Discussion The total amounts of hydrous Pu02 solubilized by Bacillus strains SD 1and MBX 1in TSB media with and without NTA at day 2 and day 6 are shown in Figure 1. Data for the solubilization of Pu02 in uninoculated controls containing TSB and TSB amended with NTA are included. In the presence of NTA, isolate SD 1solubilized 45 % of the Pu02 in 2 days and an additional 40% after 4 more days of incubation. Slightly less Pu was solubilized by B. polymyxa MBX 1. Only 1.3% of the total Pu was solubilized in the uninoculated TSB and 4.5 % by TSBNTA in the 6-day incubation period. Nitrilotriacetic acid significantly enhanced the dissolution of plutonium by both B. circulans strains SD 1 and MBX 1 (p < 0.01). The solubilization of Pu02 by B. circulans SD 1in MSM with NTA is shown in Figure 2. This figure also shows Environ. Scl. Technol., Vol. 28, No. 9, 1994
1667
1.4,
I
12
1
Ia
0.8
-; 2
06
0.4 0.2 n SD 1.NTA
MBX 1-NTA
SD 1
MBX 1
TSB-NTA
TSB
Figure 1. Solubilization of 1.3-1.6 pmol of hydrous Pu02in TSB with and without 50 mM NTA. SD 1 and MBX 1 are iron-reducing Bacillus strains; -NTA indicates nitrilotriacetic acid present; TSB is tryptic soy broth. The vertical bars represent 95 % confidence intervals. 0.7 I
I
Flgure 3. Total amount of hydrous plutonium oxide solubilized by Fe(111)-reducing Bacillus strains in 2 days. Initially, 1.3-1.6 pmol of solid PuOp present. SD 1, MBX 1, MBX 2, MBX 10, and MBX 25 are ironreducing strains of Bacillus; TSB is tryptic soy broth; -NTA indicates nitrilotriacetlcacid present. The vertical bars represent 95 Yo confidence intervals.
Table 2. Survival of Bacillus Strains MBX 2, SD 1, MBX 10, and MBX 1 in Presence of Plutonium with and without 50 mM NTAB
Bacillus strain MBX 2-NTA SD 1-NTA MBX 10-NTA MBX 25-NTA MBX 1-NTA TSB-NTA
SD 1 MBX 1 TSB
Y
SD1-NTA
E coli-NTA
SMSM-NTA
Figure 2. Solubilizationof 0.4-0.6 pmol of hydrous PuOl in MSM with 50 mM NTA. SD 1, iron-reducing Bacillus; E. coli, non-iron-reducing bacterium; -NTA, nitrilotriacetic acid present; SMSM, cell-free spent mineralsalts mediumfrom the 4dayanaerobic incubationof 8. circulans SD 1. The vertical bars represent 95% confidence intervals.
controls containing E. coli and spent medium from the anaerobic incubation of SD 1in MSM-NTA. These data demonstrate that the solubilization of hydrous Pu02 was mediated by B. circulans SD 1 in a defined medium and did not occur to a significant extent in controls containing metabolites produced by SD 1. This observation confirms and extends previous results with Mn and Fe showing that these metal-reducing Bacilli require direct physical contact with mineral particles for solubilization to occur (I). Very little plutonium was solubilized in the presence of a non-iron-reducing E. coli, suggesting that the dissolution of hydrous Pu02 was not a generalized microbial phenomenon but was mediated by the iron-reducing bacteria. Indeed, Bacillus SD 1 solubilized 91% of the PuOz while only 5% was solubilized by the E. coli. The solubilization of plutonium by several other ironreducing isolates, under anaerobic conditions, was examined. Amounts of plutonium solubilized in TSB media over a 2-day period by Bacillus strains SD 1, MBX 1, MBX 2, MBX 10,and MBX 25 are shown in Figure 3. The ability to solubilize plutonium appears to be a general characteristic of these strains of Fe(II1)-reducingbacteria. 1888 Environ. Scl. Technol., Vol. 28, No. 9, 1994
control day2 day 6 6.46 6.79 7.04 6.77 5.93
