APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2004, p. 7558–7560
0099-2240/04/$08.00?0 DOI: 10.1128/AEM.70.12.7558–7560.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 12
Resistance of Solid-Phase U(VI) to Microbial Reduction during In
Situ Bioremediation of Uranium-Contaminated Groundwater
Irene Ortiz-Bernad,* Robert T. Anderson, Helen A. Vrionis, and Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts
Received 21 June 2004/Accepted 28 July 2004
Speciation of solid-phase uranium in uranium-contaminated subsurface sediments undergoing uranium
bioremediation demonstrated that although microbial reduction of soluble U(VI) readily immobilized uranium
as U(IV), a substantial portion of the U(VI) in the aquifer was strongly associated with the sediments and was
not microbially reducible. These results have important implications for in situ uranium bioremediation
The large volumes of uranium-contaminated groundwater at
many uranium-contaminated sites precludes pump and treat
remediation strategies. In situ immobilization of uranium via
microbial reduction of soluble U(VI) to insoluble U(IV) is a
potential strategy for preventing further migration of uranium
with the groundwater (11, 12). Addition of acetate to a urani-
um-contaminated aquifer effectively stimulated the growth of
dissimilatory metal-reducing microorganisms in the family
Geobacteraceae and the removal of U(VI) from the ground-
water (1) in accordance with previous predictions from labo-
ratory incubations of uranium-contaminated sediments (4, 5,
7). Continuing field analysis has suggested that once uranium
is precipitated from groundwater, it is immobilized for sub-
stantial periods of time after the acetate additions are stopped
Speciation of uranium in subsurface sediments from a
bioremediation site. In order to further evaluate the immobi-
lization of uranium during in situ bioremediation, sediment
cores were collected from the previously described uranium
bioremediation site in Rifle, Colo. (1), from depths of 4.0 to
5.2 m by a diamond-rotosonic method (Boart Longyear, Envi-
ronmental Drilling Division, Little Falls, Minn.), 38 days into a
second summer of acetate injection. Core samples were sec-
tioned and transferred in the field to an anaerobic (N2-filled)
portable glove bag, and subsamples were placed in anaerobic
pressure tubes and packed on ice for transport back to the
laboratory. In the laboratory, sediments were extracted (1 g of
sediment per 5 ml of extractant) under anaerobic conditions in
a glove bag (83:10:7 N2-CO2-H2) with 100 mM sodium bicar-
bonate (15) for 48 h. Preliminary studies with subsurface sed-
iments from the study site demonstrated that this procedure
was sufficient to extract over 90% of the U(VI) that could be
extracted with 1 M nitric acid. U(VI) in the extract was mea-
sured with a kinetic phosphorescence analyzer (4, 15). The
sediment extractions were then aerated for 24 h to oxidize
U(IV) to U(VI) (6), and U(VI) was again measured. U(IV)
was calculated as the increase in U(VI) in the extract following
Sediments from locations in which U(VI) had been effec-
tively reduced in the groundwater contained U(IV), but most
of the uranium associated with the sediments was in the form
of U(VI) (Fig. 1). There were also substantial quantities of
U(VI) in sediments upgradient from the acetate injection
zone. This fact indicated that U(VI) adsorption was not asso-
ciated with the in situ uranium bioremediation. When a given
sediment volume is considered, U(VI) associated with the sed-
iments was ca. eightfold more abundant than U(VI) in the
Sediment incubations. In order to evaluate the apparent
persistence of U(VI) in the acetate-injection zone under
strictly anaerobic conditions, sediments (115 g) from outside
the acetate injection zone at the Rifle study site were incubated
with associated uranium-contaminated groundwater (30 ml)
under anaerobic conditions in sealed serum bottles as previ-
ously described (4, 5). These sediments, which are naturally
partially reduced, as evidenced by the presence of Fe(II), con-
tained U(IV) and well as U(VI) (Fig. 2A). If no amendments
were made to the sediments, there was an increase in soluble
U(VI) and a decline in U(VI) associated with the sediments,
presumably due to reequilibration of the sediment-bound ura-
nium with the added groundwater. At apparent equilibrium,
the soluble and sorbed U(VI) fractions contributed nearly
equally to the total U(VI) pool. Addition of acetate (2 mM) to
simulate in situ uranium bioremediation stimulated microbial
metal reduction, as was evident from an accumulation of
Fe(II) as well as a loss of U(VI) from the groundwater and an
accumulation of U(IV) in the sediments (Fig. 2B). However,
there was no loss of U(VI) from the sediments, suggesting that
the U(VI) associated with the sediments was resistant to mi-
In order to further evaluate this phenomenon, a second
study was conducted in which the concentration of U(VI) was
artificially increased with the addition of an additional 20 ?M
U(VI) (Fig. 3A). Circa 40% of the added U(VI) was lost from
solution and adsorbed onto the sediments, further demonstrat-
ing that sorption of uranium can be an important sink for
uranium in the subsurface. The addition of acetate (5 mM) to
stimulate dissimilatory metal reduction enhanced the adsorp-
* Corresponding author. Mailing address: University of Massachu-
setts, Dept. of Microbiology, Morrill Science Center IVN, Amherst,
MA 01003. Phone: (413) 577-0217. Fax: (413) 545-1578. E-mail:
tion of U(VI) relative to that with controls and resulted in
increased removal of U(VI) from the groundwater, which was
accompanied by an increase in U(IV) in the sediments (Fig.
3B). As Fe(III) was depleted and sulfate reduction became the
predominant process, the loss of soluble U(VI) and the pro-
duction of reduced U(IV) both stopped. This result is consis-
tent with previous observations from field experiments which
have suggested that Fe(III) reducers, but not sulfate reducers,
are effective in reducing soluble U(VI) to insoluble U(IV) in
contaminated aquifer sediments in the presence of acetate (1).
Implications. U(VI) adsorption onto iron oxides and hy-
droxides (2, 3, 8, 10, 16, 17) and clays (9, 13, 14) in sediments
is well known. However, previous studies of in situ uranium
bioremediation have primarily focused on the reduction of
dissolved U(VI) to insoluble U(IV) because immobilization of
contaminant uranium is of paramount importance. The results
presented here suggest that although dissimilatory metal-re-
ducing microorganisms can effectively reduce soluble U(VI),
U(VI) associated with the solid phase is not microbially reduc-
ible. The inability of microorganisms to reduce U(VI) ad-
sorbed to sediments is not a limitation during the acetate
injection phase of in situ bioremediation because this U(VI) is
already immobile. However, it does have an impact on strate-
gies for eventually extracting the immobilized uranium. For
example, it has previously been proposed that once uranium
has been immobilized in a discreet zone via U(VI) reduction,
it may be resolubilized and extracted by reoxidizing precipi-
tated U(IV) to soluble U(VI) (5). The finding that a high
proportion of the immobilized uranium is likely to be in the
form of U(VI) suggests that the oxidation of U(IV) should be
supplemented with procedures for extracting U(VI), such as
the use of bicarbonate (15), a common extraction technique for
in situ uranium mining. In a similar manner, the injection of
bicarbonate upgradient of a zone of acetate injection may
solubilize U(VI) from sediments, which may be followed by
reductive precipitation within the acetate injection zone to
remove uranium which otherwise may later serve as a source of
dissolved U(VI) in the groundwater via desorption. Further
investigations into the form of the U(VI) associated with sed-
FIG. 1. Percentages of U(VI) out of the total U of sediment cores
collected at three different depths in a uranium-bioremediation site
located in Rifle, Colo. The dashed line indicates the position of the
acetate injection gallery. P11, upgradient sediment core; P12 to P15,
downgradient cores from the point of acetate injection into the sub-
surface. The results are the means of results of five replicates, and the
standard deviations were below 10% of the means.
FIG. 2. Sediment and soluble uranium, sulfate, and ratio of Fe(II) to total acid-extractable iron (Fet) in contaminated aquifer sediments (115
g) with uranium-contaminated groundwater (30 ml) incubated with (B) or without (A) added acetate (2 mM). The results are the means of results
of triplicate sediment incubations.
VOL. 70, 2004 RESISTANCE OF SOLID-PHASE U(VI) TO MICROBIAL REDUCTION7559
iments and the mechanisms preventing the microbial reduction
of this U(VI) are warranted, as such studies may provide in-
sights into strategies for promoting the microbial reduction of
this important pool of U(VI) in uranium-contaminated
This research was supported by the Office of Science (BER), U.S.
Department of Energy, grant DE-FG02-04ER63719. I.O.-B. was the
recipient of a postdoctoral fellowship from the Secretarı ´a de Estado de
Educacio ´n y Universidades (Madrid, Spain), cofunded by the Euro-
pean Social Fund.
We thank Philip E. Long (Pacific Northwest National Laboratory),
Aaron Peacock (Microbial Insights), and Richard Dayvault (S. M.
Stoller Corporation) for core sample collection. We also thank Donald
R. Metzler (U.S. Department of Energy) for coordinating activities
conducted at the Old Rifle UMTRA site.
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FIG. 3. Sediment and soluble uranium, sulfate, and ratio of Fe(II) to total acid-extractable iron (Fet) in contaminated aquifer sediments (115
g) with uranium-contaminated groundwater (30 ml) amended with an additional 20 ?M U(VI) and incubated with (B) or without (A) added
acetate (5 mM). The results are the means of results of quintuplet extractions made on duplicate sediment incubations.
7560ORTIZ-BERNAD ET AL.APPL. ENVIRON. MICROBIOL.