Peck
Introduction
The use of Uranium (U) in many American industries has led to contamination at many
locations across the country. The prevalence of this contamination has led to increased interest in
bioremediation techniques for the reduction of certain forms of U into more stable compounds.
Of particular interest is the reduction of hexavalent U (U(VI)) into tetravalent U (U(IV)) in the
form of biogenic uraninite, UO2 + x, 0 ≤ X ≤ .25.. Due to its relative stability and insolubility,
uraninite is thought to have great potential as a means of sequestering subsurface U
contamination [1]. However, many of the properties of bioremediated uranium at contaminated
sites remain unknown as a result of its complex biogeochemistry. These properties are to be
investigated through in situ U(VI) reduction in field tests in Rifle, CO.
Typical bioremediation techniques rely on the reduction of U(VI) into U(IV) by microbes
like Shewanella[2]. Electron donor molecules like acetate are injected into the water source to
generate anoxia and thus create optimal reducing conditions. Microbes then mediate the
reduction of electron receptors like NO3-, Fe(III), SO42- and, most notably, U(VI). As uraninite is
orders of magnitude less soluble than other uranium species, it is expected to have a large impact
on the geochemistry of reduced uranium in water sources.
Such bioremediation techniques also require consideration of the adsorption of U(IV) to
biomass or minerals. Such species may have a large impact on the stability of reduced,
bioremediated uranium, and also present a possible avenue for further remediation of uranium
contamination. In particular, Fe(III) (oxyhydr-)oxides tend to be strong sorbents of uranium. Iron
redox cycling leads to structural and compositional transformations of these compounds that can
lead the release of sorbed uranium, as well as providing further long-term hosts for uranium. The
process of iron redox cycling is also driven by microbial metal reduction, further supporting the
use of stimulated bioremediation for the attenuation of uranium contamination in groundwater.
Any use of these bioremediation techniques will require a thorough understanding of the
biogeochemical processes involved. This research focuses on determining what the reduced
uranium products of acetate and bicarbonate stimulated sediment bioremediation are. Thus far,
this question has remained unanswered as a result of the extremely low concentrations of
uranium in treated sediments. However, the use of novel in-well sediment columns allows for
investigation of the products of microbial metal reduction in various conditions by permitting the
addition of uranium and other influent solutions without fear of harm to the environment.
The in-well reactor column setup used at the Rifle, CO site operates by pulling
groundwater through a glass column full of bioactive sediment from the Rifle site itself. Uranium
is amended into the influent groundwater, eliminating the previous issue of too low uranium
concentration. The sediment from the columns is then analyzed using x-ray absorption
spectroscopy (XAS). XAS is used to discern both the local and compound-scale structure of the
reduced uranium from the columns, and thus to answer the overarching question of what the
products of stimulated sediment bioremediation are. In particular, this experiment will deal with
the products of stimulated sediment bioremediation during iron and sulfate reduction.
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Materials and Methods
i. Apparatus
The primary experimental apparatus in this experiment was the Rifle in-well column
setup. The column setup, shown in Figure 1 below, consisted of a glass Kontes column filled
with sediment and containing a single inlet line on the bottom of the column and an outlet line on
top. 22 of these columns were affixed to a PVC pipe via cable ties, at depths from 22 feet below
the surface to 16 feet. At the outlet of each column, approximately eight meters of polyethylene
(PE) tubing connects to the outlet and travels up the well to the water line, where it then connects
to about 16 feet of stainless steel tubing. Stainless steel tubing is used above the water line
because of its low oxygen diffusivity. The stainless steel tubing then connects to a peristaltic
pump outside of the well. Another peristaltic pump, pumping at one tenth the rate of the effluent
pump, is used to amend uranium solution into the column. The tubing from this pump connects
to stainless steel and PE tubing just as with the effluent pump. Once at the column itself, the
influent tubing is connected to a tee that allows the influent solution to mix with the groundwater
being pulled in by the effluent pump. A meter of polyethylene tubing is used to allow the
amended solution to mix with the groundwater via diffusion. A check valve is used to prevent
any of the uranium solution from leaking into the groundwater and further contaminating the
well.
ii. Sample Preparation
Samples collected from the column reactors for analysis via XAS techniques are both
highly oxygen sensitive and radioactive, and so must be handled with extreme care. The
samples were shipped from the Rifle site in stainless steel vacuum containers, and all sample
preparation occurred within an anaerobic glove box marked as a contamination area.
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Sediment samples were transferred into centrifuge tubes via pipette, and then centrifuged for
several minutes to force the sediment to separate from any water in the sample. The sediment
was then carefully transferred into aluminum sample holders and covered with kaptan tape to
prevent any sediment from escaping while minimizing x-ray intensity loss. Once loaded into
the beam hutch, a liquid nitrogen cryostat was used to maintain a vacuum on the sample and
prevent any incursion of oxygen.
iii. X-ray Spectroscopy
Analysis of these samples relies on x-ray spectroscopic techniques. In particular, x-ray
absorption near edge spectroscopy (XANES) and x-ray absorption fine structure
spectroscopy (EXAFS) are utilized to discern the oxidation state and local molecular
structures of the reduced uranium in the samples. X-ray scattering techniques are also used to
provide nanoparticle diameters and morphologies. These well-known techniques are
combined to provide a full view of the structure of the uranium in the samples.