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Applied and Environmental Microbiology, June 2000, p. 2451-2460, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effect of Electron Donor and Solution Chemistry on
Products of Dissimilatory Reduction of Technetium by
Shewanella putrefaciens
R. E.
Wildung,*
Y. A.
Gorby,
K. M.
Krupka,
N. J.
Hess,
S.
W.
Li,
A. E.
Plymale,
J. P.
McKinley, and
J. K.
Fredrickson
Pacific Northwest National Laboratory,
Richland, Washington 99352
Received 1 July 1999/Accepted 15 February 2000
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ABSTRACT |
To help provide a fundamental basis for use of microbial
dissimilatory reduction processes in separating or immobilizing
99Tc in waste or groundwaters, the effects of electron
donor and the presence of the bicarbonate ion on the rate and extent of pertechnetate ion [Tc(VII)O4
] enzymatic
reduction by the subsurface metal-reducing bacterium Shewanella
putrefaciens CN32 were determined, and the forms of aqueous and
solid-phase reduction products were evaluated through a combination of
high-resolution transmission electron microscopy, X-ray absorption
spectroscopy, and thermodynamic calculations. When H2
served as the electron donor, dissolved Tc(VII) was rapidly reduced to
amorphous Tc(IV) hydrous oxide, which was largely associated with the
cell in unbuffered 0.85% NaCl and with extracellular particulates (0.2 to 0.001 µm) in bicarbonate buffer. Cell-associated Tc was present
principally in the periplasm and outside the outer membrane. The
reduction rate was much lower when lactate was the electron donor, with
extracellular Tc(IV) hydrous oxide the dominant solid-phase reduction
product, but in bicarbonate systems much less Tc(IV) was associated
directly with the cell and solid-phase Tc(IV) carbonate may have been
present. In the presence of carbonate, soluble (<0.001 µm)
electronegative, Tc(IV) carbonate complexes were also formed that
exceeded Tc(VII)O4 in electrophoretic
mobility. Thermodynamic calculations indicate that the dominant reduced
Tc species identified in the experiments would be stable over a range
of Eh and pH conditions typical of natural waters. Thus,
carbonate complexes may represent an important pathway for Tc transport
in anaerobic subsurface environments, where it has generally been
assumed that Tc mobility is controlled by low-solubility Tc(IV) hydrous
oxide and adsorptive, aqueous Tc(IV) hydrolysis products.
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INTRODUCTION |
Technetium (element 43) is present
in the environment principally as a result of fallout from nuclear
weapons testing, uranium enrichment, nuclear fuel processing, and
disposal after pharmaceutical use (34). During nuclear fuel
reprocessing, Tc is solubilized from spent fuels and is present in all
waste streams (10) principally as the pertechnetate anion
[Tc(VII)O4]. Over the pH and
Eh ranges typical of most groundwaters, Tc may exist
in oxidation states VII, VI, V or IV, but in the absence of strong
complexing agents Tc(VI) and Tc(V) may be expected to disproportionate
to Tc(VII) and Tc(IV) (31). The dominant oxidation state
under oxic conditions is Tc(VII), which is weakly sorbed by most soils
and subsurface sediments at near neutral pH values (15, 35).
Under anoxic conditions and in the absence of aqueous complexing agents
other than OH, Tc(IV) is largely immobile because it
forms concentration-limiting solid phases and strong surface complexes
with hydroxylated surface sites on Al and Fe oxides and clays (5,
9, 24, 31). Organisms capable of anaerobic energy metabolism,
including the dissimilatory metal-reducing bacteria (DMRB)
Shewanella putrefaciens, Shewanella alga, and
Geobacter metallireducens (16, 36) and the
sulfate-reducing bacterium Desulfovibrio desulfuricans
(17, 18), have been shown to be capable of gaining
energy for maintenance and growth by coupling the oxidation of organic
C or H2 to the reduction of Tc(VII). The microbial
reduction of Tc(VII) has been suggested as a potential mechanism for
the removal of Tc from contaminated environments or waste streams
(16, 19, 20).
In nonsulfidogenic, waste-contaminated groundwaters, the reduction of
Tc(VII) may potentially occur directly, through enzymatic reduction,
and/or indirectly via the microbial reduction of Fe(III) oxides and the
formation of aqueous Fe(II) and/or Fe(II)-containing solids (6, 8,
12), which may in turn reduce Tc(VII) (4). The
kinetics of Tc reduction and the aqueous chemistry of the environment
are critical factors in determining which of these reactions dominates,
as well as the nature of the hydrolysis and other complexation
reactions that affect the form and stability of lower oxidation states
of Tc. This information will be of key importance in developing waste
and groundwater treatment strategies and assessing the mobility of
lower oxidation states of Tc in groundwaters. However, the complex
chemistry of Tc has provided formidable challenges to understanding the
role of microorganisms in influencing its behavior under anaerobic
environmental conditions. As a first step in assessing the combined
effects of direct and indirect reduction processes on Tc speciation and
mobility in waste and groundwaters, these studies examined the
influence of electron donor and the presence of the inorganic
complexing ligand bicarbonate on the rate of enzymatic Tc(VII)
reduction by Shewanella putrefaciens CN32. The forms of
solid- and aqueous-phase reduction products were determined by
high-resolution transmission electron microscopy (TEM), X-ray
absorption spectroscopy (XAS), and solution chemistry. Thermodynamic
calculations were used to establish constraints on the solubility and
chemical speciation of reduction products over a range of pH and
Eh values commonly encountered in the subsurface.
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MATERIALS AND METHODS |
Cell preparation.
S. putrefaciens CN32, a facultative
anaerobic bacterium, was originally isolated from an anaerobic
sandstone at a depth of 250 m in the Morrison formation of
northwestern New Mexico (8). CN32 was provided through the
courtesy of D. Boone (Portland State University, Portland, Oreg.).
Cells were cultured aerobically in tryptic soy broth (100 ml) and were
incubated on a rotary shaker (100 rpm) at 30°C. The cultures were
harvested after 16 h by centrifugation (5,000 × g, 15 min, 4°C).
Bacterial reduction.
The bacterial reduction of Tc(VII) was
measured with time under anaerobic, nongrowth conditions. Bacterial
cells were washed twice with a pH 7 solution of 30 mM
NaHCO3 and 1.3 mM KCl (bicarbonate solution) or 0.85% NaCl
(saline solution) that had been previously made anoxic by sparging it
with a mixture of O2-free N2-CO2
(80:20; bicarbonate solution) or N2 (saline solution).
Bacterial cells were resuspended in vials (20 ml) containing anoxic
bicarbonate or saline solution (10 ml) and appropriate headspace gas to
a density of ~108 cells/ml. The vials were sealed with
thick butyl rubber stoppers. All subsequent preparations and
incubations were conducted at 30°C in an anaerobic glove box (Forma)
containing an atmosphere of Ar (95%) and H2 (5%) and
<104% O2. All reagents were added or
samples removed by penetrating the stopper with a needle and syringe.
Either Na lactate (10 mM) or H2 (4.5 × 104 M) was added as an electron donor. Technetium was
added as pertechnetate [NH499Tc(VII)O4] (Amersham Life
Sciences Products, Arlington Heights, Ill.) to achieve concentrations
ranging from 1.86 × 105 to 2.23 × 107 dpm/ml and 5 µM to 6 mM, depending on the experiment.
Enzymatic reduction was assumed as the loss of pertechnetate in
filtered (see below) and unfiltered subsamples as a function of time.
Pertechnetate was determined by direct extraction (33) and
liquid scintillation counting of 99Tc (0.292 MEv beta). The
pH and Eh were measured directly in the sealed vials by
insertion of calibrated electrodes (Microelectrodes, Inc., Londonderry,
N.H.). Because of potential interactions between H2 with
the Pt electrode, Eh-sensitive dyes of known redox
potential (2) were used as the standard measure of
Eh at the end of the experiments and in aliquots of
solutions with time. Three dyes (methylene blue, E0', +11
mV; resazurin, E0', 
51 mV; and phenosafranin, E0', 251 mV) were employed to bracket the Eh
values in these systems, and therefore a range in Eh values
is reported. Reduced Tc(IV) controls were prepared using
SnCl2 (11) and hydrazine (23) as reductants.
Size distribution of Tc reduction products.
The relative
size distribution (0.2, 0.01, and 0.001 µm) of Tc-containing solids
formed upon reduction was determined by syringe filtration (Omega TM
modified polyethersulfone; 13-mm Pall-Gelman Filtron) or centrifugation
(Omega TM Filtron Nanosep; Pall-Gelman) of solution aliquots in
combination with phosphorimaging or liquid scintillation counting to
determine the concentration of 99Tc.
TEM.
Reduction products of >0.2 µm were imaged and
analyzed using TEM. All samples were prepared in an anaerobic glove box
to avoid oxidation of Tc(IV) solids. Whole mounts were prepared by
placing a drop of cell suspension on a Formvar-coated Cu TEM grid.
Grids were dried and stored in a sealed vial under an anaerobic
atmosphere until they were transferred to the high vacuum of the TEM.
Unstained whole mounts were analyzed by using a JEOL JEM 2000FX TEM
apparatus, along with ancillary diagnostic equipment. Samples were
observed at a 160-KeV accelerating voltage, and images were recorded on photographic film. Elemental analyses were accomplished using an
Oxford-Link Isis energy-dispersive spectrometer (EDS) equipped with a
SiLi detector. Electron diffraction patterns were obtained from
representative solids shown to contain Tc by EDS in the
saline-H2 systems. For solid-phase identification,
diffraction patterns were digitized from photographic negatives using a
flatbed scanner. Diffraction patterns were analyzed by search-match
against the Powder Diffraction File (PDF) database (sets 1 to 48 and 70 to 85, year 1998) produced by the International Centre for Diffraction Data, Newtown Square, Pa. (Materials Data, Inc., Livermore, Calif.).
Samples of >0.2-µm solids for thin sectioning were fixed for 1 h by adding an anoxic solution of glutaraldehyde directly to the sample
to yield a final fixative concentration of 2.5%. Fixed samples were
gently mixed with an equal volume of 4% Noble agar. After solidifying
them at room temperature, the samples were cut into 1-mm cubes and
washed three times with an anoxic solution of 10 mM PIPES
[piperazine-N,N'-bis(2-ethanesulfonic acid)]
buffer (pH 7) to remove the glutaraldehyde. The samples were slowly
dehydrated using an anoxic graded ethanol series. Dehydrated samples
were infiltrated with anoxic, low-viscosity LR-White embedding
solution, placed in gelatin embedding capsules, and hardened by
incubating them at 60°C for 1 h under an anoxic atmosphere.
Embedded samples were sectioned to a 50-nm thickness inside an
anaerobic glove box using a Leica Ultracut T microtome located in the
glove box. Thin sections were placed on 200-mesh Cu TEM grids coated
with a lacey carbon film. The unstained sections were examined using a
JEOL 2010 TEM apparatus operating at a 200-kV accelerating voltage. Low-magnification and high-resolution images were digitally recorded using a 1,024 × 1,024 charge-coupled device camera (Gatan
Instruments, Pleasantville, Calif.). Lattice fringe images were
generated at high magnification to assist in the identification of
solids and their crystallinity. The d-spacings were measured directly
from the digital images and compared to those of standard Tc solids available from the PDF database.
Paper electrophoresis.
The charge and relative mobility of
reduced Tc in filtrates were evaluated by paper electrophoresis
(11) using cellulose polyacetate membrane paper (5.7 by 12.7 cm; Seprahpore; Gelman Sciences) and an electrophoresis chamber
(Gelman) in the anaerobic glove box at 60 V for up to 1 h. Two
solvent systems (30 mM NaHCO3-1.3M KCl and 10 mM HCl) were
used to differentiate reduced Tc carbonate species. Tc(VII) and Tc
reduction products were visualized and quantified by phosphorimaging
(Bio-Rad) as described by Lloyd and Macaskie (16). Ammonium
pertechnetate and the reduced Tc(IV) controls (above) served as
standards for the calculation of retention factors.
X-ray absorption spectroscopy.
The oxidation state and the
dimensionality of Tc electronic states and coordination were determined
for selected solids and filtrates by using extended X-ray absorption
fine structure (EXAFS) measurements conducted at the Stanford
Synchrotron Radiation Laboratory under dedicated operating conditions
(3.0 GeV and a current of 40 to 90 mA). Filtrates, precipitates, or
slurries (0.1 to 0.2 ml) from enzymatic reduction experiments conducted
at initial Tc concentrations of 3 to 6 mM were transferred into a
Teflon tube and sealed under anaerobic conditions. The sealed tubes
were placed in sample holders and secondary and tertiary containment cells with kapton windows, also under anaerobic conditions. The X-ray
absorption analyses were conducted with the tertiary containers in a
nitrogen glove bag to inhibit oxidation of the samples during the
collection of absorption spectra. Spectra for Tc samples were collected
at the Tc K-edge in the fluorescence and transmission geometries
simultaneously, up to a photoelectron wave vector of 13 Å1, using an energy-resolving 13-element Ge detector for
the fluorescence measurement and nitrogen-filled ion chambers for the
transmission measurement. The transmission signal of a Tc standard was
measured simultaneously with each sample to provide energy calibration. Energy calibration was made by assigning the first inflection point in
the absorption edge of the Tc standard to 21,044 eV. The absorption
spectrum was normalized by fitting polynomials through the pre- and
postedge regions. At E0, the value of the extrapolated
preedge was set to zero and the difference between the extrapolations
of the pre- and postedge polynomials was set to unity.
The EXAFS oscillations were extracted by fitting a polynomial spline
function through the postedge region and normalizing
the difference
between this approximation of the solitary-atom
EXAFS and the actual
data with the absorption decrease calculated
using the McMaster tables
(
22). Fourier transforms were taken
over photoelectron wave
vector ranges that varied on the basis
of the signal-to-noise ratio for
each sample. EXAFS nodes were
selected as endpoints to the Fourier
transform range, and a two-sigma-wide
Gaussian window was used to
dampen the EXAFS oscillations at the
endpoints. The phase shift was not
removed from the Fourier transforms,
and, consequently, the peaks in
the transform moduli appear 0.2
to 0.5 Å shorter than the actual
distance from the absorber to
the neighboring atoms. The phase and
amplitude for the Tc-oxygen
and Tc-Tc paths were calculated using the
ab initio code FEFF7.02
(
32,
37). These scattering paths
were then parameterized and
used to fit the experimentally measured
EXAFS. The MoO
2 structure
was used to approximate the metal
cation environment in TcO
2 oxide.
Citrate and
diethylenetriamine pentaacetic acid complexes of reduced
Tc were
utilized to model the Tc-O-Tc and Tc-N scattering
paths.
Thermodynamic calculations.
Equilibrium thermodynamic
calculations were conducted iteratively with the enzymatic reduction
experiments and analysis of Tc aqueous and solid phases to establish
solubility, pH, and Eh limits on Tc reduction products and
potential mobility under waste and groundwater conditions. The MINTEQA2
(version 3.11) geochemical reaction code (1) was used to
calculate the aqueous speciation, saturation indices, and solubility of
dissolved Tc for the solution compositions and conditions in the
enzymatic reduction experiments. Eh-pH diagrams were
calculated for Tc aqueous species using HSC Chemistry for Windows
(version 2.03; Outokumpu Research Oy, Pori, Finland). The thermodynamic
calculations were performed using the concentrations of dissolved Tc
measured in the <0.001-µm (pore-size) filtrates at the end of the
enzymatic reduction experiments. The thermodynamic databases in
MINTEQA2 and HSC Chemistry were supplemented with Tc thermodynamic
constants (Table 1) from Lemire and Jobe (14) to calculate aqueous speciation and solubility of
Tc(IV), which was shown by XAS to be the dominant Tc reduction product in these systems. Thermodynamic constants for Tc(V) and Tc(VI) species
and for Tc(IV) carbonate solid phases have not been published. Nordstrom and Munoz (29) and Langmuir (13)
discuss geochemical modeling techniques and Eh-pH diagrams
and their usefulness in understanding environmental behavior of metals
in aqueous systems.
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RESULTS |
Effect of electron donor and solution composition on the rate and
extent of Tc reduction.
S. putrefaciens CN32 was able to
enzymatically reduce Tc(VII), but at very different rates depending
upon the solution composition and the electron donor (Fig.
1). The Tc reduction rate [decrease in
Tc(VII)] was most rapid with H2 as the electron donor, and reduction was complete in the saline and bicarbonate solutions after 5 and 21 h, respectively. Reduction rates were lower with lactate as
the electron donor, with only 10 and 20% of the Tc(VII) reduced after
21 h in the saline and bicarbonate solutions, respectively. The
results were highly reproducible over a broad Tc(VII) concentration range (5 µM to 6 mM). The Eh values of the unfiltered
solutions after 21 h were 111 mV > Eh > 312 mV for the lactate electron donor and <312 mV for the
H2 electron donor. The pH values over the time of
incubation were 7.5 ± 0.2, 7.2 ± 0.1, and 7.0 ± 0.0 for the bicarbonate-H2, bicarbonate-lactate, and
saline-lactate treatments, respectively. The pH increased from
7.00 ± 0.1 to 7.90 ± 0.1 in the saline-H2
treatment, which was unbuffered and exhibited maximum Tc(VII)
reduction. The increase in pH resulted from the consumption of
H+ during the reduction of Tc(VII) as represented by the
following equation:
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The results of thermodynamic calculations (discussed below)
indicated that a change in pH over this range would not affect
the
chemical speciation of Tc in these systems. Controls with
or without
cells and electron donors maintained E
h values of >
49
mV
and did not exhibit Tc reduction. The quantity of Tc(VII) associated
with the cells (>0.2 µm) in the controls was <5% of the total.
Residual (0.5 M HCl extractable) Fe(II,III) associated with the
cells
before initiation of enzymatic reduction amounted to 7.69E-19
mol per
cell, or <0.006 µmol of total Fe per treatment, and was
therefore
insufficient to influence measured Tc(VII) reduction
rates.
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FIG. 1.
Reduction of Tc(VII) by S. putrefaciens CN32:
effects of electron donor, solution composition, and reaction time on
Tc distribution in filtrates. Symbols:  , total Tc in the <0.2-µm
filtrate;  , TcO4 in the <0.2-µm
filtrate;  , Tc reduction products in the 0.2- to 0.001-µm
fraction;  , Tc reduction products in the <0.001-µm filtrate. Data
are means ± the standard deviation for duplicate assays. Controls
with or without cells and electron donors did not exhibit Tc
reduction.
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Cell-associated Tc reduction products.
When H2 was
used as the electron donor in saline solutions (Fig. 1), essentially
all of the Tc(VII) was converted to a black solid phase that was
completely retained by a 0.2-µm filter, a finding suggestive of the
precipitation of Tc(IV) oxide (23). Unstained TEM images
(Fig. 2A) of cell thin sections in this
treatment (visualized by the presence of Tc) showed that Tc reduction
products formed principally within the periplasmic region and on the
outer membrane surface. Within the periplasmic region, Tc reduction products clearly formed invaginations that extended into the cytoplasm (Fig. 2A). It is not possible to determine from these observations, however, whether all Tc in the cytoplasm originated from this source.
Cells suspended in saline solution with lactate as the electron donor
exhibited similar properties (Fig. 2B). In bicarbonate solution with
H2 as the electron donor, Tc precipitates were deposited in
the periplasmic region and on the outer margins of the cells (Fig. 2C).
The outer membrane was clearly visible as an electron-transparent region between the zones of Tc deposition. Invaginations of the cell
periplasmic region were not observed in bicarbonate solutions. It is
noteworthy that Tc precipitates did not form in or on the surfaces of
cells in bicarbonate solutions when lactate was used as the electron
donor (note the faint outline of cell in Fig. 2D). The extracellular
particulates were fine grained when lactate was used as the electron
donor compared to larger aggregates which were formed when
H2 was the electron donor.
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FIG. 2.
TEM images of S. putrefaciens CN32 after
enzymatic reduction of Tc with different electron donors and solution
compositions. Panels: A, saline-H2; B, saline-lactate; C,
bicarbonate-H2; D, bicarbonate-lactate. The cells are
unstained, and the images result from the presence of reduced Tc.
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Characterization of the cell-associated Tc reduction products and
results of the thermodynamic calculations provide strong
evidence for
the formation of sparingly soluble, amorphous Tc(IV)
hydrous oxide. The
precipitated Tc (Fig.
2A, B, and C) was amorphous
or poorly
crystalline, as determined by selected area electron
diffraction and
high-resolution TEM. The limited crystallinity
of these solids did not
result in definitive diffraction patterns
that could be matched with
solids in the PDF database. The EDS
analyses showed that Tc
precipitates contained Tc and O only.
Microbially reduced Tc solids
aged under anaerobic conditions
for 6 months did not show evidence of
increased
crystallinity.
EXAFS analyses of the solids formed by bioreduction in the
saline-H
2 system indicated the presence of Tc(IV) oxide
(Fig.
3).
An E
h-pH diagram
calculated for the saline-H
2 system (Fig.
4)
using the thermodynamic constants from
Lemire and Jobe (
14)
and the detection limit concentration
of total dissolved Tc (10
6.5 mol/liter) indicated that
these solutions were oversaturated
(shaded area in Fig.
4) with respect
to hydrous TcO
2 oxide at
the E
h and pH of these
experiments. Geochemical modeling (Fig.
5) using the same thermodynamic constants
and concentration of
dissolved Tc showed that the system was less
than 2 orders of
magnitude oversaturated with respect to hydrous
TcO
2 solid. Oversaturation
would be consistent with
experimental observations (Fig.
1), which
indicated that
Tc(VII) was reduced to Tc(IV) and removed by filtration
(0.001 µm [pore size]). The modeling results also indicate that
a
neutral hydroxyl Tc(IV) complex is the predominate aqueous complex
at
the E
h (<
50 mV) and pH of these experiments. The
calculated
distribution of dissolved Tc complexes in the
saline-H
2 treatment
was approximately 58%
[Tc(IV)O(OH)
2]
20 (aq), 38%
Tc(IV)O(OH)
20 (aq), and 4%
Tc(IV)O(OH)
3.
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FIG. 3.
Fourier transform of the EXAFS of Tc products formed by
microbial reduction of TcO4 in saline
solution (>0.2-µm fraction) with H2 as the electron
donor. A low-solubility Tc(IV) oxide is formed.
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FIG. 4.
Eh-pH diagram for Tc in saline solution with
H2 as the electron donor, indicating the dominant Tc
aqueous species in solution and the Eh-pH regions (shaded
area) theoretically oversaturated with respect to Tc(IV) hydrous oxide.
The diagrams were calculated by using 5.3 × 107
mol/liter for total dissolved Tc and thermodynamic constants from
Lemire and Jobe (14).
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FIG. 5.
Calculated total concentration (solid line) of dissolved
technetium (log moles/liter) in equilibrium with amorphous hydrous
Tc(IV) oxide for different solution compositions as a function of
Eh. The vertical dotted lines represent the limiting
Eh values. The horizontal dashed lines represent the
dissolved Tc measured in the <0.001-µm filtrate at the end of the
incubation period as indicated in Fig. 1. The minimum detection limit
for total dissolved Tc is 106.5 mol/liter.
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Extracellular particulate Tc reduction products.
Total Tc in
the <0.2-µm filtrates accounted for 70, 83, and 89% of the Tc in
the bicarbonate-H2, saline-lactate, and bicarbonate-lactate systems, respectively, at the end of incubation. Thus, the major fraction of the reduction products in these treatments was not associated with the cells, which were removed by 0.2-µm filtration. A
major portion of the reduced Tc in the <0.2-µm fraction was retained by 0.001-µm filters. A much smaller quantity (<11%)
was in true solution (<0.001-µm filtrate) at the end of the
incubation period. The EXAFS analyses of the <0.2-µm filtrate
fraction of the saline-lactate, bicarbonate-H2, and
bicarbonate-lactate systems indicated the presence of Tc(IV) oxide, as
illustrated in Fig. 3. Solids from the bicarbonate-H2
system, collected on 0.01-µm and 0.001-µm filters, were black and
were identified by EXAFS as Tc(IV) oxide as illustrated in Fig. 3.
The black color of the solids from the bicarbonate-H
2 and
saline-H
2 systems contrasted with the amber and pink color
of solids
from the saline-lactate and bicarbonate-lactate systems,
respectively.
EXAFS analyses of these solids indicated that a mixture
of Tc(IV)
and Tc(VII) oxidation states was present, but there were
insufficient
quantities for identification of the Tc form. The
saline-lactate
filtrates (<0.001 µm) were calculated (Fig.
5) to be
approximately
4 orders of magnitude oversaturated with respect to
amorphous
hydrous Tc(IV) oxide over the range of E
h values
measured by the
redox indicators (
111 to
312 mV). The measured
dissolved Tc
concentration was much higher than that predicted from
equilibrium
solubility, reflecting the lack of steady-state conditions,
but
the presence of solids (Fig.
1) is consistent with oversaturation.
At the measured pH values and limiting E
h values of the
experiments
(determined colorimetrically), the concentrations of
dissolved
Tc in the <0.001-µm filtrates from the
bicarbonate-hydrogen (E
h <
312 mV) and
bicarbonate-lactate (
111 to
312 mV) systems were
calculated to be
slightly undersaturated (Fig.
6) with
respect
to hydrous Tc(IV) oxide. The geochemical modeling results (Fig.
5) indicated that these solutions were less than 0.5 orders of
magnitude undersaturated with respect to hydrous Tc(IV) oxide,
suggesting that this is the dominant solid phase controlling the
dissolved Tc concentration. Thus, Tc(IV) oxide appears to be the
dominant extracellular product common to all treatments, but the
bicarbonate-lactate system may also contain (pink) Tc(IV) carbonate
solids.
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FIG. 6.
Eh-pH diagram for Tc in bicarbonate solution
with H2 as the electron donor, indicating the dominant Tc
aqueous species in solution, and Eh-pH regions (shaded
areas) theoretically oversaturated with respect to Tc(IV) hydrous
oxide. The diagrams were calculated by using 3.91 × 104 mol/liter for total dissolved Tc, 2.97 × 104 mol/liter for total dissolved carbonate, and
thermodynamic constants from Lemire and Jobe (14).
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Extracellular soluble Tc reduction products.
In the
<0.001-µm filtrates, Tc(VII), which was determined by chemical
extraction, constituted 98, 65, and 25% of the total Tc for the
saline-lactate, bicarbonate-lactate, and
bicarbonate-H2 systems, respectively. The rate of Tc(VII)
reduction was much slower in these systems than in the
saline-H2 system, and changes in Tc speciation
were still taking place at the time of sampling. These factors,
combined with the presence of Tc(VII) at low measured Eh values in these systems, suggest that the concentrations
of Tc(VII) enzymatic reduction products had not reached steady-state conditions (Fig. 1). Assuming complete reduction of the initial Tc(VII)
and the presence of carbonate from the oxidation of lactate, the final
concentration of dissolved (<0.001-µm filtrate) Tc in the
saline-lactate treatment was calculated to be comprised of approximately 72%
[Tc(IV)O(OH)2]20 (aq), 26%
Tc(IV)OH(CO3)2, and 2%
Tc(IV)O(OH)20 (aq).
The bicarbonate solutions (<0.2- and <0.001-µm filtrates) became
progressively pink with time as Tc(VII) was reduced. Spectrophotometric
analysis of these solutions indicated that the pink color was
due to an
absorption peak at 512 nm characteristic of Tc(IV) carbonates
(
30). Concentrations of Tc in the <0.001-µm filtrates
were insufficient
for unequivocal interpretation by EXAFS, but EXAFS
verified the
presence of Tc(VII) in the saline-lactate system and
suggested
the presence of Tc(IV) in the bicarbonate systems.
Thermodynamic
calculations for both bicarbonate systems indicated
that >99.9%
of the final concentrations of dissolved Tc over
the pH and E
h range encompassing these experiments should
be comprised of the
negatively charged aqueous
Tc(IV)OH(CO
3)
2 complex (Fig.
6).
At the limiting E
h values of the experiments
(determined
colorimetrically), the calculated solubilities of
hydrous Tc(IV)
oxide (Fig.
5) in the bicarbonate-hydrogen (E
h <
312 mV) and bicarbonate-lactate (
111 to
312 mV) systems were
several orders of magnitude higher than those for the other systems,
reflecting the presence of soluble, reduced Tc carbonate
species.
Further insight into the solid and aqueous forms of Tc in these systems
was provided by paper electrophoresis, which documented
changes in Tc
speciation based on altered electrophoretic mobility
in the
<0.001-µm filtrates of bicarbonate and saline solutions
(Fig.
7). There was essentially no Tc present
in this fraction
for the saline-H
2 system. The
saline-lactate system was predominately
Tc(VII), as indicated by direct
Tc(VII) measurements, but contained
a small quantity of Tc that
exceeded Tc(VII) in electrophoretic
mobility as indicated by the gray
area extending toward the cathode.
The bicarbonate-lactate and
bicarbonate-H
2 systems also contained
at least one
component equivalent to or greater in electrophoretic
mobility than
Tc(VII). The Tc concentrations in these components,
determined by
phosphoimagery, approximated the difference between
total Tc and
Tc(VII) determined directly in the filtrates. Thus,
it is likely that
they comprise the reduced Tc in the soluble
fraction (Fig.
1) and are
the negatively charged Tc(IV) carbonate
species indicated by
spectrophotometric analysis and thermodynamic
calculations (Fig.
6).
View larger version (39K):
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|
FIG. 7.
Paper electrophoretic separation of soluble
(<0.001-µm filtrate) Tc reduction products in different solutions
after microbial reduction of TcO4. The
intensity is proportional to the 99Tc concentration. The
positive sign represents the position of the cathode.
|
|
| |
DISCUSSION |
S. putrefaciens CN32 was capable of using
H2 and lactate as electron donors in the reduction of
soluble Tc(VII) under nongrowth conditions, thus confirming previous
observations that S. putrefaciens (ATCC 8071, Hammer strain)
can reduce Tc(VII) to insoluble products of lower oxidation states
(16). The current research demonstrated that the rate of
Tc(VII) reduction by a subsurface strain of S. putrefaciens
and the products of reduction were dependent upon solution composition
and the electron donor. Bicarbonate was chosen as a naturally occurring
inorganic ligand that forms soluble and insoluble complexes with some
metals, while NaCl was used to represent dilute solutions with little
or no complexation capacity but with sufficient ionic strengths to
prevent bacterial lysis. H2 and lactate were chosen as the
electron donors because they have previously been shown to be effective
donors for metal reduction in S. putrefaciens (3)
and because they are candidate electron donors for stimulating metal
reduction in in situ and ex situ remediation processes. A summary of
the conclusions of the Tc(VII) microbial enzymatic reduction
experiments is given in Table 2.
The rate and extent of Tc reduction by S. putrefaciens CN32
was much greater when H2 was provided as an electron donor,
in both the saline and bicarbonate solutions, compared to lactate. This
phenomenon was consistent with the results of related studies in which
the rates of reduction of a variety of metals, including Cr(VI), U(VI),
and both dissolved and solid forms of Fe(III), by S. putrefaciens strains CN32, MR-1, and BrY were consistently higher
with H2 as the electron donor (Y. A. Gorby,
unpublished data). The reasons for this are unclear, but there are
several possible explanations. First, H2 is a relatively
small, diffusible molecule that can readily cross the outer membrane of
gram-negative bacteria and enter the periplasmic region, where
it can be oxidized by soluble hydrogenases or hydrogenases associated
with the cytoplasmic membrane. In contrast, lactate dehydrogenase is
typically located in the cytoplasmic membrane or cytoplasm and
transport of lactate to these sites may be rate limiting. Also,
electrons from this process feed through intermediates such as NADH
before entering the electron transport chain, adding additional steps
that can slow the process. A second possible explanation is that
electrons liberated during the oxidation of H2 enter a
separate and perhaps less complex electron transport chain than is used
when lactate is the electron donor. The enzymes and pathways involved
in H2 and lactate oxidation in S. putrefaciens
have not been thoroughly investigated. A soluble hydrogenase from
Desulfovibrio desulfuricans has been shown to transfer
electrons from H2 directly to a soluble c3 cytochrome that served as the terminal metal
reductase (21). This two-component, in vitro system
reduced a wide range of metals, including Fe(III), U(VI), and
Cr(VI). Because D. desulfuricans cannot grow with metals as
electron acceptors, the significance of this phenomenon is unknown.
However, the results do demonstrate the potential for an abbreviated
pathway for electrons during the reduction of metals with
H2 as the electron donor. The rate of Tc(VII) reduction by
D. desulfuricans also was found to be much greater when
H2 was supplied as the electron donor in comparison to
lactate (18), a result consistent with an abbreviated
electron transport pathway. Lactate dehydrogenase is presumed to
function similarly to formate dehydrogenase, which has been studied in the S. putrefaciens MR-1 (26). Electrons gained
from the oxidation of formate by formate dehydrogenase enter an
electron transport chain through a number of identified quinone
compounds. The electron transport chain, which is composed of a complex
series of Fe-S proteins and cytochromes, then serves to direct
electrons toward outer-membrane cytochromes that may function as
terminal metal reductases (20). Because of multiple enzyme
steps in formate, and presumably lactate, oxidation it might be
expected that the rate of reduction would be inherently slower than
with H2, where a system composed of a limited number
of electron transport components, possibly as few as two, catalyzes
the oxidation of H2 and the reduction of Tc(VII).
S. putrefaciens MR-1 and four other strains of S. putrefaciens localize high concentrations of membrane-bound
cytochromes (>80%) to the outer membrane when grown anaerobically
with fumarate (28). At least four distinct c-type
cytochromes that are readily reoxidized by Fe(III)-citrate have been
identified in the outer membrane of MR-1 (27). Given the
difference in standard potential, E0, values for the
Tc(VII)O4/TcO2 (solid)
(E0 = 0.75 V) and
Fe(III)-citrate/Fe(II)-citrate (E0 = 0.31 V) redox couples, the outer membrane cytochromes in
S. putrefaciens would be theoretically capable of reducing
TcO4. This, coupled with the known presence
of c-type cytochromes in the periplasm of S. putrefaciens and the association of Fe reductase activity with the
cell membrane (25), is consistent with the observation of
Tc(IV) precipitates within the periplasm, in association with the outer
cell surface, and possibly with the cell membrane. Due to the relative
insolubility of Tc(IV) in this system, solids would be expected to
precipitate at the site of reduction, and/or possibly bind to cell
components, particularly when Tc(IV) is rapidly generated in the
absence of complexing ligands such as in the saline-H2
treatment. With H2 as the electron donor in pH 7 MOPS (morpholinepropanesulfonic acid) buffer, Tc was shown to be
associated with the periphery of the cells in D. desulfuricans (18). The participation of periplasmic
hydrogenases in the reduction of Tc(VII) by S. putrefaciens
cannot be ruled out. In fact, several putative genes coding for
periplasmic hydrogenase genes, with high homology to
Desulfovibrio periplasmic hydrogenases, have been identified
from whole genome sequencing of S. putrefaciens MR-1
(M. F. Romine, personal communication). The enzymatic reduction of
metals by S. putrefaciens is clearly a complex process
involving multiple proteins that will require further investigations to elucidate the role of specific proteins in the reduction of metals and
the localization of these activities within cells. Although Tc(VII)
reduction was also rapid in bicarbonate solutions with H2
as the electron donor, and some Tc was again associated with the
periplasm and outer cell surface, there was a much greater proportion
(76%) of bioreduced Tc that was present in the fine-grained (0.2 to
0.001 µm) extracellular fraction, with lower concentrations of a
soluble (<0.001-µm filtrate) species, likely
TcIVOH(CO3)2
(Table 2). There is strong evidence from EXAFS and TEM analyses, supported by Eh and pH calculations and equilibrium
modeling, that amorphous Tc(IV) hydrous oxide was formed inside and
outside the cell (Fig. 1 and 2) in both saline and bicarbonate systems when H2 was the electron donor. This solid was
oversaturated in the extracellular solution at the pH and
Eh of the experiments and was calculated to be stable over
a broad pH and Eh range (Fig. 4 and 6).
In the lactate-bicarbonate system, the formation of pink extracellular
Tc(IV) solids was suggestive of carbonate complexes, but the
concentrations of bioreduced Tc were insufficient to develop supporting
evidence. The absence of thermodynamic data for Tc(IV) carbonate solids
(which have not been previously identified) precluded calculating their
equilibrium aqueous solubilities. In this regard, it must also be
recognized that distinctly different chemical environments may exist
within and outside the cell but they are likely closely interrelated.
For example, soluble Tc(IV) species, such as carbonate complexes, that
form in the periplasm may diffuse to the cell exterior, where
precipitation of Tc solids may be more favorable due to differences in
such factors as pH, Eh, and pCO2 derived from
lactate oxidation. Microenvironmental differences between the cell
surface-periplasm and the bulk solutions may have contributed to
differences in Fe(II) biominerals generated as a result of reduction of
hydrous ferric oxide by S. putrefaciens CN32 (7).
The biologically mediated formation of reduced Tc solids during
enzymatic reduction will clearly require further investigation if these
processes are to be used in remediation of Tc dissolved in groundwaters.
The combined evidence also demonstrates that in conjunction with
cell-associated and extracellular particulates (0.2 to 0.001 µm),
soluble (<0.001-µm filtrates) Tc(IV) carbonate complexes were formed
in the bicarbonate-H2 and bicarbonate-lactate systems. Much
remains to be learned about the form and mobility of aqueous Tc(IV)
carbonate complexes since there is uncertainty regarding the
stoichiometries, and therefore the charges, for these species (5,
30). However, the results of paper electrophoresis suggested that
these complexes were more negative than Tc(VII) as
TcO4. The results of thermodynamic
calculations were consistent with their presence in the saline-lactate
system as well. The effect of low concentrations of the soluble
carbonates (pink) and of Tc(IV) hydrous oxides (black) likely resulted
in the amber color of the saline-lactate system. Although the reduction
of Tc(VII) to Tc(IV) must entail a transfer of three electrons which
offers the opportunity for formation of the intermediate oxidation
states Tc(VI) and Tc(V), there was no evidence of their presence in
these systems.
Implications.
The results of this research have implications
for the fate and transport of Tc in the subsurface, where anoxic
conditions may develop naturally or as a result of in situ stimulation
of microbial activity. In anoxic sediments, DMRB may play an important role in directly altering the form and mobility of Tc. However, the
complex chemistry of Tc under reducing conditions requires careful
consideration of both aqueous and solid-phase products resulting from
dissimilatory reduction, which in turn will be strongly dependent upon
the electron donor and solution composition. These studies suggest that
direct bacterial dissimilatory reduction will result in rapid removal
of Tc(VII) from groundwaters through the formation of low-solubility
Tc(IV) solids, which will control Tc solubility unless Tc(IV) is
stabilized in the aqueous phase by complexing ligands such as
carbonate. The formation of highly electronegative, soluble Tc(IV)
carbonate complexes indicates that it may be necessary to reassess
current concepts of Tc transport in anaerobic, carbonate-enriched
groundwaters, where Tc mobility has been considered to be controlled
largely by the low solubility of Tc(IV) hydrous oxide. With apparent
electronegativities greater than Tc(VII)O4,
which forms only weak surface complexes with hydroxylated surface sites
on Al and Fe oxides and clays and is only minimally retarded in oxic
groundwaters, the presence of these Tc species would have clear
implications for bioremediation strategies and suggests that transport
through anaerobic aquifers needs to be considered as a possible pathway
in assessing the Tc radiation dose to humans. Ultimately, prediction of
Tc behavior in anaerobic nonsulfidogenic groundwater systems will
require a fundamental understanding of the mechanisms for direct
(enzymatic) and indirect (e.g., biogenic Fe) Tc reduction and the
factors controlling the combined effects of these process. The complex
chemistry of Tc provides formidable challenges and the potential for
misinterpretation in defining the chemical speciation and stability of
Tc reduction products over a range of environmental conditions. A
combination of rigorous analytical and modeling approaches will
continue to be required to address these fundamental questions.
| |
ACKNOWLEDGMENTS |
This research was supported by the Natural and Accelerated
Bioremediation Research Program (NABIR), Office of Biological and Environmental Research, U.S. Department of Energy (DOE). Pacific Northwest National Laboratory is operated for the DOE by Battelle Memorial Institute under contract DE-AC06-76RLO 1830.
The insights provided by T. R. Garland on the chemistry of Tc in
these systems was invaluable. We appreciate the assistance of A. Dohnalkova with the high-resolution TEM analyses. The continued support
of F. J. Wobber is greatly appreciated. We also thank David Boone
of Portland State University for providing S. putrefaciens CN32 to us from the Subsurface Microbial Culture Collection,
supported through Florida State University by DOE grant no.
DE-FG05-90ER61039.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Environmental
Science Research Center, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352. Phone: (509) 376-5680. Fax: (509) 376-9650. E-mail: r.wildung{at}pnl.gov.
| |
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Abstract
Introduction
