Transcript ppt file

Uranium Reduction by Clostridia
A.J. Francis, Cleveland J. Dodge, and Jeffrey B. Gillow
Brookhaven National Laboratory
Annual ERSD PI Meeting
April 5, 2006
Outline
• Growth of Clostridium sp. under normal culture
conditions.
• Fate of metals and radionuclides in the presence of
Clostridia.
• Bioreduction of uranium associated with nitrate,
citrate, and lepidocrocite.
• Utilization of Clostridium sp. for immobilization of
uranium at the FRC Area 3 site.
Background
• The FRC groundwater and sediment contain significant
concentrations of U and Tc and are dominated by low pH, and
high nitrate and Al concentrations where dissimilatory metal
reducing bacterial activity may be limited.
• The presence of Clostridia in Area 3 at the FRC site has been
confirmed and their ability to reduce uranium under site
conditions will be determined.
• Although the phenomenon of uranium reduction by Clostridia
has been firmly established, the molecular mechanisms
underlying such a reaction are not very clear.
- we are exploring the hypothesis that U(VI) reduction occurs
through hydrogenases and other enzymes (Matin and Francis).
• Fundamental knowledge of metal reduction using Clostridia
will allow us to exploit naturally occurring processes to
attenuate radionuclide and metal contaminants in situ in the
subsurface.
Materials and Methods
Bacterial culture: Obligate anaerobic bacteria Clostridium sp.
(ATCC 53464) capable of utilizing glucose, and a citrate
degrading bacterium Clostridium sphenoides (ATCC 19403).
Bioreduction studies: Growth medium (pH 6.8) containing glucose
and inoculated with Clostridia and U-nitrate, U-citrate or Fe-Ucoprecipitate added at the start of the experiment.
Metal speciation: Uranium: oxidation state determined in solution
by KPA and UV-vis spectrophotometry, in solid phase by
XPS and XANES, molecular association by EXAFS.
Technetium: gel filtration chromatography, gamma-counting.
Plutonium: liquid scintillation counting, XANES.
Clostridium sp.
• Strict anaerobic, spore-forming,
fermentative bacteria.
• Metal reduction
Fe3+ to Fe2+
Mn4+ to Mn2+
Tc7+ to Tc4+
U6+ to U4+
• Mobilization/Immobilization
- U(VI)-nitrate(aq)  U(IV)s
- U(VI)-citrate(aq)  U(IV)-citrate(aq)
Cell size: 1 mm x 5 mm
Serum Bottles for Culturing Anaerobic Bacteria
Anaerobic growth medium is prereduced and autoclaved. The metal-containing
solution is added through the butyl rubber stopper using a needle and syringe.
Growth of Clostridium sp.
Change in pH and Glucose Consumption
During Growth of Clostridium sp.
7
3.0
pH
5
2.0
1.5
4
1.0
pH
3
0
0.50
0.0
10 20 30 40 50 60 70 80
Hours
Acid production (mM)
Glucose
Glucose (mM)
2.5
6
2
Production of Organic Acid Metabolites
by Clostridium sp.
700
600
Butyric acid
500
400
300
Acetic acid
200
100
0
0
10 20 30 40 50 60 70 80
Hours
Clostridium sp. rapidly metabolizes glucose to acetic and butyric acids which
lowers the pH of the medium. Equimolar amounts of carbon dioxide and
hydrogen are also produced.
Toxicity effects (e.g. Pb) can be minimized by addition of iron. (Francis and
Dodge. 1987. Arch. Env. Contam. Toxicol. 16:491-498).
Metal Reduction by Clostridium sp.
Bioreduction of 1:1 Fe(III)-citrate complex
by Clostridium sp.
0.6
1:1 Fe(III):citric acid (control)
Iron in solution (mM)
0.5
1:1 Fe(II):citric acid
0.4
0.3
0.2
1:1 Fe(III):citric acid
0.1
0
0
1
2
3
4 5
Hours
6
7
8
Ferric ion was reduced to ferrous form by the bacteria and remained in
solution as the 1:1 Fe(II):citric acid complex. (Francis and Dodge. 1993.
Appl. Env. Microbiol. 59:109-114).
Bioreduction of Pertechnetate by Clostridium sp.
Pertechnetate (uninoculated control)
100
Tc (%)
Reduced Tc
99m
80
60
40
Pertechnetate
20
00
1
2
3
4 5
Hours
6
7
8
Pertechnetate (NaTcO4) added to resting cells of Clostridium sp. was rapidly reduced to Tc(IV)
by bacterial activity. The reduced Tc was associated with the biomass and in solution
complexed with >5kDa metabolites. Tc(VII) added in the presence of citrate or DTPA formed
soluble Tc(IV)-organic complexes (Francis et al. 2002. Radiochim. Acta 90:791-797).
Dissolution of Pu Species by the Activity of Clostridium sp.
100
Plutonium in solution (%)
control (<0.03 um)
control (<0.4 um)
80
inoculated (<0.03 um)
inoculated (<0.4 um)
60
40
20
0
0
50
100
Hours
150
200
Pu(IV)-nitrate was added to the growth medium and initially precipitated
from solution. Anaerobic bacterial activity solubilized the Pu.
XANES Analysis of Pu Following Anaerobic
Bacterial Activity
Normalized absorbance
2.0
1.5
1.0
Pu(IV)-nitrate
Clostridium sp.
0.5
0.0
18000
18100
Energy (eV)
18200
Comparison of the absorption edge position for the bacterially treated sample at (18.059
keV) with that for Pu(IV)-nitrate (18.062 keV) confirms the presence of Pu3+.
Uranium Reduction by Clostridia
Uranium Speciation in the Presence of Clostridium sp.
inoculated
uninoculated
The uranyl ion has a characteristic yellow color (L), while the bacteriallytreated uranium (R) has a dark color.
XPS and XANES Analysis of Uranium Following
Anaerobic Bacterial Activity
XPS analysis of the uranyl nitrate treated sample shows a 1.6 eV decrease in binding
energy to 380.6 eV compared to uranyl ion (382.0 eV); XANES spectra at the MV
absorption edge shows shift in sample absorption peak to 3550.1 eV from 3551.1 eV
for U(VI). These complementary techniques confirm bacterial reduction of uranyl ion
to U(IV). Francis et al. 1994. Environ. Sci. Technol. 28:636-639.
Mechanisms of Uranium Reduction by Clostridium sp.
Treatment
U4+detected (%)
Control (no bacteria)
none detected
Synthetic spent medium (org. acids)
none detected
Cell free spent
medium (filtered)
- spent medium (filtered/autoclaved)
Cell free
1
none detected
Heat-killed
cells
2
Resting cells (no growth)
94
Growing cells
100
210 mM as uranyl acetate was added.
Samples were analyzed at 24 -h following addition of uranium.
Reduction of uranyl nitrate was observed only in the presence of cells. The
components of the growth medium and the metabolic acids did not reduce
uranium.
Anaerobic Bacterial Reduction of Uranium
Complexed With Citric Acid
Change in UV-vis
spectrum of U(VI)-citrate
following bioreduction
confirms it was reduced
to U(IV).
0
Clostridium sp. reduced U(VI) complexed to
citric acid only in the presence of carbon source.
The reduced U remained in solution associated
with the citric acid as the U(IV)-citrate complex.
XANES analysis of the
bacterial growth
medium at the LIII
absorption edge shows
a shift in energy to
17166 eV compared to
uranyl ion (17171 eV),
confirming reduction
of U(VI) to U(IV).
Francis et al., 2002. J. Nucl. Sci. Technol. 3:935-938.
Proposed Structure for U(IV)-Citrate Complex
U
U
bacteria
electron donor
U
EXAFS analysis indicates the binuclear U(VI)-citrate complex is transformed
to a mononuclear biligand complex following reduction of U(VI) to U(IV).
Bioreduction of Uranum-Lepidocrocite Coprecipitate
by Clostridium sp.
100
Metal in solution (%)
90
80
Fe2+
70
XPS
U4+
60
50
40
30
20
UO22+
10
0
0
24
48
72
96 120 144 168 192
396
Hours
388
380
372
364
Binding Energy (eV)
EXAFS analysis has shown uranium forms an inner-sphere complex with lepidocrocite .
O
Fe
U
O
Anaerobic bacterial activity solubilized the iron as ferrous form; however, uranium was
not solubilized due to formation of U4+. (Dodge et al. 2002. Environ. Sci. Technol.
36:3504-3511).
Uranium Reduction by Clostridium sp.
in Groundwater Collected at the FRC
Field Research Center
Methodology
Groundwater: Groundwater samples FW-024-000233 and FW-026000343 from Area 3 of FRC site were obtained from Dave Watson
and were characterized using potentiometric titration, ICP-OES, and
EXAFS.
Microbial experiments:
• The ability of indigenous bacteria to reduce U was tested on “as
received” and pH 6.5 adjusted groundwater.
• Uranium reduction by log-phase Clostridium sp. was determined in
the presence of groundwater, uranium, and aluminum.
• Growth was monitored by measuring optical density (600 nm), pH,
and glucose consumption.
• Uranium speciation was determined using KPA, XPS, XANES,
and EXAFS analysis.
Chemical Characterization of FRC Groundwater FW-024
Sample
pH
FRC data
3.7
Al
Ca
Mg
U
-------------------- ppm -------------------611
1260
201
52
“as received”
3.4
471
897
155
43.8
pH adjusted
6.5
9.9
834
148
<0.5
The pH of the groundwater was acidic and major metals were Al, Ca, Mg, and U.
Adjustment of the groundwater to pH 6.5 resulted in precipitation of Al and U. Ca
and Mg were only slightly affected.
Potentiometric Titration of FRC Groundwater
Potentiometric titration of FW-024 groundwater shows presence of a
buffer region between pH 4 and 5 due to formation of Al-hydroxide
species. A whitish precipitate is formed at pH 6.5.
Fourier transform magnitude
EXAFS Analysis of pH-Adjusted Groundwater
X(k)k
3
FW-026
Synthetic FW-026
2
4
6
8 10
-1
k(Å )
12
14
16
FW-026
Synthetic FW-026
0
1
2 3 4 5 6
Radial distance (Å)
7
8
Comparison of pH-adjusted groundwater with synthetically prepared Al-U
coprecipitate shows uranyl ion present as hydrated form and not associated as
an inner-sphere complex with Al.
Effect of Indigenous Bacterial Growth on
Uranium Reduction
0.25
0.19
Total U pH 3.4
U(VI)
Reduced U
A
0.13
0.06
0
ND
Indigenous
ND
Clostridia
Indigenous+C Clostridia+C
Treatment
Uranium in solution (mM)
Uranium in solution (mM)
0.25
0.19
Total U
U(VI)
Reduced U
pH 6.5
B
0.13
0.06
0
Indigenous
Clostridia
Indigenous+C Clostridia+C
Treatment
Uranium reduction at pH 3.5 was detected only in the presence of carbon source and
indigenous or Clostridium sp. However, in the pH adjusted samples the added
Clostridium sp. and glucose reduced 62% of the uranium to U(IV).
Effect of Aluminum Addition on Growth of Clostridium sp.
5.0
1.6
Glucose
2.0
Optical Density (600 nm)
pH
4.0
pH
Glucose consumption (mM)
3.0
1.0
0.0
3.0
0
5
10
15
Al added (mM)
20
2.0
0
5
10
15
Al added (mM)
20
Optical density
1.2
0.8
0.4
0.0
0
5
10
15
Al added (mM)
20
Addition of 1 to 20 mM aluminum to 12-hour old culture of Clostridium sp. inhibited
growth of bacteria. The inhibition was most notable at >2 mM Al addition. This may
be due to toxicity or low pH of the medium.
Uranium speciation (%)
Effect of Aluminum Addition on Uranium
Reduction by Clostridium sp.
U(VI)aq
U(IV)aq
U(IV)s
100
80
60
40
20
0
0
1
2
5
10
Aluminum added (mM)
20
Even though bacterial growth was inhibited, bacterial cells were able to reduce U(VI)
to U(IV). The partitioning of uranium into the solid phase with increased Al
concentration may be due adsorption to Al hydroxide colloids.
Effect of Addition of FRC Groundwater on
Growth of Clostridium sp.
1.2
7.0
0.9
pH
6.0
groundwater addition
5.0
groundwater addition
pH
Optical Density (600 nm)
Optical density
0.6
4.0
no addition
5%
10%
25%
0.3
0.0
0
4
8
12 16
Hours
20
no addition
5%
10%
25%
3.0
24
2.0
0
4
8
12 16
Hours
20
24
Addition of varying amount of FRC groundwater to 12 hour old culture of Clostridium
sp. affected the growth of the bacteria as indicated by a decrease in optical density and
pH with increasing groundwater addition.
Uranium speciation (%)
Speciation of Uranium Following Addition of
FRC Groundwater to Clostridium sp.
100
U(VI)aq
U(red)aq
80
60
0
U(VI)aq
U(red)aq
U(IV)s
40
20
U(VI)aq
U(IV)s
U(IV)s
5
10
25
FRC Groundwater (%)
A significant amount of uranium reduction to U(IV) was observed in
up to 25% FRC groundwater.
Effect of Addition of Uranyl Nitrate on
Growth of Clostridium sp.
1.2
1.9
1.3
0.6
0.0
0.0
0.2
0.4
0.6
0.8
Uranium added (mM)
1.0
Optical density
Absorbance at 600 nm
Glucose
Optical Density (600 nm)
Glucose consumption (mM)
2.5
0.9
U addition
0.6
no U
0.10 mM U
0.25 mM U
0.50 mM U
0.3
0.0 0
4
8
12
16
20
24
Hours
Addition of uranyl nitrate to 12 hour old culture of Clostridium sp. affected the growth
as shown by decrease in optical density and glucose consumption.
Effect of Uranyl Nitrate on Uranium Reduction
by Clostridium sp.
U(VI)
aq
U(reduced)
aq
U(reduced)
s
100
80
60
40
20
0
0.1
0.25
0.5
1
Uranyl nitrate added (mM)
Although bacterial growth was inhibited, uranium reduction to U(IV) from U(VI)-nitrate
occurred in the presence of up to 1.0 mM uranyl nitrate. The reduced uranium was present
predominantly in the solid phase.
Summary
Pure culture studies:
• Clostridium sp. and Clostridium sphenoides reduced U(VI) to
U(IV), Fe(III) to Fe(II), Tc(VII) to Tc(IV), and Pu(IV) to
Pu(III).
• Uranium was reduced only in the presence of actively growing
bacterial cells indicating a direct mechanism is involved.
FRC:
• In groundwater experiments slight reduction of U (3%) was
observed at pH 3.4. At higher pH indigenous bacteria plus
glucose reduced 6% of the U, while addition of Clostridium sp.
plus glucose resulted in 62% uranium reduction.
• The primary mechanism of uranium reduction by Clostridium
sp. in FRC groundwater appears to be the result of direct
enzymatic activity.
Acknowledgements
National Synchrotron Light Source
Beamlines X11A, X19A, X23A2
SUNY at Stony Brook
Dr. Gary Halada, Materials Sciences
Department of Energy
Environmental Remediation Sciences Division
Office of Biological and Environmental Research
Office of Science