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Reduction Processes and Community Structure in Remediation of Uranium. Anthony V. Palumbo 1 , Craig C. Brandt 1, Susan M. Pfiffner 2 , Lisa A. Fagan 1 , Andrew S. Madden 1 , Tommy Phelps 1 , Jack C. Schryver 1 Meghan S. McNeilly 1 , - PowerPoint PPT Presentation
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1
OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Reduction Processes and Community Structure in Remediation of Uranium
Anthony V. Palumbo1, Craig C. Brandt1,
Susan M. Pfiffner2, Lisa A. Fagan1, Andrew S. Madden1, Tommy Phelps1,
Jack C. Schryver1 Meghan S. McNeilly1, Chris W. Schadt1 Jana R. Tarver1, Sara Bottomly2,Heath J. Mills3, Denise M. Akob3, Joel E. Kostka3
1Oak Ridge National Laboratory, E-mail: [email protected] of Tennessee, Knoxville, TN, USA
3Florida State University, Tallahassee, FL, USA
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Background
The relationships among microbial community structure, geochemistry, and metal reduction rates in subsurface sediments may be critical in the remediation of metal contaminated environments.
Many microorganisms can change the geochemical conditions so metal reduction becomes an energetically favored reaction while some microbes can directly catalyze the necessary reactions.
In the second case the composition of the community may be important but in the first it is not.
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Research Questions
Does microbial community structure affect uranium reduction rates? Are there donor-specific effects that lead to enrichment of
specific community members that then impose limits on the functional capabilities of the system?
Is the metabolic diversity of the in situ microbial community sufficiently large and redundant that bioimmobilization of uranium will occur regardless of the type of electron donor added to the system?
Other questions are addressed in the project but not in this presentation (e.g., humics, resource ratio – P).
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Goal
The overall goal is to improve our understanding of the relationships between microbial community structure, geochemistry, and metal (uranium) reduction rates.
Is uranium reduction more like hydrocarbon degradation or chlorinated solvent degradation?
Uranium
Reduction
Rate
Community
Structure
Geochemistry Addition of Humics
Addition of Different Electron Donors
Addition of Phosphate (alter C:P)
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Approach We are using triplicate laboratory microcosms for each treatment
(ph, substrate, etc) containing sediment and groundwater to address the questions.
Sediments samples were homogenized under anaerobic conditions prior to use in the microcosms.
Each microcosm used 20 g of sediment and 80 mL of groundwater from a uranium-contaminated field site (U distributes between).
Carbon substrate concentrations were adjusted to give equivalent electron donor potential.
Control (e.g,) Treatment
VI 4% water VI
IV VI 96% sediment IV VI
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
FRC Site
Samples were collected from the Environmental Remediation Sciences Program (ERSP) Field Research Center (FRC) located at Oak Ridge National Laboratory.
The site is adjacent to a former disposal pond that has been filled and is now a parking lot.
The FRC is contaminated with uranium and has high levels of nitrate and an acidic pH due to disposal of nitric acid cleaning solutions.
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Experimental Setup
The pH was adjusted using sodium bicarbonate.
Unamended controls were included in each experiment.
Microcosms were incubated in an anerobic glove bag for the smaller experiments (e.g., 15 microcosms).
In the third experiment (96 microcosms) the incubation was on the lab bench.
Glove Bag in Field Trailer
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Electron Donors Used to Influence Community Structure
Donor Formula e- Predominantly Utilized by Exp
Acetate C2H3O2 8 FeRB/acetogenic methanogens 3
Lactate C3H6O3 12 SRB/FeRB 3
Pyruvate C3H4O 10 SRB/FeRB 3
Methanol CH4O3 6 Acetogens/methanogens 1,2,3,4
Ethanol C2H6O 12 SRB/FeRB 1,2,3,4
Glycerol C3H8O3 14 Clostridia/gram positive anaerobes 3
Glucose C6H12O6 24 Clostridia/other heterotrophs 1,2,3,4
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Experiments Exp. 1 – Three electron donors & control
archived sediments and fresh groundwater methanol (20 mM), ethanol (10 mM), glucose (5 mM) and control
(no added substrate) – data not shown
Exp. 2 – Three electron donors & control fresh sediment and groundwater same donors as Exp. 1 but at twice the concentration (methanol
[40 mM], ethanol [20 mM], and glucose [10 mM]) and a control
Exp. 3 – Full factorial (next slide)
Exp. 4 – Three electron donors & humic & control methanol (20 mM), ethanol (10 mM), glucose (5 mM), ethanol plus
humic and control (no added substrate) Exp. 5. Same as 4 at ½ substrate level
plus methanol and humics
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 3 – Full Factorial Experiment
Similar design to Exp. 2 except we used a full factorial design with pH and substrate.
7 carbon substrates (and a control) Methanol (40 mM) Ethanol (20 mM) Glucose (10 mM) Acetate (30 mM) Lactate (20 mM) Pyruvate (24 mM) Glycerol (17 mM) Control (no added electron donor)
4 pHs (5.5, 6.0, 6.5, 7.0)
3 reps/treatment = 96 microcosmsGlucose (top) and Methanol Microcosms
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 2 – Nitrate and U Results
No lag times in nitrate reduction with fresh sediments.
No uranium reduction with methanol.
Glucose and ethanol (not shown) exhibited both uranium and nitrate reduction
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Nitrate and Uranium Reduction Rates
Fastest rates of U reduction with glucose.
Substantial nitrate and U reduction with ethanol.
Nitrate but no U reduction with methanol.
Electron Donor
Methanol Ethanol Glucose
U r
ate
(pp
m/d
ay)
-0.25
-0.20
-0.15
-0.10
-0.05
0.00
Nitr
ate
(p
pm
/da
y)
-120
-100
-80
-60
-40
-20
0
U Rate N Rate
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 2 – Community Structure by Hierarchical Cluster Analysis of PLFA Data
Two major clusters: (1) high U red rate (2) no U reduction
Control 2 and the fresh sediment are very different lower biomass
Control Fresh Ethanol Glucose Methanol Control
(1) High U Red. (2) No U Red.
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Treatments tend to be similar.
One control (2) is consistently different than the other two controls.
High U reduction treatments (ethanol and glucose) separate from control and methanol (also by cluster analysis).
No U Reduction
Exp. 2 – Community Structure by Principle Components Analysis of PLFA Data
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Stress in Methanol and Control Treatments
PLFA biomarkers indicate nutritional stress in the methanol treatment and the control treatments was indicated by the cyclopropyl to monounsaturated fatty acid ratio
There also appears to be a potential toxicity stress in the methanol treatment indicated by the trans/cis ratio of monounsaturated fatty acids
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 3 – Nitrate Results (averaged over pH)
Results consistent with earlier studies Nitrate reduction is rapid Differences among
substrates are small Methanol lags Glucose, ethanol, lactate
rapid
Minimal to no effect of pH (data not shown)
No uranium loss in control, methanol, or pyruvate (actual increases observed)
Time (days)
0 10 20 30 40 50 60
U (
mg
/l)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
ControlMethanol Ethanol Glucose Lactate Acetate PyruvateGlycerol
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 3 – Community Structure by PLFA
There are community differences among treatments – methanol and pyruvate cluster and don’t reduce uranium
High U Red
High U Red
Low U Red
Low U Red
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 4 – Nitrate Results
Nitrate reduction starts without a long lag
Reduction slowest for methanol
All complete by 15 days
No difference with humics
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 4 – Uranium and Sulfate
Uranium reduction lags behind nitrate reduction
Sulfate reduction lags U reduction
No uranium reduction seen for methanol
Very slow sulfate reduction with methanol
No detectable difference with ethanol + humic
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Uranium Valence by X-ray Absorption Spectroscopy of Sediments from Microcosoms Kelly and Kemner
(Adv. Photon Source at ANL) working with A. Madden
Glucose end point 83 ±8 % U(VI) 17 ±8 % U(IV)
Ethanol end point 96 ±4 % U(VI) 4 ±4 % U(IV)
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
U in solution (all) plus some in sediment is reduced
Average total solid phase U ~96 ppm
Microcosms – 4:1 liquid:solid; initially ~1.5 ppm U(aq)
Partial reduction in sediments is consistent with literature (e.g., Ortiz-Benard et al. 2004 AEM) and results reported for FRC experiments
17% for glucose 4% for ethanol – need more replicates
Moon et al. JEQ(in press)
Control Treatment
VI 6%
water VI .0%
IV 0%
VI 94%
sediment IV 17%
VI 83%
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Donor Consumption and Metabolite Formation
Primary electron donor is consumed quickly
Acetate tends to accumulate over time and persist till end of experiment
Acetate is present in high concentrations at the FRC site (S. Brooks)
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 4 – Community Structure by T-RFLP
Breaks into two major groups
Ethanol above
Glucose below
Ethanol
Glucose
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 4 – Community T-RFLP Results
EthanolActinos dominate
GlucoseMore α and β Proteobacteria
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Research Questions
Are there donor specific effects that lead to enrichment of specific community members that then impose limits on the functional capabilities of the system?
Yes – methanol (and pyruvate?) imposes limits.
Is the metabolic diversity of the in situ microbial community sufficiently large and redundant that bioimmobilization of uranium will occur regardless of the type of electron donor added to the system?
There is enough metabolic diversity to accommodate many different electron donors (e.g., glucose, ethanol, glycerol, acetate) for U reduction but perhaps not all.
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Summary Consistent results in the experiments indicating:
all substrates promoted nitrate reduction, methanol (and pyruvate) did not promote U reduction but glucose
and ethanol promoted rapid U reduction, PLFA indicated different communities with methanol T-RFLP indicated distinct differences among communities even in
treatments that promoted U reduction there appear to be limitations imposed on the community related
to some substrates (e.g. methanol).
Limited pH effects Donor levels critical (Exp. 5 data not shown) Further data and analysis of the community structure is on
going (e.g. functional gene arrays, T-RFLP, clone libraries) Additional studies will take place with glucose, ethanol, and
methanol with humics and different C/P ratios.
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Acknowledgements
This research is funded by the Environmental Remediation Science program (ERSP), Biological and Environmental Research (BER), U.S. Department of Energy.
Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy under contract DE-AC05-00OR22725.
We thank the organizers of the meeting for the opportunity to present this ongoing work.
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Maximum Loss of U Related to Substrate
Ethanol Lactate and Glucose achieve relative high rates of U reduction (and N).
Little or no reduction in control, methanol, pyruvate.
Results similar across experiments.
*Bars labeled with the same letter are not significantly different than each other
Substrate
Lo
ss o
f U
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Delta Max to 35 Delta Max to 42
AA
A A
AA
A A A
C
BB B
B
B
BC CC
C
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
U over time (averaged over pH) Increases could be
related to kinetic effects on
equilibrium in slurries, reoxidation due to
nitrate, leakage of air into
microcosms. No loss in control or
methanol. Pyruvate starts very
high and continues to increase (data not shown).
Next experiment will be incubated in anaerobic chamber and with better stoppers. Time (days)
0 10 20 30 40 50 60
U (
mg
/l)
0
1
2
3
4
5
ControlMethanol Ethanol Glucose Lacatate Acetate Glycerol
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Analytical Methods
Nitrate was measured spectrophotometrically on diluted samples using Szechrome reagents (Polysciences) in Experiment 1 and the HACH method in the second and third experiments.
A Chemchek KPA (kinetic phosphorescence analyzer) was used to measure the uranium in diluted samples from both experiments.
Measurements of pH were made with a small electrode on 1 ml samples from the microcosms.
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Rate Calculations, Statistics, and Community Structure
Reduction rates were calculated from the linear portions of the plots of loss of nitrate and uranium from solution.
SAS was used for ANOVA and PCA. We made limited measurements of
community structure at the final time point of experiment 2 using membrane lipid techniques.
Other community analysis is ongoing.
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 4 – Sulfate Results
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 5 10 15 20 25 30 35 40
Time (days)
Sul
fate
(tre
at/c
ontr
ol)
Methanol
Ethanol
Glucose
Ethanol + Humics
Sulfate reduction lags behind nitrate and U reduction
Very slow response for methanol
No detectable difference with Ethanol + Humic
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Two Views of U Loss Related to pH
Some pH effect especially between 7 and 5.
Some interactions due to differences among substrates in potential for reduction.
pH
7 6.5 6 5
Lo
ss o
f U
Bet
wee
nD
ay 7
an
d D
ay 4
2 (%
)
-200
-100
0
100
200
Lo
ss o
f U
Bet
wee
n D
ay0
and
42
(mg
/L)
-2
-1
0
1
2
Delta 7 42 Delta 0 42
AA
BB
B
B
BB
*Bars labeled with the same letter are not significantly different than each other
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OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY
Exp. 1 – Nitrate Results Ethanol
resulted in faster nitrate reduction and shorter lag time than did glucose and methanol additions.
No U reduction was evident in the methanol treatments (data not shown).