# Presentation: 522973

## 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: _[palumboav@ornl.gov](mailto:palumboav@ornl.gov)_
- 2University of Tennessee, Knoxville, TN, USA
- 3Florida State University, Tallahassee, FL, USA

## 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.

## 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).**

## 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?**

## 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 |

## 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.**

## 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.**

## 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 |

## 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 1⁄2 substrate level**
**plus methanol and humics**

## 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 microcosms**

**Glucose (top) and Methanol Microcosms**

## 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**

## 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.**

## 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.**

## 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).

**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

## 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**

## 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)**

## 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**

## Exp. 4 – Nitrate Results

**Nitrate reduction starts without a long lag**
**Reduction slowest for methanol**
**All complete by 15 days**
**No difference with humics**

## 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**

## 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)

## 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% |

## 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)**

## Exp. 4 – Community Structure by T-RFLP

**Breaks into two major groups**
**Ethanol above**
**Glucose below**

## Exp. 4 – Community T-RFLP Results

**Ethanol**
**Actinos dominate**

**Glucose**
**More ****α**** and ****β**** Proteobacteria**

## 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.

## 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.**

## 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.**

## 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**

## 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.**

## 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.**

## 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. **

## Exp. 4 – Sulfate Results

**Sulfate reduction lags behind nitrate and U reduction**
**Very slow response for methanol**
**No detectable difference with Ethanol + Humic**

## 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.**

***Bars labeled with the same letter are not significantly different than each other**

## 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).**