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Subsurface Biogeochemical Research Program

Performance Measures and Milestones: FY2005

GPRA Goal/Annual Target (SC GG 5.21.1)
Determine scalability of laboratory results in field experiments - Conduct two sets of field experiments to evaluate biological reduction of chromium and uranium by microorganisms and compare the results to laboratory studies to understand the long term fate and transport of these elements in field settings.
1st Quarter Measures Conduct monitoring at Old Rifle UMTRA experimental site and collect data on the bioreduction of uranium.
1st Quarter Results From: Long, Philip E [mailto:philip.long@pnl.gov]
Sent: Tuesday, January 11, 2005 12:35 PM
To: Kuperberg, Michael
Cc: Bayer, Paul; Anderson, Todd
Subject: ESRD First Quarter FY-2005 Performance Measure

To: Dr. J. Michael Kuperberg, Acting Director
Environmental Remediation Sciences Division
Office of Biological and Environmental Research
SC-75/Germantown Building
U.S. Department of Energy
1000 Independence Avenue, SW
Washington, D.C. 20585-1290

Subject: ERSD (now CESD) FY05 First Quarter Performance Measure

Dear Dr. Kuperberg,

In the first quarter of 2005, sampling of groundwater was conducted at an experimental field site in Old Rifle, Colorado, and samples were analyzed for U concentrations. ERSD (now CESD) FY05 First Quarter Performance Measure entitled "Conduct monitoring at Old Rifle UMTRA experimental site and collect data on the bioreduction of uranium" was successfully met by this activity.

Monitoring (sampling) of groundwater wells was done as a follow up to an in situ acetate amendment experiment designed to stimulate growth of metal-reducing bacteria such as Geobacter. Uranium concentrations in groundwater down gradient decreased in a fashion similar to a 2002 field experiment except that the 2004 field experiment was terminated prior to development of extensive sulfate reduction. The decrease in U(VI) paralleled dominance of Geobacter in groundwater samples, strongly indicating that Geobacter is responsible for enzymatic reduction of U(VI) in situ (based on research of Derek Lovley et al. in progress). Monitoring of the minigallery in the first quarter of FY-2005 shows that U(VI) rebounded by an average of 47% and a maximum of 78%. This result is interpreted to reflect the short duration of the 2004 acetate amendment (~1 month), producing less total biomass than longer experiments. Microbially mediated U(VI) reduction thus may not be sustained post-acetate injection as appears to be the case after longer experiments.

For additional information on this performance measure, please see http://www.pnl.gov/nabir-umtra/monitor.stm

PRINCIPAL INVESTIGATOR:
Philip E. Long
Phone: 509 372-6090 FAX: 509 372-6089
Pacific Northwest National Laboratory
Mail Stop K9-33; P.O. Box 999, Richland, WA 99352
E-mail:philip.long@pnl.gov

COLLABORATORS: Derek R. Lovley(1), Kelly Nevin(1), Regina O'Neil(1), C. T. Resch(2), Aaron Peacock(3), Helen Vrionis(1), Yun-Juan Chang(3), Dick Dayvault(4), Irene Ortiz-Bernad(1), Ken Williams(5), Susan Hubbard(5), Steve Yabusaki(2), Yilin Fang(2), and D. C. White(2)

1: University of Massachusetts, Amherst, MA; 2: Pacific Northwest National Laboratory, Richland, WA; 3: University of Tennessee, Knoxville, TN; 4: S. M. Stoller Corporation, U.S. Department of Energy, Grand Junction, CO; 5: Lawrence Berkeley National Laboratory, Berkeley, CA.
2nd Quarter Measures Conduct Hydrogen Releasing Compound (HRC) injection monitoring at Hanford chromium contamination site and collect data on the bioreduction of chromium.
2nd Quarter Results From: Terry C. Hazen <tchazen@lbl.gov>
Date: March 14, 2005 4:58:27 PM PST
To: Kuperberg Mike <mkupe@mailer.fsu.edu>, Bayer Paul <PAUL.BAYER@science.doe.gov>
Cc: Faybishenko Boris A <BAFaybishenko@lbl.gov>
Subject: 2005 Milestone Summary for Hanford 100H Field StudiesDear Dr. Kuperberg,

Below you will find the most recent summary of results from our Field Studies at Hanford 100H. Details, including figures, papers, and data can be found at the project website: http://www-esd.lbl.gov/ERT/hanford100h/index.html

Please let us know if you need an further information.

1. Overall Objective and Hypothesis
Overall Objective: To carry out field investigations to assess the potential for immobilizing and detoxifying chromium-contaminated groundwater using lactate-stimulated bioreduction of Cr(VI) to Cr(III) at the Hanford 100H site.

Hypothesis: Lactate (Hydrogen Release Compound—HRCTM) injection into chromium contaminated groundwater through an injection well will cause bioreduction of chromate [Cr(VI)] and precipitation of insoluble species of [Cr(III)] on soil particles, probably catalyzed at oxide surfaces, at the field scale.

2. Types of Investigations Performed
We have conducted a series of bench-scale and field-scale integrated treatability studies, including the following types of investigations:

1. Pilot field-scale biostimulation of the groundwater was conducted, using injection of 40 lbs of 13C-labeled HRC into the injection Well 699-96-45 (followed by Br-tracer injection) over the depth interval from 44 ft to 50 ft within the Hanford formation. Pumping from the monitoring well 699-96-44 started immediately after the injection on August 3, 2004, and continued for 27 days.

5. Pre- and post-HRC injection groundwater sampling was performed from 5 water samplers in each borehole. During pumping, samples of pumped water were collected and on-site measurements using the Hydrolab (DO, pH, Redox potential, electrical conductivity, and temperature) were performed. Hydrolab measurements were also conducted after the pumping was ceased.

6. Groundwater sampling was conducted initially weekly and then monthly.

7. Microbial analyses of water samples included: Acridine orange direct counts and molecular analyses—PLFA, 16S GeneChi, and Clone library, and qPCR.

8. Analytical analyses of anions in water samples included analyses of: bromide (tracer added to the injection well), chloride and phosphate (added to HRC), acetate (byproduct of HRC microbial metabolism, nitrate and sulfate (present in groundwater under background conditions

9. Analytical analyses of cations in water samples included the determination of Cr(VI), total Cr, and Fe(II) and total Fe.

10. Water samples were analyzed to determine carbon, nitrate, and oxygen isotopic compositions.

3. Main Results
We have investigated coupled hydraulic, geochemical, and microbial conditions, which are necessary to maximize the extent of Cr(VI) bioreduction and minimize the Cr(III) reoxidation in groundwater.

1. Pilot field-scale biostimulation of the groundwater shows microbial cell counts reached the maximum of 2×107 cells g-1 13-17 days after the injection. The HRC injection generated highly reducing conditions: DO dropped from 8.2 to 0.35 mg/l, Redox Potential—from 240 to -130 mV, and pH—from 8.9 to 6.5.

2. After pumping was ceased (under conditions of natural regional groundwater flow): DO, Redox, and pH began to recover to background values. High biomass in groundwater lasted for 2 months and then decreased to values even less than those under pre-HRC-injection conditions. PLFA and direct counts both indicated similar biomass changes; however, the PLFA also indicated an increase during the last 2 months at one depth in Well 45. Carbon isotope ratios of DIC decreased, but remain above background in Well 699-96-44 and within the injection interval in Well 699-96-45 until December 2004.

3. No measurable methane was detected in samples tested. No methanogens were detected by 16s rDNA or by PLFA.

4. PLFA indicated low microbial diversity under background conditions, which increased after injection and continued to increase for the first 6 weeks, followed by the decrease in the microbial diversity. A similar pattern was observed using the 16s rDNA chip analyses.

5. The isotopic composition of nitrate is consistent with that of natural background sources (not agricultural origin) with minor modification due to biodegradation. Low oxygen isotope ratios may indicate high concentrations of nitrite.

6. Geophysical investigations show that HRC products injected into groundwater can be detected using radar and seismic survey. High spatial resolution data are being used to illustrate the distribution of the HRC between the wells over time. One of the unresolved problems is the effect of changes in metal concentration on electrical conductivity. At different times, EM, radar, and seismic were sensitive to subsurface changes caused by HRC injection.

7. 13C ratios in dissolved inorganic carbon confirmed microbial metabolism of HRC. 13C ratios remain elevated (above background values) after 6 months. Increase in carbon isotope ratios of DIC in Well 44 are coincident with increases in bromide, chloride and acetate and decreases in nitrate. Chloride was determined to be from the HRC.

8. Hydrogen sulfide production was first observed after about 20 days post-injection, which corresponds with the enrichment of a Desulfovibrio species (sulfate reducer) identified using 16s rDNA microarray and monitored by direct fluorescent antibodies. DO and nitrate began to return to background concentrations two months after HRC injection, despite bacterial densities remaining high (>107 cells/ml).

9. Cr(VI) concentrations in the monitoring and pumping wells decreased significantly and remained below up-gradient concentrations even after 6 months, when reducing conditions and microbial densities had returned to background concentrations and density.
From: "Enloe, Sonia Y" <sonia.enloe@pnl.gov>
Date: April 15, 2005 4:38:57 PM PDT
To: michael.kuperberg@science.doe.gov
Cc: paul.bayer@science.doe.gov, SASeybold@lbl.gov, "Fredrickson, Jim K" <Jim.Fredrickson@pnl.gov>, todd.anderson@science.doe.gov, TCHazen@lbl.gov, "Zachara, John M" <john.zachara@pnl.gov>, "Enloe, Sonia Y" <sonia.enloe@pnl.gov>, "Ray, Douglas" <doug.ray@pnl.gov>, "Bolton, Harvey Jr" <harvey.bolton@pnl.gov>
Subject: ERSD (now CESD) FY05 Second Quarter Performance Measure for Bioreduction of Uranium

Dear Dr. Kuperberg:

In this note Dr. Jim Fredrickson and I summarize recent PNNL laboratory research on the bioreduction of uranium by a metal-reducing bacterium. Experimental procedures, measurements, and experimental observations that support the conclusions noted herein are presented in a poster that is posted on the NABIR web site. A publication is currently in preparation based on these findings. This work will be presented at the 2005 Annual NABIR Principal Investigators Meeting to be held April 18-20, 2005 in Warrenton, Virginia. We find these results to be fascinating and significant. Please let us know if you need or desire additional information on the research or its implications to subsurface uranium migration and remediation.

1. Overall Objective and Hypothesis
Overall Research Objective: To perform laboratory investigations on intra- and extra-cellular microbiologic mechanisms of uranium reduction, and to characterize the effect of these mechanisms on the localization point (periplasm, cell envelope, or bathing electrolyte), properties (e.g., size, aggregation state, protein content, etc.), and reactivity (e.g., oxidation rate) of biogenic uranite (UO2(s)). This objective provides basic scientific information in support of bioremediation concepts to eliminate mobile uranium from groundwater.

Background: Shewanella oneidensis MR-1 reduces a wide range of metals and radionuclides and produces many periplasmic and outer membrane associated c-type cytochromes that are believed to facilitate the transfer of electrons to metal ions external to the cell including solid metal oxides. Depending on condition, bioreduced U(IV) can accumulate in the MR-1 periplasm, on the cell envelope, or within the bathing electrolyte/media. The mechanisms controlling the localization point of biogenic (UO2(s)) are unclear. Genomic analysis of MR-1 has revealed that this organism possesses a functional type II protein secretion pathway (T2S) that appears to be involved in the proper localization of certain cytochromes and possibly in the localization of other proteins involved in the reduction of U(VI) and translocation of periplasmic (UO2(s)) nanoparticles to the cell surface and beyond.

Hypothesis: The T2S system: i.) facilitates uranium(IV) nanoparticle export across the outer membrane, and ii.) correctly localizes U(VI) reducing proteins (c-type cytochromes) in the outer membrane.

Methodology: To characterize the role of c-type cytochromes and the T2S in the reduction of U(VI), a series of cytochrome gene deletion and insertional mutants interrupting critical genes in the T2S pathway were constructed and characterized for phenotypic differences when using uranium as the terminal electron acceptor. Bioreduction/localization experiments were performed in batch laboratory incubations under anaerobic conditions with uranyl acetate as the uranium form. Transmission electron microscopy (TEM) with selected area diffraction (SAED) and synchrotron x-ray fluorescence microscopy were applied to study the localization, and crystallochemical character of the (UO2(s)).

2. Important Findings
A. The reduction of U(VI) by MR-1 results in extracellular accumulation of crystalline UO2(s) nanoparticles (≈5 nm), with some in association with fiber-like biostructures.

B. The T2S system is not essential for the reduction of U(VI) by MR-1, but is necessary for the extracellular localization of reduced UO2(s) nanoparticles. Mutants in the T2S pathway (gspD- or gspG-) accumulated UO2(s) in the periplasm and at the outer membrane surface. The T2S system appears to eliminate periplasmic UO2(s), thereby eliminating potential deleterious effects of its accumulation.

C. U(VI) reduction was abolished by an insertion in the c-type cytochrome maturation pathway gene ccmC, indicating a functional role for one or more c-type cytochromes in U(VI) reduction.

D. The extracellular reduction of U(VI) by MR-1 is facilitated by several c-type cytochromes (MtrA, MtrC, and OmcA) and a related membrane protein (MtrB), while other mtr deletions (mtrD-, mtrE-, or mtrF-) had little effect on U(VI) reduction.

E. In MR-1, extracellular co-localization of U with Fe and P was present on extended fiber-like biostructures; the composition of the biostructures and their high reactivity for U implied that they were lipid-containing features (e.g., lipid bilayers, membrane extensions) that contained c-type cytochromes. In contrast, only periplasmic UO2(s) was observed in MtrC-/OmcA- cells.

3. Implications
Active uranium(IV) reduction occurs within the periplasm and on extended cytochrome-containing biostructures with MR-1. These points of enzymatic reduction regulate the size of the uranite precipitates to a narrow range (e.g., 3-7 nm), for reasons as yet unknown. The T2S system appears responsible for extracellular transport of periplasmic UO2(s), as a possible detoxification response, and/or localization of U(VI)-reducing cytochromes to the outer surfaces of the cells. The association of nano-crystalline UO2(s) with the extracellular biostructures may have an important influence on their long term stability and transport in the environment. Preliminary studies indicate that this form of uranite is particularly resistant to oxidation, a beneficial attribute for bioremediation.

John M. Zachara, Ph.D.
Sr. Chief Scientist for Environmental Chemistry
Fundamental Sciences Directorate
Pacific Northwest National Laboratory
PO Box 999; MS K8-96
Richland, WA 99352
E-Mail: john.zachara@pnl.gov
Phone: (509) 376-3254
Fax: (509) 376-3650
3rd Quarter Measures Report results of Old Rifle field experiments and compare to previous laboratory studies.
3rd Quarter Results From: Long, Philip E [mailto:philip.long@pnl.gov]
Sent: Wednesday, June 29, 2005 1:43 AM
To: Kuperberg, Michael
Cc: Bayer, Paul; Anderson, Todd
Subject: ESRD Third Quarter FY-2005 Performance Measure

To: Dr. J. Michael Kuperberg
Acting Director
Environmental Remediation Sciences Division
Office of Biological and Environmental Research
SC-75/Germantown Building
U.S. Department of Energy
1000 Independence Avenue, SW
Washington, D.C. 20585-1290

Subject: ERSD (now CESD) FY05 Third Quarter Performance Measure

Dear Dr. Kuperberg,

In the third quarter of 2005, sampling and analysis of groundwater continued at the Old Rifle field site in Rifle, CO. Evaluation and publication of results is ongoing (see publication list, http://www.pnl.gov/nabir-umtra/pubs.stm). ERSD (now CESD) FY05 Third Quarter Performance Measure entitled "Report results of Old Rifle field experiments and compare to previous laboratory studies" was successfully met by these activities.

Recent analyses and data include

1) U(VI) removal rates for field experiments and laboratory incubations 2) Observation of dissolved oxygen (DO) stratification in the Old Rifle alluvial sediments during spring runoff and its relationship to increases in U(VI) concentration 3) Progress in reactive transport modeling that accounts for bioreduction of U(VI), mineral equilibria, and groundwater flux and dispersion, including the concept of adapting the reactive transport model to modeling of bottle incubations.

Overall, the results show that removal of U(VI) in field experiments is approximately an order of magnitude slower than in bottle incubations. This is expected since bottle incubations are closed systems and field experiments are open systems with flowing groundwater. The difference in U(VI) removal rates underscores the importance of field experiments for measuring key in situ rate parameters in systems undergoing bioreduction of variable redox contaminants. Monitoring of U concentrations during high spring runoff at the Rifle site has now shown a positive corrleation between rising, oxygenated groundwater and U(VI) concentration. We anticipate using lab-scale experiments to constrain the mechanistic processes involved in a way that will enable us to better predict the behavior of bioreduced U(IV) under oxidizing conditions. Finally, reactive transport modeling is beginning to match observed field data. As this research progresses we will incorporate all the significant hydrologic, geochemical, and microbial processes into the model, fully rationalizing field and laboratory experimental results.

For the full report on this performance measure, please see http://www.pnl.gov/nabir-umtra/monitor.stm

PRINCIPAL INVESTIGATOR:
Philip E. Long
Phone: 509 372-6090
FAX: 509 372-6089
Pacific Northwest National Laboratory
Mail Stop K9-33; P.O. Box 999, Richland, WA 99352
E-mail:philip.long@pnl.gov

COLLABORATORS: Derek R. Lovley(1), Kelly Nevin(1), Regina O'Neil(1), C. T. Resch(2), Aaron Peacock(3), Helen Vrionis(1), Yun-Juan Chang(3), Dick Dayvault(4), Irene Ortiz-Bernad(1), Ken Williams(5), Susan Hubbard(5), Steve Yabusaki(2), Yilin Fang(2), and D. C. White(2)

1: University of Massachusetts, Amherst, MA; 2: Pacific Northwest National Laboratory, Richland, WA; 3: University of Tennessee, Knoxville, TN; 4: S. M. Stoller Corporation, U.S. Department of Energy, Grand Junction, CO; 5: Lawrence Berkeley National Laboratory, Berkeley, CA.
4th Quarter Measures Report results of Hanford field experiments and compared to previous laboratory studies.
4th Quarter Results From: Hazen Terry C. [mailto:TCHazen@lbl.gov]
Sent: Friday, September 16, 2005 8:26 PM
To: Kuperberg, Michael
Cc: Bayer, Paul; Anderson, Todd; Faybishenko Boris A
Subject: 4th Quarter Performance measure

Dear Dr. Kuperberg,

The HRC injection done more than a year ago is still showing that it is stimulating bioactivity in keeping Cr(IV) at undetectable levels. Our most recent data shows that the bioactivity that we are seeing is from the 13C labeled lactate that we originally injected. The summary report for this quarter follows.

Kind Regards,

Terry

Terry C. Hazen, Ph. D.
Senior Staff Scientist
Head, Ecology Department
Head, Center for Environmental Biotechnology
Co-Director, Virtual Institute Microbial Stress and Survival
Earth Sciences Division, Lawrence Berkeley National Laboratory, University of California
MS70A-3317, One Cyclotron Rd. Berkeley, CA 94720
Phone: 510-486-6223, Cell: 707-631-6763
Email: TCHazen@lbl.gov URL: www-esd.lbl.gov/ECO

Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to
Cr(III) at the Hanford 100-H Area

4th Quarter FY05 - summary report

- In comparison to previous laboratory studies the electron donor (HRC) has persisted longer then expected. In the laboratory the HRC was depleted in a matter of weeks, while in the field it has persisted more than 12 months. This has kept microbial activity, especially in the less permeable area higher and Cr(VI) at undetectable levels. Previous laboratory tests also showed that the reduced Cr would stay reduced for long periods of time with only minor reoxidation, this trend is also being observed in the field.

- Field investigations conducted during the 4th quarter of FY05 aimed at the evaluation of the longevity of HRC in groundwater, which was injected on August 2, 2004.

- During the 4th quarter, investigations included collecting water samples for the determination of the microbial populations, Cr and Fe concentrations, and redox conditions in water samples collected before, during, and after the groundwater pumping test that was conducted from June 6-July 11, 2005. Groundwater was withdrawn from the monitoring well 699-96-44.

- Water samples were collected from both wells (699-96-44 and 699-96-45) along with on site measurements of pH, DO, electrical conductivity, redox potential, and temperature. Br-tracer test was conducted in conjunction with the pumping test (tracer was added to the injection well 699-96-45).

- Geophysical (seismic and radar) cross-borehole measurements were performed to attempt to delineate the spatial distribution of the zone affected by biostimulation.

- Microbial analyses of groundwater samples included: Acridine orange direct counts and molecular analyses-PLFA, 16S GeneChip, clone library, and qPCR. Analytical analyses of groundwater samples included bromide, chloride and phosphate (added to HRC), acetate (byproduct of HRC microbial metabolism, nitrate and sulfate (present in background groundwater). Analytical analyses of metals in filtered groundwater samples included Cr(VI), total Cr, and Fe(II) and total Fe.

- The initial drop in the DO concentration, redox potential, and soluble Cr(VI) and total Cr concentrations in water samples collected from all water samplers clearly indicates that one year after the HRC injection a small amount of HRC was still present in the Hanford aquifer. As pumping progressed, it is likely that some Cr- contaminated water from the surrounding area mixed with the initial HRC-effected groundwater, causing redox conditions and microbial densities to return to practically background levels. At the same time, the soluble Cr(VI) concentration increased in the pumped water and a water sample at a depth of 42 ft, but did not reach background concentrations. The soluble Cr(VI) concentration in all other water samples from both the injection and monitoring wells remained below the detectable limit (<0.28 mg/L). The total Cr concentration in the monitoring well decreased by a factor of 4 compared to that under background conditions. The preliminary analysis clearly indicates the successful effect of application of HRC for Cr(VI) bioreduction over a period of one year.

- To assess a potential effect of Cr(VI) reoxidation under field conditions, we developed a field work plan to continue, including drilling and coring of 3 new wells in the vicinity of existing wells at Hanford 100H site. These wells will then be completed for collecting water samples before and after a new HRC injection test.
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