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2003 Progress Report: Development of the Push-Pull Test to Monitor Bioaugmentation with Dehalogenating Cultures
EPA Grant Number: R828772C006Subproject: this is subproject number 006 , established and managed by the Center Director under grant R828772
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: HSRC (2001) - Western Region Hazardous Substance Research Center for Developing In-Situ Processes for VOC Remediation in Groundwater and Soils
Center Director: Semprini, Lewis
Title: Development of the Push-Pull Test to Monitor Bioaugmentation with Dehalogenating Cultures
Investigators: Field, Jennifer A. , Dolan, Mark E. , Istok, Jonathan D.
Institution: Oregon State University
EPA Project Officer: Lasat, Mitch
Project Period: September 1, 2001 through August 31, 2003
Project Period Covered by this Report: September 1, 2002 through August 31, 2003
Project Amount: Refer to main center abstract for funding details.
RFA: Hazardous Substance Research Centers - HSRC (2001)
Research Category: Hazardous Waste/Remediation
Description:
Objective:The objectives of this research project are to: (1) modify the single-well "push-pull" groundwater test as a means of obtaining quantitative information on in situ dechlorinating activity before and after bioaugmentation; (2) modify trichlorofluoroethene (TCFE) and fumarate assays to determine trichloroethene (TCE)-transformation potential for use in monitoring bioaugmentation; (3) develop methods for monitoring the transport of dehalogenating cultures during push-pull tests, and (4) evaluate the ability of push-pull tests to monitor changes in TCE-transformation potential resulting from the injection of dehalogenating cultures.
Rationale. Technologies are needed to enhance the in situ remediation of groundwater contaminated by chlorinated aliphatic hydrocarbons (CAHs), such as TCE. Bioaugmentation may be a viable alternative for remediating TCE source zones. Currently, it is difficult to assess if bioaugmentation is increasing in situ dechlorination activity. The single-well "push-pull" tests with the TCE surrogate TCFE can provide quantitative information on in situ biological activity and can be modified for use in determining the effectiveness of bioaugmentation.
Approach. Two cultures (Evanite and Point Mugu) that transform TCE to ethene will be characterized in collaboration with Dr. Semprini (See the Annual Report for R828772C001). The transport of the culture(s) will be determined during injection into anaerobic physical aquifer models (PAMs). Spatial distributions of dechlorinating activity and redox will be determined from a suite of assays conducted at sampling ports and at the injection/extraction well. Push-pull tests will be conducted at the injection/extraction well to assess changes in reductive dechlorination activity resulting from bioaugmentation.
Progress Summary:The background activity of sediment collected from a site with known indigenous reductive dechlorination activity has been characterized with respect to the kinetics of TCE, TCFE, fumarate, and succinate utilization and product formation. These four substrates were proposed for this project as substrates that could be used to assay for reductive dechlorination potential in situ. The microcosm study was used to determine the relationship between TCE and TCFE transformation rates, and product speciation when fed fumarate and succinate prior to initiating the assays in PAMs. The microcosms were operated over a period of approximately 250 days. Succinate- and fumarate-fed microcosms produced very similar results for lag times, transformation rates, and product speciation, with very similar results from triplicate microcosms at each condition. Lag-time to the onset of TCE transformation in both fumarate- and succinate-fed microcosms was about 2 weeks. The corresponding lag-time for TCFE transformation under the same conditions was about 6 weeks. TCE transformation rates, based on a first order model fit after the lag-time, were from 3.3 times (fumarate-fed) to 5 times (succinate-fed) faster than microcosms without exogenous electron donor addition. TCFE transformation rates were about 2.4 times faster than control microcosms and about 4 to 5 times slower than TCE transformation rates. TCE transformation products were cis-dichloroethene (DCE) and trans-DCE in approximately a 2:1 ratio and TCFE transformation products were cis-dichlorofluoroethene (DCFE) and trans-DCFE in approximately a 2:1 ratio as well. TCE ultimately was reduced to vinyl chloride (VC), but very little ethene was observed. TCFE was transformed into a mixture of DCFEs and CFEs, with no fluoroethene (FE) formation. CAH transformation rates were not affected by sulfate addition. From these tests, it was determined that succinate was a potential electron donor for further experiments and that TCFE transformation rates would have to be assessed in the sediments used in the PAM tests to determine the relationship to TCE rates.
The seed culture obtained from Dr. Semprini's group from their Evanite culture reactor was serially fed butanol andperchloroethene (PCE) for about 2 months and has shown complete dehalogenation of PCE to ethene. This culture will be used in future tests related to this project. A series of microcosms has been prepared with the same sediments that were used to pack the PAM and will be used to test the survivability of the bioaugmented culture under different geochemical conditions. The water phase in the microcosms consists of tap water or tap water amended with 5 percent media solution used in the culture reactor. Both lactate and butanol will be tested as fermentable substrates, and bioaugmentation doses of 0.1, 1, and 10 mL of reactor culture will be tested. The microcosms recently have been inoculated, and survivability should be assessed within about 30 days. The results will be used to determine the necessary water amendments and bioaugmentation dose for the PAM experiments.
A glass column of 5 cm in diameter and 34 cm in length has been packed with the same sediments used to pack the PAMs, and will be used to evaluate the transport characteristics of the bioaugmentation culture. A feed rate approximating the same linear average velocity to be used in the PAM will be used with an influent culture concentration approaching that found in the mother reactor (~ 25-40 mg/L protein). Effluent samples will be acquired and analyzed for Dehalococcoides sp. using group-specific polymerase chain reaction (PCR) primers and compared to influent concentrations. The Evanite culture was tested using Dehalococcoides sp. group-specific primers in PCR reactions and universal bacterial primers for terminal restriction fragment length polymorphism (T-RFLP) analyses and was found to be highly enriched in Dehalococcoides sp. Serial dilutions of the Evanite culture were extracted and analyzed using Dehalococcoides sp. group-specific PCR and dilutions down to 10-4 were detectable by this process.
The PAMs have been packed with sediment from the Hanford, WA, site, and have been saturated with oxygen-free water to produce anoxic conditions for the start of the test. Lactate solution will be added to the PAM just prior to bioaugmentation to ensure anaerobic conditions prior to bioaugmentation. Information in the literature on Dehalococcoides sp. involved in the critical step of VC transformation to ethane indicates an extreme sensitivity to oxygen, and every effort will be made to ensure anaerobic conditions in the PAM before onset of bioaugmentation.
Future Activities:We will expand our testing to limited real-time quantifiable PCR analyses to attain better enumeration of Dehalococcoides sp. within the effluent samples and for use in the PAM tests. We will complete the bromide tracer test that currently is underway on the column to determine the flow characteristics of the system.
Supplemental Keywords:bioaugmentation, groundwater, microbial activity, reductive dechlorination, ecosystem protection/environmental exposure and risk, waste, water, aquatic ecosystem restoration, chemical engineering, engineering, chemistry, physics, environmental chemistry, environmental engineering, groundwater remediation, hazardous, hazardous waste, remediation, restoration, trichloroethene, TCE, TCE degradation, advanced treatment technologies, aquatic ecosystems, aquifer remediation, aquifer remediation design, contaminated aquifers, dehalogenation, groundwater contamination, groundwater pollution, hazardous waste treatment, in situ remediation, in situ treatment, in situ bioremediation, in situ biotransformation, microbial degradation, push-pull test, VC, vinyl chloride, TCFE, trichlorofluoroethene, DCE, dichloroethene, PCE, perchloroethene, CAH, chlorinated aliphatic hydrocarbons, PAM, physical aquifer model, DCFE, dichlorofluoroethene. , Ecosystem Protection/Environmental Exposure & Risk, Water, Scientific Discipline, Waste, RFA, Remediation, Engineering, Chemistry, & Physics, Restoration, Chemical Engineering, Aquatic Ecosystem Restoration, Hazardous Waste, Environmental Engineering, Environmental Chemistry, Groundwater remediation, Hazardous, dehalogenation, bioaugmentation, microbial degradation, in-situ biotransformation, groundwater, contaminated aquifers, aquifer remediation design, reductive dehalogenation, groundwater pollution, in-situ bioremediation, aquatic ecosystems, groundwater contamination, TCE, in situ treatment, reductive dechlorination, TCE degradation, advanced treatment technologies, contaminated groundwater, hazardous waste treatment, in situ remediation, aquifer remediation
Relevant Websites:
http://wrhsrc.oregonstate.edu/ 
Progress and Final Reports:
2002 Progress Report
Original Abstract
Main Center Abstract and Reports:
R828772 HSRC (2001) - Western Region Hazardous Substance Research Center for Developing In-Situ Processes for VOC Remediation in Groundwater and Soils
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R828772C001 Developing and Optimizing Biotransformation Kinetics for the Bio- remediation of Trichloroethylene at NAPL Source Zone Concentrations
R828772C002 Strategies for Cost-Effective In-situ Mixing of Contaminants
and Additives in Bioremediation
R828772C003 Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbon Compounds with Butane-Grown Microorganisms
R828772C004 Chemical, Physical, and Biological Processes at the Surface of Palladium Catalysts Under Groundwater Treatment Conditions
R828772C005 Effects of Sorbent Microporosity on Multicomponent Fate
and Transport in Contaminated Groundwater Aquifers
R828772C006 Development of the Push-Pull Test to Monitor Bioaugmentation
with Dehalogenating Cultures
R828772C007 Development and Evaluation of Field Sensors for Monitoring
Bioaugmentation with Anaerobic Dehalogenating Cultures for In-Situ Treatment of
TCE
R828772C008 Training and Technology Transfer
R828772C009 Technical Outreach Services for Communities (TOSC) and Technical Assistance to Brownfields Communities (TAB) Programs
R828772C010 Aerobic Cometabolism of Chlorinated Ethenes by Microorganisms that Grow on Organic Acids and Alcohols
R828772C011 Development and Evaluation of Field Sensors for Monitoring Anaerobic Dehalogenation after Bioaugmentation for In Situ Treatment of PCE and TCE
R828772C012 Continuous-Flow Column Studies of Reductive Dehalogenation with Two Different Enriched Cultures: Kinetics, Inhibition, and Monitoring of Microbial Activity
R828772C013 Novel Methods for Laboratory Measurement of Transverse Dispersion in Porous Media
R828772C014 The Role of Micropore Structure in Contaminant Sorption and Desorption