Title: Western Region Hazardous Substance Research Center Project 1-OSU-03
Development of the Push-Pull Test to Monitor Bioaugmentation with Dehalogenating Cultures

Investigator: Mark E. Dolan, Jennifer Field, and Jonathon Istok, Oregon State University

Institution: Oregon State University

Research Category: Bioremediation, groundwater, bioaugmentation.

Project Period: 2001-2003

Goal: The overall goal was to modify the single-well push-pull groundwater test as a means for obtaining quantitative information on in situ dechlorinating activity before and after bioaugmentation. The specific objectives included: 1) modifying TCFE and fumarate assays to determine TCE-transformation potential for use in monitoring bioaugmentation, 2) developing methods for monitoring the transport of dehalogenating cultures during push-pull tests, and 3) evaluating 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 (e.g., trichloroethene or TCE). Bioaugmentation of anaerobic dehalogenating consortia may be a viable alternative for remediating TCE source zones. Currently, it is difficult to assess if bioaugmentation has increased in situ dechlorination activity. Single-well “push-pull” tests using TCE, the target compound, can be difficult to interpret due to background TCE concentrations and uncertain flux through the zone. However, single-well “push-pull” tests using the surrogate substrate trichlorofluoroethene, TCFE, can provide quantitative information on in situ biological activity and can be correlated to known culture transformation kinetics for both TCE and TCFE to estimate in situ TCE transformation rates. With modifications, the single-well “push-pull” tests could be used in determining the effectiveness of bioaugmentation. 

Approach: Two cultures (Evanite and Pt. Mugu) that transform TCE to ethene will be characterized in collaboration with Dr. Semprini (Developing and Optimizing Biotransformation Kinetics for the Bio-remediation of Trichloroethylene at NAPL Source Zone Concentrations). Transport and transformation tests will be conducted using a physical aquifer model (PAM) shaped like a piece of pie simulating a radial segment around a central injection/extraction well. The transport of the culture(s) will be determined during injection into the PAM that will be maintained under anaerobic conditions. The sediment used to pack the PAM will first be tested for in situ dehalogenation activities towards both TCE and TCFE. Dehalogenating microbial activity will be monitored in the PAM with and without bioaugmentation. Spatial distributions of dechlorinating activity and redox will be determined from a suite of assays conducted at sampling ports and at the injection/extraction location. Push-pull tests will be conducted at the injection/extraction well to assess changes in reductive dechlorination activity resulting from bioaugmentation.

Summary of Findings:

Stimulation of dehalogenation in PAM sediments. The background activity of sediment collected from a site with known indigenous reductive dechlorination activity was 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. A microcosm study was used to determine the relationship between TCE and TCFE transformation rates and product speciation when fed fumarate and succinate. Results obtained here will be useful when initiating the assays in PAMs. The microcosms were operated over a period of approximately 250 days. Succinate- and fumarate-fed microcosms produced 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 two weeks. The corresponding lag times for TCFE transformation under the same conditions was about six weeks and may indicate slower microbial growth using TCFE. Based on a first order model fit after the lag time, TCE transformation rates were from 3.3 times (fumarate-fed) to five times (succinate-fed) faster than microcosms without exogenous electron donor addition. TCFE transformation rates were about 2.4 times faster than control microcosms but about four to five times slower than TCE transformation rates. TCE transformation products were cis-DCE and trans-DCE in approximately a 2:1 ratio and TCFE transformation products were cis-DCFE and trans-DCFE in approximately 2:1 ratio as well. TCE was ultimately reduced to VC, but very little ethene was observed. TCFE was transformed into a mixture of DCFEs and CFEs, with no 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 assessed in the sediments used in the PAM tests to determine the relationship to TCE rates.

Bioaugmentation dose. A seed culture, obtained from Dr. Semprini’s group from their “Evanite” culture reactor, was serially fed butanol and PCE for about two months, and showed complete dehalogenation of PCE to ethene. This culture was used in other tests related to this project. A series of microcosms were prepared with the same sediments that were used to pack the PAM and were used to test the survivability of the bioaugmented culture under different geochemical conditions. The aqueous phase in the microcosms consisted of tap water or tap water amended with 5% media solution used in the culture reactor. Both lactate and butanol were tested as fermentable substrates and bioaugmentation doses of 0.1, 1, and 10 mL of reactor culture, representing culture dilutions of 0.1 to 8%, were tested. Although all of the bioaugmented microcosms survived to exhibit dehalogenation activity, microcosms receiving the highest bioaugmentation dose had dechlorination rates significantly higher than the lower doses in the same time period and microcosms with 5% media addition had faster activity than those without media. Microcosms containing 5% media that received the 10 mL culture dose and were fed butanol were able to completely transform the TCE to ethene within 96 days. This dose was used in the bioaugmentation of the PAMs.

Culture transport. A glass column of 5 cm diameter and 34 cm length was packed with the same sediments used to pack the PAMs, and was used to evaluate the transport characteristics of the bioaugmentation culture. Molecular tests using PCR reactions targeting the 16S rDNA of Dehalococcoides sp. showed efficient transport of the bioaugmentation culture through the column. Microscopic direct count of DNA-stained cells was the technique used to assess transport of the bioaugmented cells in the PAM. Background cell counts were from 1-3 *104 cells/mL before bioaugmentation and rose to 34 to 57% of the influent cell concentrations (1*107 cells/mL) throughout the PAM indicating successful transport of the bioaugmentation culture over the 34 cm length of the PAM.

PAM results. The PAM was packed with sediment and saturated with oxygen-free water to produce anoxic conditions for the start of the test. Lactate solution was added to the PAM just prior to bioaugmentation in an effort to assure anaerobic conditions prior to bioaugmentation. The PAM was bioaugmented with a solution containing approximately 1*107 cells/mL and 50 mM TCE. No activity values were obtained with the initial bioaugmentation since all of the TCE was transformed to ethene at all sample ports by the time samples were acquired 7 days later (a longer initial incubation was estimated from microcosm tests). Subsequent injections of solution containing TCE, TCFE or both TCE and TCFE were conducted to evaluate product distribution and transformation rates. Essentially complete transformation of TCE to ethene was accomplished with each injection with the maximum measured transformation of about 250 mM influent TCE and 320 mM TCFE to ethene and fluoroethene respectively within 30 days. Butanol injected as an electron donor was fermented into butyrate, propionate and, eventually, acetate. When injected alone at about 250 mM, TCE transformation rates ranged from 30- 80 mM/d with the highest activity around ports 5 and 6. When injected at the same concentration along with 320 mM TCFE, TCE transformation rates remained high at around 26-36 mM/d. TCFE rates were equally as high with estimates of 29-45 mM/d being transformed in conjunction with the TCE, indicating that inclusion of the surrogate transformational tracer TCFE should enable estimation of attainable TCE transformation rates. Repeated addition of electron donor was required over the 120 day study to provide energy for the dehalogenation reactions. The control PAM operated without bioaugmentation showed limited transformation to cis-DCE and no DCFE production during 70 days of operation.

Push-pull transformation tests. A push-pull test was initiated using the bioaugmented PAM and a solution containing TCFE and bromide as a non-reactive tracer. The solution was injected into the PAM, allowed to reside for 35 hr, and then extracted and analyzed over 25 hr. Using the non-reactive tracer to produce a mass balance on TCFE, a FE production rate of about 60 mM/d was calculated. This is on the order of the TCFE transformation rates found in the initial activity tests in the bioaugmented PAM, but is significantly higher than FE production rates calculated in those tests (~5-8 mM/d). This may in part be due to only capturing 47% of the injected TCFE in transformed products. It is expected that gas pockets trapped during the packing of the PAM provided sinks for the FE produced that were not included in the aqueous sampling and therefore were not recovered here or in the significantly longer initial transformation tests. A reactive model was used incorporating the kinetic characteristics of the bioaugmented Evanite culture to simulate transformation in the PAM. Although the results were consistent with the observed data, estimates of FE production consistently exceeded actual FE concentrations, perhaps due to partitioning of the FE into trapped gas pockets within the PAM. The ability of this dechlorinating culture to transform TCE and TCFE at appreciably similar rates allows estimation of TCE transformation rates based on single-well push pull tests conducted using TCFE as a surrogate substrate. The push-pull tests produced better results due to the shorter time-scale required for the test and the potential for FE losses associated with partitioning into a trapped gas phase.



Journal Articles

Field, J. A., J. D. Istok, L. Semprini, P. Bennett, and T. E. Buscheck. (2005). Trichlorofluoroethene: A Reactive Tracer for Evaluating the Effectiveness of In Situ Trichloroethene Remediation, Ground Water Monitoring Remediation Vol 25(2):68-77. 

Schroth, M.H., J.D. Istok. (2005). Approximate solution for solute transport during spherical-flow push-pull tests, Ground Water, 43(2), 280-284. 


Abstracts and Posters

Lee, J. H, Dolan, M.E., Istok, J., Reed R., and Field, J.A. (2004). Monitoring Bioaugmentation with Single-Well Push-Pull tests in Sediment Systems Contaminated with Trichloroethene. Fourth SETAC World Congress and 25th Annual Meeting. Portland, Oregon (November 14 – 18).



Lee, Jae-Hyuk (2006). Anaerobic Reductive Dechlorination of TCE and TCFE in TCE-Contaminated Sediments: Enhnced Bioremediation and Bioaugmentation. Ph.D., Oregon State University.