Trichloroethene (TCE) is the most frequently detected organic contaminant in groundwater, is classified as a probable human carcinogen, and exhibits toxicological effects on the human endocrine, immune, developmental, and reproductive systems. While significant research efforts have been devoted to the development of strategies for remediating TCE-contaminated groundwater, their advancement is currently hindered by limitations in current methodologies for measuring in situ reductive dechlorination rates, especially for sorbing solutes. This dissertation describes the development, evaluation, and demonstration of a method for measuring in situ reductive dechlorination rates that utilizes single-well, "push-pull" test technology. Initial field tests indicated that trichlorofluoroethene (TCFE) could be used as a surrogate for TCE in push-pull tests since (a) TCE and TCFE were transported similarly and (b) TCFE underwent reductive dechlorination by a pathway analogous to that of TCE while retaining the fluorine label. Because TCFE and TCE experienced sorption at the selected field site, a novel data analysis technique called "forced mass balance" (FMB) was developed to obtain in situ transformation rates of sorbing solutes from push-pull test data. The FMB technique was evaluated by quantifying errors in rates derived by applying FMB to push-pull test data generated by a numerical model. Results from simulated tests indicated that an example in situ rate for the reductive dechlorination of TCFE, which was obtained by applying FMB to field data, was underestimated relative to the true in situ rate by 10%. The utility of the rate-determination method presented in this dissertation was demonstrated by using it to evaluate the effectiveness of a chemical amendment, namely fumarate, at enhancing in situ reductive dechlorination rates in TCE-contaminated groundwater. Reductive dechlorination rates increased following three consecutive additions of fumarate in all five of the tested wells. The development of the rate-determination method described in this dissertation advances the state of bioremediation technology because methods for measuring in situ transformation rates are needed to both assess the potential for natural attenuation and to quantify the effects of bioremediation techniques in the field.

This research focused on the enhanced reductive dechlorination of trichloroethene (TCE) and its surrogate, trichlorofluoroethene (TCFE), using two bioremediation methods in anaerobic conditions. Two anaerobic bioremediation studies were conducted to investigate the effects of microbial communities in the presence of different electron acceptors and donors during anaerobic reductive dechlorination of TCE and TCFE. The first study was conducted in the groundwater microcosm bottles, filled with groundwater and sediments collected from Richmond site, CA. Parallel reductive dechlorination of TCE and TCFE was evaluated in the presence of fumarate and its product, succinate, while active reduction of high background concentrations of sulfate (2.5 mM) occurred. Because sulfate was assumed as a favorable electron acceptor during reductive dechlorination of chlorinated aliphatic hydrocarbons (CAHs), all microcosms receiving TCE and TCFE with substrates showed enhanced reductive dechlorination activity and even no substrate addition microcosms generated biotransformation products. From the electron mass balance calculations, more than 87.5% of electrons went to sulfate reduction and less than 10% of available electrons involved in dechlorination after sulfate reductions. After amending varying concentrations of sulfate (0 2.5 mM), no inhibition was found between reductive dechlorination of TCE and sulfate reduction. The result indicated that reductive dechlorination could be directly competed with sulfate reduction for available electrons. The second study investigated the effectiveness of in situ push-pull tests to evaluate bioaugmentation in physical aquifer models (PAMs) using dehalogenating strains to reductively dechlorinate TCE to ethene and TCFE to FE in the TCE contaminated sediments. Complete reduction of TCE to ethene occurred in less than 14 days with repeated additions of TCE (13.0 to 46.0 mg/L) and TCFE (15.0 mg/L) was completely transformed to FE in under 24 days. Increased rate and extent of dechlorination in the bioaugmented PAM compared to the nonaugmented control PAM indicated successful transport of the bioaugmented culture through the PAM. Similar transformation rates and time course of TCE and TCFE also indicated that TCFE was a bioprobe for reductive dechlorination of TCE. TCE and TCFE were transformed to cisdichloroethene (c-DCE) and cis-dichlorofluoroethene (c-DCFE) respectively at two of the three sampling ports after 50 days of incubation in the nonaugmented PAM indicating reductive dechlorination activity of indigenous microorganisms. The results showed that it is possible to increase the rate and extent of reductive dechlorination of TCE and TCFE by bioaugmentation and that push-pull tests are effective tools for detecting and quantifying these processes in situ. The third study focused on numerical modeling of the second study. The objectives of this study were (1) to evaluate a simplified method for estimating retardation factors for injected solutes and bioaugmented microorganisms using "pushpull" test injection phase breakthrough curves, (2) to identify whether bioaugmented microorganisms have kept the same transformation capacity of Evanite culture using Michaelis-Menten kinetics by the values provided by Yu et al. (2005) and to verify in situ rates of TCFE reductive dechlorination rates of push-pull tests by numerical modeling, and (3) to investigate a reasonable answer for the nonuniform recovery of ethene and FE during the activity test and the push-pull test. The bioaugmented microorganisms were effectively transported through Hanford sediment. The estimated retardation factor was 1.33. A numerical simulation predicted cell transport in the PAM as far as port 5. This was qualitatively confirmed by cell counts obtained during bioaugmentation but, cells were distributed nonuniformly. The transport test indicated that TCE and TCFE transport was relatively retarded compared to coinjected bromide tracer (retardation factors ranged from 1.33-1.62 for TCE and from 1.44-1.70 for TCFE). The modeling simulation of Michaelis-Menten kinetics for the activity test was well matched for reductive dechlorination rates for TCE and less dechlorinated ethenes using the previous published values of kmax and Ks of chlorinated ethenes by Yu et al. (2005); the model match indicated that the bioaugmented microorganisms kept the same transformation capacity as the original source, Evanite culture (Yu et al., 2005) over 4 months in the PAM. A numerical simulation resulted in the simple first order FE production rate of 1 day' using STOMP code (2002) and the value of FE production rate was in the range of the transformation rates of TCFE during the activity test. The bioaugmented PAM has caused slow loss of injected CAHs during the activity test and the push-pull test.

The project Bench-Scale Design and Testing for In Situ Chemical Reduction of Explosives Contamination in Groundwater was designed to determine if explosives-contaminated groundwater could be treated by In Situ reduction with the use of electron donating substrates. The study evaluated the ability of two substrates to develop and maintain reducing conditions in reactors simulating aquifer conditions. The source of the contaminated groundwater was from the Francis E. Warren Air Force Base located 3 miles west of Cheyenne, Wyoming; the groundwater contained the contaminant trichloroethene (TCE) which through reductive dechlorination can be degraded to unharmful products or products that can be degraded aerobically. By introducing sodium lactate and HRC (Hydrogen Release Compound) into reactors designed to simulate aquifer conditions, the study focuses on increasing the rate of contaminant removal by enhancing reductive dechlorination. The reactors were monitored for the formation of reductive dechlorination products of TCE which include cis-1,2-dichloroethylene (cis-1,2-DCE), vinyl chloride and ethene. The products were monitored by planning sampling events that were scheduled on the basis of measured reactor conditions, including pH and ORP measurements. Target contaminants of interest that were analyzed for each sample event included TCE, cis-1,2-DCE, vinyl chloride and ethene. Other non-target analytes of interest analyzed for included nitrate and sulfate. Results suggest that the increased rate of contaminant removal observed, decreasing ORP readings and accumulation of cis-1,2-DCE could be credited to reductive dechlorination.

​This volume provides a review of the past 10 to 15 years of intensive research, development and demonstrations that have been on the forefront of developing bioaugmentation into a viable remedial technology. This volume provides both a primer on the basic microbial processes involved in bioaugmentation, as well as a thorough summary of the methodology for implementing the technology. This reference volume will serve as a valuable resource for environmental remediation professionals who seek to understand, evaluate, and implement bioaugmentation.

In the late 1970s and early 1980s, our nation began to grapple with the legacy of past disposal practices for toxic chemicals. With the passage in 1980 of the Comprehensive Envir- mental Response, Compensation, and Liability Act (CERCLA), commonly known as Sup- fund, it became the law of the land to remediate these sites. The U. S. Department of Defense (DoD), the nation’s largest industrial organization, also recognized that it too had a legacy of contaminated sites. Historic operations at Army, Navy, Air Force, and Marine Corps facilities, ranges, manufacturing sites, shipyards, and depots had resulted in widespread contamination of soil, groundwater, and sediment. While Superfund began in 1980 to focus on remediation of heavily contaminated sites largely abandoned or neglected by the private sector, the DoD had already initiated its Installation Restoration Program in the mid-1970s. In 1984, the DoD began the Defense Environmental Restoration Program (DERP) for contaminated site assessment and remediation. Two years later, the U. S. Congress codified the DERP and directed the Secretary of Defense to carry out a concurrent program of research, development, and demonstration of innovative remediation technologies. As chronicled in the 1994 National Research Council report, “Ranking Hazardous-Waste Sites for Remedial Action,” our early estimates on the cost and suitability of existing techn- ogies for cleaning up contaminated sites were wildly optimistic. Original estimates, in 1980, projected an average Superfund cleanup cost of a mere $3.