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.

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 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.

Tetrachloroethylene (PCE) and trichloroethylene (TCE) are among the most frequently detected ground water contaminants at industrial sites, including many DoD facilities. Due to the high cost and uneven performance of traditional remediation technologies, monitored natural attenuation is emerging as a new technology for ground water remediation of pollutants such as these. In addition, there is growing interest in active remediation technologies that employ abiotic minerals. PCE and TCE are susceptible to reductive dechlorination by microorganisms as well as reduced minerals such as iron sulfide (FeS). Unlike biological reductive dechlorination, which often results in accumulation of harmful intermediates such as cis 1,2-dichloroethylene (cis-DCE) and vinyl chloride (VC), abiotic mineral-mediated dechlorination of PCE and TCE tends to result in complete transformation to non-toxic products such as acetylene. To more accurately apply natural attenuation and other remediation technologies, a greater understanding of the geochemical factors affecting the rates of purely abiotic reductive dechlorination of PCE and TCE is needed. Additional tools are also needed to determine whether or not abiotic reductive dechlorination is occurring at a particular site, and its relative importance compared to microbial dechlorination under a variety of geochemical conditions.

Chlorinated aliphatic hydrocarbons (CAHs), such as tetrachloroethene (PCE) and trichloroethene (TCE), are ubiquitously pollutants in aquifer sediments and groundwater due to their heavy usage in industry and inappropriate disposal in the last century. Among about 1300 NPL (National Priorities List) sites, PCE and TCE are the two most frequently detected hazardous contaminants. Engineered bioremediation, including biostimulation and bioaugmentation, is a promising technology to clean those PCE and/or TCE contaminated sites. However, in many contaminated groundwater systems and hazardous waste sites, pH can be lower than 5 to 6. And release of HCl (strong acid) from anaerobic reductive dechlorination may lower the pH of groundwater. Besides, another main source of acidity comes from the fermentation of additive electron donors such as alcohols, organic acids and etc. Decreasing pH has been proved to be detrimental to the microbes that dechlorinated PCE or TCE. We intended to enrich and isolate microorganisms, which can perform anaerobic reductive dechlorination at low pH environments, by establishing microcosms, which will be beneficial to in situ bioremediation. We also screened some existing cultures for dechlorinating activity at low pH and determined the pH tolerance of consortium BDI, which had been successfully, applied for in situ bioremediation. Besides, this study investigated and explored the effects of solids on BDI consortium under low pH conditions. Generally, various dechlorinating pure cultures and consortium BDI show highest dechlorination rates and extent at circumneutral pH. Only Sulfurospirillum multivorans among tested cultures dechlorinated PCE to cDCE at pH 5.5. The screening efforts suggest that microbes capable of dechlorination below pH 5.5 are not common. It was observed that solids play an important role for enhancing microbial activities under low pH conditions. And BDI consortium can recover from up to 8 weeks exposure to low pH conditions, although the VC-to-ethene dechlorination step was affected.

Trichloroethene (TCE) is a ubiquitous soil and groundwater contaminant. The most common bioremediation approach for TCE relies on the process of reductive dechlorination by Dehalococcoides mccartyi. D. mccartyi uses TCE, dichloroethene, and vinyl chloride as electron acceptors and hydrogen as an electron donor. At contaminated sites, reductive dechlorination is typically promoted by adding a fermentable substrate, which is broken down to short chain fatty acids, simple alcohols, and hydrogen. This study explored microbial chain elongation (MCE), instead of fermentation, to promote TCE reductive dechlorination. In MCE, microbes use simple substrates (e.g., acetate, ethanol) to build medium chain fatty acids and also produce hydrogen during this process. Soil microcosm using TCE and acetate and ethanol as MCE substrates were established under anaerobic conditions. In soil microcosms with synthetic groundwater and natural groundwater, ethene was the main product from TCE reductive dechlorination and butyrate and hydrogen were the main products from MCE. Transfer microcosms using TCE and either acetate and ethanol, ethanol, or acetate were also established. The transfers with TCE and ethanol showed the faster rates of reductive dechlorination and produced more elongated products (i.e., hexanoate). The microbial groups enriched in the soil microcosms likely responsible for chain elongation were most similar to Clostridium genus. These investigations showed the potential for synergistic microbial chain elongation and reductive dechlorination of chlorinated ethenes.