This dissertation examines the applicability of the iron-based degradative solidification/stabilization (DS/S-Fe(II)) to various chlorinated aliphatic hydrocarbons (CAHs) that are common chemicals of concern at contaminated sites. The research focuses on the transformation of 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2,2-tetrachloro-ethane (1,1,2,2-TetCA) and 1,2-dichloroehtane (1,2-DCA) by Fe(II) in cement slurries. It also investigates the degradation of 1,1,1-TCA by a mixture of Fe(II), cement and three iron-bearing phyllosilicates. Transformation of 1,1,1-TCA and 1,1,2,2-TetCA by Fe(II) in 10% cement slurries was characterized using batch reactors. Dechlorination kinetics of 1,1,1-TCA and TCE* (TCE that was produced by transformation of 1,1,2,2-TetCA) was strongly dependent on Fe(II) dose, pH and initial target organic concentration. Degradation of target organics in DS/S-Fe(II) process was generally described by a pseudo-first-order rate law. However, saturation relationships between the rate constants and Fe(II) dose or between the initial degradation rates and target organic concentration were observed. These behaviors were properly described by a modified Langmuir-Hinshelwood kinetic model. This supports the working hypothesis of this research that reductive dechlorination of chlorinated ethanes occurs on the surface of active solids formed in mixtures of Fe(II) and cement. Transformation products for 1,1,1-TCA and 1,1,2,2-TetCA in mixtures of Fe(II) and cement were identified. The major product of the degradation of 1,1,1-TCA was 1,1-DCA, which indicates that the reaction followed a hydrogenolysis pathway. However, a small amount of ethane was also observed. TCE* was rapidly produced by degradation of 1,1,2,2-TetCA and is expected to undergo β-elimination to produce acetylene. Dechlorination of 1,1,1-TCA in suspension of Fe(II), cement and three soil minerals (biotite, vermiculite, montmorillonite) was characterized using batch reactors. A first-order rate model was generally used to describe the dechlorination kinetics of 1,1,1-TCA in this heterogeneous system. The rate constants for 1,1,1-TCA in mixtures of Fe(II), cement and soil minerals were influenced by soil mineral types, Fe(II) dose and the mass ratio of cement to soil mineral. It was demonstrated that structural Fe(II) and surface-bound Fe(II) in the soil minerals affect dechlorination kinetics and the effects vary with mineral types. Furthermore, it suggests that the reductant formed from Fe(II) and cement hydration components is also effective in systems that include soil minerals.

This dissertation examines the applicability of the iron-based degradative solidification/stabilization (DS/S-Fe(II)) to various chlorinated aliphatic hydrocarbons (CAHs) that are common chemicals of concern at contaminated sites. The research focuses on the transformation of 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2,2-tetrachloro-ethane (1,1,2,2-TetCA) and 1,2-dichloroehtane (1,2-DCA) by Fe(II) in cement slurries. It also investigates the degradation of 1,1,1-TCA by a mixture of Fe(II), cement and three iron-bearing phyllosilicates. Transformation of 1,1,1-TCA and 1,1,2,2-TetCA by Fe(II) in 10% cement slurries was characterized using batch reactors. Dechlorination kinetics of 1,1,1-TCA and TCE* (TCE that was produced by transformation of 1,1,2,2-TetCA) was strongly dependent on Fe(II) dose, pH and initial target organic concentration. Degradation of target organics in DS/S-Fe(II) process was generally described by a pseudo-first-order rate law. However, saturation relationships between the rate constants and Fe(II) dose or between the initial degradation rates and target organic concentration were observed. These behaviors were properly described by a modified Langmuir-Hinshelwood kinetic model. This supports the working hypothesis of this research that reductive dechlorination of chlorinated ethanes occurs on the surface of active solids formed in mixtures of Fe(II) and cement. Transformation products for 1,1,1-TCA and 1,1,2,2-TetCA in mixtures of Fe(II) and cement were identified. The major product of the degradation of 1,1,1-TCA was 1,1-DCA, which indicates that the reaction followed a hydrogenolysis pathway. However, a small amount of ethane was also observed. TCE* was rapidly produced by degradation of 1,1,2,2-TetCA and is expected to undergo β-elimination to produce acetylene. Dechlorination of 1,1,1-TCA in suspension of Fe(II), cement and three soil minerals (biotite, vermiculite, montmorillonite) was characterized using batch reactors. A first-order rate model was generally used to describe the dechlorination kinetics of 1,1,1-TCA in this heterogeneous system. The rate constants for 1,1,1-TCA in mixtures of Fe(II), cement and soil minerals were influenced by soil mineral types, Fe(II) dose and the mass ratio of cement to soil mineral. It was demonstrated that structural Fe(II) and surface-bound Fe(II) in the soil minerals affect dechlorination kinetics and the effects vary with mineral types. Furthermore, it suggests that the reductant formed from Fe(II) and cement hydration components is also effective in systems that include soil minerals.

The industrial solvent trichloroethylene (TCE) is among the most ubiquitous chlorinated compounds found in groundwater pollution. TCE in environment can be removed by physical, chemical and biological procedures. The objective of this pilot-scale study was to apply an enhanced in situ bioremediation technology to remediate TCE-contaminated groundwater. Both aerobic and anaerobic remedial systems were evaluated at a TCE-spill site located in southern Taiwan. In the aerobic test zone, the effectiveness of air, nutrient, and sugarcane molasses injection to enhance the aerobic cometabolism on TCE degradation was evaluated. In the anaerobic test zone, the effectiveness of nutrient and sugarcane molasses injection to enhance the anaerobic reductive dechlorination on TCE degradation was also evaluated. Polymerase chain reaction was applied to analyze the gene variation in TCE-microbial degraders during the treatment process. Results from this study indicate that the aerobic TCE-degraders (type ¢º methanotrophs) and the gene of degradation enzymes (toluene monooxygenase, toluene dioxygenase, particulate methane monooxygenase) were detected after the treatment process in the aerobic test zone. Moreover, TCE concentration dropped from approximately 0.1 mg/L to below 0.05 mg/L in the aerobic test zone after six months of treatment. In the anaerobic treatment zone, Dehalococcoides (anaerobic TCE-degrader) and the gene of degradation enzyme (vcrA) were detected and a significant drop of TCE concentration was also observed. Results reveal that both the aerobic cometabolism and anaerobic dechlorination are feasible and applicable technologies to clean up TCE contaminated aquifers.

Chloroethenes such as tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene (DCE) and vinyl chloride (VC), are major contaminants in subsurface systems threatening water quality and human health. Under anaerobic conditions, PCE and TCE can be reductively dechlorinated to ethene. Recent findings indicate that members of the Dehalococcoides group are responsible for ethene formation at chloroethene-contaminated sites. Dehalococcoides species exhibit diverse dechlorination activities, but share highly similar 16S rRNA genes. Hence, additional gene targets that go beyond the 16S rRNA gene are needed to reliably detect and quantify Dehalococcoides populations involved in high rate chloroethene detoxification at contaminated sites. Dehalococcoides sp. strain BAV1 couples growth to reductive dechlorination of VC to ethene. To shed light on the genes involved in reductive dechlorination in strain BAV1, degenerate primers targeting reductive dehalogenase (RDase) genes of Dehalococcoides were designed using available sequence information. PCR amplification with these primers yielded seven putative RDase genes with genomic DNA from strain BAV1 as template. Transcription analysis identified one RDase gene possibly involved in VC dechlorination, which was named bvcA. The bvcA gene was not present in Dehalococcoides strains that failed to couple growth with reductive dechlorination of VC (i.e., Dehalococcoides isolates CBDB1, FL2 and 195). Primers specific for bvcA detected this gene in several, but not all, Dehalococcoides-containing, ethene-producing mixed cultures. Apparently, the bvcA-targeted primers do not capture the diversity of VC RDase genes. Nevertheless, a relevant target was identified, and bvcA-targeted primers are commercially applied to monitor Dehalococcoides sp. strain BAV1 and related organisms at contaminated sites undergoing bioremediation treatment. Additional RDase genes were identified in Dehalococcoides sp. strain FL2, and expression analysis was performed when FL2 was grown with cis-DCE and TCE as electron acceptors. Multiple RDase genes were transcribed with each electron acceptor. This work identified novel process-specific target genes that are useful for site assessment and bioremediation monitoring at chloroethene-contaminated sites. In particular, bvcA emerged as a relevant target for monitoring the critical detoxification step from VC, to ethene. Additionally, the RDase genes retrieved in this work form a basis for further exploration of the specific functions and regulation mechanisms involved in reductive dechlorination processes.

The findings of this study demonstrate that Dehalococcoides species are intimately involved in complete reductive detoxification of chlorinated ethenes and are widely distributed in anoxic sediments and aquifers, including non-contaminated (pristine) environments. Careful examination of enrichment culture dechlorination kinetics, 16S rRNA gene based analyses, and reductive dehalogenase gene targeted PCR approaches revealed that complete reductive dechlorination is carried out by multiple dechlorinators. Two new dechlorinating species were isolated from contaminated and non-contaminated site materials. The first new isolate, designated strain SZ, was isolated from PCE-to-ethene dechlorinating microcosms established with creek sediment. 16S rRNA gene sequence of the strain SZ indicates that the new isolate is affiliated with the genus Geobacter most closely related to G. thiogenes. Strain SZ is capable of stepwise dechlorination of PCE to cis-DCE, while the closest relatives were not able to dechlorinate PCE or TCE. Dechlorination of PCE or TCE by strain SZ was supported by acetate, hydrogen or pyruvate as electron donor. Chloroethene-dechlorinating populations have been shown to have distinct electron donor requirements. However, none of previously described chlorinated ethene degrading population can use both, acetate and hydrogen, as electron donors. PCE dechlorination by strain SZ uses both acetate and hydrogen as electron donors suggesting that the ability to versatile electron donor utilization may increase the efficiency of bioremediation approaches. Importantly, strain SZ reduced two environmental priority pollutants, PCE and U(VI) concomitantly and detected from both bio-stimulated chloroethene and uranium contaminated sites, strongly suggesting that strain SZ play a important roles in in-situ bioremediation of chloroethene and U(VI) contaminated sites. The second, a new Dehalococcoides species designated strain GT, was isolated from contaminated site materials. Strain GT uses trichloroethene (TCE), cis-DCE, 1,1-dichloroethene (1,1-DCE), and the human carcinogen vinyl chloride (VC) as growth supporting electron acceptors producing products ethene and inorganic chloride. The new isolate shares common traits of Dehalococcoides such as ampicillin resistance, strict hydrogen-dependent metabolism, and a low hydrogen consumption threshold concentration. Culture-dependent and independent, 16S rRNA gene and reductive dehalogenase gene targeted PCR approaches suggested culture purity.

Trichloroethene (TCE) and tetrachloroethene (PCE) are among the most commonly detected groundwater contaminants, and are often difficult to remediate due to their presence as dense non-aqueous phase liquids (DNAPLs) in the subsurface. The objective of this study was to assess the potential of using a passive in situ carbon/hydrogen releasing barrier system to bioremediate TCE-contaminated groundwater. The slow carbon/hydrogen releasing material would cause the aerobic cometabolism and reductive dechlorination of TCE in aquifer. The carbon/hydrogen releasing materials would release carbon when contacts with groundwater and release hydrogen after the anaerobic biodegradation of released carbon, thus cause the reductive dechlorination of TCE. Results from the microcosm study indicate that the addition of emulsified substrate, cane molasses, Simple GreenTM (a biodegradable surfactant), or lecithin would enhance the biodegradation rate of TCE under anaerobic conditions. However, addition of multivitamin would increase the bacterial population in the media but would not be able to enhance the TCE degradation rate. Results show that a significant pH drop was observed due to the production of organic acids after the aerobic biodegradation process of cane molasses and lecithin. This also caused the inhibition of microbial growth in microcosms. Results reveal that higher TCE removal efficiency was observed in microcosms with Simple GreenTM addition followed by the addition of cane molasses, lecithin, multivitamin, emulsified substrate, groundwater (without substrate addition). Results from the microcosm study indicate that the addition of emulsified substrate would enhance the biodegradation rate of TCE under anaerobic conditions. However, appearance of high nitrate concentration would inhibit the TCE degradation process due to the occurrence of denitrification. Compared with nitrate, high sulfate concentration would not have significant impact on the reductive dechlorination of TCE. Results reveal that higher TCE removal efficiency was observed in microcosms with emulsified substrate addition followed by the addition of high sulfate concentration, high nitriate concentration, groundwater (without substrate addition). Results from the gene analysis show that phenol monooxygenase, toluene monooxygenase, and toluene dioxygenase were observed in the microcosms with lecithin, cane molasses, Simple GreenTM, and emulsified substrate. This indicates that the addition of substrates would induce the potential of TCE-degrading enzyme. Addition of emulsified substrate and emulsified substrate in nitrate or sulfate-rich media would stimulate Dehalococcoides sp. to induce tceA, bvcA, and vcrA, enzymes for TCE reductive dechlorination.

Fe(II)-based degradative solidification/stabilization (Fe(II)-DS/S) technology is the modification of conventional solidification/stabilization (S/S). Inorganic pollutants are immobilized by Fe(II)-DS/S while organic pollutants are destroyed. Experimental studies were conducted to identify the active agents for Tetrachloroethylene (PCE) degradation as well as the conditions that enhance the formation of the active agents in the Fe(II)-DS/S system. PCE was chosen as a model chlorinated aliphatic hydrocarbon in this study. First, the conditions that lead to maximizing production of the active agents were identified by measuring the ability of various chemical mixtures to degrade PCE. Results showed that Fe(II), Fe(III), Ca, and Cl were the the important elements that affect degradation activity. Elemental compositions of the mixtures and the conditions affecting solid formation might be the important factors in determining how active solids are formed. Second, instrumental analyses (XRD, SEM, SEM-EDS) were used to identify minerals in chemical mixtures that have high activities. Results indicate that active agents for PCE degradation in Portland cement slurries and in cement extracts might be one of several AFm phases. However, systems without cement did not form the same solids as those with cement or cement extract. Ferrous hydroxide was identified as a major solid phase formed in systems without cement. Finally, the effect of using different types of ordinary Portland cement (OPC) on PCE degradation rate during Fe(II)-DS/S was examined and the solids were examined by instrumental analyses (XRD, SEM, SEM-EDS). Four different OPC (Txi, Lehigh, Quikrete, and Capitol) showed different PCE degradation behaviors. Pseudo first-order kinetics was observed for Capitol and Txi OPC and second-order kinetics was observed for Quikrete. In the case of Lehigh cement, pseudo first-order kinetics was observed in cement slurry and second-order kinetics in cement extract. Calcium aluminum hydroxide hydrates dominated solids made with Txi, Quikrete, and Lehigh cements and Friedel’s salt was the major phase found in solids made with Capitol cements. Fe tended to be associated with hexagonal thin plate particles, which were supposed to be a LDH.

Numerical models (GMS MODFLOW, SEAM3D, and SEAM3D Interface) were applied to simulate groundwater flow, petroleum hydrocarbon compound (PHC) transport and biodegradation, and the transport and biotransformation of chlorinated ethenes at Site FT-002 Plattsburgh Air Force Base (PAFB), NY. Site FT-002 was contaminated with waste jet fuel and chlorinated ethenes used as a fire source during fire fighting training. The results of groundwater analysis indicated that the aquifer exhibited aerobic, nitrate reducing, ferrogenic, sulfate reducing and methanogenic conditions due to the biodegradation of the PHCs. Additional groundwater analysis showed the biotransformation of TCE to DCE, VC, and ethene. A numerical model was developed to simulate and assess the extent to which reductive dechlorination and direct anaerobic oxidation were responsible for the biotransformation of the chlorinated ethenes. Reductive dechlorination accounted for the 100%, 98.3%, and 97.5% of the biotransformation of TCE, DCE, and VC respectively. Direct anaerobic oxidation accounted for 1.7% and 2.5% of the biotransformation of DCE and VC respectively. Though direct anaerobic oxidation only accounted for a small percentage of total biotransformation it was necessary to fully develop the biotransformation of the DCE and VC in the ferrogenic zone. This study focused on the mechanisms responsible for the biotransformation of chlorinated ethenes, specifically reductive dechlorination and direct anaerobic oxidation. By further defining the NAPL source and initial conditions it could be used as a tool to accurately predict the monitored natural attenuation (MNA) of the FT-002 contaminant plume. / Master of Science

The purpose of this research was to investigate the microorganisms, genetics and biochemistry of anaerobic dechlorination of chlorinated ethanes, which are common groundwater contaminants. Specifically, this project used mixed microbial cultures to study the dechlorination of 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,1-trichloroethane (1,1,1-TCA). A mixed microbial culture enriched from a contaminated multilayered aquifer in West Louisiana dechlorinated 1,2-DCA, 1,1,2-TCA, tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride (VC) to non-toxic ethene when amended with ethanol as the electron donor. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organisms Dehalobacter and Dehalococcoides spp. Denaturing gradient gel electrophoresis analysis and quantitative PCR (qPCR) with species-specific primers demonstrated that both organisms grew during the dichloroelimination of 1,2-DCA to ethene. Conversely, during the dichloroelimination of 1,1,2-TCA to VC only Dehalobacter grew, while during the reductive dechlorination of VC to ethene only Dehalococcoides grew. Further enrichment with 1,2-DCA, H2 and acetate yielded a co-culture of Dehalobacter and Acetobacterium spp. that did not dechlorinate other chlorinated ethanes or ethenes. Dehalobacter grew in the presence but not in the absence of 1,2-DCA, while Acetobacterium growth was not affected by 1,2-DCA. A novel putative Dehalobacter-associated 1,2-DCA reductive dehalogenase gene was identified and was shown to be transcribed only in the presence of 1,2-DCA. An enrichment microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States was also studied. This culture, referred to as MS, reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) when amended with methanol, ethanol, acetate and lactate. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organism Dehalobacter sp., whose growth during 1,1,1-TCA and 1,1-DCA dechlorination was confirmed by qPCR. In the presence of chlorinated ethenes, dechlorination 1,1,1-TCA by the culture MS was slowed, while dechlorination of 1,1-DCA was completely inhibited. Experiments with cell-free extracts and whole cell suspensions of culture MS suggested that chlorinated ethenes have direct inhibitory effects on 1,1,1-TCA reductive dehalogenase(s), while the inhibition of 1,1-DCA dechlorination may be due to effects on non-dehalogenase components of Dehalobacter sp. cells. Additionally, two novel reductive dehalogenase genes associated with 1,1,1-TCA reductive dechlorination were identified.

abstract: 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 use 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. / Dissertation/Thesis / Masters Thesis Civil, Environmental and Sustainable Engineering 2019