Prior to the implementation of an enhanced bioremediation pilot study for a trichloroethene (TCE) source area at an industrial site in the United Kingdom (the Site), laboratory microcosm and column studies were performed. The purpose of this column study was to determine if TCE removal rates could be increased with the addition of partitioning electron donors and bioaugmentation with KB-1® culture. Three 1-meter continuous flow columns were constructed using aquifer solids from the Site and artificial groundwater. A TCE dense non-aqueous phase liquid (DNAPL) zone was emplaced in each column. SRS™, a commercially available emulsified vegetable oil (EVO) product, and n-butyl acetate (nBA) were evaluated as partitioning electron donors, while the third column acted as an unamended control. Both nBA and SRSTM were successfully used in previous microcosm studies with high concentrations of TCE (400 and 800 mg/L) to successfully promote the reductive dechlorination of TCE to ethene. Dechlorination of TCE to cis-1,2-dichloroethene (cis-DCE) with trace amounts of vinyl chloride (VC) and ethene, as well as sulfate reduction, were observed in the SRSTM column effluent while DNAPL was present. A dissolution enhancement factor of 2.1 was calculated. The TCE source zone was depleted after approximately 300 days of column operation. Following depletion of the TCE DNAPL, high concentration (~400 mg/L) of TCE amended artificial groundwater was pumped through the column to simulate high TCE concentrations in a plume down gradient from a source zone. Dechlorination of TCE via cis-DCE and VC to ethene was observed in the column effluent along with increases in Dehalococcoides (Dhc) counts. Sulfate concentrations increased during the plume phase while dechlorination to ethene still occurred indicating that complete dechlorination to ethene was possible in the presence of sulfate. Dechlorination of TCE to cis-DCE was observed, but neither VC nor ethene was detected in the nBA Amended column. The nBA was observed to degrade in the column to butyl alcohol and acetate, neither of which partition as strongly as nBA, and were not retained in the column. A continuous addition of nBA promoted the highest amount of cis-DCE production and sulfate reduction was also observed. Once the continuous addition was stopped, dechlorination and sulfate reduction halted indicating that electron donor retention in the column was not achieved. Dehalococcoides (Dhc) concentrations did not increase in the effluent of this column. A dissolution enhancement factor of 1.2 was calculated for the nBA column.

Prior to the implementation of an enhanced bioremediation pilot study for a trichloroethene (TCE) source area at an industrial site in the United Kingdom (the Site), laboratory microcosm and column studies were performed. The purpose of this column study was to determine if TCE removal rates could be increased with the addition of partitioning electron donors and bioaugmentation with KB-1® culture. Three 1-meter continuous flow columns were constructed using aquifer solids from the Site and artificial groundwater. A TCE dense non-aqueous phase liquid (DNAPL) zone was emplaced in each column. SRS™, a commercially available emulsified vegetable oil (EVO) product, and n-butyl acetate (nBA) were evaluated as partitioning electron donors, while the third column acted as an unamended control. Both nBA and SRSTM were successfully used in previous microcosm studies with high concentrations of TCE (400 and 800 mg/L) to successfully promote the reductive dechlorination of TCE to ethene. Dechlorination of TCE to cis-1,2-dichloroethene (cis-DCE) with trace amounts of vinyl chloride (VC) and ethene, as well as sulfate reduction, were observed in the SRSTM column effluent while DNAPL was present. A dissolution enhancement factor of 2.1 was calculated. The TCE source zone was depleted after approximately 300 days of column operation. Following depletion of the TCE DNAPL, high concentration (~400 mg/L) of TCE amended artificial groundwater was pumped through the column to simulate high TCE concentrations in a plume down gradient from a source zone. Dechlorination of TCE via cis-DCE and VC to ethene was observed in the column effluent along with increases in Dehalococcoides (Dhc) counts. Sulfate concentrations increased during the plume phase while dechlorination to ethene still occurred indicating that complete dechlorination to ethene was possible in the presence of sulfate. Dechlorination of TCE to cis-DCE was observed, but neither VC nor ethene was detected in the nBA Amended column. The nBA was observed to degrade in the column to butyl alcohol and acetate, neither of which partition as strongly as nBA, and were not retained in the column. A continuous addition of nBA promoted the highest amount of cis-DCE production and sulfate reduction was also observed. Once the continuous addition was stopped, dechlorination and sulfate reduction halted indicating that electron donor retention in the column was not achieved. Dehalococcoides (Dhc) concentrations did not increase in the effluent of this column. A dissolution enhancement factor of 1.2 was calculated for the nBA column.

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.

Natural attenuation of trichloroethene (TCE) and technetium (99Tc) was studied for five consecutive seasons (from January 2002 to January 2003) in Little Bayou Creek. The stream receives ground water discharge from an aquifer contaminated by past waste disposal activities at the Paducah Gaseous Diffusion Plant (PGDP), a uranium enrichment facility near Paducah, Kentucky. Results from stream gaging, contaminant monitoring, tracer tests (with bromide, nitrate, rhodamine WT and propane) and simulation modeling indicate the TCE is naturally attenuated by volatilization and dilution, with volatilization rates related to the ambient temperature and surface discharge rate. The only apparent mechanism of 99Tc attenuation is dilution. Travel times of non-gaseous tracers were found to be similar and have highest values in October and lowest in June. It was also estimated from modeling that the transport of the solutes in the stream was mostly one-dimensional with insignificant secondary storage.

Perchloroethene and trichloroethene are among the most persistent groundwater pollutants, and Dehalococcoides is the only known species that can degrade these compounds completely to non-toxic ethene. Characterization of the reductive dehalogenase (RDase) enzymes responsible for dechlorination is important to understanding this process. A series of dechlorination assays were performed with whole cell suspensions and cell-free extracts of three Dehalococcoides-containing mixed microbial consortia to compare dechlorination kinetics and to characterize co-contaminant inhibition. Michaelis-Menten kinetic parameters Vmax and Km, as well as non-competitive inhibition coefficients for 1,1,1-trichloroethane and 1,1-dichloroethane inhibitors are reported. Secondly, blue native gel electrophoresis was developed as a method to isolate active protein complexes containing RDases. Thirdly, sources of variability in the isotopic fractionation of vinyl chloride to ethene reaction step were examined using cell-free extracts and whole-cell suspensions. Understanding the function and range of RDases are goals towards the successful application of Dehalococcoides-containing cultures to remediate contaminated sites.

Perchloroethene and trichloroethene are among the most persistent groundwater pollutants, and Dehalococcoides is the only known species that can degrade these compounds completely to non-toxic ethene. Characterization of the reductive dehalogenase (RDase) enzymes responsible for dechlorination is important to understanding this process. A series of dechlorination assays were performed with whole cell suspensions and cell-free extracts of three Dehalococcoides-containing mixed microbial consortia to compare dechlorination kinetics and to characterize co-contaminant inhibition. Michaelis-Menten kinetic parameters Vmax and Km, as well as non-competitive inhibition coefficients for 1,1,1-trichloroethane and 1,1-dichloroethane inhibitors are reported. Secondly, blue native gel electrophoresis was developed as a method to isolate active protein complexes containing RDases. Thirdly, sources of variability in the isotopic fractionation of vinyl chloride to ethene reaction step were examined using cell-free extracts and whole-cell suspensions. Understanding the function and range of RDases are goals towards the successful application of Dehalococcoides-containing cultures to remediate contaminated sites.

Chlorinated aliphatic hydrocarbons (CAHs) include tetrachloroethene (PCE), trichloroethene (TCE), and others. The industrial solvent TCE is among the most ubiquitous chlorinated compounds found in groundwater pollution. TCE in environment can be removed by physical, chemical and biological procedures. Dehalorespiration is a biological pathway from which bacteria can derive energy from the reductive dechlorination of chlorinated ethenes using hydrogen or organic acids as electron donors and yielding chloride and ethene as degradation products. Dehalorespiration can be used to remediate chlorinated ethene contaminated aquifers if an appropriate aquifer ecosystem exists including populations of dechlorinating bacteria and companion organisms that contribute to the biogeochemical environment conducive to dehalorespiration activity. Enhanced in-situ aerobic or anaerobic bioremediation of chlorinated solvents is a cost-effective, expanding technology for the clean-up of chlorinated solvent-contaminated sites. 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 bioremediation zone, the effectiveness of air, nutrient, and sugarcane molasses injection to enhance the aerobic cometabolism on TCE degradation was evaluated. Results show that the decreases in TCE concentration were observed over 204 days operating period. Up to 73¢H-99¢H of TCE removal efficiency was obtained in this treatment system. In the anaerobic test zone, the effectiveness of nutrient and sugarcane molasses injection to enhance the anaerobic dechlorination on TCE degradation was also evaluated. Results show that the decreases in TCE concentration were observed over a 193-day operating period. Up to 53¢H-91¢H of TCE removal efficiency was obtained in this treatment system. 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¢¹methanotrophs and 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. In the anaerobic treatment zone, Dehalococcoides (anaerobic TCE-degrader) and the gene of degradation enzyme (vcrA and tceA) were detected and a significant drop of TCE concentration was also observed. Based on 16S rDNA sequence analysis, samples of groundwater from aerobic/anaerobic bioremediation zone are close related to the genera of Dehalococcoides sp. MB, Dehalococcoides ethenogenes 195, Dehalococcoides sp. VS, Acidovorax sp., Alicycliphilus sp., Burkholderiales, Caulobacter sp., Caulobacter tuntrae, Caulobacter vibrioides, Comamonadaceae, Hydrogenophaga sp., Iron-reducing bacterium, Mitsuaria chitosanitabida, Rhodocyclacea, Pseudomonas sp., Rhodoferax ferrireducens, Acinetobater sp., actinomycete, Pseudomonas aeruginosa and Variovorax sp. Results reveal that both the aerobic cometabolism and anaerobic dechlorination are feasible and applicable technologies to clean up TCE contaminated aquifers. Thus, the in situ bioremediation technology has the potential to be developed into an environmentally, economically and naturally acceptable remediation technology.

abstract: Reductive dechlorination by members of the bacterial genus Dehalococcoides is a common and cost-effective avenue for in situ bioremediation of sites contaminated with the chlorinated solvents, trichloroethene (TCE) and perchloroethene (PCE). The overarching goal of my research was to address some of the challenges associated with bioremediation timeframes by improving the rates of reductive dechlorination and the growth of Dehalococcoides in mixed communities. Biostimulation of contaminated sites or microcosms with electron donor fails to consistently promote dechlorination of PCE/TCE beyond cis-dichloroethene (cis-DCE), even when the presence of Dehalococcoides is confirmed. Supported by data from microcosm experiments, I showed that the stalling at cis-DCE is due a H2 competition in which components of the soil or sediment serve as electron acceptors for competing microorganisms. However, once competition was minimized by providing selective enrichment techniques, I illustrated how to obtain both fast rates and high-density Dehalococcoides using three distinct enrichment cultures. Having achieved a heightened awareness of the fierce competition for electron donor, I then identified bicarbonate (HCO3-) as a potential H2 sink for reductive dechlorination. HCO3- is the natural buffer in groundwater but also the electron acceptor for hydrogenotrophic methanogens and homoacetogens, two microbial groups commonly encountered with Dehalococcoides. By testing a range of concentrations in batch experiments, I showed that methanogens are favored at low HCO3 and homoacetogens at high HCO3-. The high HCO3- concentrations increased the H2 demand which negatively affected the rates and extent of dechlorination. By applying the gained knowledge on microbial community management, I ran the first successful continuous stirred-tank reactor (CSTR) at a 3-d hydraulic retention time for cultivation of dechlorinating cultures. I demonstrated that using carefully selected conditions in a CSTR, cultivation of Dehalococcoides at short retention times is feasible, resulting in robust cultures capable of fast dechlorination. Lastly, I provide a systematic insight into the effect of high ammonia on communities involved in dechlorination of chloroethenes. This work documents the potential use of landfill leachate as a substrate for dechlorination and an increased tolerance of Dehalococcoides to high ammonia concentrations (2 g L-1 NH4+-N) without loss of the ability to dechlorinate TCE to ethene. / Dissertation/Thesis / Ph.D. Microbiology 2013

The current understanding of contaminant migration in fractured sedimentary rock aquifers is inadequate due to the difficulty in describing the geologic and hydrogeologic controls on contaminant fate and transport with appropriate detail. To address contamination at fractured rock sites, multiple methods focusing on different aspects of the hydrologic system are required, and particular emphasis needs to be placed on the rock matrix. This thesis shows the further development and utility of the decade-old rock core VOC method (i.e. CORETM), a rock matrix method, when used in conjunction with multiple high resolution datasets as it applies to a 100 m thick highly productive dolostone aquifer in Guelph, Ontario. The research site and surrounding area, located in the northwestern quadrant of the municipality of Guelph, was a productive zone for water supply until the early 1990s when the two closest municipal supply wells (Sacco, Smallfield) were shut down (1991, 1993 respectively) due to volatile organic compounds (VOCs) in the groundwater. Trichloroethene (TCE), a VOC, was used as a degreaser at the Guelph site and likely entered the groundwater more than 20 years ago. The thin overburden, shallow water table, relatively constant dolostone mineralogy, proximity to the UW analytical laboratory, relatively simple plume composition showing minimal degradation, and local importance make this an excellent study site for TCE fate and migration in fractured sedimentary rocks. This thesis is composed of four chapters. Chapter 1 provides a brief background to the rock core VOC method and gives the conceptual framework for the investigation. Chapter 2 focuses on the further development of the rock core VOC method by providing the field validation of a recently adapted extraction method for VOCs in rock core using microwave assisted extraction (MAE), demonstrating the importance of rapid field preservation of samples, and comparing to the industry standard purge and trap method for VOCs on solid matrices. Results indicate that the microwave assisted extraction (MAE) method typically provides equivalent or higher concentrations when compared with the shake-flask and purge and trap extraction methods, indicating more complete extraction or less loss during transfer and/or storage. The purge and trap method provided false negatives (i.e. non-detects) due to inadequate preservation, incomplete extraction, and the elevated detection limit for TCE. The necessity for field preservation was examined by comparing crushed rock samples preserved in methanol in the field to samples unpreserved in the field with a laboratory addition of methanol less than 12 hours later. Chapter 3 creates high resolution porosity and bulk density logs by using selected geophysical logging tools in combination with core derived physical properties for the purpose of calculating porewater concentrations from total contaminant mass concentrations obtained from the rock core VOC method and sample specific rock properties relevant to the conversion. This is beneficial because total mass estimates obtained from the rock core VOC method are not necessarily indicative of the groundwater concentrations given the presence of solid organic carbon controlled sorption. Chapter 4 is a demonstration of the discrete fracture network approach (Parker 2007) applied to the Guelph field site with emphasis on the insights gained through high resolution contaminant profiles generated from cored holes in or near the source area and along a transect. Together, these four chapters present a framework for investigating VOC contamination in fractured sedimentary rocks and with emphasis on evaluating recent advances in the rock core VOC methodology in a field site context.

The current understanding of contaminant migration in fractured sedimentary rock aquifers is inadequate due to the difficulty in describing the geologic and hydrogeologic controls on contaminant fate and transport with appropriate detail. To address contamination at fractured rock sites, multiple methods focusing on different aspects of the hydrologic system are required, and particular emphasis needs to be placed on the rock matrix. This thesis shows the further development and utility of the decade-old rock core VOC method (i.e. CORETM), a rock matrix method, when used in conjunction with multiple high resolution datasets as it applies to a 100 m thick highly productive dolostone aquifer in Guelph, Ontario. The research site and surrounding area, located in the northwestern quadrant of the municipality of Guelph, was a productive zone for water supply until the early 1990s when the two closest municipal supply wells (Sacco, Smallfield) were shut down (1991, 1993 respectively) due to volatile organic compounds (VOCs) in the groundwater. Trichloroethene (TCE), a VOC, was used as a degreaser at the Guelph site and likely entered the groundwater more than 20 years ago. The thin overburden, shallow water table, relatively constant dolostone mineralogy, proximity to the UW analytical laboratory, relatively simple plume composition showing minimal degradation, and local importance make this an excellent study site for TCE fate and migration in fractured sedimentary rocks. This thesis is composed of four chapters. Chapter 1 provides a brief background to the rock core VOC method and gives the conceptual framework for the investigation. Chapter 2 focuses on the further development of the rock core VOC method by providing the field validation of a recently adapted extraction method for VOCs in rock core using microwave assisted extraction (MAE), demonstrating the importance of rapid field preservation of samples, and comparing to the industry standard purge and trap method for VOCs on solid matrices. Results indicate that the microwave assisted extraction (MAE) method typically provides equivalent or higher concentrations when compared with the shake-flask and purge and trap extraction methods, indicating more complete extraction or less loss during transfer and/or storage. The purge and trap method provided false negatives (i.e. non-detects) due to inadequate preservation, incomplete extraction, and the elevated detection limit for TCE. The necessity for field preservation was examined by comparing crushed rock samples preserved in methanol in the field to samples unpreserved in the field with a laboratory addition of methanol less than 12 hours later. Chapter 3 creates high resolution porosity and bulk density logs by using selected geophysical logging tools in combination with core derived physical properties for the purpose of calculating porewater concentrations from total contaminant mass concentrations obtained from the rock core VOC method and sample specific rock properties relevant to the conversion. This is beneficial because total mass estimates obtained from the rock core VOC method are not necessarily indicative of the groundwater concentrations given the presence of solid organic carbon controlled sorption. Chapter 4 is a demonstration of the discrete fracture network approach (Parker 2007) applied to the Guelph field site with emphasis on the insights gained through high resolution contaminant profiles generated from cored holes in or near the source area and along a transect. Together, these four chapters present a framework for investigating VOC contamination in fractured sedimentary rocks and with emphasis on evaluating recent advances in the rock core VOC methodology in a field site context.