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The addition of exogenously supplied hydrogen stimulates PCP reductive dechlorination and increases bacterial growth. While research focuses mainly on pure cultures, few exist capable of aryl reductive dechlorination, and few markers exist to identify reductively dechlorinating bacteria within mixed cultures. Furthermore, most active bioremediation projects stimulate mixed cultures of native biota. This work describes a method to estimate reductively dechlorinating bacterial growth within a mixed soil culture under controlled environmental conditions. The experiments discussed in this paper were performed in a fed-batch reactor. The reactor was operated in a way to maintain environmental conditions such as pH, E[subscript H], headspace concentration, and temperature constant while substrate is allowed to degrade without the corruption of additional changes. Substrate utilization and cell growth were examined under an array of environmental conditions. This dissertation examined the correlation between hydrogen concentration and the growth rate of reductively dechlorinating bacteria. Under low hydrogen partial pressures, between 9.4 x 10������ and 2.9 x 10������ atm, the growth rate of reductively dechlorinating bacteria increased as predicted by dual Monod kinetics with respect to hydrogen and chlorophenol concentration; however, studies showed that the relationship was more complex. At higher concentrations of hydrogen, the observed growth rate of reductively dechlorinating bacteria declined. A dual Monod kinetics model with hydrogen substrate inhibition approximates experimental data. Reductive dechlorination of 2,3,4,5-tetrachlorophenol and 3,4,5-trichlorophenol were also studied. Pentachlorophenol reductive dechlorination primarily produces 3,4,5-trichlorophenol via 2,3,4,5-tetrachlorophenol. The reductive dechlorination of 2,3,4,5-tetrachlorophenol parallels that of pentachlorophenol, and the estimated growth rates based on pentachlorophenol and 2,3,4,5-tetrachlorophenol are very similar. Reductive dechlorination of 3,4,5-trichlorophenol was catalyzed by the PCP reductively dechlorinating bacterial culture after a lag period. 3,4,5-Trichlorophenol was not maintained for extended periods, and multiple additions of 3,4,5-trichlorophenol did not result in measurable growth. / Graduation date: 2001

Cometabolic biodegradation processes are potentially useful for the bioremediation of hazardous waste sites. In this study the potential application of phenol-oxidizing and nitrifying bacteria as "priming biocatalysts" was examined in the degradation of polycyclic aromatic hydrocarbons (PAHs), aryl ethers, and aromatic ethers. We observed that a phenol-oxidizing Pseudomonas strain cometabolically degrades a range of 2- and 3-ringed PAHs. A sequencing batch reactor (SBR) was used to overcome the competitive effects between two substrates and the SBR was evaluated as a alternative technology to treat mixed contaminants including phenol and PAHs. We also have demonstrated that the nitrifying bacterium Nitrosomonas europaea can cometabolically degrade a wide range polycyclic aromatic hydrocarbons (PAHs), aryl ethers and aromatic ethers including naphthalene, acenaphthene, diphenyl ether, dibenzofuran, dibenzo-p-dioxin, and anisole. Our results indicated that all the compounds are transformed by N. europaea and that several unusual reactions are involved in these reactions. In the case of naphthalene oxidation, N. europaea generated predominantly 2-naphthol whereas other monooxygenases generate 1-naphthol as the major product. In the case of dibenzofuran oxidation, 3-hydroxydibenzofuran initially accumulated in the reaction medium and was then further transformed to 3-hydroxy nitrodibenzofuran in a pH- and nitrite-dependent abiotic reaction. A similar abiotic transformation reaction also was observed with other hydroxylated aryl ethers and PAHs. We also characterized the role of AMO in the degradation of aromatic ethers. Our results indicated that aromatic ethers including anisole were transformed by both 0-dealkylation or hydroxylation reactions. This research has led to the development of a rapid colorimetric assay to detect AMO activity. / Graduation date: 1998

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.

Quantifying the effects of hydrocarbon contamination on hybrid poplar fine root dynamics provides information about how well these trees tolerate the adverse conditions imposed by the presence of petroleum in the soil. Infection by ectomycorrhizal (ECM) fungi may benefit hybrid poplar growing in contaminated soils by providing greater access to water and nutrients and possibly inducing greater contaminant degradation. The overall objectives of this research were to: 1) investigate the relationship between the varying concentrations of total petroleum hydrocarbons (TPH) and nutrients across a hydrocarbon-contaminated site, as well as interactions between these contaminants and physical and chemical soil properties; 2) quantify the effects of these properties on the spatial and temporal patterns of fine root production for Griffin hybrid poplar (<i>P. deltoids </i> x <i>P. petrowskyana</i> c.v. Griffin); and (3) quantify the effect of ectomycorrhizal colonization on hybrid poplar fine root dynamics and N and P uptake when grown in diesel contaminated soil under controlled conditions. A minirhizotron camera provides a nondestructive approach for viewing roots in situ. This camera was used in both the field and growth chamber experiments to provide the data necessary for estimating fine root production. The field study was conducted at Hendon, SK, Canada. Twelve minirhizotron tubes were distributed across the field site and facilitated quantification of fine root production in areas of varying contamination levels. Residual hydrocarbon contamination was positively correlated with soil total C and N, which may suggest that the hydrocarbons remaining in the soil are associated with organic forms of these nutrients or increased microbial biomass. Total fine root production at the site was greater in the 0- to 20-cm depth (1.27 Mg/ha) than the 20- to 40-cm depth (0.51 Mg/ha) in 2004. Fine root production was stimulated by small amounts of hydrocarbon contamination at the field site. Nonlinear regression described fine root production as increasing linearly up to approximately 500 mg/kg TPH, then remaining constant as contamination increased. This trend was most pronounced in the 0- to 20-cm soil layer, with a (r&178; = 0.915). Stimulation of fine root production in the presence of hydrocarbons has significant implications for phytoremediation. If hybrid poplar can maintain increased root production in hydrocarbon contaminated soils, the rhizosphere effect will be exaggerated and increased degradation of contaminants is likely to occur. Under controlled conditions, colonization of hybrid poplar roots by the ectomycorrhizal fungus <i>Pisolithus tinctorius</i>increased fine root production in a diesel contaminated soil (5000 mg diesel fuel/kg soil) compared to non-colonized trees growing in the same soil. Fine root production was 56.6 g/m&178; in the colonized treatment and 22.6 g/m&178; in the non-colonized treatment. In diesel contaminated/ECM colonized treatment, hybrid poplar leaf N and P concentrations after 12 wk were 23.1 and 3.6 g/kg, respectively. In diesel contaminated/non-colonized treatment, N and P concentrations were 15.7 and 2.7 g/kg, respectively. After 12 wk, 5.0&37; of the initial concentration of diesel fuel remained in the soil of the non-colonized treatment and 6.7&37; remained in the colonized treatment. Both treatments removed more contaminants from the soil than an unplanted control, which contained 8.9&37; of the initial diesel fuel concentration after 12 wk. Significantly more hydrocarbons were found sequestered in hybrid poplar roots from the colonized treatment (354.1 mg/kg) than in the non-colonized treatment (102.2 mg/kg). The results of this study indicate that hybrid poplar may be good candidates for use in phytoremediation of petroleum hydrocarbons because of the stimulation of fine root production at low levels of hydrocarbon contamination. However, colonization of hybrid poplar growing in diesel contaminated soil by <i>P. tinctorius</i> inhibited remediation of diesel fuel.

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.

Oxygen has been shown to be an effective addition of enhancing the bioremediation of petroleum hydrocarbon contamination in porous media; however, the ability to effectively deliver oxygen to petroleum hydrocarbon contaminated groundwater has proven difficult. A field and numerical modelling study was completed at a former gas station in southern Ontario, to assess the delivery of oxygen into groundwater in a fractured limestone aquifer that had been contaminated with petroleum hydrocarbons. A field investigation was completed to characterize the bedrock aquifer and the groundwater flow system. Several hydraulically active fracture zones were identified and characterized. To evaluate how dissolved oxygen would behave in this type of groundwater environment, an injection test was completed using iTi’s gPro® oxygen injection technology. About 1000 L of water containing dissolved oxygen at ~ 30 mg/L and a bromide tracer was injected over ~ 90 minutes and monitored for ~ 10 days in the injection well and in a multilevel monitoring well located 3 metres down-gradient. The oxygen concentration rose rapidly within the injection well and at two of the down-gradient monitor intervals which were aligned with the injection well via major fractures. Concentration tailing persisted in the injection well for several days following injection. The effects of biodegradation were not assessed as part of this investigation. A three-dimensional numerical model for groundwater flow and advective-dispersive transport within a discretely-fractured porous medium was calibrated to the field conditions. The simulated injection test demonstrated that oxygen rapidly filled the porous matrix surrounding the injection well and filled the local intersecting fractures. Following injection, the oxygenated groundwater in the local fractures was rapidly flushed by the natural groundwater flow, with oxygen arrivals appearing as sharp pulses in the fracture-associated breakthrough curves in the monitor well. Back diffusion of oxygen from the porous matrix into the injection well was accurately reproduced by the model. Media properties (fracture apertures, hydraulic gradient and hydraulic conductivity) were varied to assess the sensitivity of the model and to evaluate the effectiveness of the remediation technology under different conditions. The sensitivity runs demonstrated that the distribution of oxygen within the system could be significantly different with varying degrees of advective transport within the fractures and diffusion into the rock matrix which depends on the physical properties and hydrogeological conditions. Predictive simulations were then run with two different injection scenarios: a continuous injection for 1 week and a cyclic injection scenario (injection every 2 days). The same mass of oxygen was delivered in each simulation (~3 kg). The results demonstrated that the delivery of oxygen into the system (continuous or cyclic) could affect the advective transport of oxygen through the fractures and the diffusion of oxygen into the matrix. The continuous injection resulted in a maximum zone of influence (down-gradient and in the transverse direction) while maintaining high levels of oxygen within the matrix. On the other hand the cycle injection provided a more continuous supply of oxygen over time to the system. The zone of influence was reduced but diffusion into the matrix along the fractures increased, creating a more uniform zone of increased oxygen concentrations around the injection well and along the fractures. This study demonstrated that oxygen could effectively be delivered to a fractured bedrock system at levels potentially sufficient to enhance aerobic biodegradation. Additional areas requiring investigation include the behavior of oxygen during hydrocarbon biodegradation through field and modelling studies. Full scale implementation of the technology should then be considered to provide additional information with respect to the applicability of the technology to real world environments.

Best or beneficial management practices (BMPs) are often relied upon as a mitigation strategy for nitrate contamination throughout Canada. At a regional scale, reducing the quantity of nutrients applied to agricultural land is one BMP approach that has been implemented internationally. While these BMP strategies have been proven to successfully reduce the environmental impact of agriculture on water systems, the time interval between BMP implementation and a noticeable improvement in groundwater quality can be quite extensive. This lag time has been observed at the agriculturally impacted Thornton Well Field in Oxford County. Despite seven years of significant reductions in fertilizer application within the capture zone of this municipal well field, declining nitrate concentrations have yet to be observed in the production water wells. In order to accelerate nitrate reductions at the Thornton Well Field, an integrated approach, combining BMPs with a stimulated in situ denitrification strategy, was implemented. This research focused on the use of a cross-injection scheme to stimulate in situ denitrification within the production aquifer units, up-gradient of the Thornton Well Field. Briefly, this strategy involves injecting a carbon source and electron donor into a high flux aquifer zone using an injection and extraction system positioned perpendicular to the regional flow field. Through altering the geochemical conditions, the injections stimulate indigenous bacteria to reduce harmful nitrate to innocuous dinitrogen gas. The main objectives of this research included: characterizing the hydrogeologic and geochemical properties of the target aquifer; pilot scale testing of the proposed in situ denitrification system; and suggesting an approach for up-scaling to a full-scale treatment scheme capable of remediating the elevated nitrate concentrations at the Thornton Well Field. Core logging, electrical resistivity studies, several methods of hydraulic characterization, tracer testing, and three-dimensional groundwater modelling were used to quantify the physical properties of the target aquifer and to develop a hydrogeologic conceptual model of the site. The aquifer unit was found to be unconfined in the experiment vicinity, consisting of a complex system of six main hydrostratigraphic layers of sand and gravel featuring variable hydraulic conductivity (K) values. Despite the hydrogeologic complexity, the geochemical properties of the aquifer were relatively uniform with depth. Anion, cation, alkalinity, pH, dissolved oxygen, and nitrous oxide data all contributed to this conjecture. Of particular interest, however, were the elevated dissolved oxygen concentrations, which rivalled atmospheric saturation throughout the entire aquifer sequence. The background physical and chemical characterization identified two main challenges that would potentially influence the performance of the in situ denitrification process: stimulating uniform denitrification in the fast flowing, complex aquifer system and overcoming the elevated oxygen concentrations to achieve the necessary anaerobic conditions. Following the initial site characterization phase, several preliminary cross-injection experiments were designed and performed. These experiments featured an injection-extraction circulation cycle which spanned five metres and was operated normal to groundwater flow. Acetate was selected as the electron donor and carbon substrate. The first test involved a single acetate injection followed by an extensive period of groundwater sampling. Unfortunately, this initial test provided no indication of stimulated in situ denitrification. All anion, cation, and nitrous oxide concentration and isotope data collected during and following this injection remained within the range of background estimates. Following the first injection experiment, a subsequent test involving multiple, repetitive acetate injections was implemented to overcome the highly aerobic nature of the aquifer and support the growth and reproduction of denitrifying bacterial populations. The second injection phase included 19 individual injections that were operated at intervals of every day to every other day over a total period of 26 days. These injections successfully lowered the dissolved oxygen concentrations within the target aquifer to an average range of 0 to 4 mg/L. The least conductive layers featured the lowest oxygen concentrations, while the higher K layers maintained elevated oxygen concentrations. The nitrite, nitrate, and enriched NO3-15N and NO3-18O isotope data suggested a high degree of stimulated denitrification in the least conductive layers and a limited degree in the high-K layers. The lower-K units corresponding to multi-level well ports ML7-2, ML7-5, and ML7-6 achieved a 46 percent reduction in nitrate, while the layer represented by ML7-1 attained a 100 percent reduction in nitrate. Alternatively, due to the constant influx of dissolved oxygen and limited residence times, very little denitrification was observed in the fast flowing layers corresponding to ports ML7-3, ML7-4, and ML7-7. Overall, a percent reduction, in terms of nitrate mass crossing the 5-m wide treatment lens, of only eleven percent was calculated. These results clearly demonstrate that the K-profile had a significant impact on stimulating in situ bioremediation. Two major system challenges were observed, including an inability to successfully stimulate denitrification within the highly permeable layers and the generation of harmful nitrite at nearly all aquifer depths. Based on these significant challenges, it was concluded that additional experimentation is required before this remediation technique can be expanded to a full-scale in situ treatment scheme. The most significant recommendation requested the development and execution of a third injection phase, consisting of multiple, consecutive substrate injections designed to systematically test various pulsing intervals, injection concentrations, and electron donors. Despite the current limitations, this approach has great potential. It is believed that with additional research, the in situ stimulation of denitrification could be used to successfully reduce the elevated nitrate concentrations at the Thornton Well Field.

Oxygen has been shown to be an effective addition of enhancing the bioremediation of petroleum hydrocarbon contamination in porous media; however, the ability to effectively deliver oxygen to petroleum hydrocarbon contaminated groundwater has proven difficult. A field and numerical modelling study was completed at a former gas station in southern Ontario, to assess the delivery of oxygen into groundwater in a fractured limestone aquifer that had been contaminated with petroleum hydrocarbons. A field investigation was completed to characterize the bedrock aquifer and the groundwater flow system. Several hydraulically active fracture zones were identified and characterized. To evaluate how dissolved oxygen would behave in this type of groundwater environment, an injection test was completed using iTi’s gPro® oxygen injection technology. About 1000 L of water containing dissolved oxygen at ~ 30 mg/L and a bromide tracer was injected over ~ 90 minutes and monitored for ~ 10 days in the injection well and in a multilevel monitoring well located 3 metres down-gradient. The oxygen concentration rose rapidly within the injection well and at two of the down-gradient monitor intervals which were aligned with the injection well via major fractures. Concentration tailing persisted in the injection well for several days following injection. The effects of biodegradation were not assessed as part of this investigation. A three-dimensional numerical model for groundwater flow and advective-dispersive transport within a discretely-fractured porous medium was calibrated to the field conditions. The simulated injection test demonstrated that oxygen rapidly filled the porous matrix surrounding the injection well and filled the local intersecting fractures. Following injection, the oxygenated groundwater in the local fractures was rapidly flushed by the natural groundwater flow, with oxygen arrivals appearing as sharp pulses in the fracture-associated breakthrough curves in the monitor well. Back diffusion of oxygen from the porous matrix into the injection well was accurately reproduced by the model. Media properties (fracture apertures, hydraulic gradient and hydraulic conductivity) were varied to assess the sensitivity of the model and to evaluate the effectiveness of the remediation technology under different conditions. The sensitivity runs demonstrated that the distribution of oxygen within the system could be significantly different with varying degrees of advective transport within the fractures and diffusion into the rock matrix which depends on the physical properties and hydrogeological conditions. Predictive simulations were then run with two different injection scenarios: a continuous injection for 1 week and a cyclic injection scenario (injection every 2 days). The same mass of oxygen was delivered in each simulation (~3 kg). The results demonstrated that the delivery of oxygen into the system (continuous or cyclic) could affect the advective transport of oxygen through the fractures and the diffusion of oxygen into the matrix. The continuous injection resulted in a maximum zone of influence (down-gradient and in the transverse direction) while maintaining high levels of oxygen within the matrix. On the other hand the cycle injection provided a more continuous supply of oxygen over time to the system. The zone of influence was reduced but diffusion into the matrix along the fractures increased, creating a more uniform zone of increased oxygen concentrations around the injection well and along the fractures. This study demonstrated that oxygen could effectively be delivered to a fractured bedrock system at levels potentially sufficient to enhance aerobic biodegradation. Additional areas requiring investigation include the behavior of oxygen during hydrocarbon biodegradation through field and modelling studies. Full scale implementation of the technology should then be considered to provide additional information with respect to the applicability of the technology to real world environments.

Best or beneficial management practices (BMPs) are often relied upon as a mitigation strategy for nitrate contamination throughout Canada. At a regional scale, reducing the quantity of nutrients applied to agricultural land is one BMP approach that has been implemented internationally. While these BMP strategies have been proven to successfully reduce the environmental impact of agriculture on water systems, the time interval between BMP implementation and a noticeable improvement in groundwater quality can be quite extensive. This lag time has been observed at the agriculturally impacted Thornton Well Field in Oxford County. Despite seven years of significant reductions in fertilizer application within the capture zone of this municipal well field, declining nitrate concentrations have yet to be observed in the production water wells. In order to accelerate nitrate reductions at the Thornton Well Field, an integrated approach, combining BMPs with a stimulated in situ denitrification strategy, was implemented. This research focused on the use of a cross-injection scheme to stimulate in situ denitrification within the production aquifer units, up-gradient of the Thornton Well Field. Briefly, this strategy involves injecting a carbon source and electron donor into a high flux aquifer zone using an injection and extraction system positioned perpendicular to the regional flow field. Through altering the geochemical conditions, the injections stimulate indigenous bacteria to reduce harmful nitrate to innocuous dinitrogen gas. The main objectives of this research included: characterizing the hydrogeologic and geochemical properties of the target aquifer; pilot scale testing of the proposed in situ denitrification system; and suggesting an approach for up-scaling to a full-scale treatment scheme capable of remediating the elevated nitrate concentrations at the Thornton Well Field. Core logging, electrical resistivity studies, several methods of hydraulic characterization, tracer testing, and three-dimensional groundwater modelling were used to quantify the physical properties of the target aquifer and to develop a hydrogeologic conceptual model of the site. The aquifer unit was found to be unconfined in the experiment vicinity, consisting of a complex system of six main hydrostratigraphic layers of sand and gravel featuring variable hydraulic conductivity (K) values. Despite the hydrogeologic complexity, the geochemical properties of the aquifer were relatively uniform with depth. Anion, cation, alkalinity, pH, dissolved oxygen, and nitrous oxide data all contributed to this conjecture. Of particular interest, however, were the elevated dissolved oxygen concentrations, which rivalled atmospheric saturation throughout the entire aquifer sequence. The background physical and chemical characterization identified two main challenges that would potentially influence the performance of the in situ denitrification process: stimulating uniform denitrification in the fast flowing, complex aquifer system and overcoming the elevated oxygen concentrations to achieve the necessary anaerobic conditions. Following the initial site characterization phase, several preliminary cross-injection experiments were designed and performed. These experiments featured an injection-extraction circulation cycle which spanned five metres and was operated normal to groundwater flow. Acetate was selected as the electron donor and carbon substrate. The first test involved a single acetate injection followed by an extensive period of groundwater sampling. Unfortunately, this initial test provided no indication of stimulated in situ denitrification. All anion, cation, and nitrous oxide concentration and isotope data collected during and following this injection remained within the range of background estimates. Following the first injection experiment, a subsequent test involving multiple, repetitive acetate injections was implemented to overcome the highly aerobic nature of the aquifer and support the growth and reproduction of denitrifying bacterial populations. The second injection phase included 19 individual injections that were operated at intervals of every day to every other day over a total period of 26 days. These injections successfully lowered the dissolved oxygen concentrations within the target aquifer to an average range of 0 to 4 mg/L. The least conductive layers featured the lowest oxygen concentrations, while the higher K layers maintained elevated oxygen concentrations. The nitrite, nitrate, and enriched NO3-15N and NO3-18O isotope data suggested a high degree of stimulated denitrification in the least conductive layers and a limited degree in the high-K layers. The lower-K units corresponding to multi-level well ports ML7-2, ML7-5, and ML7-6 achieved a 46 percent reduction in nitrate, while the layer represented by ML7-1 attained a 100 percent reduction in nitrate. Alternatively, due to the constant influx of dissolved oxygen and limited residence times, very little denitrification was observed in the fast flowing layers corresponding to ports ML7-3, ML7-4, and ML7-7. Overall, a percent reduction, in terms of nitrate mass crossing the 5-m wide treatment lens, of only eleven percent was calculated. These results clearly demonstrate that the K-profile had a significant impact on stimulating in situ bioremediation. Two major system challenges were observed, including an inability to successfully stimulate denitrification within the highly permeable layers and the generation of harmful nitrite at nearly all aquifer depths. Based on these significant challenges, it was concluded that additional experimentation is required before this remediation technique can be expanded to a full-scale in situ treatment scheme. The most significant recommendation requested the development and execution of a third injection phase, consisting of multiple, consecutive substrate injections designed to systematically test various pulsing intervals, injection concentrations, and electron donors. Despite the current limitations, this approach has great potential. It is believed that with additional research, the in situ stimulation of denitrification could be used to successfully reduce the elevated nitrate concentrations at the Thornton Well Field.