Enhanced Reductive Bioremediation (ERB) is a sustainable remediation technology for the in situ treatment of chlorinated solvent contamination in aquifers. However, monitoring efforts employed to measure performance metrics rely on inferences of the subsurface environment from water samples collected at monitoring wells, ignoring the microbial activity that occurs at the granular level of aquifer sediment. This study compared potential bioavailable organic carbon (PBOC) and microbial diversity of two ERB sites. A two-sample t-test and a one-way ANOVA test with Tukey's HSD were performed to show differences between ERB and non-ERB samples and their degree of variability at selected geospatial locations downgradient of ERB treatment. Non-parametric multidimensional scaling (MDS) with similarity analysis was performed along with other data visualization plots to show microbial diversity. At Tinker AFB, results from the t-test showed that the PBOC concentrations from the ERB samples were statistically significantly greater than the samples without treatment (95% confidence; p-value = 0.018). For Dover AFB, results from the ANOVA with Tukey's HSD showed that there is a significant difference between the sample (DV3) collected in the ERB treatment zone to all other samples upgradient and downgradient of the ERB treatment. MDS and similarity analysis performed on relative abundance results from the Illumina MiSeq Sequencing of 16S rRNA genes showed large similarities among the samples within each site and the only observed differences occurred when comparing any sample to DV3, nearest to treatment. / Master of Science / Enhanced Reductive Bioremediation (ERB) is a sustainable remediation technology for the treatment of chlorinated solvent contamination in groundwater within the subsurface. ERB acts as a stimulant of microbial communities to accelerate remediation. Current ways to measure the success of remediation technologies use water from monitoring wells and they ignore the microbial activity that occurs within the subsurface sediment, where the water is stored. This study compared potential bioavailable organic carbon (PBOC) and microbial diversity of two ERB sites. PBOC is the amount of carbon in sediment that is consumable by bacteria. Microbial diversity are the various communities of microscopic organisms present in a sediment sample. A two-sample t-test and a one-way ANOVA test with Tukey’s HSD were performed to show differences between ERB and non-ERB samples and their degree of variability at selected geospatial locations downgradient of ERB treatment. A two-sample t-test determined if there is a statistical difference between two values. A one-way ANOVA test compared multiple values to each other and all their possible combinations. The Tukey’s HSD showed how different those values were from each other, from the ANOVA test results. Non-parametric multidimensional scaling (MDS) with similarity analysis was performed along with other data visualization plots to show microbial diversity. These visualization techniques helped determine similarities and demostrate microbial diversity. At Tinker AFB, results from the t-test showed that the PBOC concentrations from the ERB samples were statistically significantly greater than the samples without treatment (95% confidence; pvalue = 0.018). For Dover AFB, results from the ANOVA with Tukey’s HSD showed that there is a significant difference between the samples (DV3) collected in the ERB treatment zone to all other samples upgradient and downgradient of the ERB treatment. MDS and similarity analysis performed on relative abundance results from the Illumina MiSeq Sequencing of 16S rRNA genes showed large similarities among the samples within each site and the only observed differences occurred when comparing any sample to DV3, nearest to treatment.

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.

Les chloroéthènes sont les polluants majeurs des eaux souterraines et des nappes phréatiques. De par leur toxicité et leur effet cancérigène, ils représentent une préoccupation majeure pour les autorités publiques et sanitaires. La restauration des sites contaminés est possible par des techniques de dépollution biologique impliquant les microorganismes (bioremédiation microbienne). Cependant, la réussite des traitements dépend à la fois des conditions physicochimiques du site pollué et des capacités de dégradation de la microflore indigène. Ainsi, pour optimiser les processus de décontamination, l’identification et le suivi des différentes populations microbiennes sont indispensables avant et pendant le traitement. Les biopuces ADN fonctionnelles (FGA, Functional Gene Array), outils moléculaires haut débit, sont particulièrement bien adaptées pour des applications en bioremédiation. Leur élaboration nécessite de disposer de logiciels performants pour le design de sondes qui combinent à la fois une forte sensibilité, une très bonne spécificité et un caractère exploratoire, ce dernier étant indispensable pour la détection des séquences connues mais surtout de celles encore jamais décrites au sein d’échantillons environnementaux. Un nouveau logiciel, autorisant la sélection de sondes combinant tous ces critères, a été développé et nommé HiSpOD. Son utilisation pour la construction d’une FGA dédiée aux voies de biodégradation des chloroéthènes a permis d’évaluer l’effet de traitements de biostimulation sur la microflore indigène pour plusieurs sites industriels contaminés. Les données révèlent différentes associations entre microorganismes déhalorespirants qui sont fonction des paramètres environnementaux. / Chlorinated solvents are among the most frequent contaminants found in groundwater and subsurface ecosystems. Because of their high toxicity and carcinogenicity, they represent a serious risk for human health and the environment. Thus, such polluted sites need a rehabilitation treatment. Among remediation solutions, microbial bioremediation represents a less invasive and expensive alternative than physico-chemical treatments. However, the process efficiency greatly depends on the environmental conditions and the microbial populations’ biodegradation capacities. Therefore, bioremediation treatment optimization requires the identification and monitoring of such capacities before and during the treatment. Functional Gene Arrays (FGA), by profiling environmental communities in a flexible and easy-to-use manner, are well adapted for an application in bioremediation. But, constructing efficient microarrays dedicated to microbial ecology requires a probe design step allowing the selection of highly sensitive, specific and explorative oligonucleotides. After a detailed state of the art on probe design strategies suitable for microbial ecology studies, we present new software, called HiSpOD, generating efficient explorative probes for FGA dedicated to environmental applications. Finally, this bioinformatics tool was used to construct a FGA targeting most genes involved in chloroethenes biodegradation pathways which allowed the evaluation of biostimulation treatments conducted on indigenous bacterial populations for several industrial contaminated sites.

Groundwater remediation strategies have advanced toward more effective and economical remedial technologies. Monitored natural attenuation (MNA) has become accepted by federal regulatory agencies as a viable remediation strategy for contaminants under site-specific conditions. At chloroethene contaminated sites where MNA is used as a remediation strategy, microbially-mediated reductive dechlorination is typically the dominant pathway for natural attenuation. The efficacy of reductive dechlorination at sites with no anthropogenic carbon sources is often influenced by the availability of readily-biodegradable natural organic carbon along with favorable geochemical conditions for supporting microbial dehalogenation. Recent research studies have suggested that the pool of labile natural organic carbon, operationally defined as potentially bioavailable organic carbon (PBOC), may be a critical component related to sustaining reductive dechlorination at MNA sites. The objective of this study was to evaluate PBOC as a quantitative measure of the labile organic carbon fraction of aquifer sediments in relation to microbial reductive dechlorination of chlorinated solvents. In the first phase of this study, the variability of PBOC in aquifer sediments was examined among 15 chloroethene contaminated sites. Results showed that PBOC displayed considerable variability among the study sites, ranging over four orders of magnitude. Regression results demonstrated that a positive correlation existed between PBOC, solid phase total organic carbon (TOCs), and reductive dechlorination activity at the sites. Results supported that greater levels of PBOC and TOCs corresponded to higher reductive dechlorination activity at the sites. Composition results showed that 6-86% of PBOC consisted of proteins and amino acids. Results also suggested a positive relationship existed between PBOC, concentrations of potentially bioavailable organic compounds present in the aquifer system, expressed as hydrolyzable amino acids (HAA), and the natural attenuation capacity (NAC) at the sites. Higher PBOC levels were consistently observed at sites with greater NAC and levels of HAA. The results of this study suggested that the variability of PBOC in the aquifer sediments exhibited a reasonable correlation with TOCs, hydrolyzable amino acids, and chloroethene transformation among the selected sites. In the second phase of this study, the relationship between PBOC in aquifer sediments and site specific performance data was evaluated among 12 chloroethene contaminated sites. Results demonstrated that PBOC in aquifer sediments was directly correlated to independent field metrics associated with reductive dechlorination. Levels of PBOC demonstrated direct relationships with hydrogen (H2) and dissolved oxygen (DO) concentrations within the groundwater system at the selected study sites. Results also indicated that PBOC demonstrated positive relationships with reductive dechlorination activity and the natural attenuation capacity of the sites. The findings of this study suggested that the level of PBOC in aquifer sediments may be a key factor in sustaining conditions favorable for microbial reductive dechlorination. In the third phase of this study, the distribution of PBOC was investigated at a chloroethene contaminated site. PBOC was measured in surficial aquifer sediment samples collected at varying depths in the vicinity of a chloroethene plume. Results demonstrated that levels of PBOC were consistently higher in aquifer sediments with minimal chloroethene exposure relative to samples collected in the PCE-contaminated source zone. Regression results demonstrated that a statistically significant inverse correlation existed between PBOC levels and chloroethene concentrations for selected temporary wells in the contaminated source zone at the study site. Consistent with these findings, results also indicated a similar trend of increased PBOC in aquifer sediments outside the chloroethene plume relative to aquifer sediments inside the plume. Results from this study further suggested that differences in extracted carbon levels at the site for surficial aquifer sediment samples in the PCE-contaminated source zone could impact the extent of reductive dechlorination within the hydrographic unit. / Ph. D.