Site characterization methods for measuring the occurrence, magnitude, and rate of microbial mediated transformation processes were evaluated to assess the implementation of monitored natural attenuation (MNA) at chlorinated ethenes contaminated sites. A model site in Arizona, the Park-Euclid WQARF site in Tucson, was selected for the study. Field, geochemical, and compound specific carbon isotope fractionation (CSI) data confirm intrinsic biodegradation is occurring in the perched aquifer. Use of the BIOCHLOR model and a screening protocol support the potential for reductive dehalogenation found in the perched aquifer. Biotransformation of tetrachloroethene to cis-1,2-dichloroethene (cis-DCE) was achieved in microcosm studies. Transformation of cis-DCE to vinyl chloride and to ethene is, at the moment, the laboratory rate limiting step. PCR analysis established that the aquifer contains Dehalococcoides sp. and other dechlorinating microorganisms, though genes that encode for enzymes capable of achieving complete dehalogenation of the chlorinated contaminants were confirmed only in one monitoring well. The regional aquifer shows little evidence of intrinsic biodegradation. This study corroborates that CSI analysis can be used as an additional line of evidence to evaluate and verify MNA. Microbial analysis provides relevant information about the capabilities of native microbial communities to carry out reductive dehalogenation and thus, to naturally attenuate chlorinated compounds at a contaminated site. The combination of microcosm studies, CSI analysis, and bacterial DNA identification is becoming a convincing line of evidence for the assessment of MNA application to chloroethenes contaminated sites.

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / Halogenated organic compounds have had widespread and massive applications in industry, agriculture, and private households, for example, as degreasing solvents, flame retardants and in polymer production. They are released to the environment through both anthropogenic and natural sources. The most common chlorinated solvents present as contaminants include tetrachloroethylene (PCE, perchloroethene), trichloroethene (TCE), trichloroethane (TCA), and carbon tetrachloride (CT). These chlorinated solvents are problematic because of their health hazards and persistence in the environment, threatening human and environmental health. This contribution provides insight on PCE degradation at laboratory and field scale at a former dry cleaning site in Manhattan, KS. Biostimulation experiments included combinations and concentrations of the following nutrients: soy oil methyl esters (SOME), yeast extract (YE), glucose, lactate, methanol and cheese whey. Bioaugmentation studies used KB-1 bacterial consortium (commercially available culture containing Dehalococcoides). This culture is known to complete the degradation of PCE to a safe end product, ethene. Concentrations of PCE and its degradation intermediates were monitored in the gas phase of the microcosm vials. Biostimulation of the natural ground water and soil microflora did not completely degrade PCE as cis-DCE (c-DCE) accumulated in the sample. Bioaugmented microcosms containing YE and SOME created reducing conditions for KB-1 culture, resulting in ~ 90% dechlorination of PCE to methane and c-DCE. Cheese whey microcosms containing 0.05% cheese whey inhibited the KB-1 culture. This inhibition was due to a drop of pH that inhibited the culture activity. Lower concentrations of cheese whey (e.g. 0.01% to 0.025%) reduced PCE and generated methane in KB-1 augmented microcosms. Based on microcosm results, a pilot bioremediation field study was conducted for a dry cleaning site contaminated with PCE. Ground water flow threatened public water wells located 1.5 miles from the source. Concentrations of PCE in the aquifer was 15 mg/L above the maximum contaminant level of 5 µg/L. Tracer studies with potassium bromide (KBr) were conducted before, during and after the bioremediation study. Nutrient solutions prepared with YE, SOME, lactate and glucose were used for biostimulation and preconditioning of ground water prior to KB-1 injection. Nutrients were provided twice during the pilot study to supplement microbial growth and cheese whey was used. During biostimulation no degradation beyond DCE was evident. The addition of KB-1 reduced PCE and DCE concentrations in the monitoring wells of the pilot study area. Total chlorinated ethene concentrations did not reach background levels 2 years after the last nutrient addition. Tracer results showed that microbial growth decreased ground water velocity during the study, but returned to normal conditions 1 year after the last nutrient addition. In this study we were able to show that native microbial population was not able to degrade PCE to final end products. Therefore, it was necessary to introduce KB-1 culture a long with nutrients to support complete reductive dechlorination of PCE.

Dentre os contaminantes de água subterrânea de maior importância está o tetracloroeteno (PCE), o qual é altamente tóxico e potencialmente carcinógeno. As comunidades bacterianas de águas subterrâneas contaminadas com PCE e a diversidade de bactérias capazes de degradar esses organoclorados são pouco conhecidas. O objetivo deste trabalho é comparar a estrutura das comunidades de bactérias de amostras de água subterrânea em uma área contaminada com PCE e selecionar um consórcio microbiano capaz de degradar eficientemente o PCE em reator horizontal de leito fixo (RHLF). Amostras de água subterrânea de oito poços de monitoramento, instalados em uma área contaminada com PCE foram coletadas e analisadas para determinação de oxigênio dissolvido, potencial redox, condutividade elétrica, pH e concentração de tetracloroeteno, tricloroeteno, cis-dicloroeteno e cloreto de vinila (COVs). As amostras foram analisadas também para a determinação da estrutura das comunidades de bactéria por PCRDGGE e seqüenciamento de clones do gene rRNA 16S. Os parâmetros físico-químicos oscilaram consideravelmente ao longo do tempo em todos os poços de monitoramento (PM). Tetracloroeteno e tricloroeteno foram detectados apenas no PM6. As estruturas das comunidades bacterianas dos PMs analisados mostraram tanto variação temporal quanto espacial. As análises das comunidades bacterianas nos PM6 e PM8, contaminado e não-contaminado com PCE, revelaram resultados semelhantes aos obtidos por DGGE. Uma maior riqueza estimada de espécies bacterianas foi observada nas amostras do PM8, pelo menos em duas épocas de amostragem, sugerindo que a contaminação com PCE está associada com a redução da diversidade bacteriana em água subterrânea. Cultivos de enriquecimento e ensaios de degradação do PCE foram realizados utilizando-se um RHLF, o qual foi preenchido com sedimento do PM6 imobilizado em espuma de poliuretano e enriquecido com meio mineral básico suplementado com PCE. A análise das alterações nas comunidades de bactérias nos reatores foi feita por PCRDGGE e seqüenciamento parcial do gene rRNA 16S. No ensaio de degradação do PCE no RHLF foi utilizado meio com PCE suplementado ou não com lactato e acetato. Tanto pelo DGGE quanto pelo seqüenciamento, foi observada a seleção de bactérias específicas no reator. A partir das análises de seqüenciamento, essas bactérias foram identificadas como Alphaproteobacteria e Sphingobacteria. No ensaio de degradação do PCE, os parâmetros físico-químicos do meio não mostraram variações ao longo do comprimento dos reatores. As análises de COVs mostraram uma grande eficiência na degradação do PCE (98%), com um tempo de retenção de 12 horas, não havendo diferença significativa na percentagem de degradação em meio com lactato ou acetato, com relação ao controle sem fonte de carbono. No processo de degradação nenhum dos produtos da via de degradação do PCE foi detectado, o que sugere uma via alternativa de degradação do PCE, a qual ocorre em aerobiose. / Tetrachloroethene (PCE) is one of the most important contaminants of groundwater, since it is highly toxic and potentially carcinogenic. The bacterial communities of PCE contaminated groundwater and the diversity of bacteria capable of degrading this contaminant are barely known. The objective of this work is to compare the structure of bacterial communities from groundwater samples from a PCE contaminated site and select a microbial consortium capable to degrading efficiently PCE in a horizontal fixed bed reactor (HFBR). Groundwater samples from eight monitoring wells, installed in a PCE contaminated site were collected and analyzed for determination of dissolved oxygen, redox potential, electrical conductivity, pH, and concentrations of tetrachloroethene, trichloroethene (TCE), cis- and trans-dichloroethene, vinyl chloride (VOCs). The structure of the bacterial communities was determined by PCR-DGGE and 16S rRNA gene clone sequencing. The physical-chemical parameters oscillated considerately throughout time in all the monitoring wells (MW). PCE and TCE were detected only in MW6. The bacterial community structures in the groundwater from the MWs analyzed showed temporal and spatial variation. The analysis of the bacterial communities in MW6 and MW8, contaminated and non-contaminated with PCE, respectively, based on sequencing of 16S rRNA gene clones revealed results to the ones observed by DGGE. Estimated richness of bacterial species was higher in samples from MW8, at least in two sampling times, suggesting that the contamination with PCE is associated with reduction of bacterial diversity in groundwater. Enrichment cultures and PCE biodegradation assays were performed in a HFBR, which was filled with sediment from MW6 immobilized onto polyurethane foam and enriched with basic mineral medium supplemented with PCE. Shifts in bacterial community structure were analyzed using PCRDGGE and partial sequencing of 16S rRNA gene clones. In the PCE biodegradation assays in the HFBR, were performed in medium containing lactate or acetate. DGGE and 16S rRNA gene clone sequencing data suggest selection of specific bacteria in the reactor. Sequencing data showed that these bacteria belong to Alphaproteobacteria and Sphingobacteria. In the PCE biodegradation assays, media physical-chemical parameters did not show variation along the reactor length. VOC analyses showed a great efficiency in the degradation of PCE (98%) with a residence time of 12 hours in the reactor, and no significant differences were observed in the presence of lactate or acetate, as compared to the medium without a carbon source. During the biodegradation process, none of the products from the anaerobic pathway of PCE reductive dechlorination was detected, suggesting that an alternative PCE biodegradation pathway is occurring in aerobiosis.

Dentre os contaminantes de água subterrânea de maior importância está o tetracloroeteno (PCE), o qual é altamente tóxico e potencialmente carcinógeno. As comunidades bacterianas de águas subterrâneas contaminadas com PCE e a diversidade de bactérias capazes de degradar esses organoclorados são pouco conhecidas. O objetivo deste trabalho é comparar a estrutura das comunidades de bactérias de amostras de água subterrânea em uma área contaminada com PCE e selecionar um consórcio microbiano capaz de degradar eficientemente o PCE em reator horizontal de leito fixo (RHLF). Amostras de água subterrânea de oito poços de monitoramento, instalados em uma área contaminada com PCE foram coletadas e analisadas para determinação de oxigênio dissolvido, potencial redox, condutividade elétrica, pH e concentração de tetracloroeteno, tricloroeteno, cis-dicloroeteno e cloreto de vinila (COVs). As amostras foram analisadas também para a determinação da estrutura das comunidades de bactéria por PCRDGGE e seqüenciamento de clones do gene rRNA 16S. Os parâmetros físico-químicos oscilaram consideravelmente ao longo do tempo em todos os poços de monitoramento (PM). Tetracloroeteno e tricloroeteno foram detectados apenas no PM6. As estruturas das comunidades bacterianas dos PMs analisados mostraram tanto variação temporal quanto espacial. As análises das comunidades bacterianas nos PM6 e PM8, contaminado e não-contaminado com PCE, revelaram resultados semelhantes aos obtidos por DGGE. Uma maior riqueza estimada de espécies bacterianas foi observada nas amostras do PM8, pelo menos em duas épocas de amostragem, sugerindo que a contaminação com PCE está associada com a redução da diversidade bacteriana em água subterrânea. Cultivos de enriquecimento e ensaios de degradação do PCE foram realizados utilizando-se um RHLF, o qual foi preenchido com sedimento do PM6 imobilizado em espuma de poliuretano e enriquecido com meio mineral básico suplementado com PCE. A análise das alterações nas comunidades de bactérias nos reatores foi feita por PCRDGGE e seqüenciamento parcial do gene rRNA 16S. No ensaio de degradação do PCE no RHLF foi utilizado meio com PCE suplementado ou não com lactato e acetato. Tanto pelo DGGE quanto pelo seqüenciamento, foi observada a seleção de bactérias específicas no reator. A partir das análises de seqüenciamento, essas bactérias foram identificadas como Alphaproteobacteria e Sphingobacteria. No ensaio de degradação do PCE, os parâmetros físico-químicos do meio não mostraram variações ao longo do comprimento dos reatores. As análises de COVs mostraram uma grande eficiência na degradação do PCE (98%), com um tempo de retenção de 12 horas, não havendo diferença significativa na percentagem de degradação em meio com lactato ou acetato, com relação ao controle sem fonte de carbono. No processo de degradação nenhum dos produtos da via de degradação do PCE foi detectado, o que sugere uma via alternativa de degradação do PCE, a qual ocorre em aerobiose. / Tetrachloroethene (PCE) is one of the most important contaminants of groundwater, since it is highly toxic and potentially carcinogenic. The bacterial communities of PCE contaminated groundwater and the diversity of bacteria capable of degrading this contaminant are barely known. The objective of this work is to compare the structure of bacterial communities from groundwater samples from a PCE contaminated site and select a microbial consortium capable to degrading efficiently PCE in a horizontal fixed bed reactor (HFBR). Groundwater samples from eight monitoring wells, installed in a PCE contaminated site were collected and analyzed for determination of dissolved oxygen, redox potential, electrical conductivity, pH, and concentrations of tetrachloroethene, trichloroethene (TCE), cis- and trans-dichloroethene, vinyl chloride (VOCs). The structure of the bacterial communities was determined by PCR-DGGE and 16S rRNA gene clone sequencing. The physical-chemical parameters oscillated considerately throughout time in all the monitoring wells (MW). PCE and TCE were detected only in MW6. The bacterial community structures in the groundwater from the MWs analyzed showed temporal and spatial variation. The analysis of the bacterial communities in MW6 and MW8, contaminated and non-contaminated with PCE, respectively, based on sequencing of 16S rRNA gene clones revealed results to the ones observed by DGGE. Estimated richness of bacterial species was higher in samples from MW8, at least in two sampling times, suggesting that the contamination with PCE is associated with reduction of bacterial diversity in groundwater. Enrichment cultures and PCE biodegradation assays were performed in a HFBR, which was filled with sediment from MW6 immobilized onto polyurethane foam and enriched with basic mineral medium supplemented with PCE. Shifts in bacterial community structure were analyzed using PCRDGGE and partial sequencing of 16S rRNA gene clones. In the PCE biodegradation assays in the HFBR, were performed in medium containing lactate or acetate. DGGE and 16S rRNA gene clone sequencing data suggest selection of specific bacteria in the reactor. Sequencing data showed that these bacteria belong to Alphaproteobacteria and Sphingobacteria. In the PCE biodegradation assays, media physical-chemical parameters did not show variation along the reactor length. VOC analyses showed a great efficiency in the degradation of PCE (98%) with a residence time of 12 hours in the reactor, and no significant differences were observed in the presence of lactate or acetate, as compared to the medium without a carbon source. During the biodegradation process, none of the products from the anaerobic pathway of PCE reductive dechlorination was detected, suggesting that an alternative PCE biodegradation pathway is occurring in aerobiosis.

The objective of this study was to utilize an alternating anaerobic/aerobic sequence to biologically transform perchloroethylene to non-hazardous end products such as ethylene, CO2 and H2 using a single microbial consortia in a methanogenic and/or a homoacetogenic environment followed by a aerobic methanotrophic environment. Reductive dechlorination of PCE and TCE to cDCE and VC in an anaerobic environment is typically carried out by methanogens, sulfidogens, or homoacetogens but often (e.g. in-situ) leads to an accumulation of daughter compounds (cDCE, VC) which are more toxic than their parent compounds (PCE, TCE). Furthermore, PCE is resistant to degradation in aerobic environments while VC and cDCE are readily oxidized co-metabolically by aerobic methanotrophic bacteria, among others. In order to achieve complete mineralization of chlorinated solvents using a biotic system, an anaerobic/aerobic treatment strategy was investigated. This strategy has been accomplished successfully at a lab scale with anaerobic and aerobic reactors in series, and in-situ anaerobic zones with downgradient aerobic zones have been proposed in the field. In contrast, the focus of this research was to expose single mixed microbial consortia to sequential anaerobic/aerobic treatments in order to determine if reductive dechlorination could be sustained following aerobic phases of treatment. If possible this would imply that the anaerobic and aerobic zones (in-situ) or reactors (ex-situ) would not necessarily have to be spatially separated. In pure or dilute cultures where soil material is not present strict anaerobes would typically not resume metabolic activity if exposed to frequent aerobic phases of treatment. However in aquifer material or reactors with large floc/granules it might be possible due to the protection of anaerobic micro-environments as a result of diffusion limitations. Microcosms contained in sealed 120-mL serum bottles were used to generate experimental data including autoclaved abiotic controls with mercuric chloride. Inocula for these microcosms come from a several sources, including anaerobic digester sludge, soils, and contaminated aquifers. Once an experimental microcosm showed signs of reductive dechlorination, an aerobic treatment was implemented. The anaerobic phase of the microcosm was interrupted with a short duration aerobic phase. Headspace air or hydrogen peroxide addition was used to supply oxygen. Analytical data from the experiments indicated that anaerobic reductive dechlorination was readily accomplished during anaerobic phase experiments as PCE was sequentially dechlorinated to TCE and then to cDCE as reported in previous research reported by others in the literature. Additionally, a few mixed consortia microcosms showed evidence of further reductive dechlorination to VC and ethylene. During the sequential environment experiments, analytical data also indicated that reductive dechlorination also resumed after an aerobic sequence utilizing hydrogen peroxide as an oxidizer in the microcosm. No conclusive evidence was observed to indicate that aerobic degradation of cDCE during any of the aerobic phase treatments. This was probably due to the inocula not containing methanotrophs.