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.

This dissertation contains the results of two different projects. The first one is a study of the transport of protozoa pathogens Cryptosporidium parvum and Encephalitozoon intestinalis in soils. The aim of this project was to investigate the movement and retention mechanisms of these microorganisms in natural porous media. The work determined that in the case of C. parvum, the retention was primarily produced by straining and in the case of E. intestinalis the main retention mechanism was attachment. The results of C. parvum lysimeter experiment compared to the results from the 7 cm column experiments suggest that retention is proportional to the length of the column. The second study evaluated the Polymerase Chain Reaction (PCR) as a tool to identify dechlorinating bacteria in groundwater contaminated with chloroethenes. The target DNA regions to identify these microorganism were the 16s rDNA specific for dehalococcoides sp. and Desulfuromonas and DNA sequences coding for the reductive dehalogenase enzymes pceA, tceA, bvcA and vcrA. Bacteria able to transform PCE into DCE were detected in all groundwater samples. Bacteria able to transform VC into ethene were found only in one of the samples. This study shows that PCR analysis of 16s rDNA and reductive dehalogenase gene sequences together with microcosm results are useful tools to analyze the populations of reductive dechlorinators and their activity in a given site.

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.

abstract: Contamination by chlorinated ethenes is widespread in groundwater aquifers, sediment, and soils worldwide. The overarching objectives of my research were to understand how the bacterial genus Dehalococcoides function optimally to carry out reductive dechlorination of chlorinated ethenes in a mixed microbial community and then apply this knowledge to manage dechlorinating communities in the hydrogen-based membrane biofilm reactor (MBfR). The MBfR is used for the biological reduction of oxidized contaminants in water using hydrogen supplied as the electron donor by diffusion through gas-transfer fibers. First, I characterized a new anaerobic dechlorinating community developed in our laboratory, named DehaloR^2, in terms of chlorinated ethene turnover rates and assessed its microbial community composition. I then carried out an experiment to correlate performance and community structure for trichloroethene (TCE)-fed microbial consortia. Fill-and-draw reactors inoculated with DehaloR^2 demonstrated a direct correlation between microbial community function and structure as the TCE-pulsing rate was increased. An electron-balance analysis predicted the community structure based on measured concentrations of products and constant net yields for each microorganism. The predictions corresponded to trends in the community structure based on pyrosequencing and quantitative PCR up to the highest TCE pulsing rate, where deviations to the trend resulted from stress by the chlorinated ethenes. Next, I optimized a method for simultaneous detection of chlorinated ethenes and ethene at or below the Environmental Protection Agency maximum contaminant levels for groundwater using solid phase microextraction in a gas chromatograph with a flame ionization detector. This method is ideal for monitoring biological reductive dechlorination in groundwater, where ethene is the ultimate end product. The major advantage of this method is that it uses a small sample volume of 1 mL, making it ideally suited for bench-scale feasibility studies, such as the MBfR. Last, I developed a reliable start-up and operation strategy for TCE reduction in the MBfR. Successful operation relied on controlling the pH-increase effects of methanogenesis and homoacetogenesis, along with creating hydrogen limitation during start-up to allow dechlorinators to compete against other microorgansims. Methanogens were additionally minimized during continuous flow operation by a limitation in bicarbonate resulting from strong homoacetogenic activity. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2012

Polaromonas sp. strain JS666 is the only isolated bacterium capable of aerobic growth using the groundwater pollutant cis-1,2-dichloroethene (cDCE) as a sole carbon source. Its genome has a wealth of evidence of recent gene acquisition through horizontal gene transfer, and contains gene clusters predicted to encode enzymes allowing the metabolism of a wide variety of xenobiotic compounds. Culture growth using each of these hypothesized substrates was tested experimentally, and many were confirmed as sole carbon sources for strain JS666. In addition to pollutant degradation, many of these metabolic pathways have applicability in the field of biocatalysis, as does the genome-assisted pathway prediction approach to biocatalyst discovery. During (or immediately following) growth on cDCE, cultures of Polaromonas sp. strain JS666 oxidize ethene to epoxyethane at an increased rate, and also cometabolically oxidize several other chlorinated ethenes. Given the involvement of a monooxygenase in other species' 1-chloroethene (vinyl chloride) oxidation, it was hypothesized that alkene oxidation in strain JS666 was due to the activity of a monooxygenase that also was responsible for the first step in cDCE oxidation. The alkene oxidation activity of strain JS666 was investigated using gene expression analysis, proteomics, and whole-cell kinetic assays. Results of these experiments pointed to the upregulation of a cyclohexanone monooxygenase (CHMO) during growth on cDCE and during oxidation of ethene. To determine the activity of this cyclohexanone monooxygenase, its gene was cloned and heterologously expressed in an E. coli host. Our CHMO expression system exhibited activity on cyclohexanone, but not cDCE or ethene, disproving our hypothesis about its involvement in alkene oxidation. The heterologously expressed monooxygenase was also investigated for enantioselective oxidation of racemic cyclic ketones to chiral lactones, and was discovered to have very high enantioselectivity with the tested compounds. Chiral lactones and other single-enantiomer oxidation products are valuable for fine chemical synthesis, and their biocatalytic production is more environmentally sustainable and often less expensive than traditional techniques. The research described in the following chapters illustrates the many opportunities that arise when the fields of bioremediation and biocatalysis converge. The shared research goals and methods of these two areas lend themselves to interdisciplinary research, and increased communication and crossover between them should provide benefits for both environmental remediation and sustainable chemical synthesis.

Natural Attenuation Software (NAS) was developed as a screening tool to estimate remediation timeframes for monitored natural attenuation (MNA) to lower groundwater contaminant concentrations to regulatory limits, and to assist in decision-making on the level of source zone treatment in conjunction with MNA using site-specific remediation objectives. In addition, NAS facilitates the combined use of MNA with engineered remedial actions (ERAs) so that the benefits of each technology can be maximized while minimizing costs of remediation. The primary expected benefit of NAS is to increase regulatory acceptance of MNA, thereby decreasing overall remediation costs. NAS is designed for application to ground-water systems consisting of porous, relatively homogeneous, saturated media, and assumes that groundwater flow is uniform and unidirectional. NAS consists of a combination of analytical and numerical solute transport models implemented in three main interactive modules to provide estimates for: (1) target source concentration required for a plume extent to contract to regulatory limits, (2) time required for NAPL contaminants in the source area to attenuate to a predetermined target source concentration, and (3) time required for a plume extent to contract to regulatory limits after source reduction. Natural attenuation processes that NAS models include advection, dispersion, sorption, non-aqueous phase liquid (NAPL) dissolution, and biodegradation. NAS determines redox zonation, and estimates and applies varied biodegradation rates from one redox zone to the next. Recently, NAS was enhanced to include petroleum hydrocarbons, chlorinated ethenes, chlorinated ethanes, chlorinated methanes, and chlorinated benzenes, or any user-defined contaminants (e.g., heavy metals, radioisotopes), and has included the capability to model co-mingled plumes. To enable comparison of remediation timeframe estimates between MNA and specific ERAs, NAS was modified to incorporate an estimation technique for timeframes associated with pump-and-treat remediation technology for comparison to, or in conjunction with, MNA. NAS also expanded analysis tools for improved performance assessment, as well as the assessment of sustainability of natural attenuation processes over time. A Department of Defense (DoD) Environmental Security Technology Certification Program (ESTCP) demonstration was undertaken to evaluate the capability of the NAS software to provide reasonable estimates of MNA cleanup timeframes in a variety of environments and sites throughout the United States. Overall, results suggest that NAS was satisfactory in meeting performance objectives set forth in the demonstration, and that because NAS is based on sound science, it can serve as an effective tool for decision-making and data analysis at a wide range of contaminated sites and is not limited to a small subset of “simple sites” because of its simplicity. At some sites, NAS-estimated timeframes were crucial for winning regulatory acceptance of MNA, with cost-benefit analyses providing estimates of savings associated with using MNA as a final remediation strategy. / Ph. D.

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No description available.

For a numerical or analytical model to be useful it should be ensured that the model outcome matches the observations or field measurements during calibration. This process has been typically done by manual perturbation of the model input parameters. This research investigates a methodology for using non linear parameter estimation technique (the Marquardt-Levenberg technique) with the multi component reactive solute transport model SEAM3D. The reactive multi-component solutes considered in this study are chlorinated ethenes. Previous studies have shown that this class of compounds can be degraded by four different biodegradation mechanisms, and the degradation path is a function of the prevailing oxidation reduction conditions. Tests were performed in three levels; the first level utilized synthetic model-generated data. The idea was to develop a methodology and perform preliminary testing where "observations" can be generated as needed. The second level of testing involved performing the testing on a single redox zone model. The methodology was refined and tested using data from a chlorinated ethenes-contaminated site. The third level involved performing the tests on a multiple redox zone model. The methodology was tested, and statistical validation of the recommended methodology was performed. The results of the tests showed that there is a statistical advantage for choosing a subgroup of the available parameters to optimize instead of the optimizing the whole available group. Therefore, it is recommended to perform a parameter sensitivity study prior to the optimization process to identify the suitable parameters to be chosen. The methodology suggests optimizing the oxidation-reduction species parameters first then calibrating the chlorinated ethenes model. The results of the tests also proved the advantage of the sequential optimization of the model parameters, therefore the parameters of the parent compound are optimized, updated in the daughter compound model, for which the parameters are then optimized so on. The test results suggested considering the concentrations of the daughter compounds when optimizing the parameters of the parent compounds. As for the observation weights, the tests suggest starting the applied observation weights during the optimization process at values of one and changing them if needed. Overall the proposed methodology proved to be very efficient. The optimization methodology yielded sets of model parameters capable of generating concentration profiles with great resemblance to the observed concentration profiles in the two chlorinated ethenes site models considered. / Ph. D.

Nearly 250,000 sites with past and present potentially polluting activities need urgent remediation within Europe. Major pollutants include organochlorines (OCls), e.g. chlorinated ethenes (CEs) and hexachlorocyclohexanes (HCHs), mainly used as industrial solvents and pesticides, respectively. Due to improper handling and disposal, OCls contaminants are present in the soil or groundwater surrounding sites, where they have been produced or used. CEs and HCHs can undergo degradation by microorganisms indigenous to the soil or groundwater. Therefore natural attenuation (NA), relying on the in situ biodegradation of pollutants, is considered as a cost effective remediation strategy, yet it requires accurate monitoring methods. Compound specific isotope analysis (CSIA) is a powerful tool to provide information on the extent of degradation and, when combining two isotope systems (2D-CSIA), such as carbon (δ13C) and chlorine (δ37Cl), on reaction mechanisms. The diagnostic reaction-specific isotope enrichment factors (εC and εCl) were determined in laboratory experiments for the anaerobic degradation of PCE, TCE (Paper II) and α-HCH (Paper III) by mixed bacterial cultures enriched from CEs and HCHs contaminated sites, respectively. The related mechanism-specific εCl/εC ratios were calculated as 0.35 ± 0.11 (PCE), 0.37 ± 0.11 (TCE) and 0.52 ± 0.23 (α-HCH). These values are smaller than previously reported values for pure cultures. This is explained by the microbial community composition changes observed during degradation of PCE and α-HCH, which also reflect the variability of the microbial community at the field level. Furthermore, εCl/εC ratio might be bacteria specific. These values allowed the estimation of the extent of contaminant degradation at the respective study sites (Paper III and IV). Application of both isotope systems (δ13C and δ37Cl) led to comparable estimates. However the choice of representative ε values is crucial for an accurate assessment. These studies show that CSIA is useful to quantify in situ degradation of OCls contaminants and identify reaction pathways, by combining δ13C and δ37Cl. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: Manuscript.</p>