Date of Award

December 2020

Document Type


Degree Name

Doctor of Philosophy (PhD)


Environmental Engineering and Earth Science

Committee Member

David L. Freedman

Committee Member

Kevin T. Finneran

Committee Member

Sudeep Popat

Committee Member

Vijay Shankar


1,4-Dioxane is a widely distributed contaminant within the U.S. along with chlorinated solvents, specifically 1,1,1-trichloroethane (1,1,1-TCA) and other compounds. Due to its apparent recalcitrance and carcinogenicity, it is a contaminant that has raised considerable concerns because of its potential adverse effects on health. The physical and chemical properties and behavior of 1,4-dioxane create challenges for its characterization and treatment. It is highly mobile and does not readily biodegrade in the environment. Therefore, remediation for 1,4-dioxane has gained an increasing importance. The current leading technologies for remediation are energy and chemically intensive and ineffective for mass removal, whereas in situ bioremediation is a suitable alternative due to its lower overall cost for implementation.

Given the gaps in knowledge from the scientific literature, and in order to expand the understanding of 1,4-dioxane biodegradation and its potential in situ bioremediation applications, the objectives of this study include: 1) Evaluate the cometabolic biodegradation potential of high concentrations of 1,4-dioxane (i.e., > 5 mg·L-1) by propanotrophs; 2) Isolate and characterize two isolated bacteria that can metabolize 1,4-dioxane obtained from contaminated sites; 3) Evaluate the potential loss of essential genes for aerobic biodegradation of 1,4-dioxane in the presence of other substrates other than 1,4-dioxane; 4) Evaluate the movement of 1,4-dioxane degrading microbes through porous media; 5) Develop and validate a protocol to directly measure rate constants for natural biodegradation of 1,4-dioxane in groundwater using 14C-labeled 1,4-dioxane; 6) Apply the 14C-assay for actual contaminated sites; 7) Evaluate the potential for anaerobic biodegradation of 1,4-dioxane by the use of groundwater and soil samples from contaminated sites.

The first objective was accomplished by the construction of microcosms to evaluate the potential of cometabolic degradation of 1,4-dioxane, using the mixed propanotrophic culture ENV487 for one site contaminated with high levels of 1,4-dioxane (>1,000 mg·L-1). Indigenous propanotrophs were confirmed in the site using bottles supplemented with propane only. Inoculated bottles with the culture showed an enhanced degradation rate for 1,4-dioxane. Complete degradation of 1,4-dioxane was achieved in most of the bottles, and in a few cases, nutrients were needed to resume and complete the degradation process. A 1,4-dioxane degrader was confirmed in the site after adding nutrients. Overall, cometabolism of 1,4-dioxane at high concentrations by propanotrophs is a feasible method to remediate sites contaminated with this compound.

The second objective was addressed by obtaining the whole genome sequence of the isolated strains BERK-1A and BERK-1B. Whole genomes were annotated and used to compared against annotated genes from another 1,4-dioxane-degrader, Pseudonocardia dioxanivorans CB1190.

Third objective was accomplished by performing tests to estimate kinetic parameters using strain BERK-1A. Kinetic parameters were compared against the known 1,4-dioxane metabolizer P. dioxanivorans CB1190.

The fourth objective was fulfilled by developing a method to quantify rate coefficients based on product accumulation for 1,4-dioxane biodegradation, using a radio-isotope (14C) assay to confirm natural attenuation in groundwater. Pseudo-first order rate coefficients were quantified by fitting the product accumulation data to a mass balance model for 14C products.

To achieve the fifth objective, microcosms were set up with groundwater and soil from four contaminated sites at which the field data suggests that 1,4-dioxane is undergoing anaerobic biodegradation. The samples did not contain a significant amount of chlorinated solvents. Samples from one site were amended with 14C-1,4-dioxane to characterize degradation products. Amendments included Fe(III) oxide, Fe(III)-ethylene-diaminetetraacetic acid (Fe(III)-EDTA), anthraquinone disulfonate (AQDS), nitrate, sulfate and oxygen. There was no evidence to support biodegradation of 1,4-dioxane under anaerobic conditions. Further laboratory studies are needed to determine the feasibility of anaerobic biodegradation of 1,4-dioxane. Until then, aerobic treatment remains the only viable bioremediation alternative.

Aerobic biodegradation of 1,4-dioxane was demonstrated in up-, mid- and down-gradient microcosms with soil and groundwater from a site in Europe. The up-gradient location has 1,4-dioxane concentrations of ~1,500 mg·L-1, among the highest reported for an aquifer. Biodegradation required amendment with propane. Although indigenous propanotrophs were able to co-oxidize 1,4-dioxane, higher rates were achieved following bioaugmentation with the mixed propanotrophic culture ENV487. Nutrient addition was essential for stimulating biodegradation activity. First order rate coefficients were similar to ones reported for a field study of propane biosparging. Transformation yields were lower than values obtained under ideal conditions (i.e., medium instead of groundwater and soil), but were notably higher than the transformation yields for chlorinated ethenes and ethanes. Evidence for the presence of microbes cable of degrading 1,4-dioxane as a sole source of carbon and energy was obtained with the mid-gradient microcosms.

Using microcosms prepared with soil and groundwater from two contaminated sites, enrichment cultures were developed that aerobically consumed 1,4-dioxane as a major source of carbon and energy. Isolates were obtained from both enrichments; both are strains of Pseudonocardia dioxanivorans. This is the second reported pure culture of a Pseudonocardia spp. capable of metabolizing 1,4-dioxane that comes from a contaminated aquifer. Molecular tools confirmed that the two isolates are the same strain (designated BERK-1), and they are different from strain CB1190. The kinetics for growth of BERK-1 on 1,4-dioxane are similar to those of CB1190. Minimal morphological differences were observed between P. dioxanivorans strains CB1190 and BERK-1 according to cell surface analysis by SEM.

Curing of plasmids from strains CB190 and BERK-1 that carry essential genes for initiating aerobic biodegradation of 1,4-dioxane was only possible following growth in a rich medium (LB). Curing did not occur following growth on lactate and emulsified vegetable oil (EVO). This indicates that excess electron donor used to anaerobically remediate chlorinated solvent plumes will not trigger the loss of essential genes for aerobic biodegradation of 1,4-dioxane.

BERK-1 was able to move at a slightly higher rate than CB1190 through sand and silt without the aid of recirculation. This is consistent with a lower level of clumping and adherence to surfaces by BERK-1 following growth in medium. However, further tests with continuous flow columns should be implemented to corroborate these results.

The whole genome for BERK-1 was successfully assembled and provided enough differences to discern it from the well-known 1,4-dioxane degrader P. dioxanivorans CB1190. The draft genome sequence and annotation have been deposited in the DDBJ/ENA/GenBank database under the accession no. PJPW00000000. The version described in this dissertation is PJPW02000000.

A 14C assay was developed that enables quantification of first order biodegradation rate coefficients in groundwater microcosms. A method was verified for purification of 14C-1,4-dioxane by passage through an HPLC column. The 14C assay was validated with metabolic and cometabolic cultures (i.e., CB1190 and ENV487, respectively). Detection limits for rate coefficients were on the same order of magnitude, with half-lives of 43 and 33 years for CB1190 and ENV487, respectively.

Further validation of the 14C assay included the ability to block biodegradation by addition of acetylene to microcosms, or incubation in the absence of oxygen. These treatments verify the involvement of monooxygenases in aerobic biodegradation of 1,4-dioxane. This suggests it may be possible to correlate monooxygenase genes quantified in groundwater to first order rate coefficients, as has been done with aerobic co-oxidation of TCE.

Of the 49 groundwater samples evaluated, statistically significant rate coefficients were determined using the 14C assay for 15; no significant rate of degradation was observed in 34 of the samples. The median rate coefficient observed using the 14C assay was 0.0061 yr-1 (half life = 114 yr); the maximum rate coefficient was 0.096 yr-1 (half-life = 7.2 yr). These results indicate that for most of the wells examined, biodegradation of 1,4-dioxane is occurring at a relatively slow rate or not at all. There appears to be good correlation between results from the 14C assay and other lines of evidence for the occurrence of biodegradation being gathered as part of an ESTCP project.

Nutrients may be a limiting factor in the 14C assay. Adding nutrients had a significant impact on metabolic and cometabolic cultures. Nutrient limitation must therefore be considered when interpreting rate coefficients from the 14C assay based only on groundwater, i.e., in the absence of soil. For this reason, the assay may be most useful as a screening tool to help decide if it would worthwhile to resample a site and collect both soil and groundwater. Collecting soil cores is considerably more costly but may allow for determination of more accurate rate coefficients.

This is the first study to report formate as a significant soluble product from co-oxidation of 1,4-dioxane by a propanotrophic culture. This was confirmed by co-elution on an HPLC column with authentic material and application of a formate dehydrogenase assay. A pathway for co-oxidation is proposed, with ethylene glycol being a likely precursor to formate. Other presumptive products identified by co-elution on the HPLC were acetate, glycerate, glycolaldehyde, ethylene glycol, and glycolate. Although identification of formate as a product from propanotrophic co-oxidation of 1,4-dioxane is of interest, formate is not likely to serve as a useful marker for biodegradation in situ, since many microbes biodegrade formate and there are many sources of formate in addition to co-oxidation of 1,4-dioxane.

Anaerobic biodegradation of 1,4-dioxane was evaluated for seven sets of microcosms prepared with groundwater or MSM and soil from four contaminated sites. Reducing conditions were established with a variety of anaerobic electron acceptors. There was no compelling evidence for anaerobic biodegradation, even in microcosms that were incubated as long as seven years and with the benefit of using 14C-1,4-dioxane to facilitate detection of degradation products. These results are in contrast to compelling field evidence for the occurrence of anaerobic biodegradation of 1,4-dioxane.



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