The overall goal of this limited scope project was to measure and assess the extent to which 1,4-dioxane can be biodegraded by methane-oxidizing bacteria under conditions representative of a co-mingled chlorinated solvent plume. To attain this overall goal, the following specific objectives were proposed:

Conduct a preliminary screening assessment to determine the extent to which methanotrophs capable of degrading 1,4-dioxane are present at 1,4-dioxane contaminated sites;Determine the conditions, with respect to methane, dissolved oxygen, and chlorinated solvent concentrations, under which effective biodegradation of 1,4-dioxane by methanotrophs will occur;

• Determine the rates and extents of 1,4-dioxane degradation by indigenous methanotrophs;

Evaluate the overall potential for natural attenuation of 1,4-dioxane by methanotrophs in the downgradient plume following and during bioremediation of co-mingled chlorinated solvents.

For this effort, the focus initially was on the co-metabolic biodegradation of 1,4-dioxane by methanotrophs. The methane required for this process is a typical byproduct of organic substrate addition to aquifers, the bioremediation approach most commonly used for chlorinated solvents. The ability of methanotrophs to biodegrade 1,4-dioxane was evaluated in a series of laboratory batch and column experiments under geochemical conditions representative of those observed downgradient of typical solvent plumes that have undergone biological treatment via substrate addition.

The overall approach employed for this study consisted of first collecting soil from 1,4-dioxane and chlorinated solvent impacted sites. These soil samples were then used in screening experiments to determine if methane (or other short-chain hydrocarbons such as propane or ethane) oxidizing bacteria were present, and if these bacteria could facilitate the oxidation of 1,4-dioxane. After preparing enrichment cultures from selected samples, batch experiments were performed to assess the co-metabolic biodegradation kinetics of 1,4-dioxane. Finally, column experiments were performed to simulate the co-metabolic biodegradation of 1,4-dioxane in the downgradient plume.

Results obtained during the study showed that methane-oxidizing bacteria were not effective for promoting the biodegradation of 1,4-dioxane. For all conditions examined, including use of copper chelators, various nutrients, pure and enrichment cultures, and soil collected from 3 different sites, 1,4-dioxane biodegradation was not observed with methane as a cosubstrate. However, ethane- and propane-oxidizing bacteria were shown to biodegrade 1,4-dioxane. Because ethane often is present at 1,4-dioxane sites (due to dechlorination processes associated with co-mingled chlorinated solvents), our research efforts with respect to kinetic and column studies shifted to assessing ethanotrophs for biodegradation of 1,4-dioxane. To this point, ethane has rarely been considered as a co-metabolic substrate in aquifers with 1,4-dioxane or other contaminants-of-concern.

Michaelis-Menten kinetic parameters were determined for both 1,4-dioxane and ethane using a mixed culture obtained from former Myrtle Beach Air Force Base (MBAFB) in South Carolina. Regression of the model parameters Vmax and Km for 1,4-dioxane were 4.6 ± 1.7 × 10-5 mg/mg/cell/h and 0.23 ± 0.07 mg/L, respectively; Vmax and Km for ethane were 1.8 ± 0.6 ×10-3 mg/mg/cell/h and 0.064 ± 0.02 mg/L, respectively. Ethane inhibition of 1,4-dioxane biodegradation was well described by assuming the inhibition coefficient was equal to the ethane half-saturation coefficient. 

Using these regressed parameters, as well as the observed rates of ethane biodegradation in MBAFB soil, the estimated half-life for 1,4-dioxane was approximately 1.9 years. This value is in excellent agreement with published rates of 1,4-dioxane biodegradation observed in the field. In addition, results from MBAFB in this study show that ethane is present within the 1,4-dioxane plume at concentrations on the order of 20 µg/L. Others have shown ethane at concentrations on the order of 0.06 µg/L within 1,4-dioxane plumes. Thus, results of this research suggest that ethane-oxidizing bacteria, sustained by the presence of ethane at sites with co-mingled 1,4-dioxane and chlorinated solvent plumes, may be responsible for slow yet sustained 1,4-dioxane biodegradation at some DoD facilities. Further laboratory and field studies are necessary to confirm this hypothesis and to develop an improved understanding of the occurrence and types of ethane-degrading bacteria in groundwater aquifers and the enzyme(s) they utilize for degrading ethane and 1,4-dioxane, as well as their potential role in the fate of other DoD contaminants of concern.

Results attained in this limited scope study suggest that ethane-oxidizing bacteria may play a significant role in the intrinsic biodegradation of 1,4-dioxane at DoD sites. Key remaining questions include the following:

• Are all bacteria capable of biodegrading ethane also capable of degrading 1,4-dioxane, and what genes/enzymes are involved in this process?
• Are the low levels of ethane observed within comingled chlorinated solvent and 1,4-dioxane plumes really responsible for the observed 1,4-dioxane aerobic biodegradation observed at some DoD sites?
• Can this relatively slow 1,4-dioxane biodegradation by ethane-oxidizing bacteria be verified and quantified in the field?