Formatted Title

Comparison of Isobutane and Isobutene as Primary Substrates for Cometabolic Biodegradation of 1,4-Dioxane and Chlorinated Hydrocarbon Mixtures

Background/Objectives

1,4-Dioxane (1,4-D), a probable human carcinogen, has emerged as a common groundwater contaminant at military and industrial sites due to its use as a stabilizer for the chlorinated solvent 1,1,1-trichloroethane (TCA). Environmental concentrations of 1,4-D are typically low (< 1 ppm) and commonly occur with chlorinated aliphatic hydrocarbons (CAHs) including TCA, trichloroethene (TCE), and lesser-chlorinated daughter products, such as 1,1-dichloroethene (1,1-DCE). Anaerobic bioremediation, a common approach for CAHs, does not transform 1,4-D. By contrast, both 1,4-D and some CAHs can be transformed via aerobic cometabolism. This work compares isobutane and isobutene as primary substrates to induce cometabolic transformation of 1,4-D and CAH mixtures.

Approach/Activities

Isobutane and isobutene were assessed as primary substrates for the growth of bacteria in pure culture and for the stimulation of microorganisms present in groundwater from a Navy site in California contaminated with 1,4-D, TCE, and 1,1-DCE. The model isobutane-grown bacterium was Rhodococcus rhodochrous strain 21198. Mycobacterium sp. strain ELW1 was grown on isobutene. Pure cultures were evaluated in batch tests to assess cometabolic transformation of single compounds (1,4-D, chlorinated ethenes, and chlorinated ethanes) as well as contaminant mixtures. Batch microcosms were constructed with site groundwater. Observed contaminant degradation was modeled using Michaelis-Menten/Monod and first-order kinetics, including competitive inhibition and transformation capacity terms, to offer additional insight.

Results/Lessons Learned

Bacteria grown on isobutane or isobutene rapidly transformed 1,1-DCE. Both primary substrates also induced cometabolic transformation of TCE, but at slower rates than 1,1-DCE. Negligible transformation of 1,4-D, TCA, or 1,2-dichloroethane was observed by isobutene-grown bacteria, whereas all were transformed by isobutane-grown bacteria. Another important difference was that the presence of isobutene strongly inhibited cometabolic transformation of all contaminants, while the presence of isobutane did not. This allowed isobutene-utilizing bacteria to accumulate biomass and energy prior to exposure to the potential toxicity (or energy drain) associated with cometabolic transformation. Primary substrate inhibition was most important with regard to cometabolic transformation of 1,1-DCE, which imparted irreversible toxicity from its transformation to a highly reactive epoxide by both isobutane- and isobutene-grown bacteria. Inhibition by the presence of isobutene allowed for biostimulation of isobutene-utilizers in microcosms in the presence of 1,1-DCE, followed by cometabolic contaminant transformation of both 1,1-DCE and TCE. By contrast, the presence of isobutane did not inhibit 1,1-DCE cometabolism. Isobutane utilization by bacteria in site groundwater was observed only after 1,1-DCE had been sparged from the microcosms. Inhibition was also observed between contaminants, such as the inhibition of TCE degradation by the presence of 1,1-DCE. Modeling analyses offered an additional line of evidence that biostimulation with isobutane would likely not be successful in the presence of 1,1-DCE. The applicability of kinetic parameter values to model transformations by both pure cultures and uncharacterized isobutane- or isobutene-utilizing bacteria in microcosms illustrated the generalizable influence of a specific primary substrate in contaminant transformation. Overall, this work illustrated that isobutane and isobutene have potential utility as a primary substrate, but the choice depends on the contaminants present. In addition, when considering the application of cometabolic bioremediation, it is important to consider inhibition interactions between both contaminant mixtures and primary substrates as well as the formation of potentially toxic transformation products.