Gas fed bioreactors

Chlorinated solvents such as 1,1,1-trichloroethane (1,1,1-TCA) have been widely used at many Department of Defense (DoD) installations. Cyclic ethers, including 1,4-dioxane, are added as stabilizers in commercial formulations of 1,1,1-TCA to extend the working life of the active chlorinated solvent. The concentration of stabilizers can significantly increase during normal solvent use and incorrect disposal of spent solvents can result in 1,4-dioxane becoming an important co-contaminant at solvent-impacted sites. Aerobic cometabolic biodegradation processes may be reliable and cost-effective in situ remediation approaches for 1,4-dioxane and its 1,1,1-TCA-derived co-contaminants. However, understanding of the microorganisms and substrates that can promote these processes is currently limited.

The overall aim of this project is to evaluate the two simplest branched hydrocarbons (isobutane [2-methylpropane] and isobutylene [2-methylpropene]) as stimulants for the cometabolic degradation of 1,4-dioxane and its co-contaminants, including 1,1,1-TCA and its degradation products. These hydrocarbons have not been previously recognized or examined for their abilities to support cometabolic processes for cyclic ethers or chlorinated hydrocarbons. However, these compounds potentially offer considerable specificity in the types of microorganisms and enzyme activities that they can stimulate. They can also be introduced into groundwater using well-established approaches used to promote cometabolic biodegradation of compounds like trichloroethene (TCE). Preliminary data show isobutane and isobutylene support rapid cometabolic 1,4-dioxane biodegradation in two model organisms, Mycobacterium vaccae JOB5 and Mycobacterium ELW1, respectively.

Technical Approach

Genome-enabled proteomic and activity-based protein profiling approaches will be used to identify the 1,4-dioxane/branched hydrocarbon-oxidizing monooxygenases in strains JOB5 and ELW1. The major products of 1,4-dioxane degradation by these organisms will also be determined using HPLC, GC/MS, and 13C-NMR. Similar approaches will be used to determine how frequently these activities, enzymes, and products are associated with 1,4-dioxane degradation in newly isolated branched hydrocarbon-utilizing strains isolated from 1,4-dioxane-contaminanted DoD sites. Strains JOB5 and ELW1 will also be used to define the kinetic features of 1,4-dioxane biodegradation and the interactions between hydrocarbon growth substrates, 1,4-dioxane, and its 1,1,1-TCA-derived co-contaminants. Microcosms and one-dimensional column studies will be used to characterize the branched hydrocarbon-stimulated degradation of 1,4-dioxane by native and bioaugmented microorganisms. 13C-DNA-SIP and qPCR will also be used to identify and quantify the metabolically active organisms in these systems and to explore the fate of 1,4-dioxane-derived metabolites generated during 1,4-dioxane cometabolism. Single well push-pull approaches using fully deuterated (D8) 1,4-dioxane or ethene will be used to measure in situ rates of 1,4-dioxane degradation by native or bioaugmented microorganisms stimulated by isobutane and isobutylene.


This project will provide a thorough evaluation of the potential use of simple branched hydrocarbons as selective stimulants for the in situ cometabolic degradation of 1,4-dioxane and its associated contaminants. These studies can be used to develop predictable, reliable, and cost-effective biotreatment processes for 1,4-dioxane. The branched hydrocarbon stimulants evaluated in this project could also potentially be applied to the cometabolic biodegradation of other emerging contaminants of interest to DoD, including 1,2,3-trichloropropane (TCP) and N-nitrosodimethylamine (NDMA). (Anticipated Project Completion - 2021)


Bennett, K., N.C. Sadler, A.T. Wright, C. Yeager, and M.R. Hyman. 2016. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea. Applied and Environmental Microbiology, 82:2270-2279. doi.org/10.1128/AEM.03556-15.

Bennett, P., M. Hyman, C. Smith, H. El-Mugammar, M-Y. Chu, M. Nickelsen, and R. Aravena. 2018. Enrichment of Carbon-13 and Deuterium during Monooxygenase-mediated Biodegradation of 1,4-Dioxane. Environmental Science & Technology Letters, 5(3):148-153. doi.org/10.1021/acs.estlett.7b00565.

Kottegoda, S., E. Waligora, and M. Hyman. 2015. Metabolism of 2-Methylpropene (Isobutylene) by the Aerobic Bacterium Mycobacterium sp. strain ELW1. Applied and Environmental Microbiology, 81:1966-1976. doi.org/10.1128/AEM.03103-14.

Rolston, H.M., M.R. Hyman, and L. Semprini. 2019. Aerobic Cometabolism of 1,4-dioxane by Isobutane-Utilizing Microorganisms including Rhodococcus rhodochrous Strain 21198 in Aquifer Microcosms: Experimental and Modeling Study. Science of the Total Environment, 694:133688. doi.org/10.1016/j.scitotenv.2019.133688.