Gas fed bioreactors

Chlorinated solvents such as 1,1,1-trichloroethane (1,1,1-TCA) have been widely used. Cyclic ethers, including 1,4-dioxane (14D), 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 a chemical of concern at solvent-impacted sites. Aerobic cometabolic biodegradation processes may be reliable and cost-effective in situ remediation approaches for 14D and its 1,1,1-TCA-derived co-occurring chemicals. 

The overall aim of this project was to evaluate the potential for the two simplest branched hydrocarbons (isobutane [2-methylpropane] and isobutylene [2-methylpropene]) to serve as primary growth substrates for bacteria that can subsequently cometabolically degrade 14D and its associated chlorinated co-occurring chemicals. Neither of these gases had previously been studied as potential stimulants for cometabolic processes. In this project, the project team focused on model branched hydrocarbon-metabolizing bacteria and aimed to determine their chemical cosubstrate ranges, identify the monooxygenases enzymes responsible for these activities, and characterize the interactions between these substrates. The model isobutylene-metabolizing strain was Mycobacterium sp. ELW1, while the model isobutane-metabolizing strains were Mycobacterium vaccae JOB5 and Rhodococcus rhodochrous ATCC 21198.

In addition to molecular approaches, ¹³C₄-14D degradation studies were used to examine the intermediates generated during the cometabolic degradation of 14D and the eventual fate of carbon from this compound. Studies with ¹³C-NMR suggest that 2-hydroxyethoxyacetic acid is an initial metabolite of 14D degradation by isobutane-grown cells of R. rhodochrous ATCC 21198, while long term studies suggested that ¹³C₄-14D can be fully mineralized to ¹³CO₂ by this bacterium. Approaches based on ¹³CO₂ production have not been previously extensively explored but provided a more facile approach than the use of ¹⁴C-labeled compounds to determine the full kinetics of chemical degradation. This approach also potentially enabled researchers to more easily address the question as to whether cometabolically-active microorganisms obtain a carbon benefit from cometabolic processes.

In the case of Mycobacterium sp. ELW1, the project team established that isobutylene-grown cells of this strain do not degrade 14D at environmentally relevant concentrations (≤100 µg/L) although they rapidly oxidize much higher concentrations of 14D (≥100 mg/L). Isobutylene-grown cells can also oxidize chlorinated alkanes including trichloroethene and 1,1-dichloroethene (1,1-DCE), but few other common chemicals associated with 14D. Genome-enabled shotgun proteomic analyses indicate the key monooxygenase in isobutylene-grown cells is an alkene monooxygenase that is very similar to the enzyme found in the model propylene-metabolizing strain Xanthobacter autotrophicus Py2. With M. vaccae JOB5, the project team established this bacterium can readily degrade low, environmentally relevant concentrations of 14D (≤100 µg/L). Genome-enabled shotgun proteomic analyses indicate this strain concurrently expresses two different monooxygenases during growth on isobutane and other gaseous alkanes. These are a Group 6 soluble di-iron monooxygenase (SDIMO) named short chain alkane monooxygenase (SCAM) and a membrane-bound copper containing monooxygenase.

The majority of the research for this project focused on R. rhodochrous ATCC 21198. Like M. vaccae JOB5, the project team has shown R. rhodochrous ATCC 21198 grown on isobutane and other gaseous alkanes readily degrades low, environmentally relevant concentrations of 14D and can also degrade 1,1,1-trichloroethane, 1,1-DCE, 1,2-dichloroethane, cis-1,2- dichloroethene and vinyl chloride. The ability to degrade low concentrations of 14D (<100 µg/L) was also consistently observed in a wide variety of isobutane-metabolizing axenic and mixed cultures and was also consistently associated with the presence of genes encoding SCAM. Many of the strains and cultures that were characterized also possess genes that encode another SDIMO; propane monooxygenase (PrMO). Shotgun proteomics, activity-based protein profiling, and resting cell degradation studies all indicate that PrMO is only expressed at catalytically relevant levels in propane-grown cells and does not contribute to the 14D-degrading activity of isobutane-grown bacteria. In addition to identifying SCAM as a key 14D-degrading monooxygenase, the project team developed a quantitative polymerase chain reaction and a fluorescent protein detection method for this enzyme. Compound specific isotope analyses were also conducted which established the ¹³C/12C and ²H/¹H enrichments associated with 14D degradation by R. rhodochrous ATCC 21198. The enrichments observed for SCAM were very distinct from the enrichments generated by tetrahydrofuran monooxygenase which initiates 14D metabolism in Pseudonocardia dioxanivorans CB1190. The CSIA results therefore potentially allow discrimination between metabolic and cometabolic degradation of 14D. In combination, these tools now provide powerful new and established approaches for identifying and quantifying the activity of SCAM-expressing bacteria in either natural or engineered treatment of 14D in groundwater.

In a transition toward more field-based research, microcosm studies were also conducted and these demonstrated that like the pure culture studies, isobutane can promote and support the cometabolic degradation of low, environmentally relevant concentrations of 14D in groundwater systems. Similar results were obtained with microcosms bioaugmented with R. rhodochrous ATCC 21198 and native microorganisms biostimulated with isobutane. Modeling analysis showed that similar rate parameters derived in the laboratory studies could be applied to successfully model the microcosm tests for both the native and bioaugumented microcosms. The modeling successfully simulated the uptake of repeated additions of isobutane and the cometabolic degradation of 14D over a period of approximately one year. First-order transformation rate coefficients fit the transformation of 14D to low concentrations. Preliminary studies with single well push-pull tests also suggest that native aquifer microorganisms can also be stimulated using isobutane and that these microorganisms have cometabolic transformation potential as determined by the oxidation of isobutylene, a 14D surrogate, to isobutylene oxide.

This project provided 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 chemicals of concern. 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 chemicals of concern, including 1,2,3-trichloropropane and N-nitrosodimethylamine. (Project Completion - 2024)

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.