The overarching objective of this proof-of-concept project was to understand and apply potential treatment synergies to ultimately achieve biodegradation of multiple contaminants in source zones as well as distal plumes. Specifically, a microbial community was formulated to simultaneously or sequentially degrade chlorinated volatile organic compounds (CVOC) and 1,4-dioxane across changing redox environments. Some CVOCs have used 1,4-dioxane as a solvent stabilizer; consequently, 1,4-dioxane is detected as a co-occurring contaminant with CVOCs at many sites. Anaerobic biological reduction is a common remediation approach for CVOCs in groundwater, such as trichloroethene (TCE). However, under some conditions, intermediate daughter products, such as cis-1,2-dichloroethene (cDCE) and vinyl chloride, are accumulated, which may be more toxic than their parent compounds. Aerobic cometabolism of CVOCs requires additional amendments of primary substrates. 1,4-Dioxane is mainly biodegraded under aerobic conditions. The opposing redox conditions favored by CVOC- and 1,4-dioxane-degrading bacteria pose challenges for concurrent bioremediation of both contaminant classes. By combining anaerobic and aerobic microbes, CVOC transformation products are less likely to persist, and can even be biodegraded aerobically, thereby mitigating their inhibition of 1,4-dioxane-degrading bacteria.

In this research project, a microbial community was developed that was composed of the anaerobic culture, KB-1^®, and the aerobic strain, Pseudonocardia dioxanivorans CB1190 (CB1190), in a modified medium to degrade CVOCs and 1,4-dioxane. The concept of combining organisms that typically cannot survive together was initially counterintuitive. However, after rigorous trial and error, a mixed community was assembled that successfully biodegraded TCE as well as 1,4-dioxane over changing redox conditions. Additionally, aerobic degradation of cDCE was carried out by CB1190 in the presence of 1,4-dioxane.

The project team reports that CB1190 is uniquely equipped with the ability to withstand a variety of environments usually favored by anaerobic microorganisms. For example, CB1190 was able to survive over 35 days of anaerobic incubation and subsequently degrade 1,4-dioxane when oxygen became available. During the anaerobic phase, KB-1 reductively dechlorinated TCE to cDCE via tceA-coded enzymes, while CB1190 down regulated the dxmB and aldH genes, which serve as biomarkers for the degradation of 1,4-dioxane and its downstream products. However, when oxygen was supplied, the genes necessary for 1,4-dioxane biodegradation were upregulated. CB1190 degraded 1,4-dioxane even with as low as 3 mg/L dissolved oxygen. The project team demonstrated that bioaugmented CB1190 was viable and active in wells at a site previously treated with enhanced reductive dechlorination (ERD). These data confirm CB1190’s versatility and ability to withstand extended periods with limited availability of 1,4-dioxane as electron donor as well as oxygen as electron acceptor, and successfully biodegrade 1,4-dioxane in the presence of inhibitory CVOCs.

Biological treatment of mixed contaminants is often limited by the fact that certain microbes can only biodegrade a subset of compounds and are sensitive to prevailing geochemical conditions. Formulation of aerobic and anaerobic contaminant-degrading bacteria will increase the ability to remove pollutant mixtures in varying redox zones across contaminated sites. Costs associated with injecting contaminant-degrading microorganisms, carbon substrates, and nutrient amendments are among the highest considerations for in situ bioremediation methods. This coculture reduces the number of injections that would otherwise be needed to transform CVOCs as well as 1,4-dioxane into benign end products. Results from this project will help the Department of Defense with the decision process of when and how to transition from active ERD to enhanced attenuation of CVOCs as well as 1,4-dioxane. Under certain conditions, this approach could limit the cost and timeframe of the ERD phase (if already implemented) or replace ERD as the primary remedial technology. This approach of using engineered microbial communities could reduce the cost, energy, and substrates required as well as expand the number of sites where in situ bioremediation is considered to be a viable remedy for these compounds. (Anticipated Phase II Completion - 2025).

Polasko, A., A. Zulli, P. B. Gedalanga, P. Pornwongthong, and S. Mahendra. 2019. A Mixed Microbial Community for the Biodegradation of Chlorinated Ethenes and 1,4-Dioxane. Environmental Science and Technology Letters, 6(1):49-54. doi.org/10.1021/acs.estlett.8b00591.

Polasko, A.L., Y. Miao, I. Kwok, K. Park, J. O. Park, and S. Mahendra. 2021. Vinyl Chloride and 1,4-Dioxane Metabolism by Pseudonocardia Dioxanivorans CB1190. Journal of Hazardous Materials Letters, 2:100039. doi.org/10.1016/j.hazl.2021.100039.

Ramos, P., I. Kwok, J. Ngo, D. Zgonc, Y. Miao, P. Pornwongthong, J. Blotevogel, and S. Mahendra. 2022. A, B, Cs of 1,4-Dioxane Removal from Water: Adsorption, Biodegradation, and Catalysis. Current Opinion in Environmental Science and Health, 29:100386. doi.org/10.1016/j.coesh.2022.100386.

Patent

Mahendra, S. and A. Polasko. 2017. Anaerobic-Aerobic Bioremediation of Contaminated Water. US Patent, Patent No. US 62/590,030.