Monitored natural attenuation (MNA), which relies primarily on biodegradation, is renowned as a cost-effective approach to managing the large and dilute groundwater plumes formed by 1,4-dioxane (dioxane). However, the burden of proof that MNA is an appropriate solution lies in site-specific demonstration of the presence and expression of relevant biodegradation capabilities. Previous research on dioxane biodegradation has been centered on group-5 tetrahydrofuran monooxygenase (THM)-expressing bacteria. Recent studies have revealed the significant contribution of group-6 propane monooxygenase’s (PRM) expressing organisms in dioxane attenuation in the field. Particularly, compared to THM, PRM exhibits a higher affinity to dioxane, lower inhibition by chlorinated solvents, and a broader substrate range, eliciting physiological and ecological advantages at some sites.

In this proof-of-concept project, the project team will categorize group-6 PRMs into three sub-clusters based on their phylogenetic divergence. The project team aims to characterize their biodegradation kinetics and quantitatively assess their contributions to dioxane attenuation at environmentally relevant concentrations. Specific objectives include:

• characterizing dioxane biodegradation kinetics of PRMs from different sub-clusters at environmentally relevant concentrations and assessing their ability to degrade other minor components (e.g., 1,2-DCA);
• designing and validating specific biomarkers for the comprehensive and sensitive quantification of PRM coding genes from different sub-clusters;
• assessing the correlation between dioxane attenuation rates observed in groundwater microcosms and abundance of PRMs- and THMs-coding genes enumerated by the biomarker assays; and
• conducting a metastudy of previous or ongoing dioxane attenuation remediation and exploring key geochemical and microbial ecology factors for minor modifications (e.g., air sparging or nutrient addition) made to enhance the removal of dioxane and other co-occurring chemicals.

The main goal of the project is to investigate dioxane biodegradation mechanisms and kinetics by PRM-expressing microbes at environmentally relevant concentrations. To circumvent the discrepancies of host cells, the project team will employ a heterologous expression system to produce active PRMs for kinetic and substrate range assessments (Task 1). To mimic environmentally relevant conditions, dioxane degradation by both wild-type strains and transformant clones that express PRMs will be validated at an initial dosage of 10, 25, and 100 parts per billion. Primers/probe sets specific to individual PRM sub clusters will be designed by targeting the amino acid residues surrounding the active sites. The reliability, selectivity, and sensitivity of these probes will be determined using the genomic DNAs from pure cultures that express PRMs and phylogenetically relevant enzymes (Task 2). Probe validation (Task 3) involves determining whether dioxane attenuation rates in microcosms correlate with biomarker copy numbers. In Task 4, the project team will perform a metastudy of dioxane and 1,2-DCA attenuation rates at ~50 sites inclusive of a range of geochemical conditions and remedial activities that have been completed or are underway for dioxane (e.g., propane biosparging) or co-occurring chemicals (e.g., in situ bioremediation for chlorinated solvents), enabling a comparison to laboratory-derived findings achieved in Tasks 1-3.

This one-year proof-of-concept study is of critical value and relevance to the Department of Defense (DoD) because it will advance the fundamental understanding of dioxane-degrading enzymes. It will also enable the development of a diagnostic molecular tool for the prediction of PRM environmental behaviors and contributions to dioxane biodegradation naturally occurring in the field or stimulated with auxiliary substrates. The expected results will be beneficial for DoD and other stakeholders to select and design more cost-effective and efficient remedial approaches (e.g., MNA and biostimulation) to mitigate dioxane and other relatively minor components of common groundwater chemical mixtures at impacted sites. (Anticipated Project Completion - 2024)