This project builds on the important and recent discovery that, when supplemented with thiamine (vitamin B1), Rhodococcus ruber RJ01 can rapidly metabolize low dioxane concentrations (< 100 µg/L) to below 0.35 µg/L, which is the current Health Reference Level. While cometabolic strategies have been suggested for bioremediation of dilute dioxane plumes, R. ruber RJ01’s capacity to metabolically degrade such low concentrations is unprecedented and could facilitate more efficient and cost-effective dioxane bioremediation with minimal post-bioaugmentation inputs. However, as strain survival is the most common cause for bioaugmentation failures, it is necessary to develop methods for maintaining strain viability under a wide range of realistic conditions while simultaneously ensuring access to substrates required for sustained biodegradation.

To address these needs, this research project will address the following objectives:

1. Demonstrate long-term performance of R. ruber RJ01 under dilute dioxane plume conditions (i.e., survival in flow-through aquifer columns fed just 100 µg/L dioxane, and sustained dioxane degradation below maximum contaminant level if thiamine is available).
2. Develop practical strategies for in an situ supply of thiamine to R. ruber RJ01.
3. Develop and scale up encapsulation and immobilization techniques for R. ruber RJ01 and other co-amended species (or substrates) to improve RJ01 survival and sustain activity post-bioaugmentation.
4. Validate performance under a range of relevant site conditions using microcosm and flow-through aquifer column experiments.

Two key aspects of this project's approach will be to (1) ensure a continual supply of thiamine to maintain dioxane biodegradation, and (2) provide a refuge to protect RJ01 from abiotic and biotic stress. Thiamine supplementation will be assessed using both slow-release composites and co-culturing with syntrophic thiamine-producing bacteria. Immobilized or planktonic RJ01 will then be encapsulated with the thiamine source using various hydrogel formulations and surface coatings to enhance physical integrity. Various formulations will then be tested in microcosms and flow through columns to optimize performance under a wide range of environmental conditions.

Dilute dioxane plumes are found at many Department of Defense sites and present a formidable remediation cost. Bioremediation is a promising cost-effective strategy to treat these plumes. However, strains tested to date have limitations that hinder their bioremediation efficacy, particularly at low dioxane concentrations. RJ01’s reliable performance at low dioxane concentrations (<100 µg/L) is unprecedented, and this project will yield an optimized bioaugmentation process suitable for testing in a field trial that will significantly enhance dioxane bioremediation capabilities. (Anticipated Project Completion - 2027)