1,4-dioxane has been used extensively as a stabilizing agent in chlorinated solvents such as 1,1,1-trichloroethane, and it has recently emerged as a groundwater contaminant throughout the United States and elsewhere. Because of its miscibility in water, its low Henry’s Law constant, and its low octanol/water partitioning coefficient, it is poorly retarded in aquifers. As a result, this compound has the potential to create large contaminant plumes and to threaten drinking water supplies. Few treatment methods have proven successful and economically feasible for removing 1,4-dioxane from groundwater. Relatively few studies have evaluated biological degradation of 1,4-dioxane, but indigenous microorganisms typically are not able to degrade this compound. In the last several years, however, 1,4-dioxane biodegradation has been reported in both pure and mixed cultures. Little or no controlled research has been done to evaluate 1,4-dioxane degradation under redox conditions that are common in aquifers or to evaluate the effect of co-contaminants or biostimulation additives on 1,4-dioxane degradation.

The objectives of this project were to (1) evaluate biodegradation of 1,4-dioxane in environmental samples under different redox and chemical/physical conditions and different treatment regimes; (2) identify and isolate new 1,4-dioxane-degrading microbes from environmental microcosms; (3) identify the products of 1,4-dioxane biodegradation by studying degradation pathways in pure bacterial cultures; (4) confirm that the same biodegradation pathways occur in active environmental samples; and (5) identify and evaluate genes involved in 1,4-dioxane biodegradation.

Biodegradation of 1,4-dioxane in environmental samples was performed by utilizing microcosms constructed with samples from two hydrogeochemically different 1,4-dioxane-contaminated aquifers and by performing enrichment culturing with samples from these and two other sites. The 1,4-dioxane biodegradation pathways were determined by elucidating 1,4-dioxane degradation products produced by strains ENV425, ENV473, and ENV478.

Microcosms were incubated under aerobic, nitrate reducing, iron reducing, sulfate reducing, and methanogenic conditions. Some microcosms received sodium lactate, molasses, or vegetable oil to simulate anaerobic biostimulation of chlorinated solvent degradation. Likewise, some aerobic microcosms received propane or tetrahydrofuran (THF), known co-substrates for 1,4-dioxane degradation, while others received Pseudonocardia sp. strain ENV478 or Rhodococcus sp. strain ENV425 to simulate bioaugmentation. 1,4-dioxane was not degraded in any of the anaerobic microcosms (more than 400 days of incubation). Some degradation was observed in microcosms from Elkton, Maryland, that had been propane-stimulated. Degradation in these microcosms was not apparent until after 100 days of incubation, and an additional spike of 1,4-dioxane was not fully degraded. 1,4-dioxane also was degraded in microcosms augmented with strain ENV478 or strain ENV425 plus propane. Several repeat additions of 1,4-dioxane were degraded by strain ENV478, but repeated additions were not well degraded in microcosms augmented with strain ENV425 plus propane. Unfortunately, the difficulty of growing ENV478, its requirement for THF for prolonged activity, and its tendency to form dense cell clumps would likely limit its utility as a biocatalyst for in-situ treatment of 1,4-dioxane.

The first elucidation of a bacterial biodegradation pathway for 1,4-dioxane was completed. Each of the strains tested co-metabolically degraded 1,4-dioxane after growth on either propane or THF. 1,4-dioxane degradation resulted in the production of 2HEAA that was not further oxidized by the strains tested, although it was degraded by other organisms and in environmental samples. From these studies, researchers concluded that the inability to metabolize 2HEAA, and thereby generate energy to support the oxidation of 1,4-dioxane, is likely a significant contributing factor preventing biological degradation of 1,4-dioxane. Likewise, the inability of 1,4-dioxane to induce propane and THF monooxygenase genes may also contribute to the recalcitrance of 1,4-dioxane.

Extensive molecular biological analysis of 1,4-dioxane-degrading bacteria revealed that each of them produced multiple and diverse monooxygenase enzymes, many of which are induced during growth on their primary substrates. Although none of the cloned genes could be functionally expressed in heterologous host strains, evidence generated suggested that broad substrate soluble diiron monoxygenase enzymes are the most likely catalysts of co-metabolic 1,4-dioxane degradation.

Results of this study demonstrated the recalcitrance of 1,4-dioxane. Although several organisms were shown to degrade 1,4-dioxane via cometabolism during growth on propane or THF, 1,4-dioxane was not degraded in microcosms created with samples from two different aquifers regardless of the redox conditions employed. Likewise, 1,4-dioxane was not degraded in samples from two different treatment systems that had been exposed to 1,4-dioxane for extended periods. No bacteria that could grow on 1,4-dioxane were enriched or isolated from the four systems tested. Therefore, results of this study demonstrate that biological treatment and natural biological attenuation are unlikely to be successful remedial alternatives for 1,4-dioxane-contaminated sites. (Project Completed – 2007)