1,4-Dioxane (dioxane) and n-nitrosodimethylamine (NDMA) are emerging groundwater contaminants that are probable human carcinogens. Neither compound is significantly attenuated in the environment by volatilization or sorption processes, but a role for aerobic microbial processes in their removal has been observed. The objective of this SERDP project was to identify organisms, enzymes and biochemical pathways involved in the aerobic biodegradation of dioxane and NDMA, in order to develop a better understanding of the effects of bacterial degradation on the fate and persistence of dioxane and NDMA in the environment. This project focused specifically on oxygenase-catalyzed biodegradation of the targeted compounds.

Bacterial isolates were tested for degradation of NDMA or dioxane under monooxygenase-inducing conditions. The potential role of monooxygenases in contaminant removal was confirmed by exposing cells to the monooxygenase inhibitor acetylene and monitoring for loss of activity. The role of monooxygenases was also verified with molecular techniques by knocking out or heterologously expressing putative monooxygenase genes. The effect of the common co-contaminants 1,1,1-trichloroethane and 1,1-dichloroethene on dioxane degradation was tested, as was the effect of propane on NDMA degradation. Analytical chemistry techniques were used to identify the suite of transformation intermediates of dioxane metabolism. The genome of the dioxane metabolizing actinobacterium Pseudonocardia dioxanivorans strain CB1190 was sequenced, and a gene expression microarray was applied to identify genes linked to dioxane and tetrahydrofuran metabolism. Amino acid isotopomer analysis with ¹³C-labeled carbon substrates was used to demonstrate the activity of strain CB1190 metabolic pathways during dioxane and C2 substrate metabolism. Transformation of strain CB1190 with plasmid and transposomes was tested to develop a set of genetic tools for verifying the importance of particular genes in strain CB1190 metabolism.

NDMA degradation in tested actinobacteria was linked to propane degradation and in Rhodococcus jostii strain RHA1 directly to a propane monooxygenase. In two tested isolates, propane negatively impacted NDMA degradation. Monooxygenases were also linked to dioxane degradation in a number of tested bacterial isolates, and dioxane-transforming activity was found to be relatively common among monooxygenase-expressing bacteria. Both 1,1,1-trichloroethane and 1,1-dichloroethene inhibited dioxane degradation by bacterial isolates. The first complete pathway for the mineralization of dioxane was proposed based on identification of transformation intermediates. The complete sequence of the strain CB1190 genome was obtained, and both chromosomal- and plasmid-encoded genes were induced by dioxane and tetrahydrofuran (THF). Strain CB1190 was shown to use the glyoxylate carboligase pathway during dioxane metabolism, which identified how energy is obtained by dioxane degradation. The plasmid-encoded monooxygenase was demonstrated to transform both dioxane and THF. Strain CB1190’s large genome encodes for the utilization of a wide variety of carbon and nitrogen compounds, and annotation of some carbon utilization pathways was verified. The transformation of strain CB1190 was achieved, which will enable further development of a genetic system for this dioxane-metabolizing bacterium.

This work highlights the importance of monooxygenases in the degradation of both NDMA and dioxane. The induction of monooxygenases in (co)metabolizing bacteria will lead to improved methods for the removal of NDMA and dioxane from contaminated groundwater.