Identification of the Intermediates of in Vivo Oxidation of 1,4-Dioxane by Monooxygenase-Containing Bacteria

Abstract

1,4-dioxane is a probable human carcinogen and an emerging water contaminant. Monooxygenase-expressing bacteria have been shown to degrade dioxane via growth-supporting as well as cometabolic mechanisms. In this study, the intermediates of dioxane degradation by monooxygenase-expressing bacteria were determined by triple quadrupole-mass spectrometry and Fourier transform ion cyclotron resonance-mass spectrometry. The major intermediates were identified as 2-hydroxyethoxyacetic acid (HEAA), ethylene glycol, glycolate, and oxalate. Studies with uniformly labeled 14C dioxane showed that over 50% of the dioxane was mineralized to CO2 by CB1190, while 5% became biomass-associated after 48 h. Volatile organic acids and non-volatiles, respectively, accounted for 20 and 11% of the radiolabeled carbon. Although strains cometabolizing dioxane exhibited limited transformation capacities, nearly half of the initial dioxane was recovered as CO2. On the basis of these analytical results, we propose a pathway for dioxane oxidation by monooxygenase-expressing cells in which dioxane is first converted to 2-hydroxy-1,4-dioxane, which is spontaneously oxidized to HEAA. During a second monooxygenation step, HEAA is further hydroxylated, resulting in a mixture of dihydroxyethoxyacetic acids with a hydroxyl group at the ortho or para position. After cleavage of the second ether bond, small organic molecules such as ethylene glycol, glycolate, glyoxalate, and oxalate are progressively formed, which are then mineralized to CO2 via common cellular metabolic pathways. Bioremediation of dioxane via this pathway is not expected to cause an accumulation of toxic compounds in the environment.

Identification of Biomarker Genes To Predict Biodegradation of 1,4-Dioxane

“…Identification of candidate biomarkers was focused on monooxygenase and dehydrogenase genes based on earlier work describing the dioxane degradation pathway for monooxygenase-expressing bacteria (29,36) and expression of dehydrogenase genes in bacteria during utilization of monooxygenase-inducing substrates (37)(38)(39). To facilitate gene selection, an in silico analysis was performed to identify all annotated monooxygenase groups within CB1190 and its associated plasmids (35).…”

Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species

“…strain ENV478 initially grown on THF (10). HEAA and 12 additional intermediates of dioxane degradation were subsequently detected for strain CB1190, including 2-hydroxy-1,4-dioxane, 2-hydroxyethoxyacetaldehyde, 1,4-dioxane-2-one, 1,3-dihydroxyethoxyacetic acid, 2-hydroxyethoxy-2-hydroxyacetic acid, glycoaldehyde, glyoxal, and formic acid (16). Even though a dioxane degradation pathway was proposed based on these detected intermediates (16), genes likely associated with several enzymatic steps in the pathway were only recently identified (17).…”

“…HEAA and 12 additional intermediates of dioxane degradation were subsequently detected for strain CB1190, including 2-hydroxy-1,4-dioxane, 2-hydroxyethoxyacetaldehyde, 1,4-dioxane-2-one, 1,3-dihydroxyethoxyacetic acid, 2-hydroxyethoxy-2-hydroxyacetic acid, glycoaldehyde, glyoxal, and formic acid (16). Even though a dioxane degradation pathway was proposed based on these detected intermediates (16), genes likely associated with several enzymatic steps in the pathway were only recently identified (17). Transcriptional analyses showed that the putative THF monooxygenase (MO) gene cluster thmADBC in strain CB1190 was the only MO out of eight previously identified by genome sequencing (18) that was upregulated during growth on dioxane versus glycolate.…”

Genome Sequence of the 1,4-Dioxane-Degrading Pseudonocardia dioxanivoransStrain CB1190

“…P. dioxanivorans strain CB1190 (NCBI taxonomy ID 675635) was isolated from 1,4-dioxane-contaminated sludge and displays the rare ability to use 1,4-dioxane, an emerging groundwater contaminant, as a sole carbon and energy source (9,10,11,14). This strain can also grow on other ethers (e.g., 2-methyl-1,3-dioxolane and butyl methyl ether), alcohols, and benzene as the sole carbon and energy source, and it can grow autotrophically with CO 2 by using H 2 as the electron donor (9,14).…”