Enrichment with Carbon-13 and Deuterium during Monooxygenase-Mediated Biodegradation of 1,4-Dioxane

Recent technical developments have enabled the application of compound-specific isotope analysis (CSIA) of low (parts per billion) concentrations of 1,4-dioxane that are often found in groundwater at 1,4-dioxane-contaminated sites. However, to quantify 1,4-dioxane biodegradation, isotopic enrichment factors are needed to interpret the CSIA data obtained from field samples. In this study, the carbon and hydrogen isotopic enrichment factors (ε C and ε H, respectively) for 1,4-dioxane biodegradation have been determined for axenic propane- or isobutane-grown cultures of Rhodococcus rhodochrous ATCC 21198 and for tetrahydrofuran-grown cultures of Pseudonocardia tetrahydrofuranoxidans K1. The enrichment factors for propane-grown (ε C = −2.7 ± 0.3‰, and ε H = −21 ± 2‰) and isobutane-grown (ε C = −2.5 ± 0.3‰, and ε H = −28 ± 6‰) cells of strain 21198 were similar and substantially smaller than those determined for tetrahydrofuran-grown cells of strain K1 (ε C = −4.7 ± 0.9‰, and ε H = −147 ± 22‰). The presence of 1-butyne consistently inhibited both the biodegradation and isotopic fractionation of 1,4-dioxane, and this effect implicates monooxygenase enzymes in both the biodegradation and isotopic enrichment of 1,4-dioxane. Our results confirm that an increasing level of enrichment of heavier isotopes of carbon and hydrogen can be used to quantify 1,4-dioxane biodegradation and suggest CSIA can discriminate between the activities of monooxygenase-expressing bacteria expected to be prevalent in engineered, gaseous alkane-stimulated 1,4-dioxane treatment systems and those that may involve microbial metabolism of 1,4-dioxane as a natural attenuation process.