The Dioxane Biodegradation Package answers the key questions impacting the feasibility and performance of monitored natural attenuation (MNA) or enhanced bioremediation as treatment strategies: (1) What are the concentrations of contaminant degrading microorganisms (2) Is contaminant biodegradation occurring?

Package 1
Stable Isotope Probing (SIP)

An increasing number of microorganisms have been isolated that utilize dioxane as a growth supporting substrate under aerobic conditions suggesting biodegradation is a viable attenuation mechanism. In the aerobic dioxane utilizing microorganism Pseudonocardia dioxanivorans CB1190, the first step in dioxane metabolism is mediated by a dioxane/tetrahydrofuran monooxygenase (DXMO/THFMO). An aldehyde dehydrogenase (ALDH) is co-expressed with DXMO/THFMO.

Therefore, CENSUS qPCR assays have been developed to quantify the DXMO/THFMO and ALDH genes to evaluate the potential for dioxane/tetrahydrofuran (co)metabolism in environmental samples.

DXMO/THFMOInitiates aerobic metabolism of 1,4-dioxane by P. dioxanivorans CB1190. In other organisms however, DXMO/THFMO initiates metabolism of tetrahydrofuran and only co-oxidation of dioxane.
Aldehyde DehydrogenaseALDHCo-expressed with DXMO/THFMO in P. dioxanivornans CB1190.

Under aerobic conditions, dioxane is also amenable to cometabolism by several groups of organisms expressing monooxygenase genes for the metabolism of a variety of primary substrates including propane and other n-alkanes, tetrahydrofuran, and toluene. Engineered propane injection to stimulate dioxane cometabolism has been demonstrated at pilot scale in the field. CENSUS qPCR assays are available to quantify monooxygenase genes to assess the potential for cometabolism of dioxane.

Propane Monooxygenase
PPOWith addition of propane as a growth supporting substrate, aerobic propane utilizing bacteria are capable of co-oxidation of dioxane.
Ring Hydroxylating Toluene MonooxygenaseRMOWhen expressed, a group of related toluene monooxygenases (RMO, RDEG) responsible for aerobic biodegradation of BTEX also co-oxidize dioxane. More specifically, RMO quantifies a subfamily of toluene-3- and toluene-4-monooxygenase genes.
Ring Hydroxylating Toluene MonooxygenaseRDEGRDEG targets groups of toluene-2-monoxygenase genes also capable of co-oxidation of dioxane.
Small Chain Alkane MonooxygenaseSCAMOngoing research indicates that small chain alkane monooxygenases are induced by a wide variety of gaseous alkanes and are especially effective for 1,4-D cometabolism.

Stable Isotope Probing (SIP)

Stable isotope probing (SIP) is an innovative molecular biological tool that can conclusively determine whether in situ biodegradation of a specific contaminant has occurred.

Demonstrating that 1,4-dioxane biodegradation is occurring is a critical question in determining the feasibility of monitored natural attenuation (MNA). Therefore, SIP studies with 13C dioxane have been performed to conclusively determine whether dioxane biodegradation is occurring on site and to evaluate MNA as a remediation strategy.

With the SIP method, a Bio-Trap® amended with a 13C “labeled” contaminant (e.g., 13C dioxane) is deployed in an impacted monitoring well for 30 to 60 days. The 13C label serves much like a tracer which can be detected in the end products of biodegradation – microbial biomass and CO2. Following in field deployment, the Bio-Trap® is shipped to MI for analysis:  Detection of 13C enriched phospholipid fatty acids (PLFA) following in field deployment, conclusively demonstrates in situ biodegradation and incorporation into microbial biomass. Detection of 13C enriched dissolved inorganic carbon demonstrates contaminant mineralization to CO2.

Compound Specific Isotope Analysis (CSIA)

As described above, Stable Isotope Probing (SIP) is recommended to conclusively determine whether biodegradation of dioxane is occurring. However, the use of Compound Specific Isotope Analysis (CSIA) to evaluate dioxane biodegradation is an area of continuing research. Carbon isotope enrichment factors have been measured in the laboratory but are relatively small.  While additional research is needed, study suggest that dioxane cometabolism may lead to more notable hydrogen isotope (δ2H) enrichment. In addition, 2D-CSIA (δ13C and δ2H) has important applications for contaminant source distinction/delineation at sites where multiple sources may be present.