1,4-Dioxane

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.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Dioxane/Tetrahydrofuran Monooxygenase	DXMO/THFMO	Initiates 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 Dehydrogenase	ALDH	Co-expressed with DXMO/THFMO in P. dioxanivorans CB1190.
Rhodococcus ruber strain 219	RRUB219	An aerobic organism that is capable of degrading various petroleum hydrocarbons including linear, branched, and cyclic alkanes and mono- and polyaromatic hydrocarbons.  R. ruber 219 is also capable of degrading 1,4-dioxane, and in the presence of added thiamine it was able to sustain biodegradation of 1,4-dioxane to below health advisory levels.

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.

TARGET	CODE	RELEVANCE / DATA INTERPRETATION
Propane Monooxygenase	PPO	With addition of propane as a growth supporting substrate, aerobic propane utilizing bacteria are capable of co-oxidation of dioxane.
Ring Hydroxylating Toluene Monooxygenase	RMO	When 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 Monooxygenase	RDEG	RDEG targets groups of toluene-2-monooxygenase genes also capable of co-oxidation of dioxane.
Small Chain Alkane Monooxygenase	SCAM	Ongoing 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.

REFERENCE
Chen W, Hyman M. Aerobic cometabolic biodegradation of 1,4-dioxane and its associated co-contaminants. Current Opinion in Environmental Science & Health. 2023;32. https://doi.org/10.1016/j.coesh.2023.100442.
Gedalanga PB, Pornwongthong P, Mora R, Chiang SD, Baldwin B, Ogles D, Mahendra S. Identification of biomarker genes to predict biodegradation of 1,4-dioxane. Applied and Environmental Microbiology. 2014;80:3209-18. https://doi.org/10.1128/AEM.04162-13.
Gedalanga PB, Madison A, Miao Y, Richards T, Hatton J, DiGuiseppi WH, Wilson J, Mahendra S. A multiple lines of evidence framework to evaluate intrinsic biodegradation of 1,4-dioxane. Remediation. 2016;27:93-114. https://doi.org/10.1002/rem.21499.
Lan RS, Smith CA, Hyman MR. Oxidation of cyclic ethers by alkane-grown Mycobacterium vaccae JOB5. Remediation. 2013;23:23-42. https://doi.org/10.1002/rem.21364.
Lippincott D, Streger SH, Schaefer CE, Hinkle J, Stormo J, Steffan RJ. Bioaugmentation and propane biosparging for in situ biodegradation of 1,4-dioxane. Groundwater Monitoring and Remediation. 2015;35:81-92. https://doi.org/10.1111/gwmr.12093.
Mahendra S, Alvarez-Cohen L. Kinetics of 1,4-dioxane biodegradation by monooxygenase-expressing bacteria. Environmental Science & Technology. 2006;40:5435-42. https://doi.org/10.1021/es060714v.
Simmer RA, Richards PM, Ewald JM, Schwarz C, da Silva MLB, Mathieu J, Alvarez PJJ, Schnoor JL. Rapid metabolism of 1,4-dioxane to below health advisory levels by thiamine-amended Rhodococcus ruber Strain 219. Environmental Science & Technology Letters. 2021;8:975-980. https://doi.org/10.1021/acs.estlett.1c00714.