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1,4-Dioxane (14D) does not readily biodegrade in most environments. However, a variety of microorganisms have been identified that can biodegrade 14D through either direct growth-related metabolism or cometabolism. During metabolic biodegradation, microorganisms use 14D as the growth substrate, however growth is slow unless 14D concentrations are very high (>100 mg/L). During cometabolic biodegradation, an additional growth substrate must be supplied to support biomass growth and induce the appropriate 14D-degrading enzymes. Unlike metabolic degradation, cometabolic processes can reduce 14D to very low concentrations.

Related Article(s):

• 1,4-Dioxane
• Biodegradation - Cometabolic

CONTRIBUTOR(S): Dr. Shaily Mahendra and Dr. Michael Hyman

Key Resource(s):

• Advances in bioremediation of 1,4-dioxane-contaminated waters

1,4-Dioxane Biodegradation

Figure 1. Growth Rates of Two 14D Metabolizers Versus 14D Concentration

1,4-Dioxane (14D) does not readily biodegrade in most anaerobic environments. However, multiple studies have demonstrated that 14D can be biodegraded by a variety of microorganisms under aerobic conditions. A summary of microorganisms that are reported to aerobically biodegrade 14D is presented in Table 1. Under aerobic conditions 14D can be biodegraded through two physiologically distinct processes: (a) metabolism; and (b) cometabolism. Metabolism is a process in which microorganisms use the organic contaminants as a carbon and energy source to support their growth. [Cometabolism] occurs when microorganisms degrade contaminants using non-specific enzymes but do not gain carbon or energy to support growth from the degradation process.

Table 1. List of 1,4-Dioxane Degrading Microorganisms and Biodegradation Rates

Strain	Induced Enzyme	Biodegradation Rate	Reference
Metabolism
Pseudonocardia dioxanivorans CB1190	THFMO	0.19 ± 0.007 mg/hr/mg-protein	Mahendra and Alvarez-Cohen (2005, 2006)
Actinomycete CB1190*	N/A	0.33 mg/min/mg-protein	Parales et al. (1994)
Amycolata sp. CB1190*	N/A	0.038 ± 0.012 mg/hr/mg-protein	Kelley et al. (2001)
Pseudonocardia benzenivorans B5	THFMO	0.01± 0.003 mg/hr/mg-protein	Mahendra and Alvarez-Cohen (2006)
Pseudonocardia carboxydivorans RM-31	?	31.6 mg/L/hr	Matsui et al. (2016)
Afipia sp. D1	?	0.052 to 0.263 mg/hr/mg-protein	Sei et al. (2013)
Mycobacterium sp. PH-06	PrMO	2.5 mg/L/hr	Kim et al. (2009)
Acinetobacter baumannii DD1	?	2.38 mg/L/hr	Huang et al. (2014)
Xanthobacter flavus DT8	?	Similar to CB1190	Chen et al. (2016)
Cordyceps sinensis (fungus)	?	0.011 mol/day	Nakamiya et al. (2005)
Cometabolism
Mycobacterium austroafricanum JOB5	?	0.40 ± 0.06 mg/hr/mg-protein	House and Hyman (2010) Lan et al. (2013)
Rhodococcus ruber ENV425	?	10 mg/hr/g TSS	Lippincott et al. (2015); Vainberg et al. (2006)
Pseudonocardia sp. ENV478	THFMO	21 mg/hr/g TSS	Masuda et al. (2012) Vainberg et al. (2006)
Rhodococcus RR1	?	0.38 ± 0.03 mg/hr/mg-protein	Mahendra and Alvarez-Cohen (2006)
Rhodococcus jostii RHA1	PrMO	N/A	Hand et al. (2015); Li et al. (2013)
Flavobacterium	?	N/A	Sun et al. (2011)
Pseudonocardia K1	THFMO	0.26 ± 0.013 mg/hr/mg-protein	Mahendra and Alvarez-Cohen (2006)
Burkholderia cepacia G4	T2MO	0.1± 0.006 mg/hr/mg-protein	Mahendra and Alvarez-Cohen (2006)
Ralstonia pickettii PKO1	T3MO	0.31± 0.007 mg/hr/mg-protein	Mahendra and Alvarez-Cohen (2006)
Pseudomonas mendocina KR1	T4MO	0.37± 0.04 mg/hr/mg-protein	Mahendra and Alvarez-Cohen (2006)
Aureobasidium pullmans NRRL 21064	?	6-8 mg/L within a day	Patt and Abebe (1995)
Graphium sp. ATCC 58400 (fungus)	CYP	4 ± 1 nmol/min/mg dry weight (with Propane) 9 ± 5 nmol/min/mg dry weight (with THF)	Skinner et al. (2009)

Aerobic Metabolism

While several microorganisms have been isolated that can metabolize 14D, these organisms are not common. The best characterized 14D-metabolizing strain is Pseudonocardia dioxanivorans CB1190. This bacterium was originally enriched from industrial activated sludge, fed with tetrahydrofuran (THF) and then subsequently fed with 14D. The doubling time of CB1190 was about 30 hours when it was grown in ammonium mineral salts medium at 30 °C amended with 5.5 mM (484 mg/L) 14D. Degradation of 14D by strain CB1190 may be inhibited by elevated concentrations of chlorinated solvents and their degradation products, and by some metals. Inhibition of 14D degradation was strongest for 1,1-dichloroethene (1,1-DCE) followed by cis-1,2-diochloroethene (cDCE) > trichloroethene (TCE) > 1,1,1-trichloroethane (TCA). 14D biodegradation was completely inhibited by 5 mg/L 1,1-DCE. Cu(II) was the strongest metal inhibitor of 14D degradation by CB1190, causing an increase in the lag period at 1 mg/L and an order of magnitude reduction in 14D degradation rates at 10 and 20 mg/L Cu(II). 14D degradation was less sensitive to Cd(II) and Ni(II), while Zn(II) had no impact on 14D biodegradation at the maximum concentration tested (20 mg/L Zn).

An important challenge for in situ bioremediation of 14D is the slow growth of 14D metabolizers at typical groundwater concentrations. Figure 1 shows estimated growth rates for two 14D metabolizing organisms (P. dioxanivorans CB1190 and P. benzenivorans B5) computed using published kinetic parameters. At a 14D concentrations of 1000 µg/L, CB1190 and B2 have growth rates of 0.015 and 0.002 per day. At typical groundwater concentrations (<1000 µg/L), growth rates are expected to be less than the endogenous decay rate, resulting in a steady decline in the number of 14D metabolizers.

Aerobic Cometabolism

Unlike 14D-metabolizing microorganisms, 14D-cometabolizing organisms do not grow on 14D but can degrade this compound after growth on a primary, growth-supporting substrate. As cometabolically active microorganisms do not use co-substrates as either major carbon or energy sources, they can often degrade co-substrates at concentrations well below those that can be achieved by organisms that metabolize and grow on these co-substrates. A wide variety of bacteria and some fungi can cometabolically degrade 14D (see Table 1). These include model organisms that grow on primary substrates such as toluene or methane and involve well-characterized enzymes such as soluble methane monooxygenase (sMMO) or toluene monooxygenases (TxMO), respectively. Other 14D-cometabolizing strains that grow on THF, ethane, and isobutane have also been described.

Much of the research into 14D cometabolism has focused on high concentrations of 14D (≥100 mg/L), and less is currently known about the activity of specific microorganisms and their monooxygenases at lower, more environmentally relevant 14D concentrations (<100 µg/L). Despite this, many bacterial monooxygenases can concurrently oxidize multiple co-substrates, and recent field studies have demonstrated that cometabolic approaches involving either propane biostimulation and bioaugmentation or propane biostimulation alone can be highly effective treatment strategies for degrading contaminant mixtures that contain parts-per-billion concentrations of both 14D and associated chlorinated co-contaminants.

Anaerobic Biodegradation

To date, there is little evidence of metabolic or cometabolic anaerobic 14D biodegradation. In a microcosm study using samples of aquifer material from several different 14D impacted sites, there was no evidence of 14D biodegradation. However, there is one study that reported anaerobic growth of an iron-reducing bacterium on 14D. An indirect method of anaerobic 14D biodegradation exists via a microbially driven Fenton reaction. Shewanella oneidensis, an Fe(III)-reducing facultative anaerobe, is able to generate hydroxyl radicals that can then break down 14D. Under anaerobic conditions, S. oneidensis produces Fe(II) that can then interact chemically with H₂O₂ to yield HO• radicals which can oxidatively degrade 1,4-dioxane.

Molecular Biological Tools

[Quantitative polymerase chain reaction (qPCR)] quantifies the abundance of specific microorganisms and functional genes capable of degrading a particular contaminant. qPCR analyses have been employed to quantify the abundance of microorganisms that can degrade 14D through the activity of tetrahydrofuran monooxygenase (THFMO) in samples from industrial activated sludge and groundwater. However, care is warranted in the interpretation of qPCR results in other cases (e.g. propane-stimulated cometabolic 14D degradation) where the role of enzymes such as propane monooxygenase (PrMO) in 14D degradation is less clearly established. Studies also suggest that these biomarkers can be used to detect the abundance of 14D-degraders in situ and how they compete within the larger microbial community. Microbial community analyses can be an asset towards guiding treatment strategies and predicting treatment synergies. [Compound specific isotope analysis (CSIA)] refers to measurement of the isotopic signatures of individual chemical compounds and can be used to differentiate contaminant sources, delineate reaction pathways, and provide evidence of in situ contaminant degradation. CSIA methods are now available to measure isotopic fractionation of carbon and hydrogen. For example, the aerobic biodegradation of 14D by a THF-grown 14D-degrading Pseudonocardia strain exhibited an isotopic fractionation factor (ε ) for carbon (ε_c) of −4.73 ± 0.9‰ and hydrogen (ε_H) of -147 ± 22‰, respectively. Smaller ε_cand ε_H values, (-2.7 ± 0.3‰ and -21 ± 2‰, respectively) were determined for aerobic 14D degradation by a propane-grown Rhodococcus strain. As many Pseudonocardia strains use THFMO to initiate 14D degradation, CSIA, in conjunction with qPCR analyses, may be able to discriminate the roles of metabolism and cometabolism in 14D biodegradation at field sites.

Summary

14D is not readily biodegraded under ambient conditions in most environments. However, a variety of microorganisms have been identified that can biodegrade 14D through either growth-related metabolism or fortuitous cometabolism. Metabolic growth on the target pollutant is the more traditional approach for both above ground and in situ bioremediation approaches. However, approaches based on metabolic growth may not be feasible for treatment of 14D at typical concentrations. Cometabolic treatment approaches can reduce 14D to very low levels and have been demonstrated in the field. However, implementation of cometabolic treatment is more complex, and there is currently less engineering experience in the design and operation of these systems.