Bioremediation of xenobiotics including methyl tert-butyl ether

Details Bioremediation of xenobiotics including methyl tert-butyl ether Bioremediation of xenobiotics including methyl tert-butyl ether

1998-03-11

2001-02-27

Redding, David A. (Department: 1744)

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Destruction of hazardous or toxic waste

C435S254100

Reexamination Certificate

active

06194197

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the bioremediation of xenobiotic compounds, in particular the gasoline additive methyl tert-butyl ether (MTBE), by microorganisms including Graphium sp. fungi. Biofilters that incorporate such organisms and that may be used to reduce MTBE contamination in, for example, ground water, are also disclosed.
BACKGROUND OF THE INVENTION
The ether bond has recently been described as “the single most common and unifying structural feature which confers to both biological and xenobiotic compounds a high degree of resistance to biological mineralization” (White et al., 1996). This resistance occurs in ether-bonded compounds ranging from the complex natural product lignin, to simpler and widely used anthropogenic chemicals including several pesticides (Alexander, 1973), common solvents such as diethyl ether (DEE) (Alexander, 1973) and, more recently, gasoline additives such as methyl tert-butyl ether (MTBE) (Mormille et al., 1994; Yeh and Novak, 1994), tert-amyl methyl ether (TAME) and ethyl tert-butyl ether (ETBE). At present very little is known about the microbial degradation of simple alkyl ethers. For instance, there are only two reports of microorganisms utilizing DEE as a growth-supporting substrate but these reports did not investigate the possibility of growth-supporting contaminants (Heyden, 1974; Parales et al., 1994) (the importance of this issue has been recently illustrated by the observation that growth of
Ancyclobacter aquaticus
in the presence of 2-chloroethyl vinyl ether is supported by abiotic hydrolysis products of the ether rather than the ether compound itself (van den Wijngaard et al., 1993)).
Whereas DEE is used as an industrial solvent, MTBE is widely used in many modem gasoline formulations. MTBE acts as both an octane enhancer and as an oxygenating compound, thereby permitting both an elimination of alkyl-lead anti-knocking agents and a reduction in automobile carbon monoxide emissions. Current consumption of MTBE in the United States, the world's largest consumer, was recently estimated at approximately 2.0×10
10
gallons/year (Ainsworth, 1991). There is currently considerable uncertainty about the long-term human health effects of MTBE exposure. The U.S. Environmental Protection Agency has issued a drinking-water advisory for MTBE of 20-40 &mgr;g/l USEPA (1997) (Drinking Water Advisory: Consumer acceptability advice and health effects analysis on methyl tertiary-butyl ether (MtBE) Office of Water, EPA-822-F-97-009. USEPA, Washington, D.C.). Recently, MTBE has been detected in many urban groundwater supplies, most likely as the result of gasoline spills and leaking storage tanks (Squillace et al., 1996). Recent studies also indicate that MTBE is very poorly biodegradable in groundwater under a variety of redox conditions (Mormille et al., 1994; Yeh and Novak, 1994). MTBE degradation has been described for a mixed microbial culture in a pathway involving tert-butyl alcohol (TBA) (Salanitro et al., 1994). Three bacterial isolates have been reported to exhibit slow growth on MTBE and yeast extract (Mo et al., 1997) and the oxidation of MTBE by propane-oxidizing bacteria such as
Mycobacterium vaccae
has been described (Steffan et al., 1997). However, the affinity of these organisms for MTBE (or, more specifically, the K
m
of the MTBE degrading enzymes produced by these organisms) may not be sufficient to achieve the 20-40 &mgr;g/l standard set by the EPA for MTBE levels in water.
It is an objective of the present invention to provide isolated microorganisms which degrade MTBE and/or chlorinated aliphatic hydrocarbons, and which have a high affinity for these compounds.
It is a further object of this invention to provide biofilters suitable for the bioremediation of MTBE.
SUMMARY OF THE INVENTION
The present invention rests, in part, on the discovery that certain microorganisms possess the ability to co-metabolize MTBE. In one example, Graphium sp. fungus is shown to co-metabolize MTBE when grown in the presence of gaseous n-alkanes such as ethane, propane and n-butane and simple branched alkanes such as isobutane (2-methyl propane) and isopentane (2-methyl butane). It is shown that the ability of Graphium to degrade MTBE is likely attributable to the expression of a non-specific cytochrome P-450 oxygenase activity. This activity is known to be found in a number of microorganisms, including other fungi and bacteria. Accordingly, it is anticipated that a wide range of microorganisms possess the capability to co-metabolize MTBE and other gasoline additions such as TAME and ETBE. One aspect of the present invention is thus a method of selecting a microorganism capable of co-metabolizing MTBE, wherein a pure culture of the microorganism is grown in a suitable growth medium and supplied with a sufficient amount of at least one gaseous n-alkane or simple branched alkane. MTBE is then added to the growth medium, and the growth medium is then assayed to quantify MTBE degradation. The assay permits selection of MTBE degrading microorganisms. In one embodiment, this assay step is performed by detecting the presence of MTBE degradation products, such as TBA (tert-butyl alcohol) and TBF (teri-butyl formate).
Microorganisms capable of co-metabolizing MTBE may be used in methods of decontaminating media (such as soils, water or air) containing MTBE. In the simplest embodiment of this application, a pure culture of the selected microorganism is combined with the contaminated medium in the presence of a gaseous n-alkane or a simple branched alkane (or, a suitable alkane metabolite) as a primary metabolite. In other embodiments, the present invention contemplates the use of biofilters for removing MTBE from a medium. In their basic form, such biofilters comprise a surface for supporting microbial biomass, a pure culture of the selected microorganism provided on the surface to form a microbial biomass, a supply means for supplying a gaseous n-alkane or a simple branched alkane (or a metabolite thereof) to the microbial biomass, and a supply means for supplying a medium containing MTBE to the microbial biomass.
In other aspects of the present invention, microorganisms possessing cytochrome P-450 activities are shown to be capable of co-metabolizing a wide range of xenobiotic compounds. The invention provides a method of degrading a xenobiotic compound which comprises providing a pure culture of a microorganism capable of degrading the xenobiotic compound as a co-metabolite of a gaseous n-alkane or simple branched alkane, providing this culture with a suitable alkane (or a metabolite of the alkane) as a primary metabolite and then contacting the culture with the xenobiotic compound. Xenobiotic compounds which may be degraded include MTBE, TAME, ETBE, DEE, naphthalene, dibenzofuran and chlorinated aliphatic hydrocarbons such as chloroform.


REFERENCES:
patent: 5814514 (1998-09-01), Steffan et al.
Abstract, Hardison et al., Applied and Environmental Microbiology, vol. 63, No. 8, 3059-3067, 1997.
Steffan et al., Biodegradation of the Gasoline Oxygenates Methyl tert-Butyl Ether, Ethyl tert-Bultyl Ether, and tert-Amyl Methyl Ether by Propane-Oxidizing Bacteria, Applied and Environmental Microbiology, 63:4216-4222, 1997.
Davies et al., Hyphomycetes utilizing natural gas, Can. J. Microbiol., 19:81-85, 1973.
Sariaslani, Microbial Cytochromes P-450 and Xenobiotic Metabolism, Advances in Applied Microbiology, 36:133-178, 1991.
Brady et al., Metabolism of methyl tertiary-butyl ether by rat hepatic microsomes, Arch. Toxicol. (1990) 64:157-160, 1990.
Parales et al., Degradation of 1,4-Dioxane by an Actinomycete in Pure Culture, Applied and Environmental Microbiology, 60:4527-4530, 1994.
Mo et al., Biodegradation of methyl t-butyl ether by pure bacterial cultures, Appl. Microbiol Biotechnicol, 47:69-72, 1997.
Steffan et al., Biodegradation of Methyl tert-Butyl Ether (MTBE), Environmetal and General Applied Microbiology, Q-372, Apr., 1997, p. 517.
McCarty, In situ bioremediation of chlorinated solvents, Currrent Opinion in Biot

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