Methods for bioremediation by degrading toluene

Details Methods for bioremediation by degrading toluene Methods for bioremediation by degrading toluene

1998-05-04

2003-04-22

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

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

Destruction of hazardous or toxic waste

C210S600000, C210S610000, C435S243000, C435S248000, C435S252100, C435S252300, C435S252400, C435S822000

Reexamination Certificate

active

06551814

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to biological treatment of organic compounds, and particularly to the degradation of toluene and toluene analogues.
BACKGROUND
Industrial processes that use or generate toxic organic compounds (e.g., toluene, benzene, xylenes) has lead to the contamination of nearby water and land. Such compounds are among the most water soluble of all gasoline components and can also enter aquatic environments from many sources such as gasoline underground storage tanks, leaks, and spills.
Most approaches to decontamination or “remediation” involve stopping the local dumping of such compounds and transport of the waste to another area for containment. This is costly and does not eliminate the hazard.
As a remediation technology, bioremediation is considerably more attractive. Rather than merely transporting wastes, it offers the possibility of degrading toxic compounds to harmless reaction products by the use of biologicals.
Bioremediation field trials have involved both in-situ and ex-situ treatment methods. Typically, ex-situ treatment involves the transfer of contaminated waste from the site into a treatment tank designed to support microbial growth, i.e., a “bioreactor”. The reactor provides for effective mixing of nutrients and control over temperature, pH and aeration to allow optimum microbial growth.
In-situ treatment involves adding biologicals directly to the waste. This avoids the problems associated with handling (e.g., pumping) toxic compounds. However, in-situ treatment has its own problems. Unlike bioreactors, where microbial growth can be monitored and adjusted, in-situ environmental conditions are difficult to measure and control.
Fries et al., “Isolation, characterization and distribution of denitrifying toluene degraders from a variety of habitats,”
Appl. Environ. Microbiol
. 60:2802 (1994) generally indicates that biodegradation of benzene, toluene, ethylbenzene and xylenes under aerobic conditions is well known, although the availability of oxygen due to its low solubility in water and low rate of transport in soils and sediments is rate limiting. Fries et al. describes anaerobic respiration of toluene by microorganisms isolated from nature. The microorganisms could grow on 25 ppm toluene and could be fed 50 ppm toluene.
Rates have been determined at 28-30° C. with intact cells from a variety of strains. The rates vary from between 8 to 80 nmoles toluene min
−1
mg
−1
protein. A. Frazer et al., “Toluene Metabolism Under Anaerobic Conditions: A Review,”
Anaerobe
1:293 (1995).
There remains a need to develop a bioremediation procedure that can be operated economically on a commercial scale. Such a procedure must be able to degrade organic compounds with high efficiency.
SUMMARY OF THE INVENTION
This invention relates to biological treatment of organic compounds, and particularly to the degradation of toluene and toluene analogues. In one embodiment, the present invention contemplates a method of degrading compounds contained in a liquid or solid waste source, comprising the steps of: a) providing, i) a waste source comprising toluene (and/or a toluene analogue), ii) a reaction containing means, and iii) a compound selected from the group consisting of a functional, cell-free pyruvate formate lyase homologue of a toluene-degrading bacterium and a functional, cell-free pyruvate formate lyase activating homologue of a toluene-degrading bacterium; and b) reacting said homologue and said waste source in said containing means under conditions such that toluene (and/or the toluene analogue) is degraded.
It is not intended that the present invention be limited by the specific toluene-degrading bacterium. In one embodiment, said homologue is derived from an organism of the genus Thauera. In one embodiment, the organism is
Thauera aromatica.
In another embodiment, said homologue is derived from an organism of the genus Xanthomonas. In one embodiment, the organism is
Xanthomonas maltophilia.
In yet another embodiment, said homologue is derived from an organism of the genus Geobacter. In one embodiment, the organism is
Geobacter metallireducens.
In still another embodiment, said homologue is derived from members of the genus Azoarcus. In one embodiment, the organism is
Azoarcus tolulyticus.
The present invention contemplates the nucleic acid sequences (and constructs comprising said sequences) and amino acid sequences of toluene degrading enzymes as compositions of matter (as well as antibodies to such amino acid sequences). In one embodiment, the present invention contemplates a purified nucleic acid comprising DNA having the sequence as set forth in
FIGS. 12A-Y
. In one embodiment, said DNA is in a vector. In another embodiment, said vector is a bacterial plasmid. In a particular embodiment, said bacterial plasmid is in a host cell. In one embodiment, said host cell expresses a toluene-degrading enzyme.
The present invention contemplates a functional, cell-free product of the tutD gene having the amino acid sequence as set forth in
FIGS. 11A-D
. In one embodiment, said product is contained within a reaction containing means. In a preferred embodiment, said reaction containing means is a bioreactor.
It is also not intended that the present invention be limited by the precise amino acid sequence of the homologue. In one embodiment, it is encoded by the tutD gene, a nucleic acid sequence for which is shown in
FIGS. 5A-P
, and has the amino acid sequence shown in
FIGS. 7A-C
. In another embodiment, the homologue is an expanded TutD protein having the amino acid shown in
FIGS. 11A-D
and the corresponding nucleic acid sequence shown in
FIGS. 12A-Y
. In another embodiment, the homologue is encoded by the tutE gene having a nucleic acid sequence shown in
FIGS. 12A-Y
, and a corresponding amino acid sequence shown in
FIGS. 13A-B
.
Additionally, the present invention contemplates a reporter gene fusion product constructed by fusing the tutD gene in frame to a reporter such as lacZ, luxA, or green fluorescence protein. Such constructs can be used to demonstrate regulated expression in response to toluene.
In another embodiment, the present invention contemplates a reporter gene fusion product constructed by fusing the tutE gene in frame to a reporter such as lacZ, luxA, or green fluorescence protein. Such constructs can be used to demonstrate regulated expression in response to toluene.
DEFINITIONS
To facilitate understanding of the invention, a number of terms are defined below.
The term “reaction” or “chemical reaction” means reactions involving chemical reactants, such as organic compounds. A “reaction containing means” refers to anything that can contain a reaction, including but not limited to, tubes, microtiter plates, vessels, and bioreactors. It is not intended that the present invention be limited by a particular reaction containing means. U.S. Pat. No. 5,610,061, U.S. Pat No. 5,585,272, U.S. Pat. No. 5,571,705, U.S. Pat. No. 5,560,737, U.S. Pat. No. 5,057,221 and U.S. Pat. No. 5,037,551 all describe various reaction containing means (including bioreactors) and are hereby incorporated by reference.
“Initiating a reaction” means causing a reaction to take place. Reactions can be initiated by any means (e.g., mixing, heat, wavelengths of light, addition of a catalyst, etc.)
A “solvent” is a liquid substance capable of dissolving or dispersing one or more other substances. It is not intended that the present invention be limited by the nature of the solvent used.
A “waste source” can be a solid or liquid waste source (e.g., paper pulp, pulp mill effluent, sludge, wastewater, petroleum spill, etc.).
“Toluene analogues” are structural analogues of toluene. While it is not intended that the present invention be limited to particular analogues, examples include the o-, m-, and p- isomers of chlorotoluene, fluorotoluene and xylene.
A “pyruvate formate lyase homologue” is defined as a gene product from a toluene-degrading organism, said gene product comprising i) regions of identity with the pyruvate formate lyase f

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