Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Destruction of hazardous or toxic waste
Reexamination Certificate
1994-06-24
2001-07-31
Sherrer, Curtis E. (Department: 1761)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Destruction of hazardous or toxic waste
C210S606000
Reexamination Certificate
active
06268204
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to biological treatment of waste, and particularly to the degradation of organo-halides contained in liquid wastes.
BACKGROUND
Industrial processes that use or generate toxic organic compounds has lead to the contamination of nearby water and land. 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.
Bioremediation technologies being developed to deal with waste differ further in their ability to handle specific types of compounds. For example, the effluents from most pulp or paper-making operations contain lignin or its degradation products. Lignin is an extremely complex polymer, constituting up to 35% of dry wood weight. During pulping processes, cellulose fibers must be liberated from the surrounding lignin matrix so that they can be associated with one another. The resulting free lignin is highly resistant to degradation.
Chang et al. describe the degradation of the complex lignin polymer by direct addition of cultures of the white-rot fungus,
P. chrysosporium
. See U.S. Pat. No. 4,655,926, hereby incorporated by reference. They describe the induction of lignin degradation in response to carbon and nitrogen starvation.
This fungal lignin metabolism is a secondary metabolic event. Lignin degradation allows the fungus to expose the cellulose contained within the lignin matrix as its primary food source. Indeed, lignin alone will not support
P. chrysosporium
growth.
Since cellulose fibers are the primary food source, degradation of lignin by adding the fungus directly to the wood results in reduced pulp yield and an inferior pulp product. Farrell has proposed, therefore, an improvement in lignin degradation. See U.S. Pat. Nos. 4,687,745 and 4,690,895, hereby incorporated by reference. She describes the use of fungal enzymes rather than fungal cultures. The enzymes degrade lignin without degrading cellulose fibers.
The mechanism by which these enzymes degrade lignin has been investigated. Glenn et al. have characterized the enzyme manganese peroxidase. See Arch. Biochem. Biophys. 251:688 (1986). By separating the enzyme from the substrate using a membrane, they showed that Mn(III) complexed to lactate or other hydroxy acids are intermediates capable of oxidizing organic compounds. This work was confirmed by Lackner et al., Biochem. Biophys. Res. Comm. 178:1092 (1991).
Lignin is but one by-product of paper making operations. The bleaching of paper with chlorine generates effluents that are a serious health concern. Many of these compounds are known to cause cancer in humans. Most importantly, these compounds are not degraded rapidly in the natural environment.
Chang et al. teaches that chloro-organics contained in liquid waste can also be degraded by the white-rot fungus. See U.S. Pat. No. 4,554,075, hereby incorporated by reference. In the method proposed, the fungus is immersed in the liquid containing chloro-organics and periodically exposed to an oxygen enriched atmosphere. The chloro-organics are converted from aromatics to aliphatics.
Unfortunately, the rate of chloro-organic degradation is slow since the degradation activity must be induced by starvation of the organism. To avoid the necessity for this starvation step, Aust et al. teach the direct addition of fungal enzymes rather than whole organisms. See U.S. Pat. No. 4,891,320, hereby incorporated by reference. They suggest the direct addition of a fungal peroxidase. This, however, requires the continually mixing of the enzyme with hydrogen peroxide. This is difficult whether done in an open environment or a closed reactor.
Indeed, the commercial use of fungal degradation is hampered by complex technical and engineering issues. Growth of the organism and/or enzyme production may be inhibited by waste components. See generally, Mileski et al., Appl. Environ. Microbiol. 54:2885 (1988). Furthermore, the enzyme is a protein and, as such, can undergo proteolysis by any number of proteases in the waste stream, and be rendered thereby inactive. See Dosoretz et al., Appl. Environ. Microbiol. 56:3429 (1990). In sum, the degrading ability of
P. chrysosporium
is not practically maintained for a very long period of time. See Lin et al., Biotechnology and Bioengineering 35:1125 (1990).
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 deal with diverse waste streams without significant inhibition of the degradation process.
SUMMARY OF THE INVENTION
This invention relates to biological treatment of waste, and particularly to the degradation of organo-halides contained in liquid wastes. In one embodiment, the present invention contemplates a method of degrading compounds contained in a liquid or solid waste stream, comprising the steps of: a) providing, i) a reaction containing means having a semi-permeable membrane partition, ii) at least one enzyme for which the membrane is not permeable, and iii) at least one substrate for the enzyme; b) adding to the containing means on one side of the partition, the enzyme and substrate to create a reaction mixture, thereby generating a reaction intermediate for which the membrane is permeable; c) adding to the containing means on the other side of the partition, the compounds contained in said waste stream, d) applying a pressure to the reaction mixture so as to force the reaction intermediate across said membrane into the compound-containing waste stream, thereby degrading the compounds.
In another embodiment, the present invention contemplates a method of degrading compounds contained in a liquid or solid waste stream, comprising the steps of: a) providing, i) a first reaction containing means having a semi-permeable membrane partition, ii) at least one enzyme for which the membrane is not permeable, and iii) at least one substrate for the enzyme, and iv) a second reaction containing means; b) adding to the first containing means on one side of the partition, the enzyme and substrate to create a reaction mixture, thereby generating a reaction intermediate for which the membrane is permeable; c) applying a pressure to the reaction mixture so as to force the reaction intermediate across said membrane and so as to collect the reaction intermediate on the other side of the partition; and, d) adding the reaction intermediate to the second containing means wherein the compounds to be degraded are contained, thereby degrading the compounds. In one embodiment, the reaction intermediate is added in step (d) by pumping the intermediate from the first reaction containing means to the second reaction containing means.
In one embodiment said enzyme is derived from white-rot fungus. In one embodiment, the fungus is selected from the group comprising
Phanerochaete chrysosporium, Dichromitus squalens, Phlebia radiata, Lentinula edodes, Trametes versicolor, Coriolopsis occidentalis, and Rigidoporus lignosus.
It is preferred that the enzyme is a peroxi
Miller Donald James
Selman Susanne
Medlen & Carroll LLP
Promega Corporation
Sherrer Curtis E.
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