Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1996-02-09
2001-03-20
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S262000, C435S262500, C424S490000
Reexamination Certificate
active
06204049
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the formulation of microorganisms for delivery of viable inoculum of the microorganisms to an environment. This invention also relates generally to the formulation of microorganisms for delivery of viable inoculum of the microorganisms to an environment having an indigenous microflora and to a polluted environment or site. Methods of formulating viable inoculum of microorganisms for delivery of the microorganisms to a polluted environment or site, and a method for remediation of a polluted environment or site are presented, in which a suitable microorganism having the capacity to decompose a pollutant, is applied to the site in combination with a suitable carrier for the microorganism.
The invention also relates to compositions for remediating an environment or site which has been contaminated with a chemical pollutant, and to methods for delivering microorganisms in combination with a carrier for the microorganisms to an environment or site which has been contaminated with a chemical pollutant and which is subject to bioremediation. Methods are presented for delivering nutrients in combination with a carrier for the nutrients to an environment or site which has been contaminated with a chemical pollutant and which is subject to bioremediation, as are methods for delivering nutrients in combination with a microorganism and a carrier to an environment or site which has been contaminated with a chemical pollutant and which is subject to bioremediation.
Embodiments of the invention are presented relating to the biodegradation of benzo[a]pyrene, and to the fungus
Marasmiellus troyanus
. In particular,
Marasmiellus troyanus
isolate no. 216-1867 is presented, and compositions comprising
M. troyanus
isolate no. 216-1867 in combination with a carrier are presented. Yet further, the invention relates to the degradation of benzo[a]pyrene by
M. troyanus
, and the mineralization of benzo[a]pyrene by
M. troyanus
. A process for bioremediation of polluted media contaminated with benzo[a]pyrene is also shown.
2. Background of the Related Art
Chemical pollution of various media (e.g. soil, water) is a common problem worldwide which has a major economic impact at the local, national, and global levels. The remediation of sites polluted or contaminated with toxic chemicals or hazardous wastes using existing technologies is generally extremely costly, laborious and time-consuming. For example, in the U.S., Congress established a multibillion dollar fund (the Superfund) under the Comprehensive Environmental Response, Compensation, and Liability Act of 1980, 1986 and 1990 (commonly known as the Superfund Act). The fund was established to pay for cleanup of polluted sites such as hazardous and municipal waste dumps, contaminated factories and mines, and leaking underground fuel storage tanks. During the eleven years between its inception and 1991, 30,000 potential Superfund sites were surveyed. The time required for cleanup of a Superfund site by the EPA has been as long as 10 years. Remediation efforts at hazardous waste sites have been partially effective 54% of the time and completely effective only 16% of the time (Riser-Roberts, E., (1992) “
Bioremediation of Petroleum Contaminated Sites
,” pp. 1-34, CRC Press, Boca Raton, Fla.). Currently, additional sites of chemical pollution are being discovered and new sites of pollution are being created worldwide.
Pollution of the soil with toxic chemicals creates hazards to the health of humans and other organisms, and also renders the site useless for most purposes. In addition, pollutants in the soil can be leached into underlying aquifers leading to groundwater contamination. In the U.S., groundwater is used as a source of drinking water by about 120 million people, and is also widely used to irrigate food crops.
Improved technologies for remediation of polluted soil and water are urgently needed. Traditional methods for cleanup of contaminated soil has generally involved excavation of the soil, followed by treatment or containment. Currently used techniques for remediating polluted soils include, for example, the physical removal of volatile materials by aspiration (vacuum extraction) and the incineration of contaminated soil. Because of the large volumes of soil usually involved, physicochemical methods such as those exemplified above may be prohibitively expensive. An alternative approach to cleaning up sites of chemical pollution is bioremediation, in which a biological organism is used as an agent for converting the chemical pollutant to less toxic or nontoxic compounds. For example, various microorganisms have been found to detoxify a number of toxic chemical pollutants (see, for example, G. Chaudry (Ed.) “
Biological degradation & bioremediation of toxic chemicals
,” Dioscorides Press, Portland, Oreg., 1984). Biodegradation or detoxification of chemical pollutants is normally the result of one or more enzymatic reactions, including oxidation, reduction, hydrolysis, and conjugation (see, for example, D. W. Connell, & G. J. Miller, (1984) “
Chemistry & Ecotoxicology of Pollution
,” pp. 1-48 & 231-247, John Wiley & Sons, Inc., New York, N.Y.).
One factor limiting the efficacy of prior art bioremediation processes is the tendency of microorganisms to lose viability and decline in number following their introduction to the remediation site. It has been demonstrated by numerous field trials that, in general, microorganisms released into the soil tend not to spread from the point of application, and further that their numbers tend to decline over time (see, for example, J. D. van Elsas, & C. E. Heijnen, “Methods for the introduction of bacteria into the soil: A review,”
Biol. Fertil. Soils
, 10:127-133, 1990). Factors militating against the propagation and survival of microorganisms introduced into soils include: competition with other organisms for nutrients, water and space; parasitism, antibiosis and predation by other organisms; and unfavorable physicochemical parameters of the soil milieu, including sub-optimal pH, water and oxygen concentrations. In the case of polluted soils, problems associated with survival and propagation of microorganisms introduced into such soil may be exacerbated by the presence of toxic pollutants at concentrations which are inimical to microbial growth.
In an attempt to prolong the survivability of microorganisms introduced into soil, some prior art remediation techniques have incorporated increased aeration or the large-scale application of nutrients to the soil. However, this approach is expensive and, in addition, nutrients added to the soil en masse are immediately available to the soil microflora as a whole, and consequently are prone to rapid depletion.
Another approach to increasing survivability of microorganisms introduced into soil, or other environments, is to combine the microorganisms with various carrier materials. Such carriers include a variety of organic and inorganic materials, including silica, mineral oil, peat, and various gels (see, for example, D. A. Van Schreven, “Some factors affecting growth and survival of Rhizobium spp. in soil-peat cultures,”
Plant & Soil
, 32:113-130, 1970; Fravel, D. R., et al., “Encapsulation of potential biocontrol agents in an alginate-clay matrix,”
Phytopathology
, 75:774-777, 1985; W. J. Connick, Jr., “Formulation of living biological control agents with alginate,” in B. Cross & H. B. Scher (eds.) “
Pesticide formulations: innovations & developments
,” American Chemical Society, Washington, D.C., pp. 241-250, 1988).
Apart from a carrier in combination with a microorganism serving as a vehicle for the microorganism, in some cases the particular combination of a suitable carrier material with a certain microorganism provides the added advantage of preserving the microorganism, thereby allowing for storage of the microorganism in a viable state (see, for example, D. J. Daigle & P. J. Cotty, “Formulating atoxigenic
Aspergillus fl
Bennett Joan Wennstrom
Childress Adele Marie
Connick, Jr. William Joseph
Wunch Kenneth George
Kaufman Claire M.
Murray Michael L.
Spector Lorraine
The United States of America as represented by the Secretary of
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