Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2000-09-20
2001-09-04
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S262500
Reexamination Certificate
active
06284521
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to biotechnology, ecology, environment protection, and particularly to bioremediation of environment media from polychlorinated biphenyls (PBCs), and provides a new yeast strain which is able to degrade PCBs under aerobic conditions in situ in environment media.
BACKGROUND OF THE INVENTION
Introduction of chemical advances into industry and household involves many hazards of immediate and long-term chemical impact. As of now, the issue of environment pollution control has become extremely pressing.
PCBs have been manufactured since 30th and found application for the most part in electrical industry. By late 60th, it has turned out that the environment contained from 300 to 500 thousand tons PCBs (Tarasov V.V. Contamination of the Environment with Polychlorinated Biphenyls and Ways for Minimizing Their Impact, Collection of Scientific Works of RKhTY LXXV Years/Main Scientific Achievements. Moscow, 1996, p.24-41). Annual release of PCBs averages 2000 tons. PCBs penetrate into the environment as the result of failures of PCB-containing equipment and systems and due to incineration. PCBs penetration into soil is caused by equipment failures or discharges of untreated industrial sewage from plants that employ PCBs in their production run, and by utilization of sludge from irrigated fields. Owing to a high absorption power and a low degradation ability, polychlorinated biphenyls accumulate in the soil surface layer at a depth of 2-10 cm and in bed sediments. PCBs have been detected substantially in all living nature. PCB is a polytropic poison which affects essentially all body organs and systems.
According to data published by the WHO, a human appears to be the most PCB-sensitive creature. PCBs relate to substances of the first hazard group.
Worldwide practice for remediating environment media from PCBs is to use in general various physical and chemical methods.
In Germany, PCB-contaminated soil is remediated by a method developed by National Research Council, Canada. According to the method, special dispersion sodium-oil blends are introduced into old landfill sites to assist in chlorine release from PCBs with formation of common salt. (see Deckwer W.-D. Weppen P. Review of Methods for Remediating Contaminated Soil and Refused Territories Contaminated with Hazardous Wastes.—Chemie-Ingenieur-Technik, 1987, Vol.59, #6, p.457-467). Gebruder Kemmer/Jng Buro Harbauer Group (Germany) has invented a method for decontamination of PCB-contaminated soil, which involves excavation of the soil. The method includes crushing and sieving the soil, the obtained fractions being separately decontaminated with water to which surfactants are added. Flush liquid is supplied to a sewage treatment plant (Schondorf T., Munz K. H. Removal of Polychlorinated Biphenyls from Contaminated Soil.—Chem.Rosch. (Schweiz.), 1988, v.41, #45, s.18).
Kloeckner Oecotec GmbH, Germany, has invented a method for remediation of PCB-contaminated soil, involving washing the soil under a high pressure at a pilot plant. The soil in the form of pieces of up to 10 mm in diameter is ground in a conical water jet. A high pressure of 250 bar in an annular pipe and a great rate of 200-250 m/s promote a complete homogenization and separation of smallest components and hazardous substances. After the water treatment of the soil, the suspension is separated by one of the following methods: precipitation, cyclone separation, centrifugation, filtration, etc. Developed in Russia are methods for soil decontamination from PCBs with the aid of a propulsion, plasmatrons (Tarasov V. V. Contamination of the Environment with Polychlorinated Biphenyls and Ways of Minimizing Their Impact. Collection of Scientific Works of RKhTY LXXV years/Main Scientific Achievements, Moscow, 1996, p.24-41).
All of the above methods suffer a number of essential problems: they are cumbersome, require great investments and disturb the soil ecological equilibrium. Since late 70th, an ever increasing interest has been expressed in bioremediation methods. It is well known that various bacterial strains and fungi are able to degrade PCBs. They include microorganisms of Pseudomonas genus (
Pseudomonas putide
for degrading polychlorinated biphenyls. U.S. Pat. No. 4,843,009, Int.C1. C12N 1/12, Application No.866501 filed on May 23, 1986) and fungi Whit Rod Funtigus.
Phanerochaete chrysosporium
(Degradation of 4′,4′-Dichlorobiphenyl, 3,Y,4,4′-Tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-Hexachlorobiphenyl by While Rot Fungus
Phanerochaete chrysosporium
. Applied and Environmental Microbiology. 1995. Vol.61, N.11, p.3904-3909), but the bacteria of Pseudomonas genus are fastidious to nutrient medium and storage conditions. Fungi are less convenient in production, and they cause, among other things, a shift in the ecological equilibrium when employed in environment media.
SUMMARY OF THE INVENTION
It is an object of the invention to isolate a new strain of microorganisms, in particular, yeast, which is able to degrade higher PCB concentrations in environment media in situ under aerobic conditions, and which is convenient in production and employment.
The object of the invention is attained by providing a new, genetically resistant yeast strain of
Hansenulla californica
AT.
Hansenulla californica
AT yeast strain has been isolated from soil and selected as the result of long-term subculturing of separate yeast colonies on a minimal salts medium (Practicum in Microbiology, M., MGU Publishers, 1976) containing
(NH
4
)
2
HPO
4
1.5
g
KH
2
PO
4
0.7
g
NaCl
0.51
g
Mg
2
SO
4
0.8
g
distilled water
up to 1 liter
pH
7.2
in the presence of various PCB concentrations, from 100 to 400mg per a liter of a nutrient medium.
The yeast strain was selected by a PCB degradation level and velocity, and by its genetic PCB-resistance. Genetic resistance of selected strains was attained by repeated subculturing on dense nutrient media. Colonies grown on them were then subcultured on a minimal salts medium having the above composition.
As the result, a new genetically resistant yeast strain of
Hansenulla californica
AT has been obtained.
Identification of the microorganism was made in accordance with the Kreger-van Rij identifier. The strain, (identified as
Hansenulla
(Zygomilliopsis)
californica
AT), was deposited on Feb. 12, 1997, under the terms of the Budapest Treaty with the Russian National Collection of Industrial Microorganisms (Vserossiskaya kollektsiya promyshlennykh microorganismov, abbreviated as VKPM), and has the accession deposit number of VKPM Y-2284. The address of VKPM is: Russian National Collection of Industrial Microorganisms (VKPM), GNII genetika, Dorozhny proezd. 1, Moscow 113545, Russian Federation. The strain features the following culture, morphological, physiological and biochemical characters.
Culture and Morphological Characters:
The strain grows on a medium containing 5% malt extract. After incubation at 25° C. for 72 hours, a gram-stained smear contains slightly spherical cells of 2.4-6.2×3.0-8.1 &mgr;m in size. Colonies are white, opaque and circular. The yeast forms asci of zygotic origin on an acetate medium. In a liquid nutrient medium the growth takes place at a temperature of 29° C. during 72 hours at an agitator rotational speed of 500 rev/min.
Physiological Characters:
The strain is aerobic, grows at a temperature from +4° C., does not grow at a temperature of +42° C., the optimal temperature being +20° C. Grows at pH 5.0-7.5, optimal pH 6.8-7.0. Grows on rich nutrient media based on a meat infusion broth (MIB) and enzymic fish flour digest (EFFD).
Biochemical Characters:
In the Giss media, the strain assimilates saccharose, maltose, cellobiose, D-xylose, D-mannite, citric acid, lactic acid, alpha-methyl-d-glycoside as a sole carbon source. Ferments glucose for 3-5 days. Uses nitrogen of organic and inorganic origin.
Storage Media: EFFD or MIB and minimal salts media with PCB. To produce yeast biomass for degrading PCB, the strain may be incub
Borovick Roman Vladimirovich
Kapranov Vladimir Vladimirovich
Zharikov Gennady Alekseevich
Chang Randall W.
Daubenspeck Willaim C.
Gottlieb Paul A.
Lilling Herbert J.
Research Center for Toxicology and Hygienic Regulation of Biopre
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