Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-09-20
2001-09-11
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S262500, C435S829000
Reexamination Certificate
active
06287842
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to biotechnology and provides a new bacterial strain which is able to degrade polychlorinated biphenyls (PBCs) under aerobic conditions in situ in environment media.
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.
BACKGROUND OF THE INVENTON
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 high absorption power and 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, 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. (Deckwer W.-D. Weppen P. Review of Methods for Remediating Contaminated Soil and Refused Territories Contaminated with Hazardous Waste.—Chemie-Ingenieur-Technik, 1987, Vol.59, #6, p.457-467).
Gebruder Kemmer/Jng Buro Harbauer Group (Germany) has invented a method for remediation 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 the 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 poly-chlorinated biphenyls. U.S. Pat. No.4843009, Int.Cl. C12N 1/12 Application No.866501 filed on May 23, 1986) and fungi of Whit Rod Fungus
Phanerochaete chrysosporium
(Degradation of 4′,4′-Dichlorobiphenyl, 3,Y,4,4′-Tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-Hexachlorobiphenyl by the White Rot Fungus
Phanerochaele chrysosporium
. Applied and Environmental Microbiology. 1995. Vol.61, N.11, p.3904-3909), however, the bacteria of Pseudomonas genus are fastidious to nutrient media 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 microorganism strain which is capable of degrading greater PCB concentrations in environment media in situ under aerobic conditions, and which is convenient in production and employment.
Alcaligenes latus
bacterial strain TXD-13 has been isolated from soil and selected as the result of long-term subculturing of separate bacterial 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
0.5 g
Mg
2
SO
4
0.8 g
distilled water
up to 1 liter
pH
7.2
in the presence of different PCB concentrations, from 100 to 400 mg per a liter of a nutrient medium.
The 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 bacterial strain of
Alcaligenes latus
TXD-13 has been obtained. Identification was made in accordance with the Bergey identifier. The strain, (identified as Alcaligenes latus TDX-13), 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 B-7505. 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.
On a glucose/peptone medium plus a yeast extract, the strain forms clear colonies, their surface changing from bright to opaque with age. The colonies are greyish in color, changing to yellowish. When subcultured, the colonies are 2-3 mm in diameter.
On the Endo, Ploskirev medium, the culture grows in the form of smooth bright colonies. The culture is gram-negative with a smear containing small rods and cocci. In a liquid nutrient medium the culture grows at a temperature of 29° C. during 72 hours at an agitator rotation speed of 200 rev/min.
Physiological characters
The culture is aerobic, grows at a temperature from +4° C., does not grow at a temperature of +37° C., the optimal temperature being +20° C. Grows on media with pH 6.0-8.0, optimal pH 6.8-7.5. Grows on rich nutrient media based on a meat infusion broth (MIB) and enzymic fish flour digest (EFFD).
Biochemical characters
In the Smith media, the culture assimilates glycerol, fructose, D-glucose as a sole carbon source. The culture is catalase-positive and oxidase-positive. Uses nitrogen of organic and inorganic origin.
Storage media: EFFD or MIB and minimal salts media with PCB.
To produce bacterial biomass for degrading PCB, the strain may be incubated on various nutrient media containing common carbon and nitrogen sources, mineral salts.
Carbon sources which can be used in the practice of the present invention are PCB, glucose, fructose, glycerol. Nitrogen sources
Dyadischev Nikolai Romanovich
Kapranov Vladimir Vladimirovich
Zharikov Gennady Alekseevich
Chang Randall W.
Daubepspeck William C.
Gottlieb Paul A.
Lilling Herbert J.
Research Center for Toxicology and Hygienic Regulatiom of Biopre
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