RU2155803C1 - Strain of yeast hansenulla californica decomposing polychlorinated biphenyls - Google Patents

Abstract

FIELD: technical microbiology, ecology. SUBSTANCE: invention proposes the strain of yeast Hansenulla californica NT VKPM Y-2284 designated for decomposition of polychlorinated biphenyls and detoxication of environment objects. Strain is grown in media containing sources of carbon, nitrogen and mineral salts. Strain is cultured by submerged method to titer value 5.0 x 10⁶ - 1.0 x 10⁷ cells/cm³ and obtained biomass in concentration 10⁶-10⁵ cells/cm³ is used for decomposition of polychlorinated biphenyls. Strain provides decrease of polychlorinated biphenyls content in soil and water by 30-50%. EFFECT: strain indicated above, enhanced effectiveness of yeast strain. 3 tbl, 4 ex

Description

 The invention relates to biotechnology, ecology, environmental protection, for the biological treatment of environmental objects from polychlorinated biphenyls (PCBs) and is a new strain of yeast that can decompose PCBs in aerobic conditions in situ in environmental objects.

 The introduction of the achievements of chemistry in industry and human life is associated with many dangers of the direct and long-term effect of chemical exposure. Currently, the issue of combating environmental pollution has become especially acute.

 PCBs have been produced since the 30s and they have found their application mainly in the electrical industry. By the end of the 60s, from 300 to 500 thousand tons of PCBs appeared in the environment [1]. The average annual release of PCBs is 2000 tons. The release of PCBs into the environment is associated with accidents of equipment and systems containing them, as well as during waste incineration. PCBs get into the soil during equipment accidents or in the discharge of untreated industrial effluents from enterprises using it in the production cycle, as well as when using silt sediments from irrigation fields. With high absorption capacity and low decomposition ability, polychlorinated biphenyls accumulate in the soil in the surface layer at a depth of 2-10 cm and in bottom sediments. PCBs are found in almost all wildlife. PCBs - polytropic poison that affects almost all organs and systems of the body. Data published by WHO suggests that humans are the most sensitive to PCBs. PCBs are substances of the 1st hazard group.

 To clean environmental objects from PCBs in the world, mainly various physicochemical methods are used.

 In Germany, a method developed by the National Research Council (Canada) is used to sanitize soils contaminated with PCBs. According to this method, special dispersion mixtures of sodium-oil are introduced into old dumps, which contribute to the release of chlorine from PCBs with the formation of sodium chloride [2]. The Gebruder Kemmer / Jng Buro Harbauer Group of Companies (Germany) has developed a method for cleaning soil contaminated with PCBs based on excavation. The soil is crushed and sieved, the fractions are separately cleaned with water with the addition of surfactants. Wash liquid is supplied to the wastewater treatment plant [3].

 In Germany, Kloeckner Oecotec GmbH developed a method for cleaning soil contaminated with PCBs, based on washing the soil under high pressure in a pilot plant. Soil with a diameter of pieces up to 10 mm is crushed in a conical stream of water. High pressure (250 bar) in the annular pipeline and high speed (200-250 m / s) contribute to complete homogenization and separation of the smallest components and harmful substances. Suspension separation after soil treatment with water is carried out using one of the following methods: sedimentation, cyclone separation, centrifugation, filtration, etc. In Russia, a method has been developed for cleaning soil from PCBs using a rocket engine, plasmatrons [4].

 All these methods have a number of significant drawbacks: they are laborious, require large economic costs and upset the ecological balance of the soil. Since the late 70s, an increasing interest has been shown in biological methods of purification. It is known that various bacterial strains and fungi can decompose PCBs. These include microorganisms of the genus Pseudomonas (Pseudomonas putida, capable of degrading polychlorinated biphenyls. US Pat. '- Dichlorobiphinyl, 3, Y, 4, 4'- Tetrachlorobiphinyl, and 2, 2', 4, 4 ', 5, 5'- Hexachlorobiphenyl by the White Rot Fungus Phanerochaete chrysosporium. Applied and Environmental Microbiologi., 1995, vol. 61, No. 11, p. 3904-3909), but bacteria of the genus Pseudomonas are demanding on nutrient media and storage conditions. Mushrooms are less technologically advanced in production, but, among other things, they lead to a shift in the ecological balance when they are used in environmental objects.

 The basis of the invention is the task of isolating a new strain of a microorganism, in particular yeast, capable of decomposing higher concentrations of PCBs in environmental objects in situ under aerobic conditions, which is high-tech in production and use.

 This object is achieved in that a new genetically stable strain of the yeast Hansenulla californica AT is obtained.

The strain of the yeast Hansenulla californica AT isolated from the soil and selected by prolonged reseeding of individual colonies of yeast in minimal salt medium (Workshop on Microbiology.- M .: Moscow State University, 1976), containing:
(NH ₄ ) 2HPO 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 L
pH 7.2
in the presence of various concentrations of PCBs, from 100 to 400 mg per 1 liter of 30 nutrient medium.

 The yeast strain was selected according to the level and rate of decomposition of PCBs, as well as their genetic resistance to PCBs. The genetic stability of the selected strains was achieved by repeated reseeding on solid nutrient media. The colonies grown on them were then subcultured onto the minimal salt medium of the above composition.

 As a result, a new genetically resistant strain of the yeast Hansenulla californica AT was obtained.

 The identification of the microorganism was carried out in accordance with the identifier Kreger-van Rij. The strain is deposited in the All-Russian collection of industrial microorganisms (VKPM) under the number Y-2284. The strain is characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural and morphological characters. The strain grows on a medium containing 5% malt extract. After 72 h of growth at 25 ^o C, Gram stained smears contain cells of a slightly spherical shape with sizes 2.4–6.2 x 3.0–8.1 μm. Colonies of white color, opaque, round shape. Yeast forms asks of zygotic origin on an acetate medium. In a liquid medium, it grows at a temperature of 29 ^° C for 72 hours at a stirrer speed of 200 rpm.

Physiological signs. The strain is an aerobic, grows at a temperature of 4 ^o C, does not grow at a temperature at 42 ^o C, a temperature optimum of 20 ^o C, grows at a pH of 5.0-7.5, an optimum pH of 6.8-7.0. Grows on rich nutrient media based on meat peptone broth (MPB) and enzymatic hydrolyzate of fish meal (FGRM).

 Biochemical signs. The strain in Gissa media assimilates sucrose, maltose, cellobiose, D-xylose, D-mannitol, citric acid, lactic acid, alpha-methyl d-glycoside as the only carbon source. Ferments glucose on the 3-5th day. Uses nitrogen of organic and inorganic origin.

 Storage media: FGRM or MPA and minimally saline media with PCBs. To obtain the yeast biomass used for the decomposition of PCBs, the strain can be grown on various nutrient media containing commonly used sources of carbon, nitrogen, and mineral salts. PCB, glucose, sucrose, maltose are used as a carbon source. As a source of nitrogen, both organic and inorganic substances can be used. Organic nitrogen sources can be used, such as FGRM, peptone, yeast autolysate, corn extract and others. Of the inorganic ones, ammonium salts.

Depending on the composition of the media used and culturing conditions, QOL is obtained with a cell titer from 5.0 • 10 ⁶ to 1.0 • 10 ⁷ cells / cm ³ .

 The invention is illustrated by the following examples.

 Example 1

 Obtaining biomass strain.

The strain of yeast Hansenulla californica AT is grown aerobically on an MPB-based liquid nutrient medium by a deep method at a temperature of t = 29 ± 2 ^° C and a stirrer speed of 200 rpm for 72 hours. The titer of the grown culture is 1.0 • 10 ⁷ cells / cm ³ .

 Example 2

 Laboratory soil cleaning.

 Various concentrations of PCBs are added to the soil sample and the soil is thoroughly mixed.

The resulting culture fluid is diluted with tap water to a titer of 106-105-104 cells / cm ³ . A suspension of cells is aerosolized on the surface of the soil contaminated with PCBs, at a rate of 1 l / 1 m ² . The soil is maintained at room temperature for 2 months. Soil samples for analysis are taken after 0.5, 1 and 2 months.

 The concentration of PCBs in the treated soil was determined using a Crystal gas-liquid chromatograph (according to the method of “Methods for determining the microquantities of pesticides in food, feed and the environment.” T. 2.- M .: Agropromizdat, 1992.- C. 143-148) . PCBs from a soil sample are extracted with hexane and introduced into a gas-liquid chromatograph with an electron capture detector.

 The research results are presented in table. 1.

As can be seen from the table. 1, the most optimal concentration of cells in suspension for application to the soil is 105 cells / cm ³ , a concentration of 104 cells / cm ³ is ineffective.

The concentration of 106 cells / cm ³ in suspension for soil application is economically disadvantageous.

 Example 3

 Cleaning the soil in the field.

The cell suspension obtained in example 1, is aerosolized on the surface of the soil using a backpack spray at a rate of 1 l / 1 m ² . After applying the suspension, the soil is carefully digged and kept at ambient temperature for 2 months. Soil samples for analysis were taken after 1 and 2 months. The analysis was carried out in accordance with the previously cited example.

 The research results are presented in table. 2.

 As can be seen from the table. 2 data, the concentration of PCBs in soil samples is reduced by 45.3% within 2 months.

 Example 4

 Water purification.

 The cell suspension obtained in example 1, contribute to 5 l of wastewater containing PCB 25 mg / L. The suspension is made at the rate of 104 cells / liter. After applying the suspension, the water is constantly stirred at a speed of 10 rpm for 14 days at room temperature. Water samples for analysis were taken after 1 and 2 weeks. The analysis was carried out in accordance with the previously cited example.

 The research results are presented in table. 3.

 As can be seen from the table. 3 data, the concentration of PCBs in wastewater is reduced by 49% within 2 weeks.

 The most successfully proposed strain of yeast can reduce the concentration of PCBs in environmental objects by about 50%. It can be used for detoxification of environmental objects, in particular soil, water, waste electrical and chemical industries containing PCBs. In the future, it can be used to obtain various organic compounds from PCBs and wastes containing PCBs.

Sources of information
1. Tarasov V.V. Environmental pollution by polychlorinated biphenyls and ways to minimize their effects. Collection of scientific works of the Russian Chemical Technical University LXXV years / Main achievements in science. M., 1996, p. 24-41.

 2. Deckwer W.-D. Weppen P. Review of technologies for the remediation of contaminated soil and abandoned areas contaminated with hazardous waste. - Chemie-Ingenieur-Technik, 1987, vol. 59, # 6, p. 457-464.

 3. Schondorf T., Munz K.N. Removal of polychlorinated biphenyls from contaminated soil. - Chem. Rosch. (Schweiz.), 1988, v. 41, # 45, s. 18.

 4. Tarasov V.V. Environmental pollution by polychlorinated biphenyls and ways to minimize their effects. Collection of scientific works of the Russian Chemical Technical University LXXV years / Main achievements in science. M., 1996, p. 24-41.

 5. US patent N 4843009, C 12 N 1/12, publ. 1986 year

 6. Degradation of 4, 4'-Dichlorobiphinyl, 3, Y, 4, 4'-Tetrachlorobiphinyl, and 2, 2 ', 4, 4', 5, 5'- Hexachlorobiphenyl by the White Rot Fungus Phanerochaete chrysosporium. Applied and Environmental Microbiologi., 1995, vol. 61, N 11, p. 3904-3909.

Claims (1)

 Hansenulla californica yeast strain VKPM Y-2284 decomposing polychlorinated biphenyls.

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