RU2560279C1 - STRAIN OF BACTERIA Kocuria sp. - DESTRUCTOR OF CRUDE OIL AND PETROLEUM PRODUCTS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Kocuria sp., having the ability to dispose quickly crude oil and petroleum products (diesel fuel oil, motor oil, hydraulic oil, gas condensate), deposited in the RNCM under the registration number Kocuria sp. RNCM Ac-2624D and can be used to clean soil and water reservoirs contaminated with crude oil and petroleum products, under the wide temperature range from +4 to +30°C.

EFFECT: invention enables to improve the quality of cleaning soil and water from crude oil and petroleum products.

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Description

The invention relates to the field of microbiology and is a new bacterial strain of Kocuria sp. VKM Ac-2624D is a destructor of oil and oil products, which can be used to clean soil and water from oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate).

Known strains: Pseudomonas putida 36 (1. USSR author's certificate N1076446, Strain Pseudomonas putida 36, used to clean water and soil from oil and oil products. A1, C02F 3/34, B09C 1/10, C12N 1/20 C02F 3/34 , C02F 101: 32, C12N 1/20, C12R 1:40 Application: 3474745, 07/22/1982 Published: 02/28/1984 Applicant: West Siberian Petroleum Research Institute Authors: Dyadechko VN, Tolstokorova L.E., Morozova T.N.), Acinetronacter sp. HB-1 (2. Patent RU N2077579. The bacterial strain Acinetobacter species used to purify water and soil from oil and oil products. C1, C12N 1/20, C02F 3/34, B09C 1/10, C12R 1:01, C12N 1 / 20, B09C 101: 00. Application: 94043331/13, December 6, 1994. Published: 04/20/1997 Applicant: State Research Institute of Applied Microbiology Authors: Zhirkova N.A., Kobelev V.C., Kholodenko V.P. Patentee: State Research Institute of Applied Microbiology), Pseudomonas alcaligenes E7 (3. Patent RU N2134723. The bacterial strain Pseudomonas alcaligenes E7 used to clean water and soil from oil and oil Products C1, C12N 1/20, C02F 3/34, B09C 1/10, C12N 1/20, C12R 1:38. Application: 98105707/13, 03.25.1998. Published: 08.20.1999. Applicant: State Scientific Center Applied Microbiology Authors: Ermolenko Z.M., Kholodenko VP, Chugunov VA Patentee: State Scientific Center for Applied Microbiology). The disadvantage of the strains is that they utilize oil at relatively high temperatures.

The Rhodococcus erythroplis E-15 strain (4. Patent RU N2019527. A method for purifying soils from oil pollution. C1, C02F 3/34, Е02В 15/04, C09K 17/00 showed great efficiency in low positive temperatures. Application: 93017464/26 , 04/30/1993 Published: 09/15/1994 Applicant: Northern State Research and Development Geological Center Authors: Koronelli T.V., Arakelyan E.I., Komarova T.I., Ilyinsky V.V. Patentee: Northern State Research and Development Geological Center), but the biodegradation process I oil is slow, and only by the addition of NPK or NPK and sodium nitrate.

A known consortium of oil-oxidizing microorganisms in the composition of Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. PI Ky-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 with a weight ratio of 3-12 wt. % of each microorganism, and together with a suspension of a biological product, rhizotorfin is introduced into the polluted medium in an amount of 30-120 g / m3 (5. Patent RU 2191643. Method for cleaning soils from oil and oil products pollution. C1, B09C 1/10, C12N 1/20 , C12N 1/20, C12R 1:01. Application: 2001119562/13, 09.07.2001. Published: 10.27.2002 Applicant: Closed Joint-Stock Company Polyinform Authors: Saxon V. M., Kuznetsov S. A., Boykova I.V., Novikova I.I. Patentee: Closed Joint-Stock Company “Polyinform”).

The disadvantage is that when preparing a working suspension of microorganisms, the temperature is maintained at + 18 + 22 ° C, i.e. microorganisms that are part of the consortium are not capable of degradation of oil products at low positive temperatures (+ 4 ° C).

A bacterial strain “Bacillus subtilis Kolyma 7/2” (6. Patent RU 2446900 is used as a prototype. A method for purifying frozen soils from oil with spore-forming bacteria Bacillus subtilis. C2. B09C 1/10 (2006.01). Application: 2010129158/13, 13.07. 2010. Published: 04/10/2012 Authors: Neustroev M.P., Tarabukina N.P., Neustroev M.M., Sazonov N.N., Parnikova S.I., Stepanova AM Patentee: State Scientific Institution Yakutsk Scientific Agricultural Research Institute of the Russian Agricultural Academy), which has the ability to reduce the oil concentration to 0.48%, that is, the oil level from 135143 mg / kg is reduced to 645 mg / kg during the 3 months of the summer period. However, the strain degrades only oil and is not able to utilize oil products (diesel fuel, motor oil, hydraulic oil, gas condensate), including in a wide temperature range from +4 to + 30 ° С.

The objective of the invention is to expand the range of strains with the ability to utilize both oil and petroleum products (diesel fuel, motor oil, hydraulic oil, gas condensate) in a relatively short time, over a wide temperature range from +4 to + 30 ° C.

The problem is solved in that a strain of Kocuria sp. VKM Ac-2624D is a destructor of oil and oil products with a utilizing ability with respect to oil and oil products (diesel fuel, engine oil, hydraulic oil, gas condensate), promising for cleaning environmental objects from oil and oil product pollution (diesel fuel, engine oil , hydraulic oil, gas condensate) in a wide temperature range (from +4 to + 30 ° C).

The origin of the strain.

The bacterial strain Kocuria sp. VKM Ac-2624D is isolated from the frozen soil of the Shergin Mine, Yakutsk, Republic of Sakha (Yakutia), ul. Kulakovsky, 18.

The strain is identified and deposited in the All-Russian Collection of Microorganisms (VKM) of the Federal State Budgetary Institution of Science Institute of Biochemistry and Physiology of Microorganisms named after G.K. Scriabin, Russian Academy of Sciences (IBPM RAS) under registration number VKM Ac-2624D.

The resulting strain is characterized by the following features.

Morphological signs.

Gram-positive, undisputed coccoid cells located singly and in small clusters. The size of the cells is 1-2 microns in diameter.

Cultural signs.

On meat peptone agar (MPA) (wt.%): Enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2, forms paste-like shiny colonies of orange-pink color, 1-3 mm in diameter. The consistency is soft, easily removed from the surface of the agar, easily smeared.

On nutrient agar based on a fish meal hydrolyzate (wt.%): A fish meal hydrolyzate - 1.2; enzymatic peptone - 1.2; sodium chloride - 0.6; agar - 1.0; distilled water the rest; pH 7.1-7.5, forms paste-like glossy orange-colored colonies with a diameter of 1-3 mm. The consistency is soft, easily removed from the surface of the agar, easily smeared.

On the environment of Saburo (wt.%): Hydrolyzed fish meal - 1.0; casein pancreatic hydrolyzate - 1.0; yeast extract - 0.2; monosubstituted sodium phosphate - 0.2; D-glucose 4.0; agar - 1.0; distilled water - the rest; pH 6.0 ± 3, forms creamy colonies of orange-pink color, with a diameter of 1-5 mm. The consistency is soft, easily removed from the surface of the medium, easily smeared.

In meat and peptone broth (wt.%): Enzymatic peptone - 1.0, sodium chloride - 0.5, meat water - the rest; pH 7.0-7.2, causes diffuse opacification with diffusion of the orange pigment into the broth.

On the mineral environment of Münz (7. Kersten DK Morphological and cultural properties of indicator microorganisms of oil and gas imaging - Microbiology, 1963, No. 5 - S. 1024-1030) with oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate , gasoline) of the following composition: (wt.%) KNO ₃ - 0.4; MgSO ₄ · 7H ₂ O - 0.08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ - 0.06; agar - 2.0; oil or petroleum products - 1.0; distilled water - the rest, pH 7.2, grows in the form of pasty, opaque muddy colonies with a diameter of 1 mm.

Physiological and biochemical characteristics.

The strain is an obligate aerob, grows at temperatures from +4 to + 30 ° С, grows more intensively + 10 + 20 ° С. Temperature optimum + 16 + 20 ° С. It grows at pH 5.5-9.0. Optimum pH 6.0-8.0. It grows in salt broth with the addition of 0.1-2.0% NaCl.

The strain of catalase is positive, oxidase is negative. Does not ferment glucose and lactose. Ferments sorbitol. Does not absorb lysine. Absorbs ornithine. Does not consume sodium citrate and malonate. Gelatin does not hydrolyze. Starch does not decompose. Indole is negative. The Voges-Proskauer reaction is negative. Phenylalanine deoxaminase is negative. Does not ferment beta galactosidase. Lecithinase does not form. Uses petroleum hydrocarbons and oil products as an energy source.

The strain is resistant to benzylpenicillin, furazolidone, metranidazole, amoxicillin, ampicillin, amoxiclav, kaotima, pefloxacin, amosin, sensitive to mozivar, cefataxime, ceftriaxone; moderately sensitive to oxacillin, polymyxin, chloramphenicol.

The strain is not virulent, not toxic, not toxigenic (tested on white mice).

The strain can be maintained by regular reseeding (once every 10-14 days) on beveled meat and peptone agar or stored in a lyophilized state in ampoules at a temperature of + 4 ° С.

The bacterial strain can be obtained in laboratory No. 2 of the Federal State Budgetary Institution of Science, Institute of Oil and Gas Problems of the Siberian Branch of the Russian Academy of Sciences (IPGG SB RAS), at 677980, Republic of Sakha (Yakutia), Yakutsk, October, 1.

The invention is illustrated by the following examples.

Example 1. Isolation and cultivation of a strain of Kocuria sp. VKM Ac-2624D

The bacterial strain Kocuria sp. VKM Ac-2624D is isolated from the frozen soil of the Shergin Mine, Yakutsk, Republic of Sakha (Yakutia), ul. Kulakovsky, 18 by the method of accumulative cultures in the environment of Muntz with oil, followed by reseeding at MPA.

For this, 1.0 g of frozen soil from the above object was added to 250 ml of the Muntz mineral medium of the following composition: (wt.%) KNO ₃ - 0.4; MgSO ₄ · 7H ₂ O - 0.08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ - 0.06; distilled water - the rest, pH 7.2. As the only source of carbon and energy, oil was used, which was introduced into the Münz medium in an amount of 1000 mg per 1.0 dm ^{3 of} medium. Incubation was carried out in thermostatically controlled rocking conditions on the installation "UVMT-12-250" at 200 rpm and a temperature of + 20 ± 1 ° C. Bacterial growth was observed after 7 days of incubation by the formation of a turbid emulsion with an orange tint and the disintegration of the oil layer.

A pure bacterial culture was obtained by culturing under the above conditions and repeated reseeding (more than 10) of the accumulation culture in Petri dishes with MPA (wt.%): Enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2.

Further, crops were incubated under stationary conditions at various temperatures from +4 to + 30 ° C. After 72 hours, the appearance of pasty shiny orange-colored colonies with a diameter of 1-3 mm was observed on the MPA surface, which, according to the cultural-morphological and biochemical characteristics, as well as the results of the analysis of the nucleotide sequences of the 16S rRNA gene and key phenotypic characters according to the taxonomic descriptions given in Bergey's Guide (9. Bergey's Manual of Systematic Bacteriology Book Review Int. J. of Syst. Bact; July 1985, p. 408.). identified as a strain of Kocuria sp.

Example 2. Preparation of a bacterial preparation based on cells of the strain Kocuria sp. VKM Ac-2624D

The isolated colonies obtained in example 1, are subcultured on the beveled MPA and incubated for 48 hours at a temperature of + 20 ± 1 ° C. The cells are washed off with a 0.9% NaCl solution and the initial microbial suspension of the bacterial isolate is prepared with a titer of 1 × 10 ⁹ microbial cells / cm ³ according to the optical standard GISK them. AM Tarasevich.

To obtain the drug, a liquid mineral medium of the following composition is prepared (wt.%): KNO ₃ - 0.4; MgSO ₄ · 7H ₂ O - 0.08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ - 0.06; distilled water - the rest; The pH of the medium is 7.2 and cultured in flasks under rocking conditions for 48 hours, at a temperature of 20 ± 1 ° C, at 200 rpm. A liquid turbid mixture of cells of the strain Kocuria sp. VKM Ac-2624D with a titer of 1-5 × 10 ⁹ cells / cm ³ , which can be used as a preparation for processing soil contaminated with oil and oil products.

Example 3. The study of the oil-oxidizing activity of the strain Kocuria sp. VKM Ac-2624D

Cells of strain Kocuria sp. VKM Ac-2624D from mowed meat-peptone agar is washed off with 0.9% NaCl solution, the initial microbial suspension of the bacterial isolate is prepared, with a volume of 25 cm ³ , with a concentration of 1 × 10 ⁹ microbial cells / cm ³ according to the optical standard GISK them. AM Tarasevich.

The resulting bacterial isolate of the strain Kocuria sp. VKM Ac-2624D is cultivated in an aqueous medium with a mineral composition (wt.%): KNO ₃ - 0.4; MgSO ₄ · 7H ₂ O - 0.08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ - 0.06; distilled water - the rest; the pH of the medium is 7.2.

100.0 cm ^{3 of the} finished mineral medium and 1000 mg of oil are introduced into cones with a volume of 200.0 cm ³ . Flasks are seeded with cells of the strain to a concentration of 1 × 10 ⁹ cells / cm ³ . As a control, put the same flask with medium, oil and without bacteria to determine the total (natural) losses. The experiment is carried out in triplicate. Flasks are cultured at temperatures of + 4 ° C; + 20 ° C; + 30 ° C for 7 days.

Biodegradation of oil and oil products is determined by the spectrometric method using the IKN-025 concentrator (8. FR.1.31.2007.03234 MVI 02/01/117 Method for measuring the mass concentration of oil products in drinking, natural and waste waters by the IR spectrometric method using the IKN concentrator -025 ").

The experimental data show that in the aquatic environment with mineral components the proposed strain already utilizes 6.38-13.34% of oil and oil products already on the 7th day at a temperature of + 4 ° C; at a temperature of + 30 ° C - 51.9-69.25%; at a temperature of + 20 ° C - 57.78-81.46%, depending on the type of xenobiotic (table 1).

Example 4. Water purification from oil using cells of the strain Kocuria sp. VKM Ac-2624D

3.0 dm ^{3 of} tap water was added to a desiccator with a volume of 5.0 dm ³ , which had settled for one day at room temperature. 30000 mg of crude crude oil and 30.0 cm ^{3 of the} bacterial isolate of the strain Kocuria sp. VKM Ac-2624D (as in Example 1) containing 1.0-10 cells / cm ³ suspension. As a control, water is used in an amount of 3.0 dm ³ with the addition of 30,000 mg of oil and without introducing a microbial suspension of the bacterial isolate.

The experiment is carried out for 2 months, maintaining the water temperature in the range of + 15 + 20 ° C.

The results obtained (table 2) showed that in water, the destruction of oil under the influence of the strain Kocuria sp. VKM Ac-262 for 2 months amounted to 55.19%. The destruction in the control tank has not changed significantly.

Thus, the advantage of the proposed strain is that it has a high utilizing ability in relation to both oil and oil products in a relatively short time (from 7 days to 2 months), in a wide temperature range from +4 to +30 ° С, which can be used both for cleaning environmental objects from oil and oil products pollution (diesel fuel, motor oil, hydraulic oil, gas condensate).

Claims (1)

 The bacterial strain Kocuria sp. VKM Ac-2624D is a destructor of oil and oil products.