RU2564105C1 - STRAIN OF BACTERIA Exiguobacterium sp. - DECOMPOSER OF CRUDE OIL AND PETROLEUM PRODUCTS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: bacterial strain Exiguobacterium sp. ELA-6, having the ability to dispose of crude oil and petroleum products, is deposited in Federal State Institution of Science the Institute of Biochemistry and Physiology of Microorganisms of RAS under the registration number RNCM B-2813D and can be used to purify soil and water reservoirs contaminated with crude oil and petroleum products, in a wide temperature range from +4 to +30°C.

EFFECT: invention enables to reduce the time of purification of soil and water reservoirs 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 Exiguobacterium sp. VKM B-2813D 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).

The known strains are: Pseudomonas putida 36 (1. USSR author's certificate N 1076446, 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 Oil and Gas Research Institute Authors: Dyadechko VN , Tolstokorova L.E., Morozova T.N.), Acinetronacter sp. HB-1 (2. Patent RU N 2077579. 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 NA, Kobelev VS, Kholodenko V.P. Patentee: State Research Institute of Applied Microbiology), Pseudomonas alcaligenes E7 (3. Patent RU N 2134723. The bacterial strain Pseudomonas alcaligenes E7 used to clean water and soil from oil and 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 V.A. 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 N 2019527. A method for purifying soils from oil pollution. C1, C02F 3/34, Е02В 15/04, С09К 17/00 showed great efficiency at low positive temperatures. Application: 93017464 / 26, 04/30/1993 Published: 09/15/1994 Applicant: Northern State Research and Design 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 The oil fraction proceeds slowly and only with the addition of nitroammophos or nitroammophos and sodium nitrate.

A known consortium of oil-oxidizing microorganisms consisting of Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. PI Ku-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 with a weight ratio of 3-12 wt. % of each microorganism, while together with a suspension of a biological product, rhizotorfin is introduced into the contaminated medium in an amount of 30-120 g / m ² (5. Patent RU 2191643. A method for cleaning soils from contamination by oil and oil products. C1, B09C 1/10, C12N 1 / 20, C12N 1/20, C12R 1:01. Application: 2001119562/13, 07/09/2001. 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 MP, Tarabukina NP, Neustroev MM, Sazonov NN, Parnikova SI., Stepanova AM Patentee: State Scientific Institution Yakutsk Research Institute Agricultural Academy of the Russian Academy of Agricultural Sciences), 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 over 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 ° C

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, in a wide temperature range from +4 to + 30 ° C

The problem is solved in that a strain of Exiguobacterium sp. VKM V-2813D 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.

Bacterial strain Exiguobacterium sp. VKM B-2813D was isolated from a sample of a soil-gravel mixture contaminated with motor oil, selected for research at JSC AK Yakutskenergo, Republic of Sakha (Yakutia).

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 B-2813D. The resulting strain is characterized by the following features.

Morphological signs. Gram-positive sticks located singly and in small clusters. Cell size 1.3 × 0.6 μm,

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 large creamy colonies, yellow-orange in color, with a diameter of up to 3 mm. 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; pancreatic, casein 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 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.

In meat and peptone broth (wt.%): Enzymatic peptone - 1.0, sodium chloride - 0.5, meat water - the rest; pH 7.0-7.2, forms a homogeneous turbidity with agitation.

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 - P. 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 dull colonies with an orange-yellow tint, a diameter of 1-2 mm.

Physiological and biochemical characteristics.

The strain is an obligate aerob, grows at a temperature of +4 to + 30 ° C. Temperature optimum +18 + 20 ° C. It grows at pH 4.5-9.0. The optimum pH is 6.8-7.5. It grows in salt broth with the addition of 0.1-2.0% NaCl.

The oxidase-positive strain does not decarboxylate lysine and ornithine, and is not able to cleave phenylalanine. Indolenegative .. Hydrogen sulfide does not produce. Active against inositol. Urease is negative. The test with β-galactosidase is negative. Ferments sorbitol. Does not ferment lactose, glucose, sodium citrate, sodium malonate. Uses petroleum hydrocarbons and oil products as an energy source.

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

When sequencing the variable regions of a gene encoding 16S rRNA, the following assembled nucleotide sequence was obtained for the studied strain:

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 mowed meat and peptone agar or stored in a lyophilized state in ampoules at a temperature of + 4 ° C.

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 Exiguobacterium sp. VKM B-2813D

Bacterial strain Exiguobacterium sp. VKM B-2813D was isolated from a sample of a soil-gravel mixture contaminated with motor oil, selected for research at JSC AK Yakutskenergo, Republic of Sakha (Yakutia).

To do this, 1.0 g prepared for the study of the soil-gravel mixture from the above object was added to 250 ml of the 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. Oil was used as the sole source of carbon and energy, which was introduced into the mineral medium in the amount of 1000 mg per 1.0 dm ^{3 of} medium. The incubation was carried out in thermostatic 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-yellow tint and the disintegration of the oil layer.

A pure bacterial culture was obtained by culturing under the conditions listed above and repeated reseeding (more than 10) of the accumulative 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, on the surface of the MPA, the appearance of paste-like brilliant orange-colored colonies with a diameter of 1-3 mm was observed, 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 Bergi Key (8. Bergey’s Manual of Systematic Bacteriology Book Review Int. J. of Syst. Bact; July 1985, p. 408.), Identified as a strain of Exiguobacterium sp.

Example 2. Preparation of a bacterial preparation based on cells of a strain of Exiguobacterium sp. VKM B-2813D

The isolated colonies obtained in example 1, are subcultured on 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 turbid liquid mixture of cells of the strain Exiguobacterium sp. VKM B-2813D, 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 a strain of Exiguobacterium sp. VKM B-2813D

Cells of strain Exiguobacterium sp. VKM B-2813D 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 Exiguobacterium sp. VKM B-2813D is cultivated in an aqueous medium with a mineral composition (wt.%): KNO ₃ - 0.4; MgSO ₄ · 7H ₂ O - 008; 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 3 days.

Biodegradation of oil and oil products is determined by the spectrometric method using a concentrator "IKN-025" (9. FR .31.2007.03234 MVI 02/01/117 Method for measuring the mass concentration of oil products in drinking, natural and waste waters by IR spectrometric method using a concentrator " IKN-025 ").

The experimental data show that in the aquatic environment with mineral components the proposed strain already utilizes 4.19-8.76% of oil and oil products already on the 3rd day at a temperature of + 4 ° C; at a temperature of + 30 ° C - 38.76-49.46%; at a temperature of + 20 ° C - 49.78-59.4%; depending on the type of xenobiotic (table 1).

Example 4. Water purification from oil using cells of strain Exiguobacterium sp. VKM B-2813D

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. 30,000 mg of oil and 30.0 cm ^{3 of the} bacterial isolate of the strain Exiguobacterium sp. VKM B-2813D (according to example 1) containing 1.0 · 10 ⁹ 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 Exiguobacterium sp. VKM B-2813D for 2 months was 44.9%. 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 with respect to both oil and oil products in a relatively short time (from 3 days to 2 months), in a wide temperature range from +4 to +30 ° C and can be used to clean environmental objects from oil and oil products pollution (diesel fuel, motor oil, hydraulic oil, gas condensate).

Claims (1)

Bacterial strain Exiguobacterium sp . VKM B-2813D is a destructor of oil and oil products.