RU2558299C1 - STRAIN OF BACTERIA Rhodococcus sp. - DECOMPOSER OF CRUDE OIL AND PETROLEUM PRODUCTS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Rhodococcus sp. LER-12 having the ability to dispose quickly of crude oil and petroleum products (diesel fuel, motor oil, hydraulic oil, gas condensate) is deposited in the RNCM under the registration number Rhodococcus sp. RNCM Ac-2626D and can be used for purification of soils and water from contaminations with crude oil and petroleum products, in a wide temperature range from +4 to +30°C.

EFFECT: invention enables to improve the quality of purification of 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 Rhodococcus sp. LER-12 VKM Ac-2626D is a destructor of oil and oil products, which can be used to clean soil and water from oil and oil products (diesel fuel, engine oil, hydraulic oil, gas condensate).

The known strains are: Pseudomonas putida 36 (1. USSR author's certificate No. 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 BC, Kholodenko VP : State Research Institute of Applied Microbiology), Pseudomonas alcaligenes E7 (3. Patent RU Ν 2134723. The bacterial strain Pseudomonas alcaligenes E7, used to clean water and soil from oil and not 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, С09K 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 Oil Ia flows slowly and only by adding 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 Ku-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 with a weight ratio of 3-12 wt.% Of each microorganism, while risotorfin is introduced into the contaminated medium in an amount of 30-120 g / m ² (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, 07/09/2001. Published: 10.27. 2002. Applicant, Polyinform Closed Joint-Stock Company. Authors: Saxon V.M., Kuznetsov S.Α., 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).

As a prototype, a bacterial strain ((Bacillus subtilis Kolyma 7/2 "(6. Patent RU 2446900. The method of cleaning frozen soils from oil with spore-forming bacteria Bacillus subtilis. C2. B09C 1/10 (2006.01). Application: 2010129158/13, 13.07.07 .2010. Published: 04/10/2012 Authors: Neustroev M.P., Tarabukina N.P., Unsettled M.M .. Sazonov N.N., Parnikova S.I., Stepanova A.M. Patentee: State Scientific the establishment of the Yakutsk Research Institute of Agriculture of the Russian Agricultural Academy), which has the ability to reduce the oil concentration to 0.48%, that is, the oil level from 135143 m g / kg decreases 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 ° 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, over a wide temperature range from +4 to + 30 ° C.

The problem is solved in that a strain of Rhodococcus sp. LER-12 VKM Ac-2626D is a destructor of oil and oil products with a utilizing ability in relation 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 , motor oil, hydraulic oil, gas condensate) in a wide temperature range (from +4 to + 30 ° C).

The origin of the strain.

The bacterial strain Rhodococcus sp. LER-12 VKM Ac-2626D 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-2626D. The resulting strain is characterized by the following features.

Morphological signs.

Gram-positive, motionless sticks. In a one-day culture 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) forms short thick sticks. During division, a characteristic arrangement of cells at an angle to each other is observed.

Cultural signs.

On meat peptone agar (ΜΠΑ), wt.%: Enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2, forms pasty smooth colonies of creamy pink color with a diameter of 1-5 mm.

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 creamy pink colonies with a diameter of 1-5 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 creamy-pink colonies 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 turbidity.

On the mineral environment of Muntz 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 · 7Η ₂ O - 0.08; NaCl - 0.1; K ₂ NRA 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 grows at a temperature of + 4 + 30 ± 1 ° C, under aerobic conditions and weakly under anaerobic conditions. Growth optimum + 4- + 20 ± 1 ° C.

Oxidase negative, does not decarboxylate lysine, decarboxes ornithine, is not able to break down phenylalanine. Indolenegative. Hydrogen sulfide does not produce. Not active against inositol. Urease is negative. The test with β-galactosidase is negative. Does not ferment lactose, glucose, sodium citrate, sodium malonate. Uses petroleum hydrocarbons as an energy source.

It is resistant to amoxiclav, ampicillin, kaotima, amoxicillin, iffloxacin, amosin. Low resistance to chloramphenicol. It is sensitive to benzylpenicillin, mozivar, oxacillin, cefatoxime, ceftriaxone, polymyxin, furazolidone, metranidazole.

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 Rhodococcus sp. LER-12 VKM Ac-2626D

The bacterial strain Rhodococcus sp. LER-12 VKM Ac-2626D 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 ΜΠΑ.

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.%: ΚΝΟ ₃ - 0.4; MgSO ₄ · 7Η ₂ O - 0.08; NaCl - 0.1; K ₂ NRA 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 a thermostat-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 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 accumulative culture in Petri dishes with ΜΠΑ, 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 48-72 hours on the surface of ΜΠΑ, the appearance of pasty smooth creamy pink colonies with a diameter of 1-5 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 taxonomic descriptions given in the Bergi Key Book (8. Bergey’s Manual of Systematic Bacteriology Book Review Int. J. of Syst. Bact; July 1985, p. 408) were identified as a strain of Rhodococcus sp.

Example 2. Preparation of a bacterial preparation based on cells of a strain of Rhodococcus sp. LER-12 VKM Ac-2626D

The isolated colonies obtained in example 1 are subcultured on beveled ΜΠΑ 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 preparation, a liquid mineral medium of the following composition is prepared, wt.%: ΚΝO ₃ - 0.4; MgSO ₄ · 7Η ₂ Ο - 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 Rhodococcus sp. LER-12 VKM Ac-2626D, 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 Rhodococcus sp. LER-12 VKM Ac-2626D

Cells of the strain Rhodococcus sp. LER-12 VKM Ac-2626D from beveled ΜΠΑ is washed off with a 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 Rhodococcus sp. LER-12 VKM Ac-2626D is cultivated in an aqueous medium with a mineral composition, wt.%: KNO ₃ - 0.4; MgSO ₄ · 7Η ₂ Ο - 0.08; NaCl - 0.1; K ₂ HPO ₄ - 0.14; KH ₂ PO ₄ - 0.06; distilled water - the rest; pH of 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 Rhodococcus sp. LER-12 VKM Ac-2626D 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 an IKN-025 concentrator (9.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 an IKN concentrator -025 ").

The experimental data show that in an aqueous medium with mineral components of the proposed strain of Rhodococcus sp. LER-12 VKM Ac-2626D already on the 7th day at a temperature of + 4 ° C utilizes 14.0-19.8% of oil and oil products; at a temperature of + 20 ° C - 49.77-59.1%; at a temperature of + 30 ° C - 43.2-66.18%, depending on the type of xenobiotic (Table 1).

Example 4. Purification of water from oil using cells of the strain Rhodococcus sp. LER-12 VKM AC-2626D

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 Rhodococcus sp.LER-12 VKM Ac-2626D (as in Example 1) containing 1 · 10 ⁹ cells / cm ³ suspension are added to water. 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 + 15- + 20 ° C.

The results obtained (Table 2) showed that in water, the destruction of oil under the influence of the strain Rhodococcus sp. LER-12 VKM Ac-2626D for 2 months amounted to 52.55%. The destruction in the control tank has not changed significantly.

Example 5. Purification of soil from oil using cells of strain Rhodococcus sp. LER-12 VKM Ac-2626D

The ability of the strain Rhodococcus sp. LER-12 VKM Ac-2626D to activate the processes of oil degradation in permafrost soil are shown in the field experiment (table 3).

Biodegradation of oil in the soil during purification by the strain Rhodococcus sp. LER-12 VKM Ac-2626D is determined by the method of cold extraction in chloroform according to the method (10.RD 39-0147098-90 Instructions for monitoring the state of soils at the facilities of enterprises of the Ministry of Oil and Gas Industry).

For 2 months, the degradation of oil pollution in the soil treated with a bacterial preparation based on the strain: Rhodococcus sp. LER-12 VKM Ac-26260 was 53.95%; in the control variant, natural losses of oil pollution amounted to 11.54% over the same period of time.

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 7 days to 2 months), in a wide temperature range from +4 to + 30 ° C , which can be used to clean soils and water from oil and oil products pollution (diesel fuel, motor oil, hydraulic oil, gas condensate).

Claims (1)

The bacterial strain Rhodococcus sp. BKM Ac-2626D - a destructor of oil and oil products.