Strain of bacteria rhodococcus sp. - decomposer of crude oil and petroleum products

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.

3 tbl, 5 ex

 

The invention relates to the field of Microbiology and represents a new bacterial strain Rhodococcus sp. LER-12 VKM Ac-2626D - destructor of oil and oil products, which can be used for cleaning soil and water from oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate).

Known strains: Pseudomonas putida 36 (1. Copyright certificate of the USSR No. 1076446, a Strain of Pseudomonas putida 36, used for purification of water and soil from oil and oil products. A1 C02F 3/34, VC 1/10, C12N 1/20 C02F 3/34, C02F 101:32, C12N 1/20, C12R 1:40. Application: 3474745, 22.07.1982. Posted: 28.02.1984. Applicant: West-Siberian scientific-research geological oil Institute. Authors: Dyadechko V. N., Tolstokorova L. E., Morozova T. N.), Acinetronacter sp. HB-1 (2. Patent RU N 2077579. The strain of bacteria Acinetobacter species used for purification of water and soil from oil and oil products. C1, C12N 1/20, C02F 3/34, VC 1/10, C12R 1.01, C12N 1/20, B09C 101:00. Application: 94043331/13, 06.12.1994. Posted: 20.04.1997. Applicant: State scientific-research Institute of applied Microbiology. Authors: Zhirkov N. And., Kobelev B. C., Kholodenko, V. P. Patentee: State scientific-research Institute of applied Microbiology), Pseudomonas alcaligenes E7 (3. Patent RU Ν 2134723. The bacterial strains Pseudomonas alcaligenes E7, used for purification of water and soil from oil and oil products. C1, C12N 1/20, C02F 3/34, VC 1/10, C12N 1/20, C12R 1:38. W�turnout: 98105707/13, 25.03.1998. Posted: 20.08.1999. Applicant: State scientific center of applied microbilogy. Authors: Z. M. Ermolenko, V. P. Kholodenko, Chugunov V. A. Patentee: State scientific center of applied microbilogy).

The disadvantage of strains is that they recycle the oil at relatively high temperatures.

Greater efficiency at low positive temperatures showed the strain Rhodococcus erythroplis E-15 (4. Patent RU N 2019527. The method of cleaning soil from oil pollution. C1, C02F 3/34, EV 15/04, SC 17/00. Application: 93017464/26, 30.04.1993. Posted: 15.09.1994. Applicant: Northern state scientific-research and design geological center. Authors: T. V. coronelli, Arakelyan E. I., Komarov I. V., Il'inskii, V. V. Patentee: Northern state scientific-research and design geological center), but the process of oil biodegradation occurs slowly and only adding NPK or NPK and sodium nitrate.

Known consortium nefteokislyayuschey of microorganisms consisting of Pseudomonas putida PI Ko-1, Pseudomonas fluorescens P-896, Micrococcus sp. PI Ku-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 with a weight ratio of 3-12 wt.% each micro-organism, together with a suspension of a biological product in the contaminated medium is introduced risotorphine in the amount of 30-120 g/m2(5. P�tent EN 2191643. The method of cleaning soil from pollution by oil and oil products. C1, VC 1/10, C12N 1/20, C12N 1/20. C12R 1:01. Application: 2001119562/13, 09.07.2001. Posted: 27.10.2002. The applicant. Closed joint stock company "Polinfor". Authors: Saxon V. M., Kuznetsov, S. Α., Boikova, I. V., Novikova I. I. the Patentee: closed joint stock company "Polinfor").

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

As the prototype is a strain of bacteria (Bacillus subtilis Kolyma 7/2" (6. Patent RU 2446900. Method of purification of permafrost soils from oil spore-forming bacteria Bacillus subtilis. C2. VS 1/10 (2006.01). Application: 2010129158/13, 13.07.2010. Posted: 10.04.2012. Authors: M. P. Neustroev, Tarabukina N. P., Unsettled M. M.. N. Sazonov.N., Greenhouse, S. I., Stepanova, A. M., Patentee: State scientific institution Yakut research Institute of agriculture of RAAS), has the ability to reduce the concentration of oil to 0.48%, i.e. the level of oil from 135143 mg/kg is reduced to 645 mg/kg within 3 months of the summer period. However, the strain carries out the degradation of oil and is not able to dispose of the oil products (diesel fuel, m�BLR motor, the hydraulic oil, gas condensate), including in the conditions of wide temperature range from +4 to +30°C.

The object of the invention is the expansion of the range of strains that have the ability to dispose of as oil and oil 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 task is solved by the fact that the resulting strain Rhodococcus sp. LER-12 VKM Ac-2626D - destructor of oil and oil products with recycling capacity in relation to oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate), promising to clean the environment from pollution by oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate) in a wide temperature range (from +4 to +30°C).

The origin of the strain.

The strain of bacteria Rhodococcus sp. LER-12 VKM Ac-2626D extracted from frozen soil Shergin Shaft, Yakutsk, Republic of Sakha (Yakutia), Kulakovsky str., 18.

The strain was identified and deposited in all-Russian Collection of Microorganisms (VKM) of Federal state budgetary institution of science Institute of biochemistry and physiology of microorganisms them. G. K. Skryabin Russian Academy of Sciences (IBPM RAS) under the register�operating room VKM Ac-2626D. The resulting strain is characterized by the following features.

Morphological features.

Gram-positive, immobile sticks. In a one-day culture on a mineral medium Muntz (7. Kersten D. K. Morphological and cultural properties of indicator microorganisms in the oil and gas shooting-Microbiology, 1963, No. 5 Pp. 1024-1030) forms a short thick sticks. When dividing the observed characteristic arrangement of cells at an angle to each other.

Cultural characteristics.

On mesopatamia agar (ΜΠΑ), wt.%: enzymatic peptone - 1,0; sodium chloride is 0.5; agar - 1,0; water meat - the rest, pH 7,0-7,2, forms a smooth pasty colony of cremolata-pink color with a diameter of 1-5 mm.

On nutrient agar-based hydrolysate fish meal, wt.%: hydrolysate fish meal - 1,2; peptone enzymatic - 1,2; sodium chloride - 0,6; agar - 1,0; distilled water the rest; pH of 7.1 to 7.5, forms a pasty shiny colony of cremolata-pink color with a diameter of 1-5 mm. Consistency soft, easily removed from the surface of the agar, easily smeared.

On Wednesday Saburo, wt.%: hydrolysate fish meal - 1,0; pancreatic casein hydrolysate and 1.0; yeast extract and 0.2; sodium phosphate odnozameshchenny -0,2; D-glucose - 4,0; agar - 1,0; distilled water - the rest; pH 6.0±3, forms a creamy colony of cremolata pink �emetrol 1-5 mm. Consistency soft, easily removed from the surface environment, easily smeared.

In mesopatamia broth, wt.%: enzymatic peptone - 1.0, sodium chloride - 0,5, water meat - the rest; pH 7,0-7,2, causes diffuse opacity.

Mineral environment Muntz with oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate, gasoline) of the following composition, wt.%: KNO3- 0,4; MgSO4·7Η2O - 0,08; NaCl - 0,1; K2The NRA4- 0,14; KN2RO4- 0,06; agar - 2,0; oil or petroleum products - 1,0; distilled water - the rest, pH 7,2, grows in the form of paste opaque opaque colonies with a diameter of 1 mm. Physiological and biochemical characteristics.

The strain grows at temperatures between +4 and+30±1°C, in aerobic conditions and poorly under anaerobic conditions. Optimum growth+4-+20±1°C.

Oxidatively, not decarboxylase lysine, decarboxylate ornithine, not able to break down phenylalanine. Indoctrination. Hydrogen sulfide is not produced. Not active against Inositol. Urease negative. Test with β-galactosidase-negative. Not aged lactose, glucose, sodium citrate, sodium malonate. Uses as energy source for oil hydrocarbons.

Resistant Amoksiklav, ampicillin, CATIA, amoxicillin, eloxatine, amasino. Resistant to chloramphenicol. Sensitive to benzylpenicillin, Motiva�, oxacillin, Cefotaxime, Ceftriaxone, polymyxin, furazolidone, metronidazole.

Strain is not virulent, non-toxic, non toxigenic (test conducted on white mice).

The strain can be maintained by regular re-seeding (1 every 10-14 days) on stubble mesopatamia agar or be stored in a lyophilized state in the ampoule at a temperature of +4°C.

The bacterial strains can be obtained in the laboratory No. 2 Federal state budgetary institution of science Institute of oil and gas problems, Siberian branch of the Russian Academy of Sciences (IPng RAS), at the following address: 677980, Republic of Sakha (Yakutia), Yakutsk, St. October, 1.

The invention is illustrated by the following examples.

Example 1. Isolation and cultivation of the strain Rhodococcus sp. LER-12 VKM Ac-2626D

The strain of bacteria Rhodococcus sp. LER-12 VKM Ac-2626D extracted from frozen soil Shergin Shaft, Yakutsk, Republic of Sakha (Yakutia), Kulakovsky str., 18 by the method of cumulative crops on the environment Muntz oil with subsequent subculturing on ΜΠΑ.

For this purpose, 1.0 g of frozen soil with the above object introduced into 250 ml of mineral medium Muntz following composition, wt.%: ΚΝΟ3- 0,4; MgSO4·7Η2O - 0,08; NaCl - 0,1; K2The NRA4- 0,14; KN2RO4- 0,06; distilled water - the rest, pH of 7.2. As the sole source of carbon and energy used oil, �otori was made on Wednesday Muntz in the amount of 1000 1.0 mg DM 3environment. Incubation was carried out in thermostatically katalozhnyh conditions at the "WVMT-12-250" at 200 rpm and a temperature of +20±1°C. bacterial Growth was observed after 7 days of incubation on the formation of a turbid emulsion and disintegration of the layer of oil.

Pure culture of bacteria was obtained by culturing under the above conditions and reseeding of multiple (more than 10) cumulative culture on Petri dishes with ΜΠΑ, wt.%: enzymatic peptone - 1,0; sodium chloride is 0.5; agar - 1,0; water meat - the rest, pH 7,0-7,2.

Further crops were incubated under stationary conditions at different temperatures from +4 to +30°C. for 48-72 h on the surface ΜΠΑ watched the appearance of a smooth pasty colonies of cremolata-pink color with a diameter of 1-5 mm, which according to cultural, morphological and biochemical characteristics, as well as the results of the analysis of nucleotide sequences of 16S rRNA and key phenotypic features according to taxonomic descriptions in this Determiner Bergi (8. Bergey's Manual of Systematic Bacteriology Book Review Int. J. of Syst. Bact; July 1985, p. 408) identified as a strain of Rhodococcus sp.

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

Isolated colonies obtained in example 1, subcultured on beveled ΜΠΑ and incubated 48 h at temperatures� +20±1°C. Cells are washed in 0.9% NaCl solution and prepare the initial microbial suspension of bacterial isolate with a titer of 1 x 109microbial cells/cm3optical standard Institute. A. M. Tarasevich.

To obtain the drug is prepared in liquid mineral medium of the following composition, wt.%: ΚΝO3- 0,4; MgSO4·7Η2Ο - 0,08; NaCl - 0,1; K2The NRA4- 0,14; KN2RO4- 0,06; distilled water - the rest; pH 7.2 and cultured in flasks in katalozhnyh conditions for 48 h, at a temperature of 20±1°C at 200 Rev/min. liquid turbid mixture of cells of the strain Rhodococcus sp. LER-12 VKM Ac-2626D, with a titer of 1 to 5·109cells/cm3that can be used as a drug for the treatment of soil contaminated by oil and oil products.

Example 3. A study of oil-oxidizing activity of the strain Rhodococcus sp. LER-12 VKM Ac-2626D

Cells of the strain Rhodococcus sp. LER-12 VKM Ac-2626D with beveled ΜΠΑ washed with 0.9% NaCl, prepare initial microbial suspension of bacterial isolate, with a volume of 25 cm3, with a concentration of 1·109microbial cells/cm3optical standard Institute. A. M. Tarasevich.

The resulting bacterial isolate of the strain Rhodococcus sp. LER-12 VKM AC-2626D cultured in an aqueous medium of mineral composition, wt.%: KNO3- 0,4; MgSO4·7Η2Ο - 0,08; NaCl - 0,1; K2The NRA4- 0,14; KN2RO4- 0,06; di water�fillerbunny - else; pH of 7.2.

In flasks with a volume of 200.0 cm3contribute by 100.0 cm3prepared mineral medium and 1000 mg of oil. The flask was seeded with cells of the strain Rhodococcus sp. LER-12 VKM Ac-2626D to a concentration of 1·109cells/ cm3. As a control, put the same flask with medium, without oil and bacteria to determine the overall (natural) losses. The experiment is performed in three replicates. Flasks are cultured at temperatures of +4°C; +20°C +30°C for 7 days.

Biodegradation of oil and oil products is determined by spectrometry, using concentratometer "IRH-025" (9.FR. 1.31.2007.03234 MIM 01.02.117 procedure of measurement of mass concentration of oil products in drinking, natural and waste waters of the IR-spectrometric method using concentratometer "IRH-025").

The experimental data show that in aqueous medium the mineral components of the proposed strain Rhodococcus sp. LER-12 VKM Ac-2626D for 7 days at a temperature of +4°C disposes of 14.0-19.8 per cent 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 the xenobiotic (PL. 1).

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

In the desiccator volume of 5.0 DM3make a 3.0 DM3tap water that has stood overnight at room t�mperature. In water add 30000 mg of raw crude oil and 30.0 cm3bacterial isolates of the strain Rhodococcus sp.LER-12 VKM Ac-2626D (of example 1) containing 1·109cells/cm3suspension. As a control using water in an amount of 3.0 DM3with the addition of 30,000 mg of oil and without making a microbial suspension of bacterial isolate.

The experiment was carried out for 2 months, keeping the water temperature in the interval+15-+20°C.

The obtained results (tab. 2) showed that in water, destruction of oil under the influence of the strain Rhodococcus sp. LER-12 VKM Ac-2626D for 2 months was 52,55%. Destruction in the control tank did not change significantly.

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

The ability of the strain Rhodococcus sp. LER-12 VKM Ac-2626D to activate the degradation of contaminants in permafrost soils is shown in the field experiment (table. 3).

Biodegradation of oil in the soil in the cleaning process of the strain Rhodococcus sp. LER-12 VKM Ac-2626D determined by cold extraction in chloroform by the method of (10.RD 39-0147098-90 manual for monitoring the condition of soils at the sites of the enterprises of Minneftegazprom).

For 2 months the degradation of oil pollution in soil treated with a bacterial preparation on the basis of strain of: Rhodococcus sp. LER-12 VKM AC-26260 �left 53,95%; in the control variant of natural loss of oil pollution amounted 11,54% over the same period of time.

Thus, the advantage of the proposed strain is that it has a high recycling capacity in relation to 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 for cleaning soil and water from pollution by oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate).

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



 

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