RU2513702C1 - Strain of bacteria bacillus vallismortis - destructor of oil and oil products - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain of bacteria Bacillus vallismortis VKPM V-11017 is proposed - destructor of oil and oil products. Strain may within short period of time in the wide range of temperatures from +8 to +37°C degrade oil by 78.3%.

EFFECT: strain may be used to clean soil and water contaminated by oil and oil products, such as diesel fuel, motor oil, gas condensate.

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Description

The invention relates to the field of microbiology and is a new bacterial strain of bacteria Bacillus vallismortis VKPM B-11017, with the ability to quickly dispose of oil and oil products (diesel fuel, motor oil, gas condensate). The strain can be used to clean soils and water bodies contaminated with oil and oil products in a wide temperature range from +8 to + 37 ° C.

State of the art

The known strains are: Rhodococcus erythropolis, Rhodococcus luteus, Micrococcus flavus (1. Klyushnikova TM, Smirnova G.F., Kuberskaya S.L., Slabospitskaya A.T., Zalevsky BC, Kasatkina T.P., Eliseeva G.S. A .s. SU 1493666-A1. Method for purification of shale wastewater from oil products. Application 4280617, 07/09/1987. Publish. 07/15/1989); Flavobacterium tirrennicum (2. Novozhilova M.I., Popova L.E., Nedovodieva T.P., Kalamkarova L.I.A.S. SU 1409594. The bacterial strain Flavobacterium tirrennicum used to clean oil and industrial wastewater from oil and petroleum products. Application 4066152, 03.24.1986. Publish. 07.15.1988), which can decompose oil hydrocarbons.

The disadvantage of the above strains is that they carry out the destruction of oil products only in water and are not able to clean the soil from oil.

Known strains: Pseudomonas putida 36 (3. Dyadechko V.N., Tolstokorova L.E., Morozova T.N. Strain @ @ 36, used to clean water and soil from oil and oil products. AS USSR 1076446. Application 3474745, 07.22.1982. Published. 02.28.1984); Acinetronacter sp.NV-1 (4. Zhirkova N.A., Kholodenko V.P. The bacterial strain ACINETOBACTER SPECIES used to purify water and soil from oil and oil products. Patent of the Russian Federation 2077579. Application 9404331, December 6, 1994. Publish. 20.04 .1997); Acinetronacter sp.JN-2 (5. Ushkov SB, Ilyinskaya NV The strain ACINETOBACTER SPECIES used to clean water and soil from oil and oil products. Patent of the Russian Federation 2064017. Application 93009595, 02.23.1993. Publish. 20.07. 1996); Pseudomonas alcaligenes E7 (6. Ermolenko Z.M., Kholodenko V.P., Chugunov V.A. Bacterial strain PSEUDOMONAS ALCALIGENES E7 used to purify water and soil from oil and oil products. Patent of the Russian Federation 2134723. Application 98105707, 03.25.1998 Publish. 08.20.1999) and Pseudomonas alcaligenes B-1 (7. Ermolenko Z.M., Kholodenko V.P., Chugunov V.A., Kobelev BC, Akimova N.A. Bacterial strain PSEUDOMONAS ALCALIGENES B-1, used for purification of water and soil from oil and oil products. Patent of the Russian Federation 2133770. Application 98105709, 03.25.1998. Publish. 07.27.1999).

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

The strain Rhodococcus erythroplis E-15 (8. Koronelli T.V., Arakelyan E.I., Komarova T.N., Ilyinsky V.V. A method for purifying soils from oil pollution was shown to be more effective under low positive temperatures. RF patent 2019527 Application 93017464, 04/04/1993, publ. 09/15/1994), but the process of biodegradation of oil proceeds slowly and only with the addition of nitroammophos or nitroammophos and sodium nitrate.

The prototype is a bacterial strain "Bacillus subtilis Kolyma 7/2" (9. Neustroev M.P., Tarabukina N.P., Neustroev M.M., Sazonov N.N., Parnikova S.I., Stepanova AM Method method of purification of frozen soils from oil by spore-forming bacteria BACILLUS SUBTILIS. RF patent 2446900. Application 2010129158, 07/13/1010. Publish. 07/13/2010), 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 for 3 months of the summer period. However, the strain degrades only oil and only in the soil under the conditions of a model experiment and is not able to purify water from oil and dispose of oil products (diesel fuel, motor oil, gas condensate), including in a wide temperature range from +4 to +37 ° C.

The objective of the invention is to obtain a strain of bacteria with high utilizing ability in relation to both oil and oil products in a relatively short time, in a wide temperature range from +8 to + 37 ° C, which can be used to clean soils and water contaminated oil and oil products (diesel fuel, motor oil, gas condensate).

The implementation of the invention

The proposed bacterial strain Bacillus vallismortis VKPM B-11017 was isolated from oil-contaminated soil of the Eastern Siberia-Pacific Ocean trunk pipeline, 1338.5 km, Lensky district, Republic of Sakha (Yakutia), May 19, 2010. The strain was deposited by the All-Russian collection of industrial microorganisms in the Federal State Unitary Enterprise GosNIIGenetika - VKPM, 117545, Moscow, 1st Dorozhniy proezd, 1. Collection number VKPM V-11017.

The strain Bacillus vallismortis VKPM B-11017 utilizes oil and oil products (diesel fuel, motor oil, gas condensate) as the only carbon source. The process runs in a wide temperature range (from +8 to + 37 ° C).

The use of the Bacillus vallismortis strain VKPM B-11017 in vivo for cleaning frozen soil from oil, at an average soil temperature of +8 to + 14 ° C, allows to achieve 78.3% of the destruction of oil in 60 days.

The destruction of oil and oil products in water at a water temperature of + 20 ° C is 50.8%, over 2 months.

The destruction of oil and oil products in the aquatic environment with the addition of mineral components after 7 days at a temperature of + 8 ° C is 18.5 - 56.4%, at a temperature of + 20 ° C - 31.7 - 67.8%, at a temperature of +30 ° C - 44.7 - 74.0%, at a temperature of + 37 ° C - 50.0 - 84.7%, depending on the type of pollutant.

The resulting strain is characterized by the following features.

Morphological and cultural characteristics.

Gram-positive spore bacilli in smears are located singly and in short chains.

On meat and peptone agar (wt.%: Enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2) with the addition of 1% glucose grows in the form wrinkled, dry, beige colonies, after 1-3 days, the colonies turn brown, the substrate does not pigment.

On the surface of beveled meat-peptone agar without adding 1% glucose, it grows in the form of a dry, wrinkled film of beige color.

On Saburo medium (wt.%: Fish meal hydrolyzate - 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 large colonies from dirty brown to marsh brown in diameter up to 1 cm or more.

In meat and peptone broth (wt.%: Enzymatic peptone - 1.0, sodium chloride - 0.5, meat water - the rest; pH 7.0-7.2) grows in the form of a wrinkled film of beige color over the broth.

On the mineral environment of Münz (10. 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, gas condensate) 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, in the form of opaque matte gray-beige colonies with a diameter of 0.1-0.2 cm

Physiological and biochemical characteristics.

The strain grows well at temperatures from +8 to + 37 ° C, under aerobic conditions. At temperatures below + 8 ° C and under anaerobic conditions, the strain does not die immediately, but develops more slowly. Optimum growth + 30 ° C. It grows at pH 6.0 - 8.0. It grows in salt broth with the addition of 0.1-2.0% NaCl.

The strain is catalase-positive, oxidase-negative. Oxidative metabolism. Biochemical activity is poorly expressed. Does not absorb lysine and ornithine. Does not consume sodium citrate and malonate. Gelatin does not hydrolyze. Starch does not decompose. Indolene negative. Does not form hydrogen sulfide. The Voges-Proskauer reaction is negative. Phenylalanine deoxaminase is negative. Fermenting beta-galactosidase.

The strain is not virulent, not toxic, not toxigenic, not phytotoxic (the test was carried out on white mice and seeds of higher plants (oats cultivar Skakun and wheat cultivar Prilenskaya-19)).

Storage conditions:

1. In lyophilized form in ampoules at + 4 ° C.

2. The strain can be maintained by regular reseeding (1 time in 2 weeks) on the slanted MPA.

The invention is illustrated by the following examples.

Example 1. The cultivation of a strain of Bacillus vallismortis VKPM B-11017

The proposed strain of Bacillus vallismortis VKPM B-11017 was isolated from oil-contaminated soil of the Eastern Siberia-Pacific Ocean trunk pipeline, 1338.5 km, Lensky district, Republic of Sakha (Yakutia), May 19, 2010.

The cultivation of the strain was carried out in the Muntz mineral medium of the following composition (g / dm ³ ): KNO ₃ - 4.0; MgSO ₄ .7H ₂ O - 0,8; NaCl - 1.0; K ₂ HPO ₄ - 1.4; KH ₂ PO ₄ 0.6; agar - 20.0; distilled water 1.0 dm ³ ; the pH of the medium is 7.2. For this, 1.0 g of oil-contaminated soil from the above object was added to 250 ml of the Muntz mineral medium. As the only carbon and energy source used oil, which was inoculated into the medium in an amount Muntz 1000 mg per 1.0 dm ³ medium. The incubation was carried out in thermostatic rocking conditions on the installation "UVMT-12-250" at 200 rpm and a temperature of + 29 ± 1 ° C. Bacterial growth was observed after 7-10 days of incubation by the formation of a turbid whitish emulsion and the disappearance of oily stains of oil. A pure bacterial culture was obtained by culturing under the above conditions and repeated reseeding (more than 10) of the accumulative culture on Petri dishes with agar from a fish meal hydrolyzate (GRM agar) with the addition of 1% glucose. Further, crops were incubated under stationary conditions in an incubator at + 30 ° C. After 48 hours, on the surface of the timing agar, dry, beige-colored colonies appeared, which according to the cultural-morphological and biochemical characteristics, as well as according to 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 the Bergi Identifier (11. Bergey's Manual of Systematic Bacteriology Book Review Int. J. of Syst. Bact .; July 1985, p. 408), identified as a strain of Bacillus vallismortis.

Isolated colonies were subcultured on slanted meat-peptone agar. After 48 hours of incubation at a temperature of + 30 ° C, cells of the Bacillus vallismortis strain VKPM B-11017 from mowed agar were used to determine activity in the process of destruction of oil and oil products.

Example 2. The study of the oil-oxidizing activity of the bacterial strain Bacillus vallismortis VKPM B-11017

The cells of the strain Bacillus vallismortis VKPM B-11017 are washed off with beveled meat and peptone agar 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 × 109 microbial cells / cm ³ according to the optical standard GISK named after AM Tarasevich.

The obtained bacterial isolate of the strain Bacillus vallismortis VKPM B-11017 is cultivated in an aqueous medium with a mineral composition similar in composition to the Münz medium, the composition of which is described in example 1.

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. The flasks are cultured in rocking conditions on the installation "UVMT-12-250" at 200 rpm, at temperatures + 8 ° C; + 20 ° C; + 30 ° C and + 37 ° C for 7 days.

Biodegradation of oil and oil products is determined by the spectrometric method using an IKN-025 concentrator (12. 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 the aquatic environment with mineral components the proposed strain already utilizes 18.59-52.82% of oil on the 7th day at a temperature of + 8 + 37 ° C; 25.46-82.26% - diesel fuel; 28.19-50.04% - motor oil; 56.40-84.73% - gas condensate (table 1).

Table 1 The degree of utilization of oil and oil products by a strain of Bacillus vallismortis VKPM B-11017 after 7 days of cultivation Experience option t ° C Xenobiotic, mg / dm ³ Term +8 +20 +30 +37 Oil to experience 1000,0 1000,0 1000,0 1000,0 after experience 814.1 425.0 393.0 471.8 % destruction 18.59 57.5 60.7 52.82 Diesel fuel to experience 1000,0 1000,0 1000,0 1000,0 after experience 745.4 393.7 171.0 177.4 % destruction 25.46 60.63 82.9 82.26 Motor oil to experience 1000,0 1000,0 1000,0 1000,0 after experience 718.1 682.8 553.0 499.6 % 28.19 31.72 44.7 50.04 destruction Gas condensate to experience 1000,0 1000,0 1000,0 1000,0 after experience 436.0 321.2 259.4 152.7 % destruction 56.4 67.88 74.06 84.73

Example 3. Preparation of a preparation based on cells of a strain of Bacillus vallismortis VKPM B-11017

The bacterial isolate obtained by the method described in example 2 is transferred into flasks with a sterile liquid medium similar in composition to that of Stepin et al. (13. Stepin A.A., Samygin V.M., Kukhtin V.P., Vladimtseva I.V., Zhoga L.K., Kapliev V.I. Method for producing biomass of bacilli Patent of the Russian Federation 2053293. Application 5036470, 01/09/1992. Publ. 01/27/1996), g / dm ³ : casein hydrochloride hydrolyzate - 14, 0; yeast extract 3.0; MgSO ₄ · 7H ₂ O — 0.2; NaCl - 4.0; (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O - 0.5; distilled water up to 1.0 dm ³ ; pH 7.3-7.4. The medium is sterilized at 0.5 atm for 20 minutes.

Under sterile conditions, 2.0 cm ^{3 of} Perftoran emulsion (OJSC NPF Perftoran, Russia) and 10.0 cm ^{3 of} suspension of bacterial isolate are added to 100.0 cm ^{3 of the} finished medium. Cultivate in flasks on a rocking chair for 48 hours, at a temperature of 29 ± 1 ° C. A liquid turbid culture is obtained with a titer of at least 1 billion cells / cm ³ , which is used as a preparation for treating soil or water from oil pollution.

Example 4. Water purification from oil using a preparation based on cells of the strain of Bacillus vallismortis VKPM B-11017

3.0 dm ^{3 of} water and 30.0 cm ^{3 of a} preparation based on the bacterial strain Bacillus vallismortis VKPM B-11017 obtained by the above method were added to a desiccator with a volume of 5.0 dm ³ (example 3). To create an oil film, 30,000 mg of crude crude oil was added to the vessel. As a control, water was used in an amount of 3.0 dm ³ with the addition of 30,000 mg of oil and without introducing microorganisms.

The experiments were carried out for 2 months at a water temperature of + 20 ± 1 ° C.

Oil biodegradation is determined by the spectrometric method using an IKN-025 concentrator (12. 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 ").

It was found that the destruction of oil in water for 2 months amounted to 50.8%. The destruction in the control capacity for 2 months has not changed significantly (table 2).

table 2 The dynamics of the destruction of oil in water after making the drug based on the strain of Bacillus vallismortis VKPM B-11017 Experience option The amount of oil before introducing the cells of the strain, mg The amount of oil 2 months after the introduction of the cells of the strain, mg The degree of utilization of oil,% Water + oil + cells of the Bacillus vallismortis strain VKPM B-11017 30,000 14 736.2 50.80 The control 30,000 29 334.6 2.21

Example 5. Purification of soil from oil using cells of the bacterial strain Bacillus vallismortis VKPM B-11017

An experiment was carried out on the purification of permafrost soil contaminated with oil using cells of the Bacillus vallismortis strain VKPM B-11017. The experiment was performed in vivo. The average soil temperature, over the entire period of the experiment, at a horizon of 20 cm was + 8 ° C; at the horizon of 10 cm - + 14 ° C.

For the experiment, cells of the strain Bacillus vallismortis VKPM B-11017 from beveled meat and peptone agar obtained by the method described in example 1 are washed with 0.9% NaCl solution, a microbial suspension of the bacterial isolate is prepared, with a concentration of 1 × 10 ⁹ microbial cells / cm ³ optical GISK standard to them. AM Tarasevich.

The resulting suspension of cells of the strain Bacillus vallismortis VKPM B-11017 is applied to the soil contaminated with oil in an amount of 1 liter of suspension per 1 cubic meter. m oil contaminated soil. The soil is thoroughly mixed and exposed for 60 days.

For analysis, soil samples are taken before the start of the experiment and 60 days after its completion. The effectiveness of the biodegradation of oil by the proposed strain is evaluated by the gravimetric method (14. RD 39-0147098-90. Instructions for monitoring the condition of soils at facilities of enterprises of the Ministry of Oil and Gas Industry. - 1990). The research results show that the proposed strain within 60 days, at an average soil temperature of + 8 + 14 ° C, carries out oil degradation by 78.3%, while the natural loss of oil under the same environmental conditions in the soil not treated with the drug, amounted to 7.32% (table 3).

Table 3 The dynamics of oil degradation in permafrost soils treated with a suspension based on cells of the Bacillus vallismortis strain VKPM B-11017 Experience option The oil content before the introduction of the strain, mg / kg The oil content in 60 days after the introduction of the strain, mg / kg Oil destruction in 60 days,% Soil + oil + drug based on a strain of Bacillus vallismortis VKPM B-11017 3886 843 78.3 The control 13771 12762 7.32

Feasibility

Thus, the advantage of the proposed bacterial strain BACILLUS VALLISMORTIS VKPM B-11017 is that it has a high utilizing ability with respect to both oil and oil products and in a relatively short time (from 7 to 60 days), in a wide temperature range from +8 to + 37 ° C carries out oil degradation by 78.3%, while the natural loss of oil under the same environmental conditions in the soil not treated with the preparation is 7.32%. The strain can be used both for cleaning soils and for cleaning water contaminated with oil and oil products (diesel fuel, motor oil, gas condensate).

Claims (1)

The bacterial strain Bacillus vallismortis VKPM B-11017 is a destructor of oil and oil products.