Method of cleaning cryogenic soils and water environment from crude oil and petroleum products by spore-forming bacteria bacillus vallismortis

FIELD: biotechnology.

SUBSTANCE: bacterial strain Bacillus vallismortis RNCIM B-11017 is grown and a suspension is made of it, which is applied in cryogenic soil and the water environment. It is exposed under the specified parameters from 7 to 60 days and the quantitative content of crude oil and petroleum products in the cryogenic soil and water environment is determined.

EFFECT: invention enables to reduce the time of denaturation of crude oil and petroleum products and to reduce their concentration in the cryogenic soil and water environment.

3 tbl, 4 ex

 

The invention relates to the protection of the environment, in particular to methods of purification of frozen soils and the aquatic environment from oil pollution. For treatment of frozen soil and water pollution from oil contaminated substrates contribute suspension of the bacterial strain Bacillus vallismortis VKPM B-11017. The invention allows for a short time to achieve a high degree of purification of frozen soils and the aquatic environment from oil and oil products (diesel fuel, gas condensate.

The level of technology

Known strains: Rhodococcus erythropolis, Rhodococcus luteus, Micrococcus flavus (1. Klyuchnikova M., Smirnova GF, Kubersky S.L., Slabospitsky A.T., Zalewski B.C., Kasatkina, ETC., Eliseeva G.S., A.S. SU 1493666-A1. Cleaning method podslantsevyh wastewater from petroleum products. Application 4280617, 09.07.1987. Publ. 15.07.1989); Flavobacterium tirrennicum (2. Novozhilov M.I. Popova LE, Nedovodiyev ETC., A. Kalamkarov LI bacterial Strain Flavobacterium tirrennicum used to clean podslanevye and industrial wastewater from oil and oil products. A.S. SU 1409594. Application 4066152, 24.03.1986. Publ. 15.07.1988), which can decompose the hydrocarbon oil. The disadvantage of the above strains is that they carry out the degradation of petroleum products only in water and not able to clean the soil from oil.

Known strains: Pseudomonas putida 36 (3. Dyadechko V.N., Tolstokorova LE, Morozova so-CALLED. Strain @ @ 36 used for isdi water and soil from oil and oil products. A.S. USSR 1076446. Application 3474745, 22.07.1982. Publ. 28.02.1984); Acinetronacter sp.-1 (4. Zhirkov, N.A., Kobelev B.C., cholodenko's VP STRAIN of BACTERIA ACINETOBACTER SPECIES USED FOR PURIFICATION of WATER AND SOIL FROM OIL AND OIL products. RF patent №2077579. Application 94043331 from 06.12.1994. Publ. 20.041997.), Acinetronacter sp.JN-2 (5. Ushkov S. B., Elias N. STRAIN ACINETOBACTER SPECIES USED FOR PURIFICATION OF WATER AND SOIL FROM OIL AND OIL PRODUCTS. Patent RU N 2064017. Application No. 93009595 from 23.02.1993. Publ. 20.07.1996.), Pseudomonas alcaligenes E7 (6. Ermolenko SM, cholodenko VP, Chugunov V. BACTERIAL STRAIN PSEUDOMONAS ALCALIGENES E7 USED FOR PURIFICATION of WATER AND SOIL FROM OIL AND OIL products. Patent RU N 2134723, Application No. 98105707 from 25.03.1998. Publ. 20.08.1999) and Pseudomonas alcaligenes B-1 (7. Ermolenko SM, cholodenko VP, cast Irons, VA, Kobelev B.C., Akimova N.A. BACTERIAL STRAIN PSEUDOMONAS ALCALIGENES IN-1, USED FOR PURIFICATION of WATER AND SOIL FROM OIL AND OIL products. Patent RU N 2133770. Application No. 98105709 from 25.03.1998. Publ. 27.07.1999.). The disadvantage of strains is that they disposed of the oil at relatively high temperatures.

Greater efficiency at low positive temperatures showed the strain Rhodococcus erythroplis E-15 (8. There are regional laws T.V., Arakelyan AI, Komarova TI, Ilyinsky V.V. METHOD of CLEANING SOIL FROM OIL POLLUTION. Patent RU No. 2019527. Application No. 93017464 from 30.04.1993. Publ. 15.09.1994.), but the process of biodegradation of oil flows slowly and only adding n-p-K or nits is kamofsky and sodium nitrate.

Closest to the claimed method is a method of purification of permafrost soils from oil spore-forming bacteria Bacillus subtilis (9. Neustroev BTW, Tarabukina, NP, Neustroev, M., Sazonov, N., Greenhouse, S., Stepanova, A. M. PURIFICATION METHOD PERMAFROST SOILS FROM OIL SPORE-forming BACTERIUM BACILLUS SUBTILIS. Patent RU No. 2446900. Application No. 2010129158 from 13.07.2010. Publ. 20.01.2012.), having the ability to reduce the concentration of oil to 0.48%, within 3 months of the summer period. However, the strain carries out the degradation of oil in soil only in terms of the model experiment and not capable of cleaning the water environment from oil and dispose of petroleum products (diesel fuel, motor oil, gas condensate), including in conditions of wide temperature range from +8 to +37°C.

The task of the invention to provide a method of cleaning permafrost soils and the aquatic environment, not only from oil, but also from petroleum products (diesel fuel, gas condensate, oil motor) in a relatively short time, in a wide temperature range from +8 to +37°C.

The implementation of the invention

The solution of this problem is achieved by depositing contaminated by oil or oil products (diesel fuel, gas condensate, oil motor) permafrost soils and the aquatic environment of microbial drug based on the bacterial strain Bacillus vallismortis PMBC-11017.

The technical result of the invention is achieved as follows.

Use of bacterial suspension of the strain Bacillus vallismortis VKPM B-11017 with the contents of living cells in a concentration not less than 1·109CFU/ml

The bacterial strain Bacillus vallismortis VKPM B-11017 deposited in Russian national collection of non-pathogenic industrial microorganisms FSUE gosniigenetika - PMBC, 117545, , Moscow, 1st Road travel, 1. Collection number VKPM B-11017. The strain of bacteria is promising for the remediation of oil-contaminated frozen soils and soil and water from pollution by oil and oil products.

The bacterial strain may be obtained in the laboratory No. 2 of the Federal state budgetary institution of science Institute of oil and gas problems, Siberian branch of the Russian Academy of Sciences (oil and gas research Institute of RAS), 677890, Republic of Sakha (Yakutia), Yakutsk, street October, 1.

The method is illustrated by the following examples.

Example 1. Obtaining a bacterial suspension on the basis of the cells of the bacterial strain Bacillus vallismortis VKPM B-11017

The bacterial strain Bacillus vallismortis VKPM B-11017 grown on mesopatamia agar (wt.%: enzymatic peptone - 1.0; sodium chloride and 0.5; agar - 1,0; water meat rest, pH 7.0 to 7.2) at a temperature of +30 +37°C for 2 days, then washed in 0.9% NaCl at a concentration of not less than 1·109CFU/ml on the optical standard is the mouth gisk named after. A.M. Tarasevich. Thus obtained bacterial suspension used for cleaning soil and water pollution from oil and petroleum products.

Example 2. Determination of oxidizing ability of the bacterial suspension of the strain Bacillus vallismortis VKPM B-11017

Bacterial suspension of the strain Bacillus vallismortis VKPM B-11017, obtained by the method described in example 1, making mineralized aqueous medium of the following composition (g/DM3): KNO3- 4,0; MgSO4·7H2O - 0,8; NaCl to 1.0; K2NRA4- 1,4; KH2PO4- 0,6; agar - 20,0; distilled water - 1,0 DM3contaminated by oil or oil products in the amount of 1000 mg per 100,0 cm3environment, and exhibit at temperatures of +8°C; +20°C +30°C and +37°C in katalozhnyh conditions for 7 days. The biodegradation of crude oil and petroleum products are determined by a spectrometric method using concentrator "TSC-025" (10. FR. MIM 01.02.117. The method of measurement of mass concentration of petroleum products in drinking water and wastewater IR-spectrometric method using concentrator "TSC-025).

The experimental data shows that saline water environment strain of Bacillus vallismortis VKPM B-11017 7 days at a temperature of +8-(+37°C)disposes of 18,59-52,82% oil; 25,46-82,26% diesel fuel; 28,19-50,04% motor oil; 56,40-84,73% gas condensate (table 1).

Example 3. Cleaning permafrost soils from oil strain of Bacillus vallismortis VKPM B-11017

The experiment is carried out for cleaning permafrost soils contaminated with oil, using a bacterial suspension of the strain Bacillus vallismortis VKPM B-11017. The experience made in the conditions of open ecosystems on the territory of the accidental spillage of raw crude oil. The average soil temperature during the entire period of the setting of the experiment, on the horizon of 20 cm averaged +8°C; on the horizon 10 cm (+14)°C.

To experience a bacterial suspension of the bacterial strain Bacillus vallismortis VKPM B-11017, obtained by the method described in example 1, applied to the soil contaminated with oil, in the amount of 1 liter of the drug per 1 cubic meters of contaminated soil. The soil is thoroughly mixed and exhibit within 60 days.

For the analysis of soil samples taken before the start of the experiment and after 60 days after its termination. The efficiency of oil biodegradation by strain Bacillus vallismortis VKPM B-11017 appreciate the gravimetric method (11. RD 39-0147098-90. Instructions to monitor the condition of soils at the sites of enterprises Minneftegazstroy. - 1990).

The research results show that bacterial suspension of the strain Bacillus vallismortis VKPM B-11017 within 60 days, at an average soil temperature of +8-(+14°C, carries out the degradation of oil by 78.3%, while natural loss of oil under the same condition is the beautiful environment in soil, not treated, are to 7.32% (table 2).

Table 1
The degree of utilization of oil and petroleum products by a strain of Bacillus vallismortis VKPM B-11017 in saline aqueous medium after 7 days
Version of experiencet, °C
Xenobiotic, mg/DM3Period+8+20+30+37
Oilto experience1000,01000,01000,01000,0
after the experience814,1425,0393,0and 471.8
% degradation18,59of 57.560,752,82
Diesel fuelto experience1000,01000,01000,01000,0
after the experience 745,4393,7171,0177,4
% degradation25,4660,6382,982,26
Motor oilto experience1000,01000,01000,01000,0
after the experience718,1682,8553,0499,6
% degradation28,1931,7244,750,04
Gas condensateto experience1000,01000,01000,01000,0
after the experiencekm 436.0321,2259,4152,7
% degradationof 56.467,8874,0684,73
Table 2
The dynamics of degradation of oil in permafrost soils, microbial treated by drug-based cells of the bacterial strain Bacillus vallismortis VKPM B-11017
Version of experienceThe oil content to make strain, mg/kgThe amount of oil that is 60 days after application of strain, mg/kgDestruction of oil for 60 days %
Soil + oil + drug on the basis of the strain Bacillus vallismortis VKPM B-110173886843to 78.3
Control (soil + oil)1377112762to 7.32

Example 4. Water purification from oil using a strain of Bacillus vallismortis VKPM B-11017

In a desiccator, the volume of 5.0 DM3made 3,0 DM3water and 30.0 cm3bacterial suspensions of strains of Bacillus vallismortis VKPM B-11017 obtained in example 1. To create an oil film capacity was added 30 000 mg of raw crude oil. As control was used the water in an amount of 3.0 DM3with the addition of the 30 000 mg of oil and without making microorganisms.

The experiments were carried out for 60 days at a water temperature of +20±1°C. the Biodegradation of oil determined by a spectrometric method using concentrator "TSC-025" (10. FR. 1.31.2007.03234. MIM 01.02.117. The method of measurement of mass concentration of petroleum products in drinking water and wastewater IR-spectrometric method using concentrator "TSC-025).

It is established that the destruction of oil in water for 60 days amounted to 50.8 per cent (table 3).

Table 3
Dynamics of destructi and oil in the water after making the drug on the basis of the strain Bacillus vallismortis VKPM B-11017
Version of experienceThe amount of oil to make cells of strain mgThe amount of oil within 60 days after making the cells of strain mgThe degree of utilization of oil, %
Water + oil + cells of strain Bacillus vallismortis VKPM B-1101730 00014 736,250,80
Control30 00029 334,6of 2.21

Thus, the advantages of the m of the proposed method is he can be disposed of as oil and oil products in a relatively short time (from 7 to 60 days)in a wide temperature range from +8 to +37°C and can be used for purification of frozen soils, and for treatment of the water environment from oil and oil products (diesel fuel, motor oil, gas condensate). The proposed method can be used for the rehabilitation of contaminated territories in the Far North.

The method of purification of permafrost soil and water pollution from oil and oil products, characterized in that the cleaning of contaminated substrates using a bacterial suspension of the strain Bacillus vallismortis VKPM B-11017.



 

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