Method to clean permafrost soils and water environment by spore-forming bacteria bacillus atrophaeus vkpm v-10592

FIELD: biotechnologies.

SUBSTANCE: strain of bacteria Bacillus atrophaeus VKPM V-10592 is grown, and suspension is prepared from it, which is introduced into permafrost soil and water environment. Maintained at the specified parameters from 7 to 60 days, and then they determine quantity content of oil and oil products in permafrost soil and water environment.

EFFECT: invention makes it possible to reduce time for oil and oil product denaturation and to reduce their concentration in permafrost soil and water environment.

5 tbl, 6 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 and oil products. For treatment of frozen soil and water pollution from oil contaminated substrates contribute suspension of the bacterial strain Bacillus atropheus VKPM B-10592. 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).

The level of invention

Known strains of 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. cleaning Method podslantsevyh wastewater from petroleum products. A.S. SU # 1493666. Application No. 4280617 from 09.07.1987. Publ. 15.07.1989) and Flavobacterium tirrennicum (2. Novozhilov M.I. Popova LE, Nedovodiyev ETC., A. Kalamkarov SR Strain of bacteria Flavobacterium tirrennicum. A.S. SU # 1409594. Application No. 4066152 from 15.07.1986. Publ. 15.07.1988), used to clean podslanevye and industrial wastewater from oil and petroleum products.

In the first invention of improving the quality of water purification from oil products is carried out by introducing into plants with activated sludge cultures of microorganisms having high oxidizing activity, good physiological compatibility. The method consists in the fact that the Association's use, see the camping specially selected strains of bacteria RHODOCOCCUS ERYTHROPOLIS VKPM B-3078 (36A-1), RHODOCOCCUS LUTEUS VKPM B-3077 (25) and MICROCOCCUS FLAVUS VKPM B-3079 (53) in the following ratio crops: (0,5-1,5):(0,5-1,5):(0,5-1,5).

In the second case, for the treatment of industrial waters contaminated with oil, is used, a strain of bacteria Flavobacterium tirrenicum VKPM B-3369 able to more actively in comparison with the known strains of decomposing oil of different composition. When the content in 1 l medium 4 g of oil in aeration after 24 h since the introduction of strain decomposes 81.1% of paraffin, 40% methane-naphthene-aromatic, and 80.3% methane-naphthenic 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 of Pseudomonas putida 36 (3. Dyadechko V.N., Tolstokorova LE, Morozova so-CALLED. The strain Pseudomonas putida 36 used for purification of water and soil from oil and oil products. Speaker of the USSR № 1076446. Application No. 3474745 from 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. Patent RU No. 2077579. Application No. 94043331 from 06.12.1994. Publ. 20.04.1997); Acinetronacter sp.JN-2 (5. Ushkov Coll., Elias NV STRAIN ACINETOBACTER SPECIES USED FOR PURIFICATION of WATER AND SOIL FROM OIL AND OIL products. Patent RU No. 2064017. Application No. 93009595 from 23.02.1993. Publ. 20.07.1996); Pseudomonas alcaligenes E7 (6. Patent RU No. 2134723, 1999) and Pseudomonas alcaligenes B-1 (7. is 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 No. 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 2019527. Application 93017464 from 30.04.1993. Publ. 15.09.1994), but the process of biodegradation of crude oil by using the specified strain occurs slowly and only adding ammonium nitrate or ammonium nitrate and sodium nitrate is accelerated.

Close on the merits of 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 2446900. Application No. 2010 from 13.07.2010. Publ. 10.04.2012), with 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 and only in the soil conditions of the model experiment and is not capable of Khujand is indeed clean water environment from oil and dispose of petroleum products (diesel fuel), including in conditions of wide temperature range from +8 to +37°C.

The implementation of the invention

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

The solution of this problem is achieved by depositing contaminated by oil or oil products (diesel fuel) permafrost soils and the aquatic environment of microbial drug based on the bacterial strain Bacillus atropheus VKPM B-10592.

The technical result of the invention is achieved as follows.

Use a suspension of the bacterial strain Bacillus atropheus VKPM B-10592 with the contents of living cells in a concentration not less than 1·109CFU/ml

The bacterial strain Bacillus atropheus VKPM B-10592 deposited in Russian national collection of non-pathogenic industrial microorganisms FSUE gosniigenetika - PMBC, 117545, , Moscow, 1st Road travel, 1. Collection number VKPM B-10592. The strain of bacteria is promising for the remediation of oil-contaminated frozen soils and clean water environment from pollution by oil and oil products (diesel fuel).

The bacterial strain may be obtained in the laboratory No. 2 of the Federal state budgetary institution of science Institute of problems of oil and gas Sibir is anyone 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 of a strain of Bacillus atropheus VKPM B-10592

The bacterial strain Bacillus atropheus VKPM B-10592 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°C for 2 days, then washed in 0.9% NaCl at a concentration of not less than 1·109CFU/cm3optical standard gisk named after. A.M. Tarasevich. Thus obtained bacterial suspension of a strain of Bacillus atropheus VKPM B-10592 used for cleaning of frozen soils and the aquatic environment from oil and petroleum products.

Example 2. Determination of oxidizing ability of the strain Bacillus atropheus VKPM B-10592

Bacterial suspension of a strain of Bacillus atropheus VKPM B-10592 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; K2HPO4to 1.4; KH2PO4of-0.6; agar-20,0; distilled water 1,0 DM3contaminated by oil or oil products in the amount of 1000 mg per 100,0 cm3substrate, and exhibit at temperatures of +8°C; +20°C +30°C for 7 days.

The biodegradation of crude oil and petroleum products are determined by a spectrometric method using the 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").

The experimental data shows that saline water environment strain of Bacillus atropheus VKPM B-10592 after 7 days at a temperature of +8°C recycled 56.4% of oil; at +20°C to 96.5% of the oil; at +30°C - 97,13% oil (table 1).

Table 1
The degree of utilization of oil and oil products in mineralized aqueous medium by a strain of Bacillus atropheus VKPM B-10592 after 5 days
Version of experiencet°C
Xenobiotic, mg/DM3Period+8+20+30
Oilto experience1000,01000,01000,0

after the experience405,7834,32 28,64
% degradation56,4296,5697,13

Example 3. Water purification from oil strain of Bacillus atropheus VKPM B-10592

In the desiccator volume of 3.0 DM3make 3,0 DM3tap water, 30 g of raw crude oil and 30.0 cm3bacterial suspensions of strains of Bacillus atropheus VKPM B-10592, prepared according to example 1. As a control using water in an amount of 3.0 DM3with the addition of 30 g of oil and without making a bacterial suspension of a strain of Bacillus atropheus VKPM B-10592.

Table 2
The dynamics of degradation of oil in water by a strain of Bacillus atropheus VKPM B-10592
Version of experienceThe amount of oil to make cells of strain, gThe amount of oil in 2 months after making the cells of the strain, gThe degree of utilization of oil, %
Water + oil + strain of Bacillus atropheus VKPM B-10592300,471898,42
Water + oil30 300,0

The experiment is carried out within 2 months at room temperature (+20 +22°C).

The results showed that in water, the destruction of oil under the influence of the strain Bacillus atropheus VKPM B-10592 2 months amounted 98,42% (table 2).

Example 4. Cleaning permafrost soils from oil strain of Bacillus atropheus VKPM B-10592

Experiment on cleaning permafrost soils contaminated with oil, held in an open ecosystem. The soil temperature during the entire period of the setting of the experiment, at a depth of 20 cm averaged +8°C; at a depth of 10 cm to +14°C.

Bacterial suspension of a strain of Bacillus atropheus VKPM B-10592 obtained in example 1, applied to the soil contaminated with oil, based on 1 liter of suspension for 1 m2oil-contaminated soil. The soil is thoroughly mixed with a shovel and exhibit within 60 days.

The oil content in the soil is determined in accordance with RD 52.18.647-2003. (11. RD 39-0147098-90 instruction to monitor the condition of soils at the sites of enterprises Minneftegazstroy).

Table 3
The dynamics of degradation of oil in soil treated with Metarhizium strain of Bacillus atropheus VKPM B-10592
Version of experience The oil content to make strain, mg/kgThe amount of oil that is 60 days after application of strain, mg/kgDestruction of oil, %
Soil + oil + strain of Bacillus atropheus VKPM B-105921860798034356,82
Soil + oil596055616,69

The research results show that in 2 months when average soil temperature +8 +14°C, degradation of crude oil under the influence of the strain Bacillus atropheus VKPM B-10592 was 56,82% in that time, as a natural loss of oil in permafrost soil not treated with the drug, was 6.69 per cent (table 3).

Example 5. Cleaning permafrost soils from the diesel fuel strain of Bacillus atropheus VKPM B-10592

Experiment on cleaning permafrost soils contaminated with diesel fuel, conducted in an open ecosystem. The soil temperature during the entire period of the setting of the experiment, at a depth of 20 cm averaged +8°C; at a depth of 10 cm to +14°C. the Depth of oil penetration up to 15 cm

Bacterial suspension of a strain of Bacillus atropheus VKPM B-10592 obtained in example 1, applied to the soil contaminated with diesel fuel, based on 1 liter of the drug per 1 m is neftezagryaznannoi soil with depth of oil penetration up to 15 see The soil is thoroughly mixed with a shovel and exhibit within 45 days.

The oil content in the soil is determined in accordance with RD 39-0147098-90 (11. RD 39-0147098-90 instruction to monitor the condition of soils at the sites of enterprises Minneftegazstroy).

Table 4
Destruction of diesel fuel after cleaning soil strain of Bacillus atropheus VKPM B-10592
The scheme experiencepHHumidity %The content of the NP, mg/kgDestruction NP 45 days %
before cleaningafter cleaning
Soil + diesel6,9949,5258232124717,7
Soil + diesel + strain of Bacillus atropheus VKPM B-105927,0146,823420245089,53

Research results pok is said, that 45 days pollution in soil treated object fell 9.5 times the degradation of diesel fuel was 89,53% (table 4).

Example 6. Purification of mineralized water environment from diesel fuel by a strain of Bacillus atropheus VKPM B-10592

In flasks with a volume of 200.0 cm3poured into 100,0 cm3sterile tap water with mineral components of the following composition (wt.%): KNO3-0,4; MgSO4·7H2O-0,08; NaCl-0,1; K2HPO4-0,14; KH2PO4-0,06; make a bacterial suspension of a strain of Bacillus atropheus VKPM B-10592 obtained in example 1, in the amount of 10-15% by volume of water and add 1% diesel fuel.

As a control use the same flask with medium and diesel fuel, but without bacterial suspension (to determine the total (natural) losses). The experience carried out in three replications. Bulb exhibit in katalozhnyh conditions, at a temperature of +20°C for 7 days.

The oil content in the environment is determined with the aid of concentrator "TSC-025" (10. FR. 1.31.2007.03234).

The results in table 5 show that in water with mineral components at a temperature of +20°C the degradation of petroleum products when making a bacterial suspension of a strain of Bacillus atropheus VKPM B-10592 was 55,86% for 7 days.

Tab the Itza 5
The degree of utilization of diesel fuel in saline aqueous medium under the influence of the strain Bacillus atropheus VKPM B-10592
t°C +To experience mgAfter 7 days mgDestruction, %
201000,0558,6455,86
Control1000,01000,00,0

Thus, by the high degree of purification of frozen soils and water from oil and oil products offered by the method.

The advantage of the strain Bacillus atropheus VKPM B-10592 is that it has a high scavenging ability towards both oil and oil products (diesel fuel) in a relatively short time (from 7 days to 2 months) in a wide temperature range from +8 to +30°C, which can be used to clean soils and clean water environment contaminated by oil and oil products (diesel fuel).

The proposed method can be used for the rehabilitation of contaminated territories in the Far North.

The method of purification of permafrost soils and the aquatic environment from n the PTI and petroleum products (diesel fuel), characterized in that the cleaning of contaminated substrates using a bacterial suspension of a strain of Bacillus atrophaeus VKPM B-10592.



 

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