RU2513699C1 - Method to clean permafrost soils and water environment by spore-forming bacteria bacillus atrophaeus vkpm v-10592 - Google Patents

Abstract

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.

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Description

The invention relates to environmental protection, in particular to methods for cleaning permafrost soils and the aquatic environment from oil and oil products. To clean the frozen soil and water from oil and oil products, a suspension of the bacterial strain Bacillus atropheus VKPM B-10592 is added to the contaminated substrates. The invention allows for a short time to achieve a high degree of purification of permafrost soils and the aquatic environment from oil and oil products (diesel fuel).

Level of invention

Known strains of Rhodococcus erythropolis, Rhodococcus luteus, Micrococcus flavus (1. Klushnikova TM, Smirnova GF, Kuberskaya SL, Slabospitskaya AT, Zalevsky BC, Kasatkina TP, Eliseeva GS Method of cleaning of shale wastewater from petroleum products. A.S. SU No. 1493666. Application No. 4280617 from 07/09/1987. Publish. 07/15/1989) and Flavobacterium tirrennicum (2. Novozhilova MI, Popova LE, Nedovodieva TP ., Kalamkarova S.I. Bacterial strain Flavobacterium tirrennicum. A.S. SU No. 1409594. Application No. 4066152 of 07.15.1986. Publish. 07.15.1988) used for purification of oil and oil products from bilge and industrial wastewater.

In the first invention, improving the quality of water purification from oil products is carried out by introducing microorganisms with high oil-oxidizing activity and good physiological compatibility into structures with activated sludge. The method consists in using a mixture of specially selected bacterial strains RHODOCOCCUS ERYTHROPOLIS VKPM B-3078 (36a-1), RHODOCOCCUS LUTEUS VKPM B-3077 (25) and MICROCOCCUS FLAVUS VKPM B-3079 (53) in the following crop ratio as an association : (0.5-1.5) :( 0.5-1.5) :( 0.5-1.5).

In the second case, the bacterial strain Flavobacterium tirrenicum VKPM B-3369 is used to purify industrial waters contaminated with oil, which is able to decompose oil of various compositions more actively than known strains. With a content of 4 g of oil in 1 liter of medium under aeration conditions after 24 hours from the moment of administration, the strain decomposes 81.1% paraffinic, 40% methane-naphthenic-aromatic and 80.3% methane-naphthenic oil.

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 of Pseudomonas putida 36 (3. Dyadechko V.N., Tolstokorova L.E., Morozova T.N. The strain Pseudomonas putida 36 used to clean water and soil from oil and oil products. USSR AS No. 1076446. Application No. 3474745 from 07/22/1982, publ. 02/28/1984); Acinetronacter sp.NV-1 (4. Zhirkova N.A., Kobelev BC, Kholodenko V.P. ACINETOBACTER SPECIES STRAIN USED FOR WATER AND SOIL PURIFICATION FROM OIL AND OIL PRODUCTS. Patent RU No. 20740431. 1994. Published on 04/20/1997); Acinetronacter sp.JN-2 (5. Ushkov SB., Ilyinskaya N.V. ACINETOBACTER SPECIES STRAIN USED FOR WATER AND SOIL PURIFICATION FROM OIL AND OIL PRODUCTS. Patent RU No. 2064017. Application No. 93009595 from 23.02.1993. 1996); Pseudomonas alcaligenes E7 (6. Patent RU No. 2134723, 1999) and Pseudomonas alcaligenes B-1 (7. Ermolenko Z.M., Kholodenko V.P., Chugunov V.A., Kobelev BC, Akimova N.A. STRAIN OF BACTERIA PSEUDOMONAS ALCALIGENES B-1 USED FOR WATER AND SOIL PURIFICATION FROM OIL AND OIL PRODUCTS Patent RU No. 2133770. Application No. 98105709 dated 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.I., Ilyinsky V.V. METHOD FOR CLEANING SOILS FROM OIL CONTAMINATIONS showed great efficiency at low positive temperatures. Patent RU 2019527 Application 93017464 dated 04/30/1993, published on 09/15/1994), but the process of oil biodegradation using this strain proceeds slowly and only with the addition of nitroammophos or nitroammophos and sodium nitrate is accelerated.

Close essentially to the claimed method is a method of cleaning permafrost soils from oil with spore-forming bacteria Bacillus subtilis (9. Neustroev MP, Tarabukina NP, Neustroev MM, Sazonov NN, Parnikova SI, Stepanova AM METHOD FOR CLEANING FROZEN SOILS FROM OIL BY SPORING FORMING BACTERIA BACILLUS SUBTILIS Patent RU 2446900. Application No. 2010 dated 07/13/2010. Published on 04/10/2012) with the ability to reduce the oil concentration to 0.48% over a 3-year period. 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 the aquatic environment from oil and dispose of oil products (diesel fuel), including in a wide temperature range from +8 to + 37 ° C.

The implementation of the invention

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

The solution to this problem is achieved by introducing into the frozen soil and water environment of a microbial preparation contaminated with oil or oil products (diesel fuel) 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 a content of living cells in a concentration of at least 1 · 10 ⁹ CFU / ml.

The bacterial strain Bacillus atropheus VKPM B-10592 was deposited in the All-Russian collection of non-pathogenic industrial microorganisms FSUE GosNII Genetika - VKPM, 117545, Moscow, 1st Dorozhniy passage, 1. Collection number VKPM B-10592. The bacterial strain is promising for the restoration of oil-contaminated permafrost soils and purification of the aquatic environment from contamination by oil and oil products (diesel fuel).

A 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), 677890, Republic of Sakha (Yakutia), Yakutsk, ul. Oktyabrskaya, house 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 is grown on meat peptone agar (wt.%: Enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2 ) at a temperature of + 30 ° C for 2 days, then washed in a 0.9% NaCl solution at a concentration of at least 1 · 10 ⁹ CFU / cm ³ according to the optical standard GISK them. AM Tarasevich. The bacterial suspension of the Bacillus atropheus VKPM B-10592 strain thus obtained is used to clean oil and oil products from frozen soils and the aquatic environment.

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

The bacterial suspension of a strain of Bacillus atropheus VKPM B-10592, obtained by the method described in example 1, is introduced into a mineralized aqueous 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 contaminated with oil or oil products in an amount of 1000 mg per 100.0 cm ^{3 of} substrate, and exposed at temperatures of + 8 ° 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 (10. 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 a mineralized aqueous medium, the Bacillus atropheus strain VKPM B-10592 strain utilized 56.4% of the oil after 7 days at a temperature of + 8 ° C; at + 20 ° C - 96.5% of oil; at + 30 ° C - 97.13% of oil (table 1).

Table 1 The degree of utilization of oil and oil products in a mineralized aqueous medium by a strain of Bacillus atropheus VKPM B-10592 after 5 days Experience option t ° C Xenobiotic, mg / dm ³ Term +8 +20 +30 Oil to experience 1000,0 1000,0 1000,0

after experience 405.78 34.32 28.64 % destruction 56.42 96.56 97.13

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

3.0 dm ^{3 of} tap water, 30 g of crude crude oil and 30.0 cm ^{3 of a} bacterial suspension of the Bacillus atropheus VKPM B-10592 strain prepared according to Example 1 are added to a desiccator with a volume of 3.0 dm ^3. 3.0 dm ³ with 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 the destruction of oil in water by a strain of Bacillus atropheus VKPM B-10592 Experience option The amount of oil before introducing the cells of the strain, g The amount of oil 2 months after the introduction of the cells of the strain, g The degree of utilization of oil,% Water + oil + Bacillus atropheus strain VKPM B-10592 thirty 0.4718 98.42 Water + oil thirty thirty 0,0

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

The results showed that in water, the destruction of oil under the influence of a strain of Bacillus atropheus VKPM B-10592 for 2 months amounted to 98.42% (Table 2).

Example 4. Purification of permafrost soil from oil by a strain of Bacillus atropheus VKPM B-10592

An experiment on cleaning permafrost soil contaminated with oil was carried out in an open ecosystem. The temperature of the soil, over the entire period of the experiment, at a depth of 20 cm averaged + 8 ° C; at a depth of 10 cm - + 14 ° C.

The bacterial suspension of the strain Bacillus atropheus VKPM B-10592, obtained according to example 1, is applied to the soil contaminated with oil, at the rate of 1 liter of suspension per 1 m ^{2 of} oil-contaminated soil. The soil is thoroughly mixed with a shovel and exposed for 60 days.

The oil content in the soil is determined in accordance with RD 52.18.647-2003. (11. RD 39-0147098-90 Instructions for monitoring the state of soils at facilities of enterprises of the Ministry of Oil and Gas and Gas Industry).

Table 3 Dynamics of oil degradation in soil treated with a preparation based on the Bacillus atropheus strain VKPM B-10592 Experience option The oil content before the introduction of the strain, mg / kg The oil content, 60 days after the introduction of the strain, mg / kg The destruction of oil,% Soil + oil + Bacillus atropheus strain VKPM B-10592 186079 80343 56.82 Soil + oil 5960 5561 6.69

The research results show that for 2 months, at an average soil temperature of +8 + 14 ° C, the degradation of crude oil under the influence of the Bacillus atropheus VKPM B-10592 strain amounted to 56.82%, while the natural loss of oil in permafrost soil not treated with the drug, amounted to 6.69% (table 3).

Example 5. Purification of permafrost soil from diesel fuel by a strain of Bacillus atropheus VKPM B-10592

An experiment to clean permafrost soil contaminated with diesel fuel was conducted in an open ecosystem. The temperature of the soil, over the entire period of the experiment, at a depth of 20 cm averaged + 8 ° C; at a depth of 10 cm - + 14 ° C. Oil penetration depth up to 15 cm.

The bacterial suspension of the strain Bacillus atropheus VKPM B-10592, obtained according to example 1, is applied to the soil contaminated with diesel fuel, at the rate of 1 liter of the preparation per 1 m oil-contaminated soil with an oil penetration depth of 15 cm. The soil is thoroughly mixed with a shovel and exposed to within 45 days.

The oil content in the soil is determined in accordance with RD 39-0147098-90 (11. RD 39-0147098-90 Instructions for monitoring the condition of soils at the facilities of enterprises of the Ministry of Oil and Gas Industry).

Table 4 The destruction of diesel fuel after cleaning the soil with a strain of Bacillus atropheus VKPM B-10592 Experience outline pH Humidity% The content of NP, mg / kg Destruction of NP after 45 days,% before cleaning after cleaning Soil + Diesel 6.99 49.5 25823 21247 17.7 Soil + diesel fuel + Bacillus atropheus strain VKPM B-10592 7.01 46.8 23420 2450 89.53

The research results showed that after 45 days the pollution in the soil of the cleaned object decreased by 9.5 times, the destruction of diesel fuel amounted to 89.53% (Table 4).

Example 6. Purification of the mineralized aqueous medium from diesel fuel by a strain of Bacillus atropheus VKPM B-10592

100.0 cm ^{3 of} sterile tap water with mineral components of the following composition (wt.%) Are poured into cones with a volume of 200.0 cm ³ : 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; make a bacterial suspension of a strain of Bacillus atropheus VKPM B-10592, obtained according to example 1, in the amount of 10-15% of the volume of water and add 1% of 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 experiment is carried out in triplicate. The flasks are exposed in rocking conditions, at a temperature of + 20 ° C for 7 days.

The oil content in the medium is determined using a concentrator "IKN-025" (10. FR. 1.31.2007.03234).

The results shown in Table 5 show that in water with mineral components at a temperature of + 20 ° C, the degradation of petroleum products upon application of a bacterial suspension of the Bacillus atropheus strain VKPM B-10592 was 55.86% over 7 days.

Table 5 The degree of utilization of diesel fuel in a mineralized aqueous medium under the influence of a strain of Bacillus atropheus VKPM B-10592 t ° C, + Before the experiment, mg After 7 days, mg Destruction,% twenty 1000,0 558.64 55.86 The control 1000,0 1000,0 0,0

Thus, a high degree of purification of permafrost soils and water from oil and oil products of the proposed method is confirmed.

The advantage of the strain Bacillus atropheus VKPM B-10592 is that it has a high utilizing ability in relation to 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 both for cleaning soil and for cleaning water contaminated with oil and oil products (diesel fuel).

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

Claims (1)

The method of cleaning permafrost soil and water from oil and oil products (diesel fuel), characterized in that for the treatment of contaminated substrates use a bacterial suspension of a strain of Bacillus atrophaeus VKPM B-10592.