RU2763428C1 - Preparation for biodegradation of oil and oil products (oil destructor) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. The proposed drug for biodegradation of oil and oil products (oil destructor), including the association of bacteria: Bacillus atrophaeus VKM B-81, Pseudomonas spp. VKM B-892, Pseudomonas putida VKM B-1301, Arthrobacter sp. Ac-950, Microbacterium flavescens VKM Ac-1415, Bacillus megaterium VKM B-112 in a given ratio.

EFFECT: invention improves the efficiency of cleaning the soil and water area at a temperature range from -10°C to +40°C, as well as the possibility of utilizing the hydrocarbon phase with a high concentration of salts in the soil.

16 cl, 2 tbl, 4 ex

Description

The invention relates to biotechnology and ecology, namely, to compositions for cleaning soil and water bodies contaminated with oil and polycyclic aromatic hydrocarbons while restoring the physicochemical properties and natural biocenosis of soils and water areas.

The oil destructor is intended for the neutralization of oil-containing wastes and withdrawn oil-contaminated soils that have passed the cleaning stage by mechanical, physical or physico-chemical methods; localization and liquidation of spills of oil and oil products; recultivation of oil-contaminated lands; cleaning water bodies from oil film.

The use of biopreparations-oil destructors, which include strains of hydrocarbon-oxidizing microorganisms, is the most environmentally friendly way to clean and restore oil-contaminated media due to the formation of non-toxic products in the process of biodegradation of the hydrocarbon phase. Currently, a large number of biopreparations-oil destructors are known.

From the document RU 2681831 (IPC C12N 1/20, B09C 1/10, C02F 3/34, C12R 1/01, published on March 12, 2019), a preparation for the biodegradation of petroleum products is known, including the association of bacteria Bacillus megaterium VKPM B-607, Bacillus subtilis VKPM B-5328, Pseudomonas putida VKPM B-5624, Rhodococcus erythropolis VKPM AC-1269 immobilized on a glauconite-containing carrier in the amount of 10 ⁷ - 10 ¹⁰ cells/g.

From the document RU 2668789 (IPC C12N 1/26, B09C 1/10, C02F 3/34, C12R 1/01, C12R 1/06, C12R 1/40, published on 02.10.2018), a biopreparation-oil destructor is known, which is an association of oil-oxidizing soil microorganisms, containing as oil-oxidizing soil microorganisms bacteria of the genus Bacillus atrophaeus ARK-81, Pseudomonas putida ARK-1301, Rhodococcus sp. ARK-66, Artrobacter sp. ARK-950, Bacillus megaterium ARK-396, grown in separate cultivation and mixed to a total bacteria content of 15-20×10 ⁹ cells/ml.

The closest analogue of the invention is the technical solution described in the RF patent RU2516412 (IPC B01J 20/10, B01J 20/283, C02F 3/34, C02F 1/28, B09C 1/10, C12R 1/125, C12R 1/32, C12R 1/38, C12R 1/40, published 05/20/2014). From the prototype, a preparation for cleaning water and soil from oil pollution is known, containing microorganisms - oil decomposers, a sorbent, microfertilizers, and as oil destructors it contains 75-85% of the total number of cells, an association of oil-oxidizing microorganisms: Bacillus subtilis VKM B-81, Pseudomonas spp. VKM V-892, Pseudomonas putida VKM V-1301, Rhodococcus sp. VKM Ac-950, Mycobacterium flavescens VKM Ac-1415, as well as soil bacteria Agrobacteium radiobacter VKM B-1219 - 15-25% of the total number of cells, cryoprotectant - glycerol, sorbent, which is a fine dehydrated zeolite with a granule size of 0.1- 0.5 mm, powdered with Aerosil A-300 nanoparticles, microfertilizers - sodium nitrate 0.5% and potassium phosphate 0.5%, at a certain ratio of components.

Based on the prior art, the technical problem is to create an oil destructor drug that can actively and efficiently work in a wide humidity-temperature range, which will make it possible to use it in different climatic zones.

The technical result consists in increasing the efficiency of the consortium when used in a wider humidity-temperature range from -10°C to +40°C, including those with significant daily temperature differences, as well as in the possibility of utilizing the hydrocarbon phase at a high concentration of salts in the soil . The consortium has a high adaptability to extreme environmental conditions.

When carrying out the reclamation of oil-contaminated lands, this preparation can be used with humates, which improves the quality of the soil after cleaning.

To achieve the specified technical result, the preparation for the biodegradation of oil and oil products (oil destructor), including the association of bacteria: Bacillus atrophaeus VKM B-81, Pseudomonas spp. VKM B-892, Pseudomonas putida VKM B-1301, Arthrobacter sp. Ac-950, Microbacterium flavescens VKM Ac-1415, contains the bacterial strain Bacillus megaterium VKM B-112. The total content of bacterial cells in the suspension is not less than 1×10 ⁹ CFU/ml.

In a particular case, the preparation for the biodegradation of oil and oil products may additionally contain the bacteria Agrobacterium radiobacter VKM B-1219 and/or the bacteria Pseudomonas turukhanskensis VKM B-2935.

An additional bacterial strain Pseudomonas turukhanskensis VKM B-2935 enhances the phenol-oxidizing ability and also works at low ambient temperatures, which gives the prospect of its use in regions with a short thermal period.

In a particular case, the preparation for the biodegradation of petroleum products may additionally contain a cryoprotectant - glycerin, sodium nitrate and/or potassium phosphate. Moreover, sodium nitrate is contained in an amount of 0.5% wt., potassium phosphate contains 0.5% wt.

The biodegradable preparation may be in the form of an aqueous suspension with a cell titer of at least 1×10 ⁹ CFU/ml, preferably (1-3)×10 ⁹ CFU/ml.

The preparation for biodegradation can be in the form of a freeze-dried biomass of strains with a cell titer of the dehydrated product of at least 1×10 ¹¹ CFU/g, preferably (1-8)×10 ¹¹ CFU/g.

The preparation for biodegradation can be in the form of a dry form immobilized on a sorbent. As a sorbent, zeolites, or glauconites, or crushed remains of wood or peat are used.

The drug on the sorbent can be obtained by contact-sorption dehydration by immobilizing a concentrated suspension of the drug on the sorbent in a screw mixer, in a ratio of 1:10.

The preparation for the biodegradation of petroleum products may additionally contain a soil structurant, which can be used as humic substances consisting of humic acids and/or their salts.

In the preparation, bacterial strains Bacillus atrophaeus VKM B-81, Pseudomonas spp. VKM B-892, Pseudomonas putida VKM B-1301, Arthrobacter sp. Ac-950, Microbacterium flavescens VKM Ac-1415, Bacillus megaterium VKM B-112 can be in the following ratios 3:1:1:1:1:3; 2:1:1:1:1:2 or in equal proportions. In this case, the deviation from the indicated ratios up to 10% is included in the indicated ratios.

The share of cells of individual bacteria of each strain in the association is 8-15%.

As you know, phosphorus and nitrogen are vital for bacteria to grow and develop. The technical result is achieved through the use of the bacterial strain Bacillus megaterium VKM B-112. The bacterial strain Bacillus megaterium VKM B-112 is not only oil-oxidizing, but also capable of leaching (dissolving, releasing) phosphorus from lithosphere objects, making it available to plants and other bacteria. This strain of Bacillus megaterium VKM B-112 is spore-forming and is able to survive in harsh climatic conditions, as well as at large temperature fluctuations. Another soil strain of Agrobacterium radiobacter is a nitrogen fixing bacterium. These strains together are good soil structurers and help other bacteria grow and develop better in oil-contaminated soil.

The association of microorganisms can be selected so that different strains complement each other, which ensures the effectiveness of their use in a wider humidity-temperature range from -10°C to +40°C, including those with significant daily temperature differences.

Materials explaining the essence of the invention:

Table 1 - Residual content of oil products in the soil after treatment with the drug.

Table 2 - Results of laboratory studies.

The drug is obtained as follows. Microorganisms of the biological product Bacillus atrophaeus VKM B-81, Pseudomonas spp. VKM B-892, Pseudomonas putida VKM B-1301, Arthrobacter sp. Ac-950, Microbacterium flavescens VKM Ac-1415, Bacillus megaterium VKM B-112 are grown separately in a bioreactor by known methods, then a microbial association is prepared by mixing the calculated proportions of culture liquids in one container.

The preparation can be in the form of a homogeneous liquid suspension, a dry form on a sorbent, and a sublimated powder.

The number of cells of microorganisms, depending on the aggregate form of the drug: a homogeneous liquid suspension - (1-3)×10 ⁹ CFU/ml; sublimated form - (1-8)×10 ¹¹ CFU/g; dry form on the sorbent - (5-7)×10 ⁹ CFU/g.

It is possible to adapt the consortium to a specific region by introducing native microorganisms isolated from soil samples of an oil-contaminated area where reclamation is planned to be carried out into the main composition.

The finished preparation in the form of a homogeneous liquid suspension and sublimated powder in the activated state is a liquid from light brown and yellow to deep dark brown in color with a specific odor characteristic of this product.

The invention is confirmed by the following examples:

Example 1. The drug is obtained as follows:

Microorganisms of the biological product are cultivated separately in industrial fermenters for 70-300 liters. Glucose-peptone-yeast medium is used as a nutrient medium. To adapt bacteria to the destruction of hydrocarbons, 0.5 l of diesel fuel is added at the final stage of cultivation. The fermenter and nutrient medium are sterilized at 120°C for 30 minutes. The pH is maintained at 7.2 with ammonia water.

The cultivation of microbial cultures in the fermenter is carried out at a temperature of 32+1°C with a constant supply of air for aeration and rotation of the stirrer 250 rpm ^-1 for 35+2 h. rotameter, pressure-vacuum meter and balanced bridge. Cultivation is stopped when the pH reaches (7.5±0.5) units. pH.

To prepare the liquid form of the drug, all microorganisms are mixed in a separate container in the required ratios. The total amount is controlled by the method of seeding on a dense nutrient medium (GRM-agar). The number of living cells is (1-3)×10 ⁹ CFU/ml.

The necessary additives can be added to the finished mixture: sodium nitrate and potassium phosphate, as well as glycerin. The liquid form of the drug is packaged in polyethylene containers with a breathing valve with a volume of 1 to 25 liters.

The dry form of the drug is obtained by contact-sorption dehydration with immobilization of a concentrated suspension on structured prepared sorbents with a porous structure in a screw mixer. Sorbents are pre-calcined at a temperature of 350-400°C. The number of cells in the form on the sorbent - (5-7)×10 ⁹ CFU/g. With this technology of preparation of the drug, the moisture content of the product is 8-10%.

The sublimated form is produced on an industrial sublimation drying unit TG-50. The number of cells in the finished product is at least 1×10 ¹¹ CFU/g.

The form, brand and type of drugs used vary depending on the aggregate form of the contaminated environment, the remoteness of the region, the type and degree of pollution.

Example 2

Approbation of the Oil Destructor was carried out in an area with a short growing season. In the course of the tests, the combined use of the drug and the soil structurer - the humic-mineral complex "Bio-GMK" was investigated. The venue was a territory with an anthropogenic relief on the territory of one of the fields located on the territory of the Khanty-Mansiysk Okrug - Yugra, on the surface of which oil pollution and spills were noted, as well as disturbed oil-contaminated lands with a high salt content. An area of 900 m ² was allocated for the events.

The activation of the liquid concentrated form of the Oil Destructor took place within 48 hours in a two-cube container using local swamp water, which includes native oil-oxidizing microorganisms. Continuous aeration was maintained with a compressor and water temperature at 24-27°C. Ammonium nitrate (3 kg), monopotassium phosphate (2 kg) and Azofoska (3 kg) were used as mineral nutrition elements. Sugar in the amount of 10 kg was introduced as a carbon source.

The biological stage of reclamation included the introduction of the Oil Destructor and the Bio-GMK soil structurizer to the characteristic contaminated areas and the subsequent sowing of the phyto-reference mixture.

The results of laboratory studies on residual oil content are presented in table 1.

In addition to monitoring the dynamics in two marked areas, a third object was selected (the entrance to 1 area), which has a high concentration of hydrocarbons, which was also processed by the Oil Destructor.

The tests carried out have proved the effectiveness of the use of the biological product Neftedestruktor on oil-contaminated and disturbed lands, as well as the synergistic effect when it is used together with the soil structurator "Bio-GMK". The value of the oil content index on oil-contaminated and disturbed lands was achieved at a level below the established standards for the territories of the Khanty-Mansiysk Autonomous Okrug - Yugra, and a stable vegetation cover was also provided. It has been proven that the consortium, which is part of the consortium, is able to utilize the hydrocarbon phase at a high concentration of salts in the soil, which confirms its adaptability to extreme environmental conditions.

Analysis of the experimental data obtained at one of the operating fields of the Khanty-Mansiysk Autonomous Okrug - Yugra showed a high oil-oxidizing ability of the Oil Destructor (more than 85% of hydrocarbon destruction), which gives the prospect of its further use in Western Siberia.

Example 3

The test site was a territory with an anthropogenic relief on the landfill site, on the surface of which waste dumps were noted and the presence of oil products in the soil was determined (a land plot occupied by an industrial and domestic waste dump located behind the Krasnaya Etna cemetery on the territory of the Shuvalov industrial zone in the Leninsky district city of Nizhny Novgorod).

The territory of the Shuvalovskaya industrial zone has been used for more than 20 years as a landfill for solid municipal and construction waste. Over the past few years, there have been cases of focal combustion in this area.

It was decided to recultivate this site and further use it for the needs of the city. After the waste was removed from the landfill, it was necessary to carry out a set of reclamation measures to reduce the negative impact on the environment, as well as improve the landscape of the territory and prepare it for possible construction.

Taking into account the presence of oil-contaminated soils on the territory, it was proposed to use biological methods for the destruction of hydrocarbons using oil-degrading microorganisms.

At the Shuvalovskaya industrial zone, a test site with insulation was equipped and provided for pilot testing.

The sequence of pilot tests:

1) Activation of the Biological product in dry form on a sorbent for 3 hours (aerating with a compressor) with the addition of fertilizers;

2) Preparation of a solution of the humic-mineral complex "Bio-GMK", 1%;

3) Applying the required amount of the drug to each site.

Samples were taken on the day of the experiment from sites without drugs and on the day of the end of the experiment (after 45 days) from all sites.

During the entire period of pilot testing, work was carried out on loosening (aerating) and watering (if necessary) the soil for better development and vital activity of oil-oxidizing microorganisms.

Table 2 presents the results of studies obtained with the help of an accredited laboratory of industrial and environmental toxicology of the Department of Biological Research of the Research Institute of Chemistry of the UNN. N.I. Lobachevsky.

When treating oil-contaminated soil with various concentrations of oil products (from 10% to 15%) with the Oil Destructor in combination with the humic-mineral complex, a decrease in the concentration of the toxicant in the soil by 54-76% in 45 days is noted, which indicates the effectiveness of the combination of drugs used and the stimulating effect of humates during the decomposition of hydrocarbons.

Laboratory analysis for the content of mobile forms of heavy metals in the soil showed that their amount in the treated soil decreased significantly, which indicates the ability of humic substances to immobilize superecotoxicants.

The content of benzo(a)pyrene, a carcinogen, in the soil significantly decreased after the treatment of oil-contaminated soil with a Biological product in combination with an organomineral complex.

Example 4

In November 2019, the Oil Destructor was tested in the Voronovskoye settlement on the territory of NPO Volga-Ecology in the Troitsky district of Moscow. It was planned to lay the experiment for the winter and take samples in the spring of 2020 to test the effectiveness of the biological product in the off-season period, as well as the viability of microorganisms at low temperatures.

In preparation for the pilot tests, a research site was equipped.

The procedure for conducting pilot tests:

1) Activation of the sublimated form of the Oil Destructor for 1 hour (aerating with a compressor) with the addition of fertilizers;

2) Creation of model pollution at the sites (diesel fuel + fuel oil = 1:2 as a pollutant)

3) Leveling of contaminated soil;

4) Applying the required amount of the drug to each site.

In the laboratory of the Russian State University of Oil and Gas. THEM. Gubkin, studies were carried out on the residual content of oil products in the soil by the gravimetric method using an extractor.

When using the Oil Destructor, a decrease in model pollution was noted from 14.8 to 2.9%, which is more than 80% of the efficiency.

Pilot tests of the drug were carried out to determine the degrading ability, economic feasibility and reliability of the use of a biological product to reduce the time of land reclamation and reduce the cost of environmental restoration measures. The drug was tested at eight sites in different regions of Russia and showed high efficiency. Industrial tests at oil production facilities demonstrated the efficiency of the technology - within 72 hours, the degree of purification reached 94%.

The scope of the drug is great - from the disinfection of oil-contaminated natural reservoirs and soils to the neutralization of chemical industry waste. The oil destructor is able to work actively and efficiently in a wide humidity-temperature range, which allows it to be used in different regions of the world.

Table 1 - Residual content of oil products in the soil

Sample name Residual content of oil products in the soil, g/kg The effect, % Staging of the OPI 10 days 30 days 45 days Oil-contaminated area (1) 218.6 57.1 35.8 32.2 85.27 Oil-contaminated area with high salt content 92.2 42.2 21.4 12.1 86.88 Lot 1 entrance 415.4 351.3 242.4 55.5 86.64

Table 2 - Results of laboratory studies

Input control Output control sample number Content of oil products, mg/kg The result of biotesting and phytotesting sample number Content of oil products, mg/kg The result of biotesting and phytotesting 1.1* 24150 - Does not have acute and chronic toxic effects
- "practically harmless"
- belong to the 5th hazard class 1.2 5883 - Does not have acute and chronic toxic effects
- "practically harmless"
- belong to the 5th hazard class 2.1 41859 -Does not have acute and chronic toxic effects
- "practically harmless"
- belong to the 5th hazard class 2.2 18958 - Does not have acute and chronic toxic effects
- "practically harmless"
- belong to the 5th hazard class

* Approx. 1.1, 2.1 - sites untreated with a biological product with a concentration of oil products of 10% and 15%, respectively; 1.2, 2.2 - sites treated by the Oil Destructor and Bio-GMK with a concentration of oil products of 10% and 15%, respectively

Claims (16)

1. Preparation for the biodegradation of oil and oil products, including the association of bacteria: Bacillus atrophaeus VKM B-81, Pseudomonas spp. VKM B-892, Pseudomonas putida VKM B-1301, Arthrobacter sp. Ac-950, Microbacterium flavescens VKM Ac-1415, characterized in that it contains the bacterial strain Bacillus megaterium VKM B-112. 2. Preparation for the biodegradation of oil and oil products according to claim 1, characterized in that it additionally contains the bacteria Agrobacterium radiobacter VKM B-1219. 3. A preparation for the biodegradation of oil and oil products according to claim 1 or 2, characterized in that it additionally contains a bacterial strain Pseudomonas turukhanskensis VKM B-2935. 4. A preparation for the biodegradation of oil and oil products according to claim 1, characterized in that it additionally contains a cryoprotectant - glycerin. 5. A preparation for the biodegradation of oil and oil products according to claim 1, characterized in that it additionally contains sodium nitrate and/or potassium phosphate. 6. The preparation for the biodegradation of oil and oil products according to claim 5, characterized in that it additionally contains sodium nitrate in an amount of 0.5% wt. and/or potassium phosphate 0.5% wt. 7. Preparation for the biodegradation of oil and oil products according to claim 1, characterized in that the total content of bacterial cells is not less than 1×10 ⁹ CFU/ml. 8. A preparation for the biodegradation of oil and oil products according to claim 1, characterized in that it is an aqueous suspension with a cell titer of (1-3)×10 ⁹ CFU/ml. 9. Preparation for the biodegradation of oil and oil products according to claim 1, characterized in that it is made immobilized on a sorbent, with the number of cells (5-7)×10 ⁹ CFU/g. 10. A preparation for the biodegradation of oil and oil products according to claim 9, characterized in that zeolites or glauconites or crushed wood residues or peat are used as a sorbent. 11. The drug for the biodegradation of oil and oil products according to claim 9, characterized in that the drug on the sorbent is obtained by contact-sorption dehydration by immobilizing a concentrated suspension of the drug on the sorbent in a screw mixer. 12. A preparation for the biodegradation of oil and oil products according to claim 1, characterized in that it is a sublimated form with a cell titer of (1-8)×10 ¹¹ CFU/g. 13. A preparation for the biodegradation of oil and oil products according to claim 1, characterized in that it additionally contains a soil structurator. 14. A preparation for the biodegradation of oil and oil products according to claim 13, characterized in that humic substances consisting of humic acids and / or their salts are used as a soil structurer. 15. The preparation for the biodegradation of oil and oil products according to claim 1, characterized in that the strains of bacteria Bacillus atrophaeus VKM B-81, Pseudomonas spp. VKM B-892, Pseudomonas putida VKM B-1301, Arthrobacter sp. Ac-950, Microbacterium flavescens VKM Ac-1415, Bacillus megaterium VKM B-112 in the following ratios 3:1:1:1:1:3 or 2:1:1:1:1:2 or in equal ratios. 16. A preparation for the biodegradation of oil and oil products according to claim 1, characterized in that the association contains at least 10% of bacteria of each strain.