RU2529735C1 - Method of producing biopreparation for cleaning and restoring fertility of soil contaminated with petroleum products - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, particularly to microbiological methods of cleaning the environment. The method includes preparing a mixture of liquid bacterial cultures in the form of Pseudomonas fluorescens VKG RCAM00538 strains with a titre of 10^-13, KOA-4 Pseudomonas fluorescens ND-610 VNIISKHM with a titre of 10^-10, Azotobacter chroococcum AIN RCAM00539 with a titre of 10^-12 in ratio of 3:1.5:0.5-3:2:1, respectively. The method includes treating wastes from beneficiation of brown coal by adding the obtained liquid mixture of strains followed by mixing and drying the prepared mixture in not less than 2.5 days at room temperature, wherein the wastes from beneficiation of brown coal and the prepared mixture of strains are taken in the ratio of 40-50:50-60 wt %, respectively.

EFFECT: invention increases content of humic, carboxylic and amino acids and polysaccharides in the biopreparation at minimal costs and duration of production.

7 tbl, 7 ex

Description

The invention relates to biotechnology, in particular to microbiological methods for cleaning the environment, and can be used for soil formation and restoration of soil fertility, contaminated with oil and oil products.

A variety of biological products based on microorganisms and their associations used to reduce oil and oil products in soils are known. For example, a consortium of strains of microorganisms - destructors is known, consisting of 4 strains of the genus Alcaligenes and 1 strain of the genus Pseudomonas (RF patent No. 2160719, IPC C02F 3/34).

These hydrocarbon destructive consortia are composed of prokaryotic microorganisms, which limits the possibility of their use. Since these microorganisms are characterized by the absence in the cells of a real nucleus with a membrane separating it from protoplasm, which allows these microorganisms to be classified as a primitive group, i.e. the lack of the ability to pass on to acquired generations acquired signs, in this case, decompose oil. (N.F. Reimers, A.V. Yablokov "Dictionary of terms and concepts related to the protection of wildlife", ed. "Science", M., 1982, p. 98, 42).

A known method of obtaining a biological product "Biava" for soil remediation (RF patent No. 2248255, IPC B09C 1/10), which contains a porous carrier containing glassy silicophosphates of variable composition and heterogeneous structure, providing optimal conditions for the immobilization and vital activity of microorganism cells on porous structure, which contains monovalent and 2 - valence alkaline earth cations, alloying trace elements, additives - metal oxides that affect kinetics. As active microorganisms, proteolytic and amylolytic microorganisms, or actinomycetes, or nitrogen-fixing bacteria, or cellulose-degrading and humus-degrading microorganisms, or denitrification are introduced.

The known biological product has a complex production technology due to the fact that it includes the deep cultivation of microorganisms in a liquid nutrient medium, the preparation of a sterile carrier - glassy silicophosphates with through pores for immobilization and cell activity of active microorganisms on a porous structure. This increases the cost of its production and use, which limits its use over large areas.

A known method of obtaining a preparation of humic acids for soil formation. (AS of the Russian Federation No. 1213760 IPC C12N 1/00). The preparation is prepared from coal industry wastes inoculated with microorganisms, followed by cultivation of an aqueous suspension for 10-15 days before the lysis of bacterial cultures. During the preparation of the drug, phenol is introduced into the aqueous suspension twice, which is a source of organic carbon for the life of microorganisms.

However, phenol is also a bactericidal substance, leading to the lysis of microorganisms, i.e. a drug that does not contain active microflora becomes less active.

In this known solution, the long preparation of the preparation (10-15 days), as well as the limited content of live bacterial cells in the preparation in view of their lysis and the addition of phenol to the preparation as a carbon source, indicate the disadvantages of this preparation.

In addition, the preparation of a liquid preparation requires rapid use due to the fact that liquid preparations quickly deteriorate and lose their activity. In this regard, such drugs are used in the same area where they are obtained, because they cannot be transported over long distances.

A known method for producing the drug, which consists in treating coal industry wastes by adding water to them and inoculating the resulting solution with cultures of Pseudomonas aeruginosa and Bacillus megaterium, followed by exposure of the mixture at t 28-30 ° C for 10-12 days (a.s. RF No. 1077277 IPC C12N 1/00), adopted by us for the prototype. The preparation obtained by this method contains an insignificant content of humic acids (57%) and carboxylic acids (11%), while amino acids and polysaccharides are absent.

This method has the same disadvantages as the above, due to the fact that it relates to the preparation of a liquid preparation. In addition, when transported over long distances, it loses activity, which is already not very high, the activity of amino acids in it is also reduced - it is an additional food source for native microflora and does not contain polysaccharides involved in the process of structuring reclaimed soil. Coal industry wastes contain oxides of toxic metals: copper, chromium, zinc, barium, which adversely affect soil microflora and plants grown.

The technical result, the achievement of which the present invention is directed, is to increase the content of humic, carbonic, amino acids and polysaccharides in the biological product at minimal cost and time for its manufacture.

To achieve the technical result in the method of obtaining a biological product for cleaning and restoring the fertility of soil contaminated with oil, based on the filler and bacterial cultures, according to the invention, a mixture of liquid bacterial cultures is prepared, which are taken as strains of Pseudomonas fluorescens VKG RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens ND-610 VNIISCHM with a titer of 10 ^-10 , Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 , in a ratio of 3: 1.5: 0.5-3: 2: 1, respectively, and enrichment waste is processed brown coal by adding pouring the obtained liquid mixture of strains to them, followed by stirring and drying the prepared mixture for at least 2.5 days at room temperature, while brown coal enrichment waste and the previously prepared mixture of strains are taken in the corresponding ratio, wt.%: 40-50: 50-60.

The strain Pseudomonas fluorescens VKG was deposited in the collection of the State Scientific Research Institute of Agricultural Microbiology (St. Petersburg) July 05, 2011 under registration number RCAM00538,

The strain KOA-4 Pseudomonas fluorescens was deposited in the collection of the State Scientific Research Institute of Agricultural Microbiology (St. Petersburg) on August 2, 2002 under the registration number "ND-610 VNIISKHM".

The strain Azotobacter chroococcum AIN was deposited in the collection of the State Scientific Research Institute of Agricultural Microbiology (St. Petersburg) on July 05, 2011 under registration number RCAM00539.

The claimed combination of essential features makes it possible to increase the content of humic, carboxylic, amino acids and polysaccharides in the biological product at minimal cost and time for its manufacture. The use of brown coal enrichment waste eliminates the influence of harmful trace elements on the process of cleaning and restoring soil fertility.

The method is as follows.

Example 1. To obtain a biological product, brown coal enrichment waste containing oxides of the following composition was taken (%): manganese (IV) - 0.01; iron (III) - 0.82; silicon (IV) - 13.8; aluminum - 4.01; calcium - 0.19; magnesium - 0.10; phosphorus (V) - 0.03; potassium - 0.06; sodium - 0.02; sulfur - 0.36. The mass fraction of moisture in the waste and the mass loss during calcination were, respectively, (%): 73.17 and 5.93.

Brown coal enrichment waste of this composition is used without crushing, because has a fractional composition of the following content, which significantly reduces the cost of the technology for the preparation of a biological product.

2.5-3.0 mm 1.5-2.0 mm 0.5-1.0 mm 0.2-0.3 mm 0.5% 3.0% 87.0% 9.5%

To obtain the biological product, strains of Pseudomonas fluorescens VKH RCAM00538, KOA-4 Pseudomonas fluorescens ND-610 VNIISM and Azotobacter chroococcum AIN RCAM00539 were taken.

Microorganism strains were previously isolated from oil-contaminated lands of the Perm Territory. Microorganisms are adapted to the chemical composition of oil soils in natural conditions.

The strain Pseudomonas fluorescens VKH RCAM00538 was grown on a nutrient medium of the following composition (g / l):

NH ₄ Cl KH ₂ PO ₄ MgSO ₄ · 7H ₂ O NaCl FeSO ₄ · 7H ₂ O CaCO ₃ Glucose Thymol blue H ₂ O distill. pH of the medium 1,0 2.0 0.5 2.0 traces 0.5 10 0.05 Rest 7.2

1 ml of a sterile mixture of microelements was added to the nutrient medium prepared and sterilized in an autoclave at 1 atmosphere for 30 minutes. A mixture of trace elements contains in 1 liter of water, (g): H ₃ BO ₃ - 5; (NH _{4) 2} MoO ₄ - 5; KJ - 0.5; NaBr - 0.5; ZnSO ₄ · 7H ₂ O — 0.2; Al ₂ (SO ₄ ) ₃ - 0.3. The specified mixture of trace elements is pre-sterilized in an autoclave at a pressure of 0.5 atmospheres for 15 minutes. In a sterile nutrient medium of the above composition, the bacterial strain Pseudomonas fluorescens VKH RCAM00538 was inoculated in an amount of 1 ml with a titer of 10 ^-13 and placed in a thermostat at a temperature of 28 ° C. The strain in the thermostat was aerated using a FAT-mini aerator for 2 days. After 2 days, the bacterial culture of the strain Pseudomonas fluorescens VKH RCAM00538 with a titer of 10 is ready for use. Similarly grown strain with a titer of 10 ^-12 , 10 ^-10 .

The strain KOA-4 Pseudomonas fluorescens ND-610 VNIISCHM was grown on a nutrient medium of the following composition, (g / l):

NH ₄ Cl KH ₂ PO ₄ MgSO ₄ · 7H ₂ O NaCl FeSO ₄ · 7H ₂ O CaCO ₃ Glucose Thymol blue H ₂ O distilled pH of the medium 1,0 0.5 0.5 1,5 0.05 1,0 10 0.005 rest 7.0

1 ml of a sterile mixture of microelements is added to the nutrient medium prepared and sterilized in an autoclave at 1 atmosphere for 30 minutes. A mixture of trace elements contains in 1 liter of water, (g): H ₃ BO ₃ - 5; (NH _{4) 2} MoO ₄ - 5; KJ - 0.5; NaBr - 0.5; ZnSO4 0.2; Al ₂ (SO ₄ ) ₃ - 0.3. The specified mixture of trace elements is pre-sterilized in an autoclave at a pressure of 0.5 atmospheres for 15 minutes. The KOA-4 strain Pseudomonas fluorescens ND-610 VNIISCHM was inoculated into a sterile growth medium of the above composition in an amount of 1 ml with a titer of 10 ^-10 and placed in a thermostat at a temperature of 28 ° C. The strain in the thermostat was aerated using a FAT-mini aerator for 2 days. After 2 days, the bacterial culture of the strain KOA-4 Pseudomonas fluorescens ND-610 VNIISCHM with a titer of 10 ^-10 is ready for use. Similarly grown strain with a titer of 10 ^-11 , 10 ^-12 .

The strain Azotobacter chroococcum AIN RCAM00539 was grown on a nutrient medium of the following composition, (g / l):

CaHPO ₄ KH ₂ PO ₄ MgSO ₄ · 7H ₂ O NaCl FeCl ₃ CaCO ₃ K ₂ SO ₄ Glucose H ₂ O distilled pH of the medium 0.2 0.3 oh s 0.5 0.01 5,0 0.2 20,0 the rest is 7.0

1 ml of a sterile mixture of microelements is added to the nutrient medium prepared and sterilized in an autoclave at 1 atmosphere for 30 minutes. A mixture of trace elements contains in 1 liter of water, (g): H ₃ BO ₃ - 5; (NH _{4) 2} MoO ₄ - 5; KJ - 0.5; NaBr - 0.5; ZnSO ₄ 0.2; Al ₂ (SO ₄ ) ₃ - 0.3. The specified mixture of trace elements is pre-sterilized in an autoclave at a pressure of 0.5 atmospheres for 15 minutes. In a sterile nutrient medium of the above composition, the Azotobacter chroococcum AIN strain RCAM00539 was inoculated in an amount of 1 ml with a titer of 10 ^-12 and placed in a thermostat at a temperature of 28 ° C. The strain culture in the thermostat was aerated with a FAT-mini aerator for 2 days. After 2 days, the bacterial culture of the Azotobacter chroococcum AIN strain RCAM00539 with a titer of ^10-12 is ready for use. Similarly grown strain with a titer of 10 ^-11 , 10 ^-10 .

Example 2 For a biological product obtained by the inventive process taken liquid strains Pseudomonas fluorescens VCG RCAM00538 ^-13 with a titer of 10, KLA-4 Pseudomonas fluorescens ND-610 ARRIAM with a titer of 10 ^-10 and Azotobacter chroococcum ANI RCAM00539 with a titer of 10 ^-12 in a ratio of 3: 1.5: 0.5 and mixed together. The resulting liquid mixture of the above strains was poured into dry brown coal enrichment waste, while the ratio of brown coal enrichment waste and a mixture of Pseudomonas fluorescens strains VKH RCAM00538, KOA-4 Pseudomonas fluorescens ND-610 VNI-ISKhM and Azotobacter chroococcum AIN 50AM50::50 is 50:50: .%. The resulting mixture was stirred, laid out on a horizontal surface and dried at room temperature for 2 days. The dried mixture was triturated with a metal roller with a diameter of approximately 10 cm, so that no large pieces remained, as a result, a biological product with a fractional composition was obtained:

2.5-3.0 mm 1.5-2.0 mm 0.5-1.0 mm 0.2-0.3 mm 0.1% 0.8% 98.0% 1.1%

containing strains of Pseudomonas fluorescens VKH RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens ND-610 VNIISCH with a titer of 10 ^-10 , Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 . In the obtained biological product, the amount of humic, carboxylic, amino acids, polysaccharides and biomass by dry weight were determined, the content of which was (%): humic acids - 68.5, carboxylic acids - 12.6, amino acids - 2.8, polysaccharides - 3, 1 and the total biomass content is 14.9. These results are presented in table 1.

Example 3. Analogously to example 2, a biological product was prepared by the claimed method, but the ratio of brown coal enrichment waste and liquid culture mixture of Pseudomonas fluorescens VKH strains RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens ND-610 VNIISM with a titer of 10 ^-10 , Azotobacter A chrooccum RCAM00539 with a titer of 10 ^-12 was 45:55 wt.%, Respectively. Drying of the prepared mixture was carried out at room temperature for 2.5 days. To prepare a liquid mixture of cultures of strains of Pseudomonas fluorescens VKH RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens ND-610 VNIISCH with a titer of 10 ^-10 , Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 they were taken, respectively, in a ratio of 3: 2: ,5.

In the obtained biological product, the amount of humic, carboxylic, amino acids, polysaccharides and biomass by dry weight were determined, the content of which was (%): humic acids - 72.7, carboxylic acids - 15.3, amino acids - 3.8, polysaccharides - 3, 7 and the content of the total biomass of the strains is 16.1. These results are presented in table 1.

Example 4. Analogously to example 2, a biological product was prepared by the claimed method, but the ratio of brown coal enrichment waste and liquid culture mixture of Pseudomonas fluorescens VKH strains RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens ND-610 VNIISKhM with a titer of 10 ^-10 , Azotobacter A chrooccum RCAM00539 with a titer of 10 ^-12 was 40:60 in mass%, respectively. Drying of the prepared mixture was carried out at room temperature for 3 days. To prepare a liquid mixture of cultures of strains of Pseudomonas fluorescens VKH RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens ND-610 VNIISCH with a titer of 10 ^-10 , Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 they were taken, respectively, in a ratio of 3: 2: .

In the obtained biological product, the amount of humic, carboxylic, amino acids, polysaccharides and biomass by dry weight were determined, the content of which was (%): humic acids - 72.6, carboxylic acids - 15.3, amino acids - 3.6, polysaccharides - 3, 6 and the total biomass content of the strains is 16.0. These results are presented in table 1.

Example 5. Analogously to example 2, a biological product was prepared by the claimed method, but the ratio of brown coal enrichment waste and a mixture of liquid culture of Pseu-domonas fluorescens VKH strains RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens ND-610 VNIISCH with a titer of 10 ^-10 , Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 was 55:45 mass%, respectively. Moreover, to prepare a liquid mixture of the culture of the strains, they are taken in a ratio of 3: 1.0: 0.5, respectively.

Drying the waste with a mixture with liquid strains was carried out at room temperature for 3 days. In the obtained biological product, the amount of humic, carboxylic, amino acids, polysaccharides and biomass by dry weight were determined, the content of which was: humic acids - 67.7%, carboxylic acids - 10.8%, amino acids - 1.9%, polysaccharides - 2.8 % and the content of the total biomass of the strains is 11.5.0%. These results are presented in table 1.

In the process of obtaining a biological product by the claimed method, the drying time of the preparation was determined experimentally depending on its moisture content: a preparation containing brown coal sludge in an amount of 50% and a liquid mixture of strains - 50% (Pseudomonas fluorescens VKG RCAM00538, KOA-4 Pseudomonas fluorescens ND-610 VNIISKHM and Azotobacter chroococcum AIN RCAM00539), at the beginning of drying it had a humidity of 60% and dried out for 2 days. A preparation containing 45% sludge and a liquid mixture of strains - 55%, dried in 2.5 days at a moisture content of 68%, and a preparation containing sludge in an amount of 40% and the rest - a liquid mixture of strains of 60%, had a moisture content of 70% and dried in within 3 days.

Example 6 As a control, compared biological product obtained by the inventive process, based on the maximum content of humic, carboxylic, amino acids, polysaccharides and biomass Pseudomonas fluorescens VCG with a titer of 10 ^-13, KLA-4 ^-10 10 Pseudomonas fluorescens and Azotobacter chroococcum ANI RCAM00539 10 ^-10 in the obtained variants containing brown coal waste and a mixture of strains in the ratios of 45% -55%, 50% -50% and 40-60%, with the preparation obtained in a known manner. The results are presented in table 2.

In the best case scenario, at a ratio of 45: 55% of brown coal enrichment waste and a mixture of Pseudomonas fluorescens strains VKH with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens 10 and Azotobacter chroococcum AIN RCAM00539 10 in a biological product obtained by the claimed method, in comparison with the analogue, the composition of carbon and amino acids in biological products are presented in table 3.

Table 3 Biological product Production-valuable properties of drugs humic acids,% carboxylic acids,% amino acids,% polysaccharides,% microorganism biomass, mg / kg Proposed 72.7 15.3 3.8 3,7 16.1 Famous 57 11.97 - 14.0

The tests showed that in the biological product obtained by the claimed method, the content was, (%): humic acids in an amount of 72.7; carbon - 15.3; amino acids - 3.8 and polysaccharides - 3.7, with a biomass content (mg / kg) of 16.1.

In the known preparation contains only humic and carboxylic acids, (%) - 57 and 11.97, respectively. And the biomass content is 14.0 mg / kg. The drug does not contain amino acids and polysaccharides.

The quantitative content of carboxylic and amino acids in biological products is presented in table 4.

Table 4 Biological product Carboxylic acids Amino acids vinegar propionic oil valerianova Total aspartic glutamine Total Proposed 2.2 1.7 9.6 1.8 15.3 2D 1.7 3.8 Famous 1.88 0.10 8.42 1.44 11.97 - - -

As you can see, the biological product obtained by the claimed method contains significantly more carboxylic acids, such as acetic acid - 2.2%, in the known -1.88%; propionic - 1.7%, in the known 0.1%; Amino acids, such as: aspartic - 2.1%; glutamine - 1.7%, in the well-known preparation, these components are absent.

As can be seen in the biological product obtained by the claimed method, the content of humic, carboxylic, amino acids and polysaccharides is much higher at minimum cost and time for its manufacture.

Upon receipt of a biological product, by the claimed method, with the content of brown coal enrichment waste and a mixture of strains 50% and 50%; 45% and 55% experiments were conducted to determine the activity of titers of strains containing Pseudomonas fluorescens VKH RCAM00538 with a titer of 10 ^-13 , 10 ^-12 and 10 ^-11 , KOA-4 Pseudomonas fluorescens ND-610 VNIISM with a titer of 10 ^-10 , 10 ^{- 9} and 10 ^-8 and Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 , 10 ^-11 and 10 ^{-10, the} data are presented in table 5.

Table 5 Brown coal enrichment waste,% A mixture of strains,% Caption Pseudomonas fluoresce ns VKG Caption KOA-4 Pseudomonas fluoresce ns Title Azotobacter chroococcum ain Humic acids,% Carboxylic acids,% Amino acids,% Polysaccharides,% Biomass of Ps. Fluor escens VKG, KOA-4 Pseudomonas fluoresce ns and Az. chroococcum AIN,% fifty fifty 10 ^{-13 10-10} 10 ^-12 68.5 12.6 2,8 3,1 12,4 10 ^-12 10 ^{-9 10-11} 65,4 9.6 1D 2.7 13.1 ^10-11 10 ^{-8 10-10} 63.6 8.2 0.5 2D 11.6 45 55 10 ^{-13 10-10} 10 ^-12 72.7 15.3 3.8 3,7 16.1 10 ^-12 10 ^{-9 10-11} 66.3 11.1 1.9 3,1 14.8 ^10-11 10 ^{-8 10-10} 65.5 10.9 0.7 2.6 12,2

The results of the experimental verification showed that the best results were in the experiment, which included 45% of brown coal enrichment waste and 55% of a mixture of Pseudomonas fluorescens VKH RCAM00538 strains with titer U-13, KOA-4 Pseudomonas fluorescens. with a titer of 10 ^-10 Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 taken in a ratio of 3: 2: 0.5, respectively, which is seen in the content of humic, carboxylic, amino acids, polysaccharides, and in the biomass of microorganisms, the content of which is respectively % - 72.7; 15.3; 3.8; 3.7; 16.1.

Also, in the optimal variant, containing 45% brown coal sludge, a mixture of strains in an amount of 55%, the titer Pseudomonas fluorescens VKH RCAM00538, KOA-4 Pseudomonas fluorescens ND-610 VNIISM and Azotobacter chroococcum AIN RCAM00539 were tested. As a result of studies, it was found that with a titer of the above microorganisms - 10 ^-13 , 10 ^-10 and 10 ^-12 , optimal options were obtained for the content of humic, carboxylic, amino acids, polysaccharides and biomass with a ratio of strains: 3: 2: 0.5.

When the preparation contains 50% brown coal enrichment waste and 50% culture mixture of strains at titers 10 ^-13 , 10 ^-10 , 10 ^-12, respectively, of the strain Pseudomonas fluorescens VKH, KOA-4 Pseudomonas fluorescens and Azotobacter chroococcum AIN RCAM00539, taken in the ratio 3: 1.5: 0.5 - the amount of humic, carboxylic, amino acids and biomass of microorganisms, respectively, amounted to (%): 68.5; 12.6; 2.8; 3.1; 12.4. In preparations with lower titers, the amount of the above ingredients was significantly less.

Example 7. To study the possibility of using a biological product obtained by the claimed method, tests were carried out in growing vessels in laboratory conditions. The capacity of the vegetation vessels was 3 l, height 25 cm. The vessels were filled with soil contaminated with oil products. The weight of the soil during packing was 2.5 kg. The pH of the soil is 7.2.

The seeds of the cereal plant of wheatgrass were sown in the soil, 30 seeds per vessel. The seeds were treated with a dry biological product obtained by the claimed method, containing strains of Pseudomonas fluorescens VKH RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonas fluorescens with a titer of 10 ^-10 , Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 . Then, the soil treated with the biological product was poured with distilled water in an amount of 100 ml per vessel.

In parallel, experiments were conducted with the humic preparation of the analogue, i.e. at the beginning, grain seeds were sown, after which treatment with a liquid preparation of humic acids was carried out in an amount of 1 ml, diluted to 100 ml with distilled water. The results are presented in table 6.

Table 6 The biological product obtained by the method The amount of humic acids in the preparation,% The amount of carboxylic acids in the preparation,% The number of amino acids,% The amount of polysaccharides,% Height of plants in vegetation vessels, cm The amount of azotobacter in the soil, thousand / g Famous 57.0 11.0 - - 20,0 2,8 Proposed 72.7 15.3 2,8 3.8 48.7 27.4

Table 7 presents the oil content in the vegetation vessels before cleaning and after cleaning using a biological product obtained by the claimed method.

Table 7 The biological product obtained by the method The amount of oil products in the soil,% before cleaning after cleaning in a month after cleaning after 3 months Famous one hundred 75.6 12.01 Proposed one hundred 1,2

As you can see, after a month the amount of oil products with the drug obtained by the proposed method was almost 70 times less.

Tests have shown that optimal results were obtained using a biological product obtained by the proposed method. After 20 days, the height of the plants was 48.7 cm. Using the preparation obtained by the known method, the height of the plants was only 20 cm. The biological product obtained by the claimed method has a stimulating effect on the development of the azotobacter - an indicator on fertility. The amount of azotobacter increased over 20 days by 10 times compared with the content in soils using the preparation of humic acids by a known method.

Thus, the increased content of carboxylic, humic, and amino acids are good stimulants of plant growth and saprophytic microflora, polysaccharides take part in the structuring of soil, which contributes to increased soil fertility.

Therefore, the preparation technology of the claimed method provides, at minimum cost, obtaining the content of humic, carboxylic, amino acids, polysaccharides in a larger amount than the analogue, as well as the biomass of microorganism strains that exceeds the analogue biomass. At the same time, the speed of obtaining the drug is 2-3 times faster compared to the analogue in which the drug was obtained in 10-12 days. In addition, a dry biological product obtained by the claimed method retains its useful properties for one year, and the known liquid only one month.

The proposed drug can be used in the reclamation of disturbed lands in the oil, coal, iron ore and gold mining industries, as well as to increase the fertility of unproductive lands and restore the fertility of football lawns.

Claims (1)

A method of obtaining a biological product for cleaning and restoring fertility of soil contaminated with oil products, based on filler and bacterial cultures, characterized in that a mixture of liquid bacterial cultures is prepared, which are taken as strains of Pseudomonas fluorescens VKH RCAM00538 with a titer of 10 ^-13 , KOA-4 Pseudomonasas fluorescens ND-610 VNIISKhM with a titer of 10 ^-10 , Azotobacter chroococcum AIN RCAM00539 with a titer of 10 ^-12 in a ratio of 3: 1.5: 0.5-3: 2: 1, respectively, and brown coal enrichment waste is processed by adding to them the resulting liquid mixture of strains followed by m stirring and drying the prepared mixture for at least 2.5 days at room temperature, while the waste from brown coal enrichment and the previously prepared mixture of strains are taken respectively in the ratio, wt.%: 40-50: 50-60.