RU2297290C1 - Method of recultivation of the bleaching soil polluted with the oil products - Google Patents

Abstract

FIELD: biotechnology; methods of purification of the oil polluted bleaching soil.

SUBSTANCE: the invention is pertaining to the biotechnology, in particular, to purification of the bleaching earth from pollution with the oil products. The method provides for introduction into the soil of the biomass of the consortium of petroleum oxidizing strains - Bacillus brevis IB DT 5-1 and Arthrobacter species IB DT 5-3 and the biomasses of the strain of bacteria Azotobacter vinelandii IB 4 at the mass ratio of the biomasses equal to 1:1. The invention allows to activate the process of the oil products biodegradation, at that the degree of the degradation for 5 months compounds 81 %.

EFFECT: the invention ensures activation of the process of the oil products biodegradation with the degree of the degradation for 5 months making 81 percent.

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Description

The invention relates to the protection of the environment, in particular to the protection of soil resources from oil pollution.

At present, oil refining complex enterprises that use bleaching earth for contact refining of base oils (removal of resinous compounds and polycyclic aromatic hydrocarbons) are forced to store their production waste in the form of dumps, thereby aggravating the ecological situation and occupying additionally alienated territories.

There are various biological methods for the restoration of soils contaminated with oil and oil products, based on the introduction of specialized microorganisms into such soils, previously isolated from various contaminated sources or genetically modified. The main agents of biodegradation of oil and oil products are bacteria. This is explained by the fact that the use of hydrocarbons in their life activity among bacteria is quite widespread.

A number of researchers have shown that bacteria of the genus Rhodococcus are promising for the creation on their basis of preparations for biodegradation of oil and oil products polluting the soil [1, 2].

Spore-forming bacteria are also widely used to create drugs that help cleanse the soil and water from oil contamination [3-5].

The disadvantages of these drugs is the low efficiency in the reclamation of bleaching earth contaminated with oil products, the bulk of which are resinous substances and polycyclic aromatic compounds.

Closest to the proposed method is the reclamation of bleaching earth contaminated with oil products, where the Bacillus brevis consortium of oil oxidizing microorganisms IB DT 5-1 and Arthrobacter species IB DT 5-3 are used to activate the biodegradation of oil products [6].

The disadvantage of this method is the relatively low degree of biodegradation of petroleum products.

The technical task of the proposed method is to increase the efficiency of biodegradation of petroleum products contained in bleaching earth.

The problem is solved by introducing into the bleaching earth an additive containing, in addition to the biomass of a consortium of oil-oxidizing microorganisms Bacillus brevis IB DT 5-1 and Arthrobacter species IB DT 5-3, the biomass of aerobic nitrogen-fixing microorganisms Azotobacter vinelandii IB 4, made in mass ratio 1: microorganisms.

The specified strain of microorganisms Azotobacter vinelandii is known as the basis of a biological product for controlling root rot of wheat and increasing the quantity and quality of the crop [7].

Examples of specific performance.

In laboratory tests of the method of biological reclamation of bleached earth contaminated with oil products, bleached earth was taken from the dumps of OJSC Orsknefteorgsintez, the biomass of the consortium of microorganisms Bacillus brevis and Arthrobacter species, obtained by their joint fermentation, and the biomass of microorganisms v Azinobobact.

A consortium of microorganisms Bacillus brevis IB DT 5-1 and Arthrobacter species IB DT 5-3 (Lenoil biological product) is grown under aerobic conditions on a nutrient medium of the following composition, g / l: Na ₂ CO ₃ - 0.1; CaCl ₂ - 0.01; MnSO ₄ · 7H ₂ O — 0.02; FeSO ₄ · 7H ₂ O — 0.02; NaH ₂ PO ₄ - 1.5; K ₂ HPO ₄ - 1.0; MgSO ₄ · 7H ₂ O — 0.2; NH ₄ NO ₃ - 2.0; distilled water to 1000 ml. Diesel fuel is used as the sole carbon source; the fermentation process is carried out at room temperature until the microorganism titer in the culture fluid reaches 10 ⁹ CFU / ml.

The biomass of microorganisms Azotobacter vinelandii IB 4 (biological product "Azolen") is grown under aerobic conditions on a nutrient medium of the following composition, g / l: KH ₂ PO ₄ - 0.2; K ₂ HPO ₄ - 0.8; CaSO ₄ · 2H ₂ O - 0.1; MgSO ₄ · 7H ₂ O — 0.2; FeCl ₃ - 0.01; Na ₂ MoO ₄ - 0.01; yeast extract - 0.5; sucrose - 20 g; distilled water to 1000 ml. The fermentation process is carried out at room temperature until the titer of microorganisms in the culture fluid is 10 ⁹ CFU / ml.

To assess the rate of decomposition of petroleum products, an indicator of the residual content of petroleum products in bleaching earth was used.

The residual oil content was determined by spectrophotometric and weight methods [8]. In the first of these, a sample weighing 10 g was placed in a filter paper cartridge and transferred to a Soxhlet apparatus. The extraction was carried out with 120-150 ml of hexane for 2-3 hours at the boiling point of hexane (69 ° C). The hydrocarbon concentration in the sample was determined by the optical density of the extract on an SF-46 spectrophotometer at a wavelength at which this hydrocarbon mixture had a maximum absorption peak. The maximum absorption peak was determined on a SPECORD UF-VIS spectrophotometer. According to the weighing method, a sample weighing 10 g was placed in a filter paper cartridge and weighed on an electronic balance. The extracted samples together with the cartridges were dried in an oven at a temperature of 100-120 ° C and again weighed on an electronic balance. The difference in the weight of the samples before and after extraction was determined by the content of residual oil products.

The number of the main groups of microorganisms involved in the biotransformation of oil products was determined by sowing the soil suspension by dilution on solid nutrient media [9]: bacteria assimilating organic nitrogen on meat peptone agar (MPA), oligonitrophils on Ashby nutrient medium, microscopic fungi (micromycetes) - on Chapek’s environment and hydrocarbon-oxidizing microorganisms — on Tsukamura’s environment, where a sterile mixture of hydrocarbons obtained after extraction with hexane was used as a carbon source contamination of bleaching earth.

The experiments were repeated three times.

Scheme of the laboratory (model) experiment on the reclamation of bleaching earth: mineral fertilizers were applied to the bleaching earth containing 181 g / kg of residual hydrocarbons (nitrophoska at the rate of 0.25 g / kg of substrate), the Lenoil biological product was calculated for the bleaching earth and the biological product "Azolen" in a mass ratio to the biological product "Lenoil", comprising 1: 1, 1.25: 1 and 0.75: 1. The experiments were repeated three times, the duration of the experiments was 150 days. The humidity in the vessels during the entire incubation period was maintained at 60%, and the temperature was 20-25 ° C. Sampling of bleaching earth was carried out after 30 days.

The test results of the proposed method for reclamation of bleaching earth showed that the introduction of a consortium of oil-oxidizing microorganisms Bacillus brevis and Arthrobacter species with an additional addition of bacterial biomass Azotobacter vinelandii IB 4 significantly increases the efficiency of the process of biodegradation of oil products in bleaching earth (Table 1). After 150 days of incubation, the degree of reduction in the content of petroleum products by the proposed method was 81.3 wt.%. The use of the Lenoil biological product without introducing the Azolen biological product under the experimental conditions proved to be less effective compared to the proposed technical solution, and the degree of biodegradation of oil products amounted to only 69.3 wt.%. When the mass ratio of the biomass of bacteria Azotobacter vinelandii IB 4 to the biomass of the consortium of oil-oxidizing microorganisms is 0.75: 1, the efficiency of the process of biodegradation of oil products decreased, and at a ratio of 1.25: 1, the degree of biodegradation practically did not differ from the corresponding indicator achieved with the mass ratio equal to 1: 1.

The introduction of the biomass of bacteria Azotobacter vinelandii IB 4 contributes to a significant increase in the number of soil microorganisms involved in the transformation of oil products polluting the bleaching earth - heterotrophic bacteria growing on MPA, oligonitrophils, hydrocarbon-oxidizing bacteria and micromycetes (Table 2-5). During their life, the bacteria Azotobacter vinelandii IB 4 secrete various biologically active substances, including cytokinins, into the environment [7], and, apparently, the biomass of these microorganisms plays the role of an effective biological fertilizer that stimulates the development of the entire microbiota in an oil-contaminated object.

Thus, the proposed method for the biological reclamation of bleaching earth using a consortium of oil-oxidizing microorganisms Bacillus brevis IB DT 5-1 and Arthrobacter species IB DT 5-3 and the addition of bacterial biomass Azotobacter vinelandii IB 4 compared with the known one can increase the efficiency of the process of biodegradation of oil products in soil , intensify the microbiological activity of the soil.

Bibliography

1. The bacterial strain Rhodococcus erythropolis used to clean water and soil from oil and oil products: A.s. 1805097 USSR, MKI ⁵ С 02 В 3/34, Е 02 В 15/04 // Yagafarova G.G., Skvortsova I.N., Zinoviev A.P., Yagafarov I.R.

2. A method of cleaning soils from oil pollution: Pat. 2019527, Russia, MKI ⁵ C 02 F 3/34 // Coronelli T.V., Arakelyan E.I., Komarova T.I., Ilyinsky V.V.

3. The bacterial strain Bacillus sp. - Destructor of petroleum products and phenolic compounds: Pat. 1784592, USSR, MKI ⁵ C 02 F 3/34 // Astrova N.G., Moiseeva L.V., Protchenko P.Z.

4. Stabnikova E.V., Selezneva M.V., Reva O.N., Ivanov V.N. The choice of an active microorganism - a hydrocarbon destructor for cleaning oil-contaminated soils // Applied Biochemistry and Microbiology. - 1995 - T. 31, No. 5. - S.534-539.

5. The method of reclamation of soils contaminated with oil and oil products: Pat. 2077397, Russia, MKI ⁶ В 09 С 1/10, С 09 К 3/32 // Andreson R.K., Khaziev F.Kh., Deshura BC, Bagautdinov F.Ya., Boyko T.F., Novoselova E .AND.

6. The method of reclamation of bleaching earth contaminated with oil: Pat. 2237711, Russia, MKI ⁷ C 12 N 1/20 // Loginov O.N., Pilyugin V.V., Kostyuchenko V.P., Komarov S.I., Silishchev N.N.

7. The bacterial strain Azotobacter vinelandii to obtain a biological product to combat root rot of wheat and increase the quantity and quality of the crop: Pat. 2245918, Russia, MKI ⁷ C 12 N 1/20 // Loginov O.N., Pugacheva E.G., Silishchev N.N., Boyko T.F., Galimzyanova N.F.

8. Gruzdyakova R.A. Spectrophotometric determination of petroleum products in soil samples // Hygiene and sanitation. - 1993. - No. 3. - S. 73-74.

9. Zvyagintsev D.G. Methods of soil microbiology and biochemistry. - M .: Moscow State University. - 1980 .-- 223 p.

Table 1 The dynamics of changes in the degree of biodegradation of hydrocarbons in bleaching earth Experience Options The degree of biodegradation of hydrocarbons, wt.% 30 days 60 days 90 days 120 days 150 days Control - bleaching earth without biologics 5.3 6.1 13.1 13.1 15.3 Biological product "Lenoil" 32.1 39.3 43.1 55,4 69.3 Biological product "Azolen" 23,2 25,2 25.7 28.3 31.1 Joint introduction of biological products "Lenoil" and "Azolen" in a mass ratio of 1: 1 42.3 61.2 68.1 79.2 81.3 Joint introduction of biological products "Lenoil" and "Azolen" in a mass ratio of 1.25: 1 41,4 60.0 66.8 77,2 78.6 Joint introduction of biological products "Lenoil" and "Azolen" in a mass ratio of 0.75: 1 42.3 61.5 69.0 79.3 81.3

table 2 The dynamics of changes in the number of bacteria on MPA Experience Options The number of cells, thousand in 1 g of absolutely dry soil 30 days 60 days 90 days 120 days 150 days Control - bleaching earth without biologics 20 ± 1 80 ± 11 80 ± 6 90 ± 5 130 ± 8 Biological product "Lenoil" 670 ± 43 320 ± 13 590 ± 46 990 ± 104 1120 ± 82 Biological product "Azolen" 530 ± 50 160 ± 31 280 ± 35 530 ± 72 640 ± 47 Joint introduction of biological products "Lenoil" and "Azolen" 980 ± 68 2800 ± 63 1750 ± 122 1390 ± 111 1180 ± 101

Table 3 The dynamics of changes in the number of oligonitrophils in Ashby medium Experience Options The number of cells, thousand in 1 g of absolutely dry soil 30 days 60 days 90 days 120 days 150 days Control - bleaching earth without biologics 10 ± 1 180 ± 8 280 ± 50 360 ± 22 410 ± 31 Biological product "Lenoil" 40 ± 6 750 ± 92 790 ± 103 850 ± 68 900 ± 68 Biological product "Azolen" 270 ± 11 670 ± 72 550 ± 54 540 ± 48 410 ± 35 Joint introduction of biological products "Lenoil" and "Azolen" 560 ± 49 490 ± 57 580 ± 97 640 ± 38 680 ± 50

Table 4 The dynamics of changes in the number of hydrocarbon-oxidizing bacteria in the environment of Tsukamura Experience Options The number of cells, thousand in 1 g of absolutely dry soil 30 days 60 days 90 days 120 days 150 days Control - bleaching earth without biologics 15 ± 2 50 ± 3 50 ± 7 50 ± 6 30 ± 1 Biological product "Lenoil" 270 ± 48 130 ± 19 148 ± 22 176 ± 14 180 ± 20 Biological product "Azolen" 160 ± 11 180 ± 14 241 ± 21 270 ± 16 270 ± 11 Joint introduction of biological products "Lenoil" and "Azolen" 310 ± 28 200 ± 20 267 ± 17 281 ± 32 310 ± 30

Table 5 The dynamics of changes in the number of micromycetes in the Chapek environment Experience Options The number of cells, thousand in 1 g of absolutely dry soil 30 days 60 days 90 days 120 days 150 days Control - bleaching earth without biologics 15 ± 2 18 ± 2 13 ± 5 20 ± 4 13 ± 2 Biological product "Lenoil" 53 ± 3 25 ± 6 13 ± 1 33 ± 2 13 ± 4 Biological product "Azolen" 33 ± 2 65 ± 4 40 ± 5 50 ± 8 30 ± 7 Joint introduction of biological products "Lenoil" and "Azolen" 60 ± 9 65 ± 6 60 ± 8 70 ± 8 70 ± 20

Claims (1)

The method of reclamation of bleaching earth contaminated with oil products by applying an additive containing biomass of a consortium of oil-oxidizing microorganisms Bacillus brevis IB DT 5-1 and Arthrobacter species IB DT 5-3 to the soil, characterized in that the additive additionally contains a biomass of Azotobacter i 4inand microorganisms introduced in a mass ratio equal to 1: 1 to a consortium of oil-oxidizing microorganisms.