RU2376084C1 - Method of restoring soils and grounds polluted with oil and oil products, including old oil contaminations - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to biotechnology, in particular to field of environment protection. Method includes introduction of carbon-oxidising microorganisms with further periodic soil aeration with simultaneous introduction of feeding elements. In order to increase efficiency of soil purification from oil and oil products from microorganisms used is consortium of strains of oil-destructing microorganisms, possessing higher activity to heavy fractions of oil, consisting of microscopic fungus Cladosporium elegantulum Pidopliczko et Deniak IB-KF1, pseudomonads- P. putida (biovar A) IB-K1 and P. putida (biovar A) IB-K2, eight species of bacilla B. brevis IB-K3, IB-K17, B. circulans IB-K4, IB-K6, IB-K11, B. coagulans IB-K16, B. popilliae IB-K10, B. stearothermophilus IB-K19, as well as four cultures of rhodocooci: R. maris IB-K5, R. luteus IB-K7, R. maris IB-K20, R. mbropertmctus IB-K8. Soil aeration is preferably carried out with periodicity 20-25 days, and feeding elements are introduced in form of diammofoska in amount 0.25-0.5 wt %.

EFFECT: invention allows to increase efficiency of soil purification from oil and oil products.

6 dwg, 7 tbl

Description

The invention relates to the field of environmental protection and biotechnology, and in particular to methods for the restoration of soils and soils contaminated with oil and oil products.

A known method of purifying water and soil from oil, oil products and polymer additives in a drilling fluid, including the introduction of microorganisms, characterized in that the microorganisms use the association of bacterial strains Rhodococcus erythropolis BKM Ac-1339D, Bacillus subtilis BKM B-1742D and Pseudomonas putida BKM 1301 RF patent No. 2093478, publ. 10/20/1997).

The disadvantage of this method is the low degree of purification of the soil from oil pollution and the inability to restore soils with chronic oil pollution, since the known association of bacterial strains is not capable of biodegradation of hydrocarbons of heavy oil residues.

The closest to the proposed method according to the technical result is a method of restoring soils and soils contaminated with oil and oil products, including the distribution of contaminated soil on a horizontal platform, the introduction of a mixture of clean soil and sand, the application of sorbent and oil-oxidizing microorganisms in the form of the bacterial preparation Devoroil, a complex mineral fertilizers, moistening and periodic soil aeration. Treatment with a sorbent, the Devoroil preparation and complex fertilizer is carried out periodically in accordance with the microbiological parameters of the soil (RF patent No. 2322312, publ. 04/20/2008).

The disadvantage of this method is that the use of the bacterial preparation "Devoroil" does not allow for a high rate of destruction of oil hydrocarbons at the final stage of treatment, which reduces the efficiency of treatment and increases the recovery time of soils with old oil pollution.

The task of the invention is to increase the efficiency of soil cleaning from oil and oil products, including from old oil pollution.

The problem is solved in that in the method of restoring soils and soils contaminated with oil and oil products, including chronic oil pollution, by introducing hydrocarbon-oxidizing microorganisms followed by periodic aeration of the soil with the simultaneous introduction of nutrients from microorganisms, a consortium of microorganism strains - oil destructors having increased activity to heavy oil fractions, consisting of a microscopic fungus - Cladosporium elegantulum Pidopliczko et Deniak IB-KF1, pseudomonas - P.putida (biovar A) IB-K1 and P. putida (biovar A) IB-K2, eight species of B. brevis IB-K3, IB-K17, B.circulans IB-K4, IB-K6, IB- K11, B. coagulans IB-K16, B. popilliae IB-K10, B. stearothermophilus IB-K19, as well as four cultures of Rhodococcus: R.maris IB-K5, R. luteus IB-K7, R.maris IB-K20, R.rubropertinctus IB-K8.

In addition, aeration of the soil is carried out with a frequency of 20-25 days, and nutrients contribute in the form of diammofoski in the amount of 0.25-0.5% of the mass.

The use of a consortium of 15 strains of microorganisms as hydrocarbon-oxidizing microorganisms capable of decomposing oil hydrocarbons and having increased activity to heavy oil fractions allows to ensure the rate of destruction of oil pollution at the highest level due to the high utilizing ability of the consortium with respect to oil and oil products in a wide range of contents the latter in a hydrocarbon-containing substrate.

As a result of the experiments, it was found that the proposed consortium of microorganism strains retains high activity properties at all stages of the oil biodegradation process, oxidized oil fractions biodegradation, which improves the efficiency of soil cleaning by ensuring high efficiency of oil pollution destruction at the final stage of the microbiological stage of cleaning oil-contaminated soils soil. This effect is most important in the elimination of long-standing oil pollution, in which oil is degraded and is represented by heavy fractions.

Conducting aeration of the soil with a frequency of 20-25 days and the introduction of nutrients in the amount of 0.25-0.5% of the mass. is the optimal condition for maintaining the number of microorganisms at the highest possible damage, which ensures the effectiveness of the process of oil biodegradation.

As a result of the experiments, it was found that the number of microorganisms of the proposed consortium begins to decrease after 20-25 days due to a decrease in the content of nutrients - nitrogen, phosphorus, potassium, consumed as a result of their vital activity. The introduction of these elements in 20-25 days maintains the vital activity of microorganisms at a high level and, accordingly, provides increased cleaning efficiency.

A consortium of microorganism strains is isolated from soil contaminated with oil hydrocarbons of the Belebeyevsky district of the Republic of Bashkortostan and is a consortium of strains of indigenous hydrocarbon-oxidizing microorganisms adapted to this region. It is well known that with the activation of indigenous microflora, the latter is highly active in the oxidation of oil hydrocarbons. Due to the duration of the activation process, not only hydrocarbon-oxidizing microorganisms are activated, but also the entire soil pool as a whole, therefore, the possibility of activation and development of pathogenic microorganisms is not excluded, which can lead to bacteriological infection of the work area. To eliminate this drawback, the consortium and the strains included in its composition were subjected to toxicological examination.

The resulting consortium consists of 15 microorganism strains (hereinafter referred to as KShB-15 biological product).

The number of P. putida strains (Biovar A) (IB-K1 and IB-K2) is not more than 9-14% (2-3 × 10 ⁷ CFU / ml) of the total number of microorganisms in the medium of MPA, GPA and Raymond.

The number of rhodococci is about 80% (1.6-2.4 × 10 ⁸ CFU / ml) of the total number of microorganisms in the medium MPA, GPA and Raymond, where 66% (1.3-1.9 × 10 ⁸ CFU / ml ) are the strains of R.maris (IB-K5 and IB-K20), 10% (2-3 × 10 ⁷ CFU / ml) strain R. luteus IB-K7 and 4% (8 × 10 ⁶ -1 × 10 ⁷ CFU / ml) strain R.rubropertinctus IB-K8.

The number of bacilli B. brevis (IB-K3, IB-K17), B. circulans (IB-K4, IB-K6, IB-K11), B. coagulans IB-K16, B. popilliae IB-K10 and B. stearothermophilus IB -K19 is 10-15% (2-3 × 10 ⁷ CFU / ml) of the total number of microorganisms in the environment of MPA, GPA and Raymond.

The number of the fungus Cladosporium elegantulum Pidopliczko et Deniak IB-KF1 in the consortium is 0.03-0.001% (2-3 × 10 ³ CFU / ml) of the total number of microorganisms in Raymond medium.

The microorganism strains included in the consortium are characterized by the following features.

Characterization of the fungus Cladosporium elegantulum Pidopliczko et Deniak IB-KF1

Morphological and cultural characteristics. Mycelium creeping, septate, superficial, dark-colored consisting of a large number of conidiophores. Conidiophores are tree-shaped, with whorled-tree branches, erect, solitary, septate, smooth, olive-brown. Conidiophores up to 100 microns long, 3-5 microns thick. Conidia are unicellular, smooth, from oblong to ellipsoidal, in branched chains spherical, ovate and obovate, olive-brown, with a noticeable scar on one end. The apical conidia are spherical 2.7-3.0 microns, ovate and obovate 2.1-2.7 × 3.2-5.4 microns. Basal - 2.7-3.2 × 4.3-6.7 microns.

On Chapek’s solid medium at 28 ° C for 3 days, the size of the colonies is 9-14 mm, rhizoid, dark sand, the color of the spore-bearing part is brownish, the reverse side of the colonies is dark sand, the colony is translucent, soft, with a mold smell. There are many single conidiophores, more than a hundred conidia are formed on one conidiophore, the conidiophore consists of dense, branched, short chains of conidia. On the 6th day, the colony is 25-29 mm: in the center is a convex velvety tubercle, beige, opaque; the middle part of the colony is slightly transparent, tobacco-brown in color, less velvety than the center; edges translucent, dirty brown, slightly velvety. The reverse side is dark brown, the edges are tobacco brown. By 10 days of the colony, 30-35 mm, and by 15 days, 32-37 mm, weakly radially scored, the farther from the center, the more transparent, otherwise the appearance of the colony is unchanged.

Physiological and biochemical characteristics. It liquefies gelatin, grows on glycerin (Chapek with glycerin) and cellulose, oxidizes hydrocarbons of oil and diesel fuel, grows well at 28-37 ° С, weak growth at 13 ° С.

Characterization of strain P. putida (biovar A) - IB K1

Morphological and cultural characteristics. Gram-negative, moving sticks, 0.44-0.55 × 1.10-2.53 microns in size. On the MPA medium, on the first day, colonies of 1 mm are formed, round, dirty white, opaque, smooth, convex, shiny, with a smooth edge, fine-grained, soft, sticky and oily. On the second day of the colony, 3.0-4.0 mm, cream colored, pale yellow pigment is observed in the nutrient substrate. On the fifth day of the colony, 5.0-8.0 mm.

Physiological and biochemical characteristics. Forms a yellow-green fluorescent pigment diffusing into the medium. Absorbs glucose, arabinose, xylose with the release of acid. It does not absorb mannitol, sorbitol, trehalose, mesoinositis. Among amino acids, alanine, arginine is well absorbed and does not absorb valine, methionine, cysteine. It is well absorbed by phenylacetic, hippuric and nicotinic acids, creatine and does not absorb tartaric acid and geraniol. As a source of nitrogen, it assimilates both organic forms (peptone, autolysates, extracts), and mineral forms (ammonium salts, nitrates).

Oxidative. Catalopositive. Arginine dihydrolase is positive. It does not have ornithine decarboxylase. Does not form poly-β-hydroxybutyrate inclusions within cells. Levan does not form from sucrose, gelatin does not thin, starch does not hydrolyze, does not possess lecithinase and lipase activity, lactose does not ferment, does not hydrolyze lipids. Tyrosine and casein decompose.

Aerobe. It grows well in the temperature range from 13 to 28 ° C.

Characterization of strain P. putida (biovar A) - IB K2

Morphological and cultural characteristics. Gram-negative, moving sticks, 0.44-0.55 × 1.10-2.42 microns in size. On the IPA medium, on the first day, colonies of up to 1 mm are formed, round, colorless, translucent, smooth, flat, shiny, with a smooth edge, uniform, soft and oily. On the second day of the colony, 2.0-3.0 mm, slightly convex, cream in color with a slight yellowish tint, a pale yellow pigment is released on Wednesday. On the sixth day, colonies of 2.0-5.0 mm, translucent, small colonies are more transparent than large ones.

Physiological and biochemical characteristics. Forms a yellow-green fluorescent pigment diffusing into the medium. Absorbs glucose, arabinose, xylose with the release of acid. It does not absorb mannitol, sorbitol, trehalose, mesoinositis. Among amino acids, alanine, arginine is well absorbed and does not absorb valine, methionine, cysteine. It absorbs nicotinic acid, creatine well and does not absorb tartaric acid, hippuric acid and phenylacetic acid and geraniol. As a source of nitrogen, it assimilates both organic forms (peptone, autolysates, extracts), and mineral forms (ammonium salts, nitrates).

Oxidative. Catalopositive. Arginine dihydrolase is positive. It does not have ornithine decarboxylase. Does not form poly-β-hydroxybutyrate inclusions within cells. Levan does not form from sucrose, gelatin does not thin, starch does not hydrolyze, does not possess lecithinase and lipase activity, lactose does not ferment, does not hydrolyze lipids. Tyrosine and casein decompose.

Aerobe. It grows well at a temperature of 13-28 ° C.

Characterization of strain B. brevis IB-K3

Morphological and cultural characteristics. Gram-positive, movable sticks, 0.54-0.65 × 1.65-4.40 microns in size. Disputes, elliptical, terminal location, less often subterminal, slightly inflate sporangia. The spore size is 0.88-0.99 × 1.21-1.54 μm. On the MPA medium, colonies of 1.0-2.0 mm are formed, round, colorless, translucent, smooth, slightly convex, shiny, with a smooth edge, fine-grained, soft and oily. On day 3, the colonies were 2.0-4.0 mm, whitish, opaque, slightly convex - in the center there is a small tubercle, the edge is slightly wavy. On the 6th day the size of the colonies is 3.0-5.0 mm, the edges of the colony take the form of small festoons.

Physiological and biochemical characteristics. Oxidase-negative. Catalopositive. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative. Does not absorb glucose, arabinose, xylose and mannitol. When sugar is fermented, gas is not formed, acid is not released. Gelatin liquefies, casein and tyrosine decompose, has lecithinase and lipase activity. Hydrolyzes lipids (Tween 60 and Tween 80). Forms ammonia. Starch does not hydrolyze, does not use citrate and propionate, phenylalanine does not deamination, indole and hydrogen sulfide do not form. Nitrate does not restore.

Aerobe. It grows at pH 5.7-6.8. It does not grow in the presence of 5% NaCl. It grows in the temperature range from 30 to 50 ° C.

Characterization of strain B. brevis IB-K17

Morphological and cultural characteristics. Gram-positive, movable sticks, 0.77-0.88 × 1.65-5.05 microns in size. Disputes, elliptical, terminal arrangement, less often subterminal, inflate sporangia. The spore size is 0.88-1.10 × 1.65-2.20 microns. On the MPA medium, colonies for 2 days are up to 1.5 mm or less, round, fawn, shiny, opaque, smooth, slightly convex, with a smooth edge, uniform, soft, sticky. On the 3rd day, 1.0-2.0 mm, slightly radially outlined, with a slightly wavy edge. On day 9, 2.0-4.0 mm, dark cream in color, slightly convex, rough, with a slightly noticeable concentric striation, the edge is shallow.

Physiological and biochemical characteristics. Oxidative. Catalopositive. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative. Absorbs glucose with the release of acid, gas is not formed. Does not absorb arabinose, xylose and mannitol. When sugar is fermented, gas is not formed, acid is not released. Gelatin liquefies, casein and tyrosine decompose, citrate uses.

Hydrolyzes lipids (Tween 60 and Tween 80). Starch does not hydrolyze, does not possess lecithinase and lipase activity, does not use propionate, phenylalanine does not deamination, indole and ammonia do not form. Nitrate reduces to nitrite.

Aerobe. It does not grow in the presence of 5% NaCl. It grows in the temperature range from 30 to 50 ° C.

Characterization of strain B. circulans IB-K4

Morphological and cultural characteristics. Gram-positive, moving sticks, 0.44-0.56 × 1.76-3.30 microns in size. Spores, elliptical, the location is predominantly central, less often paracentral, inflate sporangia. The spore size is 0.99-1.10 × 1.65-1.98 μm. On the MPA medium, on day 2, colonies of 1.0-2.0 mm are round, dirty white, opaque, smooth, slightly convex, shiny, with a smooth edge, uniform, soft and oily. On day 3, colonies with a diameter of 1.0-3.0 mm, cream-colored, fine-grained. Small colonies are slightly convex and smooth; in large colonies also convex, a small dent appears in the center. On the 6th day of the colony, 2.0-6.0 mm, they look like on the 3rd day, only the edges become slightly wavy, in some places small teeth are observed. Over time, colonies grow into agar.

Physiological and biochemical characteristics. Oxidative. Catalopositive. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative. Absorbs glucose, arabinose, xylose and mannitol. During the fermentation of sugars, acid is released, gas is not formed. Hydrolyzes starch, lipids (Tween 60 and Tween 80), gelatin dilutes. Casein and tyrosine do not decompose. Does not possess lecithinase and lipase activity. Citrate and propionate do not use. Phenylalanine deamination does not occur. Indole and hydrogen sulfide does not form. Nitrate reduces to nitrite. Forms ammonia.

Anaerobe. It grows at pH 5.7-6.8. It grows in the presence of 5% NaCl and in the temperature range from 30 to 50 ° C.

Characterization of strain B.circulans IB-K6

Morphological and cultural characteristics. Gram-positive, movable sticks, 0.33-0.55 × 1.43-3.85 microns in size. Disputes, elliptical, the location is mainly terminal, less often subterminal, inflate sporangia. The spore size is 0.77-1.10 × 1.21-1.65 μm. On the IPA medium, colonies up to 1.0 mm or less are formed on day 2, round, colorless, transparent, smooth, convex, shiny, with a smooth edge, uniform, soft. On day 5, the colonies were 1.0-2.0 mm, fawn, translucent, smooth, shiny, with a fine-grained texture.

Physiological and biochemical characteristics. Oxidative. Catalopositive. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative. Absorbs glucose, arabinose, xylose and mannitol. During the fermentation of sugars, acid is released, gas is not formed. Hydrolyzes starch. Gelatin does not dilute, it does not decompose casein and tyrosine, it does not hydrolyze lipids (Tween 60 and Tween 80), it does not have lecithinase and lipase activity, it does not use citrate and propionate, phenylalanine does not deamination, indole and hydrogen sulfide do not form. Nitrate reduces to nitrite. Forms ammonia.

Anaerobe. It grows at pH 5.7-6.8. It does not grow in the presence of 5% NaCl. It grows in the temperature range from 30 to 40 ° C.

Characterization of strain B.circulans IB-K11

Morphological and cultural characteristics. Gram-positive, movable sticks, 0.34-0.57 × 1.98-3.52 microns in size. Disputes, elliptical, terminal arrangement, inflate sporangia. The spore size is 0.57-0.86 × 1.18-1.62 microns. On the MPA medium on the 4th day colonies of up to 1.0 mm are formed, round, cream, opaque, convex, shiny, with a slightly wavy edge, fine-grained, soft. On day 6, colonies 1.0-1.5 mm, with a smooth edge, granular. On the 15th day, 1.5-3.0 mm, round, opaque, with a dull sheen, slightly rough, large colonies have a small-lobed margin, small serrated colonies have a small flattened border, the main part of the colony is strongly convex with respect to the border, with weak concentric circles, granular, pasty, there are zones of proteolytic activity.

Physiological and biochemical characteristics. Oxidative. Catalopositive. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative. Absorbs glucose, arabinose, xylose and mannitol. During the fermentation of sugars, acid is released, gas is not formed. Hydrolyzes starch, decomposes casein. Gelatin does not dilute, does not decompose tyrosine, does not hydrolyze lipids (Tween 60 and Tween 80), does not have lecithinase and lipase activity, does not use propionate, phenylalanine does not deamination, indole and hydrogen sulfide do not form. Nitrate reduces to nitrite.

Anaerobe. It does not grow in the presence of 5% NaCl. It grows in the temperature range from 30 to 50 ° C.

Characterization of strain B. coagulans IB-K16

Morphological and cultural characteristics. Gram-positive, motile sticks, 0.33-0.55 × 1.43-3.30 microns in size. Spores, elliptical, terminal arrangement, sporangia greatly inflate. The spore size is 0.88-1.21 × 1.32-1.98 μm. On the MPA medium, colonies on day 2 1.0 mm, round, colorless, shiny, transparent, smooth, slightly convex, with a smooth edge, uniform, soft. On day 3, 1.0-1.5 mm, pale grayish, opaque, convex, with a slightly noticeable tubercle, in large colonies the margin is finely serrated, in small colonies it is even. On day 9, 1.0-4.0 mm, convex, the colony profile is curved, from the edges rises up to the central part of the colony, which is in the form of a high flattened area, coarse-grained. In large colonies, a secondary growth zone is observed in the form of a transparent veil, an irregular shape with a very wavy edge.

Physiological and biochemical characteristics. Oxidative. Catalopositive. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is positive. Absorbs glucose, arabinose, xylose and mannitol. During the fermentation of sugars, acid is released, gas is not formed. Hydrolyzes starch. Gelatin does not dilute, it does not decompose casein and tyrosine, it does not hydrolyze lipids (Tween 60 and Tween 80), it does not have lecithinase and lipase activity, it does not use propionate and citrate, phenylalanine does not deamination, indole and hydrogen sulfide do not form. Nitrate reduces to nitrite. Forms ammonia.

Anaerobe. It grows at pH 5.7-6.8. It does not grow in the presence of 5% NaCl. It grows in the temperature range from 30 to 50 ° C.

Characterization of strain B. popilliae IB-K10

Morphological and cultural signs. Gram-positive, moving sticks, 0.55-0.77 × 2.20-7.70 microns in size. Disputes, elliptical, subterminal or terminal arrangement, inflate sporangia. On the 4-day MPA medium, colonies of up to 1.0 mm are round, colorless, translucent, slightly convex and finely tuberous, glossy, with a wavy edge, which in places passes into small teeth, uniform, soft. On day 6, colonies of 1.0-1.5 mm, pale sand in color, look the same as on day 4. On the 15th day of the colony, 1.5-2.0 mm, slightly convex, with a dull sheen, translucent, a small tubercle in the center of the colony, fine-toothed edges turning into radial smoothed folds not reaching the center, soft, sticky, coarse-grained.

Physiological and biochemical characteristics. Oxidase-negative. Catalase-negative. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative. Absorbs glucose with the release of acid, gas does not emit. Does not absorb arabinose, xylose and mannitol. Casein decomposes, has lecithinase activity. It does not hydrolyze starch and lipids, gelatin does not thin, propionate does not use, deamination of phenylalanine does not occur, indole does not form. Forms ammonia.

Anaerobe. It does not grow in the presence of 5% NaCl. It grows at a temperature of 30 ° C.

Characterization of strain B. stearothermophilus IB-K19

Morphological and cultural characteristics. Gram-positive, moving sticks, 0.66-0.77 × 1.32-3.30 microns in size. Disputes, elliptical, centrally less often paracentral, slightly inflate sporangia. The spore size is 0.99-1.10 × 1.22-1.64 μm.

On the MPA medium, colonies on day 2 1.5-2.0 mm, round, with a straight edge, fawn, shiny, opaque, smooth, slightly convex, similar to “fried eggs” - “protein” slightly translucent and less convex, “yolk” elevated and opaque, uniform, soft, sticky. On the 9th day, 2.5-5.0 mm, fawn, slightly convex, small bubbles are observed on the surface in some colonies. Small colonies are smooth, with a smooth edge. Large colonies in the central part have a large, high, flat plateau with a small tubercle in the center, a falling, toothed wide margin.

Physiological and biochemical characteristics. Oxidative. Catalase-negative. The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative. Absorbs glucose with the release of acid, gas does not emit. Does not absorb arabinose, xylose and mannitol. During the fermentation of sugars, acid is released, gas is not formed. It hydrolyzes starch and lipids (Tween 60, Tween 80), decomposes casein, dilutes gelatin, possesses lecithinase and lipase activity. Propionate and citrate do not use, deamination of phenylalanine does not occur, indole, hydrogen sulfide and ammonia does not form.

Anaerobe. It does not grow in the presence of 5% NaCl. It grows in the temperature range from 30 to 55 ° C.

Characterization of the strain R.maris IB-K5

Morphological and cultural characteristics. Gram-positive, non-acid resistant. The morphogenetic cycle consists of three stages (cocci - rods - cocci). The cell size is 0.56-0.84 × 2.80-3.50 microns. The cells are motionless.

On the MPA medium on the 2nd day, colonies of 1.0-3.0 mm are formed, round, colorless, translucent, smooth, slightly convex, shiny, with a smooth edge, homogeneous, soft and oily. On day 5, colonies 3.0-5.0 mm, marbled pink, opaque.

Physiological and biochemical characteristics. It absorbs glucose, glycerin, sucrose, fructose with the release of acid and does not absorb arabinose, galactose, dulcite, inositol, xylose, lactose, mannitol, maltose, rhamnose, raffnose, sorbitol, cellobiose. Absorbs sodium salts of benzoic, butyric, lactic, pyruvic, propionic, acetic and succinic acids. Does not absorb sodium salts of tartaric, citric, salicylic, oxalic and phenylacetic acids. As a source of nitrogen, it assimilates both organic forms (peptone, autolysates, extracts), and mineral forms (nitrates), weak growth on Ashby's nitrogen-free medium. Restores nitrates to nitrites.

Oxidase-negative. Catalopositive. Hydrolyzes starch and lipids (Tween 60, Tween 80), decomposes casein, has urease activity. Gelatin does not thin, does not possess lecithinase and lipase activity. Tyrosine does not decompose. Citrate uses.

Resistant to external environmental factors: able to grow at 7% NaCl; at initial pH 5.7 and 8.0; at a temperature of 5 to 42 ° C; withstands heating at 60 ° C for 30 min and 72 ° C for 15 min; tolerant to antimicrobial agents: grows in media containing 0.003% basic fuchsin, 0.0004% crystalline violet and 0.1% phenol.

Characterization of strain R..luteus IB-K7

Morphological and cultural characteristics. Gram-positive, non-acid resistant. The morphogenetic cycle consists of three stages (cocci - rods - cocci). The cell size is 0.54-0.72 × 2.85-5.23 microns. The cells are motionless.

On the MPA medium, point colonies of less than 1.0 mm are formed, round, colorless, transparent, smooth, convex, shiny, with a smooth edge, uniform, soft. On day 9, colonies are less than 1.0 and up to 1.5 mm, round. In the GPA environment, the colonies are dotted less than 1.0 mm, round, colorless, transparent, smooth, flat, shiny, with a smooth edge, uniform, soft. On the 9th day of the colony less than 1.0 and up to 3.0 mm, round, waxy yellow, convex with a small fossa in the center of the colony.

Physiological and biochemical characteristics. It absorbs arabinose, galactose, glucose, glycerin, xylose, mannitol, sucrose, sorbitol, fructose with the release of acid and does not absorb dulcite, inositol, lactose, maltose, rhamnose, raffnose, cellobiose. Absorbs sodium salts of citric, butyric, lactic, pyruvic, acetic and succinic acids. Does not absorb sodium salts of benzoic, tartaric, propionic, salicylic, oxalic and phenylacetic acids. As a source of nitrogen, it absorbs both organic forms (peptone, autolysates, extracts), and mineral forms (ammonium, nitrates), good growth on Ashby's nitrogen-free medium. Does not reduce nitrates to nitrites.

Oxidase-negative. Catalopositive. Hydrolyzes lipids (Tween 40, Tween 60, Tween 80), has urease activity. Does not hydrolyze starch, gelatin does not thin, does not decompose casein, does not possess lecithinase and lipase activity. Tyrosine does not decompose.

Resistant to external environmental factors: able to grow at 7% NaCl; at initial pH 5.7 and 8.0; at a temperature of 10 to 37 ° C; withstands heating at 60 ° C for 30 min and 72 ° C for 15 min; tolerant to antimicrobial agents: grows in media containing 0.003% basic fuchsin and 0.0004% crystalline violet.

Characterization of the strain R.maris IB-K20

Morphological and cultural characteristics. Gram-positive, non-acid resistant. The morphogenetic cycle consists of three stages (cocci - rods - cocci). The cell size is 0.44-0.76 × 2.53-3.30 microns. The cells are motionless.

On the MPA medium, on day 2, colonies of less than 1.0 mm are round, slightly pinkish, opaque, smooth, convex, shiny, with a smooth edge, uniform, soft. On the 4th day, the colonies are 0.5-1.5 mm, flesh-pink, the center is more saturated in color, 2 types of colonies are observed: 1 - uniformly convex; 2 - the central part of the colony is more convex than the edges.

Physiological and biochemical characteristics. It absorbs glucose, glycerin, fructose with the release of acid and does not absorb arabinose, galactose, dulcite, inositol, xylose, lactose, mannitol, maltose, rhamnose, rafnose, sucrose, sorbitol, cellobiose. Absorbs sodium salts of benzoic, butyric, lactic, pyruvic, propionic, acetic and succinic acids. Does not absorb sodium salts of tartaric, citric, salicylic, oxalic and phenylacetic acids. As a source of nitrogen, it assimilates both organic forms (peptone, autolysates, extracts), and mineral forms (ammonium, nitrates), weak growth on Ashby's nitrogen-free medium. Restores nitrates to nitrites.

Oxidase-negative. Catalopositive. Hydrolyzes starch and lipids (Tween 40, Tween 60, Tween 80), decomposes casein, has urease activity. Gelatin does not thin, does not possess lecithinase and lipase activity. Tyrosine does not decompose. Citrate uses.

Resistant to external environmental factors: able to grow at 7% NaCl; at initial pH 5.7 and 8.0; at a temperature of 10 to 42 ° C; withstands heating at 60 ° C for 30 min and 72 ° C for 15 min; tolerant to antimicrobial agents: grows in media containing 0.003% basic fuchsin, 0.0004% crystalline violet and 0.1% phenol.

Characteristic strain R.rubropertinctus IB-K8

Morphological and cultural characteristics. Gram-positive, non-acid resistant. The morphogenetic cycle consists of three stages (cocci - rods - cocci). The cell size is 0.30-0.50 × 2.70-4.76 μm. The cells are motionless.

On the MPA medium, colonies appear on the 3rd day, very small and slightly noticeable. On day 4, point colonies of less than 1.0 mm, round, white, opaque, smooth, flat, dull, with a smooth edge, uniform, soft. On day 5, colonies 1.0 mm, pale orange, strongly convex colonies resemble a “steep volcano” with a wide base and a small “crater” at the apex, small smoothed folds extend from the colony's edge to its apex, with a wavy edge, fine-grained structure, growing into agar, curdled consistency. On the 17th day of the colony, 2.0-3.0 mm, rusty, the scalloped edge of the colony goes into deep folds towards the top of the colony “crater”.

Physiological and biochemical characteristics. It absorbs glucose, glycerin, mannitol, sucrose, sorbitol, fructose with the release of acid and does not metabolize arabinose, galactose, dulcite, inositol, xylose, lactose, maltose, rhamnose, rafnose, cellobiose. Absorbs sodium salts of butyric, lactic, pyruvic, propionic, acetic and succinic acids. Does not absorb sodium salts of benzoic, tartaric, citric, salicylic, oxalic and phenylacetic acids. As a source of nitrogen, it absorbs both organic forms (peptone, autolysates, extracts), and mineral forms (ammonium, nitrates), good growth on Ashby's nitrogen-free medium. Restores nitrates to nitrites.

Oxidase-negative. Catalopositive. Hydrolyzes lipids (Tween 40), has urease activity. Starch and casein do not hydrolyze, gelatin does not thin, does not possess lecithinase and lipase activity. Tyrosine does not decompose. Citrate uses.

Resistant to external environmental factors: able to grow at 7% NaCl; at initial pH 5.7 and 8.0; at a temperature of 10 to 37 ° C; withstands heating at 60 ° C for 30 min and 72 ° C for 15 min; tolerant to antimicrobial agents: grows in media containing 0.003% basic fuchsin, 0.0004% crystalline violet and 0.1% phenol.

The proposed method for the restoration of soils and soils contaminated with oil and oil products, including chronic oil pollution, is illustrated by the following graphs: figure 1 shows the dynamics of changes in the number of hydrocarbon-oxidizing microorganisms in variants of experiment 1 with the biological product Devoroil; figure 2 - dynamics of changes in the number of hydrocarbon-oxidizing microorganisms in the variants of experiment 1 with biological product KShB-15; figure 3 - dynamics of changes in the number of hydrocarbon-oxidizing microorganisms depending on the introduction of nutrients of experiment 2; figure 4 - the dynamics of the destruction of oil in the variants of experiments with oil experiment 3; figure 5 - the dynamics of the destruction of oil in the variants of experiments with oil sludge experiment 3; figure 6 - the dynamics of the destruction of oil in the variants of experiments with oil-contaminated soil (taken from the place of chronic oil pollution) experiment 3.

The effectiveness of the proposed method was confirmed experimentally.

In order to confirm the increase in cleaning efficiency with the claimed method parameters, 3 experiments were performed.

For all experiments, the soil type common for the Republic of Bashkortostan was used - gray forest medium loamy soil. Chemical, physico-chemical characteristics of this soil are presented in table 1.

The degree of soil contamination for all experiments was,% of the mass: oil - 9.27%, oil sludge - 4.07%, oil pollution soil (taken from the site of chronic oil pollution) - 7.9%.

The sample volume of each experiment was 0.5 kg.

The dosage of biological solutions in each sample was 30 ml with an initial microorganism content of 10 ⁶ cells / ml. Solutions of nutrients of various concentrations were added in the same volume.

In each experiment, variants of experiments were laid with the addition of gummi 0.05-0.08% of the mass.

The data of all experimental variants were confirmed in three repetitions.

As a nutritional supplement used mineral fertilizer - diammofoska.

Each sample was incubated at a constant temperature of 29-30 ° C.

The depth and intensity of the process of biodegradation of oil products was judged by the quantitative residual content of oil products (% wt.).

Experiment 1 was conducted to determine the optimal dosage of nutrients. The necessity of the formulation is that during experimental and field studies, with the introduction of a calculated dose of a nutrient solution, a slow development of microorganisms of the consortium was noted (hereinafter the biological product KShB-15). When introduced in the form of granules, the development of microorganisms depended on the amount of precipitation. In this regard, it was suggested that there should be an optimal dosage, which, in order to be independent of weather conditions (rainfall) in the growing season of the year (minimum period of the biological stage), should be applied in dissolved form.

The experiment includes the experimental options presented in table 2.

The results of the experiment are presented in table 3 and figure 1,2.

The analysis of the experimental results both in the version with the Devoroil biological product and the KShB-15 biological version showed that the optimal single dose of mineral nutrients (diammofosk) is 0.25-0.5% of the mass. to the mass of the cultivated soil. At lower concentrations, the growth in the number of hydrocarbon-oxidizing microorganisms quickly begins to decline, and at higher concentrations, their initial growth is significantly slowed down and only after a certain time begins to increase, but does not reach the values obtained at the optimal dosage.

Experiment 2 was carried out with the aim of determining the frequency of nutrient addition, subject to the dosage conditions for nutrients defined in experiment 1. The need for the formulation of this experiment is that in the first experiment, the dose of nutrients is determined to be less than that necessary for the entire growing season, which leads to the outcome of a certain period of time to reduce the number of hydrocarbon-oxidizing microorganisms to their background content in this type of soil.

The experiment includes the experimental options presented in table 4.

The dosage of nutrients is 0.3% by weight of the cultivated soil.

The results of the experiment are presented in table 5 and graph 3.

An analysis of the experimental results showed that the number of hydrocarbon-oxidizing microorganisms begins to decrease after 20-25 days, and when nutrients are re-added after 25 days, it is restored.

Thus, it was found that in order to maintain the number of hydrocarbon-oxidizing microorganisms at the highest possible level, the frequency of the introduction of nutrients in dissolved form should be 20-25 days.

Experiment 3 was carried out with the aim of determining the effectiveness of the destruction of oil and oil products by biological products KSHB-15 in the proposed method and Devoroil - in the prototype method. As the objects under study, options for soil contaminated with oil from the Arlansky field (sour oil), oil sludge (degraded oil containing 10% of fractions boiling up to 250 ° C), and directly soil taken from the place of old oil pollution that existed for more than 3 years are considered. The experiment was carried out under the conditions defined in experiments 1 and 2 - the dosage of nutritional experiments and the frequency of their introduction.

The experiment includes the experimental options presented in table 6.

The dosage of nutrients is 0.3% by weight of the cultivated soil. The frequency of application is 25 days.

The experimental results are presented in table 7 and graphs 4, 5, 6. Analysis of the experimental results showed that the oil degradation rate according to the proposed method using the KShB-15 biological product is higher than the prototype method using the Devoroil biological product, especially at the final stages of the experiment. This is most pronounced in variants with oil-contaminated soil and soil contaminated with oil sludge. In variants with oil, an increase in the rate of destruction by the KShB-15 biological product is explained by the fact that, as the destruction occurs, the specific content of heavy oil fractions increases, the destruction of which by a consortium of microorganism strains included in the KShB-15 biological product is more effective. The microorganism strains included in the consortium used in the method are deposited in the microorganism collection at the Institute of Biology, Ufa Scientific Center of the Russian Academy of Sciences.

Table 1 Horizon and depth, cm pH Humus,% of the mass. Absorbed Hydrolytic activity water saline common mobile % of the total Ca ⁺⁺ Mg ⁺⁺ mg equiv per 100 g of soil A ₁ 1-10 5.75 5.15 5.47 0.50 9.1 22 5 3.0 A ₂ B 32-43 6.65 5.65 1.40 0,07 5,0 22 four 2.6 B ₁ 45-55 7.10 6.15 0.63 0,03 4.8 16 3 1,2 B ₂ 60-70 8.0 6.95 0.40 0,03 7.5 fourteen 3 no Sun 90-100 8.00 7.00 0.29 0.01 3.4 eighteen 2 no S 140-150 8.20 7.20 0.22 0.01 4,5 9 2 no

table 2
Experiment Experiment 1 Options The experiments Biological products Dosage of nutrients,% mass. Experiment Options The dynamics of changes in the number of hydrocarbon-oxidizing microorganisms depending on the dosage of nutrients Devoroil 0.0025 D-D-1 0.005 D-D-2 0.05 D-D-3 0.25 D-D-4 0.5 D-D-5 1,0 D-d-6 2.0 D-d-7 4.0 D-D-8 KShB-15 0.0025 K-D-1 0,0005 K-D-2 0.05 K-D-3 0.25 K-D-4 0.5 K-D-5 1,0 K-D-6 2.0 K-D-7 4.0 K-D-8

Table 3
The dynamics of changes in the number of hydrocarbon-oxidizing microorganisms depending on the dosage of nutrients in the experimental variants of experiment 1 Experiment Options The duration of the experiments, day 0 3 6 9 12 fifteen eighteen 21 24 27 thirty 33 36 The number of microorganisms, 10 ⁴ cells / g asp D-D-1 477 496 439 323 142 eleven 5 D-D-2 493 510 456 331 118 5 one 0.23 D-D-3 486 518 610 490 318 205 56 0.93 D-D-4 585 627 701 693 619 586 573 580 501 193 27 1.4 0.62 D-D-5 569 511 617 632 577 561 584 613 584 501 418 256 201 D-d-6 86 95 113 56 48 56 94 185 217 198 236 311 273 D-d-7 fifteen eleven 27 36 120 131 98 112 196 227 254 296 301 D-D-8 0.002 0.001 0.002 0.003 0.001 0.014 0,026 0,098 0.26 1.93 15.78 102 218 K-D-1 492 411 398 276 243 121 86 29th 1.44 0.18 0.02 K-D-2 501 526 480 304 257 190 120 55,4 23.6 2.24 0,07 K-D-3 566 583 620 590 435 342 218 176 54 2,51 0.93 K-D-4 583 611 726 817 878 924 896 931 882 656 423 118 27 K-D-5 516 584 623 715 794 817 877 901 856 783 719 636 511 K-D-6 322 298 311 372 456 423 394 387 423 395 301 288 269 K-D-7 0.72 0.36 2.77 1.93 2.18 10.24 121 314 395 386 317 351 390 K-D-8 0.15 0.27 1,54 20.76 51.12 40.41 102 236 189 274 318 298 356

Table 4
Experiment Experiment 2 Options Experiment 2 Biological products Experiment Options Reapply nutrients Dynamics of changes in the number of hydrocarbon-oxidizing microorganisms depending on the frequency of nutrient application Devoroil 1-D No KShB-15 2-K No Devoroil 3-D + G Gum KShB-15 4K + G Gum Devoroil 5-D + D Diammofoska KShB-15 6-K + D Diammofoska Devoroil 7-D + D + D Diammofoska + gummy KShB-15 8-K + D + G Diammofoska + gummy

Table 5
The dynamics of changes in the number of hydrocarbon-oxidizing microorganisms depending on the frequency of nutrient application in the experimental variants of experiment 2 Experiment Experiment 2 Options The duration of the experiments, day 0 one 5 10 fifteen twenty 25 thirty 35 40 45 fifty 55 60 The number of microorganisms, 10 ⁴ cells / g asp 1-D 0.015 450 510 620 580 430 338 10.48 0.185 0.012 2-K 0.018 520 601 627 610 590 410 28.03 1.47 0,093 3-D + G 0.013 510 560 690 630 470 256 45.32 18,365 0,047 4K + G 0.011 586 634 718 690 610 460 135.26 32.15 0.056 5-D + D 0,021 430 490 540 626 612 423 586 574 536 428 19.46 0.24 0.019 6-K + D 0.019 496 621 643 630 615 510 590 601 586 517 31,2 0.36 0,027 7-D + D + D 0.018 475 543 591 682 675 461 719 710 685 601 153.2 23.14 0,043 8-K + D + G 0.014 543 670 700 690 673 528 740 701 636 620 218.6 56.12 0.058

Table 6
Experiment Options Experiment 3 Experience option The initial content of petroleum products,% mass. Option Numbers Biological product KShB-15 Oil soil 9.27 1 KOH Oil sludge soil 4.07 3 CONS Oil contaminated soil 7.9 5 CCP Biological product "Devoroil" Oil soil 9.27 1 day Oil sludge soil 4.07 3 DNS Oil contaminated soil 7.9 5 DZP Control (without introducing microorganisms) Oil soil 9.27 1 KN Oil sludge soil 4.07 3 KNSh Oil contaminated soil 7.9 5 KNS Biological product KShB-15 Oil soil + Gumi 9.27 2 KON + G Oil sludge + Gumi soil 4.07 4 CONSH + G Oil Contaminated Soil + Gumi 7.9 6 KOZP + G Biological product "Devoroil" Oil soil + Gumi 9.27 2 days + g Oil sludge + Gumi soil 4.07 4 DNS + G8 Oil Contaminated Soil + Gumi 7.9 6 DZP + G Control (without introducing microorganisms) Oil soil + Gumi 9.27 2 KN + G Oil sludge + Gumi soil 4.07 4 KZP + G Oil Contaminated Soil + Gumi 7.9 6 KZP + G

Table 7
The dynamics of oil degradation in experiment 3 Biological products The oil content,% of the mass. Experiment Options Duration, day 0 twenty 40 60 80 Devoroil 9.27 6.95 6.68 6.36 5.92 1 day KShB-15 9.27 6.74 6.63 6.15 5.72 1 KOH The control 9.27 9.16 9.11 7.57 7.23 1 KN Devoroil 9.27 6.80 6.61 5.86 5.31 2 days + g KShB-15 9.27 6.74 6.66 5.18 4.77 2 KON + G The control 9.27 9.16 9.02 7.31 7.05 2 KN + G Devoroil 4.07 3.75 3.01 2,36 1.83 3 DNS KShB-15 4.07 2.84 2.82 2.06 1.17 3 CONS The control 4.07 3.28 3.05 2.6 1.86 3 KNSh Devoroil 4.07 3,54 2.96 2.24 1.73 4 NS + G KShB-15 4.07 2.84 2.71 1.91 1.05 4 CONSH + G The control 4.07 3.88 3.82 3.2 3.16 4 KNSH + G Devoroil 7.9 6.54 6.27 5.83 4.98 5 DZP KShB-15 7.9 6.6 6.01 4.75 2.92 5 CCP The control 7.9 7.9 7.88 7.45 7.33 5 KZP Devoroil 7.9 6.54 6.27 5.36 4.02 6 DZP + G KShB-15 7.9 6.6 6.15 4.64 2,58 6 KOZP + G The control 7.9 7.89 7.8 7.12 6.25 6 KZP + G

Claims (2)

1. The method of restoration of soils and soils contaminated with oil and oil products, including chronic oil pollution, by introducing hydrocarbon-oxidizing microorganisms followed by periodic aeration of the soil with the introduction of nutrients, characterized in that a consortium of strains of oil-degrading microorganisms having microorganisms having increased activity to heavy oil fractions, consisting of a microscopic fungus - Cladosporium elegantulum Pidopliczko et Deniak IB-KF1, pseudomonas - P.putida (biovar A) IB-K1 and P. putida (biovar A) IB-K2, eight bacilli species B. brevis IB-K3, IB-K17, B. circulans IB-K4, IB-K6, IB-K11, B. coagulans IB-K16, B. popilliae IB-K10, B. stearothermophilus IB-K19, as well as four cultures of Rhodococcus: R.maris IB-K5, R. luteus IB-K7, R.maris IB-K20, R.rubropertinctus IB-K8. 2. The method according to n.1, characterized in that the aeration of the soil is carried out with a frequency of 20-25 days, and nutrients are introduced in the form of diammofoski in the amount of 0.25-0.5 wt.%.

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