LT5057B - Biological preparation for removing crude oil and petroleum product contaminants from soil and water, method for production and use thereof - Google Patents

Abstract

The present invention relates to biological preparation for removing crude oil and petroleum-product contaminants from soil and/or water, method for preparation and use thereof in different climate zones. the proposed biological preparation consistsof mixture of the following hydrocarbon-oxidising strains of micro-organisms: Pseudomonas sp. GVT 3B, Rhodococcus sp. GVT 12B, Bacillus cerus GVT 17B, Candida sp. GVT 9M, Trichoderma lignorum GVT 27M (deposited with the Collection of Microbiology-Chemistry department in Kiskenai village, Klaipeda region, LT, with corresponding deposition numbers: KMChL: B-3, B-12, B-17, M-9, M-27). Biomass of culture is cultivated in fermentation equipment in liquid organic-mineral medium using the mixture (1:1) of a drew and masut like hydrocarbon inductor. Treatment of the soil and/or water is carried out by using dry or liquid preparation, containing 3x109-5x109 live microbial cells in one g or ml. 30 g/m2 of dry biological preparation or 3 l/m2 of suspension is added

Description

The present invention relates to the field of biotechnology and environmental protection, in particular to the cleaning of soil and water contaminated by oil and oil products using a consortium of oil oxidizing microorganisms in the form of a preparation.

Oil-oxidising microorganisms (NOMs) are used to clean soil, water from oil and oil products, whether ex-situ or in-situ. NOM preparations are produced. Known preparations for soil and water treatment of petroleum products using a single NOM strain: using Pseudomonas oil-oxidizing microorganisms (USSR A.1. No. 1 076 446.1 428 809), preparation using NOM Rhodococcus erythropolis (RU patent) No. 2,019,527), a biopreparation with Actinomucor sp. (U.S. Patent No. 4,415,662), as well as a biopreparation with Acinetobacter valentis subspecies paraffinicum (RU No. 2,053,204).

The disadvantage of these preparations is that they use only one strain of NOM. Biopreparations of this type have proved to be of low activity because the degradation of petroleum products is a multi-step process and one set of enzymes available to the microorganism strain is not sufficient to destroy them.

NOM preparations of several strains are also known (U.S. Patent Nos. 3843517, 3,871,957, 3,959,127, 4,136,024, 4,288,545, 4,452,894.4,521,515, 4,535,061, 4,593,003, 4,732,680; 5,494,580; USSR a. No. Nos. 793,375, 823,314, 925,875; RU Patent No. 2,122,980).

The disadvantage of these preparations is the use of hydrophilic NO microorganisms. In a two-phase system, water-petroleum hydrocarbons hydrophilic oil-oxidizing microorganisms concentrate in the water phase. Their destructive activity is limited because they are capable of utilizing only water-soluble hydrocarbons.

The closest to the technical solution is a biopreparation consisting of a mixture of oil oxidizing microorganisms: Rhodococcus sp. BMK-AC-1500D, Rhodococcus maris BMK-AC1501 D, Rhodococcus erythropolis BMK-AC-1502D, Pseudomonas stutzeri BMK-B-1972D, Candida sp. BMK-Y-2778D, and a method for purifying water and soil from petroleum and petroleum products using said biopreparation (International Application Publication WO 95/31408). The biopreparation is used in the form of a slurry, which is sprayed on oil-contaminated soil and water.

The disadvantage of this biopreparation is its limited use and the microorganisms contained in it are slightly degradable in petroleum products when the medium (soil, water) is of low salinity. Optimal operating conditions of the biopreparation with a salinity of 5 - 10%. The microorganisms in the biopreparation slowly disintegrate heavy fraction petroleum products (fuel oil), and the biopreparation is used only in solution form.

SUMMARY OF THE INVENTION The object of the present invention is to provide a biopreparation of oil-oxidizing microorganisms, the combination of which components ensures its efficiency and advantage over other known analogous biopreparations, suitable for destruction of light and heavy petroleum product fractions and effective purification of various petroleum hydrocarbon contaminated soil and water. and to extend the potential for its use.

The essence of the problem is that the biopreparation for cleaning soil and water contaminated by petroleum and its products contains a mixture of metabolic products in exchange of hydrophilic and lipophilic microorganisms oxidizing petroleum hydrocarbons. Hydrophilic bacteria Pseudomonas sp. DVT 3B, lipophilic bacterium Rhodococcus sp. GVT 12B, Bacillus cereus GVT 17B, and micromycetes Candida sp. GVT 9M, Trichoderma lignorum GVT 27M were mixed in approximately equal parts and co-cultured.

The microorganisms that make up the biopreparation are deposited in the Collection of Microbiology-Chemistry Laboratory of Klaipėda Branch of Public Company “Soil Cleaning Technology” (Kiskėnai, Klaipėda region LT), their characteristics are:

Pseudomonas sp. DVT 3B (Collection number: KMChL: B-3) Morphological characteristics: Gram-positive, motile microorganisms; cell size -1.6 x 0.8 μ; colonies (on MPA) gray, round, 4-16 mm in diameter, wavy, soft in texture, colonies easily removed from the surface of the agarized medium. Physiological-biochemical properties: metabolism - aerobic type; absorbs acetate, propionate, butyrate; assimilates glucose, maltose, arabinose, lactose; hydrolyzes starch; absorbs ammonium and nitrate nitrogen; spread oil products; pH optimum - 6.7 - 7.3; optimal growth temperature -27 - 29 ° C.

Rhodococcus sp. GVT 12B (Collection Number: KMChL: B-12) Morphological Characteristics: Gram-positive, immobile microorganisms; rod-shaped, cell size 2.8 x 0.7 μ, well growing in mineral-organic medium (g / l: NH4Cl - 2, NaCl - 2, Na ₂ HPO ₄ -3, KH ₂ PO ₄ - 0.1; MgSO ₄ x 7 H ₂ O - 2, CaCl ₂ x 6 H ₂ O - 0.01, MnSO ₄ x 5 H ₂ O - 0.02, FeSO ₄ x 7 H ₂ O - 0.01 with acetate and yeast extract additive ); colony (on potato agar) round, 4-6 mm in diameter, smooth-edged, mucoid, soft in texture, growing well on MPA.

Physiological-biochemical properties: absorbs acetate, butyrate, ethanol, butanol, glucose, maltose, lactose, grows in mineral medium, absorbs ammonium and nitrate nitrogen; gelatine does not degrade casein, does not hydrolyze starch, degrades petroleum products and their hydrocarbons; pH optimum - 6.6 - 7.4; optimal growth temperature - 26 - 30 ° C.

Bacillus cereus GVT 17B (Collection number: KMChL: B-17) Morphological features: spore sticks, 1.5 - 5.0 μ long, spores oval (formed within 2 days on MPA), colonies (on MPA) light-colored, smooth edge, soft colonies, easily removable from the nutritional substrate.

Physiological-biochemical properties: facultative anaerobic, pigment-free, oxidative and catalase positive, Foges-Proskauer positive; absorbs citrate and propionate hydrolyses gelatin, casein starch; utilizes nitrates, ammonium salts, amino acids, peptone glucose, sucrose, decomposes petroleum products; pH optimum - 4.0 7.5; optimal growth temperature - 27 - 30 ° C.

Candida sp. GVT 9M (Collection number: KMChL: M-9) Morphological features: When grown in mineral medium, cells are usually solitary, round in size, -3-4 μ, producing no ascospores.

Physiological-biochemical properties: uptake of acetate, propionate, weak ethanol; utilizes ammonium nitrogen, petroleum products (especially hungry); pH optimum - 5.5 - 6.5; optimal growth temperature - 26 - 29 ° C.

Trichoderma lignorum GVT27M (Collection number: KMChL: M-27) Morphological characteristics: colonies grow rapidly in agarized organo-mineral medium (g / 1: NaNO ₃ - 2, KH ₂ PO ₄ - 1, MgSO ₄ - 0.5; KCl 0.5; FeSO ₄ 0.01; glucose 30; agar 40); colonies diameter of 8-9 cm within 3 days at 25 ° C; the culture is in the form of a dense micelle resembling a film; the micelle is composed of septic, strongly branched hyphae with diameters of 1.5 - (10.0 - 12.0) μ. The color of the mature colony is green and the lower part is colorless. Chlamydospora intercalary, colorless, 13 - 14 μ in diameter. Codcorns branched. Conidia spherical 3.5 - 4.5 μ diameter.

Physiological-biochemical properties: aerobic, absorbs sucrose, glucose, starch, molasses petroleum hydrocarbons; nitrogen sources - ammonium salts, amino acids, peptone plant and yeast hydrolysates; produces extracellular enzymes chitinases, proteases; has antagonistic properties against phytopathogenic fungi Fusarium, Verticillium, Alternarium. Optimal pH 5-6; optimal growth temperature - 27 - 30 ° C.

The advantage of a biopreparation is that it is stable. It does not eliminate individual association NOM cultures, since they are isolated from the petroleum product substrate, which contains a naturally occurring persistent community of these microorganisms, by interrelated metabolic exchange. The advantage of the biopreparation is that it is suitable for the destruction of light and heavy petroleum products.

Cultures absorb carbon (C) sources: petroleum hydrocarbons, sucrose, glucose, starch galactose; nitrogen (N) sources: ammonium salts, nitrates, protein hydrolysates, amino acids, peptone

The method of preparation of the biopreparation involves isolation of microorganisms from soil contaminated with oil and oil products, depositing them in fermenters and cultivating them in depth, using medium (g / liter):

NH4NO3 1.5-2.5

KH ₂ PO ₄ 0.75-1.25

K ₂ HPO ₄ 0.75 - 1.25

MgSO ₄ x 7H ₂ O 0.5

CaCl ₂ x 2 H ₂ O 0.5

FeSO ₄ x 7 H ₂ O or FeCl ₃ x 6 H ₂ O 0,01 maize extract 10 ml sucrose (or molasses) 20 inducer (1: 1 mixture of diesel fuel and fuel oil) 0,5 - 1,0, grown For a period of 3 days at a temperature of 25-29 ° C, preferably 27 ° C during cultivation, aeration of approximately 1 volume of air / 1 volume of medium / min.

The biopreparation is formulated and may be used in the form of a dry to finely dispersion particulate or suspension. It contains an active oil-oxidizing culture that uses different classes of petroleum hydrocarbons.

A method of using a dry biopreparation for soil cleaning, wherein 28 to 32 g / m ² , preferably 30, are applied to the soil to be cleaned by spreading it with a layer of 35 40 cm thick and fertilized with mineral fertilizers, preferably 3 days ago, several times during the cleaning season. g / m ² of dry biopreparation containing 3 · 10 ⁹ - 5-10 ⁹ viable NOM cells per gram, fertilized periodically, at least 4 times, with NPK fertilizer to the cleaned soil with a C: N: P ratio of 100: 5: 1 to 100: 10: 1, preferably 100: 7: 1, aerates the soil by cultivating it at least twice a week and maintains humidity between 65 and 70% of the soil water volume.

Method of applying the biopreparation slurry to soil cleaning by applying 2-4 liters / m ² , preferably 3 days before spreading to the soil to be cleaned and spreading with mineral fertilizers, preferably 3 days ago, several times during the cleaning season, preferably 3 times. 3 liters / m ² , in the form of a liquid suspension of the biopreparation NOM, containing not less than 310 ⁹ - 5 10 ⁹ viable NOM cells per milliliter, periodically, at least 4 times, with NPK fertilizer to the cleaned soil, providing C: N: A ratio of 100: 5: 1 to 100: 10: 1, preferably 100: 7: 1, is aerated by cultivating the soil at least twice a week and maintaining humidity between 65 and 70% of the water volume of the soil.

The biopreparation method for cleaning soil and water contaminated with oil and oil products is superior to the prototype because of multiple fertilization and application of the treated soil, which ensures high NOM concentration in the substrate and more intensive cleaning.

The efficiency and advantage of the cleaning method is achieved by intensive aeration of the soil to be cleaned twice a week.

The biopreparation contains active bacterial and micromycete cultures that use petroleum-fuel oil hydrocarbons as a source of carbon (C) and provide adequate mineral nutrition (NOM requires nitrogen, phosphorus, potassium and some other elements, micronutrients) and have favorable ecological conditions (pH, humidity, temperature, aeration) breaks down petroleum products to end products of decomposition - carbon dioxide and water.

A method of using a bio-preparation for treating water by treating and collecting the oil-contaminated water surface with sorbents, followed by spraying 1.5 - 2.0 liters / m ^{2 of} NOM liquid suspension of the bio-preparation containing 3 · 10 ⁹ - 510 ⁹ viable NOM cells per milliliter.

The advantage of using a biopreparation for water purification when cleaning oil-contaminated water surfaces is the combined use of cleaning agents: initially, contaminants are collected with sorbents and their residues are eliminated with NOM.

Biopreparation for oil and petroleum-contaminated soil and water treatment was obtained as follows.

Cultures of microorganisms were obtained from Vš. Soil cleaning technologies Klaipeda branch oil and soil contaminated with oil products. Cultures are saprophytic, non-pathogenic. The microorganisms were grown in fermenters by deep-water treatment. Media used (g / liter): NH4NO3 - 2; KH 2 PO 4 -1; K 2 HPO 4 - 1; MgSO ₄ x 7 H ₂ O 0.5; CaCl 2 x 2 H2O - 0.5; FeSOą χ 7 H2O or FeCl3 x 6 H2O - 0.01; corn extract -10 ml; sucrose (or molasses) - 20; inducer used diesel fuel oil (1: 1) - 1. Cultures are mixed in equal proportions, grown at 27-29 ° C. Aeration - 1 volume air / 1 volume medium / 1 min. Rearing time - 3 days. The resulting biopreparation is active over a wide temperature range. It is suitable for use at temperatures between 6 and 30 ° C, preferably between 8 and 28 ° C.

The biopreparation is prepared in the form of a suspension and a dry micro-organism preparation. In one case, the NOM suspension is immediately added to the petroleum-based primer. Alternatively, the 'LT 5057 B media NOM is concentrated. The concentrate is mixed with a protective substrate (talc, diatomite, bentonite). Dry to air dry condition.

Activity of the biopreparation is evaluated using mixed oil-oxidizing microorganisms Pseudomonas sp. GVT 3B, Rhodococcus sp. GVT 12B, Bacillus cereus GVT 17B, Candida sp. GVT 9M, Irichoderma lignorum GVT 27M - Association. NOM dry substance (with inert medium) infects (inoculates) substrate contaminated with petroleum products by a mixture of fuel oil and diesel (1: 1 ratio). The composition of fuel oil hydrocarbons is as follows: paraffins - naphthenic hydrocarbons - 15%, olefins and cyclodiolefins - 5%, alkylaromatic hydrocarbons - 1%, alkyl aromatic hydrocarbons - 4%, polyaromatic hydrocarbons - 20%, benzene resins - 30%, alcohol - 25%. Density of fuel oil (kg / m ³ ) - 956.4. The NOM preparation is used at 1% by weight of the inoculated substrate. The test substrate is enriched with NPK mineral compounds. Its humidity is maintained at 65% of the water volume of the substrate. The substrate is aerated by daily mixing. The substrate is maintained at optimum growth of NOM at 27 ° C.

The destructive activity of the product during the first 8 days was 720 - 740 mg / kg soil / day. During this time, 79 - 88% of the initial oil product mixture (diesel fuel + fuel oil, equal parts) was destroyed.

It is noteworthy that the used petroleum product contained a high amount of hard resin (up to 55%). When the substrate contains high levels of pollutant-resins, their hydrophobicity is strongly demonstrated. This makes it difficult to irrigate the soil and thus to destroy the pollutant.

After obtaining favorable results of destructive activity of the produced biopreparation, its experimental use for cleaning works was performed. It should be noted that the biopreparation is equally effective when used in both dry and slurry form.

For intensive NOM activities, the soil hydrogen ion concentration should be in the range of 5.0 - 9.0 (optimum - 7.0). Temperature: 6 - 30 ° C (optimum - 27 ° C).

The use of the biopreparation is illustrated by examples.

example. Removal of fuel oil contaminated soil using reduced NPK for soil fertilization

Chemical analysis of fuel oil contaminated soil is performed. The data (average values) are as follows: pH - 7, Organic carbon (C) - 1.5%, Airborne soil nitrogen (N, total) - 0.27%, Nitrogen (N) in liquid soil fraction - Nnh4 ( ^m i0) · 70.0; Nnq ₂ (mg / l) · 0.5; Nnq ₃ (mg / l) · 27.0; Ca, Mg carbonates - 4.92%, phosphorus (P) a single mobile, according to P ₂ O ₅ mg / 100 g ⁷ EN 5057 B, air-dry soil - 0.1; phosphorus in the liquid fraction of soil - P mineral (mg / l) 7.3; P organic soluble (mg / l) 2.6; P total soluble (mg / l).

The amounts of the main trace elements in the cleaning soil (mg / kg soil) are as follows: boron (B) - 20.0; lead (Pb) - 19.0; manganese (Mn) -220.0; Nickel (Ni) - 17.0; barium (Ba) - 150.0; vanadium (V) - 49.0; molybdenum (Mo) - 0.8; cobalt (Co) - 2.2; Copper (Cu) - 7.0; zinc - (Zn) 40.0; cadmium (Cd) less than 3.0.

None of these trace elements exceeds the permitted limit. Concentrations of important trace elements such as molybdenum, cobalt, copper, manganese are too low for NOM activities. Analytical data show that soils contaminated with 25 to 30 g of fuel oil per kg of soil, nitrogen and phosphorus contain about 50 times less than the amount needed to eliminate fuel oil pollution using NOM.

Microbiological analysis of the soil to be cleaned revealed that the initial amount of heterotrophs (growing in MPA medium) is about 2 million. colonies / gram of soil, and autochthonous oil-oxidizing bacteria - 50 - 100 thousand. colonies / gram of soil.

For normal fuel oil biodestruction, these microorganisms must be increased 50 to 100 times by means of intensifying their reproduction.

Cleaning is done this way. The contaminated soil (112 tons) is applied in a layer of 35 - 40 cm on a 200 m ² drainage base. Initial amount of pollutant - fuel oil in treated soil 21 740 mg / kg soil. The soil is fertilized four times a season (monthly in summer) with NPK fertilizer. Fertilizers used: ammonium nitrate (NH4NO3, 34% N) 25 kg / 200 m ² , or 126 g / m ² ; superphosphate (CaH ₂ PO ₄ χ H ₂ O χ 2 CaSO ₄ , 23% P ₂ O ₅ ) 14.7 kg / 200 m ² or 73.5 g / m ² ; potassium chloride (KCl) 10.5 kg / 200m ² or 52.5 g / m ² . The fertilizers used were mineral fertilizers (ammonium nitrate, superphosphate, potassium chloride) in kilograms:

I fertilization - 3.6; 2.1; 1.5

II fertilization - 7.2; 4.2; 3.0

III fertilization - 7.2; 4.2; 3.0

IV fertilization - 7.2; 4.2; 3.0.

The NOM biopreparation is added to the cleaned soil in the form of a liquid suspension - 3 liters of suspension per m ² of cleaned soil or 30 g / m ² dry NOM biopreparation. The biopreparation is applied to the soil by spraying it from the recovery machine with a high pressure pump. The biopreparation is sprayed three times during cleaning: May, June and July. The biopreparation is applied to the soil after fertilization with NPK fertilizer (third day after fertilization).

NOMs are aerobics. Their physiological-biochemical activities require oxygen. As a result, the soil was cultivated and loosened regularly - twice a week. The moisture of the soil to be cleaned is monitored ⁸ LT 5057 B. Humidity was determined by Ohaus firm MB 200 and maintained at 65 - 70% soil water volume.

Temperature is a very important factor for soil remediation. The most suitable temperature for NOM activities is 27 ° C. In the treatment area, the hottest in July, the average air temperature was 17 ° C. The soil temperature is almost equal to the air temperature. Significant fluctuations in temperature during the day - the temperature drops by a few degrees at night.

The season for effective soil biofueling lasts 120 days: from May to May. second half to early September. July is the best time for bio-cleaning. Providing the ecological conditions suitable for NOM reproduction, it amounts to 300 million lbs. ex./grame primer.

During the bio-cleaning season, 3210 mg remained on 200 m ^{2 of} a 35 cm thick soil layer containing 112 tonnes of soil which was initially contaminated with 21 740 mg / kg of fuel oil, ie 2 435 kg at the end of bio-treatment. / kg of fuel oil resins or resin 359 kg of fuel oil around 200 m ²⁾ 200 m ² 2076 kg of heavy fuel oil, or 85% of its original volume.

Paraffin-naphthenic and olefinic hydrocarbons are most likely to be degraded. They are completely decomposed within a month - 100%. In the same time, fuel oil is degraded by 30 to 35% in aromatic hydrocarbons, and resins are the most difficult to degrade in high molecular weight compounds.

example. Cleaning of fuel oil contaminated soil using increased NPK for soil fertilization

The contaminated soil, as in the first example, is applied to the drainage substrate in a layer of 35 - 40 cm. Soil contaminated with fuel oil - 21 700 mg / kg or 2 708 kg fuel oil over a total area of 230 m ² (124 800 kg contaminated soil). The soil used for cleaning NPK fertilizers is 1 m ² : 253 g of ammonium nitrate, 146 g of superphosphate and 106 g of potassium chloride. NPK fertilizers (ammonium nitrate, superphosphate, potassium chloride) were used for fertilization:

fertilization - 8.3; 4.8; 3.5;

II fertilization - 16.6; 9.6; 7.0;

III fertilization - 16.6; 9.6; 7.0;

IV fertilization - 16.6; 9.6; 7.0.

Inoculation of the NOM preparation into the soil soil cultivation (aeration), moisture regulation was performed as in the first example. The difference with the first example is the doubling of NPK mineral fertilization. In the same time as in the first example, the fuel oil from the initial 21,700 mg / kg soil to

930 mg / kg of fuel oil resin. Thus, 18 770 mg of fuel oil per kg of soil were destroyed. 2342 kg of fuel oil or 86% of its initial volume were destroyed over a total area of 230 m ² .

Data on the daily rate of fuel oil degradation (mg / g soil) showed that biodegradation up to 70 - 80 days under increased NPK fertilization is 40 - 90% faster compared to the lower NPK fertilization variant. After 80 days, biological treatment on less and more fertilized plots settles. This is due to the fact that in both cases the oil remains in the soil, which is difficult and slow to dismantle in the NOM.

It is noteworthy that at higher NPK fertilization rates the fuel oil disintegration rate reached 610 mg / kg daily in the initial disintegration stage (first month).

Biopreparation can be used to clean various water bodies (rivers, lakes, ponds) from oil products.

example. Water purification

The first step in the cleaning process is to collect the oil and its products by sorbents. It is an effective means of collecting large quantities of oil in the short term. Sorbents to be used: Joresorb sorbent, granules readily dispersed on water surface, adsorbent sorbent / petroleum 1: 6.5; Qualisorb Gold sorbent - granules, lightweight, absorbs petroleum products in a sorbent / oil ratio of 1: 2; Sphagsorb sorbent is a mossy peat product, oil is collected from the oily surface within minutes, but the highest sorption power is activated charcoal USVR-VIP, 1 kg sorbent collects 30 - 40 kg oil, sorption rate - 30 - 40 seconds.

The fresh water body (600 m ² ) is contaminated with 400 kg of diesel fuel. Activated carbon sorbent, 13 kg, was poured onto the water surface. 320 kg of diesel was collected. Water-emulsified diesel fuel (80 kg) was treated with a biopreparation suspension of 1.5 - 2.0 liters / m ² by spraying it on the water surface. 1000 liters of bio-formulation were used. Water temperature - 20 ° C. After 14 days of use, no pollutant - diesel fuel was found.

The combination of the components of the patented biopreparation ensures its efficacy and advantage over other known analogous biopreparations. It is suitable for destruction of light and heavy petroleum product fractions. The microorganisms contained in the biopreparation disintegrate paraffins - naphthenes, olefins and cyclodiolefins, alkylaromatic, polyaromatic hydrocarbons, as well as benzene resins, and the biopreparation is active over a wide temperature range, despite significant changes in the temperature of soil and water during the day and cleaning season. It is suitable for use at temperatures between 6 - 8 ° C and 28 - 30 ° C. The patented biopreparation can be effectively used to eliminate hydrocarbon pollutants released into the environment (soil, water) in emergencies in various natural climate zones.

Claims (6)

1. A biopreparation for treating soil and water contaminated by petroleum and oil products, comprising a mixture of petroleum hydrocarbon-oxidising microorganisms, characterized in that the petroleum hydrocarbon-oxidising microorganism is formed by an association of interrelated metabolites of hydrophilic and lipophilic microorganisms. 2. A biopreparation according to claim 1, characterized in that the hydrophilic bacteria forming the association of microorganisms (deposited in the Collection of Microbiology-Chemistry Laboratory of Klaipėda Branch of Public Institution “Soil Cleaning Technologies”, Kiskėnai, Klaipėda Region, LT) Pseudomonas sp. DVT 3B (reg. No. KMChL: B-3), a lipophilic bacterium of Rhodococcus sp. DVV 12B (reg. # KMChL: B-12), Bacillus cereus GVT 17B (reg. # KMChL: B-17) and micromycetes Candida sp. GVT 9M (Reg. No. KMChL: M-9), Trichoderma lignorum GVT 27M (Reg. No. KMChL: M-27) were mixed in approximately equal parts and co-cultured. 3. A process for the preparation of a biopreparation according to claims 1 and 2, characterized in that the microorganisms are isolated from the soil contaminated with oil and oil products, stored and cultivated in fermenters using medium (g / l) for cultivation: NH ₄ NO ₃ 1.5-2.5 KH ₂ PO ₄ 0.75 - 1.25 K ₂ HPO ₄ 0.75 - 1.25 MgSO ₄ x 7 H ₂ O 0.5 CaCl ₂ x 2 H ₂ O 0.5 FeSO ₄ x 7 H ₂ O or FeCl ₃ x 6 H ₂ O 0.01 maize extract 10 ml sucrose (or molasses) 20 inducer (diesel fuel oil 1: 1) 0.5 -1.0, cultivated For 3 days, maintains a temperature of 25 to 29 ° C during cultivation, preferably 27 ° C, and aerates at a ratio of approximately 1 volume of air / 1 volume of medium / min. 4. A method of using the biopreparation according to claim 1 and 2 for soil cleaning, characterized in that the soil to be cleaned, spread over a layer 35-40 cm thick and fertilized with mineral fertilizers, preferably 3 days, several times during the cleaning season, preferably 3 times, inserts 28 to 32 g / m ² , preferably 30 g / m ² , dry biopreparation containing 310 ^{4 * * * * 9} 510 ⁹ viable NOM cells per gram, cleanser primer periodically, at least 4 times, fertilizes NPK fertilizers providing a C: N: P ratio of 100: 5: 1 to 100: 10: 1, preferably 100: 7: 1, aerate the soil by cultivating it at least twice a week and maintaining humidity of 65 - 70% of the water volume of the soil. 5. A method of using the biopreparation according to claims 1 and 2 for soil cleaning, characterized in that the soil to be cleaned, applied in a layer of 35-40 cm thickness and fertilized with mineral fertilizers, preferably 3 days, several times during the cleaning season, preferably 3 times, inserts 2 - 4 liters / m ² , preferably 3 liters / m ² , in the form of a liquid suspension of the bio-formulation NOM, containing at least 3-10 ^{6 * * 9} - 5 10 ⁹ viable NOM cells per milliliter cleaned periodically during the cleaning season, at least 4 times, fertilizes with NPK fertilizer, providing a C: N: P ratio of 100: 5: 1 to 100: 10: 1, preferably 100: 7: 1, aerates the soil at least twice a week and maintains moisture 65 - 70% of the water volume of the soil. 6. A method of using the bio-preparation according to claims 1 and 2 for treating water surface contaminated with petroleum and oil products by sorbents and collecting them, characterized in that 1.5 - 2.0 liters / m ^{2 of the} bio-formulation NOM is sprayed onto the surface of the treated water. liquid suspensions containing 3 10 ⁹ to 5 10 ⁹ viable NOM cells per ml.