RU2617953C1 - Preparation for soil and water purufication from oil and oil products - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: drug for treatment of soils and water from oil and oil products contains oil, biomass hydrocarbon bacterial strain Bacillus subtilis SNBS-1, deposited in the Russian National Collection of Industrial Microorganisms under accession number VKPM B-12239, anastringent component, which is used as sodium carboxymethylcellulose and organomineral substrate booster - compositional mixture based on clinoptilolite prepared sawdust and humus components at a predetermined ratio.

EFFECT: invention improves the efficiency of purification of soil and water from the crude oil and oil products.

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Description

The invention relates to biotechnology, namely, preparations for cleaning soils and water from oil and oil products.

Currently, environmental pollution with petroleum hydrocarbons (HC) is an urgent environmental issue. The problem of rehabilitation of disturbed territories after emergency oil spills is especially relevant for the Far North, where the main oil and gas fields of the Russian Federation are operated. Being a highly organized substance, oil independently degrades very slowly, the oxidation processes of some structures are inhibited by other structures, the transformation of individual compounds proceeds along the path of acquiring stable hardly oxidized forms (see Gritsenko A.I., Akopova G.S., Maksimov V.M. Ecology. Oil and gas. - Moscow: Nauka, 1997 .-- 557 p.). Without the use of additional measures for the rehabilitation of disturbed lands, the process of self-healing of oil-contaminated soils in regions with favorable climatic conditions takes 10-25 years, while the destruction of oil and its derivatives in the North can last up to 50 years or more (see Pikovsky Yu.I. Transformation of technogenic oil flows in soil ecosystems // Restoration of oil-contaminated soil ecosystems. - M .: Nauka, 1988. - P. 7-31; Oborin AA, Kalachnikova I.G., Maslivets T.A., Bazenkova E.I. , Pleshcheva O.V., Ogloblin A.I. // Vossta the development of oil-contaminated soil ecosystems. - M .: Nauka, 1988.P. 140-159; Foght JM, Westlike DWS Bioremediation of oil spills // Spill Technol. Newsiett., 1992. - V. 17. - P. 1-10) .

Of the known solutions that can effectively cleanse the soil of northern regions from oil, the biological methods based on the intensification of the microbiological degradation of oil hydrocarbons are considered the most environmentally sound (see Koronelli T.V., Komarova T.I., Ilyinsky V.V. et al. The introduction of bacteria of the genus Rhodococcus into tundra soil contaminated with oil // Applied Biochemistry and Microbiology. 1997. V. 33. No. 2. S. 198-201).

A biological product that uses a consortium of oil-oxidizing microorganisms consisting of: Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. Has shown good efficiency in cleaning soils from oil and oil products pollution. PI Ky-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 with a weight ratio of 3-12 mass. % of each microorganism (see RU No. 2191643, IPC B09C 1/10, C12N 1/20, C12N 1/20, C12R 1/01, publ. 10.27.2002).

At the same time, rizotorfin (a fertilizer of nitrogen-fixing microorganisms) in the amount of 30-120 g / m ² is introduced into the contaminated medium for effective soil cleaning from oil pollution, which complicates the processing of oil-contaminated substrate.

The known biological product “Putidoyl” (see SU No. 1076446, IPC C02F 3/34, B09C 1/10, C12N 1/20 C02F 3/34, C02F 101/32, C12N 1/20, C12R 1/40, publ. 28.02 .1984) used to clean the soil from oil pollution. The biological product is based on a monoculture of bacteria Pseudomonas putida 36 (see Dyadechko V.N., Tolstokorova L.E., Gashev S.N. et al. On the biological reclamation of oil-contaminated sandy soils of the Middle Ob region // Soil Science. - 1990, No. 9. - S.148-151).

A disadvantage of the known solution is the complicated technology of preparation of the drug, which provides for spray drying of a live culture of bacteria, which causes injury to bacteria and, as a result, their death or loss of necessary activity due to inactivation of cells under high temperatures (+ 60 ° C). To restore the vital activity of bacteria, the authors apply a complex set of measures: heating a large amount of water (5 m ³ ) to + 18 ... + 28 ° C, mixing, aeration, and all this for a long time (16-24 hours), which in the field It’s quite difficult to complete.

A known method of cleaning the soil using a biological product "Devoroil" containing a consortium of hydrocarbon-oxidizing microorganisms Rhodococcus maris, Rhodococcus erythropolis, Pseudomonas stutzeri, Yarrowia lipolytica (see RU No. 2114071, IPC C02F 3/34, B09N 1/10, C12N10, C12 1/26, C12Q 1/02, publ. 06/27/1998). This method involves the storage of a suspension of microorganisms using NaCl, biologically active substances (vitamins) and osmoprotectors (betaine).

The disadvantage of this method is that, despite the addition of protective substances to the bacterial suspension, the number of microorganisms decreases regardless of the storage temperature.

A known method of cleaning soils and soils contaminated with oil and oil products using an oleophilic biological product based on the association of oil-oxidizing microorganisms — Rhodococcus erythropolis IEGM 708 and Rhodococcus ruber IEGM 327 and a biosurfactant — Rhodococcus, characterized in that the soil is thoroughly cultivated to reach the initial level residual oil 5-10 weight. % and the subsequent addition of an organic baking powder, an oleophilic biological product is applied in an amount of at least 10 liters per 0.5-1.0 m ^{3 of} soil / soil at least once a week for the first month and subsequently at least once a month before the end of the bioremediation cycle, in combination with periodic loosening and moistening, and additionally produce phytoremediation - sowing with perennial grasses (see RU No. 2193464, IPC B09C 1/10, C12N 1/26, C12N 1/26, C12R 1/01, publ. 27.11 .2002).

The disadvantage of the invention is the complex technology of the application of the method, involving the use of solid-liquid bioreactor and / or aerated soil pads as the initial treatment of highly contaminated soil. This method of reducing the level of pollution is time-consuming and involves disruption of the fertile soil layer (humus part of the soil profile), which is not applicable for accidents and oil spills in the Far North (including in the tundra zone). Since a prerequisite for cleaning and restoring oil-contaminated permafrost soils is the preservation of the fertile soil layer. This is explained by the fact that at the place of removing the soil cover under permafrost conditions, the formation of thermokarst defrost with the formation of funnels, dips or alas is possible, which in turn leads to the development of thermoerosion and causes great damage to the vulnerable and difficult to restore soil ecosystem of the Far North.

Known biological product "Naftox", the technology for the preparation and use of which is relatively simple. The biological product allows you to effectively clean the soil and water from oil and oil products (see RU No. 2053206, IPC С02F 3/34, C09K 3/32, B09C 1/10, B09C 101/00, published on 01.27.1996).

The disadvantage of Naftox is the short shelf life of the biological product - not more than 1 month, as well as the inability to use it in conditions of low positive temperatures (+ 5 ° C).

A biological product is known, which is an aqueous suspension of highly effective bacteria capable of purifying water and soil from oil and oil products (see RU No. 2428469, IPC C12N 1/20, C02F 3/34, C09K 3/32, B09C 1/10, publ. 10.09 .2011).

Its disadvantage is that the bacteria of the drug utilize only normal oil paraffins (C12-C18). Another disadvantage of this invention is the inability of the biological product to work in conditions of low positive temperatures (+ 5 ° C). The third disadvantage of this invention is to obtain only a liquid form of the drug, which reduces the shelf life of the biological product.

A known method of purifying soils from oil with a strain of bacteria Bacillus subtilis grown on meat and peptone agar at a temperature of + 37 ° C. The method allows for 3 months of the summer period to reduce the concentration of oil in the soil to 0.48% (see RU No. 2446900, IPC B09C 1/10, publ. 10.04.2012).

The disadvantage of this method is the use of only a liquid form of biological product, which reduces the shelf life of bacteria, as well as the fact that the strain degrades only oil and only in the soil and is not able to dispose of oil products (diesel fuel, motor oil, hydraulic oil, gas condensate), including in a wide temperature range from +5 to + 40 ° С.

Known biological product consisting of aerobic oil-oxidizing bacteria immobilized on solid substrate substrates (see RU No. 2404138, IPC C02F 3/34, C12N 1/26, publ. 20.11.2010). The biological product is highly active in relation to oil pollution. In this case, the biological product provides only for the purification of sea water and does not allow to evaluate the destructive ability of the biological product in relation to the soil. In addition, the bacterial strain Phyllobacterium myrsinacearum DKS-1, which is the basis of the biological product, is not able to develop at low positive temperatures (from + 5 ° C), which makes it unpromising for oil refining in areas with a short thermal period (see RU No. 2268934 IPC C12N 1/20, C02F 3/34, publ. 01/27/2006).

Closest to the claimed technical solution is a bacterial preparation consisting of highly active live aerobic oil-oxidizing bacteria (Mycobacterium, Pseudomonas and others) grown on solid carrier substrates with titers of 2.5-7.0 · 10 ⁹ cells / g. (see RU No. 2053205, IPC C02F 3/34, C09K 3/32, B09C 1/10, B09C 101/00, publ. 01.27.1996). As a carrier substrate for the immobilization of microorganisms use gamma-sterile peat with a pH of 6.8-7.0 in an amount of 40-45 wt. % and water in the amount of 58.95-53.25 mass. % In order to maintain oil-oxidizing activity during storage, nutrient substrates are additionally introduced into the preparation: ammonium oxalate (0.05-1.0 wt.%) And normal paraffins (1.0-1.5 wt.%).

The disadvantage of this biological product is that the biological product bacteria utilize only normal oil paraffins (C12-C18), which is 10-20% of the oil composition. The composition of oil includes, in addition to normal paraffins, naphthenes, aromatic, polyaromatic, unsaturated, saturated, isoprenoid and other hydrocarbons, all of them are more toxic to wildlife than normal hydrocarbons, and without their biodegradation, oil pollution will be negligible. In addition, the biological product contains oil-oxidizing bacteria, which require a pH of 6.8-7.0 and a large amount of water (about 60% water) to maintain their vital functions, which reduces its effectiveness over time.

The problem to which the claimed invention is directed is expressed in eliminating these drawbacks and is to expand the range of products for cleaning soils and water from oil and oil products in a wide temperature range, from +5 to + 40 ° С.

The technical effect obtained when solving the problem is expressed in the preparation of a preparation for cleaning soils and water from oil and oil products.

To solve this problem, a preparation for cleaning soils and water from oil and oil products, containing, in mass. %: oil - 0.1; the biomass of bacteria, biodestructors of oil and oil products - 1.0; a binder component of 1.0 and an organomineral substrate carrier for biodestructors - up to 100.0. At the same time, the preparation contains hydrocarbon-oxidizing bacteria Bacillus subtilis SNBS-1 as biodestructors of oil and oil products. As a binder component - sodium carboxymethyl cellulose. As an organomineral substrate, the carrier contains a composite mixture of clinoptilolite, prepared wood sawdust and humus in the ratio, mass. %: clinoptilolite - 10.0; prepared wood sawdust - 10.0; humus - 80.0.

A comparative analysis of the characteristics of the claimed solution with the signs of analogues indicates the conformity of the claimed solution to the criterion of "novelty."

The drug differs from the analogue in that it contains Bacillus subtilis SNBS-1 bacteria as biodestructors of oil and oil products, capable of developing and degrading oil and oil products under a wide temperature range (from +5 to + 40 ° С); as a solid carrier substrate for biodestructors, it contains an organomineral composition based on clinoptilolite, prepared wood sawdust and humus. As a binder component contains sodium carboxymethyl cellulose.

Characterization of the strain used to obtain the drug.

The proposed bacterial strain Bacillus subtilis SNBS-1 was isolated on February 16, 2015 from frozen soil samples contaminated with diesel fuel (diesel fuel) taken near the containers of the Momsky Distribution Zone of Sakhaenergo OJSC, Northeast Yakutia, Momsky District, Honuu Village.

The strain was identified and deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), Federal State Unitary Enterprise GosNIIgenetika, 117545, Moscow, 1st Dorozhny pr-d, 1, with assignment of a registration number to VKPM B-12239.

Initial screening using the GenBank and RDP-II database showed that the studied strain belongs to the following systematic groups: Bacteria; Firmicutes Bacilli; Bacillales; Bacillaceae; Bacillus.

According to the results of the analysis of sequencing of variable regions of genes encoding 16S rRNA (99%), as well as PCR analysis using species-specific primers, the test strain is classified as Bacillus subtilis.

The resulting strain is characterized by the following features.

Morphological features

Gram-positive spore bacilli located in random clusters.

Cultural signs

On meat peptone agar (MPA) (wt.%): Enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2, forms yellow rounded colonies with a diameter of up to 5 mm. The consistency is soft, easily removed from the surface of the agar, easily smeared.

On nutrient agar based on a fish meal hydrolyzate (wt.%): A fish meal hydrolyzate - 1.2; enzymatic peptone - 1.2; sodium chloride - 0.6; agar - 1.0; distilled water - the rest; pH 7.1 - 7.5, forms yellow rounded colonies with a diameter of up to 1-3 mm. The consistency is soft, easily removed from the surface of the agar, easily smeared.

On the environment of Saburo (wt.%): Hydrolyzed fish meal - 1.0; casein pancreatic hydrolyzate - 1.0; yeast extract - 0.2; monosubstituted sodium phosphate - 0.2; D-glucose 4.0; agar - 1.0; distilled water - the rest; pH 6.0 ± 3, forms opaque yellowish colonies with a diameter of 1-5 mm. The consistency is soft, easily removed from the surface of the medium, easily smeared.

In meat and peptone broth (wt.%): Enzymatic peptone - 1.0, sodium chloride - 0.5, meat water - the rest; pH 7.0-7.2, form a haze and film on the surface of the broth.

On the mineral environment of Münz (see Kersten D.K. Morphological and cultural properties of indicator microorganisms of oil and gas imaging. - Microbiology, 1963, No. 5. - S.1024-1030) with oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate, gasoline) of the following composition: (wt.%) KNO₃- 0.4; MgSO_four7H₂O - 0.08; NaCl - 0.1; K₂HPO_four- 0.14; Kh₂PO_four- 0.06; agar - 2.0; oil or petroleum products - 1.0; distilled water - the rest, pH 7.2, grows in the form of grayish-yellow convex colonies with a diameter of 1 mm.

Physiological and biochemical characteristics

The aerobic strain grows at a temperature of +5 to + 40 ° C, grows more intensively at + 16 ... + 37 ° C. It grows at pH 5.5–9.0. The optimum pH is 6.0–8.0. It grows in salt broth with the addition of 0.1-2.0% NaCl.

Used carbon sources: sorbitol.

Proven unused carbon sources: lactose, glucose, sodium citrate, sodium malonate. The strain is indole negative. The Voges-Proskauer reaction is negative. Phenylalanine deoxaminase is negative. Does not ferment beta galactosidase. Disposes of inositol. Product synthesized by strain: surfactants capable of degrading oil. Uses petroleum hydrocarbons and oil products as an energy source.

The strain is not toxic to higher plants. Biotesting was carried out on the seeds of common oats and oil radish.

The strain can be stored in a lyophilized state in sealed ampoules and in test tubes on beveled meat and peptone agar at a temperature of + 4 ° C.

Characteristics of the raw materials used for the preparation of an organomineral carrier substrate for immobilizing bacteria

To prepare a solid carrier substrate for the immobilization of the Bacillus subtilis SNBS-1 strain, a composite mixture of clinoptilolite, prepared wood sawdust and humus based on animal waste (cow manure) is used.

The main advantages of an organomineral carrier substrate are to increase soil fertility, improve soil structure, activate biological degradation of oil pollution, reduce the acidity of the soil while reducing the amount of traditional mineral fertilizers used up to 90%. The organomineral carrier substrate has a high biological activity due to the presence of humic and fulvic acids, which significantly increases the nutrient intake of the plant, activates the soil microflora, the synthesis of proteins and carbohydrates, chlorophyll and vitamins, and increases the resistance of plants to the effects of high doses of agrochemicals and petroleum hydrocarbons.

The adsorption and ion-exchange properties of clinoptilolite, which is part of the organomineral composition, as well as the content of a significant amount of plant nutrition elements in them, determine their use as a raw material for the production of substrates. The clinoptilolite crystal lattice is formed by tetraids with a well-developed inner surface, which provides the sorption ability of the mineral, allowing sorbing oil, oil products and other mechanical pollutants not only to the carrier surface, but also to remain sorbed substances inside the crystal lattice.

In the manufacture of the composite mixture, pre-crushed clinoptilolite of the Khonguruu deposit (Suntarsky district, Western Yakutia) with a zeolite content of 60 ... 95% and with a chemical composition of SiO ₂ 65.01-70.45% is used; TiO ₂ 0.16-0.18%; Al ₂ O ₃ 10.99-12.00%; Fe ₂ O ₃ 0.70-0.99%; MnO 0-0.02%; MgO 0.77-3.76%; CaO 0.52-3.73%; Sr O 0.14-0.24%; Na ₂ O 0.44-3.87%; K ₂ O 0.9-11.63%; H ₂ O 12.96-11.34%; P ₂ O ₅ 0.01-0.21% (see TU 2163-001-5248924-2006. Zeolite tuff (Honguruu deposit). Technical conditions).

Khongurinsky clinoptilolite may contain (mass%) as an impurity: Cd — 0.00422; Pb - 0.00280; V - 0.00190; Cu 0.00065; Mo - 0,00020; Co - 0.00010; Be - 0.00003; As - 0.00000; F is 0.00000; Hg - 0.00000; Sb - 0.00000; Sn is 0.00000; Zn - 0.00000 (see Technological passport for zeolites of the Khonguruu (Hongurin) deposit. Additive to the diet of cattle, pigs and poultry. Ministry of Agriculture and Food of the Republic of Sakha (Yakutia), Yakutsk: Firm Hongurin, 1993. - S. 3-4).

In the absence of clinoptilolite from the Honguruu deposit, the use of natural zeolite raw materials from other deposits of a similar chemical composition is allowed.

The composition of the composite mixture used wood sawdust obtained by sawing coniferous trees on cutting equipment, previously prepared in accordance with the standard (see GOST 18320-78 technological sawdust for hydrolysis. Technical conditions), and humus obtained from cow manure in accordance with standards (see GOST R 53117-2008 Organic fertilizers based on livestock waste. Specifications; GOST 26074-84 (ST SEV 2705-80). Liquid manure. Veterinary and sanitary requirements for processing, storage s, transportation and use).

Cow Manure (Cow Manure) - is a means of improving the structure of the soil and improving the ability of the soil to retain nutrients in a form accessible to plants.

According to physical and chemical indicators, cow dung should correspond to the values of table 1.

A 0.9-1.0% aqueous solution of sodium carboxymethyl cellulose (CMC) is used as a binder component and means for encapsulating the substrate carrier (see TU 6-55-39-90. Purified sodium carboxymethyl cellulose).

CMC is a white powdery or fibrous non-toxic product with the chemical formula [C ₆ H ₇ O ₂ (OH) ₃ -x (OCH ₂ COOH) x] n. It is soluble in both cold and hot water, forming a sol. An aqueous CMC solution has pseudoplasticity.

Sanitary and hygienic indicators of raw materials should be within the acceptable standards established by the requirements (see Unified Sanitary and Epidemiological and Hygienic Requirements for Products Subject to Sanitary and Epidemiological Surveillance (Control) (approved on 05.28.10 No. 299, Chapter II, Section 19)).

Technology of preparation

The technology for producing the drug includes the main stages:

1) heat treatment and grinding of the components to obtain a substrate carrier;

2) preparation of an aqueous solution of sodium carbomethyl cellulose;

3) mixing the components;

4) obtaining the biomass of bacteria Bacillus subtilis SNBS-1;

5) the immobilization of bacteria Bacillus subtilis SNBS-1 on a substrate carrier;

6) pressing the resulting mixture with removal of excess moisture and granulation with a grain size of 0.5-0.8 cm, followed by drying to achieve a residual moisture content of 3-5%;

7) packaging of the drug.

Obtaining the biomass of bacteria Bacillus subtilis SNBS-1

To obtain biomass for the immobilization of a solid support substrate, agar cultures of the Bacillus subtilis SNBS-1 strain are used.

Cultures of Bacillus subtilis SNBS-1 grown on a chamfered timing agar of industrial production, the basis of which is a fish meal hydrolyzate or meat production peptone agar (MPA) of industrial production, washed with sterile water, a suspension is prepared in the required amount. The concentration of cells in suspension should be 1⋅10 ⁹ cells / cm ³ according to the optical standard GISK them. A.M. Tarasevich. This suspension is a seed used for inoculation into a fermenter to increase the volume to the amount necessary to obtain the drug.

The preparation of seed consists of the following stages:

1. Obtaining a culture of Bacillus subtilis SNBS-1 on oblique agar.

2. Growing culture of Bacillus subtilis SNBS-1 in a small sowing machine.

3. Growing a culture of Bacillus subtilis SNBS-1 in a large sowing apparatus.

4. The accumulation of culture cultures of Bacillus subtilis SNBS-1 in a small fermenter.

The transfer of pure cultures of Bacillus subtilis SNBS-1 from one apparatus to another is carried out at the end of the logarithmic growth phase. The quality of the seed obtained is controlled by microscopy.

Fermentation of the culture of Bacillus subtilis SNBS-1

For fermentation of the Bacillus subtilis SNBS-1 culture, an F-300V type fermenter manufactured by CJSC Elite-Complex NPS is used (see Certificate of Conformity No. C RU AI 16. A. 08334 dated March 15, 2012 Ural Certification and Testing Center LLC Ural Certificate ”).

First, a mineral nutrient medium is prepared in the fermenter. For this, 240 l of clean filtered water at room temperature is poured into the fermenter, nutrients of the following composition are added (wt.% To water): KNO ₃ - 0.4; MgSO ₄ · 7H ₂ O - 0.08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ - 0.06.

Crude oil of at least 1% (by volume) is added as an energy substrate. Close the lid of the fermenter and turn on stirring. Then the pH of the medium is adjusted. To do this, a sample of the nutrient medium is taken through a rope through a syringe, the pH is measured and doses (10-20 ml) of either a 10% aqueous acid solution (HCl) or an alkali solution (NaOH) are injected through the same channel.

After that, inoculum is introduced into the fermenter in a volume of 10% of the volume of the nutrient medium, the pump is turned on and the tap is opened. In the process of cultivation, the operation of the fermenter is controlled, the set temperature, the degree of aeration and foaming, the condition of the support systems.

If necessary, a chemical defoamer (diesel fuel) is introduced into the fermenter through a tap, samples of the culture fluid are taken for control.

At the end of the cultivation process, the accumulated biomass of bacteria Bacillus subtilis SNBS-1 is poured into sterile containers and used to immobilize a solid carrier substrate.

Immobilization of Bacillus subtilis SNBS-1 on a substrate — carrier

As a carrier substrate for immobilizing the biomass of Bacillus subtilis SNBS-1 cells, a pre-prepared organomineral composition based on clinoptilolite, wood sawdust and humus in the ratio, mass is used. %: clinoptilolite - 10.0; prepared wood sawdust - 10.0; humus - 80.0.

For immobilization using liquid biomass of Bacillus subtilis SNBS-1 cells obtained by fermentation in a volume of 10% of the volume of the substrate carrier. The prepared components are mixed in a mixing device type BS-180 at 50-60 rpm. After mixing and impregnating the carrier substrate with the biomass of the Bacillus subtilis SNBS-1 cells, it is dried by the contact method at a temperature of 37 ° C to constant weight or at room temperature for 2 days. After which the drug is ready for use. The dry preparation is placed in kegs or two-layer plastic bags and sealed.

The parameters of the drug should correspond to the values of table 2.

The use of the drug is relevant in the following areas:

- the introduction of actively degraded oil selected strains of DRR (into a polluted ecosystem) and the management of this process;

- stimulation of the natural hydrocarbon-oxidizing microflora of oil-contaminated substrates involved in the biodegradation of oil pollution;

- optimization of the processes of biological degradation of oil pollution in order to achieve a high effect in the cleaning and post-treatment of oil-contaminated substrates.

Directions for use of the drug

For cleaning soil from oil pollution

When using the drug, it is necessary to take into account the content of trace elements in the soil (local geochemical background) according to the standards (see GN 2.1.7.2041. Maximum allowable concentrations (MAC) of chemicals in the soil).

The drug is applied to the soil without preliminary preparation (in the form in which it entered the object) using

- special equipment (seeders, manure spreaders, fertilizer spreaders, etc.) for large volumes of work;

- manually with small areas of oil pollution.

The drug is recommended to be applied at an average daily soil temperature of + 5 ... + 40 ° C. Permissible acidity is pH 4.5-9.0. Soil moisture in the range of 40-60%. The recommended time interval between introducing a biological product into the soil is 30-45 days. The consumption rates of the drug are presented in table 3.

After 30-45 days, re-sampling of soil samples for the content of residual amounts of oil and oil products is carried out. This analysis will determine the need for re-processing.

The quality of soil purification from oil pollution is controlled by the gravimetric method (see RD 52.18.647-2003 Determination of the mass fraction of oil products in soils. Measurement method by gravimetric method).

For cleaning water bodies from oil pollution

The drug is introduced into water bodies without preliminary preparation (in the form in which it entered the object) using:

- special equipment (seeders, manure spreaders, fertilizer spreaders, etc.) for large volumes of work;

- manually with small areas of oil pollution, at the rate of 1 kg of the drug per 0.1 cubic meter of water.

The drug is recommended to be applied at daily average water temperatures above + 5 ... + 37 ° C. Permissible acidity is pH 4.5-9.0. The recommended time interval between introductions of a biological product is 30-45 days.

After 30-45 days, re-sampling of water for the content of the residual amount of oil and oil products is carried out. This analysis will determine the need for re-processing.

The quality of water purification from oil pollution is monitored by the IR photometric method (see FR.1.31.2010.07127 MVI01.02.161 "Methodology for measuring the mass concentration of oil products in drinking, natural and waste waters by the IR spectrometric method using the oil product concentrator IKN-025"; RD 52.24 .476-2007 Mass concentration of oil products in waters. Measurement technique by IR-photometric method).

Disposal

Disposal of substandard waste and screenings of raw materials is carried out in accordance with the instructions of SanPiN 2.1.7.1322-03, requirements for environmental protection and applicable laws, as well as local authorities (see SanPiN 2.1.7.1322-03 Hygienic requirements for the disposal and disposal of production waste and consumption). Where possible, waste generation should be avoided or minimized. Disposal of an untreated drug is carried out according to the requirements for substances of hazard class 4.

Requirements for production and safety conditions

Premises for working with microorganisms must comply with the Sanitary and Epidemiological Rules (see SP 1.3.2322-08 Safety of working with microorganisms of the III-IV pathogenicity (danger) groups and pathogens of parasitic diseases).

The invention is illustrated by the following examples.

Example 1. Cleaning the soil from oil pollution using the drug.

Experiments with the use of the drug were carried out on sandy loam soil contaminated with oil. The initial content of oil products (NP) in the soil prior to application of the drug ranged from 2780 to 2960 mg / kg (see table 4).

The effectiveness of the drug was evaluated by the degree of degradation of oil pollution according to the instructions (see RD 39-0177098-015-90 "Instructions for monitoring the state of soils at the facilities of enterprises of the Ministry of Oil and Oil Industry").

For this, soil samples before and after treatment with a biological product were subjected to extraction with carbon tetrachloride in a Soxhlet apparatus, after which the extractant was removed by distillation with a reflux condenser, bringing the residue to a constant weight. The moisture content of the samples was determined by the generally accepted method (see GOST 28268-89 “Soils. Methods for determining moisture, maximum hygroscopic humidity”), after which the content of oil products was recalculated on dry soil.

It was established that the degree of biodegradation of oil and oil products in experimental samples 45 days after a single soil treatment with the obtained preparation was at a temperature of + 5 ± 1 ° C - 28.1-29.0%; at a temperature of + 20 ± 1 ° C - 71.9-72.2%; at a temperature of + 30 ± 1 ° C - 72.2-73.4%; at a temperature of + 40 ± 1 ° C - 63.6-65.2% (see table 4).

Thus, the obtained results indicate the effectiveness of the drug for cleaning soils from oil pollution.

Example 2. Water purification from oil pollution using the drug

For the experiment, 3.0 dm ^{3 of} tap water settled for 24 hours and 0.03 L of the preparation were added to a desiccator with a volume of 5.0 dm ³ . To create an oil film in the tank, 2880 mg / dm ^{3 of} oil and oil products were added. As a control used water in an amount of 3.0 DM ³ with the addition of 2880 mg / DM ³ oil and without making the drug.

The experiment was carried out for 45 days, maintaining the water temperature in the range + 15 ... + 20 ° С. The results showed that the destruction of oil for 45 days under the influence of the drug was 59.0% for the variant with oil; 62.1% - with diesel fuel; 64.6% - with kerosene; 65.3% - with motor oil; 61.8% - with hydraulic oil (see table 5).

Thus, the advantage of the drug is that, due to the combination of the unique properties of the organomineral carrier substrate and the Bacillus subtilis SNBS-1 bacterial strain immobilized on it, it has the ability to stimulate biological degradation of oil pollution in soils and water.

The method of preparation and use of the proposed drug is economically viable, since it does not require sophisticated technological equipment for its implementation, and the components of the carrier substrate are both affordable and cheap raw materials.

Table 1

Physico-chemical characteristics of cow manure

Name of indicator Normative Consistency (phase state), visual inspection Loose Mass fraction of dry matter,%, not less than 85 Mass fraction of organic matter, on dry matter, not less than fifty pH 6.0-8.5 Total nitrogen, with initial humidity, not less than 0.6 Total phosphorus (in terms of P ₂ O ₅ ), with initial humidity 0.5 Total potassium (in terms of K ₂ O), with initial humidity 0.6 The content of heavy metals, mg / kg dry matter:
- lead
- arsenic
- cadmium
- mercury
130.0
10.0
2.0
2.1 Sanitary indicative microorganisms, cells / g:
- coliforms
- enterobacteria
- index of pathogenic microorganisms
- cysts of intestinal pathogenic protozoa, ind./100 g
1-9
1-9
Are absent
Are absent

table 2

Biological and physico-chemical properties of the drug

Test Nomenclature Normative Appearance, color
Granules from brown to black with inclusions of yellow (from sawdust) and light green (from clinoptilolite) Granule size mm 0.5-0.8 The ratio of components,%:
- zeolite
- sawdust
- carboxymethyl cellulose
- biomass of biodestructors
- manure (litter)
10
10
one
one
rest Sorption capacity, kg / kg 2-3 The moisture content of the sorbent,% 3-5 Sanitary and bacteriological indicators:
- coliform bacteria index
- enterococcal index
- index of pathogenic microorganisms
- cysts of intestinal pathogenic protozoa, ind./100 g
1-9

1-9
Are absent
Are absent Oil oxidizing activity,% not less

Table 3

The consumption rate of the drug

The concentration of oil pollution in the soil The consumption of the drug, l / kg of soil % mg / kg kg / 1 kg of soil kg / 10 kg of soil kg / 100 kg of soil kg / 1000 kg of soil 1-5 10,000 - 50,000 0.01 - 0.05 0.1-0.5 1.0-5.0 10.0-50.0 5-10 50,000 - 100,000 0.05 - 0.1 0.5-1.0 5.0-10.0 50.0-100.0 10-30 10,000 - 300,000 0.1 - 0.3 1.0-3.0 10.0-30.0 100.0-300.0 30-100 300,000 - 1,000,000 0.3 - 1.0 3.0-10.0 30.0-100.0 300.0-1000.0

Table 4

The destruction of petroleum products in the soil under the influence of the drug

Experience outline The content of NP, mg / kg Degradation of NP,
% before cleaning after cleaning Temperature + 5 ± 1 ° С Soil + oil 2960 2940 0.7 Soil + oil + B.subtilis SNBS-1 2960 2720 8.1 Soil + oil + organic mineral substrate 2960 2520 14.8 Soil + oil + drug 2960 2100 29.0 Soil + diesel fuel 2920 2900 0.7 Soil + diesel fuel + B.subtilis SNBS-1 2920 2640 10.6 Soil + diesel fuel + organic mineral substrate 2920 2520 13.7 Soil + diesel fuel + preparation 2920 2100 28.1 Temperature + 20 ± 1 ° С Soil + oil 2920 2820 2.0 Soil + oil + B.subtilis SNBS-1 2920 1300 53.9 Soil + oil + organic mineral substrate 2920 2480 15.1 Soil + oil + drug 2920 820 71.9 Soil + diesel fuel 2880 2820 3.4 Soil + diesel fuel + B.subtilis SNBS-1 2880 1300 8.1 Soil + diesel fuel + organic mineral substrate 2880 2390 17.0 Soil + diesel fuel + preparation 2880 800 72,2 Temperature + 30 ± 1 ° С Soil + oil 2860 2794 2,3 Soil + oil + B.subtilis SNBS-1 2860 1150 53.8 Soil + oil + organic mineral substrate 2860 2490 12.9 Soil + oil + drug 2860 780 72.7 Soil + diesel fuel 2860 2800 2.06 Soil + diesel fuel + B.subtilis SNBS-1 2860 1220 56.4 Soil + diesel fuel + organic mineral substrate 2860 2440 14.7 Soil + diesel fuel + preparation 2860 760 73,4 Temperature + 40 ± 1 ° С Soil + oil 2800 2760 1.42 Soil + oil + B.subtilis SNBS-1 2800 1270 53.9 Soil + oil + organic mineral substrate 2800 2460 12.1 Soil + oil + drug 2800 1020 63.6 Soil + diesel fuel 2760 2700 1,1 Soil + diesel fuel + B.subtilis SNBS-1 2760 1240 54.9 Soil + diesel fuel + organic mineral substrate 2760 2400 13.0 Soil + diesel fuel + preparation 2760 960 65,2

Table 5

The dynamics of the destruction of oil in water after making the drug

Experience option The content of the NP, mg / DM ³ Destruction of NP,
% to experience after experience Control (water + oil) 2880 2740 4.86 Water + Oil + Drug 2880 1180 59.0 Water + diesel fuel + Drug 2880 1090 62.1 Water + Kerosene + Drug 2880 1020 64.6 Water + gas condensate + Drug 2880 1000 65.3 Water + motor oil + Preparation 2880 1000 65.3 Water + hydraulic oil + Preparation 2880 1100 61.8

Claims (1)

A preparation for cleaning soils and water from oil and oil products, including, mass. %: oil - 0.1, biomass of bacteria-biodestructors of oil and oil products - 1.0, a binder component - 1.0 and an organic mineral substrate-carrier for biodestructors - up to 100.0, while the preparation contains hydrocarbon-oxidizing agents as biodestructors of oil and oil products bacteria Bacillus subtilis VKPM B-12239, as a binder component - sodium carboxymethyl cellulose, as an organomineral carrier substrate - a composite mixture based on clinoptilolite, prepared wood sawdust and humus in the ratio, mass. %: clinoptilolite - 10.0, prepared wood sawdust - 10.0, humus - 80.0.