RU2615464C1 - Preparation for soils and water bodies purification from oil products - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: preparation is represented by Bacillus subtilis SNBS-1 hydrocarbon oxidizing bacteria biomass immobilized on expanded vermiculite at 1:1 ratio.

EFFECT: invention allows to reduce terms of water and soil purification from oil and oil products.

7 tbl, 6 ex

Description

The invention relates to microbiology and biotechnology, namely the production technology of preparations intended for the purification of soils and water from oil pollution.

The well-known biological product “Putidul” (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 was created on the basis of 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).

The disadvantage of the drug is the complex technology of its preparation, which provides for spray drying of a live culture of bacteria, which causes injury to the 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 soils from oil and oil products contaminants, in which a consortium of oil-oxidizing microorganisms Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. 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).

The disadvantage of this method is that, together with a suspension of a biological product, rhizotorfin (a fertilizer of nitrogen-fixing microorganisms) is introduced into the contaminated medium in an amount of 30-120 g / m ² , which complicates the processing of an oil-contaminated substrate.

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. This method involves the storage of a suspension of microorganisms using NaCl, biologically active substances (vitamins) and osmoprotectors (betaine) (see RU No. 2114071, IPC C02F 3/34, B09C 1/10, C12N 1/20, C12N 1/26, C12Q 1/02, publ. 06/27/1998).

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 bacterial preparation is known, consisting of highly active living aerobic oil-oxidizing bacteria (Mycobacterium, Pseudomonas, etc.) grown on solid carrier substrates with titers of 2.5-7.0-10 ⁹ cells / g. 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 shelf life of a biological product is 6 months at a temperature of 10-15 ° C from the moment of its manufacture (see RU No. 2053205, IPC C02F 3/34, C09K З / 32, В09С 1/10, В09С 101: 00, published on 01/27/1996 )

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.

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 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 herbs (see RU No. 2193464, IPC B09C 1/10, C12N 1/26, C12N 1/26, C12R 1:01, publ. 27.11 .2002).

However, the above method involves the use of solid-liquid phase 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 (the 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 (in liquid and dry forms) for cleaning environmental objects from hydrocarbon pollution, which is a consortium of microorganisms consisting of bacterial strains: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAU-2 VKPM V- 11370, Shinella granuli BAK-PAH-3 VKPM B-11371, taken in equal proportions (see RU No. 2535978, IPC C12N 1/20, C02F 3/34, B09C 1/10, C12R 1/01, publ. 20.12. 2014). The biological product is used in liquid form or in the form of immobilized cell biomass. To obtain an immobilized form of a biological product, the consortium is deeply cultivated on a nutrient medium to produce biomass and further immobilized cell biomass on organic or mineral carriers (including vermiculite). The biological product is highly effective in the utilization of hydrocarbon contaminants.

The invention lacks information about the technology for obtaining a dry form of a biological product, parameters and preparation conditions and its use.

The disadvantage of the liquid form of the biological product is its short shelf life (up to 60 days), which makes it economically unprofitable.

Another disadvantage is that during the cultivation of seeds for the preparation of both liquid and dry forms of the biological product, the introduction of oil into the nutrient medium is not provided, which would ensure a high concentration of microorganisms with activated enzyme biodegradation systems in the working suspension. In addition, bacterial strains in the composition: Rhodococcus qingshengii BAK-PAH-1 VKPM AC-1946, Pusillimonas ginsegisoli BAK-PAH-2 VKPM B-11370, Shinella granuli BAK-PAH-3 VKPM B-11371, which are the basis of the biological product, are not capable develop in a wide temperature range (from +5 to +40 degrees C), which makes the biological product not very promising for use in various climatic conditions.

The closest analogue to the invention is the "Method of obtaining a biological product for the purification of sea water from oil", including expanded vermiculite, which was previously immobilized biomass strain Phyllobacterium myrsinacearum VKPM B-9079, cultivated in a medium with peptone, sucrose, a source of phosphorus, potassium, magnesium , subsequent cultivation of this strain on a medium with sucrose corn extract, a source of phosphorus, potassium, magnesium, dilution of the resulting concentrate with sterile distilled water, to which dissolved (g / l) suspension concentrate of the bacterial - 100.0; molasses - 20.0; K2HPO4 - 1.5; KH2PO4 - 1.5; MgSO ₄ - 1.5, and aging for 3-5 days at 20-25 ° C (see RU No. 2404138, IPC C02F 3/34, C12N 1/26, publ. 20.11.2010).

The difference between the claimed invention from the disclosed source (see RU No. 2404138, IPC C02F 3/34, C12N 1/26, publ. 11/20/2010) is the use of the bacterial strain Phyllobacterium myrsinacearum DKS-1, which is the basis of the biological product, which includes a more complex technology and composition a nutrient medium for culturing the strain than those in the claimed invention, which complicates and makes more expensive the technology of obtaining an analogue of a biological product. And also the fact that the analogue of a biological product provides only for the purification of sea water and does not allow to evaluate the destructive ability of a biological product with respect to soil. In addition, the bacterial strain Phyllobacterium myrsinacearum DKS-1 grows at a temperature of 22 to 30 ° C, an optimum of 28 ° C, and is not able to develop at low positive temperatures (+ 5 ° C), which makes it unpromising for oil cleaning in areas with a short thermal period, which can also be confirmed by another patent for the Phyllobacterium myrsinacearum strain used for the destruction of petroleum hydrocarbons in brackish-water ecosystems, where it is confirmed that the strain is cultivated at a temperature of 22 to 30 ° C, an optimum of 28 ° C (see RU No. 2268934, C12N 1/20, C02F 3/34, publ. 01/27/2006).

The difference of the claimed invention from the disclosed sources (see RU No. 2404138, IPC C02F 3/34, C12N 1/26, publ. 11/20/2010; RU No. 2535978, IPC C12N 1/20, C02F 3/34, B09C 1/10, C12R 1/01, published on December 20, 2014) is that the claimed preparation includes vermiculite of the Inagli deposit swollen in accordance with GOST 12865-67 “Expanded Vermiculite” as a carrier substrate.

The objective of the invention is to expand the range of drugs, including a simple and inexpensive technology for their preparation and effective not only for water purification, but also for cleaning soils from oil pollution, containing hydrocarbon-oxidizing microorganisms as biodegradants of oil pollution, capable of developing and degrading oil and oil products in a wide range of conditions temperatures (from +5 to + 40 ° C), which will allow the drug to be used for cleaning soils and water in various climatic conditions.

The technical effect obtained in solving this problem is expressed in the creation of a preparation for cleaning soils and the aquatic environment from oil and oil products, including a solid carrier substrate and biomass of hydrocarbon-oxidizing bacteria immobilized on its surface. The claimed preparation may contain the remains of a nutrient medium used for the cultivation of bacteria.

To solve this problem, a preparation for cleaning soil and water from oil and oil products includes a solid carrier substrate and a biomass of hydrocarbon-oxidizing bacteria Bacillus subtilis SNBS-1 immobilized on its surface at a concentration of 1 × ^{10 9} cells / cm ³ cultivated in a mineral nutrient medium.

At the same time, vermiculite of the Inagli deposit, expanded according to GOST 12865-67 “Expanded Vermiculite”, serves as a solid carrier substrate, and a mixture of nutrient components (wt.%): 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; oil - 1.5.

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

The features of the characterizing part of the claims provide the solution to the following functional problems.

Distinctive features of the proposed drug from the analogue are: the use of vermiculite from the Inagli deposit, expanded according to GOST 12865-67 - “Expanded Vermiculite”, as a carrier substrate for bacteria and cells of the Bacillus subtilis SNBS-1 bacterial strain as an oil pollution biodestructor capable of developing and degrading oil and oil products in the soil and in water, in a wide temperature range (from +5 to + 40 ° C). The resulting preparation allows, in a relatively short time, to efficiently clean the soil and water from oil and oil products.

The bacterial strain Bacillus subtilis SNBS-1 was isolated on February 16, 2015 from frozen soil samples contaminated with diesel fuel, taken near the containers of the Momsky Distribution Zone of OJSC Sakhaenergo, North-East of 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 collection number B-12239.

Initial screening using a database of GenBank and RDP-II showed that the studied strain belongs to the following systematic groups of Bacteria; Firmicutes Bacilli; Bacillales; Bacillaceae; Bacillus, moreover, the homology with some species of the genus Bacillus is 99%.

When sequencing the variable regions of 16S rDNA, the following assembled nucleotide sequence was obtained for the studied strain:

The results of processing sequences using a computer program located on the RDB II website (Ribosomal Database Project II), designed to determine the affinity of microorganisms and build phylogenetic trees, are presented in graphical form:

S000003473 0.986 0.940 1423 Bacillus subtilis (T); DSM10; AJ276351 S000006847 0.986 0.942 1435 Bacillus mojavensis (T); IFO15718; AB021191 S000012241 0.988 0.992 1440 Bacillus vallismortis (T); DSM11031; AB021198 S000014133 0.988 0.969 1426 Bacillus atrophaeus (T); JCM9070; AB021181 S000428475 0.988 0.935 1337 Bacillus subtilis (T); NRRL B-23049; AF074970 S000734915 0.986 0.960 1389 Bacillus amyloliquefaciens (T); NBRC 15535; AB255669 S001239910 0.980 0.903 1355 Bacillus methylotrophicus (T); CBMB205; EU194897 S001576866 0.981 0.965 1433 Bacillus siamensis (T); PD-A10; GQ281299 S002234727 0.984 0.926 1374 Bacillus tequilensis (T); 10b; HQ223107 S002287710 0.980 0.887 1469 Bacillus licheniformis (T); ATCC 14580; DSM 13; CP000002 S004067890 0.986 0.976 1453 Bacillus amyloliquefaciens (T); DSM7; FN597644

According to the analysis of RDP II 16S rRNA, a phylogenetic tree was constructed with homologous strains of Bacillus subtilis SNBS-1 (see figure).

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 signs.

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 a yellow, rounded colony, 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 - P. 1024-1030) with oil and oil products (diesel fuel, motor oil, hydraulic oil, gas condensate , gasoline) of the following composition (wt.%): 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; agar - 2.0; oil or petroleum products - 1.0-1.5; 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 rapidly + 16 + 37 ° C. It grows at pH 5.5-9.0. Optimum pH 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 maintained by regular reseeding (once every 10-14 days) on beveled meat and peptone agar or stored in a lyophilized state in ampoules at a temperature of + 4 ° С.

Characterization of the substrate carrier.

According to many authors, natural mineral materials are preferable for immobilization of DRR. At the same time, the sorbent carrier used to immobilize the cells of hydrocarbon-oxidizing bacteria (BDS) should have the following basic properties: permeability, oil capacity, non-caking, porosity and flowability.

Vermiculite of the Inagli deposit, expanded in accordance with GOST 12865-67 “Expanded Vermiculite”, most fully meets these requirements.

Vermiculite is a naturally hydrated mica. It is a laminated plate of golden yellow or brown color with a bulk density of 100-200 kg / m ³ .

Hydration of the primary phlogopite begins from a depth of 100-115 m and ends with the formation of vermiculite. Minerals of the best technological quality are developed on surfaces. World production of vermiculite is about 500-600 thousand tons per year, including in Russia - about 35-40 thousand tons (see Zedgenizov V.G. Efficiency of using multi-module modifications of electric furnaces for firing vermiculite / V.G. Zedgenizov, A .I. Nizhegorodov // Building materials, 2009. - No. 12. P. 51-53 .; Ostrovsky G.M. Pneumatic transport of bulk materials in the chemical industry / G.M. Ostrovsky D .: Chemistry, 1984. - 104 p. .; Rybiev I.A. Construction materials science: textbook for construction specialties of universities / I.A. Rybiev. M .: Higher. school., 2003 .-- 701 p.).

In Russia, the most famous deposits of vermiculite ores are: Kovdorskoye (Murmansk region), Tatarskoye (Krasnoyarsk Territory), Koksharovskoye (Primorsky Territory), Buldymskoye and Potaninskoye (Chelyabinsk Region), Uluntuyskoye and Slyudyanskoye (Irkutsk Oblast), Inaglinsky (Republic of Sakha ( Yakutia)) and others. However, due to poor equipment with special equipment, the production of vermiculite and materials based on it in Russia is only beginning to develop and is still limited (see Nizhegorodov A.I. Vermiculite and vermiculite technologies: research, production, application / A.I. Nizhegorodov Irkutsk: Publishing house BusinessStroy, - 2008. - 96 p.).

Expanded vermiculite is of multifunctional value and is used in the construction industry as a tile insulation, heat and sound insulating material, porous filler for light concrete and plaster mortars, for filtering the effluent of power plants and enterprises; in agriculture - as an ameliorant for the restoration of land, etc.

As the carrier sorbent in the claimed preparation for purification of soils and water bodies from oil and oil products for immobilization of DRR, vermiculite of the Inagli deposit is used.

Vermiculite of the Inagli deposit (Aldan region, South Yakutia) is associated with the weathering crust of chrome diopside-mica metasomatites formed along the dunites of the Inaglinsky pluton (see A. Korchagin, Inaglinsky pluton and its minerals. - M., Nedra, 1996. - 160 pp.).

The chemical composition of the vermiculite of the Inagli deposit is presented in table 1.

The oil capacity of the expanded vermiculite of the Inagli field for oil and oil products is presented in table 2 (see Kondratieva N.I., Gabysheva RI, Novgorodov P.G. Vermiculites of the Inagli field (Yakutia) for cleaning water surface and soil from oil pollution // Materials I-st international conference: The value of industrial minerals in the world economy: deposits, technology, economic estimates - Moscow, January 31 - February 3, 2006 - M., GEOS, 2006. - P. 101).

Thus, the vermiculite of the Inagli deposit, expanded according to GOST 12865-67 “Expanded Vermiculite”, is a promising carrier sorbent used for immobilization of DRR cells, since it possesses the basic properties listed above, namely: permeability, oil capacity, non-caking, porosity and flowability ; It combines the properties of a carrier for microorganisms and a sorbent for oil, which ensures high efficiency and prolonged degradation of petroleum hydrocarbons.

Based on the above stated research results, a preparation was prepared for cleaning soils and water from oil pollution.

A brief description of the drug for cleaning soils and the aquatic environment from oil pollution.

The components of the drug are: a solid carrier substrate, the biomass of oil destructors immobilized on its surface, which differs from analogues in that:

- as oil destructors it contains the biomass of hydrocarbon-oxidizing bacteria (BSS) of Bacillus subtilis SNBS-1 at a concentration of 1 × 10 ⁹ cells / cm ³ , isolated from frozen soil samples contaminated with diesel fuel, by the accumulative culture method in Munz medium with oil, followed by transfer to MPA ;

- as a solid substrate substrate contains vermiculite deposits Inagli, expanded to a fraction of 0.6-20 mm with a chemical composition (%): SiO ₂ 33,28-36,53; TiO ₂ 0.58-0.64; Al ₂ O ₃ 6.21-13.00; Fe ₂ O ₃ 6.21-6.43; FeO 0.28-0.28; MnO traces ... 0.02; MgO 24.75-25.12; CaO 0.65-1.01; K ₂ O traces ... 0.09.

As an impurity, the claimed preparation may contain the remains of a nutrient medium used in the cultivation of bacteria.

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.

The technology for obtaining a drug for cleaning soils from oil pollution includes the following steps:

1. Obtaining uterine culture.

To obtain uterine cultures, agar cultures of the Bacillus subtilis strain SNBS-1 are used. This crop is a seed used for sowing in fermenters.

To obtain a uterine culture, the following mineral composition is used (mass%): KNO ₃ - 0.4; MgSO ₄ ⋅ 7H ₂ O - 0.08; NaCl - 0.1; K ₂ NRA ₄ - 0.14; KH ₂ PO ₄ - 0.06; oil - 1.0-1.5; distilled water - the rest; pH is 7.2. The amount of seed should be 5-10% of the volume of the nutrient medium.

Isolate cultures grown on mowed 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 physiological saline, and a suspension is prepared. The concentration of cells in suspension should be 1 × 10 ⁹ cells / cm ^-3 according to the optical standard GISK them. AM Tarasevich; volume - not less than 25-50 cm ³ .

2. Cultivation of seed in flasks.

A nutrient medium of the same composition (see item 1) is poured into 150 cm ³ into flasks with a capacity of 500 cm ³ , 15-25 cm ^{3 of a} bacterial suspension of the Bacillus subtilis SNBS-1 strain prepared by flushing are inoculated. 1.5 cm ^{3 of} sterile oil is added there. Crops in flasks are incubated in UVMT 12-250 for 72 hours at 180-200 rpm, at a temperature of +5 to + 29 ± 1 ° C.

3. Obtaining seed for immobilization.

The cultivation of the Bacillus subtilis strain SNBS-1, which forms the basis of the drug, is carried out on the same medium; sterile crude oil is used as the sole carbon source; the concentration of hydrocarbon-containing products in the fermentation liquid is 1.0-1.5% (by volume). The fermentation process is carried out with vigorous stirring at a temperature of + 29 ± 1 ° C for 36-48 hours.

At the end of the cultivation process, the accumulated liquid is poured into sterile containers and used to immobilize the carrier sorbent (expanded vermiculite prepared according to GOST 12865-67).

4. Immobilization of the strain of Bacillus subtilis SNBS-1 on a sorbent carrier.

The fluid produced during the cultivation of the Bacillus subtilis strain SNBS-1 (see p. 3) is mixed in an apparatus with an agitator with a carrier sorbent (expanded vermiculite) of a fraction of 0.6-20 mm, in a 1: 1 ratio. After impregnation of vermiculite expanded with seed, it is dried by contact at a temperature of 37 ° C or at room temperature for 2 days to a moisture content of not more than 3% (Table 3). The biological product is ready for use. Dry biological product is placed in kegs or two-layer plastic bags and sealed.

The parameters and characteristics of the biological product must correspond to the values of table 3.

5. Directions for use of the drug.

5.1. The use of the drug for cleaning soils 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 consumption rates of the drug are presented in table 4.

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.

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 cleaning from oil pollution is controlled by the gravimetric method (see RD 39-0177098-015-90 “Instructions for monitoring the state of soils at the facilities of enterprises of the Ministry of Oil and Gas Industry”).

5.2. The use of the drug to purify water 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 1-2 liters of the drug per 1 m ^{2 of the} water surface.

The drug is recommended to be applied at average daily water temperatures above + 5 + 40 ° 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).

6. 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.

7. Requirements for the conditions of production and safety.

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).

Work on equipment when working with microorganisms and the manufacture of a sorbent carrier should be allowed to persons who have reached the age of 18 years who have been trained.

All employees must undergo periodic medical examinations in the manner prescribed by the health authorities (see Order of the Ministry of Health of Russia dated 14.03.96 No. 90 “On the Procedure for Preliminary and Periodic Medical Examinations of Workers and Medical Regulations for Admission to the Profession”; Order of the Ministry of Health and Social Development of Russia of 08.16.04 No. 83 “On approval of lists of harmful and (or) hazardous production factors and work, during the implementation of which preliminary and periodic medical examinations (examinations) are conducted, and the procedure how to conduct these examinations (examinations) ”(as amended on May 16, 2005).

Compliance with safety requirements must be ensured by complying with the relevant approved safety instructions and rules when carrying out appropriate work.

8. Acceleration of terms of restoration (soil remediation).

The self-healing process of oil-contaminated soil ecosystems in regions with favorable climatic conditions takes 10-25 years, while the destruction of oil and its derivatives in the North is at least 50 years (see Oborin A.A., Kalachnikova I.G., Maslivets T.A. et al. Biological reclamation of oil-contaminated lands in the taiga zone // Restoration of oil-contaminated soil ecosystems: Collection of scientific papers, Moscow: Nauka, 1988. - P. 140-159).

For the accelerated reclamation of oil-contaminated soils, the following measures must be observed:

1) oil-contaminated soils must be mixed with a clean fertile soil layer or peat;

2) according to the results of agrochemical studies (if necessary) - the introduction of mineral components in the form of fertilizers that are missing in the soil in the estimated doses. Usually, in agriculture, for basic, pre-sowing and local application, when sowing or for feeding plants, nitrogen-phosphorus-potassium fertilizers are used (nitroammophores, ammofoska or azofoska). These fertilizers are universal and are used on all types of soils and under all agricultural crops. With the main application on chernozem and heavy clay soils, it is advisable to apply fertilizers in the fall, and on light soils in the spring. Application rates depend on soil fertility and average 35–45 kg per hectare;

3) increase in the frequency of soil treatment with the drug up to 3-4 times for 1 growing season;

4) loosening the soil at least 1 time per week;

5) maintaining optimal moisture and soil pH;

6) sowing of perennial oil-tolerant grasses (fescue, creeping wheatgrass, Siberian wheatgrass, meadow timothy grass, field sow thistle, beechless campfire, meadow clover).

The invention is illustrated by the following examples.

Example 1. Isolation and cultivation of a strain of Bacillus subtilis SNBS-1.

The bacterial strain Bacillus subtilis SNBS-1 was isolated from frozen soil samples contaminated with diesel fuel by the method of accumulative cultures in a Munz medium with oil, followed by reseeding on MPA.

For this, 1.0 g of frozen soil from the above object was added to 250 ml of the Muntz mineral medium of the following composition (wt.%): 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; oil - 1.0-1.5; distilled water - the rest; pH 7.2. At the first stage, the incubation was carried out under rocking conditions at 200 rpm and a temperature of + 20 ± 1 ° С. Bacterial growth was observed after 3 days of incubation by the formation of a turbid emulsion c and the disintegration of the oil layer.

A pure bacterial culture of Bacillus subtilis SNBS-1 was obtained by culturing under the conditions listed above and repeated reseeding (more than 10) of the accumulation culture on Petri dishes with meat peptone agar (MPA).

Further, crops were incubated under stationary conditions at various temperatures from +5 to + 40 ° C. After 72 hours, the appearance of pasty shiny yellow colonies with a diameter of up to 5 mm was observed on the MPA surface, which according to the cultural-morphological and biochemical characteristics, as well as the results of the analysis of the nucleotide sequences of the 16S rRNA gene and key phenotypic characters according to the taxonomic descriptions given in Bergi's determinant (see Bergey's Manual of Systematic Bacteriology Book Review Int. J. of Syst. Bact .; July 1985, p. 408) has been identified as a strain of Bacillus subtilis.

Example 2. Preparation of a bacterial suspension from cells of a strain of Bacillus subtilis SNBS-1.

The isolated colonies obtained in example 1 were subcultured on a beveled MPA and incubated for 48 hours at a temperature of + 20 ± 1 ° C. Bacillus subtilis SNBS-1 cultures grown on MPA were washed off with 0.9% NaCl; prepared the initial microbial suspension of the bacterial isolate with a titer of 1 × 10 ⁹ microbial cells / cm ³ according to the optical standard GISK them. AM Tarasevich.

To obtain a bacterial suspension, a liquid mineral medium was prepared according to Example 1 and cultured in flasks under rocking conditions for 48 hours, at a temperature of 20 ± 1 ° C, at 200 rpm. The resulting liquid mixture of cells of the Bacillus subtilis strain SNBS-1, with a titer of 1 × 10 ⁹ cells / cm ^{3 was} used to study the oil-oxidizing activity of the strain.

Example 3. The study of the oil-oxidizing activity of a strain of Bacillus subtilis SNBS-1.

Cells of the Bacillus subtilis strain SNBS-1 are washed off with 0.9% NaCl solution from beveled MPA. Prepare the original microbial suspension of the bacterial isolate, with a volume of 25 cm ³ , with a concentration of 1 × 10 ⁹ microbial cells / cm ³ according to the optical standard GISK them. AM Tarasevich.

The obtained bacterial isolate of the Bacillus subtilis strain SNBS-1 was cultured in an aqueous medium with the mineral composition of Example 1 under constant aeration.

100.0 cm ^{3 of the} finished mineral medium and 1000 mg of oil are introduced into cones with a volume of 250.0 cm ³ . Flasks are seeded with strain cells to a concentration of 1 × ^{10 9} cells / cm ³ . As a control, put the same flask with medium, oil and without bacteria to determine the total (natural) losses. The experiment is carried out in three repetitions. Flasks are cultivated at different temperatures (from + 5 ° C to + 40 ° C) for 3-5 days.

The experimental data show that in the aquatic environment with mineral components the proposed strain already utilizes 4.8-5.4% of oil and oil products already on the 3rd day at a temperature of + 5 ° C; at a temperature of + 20 ° C - 57.2-57.8%; at a temperature of + 30 ° C - 62.6-63.7%, at a temperature of + 40 ° C - 52.2-52.8%; depending on the type of xenobiotic (see table 5).

Example 4. Obtaining a preparation for cleaning soils and water bodies from oil and oil products.

To obtain a preparation for cleaning soils and water objects from oil and oil products, seed material is prepared from the Bacillus subtilis strain SNBS-1. For this, the Bacillus subtilis SNBS-1 strain grown on a chamfered timing agar of industrial production, the basis of which is a fish meal hydrolyzate, or meat-and-peptone agar (MPA) of industrial production, is washed off with 0.9% physiological saline (NaCl) or sterile water in the required volume (but not less than 25-50 cm ³ ). The concentration of cells of the strain of Bacillus subtilis SNBS-1 in seed should be 1 × 10 ⁹ cells / cm ^-3 according to the optical standard GISK them. AM Tarasevich.

The resulting seed material is introduced into flasks with a previously prepared mineral nutrient medium of the following composition (wt.%): 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; oil - 1.0-1.5; distilled water - the rest; pH is 7.2. The amount of seed should be 5-10% of the volume of the nutrient medium. Crops in flasks are incubated on the installation "UVMT 12-250" for 72 hours at 180-200 rpm

In the next step, fermentation of the grown strain of Bacillus subtilis SNBS-1 is carried out.

For this, the required volume of clean water at room temperature is poured into the F-300V brand fermenter, after which mineral elements are added to the water (wt.%, Of the water volume): 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 and oil - 1.0-1.5%.

Then the pH of the medium is adjusted to the level of 7.2. For this, a sample of the mineral medium is taken, the pH is measured and, if necessary, 10-20 ml of either a 10% aqueous acid solution (HCl) or an alkali solution (NaOH) is introduced into the container with the mineral medium.

After that, seed material is introduced into the mineral medium containing cells of the Bacillus subtilis strain SNBS-1, obtained in the UVMT 12-250 installation according to the above method, in the amount of 10% of the total volume of the mineral medium in the fermenter. Close the lid of the fermenter. The material is ready for fermentation. The fermentation process is carried out with vigorous stirring at a temperature of + 29 ± 1 ° C for 36-48 hours.

In the fermentation process, the operation of the fermenter is controlled, the set temperature, the degree of aeration and foaming, the condition of the support systems.

At the end of the work of the fermenter, the accumulated liquid containing Bacillus subtilis SNBS-1 cells is poured into sterile containers and used to immobilize the carrier sorbent (pre-prepared expanded vermiculite, according to GOST 12865-67). The process of immobilization of vermiculite with the obtained biomass is carried out at room temperature, in a device with a BS-180 type mixer, at 29 ± 1 rpm., For 30 minutes, in a ratio of 1: 1 (1 part of the biomass of Bacillus subtilis SNBS-1 cells accumulated in the fermenter for 1 part of expanded vermiculite). For example, to immobilize 1 liter of biomass, 1 liter of expanded vermiculite is needed; to immobilize 10 l of biomass of oil destructors, 10 l of expanded vermiculite is necessary; to immobilize 100 l of biomass, 100 l of expanded vermiculite is necessary, etc.). The resulting mixture was left at room temperature for 24-48 hours to fix Bacillus subtilis SNBS-1 cells on vermiculite. After that, the resulting biological product is dried by contact at a temperature of 37 ° C or at room temperature for 2 days to a moisture content of 3%. Dry biological product is placed in kegs or two-layer plastic bags and sealed. The drug is ready to use.

The parameters and characteristics of the biological product must correspond to the values of table 3.

Example 5. 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. The effectiveness of the drug was evaluated by the degree of degradation of oil pollution in accordance with the instructions RD 39-0177098-015-90.

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 ° С - 26.0-28.7%; at a temperature of + 20 ± 1 ° С - 69.1-70.0%; at a temperature of + 30 ± 1 ° C - 61.670.6-71.4%; at a temperature of + 37 ± 1 ° С - 70.8-71.2%; at a temperature of + 40 ± 1 ° С - 57.8-62.6% (see table 6).

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

Example 6. Purification of water from oil pollution using the drug.

For the experiment, 3.0 dm ^{3 of} tap water settled for one day 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 + 20 ± 2 ° C. The results showed that the destruction of oil for 45 days under the influence of the drug was 54-59% (see table 7).

Thus, the advantage of the drug is that, thanks to the combination of the unique properties of the carrier substrate (expanded vermiculite) and the Bacillus subtilis SNBS-1 bacteria immobilized on it, it has the ability to stimulate the purification of soils and water from oil and oil products.

The method of preparation and use of the proposed drug is economically viable, since it does not require complex technological equipment for its implementation. And vermiculite, which serves as a carrier substrate in this method, is both affordable and cheap raw materials, since vermiculites have a surface occurrence and are developed in an open way. In Russia, the most famous deposits of vermiculite ores are: Kovdorskoye (Murmansk region), Tatarskoye (Krasnoyarsk Territory), Koksharovskoye (Primorsky Territory), Buldymskoye and Potaninskoye (Chelyabinsk Region), Uluntuyskoye and Slyudyaninsky (Irkutsk Oblast), Inaglinsky (Republic of Sakha ( Yakutia)), etc.

Purification of soil and water from oil pollution, the proposed drug can prevent the spread of pollutants to the associated landscapes and to eliminate stains of contaminated soil, which significantly improves the sanitary and environmental condition of the disturbed area.

The proposed preparation for cleaning soil and water from oil pollution is environmentally friendly, since the basis for its preparation is natural formations - a vermiculite mineral and a non-pathogenic bacterial strain Bacillus subtilis SNBS-1, which is effective not only for water treatment, but also for cleaning soils from oil pollution, in a wide range of temperatures (from +5 to +40 degrees C, see table 5-6), which allows the drug to be used in various climatic conditions.

Claims (1)

A preparation for cleaning soils and water objects from oil and oil products, including the biomass of hydrocarbon-oxidizing bacteria Bacillus subtilis SNBS-1 immobilized on expanded vermiculite at a ratio of 1: 1.

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