RU2636343C2 - Method for production of biopreparation for soil purification from oil and oil products - Google Patents

Abstract

FIELD: ecology.

SUBSTANCE: method for production a biological preparation for soil purification from oil and oil products is proposed. The method involves mixing and culturing of Acinetobacter calcoaceticus bacteria VKPM-B-3780, Acinetobacter radioresistens VKPM-B-2838, Acinetobacter radioresistens VKPM-B-5064 strains until the optical density of the suspension is OD=0.6-0.8, concentrating to 1-5×10¹¹ cells/ml, freeze drying at a residual pressure of 30-50 Pa, heat source temperature of 40°C to a residual moisture content of 0.5-1% and packaging in a sealed container in an inert gas environment.

EFFECT: invention allows to obtain a long-term storage preparation for efficient disposal of oil contaminants.

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Description

The invention relates to biotechnology and ecology and can be used for microbiological treatment of soils contaminated with oil and oil products.

Ecologically promising are microbiological methods of purification from oil pollution.

Significant efficacy of known preparations is characterized by compositions based on microorganisms of different taxonomic groups that selectively decompose various hydrocarbon fractions. However, the inclusion in the composition of a biological product of microorganisms of various species, genus affiliation, and even organisms from different domains (bacteria and yeast) requires separate cultivation, which significantly increases the cost of the preparation. In addition, there may be competition between such organisms for food sources, as a result of which not all of the species declared may survive.

Acinetobacter bacteria are recognized as one of the most effective oil destructors. They are used as a monoculture or as part of biological products. However, many of the currently existing biological products are either not highly effective, or they are complex and expensive to manufacture. In addition, representatives of Acinetobacter (A.) with an unclear taxonomic position are often included in such preparations.

Among domestic preparations for biodegradation of petroleum products containing representatives of the genus Acinetobacter, the biological product Ekoil is known [RF No. 2191752, IPC C02F 3/34, B09C 1/10, C12N 1/26; 1999] based on the bacteria Rhodococcus sp. 56e, Mycobacterium sp. IL 4, Mycobacterium flavescens VKPM B-6000, Acinetobacter sp. HB-1. There is also a method of microbiological cleaning of objects from oil pollution [RF No. 2067993, IPC C12N 1/20, C02F 3/34, B09C 1/10; 1996] based on strains of Acinctobacter sp. (bicoccum) VKPM B-6445, or Rhodococcus sp. BKPMS -1213, or Arthrobacter sp. S-1212. According to the formulas of the above inventions, these cultures are recommended for separate use, and the use of monoculture is less effective due to the specificity of the organism used to the substrate.

For the same reason, known individual strains of the Acinetobacter genus that are capable of degrading oil hydrocarbons [RF No. 2081910, IPC C12N 1/20, C02F 3/34, B09C 1/10, 1997; RF №2081909, IPC C12N 1/20, 1997; RF №2081908 IPC C12N 1/20, B09C 1/10, C02F 3/34, 1997; RF No. 2081907, IPC C12N 1/20, C02F 3/34, B09C 1/10, 1997; RF №2077579, IPC C12N 1/20, C02F 3/34, B09C 1/10, 1997; RF №2076150, IPC C12N 1/20, C02F 3/34, B09C 1/10, 1997; RF №2064017, IPC C12N 1/20, C02F 3/34, B09C 1/10, 1996].

A known consortium of bacterial strains Acinetobacter calcoaceticus, Pseudomonas fluorescens, Alcaligenes faecalis for the destruction of petroleum products [RF No. 2107722, IPC C12N 1/20, C02F 3/34, 1998]. Known biologically active composition for purifying surface water, soil and soil from oil pollution [RF No. 2270808, IPC C02F 3/34, B09C 1/10, 2003], including the association of oil-oxidizing bacterial cultures, peat and complex mineral fertilizer. It contains Pseudoamycolata halophobica VSB-753, Kibdelosporangium aridum VSB-754, Acinetobacter oleovorum VSB-712, Rhodococcus erytropolis HX7 in a volume ratio of 1: 1: 1: 1 as an association of oil-oxidizing bacterial cultures. The well-known drug "Lessorb-bio" [RF No. 2193533, IPC C02F 3/34, B09C 1/10, C12N 1/20, 1999] based on the bacteria Mycobacterium flavescens EX-91, Mycobacterium sp IZh4, Rhodococcus sp. 56e, Acinetobacter sp. HB-1). A known method of purifying water and soil from petroleum products [RF No. 2014286, IPC C02F 3/34, EBB 15/04, 1994] based on bacteria Candida tropicalis - Mycococcus lactis or Mycococcus lactis - Mycococcus lactis, or Candida guillermondii - Acinetobacter oleovorum subsp. paraphinicum. The disadvantage of all of the above methods is that all complex preparations containing bacteria of different taxonomic groups, in preparation require separate cultivation, which complicates and increases the cost of the preparation process.

A known method of cleaning environmental objects from oil products [RF No. 2053204, IPC C02F 3/34, ЕOВВ 15/04, 1996], based on the use of bacteria Acinetobacter valentis subsp. paraffinicum - VKPM-B-6727 H-1; Acinetobacter valentis subsp. paraffinicum VKPM-B-6728 G-1; Acinetobacter valentis subsp. paraffinicum VKPM-B-6726 C-2, which are part of the same genus.

However, the examples illustrate experiments only with low concentrations of petroleum products in a liquid medium or with sand contaminated by 0.5%. The disadvantage of the consortium is its low oxidative ability. In addition, all declared species and subspecies are not validated.

A known method of producing and using a biological product based on a consortium of strains of the genus Acinetobacter (B-2838, B-5064 and B-3780) in its composition [http: //biomolecula.content/948].

The disadvantage of this method is the lack of operational limits for the cultivation of the suspension, its concentration and freeze drying.

Closest to the claimed is a consortium of bacterial strains Acinetobacter calcoaceticus, Pseudomonas fluorescens, Alcaligenes faecalis for the destruction of oil and oil products [RF №2107722, IPC C12N 1/20, C02F 3/34, C12R 1/27, C12R 1/01; 1998], in the preparation examples of which its lyophilic form is indicated.

However, the disadvantage of this method is the inability to obtain a long-term storage preparation with a high concentration of viable microorganisms, to restore the functional activity of bacterial cells and to obtain a liquid form of the preparation with a different given degree of concentration, and also to provide the necessary conditions for the functioning of oil destructive bacteria in depleted soils, which does not allow for efficient disposal oil pollution.

An object of the invention was to obtain a long-term storage preparation with a high concentration of viable microorganisms, the ability to restore the functional activity of bacterial cells and to obtain a liquid form of the preparation with a different given degree of concentration, as well as to provide the necessary conditions for the functioning of oil destructive bacteria in depleted soils, to reduce surface and interfacial tension at bioremediation, as well as solubilization and emulsification of hydrocarbons, which allows It helps to efficiently dispose of oil pollution.

The technical task of the invention is achieved by the fact that in the method of obtaining a biological product for cleaning the soil from oil and oil products, which includes mixing and culturing the bacterial strains Acinetobacter calcoaceticus VKPM-B-3780, Acinetobacter radioresistens VKPM-B-2838, Acinetobacter radioresistens VKPM-B-5064 , the creation of a consortium on their basis, it is new that cultivation is carried out until the optical density of the suspension is OD = 0.6-0.8, concentration is up to 1-5 × 10 ¹¹ cells / ml, freeze drying at a residual pressure of 30-50 Pa , heat source temperature 40 ° С to the remainder oh 0.5-1% humidity and packaging in a sealed container under an inert gas atmosphere.

The technical result of the invention is to obtain a long-term storage preparation with a high concentration of viable microorganisms, the ability to restore the functional activity of bacterial cells and to obtain a liquid form of the drug with a different predetermined degree of concentration, as well as providing the necessary conditions for the functioning of oil destructive bacteria in depleted soils, in reducing surface interfacial tension during bioremediation, as well as solubilization and emulsification of hydrocarbon Dow, which allows efficient disposal of oil pollution.

Table 1 below shows the sensitivity of the bacterial strains A. calcoaceticus B-3780, A. radioresistens B-2838, A. radioresistens B-5064 to antibiotics and Table 2 of the results of the specific rate of destruction of oil products for consortia of microorganisms obtained in various ways.

The studied strains are able to use the following hydrocarbons as the sole source of carbon and energy:

A. calcoaceticus B-3780: decane, heptane, octane, hexadecane, undecane, cyclohexane, xylene, toluene, benzene, naphthalene, phenanthrene.

A. radioresistens B-2838: decane, heptane, octane, undecane, xylene, toluene, benzene, naphthalene, phenanthrene, anthracene.

A. radioresistens B-5064: decane, heptane, octane, undecane, cyclohexane, phenanthrene, anthracene, xylene, toluene, benzene, ethylbenzene.

Antibiotic sensitivity was studied using paper disks containing standard concentrations: ampicillin, cefoxitin, ceftazidime, cefatoxime, streptomycin, gentamicin, ofloxacin, ciprofloxacin, cefazolin, erythromycin, furagin, furadonin, levomycin, tetracytin. All studied strains were resistant to levomycetin, furadonin, erythromycin, cefazolin, ofloxacin and are sensitive to tetracycline, ciprofloxacin and gentamicin. Individual strains are sensitive to ampicillin, ceftazidime and streptomycin (Table 1).

All strains have bioemulsifying activity. Moreover, A. calcoaceticus produces an extracellular highly active surfactant that exhibits high emulsifying activity against petroleum products, hexadecane, and liquid paraffin. The A. radioresistans strains produce a biosurfactant alasan, a high molecular weight complex of proteins and polysaccharides that stabilizes various oil-water emulsions, including n-alkanes with a chain length of 10 or more carbon atoms, alkyl aromatic hydrocarbons, liquid paraffin and crude oil.

The strains are not demanding on the composition of the medium and are able to quickly accumulate biomass.

The strains are synergistic in the degradation of oil hydrocarbons. So, strain B-2838 predominantly breaks down C6-C22 paraffins, it was highly active in the destruction of gasoline, diesel fuel; strain B-5064 - n-paraffins, ethanol, crude oil, and strain B-3780 is also capable of degradation of methanol and formaldehyde. The synergism of the action of the selected strains is also manifested in the combination of the high emulsifying activity of strain B-3780 and the significant oil-oxidizing ability of strains B-2838 and B-5064.

A method of obtaining a biological product for cleaning the soil from oil and oil products involves mixing and cultivating the bacterial strains Acinetobacter calcoaceticus VKPM-B-3780, Acinetobacter radioresistens VKPM-B-2838, Acinetobacter radioresistens VKPM-B-5064, creating a consortium based on them. In this case, cultivation is carried out until the optical density of the suspension is reached OD = 0.6-0.8, concentration is up to 1-5 × 10 ¹¹ cells / ml, and then freeze drying at a residual pressure of 30-50 Pa, the temperature of the heat source is 40 ° C to residual humidity of 0.5-1% and packaging in sealed containers in an inert gas environment.

Example 1

To obtain the biomass of a consortium of bacterial strains VKPM-B-7179 strain 279, VKPM-B-7152 strain 325, VKPM-B-7180 strain 404 they are grown on N10 liquid medium of the following composition: (NH ₄ ) ₂ SO ₄ - 3 g / l , KH ₂ PO ₄ - 6.5 g / l, MgSO ₄ × 7H ₂ O - 0.7 g / l, FeSO ₄ × 7H ₂ O - 12.5 mg / l, ZnSO ₄ × 7H ₂ O - 12, 5 mg / L; MnSO ₄ × 5H ₂ O - 12.5 mg / L.

As the only carbon source, n-paraffins of the Grozny oil refinery, obtained by urea dewaxing with a high content of aromatic hydrocarbons at a concentration of 1 wt. % Inoculation of the mineral medium is carried out at 0.05 optical density units of each strain in one rocking flask. Cultivation takes place at a temperature of 3-6 ° C on a rocking chair with 140 rpm, a pH of 6.8-7.0.

Example 2

The strains A. calcoaceticus B-3780, A. radioresistens B-5064 and A. radioresistens B-2838 are separately cultivated on solid nutrient medium for 24 hours at a temperature of 6 ° C. The composition of the solid medium: yeast extract - 5.0%; peptone - 15.0%; NaCl - 5.0%; agar-agar - 15%.

Further, the cultures are reseeded separately in a liquid nutrient medium composition: yeast extract - 5.0%; peptone - 15.0%; NaCl - 5.0%. Inoculum cultivation occurs at 6 ° C for 48 hours at 30 ° C.

For the manufacture of a biological product, MS nutrient medium of the following composition is used (g / l): yeast extract - 5.0; peptone - 15.0; NaCl - 5.0. Inoculate with cell suspensions of separately grown strains of A. calcoaceticus B-3780, A. radioresistens B-5064 and A. radioresistens B-2838. Suspensions are introduced into the bioreactor in a ratio of 1: 1: 1, with a total amount of 5.6% of the medium volume. Co-cultivation is carried out at a temperature of 6 ° C, constant stirring and aeration until the optical density of the suspension reaches OD = 0.6-0.8.

Next, concentration is achieved to 1-5 × 10 ¹¹ cells / ml by centrifugation or ultrafiltration, freeze drying at a residual pressure of 30-50 Pa, a temperature of a heat source of 60 ° C to a residual moisture content of 0.5-1% and packaging in sealed containers in an environment inert gas.

Example 3

Strains A. calcoaceticus B-3780, A. radioresistens B-5064 and A. radioresistens B-2838 are separately cultivated on solid nutrient medium for 24 hours at a temperature of 30 ° C. The composition of the solid medium: yeast extract - 5.0%; peptone - 15.0%; NaCl - 5.0%; agar-agar - 15%.

Further, the cultures are reseeded separately in a liquid nutrient medium composition: yeast extract - 5.0%; peptone - 15.0%; NaCl - 5.0%. Inoculum cultivation occurs at 30 ° C for 48 hours at 30 ° C.

For the manufacture of a biological product, MS nutrient medium of the following composition is used (g / l): yeast extract - 5.0; peptone - 15.0; NaCl - 5.0. Inoculate with cell suspensions of separately grown strains of A. calcoaceticus B-3780, A. radioresistens B-5064 and A. radioresistens B-2838. Suspensions are introduced into the bioreactor in a ratio of 1: 1: 1, with a total amount of 5.6% of the medium volume. Co-cultivation is carried out at a temperature of 30 ° C, constant stirring and aeration until the optical density of the suspension reaches OD = 0.6-0.8.

Then, concentration is achieved to 1-5 × 10 ¹¹ cells / ml by centrifugation or ultrafiltration, freeze drying at a residual pressure of 30-50 Pa, a temperature of a heat source of 40 ° C to a residual humidity of 0.5-1% and packaging in sealed containers in an environment inert gas.

The results of the specific rate of destruction of oil products according to the examples are shown in Table 2 (table. 2).

The resulting consortium of microorganisms allows for the efficient use of a biological product for cleaning the soil from oil and oil products according to a scheme that includes resuspension of a biological product, application of Acinetobacter calcoaceticus VKPM-B-3780, Acinetobacter radioresistens VKPM-B-2838, Acinetobacter radioresistens VKPM-B-2838, Acinetobacter 5064 Resuspension of the biological product is carried out with water at a temperature of 35 ° C, for 8-12 hours, and then salts of nitrogen, phosphorus and potassium are added as sources of nutrients, after which the suspension is inoculated with the soil bacteria Acinetobacter calcoaceticus VKPM-B-3780, Acinetobacter radioresistens VKPM-B-2838, Acinetobacter radioresistens VKPM-B-5064, creating a starting concentration of oil destructors in the range of 10 ⁸ -10 ¹⁰ cells / g of soil. After applying the microbial suspension, the soil is further mixed.

When using the proposed consortium for cleaning oil and petroleum products, the amount and composition of an aqueous solution of mineral nutritional components is selected depending on the application conditions according to the concentrations of the main nutrients that determine the development of the proposed bacterial strains, as well as taking them into account in the environment of the object being cleaned.

The advantages of the method are that:

- the implementation of co-cultivation to achieve the optical density of the suspension OD = 0.6-0.8, concentration to 1-5 × 10 ¹¹ cells / ml, freeze drying at a residual pressure of 30-50 Pa, the temperature of the heat source 40 ° C to residual moisture 0.5-1% and packaging in sealed containers in an inert gas environment allows you to get a long-term storage product with a high concentration of viable microorganisms.

- obtaining a lyophilized form of the drug allows, by subsequent resuspension of the biological product with water at a temperature of 35 ° C for 8-12 hours, to restore the functional activity of bacterial cells and to obtain a liquid form of the drug with a different given degree of concentration;

- the introduction of salts of nitrogen, phosphorus and potassium into the resuspended biological product as sources of nutrients provides the necessary conditions for the functioning of oil destructive bacteria in lunch soils;

- the possibility of creating an optimal starting concentration of oil destructors in the range of 10 ⁸ -10 ¹⁰ cells / g of soil with additional mixing of the soil allows efficient disposal of oil pollution.

Claims (1)

A method of obtaining a biological product for cleaning the soil from oil and oil products, including mixing and culturing the bacterial strains Acinetobacter calcoaceticus VKPM-B-3780, Acinetobacter radioresistens VKPM-B-2838, Acinetobacter radioresistens VKPM-B-5064, creating a consortium based on them the fact that the cultivation is carried out until the optical density of the suspension is achieved OD = 0.6-0.8, concentration is up to 1-5 × 10 ¹¹ cells / ml, freeze drying at a residual pressure of 30-50 Pa, the temperature of the heat source is 40 ° C to a residual humidity 0.5-1% and packaging in sealed containers in the environment and ertnogo gas.