RU2361686C2 - Biopreparation for cleaning soil and water from oil and oil products - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: proposed biopreparation contains aerobic oil oxidising microorganisms, mineral nourishing substrate, normal paraffins C₁₂-C₁₈ and a solid substrate-carrier. The said preparation contains aerobic oil oxidising microorganisms in form of an association of representatives of 4 species and 8 types: Bacillus cereus, B. subtilis, Actinomyces griseus, Act. glaucus, Pseudomonas fluorescens, Ps. mesentericus, Ps. denitrificans, Arthrobacter globiformis, extracted from oil from Western Siberia deposit in concentration of 2.5×10⁹ kl/g. The preparation has a substrate-carrier in form of spherosole and also contains glucose.

EFFECT: high-efficiency cleaning.

4 dwg, 4 tbl, 8 ex

Description

The invention relates to the oil industry, agriculture and the environment and can be used for the biological treatment of oil-contaminated soils and the surface of water areas.

Oil and oil products are one of the main sources of environmental pollution. Intensive research is being carried out all over the world to study the effect of oil pollution on wildlife and to search for ways to accelerate the destruction of oil polluting the soil and water. It was established that microorganisms catalyze the enzymatic decomposition of oil and oil products, which can be used to develop biological products for the purification of oil-contaminated soils and water.

It should be noted that the process of destruction of hydrocarbons (HC) of oil is carried out by microorganisms, however, the practical importance of microbial degradation is ultimately determined by the composition of the biological product, which ensures the viability of bacteria, their activity, as well as the manufacturability, storage, transportation and use.

Known bacterial drug "Putidul", designed to accelerate the decomposition of oil [A.S. SU 1076446, class C02F 3/34, 1984]. The manufacturing technology of the biological product provides for spray drying of a live bacterial culture, which causes injury to microbes and, as a result, their partial death and loss of necessary activity. 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 ° С, mixing, aeration, and all this for a long time (16-24 hours), which is quite enough to perform in the field complicated.

Known 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. As a carrier substrate for the immobilization of microorganisms, gamma-sterile peat with a pH of 6.8-7.0 in an amount of 40-45 wt.% And water in an amount of 58.95-53.25 wt.% Are used. 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 (RF patent No. 2053205, IPC С02F 3/34). 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 vital functions, which reduces its effectiveness over time, increases transport costs, and besides this, peat-based biological products drown in water, which also reduces the effectiveness of their action, especially when cleaning the oil-contaminated surface of open water bodies. A significant disadvantage of the known biological product, significantly limiting the scope of its application, is also the possibility of using it only in a narrow pH range of the medium (pH 6.8-7.0).

The objective of the invention is the creation of a highly effective dry bacterial preparation based on a solid carrier substrate, a mineral nutrient substrate and a tread that ensures the viability of sorbed bacterial hydrocarbon-oxidizing microflora with high destructive activity in a wide pH range, as well as an increase in shelf life and manufacturability.

The technical result is achieved by the creation of a biological product for the biological purification of oil-contaminated ecosystems, the components of which are: spherozole (waste from coal-fired power plants) as a sorbent carrier, biomass of hydrocarbon-oxidizing microorganisms isolated from oil and protector glucose immobilized on its surface.

A biological product for cleaning soil and water from oil and oil products, including aerobic oil-oxidizing microorganisms, mineral nutrient substrate, normal paraffins from C ₁₂ to C ₁₆ and a solid carrier substrate, characterized in that it contains Bacillus cereus, B associations as aerobic oil-oxidizing microorganisms subtilis; Actinomyces griseus, Act. glaucus; Pseudomonas fluorescens, Ps. mesentericus, Ps. denitrificans; Arthrobacter globiformis, isolated from oil from the Soviet field of Western Siberia at a concentration of 2.5 × ^{10 9} cells / g, contains spherozole as a carrier substrate and additionally contains glucose in the following ratio of components, wt.%:

Mineral nutrient substrate - 0.85-0.95;

Normal paraffins C ₁₂ -C ₁₆ - 0.6-0.7;

Glucose - 0.9-1.0;

Association of aerobic oil-oxidizing microorganisms - 0.06-0.08;

Spherozole - up to 100.

As a solid carrier substrate, spherozole (waste from thermal power plants) is used, which is hollow glass-crystalline aluminosilicate microspheres in the composition of fly ash formed during the high-temperature burning of coal at thermal power plants. The physical properties of spherozoles are shown in table 1.

The set of unique properties of the spherozole: low density (less water), small size, spherical shape, large specific surface, high hardness and melting point, chemical inertness, free flowability (flowability) make it possible to obtain an effective, lightweight, free-flowing biological product based on it It sinks in water and does not pollute the environment. Getting into contaminated soil, spherozole contributes to its structuring, increases oxygen access for soil microflora, which stimulates its oxidative processes.

The mineral nutrient substrate of the biological product consists of the following components, g: Na ₂ CO ₃ - 0.1, MgSO ₄ - 0.2, MnSO ₄ · 5H ₂ O - 0.02, KH ₂ PO ₄ - 0.5, Na ₂ HPO ₄ - 0.7, CuCl · 6H ₂ O - 0.01, NH ₄ Cl - 2.0, NaCl - 2.0.

As an aerobic oil-oxidizing microorganism, a biological product contains an association of hydrocarbon-oxidizing microorganisms in a concentration of not less than

2.5 × l0 ⁹ cell / g. The Association of microorganisms is isolated from oil from the Soviet field of Western Siberia and includes representatives of 4 genera and 8 species.

1. The genus Bacillus. Species: B. cereus, B. subtilis - spore-forming, highly resistant bacteria to adverse conditions. Aerobes, chemorganotrophs. Oxidase and catalase positive. During the oxidation of oil hydrocarbons, bio-surfactants and other metabolic products are actively formed.

2. Genus Actinomyces. Species: Act. griseus. Act. glaucus - belong to the class of radiant fungi and have a characteristic structure, spore-forming, assimilate mineral compounds, sugars and hydrocarbons. Catalase-positive, widespread in soil and water. Dispose of aromatic and saturated hydrocarbons.

3. The genus Pseudomonas. Species: Ps. fluorescens, Ps. mesentericus, Ps. denitrificans - do not form a dispute. Aerobes. Oxidase-and catalase-positive, widely distributed in contaminated soils, groundwater and open water bodies. They actively utilize a wide range of hydrocarbons, including aromatic ones.

4. Genus Arthrobacter. Species: Arth. globiformis - do not form a spore. Aerobes. Oxidase and catalase positive. They are part of the physiological group of hydrocarbon-oxidizing microorganisms, actively utilize acyclic saturated and polyaromatic hydrocarbons. Resistant to adverse conditions. Widely distributed in contaminated soil and water.

Each type of microorganism preferably oxidizes hydrocarbons of a specific structure. In the association, they are most fully capable of degrading polluting oils and oil products. The strains included in the association were selected on the basis of their ability to utilize hydrocarbons during cultivation in contact with oil in a wide pH range, which was manifested in an increase in the growth dynamics and activity of enzymes that catalyze oxidation processes.

Example 1. The incubation of the culture of Bacillus cereus with a certain initial number of cells is carried out in Erlenmeyer flasks with a capacity of 500 ml, containing 100 ml of mineral medium and oil (0.5% of the volume of medium). Instead of oil, you can use any of the liquid n-alkanes from C ₁₂ to C ₁₆ . As the mineral medium, a solution of the mineral substrate of the following composition is used, g / dm ³ : Na ₂ CO ₃ - 0.1, MgSO ₄ - 0.2, MnSO ₄ · 5H ₂ O - 0.02, KH ₂ PO ₄ - 0.5, Na ₂ HPO ₄ - 0.7 CuCl6H ₂ O - 0.01, NH ₄ Cl - 2.0, NaCl - 2.0. Since the process of destruction of oil hydrocarbons by microorganisms proceeds in the presence of oxygen, flasks with contents are placed on a rocking bath for mixing. The duration of oil incubation in contact with microorganisms is 5 days at a constant temperature of 27 ° C. Daily culture is carried out on agar media to determine the dynamics of the number of microorganisms. On the 5th day of cultivation, their number increased 100 times from 0.035 to 3.5 million cells / ml, which indicates the use of oil hydrocarbons - the only carbohydrate source of nutrition and energy. The ability of pure cultures to biodegradation of hydrocarbons is also evaluated by the increase in the activity of catalase and dehydrogenase, the oxygenase group enzymes that characterize the biological activity of microorganisms in an oil-contaminated environment. In this case, catalase activity increases from 0.21 to 0.43 ml / ml, dehydrogenase - from 0.63 to 1.3 mg / ml. The accumulated enzymes become stable catalysts for the oxidation of polluting hydrocarbons.

Example 2. The determination of the hydrocarbon-oxidizing properties of a Bacillus subtilis culture with an initial number of 0.0328 million cells / ml is carried out according to the scheme described above (example 1). On the 5th day, the number of cells increased to 4.2 million cells / ml, the activity of catalase in this case increased from 0.18 to 0.40 ml / ml, dehydrogenase from 0.50 to 1.02 mg / ml, which confirms the oxygenase activity of the Bacillus subtilis culture.

Example 3. The hydrocarbon-oxidizing properties of the culture of Actinomyces griseus are determined according to the scheme shown in example 1. The initial number of microorganisms is 0.05 million cells / ml. On the 5th day, the number increased to 4.14 million cells / ml, catalase activity - from 0.23 to 0.40 ml / ml, dehydrogenase - from 0.6 to 1.36 mg / ml, which indicates the oxidative activity of Act. griseus when cultured in contact with oil.

Example 4. The hydrocarbon-oxidizing properties of the culture of Actinomyces glaucus cells are determined according to the scheme shown in example 1. The initial number of microorganisms is 0.043 million cells / ml. On the 5th day, the number increased to 3.28 million cells / ml, catalase activity from 0.20 to 0.38 ml / ml, dehydrogenase from 0.55 to 1.09 mg / ml, which confirms the ability of Act. glaucus to utilize petroleum hydrocarbons.

Example 5. The hydrocarbon-oxidizing properties of the cell culture of Pseudomonas fluorescens is determined according to the scheme shown in example 1. The initial number of microorganisms is 0.025 million cells / ml. On the 5th day, the number increased to 3.14 million cells / ml, catalase activity from 0.18 to 0.42 ml / ml, dehydrogenase from 0.6 to 1.36 mg / ml, which indicates the oxidative activity of the culture during oil destruction.

Example 6. The hydrocarbon-oxidizing properties of the cell culture of Pseudomonas mesentericus is determined according to the scheme shown in example 1. The initial number of microorganisms is 0.064 million cells / ml. On the 5th day, the number increased to 7.19 million cells / ml, catalase activity - from 0.29 to 0.63 ml / ml, dehydrogenase - from 0.6 to 1.36 mg / ml, which indicates a high ability of the Ps culture. mesentericus to the disposal of oil.

Example 7. The hydrocarbon-oxidizing properties of the culture of Pseudomonas denitrificans cells are determined according to the scheme shown in example 1. The initial number of microorganisms is 0.052 million cells / ml. On the 5th day, the number increased to 5.8 million cells / ml, catalase activity from 0.24 to 0.5 ml / ml, dehydrogenase from 0.48 to 1.03 mg / ml, which indicates the oxidative activity of the Ps culture. denitrificans.

Example 8. The hydrocarbon-oxidizing properties of the Arthrobacter globiformis cell culture are determined according to the scheme shown in Example 1. The initial number of microorganisms is 0.0216 million cells / ml. On the 5th day, the number increased to 3.79 million cells / ml, catalase activity from 0.18 to 0.4 ml / ml, dehydrogenase from 0.6 to 1.5 mg / ml of soil, which indicates the high activity of the enzymes of the oxygenase group of the Arth culture. globiformis in the destruction of oil.

The sorption activity of hydrocarbon-oxidizing microorganisms on the surface of spherozoles was studied in model experiments under static (table 2) and dynamic (table 3) conditions in comparison with the well-known Czech-made L 40/100 silica gel sorbent.

From the results presented in table 2, it follows that the sorption activity of microorganisms on the surface of a spherozole at a pH of 3.4, 7.2 and 9.5 is comparable to the well-known expensive sorbent - silica gel, and from the results presented in table 3, it follows that the sorption activity of microorganisms at filtration through a spherozole layer is also not inferior to the sorption activity of a known sorbent.

In order to maintain the viability and catalytic activity of hydrocarbon-oxidizing microorganisms sorbed on the surface of a spherozole, the biological product contains a nutritious mineral substrate, normal paraffins and glucose as a protector that ensures cell viability in a dry biological product. The biological product is prepared as follows.

Example 9. To accumulate biomass of hydrocarbon-oxidizing microorganisms, 25 dm ^{3 of an} aqueous solution of a mineral substrate playing the role of a nutrient medium of the following composition, g / dm ^{3, is} introduced into a 50 dm ³ fermenter:

Na ₂ CO ₃ - 0.1

MgSO ₄ - 0.2

MnSO ₄ · 5H ₂ O - 0.02

KH ₂ PO ₄ - 0.5

Na ₂ HPO ₄ - 0.7

CuCl6H ₂ O - 0.01

NH ₄ Cl - 2.0

NaCl - 2.0.

The medium is inoculated with an association of microorganisms isolated from oil from the Sovetskoye field of Western Siberia and including representatives of 4 genera and 8 species. The initial concentration of microorganisms in the fermenter is 8 × 10 ⁶ cells / ml. Then 15 kg of spherozoles are introduced into the fermenter and 150 g of glucose and 100 g of hexadecane (nC ₁₆ ) or another hydrocarbon (n-alkane) are added as an energy source and an additional carbon substrate for microbial cells. Their propagation in the fermenter and sorption on the surface of the spherozole occurs at a temperature of 25-27 ° C for 5 days with constant stirring, which provides an additional flow of oxygen. Then the spherozole with immobilized microflora is separated from the liquid phase, dried in air and packaged in 5 kg paper bags. The cell concentration in 1 g of the finished biological product 2.5 × 10 ⁹ , the moisture content of the drug 3-5 wt.%, Shelf life 1 year.

The resulting preparation was tested in model experiments for the purification of oil-contaminated water and soil.

The oil concentration in the soil was 5 wt.%, In water - 3 wt.%. The drug was added at a concentration of 1.0 g / kg. During the experiment (30 days), the dynamics of the number of microorganisms was determined, the maximum of which in oil-contaminated soil for 10-12 days is 7.15 × 10 ⁹ cells / g, in water - 12.3 × 10 ⁹ cells / cm ³ (Table 4). Growth dynamics are consistent with the destructive activity of microorganisms. At the end of the experiment, the residual oil was extracted from the soil and water by chloroform extraction. Biodegradation of oil in the soil for 30 days was 75% (37.5 g), in water - 90% (27 g). The results of laboratory experiments on a model of water and soil contaminated with oil are presented in table 4.

Changes in the molecular weight distribution of acyclic saturated hydrocarbons (n-alkanes) of oil biodegraded under the conditions of the aqueous phase and soil are presented in comparison with the original oil (Fig.1, 2). Samples were analyzed by gas chromatography. The biodegradation coefficient of n-alkanes of polluting oil, determined by the formula (Pr + Ph) / (nC ₁₇ + nC ₁₈ ), is 6.0 for soil, 6.5 for water, and this indicator does not exceed 0.6 for initial pollution.

The results of model experiments clearly show that the use of the proposed biological product on the 30th day significantly reduces the level of oil pollution of water and soil by 90 and 75%, respectively.

Table 1 Physical properties of spherozoles The form spherical Color gray white True density 0.6-0.8 g / cm ³ Particle size 10-150 microns Bulk density 0.40 g / cm ³ Specific surface area 7.0 m ² / g Melting temperature 1300 ° C pH in water 6-8 Surface humidity 0.3% wt.

Table 3 Sorption of microbial cells of the genus Micrococcus under dynamic filtration conditions through columns filled with sorbents at pH 7.0 Learning tests The original number of microbes 300 million cells 180 million cells Sorbents Spherozole Silica gel Spherozole Silica gel The height of the sorbent in the column, cm 15.5 9.5 20.5 twenty Sorbent weight, g 2.5 2.5 2.5 2.5 The volume of microbial suspension for filtration, ml 10 10 10 10 Filtration rate, ml / min 0.13 0.15 0.12 0.14 The total number of cells in the microbial suspension after filter., Million cells. 5.3 4.83 5.9 0.28 The total number of sorbed cells, million cells. 294.7 295.17 174.1 179.72 Cell sorption per 1 g of sorbent, million cells / g 117.88 118.07 69.64 71.89

Claims (1)

Biological product for cleaning soil and water from oil and oil products, including aerobic oil-oxidizing microorganisms, mineral nutrient substrate, normal paraffins from C ₁₂ to C ₁₈ and a solid carrier substrate, characterized in that as an aerobic oil-oxidizing microorganisms it contains an association of 4 representatives genera and 8 species: Bacillus cereus, B. subtilis, Actinomyces griseus, Act. glaucus, Pseudomonas fluorescens, Ps. mesentericus, Ps. denitrificans, Arthrobacter globiformis, isolated from oil from the Soviet field of Western Siberia at a concentration of 2.5 · 10 ⁹ cells / g, contains spherozole as a carrier substrate and additionally contains glucose in the following ratio of components, wt.%:
Mineral nutrient substrate 0.85-0.95 Normal paraffins C ₁₂ -C ₁₈ 0.6-0.7 Glucose 0.9-1.0 Association of Aerobic Oil-Oxidizing Microorganisms 0.06-0.08 Spherozole Up to 100.

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