RU2393215C2 - Eco-biological prepration for purifying water from oil products - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used for treating water bodies contaminated with oil and oil refining products. The biological preparation is a biodestructor cell culture which is artifically immobilised on a sorbent-carrier. The sorbent-carrier is in form of regular hollow spherical particles with diametre of 30-350 mcm, wall thickness of 2-10 mcm, mainly filled with nitrogen and carbon dioxide, and having the following composition (wt %): SiO₂ - 50-60; Al₂O₃ - 25-35; Fe₂O₃ - 1.8-2.0; CaO - 1-5; MgO - 0.5-1.5; Na₂O - 0.3-1.5; K₂O - 0.2-2.9. The biodestructor is the Pseudomonas fluorescens VKPM 6844 strain. The method of obtaining this biological preparation involves submerged culturing of the Pseudomonas fluorescens VKPM 6844 strain on a fluid medium, preparation of the said sterile sorbent-carrier and surface culturing of the biodestructor on the said sorbent-carrier.

EFFECT: eco-biological preparation for purifying water from oil products enables localisation of spills owing to active bonding of oil products by the sorbent-carrier, their active recycling using the biodestructor on the water surface, which increases ecological cleanness of the cleaning after-effects.

2 cl, 2 tbl, 2 ex

Description

The invention relates to the field of biotechnology and can be used to clean natural reservoirs contaminated with oil and products of its processing.

Ecobiological product is a cell culture of a microorganism - a destructor of oil products immobilized on a sorbent carrier. Aluminosilicate microspheres [1, 2, 3, 4] are used as a carrier sorbent, which are hollow spherical particles of regular shape with a diameter of 30 to 350 μm and a wall thickness of 2 to 10 μm, the inner cavity of which is filled mainly with nitrogen and carbon dioxide , the following composition (wt.%):

SiO ₂ - 50 ÷ 60; Al ₂ O ₃ - 25 ÷ 35; Fe ₂ O ₃ - 1.8 ÷ 2.0; CaO - 1 ÷ 5; MgO - 0.5 ÷ 1.5; Na ₂ O - 0.3 ÷ 1.5; K ₂ O - 0.2 ÷ 2.9,

and as the biodestructor of petroleum products, the strain Pseudomonas fluorescens VKPM 6844 is used.

Analogs of the invention are ecobiological preparations intended for the purification of oil-contaminated water bodies: Putidul, Avalon, Ekonadin and others.

A known method of purification of water from petroleum products using ecobiological product "Econadin" (development Kozhanova GA, UA), designed to purify water from oil pollution [5]. The basis of this ecobiological product are cells of the natural strain Pseudomonas fluorescens 2-a immobilized on sphagnum peat.

Despite the high rate of oil product disposal, the disadvantage of this eco-biological product is that after adsorption of the oil film, it settles to the bottom of the reservoir, causing significant damage to benthic organisms. This method of cleaning the water surface from oil products cannot be considered effective and environmentally friendly.

The closest to the invention in terms of the essential features of the Avobalon ecobiological product (developed by I. Limbach et al., St. Petersburg), where foamed glassy metaphosphates of variable composition are used as a porous carrier, and Serracia marcescens PL- strains of oil-oxidizing microorganisms are used as biodestructors 1, Pseudomonas fluorescens biovar II 10-1, Acidovorax delafieldii 3-1, whose cells are immobilized in the pores of the carrier [6].

Despite the fact that this ecological product provides optimal conditions for the immobilization and vital activity of microorganism cells on the porous structure for a long time, the disadvantage of this environmental preparation is the low rate of utilization of oil products.

Table 1 presents data on the composition of ecobiological preparations - analogues, conditions and the effectiveness of their use [5-8].

The ecological preparations shown in Table 1, after applying them to the surface of the water contaminated with oil products, either settle to the bottom of the reservoir or have a low rate of oil-oxidizing activity.

The purpose of the invention is the creation of a new eco-biological product that can quickly localize a spill of an oil product with high hydrocarbon-oxidizing activity and the ability to stay on the water surface for a long time in the zone of maximum pollution, which allows more efficient cleaning of water bodies contaminated with oil products, compared to existing analogues.

This goal is achieved by the fact that aluminosilicate microspheres are used as a carrier sorbent, which are hollow spherical particles of regular shape with a diameter of 30 to 350 microns, with a wall thickness of 2 to 10 microns, the inner cavity of which is filled with nitrogen and carbon dioxide, having the following composition (wt.%):

SiO ₂ - 50 ÷ 60; Al ₂ O ₃ - 25 ÷ 35; Fe ₂ O ₃ - 1.8 ÷ 2.0; CaO - 1 ÷ 5; MgO - 0.5 ÷ 1.5; Na ₂ O - 0.3 ÷ 1.5; K ₂ O - 0.2 ÷ 2.9,

as a result, the sorbent carrier has a high buoyancy and oil absorption ability and allows you to create a high concentration of active microbial cells on the water-oil interface.

High hydrocarbon-oxidizing activity in relation to oil and its refined products in a wide temperature range at a high initial concentration of oil (oil products) in a polluted medium is achieved due to the use of the Pseudomonas fluorescens B-6844 strain, which actively utilizes oil hydrocarbons, as the basis for the ecobiological product.

The microorganism culture was isolated from the soils of the Ural region contaminated with fuels and lubricants in selective nutrient media containing hydrocarbons of petroleum products as the sole source of carbon nutrition. The strain is deposited in the All-Russian collection of industrial microorganisms. The strain is stored on an agarized nutrient medium (meat-peptone agar) at a temperature of (4 ± 2) ° C.

The strain Pseudomonas fluorescens B-6844 is an aerobic gram-negative small movable sticks with rounded ends, located singly or in pairs, with a size of 0.6-1.1 * 1.3-4.5 microns.

On protein media, it forms round colonies of 3.0-3.5 mm in diameter, convex, smooth, with a denser center and a flat edge, agar-colored, shiny, opaque, with time becoming matte and pink. Quickly (within 3 days) grows at a temperature of (4 ± 2) ° С; at a temperature of + 41 ° C and above there is no growth. It produces extracellular fluorescent yellow-green pigment on protein media, especially MPA with glycerol at low temperatures. Aerobe. The oxidative type of metabolism in the O / F test. Oxidase and catalase are positive. Nitrate reduction is absent. Culture does not show halophilic properties, consumes sodium citrate. Gelatin does not thin, glucose does not ferment.

The essence of the invention is to obtain ecobiological preparation by deep cultivation of cells of the strain Pseudomonas fluorescens B-6844 on a mineral medium of the following composition: K ₂ HPO ₄ - 2.5 g; KH ₂ PO ₄ - 1.0 g; FeSO ₄ · 7H ₂ O — 0.1 g; MgSO ₄ · 7H ₂ O — 0.3 g; purified water - up to 1.0 dm ³ ; glucose - 1% and oil or diesel fuel - 1%. The cultivation process is carried out in a fermenter at a temperature of (28 ± 1) ° C, pH (6.9 ± 0.1) for (15 ± 3) hours under aerobic conditions. After this, the bacterial cells are immobilized on a sorbent carrier by mixing it with a culture fluid containing microorganisms with a titer of at least 5-7 · 10 ⁹ cells · cm ^-3 . Surface cultivation of bacterial cells on a sorbent carrier is carried out at (28 ± 1) ° C for two days. The titer of microorganisms is at least 10 ¹⁰ viable cells in 1 g of ecobiological product.

The cleaning principle of the proposed method is as follows. When applying an eco-preparation to a water surface contaminated with oil products, the sorbent proposed as a carrier of bacterial cells adsorbs an oil film on itself. Due to the presence of nitrogen and carbon dioxide sorbent particles in the internal cavity, the sorbent-oil complex remains (at least 30 days) on the water surface, where the oil content is maximum, and the microbial cells Pseudomonas fluorescens B-6844, immobilized on the sorbent, actively multiply using hydrocarbons in as the only source of carbon and energy, which increases the efficiency of cleaning the reservoir from oil pollution. At the same time, the ability of the sorbent to hold oil products on the surface of the water prevents the oil film from settling to the bottom of the reservoir, which does not have a toxic effect on the growth and development of benthic organisms.

Example 1. To obtain an ecobiological product, Pseudomonas fluorescens B-6844 cells are grown in a fermenter at a temperature of (28 ± 1) ° С, pH (6.9 ± 0.1) for (15 ± 3) hours under aerobic conditions on a mineral medium of the following composition: K ₂ HPO ₄ - 2.5 g; KH ₂ PO ₄ - 1.0 g; FeSO ₄ · 7H ₂ O — 0.1 g; MgSO ₄ · 7H ₂ O — 0.3 g; purified water - up to 1.0 dm ³ ; glucose - 1% and oil or diesel fuel - 1%. After this, the bacterial cells are immobilized on a sorbent carrier by mixing it with a culture fluid containing microorganisms with a titer of at least 5-7 · 10 ⁹ cells · cm ^-3 . Surface cultivation of bacterial cells on a sorbent carrier is carried out at (28 ± 1) ° C for two days. The titer of ecobiological product is at least 10 ¹⁰ viable cells in 1 g,

where the sorbent carrier is a hollow spherical particles (microspheres) of regular shape with a diameter of 35 μm, with a wall thickness of 3 μm of the following composition:

SiO ₂ - 56.0%; Al ₂ O ₃ - 34.1%; Fe ₂ O ₃ - 1.8%; CaO - 4.0%; MgO - 1.1%; Na ₂ O - 0.9%; K ₂ O - 2.1%.

Example 2. Culture fluid Pseudomonas fluorescens B-6844 was obtained as described on the same nutrient medium as in example 1. Immobilization of bacterial cells on a sorbent carrier was carried out by mixing it with a culture fluid containing microorganisms with a titer of at least 5-7 · 10 ⁹ C. cm ^-3 . Surface cultivation of bacterial cells on a sorbent carrier was carried out at (28 ± 1) ° C for two days. The titer of ecobiological product is at least 10 ¹⁰ viable cells in 1 g,

where the sorbent carrier is a hollow spherical particles (microspheres) of regular shape with a diameter of 350 μm, with a wall thickness of 10 μm of the following composition:

SiO ₂ - 58.6%; Al ₂ O ₃ - 30.3%; Fe ₂ O ₃ - 1.9%; CaO - 3.5%; MgO - 1.5%; Na ₂ O - 1.4%; K ₂ O - 2.8%.

Example 3. Tanks with a volume of 1000 cm ^{3 were} filled with lake water and various amounts of oil were introduced (Nefteyugansk field). An eco-biopreparation was applied to the resulting oil film, which is a culture of Pseudomonas fluorescens VKPM 6844 cells immobilized on hollow spherical particles (microspheres) of regular shape with a diameter of 30 to 350 μm with a wall thickness of 2 to 10 μm, the inner cavity of which is filled mainly with nitrogen and dioxide carbon, the following composition (wt.%):

SiO ₂ - 50 ÷ 60; Al ₂ O ₃ - 25 ÷ 35; Fe ₂ O ₃ - 1.8 ÷ 2.0; CaO - 1 ÷ 5; MgO - 0.5 ÷ 1.5; Na ₂ O - 0.3 ÷ 1.5; K ₂ O - 0.2 ÷ 2.9,

based on 0.5 g of the drug per 1 ml of pollution. The tanks were installed on an open experimental site in the autumn-summer period. The ambient temperature during the test was in the range from 10 to 28 ° C. For various observation periods, the residual oil content in the samples was determined by IR spectrometry.

The top view of the oil film in the tanks made it possible to record numerous violations of its integrity, presented in the form of bright spots. Throughout the entire incubation time of the samples, the biodegradation of oil took place on the surface of the water.

The degree of degradation of oil products was calculated as the ratio of the difference between the initial and residual content of oil products in water to their initial content, expressed as a percentage. The test results are presented in table 2.

The test results allow us to conclude that the developed ecobiological product in the temperature range from 10 to 28 ° C has a high destructive ability in relation to petroleum products, including at high concentrations in the environment. Even with an initial oil content of 101.3 g · m ^{-2, the} degree of their degradation by an ecological product was significant.

As a result of the experiments, it can be stated that, in comparison with the known ecobiological products, the developed ecobiological product has a high oil-oxidizing activity, which manifests itself in a short time. Using the developed ecobiological product for cleaning oil-contaminated water surfaces can improve the ecological purity of bioremediation technologies.

INFORMATION SOURCES

1. TU 2164-001-26301532-2002 Inorganic oil adsorbents - Mixoil reagents. Effective July 1, 2002

2. Kamenshchikov F.A., Bogomolny E.I. Oil sorbents. - Moscow-Izhevsk: Institute for Computer Research, 2003.

3. Certificate for Utility Model No. 19867 Russia. ЕВВ 43/26. Microspheres / Russia.

4. Certificate for utility model No. 19269 Russia. B03D 1/00. Installation for producing microspheres from fly ash / Russia.

5. Pat. No. 2031860 Russia. C02F 3/34, EBB 15/04. The method of purification of water from oil pollution / Russia.

6. Pat. 1076446 C02F 3/34, C12N 15/00. The strain Pseudomonas putida 36, used to clean water and soil from oil and oil products / Russia.

7. Kapotina L.N., Morshchakova G.N. Biological destruction of oil and oil products polluting soil and water // Biotechnology. - 1998. - No. 1. - S.85-92.

8. Pat. No. 2174496 Russia. C02F 3/34, C12P 39/00. Biological product "Avalon" for cleaning environmental objects from oil and oil products, the method of its production / Russia.

MATERIALS EXPLAINING THE INVENTION

Table 1 The composition of ecobiological preparations, the conditions and the effectiveness of their use [5-8] Name of ecobiological product Base strains of microorganisms, additives The maximum level of pollution, g · m ^-2 Optimum application temperature, ° С The degree of degradation of petroleum products as a percentage of the initial amount Econadin Pseudomonas fluorescens 2a, peat 4,5 5-32 95-98 * Putidul Pseudomonas putida-36 one hundred thirty 65-75 * "Biodestructor" Various microorganisms, adsorbents, surfactants 10 18-25 75-95 * Avalon Serracia marcescens PL-1, Pseudomonas fluorescens biovar II 10-1, Acidovorax delafieldii 3-1, foamed glassy metaphosphates of variable composition 25 twenty 78-95 * Note: * 6 days after application
** 2 months after application

table 2 The degree of degradation of immobilized ecobiological product of oil products with their different initial content on the water surface The initial content of oil products, g · m ^-2 The degree of degradation of petroleum products in water samples for ... days, as a percentage of the initial amount 7 fourteen 28 15.6 62.1 ± 2.3 78.9 ± 1.7 90.6 ± 1.1 25.1 57.6 ± 3.2 69.5 ± 4.1 85.6 ± 4.3 48,2 41.8 ± 6.5 52.6 ± 4.4 76.5 ± 6.7 101.3 29.6 ± 3.2 42.9 ± 2.8 63.5 ± 2.3

Claims (2)

1. Ecobiological product for purification of water from oil products, which is a culture of biodestructor cells, artificially immobilized on a sorbent carrier, characterized in that it contains hollow spherical particles of regular shape with a diameter of 30 to 350 microns, with a wall thickness of 2 up to 10 μm, the inner cavity of which is filled mainly with nitrogen and carbon dioxide, having the following composition, wt.%:
SiO ₂ 50 ÷ 60 MgO 0.5 ÷ 1.5 Al ₂ O ₃ 25 ÷ 35 Na ₂ O 0.3 ÷ 1.5 Fe ₂ O ₃ 1.8 ÷ 2.0 K ₂ O 0.2 ÷ 2.9 Cao 1 ÷ 5
and as a biodestructor of petroleum products - strain Pseudomonas fluorescens VKPM 6844. 2. The method of producing an ecobiological product according to claim 1 for cleaning the surface of water bodies from oil products, including deep cultivation of a biodestructor on a liquid nutrient medium, preparation of a sterile sorbent carrier and surface cultivation of biodestructors on a sorbent carrier, characterized in that hollow is used as a sorbent carrier spherical particles of regular shape with a diameter of 30 to 350 microns, with a wall thickness of 2 to 10 microns, the inner cavity of which is filled mainly with nitrogen and carbon dioxide, with a trace the composition, wt.%:
SiO ₂ 50 ÷ 60 MgO 0.5 ÷ 1.5 Al ₂ O ₃ 25 ÷ 35 Na ₂ O 0.3 ÷ 1.5 Fe ₂ O ₃ 1.8 ÷ 2.0 K ₂ O 0.2 ÷ 2.9 Cao 1 ÷ 5
which are mixed with the culture fluid, and the culture fluid of the strain Pseudomonas fluorescens VKPM 6844 with a titer of at least 5 · 10 ⁹ cells · cm ^{-3 is} used as a biodestructor of oil products.