RU2422521C2 - Method of producing granules containing immobilised oil-oxidising microorganisms - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: oil-oxidising microorganisms are immobilised at temperature 15-30°C in a gelling medium produced of 3-5 % sodium alginate and 5 % calcium sulphate. It is followed with simultaneous homogenising and granulation of the immobilised microorganisms with a hydrophobisated silicon dioxide powder at mixer rotation speed 2000 to 3000 rpm. The granules with a hydrophobisated surface of size no more than 0.5 mm are produced.

EFFECT: produced granules as a composition represent a free-flowing powder.

1 tbl, 10 ex

Description

The invention relates to biotechnology, in particular to methods for producing immobilized oil-oxidizing microorganisms in granules with properties characteristic of a powdered biological product suitable for use in soil and water environments when cleaning the environment.

The known method [Composition for producing a carrier of immobilized microorganisms that break down hydrocarbons, and a method for producing a carrier. RF patent 2298033], consisting in obtaining a biodegradable hydrocarbon by immobilizing microorganisms on a cellulose-containing carrier.

The composition includes a carrier with immobilized cells, water repellent, emulsifier and diluent. The basis of the carrier may be sawdust, shavings, wood chips, straw, corn cobs; water-repellent media is drying oil; the emulsifier is taken from the group (polyvinyl alcohol, carboxymethyl cellulose, hydroxyethyl cellulose, ethyl cellulose, pectin, gelatin, isopropyl alcohol, butyl alcohol), and the diluent is from the group (white spirit, petroleum ether, sulfuric ether, 5-20 - aqueous dimethyl sulfoxide) . The quantitative composition (parts by weight):

cellulose base one hundred drying oil 10-20 emulsifier 0-30 diluent 10-100.

Cell immobilization is carried out by adsorption on a prepared carrier for 3 days during incubation on an orbital shaker.

The disadvantages of this method include the multi-stage and duration of the process, including the stage of grinding the microbial culture, its mixing, dissolution, heat drying and immobilization of microbial cells. In addition, the proposed method for producing a carrier requires additional measures to protect the environment, since solvents (petroleum, sulfuric ethers, white spirit, dimethyl sulfoxide) are used in the technology for its production, which represent hazardous waste upon completion of the process. The method does not allow to obtain fine powders biodestructors.

Also known is a method [A method for producing a biocatalyst in a polysaccharide carrier. USSR author's certificate No. 1742330], consisting in the use of a polymer of furcloronone, 4 g of which is placed in 100 cm ^{3 of a} 0.2% potassium chloride solution for 1 h at a temperature of 22 ° C, and then stirred for 40 minutes at a temperature of 80 ° C. The finished polymer solution is cooled to 65 ° C and mixed with a suspension of cells in equal volumes to a final polymer concentration of 1.5-3.0% and instilled into a two-phase stabilizing system consisting of liquid paraffin and 10% potassium chloride solution, cooled to a temperature of 5 ° C. The exposure time of the granules in the stabilizing system is 10 minutes. Formed spherical granules have a size of 5-11 mm

The disadvantages of this method include:

- the inability to obtain fine powder of a biodestructor;

- high temperature of the polymer solution (65 ° C), leading to the inevitable thermal inactivation of part of the microbial population when mixed with it;

- the large size of the granules of the biocatalyst and the small surface area that reduce the contact area of cells with hydrocarbons and increase the period of release and adaptation of immobilized cells in the environment of the biochemical process, which is not enough for effective destruction.

Closest to the technical nature of the claimed invention is a method [Method for producing immobilized microorganisms that destroy xenobiotics. USSR Author's Certificate No. 1705345], which consists in preparing a 4% solution of agar-agar (agarose) in physiological saline (distilled water) and mixing in equal amounts with a cell culture to a residual agar-agar concentration of 2%. After thorough mixing, this mixture is dripped through a hole with a diameter of 3-4 mm into chilled paraffin oil with a dynamic viscosity of about 400 Pa · s. In this case, granules are obtained, which are separated and washed from liquid paraffin using surfactants, and then from surfactants. The total time for the formation of granules, including washing, is 15-20 minutes.

It is common for the known and claimed invention to obtain a polysaccharide hydrogel with cells immobilized in it and its subsequent granulation.

The disadvantages of this method include:

- washing granules from oil using surfactants, on the one hand, gives the surface of the granules hydrophilic properties, which reduces their adhesion to hydrocarbons, and on the other, exposes the cells to the inactivating effect of detergent (SAS) [Samsonova A.S., Aleschenkova Z.M. , Lim B.R., Khvin S.V., Semochkina N.F. and others. Wastewater treatment from diesel fuel using immobilized microorganisms-destructors // Biotechnology, 2003, No. 4, p.83-87];

- when mixing a microbial culture with an agar-agar solution, its temperature should be above 40 ° C (at a temperature of 36 to 40 ° C, the agar-agar solution solidifies [Guide to practical exercises in microbiology, immunology and virology. Edited by M.P. .Zykova. - M .: Medicine, 1977]), and at such temperatures the inactivation of oil-oxidizing microorganisms is inevitable;

- the relatively large size of the obtained granules (3-4 mm) increases the period of release and adaptation of immobilized cells in the environment of the biochemical process due to the insignificant contact area of the cells with the substrate [Kovalenko GA, Perminova LV, Komova OV, Simakov A.V., Khomov V.V., Borovtsova O.Yu., Rudina N.A. Macro-structured hydrocarbon-containing ceramic carriers for adsorption immobilization of enzymes and microorganisms. Biocatalytic properties of adsorption invertase // Biotechnology, 2003, No. 4, p. 52-62].

The main conditions for improving the efficiency of oil-oxidizing microorganisms in the destruction of hydrocarbons are [Sinitsin AP, Rainina EI, Lozinsky VI, Spasov SD Immobilized cells of microorganisms. M .: Publishing house of Moscow State University, 1994, p.288]:

- the presence in the zone of the biochemical process of a high concentration of viable hydrocarbon-oxidizing microorganisms;

- good contact of cells (drug) with hydrocarbons.

The objective of the invention is to develop a method for producing immobilized oil-oxidizing microorganisms in granules according to the composition, which is the final product for use in soil and water environments when cleaning the environment from pollution by hydrocarbons.

The technical result is achieved due to the fact that in the method for producing granules containing oil-oxidizing microorganisms, use:

- as the basis of the hydrogel for immobilizing microorganisms, a 3 or 5% aqueous solution of sodium alginate (polysaccharide), which, when interacting with metal salts, forms a gel at temperatures that do not cause thermal inactivation of oil-oxidizing microorganisms (15-30 ° C) [Lim F. // Microencapsulation of living cells and tissues - In: Biomedical applications of Microencapsulations / Ed.F. Lim. Boca Raton, CRC Press, Inc., 1984, p. 137-154];

- as a hardener (stabilizer) of the gel, a poorly soluble calcium salt (calcium sulfate), which provides a delay (prolongation) of the gel curing process for a period of time sufficient for mechanical crushing of the gel into granules with a ratio of gelling agents in a mixture with microbial suspension (1 dm ³ ) :

for 3% sodium alginate solution - 0.36 dm ³ ;

for a 5% solution of sodium alginate - 0.15 dm ³ ;

a solution of a 5% solution of calcium sulfate - 0.06 dm ³ (0.07 dm ³ );

- to obtain granules containing immobilized oil-oxidizing microorganisms - a method of homogenizing reagents in the presence of hydrophobized highly dispersed silicon dioxide powder with a particle size of 15-25 nm in a mixer with a stirring frequency of 2000 to 3000 rpm ^-1 , with a capacity of 5 or 10 dm ³ , equipped combined mechanical stirrer and a system for introducing reagents into the cavity of the homogenizer [Makarov Yu.I. Devices for mixing bulk materials. M., 1973, p.130-132];

- to give the resulting granules the composition of the final product, which is suitable for use in soil and water environments when cleaning the environment, - hydrophobized highly dispersed silicon dioxide powder, which modifies the surface of the granules by giving them hydrophobic properties and flowability [Encapsulated solutions of neutralizing agents for chemical and biological agents. Solid-water detoxifying reagents for chemical and biological agents: Pat. USA 7030071, IPC ³ A62D 3/00, С01В 15/055. The Regents of the Univ. of California, Hoffman Dennis M., Chiu Ing Lap. No. 10/085512; declared 02/26/2002; publ. 04/18/2006; NPK 510/110. English.], [Nechaev A.P., Kochetkova A.A., Zaitsev A.N. Nutritional supplements. M., 2002, p.120-125].

The essence of the invention is as follows.

Into a suspension of oil-oxidizing microorganism cells at a temperature of 15-30 ° C, containing 0.4-1.5 billion cells / cm ³ , with constant stirring in a mixer at a stirring frequency of 2000-3000 rpm add pre-prepared 3 or 5% alginate solution sodium. The mixing process is continued for one minute (until a homogeneous mass). Then, without turning off the stirring, the prepared gel stabilizer solution (5% calcium sulfate solution) is introduced into the obtained alginate-microbial mixture and the mixing process is continued for one minute.

The resulting semi-finished product is immediately transferred to a feeder, which is connected to a mixer reagent supply system with a capacity of 5 or 10 dm ³ , filled with hydrophobized highly dispersed silicon dioxide (for example, cabosil or fluosil) with a ratio of silicon dioxide and semi-finished product (parts by weight) of 1: 2, 5. Semi-finished product with a flow rate of 300 cm ³ / min from the feeder is fed into the cavity of the mixer. Stirring is carried out at a frequency of 2000-3000 rpm for 3 minutes. After mixing is turned off, the obtained granules are kept in the homogenizer cavity for 30 minutes to complete gel curing in the granules (chemical reaction of the replacement of sodium ions by calcium ions).

The obtained granules as a composition, representing the end product of the technology used, is an easily loose powder of light beige color with a particle size of not more than 0.5 mm, containing from 280 to 640 million cells / g. The powder is not wetted by water, but is well sorbed by a hydrocarbon film of an oil product located on a water surface or in soil.

Thus, the technological process for producing granules containing immobilized microorganisms consists of three stages:

- mixing a suspension of oil-oxidizing microorganisms with gelling agents (preparation of a semi-finished product);

- dispersing the semi-finished product with silicon dioxide in the mixer to obtain granules;

- exposure of the obtained powdery product in a homogenizer to cure the gel in granules with immobilized microbial cells.

The presence of a causal relationship between the set of essential features of the claimed object and the achieved technical result is shown in the table.

Table 1 Causal relationship between the set of essential features of the claimed object and the achieved technical result Types of technical result and their dimension Actual or Estimated Indicators Explanation, due to which (distinguishing feature and / or their combination), it became possible to improve the performance of the proposed object compared to the prototype prototype the claimed object The minimum temperature of the process of immobilization of microorganisms, ° C 40 fifteen The immobilization of microorganisms in a gel based on a 4% agar-agar solution occurs at a temperature of at least 40 ° C. Immobilization in alginate-calcium gel at a concentration of sodium alginate of 3 or 5% is provided at a temperature of 15-30 ° C, which is below the threshold for the onset of thermal inactivation of oil-oxidizing microorganisms and does not affect the fluidity of the semi-finished product when producing granules The size of the granules (particles), mm 3-4 0.5. no more Obtaining granules with a size of not more than 0.5 mm is achieved due to mechanical crushing of the semi-finished product in the homogenizer in the presence of hydrophobized silicon dioxide powder at a mixing frequency of 2000 to 3000 rpm Flowability, g / s Absent 3.0, not less It is provided by the modification of the surface of the granules due to the thin shell of highly dispersed particles of silicon dioxide, preventing their adhesion The hydrophobicity of the surface of the granules, no / yes No Yes It is provided due to the formation on the surface of the granules of the shell of particles of highly dispersed hydrophobized silicon dioxide

The invention allows by mixing the microbial suspension with gelling agents (solutions of sodium alginate and calcium sulfate) and subsequent homogenization of the mixture with a hydrophobized silica powder to obtain granules with a size of not more than 0.5 mm containing immobilized oil-oxidizing microorganisms.

The possibility of carrying out the claimed invention is shown by the following examples.

The following cultures can be used as oil-oxidizing microorganisms: yeast cells of the genus Yarowia Lipolitica, bacteria: pseudomonads of the genus Pseudomonas Stutzeri, rhodococci of the genera Rhodococcus species and Rhodococcus erythropolis, which are part of the Devoroil drug association [Patent No. 21174071 22 The method of purification of soil, natural and wastewater contaminated with oil and oil products, using biological products].

The quantitative composition of the obtained granules (parts by weight):

When using a 3% solution of sodium alginate

microorganism suspension fourteen alginate solution four 5% calcium sulfate solution 7 silica one.

When using a 5% solution of sodium alginate

microorganism suspension 21 alginate solution 6 5% calcium sulfate solution 10 silica 1,5.

As equipment for the production of granules, a high-speed mixer-homogenizer with a capacity of 5 or 10 dm ³ manufactured by Biomashpribor LLC, Yoshkar-Ola, was used.

Example 1. Obtaining granules containing immobilized Rhodococcus erythropolis cells.

In a suspension of Rhodococcus erythropolis cells at a temperature of 15-20 ° C (14 parts by weight) containing 700 million cells / cm ³ , with constant stirring in a mixer at a stirring frequency of 2000 rpm add a pre-prepared 3% solution of sodium alginate (4 parts by weight). The mixing process is continued for one minute (until a homogeneous mass). Then, without turning off the stirring, the prepared gel stabilizer solution (5% calcium sulfate solution) (7 wt. H) was added to the obtained alginate-microbial mixture and the mixing process was continued for one minute.

The resulting semi-finished product is immediately transferred to a feeder, which is connected to a reagent supply system in a mixer with a capacity of 5 dm ³ filled with hydrophobized highly dispersed silicon dioxide (for example, cabosil or fluosil) (1 part by weight). Semi-finished product with a flow rate of 300 cm ³ / min from the feeder is fed into the cavity of the mixer. Stirring is carried out at a frequency of 2000 rpm for 3 minutes. After mixing is complete, the obtained granules are kept in the homogenizer cavity for 30 minutes to complete gel curing in the granules (chemical reaction of the replacement of sodium ions by calcium ions).

The resulting granules as a composition in structure are an easily loose powder (flowability 3.8 g / s) of light beige color with a particle size of not more than 0.5 mm, containing 500 million cells / g. The powder is not wetted by water, but is well sorbed by a hydrocarbon film of an oil product located on a water surface or in soil.

Example 2. Obtaining granules containing immobilized Rhodococcus erythropolis cells.

In a suspension of Rhodococcus erythropolis cells at a temperature of 25-30 ° C (14 parts by weight) containing 1.5 billion cells / cm ³ , with constant stirring in a mixer at a speed of rotation of the stirrer 2000 rpm add pre-prepared 5% alginate solution sodium (6 parts by weight). The mixing process is continued for one minute (until a homogeneous mass). Then, without turning off the stirring, the prepared gel stabilizer solution (5% calcium sulfate solution) (10 parts by weight) was added to the obtained alginate-microbial mixture and the mixing process was continued for one minute.

The resulting semi-finished product is immediately transferred to a feeder, which is connected to a reagent supply system in a mixer with a capacity of 5 dm ³ filled with hydrophobized highly dispersed silicon dioxide (for example, cabosil or fluosil) (1.5 wt.). Semi-finished product with a flow rate of 300 cm ³ / min from the feeder is fed into the cavity of the mixer. Stirring is carried out at a frequency of 2000 rpm for 3 minutes.

After mixing is complete, the obtained granules are kept in the homogenizer cavity for 30 minutes to complete gel curing in the granules (chemical reaction of the replacement of sodium ions by calcium ions).

The obtained granules as a composition of the final product in the technology used were an easily loose powder (flowability 4.5 g / s) of light beige color with a particle size of not more than 0.5 mm, containing 640 million cells / g. The powder is not wetted by water, but is well sorbed by a hydrocarbon film of an oil product located on a water surface or in soil.

Example 3. Obtaining granules containing immobilized Pseudomonas Stutzeri cells.

The preparation of granules is carried out in accordance with the procedure described in example 1, at a temperature of 22-25 ° C with the same ratio of gelling agent, gel stabilizer and silicon dioxide, using 14 wt. including cell suspension containing 400 million cells / cm ³ .

The obtained granules in structure were a light-flowing powder (flowability 3.8 g / s) of light beige color with granules no larger than 0.5 mm in size, containing 350 million cells / g. The powder is not wetted by water, but is well sorbed by a hydrocarbon film of an oil product located on a water surface or in soil.

Example 4. Obtaining granules containing immobilized cells of Pseudomonas Stutzeri

The preparation of granules containing immobilized Pseudomonas Stutzeri cells is carried out in accordance with the procedure described in example 1 only under temperature conditions and with a ratio of components of example 2.

The obtained granules in structure were a light-flowing powder (flowability 4.5 g / s) of light beige color with granules no larger than 0.5 mm in size, containing 350 million cells / g of Pseudomonas Stutzeri. The powder is not wetted by water, but is well sorbed by a hydrocarbon film of an oil product located on a water surface or in soil.

Example 5. Obtaining granules containing immobilized Yarowia Lipolitica cells

The preparation of granules containing immobilized Yarowia Lipolitica cells at a concentration of 370 million cells / cm ³ in suspension is carried out according to Example 1 at the same temperature and at the same ratio of components. The obtained granules were free-flowing powder (flowability 3.8 g / s) of light beige color with a granule size of not more than 0.5 mm. 1 g of powder contained 290 million cells.

Example 6. The preparation of granules containing immobilized Yarowia Lipolitica cells at a concentration of 370 million cells / cm ³ is carried out according to Example 1, only under temperature conditions and the ratio of components of Example 2. The obtained granules were free-flowing powder (flowability 4.5 g / c) light beige in color with a granule size of not more than 0.5 mm. 1 g of powder contained 280 million cells.

Example 7. The preparation of granules containing immobilized cells is carried out according to example 1 under the same temperature conditions and ratios of components, only as a microbial culture use the association of cultures based on Rhodococcus erythropolisa, Pseudomonas Stutzeri, Yarowia Lipolitica, suspensions of which are taken in equal volumes and in previous concentrations. The obtained granules were free-flowing powder (flowability 3.8 g / s) of light beige color with a granule size of not more than 0.5 mm.

Example 8. Obtaining granules containing immobilized cells based on the association of microorganisms prepared according to example 7, is carried out according to example 1, only under temperature conditions and the ratio of the components of example 2. Mixing is carried out at a frequency of 3000 rpm The resulting product was also a free-flowing powder (flowability 4.5 g / s) of light beige color with a granule size of not more than 0.5 mm.

Example 9. The preparation of granules containing immobilized cells is carried out in a 10 dm ³ homogenizer using the starting components and at the temperature of Example 1, only the mechanical crushing of the semi-finished product is carried out at a stirring frequency of 2000 rpm. The resulting product is a free-flowing powder (flowability 3.9 g / s) of light beige color with a granule size of not more than 0.5 mm.

Example 10. Obtaining granules containing immobilized cells is carried out in a homogenizer with a volume of 10 dm ³ using the starting components and the temperature of example 1, with a stirring frequency of 3000 rpm The resulting product is a free-flowing powder (flowability 3.8 g / s) of light beige color with a granule size of not more than 0.5 mm.

The invention allows to obtain immobilized oil-oxidizing microorganisms in granules according to the composition, which is the final product for use in soil and water environments when cleaning the environment from pollution.

Claims (1)

A method of producing granules containing immobilized oil-oxidizing microorganisms, including immobilizing microorganisms in a gelling medium, followed by granulation, characterized in that the immobilization of oil-oxidizing microorganisms at a concentration in suspension of 0.4-1.5 billion cells / cm ^{3 is} carried out at a temperature of 15-30 ° C , 3-5% sodium alginate solution and 5% calcium sulfate solution are used as gelling agents, granulation is carried out at the stage of homogenization of immobilized microorganisms hydrophobized silicon dioxide powder with the stirrer rotation speed from 2000 to 3000 rev / min and give granules with hydrophobized surface of not larger than 0.5 mm at the starting component ratio, mass parts .:
suspension of oil-oxidizing microorganisms 14-21 3-5% sodium alginate solution 4-6 5% calcium sulfate solution 7-10 silica powder 1-1.5
the resulting granules are incubated for 30 minutes to cure the gel in them.