RU2751360C1 - Method for obtaining biological products of living microorganisms with extended period of preservation of high titer of viable cells - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. The method provides for the stabilization of biological products containing living microorganisms by co-growing target cultures of microorganisms with carriers of the attached biocenosis, microcapsules made of chitosan-modified polyurea or polylactic acid.EFFECT: invention makes it possible to increase the efficiency of using biological preparations based on living microorganisms for cleaning natural and man-made objects from pollution, including petroleum products.4 cl, 4 dwg, 3 tbl, 4 ex

Description

The invention relates to the field of biotechnology and is based on the use of cultures of living microorganisms, for example, for environmental technologies for cleaning natural and man-made objects from pollution, including oil products. More specifically, the invention can be applied to stabilize biological products containing live microorganisms in order to prolong the maintenance of a high titer of living microorganisms and high technological properties of the corresponding biological products.

For the effective use of biological products containing live microorganisms, it is important that the concentration of viable cells is at a level sufficient for the manifestation of target technological properties that differ for various biotechnologies. So, to clean the environment from oil products, biological products must have a concentration of hydrocarbon-oxidizing bacteria (HCB) at least 10 ⁸ cells / g. At the same time, in all preparations, including living cells, the death of microorganisms during storage and a decrease in the concentration of microorganisms are observed. To slow down this process, such approaches are used as drying of drugs, storage at low temperatures (Sidyakina, 1985), which is most effective, but increases the cost and complicates the technological process of obtaining biological products, which is especially important for large-scale production of biological products used for environmental technologies. ... Cheaper technologies for stabilizing biological products are immobilization of microorganisms using sorbents and inclusion in gels (Efremenko, 2018). At the same time, in some cases, the shelf life of biological products should not exceed several weeks - from the moment of manufacturing (operating time) of the biological product to its use.

A known method of storing microorganisms, which consists in growing microorganisms with the subsequent addition of synthetic fibers, nylon, polypropylene, lavsan or others (RF Patent No. 2008348), in order to store microorganisms. The disadvantage of this method is the narrow focus of the method and the difficulty of using suspensions of such preparations for processing large objects.

There is a known method of increasing the duration of storage of cultures while maintaining their viability using enamine A (RF Patent No. 2291193). The disadvantage of this method is the complexity of the synthesis, low availability and cost of the used antioxidant preservative.

There are several known methods for stabilizing biological products to eliminate oil spills / contamination. All methods provide for the initial cultivation of cultures of microorganisms with subsequent technological actions (separation or concentration of biomass, introduction of additives, drying, etc.). Some methods provide for the introduction of several stabilizers into a liquid culture of the obtained microorganisms (RF patents RU 2337069 C1, RU 0099120413, RU 2365438 C2).

Some methods provide for the production of biological products in a solid state - using bulk sorbents-fillers (RF patents RU 2615464 C1, RU 2193533 C2, RU 2617953 C1).

Such approaches, with proven technological feasibility and efficiency, are characterized by technological complexity and increased cost, despite the fact that in the market (in the field of) environmental technologies there is a sector focused on the use of liquid biological products that are easy to obtain and use with minimal added value (in addition to costs for cultivation), and shelf life no more than 1-2 months.

The closest to these requirements is a method for stabilizing grown crops (RF patent RU 2365438 C2), which is a prototype of the proposed solution. In accordance with the method, additives, glycerol and mineral salts are introduced into the suspension of microorganisms grown in a liquid medium. The disadvantage of the prototype method is the requirement to store the drug at a temperature not exceeding 10 ° C, the shelf life is not more than 1.5 months, as well as the lack of comparison with the biological product obtained and stored without the introduction of stabilizers.

The indicated disadvantages - multistage and complexity of production or increased cost of the final product, storage requirements at low temperatures, lack of control (drug without stabilizers) - lacks the proposed method for stabilizing biological products containing live microorganisms.

The technical result of the proposed solution is to increase the time for maintaining a high titer of viable cells (safety) of biological products intended for storage for several weeks without additional cooling, ease of production (without the introduction of additional technological methods such as drying, pressing), which ensures the effectiveness of the use of such biological products.

The problem is solved by the described method of obtaining preparations of living microorganisms, characterized by an extended period of preservation of a high titer of living cells, including the stage of making microcapsules as a carrier of an attached biocenosis, and the stage of growing microorganisms in the presence of microcapsules.

In accordance with the proposed method can be used gram-positive and gram-negative bacteria.

In accordance with the proposed method, microcapsules made of a polymer characterized by the ability to adhere bacteria can be used as a carrier of an attached biocenosis.

In accordance with the proposed method, preferably, but not exclusively, microcapsules can be made from chitosan-modified polyurea or polylactic acid.

In accordance with the proposed method, microcapsules in an amount of 1.5-3 g / l are added to the culture of microorganisms simultaneously with the inoculum.

The above set of features included in two independent claims is not known from the prior art. Within the scope of the above set of features, the claimed technical result is achieved, which is confirmed below. Neither in the scientific, technical, nor in the patent literature was found information about the possible influence of the distinctive features of independent claims (growing of microorganisms in the presence of a carrier of an attached biocenosis, microcapsules, and the use of chitosan-modified polyurea and polylactic acid as a material for the manufacture of microcapsules) on the technical result achieved when implementing the claimed method.

FIG. 1 shows electron micrographs of typical microcapsules of polyurea modified with chitosan (A) and polylactic acid (B).

FIG. 2 shows the dynamics of the number of viable cells of different bacteria during storage with microcapsules based on polyurea with chitosan for 6.5 months.

FIG. Figure 3 shows the responses of P. aeruginosa (A) and R. erytropolis (B) cultures grown in the presence of polylactic acid microcapsules (1.5 g / L medium) to the addition of glucose (based on the rate of CO ₂ accumulation).

FIG. 4 shows the efficiency degradation various preparations paraffin oil (%) Culture R. erytropolis, stored 11 months (A) and the efficiency of degradation (oxidation) paraffins C ₁₀ -C ₃₄ (%) different drugs P. aeruginosa culture was stored for 6 months (B). Abbreviations: to-l - control culture without stabilization, MK-PMH - microcapsules from polyurea with chitosan, MK PMK - microcapsules from polylactic acid.

The authors' choice of a method for stabilizing biological products (i.e. long-term maintenance of a high titer of viable cells of microorganisms) is based on the need to combine three scientific and technological approaches: 1) the known properties of microorganisms in an immobilized state, i.e. in the form of biofilms, have increased stress resistance and survival (Efremenko, 2018, Żur et al., 2016); 2) ease of use of liquid biological products of microorganisms, i.e. in which there are dissolved and / or highly dispersed carriers of the attached biocenosis, which do not significantly change the rheological properties of the suspensions. For these purposes, microcapsules with dimensions of a fraction of a millimeter are suitable; 3) one-stage process of growing microorganisms, which simplifies and reduces the cost of production of biological products, and was not known before our work. It was also taken into account that: 1. Transfer of bacteria to a state of reduced metabolism will allow a larger amount of biomass to grow on an initially equal amount of substrate; 2. Sorption of metabolic products with inhibitory and autoregulatory properties should contribute to the prolongation of the growth phase of microorganisms, which will also contribute to the growth of a larger number of bacteria; 3. If it is possible to use the material of the capsules as a food substrate (polylactic acid), this will also contribute to the accumulation of a greater biomass of microorganisms and a longer preservation of viability.

The essence of the method and its technical feasibility are illustrated by the following examples.

Example 1. Obtaining microcapsules for use as carriers of attached biocenosis

When choosing a material for microcapsules, we proceeded from the fact that the surface of microcapsules should be (fully or partially) hydrophobic and / or bear a positive charge in order to effectively adsorb cells of microorganisms. Two types of microcapsules were synthesized: from polyurea modified with low molecular weight chitosan (MC PMC) and from polylactic acid (MC PMC).

Obtaining microcapsules from polyurea with chitosan

The shell of microcapsules is synthesized by the technology of the interfacial reaction of a prepolymer of polyurea contained in an organic phase (halocarbon) and a water-soluble polyamine. The technological process for obtaining a microencapsulated drug includes four stages:

1. Preparation of polyurea prepolymer solution

870 g of polyisocyanate resin are dissolved in 5 l of absolute n- hexane, incubated for 3 hours at room temperature and filtered without access to air. The resulting solution is evaporated in a vacuum and dried at 20 ° C in a vacuum not higher than 1⋅10 ^-3 mm Hg. for 3 hours with stirring until the hexane vapor is completely removed.

2. Preparation of an aqueous solution of the emulsifier

Dissolution of the emulsifier is carried out in a reactor with stirring at 90-95 ° C for 1-1.5 hours. Then it is brought to the working concentration with cold water, additionally stirred for 30 minutes until complete homogenization and cooled to the working temperature.

3. Preparation of the emulsion

A halogenated carbon emulsion is prepared in portions in an emulsifier to obtain droplets of a given size. The primary control is carried out using a microscope using a Goryaev camera to determine the size. The preparation time of the emulsion is no more than 10 minutes. With an increase in the holding time, the emulsion either exfoliates or gains viscosity. In both cases, the yield and quality of the capsules deteriorate.

4. Curing of the polymer

The reaction is carried out in the main reactor with stirring and heating for an optimal time.

The optimum temperature for entering the halogenated emulsion is + 30 ° C and holding at this temperature for 2-3 hours.

The optimal conditions for obtaining microcapsules from polyurea were found:

Template (microcapsule core): Dichloromethane / Dichlorofluoroethane 2: 1

Polyisocyanate: PM-200

Curing system: Triethylenetetramine / Ethylenediamine 3: 1

Polysaccharide: low molecular weight chitosan (molecular weight in the range from 1 kDa to 30 kDa, degree of deacetylation 75-95%) - 25% by weight of the hardener.

Surfactant: TWIN 80 / polyvinyl alcohol / sodium lauryl sulfate 1: 1: 0.2

Curing mode: 20-60 ° C, 6 hours.

The suspension of the microencapsulated preparation, after receiving, is filtered on a non-woven filter material under vacuum. The filter cake is washed with distilled water. The microencapsulated preparation is a 10-15% aqueous suspension of capsules 50-200 microns in size, consisting of a polyurea shell, a halocarbon content and an aqueous phase as an external filler of the material. This material is introduced into the reactor together with the OP-7 dispersant and kept in the reactor at 130 ° C for 5 hours, after which it is cooled and used to immobilize bacteria.

The obtained preparations had the following characteristics: diameter - 50-200 microns, on average 80-100 microns, the average surface area of the shells of the microcapsules (dry) 0.054 m ² / g.

Obtaining microcapsules from polylactic acid

The method includes the following four stages:

(1) Preparation of solutions of PLA in methylene chloride and emulsifiers in water.

(2) Mechanical emulsification of the PLA solution in an aqueous solution of emulsifiers to obtain a microdroplet size of 0.05-0.5 mm.

(3) Creation of increased air pressure over the microemulsion (more than 300 kPa). At the same time, air is completely dissolved in drops and solution. The subsequent decrease in the pressure in the vessel to atmospheric pressure leads to the appearance of microbubbles in the droplets and in the solution.

(4) After the transition of methylene chloride into an aqueous solution of PVA, droplets are compressed and hollow microcapsules are formed from PLA.

The optimal conditions were as follows:

Polymer solution - 5% PLA in dichloromethane, emulsifier - Tween-80 in polyvinyl alcohol 1: 1, emulsification for 10 minutes with stirring at 3000 rpm, the main reaction - for 4 hours at 35 ° C.

The resulting microencapsulated preparation is a 10-15% aqueous suspension of capsules with a size of 50-200 μm, consisting of PLA (see Fig. 1).

The resulting preparations had the following characteristics:

Diameter - 25-100 microns, on average 80 microns.

Microcapsules of both types are represented by wrinkled rounded spheres with a smooth surface (Fig. 1). In both types of MC, there are capsules with cracks (no more than 10%). "Shrinkage" is more pronounced in polyurea capsules.

The given example proves the technical feasibility of the proposed method for obtaining carriers of attached biocenosis and the relative ease of obtaining microcapsules.

Example 2. Growing cultures of microorganisms with microcapsules

Cultures of four hydrocarbon-oxidizing bacteria: gram-negative Pseudomonas aeruginosa and Acinetobacter seifertii and gram-positive Rhodococcus erytropolis and Dietsia maris, were grown in 50 ml of sterile LB medium in 250 ml flasks. Together with the inoculum, capsules of polyurea with chitosan (1.5-3 g / l) were simultaneously introduced. The cultures were incubated for 2 days at a temperature of 28 ° C with stirring on an orbital shaker (100 rpm) until the stationary phase, after which they were left at room temperature for several months. After cultivation, the surface of the capsules was covered with a layer of adhered bacteria (Table 2). In this case, the fouling area never reaches 100% of the MC surface. The cells are localized mainly in the cavities between the protruding folds of the capsules. From the shape of the cells, we can conclude that they are in an inactive state. After 3 months of storage of cultures with capsules, the picture did not change significantly (table 2). All capsules were coated with bacterial cells, and even more so than 2-day-old cultures.

The overgrowth of capsules with bacteria was studied using scanning electron microscopy on a TM-3000 microscope (Hitachi, Japan) at an accelerating voltage of 15 kV, as well as phase-contrast microscopy on an Axio Imager M2 epifluorescence light microscope (Carl Zeiss Microscopy GmbH, Germany). The growth rate of bacteria was judged by the intensity of their respiration, which causes the accumulation of CO ₂ in the closed volume of the incubation flask, with a volume of 500 ml. The CO ₂ concentration was determined on a gas chromatograph (Kristall 5000, RF). The volume of the medium and the amount of inoculum were calculated so that inhibition of the development of cultures due to lack of oxygen or accumulation of CO _{2 was} not achieved. The number of viable cells (titer of colony-forming units, CFU / ml) was determined by plating decimal dilutions of cultures on solid media.

The introduction of microcapsules into the growth medium did not affect the growth rate of bacteria, determined by the respiration of bacteria, which was tested on the cultures of P. aeruginosa and R. erytropolis. In both cases, there was no lag phase, the crops grew at the same rate and reached the same final yield.

The number of grown cells in the presence of polyurea capsules was 1.5 times higher than in the control variant (Fig. 1), which confirms our assumption about the positive effect of growing bacteria in the presence of sorbents.

Cultivation of bacteria with microcapsules from polylactic acid was carried out with bacteria P. aeruginosa and R. erytropolis (Table 3). Both cultures form dense biofilms that cover almost the entire surface of the capsules. At the same time, it was found that electron microscopic examination underestimates the number of adhered bacteria due to the presence of sample preparation. When viewing the same samples under a light microscope, it can be seen that the microcapsules are completely covered with a thick layer of bacteria (Table 4).

Table 2. Fouling of microcapsules from urea with chitosan by various bacteria. Detection using a scanning electron microscope

Time after
inoculation P. aeruginosa R. erytropolis A. seifertii D. maris 2 days

3 months

Table 3. Overgrowth of polylactic acid microcapsules by various bacteria. Detection using a scanning electron microscope

Time after
inoculation P.aeruginosa R.erytropolis 2 days

1 month

Table 4. Fouling of polylactic acid microcapsules by various bacteria. Light microscope detection

Time after
inoculation P.aeruginosa R.erytropolis 1 month

This example proves the technical feasibility of the proposed method for immobilizing all tested bacteria on carriers of the attached biocenosis, microcapsules of different types, the absence of additional operations and costs for additional technological processes.

Example 3. Survival of bacteria in the presence of microcapsules for a long time and preservation of their metabolic activity

FIG. 2 shows the curves of the content of viable cells of four bacteria in the presence of microcapsules from polyurea with chitosan. The presence of microcapsules in the growth medium and the transition of cells to the state of biofilm growth had a positive effect on the increase in the number of viable bacterial cells in their cultures during long-term storage relative to control (2-60 times). The effect of increasing the CFU titer is provided both by an increase in the number of cells in grown 2-day-old cultures, and due to a more successful survival of cells in a state of biofilm phenotype.

The effect of polylactic acid microcapsules on the titer of viable bacterial cells was difficult to assess due to the extremely high adhesion and methodological difficulties in desorption of cells from microcapsules. Thus, for R. erytropolis after 1 month of storage, the CFU titer in the experiment was 3 orders of magnitude higher than that in the control, and for P. aeruginosa there was no difference between the control and the experiment.

For this reason, we additionally studied the metabolic activity of biological products stored for 1 month in the presence of microcapsules relative to the control. The bacteria were cultured in 60 ml flasks in Raymond's medium for 9 hours (the inoculum was a culture of the stationary growth phase, or cultures stored for 30 days were added in an amount of 2% by volume for Pseudomonas and 4% for Rhodococcus). After stabilizing the level of CO ₂ in the gaseous phase (after 4 hours of incubation), was added a solution of glucose (500 l of 10% solution).

FIG. 3A, it can be seen that the P. aeruginosa culture stored for 30 days in the presence of polylactic acid microcapsules (1.5 g / L) is five times more active than in the control. For R. erytropolis, the respiratory activity of a culture stored for 30 days in the presence of microcapsules and polylactic acid is 10 times higher than that of the control culture and only 2 times lower than that of a freshly grown culture.

The above example shows the effectiveness of the proposed method to improve the survival of bacteria and their metabolic activity during long-term storage in unfavorable conditions.

Example 4. Decomposition of hydrocarbons in liquid media by cultures of bacteria stabilized using microcapsules

To confirm the suitability of the developed method for maintaining the viability of bacterial cells and their metabolic potential for increasing the efficiency of using biological products for their intended purpose, the effectiveness of the destruction of oil products by two hydrocarbon-oxidizing bacteria was tested.

The hydrocarbon oxidizing activity of R. erytropolis cultures was determined according to the certified method No. 88-16207-054-RA.RU 310657-2017 for the destruction of n-alkanes of the aliphatic fraction by GLC on a Kristall 5000.1 chromatograph (Khromatek, RF). The degree of hydrocarbon oxidizing activity (%) of the biological product was calculated from the change in the total content of n-alkanes with a carbon chain length from 10 to 30 using a special formula. All variants were placed in 500 ml flasks with 100 ml of Raymond's mineral medium with the addition of 3% oil from the Moscow Oil Refinery with the addition of 100 μl of a biological product (culture - R. erythropolis). The flasks were cultured on a shaker at 120 rpm for 4 days at 30 ° C. For comparison, biological products based on R. erytropolis were taken: 1. Culture grown for 2 days on LB medium and stored for 5 days as a positive control; 2. The same culture, stored for 11 months without stabilization; 3. The same culture, grown in the presence of microcapsules from polyurea with chitosan, also stored for 11 months. The test results are shown in FIG. 4 A. The culture kept in the presence of microcapsules showed a high activity in the degradation of paraffins - 7 times higher than the control, and only 17% lower than the freshly grown culture.

The efficiency of destruction of a mixture of C10-C34 paraffins (Enzyme, RF) by stabilized and unstabilized preparations of P. aeruginosa UOB was tested in an experiment on the growth of UOB in a Raymond liquid medium with the addition of paraffins as the only carbon source and detection of CO ₂ accumulation on a gas chromatograph (Crystal 5000 , RF). The following cultures were used: freshly grown for 2 days on LB medium; after 6 months of storage without additives (to-l); grown in the presence of microcapsules from polyurea with chitosan or from polylactic acid.

The results shown in FIG. 4B, demonstrate that after 4 days of cultivation, a freshly grown culture of the end of the exponential growth phase oxidized 1 mg / ml of paraffins, which was 10% of its initial amount (10 mg / ml). If the medium was inoculated with a culture of pseudomonads, which initially had the same cell titer, but stored for 6 months, then 6 times less hydrocarbons were oxidized during the same time. Cell stabilization with microcapsules of both types increased the efficiency of oil product destruction by 3 times compared to the control, unstabilized variant.

Thus, laboratory testing has confirmed the effectiveness of the developed approaches to increasing the CFU titer of UOB preparations for the implementation of the target process - the degradation of hydrocarbons.

Cited sources

1. Sidyakina TM Conservation of microorganisms, Pushchino, 1985.

2. Immobilized cells: biocatalysts and processes. Ed. E.N. Efremenko. M., Rior, 2018.

3. Żur J., Wojcieszyńska D., Guzik U. Metabolic Responses of Bacterial Cells to Immobilization Molecules. 2016 Jul; 21 (7): 958.

Claims (4)

1. A method of obtaining preparations of live microorganisms, characterized by an extended period of preservation of a high titer of living cells, including the stage of growing microorganisms in the presence of microcapsules obtained from polyurea modified with low molecular weight chitosan, or from polylactic acid, which are 10-15% aqueous suspension with a diameter of 80 microns and which are added in an amount of 1-3 g / l to the culture of microorganisms simultaneously with the inoculum. 2. The method according to claim 1, characterized in that the microorganisms can be represented by gram-positive bacteria. 3. The method according to claim 1, characterized in that the microorganisms can be represented by gram-negative bacteria. 4. A method according to claim 1, characterized in that the microcapsules are made of a polymer characterized by the ability to adhere to bacteria.

Images