RU2596924C1 - Method for implementation of enzymatic processes and device therefor - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: device for enzymatic processes and method for implementation of enzymatic processes using said apparatus. Apparatus comprises fermentation vessel with heat exchange jackets for liquid phase and solid-phase processes, aerator, buffer tank, transfer chamber, control device, pipelines with controlled valves and nozzles. Fermentation vessel for performance of solid-phase processes is equipped with temperature sensor, fermentation vessel for liquid-phase processes is equipped with pH sensor and temperature sensor. Fermentation vessel for liquid phase and solid-phase fermentation processes are interconnected with bypass pipeline, buffer tank is equipped with heat exchange jacket and coil. Gate chamber is equipped with bacterial filter of gate chamber supercharging and liquid level sensor. Upper cavity of gate chamber by means of pipelines with valves is interconnected with upper and lower cavity of buffer tank and bacterial filter for supercharging of gate chamber, and lower cavity is interconnected with liquid-phase fermentation tank and drain pipes fermentation products and sewer drain.

EFFECT: invention ensures intensification of fermentative processes due to optimisation of conditions of enzymatic reactions.

2 cl, 1 dwg

Description

The invention relates to the field of biotechnology, mainly to a method and apparatus for implementing fermentation processes, with the aim of intensifying processes occurring on liquid and solid nutrient media using free or immobilized microorganisms on adsorption carriers. The invention can be used in the processes of microbial synthesis, catalysis, destruction, degradation and transformation of organic compounds.

In modern biotechnology, organic compounds are processed into biologically active substances by known methods, the main of which are:

the method of "liquid phase" fermentation,

method of "solid phase" fermentation,

method of "film" fermentation.

The choice of fermentation method depends not only on the properties of biological producers, but also on the quality of the target product.

Widely known methods of liquid-phase fermentation are known, for example, described by the authors U.E. Viestur, M.Zh. Kristapsons, E.S. Bylinkina in the book “Cultivation of microorganisms”, Moscow, “Food industry”, 1980. The cultivation of microorganisms by liquid-phase fermentation is carried out according to the traditional scheme, in which a nutrient solution is introduced into the fermentation tank, the container is sealed and sterilized with water vapor at a temperature of 120 ° C in within 1 hour. After sterilization, the container is cooled to ambient temperature, and a seed culture of microorganisms is introduced into the nutrient solution. After seeding, microorganisms are cultured under the specified conditions of aeration, mixing, temperature and time of the process.

The method of liquid-phase fermentation, in contrast to solid-phase fermentation, can significantly reduce production areas, eliminate heavy unproductive labor, simplifies the mechanization and automation of production, makes it possible to switch to a continuous process. The process of liquid-phase fermentation is accompanied by a more complete consumption of nutrients, which can significantly reduce industrial waste and produce a product with a low content of impurities.

The main disadvantages of liquid phase fermentation are:

- species restriction of producers, since the maximum enzymatic activity of many microorganisms is achieved only under conditions of surface or solid phase fermentation;

- the dissolution of the feed gas in the working fluid is carried out in conditions of intense dispersion of gases into the fluid, which is accompanied by intensive foaming of the working fluid, violating the regulatory conditions of the fermentation process;

- liquid-phase processes have a significant restriction on the concentration of substances in the working medium, since high concentrations of certain nutrients have a toxic effect on microorganisms, which significantly limits the productivity of fermenters.

A known technology for the degradation of plant materials by the method of solid-phase cultivation of higher basidiomycetes, such as Tyromyces lacteus and Goriolus hirsutus (I.M. Gracheva, A.Yu. Krivova, "Technology of enzyme preparations" NPO "ELEVAR", 2000, pp. 315-317 )

Known technology is implemented according to the traditional scheme of operations. Plant materials are loaded into a container. The container is sealed and sterilized with water vapor at a temperature of 120 ° C for 1 hour. After sterilization, the container is cooled to ambient temperature and an inoculum culture of microorganisms producing enzymes degrading lignin, cellulose and starch, previously grown in a flask on a rocking chair to a stationary growth phase, is introduced into a solid substrate. After sowing, the fungus is cultured under the specified conditions of humidity, temperature, aeration and process time.

According to the results of the studies, the authors showed:

- the performance of solid-phase processes directly depends on the optimization of mass transfer indicators, maintaining the specified ventilation conditions, humidity and temperature in a solid substrate;

- an increase in humidity disturbs the loose texture of the medium, mushroom mycelium grows only from the surface, and inside it forms a zone with a low oxygen content, the growth of which is impossible, which leads to a significant decrease in the production of fermentation products due to incomplete use of the substrate. In this zone, toxic products of biomass autolysis accumulate, which leads to a significant decrease in the rate of enzymatic reactions and the productivity of solid-phase processes.

In addition to the disadvantages, solid-phase processes have advantages. So, solid-phase processes make it possible to obtain products that cannot be produced using other technologies. For example, the fruiting bodies of myxobacteria and endospores of certain Bacillus species are formed during crop growth only on a solid medium. This is due to the fact that only under these conditions the bacteria retain physical bonds between themselves and are provided with a sufficient amount of dissolved oxygen.

In biomedical practice, the method of surface (film) fermentation is widely used, used to grow the biomass of mammalian cells, which can grow only as a monolayer, being attached to a substrate or hydrophilic materials.

In lectures O.V. Bazhevich “Cell Culture”, Belarusian State University, Minsk, 2004 describes a method of thin-layer fermentation, most often used to obtain constant cell lines characterized by a high growth rate, cell density and, therefore, higher biomass yield. Cells with adhesive properties are attached to microcarriers in the form of a monolayer, which allows to realize all the advantages of liquid-phase processes.

Known publication: V.A. Bykov et al. Microbiological production of biologically active substances and preparations. (Publishing house "Higher School", 1987). Reproduction of the producer's initial culture, for biosynthesis of biologically active substances, is carried out by performing sequential procedures: first, the producer is grown on an agarized medium, then in fallopian flasks on a rocking chair and then in a sowing system. All these procedures are fragmented and performed manually, which leads to a decrease in the enzymatic activity of the producers and a violation of the aseptic conditions of the processes being implemented.

A number of biologically active products are obtained using immobilized producers by the method of transformation of organic compounds, for example, steroids. High productivity of the processes of transformation of organic compounds is achieved by immobilizing living cells of microorganisms into cellulose fibers or into polyacrylamide gels. Cells introduced into the gel multiply intensively on a nutrient medium and can be repeatedly used as polyenzyme systems.

The disadvantage of using immobilized cells, for example, in the processes of steroid transformation is the weak resistance of the gels to hydrodynamic shock effects that occur during aeration and mechanical stirring of the working fluid, which significantly increases the cost of isolating the target products.

The closest analogue of the invention is the "Apparatus for culturing cells and microorganisms", patent for utility model RU 106899 U1, Bull. No. 21, 2011, which implements the possibility of performing in one apparatus processes of liquid phase and solid phase or film fermentation.

The known apparatus provides for the implementation of fermentation processes using free and immobilized cells, liquid and solid nutrient substrates, adsorption carriers and cartridges.

In the known apparatus, a combined method of gas and liquid exchange is used, which consists in displacing the working fluid from one fermentation tank to another under the pressure of aerating air. The devices for intensive exchange of working fluid in the apparatus are: an aerator, a bottom discharge pipe, and matrices with Rashiga rings or a solid substrate mounted above the aerator in each fermentation tank.

Before carrying out the fermentation processes, the apparatus and nutrient media are sterilized, cooled and seeded with producing microorganisms previously grown in a flask on a rocking chair to a stationary growth phase.

Liquid-phase and solid-phase processes are carried out in the fermentation tanks of the apparatus in conditions of periodic liquid exchange of the working fluid.

To intensify gas exchange, the well-known “Rashig rings” are used, which, when implementing fermentation processes, are placed in matrices that are permeable to the working fluid.

Known apparatus is equipped with:

- two fermentation tanks with heat-exchange shirts, caps for sealing containers, matrices and a lower drain pipe from the tanks;

- an aerator located in the lower cavity of the fermentation tank, for dispersing the bubbles of the feed gas into the working fluid,

- a buffer tank for collecting the working fluid displaced from the fermentation tanks in the exhaust gas stream, and for venting gases into the atmosphere;

- a lock chamber for draining liquid fermentation products;

- aseptic connector for input into the fermentation tank of the inoculum and sampling;

- bacterial filters of supply and exhaust gases;

- sensors for measuring pH and temperature in the working fluid, placed in the measuring cell;

- a network of pipelines with controlled valves;

- control device.

Significant disadvantages of the known method and the apparatus implementing the method are:

- Reducing the solubility of air oxygen in the working fluid and, as a result, the enzymatic activity of producing microorganisms, caused by the fact that in the processes of liquid-phase fermentations, aeration of the working fluid is carried out alternately in one fermentation tank, then in another fermentation tank.

- Low aeration efficiency and violation of the aseptic of the fermentation process caused by foaming of the working fluid during dispersive aeration and active respiratory activity of microorganisms, when the foam in the exhaust air stream is displaced into a buffer tank, from which part of the foam is pumped back into the working fluid, and the remaining part is displaced on the bacterial filter of exhaust air.

- In solid phase fermentation processes when a solid substrate is inoculated with a suspension of fungal mycelium, it is not possible to evenly distribute seed mycelium in the volume of the solid substrate and, thus, maintain the specified gas exchange conditions and temperature, since the seed obtained in a flask on a rocking chair is a heterogeneous suspension of agglomerated biomass .

- In the surface fermentation processes, the biomass of producing microorganisms adsorbed on the multiple surfaces of the Rashig rings cannot be structured, which contributes to the clogging of the flow channels of the Rashig rings by the biomass, disruption of mass transfer processes, cell autolysis and the accumulation of toxic cell decay substances in the working fluid, which reduce the quality of cells target products.

- Matrices placed in fermentation tanks, and a strainer placed in a buffer tank, in the process of fermentation are overgrown with biomass of growing microorganisms, which leads to a violation of the technological regulations.

- The operation of the lock chamber involves preliminary procedures: steam blowing the cavity of the lock chamber and cooling the lock chamber to a predetermined temperature. These procedures are accompanied by significant losses of time and fermentation products, which are washed off from the walls of the lock chamber into the sewer in a stream of water vapor, which increases energy costs and reduces the productivity of the apparatus.

- The use of a remote measuring cell does not allow to take reliable indicators of processes occurring in fermentation tanks, which reduces the quality of management of the processes being implemented.

The above disadvantages do not allow to optimize the conditions for enzymatic reactions and, as a result, to intensify fermentation processes.

The purpose of the invention: the elimination of these disadvantages of the analogue and the intensification of fermentation processes by optimizing the conditions for enzymatic reactions in the processes of liquid-phase, solid-phase, film fermentation and transformation of organic compounds.

This goal is achieved by the fact that in the known method, including the operation of introducing into the fermentation tanks of a liquid nutrient medium, a solid substrate or cell carrier; sterilization of the apparatus; cooling; cultivating a seed culture of microorganisms; sowing a liquid nutrient medium in a fermentation tank and carrying out "liquid phase", "solid phase", "film" or "transformation" processes in the given conditions of aeration, temperature and frequency of the working fluid exchange between fermentation tanks, according to the invention:

- inoculation of a solid substrate or carriers with producing microorganisms is carried out in the full growth cycle of the cultivation of microorganisms, under conditions of combining the "liquid phase" and "solid phase" fermentation processes, with the simultaneous deposition of cells on multiple surfaces of the solid substrate or carriers;

- stabilization of the temperature of the fermentable solid substrate and the removal of metabolites formed during solid-phase fermentation is carried out automatically, when the temperature in the solid substrate deviates by 1 ° C from the set temperature of the coolant in heat-exchange jackets, by periodically immersing the solid substrate in the working fluid;

- the foam formed in fermentation processes is destroyed in the exhaust gas stream by passing a gas-foam medium through a cooled coil in the direction against gravity;

- draining the liquid fermentation products or returning the working fluid to the fermentation is carried out automatically, with each contact of the fluid entering the lock chamber with a level sensor;

- the transformation of organic compounds is carried out in a matrix, which is filled with immobilized cells, placed in the cavity of the fermentation tank for the implementation of "solid-phase" fermentation processes and, periodically, with a given frequency, immersed in a suspension of transformable substances saturated with atmospheric oxygen.

This goal is also achieved in an apparatus for the implementation of fermentation processes, containing fermentation tanks with heat exchangers for carrying out liquid-phase and solid-phase processes, an aerator, a buffer tank, a lock chamber, a control device, pipelines with controlled valves and pipes, in which, according to the invention:

- fermentation tank for carrying out solid-phase processes is equipped with a temperature sensor;

- fermentation tank for carrying out liquid-phase processes is equipped with pH and temperature sensors;

- tanks for carrying out "liquid-phase" and "solid-phase" fermentation processes reported by the bypass pipe;

- the buffer tank is equipped with a heat-exchange jacket and a coil integrated in the heat-exchange jacket to divert exhaust gases and liquid fermentation products to the buffer tank;

- the lock chamber is equipped with a bacterial filter and a liquid level sensor that provides a predetermined dose of draining the working fluid;

- the upper cavity of the lock chamber, through pipelines with valves, is in communication with the upper and lower cavity of the buffer tank and with the bacterial filter for boosting the lock chamber, and the lower cavity is connected with the fermentation tank and drain pipes of fermentation products and sewage drain.

The attached figure shows a schematic diagram of a fermentation apparatus explaining the implementation of the proposed method in fermentation processes.

The device contains:

Fermentation tank 1, heat exchange jacket 2, loading hatch 3, aerator 4, temperature sensor 5, fermentation tank 6, heat exchange jacket 7, aerator 8, pH sensor 9, temperature sensor 10, bypass pipe 11, buffer tank 12, heat exchange jacket 13, coil 14, lock chamber 15, liquid level sensor 16, bypass pipe 17, bacterial filter 18 for supply gases, bacterial filter 19 for exhaust gases, bacterial filter 20 for boosting the lock chamber, thermostat 21, piping network with controlled valves 22-37, pipe and 38-44, the device management 45.

Description of the algorithms of the method and the fermenter.

Example 1. The algorithm for the implementation of "liquid-phase" fermentation processes.

A source of prepared nutrient solution is connected to the pipe 44 of the aseptic connector.

The valves 32, 29, 22 are opened and a predetermined volume of solution is loaded into the fermentation tank 6.

After loading the solution, the fermenter is sealed and sterilized with hot steam by a known method at predetermined values of temperature, pressure and sterilization time.

At the end of the sterilization process, the valves 25, 29, 22 are opened, and the fermenter is cooled to a predetermined temperature of the fermentation process when cold water is recycled through the thermostat 21 and heat transfer shirts 2, 7.

A producing culture of microorganisms is grown in a seed flask on a rocking chair until an exponential growth phase.

The flask with the seed culture of microorganisms is connected to the nozzle 44, the valve 32 is opened, and the suspension of the seed microorganisms is introduced into the fermentation tank 6. After seeding the fermentation tank 6, the valve 32 is closed, the valves 26, 30, 31, 24 are opened, and the temperature 45 heat-exchange shirts 2, 7, controlled by the sensor 10, and the frequency of the reciprocating movement of the working fluid through the fermentation tanks 1, 6.

To move the working fluid from the fermentation tank 6 to the fermentation tank 1, close the valve 29.

Aerating air through the pipe 38 and the bacterial filter 18 enters the aerator 8 of the fermentation tank 6 and penetrates the volume of the working fluid in the form of fine bubbles, mixing and displacing the working fluid from the tank 6 into the tank 1 through the bypass pipe 11.

The working fluid entering the tank 1, as in the tank 6, is exposed to fine bubbles of aerating air leaving the aerator 4, which allows you to activate the dissolution of oxygen in the working fluid and optimize the enzymatic reactions of microorganisms.

After emptying the container 6, open the valve 29, close the valve 25 and the working fluid under the action of aeration air through the bypass pipe 11, return to the tank 6, and the process of reciprocating movement of the working fluid from one tank to another is repeated at a given frequency until the liquid phase fermentation process is completed .

The exhaust air from the fermentation tanks 1, 6 through alternately opening valves 25, 29, a coil 14, a buffer tank 12, an open valve 22, a bacterial filter 19 and a pipe 39 is discharged into the atmosphere.

In the fermentation process, as a result of fine aeration of the working fluid and the respiratory activity of microorganisms, a foam is formed, which is destroyed in the coil 14.

The destruction of the foam occurs due to condensation of moisture from the warm exhaust air flowing through the cooled coil 14 under conditions of gravitational resistance.

The exhaust air and dropping liquid obtained as a result of the destruction of the foam in the coil 14 are displaced in the exhaust air stream into the buffer tank 12 cooled by the heat exchange jacket 13.

The exhaust air, after the destruction of the foam, from the buffer tank 12 through the open valve 22, the bacterial filter 19 and the pipe 39 is discharged into the atmosphere.

Drop liquid, after the destruction of the foam, from the buffer tank 12 through the open valve 31 enters the lock chamber 15. The air in the lock chamber 15, through the pipe 17 and through the open valve 24 is discharged into the atmosphere.

The process of accumulation of fluid in the lock chamber 15 continues until contact with the level sensor 16, the signal from which:

When implementing "periodic" processes, close the valves 31, 24 and open the valves 35, 23.

Through the pipe 40, the bacterial filter 20, the valve 23, through the pipe 17, air enters the lock chamber 15, under the pressure of which liquid from the lock chamber 15 and the open valve 35 flows into the fermentation tank 6.

Valves 23, 35 are closed, valves 31, 24 are opened, and the liquid-phase fermentation process is continued until the next contact of the working fluid with the level sensor 16.

When implementing "continuous" processes, close the valves 31, 24 and open the valves 36, 23.

Through the pipe 40, the bacterial filter 20, the valve 23, air enters the lock chamber 15 through the pipe 17, under the pressure of which the liquid from the lock chamber 15 and the open valve 36 and the pipe 42 are shipped to the product consumer.

Valves 23, 36 are closed, valves 31, 24 are opened, and the liquid-phase fermentation process is continued until the next contact of the working fluid with the level sensor.

The intensification of "liquid phase" fermentation processes is:

- In optimizing the conditions for aeration of the working fluid, which is carried out simultaneously in two fermentation tanks, in the process of liquid exchange.

- In the application of a new method for the destruction of foam in the exhaust gases. This method consists in the fact that the exhaust gas and foam are passed through a vertically mounted and cooled coil in the direction from the bottom up, which creates additional gravitational resistance to the gas-liquid flow flowing through the coil, accelerates the condensation of moisture in the foam bubbles, reduces the strength of the shells of the foam bubbles and contributes to their destruction.

- In the procedure for recirculating the working fluid and draining the liquid fermentation products, which are carried out automatically, by accumulating a predetermined volume of the working fluid in the lock chamber.

- To reduce production losses of fermentation products and time due to the exclusion of preparatory procedures, steam purging and cooling of the lock chamber with each operation.

Example 2. The algorithm for the implementation of "solid-phase" processes.

A solid substrate is introduced into the fermentation tank 1 through the loading hatch 3.

A source of prepared nutrient solution is connected to the pipe 44 of the aseptic connector.

The valves 32, 29, 22 are opened and the nutrient solution is loaded into the fermentation tank 6 in a volume equal to the volume of the solid substrate in the tank 1.

After loading the solution, the fermenter is sealed and sterilized with hot steam by a known method at predetermined values of temperature, pressure and sterilization time.

At the end of the sterilization process, the valves 25, 29, 22 are opened, and the fermenter is cooled to a predetermined temperature, when cold water is recycled through the thermostat 21 and heat exchange shirts 2, 7.

A producing culture of microorganisms is grown in a seed flask on a rocking chair until an exponential growth phase.

The flask with the seed culture of microorganisms is connected to the pipe 44, the valve 32 is opened, and the suspension of the seed microorganisms is introduced into the fermentation tank 6.

After sowing the fermentation tank 6, the valve 32 is closed, the valves 26, 30, 31, 24 are opened, and on the device 45, the temperature in the heat exchange jackets 2, 7 and the frequency of the reciprocating movement of the working fluid through the fermentation tanks 1, 6 are set.

To move the working fluid from the fermentation tank 6 to the fermentation tank 1, close the valve 29.

Aerating air through the pipe 38 and the bacterial filter 18 enters the aerator 8 of the fermentation tank 6 and penetrates the volume of the working fluid in the form of fine bubbles, mixing and displacing the working fluid from the tank 6 into the tank 1 through the bypass pipe 11.

The working fluid entering the tank 1, as in the tank 6, is exposed to fine bubbles of aerating air leaving the aerator 4, which allows you to activate the dissolution of oxygen in the working fluid and optimize the enzymatic reactions of microorganisms.

After emptying the container 6, open the valve 29, close the valve 25 and the working fluid under the action of aerating air, bypass pipe 11, return to the tank 6, and the process of reciprocating movement of the working fluid from one tank to another is repeated at a given frequency until the nutrients are depleted in the working fluid.

The sowing of the solid substrate and the propagation of microorganisms in the nutrient solution is carried out simultaneously, which allows you to evenly distribute the biomass of microorganisms in the volume of the solid substrate and adapt the microorganisms to new conditions of solid-phase fermentation.

Nutrient depletion is determined by measuring the acidity of the working fluid according to the pH sensor in the tank 6.

When the set pH value is reached, the valve 25 automatically opens, and the working fluid from the tank 1 through the pipe 11 flows into the tank 6.

At this stage of the fermentation process, the working fluid in the tank 6, depending on the task, can be replaced by a solution of a given composition. The replacement of the spent fluid with a solution of a given composition of substances is carried out through the pipe 44 of the aseptic connector.

Under the created conditions, the solid substrate in tank 1 undergoes solid-state fermentation, which proceeds with the release of liquid metabolites and excess heat, which slow down the carrying out of enzymatic reactions.

An increase in substrate temperature is a critical factor that reduces the performance of the solid-state process. Thus, an increase in temperature from the optimal value by only 5 ° C almost completely stops the reproduction of the producing culture and the fermentation of the solid substrate. The temperature of the solid-state process in the region of optimal values for carrying out enzymatic reactions, for example, for P. tigrinus culture, was experimentally 29 ± 1 ° С. Based on the result, to control and maintain a given temperature regime of the solid-phase process, a temperature was selected that was 1 ° C higher than the set value.

When the temperature in the volume of the solid substrate is increased by 1 ° C relative to the set temperature in the heat-exchange jackets, the valve 29 is closed, and the working fluid from oxygenated aeration air fills all the free space in the solid substrate.

After the tank 6 is completely emptied, the valve 29 is opened, the working fluid from the tank 1 returns to the tank 6, and a temporary pause is maintained, which is determined by the next increase in temperature in the solid substrate.

The immersion of the solid substrate first in the working fluid, and then in the atmosphere of aerating air ensures the restoration of a given temperature regime and the removal of liquid metabolites, providing optimal conditions for the implementation of the solid-phase process.

The conditions for the implementation of "solid-phase" fermentation processes are applicable to the implementation of "film" processes carried out using adsorption carriers.

The intensification of "solid-phase" and "film" fermentation processes is:

- In the inoculation of a solid substrate or carrier by producing microorganisms, which is carried out simultaneously with the process of reproduction of microorganisms in a liquid nutrient medium to a predetermined pH value.

- In the application of a new method for automatic stabilization of temperature and removal of toxic metabolites from a fermentable substrate. This method consists in the fact that when the temperature in the fermented substrate is 1 ° C higher than the set value, the solid substrate or carrier is immersed either in a solution of a given temperature and saturated with oxygen from the aeration air, or into the atmosphere of the aeration air. The method provides the restoration of a given temperature regime of the "solid-phase" or "film" fermentation process and the removal of liquid metabolites from the fermented substrate.

- The possibility of replacing the spent working fluid with a new solution, without removing from the fermenter a solid substrate or carrier enriched with producing microorganisms, which allows optimizing the conditions for enzymatic reactions in "solid-phase" and "film" fermentation processes.

The method and device according to the present invention can find application for the transformation of organic compounds using immobilized cells.

Example 3. The algorithm for the implementation of the processes of transformation of organic compounds, for example, the transformation of hydrocortisone immobilized in SDS page (polyacrylamide gel), A.globiformis cells.

Granules with immobilized cells are introduced into the cavity of the matrix (container with a perforated wall).

Through the loading hatch 3, the container is placed in a fermentation tank 1.

A source of an aqueous suspension of microcrystalline hydrocortisone is connected to the pipe 44 of the aseptic connector.

The valves 32, 29, 22 are opened and a suspension of microcrystalline hydrocortisone in a volume equal to the volume of the container in the tank 1 is loaded into the fermentation vessel 6 through the pipe 44.

The fermenter is sealed, and in the heat exchange shirts 2, 7 the temperature set on the device 45 is maintained by means of a thermostat 21 and a temperature sensor 10.

The valves 25, 26, 29, 30, 22, 31, 24 are opened, and on the device 45 the frequency of the reciprocating movement of the working fluid along the fermentation tanks 1, 6 is set.

To move the working fluid from the fermentation tank 6 to the fermentation tank 1, close the valve 29.

Aerating air through the pipe 38 and the bacterial filter 18 enters the aerator 8 of the fermentation tank 6 and saturates the suspension of microcrystalline hydrocortisone in the form of fine bubbles with air oxygen and at the same time displaces it from the tank 6 into the tank 1 through the bypass pipe 11.

Saturated with air oxygen, a suspension of microcrystalline hydrocortisone entering the tank 1, as well as in the tank 6, is exposed to fine bubbles of aerating air leaving the aerator 4.

When a container with granules of immobilized cells is immersed in a suspension of hydrocortisone around the perforated wall of the container, a screen is formed in the form of a fluidized bed consisting of a suspension of hydrocortisone saturated with oxygen in the air and fine air bubbles coming from aerator 4, which contributes to the activation of hydrocortisone transformation reactions.

After emptying the container 6, open the valve 29, close the valve 25, and the hydrocortisone suspension, under the influence of aerating air, is returned to the container 6 through the bypass pipe 11, and the process of reciprocating movement of the working fluid from one container to another is repeated with a predetermined frequency of up to 98- 100% conversion of hydrocortisone to prednisone.

The intensification of the process of transformation of organic compounds is:

- In the use of a matrix made in the form of a permeable container that protects the immobilized cells from the turbulent effects of the working suspension during aeration and mixing.

- The immobilized cells are localized in the container volume and are surrounded by a “fluidized-bed” screen of the working suspension saturated with atmospheric oxygen, which makes it possible to stabilize the percentage of dissolved oxygen in the working suspension and activate the respiratory and reaction activity of the immobilized cells.

- The transformation of organic compounds is carried out under conditions that do not allow the destruction of PAAG granules and the release of free cells into a working suspension, which allows to reduce production costs at the stages of isolation of the target product and to reuse the transforming culture in stored granules.

- In the use of two containers, in one of which a container with granules of immobilized cells is placed, and in the other - a suspension of microcrystalline hydrocortisone, which can significantly reduce manual labor and automate the procedures performed when washing granules from foreign substances and regenerating cells in aseptic PAAG granules conditions.

For all the above examples, the valves 27, 28. 33, 34, 37 operate in sterilization processes according to known technology. So, before introducing 6 sterile solutions into the fermentation tank, sowing the culture of microorganisms or carrying out the sampling procedure of the working fluid, the bypass pipe 44 is sterilized. Sterilization is carried out with open valves 33, 34 through which saturated water vapor is passed. Valve 37 is opened during sterilization of the lock chamber 15 to discharge condensate into the sewer. When sterilizing the buffer tank 12, close the valve 27 for entering cold water into the jacket 13 and open the valve 28 to discharge condensate into the sewer.

Claims (2)

1. Apparatus for enzymatic processes, containing fermentation tanks with heat-exchange jackets for carrying out liquid-phase and solid-phase processes, an aerator, a buffer tank, airlock, a control device, pipelines with controlled valves and nozzles, characterized in that the fermentation tank for carrying out solid-phase processes is equipped with a sensor temperature, the fermentation tank for carrying out liquid-phase processes is equipped with a pH sensor and a temperature sensor, while the fermentation tanks for liquid and solid-phase fermentation processes are communicated by a bypass pipe, the buffer tank is equipped with a heat exchange jacket and a coil integrated in the heat exchange jacket to divert exhaust gases and liquid fermentation products into the buffer tank, the lock chamber is equipped with a bacterial filter for pressurization of the lock chamber and a liquid level sensor that provides a predetermined liquid level the dose of draining the working fluid, where the upper cavity of the lock chamber through pipelines with valves communicated with the upper and lower cavity ufernoy capacitance and bacterial filter supercharging lock chamber and the lower chamber communicates with the liquid phase of the fermentation tank and drain pipes and sewage sludge fermentation products. 2. A method for implementing enzymatic processes using the apparatus according to claim 1, comprising the steps of introducing into the fermentation tanks a liquid nutrient medium, a solid substrate, sterilizing and cooling the apparatus, growing an inoculum culture of microorganisms, inoculating microorganisms with a liquid nutrient medium in a fermentation tank and carrying out liquid phase solid-phase, film processes under specified conditions of aeration, temperature and frequency of exchange of the working fluid between the fermentation tanks, while sowing solid cleaning with producing microorganisms is carried out in a full growth cycle of propagation of microorganisms with simultaneous deposition of cells on multiple surfaces of a solid substrate, aeration is carried out continuously in two fermentation tanks, when the temperature in the volume of the fermented substrate is 1 ° C higher than the set temperature of the coolant in the heat exchange jacket, the solid substrate is immersed into the working fluid with a given temperature, then into the atmosphere of aeration air, moving the working fluid along the enzyme ion containers carried by alternate diversion of exhaust gas containers, foam, formed in fermentation processes destroy gazopennoy by passing medium through the cooling coil against gravitational forces, liquid draining fermentation products or return the working fluid in the fermentation is carried out automatically by a predetermined level of waste liquid.

Images