RU2008347C1 - Method of microorganisms cultivation and apparatus for its realization - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: method comprises cultivation in cubic content with aeration of medium using cultural fluid of foregoing generation of microorganisms at stage of their exponential development as sowing material. This fluid is supplied from working cubic content into inoculator by means of airlift with simultaneous air pulsation 10-30 Hz and following refrigeration of cultural fluid up to 5-10 C in order to except inactivation of microorganisms. Apparatus has housing with inoculator built-in in its upper part in form of cowling with bottom. Device for supply of sowing material into inoculator is a siphon, whose upper part is located in cowling cavity and lower one is at bottom of housing. Moreover siphon is equipped with air pulsator and inoculator is equipped with autonomous heat exchanger. EFFECT: rise of output of microbic mass.

Description

 The invention relates to microbiology, and in particular to methods and devices for the cultivation of microorganisms, and can be used in the medical, chemical, food and other industries when carrying out processes in aseptic conditions, when they are subject to increased requirements to prevent penetration into sterile cavities of apparatuses and communications of extraneous microflora and the environment.

 A known method of culturing microorganisms, in which part of the culture fluid is periodically removed from the main volume of the fermenter with the gradual addition of fresh medium [1].

 This method of cultivation is intermediate between periodic and continuous, it is not acceptable for all producers and does not ensure the purity of the selection of seed. After a certain number of weaning-topping generations has elapsed, the process degenerates. Pumps are used to pump the culture fluid. At the same time, the supply of oxygen to the culture fluid in the line stops, which leads to inactivation of the producers, and the effect of the pumps has a traumatic effect on the culture of the microorganism.

 A known apparatus for the cultivation of microorganisms containing a fermenter and placed in it inoculator and sowing apparatus [2].

 The disadvantage of the apparatus is the presence of a large number of valves, devices for pumping, which increases the likelihood of infection of the culture by extraneous microflora and inactivation of the producer.

 Closest to the proposed device is an apparatus for growing microorganisms containing a vertical body, an inoculator located in its upper part, heat exchange, aeration devices and means for supplying seed to the inoculator.

 The disadvantages of this apparatus are also not a sufficiently high degree of maintenance of aseptic conditions during the cultivation process and the possibility of injury to the culture of microorganisms during pumping of the culture fluid using pumps.

 The aim of the proposed method and device is to increase product yield by eliminating the inactivation of producers and increasing aseptic performance.

 To achieve this, in the known method of cultivating microorganisms in a fermenter with an inoculator placed inside the working volume, the culture fluid of the previous generation at the exponential development stage is used as seed material, supplied from the fermenter working volume to the inoculator by airlift by pulsating air supply with a frequency of 10-30 Hz under fermenter filling level with further cooling.

 To achieve this goal, in the known device for the deep cultivation of microorganisms, containing a vertical casing, an inoculator located in its upper part, heat exchange, aeration devices and means for supplying seed to the inoculator, the latter is made in the form of a siphon, the upper curved end of which is lowered into the internal cavity inoculator, and the bottom is located with a gap from the bottom of the fermenter and is connected to the pulsator via an air duct located below the filling level of the fermenter .

 From the scientific, technical and patent literature, the authors are not aware of sources containing a combination of features set forth in the formula, which allows us to conclude that the proposals meet the criterion of "significant differences".

 In FIG. 1 shows a device for the cultivation of microorganisms, a longitudinal section.

 The apparatus for growing microorganisms contains a vertical cylindrical body 1, an aeration device consisting of an air supply pipe 2 with an aerator 3, and a mixing device containing a shaft 4 with mixers 5. A shell 6 with a bottom 7 mounted to the wall is installed coaxially to the body 1 the body forming the inoculator. In the bottom 7, tubes 8 are welded for overflowing the culture fluid from the working volume of the inoculator to the working volume of the apparatus or for sampling. A vertical annular circulation partition 9 is placed between the apparatus body and the shell 6 with a gap to the bottom 7. The upper edge of the partition 9 is located below the upper edge of the shell 6. An annular aeration device (aerator) is placed between the case wall and the circulation partition 9 10. Highways 11 and 12 for supplying a nutrient medium are connected respectively to the inoculator and apparatus. The supply of the culture fluid from the working volume of the fermenter to the inoculator is carried out using a siphon 13. The siphon is connected to the pulsator 14 via an air duct 15. The air duct 15 is located below the filling level of the fermenter. The heat exchange device 16 creates an autonomous cooling system for the inoculator, the heat exchange device 17 - for the fermenter.

 The operation of the apparatus is as follows.

 After sterilization, the nutrient medium is supplied through the line 11 and the inoculator and through the line 12 to the main unit. In this case, filling the inoculator with nutrient medium takes several minutes, and filling the main apparatus lasts about 2 hours. After filling the inoculator, it is inoculated with a pure culture of microorganisms, while filling the main apparatus continues. The aerator 10 and the heat exchange device 16 are turned on, and the biosynthesis process in the inoculator begins.

By the time the maximum concentration of microorganisms in the inoculator is reached, the nutrient medium fills the required volume of the main apparatus. Bring its temperature to the optimum temperature of cultivation. After that, sampling from the inoculator is carried out in order to check for microbiological analysis and transfer of culture fluid from the inoculator to the working volume of the apparatus. Air is supplied to the bubbler and the agitator 5 is turned on. The cultivation process is carried out in the main apparatus. Upon reaching the stage of exponential development of culture in the main apparatus, the culture fluid is supplied from the working volume to the inoculator. To do this, compressed air with a frequency of 10-30 Hz is supplied using a pulsator 14 through an air pipe 15 to the siphon 13. The culture fluid from the working volume of the fermenter is ejected through the lower end of the siphon. The rise of the resulting gas-liquid mixture occurs due to the airlift effect. The inoculator culture fluid quenched to avoid inactivation temperature to 5-10 ^{° C} and stored until the next growing season, which is used as inoculum. Subsequently, the cycle repeats.

 The use of the culture fluid of the previous generation as a seed at the stage of exponential development creates an advantage over the known method, since it makes it possible to use a young culture with a high generation coefficient and high reproduction rate and allows to increase the yield of the product and improve the aseptic characteristics of the process.

 The supply of culture fluid from the working volume of the fermenter to the inoculator by airlift by means of pulsating air supply also contributes to the achievement of the goal, which is confirmed by the data of experimental studies.

 In FIG. Figure 2 shows a graph of the growth of the exoprotease activity of Bac bacteria. thuring. var kenyae when cultured according to the known method in the known device (a), according to the proposed method in the proposed device (b).

 From the graph shows that the maximum exoprosthetic activity is observed during cultivation in the known device according to the known method at 14 hours, in the proposed - at 12 hours of cultivation and is respectively 96.8 and 432.6 μg / ml.

 A spasmodic increase in the productivity of the apparatus from the frequency of the pulsating air supply is observed in the range of 10-30 Hz (table). (56) 1. Yakovlev V.I. Technology of microbiological synthesis. M.: Chemistry, 1987, p. 26.

 2. USSR author's certificate N 1161545, cl. C 12 M 1/04, 1985.

 3. Copyright certificate of the USSR N 1010122, cl. C 12 M 1/04, 1983.

Claims (2)

1. A method of cultivating microorganisms, including growing them in a fermenter vessel using an inoculator located in the working volume of the culture medium, and providing for aeration of the culture fluid, characterized in that the culture fluid of the previous generation of microorganisms is used as seed material at the stage of their exponential development by feeding of this liquid from the working volume of the fermenter to the inoculator by means of airlift with simultaneous pulsating flow aerating of air with pulsation frequency of 10 - 30 Hz under the filling level of the container and the culture liquid, followed by cooling down to a temperature of 5 - 10 ^o C to eliminate the inactivation of microorganisms.  2. An apparatus for cultivating microorganisms, comprising a vertical housing, an inoculator placed therein, an aeration device, a heat exchange device and means for supplying seed to the inoculator, characterized in that the inoculator is located in the upper part of the container and is made in the form of a shell with a bottom, and the means for supplying seed is a siphon, the upper curved section of which is located in the inner cavity of the specified shell and the bottom is located at the bottom of the fermenter with a gap, while the siphon is equipped with n pulsator supply air duct connected to it in its lower part, the inoculator equipped with autonomous heat exchange device.

Images