JPH06261741A - Method of multiplying microorganism - Google Patents

Abstract

PURPOSE:To simplify a device by adopting DO as an index of addition of a nutritive solution of glucose. CONSTITUTION:When an amount of dissolved oxygen in a medium becomes >=a set value, a nutritive solution of glucose is added to the medium. Since the glucose concentration in the medium is maintained in a fixed range, multiplication efficiency is improved. DO is rapidly and simply detected, a specific device is not required and the device is not complicated.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for growing a microorganism by fed-batch culture.

[0002]

2. Description of the Related Art In growing a microorganism, a fed-batch culture is known in which a nutrient solution is continuously or intermittently fed into a culture tank for storing a medium according to the progress of culture time. In this fed-batch culture, the concentration of the specific component in the medium can be arbitrarily controlled, and for example, the sugar concentration can be kept within a certain range suitable for the microorganism to be cultured. For this reason, it is widely adopted because the target microorganism can be efficiently proliferated.

In controlling the feeding timing of the nutrient solution in fed-batch culture, 1) the sugar concentration (particularly glucose concentration) is detected by a biosensor (glucose sensor),
A method of feeding a nutrient solution according to the detected value, 2) a method of using dissolved oxygen as an index, 3) pH value, 4) redox potential, 5) respiratory quotient (CO ₂ gas amount and O ₂ consumption amount) Ratio),
Methods such as 6) the concentration of ethanol in exhaust gas and 7) the concentration of bacterial cells have been proposed.

[0004]

In each of the above methods,
Although the method of 1) using a glucose sensor is widely used, this method has problems that the detection speed of the glucose sensor is not fast and that the device is complicated. Further, none of the other methods 2) to 7) was still satisfactory. Therefore, there has been a demand for a method that can speed up the index detection and simplify the device.

[0005]

Means and Actions for Solving the Problems The inventor has found that there is a very close relationship between the increase and decrease of glucose concentration and the increase and decrease of dissolved oxygen in the medium in which the microorganism is cultivated. Instead of the above, the present invention has been completed through repeated studies for using the dissolved oxygen amount as an index for adding a nutrient solution.

The gist of the present invention, which has been made to solve the above problems, is to add a nutrient solution to the medium when the amount of dissolved oxygen in the medium increases in the method for growing a microorganism by fed-batch culture. There is a method for multiplying microorganisms. Hereinafter, the present invention will be described in more detail.

There is no limitation on the kind of the microorganism to be proliferated in the present invention, as long as it can be fed-batch culture. As the nutrient solution, a well-known nutrient solution containing, for example, one or several kinds of glucose, yeast extract, potassium dihydrogen phosphate (KH ₂ PO ₄ ) and the like may be used, and there is no particular limitation. Further, the concentration and addition amount of the nutrient solution may be appropriately selected according to the target microorganism. For example, in the case of growing yeast, a nutrient solution containing glucose may be added so that the glucose concentration in the medium will be about 0.5% or less. This is because the glucose concentration in the medium is 0.5
This is because if it exceeds%, the amount of alcohol produced increases and the growth of bacterial cells tends to be suppressed. Therefore, it is preferable that the nutrient concentration of the medium is within a concentration range in which the production of such metabolites is suppressed as much as possible and the cells are grown. Further, various conditions such as the medium, the culture temperature, the air supply amount, and the stirring of the medium may be set according to the growth conditions of the target microorganism, and are not particularly limited.

[0008] To detect the amount of dissolved oxygen (DO) in the medium,
Known DO sensors can be used. Microorganisms in the medium grow while consuming nutrients such as glucose, but at this time, oxygen that is normally discharged and supplied into the medium as air is also consumed. When the nutrients in the medium are consumed and become insufficient, the growth of microorganisms is suppressed, so that the oxygen consumption also decreases. Therefore, excess oxygen is produced and DO in the medium is increased. By feeding the nutrient solution in accordance with this increase in DO, the nutrient concentration of the medium can be maintained within the concentration range suitable for growth. Therefore, the growth of microorganisms is not suppressed and the growth efficiency is improved.

In particular, if nutrients are fed in at the early stage of nutrient deficiency, the nutrient deficiency state can be eliminated in a short time. DO increases sharply in the early stage of this nutritional deficiency.
The rate of increase of O tends to increase, and the second derivative is positive. Therefore, when the increasing rate of DO is increasing (= the second derivative is positive), the nutrient concentration can be kept in a more preferable range by feeding the nutrient solution.

The detection of the DO value by the DO sensor is quickly performed in almost real time. Further, since the DO sensor does not require a special device configuration for detecting DO, the breeding device does not become complicated even when online control of the feeding of the nutrient solution is performed using the detected value as an index.

[0011]

Embodiments of the present invention will now be described with reference to the drawings. FIG. 1 shows a breeding apparatus 10 used in this example. A stirrer 16 driven by a motor 14 is installed in the center of the head 12a of the culture tank 12 made of stainless steel, and a plurality of stirring blades 20 provided on a stirring shaft 18 inserted into the culture tank 12 are provided. Thus, the contents of the culture tank 12 can be stirred. Further, the head 12a has an inoculation port 22 for inoculating a microorganism to be cultured, a bubble sensor 24 for detecting a foaming state in the culture tank 12, and a vent 26 for discharging gas from the culture tank 12. And the steam pipe 28 are connected to each other. Further, the nutrient solution tank 34 is attached to the feeding nozzle 30 provided on the head 12 a via the pump 32.
Are connected, and the nutrient solution from the nutrient solution tank 34 can be supplied to the culture tank 12.

The nutrient solution tank 34 is connected to an alkaline tank 38 via a ball valve 36. In addition, the nutrient solution tank 34
And the alkaline tank 38 is connected to the culture tank 1 via the solenoid valve 40.
Alkaline nozzle 42 provided on the upper part of the second body 12b
The nutrient solution and the alkaline solution can be supplied to the culture tank 12 individually or simultaneously. A defoaming liquid tank 48 is connected to the defoaming nozzle 44 provided below the alkali nozzle 42 via an electromagnetic valve 46, and the defoaming liquid can be supplied to the culture tank 12.

The body 12b of the culture tank 12 is provided with a pair of air nozzles 50 and 52 at substantially the same level as the alkali nozzle 42 and the defoaming nozzle 44, and is sterilized by a filter mechanism (not shown). It is connected to an air pipe 54 to which sterile air is supplied. The air nozzle 52
Further, it is connected to a sparger 56 installed in the culture tank 12, and the air from the air pipe 54 is supplied to the culture tank 12.
It is possible to discharge inside.

The culture tank 12 is provided with a jacket 58 from the body 12b to the bottom 12c, and the temperature is adjusted by hot water supplied from a temperature adjusting unit 60 equipped with a heater, a pump, etc. (not shown) through hot water piping 62. It is possible. In addition, water is supplied from the water pipe 64 to the jacket 58,
Steam can be supplied from the steam pipe 28 to be cooled or heated. Further, a thermometer 66, a DO sensor 68 (manufactured by Ingold Co., Ltd.) and a pH meter 70 are installed in the body 12b of the culture tank 12 so as to penetrate the jacket 58, and the temperature and the amount of dissolved oxygen in the culture tank 12 are respectively provided. And pH can be measured and output. Further, similarly, a sampling nozzle 72 is installed on the body 12b so that sampling can be performed from the inside of the culture tank 12. On the other hand, at the bottom portion 12c of the culture tank 12, a discharge nozzle 74 is installed outside the jacket 58, and the contents of the culture tank 12 can be discharged.

Although not shown, the culture tank 12 is provided with a manhole, a handhole, a level gauge, a pressure gauge, etc. Similarly, gauges, vents, drains, It is equipped with a steam trap. As shown in FIG. 2, the breeding apparatus 10 is provided with an operation panel 84 including a control mechanism 80, an autoplotter 82 and the like. The control mechanism 80 includes a well-known microcomputer (not shown), and various instructions can be input through the keys 86, the switch 88, and the like. The control mechanism 80 inputs through the keys 86 and the like, the bubble sensor 24, the thermometer 66, the DO sensor 68, p.
When the detection values of instrumentation devices such as the H meter 70 are input, arithmetic processing is performed according to a program stored in advance, and the pump 32, the solenoid valves 40 and 46, and the temperature control unit 60 are processed according to the input. Etc. can be controlled. Further, the output values of the thermometer 66 and others are transmitted to the autoplotter 82 via the control mechanism 80 and plotted on the recording paper.

Next, an operation for growing a microorganism by using the growth device 10 having the above structure will be described. First, a medium and a nutrient solution are poured into the culture tank 12, and the medium is stirred and aerated to obtain a medium having a predetermined glucose concentration. The medium is continuously agitated and aerated throughout the growth period. Medium temperature, p
After adjusting the H value, the culture tank 12 is inoculated with a microorganism.

After this, the microorganisms grow by consuming the nutrients in the medium, for example glucose. A nutrient solution containing glucose is fed in order to keep the glucose concentration, which is lowered by consumption, in an appropriate range. The timing of feeding the nutrient solution is detected by the DO sensor 68 to control the control mechanism 8
The DO value input to 0 is determined as an index. Although the fed-batch routine is shown in FIG. 3, the control mechanism 80 repeatedly executes the fed-batch routine each time a predetermined time elapses.

First, when the detection value (D _K ) of the DO sensor 68 is input, the control mechanism 80 compares the detection value (D _K ) of the DO sensor 68 with the DO setting value (D _S ) (step). Two
00), if D _K is greater than or equal to D _S , proceed to the next step 300, and if D _K is less than D _S , exit to return and wait for a predetermined time. In step 300, the control mechanism 80 operates the pump 32 for a preset time to feed a predetermined amount of nutrient solution to the culture tank 12. After that, the process returns to return and the fed-batch routine is repeatedly executed every time a predetermined time elapses. When glucose is fed when DO increases above the set value, the glucose concentration can be maintained in an appropriate range. The changes in DO and glucose concentration in this case are shown in FIG.

Further, 1) an increase rate of the detection value D _K of the DO sensor 68 per unit time is calculated, and glucose is fed when it exceeds a set value, or 2) a unit time of D _K. It is also possible to compare the increase rate per hit with the immediately preceding increase rate, and to perform glucose fed-batch control such as feeding glucose when the increase rate exceeds a set ratio.

Next, examples of microbial growth using the growth device 10 having the above-mentioned configuration will be shown together with comparative examples. In addition, in both the examples and comparative examples, the medium had the following composition. Medium composition Honjo soy sauce 10% Salt 10% Sterile water Residue (Example 1) Microorganism type: Yeast (Z.rouxii (S-012)) Initial number of cells: 4.0 × 10 ⁶ cells / ml Medium volume: 50 l (plant) Glucose concentration in bacteria 0.5%) Medium temperature: 30 ° C. Medium pH: 5.2 Aeration rate: 30 l / min Agitation: 200 rpm Nutrient solution: Glucose concentration 50%, 0.3% relative to medium
Fed-batch Growth time: 43 hours Final cell number: 2.7 × 10 ⁹ cells / ml (Comparative Example 1) Comparative Example 1 is an example of a growth experiment by batch culture, and the addition of glucose in the culture was Absent.

Type of microorganism: Yeast (Z.rouxii (S-012)) Number of initial cells: 2.5 × 10 ⁶ cells / ml Medium amount: 50 l (Glucose concentration at inoculation 2.6%) Medium temperature: 30 ° C. Medium pH: 5.2 Aeration rate: 30 l / min Stirring: 100 rpm Growth time: 43 hours Maximum cell number: 2.8 × 10 ⁸ cells / ml (18 hours after initiation) (Comparative Example 2) Comparative Example 2 is Comparative Example 1 Similarly to the above, this is an example of a growth experiment by batch culture, and DO was kept constant (3 ppm) by changing the stirring number, although glucose was not added during the culture.

Type of microorganism: Yeast (Z.rouxii (S-012)) Number of initial cells: 3.8 × 10 ⁶ cells / ml Medium amount: 50 l (Glucose concentration at inoculation 2.6%) Medium temperature: 30 ° C. Medium pH: 5.2 Aeration rate: 30 l / min Agitation: 100 to 400 rpm Growth time: 43 hours Final cell number: 5.5 × 10 ⁸ cells / ml FIG. 5 shows the culture medium in Example 1 and Comparative Examples 1 and 2 above. It is the graph which plotted the turbidity, the number of microbial cells (viable bacterium), and the alcohol concentration for every elapsed time. The number of cells of Example 1 increased even after 24 hours when the number of cells of Comparative Examples 1 and 2 almost reached the ceiling, and the final number of cells was about 5 to 10 times that of Comparative Examples 1 and 2. Has become. In addition, in Example 1, it can be seen that there is almost no increase in alcohol concentration, and glucose is effectively consumed for bacterial cell growth.

Next, examples of microbial growth (Example 2 and Comparative Example 3) when the amount of medium is 1000 l are shown. (Example 2) Microorganism type: yeast (Z.rouxii (S-012)) Initial number of cells: 6.0x10 ⁶ cells / ml Medium amount: 1000 l (glucose concentration at inoculation 0.5%) Medium temperature: 30 C. Medium pH: 5.2 Aeration rate: 500 l / min Stirring: 110 rpm Nutrient solution: glucose concentration 50%, 0.25 to medium
Fed-batch in the amount of 100%, but the total fed-batch sugar amount is 3.0% with respect to the medium Growth time: 60 hours Final cell count: 3.5 × 10 ⁸ cells / ml (Comparative Example 3) Comparative Example No. 3 is an example of a growth experiment by batch culture, and glucose was not added during the culture.

Microorganism type: Yeast (Z.rouxii (S-012)) Initial number of cells: 5.9 × 10 ⁶ cells / ml Medium amount: 1000 l (Glucose concentration at inoculation 3.5%) Medium temperature: 30 ° C. Medium pH: 5.2 Aeration rate: 500 l / min Agitation: 110 rpm Growth time: 60 hours Final cell number: 1.5 × 10 ⁸ cells / ml FIGS. 6 to 8 show the turbidity of the medium in Example 2 and Comparative Example 3 described above. 2 is a graph in which the number of bacterial cells (viable cells) and the glucose concentration are plotted for each elapsed time. Example 2
The final number of bacterial cells was about 2.3 times that of Comparative Example 3,
Example 2 has excellent microbial growth efficiency.

Although the microbial growth method of the present invention has been described above according to the embodiments, the present invention is not limited to such embodiments and can be variously implemented without departing from the scope of the present invention.

[0026]

INDUSTRIAL APPLICABILITY In the microbial growth method of the present invention, the amount of dissolved oxygen (D) is used as an index for determining the timing of fed-batch.
O) is adopted, the detection speed is fast and the apparatus can be simplified.

[Brief description of drawings]

FIG. 1 is a flow diagram showing a configuration of a breeding apparatus of an embodiment.

FIG. 2 is an explanatory diagram of an operation panel of the breeding apparatus of the embodiment.

FIG. 3 is a flowchart of fed-batch control in the control mechanism of the breeding apparatus of the embodiment.

FIG. 4 is a graph showing the relationship between the fed-batch of glucose, the change in DO, and the change in glucose concentration in the growth of microorganisms using the growth apparatus of the example.

FIG. 5 is a graph showing changes over time in yeast growth of Example 1 and Comparative Examples 1 and 2, (a) is the turbidity of the medium, (b) is the number of cells, and (c) is the alcohol concentration of the medium. Represents

FIG. 6 is a graph showing the change over time in the turbidity of the medium during yeast growth of Example 2 and Comparative Example 3.

FIG. 7 is a graph showing changes over time in the number of bacterial cells in yeast growth of Example 2 and Comparative Example 3.

FIG. 8 is a graph showing changes over time in glucose concentration of the medium during yeast growth of Example 2 and Comparative Example 3.

[Explanation of Codes] 10 ... Proliferation device, 12 ... Culture tank, 22 ... Inoculation port, 30 ... Fed-batch nozzle, 32 ... Pump, 34
... nutrient solution tank, 68 ... DO sensor.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12N 1/20 C12R 1:01) (72) Inventor Takahiko Yoshida Sanbonmatsu, Atsuta-ku, Nagoya-shi, Aichi Machi No. 1 Japan Vehicle Manufacturing Co., Ltd. (72) Inventor Kazuhiro Ozawa 53, Shinotsuka Wakamiya, Kosakai-cho, Takai-gun, Aichi Prefecture Sanbishi Co., Ltd. 53 Shinotsuka Wakamiya Sanbishi Co., Ltd.

Claims (4)

[Claims] 1. A method for growing a microorganism by a fed-batch culture, which comprises adding a nutrient solution to the medium when the amount of dissolved oxygen in the medium increases. 2. The method for growing a microorganism according to claim 1, wherein a nutrient solution is added to the medium when the rate of increase of the dissolved oxygen amount per unit time tends to increase. 3. The method for growing a microorganism according to claim 1 or 2, wherein the microorganism is yeast. 4. The method for growing a microorganism according to claim 1 or 2, wherein the microorganism is a lactic acid bacterium.