JPH0333313B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus for cultivating microorganisms, and particularly to a high-yield culturing apparatus for microorganisms suitable for fed-batch culture aiming at high yield.

Conventionally, microbial cultivation by fed-batch culture has been performed based on the pH, temperature, pressure, and dissolved oxygen concentration (hereinafter referred to as DO) of the medium.
It is carried out by continuously or intermittently supplying the substrate to the medium while maintaining the environment of the bacterial cells during cultivation under optimal conditions. The method of supplying the substrate is to estimate the preferable relationship between culture time and substrate supply rate based on past culture results for each culture, create a program for the substrate supply rate before the start of culture, and then use that program. A method of supplying the substrate continuously or intermittently is used.

However, the substrate supplied in each culture is not necessarily the same, and the activity of the bacterial cells is also not the same, so the conventional substrate supply method described above cannot always perform high-yield cultivation. Ta. For example, when culturing ethanol-utilizing bacteria and methanol-utilizing bacteria using ethanol and methanol as the main carbon sources, respectively, an excessive substrate supply rate will inhibit the growth of the bacterial cells, and conversely, an insufficient substrate supply rate will cause inhibition of bacterial growth. As a result, bacterial cell growth is suppressed. In addition, when culturing baker's yeast using sugar as the main carbon source, if the substrate supply rate becomes excessive, baker's yeast will
The supplied sugar is converted to ethanol, resulting in a decrease in sugar yield, and conversely, if the substrate supply rate is insufficient, yeast growth is suppressed and productivity decreases. As is clear from the above culture examples, the amount of bacterial cells in the medium and their activity can be quickly and accurately determined.
Based on this, it is important to appropriately control the substrate supply rate in order to achieve high-yield culture.

Conventional methods for determining the amount of bacterial cells in a culture medium include collecting a portion of the culture medium, separating the bacteria by centrifugation or filtration, drying it, and measuring the dry weight; Methods of collecting bacteria and counting the number of bacteria using a microscope, and measuring the concentration of bacteria from the turbidity of the culture medium are known. but,
The method (2) has a time problem in that it takes more than 10 hours to obtain the dry weight after collecting the culture medium, and the method (2) has a problem in accuracy due to large variations in the counted values. In the method of , it is necessary that the medium be transparent, but
In industrial cultures, many of the cultures are colored and often contain solid substances other than bacterial cells, so there are accuracy problems that make it difficult to accurately determine the bacterial yield. Therefore, at present, there is no method for quickly and accurately grasping the amount of bacterial cells in a culture medium.

On the other hand, methods using RQ (respiratory quotient) and oxygen consumption rate are conventionally known as methods for determining bacterial cell activity in a culture medium. In the method, the ventilation flow rate, exhaust flow rate, oxygen partial pressure of ventilation and exhaust,
It is necessary to measure the partial pressure of carbon dioxide quickly and accurately, and in the method described above, it is necessary to measure the ventilation flow rate, exhaust flow rate, and oxygen partial pressure of each of the ventilation and exhaust quickly and accurately. . However, the aeration flow rate is often increased or decreased to maintain a constant DO during culture;
Oxygen partial pressure and carbon dioxide partial pressure lose their balance due to ventilation and exhaust. Therefore, the RQ and oxygen consumption rate determined by this measurement value are no longer indicators of bacterial cell activity, which was their original purpose. , and there is no way to accurately grasp it.

The present invention aims to solve the above-mentioned problems, and aims to improve the oxygen consumption rate of microorganisms and the activity of microorganisms (= amount of microorganisms x average activity of all microorganisms) in the cultivation of microorganisms by fed-batch culture. There is a proportional relationship between the amount of bacterial growth and the rate of oxygen consumption of the bacterial cells.
Based on the fact that there is a proportional relationship with the amount of change in the DO of the culture medium that occurs when the oxygen transfer conditions (operating conditions related to oxygen transfer) are temporarily increased or decreased by a certain amount while maintaining the DO at a constant value, D.O.
The oxygen consumption rate and bacterial cell activity of the bacterial cells are determined from the amount of change, and based on this, the substrate supply rate is controlled according to the oxygen consumption rate of the bacterial cells (=oxygen demand rate ∝ bacterial cell activity). By doing so, the aim is to provide a high-yield culturing device for microorganisms by fed-batch culture that can always maintain optimal substrate supply conditions and maintain a high yield of microbial cells or products.

An embodiment of the present invention will be described with reference to FIGS. 1 to 5. FIG. 1 is a flow sheet of a high-yield culturing device for microorganisms according to the present invention. In FIG. 1, 1 is a culture tank, and 2 is a stirring blade for stirring a culture medium 3, which is driven by a drive device 4. 5 is a pressure measuring device in the culture tank 1, 6 is a medium PH measuring device, 7 is a medium temperature measuring device, and 8 is a medium DO measuring device. 9 is an analog/digital converter that converts each value measured by each measuring device such as the pressure measuring device 5 into a digital value, and 10 is an analog/digital converter for inputting each value converted to a digital value, and a separate numerical input device 11. This is a calculation/control/storage/input/output device (hereinafter abbreviated as the calculation device) that compares and calculates each input setting value and outputs the respective control output signal to each adjustment device. 12 is a pressure regulator, 13 is a PH regulator, 14 is a temperature regulator, 15 is a rotation speed regulator for the stirring blade 2, 16 is an aeration oxygen partial pressure regulator, 17
18 is an aeration flow rate adjustment device, and 18 is a substrate supply rate adjustment device, and each adjustment device is connected to the calculation device 10. Reference numeral 19 denotes a culture state monitoring device for monitoring the culture state, which is separately connected to the arithmetic device 10.

In the high-yield culturing apparatus for microorganisms configured as described above, the pressure inside the culture tank 1, the pH of the medium 3,
Temperature and DO are measured by a pressure measuring device 5, a PH measuring device 6, a temperature measuring device 7, and a DO measuring device 8, respectively, and these measured values are converted into digital values by an analog/digital converter 9, and then converted to a digital value by an arithmetic device 10.
These values are compared and calculated with respective set values input separately from the numerical input device 11, and respective control output signals are sent from the calculation device 10 to the pressure adjustment device 12, PH adjustment device 13, and temperature adjustment device 1.
4. The output is sent to the aeration oxygen partial pressure adjustment device 16 and the aeration flow rate adjustment device 17 respectively, and while the culture condition is monitored by the culture condition monitoring device 19, the environment of the bacterial cells being cultured is controlled to the optimum condition, and the substrate is controlled. Proper control of feed rate is accomplished as follows. In addition, the oxygen transfer conditions that are temporarily changed by a certain amount in order to understand the oxygen consumption rate include stirring blade rotation speed, culture tank pressure, aeration flow rate, aeration oxygen partial pressure, substrate supply rate, and substrate concentration. However, in this example, the case where the rotational speed of the stirring blade is used as the oxygen transfer condition will be explained.

In order to understand the oxygen consumption rate, which is proportional to the activity of bacterial cells and the amount of bacterial growth, the measurement interval was set at
At the cycle of , and during the measurement time roh, the arithmetic unit 10 outputs a control output signal to the rotational speed adjustment device 15, and the stirring blade rotational speed, which is the oxygen transfer condition, is temporarily adjusted as shown in Fig. 2. A certain amount △ from the initial setting value ha rpm
Increase only Har rpm. Due to this increase in the rotation speed of the stirring blade, DO increases to the initial setting value as shown in Figure 3.
mg/ increases and changes by △ni and △ni′mg/, respectively. As shown in Figure 4, even if the rotational speed of the stirring blade is increased by the same amount, the oxygen consumption rate changes due to changes in the growth and activity of the bacterial cells as the culture time progresses, and the rate of oxygen consumption changes accordingly. In order to change the oxygen supply rate, the amount of change in DO is changed as shown in Figure 3. The DO value that changed as shown in Figure 3 (2+△ni, ni+△ni'mg/) was measured by the DO measuring device 8.
These measured values are converted into digital values by an analog/digital converter 9, and then converted to digital values by an arithmetic unit 10.
input and memorize it. Thereafter, based on the DO value stored in the arithmetic device 10, a control output signal for the substrate supply rate is calculated by the arithmetic expression (1) and is outputted from the arithmetic device 10 to the substrate supply rate adjustment device 18. As a result, the substrate supply rate is as shown in Figure 5.
The amount of change in DO in the figure is △d3dd'mg/, that is, depending on the oxygen consumption rate, △
E, increases by △E'/h. By periodically controlling the substrate supply rate during culture, optimal substrate supply conditions can be maintained at all times.

F=C×(DO+△DO)...(1) Here, F: current substrate supply rate (/h),
C: constant, DO: initial setting value of DO (mg/), △
DO: Change in DO (mg/) When a culture experiment was conducted under the following culture conditions, the culture time was approximately 1/2 that of the conventional culture method.
A final bacterial cell concentration of 100 g/g was obtained in 8 hours.

(1) Fungal body: Baker's yeast (Saccharomyces)
cerevisiae), initial cell concentration 50 g/ (2) Medium: Glyose was adjusted to 30% concentration, urea, sodium phosphate, magnesium sulfate, sodium citrate, yeast extract, and vitamin solution were added, and the mixture was dissolved in tap water.

(3) Culture conditions: using a fermenter with a capacity of 50,
Temperature is 30℃, pH is 5, DO is 4~5mg/
It was controlled so that In addition, DO control is performed by controlling the aeration flow rate to 5 to 50/min, the aeration oxygen partial pressure to 0.21 to 1.0 atm, and the stirring blade rotation speed to 50 to 500 rpm.
The pressure was set to 0.5Kgcm ³ G.

As explained above, the present invention is capable of culturing microorganisms by fed-batch culture while controlling the pressure in the culture tank, the pH of the culture medium, temperature, and DO to optimal conditions.
The amount of change in DO in the culture medium that occurs by temporarily increasing or decreasing the oxygen transfer conditions in the culture tank by a certain amount is measured, and based on this measurement value, the oxygen consumption rate and bacterial activity of the bacterial cells can be ascertained. By controlling the substrate supply rate according to the oxygen consumption rate of the microbial cells based on this, it is possible to maintain optimal substrate supply conditions at all times during culture, which has the effect of enabling high-yield culture.

[Brief explanation of drawings]

Figures 1 to 5 are for explaining an embodiment of the present invention. Figure 1 is a flow sheet of a microorganism high-yield culturing device, and Figure 2 is a diagram showing the passage of culture time and rotation of the stirring blade. Figure 3 is a diagram showing the relationship between DO and the passage of culture time, Figure 4 is a diagram showing the relationship between stirring blade rotation speed and the amount of change in DO as the oxygen consumption rate changes, and Figure 5 is a diagram showing the relationship between DO and the passage of culture time. , is a diagram showing the relationship between the passage of culture time and the substrate supply rate. 1... Culture tank, 2... Stirring blade, 3... Culture medium, 8
...DO measuring device, 9... Analog/digital conversion device, 10... Arithmetic device, 15... Rotation speed adjusting device, 18... Substrate supply rate adjusting device.

Claims (1)

[Claims] 1 Pressure measuring device and PH measuring device that measure the pressure in the culture tank, the pH of the culture medium, temperature, and DO, respectively.
The temperature measurement device, the DO measurement device, and the measurement values and set values input from each of the measurement devices are compared and calculated, and a control signal is sent to the pressure adjustment device, PH adjustment device, temperature adjustment device, rotation speed adjustment device, ventilation oxygen. A high-yield culturing device for microorganisms, comprising a partial pressure regulating device, an aeration flow rate regulating device, and an arithmetic device that outputs output to a substrate supply rate regulating device.