JPS6018391B2 - High-yield culture method for microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to fed-batch culture aiming at high yield, and in particular,
The present invention relates to a high-yield culture method for microorganisms suitable for culturing with a large amount of fermentation heat, such as oxygen-enriched culture and culture of methanol-assimilating yeast.

Conventionally, microorganisms are cultured while maintaining the environment for the microorganisms during culture (hereinafter referred to as microbial environment) at optimal conditions, such as the pressure inside the culture pot, the pH of the medium, the temperature, and the dissolved oxygen concentration. Based on past culture results, we estimate the preferable relationship between culture time and substrate supply rate, and based on this, we create a program for the substrate supply rate before the start of culture. It is carried out either by fixed or intermittent supply.

However, in each culture, the substrates supplied are not necessarily the same, and accordingly, the activities of the microorganisms are also not the same, so it has not been possible to always perform high-yield cultures.
For example, when culturing ethanol-utilizing bacteria and methanol-utilizing bacteria using ethanol or methanol as the main carbon source, if the amount of substrate supplied becomes excessive, bacterial growth will be inhibited.
On the other hand, when it is insufficient, bacterial growth is suppressed. In addition, when culturing Paso yeast using sugar as the main carbon source, if the amount of substrate supplied becomes excessive, baker's yeast will convert the supplied sugar to ethanol, resulting in a sugar yield (bacterial cell yield relative to the amount of substrate supplied). The ratio of the amount of growth () decreases, and conversely, when it is insufficient, the growth of baker's yeast is suppressed and productivity decreases. As is clear from the above culture examples, it is important to quickly and accurately grasp the amount of microbial cells in the medium and their activity, and to appropriately control the substrate supply rate based on this information, to make the culture process more efficient. It is heavy to drive. Currently, the methods for determining the amount of bacterial cells in a culture medium include: 1) collecting a portion of the culture medium, separating the bacteria by centrifugation or filtration, drying it, and measuring the dry weight; 2) a part of the tower material; How to collect bacteria and tell the number of bacteria using a microscope,
Alternatively, a method is known in which (2) the turbidity of the medium is measured to determine the bacterial cell concentration.

However, method (■) has a time problem in that it takes more than one feeding time from collecting the culture medium to obtaining the dry weight, and method (2) has an accuracy problem in that the counted values vary widely. There is a point. In addition, method (■) requires that the culture medium be transparent, but in recreational culture, it is often colored or contains solid matter other than bacterial cells, so the bacterial cell concentration There is an accuracy problem in that it is difficult to accurately grasp the As described above, there is currently no method for quickly and accurately determining the amount of bacterial cells in a culture medium. On the other hand, currently, as a method to understand the activity of bacterial cells in the culture medium,
Methods using ■RQ (respiratory quotient) and ■oxygen consumption rate are known.

In the method (2), it is necessary to quickly and accurately measure the ventilation flow rate, exhaust flow rate, and the oxygen partial pressure and carbon dioxide partial pressure of ventilation and exhaust, respectively. In method (2), it is necessary to quickly and accurately measure the ventilation flow rate, exhaust flow rate, and oxygen partial pressure of each of the ventilation and exhaust gases. However, the aeration flow rate is often increased or decreased in order to maintain the dissolved oxygen concentration during culture at a constant value, and at this time, the oxygen partial pressure and carbon dioxide partial pressure become unbalanced due to aeration and exhaust. Therefore, there is a problem in that the RQ and oxygen consumption rate determined by these measured values are no longer indicators of the activity of the bacterial cells, which is the original purpose. As described above, there is currently no method for accurately determining the activity of bacterial cells throughout the entire culture process. The purpose of the present invention is to solve the above-mentioned problems.The present invention aims to maintain the bacterial environment in an optimal condition while adjusting the temperature of the culture medium to a set temperature range where the growth of bacterial cells is maximized. It is an object of the present invention to provide a high-yield culture method for microorganisms, which is characterized by determining the amount of microorganisms and their activity, and controlling the substrate supply rate according to the amount of microbial cells and their activity based on the temperature control state of the culture medium.

An embodiment of the present invention will be described with reference to FIGS. 1 to 6.

FIG. 1 is a flow sheet for a high-yield culture process of microorganisms according to the present invention. In FIG. 1, 1 is a culture tank, and a culture medium 2 in the culture tank 1 is agitated by the rotation of a coagulation blade 3 to perform culture. 4 is a temperature measuring device for the culture medium 2 in the culture tank 1; 5 is an analog-to-digital converter for converting the temperature value of the culture medium 2 measured by the temperature measuring device 4 into a digital value; 6 is an analog-to-digital converter 5 A calculation is performed to compare the measured temperature value converted into a digital value with the set temperature value input separately by the numerical input device 7 and output a control output signal.

Control/memory/input/output device (hereinafter abbreviated as arithmetic device), 8 is a jacket 9 provided so as to wrap the outer surface of the culture tank 1
This is a temperature control device that is installed in the middle of the pipe 10 that supplies cooling water to the jacket 9, and adjusts the amount of cooling water supplied to the jacket 9 and the temperature of the culture medium 2 based on the control output signal output from the calculation device 6. . 11 is a substrate supply rate adjusting device;
Based on the control output signal sent from the calculation device 6 to the temperature control device 8 and temporarily stored in the calculation device 6, the calculation device 6 determines the substrate supply rate by calculation, and as a result, the substrate supply rate is determined by the calculation device 6. The supply rate of the substrate to the culture tank 1 is controlled by the control output signal.

Reference numeral 12 denotes a time measuring device which measures the control time and temperature rise time in order to estimate the fermentation heat during culturing, and is connected to the arithmetic device 6.

Reference numeral 13 denotes a culture condition monitoring device (hereinafter abbreviated as “monitoring device”) for monitoring the above-mentioned control condition, which is also connected to the arithmetic device 6.

When the culture is started at a culture time of 0, the bacterial cell environment is maintained at an optimal condition by various regulating devices (not shown), and the culture progresses as the culture time passes while the culture state is monitored by the monitoring device 13. The temperature of the medium 2 in the culture tank 1 rises due to the fermentation heat.

The temperature of the culture medium 2 is measured by a temperature measurement device 4 and converted into a digital value by an analog-to-digital conversion device 5. The temperature control device 8 includes a cooling water on/off valve that turns on and off the flow of cooling water, a variable cooling water amount control valve, etc. In this embodiment, the temperature control device 8 is used as a cooling water on/off valve. Then, as shown in FIG. 2, when the digitally converted measured temperature value approaches the upper limit of the set temperature value of the medium at which the growth of bacterial cells increases, which is separately input into the numerical input device 7, the arithmetic device 6 As a result, the output control output signal opens the cooling water on/off valve as shown in Figure 3, and the piping 1
0, cooling water starts to be supplied to the jacket 9, and as a result, the temperature of the culture medium 2 begins to fall as shown in FIG. When the temperature of the culture medium 2 further decreases and approaches the lower limit of the set temperature value of the culture medium at which the growth of bacterial cells is maximized, which is separately input into the numerical input device 7, the cooling water is turned on by the control output signal from the calculation device 6. - The off valve closes and the supply of cooling water to the jacket 9 is stopped. In this way, the temperature of the culture medium 2 in the culture tank 1 is controlled by turning on and off the cooling water, so that the upper and lower limits of the set temperature value of the culture medium at which the growth of bacterial cells is maximized, which is input separately into the numerical input device 7, is controlled. controlled between. Therefore, as shown in Figure 4, in the high activity zone, where the bacterial cell activity (= bacterial weight x average activity of all bacterial cells) is high and the amount of fermentation heat per unit time is large, the cooling water on/off valve is On the other hand, in the low activity zone, where the bacterial cell activity is low and the amount of fermentation heat per unit time is low, the total time the cooling water on/off valve is open becomes longer. The total becomes shorter. From the above, the following relationship is found between the bacterial cell activity and the cooling water on/off valve opening/closing time ratio (the operating/non-operating time ratio of the temperature control device 8).

In other words, [bacterial cell activity] is [cooling water on/off valve opening/closing time ratio] ・・・・・・・・・・・・
......[11] Furthermore, the relationship between the bacterial cell activity and the substrate supply rate is expressed by the following equation.

[Substrate supply rate] is [Bacterial cell activity]...
...The following relational expression is found from [Equation 21 '1} and Equation (■).

[Substrate supply rate] [Cooling water on/off valve opening/closing time ratio]
・・・・・・・・・・・・・・・・・・
...'3' formula (From 3-, the calculation of the control output signal for the substrate supply rate is as follows. F = FI + DF
・・・・・・・・・・・・・・・・・・・・・【4｝D
F=C/DT ・・・・・・・・・・・・・・・
......[5) In formula '4) and formula '5}, F
: current substrate supply rate (so'h), FI: previous substrate supply rate (so/h), DF: substrate supply rate change group (ku'h)
), C: constant, DT: cooling water on/off valve opening/closing time ratio.

The opening/closing time ratio (DT) of the cooling water on/off valve is determined by measuring the opening/closing time of the cooling water on/off valve within the sampling interval using the time measurement layer 12, as shown in FIG.
If the total time the off-valve is open is divided by the total time the off-valve is closed, it can be expressed as the following equation. D
T=(0,1021030...)/(C.10C2
10C30...) '61 formula {
61, 0,, 02, 03, ......: Opening time of the cooling water on/off valve (min), C,, C2, C3, ...... Closing time of the cooling water on/off valve (min) It is.

As described above, based on the regularly determined cooling water on/off valve opening/closing time ratio, the arithmetic unit 6 calculates the equation '41 and the side arithmetic expression, and the control output signal is sent from the arithmetic unit 6 to the substrate. By outputting to the supply rate adjusting device 11, the substrate feeding rate can be appropriately controlled as shown in FIG. Note that the upper limit of the substrate supply rate is input in advance to the arithmetic unit 6 to prevent growth inhibition of bacterial cells due to excessive supply of substrate during each culture period. In addition to the method described above, another method for determining the opening/closing time ratio of the cooling water on/off valve is to temporarily lower the temperature of the culture medium within the temperature control range of the culture medium where the growth of bacterial cells is greatest. Another method is to measure the time it takes for the temperature to recover to the original temperature.

Next, as a result of carrying out a culture experiment using the high-yield culture method for microorganisms according to the present invention under the following culture conditions, the culture time was 7 hours, and the bacterial cell concentration was approximately 50 microorganisms per day compared to the conventional culture method. We were able to propagate it in 90 evenings, which is twice the number of days.

m Fungal body: Baker's yeast (Sacchromyces)
Cerevisae)'21 Initial conditions: 'I' bacterial cell concentration: 50/so, [〇1 Medium volume: 20
〆、Shiichi Substrate concentration: 300 t/s、Temperature in the culture tank:
30qC, 'Village PH: 5 Glue Culture conditions: 'a) DO setting value: 4.0-6.0 9/evening, [o}
pH setting value: 4.8 to 4.9 days Rotor blade rotation speed setting value:
50-40 pm, Q Fermentation temperature setting: 29-310
0, Target culture tank internal pressure setting: 0.5 kg/○, Bamboo substrate feeding speed setting: 1.0 to 5.0 k'h The present invention:
As explained above, the bacterial activity is periodically determined from the operating/non-operating time ratio of the temperature control device that adjusts the temperature of the culture medium to the set temperature range of the culture medium where the growth of bacterial cells is maximized throughout the culture period. By controlling the substrate supply rate according to the activity of the microorganisms based on the ratio of operating and non-operating time of the control device, it is possible to maintain optimal substrate supply conditions at all times during cultivation, which has the effect of cultivating microorganisms with high yield. be.

[Brief explanation of drawings]

Figures 1 to 6 explain one embodiment of the present invention. Figure 1 is a flow sheet of a high-yield culture process for microorganisms according to the present invention, and Figure 2 is a diagram showing the passage of culture time and Figure 3 shows the relationship between the controlled culture temperature and the cold week of the culture time and the opening/closing status of the cooling water on/off valve. Figure 4 shows the relationship between the culture time and the bacterial cell activity. The relationship diagram, Figure 5, is a sampling procedure diagram of the opening/closing time ratio of the cooling water on/off valve.
FIG. 6 is a diagram showing the relationship between the passage of culture time and the substrate supply rate. 1...Cultivation pot, 2...Toji, 3...Toazusa wing, 4...Temperature measurement device, 5...Analog-to-digital conversion device , 6... Arithmetic/control/memory/input/output device, 7... Numerical input device, 8
... Temperature control equipment, 9 ... Jacket, 11 ... Substrate supply rate control device, 12 ...
...Time measuring device, 13...Culture condition monitoring device. 1 figure, 2 figures, 3 figures, 4 figures, 5 figures.

Claims (1)

[Scope of Claims] 1. A method for cultivating microorganisms that is carried out while maintaining the environment for microorganisms during cultivation at optimal conditions, such as pressure in a culture tank, pH of a culture medium, temperature, dissolved oxygen concentration, etc. The bacterial cell activity is periodically determined from the operating/non-operating time ratio of the temperature regulator that adjusts the temperature of the culture medium to the set temperature range of the culture medium where the temperature is greatest. A high-yield culture method for microorganisms, characterized by controlling the substrate supply rate according to the activity level. 2. A patent claim in which the operating/non-operating time ratio of the temperature control device is determined from the time required to temporarily force the temperature of the culture medium to fall within the set temperature range of the culture medium and then return to the original temperature. A high-yield culturing method for the microorganism according to item 1.