JPH0697990B2 - Microbial culture method and its equipment - Google Patents

Description

The present invention mainly relates to a semi-batch microorganism culture method for optimally culturing microorganisms based on a feed back control method and an apparatus therefor.

In a microbial culture plant, if the feeding amount of the substrate that is the nutrient source can be automatically controlled, labor saving of the operating staff managing the culture, stabilization of the process, stable and improved product quality can be expected. . By the way, compared with a chemical reaction system and the like, a microorganism culture system is greatly different from a chemical reaction system in that the microorganism has an autoregulatory function and exerts adaptability to the environment. Therefore, it is necessary to develop an automatic control system that is different from the chemical reaction system that takes into account the characteristics of individual microorganisms.However, due to the complexity of such a system, it is necessary to automatically adjust the substrate feed rate for microbial culture systems. Application examples for control are
Very few, and many research cases.

Next, the above circumstances will be described in more detail. As examples of conventional research, the followings were found regarding baker's yeast.

1) A sugar fed amount is controlled so that the concentration of ethanol in the exhaust gas is kept constant in a baker's yeast culture system.

2) Targeting the quotient of respiratory quotient for the baker's yeast culture system,
The sugar feed rate is controlled so that this falls within a certain fixed range.

3) For a baker's yeast culture system, the concentration of ethanol in the exhaust gas is measured, and the amount of sugar fed is controlled according to the production and assimilation state of ethanol.

However, these research examples are based on on / off control, and it is difficult to perform advanced control such that the control (target) amount such as the ethanol concentration or the respiratory quotient is kept constant. Therefore, we developed an alcohol sensor by the tubing method using porous Teflon,
It has become possible to study continuous measurement of ethanol concentration in the culture broth with an extremely rapid response speed, and by using a sensor with such excellent response characteristics, feedback control by the PID method becomes possible. ,
We were able to achieve high controllability such that the ethanol concentration was kept constant. Here, the PID is used as an abbreviation that means an adjuster equipped with three types of control of P (proportional), I (integral), and D (differential), which are adjustment calculation functions in the feedback control system. The feed back control is a correction operation based on the deviation between the target value and the controlled amount.
Generally, the manipulated variable is determined by a combination of a proportional term, an integral term, and a derivative term of the deviation. Since the gain of each term is adjusted by each parameter, optimum adjustment of control parameters is important to obtain optimum controllability. If the control system studied above is to be applied to a semi-batch culture system in a factory where various fluctuations are expected, it cannot be applied due to the following drawbacks. That is, in the semi-batch culture system, the bacterial cell concentration, for example, the concentration of baker's yeast is increased, the concentration of each product is increased or the inorganic salts initially charged, the concentration of vitamins, etc. It changes depending on the culture time. Due to that, the optimum value of the control parameter of the feed back control method such as PID method, which improves the dynamic characteristics of the controlled amount of the culture system, changes with the passage of the culture time, and as a result, the control parameter is changed to the culture time. The control characteristics were not good with the feed back control which was fixed to a constant value all the time.

Therefore, by applying a disturbance every appropriate time during culturing,
It was considered that the optimum control parameter was sequentially indexed and the feedback control operation was performed according to the index. However, in the culture system of microorganisms, giving such an artificial disturbance during the culture causes the microorganism to lose its activity,
The property was often changed and unexpected behavior was exhibited, which was not preferable.

Therefore, as a result of diligent research, the inventors have found that there is a certain relationship between the predetermined state quantity B of the culture system and the optimum control parameter during the culture, and, in advance, this relationship was experimentally determined. By obtaining in advance, the optimum control parameter corresponding to this predetermined state amount B detected during the culture is calculated by calculating with a microcomputer, etc.
We succeeded in optimally controlling the feed back without any adverse effects such as disturbances.

The present invention has been completed against the background of the above technical idea, and an object of the present invention is to provide an excellent microbial culture method and an apparatus for the same, which have solved the drawbacks of the conventional microbial culture methods.

The present invention will be described below with reference to the drawings showing an embodiment thereof. In order to make the explanation easier to understand, glucose is used as a substrate (nutrient source), baker's yeast is cultivated as a microorganism, the PID method is adopted as the feedback control method, and the state quantity A of interest is the ethanol concentration. Also, the state quantity B selected as being related to the optimum control parameter is the baker's yeast concentration X and the added glucose concentration.
I decided to use Sin. In this case, the reason for paying attention to the ethanol concentration is that by keeping the ethanol concentration constant as is well known, baker's yeast can be cultivated in high yield and in high quality in a short time.

First, the microorganism culture device according to the present invention (hereinafter referred to as the device of the present invention) will be described.

FIG. 1 is a simplified culture system diagram of the device of the present invention, in which 1 is a culture tank for culturing baker's yeast. On the upper part of the culture tank 1, a tip of a pipe 2 for flowing glucose as a substrate into the culture tank 1 is attached.
The other end of is connected to a glucose tank 3 that stores glucose. A pump 4 for supplying glucose to the culture tank 1 is installed in the middle of the pipe 2, and an operation unit 5 such as a diaphragm valve is provided. At the upper part of the culture tank 1, the concentration of ethanol in the culture solution is detected.
The alcohol sensor 6 by the tubing method using porous Teflon is attached. Reference numeral 7 is a setter for setting a target value of the ethanol concentration to be maintained constant, and 8 is a target value signal output from the setter 7 and an ethanol concentration signal output from the alcohol sensor 6. , And outputs the difference as a deviation signal ε. At the upper part of the culture tank 1, a cell concentration sensor 9 for detecting the cell concentration in the culture tank 1, that is, the concentration X of the culture baker's yeast in the present embodiment, is vertically mounted. 10 is a baker's yeast concentration X and a glucose concentration Sin supplied, and a control parameter Pr so that the control kinetics of the ethanol concentration as a controlled amount of the above-mentioned culture system becomes good.
This is a memory circuit that stores the relationship between (proportional band), Ti (integral time), and Td (differential time) that has been obtained in advance as described below, for example. The above method is a culture system in the case of a predetermined baker's yeast concentration Xs, glucose concentration Sin s, applying the Ziegler-Nichols response method to add step disturbance, in which case the optimal control of the dynamic characteristics of the controlled variable is optimum. Parameters were obtained experimentally, and in the case of other baker's yeast concentration X and glucose concentration Sin, the Ziegler-N
By the ichols method, the optimum control parameters that give the optimum dynamic characteristics are obtained, and in this way, the optimum control parameters Pr and T for the values of various baker's yeast concentrations X and glucose concentrations Sin are obtained.
The values of i and Td and the relationship are obtained. Here, the good control dynamic characteristics mean that the ethanol concentration quickly converges to the target value. In the case of the present embodiment, the above relationship is as simple as the following expression (1).

Here, Xs and Sin s are the predetermined values, and the optimum Pr value in that case is Prs. In the case of this embodiment, Ti and Td have nothing to do with X and Sin. So Ti,
Td may be fixed. Reference numeral 11 denotes an optimum parameter calculation circuit, which inputs the output signal X from the bacterial cell concentration sensor 9 and the output signal from the storage circuit 10 and outputs the baker's yeast concentration X and the culture tank 1 at that time. The optimum control parameters Pr, Ti, Td in the case of the supplied glucose concentration Sin (which is constant for each glucose tank 3 and is known in advance) are calculated based on the relational expression (1). It is a circuit to do. 12 inputs the signal output from the optimum parameter calculation circuit 11 and the deviation signal ε output from the comparison / subtractor 8, and according to the following equation (2), This is a feedback back arithmetic circuit that performs a PID feedback back calculation and calculates a control operation signal Z. The control operation signal Z is input to the operation unit 5, and the operation unit 5 adjusts the valve opening accordingly. In FIG. 1, 13 is a culture tank 1.
Is a stirring blade for stirring the culture solution, and 14 is an exhaust gas pipe of the culture tank 1.

FIG. 2 is a simplified culture system diagram of the device of the present invention in which a feedforward-like control function is added to the device of the present invention shown in FIG. A control operation signal Z output from the feedback back calculation circuit 12 and an output signal from the fungus body concentration sensor 9 are input to 15 and the control operation signal Z ′ is calculated according to the following equation (3).
Is a feedforward addition circuit for calculating

this Is added to the feedback control operation signal Z,
For example, when a large change occurs in the baker's yeast concentration X,
The ethanol concentration can be controlled with better controllability. Here, μ is the specific growth rate, Y is the yield of sugar, and V is
Is the culture fluid volume, and a and b are constants.

Next, the method for culturing a microorganism according to the present invention (hereinafter referred to as the method of the present invention) will be described by taking the above-mentioned apparatus of the present invention as an example.

In the method of the present invention, the relationship between the baker's yeast concentration X and the glucose concentration Sin and the control parameter is determined in advance by the step response method or the like so that the ethanol concentration in the culture solution is optimally controlled. Is stored in the memory circuit 10. Next, the baker's yeast concentration X in the culture tank 1 in operation and the concentration Sin of glucose supplied to the culture tank 1 are calculated. As a method of obtaining the baker's yeast concentration X, a method of indirectly estimating it from the total amount of glucose supplied to the culture tank 1 or a baker's yeast concentration sensor 9 as shown in FIG.
There is a method of automatically detecting directly. As a method for obtaining the glucose concentration Sin, a method using a densitometer attached to the pipe 2 for supplying glucose to the culture tank 1 or a method of storing a glucose solution having a constant known concentration in the glucose tank 3 is used. is there. Next, in the optimum parameter calculation circuit 11, the optimum parameter P corresponding to the obtained baker's yeast concentration X and glucose concentration Sin is obtained.
r, Ti, Td are stored in the memory circuit 10 and the relational expression (1) is stored.
Calculate based on. On the other hand, the ethanol concentration of the culture solution output from the arcose sensor 6 and the target ethanol concentration are subtracted by the comparison subtractor 8 to calculate the deviation ε thereof. Next, in the feedback back arithmetic circuit 12, the deviation ε
Based on the optimum control parameters Pr, Ti, Td and the equation (2), the control operation signal Z is calculated and output. Finally, according to the control operation signal Z, the valve opening degree of the operating portion 5 is adjusted to control the glucose supply amount. It should be noted that, even when the baker's yeast concentration X changes drastically, it is possible to control with good control dynamic characteristics, so that the term for the feed forward is used. It is also possible to add a value calculated based on the addition of the above to the control operation signal Z as shown in the equation (3) and adjust the operation unit 5 according to the sum Z ′. The amount of glucose supplied may be controlled by adjusting the rotation speed of the pump 4.

Next, the result of controlling the culture of the microorganisms carried out by the method of the present invention is shown in the center of FIG.

The conditions for the use were a 10-jar fermenter, a culture temperature of 33 ° C., a pH of 4.7, and a dissolved oxygen concentration of 3 ppm or more. For comparison, the control result of the conventional example in which the control parameters are fixed is shown in the center of FIG. Other conditions are the same as those of the embodiment of the method of the present invention. Here, in Fig. 3 and 4, T ₁ was the start point of automatic control (before that, it was manually stabilized), T ₂ and T ₅ were pulsed with 10 cc of 10% glucose aqueous solution as disturbances, respectively. It's time. The horizontal axis represents time and the vertical axis represents ethanol concentration. In FIGS. 3 and 4, a graph (horizontal axis time, vertical axis%) of one of the control parameters, Pr (proportional band), is shown above the graph showing changes in ethanol concentration, and below it. Shows a graph of baker's yeast concentration X (horizontal axis time, vertical axis g /). In the graph of baker's yeast concentration X in FIG. 3, the solid line is the baker's yeast concentration X value estimated from the total amount of glucose added to the culture tank 1. Black dots are actually measured values of the baker's yeast concentration X.

As is clear from FIGS. 3 and 4, even when a disturbance is applied, the fluctuation of the ethanol concentration is attenuated more quickly by the method of the present invention. As a result, the baker's yeast can be cultivated quickly and in good yield.

Next, an example in which the device of the present invention and the method of the present invention are applied when amino acids are produced using ethanol as a substrate (nutrient source) and ethanol-utilizing yeast as a microorganism will be described. In this case, the state quantity A of interest as the control quantity
As a result, yeast was produced by selecting the ethanol concentration and maintaining the ethanol concentration at 1000 ppm in the first half of the culture (20 hours).
The amino acid content will be increased by increasing it to ppm. That is, in this embodiment, the control amount changes stepwise. Further, as the state quantity B related to the optimum control parameter, attention is paid to the ethanol-assimilating yeast concentration X ′, the culture solution volume V and the yeast activity α. FIG. 5 is a view showing the culture system of this embodiment, and the setter 7'is a setter having a program function capable of switching the target value of the ethanol concentration to be controlled in the first half and the second half of the culture. Further, 16 is a liquid meter for detecting the liquid level V by detecting the position of the liquid surface of the culture tank 1. It has been found that the yeast activity α can be determined from the ethanol concentration P. The following equations (4) and (5) are obtained as a result of previously obtaining the relationships among the above-mentioned ethanol-assimilating yeast concentration X ', the culture solution amount V, the yeast activity α and the optimum control parameters Pr, Ti, Td.

Pr = f ₁ (α · V · X ′) (4) Td = f ₂ (α · V · X ′) (5) Ti may be fixed. Therefore, it is stored in the memory circuit 10 '. In the culture system in operation, the concentration X'of ethanol-assimilating yeast, the amount V of the culture broth and the yeast activity α are detected, and based on the above equations (4) and (5), the optimum parameter calculation circuit 11 uses the optimum control parameters. Then, the feedback control is calculated and controlled in the same manner as in the case of culturing baker's yeast. The results of carrying out the examples for amino acid production are shown in the lower part of FIG. The conditions for the use were that a 10 jar fermenter was used, pH was 4.5, culture temperature was 30 ° C., and dissolved oxygen concentration was 1 ppm or more. Control parameter Pr is 45% for comparison
The control result of the conventional example in which Td is fixed at 2.5 (min)
Shown at the bottom of the figure. Other conditions are the same as those in the example of the method of the present invention. In the figure, T ₁ shows the time when automatic control was started, and T ₂ shows the time when 20 cc of 10% ethanol aqueous solution was added as a disturbance. The horizontal axis represents time and the vertical axis represents ethanol concentration. In addition, in FIGS. 6 and 7,
The control parameter Pr (%) is set above the ethanol concentration.
Further, the control parameter Td (min) is shown above. As is clear from FIGS. 6 and 7, the method according to the present invention is more effective against disturbance.
The control characteristic of the ethanol concentration is far better, and the control characteristic of the present invention is also better by changing the setting of the target value of the ethanol concentration.

The device of the present invention and the method of the present invention are not limited to the above-mentioned cases, and the microorganisms to be cultured can be applied to bacteria, other yeasts, protozoa, etc. Further, as the substrate, sugar, molasses, glucose, alcohol, organic acid (acetic acid) and others as a carbon source, and a mixture of a carbon source and an inorganic salt in a fixed ratio can be considered. As the state quantity A to be focused on as a control quantity, the substrate concentration (ethanol, sugar, etc.) or the product concentration (ethanol, acetic acid, etc.) in the culture solution
Etc. may be used, and the concentrations of O ₂ , CO ₂ and ethanol in the exhaust gas may be used. Further, the control parameter to be changed may be all or any combination of Pr, Ti and Td or any one. As the state quantity B for obtaining a constant relationship with the optimum control parameter, the yeast concentration X,
Not limited to X ', glucose concentration Sin, etc., it is an arbitrary state quantity of the culture system. Further, the yeast concentration X can be estimated from the time integrated amount of the added base mass (unit time), and the added base mass can be determined in advance. Therefore, practically, the yeast concentration X and the optimum control parameter are set. Instead of the relationship with, the relationship between the time T and the optimum control parameter may be obtained in advance.

In addition, the setter 7, 7 ', the comparison subtractor 8, the storage circuit 10, 1
0 ', the optimum control parameter calculation circuit 11, the feed back calculation circuit, the feed forward addition circuit, etc. may be partially or entirely configured by means of a computer such as a microcomputer having the functions of the above circuits and devices. .

As is clear from the above description, the present invention adds disturbance or the like by changing parameters such as Pr, Ti, and Td based on the measured state quantity B in a culture system of a microorganism that is very difficult to control. Without it, the controlled variable can be controlled automatically with extremely good controllability.
In addition, it is an excellent invention that enables high-quality culture with high yield.

[Brief description of drawings]

The drawings are all for explaining the embodiments of the device of the present invention and the method of the present invention. FIG. 1 is a simplified culture system diagram of the device of the present invention, and FIG. 2 is a simplified culture system diagram of the device of the present invention. FIG. 3 is a graph showing the results of the method of the present invention (horizontal axis time, vertical axis ethanol concentration, etc.), FIG. 4 is a graph showing the results of the conventional method, horizontal axis time, vertical axis ethanol concentration, etc.), FIG. 5 is a simplified culture system diagram of the device of the present invention, FIG. 6 is a graph showing the results of other implementations of the method of the present invention (horizontal axis time, ethanol concentration on the vertical axis), and FIG. 7 is the results of other implementations of the conventional method. It is a graph (horizontal axis time, vertical axis ethanol concentration) shown. 1 ... Culture tank, 3 ... Glucose tank, 5 ... Operation part, 6 ... Alcohol sensor, 7,7 '... Setting device, 8
…… Comparison / subtractor, 9 …… Bacteria concentration sensor, 10,10 ′ ……
Storage circuit, 11 ... Optimal parameter calculation circuit, 12 ... Feed back calculation circuit, 15 ... Feed forward addition circuit, X ... Baker's yeast concentration, X '... Ethanol-assimilating yeast concentration, ε ... Deviation , Z, Z '... Control operation signal

Claims (6)

[Claims] 1. A microbial culture method for culturing a microorganism by adjusting the amount of a substrate to be added to a culture medium based on feed back control in which the ethanol concentration of a microbial culture system is a controlled amount. A predetermined functional relationship between the proportional band Pr of control parameters used for the feedback control calculation, the integration time Ti and the differential time Td, and the baker's yeast concentration X and the added glucose concentration Sin of the culture system that are favorable. The following formula (1), formula (2), formula (3), formula (4) and formula (5) are obtained in advance by experiments and stored in the storage means, and formula (1) Where Xs is the predetermined value of baker's yeast concentration X Sin s is the predetermined value of added glucose concentration Sin Prs is the optimum value of proportional band Pr when X is Xs and Sin is Sin s Equation (2) However, Z is the control operation signal value. Ε is the deviation between the detected ethanol concentration value and the target value. However, Z'is the control operation signal value to which the control operation value by the feedforward control calculation is added, μ is the specific growth rate, Y is the yield of sugar, V is the culture solution volume a, b is a constant formula (4) Pr = f ₁ (α · V · X ′) where α is yeast activity V is culture volume X ′ is ethanol-assimilating yeast concentration Formula (5) Td = f ₂ (α · V · X ′), culture in operation The baker's yeast concentration X and the added glucose concentration Sin of the system are measured, the optimum parameter values corresponding to the baker's yeast concentration X and the added glucose concentration Sin are calculated, while the ethanol concentration of the culture system is detected. , The deviation ε between the detected value of the ethanol concentration in the previous period and the target value is calculated, and the feedback control calculation is executed based on the deviation ε and the optimum parameter value to calculate the optimum control operation signal value Z, and the control operation is performed. Operate the operating part of the culture system according to the signal value Z Microbial culture method characterized by culturing by connexion microorganisms that optimally adjust the amount. 2. The operation of the operation section is performed according to a value obtained by adding a control operation signal value by a feed forward control calculation based on a fungus body concentration to a control operation signal value by the feed back control calculation. The method for culturing a microorganism according to claim 1, which is characterized in that. 3. The microorganism is baker's yeast, and the substrate is glucose.
Item or the method for culturing a microorganism according to Item 2. 4. A culture tank for a microorganism, a pump for supplying a substrate to the culture tank from a tank for a substrate through a pipe, an operation unit provided in the middle of the pipe for adjusting the substrate supply amount, and a predetermined location of the microorganism culture tank. A sensor for directly and automatically detecting the baker's yeast concentration X and the added glucose concentration Sin of the culture system attached to the culture system, and the baker's yeast concentration X and the added glucose concentration Sin of the culture system so as to improve the dynamic characteristics of the controlled amount. , Equation (1), Equation (2), Equation (3), Equation (4) and Equation (5), which are predetermined functional relationships among the proportional band Pr of the control parameter, the integral time Ti and the differential time Td. ) Is stored in advance as a storage means (1) Where Xs is the predetermined value of baker's yeast concentration X Sin s is the predetermined value of added glucose concentration Sin Prs is the optimum value of proportional band Pr when X is Xs and Sin is Sin s Equation (2) However, Z is the control operation signal value. Ε is the deviation between the detected ethanol concentration value and the target value. However, Z'is the control operation signal value to which the control operation value by the feedforward control calculation is added, μ is the specific growth rate, Y is the yield of sugar, V is the culture solution volume a, b is a constant formula (4) Pr = f ₁ (α · V · X ′) where α is yeast activity V is culture volume X ′ is ethanol-assimilating yeast concentration Formula (5) Td = f ₂ (α · V · X ′), from the storage means Output signal from the sensor and the output signal from the sensor, the optimum parameter calculation means for calculating the optimum control parameter, the control amount sensor attached to the culture tank to detect the control amount, the target control amount is set and output Setting means, comparing and subtracting means for inputting and comparing the output signal from the setting means and the output signal from the control amount sensor and outputting the difference, and the output signal from the comparing and subtracting means and the optimum parameter calculating circuit Input the output signal from Microbial culture apparatus characterized by a control operation signal performs fed back control operation and a fed back operation means for controlling an output and amount of substrate into the substrate adjustment operating unit based on. 5. A control operation signal obtained by adding a control operation signal obtained by a feedforward control operation based on a fungus body concentration to a control operation signal obtained by a feedback control operation, which is input to the operation section. The microbial culture device according to claim 4. 6. The microorganism according to claim 4, which is baker's yeast, and the substrate is glucose.
Item or the microorganism culture apparatus according to Item 5.