JPH0576346A - Method for controlling concentration of carbon source in aerobic culture of microorganism and apparatus therefor - Google Patents

Abstract

PURPOSE:To control the carbon source concentration in a culture tank at a low level by measuring the increase of pH and dissolved oxygen concentration of the culture liquid in a tank during the period to stop the addition of a carbon source between an addition stage and the subsequent addition stage and determining the feeding rate of carbon source in the subsequent addition period based on the measured values. CONSTITUTION:The initial addition of a carbon source feeding liquid is carried out at a prescribed feeding rate for a prescribed period and the following addition is started by using the increase of pH and the dissolved oxygen concentration as an index. The feeding rate of the liquid is determined based on the length of the stop period and the feeding rate in the addition period before the stop period in such a manner as to be low when the stop period is long and to be high when the period is short. The addition is continued for a prescribed period.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for controlling a carbon source concentration in aerobic culture of microorganisms, more specifically, an aerobic fed-batch culture of microorganisms in which a carbon source feed solution is intermittently added to a culture tank. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for automatically controlling a substrate carbon source in a culture tank during feed culture to a low concentration using a computer in continuous culture for culturing cells and recycling, and an apparatus suitable for carrying out such a method.

[0002]

2. Description of the Related Art Microorganisms are preferred for the purpose of fermentative production of various substances by using microorganisms (for example, fermentation of various amino acids and nucleic acid-related substances) and production of microbial cells themselves (for example, production of yeast cells). It is air-cultured. Aerobic culture of microorganisms is industrially performed by so-called fed-batch culture, continuous culture, or continuous reuse of bacterial cells, using a carbon source such as sugar as a main raw material.

By the way, in such a culturing method, the substrate inhibiting effect of the raw carbon source is prevented, or the raw carbon is effectively utilized for the culturing to minimize the residual carbon source such as sugar in the culturing solution. By reducing the level to a low level, the loss of raw material carbon source can be reduced, the separation of the product from the final culturing solution can be facilitated, and the environmental pollution due to the residual carbon source contained in the waste liquid after the product separation can be prevented. For the purpose, it is necessary to keep the carbon source (substrate) concentration in the culture tank at a low level during the feed culture. Especially in continuous culture and continuous reuse of bacterial cells, it is continuously discharged during the feed culture. The product is separated from the culture broth to be used, but the outflow of sugar and other carbon sources to this separation process can be minimized, and the effect of the carbon source on the separation process can be made almost zero. Is needed It is necessary to prevent the raw materials of the loss to. Further, it is strongly desired that the carbon source concentration analysis work can be eliminated in such a culture method, and the stable automatic control of the carbon source concentration can be performed without any human supervision.

Conventionally, as a method of maintaining a low concentration of a carbon source such as sugar during feed culture, oxygen consumption, carbon dioxide emission, pH, amount of by-products produced, amount of added ammonia, etc. are used as independent indicators. Then, while adding a carbon source such as sugar in an amount obtained by multiplying them by a previously obtained proportionality coefficient,
Although there are methods of controlling the carbon source concentration, these methods cannot accurately estimate the microbial activity during culture, and thus may not be able to be successfully controlled when the activity changes abnormally. Therefore, it is not possible to control the carbon source concentration in the culture at a low level (for example, 3 g / l or less).

Further, there is a method of detecting depletion of carbon source in a culture tank during culturing only by the dissolved oxygen concentration.
(A) Sensor reliability is low, (b) When the aeration and agitation state (number of agitation, aeration amount) is changed, the dissolved oxygen concentration state may fluctuate significantly, and carbon source depletion may be erroneously detected. It is not practical because the internal carbon source concentration cannot be controlled well.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to determine the rate of carbon source assimilation of sugars and the like of feed microorganisms in aerobic fed-batch culture, continuous culture and bacterial cell reuse continuous culture. It is possible to accurately estimate and control the carbon source concentration of the culture solution in the culture tank at an appropriate feed rate of the carbon source feed solution, and it is possible to reliably and reliably detect the depletion of the carbon source. It is an object of the present invention to provide a method capable of detecting depletion of a carbon source without being affected by conditions and having other advantages, and an apparatus therefor.

[0007]

Means for Solving the Problems As a result of intensive research aimed at solving the above-mentioned problems, the present inventor has found that in the aerobic culture of microorganisms by fed-batch culture, continuous culture or bacterial cell reuse continuous culture, sugars, etc. When the carbon source is intermittently added to the culture tank,
Using the increase in pH and the increase in dissolved oxygen concentration, which occur when the carbon source in the tank culture medium is depleted, during the addition stop period between one addition phase and the next addition phase as an index, the subsequent addition phase It was found that the above-mentioned problems can be solved by determining the feed rate of the carbon source feed liquid and by using a computer, and based on such knowledge, the present invention was completed.

That is, according to the present invention, in the aerobic fed-batch culture, continuous culture or microbial cell recycling continuous culture in which the carbon source feed solution is intermittently added to the culture tank, the feed solution is initially added in the feed culture. Is performed by adding the feed solution at a previously determined feed rate for a certain period of time. The second and subsequent additions are performed when the carbon source (substrate) in the culture tank is depleted during the addition stop period preceding a certain addition period. When the increase in pH or the increase in dissolved oxygen concentration that occurs is detected by a computer, the time of the addition stop period and the feed rate of the feed liquid in the addition phase preceding the addition stop period are used to determine the addition stop period. Do this by adding for a period of time at a computer-calculated feed rate such that the longer the time, the smaller the time and the shorter the time the larger. In the aerobic culture of microorganisms, the method is to control the substrate concentration in the culture tank so that it is always kept at a low level, and (i) a sensor and aeration for detecting the pH and dissolved oxygen concentration of the culture solution in the tank. A culture vessel equipped with a stirrer and adapted to receive a feed solution via a flow controller; and (ii) a computer equipped with two signal converters, and (a) a pH sensor and a pH sensor with a dissolved oxygen concentration sensor. A sensor is connected so that the detected value of the dissolved oxygen concentration is input to the computer via one of the signal converters, and (b) the feed liquid feed speed set value calculated by the computer is supplied to the other signal converter. A device for performing the above method, characterized in that it is communicatively connected via a flow control device.

The present invention will be sequentially described below.

(Description of Method) First, the method according to the present invention will be described.

For the purpose of fermentative production of various substances and production of microbial cells themselves, a feed liquid containing a substrate carbon source such as sugar is continuously (here, "continuously" is a broad sense). It is well known that microorganisms are aerobically fed-batch culture, continuous culture or continuous reuse of bacterial cells while being intermittently added to a culture tank containing ". Fed-batch culture, continuous culture and bacterial cell reuse continuous culture in the present invention are also well-known fed-batch methods, continuous culture and bacterial cell reuse continuous culture, except for the method of adding the feed solution in the feed culture period described below. It can be carried out according to.

The first addition of the feed solution in the feed culture period is usually started when the concentration of the substrate carbon source such as sugar in the culture solution in the main culture preceding the feed culture reaches a certain low level. Is. The feed rate of the feed solution obtained in advance is obtained in advance through preliminary experiments, and it is usually the same as the consumption rate of the substrate in the main culture at the start of the feed culture, that is, at the start of the first feed in the feed culture. Is. The fixed time in the addition of the feed solution is an arbitrary time selected within the range of time (usually in the range of 10 minutes to 24 hours) in which the activity of consuming carbon sources such as sugars of microorganisms does not significantly change.

When the substrate of the culture solution is depleted after the completion of the initial addition, both the pH and the dissolved oxygen concentration of the culture solution increase.
When this increase is detected by the computer through the pH sensor and the dissolved oxygen concentration sensor provided in the culture tank, the substrate feed solution is added to the culture tank via the sensor. The instructions are given and the second addition of feed liquid is started. The increase in pH and the increase in dissolved oxygen concentration do not always occur simultaneously. When there is a time difference between the two increases, the second addition is started at the earlier detected increase. Each of the pH sensor and the dissolved oxygen concentration sensor must be frequently broken or calibrated. Therefore, the detection reliability of the depletion of the substrate can be remarkably improved by using the respective sensors at the same time instead of using them individually.

The feed rate in the second addition of the feed liquid is determined from the length (period) of the addition stop period between the first addition and the second addition and the feed rate of the first addition, and the period of the addition stop period. Is longer and smaller and shorter, in other words, the substrate feed rate and substrate consumption rate are balanced to maintain the desired low substrate substrate concentration in the fermentor. It is the speed obtained by calculating. This calculation is performed by a computer, but a program for such calculation can be easily created by those skilled in the art. An example of such a program is shown in Example 1 below.

The fixed time during which the second addition is carried out is
It is determined in the same manner as the fixed time of the first addition. The first addition period and the second addition period do not have to be the same length of time, as long as they are selected within a time range in which the activity of consuming a carbon source (substrate) such as sugar of a microorganism does not change significantly. The addition period of each time is selected from the time such that the substrate consuming activity of the microorganism is not largely changed and the substrate concentration in the culture tank during the culture can be maintained at a low level.

The start timing, feed rate and addition period of the third and subsequent feed liquid additions are determined in the same manner as those for the second addition.

Thus, the second and subsequent additions are carried out at a carbon source (substrate) in the culture tank at the addition stop phase preceding a certain addition phase.
From the time of the addition stop period and the feed rate of the feed liquid in the addition phase preceding the addition stop period and when the increase in pH or the increase in dissolved oxygen concentration that occurs when the addition is depleted is detected by a computer. It is carried out by adding for a certain period of time at a feed rate calculated by the computer so that the period of the addition stop period is longer when the period is longer and shorter when the period is shorter.

In this way, it becomes possible to set the feed rate of the feed liquid of the substrate carbon source such as sugar and other addition conditions while sequentially checking the activity of the microorganisms,
The substrate concentration in the culture tank can easily be 5 g / l or less, and further 3 g / l
It is possible to keep the level as low as the following.

(Description of Apparatus) Next, the apparatus according to the present invention will be described.

In other words, the apparatus of the present invention uses a pH sensor such as a pH electrode attached to a culture tank and a dissolved oxygen sensor such as a dissolved oxygen electrode to transmit signals to the computer. It has a function to detect the depletion of the substrate carbon source and calculate the feed rate of the carbon source feed solution, and to transmit the calculated feed rate value to the flow rate controller of the feed solution installed in the carbon source addition line of the culture tank. It is a device.

As is well known, pH meters and dissolved oxygen meters are subject to frequent failure and must be calibrated frequently. According to the present invention, the reliability of detection of depletion of a carbon source such as sugar can be significantly improved by using each sensor in combination, rather than using them individually. Of course, even if each sensor is used alone, the reliability is lowered but the effect is exhibited.

[0022]

EXAMPLES The present invention will be further described below with reference to examples.

The apparatus used in each example is shown in FIG. 2, which is the same as the apparatus shown in FIG.
The culture tank shall be a small glass culture tank.
The H sensor is used as a pH electrode, the dissolved oxygen concentration sensor is used as a dissolved oxygen electrode, and the computer is used as a personal computer.
(1) is an A / D converter, the signal converter (2) is a D / A converter, and the flow control device is a feed pump.

Example 1 (L-by continuous culturing for reuse of bacterial cells
Fermentation production of glutamic acid) glucose 30 g / l, KH ₂ PO ₄ 1 g / l, MgSO _4.
4 · _7aq 0.4g / l, urea 4g / l, FeSO ₄ · 7
30 ml of an aqueous medium containing 20 mg / l of aq, 20 mg / l of MnSO ₄ .4 aq, 5 ml / l of soybean protein acid hydrolyzate, and 300 μg / l of biotin was placed in a 500 ml shake flask at 10 ° C at 115 ° C.
Heat sterilized for 1 minute. This is cooled to room temperature, and Brevibacterium lactfermentum (Brevibacterium lact
ofermentum) ATCC 13869 was inoculated and seed-cultured at 30 ° C. for 24 hours.

[0025] 30 ml of this seed culture and 80% sugar cane sugar as sugar
270 ml of an aqueous main culture medium containing g / l, KH ₂ PO ₄ at 1 g / l and soybean protein acid hydrolyzate at 10 ml / l was placed in a pre-sterilized 1 liter small glass culture tank and kept at 30 ° C., 300 ml / min of sterilized air was aerated, and stirring was started while maintaining pH at 7.5 with NH ₃ gas (main culture start).

As shown in FIG. 2, the small glass culture tank is
Connected to a 16-bit personal computer. PH detected by pH electrode and dissolved oxygen electrode inserted in the culture tank
And the analog signal of the dissolved oxygen concentration is taken into the personal computer through the A / D converter incorporated in the personal computer. Aqueous feed medium (feed liquid) after being set and calculated on a personal computer
The feed speed of D /
It is sent to the feed pump as an analog signal via the A converter.

Simultaneously with the start of the main culture, the feed rate of the first (first) feed solution to the personal computer is 30 ml.
/ H, detection value of sugar substrate depletion due to increase of pH 7.7, detection value of sugar substrate depletion due to increase of dissolved oxygen concentration 20% (normal value of dissolved oxygen concentration is 1 to 10%), feed liquid feed time 3 hours did.

Five hours after the start of the main culture, polyoxyethylene sorbitan monopalmitate was added at a concentration of 0.2% by weight for the purpose of inhibiting the growth of the bacteria and producing glutamic acid, and the main culture was continued for another 5 hours. After that, make the personal computer controllable (automatic operation),
Pre-set feed rate of 30 for initial feed addition
Started at ml / h. The feed solution added to the culture tank was 180 g / l of sugar cane sugar as sugar and soy protein hydrolyzate 5
ml / l, containing 0.2% by weight of polyoxyethylene sorbitan monopalmitate.

At the same time, the culture solution was passed through a flat membrane microfilter preliminarily sterilized by heating at a rate of (feed solution feed rate + 20) ml per hour to separate 20 ml of the cell-containing solution and the filtrate. The cell-containing liquid was returned to the culture tank. L-glutamic acid was obtained from the filtrate containing no cells by the crystallization method.

After starting the addition of the feed liquid for the first time, the computer enters the automatic operation state, and once the preset feed liquid addition time (3 hours) has passed, the addition of the feed liquid is automatically interrupted once according to the set conditions. did. With the suspension of the addition, the sugar concentration in the culture tank decreases as shown in part A of FIG. 4, and when it reaches almost 0 g / l, the increase in pH and the increase in dissolved oxygen concentration occur almost at the same time.

The computer immediately sets the second value when either the pH or the dissolved oxygen concentration reaches the substrate depletion detection value first.
Calculate the feed rate setting value of the feed liquid of the second addition,
The set value is output to the feed pump, and the feed liquid is added again at the set speed for 3 hours. After that, by repeating this operation, the sugar concentration in the culture tank is 0 to 2 g.
/ L was controlled accurately.

The feed liquid feed speed set value after the second addition is calculated from the time (τ) at which the feed liquid addition is stopped and the feed speed set value (υ) immediately before the feed liquid addition is stopped as follows. Set by rules.

If τ ≦ 10 minutes, set the feed rate of the new feed liquid to 1.1υ. Here, υ is the feed rate in the immediately preceding addition period.

If 10 minutes <τ ≤ 30 minutes, set a new feed rate to υ.

If 30 minutes <τ ≤ 1 hour, set a new feed liquid feed rate to 0.9υ.

If 1 hour <τ ≤ 2 hours, set a new feed liquid feed rate to 0.8υ.

However, the coefficient of υ and τ may differ depending on the culture conditions such as the culture method and the type of substrate, the performance of the strain used, and the like. In addition, the number of rules does not matter even if it is appropriately increased or decreased. By the way, it was confirmed by the preliminary experiment that τ> 2 hours did not occur in the culture in this example. In addition, in this embodiment, the feed liquid was added for 3 hours each time, but as described above,
It is not necessary to make this addition time constant as long as the activity of the bacterium does not change significantly.

By continuing this culture for 80 hours, L-
132 g of glutamic acid was obtained. Further, after the start of automatic operation, as shown in FIG. 5, the sugar concentration could be controlled completely automatically and stably within 2 g / l.

Comparative Example 1 (Conventional Method) (The main point of the conventional method is a method of using NH ₃ for pH adjustment and adding sugar (feed solution) in proportion to this). The seed culture obtained in the same manner and 270 ml of the same main culture medium as in Example 1 were placed in a pre-sterilized 1-liter small-sized glass culture tank, kept at 30 ° C., and sterilized air was aerated at 300 ml per minute, Stirring was started while maintaining the pH at 7.5 with NH ₃ gas. (Main culture start).

As shown in FIG. 3, the small glass culture tank is
Connected to a 16-bit personal computer.

Five hours after the start of the main culture, polyoxyethylene sorbitan monopalmitate was added to a concentration of 0.2%, the main culture was continued for another 5 hours, and then the personal computer was put in a controllable state.

An analog signal from a mass flow meter connected to the NH ₃ gas line is taken into the personal computer through an A / D converter built in the personal computer, and an amount proportional to the amount of NH ₃ added every hour inside the personal computer. After calculating the feed liquid feed rate of the above, it was programmed to send it as an analog signal to the feed pump through the D / A converter incorporated in the personal computer.

As a result, as shown in FIG. 6A, the ratio of the feed rate of NH _{3 to} the feed rate of the feed solution changed when the cell growth phase entered the glutamic acid production phase during culture, and the sugar concentration was changed. Because the controllability of
The proportional coefficient was changed at the 13th hour. In addition, since the proportional coefficient is unstable even after the change, the controllability of the sugar concentration in the culture tank is not better than that in Fig. 5, and the residual sugar concentration (sugar concentration in the culture tank)
Was checked 5 times. Further, due to such instability, the residual sugar concentration could not be lowered so much as to avoid the residual sugar concentration becoming 0 g / l during the culture.

The amount of L-glutamic acid produced, which was 120 g obtained by continuing this culture for 80 hours, was the same as in Example 1.
It was lower than 32g.

Table 1 summarizes the comparison between Example 1 and Comparative Example 1.

[0046]

[Table 1]

Example 2 (Fermentative production of L-phenylalanine by fed-batch culture) Example 1 except that the continuous cell-reuse culture was changed to fed-batch culture.
Brevibacterium lactophenylmentum according to
L-phenylalanine was fermentatively produced using (Brev.lactofermentum) FERM BP-1071.

However, the medium, the incubator, the culture method, the culture time course, and the feed rate control method of the feed solution were as follows.

[0049]

[Table 2]

[0050]

[Table 3]

(2) Incubator, culturing method; (a) Seed culture: 30 ml of a seed culture medium was placed in a 500 ml flask and shake-cultured at 30 ° C.

(B) Main culture: 30 ml of a seed culture and 270 ml of a main culture medium were placed in a 1-liter small-sized glass-made culture tank, and aerated culture was carried out at 30 ° C. (aeration volume was 150 ml per minute). The pH is NH ₃
It was controlled to 7.5.

(3) Culture time; (a) Seed culture: 48 hours (b) Main culture: 96 hours (c) Feed start time: 35 hours after start of main culture (4) Feed liquid feed rate control method; Same procedure as in Example 1, but with 10 ml initial feed rate
/ H.

Comparative Example 2 (conventional method) The pH was adjusted using NH _3, and the feed rate of the feed solution was controlled using the pH as an index. That is, p
When H rises above the set value, increase the feed speed by 5%, p
If H did not rise for 5 hours, the feed rate was reduced by 10%.

Table 2 shows a summary of comparison between Example 2 and Comparative Example 2.

[0056]

[Table 4]

Example 3 (Production of yeast cells by fed-batch culture) Example 1 except that the continuous cell-reuse culture was changed to fed-batch culture.
According to Saccharomyces cerevisiae (Saccharomyce
cerevisiae) CBS1523 was used to produce yeast cells.

However, the medium, incubator, culturing method and feed rate control method of the feed solution were as follows.

[0059]

[Table 5]

[0060]

[Table 6]

(2) Incubator, culturing method; (a) Seed culture: 30 ml of a seed culture medium was placed in a 500 ml flask and shake-cultured at 30 ° C. for 24 hours.

(B) Main culture: A seed culture solution (30 ml) and a main culture medium (270 ml) were placed in a 1-liter small glass-made culture tank, and the mixture was maintained at 30 ° C. for 10 minutes.
Aeration culture was carried out for an hour (aeration rate was 150 ml / min). In addition, pH
Was controlled to 6.5 with NH ₃ . In addition, addition of the feed solution (medium) was started 10 hours after the start of the main culture.

(3) Feed liquid feed rate control method: The same method as in Example 1 was used, but the initial feed rate was 5 ml / h.

Comparative Example 3 (Conventional Method) The feed rate of the feed solution was controlled by using the by-product ethanol production rate (measuring the ethanol gas in the exhaust gas) as an index. That is, when the ethanol production rate was higher than the set value, the feed rate was decreased, and when it was lower than the set value, the feed rate was increased.

Table 3 summarizes the comparison between Example 3 and Comparative Example 3.

[0066]

[Table 7]

Example 4 (Fermentative production of L-threonine by fed-batch culture) Example 1 except that the continuous reuse of bacterial cells was changed to fed-batch culture.
According to Brevibacterium flavum (Brev. Favum)
L-threonine was fermentatively produced using FERM BP-1173.

However, the medium, incubator, culturing method, culturing time course, and feed rate control method of the feed solution were as follows.

[0069]

[Table 8]

[0070]

[Table 9]

(2) Incubator, culturing method; (a) Seed culture: 30 ml of a seed culture medium was placed in a 500 ml flask and shake-cultured at 30 ° C.

(B) Main culture: A seed culture solution (30 ml) and a main culture medium (270 ml) were placed in a 1-liter small-sized glass culture tank, and aerated culture was carried out at 30 ° C. (aeration volume was 150 ml / min). In addition, pH is NH
_It was controlled to 7.5 at ₃ .

(3) Culture time; (a) Seed culture: 40 hours (b) Main culture: 100 hours (c) Feed start time: 20 hours after start of main culture (4) Feed liquid feed rate control method; Same method as in Example 1, but with an initial feed rate of 1.5
It was set to ml / h.

Comparative Example 4 (conventional method) The feed of the feed liquid was controlled using pH as an index. That is, if the pH rises above the set value, 0.5m
It was added at a constant rate using an on / off timer at a rate of l / min.

Table 4 summarizes the comparison between Example 4 and Comparative Example 4.

[0076]

[Table 10]

Example 5 (Fermentative production of guanosine by fed-batch culture) Example 1 except that the continuous cell-reuse culture was changed to fed-batch culture.
According to Bacillus subtilis F
Guanosine was fermentatively produced using ERM BP-3601.

However, the medium, incubator, culturing method, culturing time course, and feed rate control method of the feed solution were as follows.

[0079]

[Table 11]

[0080]

[Table 12]

(2) Incubator, culturing method; (a) Seed culture: 30 ml of a seed culture medium was placed in a 500 ml flask and shake-cultured at 30 ° C.

(B) Main culture: A seed culture solution (30 ml) and a main culture medium (270 ml) were placed in a 1-liter small-sized glass culture tank, and aerobically cultured at 30 ° C. (aeration rate was 200 ml / min). In addition, pH is NH
_It was controlled to 6.5 at ₃ .

(3) Culture time; (a) Seed culture: 30 hours (b) Main culture: 150 hours (c) Feed start time: 50 hours after start of main culture (4) Feed rate control method of feed solution; Implementation Same procedure as in Example 1, but with an initial feed rate of 1 ml
/ h.

Comparative Example 5 (conventional method) The pH was adjusted using NH _3, and the feed rate of the feed liquid was controlled using the pH as an index. That is, p
When H rises above the set value, increase the feed speed by 5%, p
If H did not rise for 5 hours, the feed rate was reduced by 10%.

Table 5 summarizes the comparison between Example 5 and Comparative Example 5.

[0086]

[Table 13]

[0087]

INDUSTRIAL APPLICABILITY According to the present invention, the carbon source (substrate) concentration in the culture tank can be lowered (for example, the sugar (carbon source) concentration can be easily reduced to 5
g / l or less, or even 3 g / l or less), and thus the substrate inhibitory effect can be prevented, and especially in continuous culture and bacterial cell recycling continuous culture, Can be prevented and the yield and production rate can be improved (1-30% and 1-30%, respectively),
In continuous culture and bacterial cell reuse continuous culture, analysis of substrate concentration can minimize the outflow of substrates such as sugar to the product separation process and can have almost no effect on the separation process. It has become possible to obtain merits such as eliminating the work at all and being able to stably and automatically control the substrate concentration without any human supervision.

[Brief description of drawings]

FIG. 1 shows an example of the device of the present invention.

FIG. 2 shows the apparatus used in Example 1.

FIG. 3 shows an apparatus used in Comparative Example 1.

FIG. 4 shows the situation of feed culture in Example 1.

FIG. 5 shows the control status of sugar concentration in Example 1 as well.

FIG. 6 shows a culture situation in Comparative Example 1.

Front page continuation (72) Inventor Keishi Shimazaki 450 Morotomi, Morotomi-cho, Saga-gun, Saga Ajinomoto Co., Inc. Kyushu Factory

Claims (2)

[Claims] 1. In aerobic fed-batch culture, continuous culture or continuous microbial cell reuse culture in which a carbon source feed solution is intermittently added to a culture tank, the initial addition of the feed solution is a previously determined feed. The feed solution is added at a constant rate for a certain period of time, and the second and subsequent additions are the carbon sources (substrates) in the culture tank during the addition stop phase that precedes a certain addition phase.
From the feed rate of the feed liquid in the addition phase, which starts when the increase in pH or the increase in dissolved oxygen concentration that occurs when oxygen is depleted is detected by the computer and the addition stop period precedes the addition stop period. Substrate concentration in culture tank in aerobic culture of microorganisms, characterized in that the addition is carried out for a certain period of time at a feed rate calculated by the computer so that it becomes smaller if the duration of the addition suspension period is longer and larger if it is shorter. How to control to always keep low level automatically. 2. A culture tank comprising: (i) a sensor for detecting the pH and the dissolved oxygen concentration of the culture solution in the tank and an aeration and stirring device, and the feed solution being received through a flow control device; and (ii) 2 A sensor having a signal converter, and (a) a sensor for inputting the detected values of pH and dissolved oxygen concentration by the pH sensor and the dissolved oxygen concentration sensor to the computer through one signal converter, respectively. And (b) the computer-calculated feed liquid feed speed setpoint is connected so as to be transmitted to the flow control device via the other signal converter. Apparatus for performing the method.