JPS63296700A - Method for controlling concentration of culture fluid - Google Patents

Abstract

PURPOSE:To enable accurate measurement without requiring calibration of a concentration of a component which is an object for measurement in a culture fluid during cultivation, by previously adding the component which is the object for measurement in a control target concentration to a dialytic solution fed to a dialyzer. CONSTITUTION:A dialytic solution is fed to a dialyzer 4 in a cultivation tank 1 and the concentration of a component which is an object for measurement contained in a dialyzate discharged from the dialyzer is measured to control the concentration of the component in the culture fluid on the basis of the measured results. In the process, the component in a control target concentration is previously added into the dialytic solution. The osmotic pressure of the dialytic solution is preferably adjusted to almost the same value as that of the culture fluid. For that purpose, e.g. sodium chloride or potassium chloride, is added.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] In the fermentation industry, the concentration of substrates, etc. in the culture solution is generally controlled throughout the culture. The present invention improves this concentration control method.

[Conventional technology]

Recently, biosensors such as enzyme electrodes have been widely used to measure the concentration of substrates and the like in culture fluids due to their high specificity and ease of measurement. If you attempt to insert this biosensor directly into a culture tank for measurement, it will be difficult to measure the heat generated by steam sterilizing the culture tank before use.
It is inactivated by 120°C). Also, the pH suitable for measurement
Since the area may not match the culture solution, a portion of the culture solution was extracted from the culture tank, diluted, pH adjusted, etc., and then used for measurement. However, troubles such as slime adhering to the piping for drawing out the culture solution and clogging caused by air bubbles during aeration and agitation occurred, so a method was developed in which a dialyzer was installed inside the culture tank and the components to be measured were extracted through the dialysis membrane (D, W ,Z
abriskie, A.E., Humphrey, B.
eotech.

Bioeng, 20.1295 (1978)).

[Problem that the invention seeks to solve]

The method using dialysis is based on the premise that the dialysis rate, that is, the ratio between the concentration of the component to be measured contained in the dialysate flowing out of the dialyzer and the concentration of the culture solution, is constant. However, this dialysis method has a problem in that the dialysis rate of the component to be measured changes depending on the aeration and stirring conditions during culture and the contamination of the dialysis membrane. Therefore, in order to eliminate the above-mentioned influence, methods were considered such as increasing the area of the dialysis membrane or circulating the dialysis fluid through the dialyzer to ensure sufficient dialysis. This was not practical as it took too much time.As a result, a method has been adopted in which the culture solution is extracted separately, its concentration is measured, and the measurement system is calibrated based on this, but this method does not require frequent calibration. It was troublesome because there were a lot of things.

[Means for solving problems]

As a result of various studies to solve these problems, the present inventors achieved this objective by adding the component to be measured at a controlled target concentration in advance to the dialysis fluid sent to the dialyzer. I was able to do that.

That is, the present invention sends a dialysis solution to a dialyzer installed in a culture tank, measures the concentration of a component to be measured contained in the dialysate flowing out from the dialyzer, and performs culture based on the results. The present invention relates to a culture concentration control method for controlling the concentration of the component in a solution, characterized in that the component to be measured at a control target concentration is added in advance to the dialysis fluid.

Needless to say, there are no particular restrictions on the type, shape, size, etc. of the culture tank in which the dialyzer is installed. The dialyzer may be installed directly within the culture tank, or may be installed outside the culture tank via piping. A normal dialyzer may be used, and an appropriate dialysis membrane is selected depending on the component to be measured.

The component to be measured is usually a substrate, such as glucose,
These include sucrose, other sugars, alcohols such as methanol and ethanol, and organic acids. However, the method of the present invention is not limited to these,
It is also applicable to materials other than substrates.

Examples of cases in which substrate concentration control is particularly necessary include the following. The first case is when the growth of microorganisms is inhibited when the substrate concentration exceeds a certain value.

If the substrate concentration is too low, the growth rate will decrease, so
It is necessary to keep the substrate concentration at an optimal value to ensure smooth cultivation.

Next, if the substrate concentration becomes too high, the production efficiency of the target product decreases or by-products are generated.

If the substrate concentration decreases, the production rate of the target product decreases, so it is necessary to control the substrate concentration to an optimal value. The method of the present invention is particularly effective in these cases.

The method of the present invention is characterized in that the component to be measured is added in advance to the dialysis fluid sent to the dialyzer at a controlled target concentration.

The allowable range of this concentration is determined according to the allowable range of concentration control and the like.

It is preferable that the dialysis solution has the same osmotic pressure as that of the culture solution, and for this purpose, salts such as sodium chloride, potassium chloride, etc. are preferably added. The type of salts is not particularly limited;
It may also be a salt of an organic acid. It is also preferable to add culture medium components other than the measurement target components to the dialysis solution in the same composition as the culture medium. Although the dialysis fluid may be sent to the dialyzer only during measurement, it is normally sent constantly.

The means for measuring the concentration of the dialysate flowing out from the dialyzer may be any known method, and is selected depending on the component to be measured. When the component to be measured is a substrate, biosensors such as enzyme electrodes and microbial electrodes are generally preferably used, but other methods such as gas chromatography, column chromatography, and colorimetry can also be used.

In the method of the present invention, the degree of isolation between the concentration of the component to be measured in the dialysate and the concentration of the culture solution is measured, and the concentration of the component in the culture solution is controlled accordingly. If the component to be measured is a substrate, the supply is stopped if the concentration is excessive, and the supply is stopped if the concentration is insufficient. If the component to be measured is a product, etc., and its concentration is excessive, wait for it to be removed from the culture solution using an ion exchange membrane or dialyzer.
On the other hand, if the concentration is low, measures can be taken to promote production. Therefore, it is preferable that the sensor for measuring the component to be measured in the dialysate is capable of sending commands to the above-mentioned control means, such as the supply valve.

The method of the present invention can be used to control the products and by-products of the culture solution, and also to control the concentration of raw material components in bioreactors using immobilized enzymes and the like.

[Effect]

In the method of the present invention, the degree of isolation of the concentration of the culture fluid relative to the concentration of the component to be measured in the dialysate fluid sent to the dialyzer is measured. In other words, if the control target concentration (the concentration of the component to be measured in the dialysis fluid) is Co, and the component to be measured in the dialysate flowing out from the dialyzer is Ci, then if the concentration of the component to be measured in the culture fluid is higher, the culture fluid Since the component diffuses into the dialysis solution through the dialysis membrane, Ci
>Co, and in the opposite case, Ci<Co. However, strictly speaking, this relationship also depends on the osmotic pressure and the concentration of other components, so it is preferable that these also match.

〔Example〕

A dialyzer 4 is provided inside an 11-jar fermentor 1 made of glass and having a stirrer 2 and a ventilation pipe 3, using an apparatus as shown in FIG.

The dialyzer is equipped with an acetylcellulose membrane having a thickness of 17 μm, a diameter of 2 IIn, and a length of 7 cm as a dialysis membrane.

Dialysis fluid is continuously supplied to this dialyzer 4 at a constant flow rate by a metering pump 5, and the dialysate flowing out from the dialyzer 4 is sent via a switching valve 9 to an existing enzyme electrode 6 that uses alcohol oxidase. There, the concentration of the component to be measured is measured and then discarded. The switching valve 9 is activated at each measurement time, and the enzyme electrode 6 is normally discarded without passing through it.

A signal from the enzyme electrode 6 is input to a control device 7, and a valve 8 of a substrate solution injection pipe is opened and closed in accordance with the command.

Using such an apparatus, the culture tank 1 is filled with a medium containing methanol at various concentrations with the following composition, and the dialyzer 4 is filled with a medium containing the same composition as this medium with a methanol concentration of 0.7%.
was used as the dialysis fluid.

FIG. 2 shows the results of measuring the methanol concentration of the dialysate flowing out from the dialyzer 4 using the enzyme electrode 6. As shown in the figure, when the methanol concentration of the culture medium exceeds 0.7%, the methanol concentration of the dialysate flowing out from the dialyzer also exceeds 0.7%, and conversely, when it is less than 0.7%, The concentration of this dialysate is also 0.7%. A similar relationship was obtained when seeding was measured under aeration and agitation conditions and when the degree of contamination of the dialysis membrane progressed.

Next, Hansenula yeast was cultured using methanol as a substrate. The medium with the above composition was used, and 20% methanol was used as the substrate solution added during the culture. Continuous culture was performed under the conditions shown below, and 0.7% was added to dialyzer 4.
A solution containing methanol and having the same composition as the above medium was flowed.

Loading amount: 600 d Aeration amount: 300 d/min Stirring number: 50 Or, p, m Temperature: 37° C. pH: 5,0 A solution having the same composition as the medium containing 0.7% methanol was flowed into the dialyzer 4. The methanol concentration of the dialysate flowing out from the dialyzer 4 is measured every 10 minutes, and when the concentration is 0.
When the concentration was 7% or less, the substrate solution was put into the culture tank 1. The feeding method was such that the control device 7 opened the valve 8 at fixed time intervals for a fixed period of time so that the substrate solution was gradually added to the culture solution in fixed amounts. The methanol concentration of the culture solution is 0.
．． When the concentration exceeds 7%, valve 8 closes to stop supplying the substrate solution.

As a result, the methanol concentration of the culture solution was reduced to 0.7% ± 0.2
Hansenula yeast that could be controlled in % grew at a good rate.

〔Effect of the invention〕

By the method of the present invention, the concentration of the component to be measured in the culture solution can be accurately determined during culture without any particular calibration, and the component to be measured, such as the substrate, can be kept constant at a control target concentration.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the configuration of an example of a device used in the control method of the present invention, and FIG. 2 shows the methanol concentration of the dialysate flowing out from the dialyzer and the methanol concentration of the culture solution using this device. FIG. 3 is a diagram showing the results of measuring the relationship with concentration. 1...Culture tank 4...Dylyzer patent applicant Representative of Biofunctional Chemical Crystallization Manufacturing Technology Research Association Patent attorney Kuninaka Masami Haka 1 person Figure 1 Figure 2 Tot, Next, Metal concentration nine (See

Claims (2)

[Claims] (1) Send the dialysis fluid to a dialyzer installed in the culture tank, measure the concentration of the component to be measured contained in the dialysate flowing out from the dialyzer, and use the results to determine the concentration of the target component in the culture fluid. A method for controlling the concentration of a culture solution, characterized in that the component to be measured at a control target concentration is added in advance to the dialysis solution. (2) The culture solution concentration control method according to claim 1, wherein the osmotic pressure of the dialysis solution is approximately the same as that of the culture solution.