JPS6224071B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method and apparatus for producing a continuous microbial culture product, and more specifically, a culture reactor equipped with a thin tube made of an ultrafiltration membrane and equipped with an inlet and an outlet. The present invention relates to a continuous microbial culture device for obtaining a cultured product, which includes an external filtration pump, an ultrafiltrate stirring container, and the like, and a method for producing a microbial cultured product. BACKGROUND TECHNOLOGY Conventionally, there have been two types of microbial culture methods: batch type and continuous type, of which batch type has been widely used. This batch-type device is an Erlenmeyer flask that is stirred on a rotating disk, or a batch tank that is equipped with a stirrer.Such devices are filled with culture solution once, cultured microorganisms, and then undergo filtration and centrifugation. Therefore, it was a culturing device for separating and collecting culture products. However, this method is a non-propagation-linked type of culture in which product production is not accompanied by the growth of microorganisms, so-called fermentation type culture, and in culture that involves a ripening operation, the amount of nutrients input for each batch must be managed over time. However, due to the nature of the batch type, it is difficult to manage the operation safely, which causes a decline in product quality. In contrast to the batch type, the continuous type allows the culture tank to be maintained in a normal state, so there are fewer temporal fluctuations and the quality of the product is stable. Therefore, a culture method in which microorganisms are grown and matured continuously is desirable. For this kind of method, Japanese Patent Publication No. 11291, which uses a parallel type continuous culture tank, and Japanese Patent Publication No. 45-20552, which uses a multi-stage continuous culture tank, are known. Kimostart is known as a device. KimoStart is a continuous operation using a batch culture tank, in which the culture solution is continuously pumped into the tank under sterile conditions, and the culture solution containing microorganisms is pumped at the same flow rate as the feed solution. This method allows the volume of the culture solution in the culture tank to be maintained at a constant level while being drained. The problem with inventions using parallel continuous culture tanks, inventions using multi-stage continuous culture tanks, and inventions using circulating continuous culture devices as described above is that many culture tanks are required for industrial implementation. Since parallel cultivation or multi-stage continuous culture tanks must be used, the cost of industrial equipment is considerable, and the supplied culture solution is easily contaminated by bacteria due to direct contact with cultured microorganisms. However, as the microorganisms in the culture tank flow out together with the culture solution through the outlet, the amount of culture solution supplied cannot be increased above a certain level, resulting in a considerable drop in efficiency. As mentioned above, in order to prevent microorganisms from flowing out with the effluent, research on fixing microorganisms to membranes has been carried out in laboratories such as Stanford University and the University of Berkeley in the United States over the past few years. According to the results, by culturing microorganisms on one side of the ultrafiltration membrane and passing the medium through the other side of the membrane, nutrients in the medium are selectively permeated through the membrane. Therefore, microorganisms are cultured and multiplied by ingesting the permeated nutrients. Problems to be Solved by the Invention However, in this method, the culture solution is blocked by an ultrafiltration membrane, so it is possible to obtain the effect that it does not mix with microorganisms and leak out. However, it is not used in practice because as the microorganisms are isolated and cultured on the membrane, the resistance of the medium to the transfer of nutrients increases, resulting in large losses in the production of the culture product. Furthermore, since the microorganisms are surrounded by a membrane, once they grow above a certain density they will no longer grow, which will gradually reduce the production rate of the cultured product. Means for Solving the Problems The present inventors conducted research to solve the above-mentioned drawbacks, and as a result, they came up with the invention of a continuous culture device that effectively produces ultrafiltration through a membrane. The present invention not only prevents microorganisms from flowing out with the culture solution, but also reduces the diffusion resistance associated with the diffusion of nutrients, reducing wasted nutrients, and achieves the outstanding effect of separating and harvesting the microorganisms and culture products. be able to. The present invention will be explained in detail below with reference to the accompanying drawings. In FIG. 1, a thin tube 1 is a thin tube made of polypropylene fiber or polysulfone membrane, and this membrane serves as an ultrafiltration membrane that diffuses nutrients and culture products in the culture medium. After collecting these thin tubes to form a bundle, both ends were fixed with epoxy resin 2, and then cut to expose the thin tube holes. Put this fixed thin tube bundle into a glass tube with two ports 4 and 5,
A culture medium inlet 6 is formed so that the culture medium is supplied through the capillary hole exposed on one epoxy surface, and a culture solution outlet 7 is formed on the other side so that the culture solution flows out.
to create culture reactor 3. FIG. 2 shows a continuous culture apparatus of the present invention which enables continuous operation by connecting peripheral equipment to the culture reactor shown in FIG. 1. A sterilized culture medium is supplied to the culture reactor 3 from a container 12 filled with glass wool with a filter 13 through an ultrafiltration pump 14, and microorganisms are introduced into the reactor 3 from the container 8 through a line 11 and port 4. be inoculated. Another pump 15 on the outlet side has the role of regulating the amount of culture solution coming out from the outlet 7 and flowing into the container 17. At this time, pump 1
The amount of culture fluid supplied through the ultrafiltration pump 4 is adjusted to be greater than the amount of outflow from the ultrafiltration pump 15, thereby creating a pressure difference between the inside and outside of the membrane. The productivity of the cultured product changes depending on the pressure difference induced by the pumps 14 and 15, and according to the results of this experiment, the flow rate of the pump 15 is lower than that of the pump 14.
A flow rate of 0.5 to 0.6 shows the highest productivity.
Due to this pressure difference, as the medium is ultrafiltered, the diffusion resistance is lowered, nutrients are easily and sufficiently taken up by the microorganisms, and some of the culture products produced are re-tubulated due to the concentration difference between the membranes. Diffusion inward, resulting in a culture product free of microorganisms. The liquid consisting of the microorganisms, nutrients and culture products outside the tube is then injected into the container 8 by means of a pump 16 through the outlet 5. If the microorganisms are aerobic, the microorganisms are stirred using the stirrer 10 while injecting air through the air supply port 9, and if the microorganisms are anaerobic, the microorganisms are stirred with the air supply port 9 blocked.
Furthermore, the liquid composed of the microorganisms to be ultrafiltered, unreacted medium, and culture products is stored in a container 19 by a vacuum pump 20, and the portion containing excess microorganisms is again passed through the line 11 to the reactor inlet 4. It is collected again into the culture reactor 3 and reused. As a result, only cultured products free of microorganisms can be obtained in the container 17. EXAMPLES AND EFFECTS OF THE INVENTION Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the present invention is not limited to the following examples. Example 1 The effect of the apparatus of the present invention was confirmed by the following alcohol fermentation. The capacity of the reactor is 37ml, and the flow rate of the culture solution at the inlet is
9.2ml/hr., and the flow rate at the outlet of the culture reactor is 3.8
ml/hr., and the ultrafiltration rate was 5.4 ml/hr.
The solution in reactor 3 was recycled at a flow rate of 83 ml/min. Under these conditions, a liquid medium with a glucose concentration of 100 g/g was injected into the reactor at 30°C, and alcohol fermentation was carried out. On the other hand, using the known chymostat shown in Fig. 3,
℃, the concentration of glucose is as above, i.e.
Alcohol fermentation was carried out by injecting 100 g/liquid medium into the reactor. The time taken for each of the above devices to come out of the incubator (cultivation time) was measured as 4 hours. The results are as follows.

[Table] * However, the concentration in the device of the present invention is the average of the outlet concentrations at two locations in order to compare with the concentration in chymostat.
Example 2 The same procedure as in Example 1 was carried out except that the outlet flow rate of the culture reactor was 6.1 ml/hr., the ultrafiltration rate was 3.1 ml/hr., and the ratio of the ultrafiltration rate to the outlet flow rate was lowered. The results are as follows.

[Table] Example 3 The procedure was the same as in Example 1 except that the outlet flow rate of the culture reactor was 3.0 ml/hr. The ultrafiltration rate was 6.2 ml/hr. and the ratio of the ultrafiltration rate to the outlet flow rate was increased. did. The results are as follows, and there is no difference from Example 1.

[Table] As can be seen from the above results, under the same conditions, the incubator of the present invention had an outstanding effect of ingesting more glucose in the medium and producing more culture products than with Kymostat. It will be done.

[Brief explanation of the drawing]

Figure 1 is a detailed diagram of a microbial culture reactor made by assembling thin tubes, Figure 2 is a schematic diagram of the assembly of the continuous culture device of the present invention, and Figure 3 is a schematic diagram of the assembly of a known chymostat. The symbols mean the following. 1
...Thin tube, 3...Culture reactor, 4, 5...Port, 6...Medium inlet, 7...Culture solution outlet, 10
...Ultrafiltrate stirrer, 14... Ultrafiltration pump, 15... Ultrafiltration pump.

Claims (1)

[Claims] 1. With the pressure of the ultrafiltration pump 14 being higher than that of the pump 15, the sterilized medium is sent through the inlet 6 of the biological culture reactor 3 into the reactor internal capillary 1, and passes through this capillary membrane. A method for producing a microbial culture product, in which the medium is sufficiently ingested by the microorganisms injected into port 4, and then diffused into the capillary again due to the concentration difference between the membranes inside the reactor, so that no microorganisms exist. 2. The method according to item 1 above, wherein the ultrafiltration flow rate due to the pressure difference between the pumps 14 and 15 is 1:0.5 to 0.6 of the pump 14 flow rate. 3. The method according to item 1 above, wherein the culture product containing microorganisms inside the reactor 3 is collected through the port 5 into the ultrafiltration stirring device 8 and reused as the injected microorganism. 4 A bundle of thin tubes made of ultrafiltration membrane is installed inside,
microbial culture reactor 3 with ports 6, 7;
A microorganism continuous culture device comprised of ultrafiltration pumps 14, 15 and ultrafiltrate stirring devices 8, 9, and 10.