JPH0595778A - Porous separation membrane integrated type incubator equipped with stirrer - Google Patents

Abstract

PURPOSE:To obtain the subject apparatus capable of efficiently producing cells at a high density for a long period by equipping a stirred type incubator or a bioreactor for a microorganism, a cell or an immobilized enzyme with a filtering and separating membrane for filtering and separating a culture solution or a reactional solution parallel to the culture or reaction. CONSTITUTION:A stirred type incubator or a stirred type bioreactor is composed of an outer vessel 2 having a culture solution taking out pipe 7 and an aeration pipe 12 and an upper lid 3 equipped with a substrate solution feed pipe 5, a culture solution circulating feed pipe 6 and an aeration pipe 11 and has a stirrer 4 in the interior. A ceramic porous cylinder 1 as a porous separation membrane for filtering and separating a culture solution or a reactional solution is installed in this incubator or bioreactor and its interior is separated into a culturing part or a reactional part and a filtering part by the partition wall (porous cylinder) 1 composed of the porous separation membrane. Thereby, a microorganism, a cell or an immobilized enzyme is filled and cultured or reacted to maintain the cell or immobilized enzyme at a high density in the culture or reactional solution. As a result, the product is continuously produced with a high efficiency.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus comprising a stirring type incubator or a stirring type bioreactor equipped with a porous separation membrane, which is suitable for carrying out culture or reaction and filtration separation in parallel.

[0002]

2. Description of the Related Art Conventionally, a stirring type culture device or a stirring type bioreactor for storing microorganisms, cells or immobilized enzymes and a filtration and separation device for a culture solution or a reaction solution have been used independently. Alternatively, it was necessary to collect the culture solution or the reaction solution from the bioreactor and transport it to the separation device. Therefore, the culturing or reaction step and the separation step are batch-wise and inefficient.

On the other hand, there is a method of slightly holding the cells inside the tower-type ceramic separation membrane in the anaerobic fermentation process and carrying out the fermentation and the separation of the culture solution in parallel, but it is impossible to stir and mix the culture solution in the tower. Therefore, the mixed state of the substrate and the cells became non-uniform, the culture rate was low, and it was difficult to increase the cell density.

On the other hand, in the case of aerobic cells which require a large amount of oxygen, it was not possible to increase the cell density inside the membrane due to lack of dissolved oxygen.

Further, in the conventional separation and filtration process using a membrane, the inside of the pores of the membrane are easily filled with microorganisms, cells, immobilized enzymes and the like, so that a large amount of separation membranes must be used.

Further, when clogging occurs, the membrane is regenerated by back-flowing a large amount of cleaning liquid.
Clogged microorganisms, cells, immobilized enzymes, etc. were washed away by this washing process, making long-term continuous operation difficult.

[0007]

Under the above circumstances, the present invention allows microorganisms, cells, or immobilized enzymes to grow or be retained for a long period of time in a culture vessel or bioreactor and to be cultured by a porous separation membrane. The object of the present invention is to provide an apparatus for continuously and efficiently separating a liquid or a reaction liquid from a microorganism, a cell or an immobilized enzyme.

[0008]

Means for Solving the Problems As a result of intensive studies to develop an apparatus having the above-mentioned preferable properties, the present inventors have installed a stirring type incubator or a stirring type bioreactor with a porous separation membrane. As a result, they have found that the object can be achieved, and have completed the present invention based on this finding.

That is, the present invention provides a porous separation for performing filtration separation of a culture solution or a reaction solution in parallel with a culture or a reaction in a stirring type incubator or a stirring type bioreactor for microorganisms, cells or immobilized enzymes. A device provided with a membrane is provided.

The porous separation membrane used in the present invention is not particularly limited, and a culture solution or reaction solution obtained by culturing or reacting a microorganism, a cell or an immobilized enzyme in a stirring type incubator or a stirring type bioreactor is used as a microorganism. As long as it has a function of separating and filtering from cells or immobilized enzyme, for example, a porous ceramic membrane, a porous glass membrane, a porous organic polymer membrane, a metal fiber knitted body, a non-woven fabric or the like can be used. Of these, a ceramic porous membrane is particularly suitable. The microorganisms, cells and immobilized enzymes are not particularly limited, but examples thereof include yeast, Escherichia coli, actinomycetes and immobilized glucose isomerase.

As the stirring and mixing method, a conventionally known method such as a stirring shaft, a stirring rod, a stirring plate, a stirring blade, a stirring magnet, a ventilation pipe, and a liquid circulation pump can be used. Therefore, a method using a stirring blade is preferable.

As the equipment structure of the porous separation membrane, it is preferable that the inside of the incubator or the bioreactor is a microorganism,
It is preferable that the cell or immobilized enzyme has a double structure having a partition structure so as to divide the outside into a culture filtrate part or a reaction filtrate part as a holding part for the enzyme. In the case of this partition structure, the holding part is provided with a stirring and mixing function. By using a device having a double structure with this porous separation membrane as a partition, the filtration separation step can be carried out while stirring the culture solution or reaction solution, and the microorganisms, cells or immobilized enzyme can be separated by the porous separation by stirring. Since it is possible to prevent the infiltration into the pores of the membrane, it is possible to prevent clogging of the porous separation membrane and obtain high filtration efficiency for a long time.

Further, in the device having the double structure,
A ventilation pipe is introduced into both the holding part inside the porous separation membrane and the filtrate part outside the porous separation membrane, and the pressure difference between the inside and outside of the porous separation membrane is adjusted by adjusting the ventilation flow rate of both. However, it is also possible to prevent clogging of the porous membrane.

Further, since the filtration and separation steps of the culture solution are performed in parallel with stirring or aeration stirring, not only anaerobic cells but also aerobic cells can be well grown, and the cell density in the incubator is set to the maximum value. Can be increased up to. Therefore, when the useful substance is produced inside the cells, the excess culture solution is continuously separated from the holding part, whereby the cells as a production means can be cultured to an extremely high density, and the culture efficiency is improved. Can be increased.

Further, by using the incubator or bioreactor according to the present invention, useful substances secreted or produced by microorganisms, cells or enzymes in the culture solution or reaction solution can be continuously recovered. Since the cells remain inside the culture medium, the high cell density can be maintained for a long time, and the target substance can be continuously and efficiently produced.

Further, even when microorganisms or cell growth inhibitory substances are produced in the medium, the inhibitory substances can always be removed by filtration separation through a porous separation membrane, and a fresh medium can be supplied, so that cell growth is prevented. Can be promoted.

Further, in the present invention, the ventilation pressure is adjusted to intermittently cause the filtrate to flow backward to eliminate clogging, and the filtration or separation of the culture solution or the reaction solution can be performed with high efficiency for a long period without replacing the porous membrane. It can be performed.

[0018]

EFFECTS OF THE INVENTION According to the apparatus of the present invention, stirring or aeration stirring is carried out while the microorganism, cell or immobilized enzyme is held in the incubator or bioreactor, and the culture solution or reaction solution is mixed with the microorganism, cell or immobilization. It is possible to efficiently and continuously carry out the separation from the enzyme. Furthermore, the cells in the culture medium can be maintained at an extremely high density for a long period of time, and the product can be continuously produced with high efficiency.

[0019]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Cultivation of yeast by an incubator equipped with a stirring blade inside an inner cylinder made of a porous ceramic separation membrane and a ventilation tube both inside and outside the inner cylinder will be described.

The incubator shown in FIG. 1 was used. In FIG. 1, reference numeral 1 denotes a bottomed cylindrical body composed of a ceramic porous separation membrane having an average pore diameter of 5 μm (hereinafter referred to as a ceramic porous cylinder).
The outer tub 2 is provided concentrically with it and has a sufficient space. The outer tubs 2 are joined to each other at the top to maintain airtightness, and their upper ends are joined to the upper lid 3. A stirring shaft is vertically provided in the ceramic porous cylinder from the central portion of the upper lid, and stirring blades 4 are attached to the stirring shaft.

A substrate liquid supply pipe 5, a culture liquid circulation supply pipe 6 and a ventilation pipe 11 are piped into the ceramic porous cylinder on the upper lid 3, and a pH electrode, a dissolved oxygen (hereinafter referred to as DO) electrode, and a temperature are provided. Electrodes, liquid level electrodes (neither shown)
Is attached. These electrodes may be attached to the outer tank 2. The outer tank 2 has a culture solution extraction tube 7 and a ventilation tube 1
2 is piped. The extracted culture fluid is switched to the take-out pipe 9 side or the culture fluid circulation supply pipe 6 side by the switching valve 8, and the culture fluid circulated to the culture fluid circulation supply pipe 6 side is fed by the culture fluid circulation pump 10. It Ventilation to each ventilation pipe is performed via the flow rate control valves 13. Further, by covering the upper part of the ceramic porous cylinder to a position lower than the culture liquid surface in advance, a pressure difference is generated by adjusting the inside / outside ventilation, and the filtrate is caused to flow backward to eliminate the clogging.

The rotation speed of the stirring blade was adjusted by the upper stirring drive system within a rotation speed range of not more than 1000 rotations per minute. From the ventilation tube, a mixed gas of oxygen and air was supplied at a flow rate not exceeding 3 l / min. The DO concentration in the culture broth was controlled by adjusting the rotating speed of the stirring blade and the partial pressure of aerated oxygen so that the optimum conditions for yeast growth could be maintained at all times.

The yeast was cultivated inside the ceramic porous cylinder of this incubator while aerating and stirring. Yeast was inoculated into a medium containing 10% by weight glucose and 5% by weight yeast extract powder, and the mixture was maintained at 30 ° C. and pH 5 and cultured with aeration and stirring. A substrate solution containing 20% by weight of glucose was automatically supplied by a pump by the DO stat method utilizing the phenomenon that the DO concentration rises when glucose is deficient. At this time, when the filtrate was withdrawn at the same flow rate as the supply liquid, glucose was hardly detected in the filtrate. By intermittently supplying the substrate and repeatedly filtering the culture solution, the cell concentration in the culture solution on the 5th day of culture was 15% by weight on a dry basis.
Reached

Example 2 The control valve 13 is switched intermittently (every 1 hour), and the ventilation inside the ceramic porous cylinder is stopped temporarily (for 10 minutes) to vent the air from the outside, whereby the ceramic porous The pressure was switched between the inside and outside of the cylinder, and 50 ml / min of the culture solution was pressed into the ceramic porous cylinder from the outside to the inside, and the same procedure as in Example 1 was carried out. The cell concentration rapidly increased, and the cell concentration reached 18% by weight on the dry basis on the 5th day of culture.

Comparative Example 1 Example 1 was repeated except that the incubator of Example 1 was replaced with an incubator having a structure in which a ceramic porous tube was omitted.
When carried out exactly in the same manner as above, the cell concentration in the culture medium on the 5th day of culture was only 1% by weight or less on a dry basis.

Comparative Example 2 The same procedure as in Example 1 was carried out except that stirring was not carried out. As a result, the cell concentration in the culture solution on the 5th day of culture was only 2% by weight on a dry basis.

[Brief description of drawings]

FIG. 1 is a schematic diagram of an incubator.

[Explanation of symbols]

 1 Ceramic Porous Cylinder 2 Outer Tank 3 Upper Lid 4 Stirring Blade 5 Substrate Liquid Supply Pipe 6 Culture Liquid Circulation Supply Pipe 7 Culture Liquid Extraction Pipe 8 Switching Valve 9 Extraction Pipe 10 Culture Liquid Circulation Pump 11, 12 Vent Pipe 13 Flow Control Valve

Claims (3)

[Claims] 1. A stirring type incubator or a stirring type bioreactor for microorganisms, cells or immobilized enzymes is equipped with a porous separation membrane for filtering and separating a culture solution or a reaction solution in parallel with culture or reaction. A device consisting of 2. An apparatus characterized in that the inside of a stirring type incubator or a stirring type bioreactor for microorganisms, cells or immobilized enzymes is separated into a culture section or a reaction section and a filtrate section by a partition wall made of a porous separation membrane. .. 3. The apparatus according to claim 2, wherein clogging of the porous separation membrane is eliminated by utilizing a pressure difference due to ventilation.