JPH0695930B2 - Bubbling type bioreactor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a bioreactor for activating proliferation and efficiently accumulating products by bubbling air directly into a module.

[Conventional technology]

The supply of oxygen is essential for the culture and growth of cells such as aerobic microorganisms. Since the hollow fiber module has a large contact area between the cell solution and the substrate solution, high proliferation efficiency can be obtained. Conventionally,
In a bioreactor using a hollow fiber module, a method of bubbling a medium has been used. As the hollow fiber, a polymer material having a pore size such that cells cannot pass through and a substrate solution can leach out, for example, a porous hollow fiber module such as polysulfone and polyvinylidene fluoride is used.

[Problems to be solved by the invention]

However, the site where cell growth and substance production reaction occur is inside the module, and bubbling of the medium is indirect. It is considered that the cell proliferation efficiency can be increased by bubbling the cells in direct contact with the substrate and proliferating in the module.

[Means for solving problems]

An object of the present invention is to solve the above-mentioned problem, and its constitution is to provide a cell liquid supply pipe and a cell liquid discharge pipe on the side wall, and to arrange a large number of porous hollow fibers in a reaction pipe having both ends closed in parallel with the reaction pipe. In addition, both ends were opened to accommodate in a header provided at the end of the reaction tube, and a porous hollow tube for air supply was penetrated into the module that guides the opening to each of the inlet and outlet collecting tubes. In addition to the above constitution, ceramics are used as the porous hollow fiber and the porous hollow tube.

The hollow fiber according to the present invention is required to be a porous material that allows the substrate solution to leach out to the outside and to have a pore size that cells such as bacteria cannot pass through. Specifically, if the pore size is about 0.1-0.2 μm and the cells are large and prevent passage of cells,
It does not matter if it is more than this. Polymer materials such as porous polysulfone and polyvinylidene fluoride are used as the material of the hollow fiber, but when ceramics are used, it is used for normal sterilization 121 ° C, 1 Kg / cm ² , 15 minutes It can withstand autoclave sterilization conditions and can be used repeatedly. As the ceramic, a sintered body such as alumina, alumina-silica or silica is used.

The module accommodates such a hollow fiber in a reaction tube whose both ends are closed, in parallel with the reaction tube, with both ends opened in the header at the end of the reaction tube, and the openings are provided in the inlet and outlet collecting tubes, respectively. It is a guide.

The module according to the present invention is provided with a porous hollow tube penetrating the reaction tube. The hollow tube may have a pore size capable of bubbling in water at a relatively low pressure of about 1.1 to 1.5 atm, specifically about 5 to 25 μm, preferably about 10 to 20 μm.
Is. The material may be a polymeric material such as porous polysulfone or polyvinylidene fluoride, but ceramics are preferable in terms of strength and durability. As ceramics,
Generally, a sintered body such as alumina, alumina-silica or silica is used. Since air is supplied to the hollow tube and the bubbled air is sent to the cell tank together with the cell liquid, it is preferable to attach an exhaust valve and a pressure gauge to the cell tank.

[Action]

The present invention uses a hollow tube to directly bubble air in a module in which cells and substrates come into direct contact, so that the necessary air is sufficiently supplied to a necessary site, high growth efficiency is obtained, and efficient production is achieved. You can get living things.

〔Example〕

FIG. 1 is a schematic diagram showing the bioreactor of the present invention.
Reference numeral 1 is a module in which headers 3 and 4 are provided at both ends of the reaction tube 2. 5 is a hollow fiber, which contains alumina as a main component,
An inner diameter of 0.5 mm, an outer diameter of 1 mm, a separated particle diameter of 0.1 μm, and a length of 90 mm were used. A hollow tube 6 made of ceramics and having a hole diameter of about 15 μm was passed through the central portion of the module. Hollow tube has a particle size of 10
It was obtained by sintering by a dry-wet method using a raw material powder of ˜20 μm. At the end of the hollow tube 6, the hollow fibers are bound together around the hollow tube with an adhesive to open the end of the hollow fiber 5 in the header, and the substrate discharge port 7 and the header provided on the side wall of the header 3 A substrate tank 9 is provided between the substrate supply ports 8 provided on the side wall of the substrate 4, and is circulated by a pump.

Reference numeral 10 denotes a cell liquid supply pipe provided on the side wall of the reaction tube, a cell tank 12 is provided between the cell liquid supply pipe 11 and a cell liquid discharge pipe 11 provided on the side wall of the reaction tube, and the cell liquid is circulated by a pump. Reference numeral 13 is a pressure gauge provided above the cell tank 12, and 14 is an exhaust valve. 15 is header 3
Is an air supply port that penetrates the header 4 to supply air to the hollow pipe 6, and 16 is an air discharge port that penetrates the header 4 and discharges air from the hollow pipe 6. Reference numeral 17 is a support frame for fixing the hollow tube 6, and 18 is an adhesive agent that airtightly adheres the hollow fiber 6 and serves as a barrier between the reaction tube 2 and the spaces of the headers 3 and 4.

Bacillus subtilis was cultivated while bubbling 2 atmospheres of sterile air using the above apparatus. The medium composition was as follows.

(NH ₄ ) ₂ SO ₄ 1 g KH ₂ PO ₄ 10 g MgSO ₄ .7H ₂ O 0.1 g sodium citrate 0.5 g KOH 1.94 g was added with distilled water to a total amount of 1000 cc. (PH 7.0) A 2% starch solution was used as a substrate. The relationship between culture time and cell number is shown in FIG. Further, amylase was measured according to a conventional method, and the relationship between the amount of amylase produced and the number of culture days is shown in FIG.

As a comparative example, an experiment was conducted in the same manner as in the example except that the hollow tube 6 was not bubbled, and the results are shown in FIGS. 2 and 3.

〔effect〕

According to the present invention, the cell growth rate and the substance production rate are increased, and the culture efficiency is increased. If the hollow fiber or hollow tube material is made of ceramics, the durability will be improved and it will be possible to use it repeatedly.

[Brief description of drawings]

The drawings show examples of the present invention, FIG. 1 is a schematic diagram showing a bioreactor, FIG. 2 is a graph showing the relationship between cell number and culture time, and FIG. 3 is a relationship between amylase production and culture days. It is a graph which shows. In the drawing, reference numeral 1 is a module, 2 is a reaction tube, 3 and 4 are headers, 5 is a hollow fiber, 6 is a hollow tube, 7 is a substrate outlet, 8 is a substrate supply port, 9 is a substrate tank, and 10 is a cell. A liquid supply port, 11 is a cell liquid discharge port, 12 is a cell tank, 14 is an exhaust valve, 15 is an air supply port, 16 is an air discharge port, 17 is a support frame, and 18 is an adhesive.

Claims (4)

[Claims] 1. A header in which a cell liquid supply pipe and a cell liquid discharge pipe are provided on a side wall, and a large number of porous hollow fibers are provided in a reaction tube whose both ends are closed in parallel with the reaction tube and both ends are provided at the end portions of the reaction tube. A bubbling type bioreactor in which a porous hollow tube for air supply is penetrated into a module which is opened and stored in a module and which guides the opening to the inlet and outlet collecting tubes. 2. The bubbling-type bioreactor according to claim 1, wherein the hollow tube has a pore size of about 5 to 25 μm. 3. A cell liquid supply pipe and a cell liquid discharge pipe are provided on a side wall, and a large number of porous ceramic hollow fibers are provided in a reaction tube whose both ends are closed in parallel with the reaction tube and both ends are provided at the end portions of the reaction tube. A bubbling-type bioreactor in which a ceramic porous hollow tube for air supply is penetrated into a module which is opened and stored in a header and leads the opening to the inlet and outlet collecting tubes. 4. The bubbling type bioreactor according to claim 3, wherein the hollow tube has a pore size of about 5 to 25 μm.