JPH0779677B2 - Incubator - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture device for culturing microorganisms and / or cells, and more particularly to a rotating drum type culture device suitable for culturing photosynthetic microorganisms and / or animal / plant cells or protoplasts.

[0002]

2. Description of the Related Art Conventionally, in a culturing apparatus for microorganisms and / or cells, particularly a liquid culturing apparatus, a fixed tank is provided and a propeller-type stirring device, a magnetic stirrer, and an air lift system are used for the purpose of gas supply and medium mixing. Draft tubes and the like have been used. Further, in the batch-type culture device, a device for performing mixing and gas-liquid contact by vibrating or rotating like a shaker such as a mono-type or reciprocating type shaker or a roller bottle is used. However, these are limited to small devices.

[0003]

[Problems to be Solved by the Invention] In industrialization,
Although large-scale culture and precise control of production (growth) amount are required, the conventional fixed tank system has the following drawbacks. (1) Due to various obstacles such as air diffuser, agitator, various sensors, draft tube, etc. in the culture device, a lot of stagnation occurs in the flow of the liquid in the tank, and the cultured bacterial cells enter there. , Algal cells or cells adhere to the surface. (2) Even if an air diffuser or a stirrer is used, the gas dissolution efficiency is not always high, and a large amount of gas must be supplied. (3) It is difficult to apply a method of causing a strong shear flow by a stirring device to a microorganism having weak strength or cell culture. Therefore, it is an object of the present invention to provide a culture device which has improved the above-mentioned problems, prevents non-uniformity in the tank, and has less gas-liquid contact efficiency with less damage to cultured cells.

[0004]

In order to solve the above-mentioned problems, in the present invention, in a culture device for culturing microorganisms and / or cells, etc., a rotary culture vessel having a fixed connection portion at least at one end, and the culture are provided. It has a driving device for rotating the container while holding it in the horizontal axis direction, and a liquid inlet and an outlet, a gas inlet and an outlet, and a light inlet are respectively provided in the fixed connection part, and these are provided in the culture container. Communicated with
In addition, the solid-liquid separation device is opened from the liquid outlet and the liquid flow
A liquid circulation mechanism for circulating a part of the culture liquid to the inlet, and the gas
Circulate a part of the gas discharged from the outlet to the gas inlet.
And a gas circulation mechanism . In the device, the liquid outlet has a front
With an outflow pipe that opens upward in the culture container,
To arrange the outlet pipe so that the height can be adjusted up and down.
Good.

Here, the culture vessel may be of any rotating shape, and can be appropriately selected from a cylindrical shape, a truncated cone shape, a spherical shape, a barrel shape, a bottle shape and the like. Further, the fixed connecting portion may be installed at one end or both ends of the rotating shaft of the culture container. When the volume of the culture container is large, other components, including the light diffuser, can be provided at the fixed connection parts at both ends of the incubator, and each component is also advantageous in terms of strength. Is.

The fixed connecting portion may be self-supporting or connected to a driving device. The fixed connection part is a connection part of inflow and outflow inside and outside the culture container, and from this, a flow means such as a light diffuser, an air diffusing pipe, a liquid supply pipe and an overflow pipe is projected into the incubator. Further, it is also a support site, and an object to be fixedly positioned in the rotating incubator, for example, a wiper (scraper) for removing adhered substances, various sensors, and the like can be made to protrude from the fixed connecting portion as necessary.

The amount of the liquid poured into the culture container of the present invention can be freely adjusted by moving the position of the mixed culture liquid outflow pipe up and down, and the operation is performed even under the condition that the volumetric efficiency is almost 100%. However, in order to maintain the gas dissolution efficiency at a high level, the volume efficiency is usually 50 to 50%.
It is preferable to set and operate in the range of 70%. Further, the liquid discharged from the device (mixture of bacterial cells) is separated into a bacterial cell and a culture-finished liquid by a solid-liquid separation device. Trace components remain. Therefore, it is recommended to combine the residual culture solution with the undiluted solution supplied to the equipment to reuse the residual components repeatedly .
Yes.

Gases are supplied from the outside of the device through an arbitrary sterilizing means by a blower or the like, and aeration tubes are used to aerate each part of the rotating culture container so as to be uniform, but particularly fragile microorganisms, cells and protoplasts are used. For the above, the diffuser may be diffused from the gas-liquid interface without using an air diffuser. The gas supply method may be transient, but its utilization efficiency is limited. Therefore, in the present invention, also for the purpose of further enhancing the agitation of the solution in the device by aeration, the exhaust gas from the device is connected to the gas supply pipe and repeatedly used, and the cost can be reduced at the same time. It goes without saying that the final exhaust gas is discharged to the outside of the system through the sterilization process.

Further, in order to improve the gas dissolution efficiency, the pressure in the container can be raised to raise the partial pressure of the gas in the liquid, but the incubator requires a container made of a material having sufficient strength. Therefore, an opaque metal container is used even for photosynthetic microorganisms or cells. In this case, it is necessary to provide a light supply means into the container as described later. Photosynthetic bacteria, microalgae or light diffusers used in photoculture of plant cells or protoplasts are those that can disperse artificial light and / or sunlight transmitted by an optical fiber in a culture container at high density, and uniformly. The material is quartz glass, PMMA (polymethylmethacrylate),
Other known materials can be applied.

Further, a method of forming grooves or scratches on the surface of a rod of PMMA or other resin, or dispersing or enclosing various kinds of particulate matter that diffusely reflects light in the various light diffusers described above. Should be adopted. In this case, in order to evenly diffuse the light in the liquid in the apparatus, it is preferable that the density of scratches, grooves or fine particles on the surface be increased as the distance from the connection point increases. Further, it is preferable that various light diffusers are protected by forming a protector with a strong transparent material.

[0011]

In the culture device of the present invention, a mixture of cells or protoplasts and / or microorganisms and a liquid medium (hereinafter,
The mixed solution = abbreviated as ML) wets the inner wall of the culture vessel to form a thin film. Therefore, the gas dissolution efficiency is high, and the culture is less likely to be damaged. However, an air diffuser may be provided in the tank for microorganisms and the like that are relatively resistant to physical stress. Since the tank itself rotates, the culture does not accumulate in a part of the tank and the ML state can be set uniformly. Further, since the device of the present invention can be configured as a continuous culture system, the growth of the culture can be controlled as a chemostat, turbidostat or the like by adjusting the inflowing substrate, the dilution ratio and the like. In addition, due to the structure in which the gas body and the rest of the substrate supplied as a liquid are returned to improve the utilization efficiency, inexpensive and resource-saving culture can be performed.

As described above, the present invention has a wide industrial application range as a precisely controllable reactor capable of mass-culturing even a delicate culture object and constituting a continuous culture system. As an application example, it is used as a culture tank for microorganisms (such as transduced Escherichia coli and marine bacillus) that produce physiologically active substances (growth factors, enzymes, antibiotics, etc.), such as bacteria, yeast, actinomycetes, and Examples of the treatment device using assimilating microorganisms include a culture device for isolated cells and viruses. Further, since it can be applied to culture under pressure, it can also be applied as a reactor for pressure-resistant microorganisms in the deep sea. Further, since a light guide and a light diffuser can be installed in the culture of the photosynthetic microorganism, the photosynthetic cell or chloroplast, the culture can be performed more effectively.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below with reference to the drawings, but the present invention is not limited to these. Example 1 This example describes the case of culturing a photosynthetic microorganism by using a device having a cylindrical shape and supporting connection portions arranged at both ends. FIG. 1 shows a photosynthetic microorganism culture device (hereinafter referred to as a culture device).
FIG. FIG. 2 is a schematic flow diagram illustrating the flow of liquid and gas in the device of FIG. In FIG. 1, 1 is a rotary culture container, and 2 is a fixed connection part. The culture container 1 and the fixed connection part 2 are rotatably connected so as not to leak. The culture container 1 is driven by a driving device 4 (the rotation speed is indicated by a tachometer of 5), and the rotating roll 3 rotates in contact with the drive device 4 and transmits the rotation to the culture container 1. The slow rotation of the culture vessel 1 causes the M in the apparatus to rotate.
As for L, a film-like thin film is formed along the inner wall of the culture vessel, and the gas supplied from the gas blowing pipe 8 and the air diffusing pipe 9 is used very effectively.

The fixed connecting portion 2 has a gas blowing pipe 8, an air diffusing pipe 9, a gas discharging pipe (gas circulating pipe) 8 '(FIG. 2) 8', and a plurality of light diffusers 7 (usually protected by a sheath 6). Fig. 3 shows a vertical sectional view of the fixed shaft. A wiper 11 for mechanically peeling off deposits on the inner wall of the culture vessel is installed, and a raw water inflow pipe 15 and an ML outflow pipe 16 are installed as shown in FIG. The volume of the solution in the culture device (volumetric efficiency) can be freely adjusted by increasing or decreasing the height of 16. In addition, as shown in FIG.
Depending on A and B), the number of light diffusers for introducing light energy into the culture device can be appropriately increased or decreased.

In FIG. 2, an appropriate amount of photosynthetic bacteria, microalgae or specific cells (hereinafter referred to as culture) is inoculated into the culture container 1 and is suitable for the growth of each microorganism and / or cells from the stock solution inflow pipe 15. The culture solution is continuously infused. The culture vessel 1 rotates at a slow speed as described above, and the supply gas is efficiently absorbed in the gas body and liquid supplied from the air diffusing tube 9 and in the ML which is carried in a film form on the inner wall of the apparatus. Further, the stirring and mixing of ML in the device is performed slowly not only by the rotation of the culture container but also by the baffle effect of a plurality of light diffusers installed in the device, so that there is no impact on the culture. .

The ML that has grown in the required amount of culture is the ML outflow tube 1
Although it is taken out of the apparatus by 6, it is separated into a culture solution 17 and a separation solution 18 by a solid-liquid separation apparatus 20 composed of a so-called membrane such as a ceramic filter or a membrane filter. It should be noted that means such as precipitation and centrifugation may be used for the separation of the culture. The culture 17 can be effectively used for producing useful substances or for other purposes. The separated liquid 18 is returned to the raw water inflow pipe 15 for the above-mentioned purpose, and the remaining active ingredient is repeatedly used for culture in the culture device.

The light energy necessary for culturing the photosynthetic microorganisms and / or cells converts sunlight 19 into sunlight concentrator 12.
And / or the artificial light from the artificial light generating device 13 is connected to the light diffuser 7 mounted in the device by the optical transmission fiber 14 to disperse the light uniformly from the plurality of light diffusers in the liquid at high density. , Supplied. On the other hand, the gas body necessary for culture is sent to the air diffuser pipe in the device through the gas blowing pipe 8 through the sterilization process (not shown) by the blower 21 and is supplied to the bacterial cells, but the exhaust gas from the device is discharged. Is connected to the intake side of the blower 9 via the gas circulation pipe 8 ',
It is repeatedly used by various microorganisms. The surplus (generated) gas is discharged from the surplus gas exhaust pipe 8 ″ to the outside of the system through a sterilization process (not shown).

[0018]

According to the present invention, with the above-described detailed structure, the following operational effects can be obtained. (1) By adopting a horizontal rotary drum type culture device having a circular cross section, the liquid inside the device becomes a film-like thin film in the gas phase portion of the inner wall of the device, and the supply gas is effectively moved to the liquid side. It is possible to mass-culture microorganisms or cells with a much smaller amount of gas. (2) Since the tank itself rotates, there is no need for a mechanical stirring device for mixing gas-liquid and bacterial cells in the culture device, which does not give strong shearing force to the culture and damages the cells. Since it is not given, it is suitable for large-scale culture of delicate objects. (3) Substrates or gas bodies necessary for culturing microorganisms or cells are repeatedly used by returning or circulating, so that the substrates can be effectively used. (4) It can be used as a continuous culture device and the dilution rate
It is possible to easily finely adjust the culture conditions such as.

[Brief description of drawings]

FIG. 1 is a cross-sectional explanatory view of a photosynthetic microorganism culture device of the present invention.

FIG. 2 is a schematic flow diagram illustrating the flow of liquid and gas in the device of FIG.

FIG. 3 is a vertical sectional view of a fixed shaft.

[Explanation of symbols]

1: Culture vessel, 2: Fixed connection part, 3: Rotating roll, 4:
Drive device, 5: tachometer, 6: sheath tube, 7: optical fiber (light diffuser), 8: gas suction tube, 8 ': gas circulation tube,
9: Air diffuser, 10: Water level, 11: Wiper, 15: Stock solution inflow tube, 16: Bacterial cell mixture (ML) outflow tube

Claims (2)

[Claims] 1. A culture device for culturing microorganisms and / or cells, comprising a rotary culture container having a fixed connection portion at least at one end, and a drive device for rotating the culture container while holding the culture container in a horizontal axis direction. , A liquid inlet and an outlet, a gas inlet and an outlet, and a light inlet are respectively provided in the fixed connection part, and these are communicated with the inside of the culture vessel, and the liquid outlet is connected via a solid-liquid separation device. Liquid inflow
A liquid circulation mechanism for circulating a part of the culture liquid to the mouth, and the gas flow
Circulate a part of the gas discharged from the outlet to the gas inlet
A culture apparatus having a gas circulation mechanism . 2. The liquid outlet is located above the culture container.
Has an outlet pipe that opens in the direction of
Claims that are arranged so that they can be adjusted up and down
Item 1. The culture device according to item 1.