JPH0530957A - Apparatus for culturing - Google Patents

Abstract

PURPOSE:To obtain the subject device for mass cluturing capabl of being readily operated by installing a vibration pump in a circulating device in a culture solution system and arranging a pipeline in a specific state in the circulating device for tlpe culture solution and a culture gas. CONSTITUTION:A vibration pump 4 is installed in a circulating device 3 for a solution system and a gas-liquid mixed fluid of a culture solution and a culture gas is jetted from a jetting port 5 to produce a vortex. The culture solution is then taken out of a suction port 2 in the central part of the vortex and, together with the circulating gas, as a gas-liquid mixed fluid introduced into a culture tank 1.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for culturing microorganisms and / or cells. Particularly, the flow of liquid or mechanical action prevents adhesion of bacterial cells, algal cells, animal or plant cells, etc. to the inner wall of the culture tank or the sensors in the culture tank, and mass-cultures microorganisms and / or cells at high speed and with high efficiency. The present invention relates to a culturing device.

[0002]

2. Description of the Related Art A conventional culture apparatus is an air diffuser for supplying air or the like necessary for culture into a culture tank, and a stirring apparatus for uniformly mixing culture solution in the tank to enhance gas dissolution efficiency. It is the mainstream that is provided. In addition, an air lift system has been developed in which a draft tube is provided in the culture tank and the culture liquid in the tank is caused to flow by the force of air blown into the tube.

Currently, there are various types of culture devices,
The conventional culture device has no means for supplying the gas-liquid mixed fluid to the culture tank without providing a special gas-liquid mixing means outside the culture tank and without giving a strong shear flow in the culture liquid supply pump. Therefore, a gas such as air necessary for culture has been supplied by installing an air diffuser in the culture tank and by providing a stirring means for giving a strong shear flow to the culture solution in the culture tank.

However, these conventional culture devices have the following technical problems, and various researches are being carried out at each research institute in order to improve these problems.

(Technical problems) 1. Since various equipment such as an air diffuser, agitator, and sensors exist in the culture tank, a large amount of stagnation occurs in the flow of the culture solution in the culture tank, and the cultured cells or cells adhere to it in a concentrated manner. However, it is a significant obstacle to obtaining highly accurate experimental data.

2. The method of causing a strong shear flow in the culture solution with a stirring device in order to prevent adhesion is not suitable for cells having low strength.

[0007]

The above-mentioned conventional culture device is not provided with means for solving the big problem of adhesion of bacterial cells, algal cells or cells. The present inventors provide a novel culturing apparatus that improves these problems of the conventional culturing apparatus, enables significantly large-scale culturing as compared with the conventional culturing apparatus, and is significantly easy to operate. Is what you are trying to do.

[0008]

Means and Actions for Solving the Problems The above problems are as follows: 1) In a culture device equipped with at least a culture tank and a circulation device having a culture solution system and a culture gas system, at least a vibration pump in the culture solution system circulation device. The culture solution and the culture gas in the culture tank are jetted as a gas-liquid mixed fluid from the wall surface of the lower part of the culture tank by the vibration pump, and the culture solution is drawn out from the center of the vortex above the culture solution in the culture tank. It is characterized in that a circulation device for the culture solution system and the culture gas system is installed so that it is introduced into the culture tank at high speed again as a gas-liquid mixed fluid together with the circulating gas, and a vortex is caused in the culture solution in the culture tank. And a culture device.

2) A carrier having a specific gravity heavier than water is mixed in the culture solution in the culture tank and circulated with the culture solution in a circulation device,
The culture device according to 1) above, which removes bacterial cells and the like adhering to the inner wall of the culture tank.

3) A sponge-like carrier having a size that does not enter the circulation device is mixed into the culture solution in the culture tank to circulate in the culture tank to remove bacterial cells and the like adhering to the inner wall of the culture tank. It is achieved by the culture device according to 1) above.

In the present invention, the vibration pump installed in the circulation device of the culture device is, for example, Japanese Patent Publication No. 3-13439 and "Piping and Device" April issue (1991) 31-36.
It is a pump based on the pumping effect that effectively utilizes the resonance phenomenon of the fluid in the pipe by vibrating the cylindrical straight pipe with the spring valve arranged at the end on the page in the pipe axial direction. The present invention is the first to be incorporated.

By adopting this innovative vibration pump as the circulation device of the culture device and improving the arrangement of the circulation device for the culture solution system and the culture gas system, it is not necessary to install a stirring device or an air diffusing tube from the culture tank. If necessary, a high-speed flow is caused in the culture solution in the culture tank to prevent the adherence of bacterial cells to the culture tank wall surface and various sensors, and more preferably by mixing a carrier with the culture solution, the culture tank wall surface and various sensors The problem was solved by removing the bacterial cells and the like attached to the species.

When installing the vibration pump in the circulation device of the culture device, at least installing the vibration pump in the culture liquid circulation device means the culture liquid system and the culture gas system of the culture device circulation device. The two circulation devices are connected to one culture system and enter the culture tank, but the position where the culture gas circulation device is connected to the culture liquid circulation device may be before or after the vibration pump, and may be connected before or after the vibration pump. Means that it may be.

The culture solution circulated in the culture solution circulation apparatus is usually circulated as a gas-liquid mixed fluid, and the gas is efficiently dissolved in the culture solution by the vibration pump. If culture gas is added at the rear of the vibration pump, it will be dispersed in the liquid at the time of jetting and become a gas-liquid mixed fluid, and when the gas-liquid mixed fluid circulates and reaches the vibration pump, the gas in the culture liquid will be better. Will be dissolved.

The performances that should be provided as a liquid circulation pump installed in the culture device are as follows: 1) The culture liquid in the culture tank can be stirred uniformly and strongly as much as possible. 2)
Air and carbon dioxide (CO) necessary for culturing cells and cells
₂ ) To be able to efficiently dissolve enriched air and / or carbon dioxide into the culture solution. 3) Do not damage the cells and cells during stirring. And 4) The liquid circulation device can be sterilized.

As the pump for circulating the liquid, conventionally, for example, a roller tube pump, a diaphragm pump and the like can be mentioned. The roller tube pump has a feature that the tube used for liquid feeding can be sterilized by autoclave, but the amount of liquid that can be processed is small, and since pressure operation is included during circulation operation, there is a concern that it will damage cells and cells. There are some problems. In addition, the diaphragm pump can uniformly and strongly agitate a large amount of culture solution, but since the pump itself cannot be autoclaved, it must be sterilized by passing steam separately through the liquid contact part. There is a problem that complete sterilization cannot be performed because there are two veneer valves. Furthermore, these roller tube pumps and diaphragm pumps do not have a positive action for dissolving the culture gas such as air in the culture solution inside the pumps.

The vibration pump of the present invention is a completely new type of pump based on the pumping effect that applies the resonance phenomenon of the fluid in the pipe as described above, and is the optimum pump for the culture device as described below. Excellent performance as a circulation device.

That is, the vibration pump can be completely sterilized because the pump itself can be autoclaved, and the internal structure is easy to clean because of its simple structure. It is possible to circulate a large amount of culture solution, and there is no fear of damaging the cells and cells during the circulation operation.

Further, in the vibration pump, the culture solution and the culture gas are sufficiently agitated and mixed in the pump, so that there is an action not seen in the other conventional pumps. That is, a gas-liquid mixed fluid of a culture solution and a culture gas consisting of air and carbon dioxide necessary for culturing cells, cells, etc., flowing in the circulation device is sufficiently agitated and mixed in a pump to form a culture gas of the culture solution. It is characterized in that the lysis is sufficiently performed while it reaches the culture tank from the pump.

However, in the case of the main culturing apparatus, the reason why the culturing gas consisting of air and carbon dioxide is dissolved more efficiently in the culturing solution than in the case of other culturing apparatuses is due to the performance of the present vibration pump, A stable vortex flow is generated in the liquid in the culture tank by jetting the gas-liquid mixed fluid into the improved culture tank of the present invention and inhaling from the center of the vortex of the fluid generated in the tank, and The circulation method also depends on the method of deploying the circulation device of the present invention in which the culture solution is repeatedly introduced into the culture tank.

Further, the vibration pump plays a major role in solving the important problem of preventing bacterial cells, algal cells or cells from adhering to the culture device. That is, as described above, the vibration pump is capable of circulating a large amount of the culture solution, and because the air and carbon dioxide necessary for culturing cells and cells are dissolved in the culture solution very efficiently. The culture device of the present invention does not require a stirring device or an air diffuser to be installed in the device, so that the structure in the culture tank can be made extremely simple. At the same time, since the structure of the vibration pump and the auxiliary circulation line is extremely simple, the culture device of the present invention can have very few places where cells, algae, cells or the like adhere to the device.

In the liquid circulation device installed in the culture apparatus of the present invention, a carrier having a specific gravity higher than that of water is mixed into the culture liquid in the culture tank by using a vibration pump, and is circulated together with the culture liquid in the liquid circulation device. Therefore, the cells, algal cells, cells and the like attached to the device can be removed by these carriers.

In order to solve the important problem of not adhering bacterial cells, algae, cells or the like to the culture device, in addition to the adoption of a vibration pump, a circulation device for the culture solution system and the culture gas system of the present invention is provided. Improvement was also an important factor. That is, a gas-liquid mixed fluid consisting of a culture solution and gas is ejected from the wall surface of the lower part of the culture tank, preferably in the tangential direction of the inner wall of the culture tank, and the culture solution is ejected from the center of the vortex above the culture solution in the culture tank. It is drawn out and passed through a culture fluid system circulation device as a gas-liquid mixed fluid together with a circulating gas, and is again introduced into the culture vessel at a high speed, and a culture fluid system is used so as to cause a swirl in the culture fluid in the culture vessel. The task could be achieved by providing a circulation device with the culture gas system. The repeated introduction of the gas-liquid mixed fluid into the culture tank contributes to the very efficient dissolution of air and carbon dioxide required for the culture into the culture solution.

With the characteristic circulation device of the present invention including the adoption of the above-mentioned innovative vibration pump, and the size of the culture liquid in the culture tank in which a carrier having a specific gravity higher than that of water is mixed or the liquid does not enter the circulation device. By mixing the sponge-like carrier with the cells and removing the cells, algae, cells, etc. adhering to the inside of the culture tank, an important problem regarding the culture apparatus was solved.

Here, specific examples of the carrier having a specific gravity heavier than water to be mixed in the culture solution include artificial sponge and sand grains in addition to the sponge-like carrier. Further, the carrier having a specific gravity of 1 to 2 is preferably used.

[0026]

DESCRIPTION OF SPECIFIC EMBODIMENTS AND OPERATION The features of the present invention and the excellent operation thereof will be described in more detail below with reference to FIGS. However, the present invention is not limited to the following specific description.

FIG. 1 is an explanatory view of the culture device of the present invention. A culture solution and fungi, algae or cells to be cultured are charged into the culture tank 1, and necessary nutrient components are added from the medium supply pipe 8. Air required for culture from the culture gas supply pipe 7,
When the carbon dioxide-enriched air and / or carbon dioxide is supplied, these gases are supplied to the culture solution through the gas system circulation device 6. The culture liquid circulates in the culture tank 1 and the liquid circulation device 3 through the liquid circulation device 3. At the same time, the culture gas circulates in the gas system circulation device 6. In the vibration pump 4, the culture solution from the liquid circulation device 3 and the culture gas from the gas circulation device 6 are mixed to form a gas-liquid mixed fluid. The gas-liquid mixed fluid is jetted from the jet outlet 5 in the tangential direction of the inner wall of the culture tank 1, rises in the culture tank 1 in a vortex shape, and the gas mixture of the culture solution above the culture tank 1 and opening at the center of the culture solution vortex The culture solution is drawn out from the suction port 2 and returns to the culture tank 1 at high speed again as a gas-liquid mixed fluid together with the circulating gas by the vibration pump, and then circulates in the culture apparatus.

By squirting the culture solution in the tangential direction of the inner wall of the culture tank 1 and sucking the culture solution from the opening at the center of the vortex in the upper part of the culture tank 1, a swirl flow is generated in the culture tank 1. The measurement of the inner wall surface of the culture tank 1 and the measurement inserted into the culture tank 1 because a water flow always occurs around the inner wall surface of the culture tank 1 and a measuring sensor (not shown) inserted in the culture tank 1. It is possible to prevent adherence of cultured bacterial cells to the sensor for use. Air, carbon dioxide-enriched air, and / or carbon dioxide necessary for culture are continuously or intermittently introduced into the gas system circulation device 6, and the same amount of gas is discharged from the gas discharge pipe 10. Since air, carbon dioxide-enriched air, and / or carbon dioxide are repeatedly introduced into the culture tank 1, the dissolution efficiency of the culture gas in the culture solution is significantly enhanced.

The culture solution withdrawn from the suction port 2 passes through the liquid system circulation device 3, and together with the circulation gas of the gas system circulation device 6 as a gas-liquid mixed fluid by the vibration pump 4 at a higher speed than the jet port 5 in the culture tank. Will be introduced to. At this time, it is efficient to connect the gas system circulation device 6 to the suction side of the vibration pump 4, but it may be connected to the outlet side of the pump 4. The cultured bacterial cells, algal cells, cells or the like are taken out from the culture solution outflow pipe 9.

FIG. 2 is an explanatory view of the water flow ejected from the ejection port 5 in the tangential direction of the inner wall of the culture tank 1. Culture tank 1
The flow velocity of the culture solution in the tangential direction is 50 in order to prevent adherence of cultured bacterial cells, algal cells or cells to the inner wall or sensors.
It is preferably cm / sec or more. In addition, the flow rate may be changed periodically in order to further enhance the adhesion prevention effect.

In FIG. 3, a carrier 11 having a higher specific gravity than water is mixed in the culture solution and circulated with the culture solution in the liquid circulation device 3,
FIG. 3 is a schematic diagram for explaining a culture device for removing adhered substances such as cultured bacterial cells to the inner wall of the culture tank 1 and sensors.

Since the specific gravity of the carrier 11 is heavier than that of water, the carrier 11 is pressed against the inner wall of the culture tank 1 by the centrifugal force generated by the vortex flow and ascends the wall surface, thereby exerting the effect of removing the deposits by the mechanical rubbing action.

The vibration pump 4 is capable of feeding even a liquid containing particles, and exhibits an excellent effect for the purpose of the present invention. However, for this purpose, the diameter of the particles of the carrier 11 is preferably 2 mm or less.

FIG. 4 shows that a sponge-like carrier having a size that cannot be put in the liquid circulation device 3 is mixed in the culture solution to attach substances such as cultured cells to the inner wall of the culture tank 1 and sensors. FIG. 3 is a schematic diagram for explaining a culture device when removing a bacterium.

A screen is provided at the screen-equipped suction port 12 of the culture solution, which is located at the upper part of the culture tank 1 and opens at the center of the vortex of the culture solution, to prevent the carrier from flowing into the liquid circulation device 3. As described above, the vibration pump 4 gives a pulsating flow to the culture solution by the pumping principle, so that it is possible to prevent the sponge-like carrier from being pressed against the screen of the suction port 12 and blocking it. The carrier migrates from the upper part to the lower part of the culture tank 1 at the center of the vortex flow, and is again pressed against the inner wall of the culture tank 1 by the centrifugal force generated by the vortex flow to rise the wall surface and exert the effect of removing the deposits by the mechanical rubbing action To do.

In order to further enhance the action of being pressed against the inner wall of the culture tank 1 by the centrifugal force generated by this vortex, the specific gravity of the carrier used here is made larger than that of water, and the normally precipitated carrier is periodically removed. It is also possible to increase the flow velocity of the jet of the culture solution to make it float and exert a mechanical rubbing action.

[0037]

[Examples] A culture experiment of a general green alga, Chlorella pyenoidosa, was carried out with a culture tank scale of 2 liters. A) The maximum flow rate of 3 liters / min.
In the April issue (1991) pages 31 to 36, the culturing apparatus of the present invention shown in FIG. 5 in which a vibrating pump 4 described as a four-stage movable permanent magnet pump is installed, and B) an air diffusing tube 4 and an agitator 3. The culture was performed using the equipped culture apparatus of the comparative example shown in FIG.

The culture gas used was air mixed with 5% carbon dioxide, the pH of the culture solution was 8.0, the temperature of the culture solution was 25 ° C., and light was emitted from the outside of the tank using a metal halide lamp (on the outer wall surface of the culture tank). Batch culture was carried out for 3 weeks under irradiation (under an illumination of 5000 lux).

The MC medium shown below was used as the medium. KNO ₃ 1.25 g MgSO ₄ 1.25 g KH ₂ PO ₄ 1.25 g A ₅ Solution 1 ml FeSolution 1 ml Distilled water 1 liter (Example 1) In FIG. 5, the culture solution in the culture tank was circulated using a vibration pump. flow rate was circulated in the (Q _L) 2 Rittoru / min. The culture gas was circulated at (Q _G ) 1 liter / min, and the solenoid valve 13 was switched for 5 minutes for 30 minutes to supply fresh gas at 1 liter / min.

In the device of the present invention, Maximum growth rate 0.20 g / hour · liter, Final suspended cell concentration: 7.0 g / liter was gotten.

The amount of algal cells attached to the wall surface was about 0.5 g in total. (Comparative example 1) In FIG.
It was supplied at liter / min.

In the apparatus of this comparative example, Maximum growth rate 0.12g / hour ・ liter, Final suspended cell concentration 2.0 g / l was gotten.

The amount of algal cells on the wall surface was about 3.0 g in total.

[0044]

【The invention's effect】

1) The use of the vibration pump of the present invention and the arrangement of the channels of the circulation device for the culture solution and the gas cause a swirling flow of the culture solution in the culture tank, and culture on the inner wall of the culture tank and various sensors in the tank. It was possible to prevent the adherence of bacterial cells.

2) By using the vibration pump of the present invention and arranging the conduits of the circulation device for the culture solution and gas, the conduits of the circulation device for the gas-liquid mixed fluid consisting of the culture solution and the culture gas are provided. As a result of repeated circulation to the inside, air, carbon dioxide-enriched air and / or carbon dioxide are sufficiently dissolved in the culture solution, the culture apparatus of the present invention does not require a stirring device or an air diffusing tube in the tank. The device was simple, and the adherence of cultured bacterial cells to the inner wall of the culture tank and various sensors in the tank could be prevented.

3) The vibration pump used in the present invention can be sterilized by autoclave, and the structure of the pump and the attached pipeline are simple, so that it is easy to wash, and even if bacteria or algae are passed through the pump. It is suitable for pumps for culture devices because there is no risk of damage to them.

4) In the vibration pump used in the present invention,
Since the culture solution and the gas are sufficiently stirred and mixed, the gas is well dissolved in the culture solution by the time the gas-liquid mixed fluid reaches the culture tank.

5) In the vibration pump used in the present invention,
Since a large flow rate can be secured even if the culture solution contains a fine carrier, the carrier with a higher specific gravity than water is mixed into the culture solution and circulated in the culture tank to culture bacteria on the inner wall of the culture tank and various sensors in the tank. The deposits such as the body could be removed.

6) A sponge-shaped carrier having a size that does not enter the circulation device in the culture solution is mixed with the culture solution and circulated in the culture tank to culture the bacteria on the inner wall of the culture tank and various sensors in the tank. The deposits such as the body could be removed.

[Brief description of drawings]

FIG. 1 is an explanatory view of a culture device of the present invention.

FIG. 2 is an explanatory diagram relating to a water flow ejected in the tangential direction of the inner wall of the culture tank of the device of the present invention.

FIG. 3 is an explanatory diagram of a culture device in which a carrier having a higher specific gravity than water is mixed with the culture solution and circulated in the culture solution.

FIG. 4 is an explanatory diagram of a culture device in which a sponge-like carrier having a size that does not enter the circulation device in the culture medium is mixed with the culture medium and circulated.

FIG. 5 is an explanatory diagram of a culture device used in Example 1.

FIG. 6 is an explanatory diagram of a culture device used in Comparative Example 1.

[Explanation of symbols]

1 Culture Tank 2 Suction Port 3 Liquid Circulation Device 4 Vibration Pump 5 Jet 6 Gas System Circulation Device 7 Culture Gas Supply Pipe 8 Medium Supply Pipe 9 Culture Liquid Outflow Pipe 10 Gas Discharge Pipe 11 Carrier 12 Suction Port with Screen 13 Liquid Flow arrow 14 solenoid valve 15 Comparative example gas discharge pipe Q _L broth circulation flow rate Q _G incubated gas circulation flow rate of the culture gas supply pipe 20 Comparative example diffusing pipe 19 Comparative example fermenter 16 magnetic stirrer 17 stirrer 18 showing the

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yu Umeda             4-2-1 Honfujisawa, Fujisawa City, Kanagawa Prefecture             Inside the EBARA Research Institute (72) Inventor Yasunori Toya             4-2-1 Honfujisawa, Fujisawa City, Kanagawa Prefecture             Inside the EBARA Research Institute (72) Inventor Tadashi Adachi             4-2-1 Honfujisawa, Fujisawa City, Kanagawa Prefecture             Inside the EBARA Research Institute (72) Inventor Etsuko Takizawa             4-2-1 Honfujisawa, Fujisawa City, Kanagawa Prefecture             Inside the EBARA Research Institute

Claims (3)

[Claims] 1. A culture device comprising a circulation device having at least a culture tank and a culture solution system and a culture gas system,
A vibration pump is installed in at least the culture liquid circulation device, and the culture liquid and the culture gas in the culture tank are jetted from the wall surface of the lower portion of the culture tank by the vibration pump as a gas-liquid mixed fluid to culture in the culture tank. The culture solution is drawn out from the center of the vortex above the liquid and is again introduced into the culture tank at a high speed as a gas-liquid mixed fluid together with the circulating gas, so that the culture solution in the culture tank and the culture gas can be swirled. A culture device comprising a circulation device with a system. 2. The culture medium in the culture tank is mixed with a carrier having a specific gravity heavier than water, and is circulated together with the culture solution in the circulation device to remove bacterial cells and the like adhering to the inner wall of the culture tank. The culture device according to claim 1. 3. A bacterial cell or the like that is circulated in the culture tank by admixing a sponge-like carrier having a size that does not fit in the circulation device into the culture solution in the culture tank and adhering to the inner wall of the culture tank. The culture device according to claim 1, wherein the culture device is removed.