JP2661848B2 - Culture method and culture device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved method for culturing microorganisms or cells and a culturing apparatus used for the method. More specifically, the present invention provides a method for efficiently recovering a liquid containing a product through a microbial or cell culture solution without causing clogging of the porous separation membrane from the culture solution while performing the culture, and The present invention relates to a culture apparatus having a function for preventing clogging of a porous separation membrane used for this method and a function for obtaining high aeration efficiency.

[0002]

2. Description of the Related Art An incubator or a bioreactor used for culturing microorganisms or cells is usually provided with a filtration / separation device for culturing microorganisms or cells at a high density or obtaining a desired product. . However, in a conventional incubator or bioreactor equipped with such a filtration / separation apparatus, the culturing step and the separation / purification step are independent of each other, and the accompanying equipment required for the filtration / recovery step of the culture solution is required. Therefore, there are problems that the equipment cost is high and the filtration / recovery operation is complicated.

Further, a porous separation membrane is generally used for the filtration / separation apparatus. In this case, however, a decrease in filtration flow rate and filtration efficiency due to clogging of the membrane is a serious problem. Conventionally, in incubators and bioreactors equipped with filtration / separation devices, in order to address this problem, after suspending or terminating the cultivation after a certain period of use, backwashing or chemical treatment is used. Such a method is extremely disadvantageous in efficiency.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves such problems in the prior art, and allows a porous separation membrane to pass through a culture solution of microorganisms or cells without causing clogging while culturing. An object of the present invention is to provide a method for efficiently collecting a liquid containing a product, and a culture apparatus used in the method.

[0005]

Means for Solving the Problems The present inventors have developed a method for efficiently separating and recovering a culture product while preventing clogging by using a porous separation membrane from the culture solution without interrupting the culture. As a result of diligent research to achieve this, a plurality of porous membrane cell tubes, which are connected to a filtrate extraction tube and an air supply tube that can be switched by a switching valve and are used for both air supply and filtrate extraction, are used regularly. By switching, by removing the clogging substance deposited in the capillary at the time of removing the filtrate by aerating from the opposite side of the porous separation membrane that is in contact with the culture solution during the air supply, They have found that the object can be achieved, and have completed the present invention based on this finding.

That is, the present invention relates to a method for culturing a microorganism or cell culture while aerating the culture medium and taking out the product liquid. By periodically switching between the two by using the liquid extraction means, air supply and production liquid extraction are performed without interrupting the culture, and the clogging substances deposited in the pores of the porous separation membrane are removed. A culture method and an incubator or a bioreactor to be disposed therein, and a plurality of porous membrane thin tubes combined with a plurality of air supply and filtrate removals,
A culturing system comprising a nutrient gas supply tube and an air supply tube connected to the porous membrane thin tube and connected to the air supply tube via a control valve and a sterilization filter, and a filtrate removal tube and an air supply tube which are mutually switched by a switching valve. An apparatus is provided.

There is no particular limitation on the porous separation membrane used in the present invention, and a porous ceramic membrane having a function of filtering and separating a culture solution or a reaction product from microorganisms or cells can be used.
There are a porous glass film, an organic polymer film, a metal nonwoven fabric, and the like, and a tube, a hollow fiber, a spiral, or the like can be used as long as the film can be ventilated. Among these membranes, tubular ceramic membranes are relatively suitable. The microorganisms and cells are not particularly limited, and include yeast, bacteria, fungi, actinomycetes, animal cells, plant cells, and the like.

[0008] These porous separation membranes are disposed in a conventional incubator or bioreactor conventionally used for culturing microorganisms or cells, and a liquid containing the product separated by the porous separation membranes. Flows out of the incubator by a suction method or the like and is collected.

A vent outlet is connected to the filtrate outlet of the porous separation membrane, and when the filtration efficiency is reduced, the filtration operation is performed in the opposite direction to the filtration operation, that is, in contact with the culture solution of the porous separation membrane. By injecting a clean gas that has passed through a sterilization filter from the opposite side, the clogging substance deposited in the pores of the membrane is removed, thereby ensuring the same filtration efficiency as in the initial operation. And a high filtration flow rate can be maintained for a long time.

[0010] Further, the clean gas used for the operation of removing the substance causing clogging of the porous separation membrane becomes micro-dispersed gas bubbles after permeation through the membrane, which increases the contact area with the culture and increases the gas content in the culture solution. It has the effect of increasing the dispersion and dissolution efficiency. As a result, the efficiency of aeration to microorganisms and cells is increased, and the culture efficiency is increased. It is desirable to supply nutrients necessary for cultivation in gaseous form, and select air, oxygen, carbon dioxide, etc., depending on the type of microorganisms and cells, and adjust the gas composition ratio. Thus, both aerobic culture and anaerobic culture can be performed.

The porous separation membrane disposed in the incubator or bioreactor is used for both aeration and filtration operations.
A hollow-tube type porous membrane thin tube is most suitable. By performing a filtration operation when the amount of the culture solution is increased and performing an aeration operation during other periods, clogging can be intermittently eliminated and high filtration efficiency can be maintained for a long period of time. Further, when two or more sets of separate and independent porous membrane thin tubes are provided, by alternately performing the operation of switching between the aeration operation and the filtration operation,
This is advantageous because the continuous aeration operation and the continuous filtration operation can be continued.

[0012] Further, using a water level meter or a liquid level sensor in the incubator or the bioreactor, the filtration operation is performed only when the amount of the culture solution inside becomes excessive, and the filtration is stopped or used for the aeration operation in other periods. By doing so, unnecessary filtration operations can be prevented, and the causes of clogging can be reduced. At the same time, in the filtration culture process, it is possible to always maintain the culture solution volume almost constant, so it becomes easy to estimate the bacterial mass and nutrient status inside the incubator,
A long-term continuous operation can be efficiently performed.

Next, the culture apparatus of the present invention used in such a culture method will be described with reference to the accompanying drawings.
FIG. 1 is a schematic view of an example of the culture apparatus of the present invention, in which two porous membrane thin tubes 4 are disposed in one of the incubators having a constant temperature water jacket 2 for temperature control and a stirring blade 3. Have been.
The porous membrane thin tube is connected to a filtrate extraction pipe 6 via a switching valve 5 and to an air supply pipe 8 via a switching valve 7. Air pipe 8
Is connected to a nutrient gas supply pipe 10 and an air supply pipe 11 via a sterilization filter 9.

The incubator 1 is provided with a DO sensor, a pH sensor, a carbon dioxide meter, a temperature sensor (all not shown), and a liquid level sensor 12. The filtrate is taken out from the pump (not shown) in conjunction with the liquid level sensor 12.
As a result, the liquid flows out of the incubator 1 through the control valve 13, and the liquid volume in the incubator is always kept constant. A substrate supply pump (not shown) works in conjunction with a pH sensor, a DO sensor, and a carbon dioxide meter, and supplies a substrate by a pH stat, DO stat, CO ₂ stat method, or the like when a nutrient source in the incubator is insufficient.

The mixing ratio of air, oxygen, carbon dioxide, and nitrogen supplied to the incubator is adjusted by a flow rate control valve 14, passes through an air supply pipe 8 through a sterilization filter 9, and forms two porous membranes. The liquid is supplied to the thin tube 4 alternately. The switching of the ventilation path is performed by receiving information from the liquid level sensor 12 and using the timer 15.
Can be implemented. In parallel with this, switching of the filtrate extraction path is performed.

FIG. 2 is a graph showing an example of the relationship between the culture time and the filtration flow rate in the case of the filtration / aeration switching operation and the case of only the filtration operation without aeration using the above-mentioned culturing apparatus. In FIG. 2, the circles indicate the case where the ceramic capillary membrane A is used.
The triangles indicate the case where the ceramic capillary membrane B was used. When the filtration / ventilation switching operation is performed, the filtration flow rate decreases slightly with the passage of time. However, when only the filtration operation is performed, the filtration flow rate rapidly decreases with the passage of time.

[0017]

According to the present invention, separation of waste products or useful products in a culture solution is continuously performed at a high filtration flow rate for a long period of time while microorganisms and cells are kept in an incubator or a bioreactor. be able to. Therefore, the apparatus of the present invention that solves the problem of clogging, which is the biggest problem in the conventional membrane separation process, has a great effect on industrial development.

[0018]

Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Example 1 Using the culture apparatus shown in FIG. 1, the experiment was carried out by the method described above. The capacity of the incubator is 1 liter, and the stirring blade 3 rotates in the range of 0 to 800 rpm by an upper stirring drive system. The two porous membrane thin tubes are made of ceramic having an average pore diameter of 5 μm, and have an inner diameter of 7 mm and an outer diameter of 10 mm. Using this incubator, baker's yeast was cultured with aeration and stirring.

A baker's yeast was inoculated into a medium containing 50 g / liter of glucose, 1 g / liter of yeast extract, and 2 g / liter of polypeptone, and cultured under aeration and stirring at 30 ° C. and pH 5.0. CO _{2 in} the incubator when glucose is insufficient
A substrate liquid containing 300 g / liter of glucose was supplied by automatic supply of a pump by a CO ₂ stat method utilizing a phenomenon of a decrease in the amount of generated gas.

When the switching time of the aeration and filtration paths was set every 6 hours, the cell concentration was 60 g by the third day of the culture.
Per liter. At this time, the filtration flow rate was maintained at 80% of the initial flow rate. No residual glucose was detected in the filtrate, and the yeast cell separation rate was 98%.
And was good.

Example 2 In the culture apparatus of Example 1, the time for switching the aeration and filtration paths was set every hour, and filtration culture was performed.
The filtration flow was maintained at about 95% of the initial flow. A remarkable effect of preventing clogging by ventilation was observed.

Comparative Example 1 In the culture apparatus of Example 1, filtration culture was performed without switching the aeration and filtration paths. On the third day of culture, the filtration flow rate of the ceramic membrane tube used for filtration was lower than the initial flow rate. It dropped to about 25%.

Comparative Example 2 In the culture apparatus of Example 1, when air was directly aerated without passing through a porous ceramic tube in the aeration path, the oxygen consumption efficiency was reduced, and the yield of bacterial cells with respect to the consumed glucose was determined in Example 1. It was only 95% of the case of 1.

Reference Example In Example 2, except that the filtrate was removed in conjunction with the substrate supply pump without using the liquid level sensor, the same procedure as in Example 2 was carried out. The liquid volume was reduced to 80% of the initial liquid volume, and the cell growth rate was similarly reduced.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of one example of a culture device of the present invention.

FIG. 2 is a graph showing an example of the effect of preventing clogging of a porous separation membrane according to the method of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Incubator 2 Temperature controlled water jacket for temperature control 3 Stirrer blade 4 Porous membrane thin tube 5, 7 Switching valve 6 Filtrate extraction tube 8 Air supply tube 9 Bactericidal filter 10 Nutrient source gas supply tube 11 Air supply tube 12 Liquid level sensor 13 , 14 control valve

 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-62-244413 (JP, A) JP-A-62-23412 (JP, A) JP-A-4-74526 (JP, U) JP-A-4-234 87800 (JP, U) Actually open 1987-87720 (JP, U)

Claims (3)

(57) [Claims] 1. A method for aerating a culture solution of microorganisms or cells and culturing while taking out a product solution, wherein the air supply means and the product solution take-out means which are in contact with the culture solution via a porous separation membrane, respectively. A culturing method characterized in that by periodically switching between the two, air is supplied and product liquid is taken out without interrupting the culturing, and a clogging substance deposited in the pores of the porous separation membrane is removed. . 2. An incubator or bioreactor, a plurality of porous membrane capillaries provided therein for both air supply and filtrate removal, and filters connected to the porous membrane capillaries and switched by a switching valve. A culture device comprising a nutrient gas supply pipe and an air supply pipe connected to an air supply pipe via a liquid extraction pipe and an air supply pipe, a control valve, and a sterilization filter. 3. The culturing apparatus according to claim 2, further comprising a water level gauge, wherein the culture operation is started and stopped while monitoring the liquid level.