JP3122050B2 - Culture and reaction method and apparatus for microorganisms, cells or immobilized enzymes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an improvement in a method for culturing or reacting microorganisms, cells or immobilized enzymes (hereinafter referred to as "microorganisms or the like"), and a culturing apparatus or reaction apparatus (hereinafter referred to as ""Bioreactor"). More specifically, the present invention provides a method for efficiently recovering a product or a reaction solution through a porous separation membrane without causing clogging of the porous separation membrane by the culture solution or the reaction solution when culturing or reacting a microorganism or the like. And a bioreactor having a function of preventing clogging of a porous separation membrane used in the method.

[0002]

2. Description of the Related Art Bioreactors used for culturing or reacting microorganisms, cells, or immobilized enzymes usually include "filtration separation" for culturing microorganisms or cells at a high density or separating and recovering a reaction solution. A device is attached. However, in a conventional bioreactor provided with such a filtration / separation device, the culturing or reaction step and the separation step are independent of each other, so that separation equipment costs are high and filtration / recovery operations are complicated. There was such a problem.

[0003] Conventional bioreactors in which a filtration separation membrane is disposed inside a bioreactor usually have a porous separation membrane from the side opposite to the side in contact with the culture solution in order to prevent clogging. Complicated equipment for intermittent ventilation and water supply is required, which causes a problem that equipment cost for separation is high.

Further, conventional bioreactors used for culturing or reacting microorganisms, cells or immobilized enzymes at a high density include a filtration and separation apparatus for recovering a reaction solution.
Stirring device for culture solution or reaction solution, temperature sensor, pH
Since complicated equipment for disposing electrodes, liquid level sensors, and the like was required, the apparatus was large-scale and expensive. Therefore, such a conventional bioreactor is extremely inconvenient for culturing or reacting a large number of microorganisms, cells, or immobilized enzymes in parallel at a high density, and separating and recovering the culture solution or the reaction solution by filtration. .

[0005]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems in the prior art, and it has been proposed that a culture solution or a reaction solution of a microorganism, a cell, or a reaction solution of an immobilized enzyme be more porous while being cultured or reacted. It is an object of the present invention to provide a method for efficiently collecting a reaction solution through a separation membrane without causing clogging of the separation membrane. Further, the present invention provides a simple device which is inexpensive, does not require complicated operations, and which is small in size and can achieve a reaction efficiency comparable to that of a conventional large-scale and complicated incubator or bioreactor. The purpose is to:

[0006]

According to the present invention, in order to achieve the above object, in the method for culturing and reacting a microorganism, a cell or an immobilized enzyme according to claim 1, a porous membrane thin tube is disposed inside. A microorganism or a cell, wherein the reaction solution is recovered by shaking the bioreactor to remove clogging substances deposited in pores of the membrane in contact with the culture solution. Alternatively, a method for culturing and reacting the immobilized enzyme is provided.

According to a second aspect of the present invention, there is provided an apparatus for culturing and reacting a microorganism, a cell or an immobilized enzyme, wherein the porous membrane tubule for culturing a microorganism, a cell or an immobilized enzyme is obtained from the porous membrane tubule. Shaking is provided with a filtrate taking-out pipe connected to the porous membrane thin tube for taking out the filtrate to the outside of the container, and a water sending pipe connected to the porous membrane thin tube for sending water to the porous membrane thin tube. The present invention provides a bioreactor characterized in that a reaction solution is recovered while removing a clogging substance deposited in pores on the side of the porous membrane tubule in contact with the culture solution.

According to a third aspect of the present invention, in the method of culturing and reacting a microorganism, a cell or an immobilized enzyme, the bioreactor according to the second aspect is shaken to allow the filtrate to penetrate into the pores of the porous membrane, or water or an aqueous solution. And a method for culturing and reacting, which promotes the penetration of a microorganism.

According to a fourth aspect of the present invention, in the method for culturing and reacting a microorganism, a cell, or an immobilized enzyme, the filtrate from the side of the porous membrane provided in the bioreactor from the side in contact with the culture solution of the porous membrane tubule. The culture and reaction method is characterized in that continuous filtration is performed while maintaining the level of the culture or reaction solution at the initial level by transferring the filtrate to the outside of the bioreactor by sucking in and shaking. provide.

[0010]

According to the first aspect of the present invention, a liquid containing a product is recovered from a reaction solution of a microorganism, a cell, or an immobilized enzyme through a porous separation membrane thin tube while culturing or reacting. Then, by shaking the bioreactor in which the porous membrane tubule is disposed, the membrane surface on the side of the porous membrane tubule in contact with the culture solution or the reaction solution is shaken, so that the eye is deposited in the tubule. The clogging substance is removed.

[0011] These porous separation membranes are disposed in a conventional shaking incubator or shaking bioreactor used for culturing microorganisms or cells or reacting with immobilized enzymes. Liquid containing filtered product (filtrate)
Is taken out of the bioreactor through a take-out tube and collected by gravity or suction.
When the filtrate is dropped and collected by gravity, it is only necessary to attach a drain pipe for the filtrate, which is a cheaper and simpler method.

When the reaction solution scattered by shaking comes into contact with the porous membrane, only the filtrate is sucked from the reaction solution by capillary action, and further, the filtrate in the thin capillary of the porous membrane moves out of the bioreactor by shaking. Auxiliary propulsion, such as suction or compression, found in conventional filtration operations may be eliminated.

Therefore, the water level inside the bioreactor can be maintained almost constant by the installation position of the porous membrane tubing, so that the filtrate can be obtained in proportion to the feed rate of the raw material liquid, and the operation of the continuous reaction is extremely easy. Becomes

The porous separation membrane capillary disperses microorganisms, cells, or immobilized enzymes in a culture solution or reaction solution by shaking a bioreactor provided with the membrane, and forms a mixture with the culture solution of the membrane. The clogging substance deposited in the contacting pores can be removed by washing with the reaction solution. As a result, the same filtration efficiency as in the initial stage of operation can be secured, and a high filtration flow rate can be maintained for a long period of time.

Also, by intermittently supplying water from the side of the porous separation membrane tubule opposite to the side in contact with the culture solution (inside of the porous tubule membrane), the clogging substance attached to the pores is washed away. And can be drained. There are two ways to connect the water pipe: one to connect directly to the porous separation membrane, and the other to connect to the filtrate outflow pipe. Thereby, the filtration operation and the water supply can be continuously repeated.

The liquid intermittently supplied from the inside of the porous capillary membrane is not limited to water, but may be a part of a filtrate, a solution containing a nutrient of microorganisms or cells, or a raw material for the reaction. And the like can be supplied.

By shaking the bioreactor,
Since the penetration of the filtrate into the inside of the porous capillary membrane can be promoted, a high filtration flow rate can be obtained by shaking.

Further, by shaking the bioreactor, penetration of the liquid intermittently fed from the inside of the porous membrane thin tube is promoted, so that the same filtration efficiency as in the initial stage of operation can be secured for a long period of time. Thus, the filtration operation can be continuously performed at a high filtration flow rate.

[0019] Further, since the high reaction rate can be maintained while maintaining the liquid volume in the bioreactor almost constant by the installation position of the porous membrane tubule, no complicated operation or equipment is required. It is easy to simplify the device.

Since the bioreactor can maintain a high filtration flow rate for a long time, the exchange rate of the culture solution or the reaction solution can be increased. Thereby, the reaction rate of the microorganism, cell or immobilized enzyme can be kept high for a long time.

Further, by shaking the bioreactor, the effect of stirring and mixing the culture solution or the reaction solution is enhanced. Thereby, the reaction rate of the microorganism, cell or immobilized enzyme can be kept high.

Further, by continuously supplying a fresh culture solution or a raw material solution into the bioreactor, the pH of the culture solution or the reaction solution, the nutrient source, and the concentration of the raw material are constantly maintained optimally, and complicated Operation and a measurement device are not required, and therefore, miniaturization of the reaction device is remarkably facilitated, and a large-scale reaction in a small bioreactor can be continuously performed.

[0023]

Next, the bioreactor of the present invention used in such a culture method or reaction method will be described with reference to the drawings. FIG. 1 is a schematic view of an incubator of the present invention, in which a single porous membrane tube 2 is disposed in a shaking incubator 1 made of a glass flask. This porous membrane thin tube is connected to a filtrate discharge tube 3 and a water supply tube 4. The filtrate taking-out tube 3 is connected to a liquid sending pump 5, and by driving this pump, the culture filtrate separated through the porous membrane thin tube 2 is taken out of the incubator. The liquid sending pump 5 is not always necessary, and can separate the filtrate by natural fall. In addition, the water supply pipe 4 is connected to the liquid supply pump 6 to supply water or an aqueous solution containing a part of the filtrate or nutrient components from the inside of the porous membrane thin tube 2 to the outside culture medium, thereby causing clogging of the membrane. Facilitates separation of material from tubules.

The incubator 1 is constantly shaken by the agitating shaker 9, which promotes the diffusion of nutrient components into the culture solution. A vent plug 7 is attached to the top of the incubator 1 to promote oxygen supply to the inside of the incubator and gas exchange by releasing fermentation gas from the inside of the incubator to the outside. Further, a multi-purpose port 8 is provided in the incubator to supply a nutrient source and take out a sample for analysis of a culture solution, or used as a mounting port for a temperature sensor or a pH sensor.

It is desirable that the porous membrane tubule 2 inside the incubator 1 is disposed above the surface of the culture solution in the stationary state and at a height soaked in the culture solution that swings when the incubator is shaken. Thus, even when the microbial cells or cells become dense, the cells or cells are prevented from aggregating and settling, and the flowing culture solution is clogged from the surface of the porous membrane tubule 2 which comes into contact with the culture solution. The causative substances are washed away, and clogging of the membrane is prevented.

FIG. 2 is a graph showing an example of the relationship between the culturing time and the filtration flow rate when the above-mentioned incubator is used for the shaking / filtration operation and when only the filtration is performed without shaking. Data are shown for two cases: when a shaking / filtration operation is performed, when water is fed halfway (when backwashing is performed), and when water is continuously filtered (when backwashing is not performed). When the filtration operation is performed without shaking, the filtration flow rate decreases rapidly with time, but when the shaking / filtration operation is performed, the filtration flow rate decreases slightly. Further, when water is supplied from the inside of the porous membrane thin tube and backwashed on the way, a further decrease in filtration flow rate is prevented.

[0027]

Example 1 A description will be given of cultivation of yeast by a glass incubator provided with a porous ceramic thin tube. In this example, the incubator (bioreactor) shown in FIG. 1 was used. In FIG. 1, reference numeral 2 denotes a ceramic porous tube having an average pore diameter of 0.5 μm, which has an inner diameter of 8 mm and an outer diameter of 11 mm. This porous membrane thin tube is connected to a filtrate discharge tube 3 and a water supply tube 4. The capacity of the incubator is 500 ml, and the stirring and shaking table 9 is rotated or reciprocated in the range of 0 to 200 rpm.

Glucose 50 g / l, yeast extract 5 g /
1, 200 ml of medium containing 5 g / l of polypeptone,
Inoculated with baker's yeast from the vent opening 7 of FIG.
At 5, the cells were reciprocally shake-cultured at 120 rpm. After culturing for one day (24 hours), glucose 100 g /
1, a substrate solution containing 10 g / l of yeast extract and 10 g / l of polypeptone was supplied by a liquid sending pump 10.

When the culture filtrate was separated from the porous membrane tubule 2 simultaneously with the inflow of the substrate solution, the bacterial cell concentration could be increased to 170 g / l by the sixth day of culture. The filtration flow rate was maintained at 90% of the initial filtration capacity, no residual glucose was detected in the filtrate, and the yeast cell separation rate was 99%.
And was good.

[0030]

Example 2 In the bioreactor of Example 1, the supply of filtration and backwash water was switched using a timer. The washing water is supplied to the water pipe 4 for 1 minute every 10 minutes of the filtration.
, The concentration of the bacterial cells was increased to 200 g / l by the sixth day of the culture. The filtration flow rate was maintained at 96% of the initial filtration capacity, and a remarkable effect of preventing clogging by the washing water was observed.

(Comparative Example 1) In the culture apparatus of Example 1, filtration cultivation was performed without stirring and shaking. By the 6th day of cultivation, the bacterial cell concentration was limited to 30 g / l, and the filtration flow rate was low. Reduced to 25% of filtration capacity.

(Comparative Example 2) In the culturing apparatus of Example 1, culturing was performed by adding a substrate solution without performing filtration. By the sixth day of the culturing, the bacterial cell concentration was only 16 g / l.

(Comparative Example 3) In the culturing apparatus of Example 1, culturing was carried out without supplying and filtering the substrate solution. By the 6th day of the culturing, the bacterial cell concentration was only 7 g / l.

(Reference Example) In the culture apparatus of Example 1,
When culturing was carried out using an incubator in which the porous membrane tubule 2 was disposed at a position 5 mm from the bottom of the incubator 1, bacterial aggregates settled in the vicinity of the porous membrane tubule, and cultivation was performed for 6 days. By the eyes, the cell concentration remained at 145 g / l, and the filtration flow rate was reduced to 75% of the initial filtration capacity.

There is no particular limitation on the porous membrane tubule used in the present invention, and a porous ceramic membrane or a porous glass membrane having a function of filtering and separating a culture solution or a reaction solution from microorganisms, cells or immobilized enzymes. , 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 it can be disposed inside the incubator or the bioreactor. Among these membranes, tubular ceramic membranes are relatively suitable. In addition, there is no particular limitation on the microorganisms and cells to be cultured or reacted, yeast, bacteria,
Molds, actinomycetes, protists, animal cells, plant cells, and the like. The immobilized enzyme and the immobilization method are not particularly limited, and examples thereof include glucose isomerase immobilized on a calcium alginate carrier, and protease immobilized on porous glass.

[0036]

As described above, according to the method of the present invention, a culture solution or a reaction solution is mixed with a microorganism, a cell, or an immobilized enzyme in a culture vessel or a bioreactor,
Separation from cells or immobilized enzymes can be performed efficiently and continuously for a long period of time. Further, microorganisms or cells in the culture solution can be increased to an extremely high density. In addition, this can increase the reaction rate and continuously produce the product with high efficiency.

The apparatus of the present invention has a remarkable effect on miniaturization and simplification of the bioreactor. That is, the complicated operation used in the conventional bioreactor is not required, the aqueous solution as the reaction raw material is continuously supplied, and the filtrate is separated through a porous membrane thin tube to increase the microorganisms and cells to a high density. As a result, the same reaction efficiency as when a large bioreactor is used is obtained. Such a bioreactor is not only very effective as a small high-speed wastewater treatment device, but also simultaneously cultivates microorganisms and cells having many different properties using a large number of small incubators,
It is possible to provide an inexpensive culture technique for obtaining a large amount of different products in a short period of time.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the bioreactor of the present invention.

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

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Glass shaking incubator 2 Porous membrane thin tube 3 Filtrate discharge pipe 4 Water supply pipe 5 Filtrate discharge liquid feed pump 6 Washing water liquid feed pump 7 Vent plug 8 Multipurpose port 9 Stirrer shaker table 10 Substrate liquid liquid feed pump 11 Substrate aqueous solution Storage bottle 12 Filtrate storage bottle 13 Washing water storage bottle 14 Rubber stopper

Claims (4)

(57) [Claims] 1. A method for culturing and reacting a microorganism, a cell or an immobilized enzyme, the method comprising shaking a bioreactor in which a porous membrane capillary is disposed to form a membrane on the side in contact with the culture solution of the membrane. A method for culturing and reacting microorganisms, cells or immobilized enzymes, wherein a reaction solution is recovered while removing a clogging substance deposited in pores. 2. A device for culturing and reacting microorganisms, cells or immobilized enzymes, comprising a porous membrane thin tube for culturing microorganisms, cells or immobilized enzyme, and a filtrate obtained from the porous membrane thin tube. A filtrate take-out tube connected to the porous membrane thin tube for taking out to the outside; and a water supply tube connected to the porous membrane thin tube for sending water to the porous membrane thin tube. A bioreactor characterized in that a reaction solution is recovered while removing a clogging substance deposited in pores on a side of a membrane of a membrane in contact with a culture solution. 3. A method for culturing and reacting a microorganism, a cell or an immobilized enzyme, wherein the bioreactor according to claim 2 is shaken to infiltrate a filtrate into a porous thin tubule, or to remove water or an aqueous solution. A culture and reaction method characterized by promoting penetration. 4. A method for culturing and reacting a microorganism, a cell, or an immobilized enzyme, wherein a filtrate is sucked from a porous membrane thin tube provided in a bioreactor from a side of the porous membrane thin tube in contact with a culture solution. A culturing and reaction method, wherein a continuous filtration is carried out while moving the filtrate to the outside of the bioreactor by shaking to maintain the level of the culture solution or the reaction solution at the initial level.