JP2984748B2 - Method for culturing microorganisms using air-permeable membrane - Google Patents

Abstract

PURPOSE:To continuously and efficiently obtain a highly active enzyme liquid and a high concentration fungi liquid by culturing a microorganism using an air-transmissible membrane. CONSTITUTION:A liquid culture medium containing a microorganism other than mushrooms is fed through a pump 2 to a microorganism culturing system 3 in which an air-transmissible membrane such as a cloth or a sheet consisting of silicone and waterproofing-finished fiber is wound and then fed through a concentration-measuring equipment 3 and pump 4 to a culture liquid- exchanging and concentrating system 5. The microorganism is recovered and then the culture medium is circulated to liquid storage tank 6.

Description

The present invention relates to a method for culturing microorganisms using a gas permeable membrane,
More specifically, the present invention relates to a method for culturing microorganisms, which comprises continuously culturing microorganisms while supplying oxygen from a gas permeable membrane made of silicon. In the present invention, the term "microorganism" refers to a microorganism other than mushrooms and algae, and includes bacteria, yeasts, molds, actinomycetes, and the like.

[Problems to be Solved by the Prior Art and the Invention] Speaking of the microorganism culturing method, it is usually a solid culture using a bran, a soybean, or the like, and a liquid culture using a flask or a jar fermenter. In particular, liquid culture is widely used industrially because large-scale culture is possible, and there is also a continuous culture system in which sterilized air is blown and a sterilized medium is continuously supplied. However, these methods require air blowing and stirring for oxygen supply, and it is not easy to remove the culture solution.

Generally, when thermophilic bacteria are cultured at a high temperature, the growth of the bacteria is poor, the productivity of enzymes and the like is low, and it is difficult to put them to practical use. Therefore, in the conventional culture of thermophilic fungi, the enzyme agent produced is expensive, and at present it is almost impossible to apply it to the food industry.

[Means for Solving the Problems] The present inventors have tried to reduce the production cost of thermophilic enzyme-producing bacteria, and furthermore, the production cost of thermophilic enzymes by increasing the efficiency and continuity of cultivation. The present inventors have found that microorganisms can be continuously produced efficiently by using a gas permeable membrane, and have completed the present invention.

Although various air-permeable membranes can be used in the method of the present invention, those which can be autoclaved by passing oxygen or carbon dioxide gas without passing water (which may or may not pass water vapor) and having high mechanical strength can be used. Is desirable.

Oxygen permeability of polyethylene is 25 ℃ even at 30μm thickness
, The maximum is 6,000 cc / m ² · day · atm, and the carbon dioxide permeability is 3 to 4 times this value. However, the permeability of recently developed silicon is 365,0 with oxygen even at a thickness of 100 μm.
^{00cc / m 2 · day · atm} , 1,165,000cc / m 2 · day · in carbon dioxide
Atm also reaches. Therefore, the present inventors have studied diligently about the microorganism cultivation with the present material and various gas permeable membranes, and by using the gas permeable membrane, the microorganism cultivation is more efficient than in the past, and the highly active enzyme solution and the high concentration bacterial solution are continuously obtained. Thus, the present invention has been completed.

 The present invention is described below.

The present invention relates to a method for culturing microorganisms using a tubular silicon gas permeable membrane. Specifically, (1) a microorganism characterized by culturing microorganisms (excluding mushrooms and algae) by continuously supplying an inoculation medium and / or a liquid medium into a tube of a tubular silicon gas-permeable membrane ( Culture method (excluding mushrooms and algae).

(2) A method for culturing microorganisms (excluding mushrooms and algae), which comprises using a tubular silicon gas-permeable membrane fixed to the outer surface of a substrate.

(3) A method for culturing microorganisms (excluding mushrooms and algae), which comprises culturing the culture solution while concentrating the culture solution in a tube made of a tubular silicon gas-permeable membrane.

(4) A method for culturing microorganisms (excluding mushrooms and algae), wherein the culture solution after completion of the culturing is passed through a tube of a gas-permeable silicon membrane and concentrated.

Among the breathable membranes, silicon is currently the most suitable material for the method of the present invention, and the breathability can be changed to a desired degree depending on the processing method, and the mechanical strength is high. Cheap. Further, it is a very useful material that can be processed into a sheet shape, a tubular shape, and other various shapes. Recently, this material has been processed into a thin tube and used for an artificial lung, which utilizes the breathability of the material.
The method of the present invention uses this property for culturing microorganisms. There is no example of using this material for culturing microorganisms, and there is no example of successful culturing. Further, in the method of the present invention, the material is not limited to silicon, but any material having excellent air permeability, such as a material obtained by waterproofing the surface of cellulose, ceramics, or the like, can be used.

Since the silicon material has a hydrophobic surface, it can easily pass through the culture medium, and can pass through even high-concentration culture medium.This culture system has a remarkably excellent air permeability. pass.

The shape of silicon is not particularly limited as long as it is suitable for culturing microorganisms, but a tubular shape is convenient.

The size of the tube is appropriately selected depending on the thickness of the tube. For example, a tube diameter of 3.0 × 3.2 mm for a thickness of 100 μm and a size of 3.6 × 4.0 mm for a thickness of 200 μm are appropriate. 300 × 3 for flat membrane
Using a 00 mm square, the bottom is reinforced with a mesh.

When a 200 μm tube is used, as shown in FIG. 1, the tube is wound around a cylinder on the outer surface of the substrate, or placed horizontally and / or vertically. When the pipes are arranged on a flat plate, a space can be provided little by little in consideration of the efficiency of ventilation, and the pipes can be stacked in ten or more stages. Basically, the culture solution is circulated or passed through a long tube provided in such a manner.
It is convenient to attach a storage tank that can keep the liquid level constant to the auxiliary medium tank. The storage tank is preferably set so that the amount of liquid in the system is kept constant and a fresh medium can always be supplied.

In addition, in the method of the present invention, a long tube of 20 m or more is used, or under a dry condition, the culture solution is extremely concentrated, and it becomes difficult to pass the solution, or the growth rate of bacteria may be reduced. In addition, a fresh medium (medium concentration is arbitrarily selected) supply device provided with a backflow stopper may be provided.

Furthermore, depending on the type of microorganism and the culturing conditions, the growth inhibitory substance may accumulate over the culturing time and the growth rate may decrease. In such a case, an ordinary filtration material and a dialysis membrane that allow other components to pass therethrough without passing through an ultrafiltration membrane or a bacterium to an appropriate portion of the system are set, and continuous culture is performed while removing growth inhibitors. According to this method, various substances produced by microorganisms can be continuously taken out.

In the main culture method using a silicon tube, the length and flow rate of the tube need only be adjusted and set.

The concentration of the initial bacterial solution and the ratio of the amount of the cultured bacterial solution to be withdrawn may be arbitrarily selected depending on the culture rate and other conditions. Usually, 2 to 10% of the inoculum solution is added to a fresh medium, and the starting solution is prepared. For example, the culture is completed in two days.

A part of the culture termination solution may be returned to the starting tank as the next inoculum, a fresh medium may be added, and the culture may be continued.

If the flow rate and the length of the tube are adjusted so that the culture is completed at the end of the tube, it is sufficient to simply pass through the tube. Withdraw 90% of the culture, add fresh medium and continue culturing.

The concentration of the culture solution is measured by a spectrophotometer, and when the absorbance reaches a certain level or more, the medium can be automatically withdrawn and set to add a fresh medium.

As described above, the type of microorganism may be other than mushrooms and algae, and can be efficiently applied to, for example, thermophilic bacteria of the genus Thermus and Bacillus, such as stearothermophilus. Pseudomonas, lactic acid bacteria, Streptomyces, photosynthetic bacteria, and many other types, and the method of the present invention can be applied to any of them. Further, two or more of these can be mixed and cultured. Furthermore, the method of the present invention can be applied to cell and / or tissue culture of animals and plants.

EXAMPLES Next, the present invention will be described in more detail and specifically with reference to Examples, but it should not be construed that the invention is limited thereto.

Example 1 A thermophilic bacterium belonging to the genus Bacillus KF9-10 (FERM P-8718) which produces cyclodextrin synthase was used as a strain.
Using bran, ammonium sulfate, charcoal medium as a medium, 65 ° C,
The cells were cultured at a flow rate of 4 ml / min. The system has a shape as shown in Fig. 1, a silicon tube 10m with a thickness of 200μm and a diameter of 3.6 x 4.0mm.
Set on an acrylic plate, 200 m
The liquid level is set to 1 cm or less in the storage tank of l. As a result of inoculating the inoculum into the entire system (excluding the medium supply device) and circulating culture using a peristaltic pump, 0.5 T was obtained in 2 days.
A culture solution having a cyclodextrin synthase activity of HU / ml was obtained. As a control, place 1 in a 500 ml Erlenmeyer flask.
The result of culturing in a medium containing 00 ml was 0.01 to 0.05 THU / ml.

Example 2 1T, except that a silicon tube 10 m having a thickness of 100 μm and a diameter of 3.6 × 4.0 mm was set on an acrylic plate.
A solvent solution having an enzyme activity of HU / ml was obtained.

Example 3 As a result of culturing in a water vapor saturation chamber in the same manner as in Example 1 to prevent excessive drying, a culture solution having an enzyme activity of 0.8 THU / ml was obtained. In addition, as a result of circulating culturing with a 1/2 dilution medium replenishing device in the middle of the tube, the activity was 1.2 THU / m
l rose.

Example 4 A silicon tube 230 m having a thickness of 200 μm and a tube diameter of 3.6 × 4.0 mm was set on an acrylic plate, and a space of 5 to 10 cm was taken to form 5 stages, and each stage was used for normal culture. As a result of culturing at a flow rate of 2 ml / min with a diluted medium supply device containing a medium having a concentration of / 10, a culture solution having an enzyme activity of 0.5 THU / ml was continuously obtained after 2 days of culture. When this culture solution was passed through the same silicon tube in a dry state, it was concentrated to 2/4 at 2 m.

[Effects of the Invention] According to the method of the present invention, culturing in a jar fermenter, which has been mainly used in culturing aerobic bacteria, becomes more efficient, and continuity is greatly simplified. Widely applicable to the production of enzymes and useful substances. In particular, when cultured at high temperature, the amount of dissolved oxygen is small,
It seems that in many cases, the ability to produce microorganisms cannot be sufficiently exhibited.However, if the method of the present invention is used, oxygen can be supplied if necessary, and the production of useful substances such as enzymes is stabilized,
Further, the productivity is increased. In addition, the main culturing method can be easily combined with various devices, and if an ultrafiltration membrane is combined for continuous production of a useful substance, the medium can be conveniently supplied.

Furthermore, the method of the present invention can be applied to photosynthetic bacteria, for example, continuous culture can be performed while irradiating light.

[Brief description of the drawings]

FIG. 1 shows the entire system in which a silicon tube is set in a cylindrical shape. This system mainly consists of a culture tube, a storage tank (reservoir tank), a medium or water supply device (auxiliary medium tank), a concentration or dialysis device (medium exchange, concentration system), and an automatic concentration sensor, which can be combined as necessary. Can be.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shinichi Kunizaki 16-16-16 Futaba-cho, Ibaraki-shi, Osaka (72) Inventor Kyoichi Sugawa 1-1-1 Himurocho, Takatsuki-shi, Osaka (56) Reference Document JP-A-51-110089 (JP, A) JP-A-56-61988 (JP, A) JP-A-52-122687 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 1/00-7/08 C12M 1/00-3/10

Claims (4)

(57) [Claims] 1. A microorganism (mushroom) characterized by culturing microorganisms (excluding mushrooms and algae) by continuously supplying an inoculation medium and / or a liquid medium into a tube of a tubular silicone gas permeable membrane. Cultivation method (excluding species and algae). 2. The culture method according to claim 1, wherein a tubular silicon gas-permeable membrane is used by fixing it to the outer surface of the substrate. 3. The culturing method according to claim 1, wherein the culturing is performed while concentrating the culture solution in a tube of a tubular silicon gas permeable membrane. 4. The culture method according to claim 1, wherein the culture solution after completion of the culture is passed through a tubular gas-permeable silicon membrane to be concentrated.