JPH0657143B2 - Module for cell concentration and separation - Google Patents

Description

TECHNICAL FIELD The present invention relates to a module for concentrating and separating bacterial cells, which can be suitably used for concentrating and separating microorganisms or bacterial cells (hereinafter referred to as bacterial cells). This module for concentrating and separating bacterial cells is used in the food industry, for example, separation of yeast in wine, separation of acetic acid bacteria in vinegar, concentration during cultivation of lactic acid bacteria, vitamin B.
It can be used for filtration culture during fermentation production of ₁₂ .

[Prior Art] In recent years, attempts have been made to utilize polymer membranes for the fermentation industry. In addition to the utilization of the membrane for microbial reaction, examples of its utilization include separation and concentration of fermentation products.

Regarding the separation of such fermentation products, conventionally, there has been known a method of carrying out continuous culture while increasing the bacterial concentration by using an ultrafiltration device as described in JP-A-52-82779. .

On the other hand, examples of the porous hollow fiber include Japanese Patent Publication No. 56-5.
A porous polypropylene hollow fiber as described in Japanese Patent No. 2123 and a method for producing the same are known. This publication describes a hollow fiber membrane (hollow fiber) made of polypropylene.
The peripheral wall portion has a thickness of less than 40 μm, a large number of minute holes connected to each other are present in the peripheral wall portion, and the radius distribution curve of the minute holes is 200 to 1200.
It is described that one having at least one local maximum within the range of Å is described, and in this case, it is possible to obtain a hollow fiber membrane characterized by gas permeability.

[Problems to be Solved by the Invention] However, in the method of concentrating and separating bacterial cells using an ultrafiltration device, the mechanical properties of the ultrafiltration membrane are a problem, and it is easy to cause damage to the membrane and Since it has poor heat resistance and chemical resistance, it has drawbacks such as difficulty in cleaning, sterilization and sterilization of the membrane and easy occurrence of microbial contamination.

On the other hand, in the conventionally known hollow fiber membrane (hollow fiber), the pores in its peripheral wall are extremely heterogeneous, and the pore shape distribution is large,
For example, when viewed as a cell separation membrane for separating microorganisms or cells, it has a large defect in selective separation ability. Further, in the case of the hollow fiber membrane, it is difficult for the bacterial cells to be adsorbed and permeate, and improvement in this point has been desired.

When a polyolefin such as polypropylene is stretched and fibrillated to be porous, in the conventional method, a portion forming a relatively thick and meandering network having a non-uniform shape and size, and a hollow fiber membrane between the meshes. The holes are formed by relatively thin fibrils (hereinafter referred to as microfibrils) that run substantially parallel to the length direction.

As described above, the shape and size of the pores obtained by the known method are completely non-uniform, and the above-mentioned relatively thick mesh-like portion forms a closed circuit. In other words, this relatively thick mesh is formed. The part to be formed is in the length direction of the hollow fiber membrane,
It runs in all directions, and forms a continuous closed circuit, that is, a mesh of various sizes with a dimension substantially the same as the length of the microfibrils.

Therefore, the length of the microfibrils also varies depending on the location, in other words, the size of the pores has a wide distribution, and as it is, when used for concentrating and separating bacterial cells in a microbial reactor, etc., It is not possible to achieve selective resolution.

[Means for Solving the Problems] Therefore, the inventors of the present invention continued to diligently study a method for making the pore shape of the porous polyolefin hollow fiber membrane constant and a means for preventing adsorption of bacterial cells, and as a result, at low temperature. , Ie −
Succeeding in forming a porous hollow fiber membrane in an extremely specific fibril state by stretching at 60 ° C. or lower, preferably −150 ° C. or lower, and coating the peripheral wall portion with a specific substance The present invention has been achieved by finding that it is extremely easy to pass through.

That is, according to the present invention, there is provided a porous hollow fiber substrate of polyolefin, the peripheral wall portion of which is a group of relatively thick rods running substantially at right angles to the length direction of the hollow fiber substrate, and between the rods. And a group of fine fibrils running in the length direction of the hollow fiber substrate and connected between the rods,
A strip-shaped micropore group is formed by these rod groups and microfibril groups, and a porous hollow fiber membrane in which at least a part of the peripheral wall surface and the micropore inner surface is coated with glycerin fatty acid ester is used. Used as a membrane for concentration and separation of bacterial cells, a plurality of the bundles are bundled, and both ends of the hollow fiber membrane are embedded in a polymer polymer partition wall in an open state, and the both ends of the hollow fiber membrane are liquid-loaded into a housing by the partition wall. Provided is a module for concentrating and separating bacterial cells, which is characterized by being tightly sealed.

A preferred embodiment of the hollow fiber substrate used in the present invention is such that the rod group runs substantially at right angles to the length direction of the hollow fiber substrate, and the average length () of the fine fibrils formed between the rod groups is 3
With a length more than double, the rod forms a closed circuit (first
(See FIGS. 2, 2 and 3), preferably 5 times or more of the average length () of the microfibrils, more preferably 10
It is characterized by forming a closed circuit with a length more than double. Here, the average length () of the minute fibrils is represented by the average of the lengths of 20 arbitrary minute fibrils around any one point on the rod.

In the present invention, various molding conditions are carefully examined to form a relatively thick rod-shaped portion only in a direction substantially at right angles to the length direction of the hollow fiber substrate. In other words, this relatively thick rod is formed. The shape is a special hollow fiber substrate which is not formed in the length direction of the hollow fiber substrate.

By forming in this way, the number of pores per same area can be increased by 20 to 30%, the strength can be maintained, and the porosity can be greatly improved. Therefore, the separation of bacterial cells can be selectively performed. I was able to do it.

A preferred embodiment of the hollow fiber substrate used in the module of the present invention has a peripheral wall thickness of 50 to 150 μm, particularly 50.
˜100 μm, inner diameter 250-1000 μm, especially 27
0 to 400 μm, the pore size is 0.2 to 1.0 μm, especially 0.2 to 0.5 when the pore size is measured by the bubble point method.
It is in the range of μm.

The porous hollow fiber membrane used in the module for concentration and separation of bacterial cells of the present invention is formed by coating at least a part of the peripheral wall surface of the hollow fiber substrate and the inner surface of the micropores thereof with glycerin fatty acid ester. is there.

Examples of the fatty acid component of the glycerin fatty acid ester include caprylic acid, capric acid, lauric acid, palmitic acid, stearic acid, oleic acid, behenic acid, and the like.
As the glycerin fatty acid ester produced by the reaction of these acid components and glycerin, any one of monoglyceride, diglyceride and triglyceride or a mixture thereof is used.

When the hollow fiber membrane having the physical properties as described above is used in a module for concentrating and separating bacterial cells, it is excellent in permeability of microorganisms or bacterial cells, and its selective concentrating separation can be efficiently performed, and moreover, the Of these, the use of polypropylene makes it possible to perform steam sterilization under high temperature and high pressure because it has excellent heat resistance, making it easy to disinfect and sterilize the membrane, which has always been a problem in microbial reactors. It is possible to prevent microbial contamination.

In the hollow fiber substrate used in the module of the present invention, preferably, the average length of the microfibrils starting from any one point on the rod () (the average length of any 20 microfibrils around the origin is the average length). The length of the rod is three times or more, and the rod forms a closed circuit.
In other words, this means that the length of the microfibrils is substantially constant over the above range.

The term "rod" as used in the present invention means the form exhibited on the outer wall surface of the hollow fiber substrate (see Fig. 1 and Fig. 2), and does not particularly mean "rod-shaped" but hollow. In the cross section of the fiber substrate, FIG. 3 is an electron micrograph showing a part of the hollow fiber substrate together with its cut surface. Specifically, the upper part of the hollow fiber substrate shown in FIG. 3 is hollow. The cross section of the fiber base is shown. The right part of the lower half of the hollow fiber base shown in the figure shows the vertical cross section of the hollow fiber base, and the left part of the lower half of the hollow fiber base shown in the same figure. Shows the inner wall surface of the hollow fiber substrate) and has the form shown in the upper part of the hollow fiber substrate. Therefore, the "thickness of the rod" referred to in the present invention is also the "thickness" (thickness) exhibited on the outer wall surface of the hollow fiber substrate.
Means As is apparent from FIG. 3, the “rod” has the same form as the outer wall surface in the inner wall surface and the vertical cross section of the hollow fiber base.

Further, the density of the fine fibrils (fine reticulated fibrils) is determined by taking the width of the average length () of the fibrils on the rod starting from any one point on the rod on the outer or inner surface of the peripheral wall of the hollow fiber substrate of the present invention. It is preferable that the number of fine fibrils within the range of d above is 3 or more and 30 or less.

As shown in FIG. 3, in the wall portion constituting the peripheral wall of the hollow fiber base, fine microfibrils run between rods running in a substantially parallel shape. That is, in FIG. 3, the rod does not form a closed circuit over 50d or more with respect to the average length () of the microfibrils. This shows that the porosity is dramatically improved, in other words, the performance of the same film area is dramatically improved.

Such a porous hollow fiber substrate can be manufactured by using a known stretching means at the low temperature as described above.

Next, a method for producing the porous hollow fiber membrane used in the present invention will be described.

The glycerin fatty acid ester described above is dissolved in a solvent in which the glycerin fatty acid ester is soluble to a concentration of 0.1 to 10% by weight. As the solvent, alcohols such as methanol and ethanol, acetone, benzene, toluene, xylene, chloroform and the like are preferably used.

Then, the porous hollow fiber substrate described above is dipped in the obtained glycerin fatty acid ester solution to sufficiently spread the glycerin fatty acid ester solution in the micropores of the porous hollow fiber substrate, and then dried to form a solvent. To remove. As the drying means, air drying, reduced pressure drying, heat drying and the like are appropriately used depending on the type of solvent.

Since the porous hollow fiber membrane formed by coating with the glycerin fatty acid ester thus obtained has sufficient hydrophilicity, it is extremely permeable to microorganisms, bacteria and the like.

Further, the module for concentrating and separating bacterial cells of the present invention, which uses the above-mentioned porous hollow fiber membrane as a membrane for concentrating and separating bacterial cells, condenses a plurality of porous hollow fiber membranes and opens both ends of this hollow fiber membrane. It is embedded in a partition wall of a high molecular weight polymer (so-called potting material) in this state, and both ends of the porous hollow fiber membrane are liquid-tightly sealed in the housing by the partition wall. A polyurethane resin or the like is generally used as the potting material forming the partition wall.

As the polyolefin used for producing the porous hollow fiber membrane in the present invention, a crystalline polyolefin such as polyethylene, polypropylene, poly-4-methylpentene-1 is used, and polyethylene and polypropylene are particularly preferable. Used. Further, polypropylene is preferable when heat resistance is particularly required.

Next, an example of the case of concentrating and separating bacterial cells using the module of the present invention will be described based on the schematic flow chart shown in FIG.

In the culture tank 10, a predetermined medium is put, a predetermined bacterial cell is inoculated, and culture is performed with or without aeration. The culture broth produced in the culture tank 10 is then divided into a product such as alcohol and the concentrated bacterial cells in the bacterial cell concentrating / separating module 11 of the present invention or the bacterial cell concentrating / separating unit 11 equipped with the module. The concentrated bacterial cells are introduced into the gas exchanger 12. In the gas exchanger 12, an action such as supplying oxygen to an aerobic microbial cell such as yeast is performed, and the microbial cell is returned to the culture tank 10.

The method of measuring the pore size by the bubble point method will be described below.

The bubble point method is based on the ASTM (American Standerd Tes
t Method), the maximum pore size of the porous material (in this case, the hollow fiber membrane) is determined.

That is, the pressure due to air is gradually applied to the inside of the hollow fiber of the hollow fiber membrane wet with the solvent, and the maximum pore size is obtained by the following formula from the pressure when bubbles first come out to the outside of the hollow fiber.

r = 2σ / p where r is the radius of the maximum pore size (cm) and p is the pressure (dyne / c
m) and σ are surface tension (dyne / cm).

The term "pore size" as used in the present invention means not the maximum pore size but the pore size obtained from the pressure at which bubbles are generated all at once.

[Examples] Hereinafter, the present invention will be described in more detail based on Examples, but it will be apparent that the present invention is not limited to these Examples.

(Example 1) Polypropylene (trade name: UBE-PP-J109G
Ube Industries, Ltd., MFI-9g / 10 minutes) diameter 30
Using a circular slit nozzle of mm, melting by a conventional method,
The hollow fiber substrate was spun and spun at a winding speed of 116 m / min.

After heat treating this hollow fiber substrate at 160 ° C. for 5 minutes,
It is introduced into a low temperature bath (liquid nitrogen) at -196 ° C and stretched by 15%, which is subsequently treated at a temperature of 150 ° C for 45 seconds for heat setting, and further stretched by 300% in a heating medium at 135 ° C. 80% at the same temperature after fibrillation
Heat treatment was performed by shrinking (based on that before stretching by 300%).

An electron micrograph of the outer wall surface of the obtained hollow fiber substrate is shown in FIG.

This hollow fiber substrate has an inner diameter of 320 μm and a peripheral wall thickness of 5
It was 5 μm and the pore diameter was 0.25 μm (measured by the bubble point method).

Next, the hollow fiber substrate was first prepared from 2. Oleic acid monoglyceride (Exel O-95N, manufactured by Kao Corporation).
After immersing in a 5 wt% acetone solution, it was air-dried until the acetone disappeared.

Using the porous hollow fiber membrane thus obtained, a module for concentrating and separating bacterial cells having a membrane area of 1.4 m ² was prepared, and its separation performance test was conducted.

In the culture tank 10 of FIG. 4, a medium (pH 7.0) composed of minerals and methanol-assimilating bacteria was sterilized at 120 ° C. for 15 minutes and then cooled, and 0.2% by volume of sterile methanol was added to the medium to culture the medium. Was prepared. This medium was adjusted to pH 7 with ammonia and aerated with stirring at 36 ° C.

Continuous culture was carried out for an average residence time of 5 hours to obtain a bacterial concentration of 21.
0 g / medium was prepared.

This culture solution was passed through the microbial cell concentration / separation module 11 to separate into a concentrated culture solution and a permeate.

The bacterial concentration of the concentrated culture was 73.5 g /. This is a 3.5-fold enrichment.

In this way, the concentrated culture solution was continuously collected, and the permeate was circulated in the culture tank 10. When the cells are concentrated by the microbial cell concentration / separation module 11 as described above, finally 200.
A concentrated solution with a bacterial concentration of 5 g / was obtained. This was a 9.5-fold enrichment. The concentrated solution thus obtained is dried and
The bacterial cell-containing composition having the quality shown in the table was obtained at a yield of 69.6% based on methanol.

[Effects of the Invention] As described above, according to the module for concentrating and separating bacterial cells of the present invention, since the porous hollow fiber membrane having the above-described specific structure is used, bacterial cells in a microbial reaction device or the like can be removed. The cells can be selectively concentrated and separated, and the permeability of bacterial cells can be improved.

[Brief description of drawings]

FIG. 1 is an electron micrograph showing the shape of some fibers on the outer wall surface of the porous hollow fiber substrate used in the module for concentrating and separating bacterial cells of the present invention, and FIG. 2 is the outer wall surface of the porous hollow fiber substrate. An electron micrograph showing a further enlarged view of the shape of some fibers of
FIG. 3 is an electron micrograph showing the shape of some fibers of the porous hollow fiber substrate together with the cut surface thereof. FIG. 4 is a schematic flow chart showing an example of the case of concentrating and separating bacterial cells using the module of the present invention. 10 ... Culture tank, 11 ... Module for concentrating and separating bacterial cells / bacterial cell concentrating separator, 12 ... Gas exchanger.

Claims (1)

[Claims] 1. A porous hollow fiber substrate of polyolefin, the peripheral wall portion of which is a group of relatively thick rods running substantially at right angles to the length direction of the hollow fiber substrate, and the hollow portion between the rods. The rod base and the microfibril group form a strip-shaped micropore group that runs in the length direction of the thread base and is connected between the rods. At least a part of the inner surface of the micropores is used as a membrane for concentrating and separating bacterial cells using a porous hollow fiber membrane coated with glycerin fatty acid ester.
A plurality of the hollow fiber membranes are bundled, and both ends of the hollow fiber membrane are embedded in a polymeric polymer partition wall in an open state, and both ends of the hollow fiber membrane are liquid-tightly sealed in a housing by the partition wall. Module for concentrating and separating cells.