JPH05236938A - Method for separating cell from culture solution - Google Patents

Abstract

PURPOSE:To completely separate cells by a high flux in separating cells from a culture solution by using a specific turntable-like membrane separating device by using a suspension prepared by adding water-insoluble fine particles having specific particle diameters to a culture solution to reduce viscosity of the culture solution. CONSTITUTION:In separating cells from a culture solution by using a turntable- like membrane separating device having a cell separating membrane made of ceramics, water-insoluble fine particles (preferably perlite) having 0.3-40mum (preferably 5-30mum) average particle diameter are added to the culture solution to give a suspension, which is used to carry out the objective cell separation.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an efficient method for separating bacterial cells from a culture solution. More specifically, it relates to a method for separating bacterial cells from a culture solution with complete and high permeation flux.

[0002]

2. Description of the Related Art A membrane separator using a rotating plate membrane is disclosed in Japanese Utility Model Laid-Open No. 62-130703.
The method for separating bacterial cells using the apparatus is disclosed in the 23rd Autumn Meeting of the Chemical Engineering Society, Abstracts (1990). However, although the bacterial cells can be completely separated by this method, the membrane permeation rate of the culture solution is often low because the occluding factor such as a polymer substance in the culture solution blocks the membrane. In addition, as a general method, after adding a flocculant to the culture medium, the culture medium is filtered by a known pressure filter, a vacuum filter or the like, and then the cells are further separated by microfiltration. Are known.
However, in this method, although it is possible to completely separate the bacterial cells while improving the decrease in the membrane permeation flux, it takes time because it involves multiple steps, and the recovery rate of useful substances in the culture solution is high because there are many steps. There are problems such as deterioration, and the efficiency of the entire process cannot be said to be good.

As described above, in the conventional method for separating bacterial cells from a culture solution, the efficiency of the whole separation process cannot be said to be sufficient due to the reduction of the membrane permeation flux and the complication of the separation process. Improvement was strongly desired. The present invention has been made to solve such problems. That is, the object of the present invention is to improve the microbial separation of the suspension by adding water-insoluble fine particles to the culture medium to be separated to form a suspension,
Then, the suspension is treated with a separation device using a rotating plate-like membrane as described above to provide a method for efficiently and simply separating bacteria from a culture solution at a high permeation flux. is there.

[0004]

Means for Solving the Problems The present inventors have added water-insoluble fine particles to a culture solution to prepare a suspension, and separating cells from the suspension using a specific membrane separation device. As a result of intensive studies, the present invention has been completed.

That is, the gist of the present invention is to separate water-insoluble fine particles having an average particle size of 0.3 to 40 μm in a method for separating bacterial cells from a culture solution using a rotating plate membrane separator having a bacterial cell separation membrane. The present invention relates to a method for separating bacterial cells, which comprises using a suspension obtained by adding it to a culture solution.

The culture medium which is the target of the separation method of the present invention is
For example, it may be a culture of microorganisms such as bacteria and yeast, or may be a culture of animal or plant cells. These culture solutions often produce polymeric separation inhibitors and the like due to cell self-digestion and the like, because these inhibitors adhere to or accumulate on the separation medium when cells are separated from the culture solution. It may be difficult to obtain a sufficient permeation flux even if the above rotary plate-shaped membrane separation device is used.

In the present invention, water-insoluble fine particles are added to such a culture solution in advance to form a suspension, and the suspension is used for cell separation. The material of the fine particles to be added is not particularly limited as long as it is insoluble in water, and may be, for example, an inorganic substance such as silica or alumina, or an organic substance such as cellulose or plastic resin. Among these, an inorganic substance composed of silica, alumina or the like is preferred from the viewpoints of easy availability, handleability, processability of waste slurry containing bacterial cells, and the like. As specific water-insoluble particles, perlite and kieselguhr are preferable.

The average particle size of the water-insoluble fine particles used is
It is usually 0.3 to 40 μm, preferably 1 to 30 μm, and more preferably 5 to 25 μm. If the average particle size is smaller than 0.3 μm, there is a risk that the fine particles themselves may be clogged in the pores of the rotary plate film, which is not preferable. Further, if the average particle diameter is larger than 40 μm, the surface area of the fine particles per unit mass is reduced, and the adsorption and trapping effect of the separation inhibitor is reduced, which is not preferable. The particle size distribution is not particularly limited, and may be monodisperse or have a wide particle size distribution.

The shape of the water-insoluble fine particles used is not particularly limited and may be spherical, angular or amorphous. Bulk density is usually 0.02 g / cc to 0.6 g / cc
However, it is more effective to suspend it well in the culture solution, so it is desirable that it is as small as possible, and it is 0.05 g / cc.
~ 0.4 g / cc is preferred. The concentration of the water-insoluble fine particles added is usually 0.05 to 3.0% by weight of the suspension to be separated, and more preferably 0.1 to 2.0% by weight. At this time, the amount can be increased for the purpose of improving the permeation flux depending on the poor properties of the culture solution. Further, it is desirable to stir the culture solution during filtration so that the fine particles are well suspended, and for the same purpose, a dispersion liquid in which the fine particles are previously dispersed may be used for the same purpose.

The rotary plate-shaped membrane separator having a microbial cell separation membrane used in the present invention is a membrane separator using a rotary plate-shaped membrane, and is disclosed in, for example, Japanese Utility Model Publication No. 62-130703. The thing of a disclosure is mentioned. In this device, since the membrane itself rotates, the membrane surface flow velocity for separating the gel layer (layer where the membrane blocking substance is deposited) formed on the membrane surface and the operating pressure that is the driving force for membrane permeation are independent. Can be controlled. By enabling independent control in this way, it has become possible to perform microbial cell separation of the culture broth quite well, which was difficult with a conventional membrane separation device such as a hollow fiber module type. That is, in a conventional membrane separation device such as a hollow fiber module type, since the membrane surface flow velocity and the transmembrane pressure difference are obtained by circulating the culture solution in the module, the membrane surface flow velocity and the transmembrane pressure difference increase and decrease at the same time. To do. Therefore, even if the membrane surface velocity is blindly increased, the transmembrane pressure difference of the module is also increased, the gel layer is consolidated in the vicinity of the membrane, and the membrane permeation flux and the recovery rate of useful substances in the culture solution are increased. descend. On the other hand, when the flow velocity on the membrane surface is reduced to reduce the transmembrane pressure difference, the gel layer peeling effect is reduced, and in this case also, the membrane permeation flux and the recovery rate of useful substances in the culture solution are reduced. Further, in the rotary plate membrane separator according to the present invention, it is not necessary to increase the discharge rate of the circulation pump to secure the membrane surface flow rate as in the hollow fiber module type, so that the lysis due to the mechanical force of the pump is less likely to occur. It has the advantage that membrane occlusion due to deterioration of properties does not occur.

The pore size of the rotary plate-shaped membrane used in the rotary plate-shaped membrane separator of the present invention is usually 0.1 to 0.8 μm, preferably 0.1 to 0.3 μm. If the pore size is smaller than 0.1 μm, the permeation flux of the membrane becomes insufficient,
If it is larger than 0.8 μm, the ability to separate cells will be insufficient. The material of the film is preferably ceramic such as alumina, titania, zirconia. Since the ceramic membrane has excellent chemical resistance and heat resistance and can perform strong membrane cleaning, membrane regeneration is easy. Also, although it cannot be washed as strongly as with ceramics, it can be used as membrane materials such as polyolefin, polysulfone, polyacrylonitrile,
An organic film such as polyether sulfone may be used. The operating temperature is usually 0 to 40 ° C, preferably 5 to 30 ° C. If the operating temperature is lower than 0 ° C., the viscosity of the culture solution becomes large and the membrane permeation flux decreases, and if it exceeds 40 ° C., the property of the culture solution deteriorates, which is not preferable.

The operating average pressure in the present invention is usually 3.
0 kg / cm ² or less, preferably 0.5 to 2.0
It is kg / cm ² . The minimum operating pressure is the value required to overcome the centrifugal force due to the rotation of the membrane and allow the filtrate to permeate, and the operating pressure is determined by the number of membrane rotations. Also,
When the operating pressure exceeds 3.0 kg / cm ² , the gel layer is densified on the membrane surface, which causes membrane clogging and lowers the permeation flux, which is not preferable. Further, the method of applying the transmembrane pressure difference may be a stock solution side pressure type, a permeate side pressure reducing type, or a combination of both. The film surface speed of the rotating plate-like film is determined by the film rotation speed, but is 0.5 to 20 m / s at the outer peripheral part of the film surface.
Is appropriate.

[0013]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

Example 1 A strain of the genus Bacillus was cultured under the following conditions, and the bacterial cells were separated from the culture broth by the method of the present invention (composition: wt%).
The medium is 3% meat extract (OXOID), 0.05% yeast extract (DIFCO), 0.1% KH ₂ PO ₄ ,
KCl 1%, glucose 0.5%, fructose 0.5
% And Na ₂ CO ₃ 0.5% were mixed and dissolved. Three
Charge 18 liters of this medium into a 0 liter culture tank, and
180 ml of the seed culture solution that had been shaken for 4 hours was added. Cultivation is performed at a temperature of 33 ° C. and a supply air flow rate of 9 Nl /
It was carried out by holding for 30 minutes under the conditions of a minute and a stirring speed of 400 rpm. The cell concentration after culturing is the wavelength 6 of the culture solution.
When measured by turbidity at 00 nm, the value was 40 in terms of absorbance.

10 liters of the culture solution obtained by the above method was placed in a container and 0.5% of perlite having an average particle size of 17 μm was added.
A suspension was prepared by adding so as to have a concentration of%. The structure of the membrane separator used will be described below with reference to FIG. The rotating plate-shaped film 9 has a diameter of 230 mm and an effective film area 708c.
It is a disc-shaped membrane made of ceramics having a nominal pore diameter of 0.1 μm and a m ² . The liquid tank 1 in which the rotating plate-shaped film 9 is installed is made of stainless steel and has an internal volume of 1.1 liter. The rotating plate-shaped membrane 9 is attached to the membrane rotating motor 12 so as to be integrated with the hollow rotating shaft 10 that is interlocked via the belt 13, and the membrane rotation speed can be continuously changed from 0 to 1000 rpm. You can The hollow rotary shaft 10 has a pressure reducing pump 14
Is connected, and the pressure can be reduced to 5 to 760 Torr. The solution that has passed through the rotating plate-shaped membrane 9 is rotated by the rotating shaft 10.
Is introduced into the hollow portion 11 of the
Liquid is collected in a glass-made collecting container 15 connected midway.

Using the above membrane separator, 10 liters of the above suspension was introduced into the liquid tank of the membrane separator by a roller pump and circulated. The suspension was ice-cooled and kept at 15 ° C. Subsequently, the film rotation speed was set to 600 rpm (the film surface speed of the film outer peripheral portion was 7.2 m / s), and the permeation side was set to 380 Torr.
The pressure was reduced to (differential membrane pressure 0.5 kg / cm ² ) and separation operation was performed to obtain 8 liters of permeate. At this time, the flow rate of the permeate was measured and the presence or absence of bacterial cells in the permeate was examined by the colony counting method.

Comparative Example 1 The same operation as in Example 1 was carried out except that pearlite was not added. At this time, the flow rate of the permeate was measured and the presence or absence of bacterial cells in the permeate was examined by the colony counting method.

From the above results, the average permeation flux with and without addition of perlite was determined. FIG. 2 shows the result obtained by stippling the permeated liquid amount per unit membrane area of the rotary plate-shaped membrane. As is clear from FIG. 2, when pearlite was added as in Example 1, the average permeation flux was dramatically improved as compared with the case where pearlite was not added as in Comparative Example 1. In addition, the presence or absence of bacterial cells in the permeated liquid was examined by the colony counting method, but no bacterial cells were detected.

[0019]

According to the present invention, the viscosity of the culture solution is lowered by adding the above-mentioned water-insoluble fine particles to the culture solution prior to the separation of the bacterial cells to form a suspension (the separation inhibitor is adsorbed and captured by the fine particles. It is considered that the fine particles collide with the gel layer and separate the fine particles, so that the gel layer is not easily formed on the film surface. Therefore, by using the rotating plate-shaped membrane separation device of the present invention, it becomes possible to completely separate the bacterial cells with a high permeation flux even in a culture solution containing a large amount of a separation inhibitor. As a result, the apparatus can be made compact, and the bacterial cells can be separated from the culture medium in one step, so that the recovery efficiency of useful substances such as enzymes present in the culture medium can be improved. ..

[Brief description of drawings]

FIG. 1 shows an example of a block diagram of a rotary plate membrane separator according to the present invention.

FIG. 2 shows the results of stippling the average permeation fluxes obtained in Example 1 and Comparative Example 1 with respect to the permeated liquid amount per unit area of the rotating plate-shaped membrane.

[Explanation of symbols]

 1 Liquid Tank 2 Suspension 3 Stirring Rod 4 Stirring Motor 5 Circulation Pump 6 Suspension Inlet 7 Suspension Outlet 8 Liquid Tank for Rotating Plate Membrane 9 Rotating Plate Membrane 10 Rotating Shaft 11 Hollow Section 12 For Membrane Rotation Motor 13 Belt 14 Decompression pump 15 Liquid collection container 16 Exhaust 17 Surface dense layer 18 Internal sponge layer

Claims (3)

[Claims] 1. A method for separating cells from a culture solution using a rotating plate membrane separator having a cell separation membrane, wherein water-insoluble fine particles having an average particle size of 0.3 to 40 μm are added to the culture solution. A method for separating bacterial cells, which comprises using the obtained suspension. 2. The method for separating bacterial cells according to claim 1, wherein the material for the bacterial cell separation membrane is ceramic. 3. The water-insoluble fine particles have an average particle size of 5 to 30 μm.
2. The method for separating bacterial cells according to claim 1, which is perlite.