JPH10304868A - Process for separating microbial cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a process for the separation of microbial cells and the recovery of valuable materials from cultured liquid of microorganism without generating noncombustible waste and enabling the automation and labor-saving modification by coagulating the microbial cells with a polymeric coagulant and filtering the liquid containing the valuable materials by a membrane- filtration method. SOLUTION: Valuable materials and microbial cells are separated from a cultured liquid of microorganism, e.g. a cultured liquid containing the microbial cells and the valuable materials and generated in the microbial production of enzymes, amino acids, peptides, constituent substances, etc., by coagulating the microbial cells with a polymeric coagulant and filtering the liquid containing the valuable materials by a membrane-filtration method. The polymeric coagulant to be used in the coagulation process is preferably a cationic or ampholytic polymeric coagulant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for separating cells. More specifically, the present invention provides a method for separating cells which can be automated and labor-saving without generating non-combustible waste, and which can efficiently separate cells of a microorganism culture solution and recover valuable resources. About the method.

[0002]

2. Description of the Related Art When valuable resources are produced by culturing microorganisms, a step of separating valuable cells from cells is required. Conventionally, a drum filter or a press filter has been used in separating cells from a microorganism culture solution. However, since the filterability is poor, a large amount of diatomaceous earth has been used as a filter aid or a precoat agent. In this method, there were a problem of disposing of cells containing diatomaceous earth and a problem of carcinogenicity of diatomaceous earth. As a method for separating cells without using diatomaceous earth, Japanese Patent Publication No. 4-6345 proposes a method for separating cells by gravity filtration and pressing after coagulation of the cells. However, since the microbial culture solution has a low fiber content and contains a lot of viscous substances, the aggregates have a high moisture content and a high viscosity, and clogging of the filter cloth occurs, which makes practical application difficult. In addition, although treatment by a microfiltration method of a microorganism culture solution has been studied, there is a problem that a permeation flux sufficient for practical use cannot be obtained because cells are clogged in a membrane.

[0003]

SUMMARY OF THE INVENTION According to the present invention, automation and labor saving are possible without generating non-combustible waste.
It is an object of the present invention to provide a method for separating cells which can efficiently separate cells from a microorganism culture solution and recover valuable resources.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, after agglutinating cells from a microorganism culture solution with a polymer coagulant, valuable cells were obtained by membrane filtration. It has been found that by filtering a liquid containing a substance, a high membrane permeation flux can be maintained and a valuable substance can be efficiently recovered, and the present invention has been completed based on this finding. That is, in the present invention, in the step of (1) separating valuable resources and bacterial cells from a microorganism culture solution, after the bacterial cells are aggregated with a polymer flocculant, the liquid containing the valuable resources is filtered by a membrane filtration method. And a method for separating bacterial cells, characterized in that: Further, as a preferred embodiment of the present invention,
(2) The bacterial cell separation method according to (1), wherein the aggregate of bacterial cells caused by the polymer flocculant is precipitated, and the supernatant is filtered by a membrane filtration method. And (4) the method for separating cells according to item (3), wherein the method of passing water in the cross-flow filtration is intermittent water flow. .

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention comprises producing a microorganism from a culture broth, for example, a culture broth containing cells and valuables generated during production of enzymes, amino acids, peptides, antibiotics and the like by the cells. It can be applied to the step of separating and recovering the valuable resources from the cells. In the method of the present invention, the polymer flocculant used for the flocculation treatment of the microorganism culture solution is not particularly limited, but usually, a cationic polymer flocculant or an amphoteric polymer flocculant can be suitably used. The cationic polymer flocculant to be used is not particularly limited, and examples thereof include polyethyleneimine, polyvinylamine, polyvinylamidine, poly (meth) allylamine, polydiallylammonium halide, polyaminoalkyl methacrylate, and chitosan. The amphoteric polymer flocculant to be used is not particularly limited. For example, the amphoteric polymer flocculant having a structure in which an anionic group such as a carboxyl group or a sulfone group is further introduced into the above-described cationic polymer flocculant. Agents and the like. In the method of the present invention, an anionic polymer flocculant can be further added for the purpose of coarsening the cell flocculent formed by the cationic polymer flocculant or the amphoteric polymer flocculant. The anionic polymer flocculant used is not particularly limited, and examples thereof include sodium polyacrylate and carboxymethyl cellulose. By adding a polymer flocculant to the microorganism culture solution and performing the flocculation treatment of the cells, the separation of the cells can be facilitated, and the permeation flux of the membrane can be maintained and improved.

In the method of the present invention, there is no particular limitation on the porous separation membrane to be used.
μm microfiltration (MF) membrane, pore size 0.002-
An ultrafiltration membrane having a thickness of 0.1 μm can be used. The material of the separation membrane is not particularly limited, and examples thereof include polyethylene, polypropylene, cellulose acetate, polyamide, polycarbonate, polyacrylonitrile, polysulfone, and polytetrafluoroethylene. There is no particular limitation on the shape of the separation membrane, for example,
Flat membranes, hollow fibers, spiral windings, tubes, plates and the like can be mentioned. In the method of the present invention, it is preferred that after the cells are aggregated with a polymer flocculant, the aggregates are sedimented, most of them are separated, and the supernatant is filtered by a membrane filtration method. If supplied to the membrane while containing the entire amount of aggregates, some of the flocs of the aggregates will be broken, and the generated fine particles may block or contaminate the membrane surface, reducing the permeation flux, By supplying the supernatant to the membrane, a high permeation flux can be obtained stably without the risk of clogging and contamination of the membrane, and the separation property by the membrane can be improved. To separate most of the aggregates, they can be separated by sedimentation or centrifugation.However, even if a portion of the aggregates is contained in the supernatant, there is little problem. In the filtration, a screen is provided inside the circulation tank of the circulation line, a culture solution that has been aggregated is introduced below the screen to settle the aggregates, and then the supernatant is supplied from above the screen to the membrane device by a pump. Is preferred. The concentrate separated by the membrane is circulated through the circulation line to the circulation tank and the membrane, but most of the aggregates are blocked by the screen and retained in the circulation tank. Agglomerates held in the circulation tank are repeatedly washed with circulating water and fresh water supplied as needed, and valuable materials held in the gaps of the aggregates are washed out and supplied to the membrane. As a result, the recovery rate of valuable resources can be increased.

[0007] In the method of the present invention, it is preferable that the membrane filtration method is cross-flow filtration. There are no particular restrictions on the operating conditions of the cross-flow filtration, but the linear flow velocity on the membrane surface is 0.5.
It is preferably from 5 to 5 m / s, more preferably from 1 to 3 m / s. If the linear flow velocity on the membrane surface is low, there is a possibility that the gel layer will be excessively deposited on the membrane surface or the membrane will be contaminated, and the permeation flux will be reduced. If the linear flow velocity on the membrane surface is too fast, it may be difficult to obtain a permeation flux corresponding to the energy consumption. The operating pressure is usually 0.2 to 10 k
g / cm ² , and can be appropriately selected depending on the pore size of the filtration membrane and the concentration of the liquid. In the cross-flow filtration, of the stock solution supplied to the membrane device, a filtrate is transferred to a valuable resource recovery tank, and a concentrated solution is returned to a stock solution tank to form a circulation filtration system. At this time, in order to increase the recovery rate of the valuables-containing liquid, the concentrate can be appropriately watered. The supply of liquid to the membrane device
Aspirating the supernatant of the agglomerated microorganism culture,
Preferably, the concentrate is returned into the sedimented sediment. During the membrane separation operation by cross-flow, a screen having an opening of about 0.1 to 5 mm is provided between the settled aggregate and the supernatant to prevent the settled aggregate from being redispersed by convection of circulating water. It is preferable to prevent the settled aggregate from migrating to the supernatant liquid.

In the method of the present invention, it is preferable that the water flow in the cross-flow filtration is intermittent water flow. In the intermittent water supply, after the water is passed through the membrane device for a certain period of time, the operation of stopping the water supply and restarting the water supply is repeated. The time for one water passage is preferably 30 seconds to 30 minutes, and more preferably 1 to 5 minutes. Also,
It is preferable that the stop time of one flow of water is 0.5 to 10 seconds. By setting the water passing method to intermittent water passing, the permeation flux of the membrane can be maintained in a high state, and the efficiency can be significantly improved. It is considered that by stopping the flow of water, the consolidation of the gel layer formed on the membrane surface is reduced, and the excess gel layer is separated at the time of re-flowing water, so that the permeation flux is maintained at a high level. The intermittent water supply can be performed by starting and stopping a pump, or by switching a flow path. In many cases, the intermittent water flow by switching the flow path is easier to control. FIG. 1 is a process flow chart of an embodiment of the method of the present invention. In this embodiment, the processing is performed in a batch mode. After adding a polymer flocculant to the microbial culture solution to flocculate the cells, a circulation tank 2 provided with a screen 1
From the bottom. The aggregated bacterial cells 3 settle down in the circulation tank, and floating in the supernatant liquid is prevented by the screen in the liquid. The supernatant liquid in the circulation tank is sent to the cross flow type membrane cell 6 via the flow meter 5 by the pump 4. A pressure gauge 7 is provided in the membrane cell. The filtrate from the membrane cell is stored in a valuable resource recovery tank 8. The required linear flow rate in the membrane cell is ensured by returning the concentrated liquid flowing out of the membrane cell to the circulation tank via the circulation line 9. A three-way cock 10 and a bypass 11 are provided in the circulation line, and intermittent water flow to the membrane cell is enabled by switching the three-way cock whose time is set. A water supply line is attached to the circulation tank, and water is added by a pump 12 interlocked with a liquid level gauge in the circulation tank. ADVANTAGE OF THE INVENTION According to the method of this invention, microbial cell isolation of a microorganism culture solution and collection | recovery of a valuable resource can be performed efficiently, and automation of microbial cell isolation and labor saving become possible. Further, according to the method of the present invention, without generating non-combustible waste such as diatomaceous earth,
The cells can be separated.

[0009]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention. Example 1 Solid content concentration 11% by weight, pH 6.2, electric conductivity 4.3 mS
/ Liter of a protein-containing microbial culture solution containing 2 l / cm
The solution was transferred to a 5 liter tank of a membrane filtration device, 0.2 liter of a 10% by weight aqueous solution of polyethyleneimine was added, and the mixture was stirred for 30 seconds. Next, 1 liter of a 0.2% by weight aqueous solution of sodium polyacrylate was added and stirred to form a flocculent floc. Further, 10 liters of ion-exchanged water was added and stirred, and the flocculant was sedimented. . A wire mesh having an aperture of 0.5 mm was provided between the settled floc and the supernatant. The wire mesh was installed at a position 2/3 from the water level of the tank water level. The membrane filtration device and the tank were connected, and the concentrated liquid of the membrane filtration device was returned to the tank again. The pump suction port for supplying the liquid to the membrane filtration device was provided in the supernatant, the return port for the concentrated liquid was provided at the lower part of the tank, and the water supply port was also provided at the lower part of the tank. The operation of the membrane filtration device is performed using a flat membrane cell at a liquid temperature of 10 ° C and a filtration pressure of 0.5 kg / c.
m ² , and the linear velocity of the film surface was 2.0 m / s. As the flat membrane, a polyolefin membrane [manufactured by Nitto Denko Corporation] having a pore diameter of 0.4 μm and a size of 4 cm × 30 cm was used. When 10 liters of the filtrate was collected, 10 liters of ion-exchanged water was added to the tank. Further, when 10 liters of the filtrate was collected, 10 liters of ion-exchanged water was added to the tank. The membrane permeation flux was 831 l / m ² / hr immediately after the start of the treatment, 84 l / m ² / hr after 1 hour, 71 l / m ² / hr after 2 hours, and 67 l / m after 3 hours. ² / hr. A total of 27 liters of filtrate was collected by membrane filtration.
The total protein recovery in the filtrate was 92.8% by weight. Example 2 The same microorganism culture solution as in Example 1 was subjected to a coagulation treatment in the same manner as in Example 1, and set in a membrane filtration device. The operation of the membrane filtration device was performed using the same flat membrane cell as in Example 1 at a liquid temperature of 10 ° C., a filtration pressure of 0.5 kg / cm ² , and a linear velocity of the membrane surface of 2.0 m / s. In Example 1, while water was continuously supplied, in this example, pump water was supplied for 1 minute, and intermittent water supply for 5 seconds was repeated. When 10 liters of the filtrate was collected, 10 liters of ion-exchanged water was added to the tank.
Further, when 10 liters of the filtrate was collected, 10 liters of ion-exchanged water was added to the tank. The membrane permeation flux was 837 liters / m ² / hr immediately after the start of treatment and 6 hours after 1 hour.
79 l / m ² / hr, 663 l / h after 2 hours
m ² / hr, 665 l / m ² / hr after 3 hours. A total of 27 liters of filtrate was collected by membrane filtration.
The total protein recovery in the filtrate was 93.2% by weight. Comparative Example 1 The same microbial culture solution as in Example 1 was subjected to membrane filtration without agglutination treatment. For 10 liters of microbial culture,
10 liters of ion-exchanged water was added, and set in the same microfiltration apparatus as in Example 1. However, a wire mesh for suppressing the dispersion of the flocculated floc was not necessary, and was removed. The operation of the membrane filtration device was performed using the same flat membrane cell as in Example 1, a liquid temperature of 10 ° C., a filtration pressure of 0.5 kg / cm ² , and a linear velocity of the membrane surface of 2.0 m.
/ S. The membrane permeation flux was 54 l / m ² / hr immediately after the start of the treatment, 31 l / m ² / hr after 1 hour, and 2 l / m ² / hr.
The time was 22 liters / m ² / hr after 3 hours and 12 liters / m ² / hr after 3 hours. In this comparative example, addition of ion-exchanged water during operation and measurement of the recovered protein were not performed. Changes in the membrane permeation flux during the operation of Examples 1 and 2 and Comparative Example 1 are shown in Table 1 and FIG.

[0010]

[Table 1]

As can be seen from Table 1 and FIG. 2, the permeation flux in membrane filtration can be improved by aggregating the protein-containing microorganism culture solution. In particular, in Example 2 in which the intermittent water flow was performed, the permeation flux was less reduced and the permeation flux was dramatically improved as compared with the time when the processing was started.

[0012]

According to the method of the present invention, it is possible to efficiently separate cells from a microorganism culture solution and recover valuable resources. Further, according to the method of the present invention, it becomes possible to automate the cell separation and save labor, and further, non-combustible waste such as diatomaceous earth is not generated.

[Brief description of the drawings]

FIG. 1 is a process flow chart of an embodiment of the method of the present invention.

FIG. 2 is a graph showing changes in membrane permeation flux during operation.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Screen 2 Circulation tank 3 Aggregated bacteria 4 Pump 5 Flow meter 6 Membrane cell 7 Pressure gauge 8 Valuables recovery tank 9 Circulation line 10 Three-way cock 11 Bypass 12 Pump

Claims (1)

[Claims] In the step of separating valuable resources and cells from a microorganism culture solution, the method comprises the steps of: aggregating the cells with a polymer flocculant; and filtering the liquid containing the valuable substances by a membrane filtration method. Cell separation method.