JPH0462715B2 - - Google Patents

Description

[Detailed Description of the Invention] (a) Industrial Application Field This invention relates to a bioreactor. More specifically, the present invention relates to an industrial bioreactor that can easily and continuously produce various organic substances such as food materials and pharmaceuticals by applying the specific functions of microorganisms, cells, or enzymes.

(b) Conventional technology Batch fermentation has traditionally been used as a method for producing various food materials and pharmaceuticals using enzymes, microorganisms, and cells, but the fermentation time is long and the enzymes, microorganisms, and cells are disposable. In addition to being expensive, there are many disadvantages such as high BOD of wastewater and complicated purification process of the product, resulting in high costs.

In this regard, enzymes, microorganisms, cells, etc. can be chemically bonded or physically adsorbed to suitable carriers, or enclosingly immobilized in gels such as carrageenan or calcium alginate, and the carriers or gel particles can be packed into a reactor. Alternatively, in order to prevent the flow of a bioreactor (carrier packed bed or carrier fluidized bed type bioreactor) in which the raw material substrate liquid is passed through by fluidization, or the flow of these immobilized enzymes, microorganisms, or cells into the product liquid, A bioreactor (membrane bioreactor) has been proposed in which a raw material solution is sent through a dialysis membrane or a hollow fiber membrane to a layer of immobilized enzymes and bacterial cells, and the product is taken out through the same membrane. It has reached the point where

(c) Problems to be Solved by the Invention However, in continuous production using the membrane-type bioreactor, the reactivity of the reactor is dominated by substrate diffusion and product diffusion through the membrane, making it difficult to achieve high reaction efficiency. The problem is that it is difficult to obtain. On the other hand, in the case of a membrane bioreactor, in which the raw material substrate liquid is forcibly supplied to a layer of immobilized enzymes, immobilized microorganisms, or cells, and a liquid containing the product is obtained outside the membrane by a pressure difference across the membrane, the reaction Although high reaction efficiency can be obtained initially, during long-term use, enzymes, microorganisms, cells, etc. form a high-density gel layer on the membrane surface, increasing pressure loss through the membrane and causing raw materials and products to be contained. There was a problem that the permeation of the liquid was inhibited, and in severe cases, clogging occurred and the function as a bioreactor was lost.

This invention was made in view of these problems, and even when used for a long time, enzymes and
The purpose is to provide a bioreactor that can efficiently produce the desired product without forming a high-density gel layer of microorganisms, cells, etc., and without being adversely affected by the growth of microorganisms in the case of proliferating bacterial cells. .

(d) Means and effects for solving the problem Thus, according to the present invention, there is provided a biological system that is configured to produce a predetermined organic substance by passing a raw material liquid through a layer holding microorganisms, cells, enzymes, etc. In the reactor, microorganisms in the retention layer are permeable to the raw material liquid,
A retaining layer for microorganisms, a cell culture solution, or an enzyme solution is set between two porous membranes that do not permeate cells or enzymes, and a circulation channel for the culture solution or enzyme solution is attached to the retaining layer. A bioreactor is provided.

The most distinctive feature of this invention is that a circulating layer of a microorganism or cell culture solution or an enzyme solution is used in the reaction section of the bioreactor. In this reaction section, the culture solution and enzyme solution are circulated, so even if microorganisms and cells multiply significantly, the porous membrane that sets up the circulation layer will not be clogged, and the permeation of raw materials and produced organic substances will not occur. The properties will be maintained in good condition for a long period of time. Furthermore, the density of enzymes, microorganisms, cells, etc. in the reaction section can be easily controlled from the outside.

The porous membrane used in the present invention has a semipermeable membrane function that allows the raw material liquid to pass through but not the enzymes, microorganisms, or cells in the retention layer. Here, "permeate the raw material liquid" means that it can sufficiently permeate at least the raw material intended for conversion. Although such a porous membrane depends on the type of microorganisms in the retention layer and the type of raw materials, it is usually preferable to use one having a pore diameter of 0.01 to 0.5 μm. However, in the case of enzymes, the thickness is preferably 0.001 to 0.01 μm. If the pore diameter is below this lower limit, pressure loss due to the membrane itself increases, which is not preferable.
Specific examples of these include cellulose acetate, regenerated cellulose, polypropylene, polycarbonate,
Examples include films such as Teflon and polyacrylonitrile, with a suitable thickness of 0.01 to 0.5 mm.
0.01-0.1 mm is preferable.

The above two porous membranes are supported on a net-like or porous support (e.g., punching board, sintered metal porous material, porous resin plate) and placed in a container equipped with a liquid inlet and a liquid outlet. The bioreactor of the present invention is constructed by arranging membranes facing each other and circulating a culture solution of microorganisms or cells or an enzyme solution between the porous membranes. The flow rate of the circulating culture solution and enzyme solution is
It is necessary to set the pressure to a level that does not impede the permeability of the raw material liquid and produced organic substances that permeate in the thickness direction through the porous membrane and the circulating liquid layer. For example, the liquid pressure of the raw liquid to the porous membrane must be set to 0.5. When setting the pressure to 2 kgf/cm ² , it is appropriate to apply the same internal pressure and set the flow rate of the culture solution to 0.5 to 10 m/sec.

The enzyme, microorganism, or cell used is determined by the raw material solution and the desired organic substance produced. Combinations of these raw material liquids/microorganisms/produced organic substances include glucose/lactic acid bacteria/L-lactic acid, glucose/propionic acid bacteria/propionic acid, glucose + ammonia/inosinic acid producing bacteria/inosinic acid,
Glucose + Ammonia / Glutamic acid producing bacteria /
Examples include glutamic acid. Typical combinations of raw material solution/enzyme/organic substance produced include lactose/galactosidase/glucose and galactose, glucose/glucose isomerase/fructose, and the like.

In addition, it is preferable to select materials for the containers that make up the bioreactor that can be sterilized by autoclaving and have excellent contact resistance, such as stainless steel.
It is preferable to select SUS 304, SUS 316, SUS 316L, etc.

(E) Example Reference numeral 1 in FIG. 1 is a diagram illustrating the configuration of an example of a bioreactor of the present invention. In the figure, the bioreactor 1 has a stainless steel top lid 5 with an inlet 51 and an outlet 52 for the substrate circulating fluid, an inlet 44 and an outlet 45 for the culture circulating fluid, and an outlet 71 and 72 for the permeate 7 containing produced organic substances. A packing material 8 (made of butadiene rubber, thickness 0.5 mm), a porous membrane 2 (polypropylene membrane filter, average pore diameter 0.2 μm, thickness 0.1 mm), and a support 21 (thickness 0.4 mm) are placed between the lower lid 10 and the lower lid 10. (stainless steel needle punch plate), spacer 9 (made of Teflon,
0.8 mm thick), porous membrane 2', support 21', and packing material 8' are installed in this order, and microorganisms 31 are placed in the gap defined by these two porous membranes and the spacer.
A circulating culture solution layer 3 (retention layer) is set. The inoculation into the bioreactor 1 is carried out using the two pots 42 and 43 and the culture circulating fluid pump 41.
This is carried out by introducing the seed culture solution 32 into the culture circulation channel 4 using a .

In addition, in the figure, 6 is a raw material substrate liquid, 61 is a pump for introducing the raw material substrate liquid, 62 is a pot for extracting the raw material liquid,
Reference numeral 63 indicates a pressure gauge, 64 indicates a pressure adjustment valve, 73 indicates a permeate flow path switching cock, and 74 indicates a valve.

The bioreactor 1 was operated under the following conditions.

Microorganisms: Streptococcus faecalis species Culture medium: Glucose (2%), polypeptone (1%), yeast extract (1%), monobasic potassium phosphate (0.1%), dibasic potassium phosphate (3.5%)
Aqueous solution (PH7.5) Raw material substrate solution: glucose (2%), polypeptone (0.1%), yeast extract (0.1%), monobasic potassium phosphate (0.68%), dibasic potassium phosphate (1.7
%) aqueous solution (PH7.2) Flow rate of raw material substrate liquid into bioreactor: 2.1
6ml/hr. for Kg/ ^cm2 , 12ml/hr. for 3.3Kg/ ^cm2 . Flow rate of culture circulating fluid: 250ml/hr. Temperature: 37℃ Effective membrane area: ^59cm It was used after being sterilized in an autoclave at 120°C for 15 minutes.

This result is: Conversion efficiency (lactic acid production capacity per membrane area): 73
g/m ²・hr. Pressure change and clogging: Bacterial count at start of operation/8×10 ⁸ cells/ml = 1Kg/cm ²
When the flow rate of the permeate was 10/ ^m2・hr., the number of bacteria after the second day was 6× ¹⁰⁹ cells/ml and 3.3Kg/ ^cm2 ,
The flow rate of the permeate was 2/m ² ·hr.

On the other hand, in a membrane bioreactor configured in the same way except for not having a circulation flow path, the lactic acid production capacity per membrane area was 11.3 g/m ² hr under the same conditions.
It was found that the bioreactor of this invention is excellent for long-term use.

(F) Effects of the Invention In the bioreactor of the present invention, a decrease in the permeability of the porous membrane due to proliferation of microorganisms and cells, gelation of enzymes, etc. is prevented. Therefore, it exhibits excellent reaction activity over a long period of time.
Furthermore, since it is equipped with a circulation channel for culture solution and enzyme solution, it is easy to control the activity and proliferation of enzymes, microorganisms, cells, etc. from the outside.

[Brief explanation of the drawing]

FIG. 1 is a configuration explanatory diagram showing an embodiment of the bioreactor of the present invention. 1...Bioreactor, 2...Porous membrane, 3
...Culture circulation liquid layer, 4...Culture circulation channel.

Claims (1)

[Scope of Claims] 1. In a bioreactor configured to produce a predetermined organic substance by passing a raw material solution through a retention layer for microorganisms, cells, enzymes, etc., the retention layer is permeable to the raw material solution. microorganisms inside,
A holding layer for a culture solution of microorganisms or cells or an enzyme solution is set between two porous membranes that do not permeate cells or enzymes, and a circulation flow path for a circulating substrate liquid is provided on one of the porous membranes. A substrate culture circulating fluid inlet is provided at one end of the holding layer to form a substrate circulating fluid inlet, and a substrate circulating fluid outlet is provided at the other end of the retaining layer, and a culture circulating fluid is provided at one end to form a circulating channel for the culture circulating fluid within the holding layer. A bioreactor characterized in that an outlet and a culture circulating fluid inlet are provided at the other end, and a liquid outlet for a permeate containing a produced organic substance is provided at the remaining porous membrane side of the porous membrane.