JPH0528594B2 - - Google Patents

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to bioreactors.

[Conventional technology]

In order to stabilize enzyme activity and enable repeated use despite being water-soluble, technology has been developed to make enzymes insoluble in water using physical or chemical methods and use them in the form of solid catalysts. It is used in the industrial production of L-amino acids, isomerized sugar, 6-aminopenicillanic acid, malic acid, etc.

If the immobilized enzyme method is further advanced and an immobilized microorganism in which live microbial cells containing an enzyme are immobilized is used, the enzyme extraction and purification operation can be omitted.

Furthermore, immobilized microorganisms have advantages such as good stability during immobilization or manipulation, advantageous use in multi-step enzyme systems, and ability to supply coenzymes, ATP, and the like.

Furthermore, a method of immobilizing living microorganisms can be used to produce secondary metabolites, and it is also possible to improve the yield by using immobilized growing microorganisms.

In order to immobilize such microbial cells, the establishment of immobilization technology is fundamentally important, and carrier binding methods,
The crosslinking method and the entrapment method are known, and the processes have been implemented as stirred bed type, fluidized bed type, and membrane type bioreactors.

For example, Ichiro Chibata, Tetsuya Tosa, Oil Chemistry, 3414
(1982), Tatsuo Sumino, Yuki Nakamura, Yasutomo Otake, Naomichi Mori, PPM, No. 6, 28 (1987), Saburo Fukui (ed.), "Microorganisms as biocatalysts", Kyoritsu Shuppan (1979).

[Problem to be solved by the invention]

In aerobic fermentation production technology, the rate of diffusion of nutrients and enzymes influences the rate of production of the desired product.

However, the utilization efficiency of nutrient sources is generally low, and the viscosity of the culture solution increases due to bacterial cell proliferation and product production, inhibiting biosynthetic reactions and reducing the production rate of the desired product.

In the case of filamentous fungi, shortages of nutrients and oxygen are particularly likely to occur due to the formation of bacterial masses with a three-dimensional network structure and thickening of the culture solution.

In addition, there is a lot of room for improvement, as it takes time and effort to separate the product from the culture solution.

On the other hand, technology has been developed to apply microbial cells immobilized in gel and proliferating microbial cells to bioreactors, and improvements in the productivity of alcohol fermentation have been reported.

However, it requires complicated operations such as preparing a gel containing the bacterial cells, adjusting the size and strength of the gel, and sterilizing it. In addition, the gel is destroyed due to the proliferation of viable bacterial cells, and furthermore, the resulting turbidity with the culture solution makes it difficult to recover the product.

[Means to solve the problem]

In order to eliminate the drawbacks of the conventional immobilization techniques for microorganisms, the present invention has been made through intensive research into an immobilization method that is inexpensive, durable, simplifies processes and operations, improves productivity, and is highly stable. As a result, it was completed.

That is, in the bioreactor of the present invention, a fabric carrying bacterial cells is housed in a bag-like material made of a flat polymer membrane having pores with a diameter of 2 μm or less, and the bag-like material is stacked in a container via a spacer. Then, a bacterial inflow tube and a bacterial cell outflow tube with their tips sealed are inserted into the container through the bag-like object and the spacer, and the bacterial cell inflow tube and the bacterial cell outflow tube penetrate the bag-like object. Microbial cell inflow pores and microbial cell outflow pores are provided in the portion to communicate the microbial cell inflow pipe and the bag-like object, and the microbial cell outflow pipe and the bag-like object, and a medium inflow pipe is provided in the container. , a culture medium outflow pipe, an air supply pipe, and an air discharge pipe are provided to form a culture medium and air flow path in a direction parallel to the laminated bag-like material.

As the polymer flat membrane in the bioreactor of the present invention, membranes that are generally used as microfiltration membranes or ultrafiltration membranes are used, and the materials thereof include acetylcellulose, polyamide, polyvinyl alcohol, polysulfone, polyethersulfone, These include polyethylene and polypropylene. In particular, in order to prevent permeation of bacteria, it is preferable that the pore diameter is 2 μm or less. In addition, fabrics such as textile fabrics, knitted fabrics, non-woven fabrics, etc. are inserted inside the bag as a support for the bacteria in order to propagate and immobilize the bacteria, secure space, and provide sufficient ventilation in the case of aerobic bacteria. It is preferable to do so.

Note that the term "microbial cells" as used herein means microorganisms such as enzymes and bacteria.

In the bioreactor of the present invention, the bag containing the fabric supporting bacterial cells is prepared by preparing and culturing useful microorganisms by a normal fermentation method, and extracting useful enzymes from the prepared bacterial cells. It can be produced by enclosing it in a bag-like material, or by inoculating seed bacteria into a bag-like material to prepare bacterial cells.

Next, the bioreactor of the present invention will be explained based on the embodiment shown in the drawings.

As shown in FIG. 1, in a bioreactor 1, bags 3 and spacers 4 are alternately stacked in a container 2. The bag 3 is formed of a flat polymer membrane, and contains a support 6 carrying bacterial cells therein.

As the spacer 4, a belt-shaped plastic sheet, for example, is used in order to effectively circulate the culture medium between the bag-shaped flat polymer membranes surrounding the bacterial cells.
Alternatively, a mesh may be used to eliminate concentration polarization at the membrane interface, and a combination of a mesh and a plastic strip may be used.

The container 2 is provided with an inflow pipe 7 and an outflow pipe 8 for the medium, and a supply pipe 9 and a discharge pipe 10 for air or oxygen, forming a medium and air flow path parallel to the laminated bag-like material 3. .

Air (oxygen) may be supplied by simply bubbling, but if the air supply pipe 9 is made of a hydrophobic porous membrane, the amount of ventilation can be adjusted.

If the bacteria supported on the support 6 are anaerobic, there is no need to supply air.

The supply of the medium is regulated by the shape of the spacer and the medium supply pump.

In such a bioreactor, the bacterial cells are confined within the bag, so the culture medium can always be kept in a clear state.

If the metabolic product is soluble in the medium solvent and has a size that allows it to pass through the pores of the membrane, it can be subjected to the separation process without going through the sterilization process, and it can be increased by mixing the bacteria and the medium. It can also prevent stickiness.

In addition, if the activity of the bacterial cells weakens due to continuous flow of the medium, it is possible to reactivate the bacteria by flowing nutrients such as glucose instead of the medium.
The membrane sheet containing bacterial cells can also be replaced with a new one.

Furthermore, in the present invention, a bacterial inflow tube 11 and a bacterial cell outflow tube 12 with sealed tips are inserted into the container 2 through the bag-like material 3 and the spacer 4,
Pores 12A and 12B for the inflow and outflow of bacterial cells are formed in the portion where the bacterial body inflow pipe 11 and the bacterial cell outflow pipe 12 penetrate the bag-like material 3. The material 3 and the bacterial cell outflow tube 12 are in communication with the bag-like material 3, respectively. In addition, the bacterial inflow pipe 11 and the outflow pipe 12 of the bag-like material 3 and the spacer 4
A gasket 13 is provided at the penetrating portion to prevent bacterial cells from leaking out.

Next, the action of the bioreactor of the present invention, ie, the method for separating bacterial cell metabolic products, will be explained.

FIG. 2 shows the case where the bioreactor of FIG. 1 is used.

The temperature of the culture medium is controlled in the culture medium tank 14, and the medium is supplied to the bioreactor 1 via the medium inflow pipe 7 by a pump 15. At the same time, the air supply pipe 9
The required amount of air flows from the spacer 4 (Figure 1) to the medium containing sufficient air (oxygen).
It flows through the gap, and during that time, due to the concentration gradient with the inside of the bag-like material 3, the amount necessary for bacterial activity passes through the polymer flat membrane 5 constituting the bag-like material 3 and is taken into the bag-like material 3. Moreover, in the present invention, the culture medium and air flow paths are parallel to the bag-like material, so compared to filtration type reactors with flow paths set perpendicular to the membrane, or reactors with complicated configurations or fine hollow fiber bundles. Substrate and air can be supplied without difficulty, and there is no pressure loss of the reflux liquid containing the target product, allowing smooth reflux. It should be noted that with an aerobic production catalyst, it is necessary to be careful that the aeration rate is rate-limiting for the production of the target substance. If the metabolic product is small enough to pass through the polymer flat membrane 5, it will pass through the membrane 5 and be dissolved in the medium.

In general, with conventional technology, the reaction is inhibited when the concentration of metabolic products becomes high, and in reactors that use a filtration method and the culture medium flow path is designed perpendicular to the membrane, the passage of the substrate through the catalyst layer is a one-shot pass, resulting in a high-speed reaction. With the exception of , the production of products whose chemical structure differs significantly from that of the substrate, such as metabolic products, is significantly inhibited.

In the bioreactor of the present invention, the flow path of the medium and air is set parallel to the bag-like object, so it is easy to control the contact time between the substrate and the biocatalyst, and the medium is not mixed with the bacterial cells. If separation of metabolic products in the medium is required, it can be directly sent to the separation step. The culture medium that has undergone the separation process can be circulated to the culture medium tank 14 again after its concentration is adjusted.

Therefore, the concentration of metabolic products in the medium sent to the bioreactor can always be kept at a constant value of 0 or close to 0, and the reaction is not inhibited and the yield of metabolic products is increased. If the activity of the bacteria weakens and production efficiency decreases, the activity of the bacteria can be reactivated by flowing nutrients from other culture tanks, allowing continuous use.

Furthermore, in the bioreactor of the present invention, the reaction can proceed continuously while replacing the bacterial cells.

That is, the circulation and separation of the culture medium are the same as described above, but when the activity of the bacteria becomes weak, the bacteria in the active state are stored in the bioreactor 1 from the bacteria tank 16 by the pump 17. If it is fed into a bag made of a polymeric flat membrane, the reaction will proceed quickly again.

In addition, even if metabolic products cannot be extracted from the culture medium, it is possible to circulate the bacteria and extract the bacteria and metabolic products as a concentrated solution that does not contain a large amount of the culture medium composition.If this is subjected to a separation process, efficient metabolism can be achieved. Products can be recovered, and reaction inhibition by metabolic products can also be prevented.

FIG. 3 shows a case in which two bioreactors 1 shown in FIG. 1 are combined, and bacteria can be activated on the bioreactor 1 side, and metabolic products can be produced on the 1' side.

In addition, when both 1 and 1' are used for producing metabolic products, the bacteria may be activated in the cell tank 16. At this time, the media must be the same or must not affect each other even if mixed.

However, if bacteria that do not contain the medium composition are circulated through the separation step, or if the bacteria
There is no problem if it is discarded as is from ''.

〔Effect of the invention〕

As described above, the biocatalyst and bioreactor of the present invention can provide the following effects.

(b) Since the culture medium and bacterial cells are not mixed, the separation process can be simplified.

(b) Since metabolic products can always be taken out, reaction inhibition is less likely to occur.

(c) Since bacteria can be reactivated, long-term use is possible.

(d) The reaction tank is compact, making it possible to downsize the equipment and system.

(E) Even if bacteria should get mixed into the culture medium, the bacteria will not mix with each other.

(f) By using different membranes depending on the type of bacterial cells and culture medium used, it is possible to control the reaction rate based on the permeation rate, diffusion rate, and surface area of the membrane.

(g) Management is easy because conditions such as medium circulation speed and air supply speed can be controlled independently.

(h) Since the biocatalyst is immobilized on the bag-like membrane sheet, the culture medium can be uniformly supplied to the immobilized biocatalyst, and metabolic products that can permeate the membrane can be easily taken out.

(li) In other words, the biocatalyst and bioreactor of the present invention have a large surface area, and there is no overcrowded growth of bacterial cells or the formation of bacterial clumps, so nutrients and oxygen can be supplied sufficiently, and metabolites can be easily extracted. in,
It is durable and can be used repeatedly over a long period of time, and it is highly productive because it can use not only proliferating cells but also dormant cells and enzymes. Sterilization is not required and complicated gel preparation operations are not required.

(j) The reaction can proceed continuously while replacing the bacterial cells.

(l) If the metabolic products cannot be taken out to the medium side, circulating the bacterial cells through the bacterial inflow tube and the bacterial cell outflow tube will allow the concentrated liquid of the bacterial cells and metabolic products to be extracted efficiently. Metabolic products can be recovered.

(E) Since the culture medium and air flow path are formed in parallel to the stacked bag-like objects, there is no pressure loss in the reflux liquid containing the target product, and the contact time between the culture medium and the biocatalyst can be easily controlled. It is.

[Brief explanation of drawings]

Figure 1 is a schematic diagram showing an example of the bioreactor of the present invention, Figure 2 is a process diagram showing the action of the bioreactor shown in Figure 1, and Figure 3 is a combination of the bioreactors shown in Figure 1. It is a process diagram showing the effect of time. DESCRIPTION OF SYMBOLS 1... Bioreactor, 2... Container, 3... Biocatalyst, 4... Spacer, 5... Bag-like material, 7... Medium inflow pipe, 8... Medium outflow pipe.

Claims (1)

[Claims] 1 A fabric carrying bacterial cells is housed in a bag made of a flat polymer membrane having pores with a diameter of 2 μm or less, and the bag is stacked in a container via a spacer, and the tip is sealed. A cell inflow tube and a cell outflow tube are inserted into the container through the bag-like material and the spacer, and a cell inflow tube is inserted into the portion where the cell body inflow tube and cell cell outflow tube penetrate the bag-like material. Holes and microbial cell outflow pores are provided to communicate the bacterial cell inflow tube and the bag-like object, and the bacterial cell outflow tube and the bag-like object, and a medium inflow tube, a medium outflow tube, and air supply to the container. A bioreactor characterized in that a tube and an air discharge tube are provided to form a medium and air flow path in a direction parallel to the laminated bag-like material.