JPH01215276A - Bioreactor - Google Patents

Abstract

PURPOSE:To keep an immobilization carrier in a proper suspended state without damaging the carrier by separating a liquid flow generation mechanism to effect the agitation and flow of a reaction liquid from the residence zone of an immobilization carrier. CONSTITUTION:A cylindrical inner cylinder 8 opened at the top and the bottom is inserted into an outer cylinder 7 and an axial flow impeller 12 is placed in the inner cylinder 8. A residence zone 2 to retain an immobilization carrier 29 and partitioned at the top and the bottom with porous plates 28 is provided in a space between the outer circumference of the inner cylinder 8 and the inner circumference of the outer cylinder 7. The liquid flows from the bottom upward in the residence zone 2 by the liquid flow generated by the axial flow impeller 12 to effect the flotation of the immobilization carrier.

Description

Detailed Description of the Invention (A) Field of Application The present invention relates to a biological reaction device (hereinafter referred to as a bioreactor) using an immobilized carrier, and more specifically, a bioreactor that can improve reaction efficiency without damaging the immobilized carrier. The present invention also relates to a bioreactor that can complete a reaction without scale error even in a large bioreactor.

(Prior art) Conventionally, bioreactors using immobilized carriers are those in which the immobilized carrier is suspended in a reaction solution filled in a container to carry out the reaction, or those in which the immobilized carrier is tightly packed in a column or the like. A type of gas-solid-liquid contact method in which the reaction liquid is circulated through the column using an external pump,
etc. are known.

However, for example, in the conventional immobilized carrier floating method, the floating immobilized carriers collide with each other during operations such as stirring and mixing the reaction solution, and the immobilized carriers are damaged.
immobilized enzyme.

There was a problem in that microorganisms and the like were often separated.

In addition, methods in which the immobilized carrier is packed in a column or the like and gas or reaction liquid is passed through it have poor power efficiency, and the inside of the bioreactor is difficult to achieve a complete mixing tank state, so it is difficult to use in a laboratory. There was a problem in that it was difficult to adopt a scale-up method of analyzing the reaction results at the level and applying them to large-scale industrial equipment. Furthermore, this column packed type device has poor gas-liquid contact efficiency, so
They typically require large amounts of air aeration or large-capacity agitators, which can lead to various problems such as foaming and entrainment.

(Problems to be Solved by the Invention) The present invention aims to solve the problems in the conventional bioreactor described above.

It is an object of the present invention to provide a bioreactor with a novel configuration in which an immobilized carrier on which an enzyme is immobilized can be maintained in a suitable floating state, and a reaction solution can be brought into sufficient flow contact with the immobilized carrier in the floating state.

Another object of the present invention is to provide a bioreactor having a structure that makes it easy to design a scaled-up industrial-scale device based on analysis results of laboratory-level tests.

Yet another object of the present invention is to provide a bioreactor in which biologically suitable environmental conditions 1 reaction conditions can be easily selected and controlled.

(Means for Solving the Problems) In order to achieve the above objectives, the bioreactor of the present invention, which was created as a result of intensive research, is characterized by a vertical cylindrical inner cylinder that is open at the top and bottom. ,20Enough t around the inner cylinder
Enzymes or microorganisms are immobilized between the outer cylinder as the outer shell of the reaction tank, which is surrounded by a gap between t311 and filled with liquid to the point where the inner cylinder is sufficiently submerged, and the outer cylinder and the inner cylinder. The immobilization carrier retaining part is provided to be able to retain and retain the immobilized carrier in a floating state, and the filling liquid is caused to flow in the axial direction of the inner cylinder within the cylinder of the inner cylinder. and liquid flow generation means for generating a vertical circulation flow rotating from the inside of the inner cylinder to the outside of the bay by the liquid flow.

In the operation of the bioreactor of the present invention, the pressure inside the outer cylinder is set to a gauge pressure of 5 kg/crr?・It must operate at less than G. b. The ratio of the inner diameter of the outer cylinder to the inner diameter d of the inner cylinder is d/D = 0.
2 to 0.6, and the ratio of the inner diameter d of the C0 inner cylinder to the axial length l of the inner cylinder is A/d≧6. many. Also, when biological reactions require aerobic conditions, it is preferable to provide greater oxygen dissolution in the liquid. For this purpose, it is appropriate to operate the apparatus under the conditions a.

Although the pressure conditions inside the outer cylinder (inside the device) are not an essential problem in the bioreactor of the present invention,
rr? - If the pressure exceeds G, there are problems such as the inability to use general-purpose air compressors, culture fluid delivery pumps, etc.

Also, d/D is the ratio of the inner diameter of the outer cylinder to the inner diameter d of the inner cylinder.
If it is less than 0.2, there is a problem that the inner cylindrical flow path becomes narrow and the discharge flow rate becomes small due to resistance.On the other hand, if it is more than 0.6, the impeller built in the inner cylinder becomes large and there is a problem that it is not economical. Therefore, it is often preferable for the device to be constructed within the above size range.

Furthermore, 1/d is the ratio of the inner diameter d of the inner cylinder to the axial length of the inner cylinder.
If it is less than 6, it is not preferable because there are problems such as the required capacity of the immobilization carrier becomes small and the amount of dissolved oxygen decreases when air is blown into the carrier.

In the present invention, the liquid filled in the reaction tank is a nutrient source or raw material necessary for microorganisms to maintain life and actively produce metabolites.

In addition, as a liquid flow generation means for generating a flow of the filled reaction liquid in the axial direction of the inner cylinder, a preferable example is one in which an axial flow pump is disposed within the inner cylinder. can.

The retention area of the immobilized carrier formed between the inner cylinder and the outer cylinder is, for example, a porous plate or net that prevents the carrier from passing through but allows the liquid to flow freely between the inner cylinder and the outer cylinder. It can be formed by arranging, etc.

In the present invention, various known immobilization carriers supporting microorganisms or enzymes can be used, such as agar media used for culturing microorganisms, bead-shaped carriers made of synthetic resin, etc. can.

In the retention section of the immobilized carrier, it is preferable to keep the immobilized carrier in a floating state so that the mixing characteristics in the reaction tank are completely mixed. A circulating flow in the vertical direction that generates a flow in the axial direction of the inner cylinder, thereby rotating from the inside of the inner cylinder to the outside of the cylinder, is provided as an upward liquid flow in the above-mentioned retention part. Good.

(Function) By having the above-described structure, the present invention can effectively ensure the frequency of contact between the immobilization carrier and the culture solution, and promote biological or biochemical reactions. Furthermore, it is possible to continue a stable and sustained reaction without damaging the immobilization carrier.

Furthermore, the mixing characteristics in the reaction tank can be obtained as a state of complete mixing, which facilitates application to scale-up of the device based on control of environmental factors and analysis of reaction results.

(Example) The present invention will be described below based on embodiments shown in the drawings.

Figure 1 conceptually shows the configuration of the bioreactor according to the present invention, and Figure 2 shows the outline of the device when two bioreactors are configured in series. .

In the example shown in these figures, a cylindrical inner cylinder 8 that is open up and down is fixedly installed inside the outer cylinder 7 forming the outer leg of the reaction tank of the bioreactor 1 as shown in the figures. ,
The inside of the inner cylinder 8 (hereinafter referred to as the inner passage 33)
An axial flow impeller 12 as a liquid flow generating device is installed at. By driving this axial flow impeller 12, a liquid flow that circulates up and down as indicated by arrows in the figure is generated in the reaction tank.

The reaction tank is maintained at an environmental temperature suitable for carrying out biological or biochemical reactions by a constant temperature device 4.

Between the outer surface of the inner tube 8 and the inner surface of the outer tube 7, there is defined a retention section 2 for retaining and retaining a microorganism (or enzyme) immobilization carrier 29, which is partitioned vertically by a porous plate 28. The liquid flows from the bottom to the top in the retention section 2 due to the liquid flow generated by the axial flow impeller 12,
As a result, the immobilized carrier floats and a completely mixed state is achieved. Therefore, good contact between the immobilization carrier (therefore, the immobilized microorganisms, etc.) and the filling liquid is ensured.

Note that 3 is a stock solution inflow pipe, and 4 is a reaction liquid delivery pipe. Further, 30 indicates a pH adjustment liquid injection pipe provided to maintain the pH state of the liquid in the reaction tank to an optimum environment for microorganisms and the like.

According to the bioreactor having the above configuration outline, the passage between the outer cylinder and the inner cylinder in the reaction tank (hereinafter referred to as the outer passage 32
The immobilized carriers, such as microorganisms, held in the retention part are held in a floating manner in the ring-shaped gap between the outer cylinder and the inner cylinder, and are suspended by the liquid flow rising from below and completely mixed. The effect of this is that good contact between the liquid and microorganisms is achieved.

Figure 2 shows a configuration in which three of the above-mentioned single-tank bioreactors are arranged in series. According to this format, biological (or Not only is it possible to carry out biochemical reactions, but it is also possible to combine reaction vessels filled with different types of microorganisms, etc., as necessary.

Fig. 3 shows the specific configuration of the device conceptually shown in Fig. 1 above, Fig. 4 is an enlarged view of the structure of the liquid flow generating device of the same device, and Fig. 5 The figure is an enlarged view of the upper part of the inner cylinder.

The bioreactor 1 of the example shown in these figures has a vertical cylindrical inner tube 8 that is concentric with the outer tube and opened up and down inside a reaction tank consisting of a vertical cylindrical outer tube. It is fixedly supported by the outer cylinder via support/baffle plates 9 which are arranged intermittently in the circumferential direction of the inner cylinder.

The inner cylinder 8 of this example is provided with a jaw part 10.11 formed in a bell mouth shape (or cone shape) toward the open end side at the upper and lower open parts, and the jaw part 10.11 is formed in a bell mouth shape (or conical shape) to prevent the liquid flow through the inner cylinder. It is designed to make the flow smooth.

Reference numeral 12 denotes an axial flow impeller disposed at the lower part of the inner cylinder 8, which is rotated by a motor 15 outside the tank in the direction of the arrow in FIG. It is designed to generate a flow. The axial flow impeller 12 also includes blades 13 having a flat cross section, and a hemispherical cap 14 is attached to the inflow side of the boss to allow liquid to flow smoothly through the flow blades. Note that the gap δ between the inner wall surface of the inner cylinder 8 and the rotating tip of the impeller blade (see Fig. 4)
is preferably made as small as possible within the allowable range in mechanical design in order to prevent backflow of liquid, and specifically, the inner diameter d of the inner cylinder 8 and the outer diameter da of the impeller blade 13. The ratio of d/d a≦1.01 is preferably selected.

The lower part of the inner cylinder 8 that surrounds the impeller has a structure in which a part prepared separately from the upper part (for example, the part indicated by reference numeral 8' in FIG. 4) is integrated by bolting or the like via a packing 18. This is because by doing so, only the lower part of the inner cylinder in which the impeller blades 13 are housed can be precision-machined, thereby facilitating the machining of the device. Note that the member indicated by the reference numeral 17 in FIG. 4 is a strut member for supporting the inner cylinder, which is disposed intermittently in the circumferential direction of the inner cylinder.

Reference numeral 20 denotes an air diffuser pipe, which is arranged at an appropriate location near the top of the inner cylinder and blows out air from a large number of air diffusers. This is used when biological (or biochemical) reactions need to be carried out in an aerobic atmosphere.

Reference numeral 21 denotes a rectifying plate disposed above the axial flow impeller 12 within the inner cylinder 8. This baffle plate is provided to prevent vortices that tend to occur in this type of device and reduce pump efficiency.

Reference numeral 3 designates a stock solution inflow pipe connected to a part of the upper part of the reaction tank, and 5 designates a reaction liquid delivery pipe connected to a part of the lower part of the reaction tank.

Note that 22 is a degassing pipe installed on the ceiling of the reaction tank,
It is opened to the atmosphere via a throttle valve 23. Then, the liquid outflow amount is given by pressurization by liquid feeding pressure from a liquid feeding pump (not shown) connected to the upstream side of the stock solution inflow pipe 3, and by the restriction of a throttle valve (not shown) connected to the downstream side of the delivery pipe. , and the balance of the throttle valve 23 connected to the degassing pipe 22, the pressure state inside the reaction tank is 5 kg/cr as mentioned above.
It is possible to set it to an appropriate value below rl-G.

Reference numeral 28 denotes a ring-shaped porous plate for forming a retention part for retaining and retaining the immobilization carrier 29, and is arranged as a partition at the upper and lower positions between the outer cylinder flap and the inner cylinder 8. has been done. This porous plate has a large number of suitable pores formed therein to prevent the immobilized carrier from flowing through it, but to allow the liquid to flow smoothly therethrough.

In the bioreactor of this example, manometers 24 and 25 are installed to detect the pressure conditions at appropriate locations inside the outer cylinder 7 and inside the inner cylinder 8, as shown in FIG. The manometer 24 is installed to detect the pressure at the same height as the inner cylinder upper jaw part 10 of the outer passage 32, and the manometer 24 is
5 is installed in the inner passage of the inner cylinder 8 so as to detect the pressure at a position downward from the upper end by the length dimension of the inner diameter d of the inner cylinder.

In the bioreactor with the above configuration, the points to keep in mind when carrying out biological or biochemical reactions are, firstly, the promotion of the biochemical reaction, and secondly, ensuring a sufficient amount of be.

In order to suitably realize these points in the bioreactor of this example, for example, the axial flow impeller 12 in FIG.
A configuration is preferably adopted in which the air diffuser tube 20 is arranged in the inner cylinder 8 where the liquid flows quickly, and the air diffuser holes are opened in the direction of the impeller. As a result, the air introduced into the liquid does not simply float to the surface, but quickly undergoes a stirring action and dissolves in the liquid, thereby making it possible to maintain a large amount of dissolved oxygen. Further, in order to increase the amount of dissolved oxygen, the inside of the reaction tank is preferably pressurized.

In addition, in order to improve the efficiency of contact between the microorganisms, etc. immobilized on the carrier and the liquid, it is preferable to circulate the liquid smoothly. The degree of opening of the bell mouth, etc. at the end of the inner cylinder (or a conical shape) is determined by the distance between the lower end of the inner cylinder and the bottom of the reaction tank, and the distance h between the upper end of the inner cylinder and the liquid level. of,
It is often preferable to set the inner diameter d of the inner cylinder 8 to 0.37 times or more. If it is 0.37 times or less, there is a risk that a large pressure loss will occur when the circulating flow rate passes through that part. There are problems such as

In addition, in order to maximize efficiency and save power while ensuring maximum flow rate, the impeller 12 in the bioreactor configured as shown in Fig. 3 is designed as a high-efficiency axial flow impeller, and the specific speed (rpm - J (m”/win)
/m'') is preferably 900 to 3000 (having a D-type cross section).

Example 1 (Preparation of lactic acid from sweet turgsum juice) (Preparation of immobilization carrier (gel beads)) Lactobacillus casei Variety casei (Lac
tobacJllus casej var case
i) in Brix (Br1gg5) medium at 37°C for 2
After culturing anaerobically for 0 hours, add 2.5 volumes of sodium alginate to 1 volume of the culture solution, and add 9 volumes of sodium alginate and 5% calcium chloride.
It was added dropwise to the solution to form spherical gel beads with a diameter of 3 to 4 mm.

(Device specifications) The bioreactor shown in Fig. 4 was configured as shown below with the specifications shown in Fig. 1.

Capacity of 1 tank: 800aj! Gel piece capacity 150mA Gel piece emptying rate 0.77 Gel bead diameter 3~4mlφ Axial flow pump: Göttingen type 3 blades Blade diameter 40mm Rotation speed 0~1100rp Operating conditions: Residence time 6. Lactic acid was produced under Ohr H7 temperature conditions of 35° C. or higher, and the results are shown in Table 1 below.

Table 1 The above results are shown in FIG. 6 as a relationship between the circumferential speed of the axial flow pump and the L-lactic acid production rate.

Since the circumferential speed and discharge volume of an axial flow pump are proportional to each other according to the pump similarity law, the number of times of contact between the gel beads and the culture medium is proportional, and from Table 1 and Figure 6 above, the L-lactic acid production rate is It can be seen that quantitative control is possible in approximately proportion to the circumferential speed of the axial pump.

Example 2 (Production of L-lactic acid from sweet turgid juice) Compared to Example 1 above, production of L-lactic acid was carried out under the same conditions as in Example 1 except that the equipment specifications were changed to the dual system shown in Figure 2. I did it.

The results for cattle are shown in Table 2 below in comparison with Example 1.

Table 2 As shown in Table 2 above, when the reaction conditions during the reaction were maintained, the same performance could be obtained over a duration of 400 h without any change in the production rate of lactic acid. Ta.

This shows that scale-up can be easily done if the performance is understood by testing a single oak.

This means that equipment design is extremely easy compared to the conventional method, which required pilot tests and commercial tests at each stage even after confirming the performance test with Jarfermenta. Combined with the advantage that equipment design on a large scale can be fully confirmed at the experimental quality level, its usefulness is extremely large.

Example 3 (Yeast: Satucharomyces luxii
s rouxii): production of acetic acid using immobilized bacterial cells) Saccharomyces rouxii (Danmu s rouxii) was cultured in YM-medium, and spherical gel beads were added to 1 volume of the culture solution in the same manner as in Example 1. adjusted.

Next, glucose 3%, ammonium sulfate 0.5%, dipotassium phosphate 0.085%, potassium phosphate 0.0
Medium (p) consisting of 15% magnesium sulfate, 0.05% magnesium sulfate, and trace components such as vitamins and inorganic ions.15.
5), acetic acid was produced using the equipment specifications of Example 1 while blowing air from the aeration pipe 20. The results are shown in Table 3 below and FIG.

Table 3 Generally, in bioreactors using aerobic bacteria, respiration is often affected by dissolved oxygen (Do).

In particular, in immobilized reaction systems, the immobilizing material inhibits the reaction
'The tendency to be rate-limited by IIA degrees will become even greater.

By the way, the results of Example 3 shown in Table 3 and Figure 7 above show that when acetic acid is produced using immobilized yeast cells and the pressure inside the reaction tank is changed, the pressure state It can be seen that acetic acid is produced approximately proportionally.

This is because when looking at the relationship between dissolved oxygen concentration Do and pressure, the concentration gradient ΔC of DO with respect to that pressure is given by ΔC=C5-CI, where Cs is saturated.

C1-Driving.

According to the formula, Cs increases in proportion to the pressure inside the reaction tank, so as a result, the oxygen supply capacity becomes ■ pressure, and it is inevitable that an amount of oxygen can be supplied into the bioreactor according to the pressure. This means that the rate of acetic acid production is proportional to pressure.

The results of Example 3 show that the production volume can be increased in the same bioreactor by increasing the pressure inside the reaction tank. Since the amount of dissolved oxygen can be increased by setting the relationship between the impeller and the air blowing position appropriately, it is not necessary to increase the blowing strength, or the degree of necessity can be reduced. This shows that the foaming and droplet entrainment that had previously occurred have been reduced as much as possible, making it extremely useful as an industrial device.

(Effects of the Invention) As described above, the bioreactor according to the present invention is divided into a liquid flow generation mechanism that stirs and flows a reaction solution through an immobilized carrier on which microorganisms and enzymes are immobilized, and a retention area for the immobilized carrier. Therefore, it is possible to maintain the immobilized carrier in an appropriate floating state without damaging it and carry out a stable reaction over a long period of time, and also to allow the reaction solution to flow sufficiently into contact with the immobilized carrier in the floating state. The effect of high reaction volumetric efficiency can be obtained by using a large-sized container.

In addition, the flow characteristics of the liquid in the reaction tank are governed by the law of similarity of an axial flow pump, which is a liquid flow generator that generates an axial liquid flow in an inner cylinder, so experiments using a small jar fermentor were conducted. It is easy to design a scaled-up device based on the analysis results of laboratory-level tests, and it also has the effect that it is easy to design an industrial-scale device and can be performed precisely.

Furthermore, the mixing characteristics in the reaction tank according to the present invention are that it is a complete mixing tank with a circulating mixed flow, and it is easy to adjust reaction control factors to the same conditions, and select microbiologically suitable reaction conditions. It also has the effect of being suitable for

[Brief explanation of the drawing]

Figure 1 conceptually shows the configuration of the bioreactor according to the present invention, and Figure 2 shows the outline of the device when two bioreactors are configured in series. . Fig. 3 shows the specific configuration of the device conceptually shown in Fig. 1 above, Fig. 4 is an enlarged view of the structure of the liquid flow generating device of the same device, and Fig. 5 The figure is an enlarged view of the upper part of the inner cylinder. Figure 6 is a diagram showing the relationship between the lactic acid production rate and the rotation speed of the axial flow impeller in Example 1, and Figure 7 is a diagram showing the relationship between the acetic acid production rate and the pressure inside the reaction chamber in Example 3. be. 1: Bioreactor 2: Retention part (gap) 3: Inflow pipe
4: Constant temperature device 5: Delivery pipe 6: Throttle valve 7: Outer cylinder 8: Inner cylinder 9: Support and baffle plate 10. jl: Jaw 12: Axial flow impeller 13: Blade 14: Hemispherical cap 15: Motor 16 two-rotation shaft 17: Support member 18: Packing 19: Air diffuser hole 20: Air diffuser pipe 21: Straightening plate 22: Deaeration pipe 23: Throttle Valve 24, 25: Manometer 26: Outer passage 27: Inner passage 28: Support plate 29: Immobilization carrier 30: pH adjustment liquid injection tube

Claims (1)

[Claims] 1. A vertical cylindrical inner cylinder that is open at the top and bottom, surrounding this inner cylinder with a sufficient gap, and filled with liquid to the point where the inner cylinder is sufficiently submerged. an outer cylinder as an outer shell of a reaction tank, and an immobilization carrier retaining part provided between the outer cylinder and the inner cylinder so that the immobilization carrier on which enzymes or microorganisms are immobilized can be retained in a floating state. , causing the filling liquid to flow in the axial direction of the inner cylinder within the cylinder of the inner cylinder, and this liquid flow in the axial direction of the inner cylinder generates a vertical circulation flow that rotates from the inside of the inner cylinder to the outside of the cylinder. A biological reaction device characterized by comprising: a liquid flow generating means for generating a liquid flow. 2. The biological reaction device according to claim 1, wherein the generated liquid flow flows from below to above within the retention section of the immobilization carrier.