JPH0361431B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a continuous fermentation process carried out using immobilized growing microorganisms. More specifically, when producing useful substances by a continuous fermentation method using at least two reactors, the immobilized growing microorganisms in at least one reactor whose production activity has decreased are reactivated to achieve continuous production and reactivation. It relates to a method of performing processing simultaneously.

BACKGROUND OF THE INVENTION Research has been conducted on methods for continuous production of useful substances such as alcohols, organic acids, amino acids, antibiotics, steroids, enzyme proteins, hydrogen, and methane by continuous fermentation using immobilized growing microorganisms. In this method, a reactor is filled with immobilized microorganisms that are immobilized on a carrier such as a natural polymer or synthetic resin, and a medium is supplied to the reactor to allow the immobilized microorganisms to grow while continuously producing useful substances. This is the way to do it. When using this method, a large amount of microorganisms can be immobilized, as well as continuous microbial growth and moderate metabolism due to partial leakage, which suppresses the decrease in the number of immobilized microorganisms and maintains high enzyme activity. Therefore, the production rate of the substance is much faster than in normal fermentation methods, and there is no risk of so-called washout. Therefore, the fermentation time can be significantly shortened, and the reactor can be made smaller and more efficient.

However, when production lasts for a long time, the death rate of immobilized microorganisms exceeds the growth rate due to lack of nutrients in the culture medium, inhibition by products, changes in pH, etc., and the number of microorganisms gradually decreases. A phenomenon occurs in which the production activity of . It is known that when immobilized microorganisms in such a state are cultured again in a medium suitable for growth, they will proliferate and their production activity will be restored.

However, such reactivation treatment requires temporary interruption of continuous production, reducing production efficiency.

The present inventors have completed the present invention as a result of intensive research to develop a method that can perform reactivation treatment without interrupting production.

That is, the present invention provides a method for producing useful substances by a continuous fermentation method using an apparatus in which at least two reactors filled with immobilized growing microorganisms are connected, and the production activity of the immobilized microorganisms in each reactor is reduced. Continuous fermentation using immobilized proliferating microorganisms, characterized in that the interconnection state of reactors is changed to perform continuous fermentation production while constantly reactivating at least one reactor whose production activity has decreased, depending on the degree of Regarding the law.

As described above, in the present invention, since the reactivation treatment of immobilized microorganisms with reduced production activity and the continuous fermentation production are carried out in parallel, there is no interruption in production as a whole. In addition, by increasing the number of reactors used, products can be obtained steadily over a long period of time.

The method of the present invention can be applied to a reactor in which reactors are connected in parallel or in series. Moreover, conventional reactors such as a complete mixing tank type, a fluidized bed type, and a packed tower type can be used as a reactor as they are, and the culture medium may be distributed by either a downward flow type or an upward flow type. The number of reactors is usually 2
~10 pieces is appropriate, but the type of reactor, type of immobilized growing microorganism, type of supply medium, supply rate of medium, type of product, reaction temperature, amount of aeration, number of stirring,
The appropriate number may be determined depending on the pH of the culture medium, etc.

Next, embodiments in which the method of the present invention is applied to multi-stage serial type and multi-stage parallel type continuous fermentation apparatus will be described, but the present invention is not limited only to such embodiments.

Multi-stage series continuous fermentation uses a device consisting of at least two reactors filled with immobilized growing microorganisms connected in series, and a production medium is continuously supplied to at least the first reactor to produce useful substances. When producing a reactor, the state of connection between the reactors is sequentially changed so that the reactor in the last stage becomes the first reactor after a predetermined period of time has elapsed.

When using such a multistage series continuous fermentation method,
Since the last stage reactor with reduced production activity becomes the first reactor after a predetermined period of time has passed, not only can the immobilized microorganisms that are becoming extinct be propagated, but also the reactor can be used for continuous production. Reactivation processing and continuous production can be performed at the same time without leaving the reactor idle during reactivation, and continuous production of useful substances can be performed stably and efficiently over a long period of time.

For example, using a device in which n reactors No. 1 to No. n are connected in series such that the No. 1 reactor is the first and the No. n reactor is the final stage, the first reactor is connected to the production medium. When the product is taken out from the final stage reactor, the immobilized microorganisms in the final stage reactor (No. production activity decreases. On the other hand, in the first reactor, the substrate and other nutrients in the culture medium are abundant, and the concentration of products is low, so microorganisms are provided with an environment in which they can sufficiently proliferate. Therefore, when the immobilized microorganisms in the final stage reactor (No. n) weaken and begin to die, the supply of culture medium is switched from the No. 1 reactor to the No. A reactor is used, and the connection states of other reactors are sequentially shifted by one stage, that is, the second stage is No. 1, the third stage is No. 2, and the final stage is No. n-1. By switching the state of connection between the reactors, the immobilized microorganisms in the No. No. reactor that were dying out can grow again and regain production activity. By repeating this switching operation, useful substances can be stably produced over a long period of time without interrupting continuous production.

The timing of switching the connection state varies depending on the number of stages, the growth rate and death rate of the microorganism used, the production activity, the substrate concentration in the medium, etc., and may be determined according to the specific conditions during production. For example, once the microorganisms to be used, the number of stages, and the substrate concentration in the medium are determined, the number of immobilized microorganisms in the final stage reactor during production and/or the concentration of the product in the reaction completion solution are detected and the concentration is determined to be below a predetermined level. When this happens, a switching operation may be performed, or a preliminary experiment may be conducted under the same conditions, and the switching timing may be set based on the results. Switching may be performed regularly or irregularly.

The number of stages, that is, the number of reactors, is not particularly limited as long as it is two or more, but as the number of stages increases, the switching operation must be performed more frequently, but the concentration of the product in the reaction finished liquid immediately after switching is less likely to decrease. On the other hand, when the number of stages is small, the product concentration in the reaction-completed liquid immediately after switching is greatly reduced, but the switching interval can be made longer.

When a packed tower type or fluidized bed type reactor is used as the reactor in this multi-stage serial continuous fermentation method, if a reflux tube is attached to circulate the medium, it can approach a complete mixing tank type and increase production efficiency.

A training tank filled with a carrier that adsorbs and holds microorganisms may be provided downstream of the reaction liquid outlet of the reactor in order to adsorb and hold the grown microorganisms contained in the reaction effluent. Examples of carriers used include sponges, fine metal nets, saran nets, glass beads, and synthetic resin beads. When a ripening tank is provided, not only can microorganisms be removed from the effluent, but also the retained microorganisms can further convert the substrate into product.

Depending on the case, a small amount of air (oxygen) may be passed through the reactor to promote the growth of microorganisms.

Next, the multi-stage serial continuous fermentation method will be explained based on the drawings. FIG. 1 is a schematic block diagram of a multi-stage serial production device using one type of production medium.
A, B, and C are all reactors, and a production medium supply line 10 is connected to these reactors, and they are interconnected by a connecting line 11 that sequentially supplies the reaction liquid of the preceding reactor to the next reactor. ing. In addition, each reactor A, B, and C have an on-off valve 13 and a reaction-finished liquid discharge line 12, respectively.
They are connected via a, 13b, and 13c. Production medium supply line 10 and each reactor A, B, C
On-off valves 14a, 14b, and 14c are provided between the reactors A and B, and an on-off valve 15a is provided on the connection line between reactors A and B, and an on-off valve 15b is provided on the connection line between reactors B and C. Opening/closing valves 15c are provided in the connecting lines between reactors C and A, respectively.

Using this device, the on-off valve 14a of the production medium supply line 10 is opened, the on-off valves 14b and 14c are closed, the on-off valves 15a and 15b of the connection line 11 are opened and the on-off valve 15c is closed, and the reaction finished liquid discharge line 1
When the on-off valves 13a and 13b of No. 2 are closed and the on-off valve 13c is opened, the production medium is supplied to the reactor A, passes through the reactor B, and the reaction finished liquid is obtained from the reactor C.

When the reactors are connected in series in this manner, the first-stage reactor A has an environment in which the culture medium is rich in nutrients and has a low substrate concentration, so that the immobilized microorganisms can sufficiently grow. On the other hand, since the immobilized microorganisms in the final stage reactor C are exposed to a high concentration of the product, the growth of the microorganisms is inhibited, and as a result, the death rate exceeds the growth rate and the number of immobilized microorganisms decreases. A decrease in production activity occurs.

When the production activity falls below a predetermined level, the on-off valve 1
3c, 14a, 15b, and open/close valves 13b, 1
Just open 4c and 15c. This switching causes reactor C to become the first stage and reactor B to become the last stage. As described above, the immobilized microorganisms in the first reactor are in an environment suitable for growth and are reactivated. By sequentially switching the final stage reactor to the first reactor, the immobilized microorganisms can be reactivated simultaneously with continuous production. In this way, the multi-stage series continuous fermentation method does not particularly require reactivation and growth media, and the immobilized microorganisms in the reactivated state are also involved in production, so production efficiency is extremely high. It gets expensive.

In fermentations where not only the above-mentioned product inhibition but also substrate inhibition is observed, a medium with a low substrate concentration is supplied in the first stage to create an environment that further promotes growth, and after the substrate is consumed, A multi-point culture medium supply system may be adopted in which medium with a high substrate concentration is supplied in appropriate amounts. An embodiment in which the method of the present invention is applied to such a system will be described based on FIG. 2. In FIG. 2, parts with the same reference numerals as in FIG. 1 indicate the same parts as in FIG. 1.

A medium with a low substrate concentration is supplied from a supply line 10, and a medium with a high substrate concentration is supplied from a supply line 16. Supply line 16 connects each reactor A, B, C.
and are connected via on-off valves 17a, 17b, and 17c, respectively.

At the start of production, a low substrate concentration medium is supplied to reactor A, a high substrate concentration medium is supplied to reactor B, and each on-off valve is opened and closed so that the reaction completion liquid is taken out from reactor C. That is, the on-off valves 13a, 13b,
14b, 14c, 15c, 17a, 17c are closed, on-off valves 13c, 14a, 15a, 15b, 1
Open 7b.

When the production activity of the immobilized microorganisms in reactor C falls below a predetermined level, the on-off valve is switched to make reactor C the leading reactor. That is, the on-off valves 13c, 14a, 15b, and 17b are closed, and the on-off valves 13b, 14c, 15c, and 17a are opened. Change the connection state like this, i.e. change the first reactor from A→C→B→A..., change the second stage reactor from B→A→C→B..., and change the last stage reactor to C.
→B→A→C... When switching from B to A to C is performed depending on the degree of decrease in production activity of immobilized microorganisms in the final stage reactor, reactivation treatment of microorganisms where product inhibition and substrate inhibition are problems is necessary. can be done efficiently.

Next, an embodiment in which the method of the present invention is applied to a multistage parallel continuous fermentation method will be described based on the drawings.

FIG. 3 shows a schematic block diagram of a multistage parallel continuous fermentation apparatus. A, B, and C are all reactors, and are connected in parallel by a production medium supply line 21 and a reaction liquid discharge line 22. A growth medium supply line 23 is further connected to each reactor A, B, and C, and a growth medium discharge line 24 is also connected thereto. The production medium supply line 21 and the growth medium line 23 are provided with on-off valves 25a, 25b, 25c and 26a, respectively.
26b and 26c are provided, and the supply of culture medium to each reactor can be switched arbitrarily.

Continuous fermentation is started by supplying production medium from line 21 to reactors A, B, and C.
The immobilized microorganisms in each reactor maintain their production activity above a certain level for a certain period of time, but then their production activity gradually decreases. When the production activity of the immobilized microorganisms in all the reactors or in any one reactor falls below a predetermined level, the reactor filled with the immobilized microorganisms whose production activity has decreased is switched to reactivation treatment. here,
Assuming that the reactor is reactor A, the on-off valves 25a, 25b, 25c are in the open state.
Of these, the on-off valve 25a is closed to stop the supply of the production medium to the reactor A, and the on-off valve 26a of the on-off valves 26a, 26b, and 26c which are in the closed state is opened to supply the growth medium to the reactor A. is supplied to reactivate the immobilized microorganisms in reactor A. Growth medium effluent exits line 24. In this state, reactors B and C are used as they are for continuous production.

When the reactivation process of the immobilized microorganisms in the reactor A is completed, the on-off valve 26a is closed and the on-off valve 25a is opened to restart production. On the other hand, reactors whose production activity is decreasing at that time, such as reactor B
The reactor B is reactivated by closing the on-off valve 25b and opening the on-off valve 26b.

In this way, the on-off valves 25a, 25b, 25c of the production medium supply line 21 and the on-off valves 26a, 26b, 26c of the growth medium supply line 23 are opened and closed according to the degree of decrease in the production activity of the immobilized microorganism in the reactor. However, stable and long-term continuous production is possible by always sequentially reactivating at least one reactor and using the remaining reactors for continuous production.

The growth medium varies depending on the immobilized microorganism used, and a medium with optimal conditions for the growth of the microorganism may be selected. Such conditions include, for example, nutrient concentration, dissolved oxygen concentration, pH, temperature, and the like.

Switching from continuous production to reactivation treatment and vice versa may be accomplished by measuring the number of immobilized microorganisms and/or product concentration. The degree of production activity varies depending on the type of immobilized microorganism used, the type of supply medium, the supply rate of the medium, the type of product, the reaction temperature, the amount of aeration, the number of stirring, the pH of the medium, etc., and depends on the specific system. The switching timing may be set as appropriate. Alternatively, the optimum switching timing may be determined through preliminary experiments, and each reactor may be sequentially reactivated periodically or irregularly at the switching interval.

As the immobilized microorganism that can be used in the method of the present invention, any microorganism that can be used for continuous fermentation can be employed. Preferred immobilized growing microorganisms include, for example, sulfate group-containing polysaccharide gels such as agar gel, carrageenan gel, and furcerelan gel, alginate alkaline earth metal salt gel (e.g., calcium alginate), polyvinyl alcohol gel, polyacrylic acid amide gel (e.g. N,N'-lower alkylene-polymer or copolymer of one or two monomers selected from bis(acrylamide), bis(acrylamide methyl) ether, and acrylamide), cellulose succinate gel, casein gel, and other gel carriers. Various types of microorganisms encapsulated therein may be mentioned, and those encapsulated in carrageenan gel or calcium alginate gel are particularly preferred. The amount of microorganisms included in the gel is not particularly limited, but it is generally preferable that the amount is equivalent to 0.01 to 10 platinum loops per 100 g (wet weight) of the gel, and the shape of the gel is 1 mm to 1 mm thick. Preferably, it is shaped into 5 cm granules, cubes, threads or membranes.

Conventionally known methods can be used to prepare these immobilized and grown microorganisms, and even if sulfate group-containing polysaccharide gel and alginate-immobilized microorganisms are
Polyvinyl alcohol gel was prepared using the method described in Publication No. 18867 on Paper at 5thInt.
Acrylamide gel-immobilized microorganisms can be obtained by the method described in Ferment.
Microbiol. 27 , 878 (1974), etc., and cellulose succinate gel or casein gel-immobilized microorganisms were prepared using J. Solid-Phase.
It can be suitably prepared by the method described in Biochem. 2 , 225 (1977). The production medium and/or growth medium used in the present invention is not particularly limited as long as it contains nutrients and substrates necessary for the growth of microorganisms. , may be used in appropriate combinations depending on the type of microorganism that uses the vitamin.

Next, the method of the present invention will be explained with reference to Examples, but the present invention is not limited to these Examples.

In addition, in order to specify the reactor used in each example, for convenience, the first reactor in the first connected state is designated as No. 1, and hereinafter No. 2, No. 3... No. n
It is numbered.

Example 1 70ml filling tower type reactor (diameter 4cm, height
Ethanol was continuously produced using a multi-stage series ethanol production device in which two 5.5 cm cylinders were connected in series (see Figure 1 for the connection state).

As an immobilized microorganism, a loopful of yeast No. 7 of the Association was added to 100 ml of a 5% carrageenan aqueous solution at 37°C, mixed, and this mixture was dropped into 500 ml of a 2% potassium chloride aqueous solution from a nozzle to form a 4 mm diameter tube. A spherical gel was used.

Fill 20ml of this spherical gel into two reactors, and prepare a molasses aqueous solution (PH5.0) containing 10% reducing sugar.
Pre-growth was carried out by feeding the cells at a flow rate of 40 ml/hr at 30°C for 48 hours. During this time, air was passed through at a flow rate of 4/hr.

After preliminary growth, the two reactors were connected in series, and an aqueous molasses solution (PH5.0) containing 20% reducing sugar was supplied to the first reactor (No. 1) at 30°C and at a flow rate of 13 ml/hr. The reaction completed liquid was obtained from the reactor in .2. Five days after the start of production, the connection state of the reactor was changed and No.
The molasses aqueous solution was supplied to the No. 2 reactor, and the reaction completed liquid was obtained from the No. 1 reactor. When this switching was repeated every 5 days, a completed reaction solution with an ethanol concentration of 80 to 85 mg/ml was stably obtained for more than 3 months.

Note that each column refluxed the reaction solution at a flow rate of 300 ml/hr.

As for the timing of switching, we decided to take 5 days as the reference point, since preliminary experiment results showed that the concentration would drop to 80 mg/ml or less in about one week.

Example 2 70ml fluidized bed reactor (bottom bottom 3.2cm, top bottom
Ethanol was continuously produced using a device in which four inverted conical units (4.8 cm and 5.5 cm high) were connected in series (see Figure 2 for the connection state). The same immobilized microorganisms as in Example 1 were used, and 20 ml of the microorganisms were filled into each reactor, and after preliminary growth in the same manner as in Example 1, four reactors were connected in series.

The culture medium is supplied by supplying a molasses aqueous solution (PH5.0) containing 10% reducing sugar to the first reactor at a flow rate of 20 ml/hr, and supplying a molasses aqueous solution (PH5.0) containing 40% reducing sugar to the second reactor. 0) at a flow rate of 5 ml/hr,
A multi-point culture medium supply system was adopted in which the reaction-completed solution was obtained from the fourth stage reactor. Note that the first reactor and the second stage reactor refluxed the reaction solution at a flow rate of 300 ml/hr.

Check the connection status of the reactor every two days at No. 1 (top) →
No.2 → No.3 → No.4, No.4 (first) → No.1 → No.2 →
No. 3, No. 3 (first) → No. 4 → No. 1 → No. 2, etc. When switching as follows, the reaction temperature was 80 to 85 at 30℃.
A reaction-finished solution with an ethanol concentration of mg/ml was stably obtained from the fourth stage reactor for more than 3 months.

In addition, the switching timing was determined based on the results of preliminary experiments, based on 2 days, which is the limit at which the viable number of immobilized microorganisms in the last stage reactor does not decrease.

In addition, in this example, if the switching is not performed, the ethanol temperature will decrease to 75% in one week.
mg/ml, decreased to 70 mg/ml in 2 weeks and 40 mg/ml in 1 month.

Example 3 Immobilized microorganisms were prepared, pre-propagated, and reactors were connected in the same manner as in Example 1, except that four fluidized bed reactors of the same type as in Example 2 were used.

The culture medium was supplied only to the first reactor with a molasses aqueous solution (PH5.0) containing 20% reducing sugar at 30% concentration.
The reaction was carried out by supplying at a flow rate of ml/hr, and the reaction completed liquid was obtained from the fourth stage.

The connection state of the reactor was changed every day in the same manner as in Example 2. In addition, air was supplied only to the first reactor at a rate of 250 ml/hr.

As a result, a reaction completed solution with an ethanol concentration of 80 to 85 mg/ml was obtained stably for more than 3 months.

Note that the switching timing was determined based on the results of preliminary experiments, based on one day, which is the limit at which the viable number of immobilized microorganisms in the last stage reactor does not decrease.

Example 4 10 ml of carrageenan gel immobilized with Kyokai No. 7 yeast, which was prepared in the same manner as in Example 1, was placed in a sarannet basket and placed in a 30 ml complete mixing tank reactor (diameter 3 cm, height 4.2 cm). (cylindrical) were filled into 8 pieces. A molasses aqueous solution (PH5.0) containing 10% reducing sugar was supplied to each reactor at 30°C at a flow rate of 20 ml/hr for 48 hours, and pre-growth was performed while stirring with a magnetic stirrer.

After preliminary growth, eight reactors were connected in series (see Figure 2 for the connection state), and a molasses aqueous solution (PH5.0) containing 15% reducing sugar was poured into the first reactor at a rate of 12 ml/hr.
A molasses aqueous solution (PH5.0) containing 40% reducing sugar was added at 1.1 ml/hr in the second and third stages, and a molasses aqueous solution (PH 5.0) containing 40% reducing sugar was added in the fourth and fifth stages.
5.0) was supplied at 2.2 ml/hr.

When the connection status of the reactors was switched every day so that the last stage reactor became the first reactor, the reaction finished liquid with an ethanol concentration of 95 to 100 mg/ml was stable for more than 3 months at a reaction temperature of 27 °C. I got it.

The switching timing is determined from 1 to 1 based on the results of preliminary experiments.
I found out that it would be less than 95 mg/ml in 2 days, so 1
Determined based on the day.

Example 5 Association No. 7 yeast with 2% glucose and 0.5 peptone
%, yeast extract 0.3%, malt extract included, PH5.0
Preculture was carried out in conditioned medium 2 at 30° C. for 24 hours. Add the obtained preculture solution to 5% carrageenan aqueous solution.
20 were mixed at 43 to 44° C., and this mixed solution was dropped from a nozzle into a 2% potassium chloride aqueous solution 100 to prepare a spherical gel with a diameter of 4 mm. By repeating this operation, the resulting gel was filled with 700 volumes each of 200 gels (cylindrical shape with a diameter of 0.7 m and a height of 1.8 m).
A molasses aqueous solution (PH5.0) containing 15% reducing sugar was supplied at 27℃ for 48 hours at a flow rate of 150/hr, and air was aerated at a rate of 1.5 Kl/hr. Pregrowth was performed by refluxing at hr.

After preliminary growth, two reactors were connected in series (see Figure 1), and the first reactor was charged with a molasses aqueous solution (PH5.0) containing 0.1% (NH ₄ ) ₂ SO ₄ and 19% reducing sugar at 27°C. ℃ and a flow rate of 150/hr. In addition, air was vented to the first reactor at 7.5 Kl/hr and to the second stage reactor at 1.5 Kl/hr, and the reaction liquid was refluxed at 200 Kl/hr in each reactor.
A 700 volume aging tank filled with 200 10 cm square urethane sponges was provided downstream of the outlet after the reaction was completed.

When the mutual connection state of the reactors was changed every 1 to 3 days, a reaction-completed solution with an ethanol concentration of 80 mg/ml was stably obtained at a reaction temperature of 27°C for more than 3 months.

Note that the switching timing was determined based on the results of preliminary experiments, based on 1 to 3 days, depending on the ethanol concentration in the effluent and the decrease in the viable number of immobilized microorganisms.

Example 6 L-arginine was continuously produced using a continuous fermentation apparatus shown in FIG. 3 in which three reactors were connected in parallel. As the reactor, a 70 ml bubble tower type reactor (inverted conical shape with a lower base of 3.2 cmφ, an upper base of 4.8 cmφ, and a height of 5.5 cm) was used.

As the immobilized microorganism, one platinum loop of Serratia marsetuses AT-531 (Feikoken Bacterium No. 6051) was used.
Add to 100 ml of 3% carrageenan aqueous solution and mix at 40℃, and mix this mixture with 500 ml of 2% potassium chloride aqueous solution.
Each reactor was filled with 20 ml of spherical gel by dropping it from a nozzle into a spherical gel having a diameter of 4 mm.

While aerating oxygen gas at 10/hr each reactor,
Pre-growth in 3 reactors with 3% sugar blocking, 1% ammonium fumarate, 0.3% urea, 0.1 yeast extract.
%, corn staple liquor 0.1%, dipotassium phosphate 0.1%, magnesium sulfate 0.035% growth medium (PH 7.0) at 30°C at a flow rate of 15 ml/hr.
This was done by feeding for 24 hours.

Then, by switching the on-off valve, the growth medium was directly supplied to reactor A, and to reactors B and C, 5% sugar blocking, 2% ammonium fumarate, and 1.5 urea were added.
%, yeast extract 0.1%, cornstarch liquor
A production medium (PH7.0) containing 0.1% dipotassium phosphate, 0.1% magnesium sulfate, and 0.035% magnesium sulfate was supplied at 30° C. for 24 hours at a flow rate of 7 ml/hr. After 24 hours, the on-off valve was switched to supply the growth medium to reactor B and the production medium to reactors A and C. Oxygen gas was bubbled through each reactor at 10/hr. From then on, switch the on-off valve every 48 hours and change the growth medium from C→A→
A reactivation treatment was performed by supplying B in this order. As a result, 10 mg/ml of L-arginine was stably obtained for one month by supplying the production medium at a rate of 14 ml/hr.

The timing for switching to the reactivation treatment was based on the results of preliminary experiments when the amount of L-arginine in the reaction-completed solution became 10 mg/ml or less.

Example 7 70 ml filling tower type reactor (diameter 4 cm, height
Ethanol was continuously produced using a multi-stage serial continuous fermentation device in which five 5.5 cm cylinders were connected in series (see Figure 2 for the connection state).

As the immobilized microorganism, one platinum loop of Yeast No. 7 of the Association was heated at 37°C in a 2% sodium alginate aqueous solution.
ml and mixed, and this mixture was dropped from a nozzle into 500 ml of a 0.1 M calcium chloride aqueous solution to form a spherical gel with a diameter of 1 to 3 mm.

20 ml of this spherical gel was filled into five packed tower reactors, and a molasses aqueous solution (PH5.0) containing 10% reducing sugar was supplied at a flow rate of 40 ml/hr at 30°C for 48 hours for preliminary growth. After this, the reactors were connected in series.

Next, a molasses aqueous solution (PH5.0) containing 10% reducing sugar was supplied to the first reactor at a flow rate of 24 ml/hr.
40% each for second and third stage reactors
3ml/hr of molasses aqueous solution (PH5.0) containing reducing sugar
The reaction-completed liquid was obtained from the fifth stage reactor.

The connection state of the reactor was changed every 24 hours as follows. In other words, if the five reactor numbers are No. 1 to No. 5, the first connection is No. 1.
(1st row) → No.2 → No.3 → No.4 → No.5, then No.5 (1st row) → No.1 → No.2 → No.3 → No.4,
Changed as No. 4 (1st row) → No. 5 → No. 1 → No. 2 → No. 3...

As a result, 80 to 85 mg/ml of ethanol was stably obtained for more than 3 months at a reaction temperature of 30°C.

Based on the results of preliminary experiments, the switching timing was set so that the ethanol concentration would not fall below 80 mg/ml.

If the switch is not made, the ethanol concentration will be 75 mg/ml after one week and 70 mg/ml after two weeks.
mg/ml, and one month later it was below 40 mg/ml.

Example 8 L-lactic acid was continuously produced using a serial continuous fermentation apparatus in which four packed tower reactors of the same type as the reactor used in Example 7 were connected in series as shown in FIG.

As immobilized microorganisms, Streptococcus
A platinum loop of Lacteis AHU1192 was added to 100 ml of a 5% carrageenan aqueous solution at 40°C, mixed, and this mixture was dropped into 500 ml of a 2% sodium chloride aqueous solution from a nozzle to form a spherical gel with a diameter of 4 mm. Each reactor was filled with 20 ml.

Pre-growth was carried out by feeding a medium containing 3% lactose, 2% yeast extract, 1% peptone and 0.5% dipotassium phosphate (PH 7.0) at a flow rate of 15 ml/hr at 37°C for 72 hours. I did it.

Next, four reactors (No.1~
The medium was supplied to the No. 1 (first stage) reactor of No. 4) at a flow rate of 7 ml/hr at 45°C, and
Stage) Reaction completed liquid was obtained from the reactor. Thereafter, the connections between the reactors were switched every 24 hours as follows. In other words, No. 4 (first row) → No. 1 → No. 2 → No.
3. No. 3 (1st row) → No. 4 → No. 1 → No. 2, etc.

As a result, 20 to 25 mg/ml of L-
Lactic acid was stably obtained for more than one month.

Based on the results of preliminary experiments, the switching timing was set so that the lactic acid in the reaction effluent did not fall below 20 mg/ml.

[Brief explanation of drawings]

1 to 3 are schematic block diagrams of embodiments of continuous fermentation equipment used to carry out the method of the present invention. (Main symbols in the drawing), 10, 21: Production medium supply line, 11: Connection line, 12, 22: Reaction finished liquid discharge line, 16: High substrate concentration medium supply line, 23: Growth medium supply line, 24 : Growth medium discharge line, A, B, C: Reactor.

Claims (1)

[Scope of Claims] 1. When producing useful substances using two or more reactors by a continuous fermentation method using immobilized growing microorganisms, continuous fermentation is performed depending on the degree of decrease in the production activity of the immobilized microorganisms in each reactor. A continuous fermentation method using immobilized growing microorganisms, characterized in that the interconnection state of reactors is changed to perform continuous fermentation production while reactivating at least one reactor whose production activity has decreased. 2 At least two reactors filled with immobilized growing microorganisms are connected in series, and a production medium is continuously supplied to at least the first reactor. The method according to claim 1, characterized in that the interconnection state of the reactors is sequentially changed so that the reactor in the stage becomes the first reactor. 3. The method according to claim 1, wherein the medium is supplied to at least two reactors. 4. The method according to claim 1, wherein the reactor is a packed column type, fluidized bed type, or complete mixing tank type reactor. 5. The method according to claim 1, wherein an aging tank filled with a carrier capable of adsorbing and retaining microorganisms is provided downstream of the reaction liquid outlet of the reactor. 6. The method according to claim 1, wherein the degree of reduction in production activity is determined based on the killing rate of the immobilized proliferating microorganism. 7. The method according to claim 1, wherein the degree of reduction in production activity is determined based on the concentration of the product.