JPS5830030B2 - Fermentation method and fermentation equipment - Google Patents

Abstract

PURPOSE:In the aerobical cultivation of microorganisms by counter-currently contacting the oxygen-containing gas with the cultivation medium, the efficiency of the liquid/gas contact is improved by dividing the fermentation column with perforated plates.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing microorganisms aerobically by bringing a medium into countercurrent contact with an oxygen-containing gas, and to an apparatus directly used for carrying out the method.

In recent years, the global shortage of proteins has become chronic, and this trend is expected to increase in the future, and research is actively being conducted on the large-scale industrial production of new proteins using fermentation methods. .

In order to commercialize protein production using this fermentation method,
From the viewpoint of resource and energy conservation, there is a need to fundamentally improve conventional fermentation methods, and it is also necessary to be able to easily scale up the equipment.

The present invention provides a fermentation method and a fermentation apparatus that can meet these demands, and particularly provides a fermentation apparatus characterized by extremely low power consumption required for producing microbial proteins.

Conventional culture tanks do not have an internal structure that allows them to divide the tank into two or more smaller tanks containing culture solutions with different microbial concentrations, so the culture solution inside the tank is almost homogeneous. It has become.

However, in such a culture tank, it is not easy to increase the concentration of microorganisms in the culture solution in order to make it easier to separate the microorganisms from the culture solution taken out from the culture tank.

In other words, by increasing the concentration of microorganisms, the physical properties of the culture solution change, reducing the oxygen absorption capacity and increasing the amount of oxygen required by the microorganisms per culture solution, resulting in an oxygen-deficient state for the microorganisms in the culture tank. As a result, the activity of microorganisms decreases, which actually worsens the productivity of the culture tank.

Therefore, in a conventional culture tank, only a culture solution with a concentration of microorganisms corresponding to the oxygen absorption capacity of the culture tank can be taken out.

In order to increase the concentration of microorganisms, it is necessary to increase the oxygen absorption capacity, and to do so, a considerable amount of power must be input.

The most commonly used culture tank is the 1st one.
As shown in the figure, there is an aeration stirring tank that improves gas-liquid contact between the culture medium and the gas supplied from the ventilation pipe 26 by rotating the stirring blades 25.

This requires a large amount of power due to aeration and stirring, and it is difficult to increase the size of the device, so it is not necessarily the most suitable culture tank for the production of microbial proteins, and the development of a new culture tank is desired.

Recently, as a culture tank for rationally carrying out this type of fermentation, a culture tank that uses only ventilation through the ventilation pipe 27 without mechanical stirring, as shown in Fig. 2, has been attracting attention. Bubble columns do not have mechanical stirring, so their oxygen absorption capacity is quite low.

Therefore, in order to increase the oxygen absorption capacity, a tank was devised in which a ventilation pipe 28 and a multi-stage perforated plate 29 were provided in the tank as shown in Fig. 3 (Japanese Patent Publication No. 45-20552, No. 47-23591, Japanese Unexamined Patent Publication No. 48-57262).

Simply providing a perforated plate in these bubble columns has a rather small effect on improving the oxygen absorption capacity.

The reason for this is that in order to improve the mixing of the culture solution in the metal body in the tank, both gas and liquid must pass through the perforated plate. Since it is necessary to reduce the pressure loss of the driftwood passing through the board, the effect of increasing the oxygen absorption capacity of the perforated board is not increased.

In other words, in order to improve the oxygen absorption capacity of such a perforated plate, if the porosity of the perforated plate is reduced and the resulting pressure loss is increased, a gas layer is formed under the perforated plate, and as a result, , the liquid can no longer pass through the perforated plate.

At this time, each space divided by the perforated plate becomes independent, and the microbial concentration of the culture solution in each space becomes different.

For this reason, the purpose of improving the mixing of the culture solution in the metal body in the tank cannot be achieved. Therefore, it is not possible to improve the oxygen absorption capacity by reducing the porosity of the perforated plate and increasing the pressure loss. Since this becomes difficult, the porosity of the perforated plate must remain large.

When performing continuous culture using a bubble column separated by such a perforated plate, a new culture medium is passed through from the bottom (or top), but the culture solution passes through the pores of the perforated plate at the same time as oxygen-containing gas. Therefore, it is presumed that the gas-liquid contact efficiency when gas passes through the pores of the porous plate deteriorates.

In order to improve the gas-liquid contact efficiency of the perforated plate, it is important to reduce the porosity, increase the pressure loss in the perforated plate, and at the same time allow only gas to pass through the pores of the perforated plate.

The present inventors conducted basic research to improve the shortcomings of conventional perforated plate bubble column culture tanks, and found that by providing a communication tube through which the culture solution is transferred to the perforated plate, the gas-liquid contact efficiency was significantly improved. At the same time, it was possible to increase the stability of the flow of the culture medium in each space separated by the perforated plate.

Thus, by using the fermentation apparatus of the present invention, a method for efficiently carrying out continuous culture of microbial proteins was completed.

The configuration of the present invention will be explained in detail with reference to FIGS. 4 and 5.

A porous separation plate 2 is disposed within the culture tank body 1 to form two or more tanks (spaces) in the vertical direction.

The porous separation plate 2 has communication pipes 3 that serve as passages for culture fluid between various spaces (spaces), and a large number of holes 4 that allow oxygen-containing gas to pass through at high speed.

Then, culture solution gas-liquid separators 5 and 8 are provided in the culture solution pipes 17 and 20 provided outside the culture tank from the upper and middle tanks (spaces), and further from the gas-liquid separator 5.8. Pipes 18 and 21 that return the separated gas to the original tank (space) and pipes 19 and 22 that return the culture solution from which the gas has been removed to the tank (space) below are provided. Coolers 6 and 9 are provided to remove fermentation heat, and pumps 7 and 10 are provided to help transfer the culture solution.

At the bottom of the culture tank body 1, ventilation equipment 1 for gas containing oxygen is provided.
1, a medium supply pipe 12 is provided at the top, and substrate supply pipes 13, 14, and 15 are provided in appropriate tanks (spaces).

In the above device, the normal culture medium is supplied to the upper tank (space) through the supply pipe 12, and the gas containing oxygen is supplied to the lowermost tank (space) through the ventilation pipe 16, separated by the porous separator plate 2. Gas and liquid are brought into contact in various (spaces), and the substrate is pipe 13.
, 14, 15, etc., to the necessary tank (space) and culture the microorganisms aerobically.

Furthermore, if necessary, by connecting the two tanks (spaces) with a pipe and circulating the culture solution, the two tanks (spaces) can be
and homogenize the culture solution in the tank (space) between them.

In this example, various culture solutions connected by pipes 17 and 19 are homogenized, and further below, culture solutions of various types (spaces) connected by pipes 20 and 22 are made uniform.

In this way, two types of culture tanks with different compositions are formed within the culture tank body.

The porous separation plates 2 installed in the fermentation apparatus of the present invention are installed horizontally at necessary locations on the culture tank body 1 to separate each stage.
It has pores 4 through which oxygen-containing gas can move from the bottom to the top, and the porosity of this porous separation plate 2 is extremely small compared to conventional ones.

Therefore, a gas layer is formed at the bottom of the porous separation plate 2, and only the gas (however, a small amount of liquid contained in the gas layer at the bottom of the porous separation plate is entrained) is allowed to pass through the holes 4.

Therefore, it is possible to have different liquid qualities on the upper and lower sides of the porous separation plate 2, and since the porosity is extremely small compared to conventional ones, the flow rate of gas passing through the holes 4 is extremely high. As a result, mass transfer between gas and liquid becomes extremely large.

As a result, sufficient oxygen necessary for the growth of microorganisms can be supplied.

The communication pipe 3 has the function of enabling the transfer of culture solution between the tanks (spaces) separated by the porous separation plate 2, and is a pipe that directly communicates the culture solution above and below the porous separation plate. In order to prevent gas from flowing into the communicating tube 3 from the gas layer formed under the porous separator plate, the communicating tube 3 must have no openings in this gas layer.

The culture solution may flow in either direction up or down in the communication tube.
In this example, when the purpose is to equalize the liquid quality between tanks (spaces), the flow is from bottom to top, and when the purpose is to separate the liquid quality between tanks (spaces), the flow is from top to bottom. Let it flow.

Next, we will discuss the culture fluid circulation system.

A large number of tanks (spaces) are formed by the porous separation plate 2.

In other words, since the oxygen absorption capacity can be increased by the porous separation plates 2, it is preferable to provide many porous separation plates 2 in the culture tank body 1, so that a large number of independent tanks (spaces) are provided. It is formed.

However, it is also possible to independently control microorganism concentration, dissolved oxygen concentration, culture temperature, various nutrient source concentrations, substrate concentration, pH, etc. for each type (space), and to make the entire culture tank a uniform culture system. It is also possible to use a culture fluid circulation system to combine many tanks (spaces) into one group with the same fluid quality.

In this example, two groups are formed. That is, the upper group is connected by pipes 17 and 19, and the lower group is connected by pipes 20 and 22. Since these have exactly the same function, the culture solution circulation system for the upper group is different. I will explain.

The gas-liquid separator 5 is for separating air bubbles in the culture solution flowing from the tank (space) through the pipe line 17 to facilitate the transfer of the culture solution within the pipe line 17.19. , the separated gas returns to the original tank through the conduit 18.

The cooler 6 provided in the pipe line 19 is for removing fermentation heat.
This does not necessarily have to be provided in the pipe line 19, and a cooling pipe may be provided in the culture tank main body 1 instead.

Although the pump 7 provided in the conduit 19 is for facilitating the transfer of the culture solution through the conduit 19, this is not necessarily necessary.

That is, the difference in density between the apparent density of the culture solution in the culture tank and the culture solution in the pipe line 19 allows the culture solution to flow down in the pipe line 19 .

The ventilation equipment 11 is a ring type porous nozzle sparger used in general fermentation equipment, and is usually installed at the bottom of the culture tank body.

When culturing is carried out using the above fermentation apparatus, the medium nutrient source is mainly supplied to one of the upper tanks (spaces) [in this example, the top tank (space)], and the lower tank ( The gas containing oxygen is supplied to the lowest tank (space) and flows through a porous separator plate. It sequentially passes through the upper tanks and flows out from the uppermost tank (space) through the pipe 24.

When gas passes through the porous separation plate at a high flow rate giving a pressure loss of 10 centimeters of water or more, the gas comes into intense contact with the culture solution on the separation plate, promoting fermentation.

At this time, a gas layer of a size corresponding to the pressure loss of the fluid passing through the porous separator plate and the communication pipe is formed below the porous separator plate. Entrainment of liquids is allowed) can pass through.

The microorganism concentration in the culture solution in the lower tank (space) group is growing.

Even if the concentration of microorganisms is made higher at the bottom of the culture tank in this manner, the oxygen absorption capacity at the bottom is greater, so it is possible to prevent a decrease in the activity of microorganisms due to oxygen deficiency.

In other words, at the bottom of the culture tank, there is a lot of unused oxygen in the gas and the static pressure is high, so the partial pressure of oxygen in the gas increases, so the oxygen absorption capacity is much better than at the top of the culture tank. .

In this way, by creating a distribution in the concentration of microorganisms within the culture tank main body, increasing the concentration at the bottom, and allowing the microorganisms to flow out, it is possible to flow out a culture solution with a higher concentration than in a conventional culture tank.

In this example, the culture tank woody plants are divided into two tank (space) groups, but they may be divided into more tank (space) groups.
Also, although one tank (space) group is formed by three tanks (spaces), it is also possible for each type of tank (space) to have the same effect as one tank (space).

The fermentation apparatus of the present invention is completely new with these characteristics, and its advantages include the following.

(1) By passing gas through the porous separation plate at a high flow rate, (a) contact between gas and liquid becomes intense, greatly promoting fermentation.

(b) Since a gas layer is formed under the porous separator,
Only gas (however, a trace amount of liquid contained in the gas layer below the porous separator is entrained) passes through the porous separator.

(2) A communication tube connects the upper and lower tanks (spaces) separated by a porous separation plate, allowing the transfer of the culture solution.

(3) By connecting tanks (spaces) and tanks (spaces) with conduits to form a tank (space) group, (a) the number of tanks (spaces) with different liquid qualities can be reduced, and equipment and more economical in terms of operation.

(b) A cooler can be provided in this conduit, which is convenient for removing fermentation heat.

(C) The apparent density difference between the culture tank body and this conduit can be used to promote circulation flow to improve the mixing of liquids in the tank (space) group.

(4) By creating a microbial concentration distribution within the culture tank body and increasing the concentration at the bottom, (a) it is possible to flow out a culture solution with a higher microbial concentration than in conventional culture tanks;

(q) Since the gas used in the lower part is sequentially used in the upper tank (space), the gas usage efficiency is good.

(C) Since the concentration of microorganisms is lower in the upper part, the oxygen demand is also lower, and the liquid quality has lower oxygen absorption resistance, so the gas used in the lower part and with a lower oxygen concentration can be fully utilized for fermentation. can.

Examples of fermentation of microbial proteins using the fermentation apparatus of the present invention are shown below as examples, but these examples are not intended to limit the present invention in any way.

Example 1 Having the structure shown in Fig. 4, inner diameter 50CrIL1 height 30
0crrL1 capacity 7007, 5-stage opening ratio 0.55%
A porous separation plate is provided, and a new pipe line is provided to connect the discharge side of the pump T to the discharge side of the pump 10.
1.5% (by volume) of methanol and 0.0% of urea were added to the culture device using a culture device in which the entire discharge amount of 1.5% (volume) was sent to the bottom of the column.
10% (weight, same below), ammonium sulfate 0.30%, phosphoric acid 2
Potassium 0.70%, monopotassium phosphate 0.20%, magnesium sulfate 0.05%, ferrous sulfate 0.003%,
Manganese sulfate 0.004%, salt 0.01%, calcium chloride 0.01%, yeast extract 0.05%, pH 7.0
Add the initial culture solution 5007 of methanol-assimilating microorganism Aeromonas methanophilum (Ae r O: m O
na smethanophilum R-1014,
(Feikoken Toyori No. 2808) was inoculated.

Ventilation line speed 25 Crr from the ring-shaped sparger at the bottom of the tank
Culture was started by aerating air at L/Sec.

The temperature was maintained at 35°C±0.7°C using an external heat exchanger, and the pH of the culture solution was maintained at 6.8±0.2 with ammonia, and the culture was carried out for >20 hours.

Methanol was added at 1.5% (volume) to the initial medium, but as the culture progressed, methanol was added successively.
6.3% (volume) was added by the end of the culture.

The seedling concentration in the culture solution was 26.89/IJ.

Example 2 Into the culture apparatus used in Example 1, 6% molasses (by weight), 0.15% urea, 0.3% ammonium sulfate, and 0.2% monopotassium phosphate were added.
, 0.05% magnesium sulfate, pH 6.0 initial culture solution 5001 was added, and Torula yeast (Cand 1dout
ilis) was inoculated.

Linear velocity of ventilation from the ring sparger at the bottom of the tank: 21cm/se
Culture was started by aerating air at step c.

The temperature was maintained at 30°C ± 0.7°C with an external heat exchanger, and the pH of the culture solution was maintained at 5.5 ± 0.2 with ammonia. After culturing for >14 hours, the culture was completed. The bacterial cell concentration in the liquid was 15.4 g/13.

Example 3 The methanol-assimilating microorganism Aeromonas methanophyllum R-1014 was cultured for 16 hours in exactly the same manner as in Example 1 to obtain a culture system with a bacterial cell concentration of 22.1 g/l.

To this, methanol 5.0% (volume), ammonium sulfate 0.1% (weight, same below), dipotassium phosphate 0.70%, monopotassium phosphate 0.20%, magnesium sulfate 0.05%,
Ferrous sulfate 0.003%, manganese sulfate 0.004%,
A continuous supply medium containing O.O.I.% sodium chloride, 0.01% calcium chloride, 0.05% yeast extract, and pH 7.0 was continuously supplied from the top of the tower, and the culture solution was allowed to flow out from the bottom of the tower.

When continuous culture was continued with the average residence time of the supply medium set to 5 hours, from around 15 hours after the start of continuous culture,
A stable culture solution with a bacterial concentration of 21±0.69/73 was flowed out, and the culture remained stable for 10 days thereafter.

Example 4 Example 3 was continued as it was, the pipe connecting the discharge side of pump 7 and the discharge side of pump 10 was closed, and as shown in the present invention, different bacterial cell concentrations were obtained in one culture tank. It is now possible to form two parts.

The feeding medium was set so that the methanol concentration was changed to 2.5% (volume) and the other concentrations were the same as in Example 3, and the medium was continuously fed from the top of the column, and the average residence time was 6 hours. .

Furthermore, pure methanol is fed to the bottom of the column at a rate of 14% per minute. ! It was supplied at a flow rate of i2.

The pH and temperature of the culture solution were maintained at the same values by the same method as in Example 3, but the top and bottom of the column were separated and therefore controlled separately.

When continuous culture was continued in this way, from around 20 hours onwards, the bacterial concentrations at the top and bottom of the tower decreased to 11.
The bacteria concentration stabilized at ±0.5fl/11 and 25±1.0g/l, and a stable culture solution with a bacterial concentration of 25±Log/13 flowed out from the main culture device.

Continuous culture was continued for 7 days after that, and the culture remained stable during that time.

[Brief explanation of the drawing]

Figure 1 is an explanatory diagram of a conventional aeration stirring tank, Figure 2 is an explanatory diagram of a conventional simple bubble column culture tank with only aeration, and Figure 3 is an illustration of a conventional bubble column culture tank equipped with a perforated plate. FIG. 4 is a side view of an apparatus showing an example of the implementation of the present invention, and FIG. 5 is a plan view of a porous separator plate. 1... Culture tank body, 2... Porous separation plate, 3... Communication pipe, 4... Hole, 5, 8
...gas-liquid separator, 11...ventilation equipment,
12... Culture medium supply pipe, 16... Ventilation pipe, 17, 18, 19, 20, 21. 22... Conduit.

Claims (1)

[Scope of Claims] 1. Two or more parts with different bacterial cell concentrations are formed in one culture tank, and a culture medium is supplied to the top of the tank, and a gas containing oxygen is supplied to the bottom of the tank to separate both parts. A continuous fermentation method characterized by countercurrent contact and aerobic cultivation of microorganisms mixed in advance in a culture tank. 2. The topmost part of a culture tank, which has several stages of separation plates having communication pipes for passing the culture solution and holes for passing oxygen-containing gas at high speed, and two or more spaces in the vertical direction. A fermentation device with a culture medium supply pipe at the top and an oxygen-containing gas supply pipe at the bottom. 3. The topmost part of a culture tank that is equipped with several stages of separation plates having communication pipes for passing the culture solution and holes for passing oxygen-containing gas at high speed, and forming two or more spaces in the vertical direction. A culture medium supply pipe is provided at the top, a gas supply pipe containing oxygen is provided at the bottom, and a pipe is formed outside the culture tank to circulate the culture medium between the two spaces or the space between these spaces. A fermentation device made of