JPH06253824A - Method for culturing microbe - Google Patents

Abstract

PURPOSE:To obtain an air bubble column-type culture tank capable of culturing mould or Actinomycetes, by mounting meshes inside a draft tube installed in a culture tank and by equipping the bottom of the tank with a fine bubble generator. CONSTITUTION:A draft tube 2 is installed inside a culture tank 1, an appropriate number of meshes 4, 5... are mounted inside the draft tube, and air or oxygen- rich air is blown from a fine bubble generator 3. With this scheme, oxygen- moving capacity factor can be raised even in the case of relatively low aerating linear velocity; thereby, even mould or Actinomycetes can be cultured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for cultivating fungi and actinomycetes whose fungi are filamentous.
To a technique for producing useful substances by culturing the above microorganisms in a bubble column culture tank having a high oxygen transfer capacity coefficient (KLa) having a draft tube equipped with a plurality of meshes.

[0002]

2. Description of the Related Art Organic acids such as citric acid and itaconic acid, which are used in the fields of food and chemical industry, and various antibiotics which are pharmaceuticals are produced by a fermentation method by culturing fungi or actinomycetes. Generally, an aeration and stirring type culture tank is used as a method for culturing these microorganisms. However, the aeration-stirring type culture tank requires a large amount of power for stirring, the internal structure is complicated and the inside of the device is insufficiently cleaned, and is easily contaminated with various bacteria. There is a risk of contamination, especially when culturing fungi and actinomycetes whose filamentous cells are filamentous, the hyphae are cut by the shearing force of the stirring blade, and the mycelial cells are easily damaged. It has drawbacks.

On the other hand, the bubble column type culture tank is
There are merits such as no shaft seal that easily causes contamination of various bacteria, simple overall structure, and no need of energy required for stirring.To date, development of various types of bubble column culture tanks has been carried out. Although it has been tried, it has not yet been put to practical use for culturing a microorganism whose cells are filamentous.

[0004]

Generally, in the large-scale production of useful substances by the fermentation method, it is unavoidable to enlarge the size of the culture tank in order to reduce the production cost. In this sense, if fermentative production of molds in a bubble column culture tank is possible, in addition to the above advantages, not only the energy required for mechanical stirring becomes unnecessary, but also the Joule heat generated by mechanical stirring is removed. Therefore, it is possible to reduce the utility cost for cooling. Furthermore, since there is no shaft sealing part, the risk of contamination by various bacteria can be reduced,
It can be expected to increase the productivity of substance manufacturing. However, despite a lot of research, a culture tank that can be put to practical use has not yet been developed. For this reason, there is one problem in the supply of oxygen into the culture solution. The bubble column-type culture tank has a smaller KLa than the aeration-agitation-type culture tank. If the air-flow linear velocity is increased to raise KLa, foaming becomes severe and the culture cannot be continued. As a method of lowering the aeration velocity and increasing KLa, a bubble column culture tank with a perforated plate inside has been developed. It becomes impossible to continue the culture.

According to the present invention, by installing a mesh inside the draft tube, a high KLa can be achieved even at a relatively low air-flow velocity, and continuous culture operation can be performed without causing foaming or clogging, and it is possible to obtain mold and mold. It is intended to provide a culturing method using a bubble column type culture tank capable of culturing actinomycetes.

[0006]

According to the present invention, a draft tube is internally provided in a culture tank, and one or a plurality of meshes are installed inside the draft tube and air or oxygen-enriched air is blown therein. Using a bubble column-type culture tank having a fine bubble generator provided at the bottom of the culture tank for culturing fungi or actinomycetes whose filaments are filamentous and performing fermentation production of useful substances A method of culturing a body is provided.

The culture method of the present invention will be described in more detail.

The bubble column type culture tank of the present invention has a double tube structure in which a draft tube is installed inside, as shown in the schematic diagram of FIG.
A mesh is mounted inside the draft tube. In FIG. 1, meshes are attached to the draft pipe at two positions, an inlet and an outlet. A micro-bubble generator (sparger) is provided at the bottom of the culture tank in FIG. Figure 1
Other devices and structures necessary for culture are omitted for simplicity of explanation.

The perforated plate used in the conventional bubble tank has a hole diameter of 1 to 4 mm and an opening ratio of 1 to 10%, and acts as a baffle plate against blown bubbles to KLa.
However, clogging occurred in the case of culturing fungi or actinomycetes whose filaments were filamentous due to the low pore size and opening ratio.

In the present invention, by installing a mesh (mesh) inside the draft tube, KLa can be sufficiently increased even at low air-flow linear velocity, and culturing can be carried out without causing cell clogging. It becomes possible.

In the present invention, the number of meshes (mesh or mesh) provided in the draft tube is 1 or more, preferably 2 to 4. The mesh is installed so that its surface is substantially vertical (that is, horizontal) to the vertical direction (the flow direction of bubbles) of the draft tube. The mesh preferably has an average area (mesh size) of one section of the lattice image of 0.1 cm ² or more and an aperture ratio of 30% or more. A more preferable lattice screen area (mesh size) is 0.1 cm ² or more, and a particularly preferable range is 0.15 to ⁵ cm ² . On the other hand, the aperture ratio is preferably 50% or more, and particularly preferably in the range of 60 to 90%.

The mesh lattice may have any shape such as a rectangle, a rhombus and a circle, but a rectangle is common.
The mesh size, aperture ratio, and lattice shape of the mesh are appropriately determined depending on the type of cells to be cultured and the culture conditions. Although the material of the mesh is not particularly limited, metals, synthetic polymers such as nylon, natural fibers and the like can be used, but if the culture solution becomes acidic, it must be a material that is not attacked by acid.

In the present invention, the fine bubble generator (sparger) for blowing air or oxygen-enriched air provided at the bottom of the culture tank into the culture tank can be appropriately used by a method generally used for culturing microorganisms. As the bubbles are smaller, KLa can be increased, so a sintered metal (stainless steel) filter or a ceramic filter that can create fine bubbles can be used.

When the bubble column type culture tank of the present invention is used, the aeration linear velocity (Vs) and the oxygen transfer capacity coefficient (KL
The relationship with a) is shown in FIG. In Fig. 2, □ mark is the result when the mesh is installed in two places, the upper part and the lower part of the draft pipe as shown in Fig. 1, and the △ mark is the mesh only in the lower part of the draft pipe (only mesh 1). ) The result when the device is installed. On the other hand, in FIG. 2, the result when the mesh is not attached at all is also shown by a circle.

From FIG. 2, when the mesh is not attached, V
Flooding occurs when s exceeds 1 cm / sec. K
Although La decreases, it is understood that when a mesh is installed in the draft tube, a high KLa can be obtained at a Vs as low as an ordinary perforated plate (pore diameter 2 mm, opening ratio 3%). This means that by inserting a mesh, not only oxygen can be efficiently dissolved in the medium and oxygen can be supplied to the cultured cells, but also the clogging of the cells found in the conventional bubble column type culture tank. It solves the problem. KLa was measured by the sulfite oxidation method. The effect of this mesh is not only to increase KLa but also to prevent the formation of filamentous fungi and pellet formation, which is a characteristic of actinomycetes, and to increase the growth effect of bacterial cells.

The culture method of the present invention can be applied to any of the conventional batch culture method, semi-batch method, repeated batch culture method, continuous culture method, etc. using the bubble column type culture tank.

An example of a culture system using the bubble column type fermenter according to the present invention is shown in FIG. In FIG. 3, air or oxygen-enriched air is blown into the double-tube draft tube 2 through the bubble generator 3. The liquid (medium) that has risen inside the draft tube passes through the outside of the draft tube and drops to the lower part of the tank, with bubbles disappearing in the upper part of the culture tank. The temperature of the medium can be controlled by circulating hot water, cold water, or the like through the jacket 15. Part of the exhaust gas is condensed by the condenser 10 and returned to the tank. If necessary, the volume of the evaporated water can be kept constant by appropriately adding sterilized water. If foaming occurs during the culture, the level sensor 16 and the pump 9
The antifoaming agent is added to the medium from the antifoaming agent storage tank 8 in association with the above. The temperature, pH and dissolved oxygen level of the culture solution are detected and controlled by the respective sensors 11, 12 and 13 so that the culture tank is always operated in the highest state. Further, if necessary, online control can be performed using these pieces of information.

[0018]

EXAMPLES The present invention will be specifically described below with reference to examples and comparative examples, but the present invention is not limited thereto.

The composition of the medium used in Examples and Comparative Examples is shown in Table 1 below.

[0020]

[Table 1]

In the bubble column culture tank shown in FIG. 3 used in Example 1, the mesh used was mounted on the upper and lower parts of the draft tube, and the area (mesh size) of one section of the mesh was It is 1 cm ² , the opening ratio is 79%, the inner diameter of the draft tube is 5 cm, and the mesh distance between the upper part and the lower part is 28 cm.

Example 1 Aspergillus was placed in an Erlenmeyer flask containing 100 ml of the seed culture medium shown in Table 1.
One platinum loop of terreus KO-3152 strain was inoculated from a slant (agar medium) and cultured at 30 ° C. for 2 days to obtain a seed culture. Next, 2 liters (L) of "production medium" shown in Table 1 was placed in a 5 L flask and sterilized by autoclave at 120 ° C for 15 minutes, placed in the bubble column-type culture tank shown in Fig. 3, and 200 ml of the inoculum solution was added. Was added and cultured at 40 ° C. and an aeration rate of 4 L / min (2 VVM). The culture results are shown in Table 2 below. Cell weight is dry weight, sugar is phenol-sulfuric acid method and itaconic acid (ITA) is improved brom method (edited by the Chemical Society of Japan, Vol. 25, Experimental Chemistry Course, 1958).
Page 56). In addition, the yield was expressed as a percentage of the produced itaconic acid with respect to the consumed reducing sugar. 41.9 g / L of itaconic acid was accumulated 5 days after the start of the culture, and the yield of sugar to sugar was 47.2% at this point, and the maximum itaconic acid production rate up to this point was 0.68 g / L.
It was / hr.

[0023]

[Table 2]

Comparative Example-1 An inoculum and a medium were prepared in the same manner as in Example-1, and an ordinary perforated plate (pore size: 3 mm, opening ratio: 3) was used instead of the mesh.
%) Example using a bubble column type fermenter equipped with two sheets
Culture was performed under the same conditions as in 1. On the second day after the start of the culture, the perforated plate was clogged and ventilation was impossible, and the culture could not be continued.

Example-2 200 ml of the inoculum solution prepared by the same method as in Example-1 was
"Seed culture medium" containing 100 g of sterilized Celite 545
5L jar fermenter with 2L (Maruhishi MD30
Type 0) was inoculated and cultured at 30 ° C. for 3 days. Then, the culture solution was aseptically filtered to separate cell-adhered celite, which was suspended in 2 L of "production medium", transferred to a bubble column type culture tank, and cultured under the same conditions as in Example-1. The amount of itaconic acid produced on the 5th day of culture was 45.5 g / L, and the yield based on sugar was 50.5%.
It was shown that the bubble column type culture tank can also be used as a packed bed type bioreactor using immobilized cells.

Example 3 Except that oxygen-enriched air (air: pure oxygen = 3: 1, aeration amount: 1 VVM) was used instead of air as a method for supplying oxygen.
Culturing was performed under the same method and conditions as in Example-1. The production of itaconic acid on the 5th day of the culture was 36.4 g / L, and the yield based on sugar was 52.0%, which was almost the same as when normal air was used.

Example-4 200 ml of the inoculum solution prepared by the same method as in Example-1
"Inoculum medium" sterilized by autoclaving at 20 ℃ for 15 minutes
This bubble column type fermenter containing 2 L was inoculated and cultured at 30 ° C. for 3 days (aeration volume 3 VVM). After completion of the culture, aeration was stopped, the bacterial cells were allowed to settle, the supernatant was discharged, and 2 L of "production medium" was newly added to this and cultured at 40 ° C. (aeration volume 2 VV
M). In addition, sterilizing water was intermittently added in order to keep the liquid surface constant. The itaconic acid concentration was quantified on the 3rd and 5th days after the start of the culture. After culturing for 5 days, aeration was stopped and the bacterial cells were allowed to settle, and fresh medium was again added to this, and batch culture was repeated. This operation was repeated 7 times, and the production amount of itaconic acid was shown in Fig.
It was shown to. 50/50 of itaconic acid in any culture
Accumulation of L or more was observed.

[0028]

EFFECTS OF THE INVENTION According to the present invention, fungi such as mold and actinomycetes in the bubble column type culture tank, which have been considered difficult, are filamentous.
In addition, it becomes possible to cultivate microorganisms that are easy to form pellets, the fermentation tank cost, energy and utility costs are lower than those of ordinary agitation-type culture tanks, and it is possible to reduce the production cost of fermentation products. Can be increased.

[Brief description of drawings]

FIG. 1 shows a schematic diagram of a bubble column culture tank.

FIG. 2 shows a relationship between aeration linear velocity (Vs) and oxygen transfer capacity (KLa) when a bubble column type culture tank is used.

FIG. 3 shows an example of a culture system using a bubble column culture tank.

FIG. 4 shows the number of times the culture operation was repeated and the amount of itaconic acid (ITA) produced in Example-4.

[Explanation of symbols]

 1: Culture tank 2: Draft tube 3: Micro bubble generator (sintered metal) 4: Mesh (2) 4 ': Plan view of mesh (2) 5: Mesh (1) 5': Plan of mesh (1) Figure 6: Sterilized water storage tank 7: Pump 8: Antifoam storage tank 9: Pump 10: Condenser 11: Temperature sensor 12: pH sensor 13: Dissolved enzyme sensor 14: Recorder 15: Jacket 16: Level sensor 17: Air (or oxygen) Enriched air) 18: Flow meter

Claims (1)

[Claims] 1. A draft tube is provided inside the culture vessel, and one or a plurality of meshes are attached to the inside of the draft tube and provided at the bottom of the culture vessel for blowing air or oxygen-enriched air. A method for culturing a fungus body, which comprises fermentatively producing a useful substance by culturing a fungus or actinomycete having a filamentous fungus in a bubble tower type culture tank having a fine bubble generator.