Culture method for aerobic microorganisms and incubator

Abstract

To solve a problem that a method of applying physical impact such as stirring and shaking on culturing aerobic microorganisms has been used, however such a method causes stress on microorganisms and consequently highly efficient culture cannot be performed.SOLUTION: A culture method for aerobic microorganisms includes: a step of filling microorganisms and culture medium into a bag being an incubator which is formed of a film having gas permeability; a step of sealing the incubator in a liquid-tight manner; and a step of mounting the incubator on a mesh-like support. In the culture method for aerobic microorganisms, microorganisms and culture medium are filled into the bag manufactured of a film having gas permeability, and stationary culture is performed in the state in which a gas-exchangeable area is increased, so that stress is not applied to the microorganisms, and effective culture can be performed.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and an incubator for culturing aerobic microorganisms using a liquid medium, typically for culturing on a relatively small scale in various laboratories. [Background technology]

[0002] When culturing aerobic microorganisms in liquid media, generally, one or both of the following are performed: forced aeration using a compressor or mechanical agitation including shaking. For example, culture vessels such as jar fermenters used for large-scale cultivation have a function of agitating while aerating.

[0003] However, in these devices, mechanical aeration and stirring operations inevitably cause physical stress to the microorganisms being cultured, so the microorganisms are constantly affected by this, and therefore the microorganisms cannot demonstrate the performance they would have if they were not under physical stress.

[0004] Patent Document 1 proposes supplying air into a jar fermenter using a fine bubble generator without placing stress on the aerobic microorganisms being cultured.

[0005] On the other hand, for relatively small-scale culture, rotary or reciprocating shakers are mainly used, which, in addition to the physical stress mentioned above, have the following problems:

[0006] First, if the heat generated by mechanical aeration and agitation is undesirable for the microorganisms, additional power is required to cool the culture medium. Second, mechanical aeration and agitation increases the risk of contamination. Third, a structure to prevent the risk of contamination is required. Fourth, a device for mechanical aeration and agitation is required.

[0007] A proposal to solve these problems is believed to require confining the culture medium and the microorganisms in an airtight space and leaving them to stand until the culture is complete. Patent Document 2 discloses a proposal to culture photosynthetic microorganisms by sealing the culture medium and the microorganisms in a three-layered bag called a reactor and floating them in a solvent with a higher specific gravity than the culture medium. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent Publication No. 2021-58816 [Patent Document 2] International Publication No. 2008 / 153202 Summary of the Invention [Problem to be solved by the invention]

[0009] Patent Document 2 is composed of three layers: a culture solution storage bag, a hydrogen gas barrier bag, and a covering bag that covers them, and is floated in a liquid that has a higher specific gravity than the culture solution, so even if air can be taken in from the surface that comes into contact with the air, the supply of oxygen is insufficient for culturing aerobic microorganisms. Also, no particular consideration is given to the replacement of the culture medium.

[0010] The present invention has been conceived in view of the above-mentioned problems, and provides a culture method and culture vessel that enable aerobic microorganisms to be cultured without subjecting them to physical stress such as stirring or shaking, and further allows the addition of new culture medium. [Means for solving the problem]

[0011] More specifically, the method for culturing aerobic microorganisms according to the present invention comprises the steps of: A step of filling a bag formed of a gas-permeable film to serve as a culture vessel with microorganisms and a culture medium; sealing the incubator liquid-tight; The method is characterized by including a step of placing the culture vessel on a mesh support.

[0012] The incubator according to the present invention further comprises: A bag formed of a gas-permeable film and enclosing aerobic microorganisms and a culture medium; The bag is characterized by having a support on which the bag is placed. Effect of the Invention

[0013] The incubator according to the present invention is a bag-shaped incubator made of a film material with high oxygen permeability, and does not have a structure such as a tube for direct flow or gas exchange of the culture solution, so that the volume of the culture solution that directly contacts the inner wall of the container can be made relatively large compared to the total volume of the culture solution. Therefore, by supplying sufficient oxygen to the microorganisms being cultured and culturing them in a stationary state, the physical stress applied to the microorganisms being cultured can be significantly reduced, and metabolic productivity can be improved.

[0014] In addition, the permeable resin film is made into a bag shape and divided into liquid-tight compartments using clips. Once cultivation has progressed in one compartment, the clips can be removed and new culture medium and cultivation space can be provided without destroying the liquid-tightness, allowing cultivation to continue without stressing the microorganisms. [Brief description of the drawings]

[0015] [Figure 1] FIG. 2 is a diagram showing a state in which a first compartment and a second compartment are provided in the incubator according to the present invention. [Diagram 2] 1 is a diagram showing the process of producing an incubator according to the present invention, in which (a) shows the film rolled up, and (b) shows the film sealed at one end to form a bag. [Diagram 3] This is a diagram showing the first compartment formed by placing microorganisms and culture medium in the compartment and sealing it with a clip. [Figure 4] FIG. 13 is a diagram showing a state in which a bag is placed on a mesh support. [Diagram 5] 1 shows the results of peroxidase production using recombinant E. coli, where (a) shows peroxidase activity and (b) shows the amount of peroxidase activity per cell. [Figure 6] 1 shows the results of peroxidase production using recombinant B. chosinensis, where (a) shows peroxidase activity and (b) shows the amount of peroxidase activity per cell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The culture method and culture vessel according to the present invention will be described below with reference to the drawings and examples. Note that the following description illustrates one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description can be modified without departing from the spirit of the present invention. Note that microbial activity refers to the number of proliferations or the amount of product per unit time.

[0017] An outline of the incubator according to the present invention is shown in Figure 1. The incubator 1 is composed of a bag 10, a clip 12 capable of dividing the bag 10, and a support 20 shown in Figure 4. A gas-permeable film 9 can be suitably used for the bag 10. From the viewpoint of gas permeability, polymethylpentene can be suitably used. This is because it supplies sufficient oxygen to the aerobic microorganisms sealed therein.

[0018] Please refer to Fig. 2(a). The film 9 can be processed into a bag shape by forming it into a cylindrical shape and welding the side surface portion 10s and one end 10a. The bag 10 is shown in Fig. 2(b). The other end 10b of the bag 10 is open. Culture medium and microorganisms, etc. are introduced from here.

[0019] It is desirable that the thickness of the film 9 constituting the bag 10 is as thin as possible. For example, a thickness of 1 μm to 500 μm can be used. If it is too thin, it will not have enough strength, and if it is too thick, gas permeability will decrease, and the supply of oxygen to aerobic microorganisms will be insufficient or will not be possible.

[0020] Moreover, it is preferable that the film 9 is transparent, because when the bag 10 is made, the progress of the culture inside the bag 10 can be visually confirmed.

[0021] Please refer to Fig. 3. There is no particular limitation on the configuration of clip 12 as long as it can divide bag 10 into a liquid-tight state and further open it. Fig. 1 shows clip 12 that can divide bag 10 into a liquid-tight state by pinching the middle of bag 10 with upper and lower bars 12a, 12b. However, bag 10 may also be divided by tying the middle of bag 10 with something like rubber or string. Therefore, in the present invention, clip 12 also includes restraining means such as rubber or string.

[0022] The bag 10 is filled with culture medium, microorganisms are added, and only the filled portion is liquid-tightly sealed with clip 12. The portion filled with the microorganisms and culture medium is referred to as the first section 10A. The thickness of the thickest portion of first section 10A is referred to as thickness t1. Here, the clip 12 that created first section 10A is referred to as clip 12α. Then, only the culture medium is poured into the portion adjacent to first section 10A, and the portion is sealed again with clip 12. Figure 1 shows this state.

[0023] Referring again to FIG. 1, a section in which only the culture medium is enclosed is created next to the first section 10A. This section is called the second section 10B. The thickness of the thickest section of the second section 10B is called thickness t2. The second section 10B is again sealed with a clip 12. This clip 12 is called clip 12β. Therefore, the incubator 1 is formed by dividing a bag 10 formed of a gas-permeable film 9 with clips 12, and is formed of a first section 10A in which microorganisms and the culture medium are enclosed, and a second section 10B in which only the culture medium is enclosed. Note that the present invention does not exclude the case in which only the first section 10A is enclosed.

[0024] In the culture method of the present invention, the microorganisms and the culture medium are cultured by simply leaving the bag 10 containing them stationary. Therefore, oxygen needs to pass from the surface of the film 9 to the center of the culture medium. Therefore, at least the thickness t1 of the first section 10A needs to be set to a certain value or less. The thickness t1 of the first section 10A varies depending on the microorganisms to be cultured and the thickness of the film 9 used, but it is preferable that it is 20 mm or less. The lower limit of the thickness t1 of the first section 10A may be at least twice the thickness of the film 9. It is preferable that the second section 10B is also set to the same thickness t2.

[0025] Moreover, the second compartment 10B may have an area (volume) smaller or larger than that of the first compartment 10A. According to this method, after culturing in the first compartment 10A, it is easy to perform operations such as providing a larger amount of medium and culture space, replenishing new substrates, newly supplying physiological activity such as hormones, and adsorbing substances secreted by the microorganisms during culturing by making an absorbent for a specific substance present in the second compartment 10B. These are expected to increase the amount of cells obtained in one culture, increase the productivity of useful substances by providing new conditions to the microorganisms and regulating their metabolism, reduce substances secreted by the microorganisms that have adverse effects on the microorganisms, and facilitate the recovery of secreted useful substances.

[0026] Please refer to Fig. 4. The incubator 1 prepared as in Fig. 1 is placed on a support 20. In Fig. 4, the incubator 1 is placed on a mesh support 20 in order to use as much area as possible of the entire surface of the bag 10 for gas exchange. The support 20 is made of a breathable material or has a shape such that gas exchange is not hindered in the area where the bag 10 is projected, and it is desirable to increase the surface area of ​​the bag 10 exposed to the gas phase.

[0027] The support 20 is preferably one that can be placed horizontally when the bag 10 containing the culture medium and the microorganisms is placed thereon. This is because the bag 10 is not moved during the culture period, and therefore mechanical strain on the bag 10 is minimized.

[0028] Moreover, if the opening of the support 20 is too large, the bag 10 will sag and drop from the opening. It is desirable to determine the shape of the opening of the support 20 so that the partial drop portion (lowest portion) of the bag 10 is less than 20 mm from the top.

[0029] Since the purpose of the support 20 is to increase the area of ​​the underside of the bag 10 that comes into contact with the air, the support 20 may have a mesh, lattice, or pinholder shape, or a combination of other geometric and random openings. A support 20 having openings formed therein in this manner is referred to as a mesh support 20 herein. The ratio of the area of ​​the support 20 that is not in contact with the underside of the bag 10 to the total area of ​​the backside of the bag 10 to the area of ​​the support 20 that is in contact with the underside of the bag 10 to support the bag 10 is referred to as the opening rate. The opening rate of the mesh support 20 is preferably 50% or more.

[0030] In addition, it is preferable that a space large enough for air to pass through is provided below the support 20. In addition, the support 20 may be in the form of a shelf on which a plurality of bags 10 can be placed.

[0031] The method for culturing aerobic microorganisms according to the present invention is carried out as follows. First, a bag 10 is made using a gas-permeable film 9. To seal the bag, the film can be welded, glued, clipped, or a combination of these methods. As described in FIG. 2, the bag 10 can be formed by making a cylindrical body and sealing one end 10a liquid-tightly, or the bag 10 can be made by making a cylindrical body and first sealing one end 10a with a clip 12. Alternatively, two gas-permeable films 9 can be stacked and the three sides of the two films can be welded or glued together.

[0032] The culture medium and aerobic microorganisms are placed in this state in bag 10, and then clips 12α are used or the bag is welded or glued to seal the bag liquid-tight. At this time, it is preferable that no air enters the sealed space. Because gas exchange takes place with the liquid, if air enters, the area for gas exchange will be substantially reduced or the liquid thickness will become uneven, resulting in reduced reproducibility. The compartment in which the aerobic microorganisms and culture medium are sealed is the first compartment 10A.

[0033] Next, the culture medium alone is placed in the adjacent portion of the first section 10A via clip 12α, and the other end 10b of the bag 10 is sealed with clip 12β or by welding or bonding to form the second section 10B. The portion containing only the culture medium is the second section 10B. In this state, the bag is placed on the support 20.

[0034] Cultivation is carried out by placing the bag 10 containing the culture medium and the microorganisms on the support 20 and leaving it as it is. The humidity around the bag 10 is preferably 80% to 100%. This is because a gas-permeable film also allows water molecules to pass through, so if the method of the present invention involves simply placing the microorganisms and culture medium in the bag 10 made of a gas-permeable film and leaving it to stand, as in the present invention, the moisture in the culture medium will gradually escape if the surrounding humidity is low.

[0035] The progress of the culture can be determined by the degree of growth of the aerobic microorganisms and the amount of product, but can also be determined by the transmittance and time. When the degree of growth is determined by turbidity, it can be observed through the film 9. After a certain period of culture has elapsed, the clip 12α between the first compartment 10A and the second compartment 10B is removed. The medium in the second compartment 10B then mixes with the medium in the first compartment 10A, providing the microorganisms with new components.

[0036] In addition, if the number of microorganisms is increasing, the space that they can occupy increases by the second compartment 10B. In this way, by obtaining new medium and culture space as the culture progresses, the microorganisms can be cultured with even less stress.

[0037] The second section 10B may contain components other than the medium, because the type of nutrients provided can be changed as the culture progresses.

[0038] As described above, in the method for culturing aerobic microorganisms according to the present invention, the microorganisms and culture medium sealed in the bag 10 are simply left to stand, so no physical stress is imposed on the microorganisms. Furthermore, as the culture progresses, new culture medium and culture space can be supplied simply by removing the clip 12, so the operation can be carried out with almost no stress.

[0039] In addition, in the case of microorganisms that tend to form clumps during cultivation, such as adventitious roots of plants and hyphae of filamentous fungi, the clumps of the microorganisms are periodically loosened through the film 9 and are constantly dispersed in the medium, thereby preventing a decrease in cultivation efficiency due to the interruption of the supply of substrate to the inside of the microbial clumps. EXAMPLES

[0040] Example 1 <Cultivation of recombinant E. coli that produces peroxidase> A bag-shaped container measuring 4 cm in length and 7 cm in width was made using a 50 μm-thick TPX film. The recombinant E. coli used was a strain that was inducible with IPTG and had a plasmid with a plant-derived peroxidase gene expression cassette linked to it.

[0041] The cells were cultured in a 500 mL flask containing 100 mL of TB medium at 37°C with shaking, and the turbidity (OD 600 When the pH reached 2.1, 2 mL of the culture was placed in the container, IPTG was added to a final concentration of 0.05 mM, and the opening was then fused to completely seal the container. The container was then left to stand at 37°C for 16 hours (TPX group). The TPX group corresponds to Example Sample 1-1 of the present invention.

[0042] As a control sample 1-1, a low-density polyethylene film (whose oxygen permeability is said to be about 1 / 8 that of TPX) was used for static culture in the same manner as in the case of the TPX film (LDPE group).

[0043] As control sample 1-2, 2 mL of the culture medium was placed in a polypropylene conical tube having an outer diameter of 30 mm and a length of 115 mm, and similarly IPTG was added thereto, followed by allowing to stand at 37° C. for 16 hours (ST group).

[0044] Control sample 1-3 was cultured by conventional shaking. 2 mL of culture medium was placed in a polypropylene conical tube with an outer diameter of 30 mm and a length of 115 mm, and cultured at 200 rpm and 37°C for 16 hours using a shaking incubator (200R-T group).

[0045] Figure 5(a) shows the total peroxidase activity per culture medium (U / mL: vertical axis), and Figure 5(b) shows the total peroxidase activity per cell (U / OD 600 10: vertical axis). The horizontal axis indicates the sample type.

[0046] The results of the significance test showed that there was no difference between the total peroxidase activity per culture medium in the TPX and 200R-T groups (Figure 5(a)), but the total peroxidase activity per cell was significantly higher in the TPX group (Figure 5(b)). Note that the high value in the LDPE group in Figure 5(b) is due to the small amount of cells, which results in low production efficiency. Therefore, the present invention achieved a production efficiency in static microbial culture that is equal to or higher than that of conventional methods. This can be said to be the result of static culture being performed under conditions of sufficient oxygen supply, resulting in stress-free culture and increased activity efficiency.

[0047] Example 2 <Cultivation of recombinant Brevibacillus chosinensis producing peroxidase> We used recombinant B. chosinensis that secretes and produces plant-derived peroxidase. B. chosinensis is more sensitive to a decrease in oxygen concentration than E. coli shown in Example 1. A bag-shaped container measuring 4 cm in length and 7 cm in width was made using TPX film with a thickness of 50 μm.

[0048] B. chosinensis was cultured in a 500 mL flask containing 100 mL of MT medium at 37°C for 16 hours with shaking, and 2 mL of the culture was dispensed into the container and the opening was fused to completely seal it. The container was left to stand at 37°C for 72 hours (TPX group). The TPX group corresponds to the example sample 2-1 of the present invention.

[0049] As a control sample 2-1, a low-density polyethylene film was used for static culture in the same manner as the TPX film (LDPE group).

[0050] As a control sample 2-2, 2 mL of the culture medium was placed in a polypropylene conical tube having an outer diameter of 30 mm and a length of 115 mm, and allowed to stand at 37° C. for 72 hours (ST group).

[0051] Control sample 2-3 was cultured on a shaking table. 2 mL of culture solution was placed in a polypropylene conical tube with an outer diameter of 30 mm and a length of 115 mm, and cultured on a shaking table at 180 rpm and 37°C for 72 hours (180R-T group).

[0052] Figure 6(a) shows the total peroxidase activity in the medium (U / mL: vertical axis), and Figure 6(b) shows the total peroxidase activity per cell (U / OD 600 6(a) and 6(b), the results are shown in Table 1. In both graphs, the horizontal axis indicates the sample type. In both Fig. 6(a) and Fig. 6(b), the results of the present invention (TPX) were significantly higher. Therefore, the present invention achieved a production efficiency in static microbial culture that was higher than that of conventional methods. This can be said to be the result of static culture being performed under conditions of sufficient oxygen supply, resulting in stress-free culture and increased activity efficiency. [Industrial Applicability]

[0053] The present invention can be suitably used for culturing microorganisms, and is particularly suitable for small-scale culturing. [Explanation of symbols]

[0054] 1 incubator 9. Film 10 bags 10a (one end of bag) 10b (other end of bag) 10s The part of the bag that touches the side 10A Section 1 10B Section 2 t1 (first section) thickness t2 (first section) thickness 12 clips 12α Clip 12β Clip 12a, 12b (clip) top and bottom bars 20 Support

Claims

1. A step of filling a bag formed of a gas-permeable film to serve as a culture vessel with microorganisms and a culture medium; sealing the incubator liquid-tight; A method for culturing microorganisms, comprising the step of placing the culture vessel on a mesh support.

2. Prior to the step of filling the microorganism and medium, Dividing the incubator into at least two or more compartments with a removable clip; The step of filling the microorganism and the medium is a step of filling one of the compartments with the microorganism and the medium, filling the culture medium into a compartment adjacent to the first compartment via the clip; After the step of placing on the support, 2. The method for culturing a microorganism according to claim 1, further comprising a step of removing the clip when the culture has progressed.

3. In the step of dividing into two or more sections, Creating sections with different sizes of adjacent sections via the clip, 3. The method for culturing microorganisms according to claim 2, wherein the step of filling the microorganisms and the medium is a step of filling the smaller one of the compartments.

4. 3. The method for culturing microorganisms according to claim 2, wherein in the step of filling the compartment adjacent to the first compartment with the medium via the clip, components other than the medium are filled in addition to the medium.

5. A bag formed of a gas-permeable film and enclosing aerobic microorganisms and a culture medium; An incubator having a support on which the bag is placed.

6. 6. The incubator according to claim 5, further comprising a removable clip for dividing the bag into at least two or more compartments so that each compartment is liquid-tight.

7. 7. The incubator according to claim 6, wherein the plurality of compartments adjacent to each other via the clips include a portion where compartments of different sizes are adjacent to each other.

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