Microbial culture device

Abstract

To provide a microbial culture device that allows easy cultivation of microorganisms.SOLUTION: A microbial culture device includes a three-layer laminated structure having a layered culture section for cultivating microorganisms, and at least one of a layered nutrient supply section for supplying nutrients to the culture section and a layered environmental component supply section for supplying environmental components to the culture section, which are arranged on a first surface of the culture section and a second surface opposite the first surface.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to an apparatus and method for culturing microorganisms, and more particularly to an apparatus and method for culturing microorganisms that can realize a variety of culture conditions and enable the acquisition of a variety of difficult-to-cultivate microorganisms. [Background technology]

[0002] Target microorganisms are collected by pure culture of microorganisms. This pure culture has generally been carried out using the agar plate surface smear method. In this method, a group of microorganisms collected from the environment is smeared on a solid medium prepared in a petri dish and cultured. However, many microorganisms do not grow using this method, and it is said that currently only about 1% of the microorganisms in the environment can be cultured. The reasons for this are thought to be as follows: (a) Because the culture environment is closed, excess substances produced by the microorganisms cannot be discharged from the system, resulting in the accumulation of metabolic products of the microorganisms and environmental components, which inhibit the growth of the microorganisms. (b) It is difficult to maintain the nutrient concentrations required for growth of the target microorganism in solid media.

[0003] Therefore, culture techniques have been proposed, such as those shown in Patent Documents 1 and 2. In Patent Document 1, culture is carried out while continuously supplying a liquid medium, while in Patent Document 2, culture is carried out by placing a solid medium containing microorganisms in a natural environment. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-86654 [Patent Document 2] U.S. Patent No. 7,011,957 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the method of Patent Document 1 only controls the medium components, making it impossible to realize a variety of culture conditions, while the method of Patent Document 2 supplies environmental components from the natural environment, making it impossible to realize stable culture conditions.

[0006] An object of the present invention is to provide a microbial culture device and a microbial culture method that can realize a variety of culture conditions and enable the acquisition of a variety of difficult-to-culture microorganisms. [Means for solving the problem]

[0007] The microbial culture device of the present invention is a layered culture section for culturing microorganisms; At least one of a layered nutrient supply section that supplies nutrients to the culture section and a layered environmental component supply section that supplies environmental components to the culture section, the layered nutrient supply section being arranged on a first surface of the culture section and a second surface opposite to the first surface; a three-layer laminate structure having The culture unit holds a microorganism-containing culture medium, The nutrient supply holds a nutrient-containing material or is adapted to pass a nutrient-containing gas or a nutrient-containing liquid through it; the environmental component supply unit holds an environmental component-containing material or allows an environmental component-containing gas or an environmental component-containing liquid to flow; The culture unit includes a first frame body that surrounds a first internal space, The nutrient supply unit includes a second frame body that surrounds a second internal space, the environmental component supply unit includes a third frame body that surrounds a third internal space, the first frame body is capable of holding a microorganism-containing culture medium in the first internal space, The second frame body is capable of holding a nutrient-containing material in the second internal space, or is capable of circulating a nutrient-containing gas or a nutrient-containing liquid in the second internal space, the third frame body is capable of holding an environmental component-containing material in the third internal space, or capable of circulating an environmental component-containing gas or an environmental component-containing liquid in the third internal space, The first frame body, the second frame body, and the third frame body can be connected to each other in a stacked state, The first frame body, the second frame body, and the third frame body are detachable from one another, Further comprising a base and a lid, the first frame body, the second frame body, and the third frame body each have a first fitting portion and a second fitting portion, the base has the first fitting portion, the lid body has the second fitting portion, and the first fitting portion and the second fitting portion can be fitted together, In the three-layer laminated structure, The culture unit is provided so as to be replaceable with another culture unit, or The nutrient supply unit is provided so as to be replaceable with another nutrient supply unit or a supply unit of an environmental component, or The environmental component supply unit is provided so as to be replaceable with another environmental component supply unit or a nutrient supply unit. It is characterized by the following.

[0008] The present invention also includes the following aspects.

[0009] The microorganism culture device according to the first aspect of the present invention comprises: a layered culture section for culturing microorganisms; At least one of a layered nutrient supply section that supplies nutrients to the culture section and a layered environmental component supply section that supplies environmental components to the culture section, the layered nutrient supply section being arranged on a first surface of the culture section and a second surface opposite to the first surface; The present invention is characterized by including a three-layer laminate structure having:

[0010] A second aspect of the present invention relates to a method for cultivating microorganisms, which uses a microorganism cultivating device in which the nutrient supplying unit 2 is configured to circulate a nutrient-containing gas or a nutrient-containing liquid, or the environmental component supplying unit 3 is configured to circulate an environmental component-containing gas or an environmental component-containing liquid, The method includes at least one of a nutrient changing process for changing at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid circulated through the nutrient supply section, and an environmental component changing process for changing at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid circulated through the environmental component supply section.

[0011] A third aspect of the present invention provides a method for cultivating microorganisms, which uses a microorganism cultivating device in which a nutrient supply unit is replaceable with another nutrient supply unit or an environmental component supply unit, or in which an environmental component supply unit is replaceable with another environmental component supply unit or a nutrient supply unit, The method includes at least one of a nutrient exchange step in which the nutrient supply unit is exchanged with another nutrient supply unit or with an environmental component supply unit, and an environmental component exchange step in which the environmental component supply unit is exchanged with another environmental component supply unit or with a nutrient supply unit.

[0012] A fourth aspect of the present invention is a method for cultivating microorganisms, which uses a microorganism cultivating device in which the nutrient supplying unit 2 is configured to circulate a nutrient-containing gas or a nutrient-containing liquid, or the environmental component supplying unit 3 is configured to circulate an environmental component-containing gas or an environmental component-containing liquid, and the cultivating unit 1 is further equipped with one or more sensors for detecting the culture state; The method includes a monitoring step in which the culture state of the culture section is detected and monitored by a sensor, and further includes at least one of a nutrient changing step in which, based on the monitoring results, at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid to be circulated to the nutrient supply section is changed, and an environmental component changing step in which at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid to be circulated to the environmental component supply section is changed.

[0013] A fifth aspect of the present invention provides a method for cultivating microorganisms, which uses a microorganism cultivating device in which the nutrient supplying unit 2 is replaceable with another nutrient supplying unit or an environmental component supplying unit, or the environmental component supplying unit 3 is replaceable with another environmental component supplying unit or a nutrient supplying unit, and the culture unit 1 is equipped with one or more sensors for detecting the culture state; The method includes a monitoring step of detecting and monitoring the culture state of the culture section using a sensor, and further includes at least one of a nutrient exchange step of replacing the nutrient supply section with another nutrient supply section or with an environmental component supply section based on the monitoring results, and an environmental component exchange step of replacing the environmental component supply section with another environmental component supply section or with a nutrient supply section. [Effects of the Invention]

[0014] According to the microorganism culturing apparatus of the present invention, microorganisms can be cultured within the through-holes of the perforated plate, the microorganisms can be cultured easily, and furthermore, a very compact microorganism culturing apparatus can be realized.

[0015] According to the first aspect of the microorganism culture device of the present invention, nutrients can be supplied from the nutrient supply unit and / or environmental components can be supplied from the environmental component supply unit to the culture unit, allowing microorganisms to be cultured in the culture unit. Moreover, since the device can be constructed using a three-layer laminated structure, the device configuration can be simplified. Therefore, the culture operation can be easily performed, making it possible to realize a variety of culture conditions and obtain a variety of difficult-to-culture microorganisms.

[0016] According to the microorganism culturing methods of the second and third aspects of the present invention, the culture conditions for the culture section can be easily changed, so that a variety of culture conditions can be realized, making it possible to obtain a variety of difficult-to-cultivate microorganisms.

[0017] According to the microbial culture methods of the fourth and fifth aspects of the present invention, the culture conditions for the culture section can be easily changed based on the monitoring results even during the culture, making it possible to easily achieve optimal culture conditions and obtain a variety of difficult-to-culture microorganisms. [Brief explanation of the drawings]

[0018] [Figure 1] 1 is a schematic cross-sectional view showing a first example of the basic form of a three-layer laminated structure included in the microorganism-culture device of the present invention. [Figure 2] 1 is a schematic cross-sectional view showing a second example of the basic form of a three-layer laminated structure included in the microorganism-culture device of the present invention. [Figure 3] 1 is a schematic cross-sectional view showing a third example of the basic form of a three-layer laminated structure included in the microorganism-culture device of the present invention. [Figure 4] 10 is a schematic cross-sectional view showing a modified example of a three-layer laminated structure. [Figure 5] 10 is a schematic cross-sectional view showing another modified example of a three-layer laminated structure. [Figure 6] 1 is a schematic cross-sectional view showing a first example of the basic form of the microorganism culture apparatus of the present invention. [Figure 7] 1 is a schematic cross-sectional view showing a second example of the basic form of the microorganism culture apparatus of the present invention. [Figure 8] 1 is a schematic cross-sectional view showing a third example of the basic form of the microorganism culture apparatus of the present invention. [Figure 9] 1 is a perspective view showing a microorganism culture apparatus according to a first embodiment of the present invention. [Figure 10] XX cross-sectional view of FIG. 9.

[0019] [Figure 11] 10 is a cross-sectional view taken along the line XI-XI in FIG. 9. [Figure 12] FIG. 10 is an exploded perspective view of the microorganism culture device of FIG. 9. [Figure 13] FIG. 11 is a partially enlarged view of FIG. [Figure 14] FIG. 4 is a perspective view showing a microorganism culture apparatus according to a second embodiment of the present invention. [Figure 15] FIG. 15 is a view taken along the arrow XV in FIG. [Figure 16] 16 is a plan view taken along the arrow XVI in FIG. 15. [Figure 17] 17 is a cross-sectional view taken along the line XVII-XVII in FIG. 16. [Figure 18]FIG. 18 is a perspective view of the cross section shown in FIG. 17. [Figure 19] 18 is a schematic diagram of the cross section shown in FIG. 17. [Figure 20] FIG. 1 is a perspective view of a jacket serving as a culture section.

[0020] [Figure 21] FIG. 21 is a view taken along arrow XXI in FIG. 20. [Figure 22] 22-XXII cross-sectional view of FIG. 21. [Figure 23] FIG. 1 is a perspective view of the jacket, which is the nutrient supply section. [Figure 24] 24 is a view taken along arrow XXIV in FIG. 23. [Figure 25] 25 is a cross-sectional view taken along the line XXV-XXV of FIG. 24. [Figure 26] FIG. [Figure 27] FIG. 27 is a view taken along arrow XXVII in FIG. 26. [Figure 28] FIG. [Figure 29] 29 is a view taken along the arrow XXIX in FIG. 28. [Figure 30] This is a cross-sectional view taken along the line XXX-XXX in Figure 29.

[0021] [Figure 31] FIG. [Figure 32] This is a cross-sectional view of Figure 31 taken along the line XXXII-XXXII. [Figure 33] FIG. 10 is a perspective view of a modified example of a jacket serving as a culture section. [Figure 34] FIG. 34 is a view taken along arrow XXXIV in FIG. 33. [Figure 35] This is a cross-sectional view of Figure 34 taken along the line XXXV-XXXV. [Figure 36] FIG. 34 is an enlarged cross-sectional perspective view of the jacket of FIG. 33. [Figure 37] 10 is a schematic cross-sectional view showing a microorganism culture apparatus according to a third embodiment of the present invention. [Figure 38] FIG. 38 is a perspective view showing an apparatus used to fabricate the device of FIG. 37. [Figure 39] FIG. 10 is a perspective view showing an apparatus used to fabricate a microorganism culture apparatus according to a fourth embodiment of the present invention. [Figure 40] FIG. 10 is a diagram showing the results of the first example. [Figure 41] 1 is a schematic cross-sectional view showing a microorganism culture device used in a second example. [Figure 42] FIG. 10 is a diagram showing the results of the second example. DETAILED DESCRIPTION OF THE INVENTION

[0022] First, the basic configuration of the microbial culture apparatus and microbial culture method of the present invention will be described.

[0023] [Basic form] <Microbial culture device> The microorganism-cultivating device of the present invention is characterized by including a three-layer laminated structure.

[0024] (Three-layer laminated structure) As shown in FIGS. 1 to 3, the three-layer laminated structure includes the following three types of configurations.

[0025] (1) The three-layer laminated structure 10 shown in Figure 1 has a layered culture section 1, a layered nutrient supply section 2 arranged on a first surface 11 of the culture section 1, and a layered environmental component supply section 3 arranged on a second surface 12 of the culture section 1.

[0026] (2) The three-layer laminated structure 10 shown in Fig. 2 has a layered culture section 1 and layered nutrient supply sections 2 arranged on a first surface 11 and a second surface 12 of the culture section 1. In this configuration, it is preferable that the nutrient supply section 2 arranged on the first surface 11 and the nutrient supply section 2 arranged on the second surface 12 differ in at least one of the type and concentration of nutrients they supply.

[0027] (3) The three-layer laminated structure 10 shown in Fig. 3 has a layered culture section 1 and layered environmental component supply sections 3 arranged on a first surface 11 and a second surface 12 of the culture section 1. In this configuration, it is preferable that the environmental component supply section 3 arranged on the first surface and the environmental component supply section 3 arranged on the second surface differ in at least one of the type and concentration of the environmental component they supply.

[0028] It is preferable that the three-layer laminated structure 10 has a membrane filter between each layer.

[0029] The three-layer laminated structure 10 also includes a configuration in which two or more culture sections 1 are laminated, as shown in Fig. 4. This is because two or more culture sections 1 laminated together can be regarded as a single culture section.

[0030] Additionally, the three-layer laminate structure 10 also includes a laterally laminated configuration, as shown in FIG.

[0031] (Cultivation Department) The culture section is adapted to culture microorganisms and has a culture medium in which the microorganisms are seeded, i.e., a microorganism-containing culture medium. As the culture medium, for example, agar or the like can be used.

[0032] The culture unit preferably includes one or more sensors for detecting the culture state. The sensors are selected from a temperature sensor, a pH sensor, and a gas concentration sensor. Furthermore, the culture unit preferably includes one or more stimulus applying units for applying external physical stimuli to the culture unit. The stimulus applying units are selected from a light irradiation unit, a heating unit, an electromagnetic wave irradiation unit, and an ultrasonic vibration unit. For example, as shown in FIG. 1, a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator (ultrasonic vibration unit) 43 are provided in the culture unit 1.

[0033] In the three-layer laminated structure, the culture section is provided so as to be replaceable with another culture section, which is a culture section that differs from the original culture section in at least one of the type of microorganism and the type or concentration of the culture medium.

[0034] (Nutrient Supply Department) The nutrient supply unit supplies nutrients to the culture unit. The nutrients are substrates (growth factors) such as nutrients necessary for the growth of microorganisms. The nutrients are supplied in a solid, liquid, or gaseous state, i.e., as a nutrient-containing material, nutrient-containing liquid, or nutrient-containing gas.

[0035] When supplying a nutrient-containing liquid or a nutrient-containing gas, the nutrient supply unit preferably has an inlet and outlet passages for circulating the liquid or gas. In this case, the inlet and outlet passages are preferably openable and closable. As the opening and closing means, a mechanism having detachable plugs for closing the inlet and outlet passages can be used. In this case, the nutrient-containing material can be used by closing the inlet and outlet passages.

[0036] In the three-layer laminated structure, the nutrient supply section is provided so as to be replaceable with another nutrient supply section or an environmental component supply section, where the other nutrient supply section is a nutrient supply section that is different from the original nutrient supply section in at least one of the type and concentration of nutrients.

[0037] (Environmental component supply department) The environmental component supply unit supplies environmental components to the culture unit. Environmental components are environmental factors that allow microorganisms to grow in conditions close to the natural environment. The environmental components are supplied in a solid, liquid, or gaseous state, i.e., as an environmental component-containing material, an environmental component-containing liquid, or an environmental component-containing gas. For example, soil can be used as is as the environmental component-containing material. For example, seawater can be used as is as the environmental component-containing liquid.

[0038] When supplying an environmental component-containing liquid or gas, the environmental component supply unit preferably has an inlet and outlet passages for circulating the liquid or gas. In this case, the inlet and outlet passages are preferably openable and closable. As the opening and closing means, a mechanism having detachable plugs for closing the inlet and outlet passages can be used. In this case, the environmental component-containing material can be used by closing the inlet and outlet passages.

[0039] In the three-layer laminated structure, the environmental component supplying section is provided so as to be replaceable with another environmental component supplying section or a nutrient supplying section, where the other environmental component supplying section is an environmental component supplying section that is different from the original environmental component supplying section in at least one of the type and concentration of environmental factors.

[0040] (Action and effect) The microorganism culture device of the present invention can achieve the following effects. (a) Since nutrients can be supplied to the culture section 1 from the nutrient supply section 2 and / or environmental components can be supplied from the environmental component supply section 3, microorganisms can be cultured in the culture section 1.

[0041] (b) Since the device can be constructed using the three-layer laminated structure 10, the device configuration can be simplified.

[0042] (c) The culture unit 1 holds a microorganism-containing culture medium, and the nutrient supply unit 2 holds a nutrient-containing material or circulates a nutrient-containing liquid or nutrient-containing gas, and / or the environmental component supply unit 3 holds an environmental component-containing material or circulates an environmental component-containing liquid or environmental component-containing gas, so that the culture operation is easy.

[0043] (d) In the case of including the three-layer laminated structure 10 of Fig. 2, if the type and / or concentration of nutrients supplied by the nutrient supplying section 2 on the first surface 11 side and the nutrient supplying section 2 on the second surface 12 side are different, different nutrient conditions can be applied to the culture section 1 from both sides. Therefore, two types of culture conditions can be set at once.

[0044] (e) In the case of including the three-layer laminated structure 10 of Fig. 3, if the environmental component supplying unit 3 on the first surface 11 side and the environmental component supplying unit 3 on the second surface 12 side supply different types and concentrations of environmental components, different environmental component conditions can be applied to both sides of the culture unit 1. Therefore, two types of culture conditions can be set at once.

[0045] (f) By providing a membrane filter between each layer, microbial contamination between each layer can be prevented.

[0046] (g) By replacing the culture section 1 with another culture section, it is possible to easily select a microorganism-containing medium that is suitable for the culture conditions that have already been set.

[0047] (h) When the nutrient supply unit 2 circulates a nutrient-containing liquid or gas, at least one of the type and concentration of the nutrients supplied can be changed during the process. Therefore, the culture conditions for the culture unit 1 can be easily changed during the culture operation, and a variety of culture conditions can be realized.

[0048] (i) When the environmental component supply unit 3 circulates an environmental component-containing liquid or gas, at least one of the type and concentration of the environmental component being supplied can be changed during operation. Therefore, the culture conditions for the culture unit 1 can be easily changed during operation, and a variety of culture conditions can be realized.

[0049] (j) By replacing the nutrient supply unit 2 with another nutrient supply unit or with an environmental component supply unit, the culture conditions for the culture unit 1 can be easily changed, and a variety of culture conditions can be realized.

[0050] (k) By replacing the environmental component supply unit 3 with another environmental component supply unit or with a nutrient supply unit, the culture conditions for the culture unit 1 can be easily changed, and a variety of culture conditions can be realized.

[0051] (l) The culture state of the culture unit 1 can be detected by a sensor. Therefore, it is easy to determine whether the culture conditions are appropriate.

[0052] (m) By monitoring the detection results by the sensor, the culture conditions for the culture unit 1 can be easily changed based on the monitoring results, as described in (h) to (k) above. Therefore, even during the culture process, optimal culture conditions can be easily achieved, making it possible to obtain a variety of difficult-to-culture microorganisms.

[0053] (n) The stimulating unit can impart a physical stimulus to the culture unit 1 from the outside, thereby activating the culture of microorganisms.

[0054] (Device type) The microorganism culturing device may be, for example, one of the four types shown below, but is not limited to these.

[0055] [1] Three-layer integrated device The three-layer integrated device has a configuration in which the three-layer laminated structure 10 is housed in a single case 51, as shown in the cross-sectional schematic diagram of Figure 6, for example. This allows for the realization of a very compact device. The three-layer laminated structure 10 may have a circular, triangular, rectangular, or other polygonal shape in plan view.

[0056] The nutrient supplying section 2 preferably has an inlet channel 27 and an outlet channel 28 that can be opened and closed. The environmental component supplying section 3 preferably has an inlet channel 37 and an outlet channel 38 that can be opened and closed.

[0057] The culture unit 1 preferably includes a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. Both sensors 41, 42 are provided to detect the culture state of the microorganisms in the culture unit 1. The detection results by both sensors 41, 42 are preferably configured to be monitored by an external device (not shown). The ultrasonic oscillator 43 is provided to apply ultrasonic vibrations to the microorganism-containing medium held in the culture unit 1. The operation of the ultrasonic oscillator 43 is preferably configured to be controlled by an external device.

[0058] Although the three-layer laminated structure 10 in FIG. 6 has the configuration in FIG. 1, it may also have the configurations in FIGS. 2 to 5, etc.

[0059] [2] Jacket-type device 7, the jacket-type device has a three-layer laminated structure 10, which is made up of a culture section 1, a nutrient supply section 2, and an environmental component supply section 3, each of which is configured by a frame body having a frame main body that surrounds an internal space. The planar shape of the three-layer laminated structure 10 can be a circle, a triangle, a rectangle, or any other polygon.

[0060] That is, the culture section 1 has a first frame body 14 surrounding the first internal space 13, the nutrient supply section 2 has a second frame body 24 surrounding the second internal space 23, and the environmental component supply section 3 has a third frame body 34 surrounding the third internal space 33, the first frame body 14 is capable of holding a microorganism-containing culture medium in the first internal space 13, the second frame body 24 is capable of holding a nutrient-containing material in the second internal space 23, or is capable of circulating a nutrient-containing gas or nutrient-containing liquid through the second internal space 23, and the third frame body 34 is capable of holding an environmental component-containing material in the third internal space 33, or is capable of circulating an environmental component-containing gas or environmental component-containing liquid through the third internal space 33, and the first frame body 14, the second frame body 24, and the third frame body 34 can be connected to each other in a stacked state. Furthermore, it is preferable that the first frame body 14, the second frame body 24, and the third frame body 34 are detachable from one another. Furthermore, it is preferable that the second frame body 24 has an inlet channel 27 for allowing fluid to flow into the second internal space 23 and an outlet channel 28 for allowing fluid to flow out from the second internal space 23, and that the third frame body 34 has an inlet channel 37 for allowing fluid to flow into the third internal space 33 and an outlet channel 38 for allowing fluid to flow out from the third internal space 33. In this case, it is preferable that the inlet channels 27, 37 and outlet channels 28, 38 of the second frame body 24 and the third frame body 34 are each configured to be openable and closable, and that the second frame body 24 is capable of holding nutrient-containing material in the second internal space 23 when both the inlet channel 27 and the outlet channel 28 are closed, and that the third frame body 34 is capable of holding environmental component-containing material in the third internal space 33 when both the inlet channel 37 and the outlet channel 38 are closed.

[0061] Thus, in the jacket-type device, the culture section 1 consists of a single jacket having a first frame body 14 configured to hold a microorganism-containing culture medium in the first internal space 13; the nutrient supply section 2 consists of a single jacket having a second frame body 24 configured to hold a nutrient-containing material in the second internal space 23, or a single jacket configured to circulate a nutrient-containing gas or nutrient-containing liquid through the second internal space 23; and the environmental component supply section 3 consists of a single jacket having a third frame body 34 configured to hold an environmental component-containing material in the third internal space 33, or a single jacket configured to circulate an environmental component-containing gas or environmental component-containing liquid through the third internal space 33. In other words, the jacket-type device is constructed by stacking jackets. It is preferable to place a membrane filter between the jackets.

[0062] Furthermore, the culture unit 1 preferably includes a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. Both sensors 41, 42 are provided to detect the culture state of the microorganisms in the culture unit 1. The detection results by both sensors 41, 42 are preferably configured to be monitored by an external device (not shown). The ultrasonic oscillator 43 is provided to apply ultrasonic vibrations to the microorganism-containing medium held in the culture unit 1. The operation of the ultrasonic oscillator 43 is preferably configured to be controlled by an external device.

[0063] Although the three-layer laminated structure 10 in FIG. 7 has the configuration in FIG. 1, it may also have the configurations in FIGS. 2 to 5, etc.

[0064] [3] Printing layer type device In the printed device, as shown in the cross-sectional schematic diagram of Fig. 8, the culture section 1, nutrient supply section 2, and environmental component supply section 3 that make up the three-layer laminated structure 10 are each formed by printing. The planar shape of the three-layer laminated structure 10 can be a circle, a triangle, a rectangle, or any other polygon.

[0065] That is, the culture unit 1 has a first printed layer 15 on which a microorganism-containing culture medium is printed, the nutrient supply unit 2 has a second printed layer 25 on which a nutrient-containing material is printed, and the environmental component supply unit 3 has a third printed layer 35 on which an environmental component-containing material is printed. It is preferable that a membrane filter 40 be disposed between each layer.

[0066] The printing can be performed using a dispenser. For example, when forming the second printing layer 25, the nutrient-containing material is made into a paste and then applied by a dispenser.

[0067] The first printed layer 15 preferably includes a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. Both sensors 41, 42 are provided to detect the culture state of the microorganisms in the first printed layer 15. The detection results by both sensors 41, 42 are preferably configured to be monitored by an external device (not shown). The ultrasonic oscillator 43 is provided to apply ultrasonic vibrations to the microorganism-containing culture medium that is the first printed layer 15. The operation of the ultrasonic oscillator 43 is preferably configured to be controlled by an external device.

[0068] Although the three-layer laminated structure 10 in FIG. 8 has the configuration in FIG. 1, it may also have the configurations in FIGS. 2 to 5, etc.

[0069] [4] Thin film type device The thin-film device differs from the printed-type device in that it includes a thin film instead of a printed layer, but otherwise is the same. Specifically, it includes a first thin film 16, a second thin film 26, and a third thin film 36. The first thin film 16 is formed by doctor-blading a microorganism-containing culture medium, the second thin film 26 is formed by doctor-blading a nutrient-containing material, and the third thin film 36 is formed by doctor-blading a material containing environmental components. It is preferable that a membrane filter 40 be disposed between each thin film.

[0070] Thinning by doctor blade can be performed, for example, as follows: For example, to produce the second thin film 26, the nutrient-containing material is slurried, placed on a carrier film, and then formed into a thin film of a predetermined thickness using a blade, followed by drying.

[0071] <Microbial culture method> The microbial culture method of the present invention can achieve a variety of culture conditions when culturing microorganisms using the above-mentioned microbial culture apparatus.

[0072] (1) The first microbial culture method of the present invention includes at least one of a nutrient changing step of changing at least one of the type and concentration of a nutrient-containing gas or nutrient-containing liquid circulated through the nutrient supply unit 2, and an environmental component changing step of changing at least one of the type and concentration of an environmental component-containing gas or environmental component-containing liquid circulated through the environmental component supply unit 3. This method can be performed using a microbial culture device in which the nutrient supply unit 2 is configured to circulate a nutrient-containing gas or nutrient-containing liquid, or the environmental component supply unit 3 is configured to circulate an environmental component-containing gas or environmental component-containing liquid.

[0073] This method makes it possible to easily change the culture conditions for the culture section 1. Therefore, it is possible to realize a variety of culture conditions and obtain a variety of difficult-to-culture microorganisms.

[0074] (2) The second microbial culture method of the present invention includes at least one of a nutrient exchange step in which the nutrient supplying unit 2 is replaced with another nutrient supplying unit or an environmental component supplying unit, and an environmental component exchange step in which the environmental component supplying unit 3 is replaced with another environmental component supplying unit or a nutrient supplying unit. This method can be performed using a microbial culture device in which the nutrient supplying unit 2 is provided so as to be replaceable with another nutrient supplying unit or an environmental component supplying unit, or the environmental component supplying unit 3 is provided so as to be replaceable with another environmental component supplying unit or a nutrient supplying unit.

[0075] This method makes it possible to easily change the culture conditions for the culture section 1. Therefore, it is possible to realize a variety of culture conditions and obtain a variety of difficult-to-culture microorganisms.

[0076] (3) A third microbial culture method of the present invention includes a monitoring step of detecting and monitoring the culture state in the culture unit 1 using a sensor, and further includes at least one of a nutrient changing step of changing at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid to be circulated through the nutrient supply unit 2 based on the monitoring results, and an environmental component changing step of changing at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid to be circulated through the environmental component supply unit 3. This method can be performed using a microbial culture device in which the nutrient supply unit 2 is configured to circulate a nutrient-containing gas or nutrient-containing liquid, or the environmental component supply unit 3 is configured to circulate an environmental component-containing gas or environmental component-containing liquid, and the culture unit 1 is further equipped with one or more sensors for detecting the culture state.

[0077] According to this method, the culture conditions for the culture section 1 can be easily changed based on the monitoring results even during the culture. Therefore, optimal culture conditions can be easily achieved, making it possible to obtain a variety of difficult-to-culture microorganisms.

[0078] (4) A fourth microbial culture method of the present invention includes a monitoring step of detecting and monitoring the culture state in the culture unit 1 using a sensor, and further includes at least one of a nutrient exchange step of replacing the nutrient supply unit 2 with another nutrient supply unit or an environmental component supply unit based on the monitoring results, and an environmental component exchange step of replacing the environmental component supply unit 3 with another environmental component supply unit or a nutrient supply unit. This method can be performed using a microbial culture device in which the nutrient supply unit 2 is replaceable with another nutrient supply unit or an environmental component supply unit, or the environmental component supply unit 3 is replaceable with another environmental component supply unit or a nutrient supply unit, and the culture unit 1 is further equipped with one or more sensors for detecting the culture state.

[0079] According to this method, the culture conditions for the culture section 1 can be easily changed based on the monitoring results even during the culture. Therefore, optimal culture conditions can be easily achieved, making it possible to obtain a variety of difficult-to-culture microorganisms.

[0080] Next, specific embodiments of the microbial culture apparatus and microbial culture method of the present invention will be described.

[0081] [First embodiment] 9 is a perspective view showing a microorganism culture device according to a first embodiment of the present invention. This microorganism culture device 100A has a configuration in which one three-layer laminated structure 10 is housed in one case 51, that is, it is a "three-layer integrated device."

[0082] Fig. 10 is a cross-sectional view taken along line XX in Fig. 9. Fig. 11 is a cross-sectional view taken along line XI-XI in Fig. 9. Fig. 12 is an exploded perspective view of the microorganism culture apparatus 100A in Fig. 9. As shown in Fig. 12, the microorganism culture apparatus 100A has a case 51, a perforated plate 52 and its mounting member 53, a cover body 54 and its sealing member 55.

[0083] The case 51 is a thin box-like body, and a first recess 56, a second recess 57, and the like are press-molded on the surface. The first recess 56 has a depth D1. An outer peripheral edge 561 of the first recess 56 is located near and along the peripheral edge 511 of the surface of the case 51. The second recess 57 has a depth D2. The second recess 57 is formed in the first recess 56 and in the center of the case 51 in the longitudinal direction X, so as to have a rectangular shape in a plan view. D2>D1, and D2 is approximately half the thickness of the case 51.

[0084] A concave mounting member 53 is fitted into the second recess 57. The second recess 57 is closed by the perforated plate 52 by fixing the perforated plate 52 to a peripheral frame 531 of the mounting member 53 with screws 521. The perforated plate 52 has a large number of through holes 522. With the perforated plate 52 closing the second recess 57, the surface of the perforated plate 52 is flush with the bottom surface of the first recess 56.

[0085] The first recess 56 has an inlet channel 564 for allowing fluid to flow into the first recess 56, and an outlet channel 565 for allowing fluid to flow out of the first recess 56. The inlet channel 564 extends upstream from the center of the first recess 56 in the width direction Y on the upstream side of the first recess 56, penetrates the upstream side surface 513 of the case 51, and is connected to the cylindrical body 567. The outlet channel 565 extends downstream from the center of the first recess 56 in the width direction Y on the downstream side of the first recess 56, penetrates the downstream side surface 514 of the case 51, and is connected to the cylindrical body 567.

[0086] The first recess 56 has a flow straightening rib 58 on a bottom surface 568 upstream of the second recess 57, and a flow straightening rib 59 on a bottom surface 569 downstream of the second recess 57. The flow straightening rib 58 includes a front-stage rib 581 provided to evenly distribute the fluid flowing in from the inlet channel 564 in the width direction Y, and a rear-stage rib 582 that rectifies the fluid distributed in the width direction Y along the length direction X. A large number of rear-stage ribs 582 are provided at equal intervals along the width direction Y. The flow straightening rib 59 includes a large number of ribs 591 provided in the same manner as the rear-stage ribs 582.

[0087] A seal groove 551 is formed around the first recess 56. The cover body 54 is fixed to the surface of the case 51 with screws 511 while pressing down from above on the seal member 55 fitted into the seal groove 551. The cover body 54 seals the first recess 56 and the second recess 57.

[0088] The second recess 57, which is closed by the perforated plate 52, contains an environmental component-containing material. Each of the numerous through-holes 522 in the perforated plate 52 is filled with a culture medium in which microorganisms have been seeded, i.e., a microorganism-containing culture medium. Since the environmental component-containing material and the microorganism-containing culture medium are in contact with each other, the microorganisms in the perforated plate 52 are supplied with environmental components. Furthermore, a nutrient-containing liquid is introduced into the first recess 56 from the inlet channel 564, flows over the surface of the perforated plate 52, and is then discharged from the outlet channel 565. In other words, since the nutrient-containing liquid and the microorganism-containing culture medium are in contact with each other, the microorganisms in the perforated plate 52 are supplied with nutrients. Therefore, the microorganism culture device 100A includes a three-layer laminated structure 10 in a case 51, the three-layer laminated structure 10 having a layered environmental component supply section formed by storing an environmental component-containing material in the second recess 57, a layered culture section formed by filling the through-holes 522 of the porous plate 52 with a microorganism-containing medium, and a layered nutrient supply section formed by circulating a nutrient-containing liquid through the first recess 56. Note that membrane filters are disposed between the environmental component supply section and the culture section, and between the nutrient supply section and the culture section.

[0089] As shown in Fig. 13, the perforated plate 52 is provided with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. Both sensors 41, 42 are provided to detect the culture state of the microorganisms in each of the through-holes 522. The detection results by both sensors 41, 42 can be monitored by an external device (not shown). The ultrasonic oscillator 43 is provided to apply ultrasonic vibrations to the microorganism-containing culture media in all of the through-holes 522. The operation of the ultrasonic oscillator 43 is controlled by the external device.

[0090] Such a microorganism-culture apparatus 100A can exhibit the following effects. (a) Environmental components can be supplied from below and nutrients can be supplied from above to the microorganisms in the through-holes 522 of the perforated plate 52, so that the microorganisms can be cultivated in the through-holes 522.

[0091] (b) Microorganisms can be cultured simply by passing a nutrient-containing liquid through the first recess 56. This makes it possible to easily culture microorganisms, thereby improving the possibility of obtaining difficult-to-culture microorganisms.

[0092] (c) Since only one three-layer laminated structure 10 capable of cultivating microorganisms is provided in the case 51, a very compact microorganism cultivating device can be realized.

[0093] (d) At least one of the type and concentration of the nutrient-containing liquid flowing through the first recess 56 can be changed (nutrient changing step). Therefore, a variety of culture conditions can be easily realized, and the process of selecting culture conditions suitable for the microorganism can be easily performed.

[0094] (e) The temperature sensor 41 and / or pH sensor 42 can detect and monitor the culture state of the microorganisms in the through-hole 522 (monitoring step). Therefore, the culture state can be determined quickly and accurately.

[0095] (f) Based on the monitoring results, at least one of the type and concentration of the nutrient-containing liquid flowing through the first well 56 can be changed (nutrient changing step). Therefore, culture conditions suitable for the microorganisms can be easily achieved.

[0096] (g) The ultrasonic oscillator 43 can apply vibrations to the microorganisms in the through-holes 522, thereby activating the culture of the microorganisms, thereby improving the culture efficiency.

[0097] [Modification of the first embodiment] The microorganism culture apparatus 100A of the first embodiment can adopt any of the following modifications.

[0098] (1) The first recess 56 is configured to allow nutrient-containing gas to flow through it.

[0099] (2) The first recess 56 is configured to allow an environmental component-containing liquid or an environmental component-containing gas to flow therethrough.

[0100] (3) The second recess 57 contains a nutrient-containing material.

[0101] (4) The second recess 57 contains a nutrient-containing material, and the first recess 56 is configured to allow an environmental component-containing liquid or an environmental component-containing gas to flow through it.

[0102] [Second embodiment] Fig. 14 is a perspective view showing a microorganism culture apparatus according to a second embodiment of the present invention. This microorganism culture apparatus 100B is a "jacket-type apparatus." Fig. 15 is a view taken along arrow XV in Fig. 14. Fig. 16 is a view taken along arrow XVI in Fig. 15 (plan view). Fig. 17 is a cross-sectional view taken along line XVII-XVII in Fig. 16. Fig. 18 is a perspective view of the cross section shown in Fig. 17.

[0103] The microorganism culture apparatus 100B is constructed by stacking seven jackets 61 to 67. Jacket 61 is stacked on a base 60, and jacket 67 is closed with a lid 68. As shown in the cross-sectional schematic diagram of Figure 19, in this apparatus 100B, jacket 61 is an environmental component supply section 3, jackets 62 to 64 and 66 are a culture section 1, and jackets 65 and 67 are a nutrient supply section 2. This apparatus 100B is equipped with two sets of three-layer laminated structures 10A and 10B; that is, by regarding jackets 62 to 64 as one culture section, it is equipped with a three-layer laminated structure 10A consisting of jackets 61 to 65, and a three-layer laminated structure 10B consisting of jackets 65 to 67.

[0104] As shown in Figures 20 to 22, the jacket 62, which is the culture unit 1, includes an annular first frame body 14 that surrounds the first internal space 13. Figure 20 is a perspective view of the jacket 62. Figure 21 is a view taken along arrow XXI in Figure 20. Figure 22 is a cross-sectional view taken along line XXII-XXII in Figure 21. The first frame body 14 has an outer fitting portion 142 with an internal thread 141 at its lower part, and an inner fitting portion 144 with an external thread 143 at its upper part. The outer fitting portion 142 has dimensions that allow it to be externally fitted into the internal fitting portion 144. The internal fitting portion 144 has dimensions that allow it to be internally fitted into the outer fitting portion 142. The first internal space 13 includes an internal space 131 surrounded by the external fitting portion 142, and an internal space 132 other than the internal space 131. The first frame body 14 holds a microorganism-containing culture medium in the internal space 132. Note that the microorganism-containing culture medium is not shown in Figures 20 to 22. The jackets 63, 64, and 66 have the same configuration as the jacket 62.

[0105] The jacket 65, which is the nutrient supply unit 2, has a second annular frame body surrounding the second internal space. In the present device 100B, the jacket 65 has the same configuration as the jacket 62, except that the nutrient-containing material is held in the internal space 132.

[0106] As shown in Figures 23 to 25, the jacket 67, which is the nutrient supply unit 2, has an annular second frame body 24 that surrounds the second internal space 23. Figure 23 is a perspective view of the jacket 67. Figure 24 is a view seen from the arrow XXIV in Figure 23. Figure 25 is a cross-sectional view taken along the line XXV-XXV in Figure 24. The second frame body 24 has an outer fitting portion 242 with an internal thread 241 at its lower part, and an inner fitting portion 244 with an external thread 243 at its upper part. The outer fitting portion 242 has dimensions that allow it to be externally fitted into the internal fitting portion 244. The internal fitting portion 244 has dimensions that allow it to be internally fitted into the outer fitting portion 242. The second internal space 23 has an internal space 231 surrounded by the external fitting portion 242, and an internal space 232 other than the outer fitting portion 242. Furthermore, the second frame body 24 has an inlet channel 27 for allowing fluid to flow into the internal space 232 and an outlet channel 28 for allowing fluid to flow out of the internal space 232. The fluid is a nutrient-containing liquid or nutrient-containing gas. Also, cylindrical bodies 691 and 692 shown in FIG. 26 are connected to the inlet channels 27 and 28, respectively, and protrude radially outward. FIG. 27 is a view taken along arrow XXVII in FIG. 26. The inlet channels 27 and 28 can be closed by inserting a plug (not shown) in place of the cylindrical body 691. This allows the inlet channels 27 and 28 to be opened and closed.

[0107] The jacket 61, which is the environmental component supply unit 3, has an annular third frame body that surrounds the third internal space. In the present device 100B, the jacket 61 has the same configuration as the jacket 67. However, the internal space 232 is configured to allow an environmental component-containing liquid or an environmental component-containing gas to circulate.

[0108] 28 to 30 show the base 60. Fig. 28 is a perspective view of the base 60. Fig. 29 is a view seen from the arrow XXIX in Fig. 28. Fig. 30 is a cross-sectional view taken along the line XXX-XXX in Fig. 29. The base 60 is an annular plate, and has an internal fitting portion 602 with an external thread 601 on the top. The internal fitting portion 602 has dimensions that allow it to be internally fitted into the external fitting portion 142 of the first frame main body 14 and the external fitting portion 242 of the second frame main body 24, respectively.

[0109] Figures 31 and 32 show the lid body 68. Figure 31 is a plan view of the lid body 68. Figure 32 is a cross-sectional view taken along line XXXII-XXXII of Figure 31. The lid body 68 is an annular plate, and has an outer fitting portion 682 with an internal thread 681 at its bottom. The outer fitting portion 682 has dimensions that allow it to be externally fitted onto the internal fitting portion 141 of the first frame body 14 and the internal fitting portion 241 of the second frame body 24, respectively.

[0110] The jacket 61 is connected to the base 60 by threading the outer fitting portion 242 into the inner fitting portion 602 of the base 60, thereby stacking the jackets. Similarly, the jackets 62 to 67 are connected to the base 60 by threading their outer fitting portions into the inner fitting portions of the jackets located below, thereby stacking the jackets. The lid 68 is connected to the jacket 67 by threading the outer fitting portion 682 into the inner fitting portion 244 of the jacket 67. A membrane filter 40 is disposed between the upper and lower jackets to separate them. An O-ring 401 (FIG. 17) seals the gap between the jackets. The device 100B thus includes jackets 61 to 67 with a seven-layer structure.

[0111] Furthermore, as shown in FIG. 19, jackets 62-64, 66, which are the culture unit 1, are each provided with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. The temperature sensor 41 and the pH sensor 42 are arranged to detect the temperature and pH of the microorganism-containing culture medium held in the first internal space 13, and are connected to an external device (not shown) by passing through the first frame body 14 from the inside to the outside. The external device can monitor the temperature and pH of the microorganism-containing culture medium via both sensors 41, 42. The ultrasonic oscillator 43 is arranged inside the first frame body 14 so as to apply vibrations to the microorganism-containing culture medium held in the first internal space 13. The operation of the ultrasonic oscillator 43 is controlled by the external device.

[0112] Such a microorganism-culture apparatus 100B can exhibit the following effects. (a) By circulating an environmental component-containing liquid through jacket 61, the environmental components can be supplied from below and nutrients can be supplied from above to the microorganisms in jackets 62 to 64, allowing the microorganisms to be cultured within jackets 62 to 64. Furthermore, by circulating a nutrient-containing liquid through jacket 67, nutrients can be supplied from both below and above to the microorganisms in jacket 66, allowing the microorganisms to be cultured within jacket 66.

[0113] (b) The two sets of three-layer laminated structures 10A and 10B have different culture conditions, allowing two different culture conditions to be implemented. This improves the efficiency of the work of selecting culture conditions, thereby increasing the possibility of obtaining difficult-to-culture microorganisms.

[0114] (c) In jackets 62 to 64, the culture section has a three-layer structure, and therefore the culture conditions are different in each layer. For example, the concentration of the supplied environmental components is highest in jacket 62 and lowest in jacket 64. Also, the concentration of the supplied nutrients is highest in jacket 64 and lowest in jacket 62. Therefore, the efficiency of the work of selecting culture conditions can be improved, and therefore the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0115] (d) Microorganisms can be cultured simply by passing an environmental component-containing liquid through jacket 61 and a nutrient-containing liquid through jacket 67. This makes it easy to culture microorganisms, thereby improving the possibility of obtaining difficult-to-culture microorganisms.

[0116] (e) Since the device can be assembled simply by connecting the jackets to each other, productivity of the device can be improved.

[0117] (f) The jacket can be easily removed by disconnecting it, and a new jacket can be connected in its place. That is, the jacket can be easily replaced. Therefore, the culture conditions can be easily changed, the efficiency of the culture condition selection process can be improved, and the possibility of obtaining difficult-to-culture microorganisms can be increased. For example, jacket 65 and / or jacket 67, which are nutrient supplying parts, can be replaced with another jacket that is a nutrient supplying part, or with another jacket that is an environmental component supplying part (nutrient replacement step). Also, jacket 61, which is an environmental component supplying part, can be replaced with another jacket that is a environmental component supplying part, or with another jacket that is a nutrient supplying part (environmental component replacement step).

[0118] (g) By increasing the number of jackets, the number of three-layer laminate structures can be increased. This allows different culture conditions to be set for each three-layer laminate structure. This improves the efficiency of the work of selecting culture conditions, thereby increasing the possibility of obtaining difficult-to-culture microorganisms.

[0119] (h) At least one of the type and concentration of the nutrient-containing liquid circulating through jacket 65 and / or jacket 67 can be changed (nutrient changing process). Also, at least one of the type and concentration of the environmental component-containing liquid circulating through jacket 61 can be changed (environmental component changing process). Therefore, a variety of culture conditions can be easily realized, and the process of selecting culture conditions suitable for the microorganism can be easily performed.

[0120] (i) The temperature sensor 41 and / or pH sensor 42 can detect and monitor the culture state of the microorganisms in the jackets 62 to 64, 66 (monitoring step). Therefore, the culture state in each jacket can be determined quickly and accurately.

[0121] (j) Based on the monitoring results, at least one of the type and concentration of the nutrient-containing liquid circulating through jacket 65 and / or jacket 67 can be changed (nutrient changing step), and at least one of the type and concentration of the environmental component-containing liquid circulating through jacket 61 can be changed (environmental component changing step). Therefore, even during the cultivation, culture conditions suitable for the microorganisms can be easily achieved.

[0122] (k) The culture can be activated by applying vibrations to the microorganisms in the jacket using the ultrasonic oscillator 43. This improves the culture efficiency.

[0123] [Modification of the second embodiment] The microorganism culture apparatus 100B of the second embodiment can optionally employ the following modifications.

[0124] (1) In the jacket 62 shown in Figures 33 to 35, the first internal space 13 is formed of a large number of through-holes 522. Figure 33 is a perspective view of the jacket 62. Figure 34 is a view seen from the arrow XXXIV in Figure 33. Figure 35 is a cross-sectional view taken along the line XXXV-XXXV in Figure 34. The jacket 62, which is the culture section 1, is made of a plate having an outer fitting portion 142, and the plate has an external thread 143 formed therein and a large number of through-holes 522 formed therein. In this jacket 62, all of the through-holes 522 are filled with a microorganism-containing culture medium.

[0125] (2) As shown in Figure 36, a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43 are provided in a jacket 62 having a large number of through-holes 522. These are provided for each of the through-holes 522. The temperature sensor 41 and the pH sensor 42 are disposed inside the jacket 62 so as to detect the temperature and pH of the microorganism-containing medium filled in the through-holes 522, and are connected to an external device (not shown). The external device can monitor the temperature and pH of the microorganism-containing medium via both sensors 41 and 42. The ultrasonic oscillator 43 is disposed inside the jacket 62 so as to apply vibrations to the microorganism-containing medium filled in the through-holes 522.

[0126] (3) The connection between the jackets is not limited to a screw mechanism using an internal thread and an external thread, but may be, for example, a slide fitting mechanism, a concave-convex fitting mechanism, or an external connecting member.

[0127] (4) The number of jacket layers is not limited to seven, and may be one or more triple-layer laminate structures. In addition, when there are two or more triple-layer laminate structures, adjacent triple-layer laminate structures may share a jacket such as jacket 65.

[0128] (5) The internal space 132 of the jacket 62, which is the culture section 1, may be a space that is partitioned arbitrarily in the horizontal direction, or may be a space that is partitioned arbitrarily in the vertical direction.

[0129] [Third embodiment] 37 is a schematic cross-sectional view showing a microorganism-culture apparatus according to a third embodiment of the present invention. This microorganism-culture apparatus 100C is a "printed layer type apparatus."

[0130] The microorganism culture device 100C is configured by laminating a first printed layer 71 to a sixth printed layer 76 on a base 70. The planar shape of each printed layer can be a circle, a triangle, a rectangle, or any other polygon.

[0131] The first printed layer 71 is the environmental component supply section 3, which is formed by printing an environmental component-containing material. The second printed layer 72, the third printed layer 73, and the fifth printed layer 75 are the culture section 1, which is formed by printing a microorganism-containing medium. The fourth printed layer 74 and the sixth printed layer 76 are the nutrient supply section 2, which is formed by printing a nutrient-containing material. Therefore, the present device 100C has two sets of three-layer laminated structures 10C and 10D: the three-layer laminated structure 10C consisting of the first printed layer 71 to the fourth printed layer 74, and the three-layer laminated structure 10D consisting of the fourth printed layer 74 to the sixth printed layer 76. Note that a membrane filter 40 is disposed between each layer.

[0132] The microorganism culture device 100C is produced using the device shown in Fig. 38. This device 9 is a dispenser having a multi-needle 91. The device 9 forms a printing layer by discharging a printing material stored in a tank 92 from the multi-needle 91 while moving the multi-needle 91. The printing material is a paste-like microorganism-containing culture medium in the case of the culture unit 1, a paste-like nutrient-containing material in the case of the nutrient supply unit 2, and a paste-like environmental component-containing material in the case of the environmental component supply unit 3. In the case of this device 100C, first, a paste-like environmental component-containing material is ejected onto a base 70 to form a first printed layer 71, then a membrane filter 40 is placed on top of that, a paste-like microorganism-containing culture medium is ejected onto that to form a second printed layer 72, then a membrane filter 40 is placed on top of that, a paste-like microorganism-containing culture medium is ejected onto that to form a third printed layer 73, then a membrane filter 40 is placed on top of that, a paste-like nutrient-containing material is ejected onto that to form a fourth printed layer 74, then a membrane filter 40 is placed on top of that, a paste-like microorganism-containing culture medium is ejected onto that to form a fifth printed layer 75, then a membrane filter 40 is placed on top of that, and a paste-like nutrient-containing material is ejected onto that to form a sixth printed layer 76.

[0133] Furthermore, the second printed layer 72, the third printed layer 73, and the fifth printed layer 75, which are the culture sections, are provided with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43, respectively. The temperature sensor 41 and the pH sensor 42 are arranged to detect the temperature and pH of the microorganism-containing medium in each printed layer, and are connected to an external device (not shown). The external device is capable of monitoring the temperature and pH of the microorganism-containing medium via the sensors 41 and 42. The ultrasonic oscillator 43 is arranged so as to impart vibrations to the microorganism-containing medium in each printed layer. The operation of the ultrasonic oscillator 43 is controlled by the external device.

[0134] Such a microorganism-culture apparatus 100C can exhibit the following effects. (a) Environmental components can be supplied from below and nutrients can be supplied from above to the microorganisms in the second printed layer 72 and the third printed layer 73, so that the microorganisms can be cultivated in the second printed layer 72 and the third printed layer 73. Furthermore, nutrients can be supplied to the fifth printed layer 75 from both below and above, so that the microorganisms can be cultivated in the fifth printed layer 75.

[0135] (b) The two sets of three-layer laminated structures 10C and 10D have different culture conditions, allowing two different culture conditions to be implemented. This improves the efficiency of the work of selecting culture conditions, thereby increasing the possibility of obtaining difficult-to-culture microorganisms.

[0136] (c) Because the culture section has a two-layer structure in the second printed layer 72 and the third printed layer 73, the culture conditions are different in each layer. For example, the concentration of the supplied environmental components is higher in the second printed layer 72, and the concentration of the supplied nutrients is higher in the third printed layer 73. This improves the efficiency of the work of selecting culture conditions, thereby increasing the possibility of obtaining difficult-to-culture microorganisms.

[0137] (d) Microorganisms can be cultured simply by forming a printed layer to construct a three-layer laminated structure, which makes it easy to culture microorganisms. This increases the possibility of obtaining difficult-to-culture microorganisms.

[0138] (e) Since assembly can be achieved simply by forming the printing layer, productivity of the device can be improved.

[0139] (f) By increasing the number of printed layers, the number of three-layer laminate structures can be increased. The culture conditions can then be varied for each three-layer laminate structure. This improves the efficiency of the work of selecting culture conditions, thereby increasing the possibility of obtaining difficult-to-culture microorganisms.

[0140] (g) The temperature sensor 41 and / or pH sensor 42 can detect and monitor the culture state of the microorganisms in the second printed layer 72, the third printed layer 73, and the fifth printed layer 75 (monitoring step). Therefore, the culture state in each printed layer can be determined quickly and accurately.

[0141] (h) The culture can be activated by applying vibrations to the microorganisms in the second printed layer 72, the third printed layer 73, and the fifth printed layer 75 using the ultrasonic oscillator 43. This improves the culture efficiency.

[0142] [Modification of the third embodiment] The microorganism culture apparatus 100C of the third embodiment can optionally employ the following modifications.

[0143] (1) The number of stacked printed layers is not limited to six, and may be one or more triple-layer laminate structures. In addition, when there are two or more triple-layer laminate structures, adjacent triple-layer laminate structures may share a printed layer, such as the fourth printed layer 74.

[0144] (2) The printing layer may be formed not only by the method using a dispenser but also by the following method. (2-1) Screen printing method. (2-2) The printing material is applied onto a PET film using a coater, and then punched out to the desired size using a die to form a laminate.

[0145] [Fourth embodiment] The microorganism culture device of the fourth embodiment of the present invention is a "thin film type device." This microorganism culture device 100D has the same configuration as the "printed layer type device" of the third embodiment shown in Fig. 37, but has a thin film instead of a printed layer.

[0146] The microorganism culture device 100D is configured by stacking a first thin film 71A to a sixth thin film 76A on a base 70A. The planar shape of each thin film may be a circle, a triangle, a rectangle, or any other polygon.

[0147] The first thin film 71 is the environmental component supply unit 3, which is made of a thin film of an environmental component-containing material. The second thin film 72, the third thin film 73, and the fifth thin film 75 are the culture unit 1, which is made of a thin film of a microorganism-containing medium. The fourth thin film 74 and the sixth thin film 76 are the nutrient supply unit 2, which is made of a thin film of a nutrient-containing material. Therefore, the present device 100C includes two sets of three-layer laminated structures 10E and 10F: the three-layer laminated structure 10E consisting of the first thin film 71 to the fourth thin film 74, and the three-layer laminated structure 10F consisting of the fourth thin film 74 to the sixth thin film 76. A membrane filter 40 is disposed between each layer.

[0148] The microorganism culture device 100D is fabricated using the device shown in Figure 39. This device 9A is used to perform the doctor blade method. Thinning by the doctor blade method can be performed, for example, as follows. For example, when forming the fourth thin film 74, the nutrient-containing material is made into a slurry, placed on a carrier film 93, and formed into a thin film of a predetermined thickness using a blade, followed by drying. Each thin film is formed in this manner. Then, each thin film is stacked with a membrane filter 40 interposed therebetween. This allows the microorganism culture device 100D to be fabricated.

[0149] Furthermore, the second thin film 72, the third thin film 73, and the fifth thin film 75, which constitute the culture unit 1, are each provided with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. The temperature sensor 41 and the pH sensor 42 are positioned to detect the temperature and pH of the microorganism-containing medium in each thin film, and are connected to an external device (not shown). The external device is capable of monitoring the temperature and pH of the microorganism-containing medium via the sensors 41 and 42. The ultrasonic oscillator 43 is positioned so as to impart vibrations to the microorganism-containing medium in each thin film. The operation of the ultrasonic oscillator 43 is controlled by the external device.

[0150] Such a microorganism-culture device 100D can exert the same effects as the "printed layer type device" of the third embodiment.

[0151] Next, specific examples of the present invention will be described.

[0152] [First Example] The microorganism culture apparatus 100A of the first embodiment (FIG. 9) was used.

[0153] (Configuration of each part) Nutrient Supply Department R2A medium (Nihon Pharmaceutical Co., Ltd.) 3.2g / L

[0154] ·Cultivation Department R2A medium (Nihon Pharmaceutical Co., Ltd.) 3.2g / L Agar powder (Nacalai Tesque, Inc.) 15g / L The above agar solution was autoclaved (121°C / 20 minutes), and when it reached around 60°C, the diluted soil extract was added, stirred, and then filled into each of the through-holes 522 in the culture section. The diluted soil extract was prepared by adding 15 mL of pure water to 5 g of soil and stirring, leaving it for 1 hour, and then serially diluting the supernatant, staining it with DAPI, and counting the number of microorganisms under a microscope, adjusting the concentration so that one microorganism would fit into each of the through-holes 522 in the culture section.

[0155] ·Environmental material supply department Powdered agar (Nacalai Tesque, Inc.) 15g / L 95 mL of the above-mentioned agar aqueous solution is autoclaved (121°C / 20 minutes), and when the temperature reaches around 60°C, 5 mL of soil extract is added, stirred, and then poured into the second recess 57. The soil extract was prepared by adding 15 mL of pure water to 5 g of soil, stirring, and leaving it for 1 hour, after which 5 mL of the supernatant was taken.

[0156] A membrane filter VCWP (Merck Millipore, 0.1 μm) was placed between the culture section and the environmental substance supply section.

[0157] (Culture work) The culture was carried out by continuously supplying R2A medium to the nutrient supply section for one week.

[0158] (Analysis work) After cultivation, colonies formed in the culture were collected and genetic analysis was performed at Techno Suruga Lab Co., Ltd. Homology analysis was performed on approximately 600 bases in the V1 to V4 regions of 16S rDNA, a simple molecular phylogenetic tree was created, and the species was identified. The homology rate indicates the degree of identity of the base sequence, and a new species was determined when the homology rate was lower than 98%. DNA extraction: Achromopeptidase (Fujifilm Wako Pure Chemical Industries, Ltd.) PCR amplification: PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) Base sequence determination: ChromasPro1.7 (Technelysium) Database DB-BA12.0 (manufactured by Techno Suruga Lab Co., Ltd.) International Nucleotide Sequence Database Search date: March 15, 2018

[0159] (Comparative Example) Colonies were generated by agar plate surface smearing and similar genetic analysis was performed.

[0160] (Analysis results) Figure 40 shows the results of genetic analysis. In Figure 40, A indicates 100-98% known species, B indicates 98-94% new species, C indicates 94-91% new genera / families, and D indicates less than 91% new orders. According to this example, approximately 45% of the microorganisms obtained were equivalent to new species, and new microorganisms at the new order and genus level were also obtained. Therefore, it was confirmed that this method is very effective in obtaining difficult-to-culture microorganisms.

[0161] [Second Example] The microorganism culture apparatus 100B of the second embodiment was used, except that the jacket 65 has the same configuration as the jacket 67, as shown in FIG.

[0162] (Configuration of each part) Nutrient supply unit (jacket 67) ·Substrate A solution R2A medium (Nihon Pharmaceutical Co., Ltd.) 0.32g / 100mL Pure water 100mL

[0163] Nutrient supply unit (jacket 65) ·Substrate B solution R2A medium (Nihon Pharmaceutical Co., Ltd.) 0.032g / 100mL Pure water 100mL

[0164] Environmental component supply unit (Jacket 61) Soil extract The ratio was 5g of soil to 15g of pure water.

[0165] Incubation section (jackets 62, 63, 64, 66) Agar powder (Nacalai Tesque, Inc.) 1.5g / 100mL Pure water 95mL The above agar solution was autoclaved (121°C / 20 minutes), and when it reached approximately 60°C, 5 mL of diluted soil extract was added. After stirring, the diluted soil extract was filled into the inner space of the jacket (the incubation section). The diluted soil extract was prepared by adding 15 mL of pure water to 5 g of soil, stirring, leaving it for 1 hour, and then serially diluting the supernatant 10,000 times. Between each section, a membrane filter VCWP (0.1 μm, manufactured by Merck Millipore) was placed.

[0166] (Culture work) Cultivation was carried out by continuously flowing substrate A liquid through the nutrient supply section (jacket 67), substrate B liquid through the nutrient supply section (jacket 65), and soil extract liquid through the environmental component supply section (jacket 61) for one week.

[0167] (Analysis work) After cultivation, colonies formed in the culture were collected and genetic analysis was performed at Techno Suruga Lab Co., Ltd. Homology analysis was performed on approximately 600 bases in the V1 to V4 regions of 16S rDNA, a simple molecular phylogenetic tree was created, and the species was identified. The homology rate indicates the degree of identity of the base sequence, and a new species was determined when the homology rate was lower than 98%. DNA extraction: Achromopeptidase (Fujifilm Wako Pure Chemical Industries, Ltd.) PCR amplification: PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) Base sequence determination: ChromasPro1.7 (Technelysium) Database DB-BA12.0 (manufactured by Techno Suruga Lab Co., Ltd.) International Nucleotide Sequence Database Search date: March 15, 2018

[0168] (Comparative Example) Colonies were generated by agar plate surface smearing and similar genetic analysis was performed.

[0169] (Analysis results) Figure 42 shows the results of genetic analysis. In Figure 42, A indicates 100-98% known species, B indicates 98-94% new species, C indicates 94-91% new genera, and D indicates less than 91% new order. According to this example, approximately 40% of the microorganisms obtained were equivalent to new species, and new microorganisms at the new order and new genus level were also obtained. Therefore, it was confirmed that this method is very effective in obtaining difficult-to-culture microorganisms.

[0170] [Third Example] The microorganism culture apparatus 100C (FIG. 37) of the third embodiment was used.

[0171] (Configuration of each part) Nutrient supply section (6th printing layer 76) ·Substrate A layer R2A medium (Nihon Pharmaceutical Co., Ltd.) 0.32g / 100mL Agar powder (Nacalai Tesque, Inc.) 1.5g / 100mL Pure water 100mL The substrate paste was obtained by autoclaving (121°C / 20 minutes).

[0172] Nutrient supply section (fourth printed layer 74) ·Substrate B layer R2A medium (Nihon Pharmaceutical Co., Ltd.) 0.032g / 100mL Agar powder (Nacalai Tesque, Inc.) 1.5g / 100mL Pure water 100mL The substrate paste was obtained by autoclaving (121°C / 20 minutes).

[0173] ·Environmental component supply section (first printing layer 71) Agar powder (Nacalai Tesque, Inc.) 1.5g / 100mL Pure water 95mL The above agar solution was autoclaved (121°C / 20 minutes), and when the temperature reached around 60°C, 5 mL of soil extract was added and stirred to obtain a soil paste. The soil extract was prepared by adding 15 mL of pure water to 5 g of soil and stirring, leaving it for 1 hour, and then 5 mL of the supernatant was removed.

[0174] ·Culture section (2nd, 3rd, 5th printing layer 72, 73, 75) Agar powder (Nacalai Tesque, Inc.) 1.5g / 100mL Pure water 95mL The above agar solution was autoclaved (121°C / 20 minutes), and when the temperature reached approximately 60°C, 5 mL of diluted soil extract was added and stirred to obtain a culture paste. The diluted soil extract was prepared by adding 15 mL of pure water to 5 g of soil, stirring, leaving it for 1 hour, and then serially diluting the supernatant 10,000 times.

[0175] (Formation of printing layer) Each layer was formed using a screw-type dispenser Quspa Ms (manufactured by Shinwa Co., Ltd.). The following multi-needle was used as the needle. The needle tip and syringe were heated to 60°C with a heater to keep each paste in a molten state. The paste was applied to a thickness of approximately 1 mm with a gap of 1.2 mm. It solidified when left at room temperature. 21G (inner diameter 0.51mm) x 8 wires, pitch 0.93mm Between each layer, a membrane filter VCWP (0.1 μm, manufactured by Merck Millipore) was placed.

[0176] (Culture work) After forming a laminate consisting of the printed layers, it was placed in a sterilized petri dish and cultured for one week. [Industrial Applicability]

[0177] The microbial culture apparatus of the present invention is highly useful industrially because it makes it possible to obtain a variety of difficult-to-culture microorganisms. [Explanation of symbols]

[0178] 100A~C Microbial culture device 10A~F Three-layer laminated structure 1 Culture department 11 1st surface 12 Second surface 13 1st interior space 14 First Frame Body 15 1st printing layer 16 First thin film body 2. Nutrient Supply Section 23 Second interior space 24 Second frame body 25 2nd printing layer 26 Second thin film body 27 Inflow channel 28 Outflow channel 3 Environmental component supply department 33 Third internal space 34 Third Frame Body 35 3rd printing layer 36 Third thin film body 37 Inflow channel 38 Outflow channel 41 Temperature Sensor 42 pH sensor 43 Ultrasonic oscillator (ultrasonic vibration part) 51 cases

Claims

1. a layered culture section for culturing microorganisms; At least one of a layered nutrient supply section that supplies nutrients to the culture section and a layered environmental component supply section that supplies environmental components to the culture section, the layered nutrient supply section being arranged on a first surface of the culture section and a second surface opposite to the first surface; a three-layer laminate structure having The culture unit holds a microorganism-containing culture medium, The nutrient supply holds a nutrient-containing material or is adapted to pass a nutrient-containing gas or a nutrient-containing liquid through it; the environmental component supply unit holds an environmental component-containing material or allows an environmental component-containing gas or an environmental component-containing liquid to flow; The culture unit includes a first frame body that surrounds a first internal space, The nutrient supply unit includes a second frame body that surrounds a second internal space, the environmental component supply unit includes a third frame body that surrounds a third internal space, the first frame body is capable of holding a microorganism-containing culture medium in the first internal space, The second frame body is capable of holding a nutrient-containing material in the second internal space, or is capable of passing a nutrient-containing gas or a nutrient-containing liquid through the second internal space, the third frame body is capable of holding an environmental component-containing material in the third internal space, or is capable of circulating an environmental component-containing gas or an environmental component-containing liquid in the third internal space, The first frame body, the second frame body, and the third frame body can be connected to each other in a stacked state, The first frame body, the second frame body, and the third frame body are detachable from one another, Further comprising a base and a lid, the first frame body, the second frame body, and the third frame body each have a first fitting portion and a second fitting portion, the base has the first fitting portion, the lid body has the second fitting portion, and the first fitting portion and the second fitting portion are fitable with each other, In the three-layer laminated structure, The culture unit is provided so as to be replaceable with another culture unit, or The nutrient supply unit is provided so as to be replaceable with another nutrient supply unit or a supply unit of an environmental component, or The environmental component supply unit is provided so as to be replaceable with another environmental component supply unit or a nutrient supply unit. A microbial culture device characterized by:

2. In the three-layer laminated structure, The nutrient supply unit is disposed on the first surface of the culture unit, and the environmental component supply unit is disposed on the second surface of the culture unit. The microbial culture device according to claim 1.

3. In the three-layer laminated structure, The nutrient supply unit is disposed on the first surface and the second surface of the culture unit. The microbial culture device according to claim 1.

4. The nutrient supplying section arranged on the first surface and the nutrient supplying section arranged on the second surface are different in at least one of the type and concentration of nutrients to be supplied. The microbial culture device according to claim 3.

5. In the three-layer laminated structure, The environmental component supply unit is disposed on the first surface and the second surface of the culture unit. The microbial culture device according to claim 1.

6. the environmental component supply unit arranged on the first surface and the environmental component supply unit arranged on the second surface differ in at least one of the type and concentration of the environmental component to be supplied; The microbial culture device according to claim 5.

7. The culture unit is equipped with one or more sensors for detecting the culture state. The microorganism culture device according to any one of claims 1 to 6.

8. The sensor is selected from a temperature sensor, a pH sensor, and a gas concentration sensor. The microbial culture device according to claim 7.

9. One or more types of stimulus applying units that apply physical stimuli to the culture unit from the outside are attached to the culture unit. The microbial culture device according to claim 1.

10. The stimulus applying unit is selected from a light irradiating unit, a heating unit, an electromagnetic wave irradiating unit, and an ultrasonic vibration unit. The microorganism culture device according to claim 9.

11. the second frame body has an inflow path for allowing a fluid to flow into the second internal space and an outflow path for allowing a fluid to flow out of the second internal space, the third frame body has an inflow path for allowing a fluid to flow into the third internal space and an outflow path for allowing a fluid to flow out of the third internal space, The inflow path and the outflow path of the second frame body and the third frame body are configured to be openable and closable, respectively; the second frame body is capable of holding a nutrient-containing material in the second internal space when both the inlet and outlet channels are closed, the third frame body is capable of holding an environmental component-containing material in the third internal space with both the inlet channel and the outlet channel closed. The microbial culture device according to claim 1.

12. A method for culturing microorganisms using the microorganism culturing apparatus according to claim 1, comprising: The method includes at least one of a nutrient changing step of changing at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid to be circulated in the nutrient supply unit, and an environmental component changing step of changing at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid to be circulated in the environmental component supply unit. A method for culturing microorganisms, comprising:

13. A method for culturing microorganisms using the microorganism culturing apparatus according to claim 1, comprising: The method includes at least one of a nutrient exchange step of exchanging the nutrient supply unit with another nutrient supply unit or an environmental component supply unit, and an environmental component exchange step of exchanging the environmental component supply unit with another environmental component supply unit or a nutrient supply unit. A method for culturing microorganisms, comprising:

14. A method for culturing microorganisms using the microorganism culture apparatus according to claim 7, which is dependent on claim 1, a monitoring step of detecting and monitoring the culture state of the culture section using a sensor; At least one of a nutrient changing process, in which at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid to be circulated through the nutrient supply unit is changed based on the monitoring results, and an environmental component changing process, in which at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid to be circulated through the environmental component supply unit is changed; Contains, A method for culturing microorganisms, comprising:

15. A method for culturing microorganisms using the microorganism culture apparatus according to claim 7, which is dependent on claim 1, a monitoring step of detecting and monitoring the culture state of the culture section using a sensor; At least one of a nutrient exchange step of exchanging the nutrient supply unit with another nutrient supply unit or an environmental component supply unit based on the monitoring result, and an environmental component exchange step of exchanging the environmental component supply unit with another environmental component supply unit or a nutrient supply unit; Contains, A method for culturing microorganisms, comprising:

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