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 a microorganism culturing apparatus and a microorganism culturing method that can realize various culturing conditions and enable the acquisition of various difficult-to-culture microorganisms.

Background Art

[0002] The collection of target microorganisms is carried out by pure culturing of microorganisms. This pure culturing has generally been conventionally performed by the agar plate surface smearing method. In this method, a group of microorganisms collected from the environment is smeared and cultured on a solid medium prepared in a petri dish. However, in this method, there are many microorganisms that do not grow, and it is said that currently, the microorganisms that can be cultured are about 1% of the microorganisms in the environment. The causes are considered as follows. (a) Since the culture environment is closed, excessive product substances from microorganisms cannot be discharged outside the system. As a result, metabolic products and environmental components of microorganisms accumulate and inhibit the growth of microorganisms. (b) It is difficult to maintain the nutrient concentration required for the growth of the target microorganisms in a solid medium.

[0003] Therefore, culture techniques such as those disclosed in Patent Documents 1 and 2 have been proposed. In Patent Document 1, culturing is performed while continuously supplying a liquid medium. In Patent Document 2, culturing is performed by placing a solid medium containing microorganisms in the natural environment.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, the method described in Patent Document 1 only controls the culture medium components, making it impossible to achieve diverse culture conditions. Furthermore, the method described in Patent Document 2 supplies environmental components from the natural environment, making it impossible to achieve stable culture conditions.

[0006] The present invention aims to provide a microbial culture apparatus and a microbial culture method that can realize diverse culture conditions and enable the acquisition of diverse difficult-to-culture microorganisms. [Means for solving the problem]

[0007] The microbial culture apparatus of the present invention is A layered culture section for culturing microorganisms, At least one of a layered nutrient supply unit for supplying nutrients to the culture unit and a layered environmental component supply unit for supplying environmental components to the culture unit are arranged on the first surface of the culture unit and the second surface opposite the first surface, It includes a three-layer laminated structure having, The culture section holds a culture medium containing microorganisms. The nutrient supply unit holds a nutrient-containing material, or is configured to circulate a nutrient-containing gas or nutrient-containing liquid. The aforementioned environmental component supply unit holds an environmental component-containing material, or is configured to circulate an environmental component-containing gas or environmental component-containing liquid. The culture section comprises a first frame body surrounding a first internal space, The nutrient supply unit comprises a second frame body surrounding the second internal space, The aforementioned environmental component supply unit includes a third frame body that surrounds the third internal space, The first frame body is capable of holding a microbial 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 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 is capable of circulating an environmental component-containing gas or 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 mutually detachable. It further comprises 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 a first fitting portion, the lid has a second fitting portion, and the first fitting portion and the second fitting portion are interlocking. In the aforementioned three-layer laminated structure, The culture section is provided to be replaceable with another culture section, or The nutrient supply unit is provided in a manner that allows it to be replaced with another nutrient supply unit, or with an environmental component supply unit, or The aforementioned environmental component supply unit is provided in a manner that allows it to be replaced with another environmental component supply unit, or with a nutrient supply unit. It is characterized by the following.

[0008] Furthermore, the present invention also includes the following embodiments.

[0009] A microbial culture apparatus according to the first aspect of the present invention is A layered culture section for culturing microorganisms, At least one of a layered nutrient supply unit for supplying nutrients to the culture unit and a layered environmental component supply unit for supplying environmental components to the culture unit are arranged on the first surface of the culture unit and the second surface opposite the first surface, It is characterized by including a three-layer laminated structure having [a specific characteristic].

[0010] A second aspect of the present invention is a microbial culture method that uses a microbial culture apparatus in which a nutrient supply unit 2 is configured to circulate a nutrient-containing gas or nutrient-containing liquid, or an environmental component supply unit 3 is configured to circulate an environmental component-containing gas or environmental component-containing liquid. The system includes at least one of the following steps: a nutrient modification step, which involves changing at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid circulated in the nutrient supply section; and an environmental component modification step, which involves changing at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid circulated in the environmental component supply section.

[0011] A third aspect of the present invention is a microbial culture method that uses a microbial culture apparatus in which a nutrient supply unit is provided in a manner that allows it to be replaced with another nutrient supply unit or with an environmental component supply unit, or in which an environmental component supply unit is provided in a manner that allows it to be replaced with another environmental component supply unit or with a nutrient supply unit. The device includes at least one of the following steps: a nutrient exchange step of exchanging a nutrient supply unit for another nutrient supply unit or for an environmental component supply unit; and an environmental component exchange step of exchanging an environmental component supply unit for another environmental component supply unit or for a nutrient supply unit.

[0012] A fourth aspect of the present invention relates to a microbial culture method that uses a microbial culture apparatus in which a nutrient supply unit 2 is configured to circulate a nutrient-containing gas or nutrient-containing liquid, or an environmental component supply unit 3 is configured to circulate an environmental component-containing gas or environmental component-containing liquid, and furthermore, a culture unit 1 is equipped with one or more sensors for detecting the culture state. The system includes a monitoring step in which the culture state of the culture section is detected and monitored by a sensor, and further includes a nutrient modification step in which at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid supplied to the nutrient supply section is changed based on the monitoring results, and at least one of the environmental component modification step in which at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid supplied to the environmental component supply section is changed.

[0013] The microorganism culture method according to the fifth aspect of the present invention is such that the nutrient supply unit 2 is provided so as to be replaceable with another nutrient supply unit or with an environmental component supply unit, or the environmental component supply unit 3 is provided so as to be replaceable with another environmental component supply unit or with the nutrient supply unit. Further, the culture unit 1 is provided with one or more sensors for detecting the culture state, and uses a microorganism culture device. It includes a monitoring step of detecting and monitoring the culture state of the culture unit by a sensor, and based on the monitoring result, a nutrient exchange step of replacing the nutrient supply unit with another nutrient supply unit or with the environmental component supply unit, and an environmental component exchange step of replacing the environmental component supply unit with another environmental component supply unit or with the nutrient supply unit, and further includes at least one of them.

Advantages of the Invention

[0014] According to the microorganism culture device of the present invention, microorganisms can be cultured in the through-holes of the porous plate, and moreover, the culture of microorganisms can be easily carried out, and furthermore, a very compact microorganism culture device can be realized.

[0015] According to the microorganism culture device of the first aspect of the present invention, nutrients can be supplied from the nutrient supply unit to the culture unit, and / or environmental components can be supplied from the environmental component supply unit, so that microorganisms can be cultured in the culture unit. Moreover, since the device can be constituted by a three-layer laminate structure, the device configuration can be simplified. Therefore, the culture operation can be easily carried out, so that various culture conditions can be realized and various difficult-to-culture microorganisms can be obtained.

[0016] According to the microorganism culture methods of the second and third aspects of the present invention, the culture conditions for the culture unit can be easily changed, so that various culture conditions can be realized and various difficult-to-culture microorganisms can be obtained.

[0017] According to the microorganism culture methods of the fourth and fifth aspects of the present invention, even during the culture, the culture conditions for the culture unit can be easily changed based on the monitoring result, so that the optimal culture conditions can be easily realized and various difficult-to-culture microorganisms can be obtained. [Brief explanation of the drawing]

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

[0019] [Figure 11] This is a cross-sectional view taken along line XI-XI in Figure 9. [Figure 12] Figure 9 is a disassembled perspective view of a microbial culture apparatus. [Figure 13] This is a partially enlarged view of Figure 10. [Figure 14] This is a perspective view showing a microbial culture apparatus according to a second embodiment of the present invention. [Figure 15] This is a view from the XV arrow in Figure 14. [Figure 16] This is a view (plan view) from arrow XVI in Figure 15. [Figure 17] This is a cross-sectional view taken along line XVII-XVII in Figure 16. [Figure 18]Figure 17 is a perspective view of the cross-section shown. [Figure 19] Figure 17 is a schematic diagram of the cross-section shown. [Figure 20] This is a perspective view of the jacket, which serves as the culture section.

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

[0021] [Figure 31] This is a plan view of the lid. [Figure 32] This is a cross-sectional view taken along line XXXII-XXXII in Figure 31. [Figure 33] This is a perspective view of a modified version of the jacket, which serves as the culture section. [Figure 34] This is a view from arrow XXXIV in Figure 33. [Figure 35] This is a cross-sectional view taken from XXXV-XXXV in Figure 34. [Figure 36] Figure 33 is an enlarged cross-sectional perspective view of the jacket. [Figure 37] This is a schematic cross-sectional view showing a microbial culture apparatus according to a third embodiment of the present invention. [Figure 38] This is a perspective view showing the equipment used to manufacture the device shown in Figure 37. [Figure 39] This is a perspective view showing the apparatus used to manufacture the microbial culture apparatus according to the fourth embodiment of the present invention. [Figure 40] This figure shows the results of the first embodiment. [Figure 41] This is a schematic cross-sectional view showing the microbial culture apparatus used in the second embodiment. [Figure 42] This figure shows the results of the second embodiment. [Modes for carrying out the invention]

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

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

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

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

[0026] (2) The three-layer laminated structure 10 shown in Figure 2 has a layered culture section 1 and layered nutrient supply sections 2 arranged on the first surface 11 and the 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 types and concentrations of nutrients supplied.

[0027] (3) The three-layer laminated structure 10 shown in Figure 3 comprises a layered culture section 1 and layered environmental component supply sections 3 arranged on the first surface 11 and the 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 types and concentrations of environmental components supplied.

[0028] Furthermore, it is preferable that the three-layer laminated structure 10 includes a membrane filter between each layer.

[0029] Furthermore, the three-layer laminated structure 10 also includes configurations in which two or more culture sections 1 are stacked, as shown in Figure 4. This is because two or more stacked culture sections 1 can be considered as a single culture section.

[0030] Furthermore, the three-layer laminated structure 10 also includes a configuration in which layers are stacked in the lateral direction, as shown in Figure 5.

[0031] (Cultivation Department) The culture section is designed for culturing microorganisms and contains a culture medium in which microorganisms are seeded, i.e., a microorganism-containing medium. For example, agar can be used as the culture medium.

[0032] The culture unit preferably includes one or more sensors for detecting the culture state. These sensors are selected from a temperature sensor, a pH sensor, and a gas concentration sensor. Furthermore, the culture unit preferably includes one or more stimulation units for applying physical stimulation to the culture unit from the outside. These stimulation 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 Figure 1, the culture unit 1 is provided with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator (ultrasonic vibration unit) 43.

[0033] In a three-layer laminated structure, the culture section is provided to be interchangeable with another culture section. The other culture section is one in which at least one of the following—the type of microorganism and / or the type or concentration of the culture medium—is different from the original culture section.

[0034] (Nutrient supply section) The nutrient supply unit is designed to supply nutrients to the culture unit. Nutrients are substrates (growth factors) such as nutrients necessary for the growth of microorganisms. Nutrients are supplied in solid, liquid, or gaseous form, that is, as nutrient-containing material, nutrient-containing liquid, or nutrient-containing gas.

[0035] When supplying nutrient-containing liquids or gases, the nutrient supply unit preferably has an inlet and an outlet for circulating them. In this case, it is preferable that the inlet and outlet are designed to be openable and closable. As a means of opening and closing, a mechanism can be employed that has detachable stoppers for closing the inlet and outlet. In this case, the nutrient-containing material can be used by closing the inlet and outlet.

[0036] In the three-layer laminated structure, the nutrient supply unit is provided in a manner that allows it to be replaced with another nutrient supply unit, or with an environmental component supply unit. Another nutrient supply unit is a nutrient supply unit in which at least one of the type and concentration of nutrients differs from that of the original nutrient supply unit.

[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 their natural environment. Environmental components are supplied in solid, liquid, or gaseous form, i.e., as environmental component-containing material, environmental component-containing liquid, or environmental component-containing gas. As an environmental component-containing material, for example, soil can be used as is. As an environmental component-containing liquid, for example, seawater can be used as is.

[0038] When supplying an environmental component supply unit, it is preferable that it has an inlet and outlet passage for circulating an environmental component-containing liquid or gas. In this case, it is preferable that the inlet and outlet passages are provided to be openable and closable. As the means for opening and closing, a mechanism can be employed that has detachable plugs for closing the inlet and outlet passages. 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 supply unit is provided in a manner that allows it to be replaced with another environmental component supply unit, or with a nutrient supply unit. Another environmental component supply unit is one in which at least one of the types and concentrations of environmental factors differs from that of the original environmental component supply unit.

[0040] (Effects and Benefits) The microbial culture apparatus of the present invention can exhibit the following effects. (a) 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, so that microorganisms can be cultured in the culture section 1.

[0041] (b) The device can be constructed using a three-layer laminated structure 10, thus simplifying the device configuration.

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

[0043] (d) When the three-layer laminated structure 10 of Figure 2 is included, if at least one of the types and concentrations of nutrients supplied by the nutrient supply section 2 on the first surface 11 side and the nutrient supply section 2 on the second surface 12 side is different, then 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) When the three-layer laminated structure 10 shown in Figure 3 is included, if at least one of the types and concentrations of environmental components supplied by the environmental component supply unit 3 on the first surface 11 side and the environmental component supply unit 3 on the second surface 12 side are different, then different environmental component conditions can be applied to the culture unit 1 from both sides. Therefore, two types of culture conditions can be set at once.

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

[0046] (g) By replacing culture section 1 with another culture section, it is possible to easily select a microbial culture medium suitable for the already established culture conditions.

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

[0048] (i) When the environmental component supply unit 3 distributes an environmental component-containing liquid or gas, at least one of the type and concentration of the supplied environmental component can be changed midway through the process. Therefore, the culture conditions for the culture unit 1 can be easily changed during operation, enabling the realization of diverse culture conditions.

[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 culture section 1 can be detected by a sensor. Therefore, it is easy to determine whether the culture conditions are suitable.

[0052] (m) By monitoring the detection results from the sensor, the culture conditions for culture section 1 can be easily changed based on the monitoring results, as described in (h) to (k) above. Therefore, even in the middle of 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 stimulation unit can apply physical stimulation to the culture unit 1 from the outside, thereby activating the culture of microorganisms.

[0054] (Device type) For example, the following four types of microbial culture devices can be used, but are 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 within a single case 51, as shown in Figure 6, which is a schematic cross-sectional view. This makes it possible to realize a very compact device. The plan view shape of the three-layer laminated structure 10 can be a circle, triangle, square, or other polygon.

[0056] It is preferable that the nutrient supply unit 2 has an openable / closable inlet passage 27 and an outlet passage 28. It is also preferable that the environmental component supply unit 3 has an openable / closable inlet passage 37 and an outlet passage 38.

[0057] The culture unit 1 preferably includes a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. Both sensors 41 and 42 are provided to detect the culture state of microorganisms in the culture unit 1. It is preferable that the detection results from both sensors 41 and 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 medium held in the culture unit 1. It is preferable that the operation of the ultrasonic oscillator 43 is controlled by an external device.

[0058] Although the three-layer laminated structure 10 in Figure 6 has the configuration shown in Figure 1, it may also have configurations such as those shown in Figures 2 to 5.

[0059] [2] Jacket type device In the jacket-type apparatus, as shown in Figure 7, a schematic cross-sectional view, the culture section 1, nutrient supply section 2, and environmental component supply section 3, which constitute the three-layer laminated structure 10, are each composed of a frame body with a frame main body surrounding the internal space. The plan view shape of the three-layer laminated structure 10 can be circular, triangular, quadrilateral, or other polygonal.

[0060] In other words, the culture unit 1 is equipped with a first frame body 14 surrounding a first internal space 13, the nutrient supply unit 2 is equipped with a second frame body 24 surrounding a second internal space 23, and the environmental component supply unit 3 is equipped with a third frame body 34 surrounding a 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 nutrient-containing material in the second internal space 23, or allowing nutrient-containing gas or nutrient-containing liquid to flow through the second internal space 23, and the third frame body 34 is capable of holding environmental component-containing material in the third internal space 33, or allowing environmental component-containing gas or environmental component-containing liquid to flow through the third internal space 33. 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 mutually detachable. Furthermore, it is preferable that the second frame body 24 has an inlet passage 27 for introducing fluid into the second internal space 23 and an outlet passage 28 for introducing fluid out of the second internal space 23, and that the third frame body 34 has an inlet passage 37 for introducing fluid into the third internal space 33 and an outlet passage 38 for introducing fluid out of the third internal space 33. In that case, it is preferable that the inlet passages 27, 37 and outlet passages 28, 38 of the second frame body 24 and the third frame body 34 are 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 passage 27 and the outlet passage 28 are closed, and the third frame body 34 is capable of holding environmental component-containing material in the third internal space 33 when both the inlet passage 37 and the outlet passage 38 are closed.

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

[0062] Furthermore, it is preferable that the culture unit 1 is equipped with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. Both sensors 41 and 42 are provided to detect the culture state of microorganisms in the culture unit 1. It is preferable that the detection results from both sensors 41 and 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 medium held in the culture unit 1. It is preferable that the operation of the ultrasonic oscillator 43 is controlled by an external device.

[0063] Although the three-layer laminated structure 10 in Figure 7 has the configuration shown in Figure 1, it may also have configurations such as those shown in Figures 2 to 5.

[0064] [3] Printing layer type device In the printing-type apparatus, as shown in Figure 8, a schematic cross-sectional view, the culture section 1, nutrient supply section 2, and environmental component supply section 3, which constitute the three-layer laminated structure 10, are each formed by printing. The plan view shape of the three-layer laminated structure 10 can be circular, triangular, quadrilateral, or other polygonal.

[0065] Specifically, the culture unit 1 comprises a first printed layer 15 on which a microorganism-containing culture medium is printed, the nutrient supply unit 2 comprises a second printed layer 25 on which a nutrient-containing material is printed, and the environmental component supply unit 3 comprises a third printed layer 35 on which an environmental component-containing material is printed. Preferably, a membrane filter 40 is placed between each layer.

[0066] 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 using 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 and 42 are provided to detect the culture state of microorganisms in the first printed layer 15. It is preferable that the detection results from both sensors 41 and 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 medium, which is the first printed layer 15. It is preferable that the operation of the ultrasonic oscillator 43 is controlled by an external device.

[0068] Although the three-layer laminated structure 10 in Figure 8 has the configuration shown in Figure 1, it may also have configurations such as those shown in Figures 2 to 5.

[0069] [4] Thin film type device The thin-film type apparatus differs from the printing type apparatus in that it has a thin film instead of a printed layer, but is otherwise the same. Specifically, it comprises a first thin film 16, a second thin film 26, and a third thin film 36. The first thin film 16 is formed by thinning a microbial culture medium using the doctor blade method, the second thin film 26 is formed by thinning a nutrient-containing material using the doctor blade method, and the third thin film 36 is formed by thinning an environmental component-containing material using the doctor blade method. Preferably, a membrane filter 40 is placed between each thin film.

[0070] Thinning using the doctor blade method can be carried out, for example, as follows: For example, when producing a second thin film 26, the nutrient-containing material is made into a slurry, placed on a carrier film, and then cut into a thin film of a predetermined thickness using a blade, and then dried.

[0071] <Microbial culture method> The microbial culture method of the present invention enables the realization of diverse culture conditions when culturing microorganisms using the microbial culture apparatus described above.

[0072] (1) The first microbial culture method of the present invention includes at least one of the following: a nutrient modification step, in which at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid circulated through the nutrient supply unit 2 is changed; and an environmental component modification step, in which 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 unit 3 is changed. This method can be carried out using a microbial culture apparatus 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 allows for easy modification of the culture conditions in culture section 1. Therefore, it is possible to achieve diverse 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 the following: a nutrient exchange step of exchanging the nutrient supply unit 2 for another nutrient supply unit or for an environmental component supply unit; and an environmental component exchange step of exchanging the environmental component supply unit 3 for another environmental component supply unit or for a nutrient supply unit. This method can be carried out using a microbial culture apparatus in which the nutrient supply unit 2 is provided in a manner that allows it to be exchanged for another nutrient supply unit or for an environmental component supply unit, or the environmental component supply unit 3 is provided in a manner that allows it to be exchanged for another environmental component supply unit or for a nutrient supply unit.

[0075] This method allows for easy modification of the culture conditions in culture section 1. Therefore, it is possible to achieve diverse culture conditions and obtain a variety of difficult-to-culture microorganisms.

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

[0077] This method allows for easy modification of the culture conditions for culture section 1 based on monitoring results, even during the culturing process. Therefore, optimal culture conditions can be easily achieved, enabling the acquisition of a diverse range of difficult-to-culture microorganisms.

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

[0079] This method allows for easy modification of the culture conditions for culture section 1 based on monitoring results, even during the culturing process. Therefore, optimal culture conditions can be easily achieved, enabling the acquisition of a diverse range 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] Figure 9 is a perspective view showing a microbial culture apparatus according to the first embodiment of the present invention. This microbial culture apparatus 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 apparatus".

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

[0083] Case 51 is a thin box-shaped body with a first recess 56 and a second recess 57 pressed into its surface. The first recess 56 has a depth D1. The outer edge 561 of the first recess 56 follows the peripheral edge 511 of the surface of case 51 in the vicinity of the peripheral edge 511. The second recess 57 has a depth D2. The second recess 57 is formed inside the first recess 56 and in the center of the case 51 in the longitudinal direction X, in a rectangular shape in plan view. D2 > D1, and D2 is approximately half the thickness of case 51.

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

[0085] The first recess 56 has an inlet passage 564 for introducing fluid into the first recess 56 and an outlet passage 565 for releasing fluid from the first recess 56. The inlet passage 564 extends upstream from the center of the first recess 56 in the width direction Y, passes through the upstream side surface 513 of the case 51, and is connected to the cylindrical body 566. The outlet passage 565 extends downstream from the center of the first recess 56 in the width direction Y, passes through 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 its bottom surface 568 upstream of the second recess 57, and a flow-straightening rib 59 on its bottom surface 569 downstream of the second recess 57. The flow-straightening rib 58 includes a front rib 581 provided to evenly distribute the fluid flowing in from the inlet passage 564 in the width direction Y, and a rear rib 582 that straightens the fluid, which has been distributed in the width direction Y, so that it flows along the length direction X. Numerous rear ribs 582 are provided at equal intervals along the width direction Y. The flow-straightening rib 59 includes numerous ribs 591 provided similarly to the rear 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 holding down the seal member 55 fitted into the seal groove 551 from above. The cover body 54 seals the first recess 56 and the second recess 57.

[0088] The second recess 57, which is sealed by the perforated plate 52, contains an environmental component-containing material. In addition, each of the numerous through-holes 522 of 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, the microorganisms on the perforated plate 52 receive a supply of environmental components. Furthermore, a nutrient-containing liquid flows into the first recess 56 from the inflow passage 564, flows across the surface of the perforated plate 52, and flows out from the outflow passage 565. In other words, since the nutrient-containing liquid and the microorganism-containing culture medium are in contact, the microorganisms on the perforated plate 52 receive a supply of nutrients. Therefore, the microbial culture apparatus 100A is equipped with a three-layer laminated structure 10 inside the case 51, which includes a layered environmental component supply section in which environmental component-containing material is housed in a second recess 57, a layered culture section in which a microbial culture medium is filled into the through-holes 522 of a porous plate 52, and a layered nutrient supply section in which nutrient-containing liquid flows through a first recess 56. Membrane filters are placed between the environmental component supply section and the culture section, and between the nutrient supply section and the culture section.

[0089] As shown in Figure 13, the perforated plate 52 is equipped with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. Both sensors 41 and 42 are provided to detect the culture state of microorganisms in each through-hole 522. The detection results from both sensors 41 and 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 medium in all the through-holes 522. The operation of the ultrasonic oscillator 43 is controlled by an external device.

[0090] This type of microbial culture apparatus 100A can exhibit the following effects: (a) Microorganisms in the through-holes 522 of the perforated plate 52 can be cultured in the through-holes 522 because environmental components can be supplied from below and nutrients can be supplied from above.

[0091] (b) Microorganisms can be cultured simply by flowing a nutrient-containing liquid through the first recess 56, making microbial culture easy to perform. Therefore, the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0092] (c) Since the case 51 contains only one three-layered structure 10 capable of culturing microorganisms, a very compact microbial culture 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 change step). Therefore, diverse culture conditions can be easily realized, and the process of selecting culture conditions suitable for microorganisms can be easily performed.

[0094] (e) The temperature sensor 41 and / or pH sensor 42 can detect and monitor the culture state of 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 recess 56 can be changed (nutrient change step). Thus, suitable culture conditions for microorganisms can be easily achieved.

[0096] (g) The ultrasonic oscillator 43 can impart vibrations to the microorganisms in the through-hole 522, thereby activating the culture of the microorganisms. Therefore, the culture efficiency can be improved.

[0097] [Modified version of the first embodiment] The microbial culture apparatus 100A of the first embodiment can be modified in any way as follows.

[0098] (1) Nutrient-containing gas is to be circulated through the first recess 56.

[0099] (2) The first recess 56 is configured to allow a liquid or gas containing environmental components to flow through it.

[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 a liquid or gas containing environmental components to flow through it.

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

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

[0104] The culture section 1, jacket 62, comprises an annular first frame body 14 surrounding a first internal space 13, as shown in Figures 20 to 22. 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 internal threads 141 at its lower end, and an inner fitting portion 144 with external threads 143 at its upper end. The outer fitting portion 142 has dimensions that allow it to fit onto the inner fitting portion 144. The inner fitting portion 144 has dimensions that allow it to fit onto the outer fitting portion 142. The first internal space 13 comprises an internal space 131 surrounded by the outer fitting portion 142, and the remaining internal space 132. The first frame body 14 holds a microorganism-containing culture medium within the internal space 132. Note that the illustration of the microorganism-containing culture medium is omitted in Figures 20 to 22. Jackets 63, 64, and 66 have the same configuration as jacket 62.

[0105] The nutrient supply unit 2, jacket 65, comprises a ring-shaped second frame body surrounding the second internal space. In this device 100B, jacket 65 has the same configuration as jacket 62. However, nutrient-containing material is held within the internal space 132.

[0106] The nutrient supply unit 2, the jacket 67, comprises an annular second frame body 24 surrounding the second internal space 23, as shown in Figures 23 to 25. Figure 23 is a perspective view of the jacket 67. Figure 24 is a view taken along arrow XXIV in Figure 23. Figure 25 is a cross-sectional view taken along line XXV-XXV in Figure 24. The second frame body 24 has an outer fitting portion 242 with internal threads 241 at its lower end, and an inner fitting portion 244 with external threads 243 at its upper end. The outer fitting portion 242 has dimensions that allow it to fit onto the inner fitting portion 244. The inner fitting portion 244 has dimensions that allow it to fit onto the outer fitting portion 242. The second internal space 23 comprises an internal space 231 surrounded by the outer fitting portion 242, and the remaining internal space 232. Furthermore, the second frame body 24 has an inlet passage 27 for introducing fluid into the internal space 232 and an outlet passage 28 for releasing fluid from the internal space 232. The fluid is a nutrient-containing liquid or nutrient-containing gas. Also, the cylindrical bodies 691 and 692 shown in Figure 26 are connected to the inlet passages 27 and 28, respectively, and protrude radially outward. Figure 27 is a view from arrow XXVII in Figure 26. The inlet passages 27 and 28 can be closed by inserting a plug (not shown) in place of the cylindrical body 691. In this way, the inlet passages 27 and 28 can be opened and closed.

[0107] The jacket 61, which is the environmental component supply unit 3, has an annular third frame body surrounding the third internal space. In this device 100B, the jacket 61 has the same configuration as the jacket 67. However, an environmental component-containing liquid or gas flows through the internal space 232.

[0108] Figures 28 to 30 show the base 60. Figure 28 is a perspective view of the base 60. Figure 29 is a view taken along arrow XXIX in Figure 28. Figure 30 is a cross-sectional view taken along line XXX-XXX in Figure 29. The base 60 is an annular plate and has an internal fitting portion 602 with an external screw 601 at its upper part. The internal fitting portion 602 has dimensions that allow it to be fitted into the external fitting portion 142 of the first frame body 14 and the external fitting portion 242 of the second frame body 24, respectively.

[0109] Figures 31 and 32 show the lid 68. Figure 31 is a plan view of the lid 68. Figure 32 is a cross-sectional view taken along line XXXII-XXXII of Figure 31. The lid 68 is an annular plate and has an external fitting portion 682 with internal threads 681 at its lower end. The external fitting portion 682 has dimensions that allow it to be 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] Jacket 61 is connected to the base 60 by screwing its outer fitting portion 242 into the inner fitting portion 602 of the base 60, and is therefore stacked. Similarly, jackets 62 to 67 are connected by screwing their outer fitting portions into the inner fitting portions of the jackets below them, and are therefore stacked. The lid 68 is connected to jacket 67 by screwing its outer fitting portion 682 into the inner fitting portion 244 of jacket 67. A membrane filter 40 is positioned between the upper and lower jackets to separate them. The spaces between each jacket are sealed by an O-ring 401 (Figure 17). Thus, the device 100B is equipped with a seven-layer structure of jackets 61 to 67.

[0111] Furthermore, as shown in Figure 19, the culture section 1, consisting of jackets 62-64 and 66, is equipped with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43, respectively. The temperature sensor 41 and pH sensor 42 are positioned 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 penetrating the first frame body 14 from the inside to the outside. The external device is capable of monitoring the temperature and pH of the microorganism-containing culture medium via both sensors 41 and 42. The ultrasonic oscillator 43 is positioned inside the first frame body 14 so as to impart vibration 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 microbial culture apparatus 100B can exhibit the following effects: (a) By circulating a liquid containing environmental components through jacket 61, microorganisms in jackets 62-64 can be supplied with environmental components from below and nutrients from above, thus enabling the cultivation of microorganisms within jackets 62-64. Furthermore, by circulating a liquid containing nutrients through jacket 67, microorganisms in jacket 66 can be supplied with nutrients from both below and above, thus enabling the cultivation of microorganisms within jacket 66.

[0113] (b) It has two sets of three-layered structures 10A and 10B, and since the culture conditions in each are different, two different culture conditions can be implemented. Therefore, the efficiency of the culture condition selection process can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved.

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

[0115] (d) Microorganisms can be cultured simply by circulating a liquid containing environmental components through jacket 61 and a liquid containing nutrients through jacket 67, thus making microbial culture easy to perform. Therefore, the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0116] (e) The equipment can be assembled simply by connecting the jackets together, thus improving productivity.

[0117] (f) The jackets can be easily removed by uncoupling them, and a different jacket can be newly attached as a replacement. In other words, the jackets can be easily replaced. Therefore, the culture conditions can be easily changed, the efficiency of the culture condition selection process can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved. For example, the nutrient supply jacket 65 and / or jacket 67 can be replaced with another nutrient supply jacket or with an environmental component supply jacket (nutrient exchange process). Also, the environmental component supply jacket 61 can be replaced with another environmental component supply jacket or with a nutrient supply jacket (environmental component exchange process).

[0118] (g) The number of three-layered structures can be increased by increasing the number of jackets. Then, different culture conditions can be applied to each three-layered structure. Therefore, the efficiency of selecting culture conditions can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved.

[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 change step). Also, at least one of the type and concentration of the environmental component-containing liquid circulating through jacket 61 can be changed (environmental component change step). Therefore, a variety of culture conditions can be easily realized, and the process of selecting culture conditions suitable for microorganisms can be easily performed.

[0120] (i) The temperature sensor 41 and / or pH sensor 42 can detect and monitor the culture state of microorganisms in jackets 62-64 and 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 change 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 change step). Therefore, suitable culture conditions for microorganisms can be easily achieved even in the middle of cultivation.

[0122] (k) By applying vibrations to the microorganisms inside the jacket using the ultrasonic oscillator 43, the culture can be activated. Therefore, the culture efficiency can be improved.

[0123] [Modified version of the second embodiment] The microbial culture apparatus 100B of the second embodiment can be modified in any way as follows.

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

[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 numerous through-holes 522. These are provided in each of the through-holes 522. The temperature sensor 41 and the pH sensor 42 are positioned inside the jacket 62 to detect the temperature and pH of the microbial medium filled in the through-holes 522 and are connected to an external device (not shown). The external device is able to monitor the temperature and pH of the microbial medium via both sensors 41 and 42. The ultrasonic oscillator 43 is positioned inside the jacket 62 to impart vibration to the microbial medium filled in the through-holes 522.

[0126] (3) The connection between jackets is not limited to a screw mechanism using internal and external threads, but can also be a slide fitting mechanism, a groove-and-groove fitting mechanism, or an external connecting member.

[0127] (4) The number of layers of jackets is not limited to seven; it is sufficient to form one or more three-layer laminated structures. Furthermore, if there are two or more three-layer laminated structures, adjacent three-layer laminated 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 arbitrarily partitioned in the horizontal direction, or a space arbitrarily partitioned in the vertical direction.

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

[0130] The microbial culture apparatus 100C is constructed by stacking the first to sixth printed layers 71 to 76 on a base 70. The planar shape of each printed layer can be circular, triangular, square, or other polygonal.

[0131] The first printed layer 71 is an environmental component supply unit 3, which is constructed by printing an environmental component-containing material. The second printed layer 72, the third printed layer 73, and the fifth printed layer 75 are culture units 1, which are constructed by printing a microorganism-containing culture medium. The fourth printed layer 74 and the sixth printed layer 76 are nutrient supply units 2, which are constructed by printing nutrient-containing material. Therefore, the device 100C comprises two sets of three-layer laminated structures 10C and 10D, namely, a three-layer laminated structure 10C consisting of the first to fourth printed layers 71 to 74, and a three-layer laminated structure 10D consisting of the fourth to sixth printed layers 74 to 76. A membrane filter 40 is placed between each layer.

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

[0133] Furthermore, the culture sections, the second printing layer 72, the third printing layer 73, and the fifth printing layer 75, are each equipped with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43, respectively. The temperature sensor 41 and pH sensor 42 are positioned to detect the temperature and pH of the microbial medium in each printing layer and are connected to an external device (not shown). The external device can monitor the temperature and pH of the microbial medium via both sensors 41 and 42. The ultrasonic oscillator 43 is positioned to impart vibration to the microbial medium in each printing layer. The operation of the ultrasonic oscillator 43 is controlled by the external device.

[0134] Such a microbial culture apparatus 100C can exhibit the following effects: (a) Microorganisms in the second and third printed layers 72 and 73 can be supplied with environmental components from below and nutrients from above, so microorganisms can be cultured in the second and third printed layers 72 and 73. In addition, nutrients can be supplied to the fifth printed layer 75 from both below and above, so microorganisms can be cultured in the fifth printed layer 75.

[0135] (b) It has two sets of three-layered structures 10C and 10D, and since the culture conditions in each are different, two different culture conditions can be implemented. Therefore, the efficiency of the culture condition selection process can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0136] (c) In the second and third printed layers 72 and 73, the culture area has a two-layer structure, so the culture conditions in each layer are different. For example, the concentration of supplied environmental components is higher in the second printed layer 72, and the concentration of supplied nutrients is higher in the third printed layer 73. Therefore, the efficiency of selecting culture conditions can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0137] (d) Microorganisms can be cultured simply by forming a printed layer to create a three-layer laminated structure, making microbial culture easy to perform. Therefore, the possibility of obtaining difficult-to-culture microorganisms can be improved.

[0138] (e) The device can be assembled simply by forming a printed layer, thus improving the productivity of the device.

[0139] (f) The number of three-layer laminated structures can be increased by increasing the number of printed layers. Then, different culture conditions can be applied to each three-layer laminated structure. Therefore, the efficiency of selecting culture conditions can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be improved.

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

[0141] (h) By applying vibrations to the microorganisms in the second printing layer 72, the third printing layer 73, and the fifth printing layer 75 using the ultrasonic oscillator 43, the culture can be activated. Therefore, the culture efficiency can be improved.

[0142] [Modified version of the third embodiment] The microbial culture apparatus 100C of the third embodiment can be modified in any way as follows.

[0143] (1) The number of printed layers is not limited to six; it is sufficient to form one or more three-layer laminated structures. Furthermore, if there are two or more three-layer laminated structures, adjacent three-layer laminated structures may share a printed layer, such as the fourth printed layer 74.

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

[0145] [Fourth Embodiment] The microbial culture apparatus of the fourth embodiment of the present invention is a "thin film type apparatus." This microbial culture apparatus 100D has the same configuration as the "printed layer type apparatus" of the third embodiment shown in Figure 37, but instead of a printed layer, it is equipped with a thin film.

[0146] The microbial culture apparatus 100D is constructed by stacking the first thin film layers 71A to the sixth thin film layers 76A on a base 70A. The planar shape of each thin film layer can be circular, triangular, square, or other polygonal.

[0147] The first thin film 71 is an environmental component supply unit 3, which is constructed by forming 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 culture units 1, which are constructed by forming thin films of a microorganism-containing culture medium. The fourth thin film 74 and the sixth thin film 76 are nutrient supply units 2, which are constructed by forming thin films of nutrient-containing material. Therefore, the apparatus 100C is equipped with two sets of three-layer laminated structures 10E and 10F, namely, a three-layer laminated structure 10E consisting of the first to fourth thin films 71 to 74, and a three-layer laminated structure 10F consisting of the fourth to sixth thin films 74 to 76. A membrane filter 40 is placed between each layer.

[0148] The microbial culture apparatus 100D is fabricated using the apparatus shown in Figure 39. This apparatus 9A is for performing the doctor blade method. Thinning using 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 the carrier film 93, and then cut into a thin film of a predetermined thickness using a blade and dried. Each thin film is formed in this manner. Then, each thin film is stacked with a membrane filter 40 in between. This allows the microbial culture apparatus 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 section 1, are each equipped 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 positioned to detect the temperature and pH of the microbial culture 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 microbial culture medium via both sensors 41 and 42. The ultrasonic oscillator 43 is positioned to impart vibration to the microbial culture medium in each thin film. The operation of the ultrasonic oscillator 43 is controlled by the external device.

[0150] Such a microbial culture apparatus 100D can exhibit the same effects and advantages as the "printed layer type apparatus" of the third embodiment.

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

[0152] [First Embodiment] The microbial culture apparatus 100A of the first embodiment (Figure 9) was used.

[0153] (Composition of each part) • Nutrient supply department • R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 3.2 g / L

[0154] ·Cultivation Department • R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 3.2 g / L Agar powder (manufactured by Nakalai Tesque Co., Ltd.) 15g / L The above agar aqueous solution was autoclaved (121°C / 20 minutes), and when it reached approximately 60°C, the soil extract dilution was added and, after stirring, packed into each through-hole 522 of the culture section. The soil extract dilution was prepared by adding 15 mL of pure water to 5 g of soil, stirring, letting it stand for 1 hour, and then successively diluting the supernatant. DAPI staining was performed, and the number of microorganisms was counted under a microscope. The concentration was adjusted so that one microorganism could be placed in each through-hole 522 of the culture section.

[0155] ·Environmental material supply department • Agar powder (manufactured by Nakalai Tesque Co., Ltd.) 15g / L • 95 mL of the above agar solution is autoclaved (121°C / 20 minutes). When the temperature reaches approximately 60°C, 5 mL of soil extract is added and, after stirring, injected into the second recess 57. The soil extract was prepared by adding 15 mL of pure water to 5 g of soil, stirring, and taking 5 mL of the supernatant after letting it stand for 1 hour.

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

[0157] (Culture work) In the nutrient supply section, R2A medium was continuously supplied for one week to perform the cultivation.

[0158] (Analysis work) After culturing, the colonies generated in the culture area were collected and genetic analysis was performed at Techno Suruga Labs Co., Ltd. Homology analysis was conducted on approximately 600 base pairs in the V1-V4 region of 16S rDNA, a simplified molecular phylogenetic tree was constructed, and the species was identified. The homology rate indicates the degree of sequence agreement; a homology rate lower than 98% was considered a new species. • DNA extraction using achromopeptidase (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) • PCR amplification using PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.) • Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) • Base sequencing: ChromasPro1.7 (Technelysium) • Database DB-BA12.0 (manufactured by Techno Suruga Lab Co., Ltd.) International DNA sequencing database Search date: March 15, 2018

[0159] (Comparative example) Colonies were generated using the agar plate surface smear method, and similar genetic analysis was performed.

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

[0161] [Second Example] The microbial culture apparatus 100B of the second embodiment was used. However, as shown in Figure 41, the jacket 65 has the same configuration as the jacket 67.

[0162] (Composition of each part) • Nutrient supply unit (Jacket 67) ·Substrate A solution • R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.32g / 100mL Pure water 100mL

[0163] • Nutrient supply unit (Jacket 65) ·Substrate B solution • R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.032g / 100mL Pure water 100mL

[0164] • Environmental component supply unit (Jacket 61) • Soil extract • Prepared by mixing 5g of soil with 15g of pure water.

[0165] • Culture section (Jackets 62, 63, 64, 66) • Agar powder (manufactured by Nakalai Tesque Co., Ltd.) 1.5g / 100mL Pure water 95mL The above agar aqueous solution was autoclaved (121°C / 20 minutes), and when it reached approximately 60°C, 5 mL of soil extract dilution was added and stirred before filling the internal space of the jacket, which served as the culture section. The soil extract dilution was prepared by adding 15 mL of pure water to 5 g of soil, stirring, letting it stand for 1 hour, and then sequentially diluting the supernatant 10,000 times. • Membrane filters VCWP (Merck Millipore K.K., 0.1 μm) were placed between each section.

[0166] (Culture work) Substrate A solution was continuously supplied to the nutrient supply unit (jacket 67), substrate B solution to the nutrient supply unit (jacket 65), and soil extract to the environmental component supply unit (jacket 61) for one week during cultivation.

[0167] (Analysis work) After culturing, the colonies generated in the culture area were collected and genetic analysis was performed at Techno Suruga Labs Co., Ltd. Homology analysis was conducted on approximately 600 base pairs in the V1-V4 region of 16S rDNA, a simplified molecular phylogenetic tree was constructed, and the species was identified. The homology rate indicates the degree of sequence agreement; a homology rate lower than 98% was considered a new species. • DNA extraction using achromopeptidase (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) • PCR amplification using PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.) • Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) • Base sequencing: ChromasPro1.7 (Technelysium) • Database DB-BA12.0 (manufactured by Techno Suruga Lab Co., Ltd.) International DNA sequencing database Search date: March 15, 2018

[0168] (Comparative example) Colonies were generated using the agar plate surface smear method, and similar genetic analysis was performed.

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

[0170] [Third Embodiment] The microbial culture apparatus 100C of the third embodiment (Figure 37) was used.

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

[0172] • Nutrient supply section (4th printing layer 74) ·Substrate B layer • R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.032g / 100mL Agar powder (manufactured by Nakalai Tesque Co., Ltd.) 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 (manufactured by Nakalai Tesque Co., Ltd.) 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 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, stirring, letting it stand for 1 hour, and then taking 5 mL of the supernatant.

[0174] ·Culture section (2nd, 3rd, 5th printing layer 72, 73, 75) • Agar powder (manufactured by Nakalai Tesque Co., Ltd.) 1.5g / 100mL Pure water 95mL The above agar aqueous solution was autoclaved (121°C / 20 minutes), and when it 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, letting it stand for 1 hour, and then sequentially diluting the supernatant 10,000 times.

[0175] (Formation of the printed 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 with a gap of 1.2 mm and a thickness of approximately 1 mm. It solidified when left at room temperature. 21G (inner diameter 0.51mm) x 8 pieces, pitch 0.93mm A membrane filter VCWP (Merck Millipore K.K., 0.1 μm) was placed between each layer.

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

[0177] The microbial culture apparatus of the present invention has great industrial value because it enables the acquisition of a wide 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. Main body of the first frame 15 1st printing layer 16 First thin film body 2. Nutrient Supply Section 23 Second interior space 24. Main body of the second frame 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. Main body of the 3rd frame 35 3rd printing layer 36 Third thin film body 37 Inflow channel 38 Outflow channel 41 Temperature sensor 42 pH sensors 43. Ultrasonic oscillator (ultrasonic vibration unit) 51 cases

Claims

1. A layered culture section for culturing microorganisms, At least one of a layered nutrient supply unit that supplies nutrients to the culture unit and a layered environmental component supply unit that supplies environmental components to the culture unit are arranged on the first surface of the culture unit and the second surface opposite the first surface, It includes a three-layer laminated structure having, The culture section holds a culture medium containing microorganisms. The nutrient supply unit holds a nutrient-containing material, or is configured to circulate a nutrient-containing gas or nutrient-containing liquid. The aforementioned environmental component supply unit holds an environmental component-containing material, or is configured to circulate an environmental component-containing gas or environmental component-containing liquid. The culture section comprises a first frame body surrounding a first internal space, The nutrient supply unit comprises a second frame body surrounding the second internal space, The aforementioned environmental component supply unit includes a third frame body that surrounds the third internal space, The first frame body is capable of holding a microbial 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 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 is capable of circulating an environmental component-containing gas or 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 mutually detachable. It further comprises 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 a first fitting portion, the lid has a second fitting portion, and the first fitting portion and the second fitting portion are interlocking. In the aforementioned three-layer laminated structure, The culture section is provided to be replaceable with another culture section, or The nutrient supply unit is provided in a manner that allows it to be replaced with another nutrient supply unit, or with an environmental component supply unit, or The aforementioned environmental component supply unit is provided in a manner that allows it to be replaced with another environmental component supply unit, or with a nutrient supply unit. A microbial culture apparatus characterized by the following features.

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

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

4. The nutrient supply unit located on the first surface and the nutrient supply unit located on the second surface differ in at least one of the types and concentrations of nutrients they supply. The microbial culture apparatus according to claim 3.

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

6. The environmental component supply unit located on the first surface and the environmental component supply unit located on the second surface differ in at least one of the types and concentrations of environmental components supplied. The microbial culture apparatus according to claim 5.

7. The culture unit is equipped with one or more sensors for detecting the culture state. A microbial culture apparatus 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 apparatus according to claim 7.

9. One or more stimulating units for applying physical stimuli from the outside are attached to the culture unit. The microbial culture apparatus according to claim 1.

10. The stimulation unit is selected from a light irradiation unit, a heating unit, an electromagnetic wave irradiation unit, and an ultrasonic vibration unit. The microbial culture apparatus according to claim 9.

11. The second frame body has an inlet passage for introducing fluid into the second internal space and an outlet passage for introducing fluid from the second internal space. The third frame body has an inlet passage for introducing fluid into the third internal space and an outlet passage for introducing fluid from the third internal space. The inlet passage and outlet passage of the second frame body and the third frame body are configured to be openable and closable, The second frame body is capable of holding nutrient-containing material in the second internal space when both the inlet passage and the outlet passage are closed. The third frame body is capable of holding environmental component-containing material in the third internal space when both the inlet passage and the outlet passage are closed. The microbial culture apparatus according to claim 1.

12. A method for culturing microorganisms using the microbial culture apparatus described in claim 1, The system includes at least one of the following steps: a nutrient modification step, which involves changing at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid circulated in the nutrient supply unit; and an environmental component modification step, which involves changing at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid circulated in the environmental component supply unit. A method for culturing microorganisms, characterized by the following features.

13. A method for culturing microorganisms using the microbial culture apparatus described in claim 1, The system includes at least one of the following steps: a nutrient exchange step of exchanging the nutrient supply unit for another nutrient supply unit or for an environmental component supply unit, and an environmental component exchange step of exchanging the environmental component supply unit for another environmental component supply unit or for a nutrient supply unit. A method for culturing microorganisms, characterized by the following features.

14. A method for culturing microorganisms, comprising culturing microorganisms using a microbial culture apparatus according to claim 7, which is dependent on claim 1, A monitoring step in which the culture state of the culture section is detected and monitored by a sensor, Based on the monitoring results, a nutrient modification step is performed to change at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid circulated in the nutrient supply unit, and at least one of the environmental component modification steps is performed to change at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid circulated in the environmental component supply unit. Includes, A method for culturing microorganisms, characterized by the following features.

15. A method for culturing microorganisms, comprising culturing microorganisms using a microbial culture apparatus according to claim 7, which is dependent on claim 1, A monitoring step in which the culture state of the culture section is detected and monitored by a sensor, Based on the monitoring results, a nutrient exchange step of replacing the nutrient supply unit with another nutrient supply unit or with an environmental component supply unit, and an environmental component exchange step of replacing the environmental component supply unit with another environmental component supply unit or with a nutrient supply unit, at least one of these, Includes, A method for culturing microorganisms, characterized by the following features.

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