Microorganism culture device and microorganism culture method

Abstract

The present invention is characterized by comprising a three-layer laminated structure having: a layered culture part (1) for culturing microorganisms; a layered nutrient supply unit (2) that is disposed on the first surface (11) of the culture unit (1) and supplies a nutrient to the culture unit (1); and a layered environmental component supply unit (3) that is disposed on the second surface (12) and supplies environmental components to the culture unit (1).

Description

[0001] This application is a divisional application of patent application 202080024024.4 (international application date: March 4, 2020, name of invention: microorganism culture device and microorganism culture method). Technical Field

[0002] The present invention relates to a device and method for culturing microorganisms, and in particular to a device and method for culturing microorganisms that can realize various culturing conditions and make it possible to obtain various difficult-to-cultivate microorganisms. Background Art

[0003] The target microorganism is taken by pure culture of microorganism. In the past, such pure culture was usually carried out by agar plate surface smearing method. In this method, the microorganism group taken from the environment is smeared on a solid culture medium made in a culture dish and cultured. However, there are also many microorganisms that cannot be cultivated by this method. It is now believed that the microorganisms that can be cultivated are about 1% of the microorganisms in the environment. The reason can be considered as follows.

[0004] (a) Since the culture environment is closed, the excess substances produced by the microorganisms cannot be discharged outside the system. As a result, the microbial metabolites and environmental components will accumulate, hindering the growth of the microorganisms.

[0005] (b) It is difficult to maintain the nutrient concentration required for the cultivation of target microorganisms in solid culture media.

[0006] Therefore, there have been proposed culture techniques such as those disclosed in Patent Documents 1 and 2. In Patent Document 1, culture can be performed while continuously supplying a liquid culture medium. In Patent Document 2, a solid culture medium containing microorganisms is placed in a natural environment for culture.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2016-86654

[0010] Patent Document 2: U.S. Patent No. 7011957 Summary of the invention

[0011] However, the method of Patent Document 1 merely controls the medium components, and therefore cannot achieve various culture conditions. In addition, the method of Patent Document 2 supplies environmental components in the natural environment, and therefore cannot achieve stable culture conditions.

[0012] The object of the present invention is to provide a microorganism culture device and a microorganism culture method, which can realize various culture conditions and make it possible to obtain various difficult-to-culture microorganisms.

[0013] A microorganism culture device according to a first aspect of the present invention is characterized by comprising a three-layer stacked structure having:

[0014] a layered culture portion for culturing microorganisms, and

[0015] At least one of a layered nutrient supplying portion for supplying nutrients to the culture portion and a layered environmental component supplying portion for supplying environmental components to the culture portion, which are arranged on a first surface of the culture portion and a second surface opposite to the first surface.

[0016] In the microorganism cultivation method of the second aspect of the present invention, a microorganism cultivation device is used in which a nutrient-containing gas or a nutrient-containing liquid is circulated in the nutrient supply section 2 or a gas or a liquid containing an environmental component is circulated in the environmental component supply section 3.

[0017] Furthermore, it includes at least one of a nutrient changing process and an environmental component changing process, wherein the nutrient changing process is to change at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid circulating in the nutrient supply section, and the environmental component changing process is to change at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid circulating in the environmental component supply section.

[0018] In the microorganism cultivation method of the third aspect of the present invention, a microorganism cultivation device is used in which a nutrient supply unit is configured to be exchangeable with other nutrient supply units or with an environmental component supply unit, or an environmental component supply unit is configured to be exchangeable with other environmental component supply units or with a nutrient supply unit.

[0019] Furthermore, it includes at least one of a nutrient exchange process and an environmental component exchange process, wherein the nutrient exchange process is to exchange the nutrient supply part with other nutrient supply parts or with the environmental component supply part, and the environmental component exchange process is to exchange the environmental component supply part with other environmental component supply parts or with the nutrient supply part.

[0020] In the microorganism cultivation method of the fourth aspect of the present invention, a microorganism cultivation device is used, in which a nutrient-containing gas or a nutrient-containing liquid is circulated in the nutrient supply section 2, or a gas or a liquid containing an environmental component is circulated in the environmental component supply section 3, and the cultivation section 1 is equipped with one or more sensors for detecting the cultivation state.

[0021] Furthermore, it includes a monitoring process of detecting and monitoring the culture state of the culture section using a sensor, and also includes at least one of a nutrient changing process and an environmental component changing process, wherein the nutrient changing process is to change at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid flowing in the nutrient supply section based on the monitoring result, and the environmental component changing process is to change at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid flowing in the environmental component supply section based on the monitoring result.

[0022] In the microorganism cultivation method of the fifth aspect of the present invention, the following microorganism cultivation apparatus is used: the nutrient supply unit 2 is set to be exchangeable with other nutrient supply units or exchangeable with environmental component supply units, or the environmental component supply unit 3 is set to be exchangeable with other environmental component supply units or exchangeable with nutrient supply units, and the cultivation unit 1 has one or more sensors for detecting the cultivation state;

[0023] Furthermore, it includes a monitoring process of detecting and monitoring the culture status of the culture unit using a sensor, and also includes at least one of a nutrient changing process and an environmental component changing process, wherein the nutrient exchanging process is to exchange the nutrient supply unit with other nutrient supply units or with the environmental component supply unit based on the monitoring result, and the environmental component exchanging process is to exchange the environmental component supply unit with other environmental component supply units or with the nutrient supply unit based on the monitoring result.

[0024] According to the microorganism culture device of the first embodiment 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 to the culture unit, so that microorganisms can be cultured in the culture unit. In addition, the device can be constructed by a three-layer stacked structure, so the device structure can be simplified. Therefore, the culture operation can be simply performed, various culture conditions can be achieved, and it is possible to obtain various difficult-to-culture microorganisms.

[0025] According to the microorganism culturing method of the second and third aspects of the present invention, the culturing conditions for the culturing section can be easily changed, so that various culturing conditions can be realized, making it possible to obtain various difficult-to-cultivate microorganisms.

[0026] According to the microorganism cultivation method of the fourth and fifth aspects of the present invention, the cultivation conditions for the cultivation part can be easily changed based on the monitoring results even in the middle of the cultivation. Therefore, the optimal cultivation conditions can be easily achieved, making it possible to obtain various difficult-to-cultivate microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic cross-sectional view showing a first example of the basic form of a three-layer stacked structure included in the microorganism culture device of the present invention.

[0028] Figure 2 It is a schematic cross-sectional view showing a second example of the basic form of the three-layer stacked structure included in the microorganism culture device of the present invention.

[0029] Figure 3 It is a schematic cross-sectional view showing a third example of the basic form of the three-layer stacked structure included in the microorganism culture device of the present invention.

[0030] Figure 4 It is a schematic cross-sectional view showing a modified example of the three-layer stacked structure.

[0031] Figure 5 It is a schematic cross-sectional view showing another modified example of the three-layer stacked structure.

[0032] Figure 6 It is a schematic cross-sectional view showing a first example of the basic form of the microorganism culture device of the present invention.

[0033] Figure 7 It is a schematic cross-sectional view showing a second example of the basic form of the microorganism culture device of the present invention.

[0034] Figure 8 It is a schematic cross-sectional view showing a third example of the basic form of the microorganism culture device of the present invention.

[0035] Fig. 9 It is a perspective view showing the microorganism culture device according to the first embodiment of the present invention.

[0036] Fig.10 for Fig. 9 XX cross-section diagram.

[0037] Fig.11 for Fig. 9 XI-XI section view.

[0038] Fig.12 for Fig. 9 An exploded perspective view of a microbial culture device.

[0039] Fig.13 for Fig.10 A partial enlarged view of .

[0040] Fig.14 It is a perspective view showing a microorganism culture device according to a second embodiment of the present invention.

[0041] Fig.15 for Fig.14 XV direction view.

[0042] Fig.16 for Fig.15 XVI view (plan view).

[0043] Fig.17 for Fig.16 Section XVII-XVII of the.

[0044] Fig.18 for Fig.17 A perspective view of the cross section shown.

[0045] Fig.19 for Fig.17 Schematic diagram of the cross section shown.

[0046] Fig. 20 This is a three-dimensional view of a jacket serving as a culture unit.

[0047] Fig.21 for Fig. 20 XXI direction view.

[0048] Fig. 22 for Fig.21 Section XXII-XXII of .

[0049] Fig.23 This is a three-dimensional view of a jacket serving as a nutrient supply unit.

[0050] Fig.24 for Fig.23 View from the XXIV direction.

[0051] Fig.25 for Fig.24 XXV-XXV section view.

[0052] Fig.26 It is a partial cross-sectional view of the cylinder.

[0053] Fig. 27 for Fig.26 View from the XXVII direction.

[0054] Fig.28 A three-dimensional image of the base.

[0055] Fig.29 for Fig.28 View in the XXIX direction.

[0056] Fig.30 for Fig.29 XXX-XXX cross-sectional view.

[0057] Fig.31 This is a plan view of the cover.

[0058] Fig.32 for Fig.31 Section XXXII-XXXII of FIG.

[0059] Fig.33 This is a perspective view of a modified example of the jacket serving as the culture section.

[0060] Fig.34 for Fig.33 View from direction XXXIV.

[0061] Fig.35 for Fig.34 XXXV-XXXV section view.

[0062] Fig.36 for Fig.33 An enlarged sectional stereogram of the jacket.

[0063] Fig.37 It is a schematic cross-sectional view showing a microorganism culture device according to a third embodiment of the present invention.

[0064] Fig.38 To express in order to make Fig.37 A three-dimensional diagram of a device used in a device.

[0065] Fig.39 It is a perspective view showing a device used to produce the microorganism culture device according to the fourth embodiment of the present invention.

[0066] Fig.40 It is a figure which shows the result of the 1st Example.

[0067] Fig.41 It is a schematic cross-sectional view showing the microorganism culture device used in the second embodiment.

[0068] Fig.42 It is a figure which shows the result of the 2nd Example.

[0069] Description of Reference Numerals

[0070] 100A~C Microbial Culture Device

[0071] 10A~F three-layer laminated structure

[0072] 1. Training Department

[0073] 11 Surface 1

[0074] 12 Surface 2

[0075] 131st Interior Space

[0076] 14 Frame 1 main body

[0077] 151st printing layer

[0078] 16 1st film

[0079] 2Nutrient supply unit

[0080] 23. The 2nd Internal Space

[0081] 24 Frame 2 main body

[0082] 25 2nd printing layer

[0083] 26 2nd film body

[0084] 27 Inflow Road

[0085] 28 outflow

[0086] 3Environmental Component Supply Department

[0087] 33 The 3rd Internal Space

[0088] 34 Frame 3 main body

[0089] 35 3rd printing layer

[0090] 36Third film body

[0091] 37 Inflow Road

[0092] 38 outflow

[0093] 41 Temperature Sensor

[0094] 42pH Sensor

[0095] 43 Ultrasonic oscillator (ultrasonic vibration part)

[0096] 51 Shell DETAILED DESCRIPTION

[0097] First, the basic aspects of the microorganism cultivation device and microorganism cultivation method of the present invention will be described.

[0098] [Basic form]

[0099] <Microorganism culture device>

[0100] The microorganism culture device of the present invention is characterized by comprising a three-layer stacked structure.

[0101] (Three-layer laminated structure)

[0102] like Figure 1 to Figure 3 As shown, the three-layer laminated structure includes the following three structures.

[0103] (1) Figure 1 The three-layer stacked structure 10 shown includes a layered culture section 1 , a layered nutrient supply section 2 disposed on a first surface 11 of the culture section 1 , and a layered environmental component supply section 3 disposed on a second surface 12 of the culture section 1 .

[0104] (2) Figure 2The three-layer stacked structure 10 shown has a layered culture section 1, and a layered nutrient supply section 2 arranged on a first surface 11 and a second surface 12 of the culture section 1. In this configuration, the nutrient supply section 2 arranged on the first surface 11 and the nutrient supply section 2 arranged on the second surface 12 preferably have at least one of the type and concentration of nutrients supplied to be different.

[0105] (3) Figure 3 The three-layer stacked structure 10 shown has a layered culture section 1, and a layered environmental component supply section 3 arranged on a first surface 11 and a second surface 12 of the culture section 1. In this configuration, the environmental component supply section 3 arranged on the first surface and the environmental component supply section 3 arranged on the second surface preferably differ in at least one of the type and concentration of the environmental component supplied.

[0106] In addition, the three-layer laminated structure 10 preferably includes a membrane filter between each layer.

[0107] In addition, the three-layer laminated structure 10 is, for example, Figure 4 As shown in the figure, it includes a structure in which two or more layers of culture parts 1 are stacked. This is because the culture parts 1 stacked in two or more layers can be regarded as a single culture part.

[0108] Furthermore, the three-layer laminated structure 10 is as follows Figure 5 As shown, it includes a structure stacked in the horizontal direction.

[0109] (Cultivation Department)

[0110] The culture part is used to culture microorganisms and has a culture medium for inoculating microorganisms, that is, a culture medium containing microorganisms. As the culture medium, for example, agar or the like can be used.

[0111] The culture section preferably has one or more sensors for detecting the culture state. The sensors may be selected from temperature sensors, pH sensors, and gas concentration sensors. Furthermore, the culture section preferably has one or more stimulation imparting sections for imparting external physical stimulation to the culture section. The stimulation imparting sections may be selected from light irradiation sections, heating sections, electromagnetic wave irradiation sections, and ultrasonic vibration sections. For example, Figure 1 As shown, the culture section 1 is provided with a temperature sensor 41 , a pH sensor 42 , and an ultrasonic oscillator (ultrasonic vibration section) 43 .

[0112] In the three-layer stacked structure, the culture section may be provided to be exchangeable with another culture section. The other culture section refers to a culture section in which at least one of the type of microorganisms and the type or concentration of the culture medium is different from that of the original culture section.

[0113] (Nutrient supply unit)

[0114] The nutrient supply unit supplies nutrients to the culture unit. Nutrients refer to substrates (cultivation factors) such as nutrients necessary for the cultivation of microorganisms. Nutrients are supplied in a solid, liquid, or gaseous state, that is, as a nutrient-containing material, a nutrient-containing liquid, or a nutrient-containing gas.

[0115] The nutrient supply unit preferably has an inflow path and an outflow path for circulating the nutrient-containing liquid or nutrient-containing gas when supplying the nutrient-containing liquid or nutrient-containing gas. In this case, the inflow path and the outflow path are preferably set to be openable and closable. As the opening and closing means, the following mechanism can be adopted: a plug that can be freely disassembled and used to close the inflow path and the outflow path. In this case, the nutrient-containing material can be used by closing the inflow path and the outflow path.

[0116] In the three-layer stacked structure, the nutrient supply unit is provided so as to be exchangeable with other nutrient supply units or with environmental component supply units. Other nutrient supply units are nutrient supply units that are different from the original nutrient supply unit in at least one of the nutrient type and concentration.

[0117] (Environmental Component Supply Department)

[0118] The environmental component supplying unit supplies the environmental component to the culture unit. The environmental component refers to an environmental factor used to cultivate microorganisms in a state close to the natural environment. The environmental component is supplied in a solid, liquid or gaseous state, that is, as an environmental component-containing material, an environmental component-containing liquid or an environmental component-containing gas. As the environmental component-containing material, for example, soil can be used directly. As the environmental component-containing liquid, for example, seawater can be used directly.

[0119] When the environmental component supply unit supplies an environmental component-containing liquid or an environmental component-containing gas, it is preferred that the supply unit has an inflow path and an outflow path for circulating the liquid or the gas. In this case, the inflow path and the outflow path are preferably configured to be openable and closable. As the opening and closing means, the following mechanism can be used: a plug that can be freely disassembled and used to close the inflow path and the outflow path. In this case, the environmental component-containing material can be used by closing the inflow path and the outflow path.

[0120] In the three-layer stacked structure, the environmental component supply unit is provided so as to be exchangeable with other environmental component supply units or nutrient supply units. The other environmental component supply unit is an environmental component supply unit having at least one of the type and concentration of the environmental component different from that of the original environmental component supply unit.

[0121] (Effect)

[0122] According to the microorganism culture device of the present invention, the following effects can be achieved.

[0123] (a) Nutrients can be supplied from the nutrient supply unit 2 to the culture unit 1 and / or environmental components can be supplied from the environmental component supply unit 3 to the culture unit 1 , so that microorganisms can be cultured in the culture unit 1 .

[0124] (b) Since the device can be constructed by the three-layer stacked structure 10, the device configuration can be simplified.

[0125] (c) While the culture section 1 maintains a culture medium containing microorganisms, the microorganisms can be cultured in the culture section 1 by only maintaining a nutrient material in the nutrient supply section 2 or circulating a nutrient liquid or a nutrient gas in the nutrient supply section 2, and / or maintaining an environmental component material in the environmental component supply section 3 or circulating an environmental component liquid or a gas in the environmental component supply section 3, thereby facilitating the culture operation.

[0126] (d) Figure 2 In the case of the three-layer stacked structure 10, when at least one of the type and concentration of nutrients supplied by the nutrient supplying section 2 on the first surface 11 side and the nutrient supplying section 2 on the second surface 12 side is different, different nutrient conditions can be applied to the culture section 1 from both sides. Therefore, two culture conditions can be set at once.

[0127] (e) Figure 3 In the case of a three-layer laminated structure 10, when at least one of the type and concentration of the environmental component supplied by the environmental component supplying section 3 on the first surface 11 side and the environmental component supplying section 3 on the second surface 12 side is different, different environmental component conditions can be applied to the culture section 1 from both sides. Therefore, two culture conditions can be set at once.

[0128] (f) By providing a membrane filter between each layer, contamination of microorganisms between each layer can be prevented.

[0129] (g) By exchanging the culture section 1 with another culture section, a microorganism-containing culture medium suitable for the set culture conditions can be easily selected.

[0130] (h) When a nutrient-containing liquid or nutrient-containing gas is circulated in the nutrient supply unit 2, at least one of the type and concentration of the supplied nutrients can be changed midway. Therefore, the culture conditions of the culture unit 1 can be easily changed during the culture operation, and various culture conditions can be realized.

[0131] (i) When the environmental component-containing liquid or the environmental component-containing gas is circulated in the environmental component supply unit 3, at least one of the type and concentration of the supplied environmental component can be changed during the operation. Therefore, the culture conditions of the culture unit 1 can be easily changed during the operation, and various culture conditions can be realized.

[0132] (j) By exchanging 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, thereby realizing various culture conditions.

[0133] (k) By exchanging 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, thereby realizing various culture conditions.

[0134] (1) The culture state of the culture section 1 can be detected by the sensor. Therefore, the suitability of the culture conditions can be easily determined.

[0135] (m) By monitoring the detection results of the sensor, the culture conditions of the culture unit 1 can be easily changed as described in (h) to (k) above based on the monitoring results. Therefore, even in the middle of the culture operation, the optimal culture conditions can be easily achieved, making it possible to obtain various difficult-to-culture microorganisms.

[0136] (n) Since a physical stimulus from the outside is applied to the culture section 1 by the stimulus applying section, the culture of the microorganisms can be activated.

[0137] (Device Type)

[0138] As the microorganism culture apparatus, for example, the following four types can be adopted, but the present invention is not limited thereto.

[0139] [1] Three-layer integrated device

[0140] In the three-layer integrated device, for example, as shown in the cross-sectional diagram Figure 6 As shown, the three-layer stacked structure 10 is contained in one housing 51. This makes it possible to realize a very compact device. The three-layer stacked structure 10 may be circular, triangular, quadrilateral, or other polygonal in plan view.

[0141] The nutrient supply unit 2 preferably has an openable and closable inflow path 27 and an outflow path 28. In addition, the environmental component supply unit 3 preferably has an openable and closable inflow path 37 and an outflow path 38.

[0142] The culture section 1 preferably includes a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. The two sensors 41 and 42 are provided in a manner to detect the culture state of the microorganisms in the culture section 1. The detection results of the two sensors 41 and 42 can preferably be monitored in an external device (not shown). The ultrasonic oscillator 43 is provided in a manner to impart ultrasonic vibration to the microorganism-containing culture medium held in the culture section 1. The operation of the ultrasonic oscillator 43 is preferably controlled by an external device.

[0143] It should be explained that Figure 6 The three-layer laminated structure 10 has Figure 1 but can also have Figure 2 to Figure 5 Etc. composition.

[0144] [2] Jacketed device

[0145] In the jacketed device, as shown in the cross-sectional diagram Figure 7 As shown, the culture part 1, nutrient supply part 2 and environmental component supply part 3 constituting the three-layer stacked structure 10 are respectively constituted by a frame having a frame body surrounding the internal space. It should be noted that the plan view shape of the three-layer stacked structure 10 can be circular, triangular, quadrilateral, or other polygonal.

[0146] That is, the culture part 1 includes a first frame body 14 surrounding the first internal space 13, the nutrient supply part 2 includes a second frame body 24 surrounding the second internal space 23, and the environmental component supply part 3 includes a third frame body 34 surrounding the third internal space 33; the first frame body 14 can hold the microorganism-containing culture medium in the first internal space 13, the second frame body 24 can hold the nutrient-containing material in the second internal space 23, or allow the nutrient-containing gas or nutrient-containing liquid to flow in the second internal space 23, and the third frame body 34 can hold the environmental component-containing material in the third internal space 33, or allow the environmental component-containing gas or environmental component-containing liquid to flow in 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, the first frame body 14, the second frame body 24, and the third frame body 34 are preferably detachable from each other. Furthermore, the second frame body 24 preferably has an inflow path 27 for allowing the fluid to flow into the second internal space 23, and an outflow path 28 for allowing the fluid to flow out of the second internal space 23; the third frame body 34 preferably has an inflow path 37 for allowing the fluid to flow into the third internal space 33, and an outflow path 38 for allowing the fluid to flow out of the third internal space 33. In this case, it is preferred that the inflow paths 27, 37 and the outflow paths 28, 38 of the second frame body 24 and the third frame body 34 are each configured in an openable and closable manner, and the second frame body 24 can hold the nutrient-containing material in the second internal space 23 in a state where the inflow path 27 and the outflow path 28 are simultaneously closed, and the third frame body 34 can hold the environmental component-containing material in the third internal space 33 in a state where the inflow path 37 and the outflow path 38 are simultaneously closed.

[0147] Thus, in the jacket type device, the culture part 1 is formed by a jacket having a first frame body 14 that holds the microorganism-containing culture medium in the first internal space 13; the nutrient supply part 2 is formed by a jacket having a second frame body 24 that holds the nutrient-containing material in the second internal space 23, or a jacket having a structure that allows the nutrient-containing gas or nutrient-containing liquid to flow in the second internal space 23; the environmental component supply part 3 is formed by a jacket having a third frame body 34 that holds the environmental component-containing material in the third internal space 33, or a jacket having a structure that allows the environmental component-containing gas or environmental component-containing liquid to flow in the third internal space 33. That is, the jacket type device can be constructed by stacking jackets. It should be noted that membrane filters are preferably arranged between the jackets.

[0148] Furthermore, the culture section 1 is preferably provided with a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. The two sensors 41, 42 are provided in a manner to detect the culture state of the microorganisms in the culture section 1. The detection results of the two sensors 41, 42 can preferably be monitored in an external device (not shown). The ultrasonic oscillator 43 is provided in a manner to impart ultrasonic vibration to the microorganism-containing culture medium held in the culture section 1. The operation of the ultrasonic oscillator 43 is preferably controlled by an external device.

[0149] It should be explained that Figure 7 The three-layer laminated structure 10 has Figure 1 but can also have Figure 2 to Figure 5 Etc. composition.

[0150] [3] Printing layer type device

[0151] In a printing type device, as shown in a cross-sectional schematic diagram Figure 8 As shown, the culture part 1, nutrient supply part 2 and environmental component supply part 3 constituting the three-layer stacked structure 10 are respectively formed by printing. It should be noted that the plan view shape of the three-layer stacked structure 10 can be circular, triangular, quadrilateral or other polygonal.

[0152] That is, the culture unit 1 includes a first printed layer 15 formed by printing a microorganism-containing culture medium, the nutrient supply unit 2 includes a second printed layer 25 formed by printing a nutrient-containing material, and the environmental component supply unit 3 includes a third printed layer 35 formed by printing an environmental component-containing material. It should be noted that a membrane filter 40 is preferably disposed between each layer.

[0153] Printing can be performed using a dispenser. For example, when forming the second printed layer 25, the nutrient-containing material is made into a paste and then applied using a dispenser.

[0154] The first printed layer 15 preferably includes a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43. The two sensors 41 and 42 are provided so as to detect the culture state of the microorganisms in the first printed layer 15. The detection results of the two sensors 41 and 42 can preferably be monitored by an external device (not shown). The ultrasonic oscillator 43 is provided so as to impart ultrasonic vibration to the microorganism-containing culture medium held in the first printed layer 15. The operation of the ultrasonic oscillator 43 is preferably controlled by an external device.

[0155] It should be explained that Figure 8 The three-layer laminated structure 10 has Figure 1 but can also have Figure 2 to Figure 5 Etc. composition.

[0156] [4] Thin film device

[0157] The film type device is similar to the printing type device except that it has a film body instead of a printing layer. That is, it has a first film body 16, a second film body 26, and a third film body 36. The first film body 26 is formed by forming a microorganism-containing culture medium into a film by a scraper method, the second film body 26 is formed by forming a nutrient-containing material into a film by a scraper method, and the third film body 36 is formed by forming a material containing environmental components into a film by a scraper method. It should be noted that a membrane filter 40 is preferably arranged between each film body.

[0158] Thin film formation using a doctor blade method can be performed as follows, for example: when preparing the second thin film body 26, the nutrient-containing material is made into a slurry, placed on a carrier film, formed into a thin film of a predetermined thickness using a doctor blade, and dried.

[0159] <Microbial culture method>

[0160] The microorganism culturing method of the present invention can realize various culturing conditions in culturing microorganisms using the above-mentioned microorganism culturing device.

[0161] (1) The first microorganism culture method of the present invention includes at least one of a nutrient changing step and an environmental component changing step, wherein the nutrient changing step is to change at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid flowing in the nutrient supply unit 2, and the environmental component changing step is to change at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid flowing in the environmental component supply unit 3. The method can be performed using a microorganism culture device that allows the nutrient-containing gas or nutrient-containing liquid to flow in the nutrient supply unit 2, or allows the environmental component-containing gas or environmental component-containing liquid to flow in the environmental component supply unit 3;

[0162] According to this method, the culture conditions of the culture section 1 can be easily changed. Therefore, various culture conditions can be realized, making it possible to obtain various difficult-to-culture microorganisms.

[0163] (2) The second microorganism culture method of the present invention includes at least one of a nutrient exchange step and an environmental component exchange step, wherein the nutrient exchange step is to exchange the nutrient supply unit 2 with other nutrient supply units or with the environmental component supply unit, and the environmental component exchange step is to exchange the environmental component supply unit 3 with other environmental component supply units or with the nutrient supply unit. This method can be performed using the following microorganism culture device, that is, the nutrient supply unit 2 is set in a manner that can be exchanged with other nutrient supply units or with the environmental component supply unit, or the environmental component supply unit 3 is set in a manner that can be exchanged with other environmental component supply units or with the nutrient supply unit;

[0164] According to this method, the culture conditions of the culture section 1 can be easily changed. Therefore, various culture conditions can be realized, making it possible to obtain various difficult-to-culture microorganisms.

[0165] (3) The third microorganism 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 changing step and an environmental component changing step, wherein the nutrient changing step is to change at least one of the type and concentration of the nutrient-containing gas or nutrient-containing liquid flowing in the nutrient supply section 2 based on the monitoring result, and the environmental component changing step is to change at least one of the type and concentration of the environmental component-containing gas or environmental component-containing liquid flowing in the environmental component supply section 3 based on the monitoring result. This method can be performed using a microorganism culture device in which a nutrient-containing gas or a nutrient-containing liquid is circulated in the nutrient supply section 2, or an environmental component-containing gas or an environmental component-containing liquid is circulated in the environmental component supply section 3; and further, the culture section 1 is provided with one or more sensors for detecting the culture state.

[0166] According to this method, even during the culture, the culture conditions of the culture section 1 can be easily changed based on the monitoring results. Therefore, the optimal culture conditions can be easily achieved, making it possible to obtain various difficult-to-culture microorganisms.

[0167] (4) The fourth microorganism 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 change step and an environmental component change step, wherein the nutrient exchange step is to exchange the nutrient supply section 2 with other nutrient supply sections or with the environmental component supply section based on the monitoring result, and the environmental component exchange step is to exchange the environmental component supply section 3 with other environmental component supply sections or with the nutrient supply section based on the monitoring result. This method can be performed using the following microorganism culture device, that is, the nutrient supply section 2 is set in a manner that can be exchanged with other nutrient supply sections or with the environmental component supply section, or the environmental component supply section 3 is set in a manner that can be exchanged with other environmental component supply sections or with the nutrient supply section; and further, the culture section 1 has one or more sensors for detecting the culture state.

[0168] According to this method, even during the culture, the culture conditions of the culture section 1 can be easily changed based on the monitoring results. Therefore, the optimal culture conditions can be easily achieved, making it possible to obtain various difficult-to-culture microorganisms.

[0169] Next, specific embodiments of the microorganism cultivation device and microorganism cultivation method of the present invention will be described.

[0170] [First embodiment]

[0171] Fig. 9 100A is a perspective view showing a microorganism cultivation device according to a first embodiment of the present invention. The microorganism cultivation device 100A has a structure in which one three-layer stacked structure 10 is housed in one housing 51 , that is, a “three-layer integrated device”.

[0172] Fig.10 for Fig. 9 XX cross-section diagram. Fig.11 for Fig. 9 XI-XI section view. Fig.12 for Fig. 9 The microorganism cultivation device 100A is an exploded perspective view of the microorganism cultivation device 100A. Fig.12 As shown, it includes a housing 51 , a perforated plate 52 and its mounting member 53 , a cover 54 and its sealing member 55 .

[0173] The housing 51 is a thin box body, and the first recess 56 and the second recess 57 are formed by pressing on the surface. The first recess 56 has a depth D1. The outer peripheral edge 561 of the first recess 56 is near the peripheral edge 511 of the surface of the housing 51 and along the peripheral edge 511. The second recess 57 has a depth D2. The second recess 57 is inside the first recess 56 and forms a plane in the center of the longitudinal direction X of the housing 51. Figure 4D2>D1, D2 is approximately half of the thickness of the housing 51.

[0174] The concave mounting member 53 is embedded in the second recess 57. In the second recess 57, the porous plate 52 is fixed to the peripheral frame 531 of the mounting member 53 by screws 521, thereby plugging the porous plate 52. The porous plate 52 has a plurality of through holes 522. The porous plate 52 is in a state of plugging the second recess 57, and is flush with the bottom surface of the first recess 56.

[0175] The first recess 56 has an inflow passage 564 for allowing the fluid to flow into the first recess 56 and an outflow passage 565 for allowing the fluid to flow out of the first recess 56. The inflow passage 564 extends upstream from the center of the first recess 56 in the width direction Y on the upstream side of the first recess 56, penetrates the upstream side surface 513 of the housing 51, and is connected to the cylinder 566. The outflow passage 565 extends downstream from the center of the first recess 56 in the width direction Y on the downstream side of the first recess 56, penetrates the downstream side surface 514 of the housing 51, and is connected to the cylinder 567.

[0176] The first recess 56 has a rectifying rib 58 on a bottom surface 568 closer to the upstream side than the second recess 57, and a rectifying rib 59 on a bottom surface 569 closer to the downstream side than the second recess 57. The rectifying rib 58 includes a front rib 581 provided so as to evenly divide the fluid flowing in from the inflow path 564 in the width direction Y, and a rear rib 582 for rectifying the fluid divided in the width direction Y along the length direction X. A plurality of rear ribs 582 are provided at equal intervals along the width direction Y. The rectifying rib 59 includes a plurality of ribs 591 provided similarly to the rear rib 582.

[0177] A sealing groove 551 is formed around the first recess 56. The cover 54 is fixed to the surface of the housing 51 by the screw 511 in a state where the sealing member 55 fitted into the sealing groove 551 is pressed from above. The cover 54 seals the first recess 56 and the second recess 57.

[0178] Then, the environmental component-containing material is contained in the second recess 57 blocked by the porous plate 52. In addition, each of the plurality of through holes 522 of the porous plate 52 is filled with a culture medium inoculated with microorganisms, that is, a culture medium containing microorganisms. Since the environmental component-containing material is in contact with the culture medium containing microorganisms, the microorganisms on the porous plate 52 are supplied with environmental components. In addition, the nutrient-containing liquid flows from the inflow path 564 into the first recess 56, flows on the surface of the porous plate 52, and flows out from the outflow path 566. That is, since the nutrient-containing liquid is in contact with the culture medium containing microorganisms, the microorganisms on the porous plate 52 are supplied with nutrients. Therefore, the microorganism culture device 100A has a three-layer stacked structure 10 in the housing 51, and the three-layer stacked structure 10 has a layered environmental component supply part configured by accommodating the environmental component-containing material in the second recess 57, a layered culture part configured by filling the microorganism-containing culture medium in the through holes 522 of the porous plate 52, and a layered nutrient supply part configured by allowing the nutrient-containing liquid to flow in the first recess 56. It should be noted that a membrane filter may be arranged between the environmental component supply part and the culture part, and between the nutrient supply part and the culture part.

[0179] In the porous plate 52, Fig.13 As shown in the figure, a temperature sensor 41, a pH sensor 42 and an ultrasonic oscillator 43 are provided. The two sensors 41 and 42 are provided in such a manner as to detect the culture state of the microorganisms in each through hole 522. The detection results of the two sensors 41 and 42 can be monitored in an external device (not shown). The ultrasonic oscillator 43 is provided in such a manner as to impart ultrasonic vibration to the culture medium containing microorganisms held in all the through holes 522. The operation of the ultrasonic oscillator 43 is controlled by an external device.

[0180] Such a microorganism cultivation apparatus 100A can achieve the following effects.

[0181] (a) Environmental components can be supplied from the bottom and nutrients can be supplied from the top to the microorganisms in the through holes 522 of the porous plate 52 , so that the microorganisms can be cultured in the through holes 522 .

[0182] (b) Since microorganisms can be cultured simply by flowing the nutrient-containing liquid through the first recess 56, the culture of microorganisms can be performed easily. Therefore, the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0183] (c) Since only one three-layer stacked structure 10 capable of culturing microorganisms is provided in the housing 51, a very compact microorganism culturing device can be realized.

[0184] (d) At least one of the type and concentration of the nutrient-containing liquid flowing through the first recess 56 can be changed (nutrient changing step). Therefore, various culture conditions can be easily realized, and the selection operation of culture conditions suitable for microorganisms can be easily performed.

[0185] (e) The culture state of the microorganisms in the through-hole 522 can be detected and monitored by the temperature sensor 41 and / or the pH sensor 42 (monitoring step). Therefore, the culture state can be determined quickly and accurately.

[0186] (f) Based on the monitoring result, at least one of the type and concentration of the nutrient-containing liquid flowing through the first recess 56 can be changed (nutrient changing step). Therefore, culture conditions suitable for microorganisms can be easily achieved.

[0187] (g) The ultrasonic oscillator 43 can be used to impart vibration to the microorganisms in the through-hole 522, thereby activating the culture of the microorganisms. Therefore, the culture efficiency can be improved.

[0188] [Modification of the first embodiment]

[0189] The microorganism culture apparatus 100A according to the first embodiment can arbitrarily adopt the following modified examples.

[0190] (1) The nutrient-containing gas is caused to flow through the first recess 56 .

[0191] (2) The liquid containing environmental components or the gas containing environmental components is caused to flow through the first recess 56 .

[0192] (3) The nutrient-containing material is accommodated in the second recess 57 .

[0193] (4) The nutrient-containing material is accommodated in the second recess 57 , and the environmental component-containing liquid or environmental component-containing gas is allowed to flow in the first recess 56 .

[0194] [Second embodiment]

[0195] Fig.14 1 is a perspective view showing a microorganism culture apparatus according to a second embodiment of the present invention. This microorganism culture apparatus 100B is a "jacket type apparatus". Fig.15 for Fig.14 XV direction view. Fig.16 for Fig.15 XVI view (plan view). Fig.17 for Fig.16 Section XVII-XVII of the. Fig.18 for Fig.17 A perspective view of the cross section shown.

[0196] The microorganism culture device 100B is constructed by stacking seven jackets 61 to 67. The jacket 61 is stacked on the base 60, and the jacket 67 is closed by the cover 68. Fig.19 As shown, in the main device 100B, the jacket 61 is the environmental component supply unit 3, the jackets 62 to 64 and 66 are the culture unit 1, and the jackets 65 and 67 are the nutrient supply unit 2. The main device 100B includes two sets of three-layer stacked structures 10A and 10B, that is, by regarding the jackets 62 to 64 as one culture unit, the device 100B includes a three-layer stacked structure 10A composed of the jackets 61 to 65 and a three-layer stacked structure 10B composed of the jackets 65 to 67.

[0197] The jacket 62 of the culture unit 1 is as follows Figure 20 to Figure 22 As shown in the figure, a first frame body 14 having a ring shape surrounding the first internal space 13 is provided. Fig. 20 It is a three-dimensional view of the jacket 62. Fig.21 for Fig. 20 XXI direction view. Fig. 22 for Fig.21 XXII-XXII cross-sectional view. In the first frame body 14, there is an outer embedding portion 142 having an inner thread 141 at the lower portion, and an inner embedding portion 144 having an outer thread 143 at the upper portion. The outer embedding portion 142 has a size that allows it to be embedded in the inner embedding portion 144. The inner embedding portion 144 has a size that allows it to be embedded in the outer embedding portion 142. The first internal space 13 has an internal space 131 surrounded by the outer embedding portion 142, and an internal space 132 outside of the outer embedding portion 142. Then, the first frame body 14 holds the microorganism culture medium in the internal space 132. It should be noted that Figure 20 to Figure 22 The illustration of the culture medium containing microorganisms is omitted. The jackets 63, 64, and 66 also have the same structure as the jacket 62.

[0198] The jacket 65 as the nutrient supply unit 2 includes a second annular frame body surrounding the second internal space. In the main device 100B, the jacket 65 has the same structure as the jacket 62. The nutrient-containing material is held in the internal space 132.

[0199] The jacket 67 as the nutrient supply unit 2 is as follows Figure 23 to Figure 25 As shown in the figure, an annular second frame body 24 surrounding the second internal space 23 is provided. Fig.23 It is a three-dimensional view of the jacket 67. Fig.24 for Fig.23 View from the XXIV direction. Fig.25 for Fig.24XXV-XXV cross-sectional view. In the second frame body 24, there is an outer embedding portion 242 having an inner thread 241 at the lower portion, and an inner embedding portion 244 having an outer thread 243 at the upper portion. The outer embedding portion 242 has a size that allows it to be externally embedded in the inner embedding portion 244. The inner embedding portion 244 has a size that allows it to be internally embedded in the outer embedding portion 242. The second internal space 23 has an internal space 231 surrounded by the outer embedding portion 242, and an internal space 232 other than the outer embedding portion 242. Furthermore, the second frame body 24 has an inlet path 27 for allowing a fluid to flow into the internal space 232, and an outlet path 28 for allowing the fluid to flow out of the internal space 232. The fluid is a nutrient-containing liquid or a nutrient-containing gas. In addition, Fig.26 The cylindrical bodies 691 and 692 shown are connected to the inflow passages 27 and 28, respectively, and protrude in the radial direction toward the outside. Fig. 27 for Fig.26 XXVII arrow view. The inflow passages 27 and 28 can be closed by inserting a plug (not shown) instead of the cylinder 691. Thus, the inflow passages 27 and 28 can be opened and closed.

[0200] The jacket 61 as the environmental component supply unit 3 includes a third annular frame body surrounding a third internal space. In the main device 100B, the jacket 61 has the same structure as the jacket 67. The internal space 232 is circulated with an environmental component-containing liquid or an environmental component-containing gas.

[0201] Figure 28 to Figure 30 A base 60 is shown. Fig.28 It is a three-dimensional view of the base 60. Fig.29 for Fig.28 View in the XXIX direction. Fig.30 for Fig.29 The base 60 is an annular plate having an inner fitting portion 602 having an outer thread 601 at the upper portion. The inner fitting portion 602 has a size capable of being embedded in the outer fitting portion 142 of the first frame body 14 and the outer fitting portion 242 of the second frame body 24.

[0202] Fig.31 and Fig.32 The cover 68 is shown. Fig.31 It is a plan view of the cover body 68. Fig.32 for Fig.31 The cover body 68 is an annular plate body, and has an outer fitting portion 682 having an inner thread 681 at the lower portion. The outer fitting portion 682 has a size that allows it to be fitted onto the inner fitting portion 141 of the first frame body 14 and the inner fitting portion 241 of the second frame body 24, respectively.

[0203] Then, the jacket 61 is externally embedded and connected to the base 60 by threading the external embedding portion 242 to the internal embedding portion 602 of the base 60, so that it can be stacked. In addition, the jackets 62 to 67 are also externally embedded and connected by threading the external embedding portion to the internal embedding portion of the jacket located below, so that they can be stacked. Moreover, the cover body 68 is externally embedded and connected to the jacket 67 by threading the external embedding portion 682 to the internal embedding portion 244 of the jacket 67. It should be noted that the membrane filter 40 is arranged between the upper and lower jackets in a manner of separating them. In addition, each jacket is separated by an O-ring 401 ( Fig.17 Thus, the main device 100B includes the jackets 61 to 67 having a seven-layer structure.

[0204] Furthermore, if Fig.19 As shown, a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43 are respectively provided in the jackets 62 to 64, 66 as the culture part 1. The temperature sensor 41 and the pH sensor 42 are arranged in a manner to detect the temperature and pH of the microorganism-containing culture medium held in the first internal space 13, penetrate the first frame body 14 from the inside to the outside, and are connected to an external machine (not shown). The external machine can monitor the temperature and pH of the microorganism-containing culture medium via the two sensors 41 and 42. The ultrasonic oscillator 43 is arranged inside the first frame body 14 in a manner that can 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 an external machine.

[0205] Such a microorganism culture device 100B can achieve the following effects.

[0206] (a) By circulating the liquid containing environmental components in the jacket 61, environmental components can be supplied from the bottom and nutrients can be supplied from the top to the microorganisms in the jackets 62 to 64, so that the microorganisms can be cultured in the jackets 62 to 64. In addition, by circulating the liquid containing nutrients in the jacket 67, nutrients can be supplied from the bottom and the top to the microorganisms in the jacket 66, so that the microorganisms can be cultured in the jacket 66.

[0207] (b) There are two sets of three-layer stacked structures 10A and 10B, and the culture conditions of each set are different, so two types of culture conditions can be implemented. Therefore, the efficiency of the culture condition selection operation can be improved, and the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0208] (c) In jackets 62 to 64, the culture section has a three-layer structure, so the culture conditions in each layer are different. For example, the concentration of the supplied environmental component is the highest in jacket 62 and the lowest in jacket 64. In addition, the concentration of the supplied nutrients is the highest in jacket 64 and the lowest in jacket 62. Therefore, the efficiency of the selection operation of the culture conditions can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0209] (d) Microorganisms can be cultured simply by circulating the liquid containing environmental components in the jacket 61 and the liquid containing nutrients in the jacket 67. Therefore, the culture of microorganisms can be performed simply. Therefore, the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0210] (e) The assembly can be performed simply by connecting the jackets to each other, and therefore, the productivity of the apparatus can be improved.

[0211] (f) The jacket can be easily disassembled by disconnecting, and can also be reconnected to other jackets as a replacement. That is, the jacket can be easily exchanged. Therefore, the culture conditions can be easily changed, the efficiency of the selection operation of the culture conditions can be improved, and therefore, the possibility of obtaining difficult-to-culture microorganisms can be improved. For example, the jacket 65 and / or the jacket 67 as the nutrient supply part can be exchanged with the jacket as other nutrient supply parts, or with the jacket as the environmental component supply part (nutrient exchange process). In addition, the jacket 61 as the environmental component supply part can be exchanged with the jacket as other environmental component supply parts, or with the jacket as the nutrient supply part (environmental component exchange process).

[0212] (g) By increasing the number of jackets, the number of three-layer stacked structures can be increased. Then, the culture conditions can be different in each three-layer stacked structure. Therefore, the efficiency of the selection operation of the culture conditions can be improved, and therefore, the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0213] (h) At least one of the type and concentration of the nutrient-containing liquid flowing through the jacket 65 and / or the jacket 67 can be changed (nutrient changing step). In addition, at least one of the type and concentration of the environmental component-containing liquid flowing through the jacket 61 can be changed (environmental component changing step). Therefore, various culture conditions can be easily realized, and the selection operation of culture conditions suitable for microorganisms can be easily performed.

[0214] (i) The culture state of the microorganisms in the jackets 62 to 64 and 66 can be detected and monitored by the temperature sensor 41 and / or the pH sensor 42 (monitoring step). Therefore, the culture state in each jacket can be determined quickly and accurately.

[0215] (j) Based on the monitoring results, at least one of the type and concentration of the nutrient-containing liquid flowing through the jacket 65 and / or the jacket 67 can be changed (nutrient changing step), and at least one of the type and concentration of the environmental component-containing liquid flowing through the jacket 61 can be changed (environmental component changing step). Therefore, even in the middle of the culture, it is possible to easily achieve culture conditions suitable for the microorganisms.

[0216] (k) The ultrasonic oscillator 43 can be used to vibrate the microorganisms in the jacket, thereby activating the culture. Therefore, the culture efficiency can be improved.

[0217] [Variation of the Second Embodiment]

[0218] The microorganism culture apparatus 100B according to the second embodiment can arbitrarily adopt the following modified examples.

[0219] (1) Figure 33 to Figure 35 In the illustrated jacket 62 , the first internal space 13 is formed by a plurality of through holes 522 . Fig.33 It is a three-dimensional view of the jacket 62. Fig.34 for Fig.33 View from direction XXXIV.

[0220] Fig.35 for Fig.34 The jacket 62 as the culture part 1 is composed of a plate body having an outer embedded part 142, and the plate body is formed with an outer thread 143 and a plurality of through holes 522. In the jacket 62, all the through holes 522 are filled with a culture medium containing microorganisms.

[0221] (2) Fig.36 As shown, a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43 are provided in a jacket 62 having a plurality of through holes 522. They are provided in each through hole 522. The temperature sensor 41 and the pH sensor 42 are arranged inside the jacket 62 in such a manner as to detect the temperature and pH of the microorganism-containing culture medium filled in the through hole 522, and are connected to an external device (not shown). The external device can monitor the temperature and pH of the microorganism-containing culture medium via the two sensors 41 and 42. The ultrasonic oscillator 43 is arranged inside the jacket 62 in such a manner as to impart vibration to the microorganism-containing culture medium filled in the through hole 522.

[0222] (3) The connection between the jackets is not limited to a threaded connection mechanism using internal and external threads, and for example, a sliding fitting mechanism, a concave-convex fitting mechanism, or an external connection member may be used.

[0223] (4) The number of jackets stacked is not limited to 7, and one or more three-layer stacked structures may be provided. In addition, when there are two or more three-layer stacked structures, adjacent three-layer stacked structures may share a jacket such as the jacket 65 .

[0224] (5) The internal space 132 of the jacket 62 as the culture section 1 may be a space arbitrarily divided in the lateral direction, or a space arbitrarily divided in the vertical direction.

[0225] [Third Embodiment]

[0226] Fig.37 100C is a schematic cross-sectional view showing a microorganism cultivation device according to a third embodiment of the present invention. This microorganism cultivation device 100C is a "printed layer type device".

[0227] The microorganism cultivation device 100C is configured by stacking the first to sixth printed layers 71 to 76 on a base 70. The plan view shape of each printed layer may be circular, triangular, quadrilateral, or other polygonal.

[0228] The first printed layer 71 is an environmental component supply unit 3 formed by printing a material containing environmental components. The second printed layer 72, the third printed layer 73 and the fifth printed layer 75 are a culture unit 1 formed by printing a culture medium containing microorganisms. The fourth printed layer 74 and the sixth printed layer 76 are a nutrient supply unit 2 formed by printing a material containing nutrients. Therefore, the main device 100C includes two sets of three-layer stacked structures 10C and 10D, namely, a three-layer stacked structure 10C composed of the first printed layer 71 to the fourth printed layer 74, and a three-layer stacked structure 10D composed of the fourth printed layer 74 to the sixth printed layer 76. It should be noted that a membrane filter 40 is arranged between each layer.

[0229] Microorganism Cultivation Device 100C Use Fig.38The device 9 is a dispensing machine having a multi-needle 91. The device 9 moves the multi-needle 91 while discharging the printing material stored in the tank 92 from the multi-needle 91, thereby forming a printed layer. The printing material refers to a paste-like microorganism-containing culture medium in the case of the culture part 1, a paste-like nutrient-containing material in the case of the nutrient supply part 2, and a paste-like environmental component-containing material in the case of the environmental component supply part 3. In the case of the main device 100C, first, a paste-like environmental component-containing material is discharged on the base 70 to form a first printed layer 71, a membrane filter 40 is placed thereon, a paste-like microorganism-containing culture medium is discharged thereon to form a second printed layer 72, the membrane filter 40 is placed thereon, a paste-like microorganism-containing culture medium is discharged thereon to form a third printed layer 73, the membrane filter 40 is placed thereon, a paste-like nutrient-containing material is discharged thereon to form a fourth printed layer 74, the membrane filter 40 is placed thereon, a paste-like microorganism-containing culture medium is discharged thereon to form a fifth printed layer 75, and the membrane filter 40 is placed thereon, a paste-like nutrient-containing material is discharged thereon to form a sixth printed layer 76.

[0230] Furthermore, a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43 are provided in the second printed layer 72, the third printed layer 73, and the fifth printed layer 75 as the culture part, respectively. The temperature sensor 41 and the pH sensor 42 are configured in a manner to detect the temperature and pH of the microorganism-containing culture medium of each printed layer, and are connected to an external machine (not shown). The external machine can monitor the temperature and pH of the microorganism-containing culture medium via the two sensors 41 and 42. The ultrasonic oscillator 43 is configured in a manner to impart vibration to the microorganism-containing culture medium of each printed layer. The operation of the ultrasonic oscillator 43 is controlled by an external machine.

[0231] Such a microorganism cultivation apparatus 100C can achieve the following effects.

[0232] (a) Environmental components can be supplied from the bottom and nutrients can be supplied from the top to the microorganisms in the second printed layer 72 and the third printed layer 73, so that the microorganisms can be cultured in the second printed layer 72 and the third printed layer 73. In addition, nutrients can be supplied from the bottom and the top to the fifth printed layer 75, so that the microorganisms can be cultured in the fifth printed layer 75.

[0233] (b) There are two sets of three-layer stacked structures 10C and 10D, and the culture conditions of each set are different, so two types of culture conditions can be implemented. Therefore, the efficiency of the culture condition selection operation can be improved, and the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0234] (c) In the second printed layer 72 and the third printed layer 73, the culture part has a two-layer structure, so the culture conditions in each layer are different. For example, the concentration of the supplied environmental component is higher in the second printed layer 72, and the concentration of the supplied nutrients is higher in the third printed layer 73. Therefore, the efficiency of the selection operation of the culture conditions can be improved, and thus the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0235] (d) Since microorganisms can be cultured simply by forming a printed layer to form a three-layer laminated structure, the culture of microorganisms can be performed easily. Therefore, the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0236] (e) Since assembly can be performed simply by forming a printed layer, the productivity of the device can be improved.

[0237] (g) By increasing the number of printed layers, the number of three-layer stacked structures can be increased. Then, the culture conditions can be different in each three-layer stacked structure. Therefore, the efficiency of the selection operation of the culture conditions can be improved, and thus, the possibility of obtaining difficult-to-culture microorganisms can be increased.

[0238] (g) The culture state of microorganisms in the second printed layer 72, the third printed layer 73, and the fifth printed layer 75 can be detected and monitored by the temperature sensor 41 and / or the pH sensor 42 (monitoring step). Therefore, the culture state in each printed layer can be determined quickly and accurately.

[0239] (h) The ultrasonic oscillator 43 provides vibration to the microorganisms in the second printed layer 72, the third printed layer 73, and the fifth printed layer 75, thereby activating the culture. Therefore, the culture efficiency can be improved.

[0240] [Variation of the third embodiment]

[0241] The microorganism cultivation apparatus 100C according to the third embodiment can arbitrarily adopt the following modified examples.

[0242] (1) The number of printed layers is not limited to 6, and one or more three-layer stacked structures may be formed. In addition, when there are two or more three-layer stacked structures, adjacent three-layer stacked structures may share a printed layer such as the fourth printed layer 74 .

[0243] (2) The printed layer may be formed by the following method without being limited to the method using a dispenser.

[0244] (2-1) Screen printing method.

[0245] (2-2) The printed material is applied onto a PET film using a coater and then punched into a desired size using a mold to form a laminate.

[0246] [Fourth embodiment]

[0247] The microorganism cultivation device according to the fourth embodiment of the present invention is a "film-type device". Fig.37 The "printed layer type device" of the third embodiment shown has the same structure, but includes a film body instead of the printed layer.

[0248] The microorganism culture device 100D is configured by stacking the first to sixth thin film bodies 71A to 76A on a base 70A. The plan view shape of each thin film body may be circular, triangular, quadrilateral, or other polygonal shapes.

[0249] The first film body 71 is an environmental component supply unit 3 formed by thinning a material containing environmental components. The second film body 72, the third film body 73 and the fifth film body 75 are a culture unit 1 formed by thinning a culture medium containing microorganisms. The fourth film body 74 and the sixth film body 76 are a nutrient supply unit 2 formed by thinning a material containing nutrients. Therefore, the main device 100C includes two sets of three-layer stacked structures 10E and 10F, namely, a three-layer stacked structure 10E formed by the first film body 71 to the fourth film body 74, and a three-layer stacked structure 10F formed by the fourth film body 74 to the sixth film body 76. It should be noted that a membrane filter 40 is arranged between each layer.

[0250] Microbial Culture Device 100D Use Fig.39 The device 9A is made by the device shown in FIG. This device 9A is used to implement the scraper method. The film formation using the scraper method can be performed as follows. For example, in the case of forming the fourth film body 74, the nutrient-containing material is made into a slurry, placed on the carrier film 93, and made into a film of a specified thickness using a scraper, and then dried. By this method, each film body is formed. Then, the membrane filter 40 is inserted between each film body and stacked. In this way, the microorganism culture device 100D can be manufactured.

[0251] Furthermore, a temperature sensor 41, a pH sensor 42, and an ultrasonic oscillator 43 are provided in the second film body 72, the third film body 73, and the fifth film body 75 as the culture section 1, respectively. The temperature sensor 41 and the pH sensor 42 are arranged in a manner to detect the temperature and pH of the microorganism-containing culture medium of each film body, and are connected to an external machine (not shown). The external machine can monitor the temperature and pH of the microorganism-containing culture medium via the two sensors 41 and 42. The ultrasonic oscillator 43 is arranged in a manner to impart vibration to the microorganism-containing culture medium of each film body. The operation of the ultrasonic oscillator 43 is controlled by the external machine.

[0252] Such a microorganism cultivation device 100D can exert the same operational effects as the "printed layer type device" of the third embodiment.

[0253] Next, specific examples of the present invention will be described.

[0254] [First embodiment]

[0255] Using the microorganism cultivation device 100A ( Fig. 9 ).

[0256] (Composition of each department)

[0257] Nutrient supply department

[0258] ·R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 3.2 g / L

[0259] ·Cultivation Department

[0260] ·R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 3.2 g / L

[0261] Agar powder (Nacalai Tesque Co., Ltd.) 15 g / L

[0262] The agar aqueous solution was treated by autoclaving (121°C / 20 minutes), and a soil extract dilution was added at about 60°C, and the solution was stirred and filled into each through hole 522 of the culture part. In addition, for the soil extract dilution, 15 mL of pure water was added to 5 g of soil and stirred, and the supernatant after standing for 1 hour was diluted successively, stained with DAPI, and the number of microorganisms was counted by microscopic observation, and the concentration was adjusted to allow one microorganism to enter each through hole 522 of the culture part.

[0263] Environmental Material Supply Department

[0264] Agar powder (Nacalai Tesque Co., Ltd.) 15 g / L

[0265] 95 mL of the agar aqueous solution was treated by autoclave (121°C / 20 minutes), 5 mL of soil extract was added at about 60°C, and after stirring, it was poured into the second recess 57. In addition, for the soil extract, 15 mL of pure water was added to 5 g of soil and stirred, and 5 mL of the supernatant was separated from the soil extract after standing for 1 hour.

[0266] A membrane filter VCWP (manufactured by Merck Millipore Co., Ltd., 0.1 μm) was placed between the culture section and the environmental substance supply section.

[0267] (Training assignment)

[0268] In the nutrient supply section, R2A medium was continuously circulated for 1 week to perform culturing.

[0269] (Analysis Assignment)

[0270] After the culture, the colonies generated in the culture section were recovered and subjected to gene analysis at Techno Suruga Lab Co., Ltd. Homology analysis was performed on approximately 600 bases in the V1 to V4 region of 16S rDNA, and a simple molecular phylogenetic tree was prepared for species identification. The homology rate indicates the degree of consistency of the base sequence, and when the homology rate is less than 98%, it is judged to be a new species.

[0271] DNA extraction colorless peptidase (Achromopeptidase) (Fuji Film Wako Pure Chemical Industries, Ltd.)

[0272] PCR amplification PrimeSTARHS DNA Polymerase (manufactured by Takara Bio Inc.)

[0273] · Cycle Sequencing BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)

[0274] ·Base sequencing ChromasPro1.7 (Technelysium)

[0275] Database DB-BA12.0 (manufactured by Techno Suruga Lab Co., Ltd.)

[0276] International Base Sequence Database

[0277] Search date: March 15, 2018

[0278] (Comparative Example)

[0279] Colonies were generated by the agar plate surface smear method and the same gene analysis was performed.

[0280] (Analysis results)

[0281] Fig.40 Indicates the results of gene analysis. Fig.40 In the figure, A represents known species 100-98%, B represents new species 98-94%, C represents new genus and new family 94-91%, and D represents new order less than 91%. According to this embodiment, about 45% of the obtained microorganisms correspond to new species, and new microorganisms at the level of new order and new genus can also be obtained. Therefore, it can be confirmed that it is very effective in obtaining difficult-to-cultivate microorganisms.

[0282] [Second embodiment]

[0283] The microorganism culture device 100B of the second embodiment is used. Fig.41 As shown, the jacket 65 and the jacket 67 have the same configuration.

[0284] (Composition of each department)

[0285] Nutrient supply unit (jacket 67)

[0286] ·Matrix A solution

[0287] ·R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.32 g / 100 mL

[0288] 100 mL pure water

[0289] Nutrient supply unit (jacket 65)

[0290] ·Base B solution

[0291] ·R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.032 g / 100 mL

[0292] 100 mL pure water

[0293] Environmental component supply unit (jacket 61)

[0294] Soil extract

[0295] Prepare at a ratio of 15 g of pure water to 5 g of soil.

[0296] ·Cultivation section (jackets 62, 63, 64, 66)

[0297] Agar powder (Nacalai Tesque Co., Ltd.) 1.5 g / 100 mL

[0298] 95mL pure water

[0299] The agar aqueous solution was treated by autoclaving (121°C / 20 minutes), 5 mL of soil extract diluent was added at about 60°C, and after stirring, it was filled into the inner space of the jacket as the culture part. The soil extract diluent was prepared by adding 15 mL of pure water to 5 g of soil and stirring, and then the supernatant after standing for 1 hour was diluted to 10,000 times by successive dilution.

[0300] A membrane filter VCWP (manufactured by Merck Millipore Co., Ltd., 0.1 μm) was placed between each section.

[0301] (Training assignment)

[0302] The culture was performed by continuously circulating the substrate A solution in the nutrient supply part (jacket 67), the substrate B solution in the nutrient supply part (jacket 65), and the soil extract solution in the environmental component supply part (jacket 61) for one week.

[0303] (Analysis Assignment)

[0304] After the culture, the colonies generated in the culture section were recovered and subjected to gene analysis at Techno Suruga Lab Co., Ltd. Homology analysis was performed on approximately 600 bases in the V1 to V4 region of 16S rDNA, and a simple molecular phylogenetic tree was prepared for species identification. The homology rate indicates the degree of consistency of the base sequence, and when the homology rate is less than 98%, it is judged to be a new species.

[0305] DNA extraction colorless peptidase (manufactured by Fuji Membrane Wako Pure Chemical Industries, Ltd.)

[0306] PCR amplification PrimeSTARHS DNA Polymerase (manufactured by Takara Bio Inc.)

[0307] · Cycle Sequencing BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)

[0308] ·Base sequencing ChromasPro1.7 (Technelysium)

[0309] Database DB-BA12.0 (manufactured by Techno Suruga Lab Co., Ltd.)

[0310] International Base Sequence Database

[0311] Search date: March 15, 2018

[0312] (Comparative Example)

[0313] Colonies were generated by the agar plate surface smear method and the same gene analysis was performed.

[0314] (Analysis results)

[0315] Fig.42 Indicates the results of gene analysis. Fig.42 In the figure, A represents 100-98% of known species, B represents 98-94% of new species, C represents 94-91% of new genus, and D represents less than 91% of new order. According to this embodiment, about 40% of the obtained microorganisms correspond to new species, and new microorganisms at the level of new order and new genus can also be obtained. Therefore, it can be confirmed that it is very effective in obtaining difficult-to-cultivate microorganisms.

[0316] [Third embodiment]

[0317] Using the microorganism cultivation device 100C ( Fig.37 ).

[0318] (Composition of each department)

[0319] Nutrient supply unit (sixth printed layer 76)

[0320] Matrix A layer

[0321] ·R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.32 g / 100 mL

[0322] Agar powder (Nacalai Tesque Co., Ltd.) 1.5 g / 100 mL

[0323] 100 mL pure water

[0324] The mixture was treated in an autoclave (121°C / 20 minutes) to obtain a matrix paste.

[0325] Nutrient supply unit (fourth printed layer 74)

[0326] Matrix B layer

[0327] ·R2A culture medium (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.032 g / 100 mL

[0328] Agar powder (Nacalai Tesque Co., Ltd.) 1.5 g / 100 mL

[0329] 100 mL pure water

[0330] The mixture was treated in an autoclave (121°C / 20 minutes) to obtain a matrix paste.

[0331] Environmental component supply unit (first printed layer 71)

[0332] Agar powder (Nacalai Tesque Co., Ltd.) 1.5 g / 100 mL

[0333] 95mL pure water

[0334] The agar aqueous solution was treated by autoclaving (121°C / 20 minutes), 5 mL of soil extract was added at about 60°C, and stirred to obtain a soil paste. In addition, for the soil extract, 15 mL of pure water was added to 5 g of soil and stirred, and after standing for 1 hour, 5 mL of supernatant was separated.

[0335] ·Cultivation section (second, third, and fifth printed layers 72, 73, and 75)

[0336] Agar powder (Nacalai Tesque Co., Ltd.) 1.5 g / 100 mL

[0337] 95mL pure water

[0338] The agar aqueous solution was treated by autoclaving (121°C / 20 minutes), and 5 mL of soil extract dilution was added at about 60°C, and stirred to obtain a culture paste. The soil extract dilution was prepared by adding 15 mL of pure water to 5 g of soil and stirring, and the supernatant after standing for 1 hour was diluted to 10,000 times by successive dilutions.

[0339] (Formation of Printing Layer)

[0340] Each layer was formed using a screw dispenser Quspa Ms (manufactured by Shinwa Co., Ltd.). The following multi-needle was used as a needle. The needle tip and the syringe were heated to 60°C by 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 about 1 mm. It solidified if left at room temperature.

[0341] ·21G (inner diameter 0.51mm)×8, spacing 0.93mm

[0342] A membrane filter VCWP (manufactured by Merck Millipore Co., Ltd., 0.1 μm) was placed between the layers.

[0343] (Training assignment)

[0344] After the laminate consisting of the printed layers was formed, it was placed in a sterilized culture dish and cultured for one week.

[0345] Industrial Applicability

[0346] The microorganism cultivation device of the present invention can make it possible to obtain various difficult-to-cultivate microorganisms, and therefore has extremely high industrial utilization value.

Claims

1. A microorganism cultivation device, characterized in that: have: The housing has a first recessed portion with a depth of D1 on its surface and a second recessed portion with a depth of D2 provided on the bottom surface of the first recessed portion, and D2>D1. a porous plate, which is flush with the bottom surface of the first recess in a state of plugging the second recess and has a plurality of through holes, an inflow passage for allowing the fluid to flow into the first recessed portion on the upstream side of the second recessed portion, an outflow passage for allowing the fluid to flow out from the first recessed portion on the downstream side of the second recessed portion, and an upstream side rectifying rib provided at the first recessed portion on the upstream side of the second recessed portion; The second recess contains a material containing environmental components or a material containing nutrients. The plurality of through holes of the multi-well plate are filled with a culture medium containing microorganisms, The structure is such that the nutrient-containing liquid or the environmental component-containing liquid flows in from the inlet passage, flows through the surface of the porous plate, and flows out from the outlet passage.

2. The microorganism culture device according to claim 1, wherein: The multi-hole plate has a plurality of through holes provided at positions overlapping with the second recessed portion when viewed from the depth direction of the second recessed portion.

3. The microorganism culture device according to claim 1 or 2, wherein: It further includes a downstream-side rectifying rib provided on the downstream side of the second recessed portion.

4. The microorganism culture device according to claim 1 or 2, wherein: The inflow path is provided in the housing.

5. The microorganism culture device according to claim 1 or 2, wherein: The outflow passage is provided in the housing.

6. The microorganism culture device according to claim 1 or 2, wherein: The porous plate is provided with a temperature sensor, a pH sensor and an ultrasonic oscillator.

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