JP2017063764A - Methods for isolating and culturing microorganisms and aggregates-containing solutions - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide improved efficiency of isolation and culture of microorganisms as well as improved isolation-culture efficiency of hardly-culturable microorganisms (new species).SOLUTION: The invention provides a method for isolating and culturing microorganisms comprising the steps of: mixing a hydrogel solution, a solution containing microorganisms, a culture medium and an aqueous phase surfactant to prepare a four component mixture solution; forming a W/O emulsion by pouring the four component mixture solution into an oil with stirring; cooling the emulsion-containing solution to 0°C or higher but not higher than 4°C while stirring to form hydrogel particulates; further stirring the solution containing the hydrogel particulates to form an aggregate-containing solution 14 that contains aggregate 41 in oil 31 formed by aggregation of hydrogel particulates 42 that contain microorganisms 21-24, culture medium 32 and aqueous phase surfactant 34; and incubating the aggregate-containing solution at a certain temperature for a certain period of time, thereby the microorganisms are isolated and cultured in respective hydrogel particulates.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a method for separating and culturing microorganisms and an aggregate-containing solution. In particular, the present invention relates to a method for separating and culturing microorganisms that can be cultivated even for difficult-to-cultivate microorganisms that are difficult to separate and culture by agar culture.

  There are a large number of microorganisms (bacteria and archaea) on the earth. However, many of them are unknown / unresolved / unused. The method of acquiring a single type of microorganism and culturing it purely (hereinafter referred to as the “separation culture method”) originates from the technology of 150 years ago, but has not progressed so far and exists in the environment. Many microorganisms to be isolated cannot be isolated and cultured.

  Even from the few microorganisms that could be isolated and cultured so far, mankind has benefited so far. For example, 60 to 70% of commercially available pharmaceuticals are compounds derived from microorganisms. Specifically, mevastatin, an HMG-CoA reductase inhibitor, has been discovered from a kind of blue mold (Penicillium citrinum) (Non-patent Document 1). In addition, compounds derived from bacteria (such as actinomycetes) such as tacrolimus (derived from actinomycetes) have also been discovered. Therefore, if a new kind of difficult-to-cultivate microorganisms that cannot be separated and cultured by conventional methods can be discovered, it may be useful in the related industries of medicine, pharmacy, and biology.

  A typical example of the conventional method is a plate culture method (hereinafter also referred to as an agar culture method). FIG. 1 is a process diagram and a photograph showing an example of a method for agar culture. As shown to Fig.1 (a), first, an agar medium is provided in a petri dish. Next, microorganisms are cultured on the surface, and then the microorganisms are cultured. As a result, colonies are formed as shown in FIG. FIG.1 (c) is a photograph which shows an example of the state in which the colony was formed.

In this conventional method, the culture is performed under conditions that are significantly different from those in the environment where the microorganisms actually live. In particular, it is necessary to isolate one inoculated cell, and the inoculation density does not exceed 10 ³ cells / ml, which is extremely low compared to the environment. Therefore, there is a problem that many microorganisms in the environment cannot be separated and cultured.

  Another example is a limiting dilution liquid culture method. In this method, the inoculation density usually does not exceed 100 cells / ml, and is extremely low compared to the environment, and many microorganisms in the environment cannot be separated and cultured.

  In recent years, a new isolation culture method has been proposed. One example is an in situ (in the real environment) separation and culture method. Non-patent documents 2 to 4 describe a separation culture method (in situ culture) in which a culture apparatus is installed in a real environment using a device capable of exchanging dissolved substances with the outside through a membrane and interacting with microorganisms. Is disclosed. However, it is possible to install a culture device in the actual environment and achieve the same bacterial cell density as in the environment, so the culture efficiency can be increased compared to the conventional method, but it must be installed on site (in the actual environment) There is. There is also a problem that the operation is complicated compared to the conventional method. Patent Document 1 relates to “a method and kit for isolation and culture of microorganisms”. A method is described in which cells are encased in gel microparticles and then introduced into the environment and cultured (in situ culture). This also has a problem that the operation is complicated as compared with the conventional method.

  As another example, there is a culture method using gel fine particles. Non-Patent Documents 5 and 6 disclose methods of using gel fine particles for culturing microorganisms. This method is said to be a high-throughput culture method capable of simultaneously separating and culturing a large number of strains. However, since this method is a structure in which microorganisms are cultured in gel microparticles dispersed in an aqueous phase (in a hydrogel), the cell density per unit volume cannot be increased, which is extremely high compared to the environment. Low density. Therefore, many microorganisms in the environment cannot be separated and cultured. In particular, it is impossible to isolate and culture microorganisms that start to grow depending on the cell density.

  Patent Document 2 relates to “a method for culturing difficult-to-cultivate microorganisms”. A method is disclosed in which microorganisms are attached to the roots of sterile duckweed plants, cultured in a liquid medium, and separated. This culture method is effective for microorganisms existing near duckweed roots from the viewpoint of environmental simulation, but is theoretically ineffective for microorganisms living in other environments. In other words, this is an effective method only for extremely limited microorganisms. Therefore, this culturing method has a disadvantage that is not general.

  Non-Patent Document 7 discloses "Escherichia coli encapsulation in a water-in-oil emulsion using an on-chip drop generator and its culture-Possibility of isolation of single species bacteria". It describes a structure in which an oil phase surfactant is added to an emulsion to increase the dispersibility of gel particles in the oil phase. This method cannot increase the culture efficiency.

  As described above, so far, there is no effective method for separating and culturing microorganisms that can replace the agar culture method.

JP 2010-41967 A JP 2009-195124 A

Endo A, Kuroda M, Tsujita Y (1976). "ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum". J Antibiot 29 (12): 1346-1348. Kaeberlein et al. Science 2002 Aoi et al. , Apple. Environ. Microbiol. 2009, Sizova et al. , Appl. Environ. Microbiol. 2012 Zengler et al. Proc Natl Acad Sci USA. 2002, Manome et al. , FMES Microbiol. Let. , 2001 http: // www. on-chip. co. jp / app_cat / app06 /

  It is an object of the present invention to provide a method for separating and cultivating microorganisms and an aggregate-containing solution with improved efficiency of separating and culturing microorganisms. It is another object of the present invention to provide a method for separating and culturing microorganisms and an aggregate-containing solution with improved separation and cultivation efficiency of difficult-to-cultivate microorganisms (new species).

In view of the above circumstances, the present inventor examined the reason why there are many microorganisms that cannot be cultured by the agar culture method. First, we examined the results of many experiments so far. In the conventional agar culture method, if the inoculation density is increased within a range below a certain inoculation density, more types of microorganisms can be separated. By cultivating the cells, the researchers have increased the cell-cell interaction, so that many microorganisms in the environment fall into a dormant state at low cell density, but the cell-cell interaction awakens from dormancy. It was assumed that growth could be started and culture efficiency could be increased. In addition, when the inoculation density is increased to 10 ³ cells / ml or more in the conventional method, various types of microorganisms that have proliferated are mixed together, making it impossible to obtain a pure strain (single type of microorganism). However, it was assumed that if the cells were isolated, mixing of microorganisms could be prevented. As a result, it was hypothesized that a large number of pure strains could be obtained while the inoculation density could be increased without increasing the inoculation-derived microorganisms, which was not possible with the conventional method, and the culture efficiency was increased.

  As a result of trial and error based on the hypothesis, the present inventor found that “the aggregate of gel particles containing the medium in the oil phase is formed, and the microorganism is cultured in the gel particles of the aggregate” I came up with a composition. As a result of repeated experiments and trial and error on specific conditions, “(1) agglomerates of hydrogel particles are formed in the oil phase, and microorganisms are isolated and stored in each hydrogel particle. The interaction between cells can be strengthened without losing contact between microorganisms, and the culture efficiency can be enhanced. (2) The structure in which the microorganisms are isolated and stored in the hydrogel fine particles can (3) The cell density per unit volume is comparable to that of the environment due to the formation of aggregates that segregate and store microorganisms in the hydrogel particles. That can be increased to a level that allows the microorganism to grow, and that the environment in which microorganisms grow can be simulated. (5) A hydrogel having only a surfactant for the aqueous phase and no surfactant for the oil phase so that each gel fine particle is not dispersed and the aggregate is stably held in the oil. (6) The structure in which microorganisms are isolated and stored in the hydrogel microparticles allows the microorganisms to be separated and isolated in the hydrogel microparticles, and the microorganisms cannot move between the hydrogels. Therefore, it is possible to purely separate and culture a single type of microorganism in each hydrogel microparticle, and (7) a configuration in which the microbe is cultured in an agglomerate that isolates and stores the microbe in the hydrogel microparticle, thereby increasing the high level. Achieving high throughput and improving the culture efficiency of microorganisms. (8) Micro-organisms in aggregates that isolate and store microorganisms in hydrogel particles. The configuration of culturing, the conventional method can also be separated cultured in flame cultivation microorganisms was difficult to separate the culture, and found "that can increase the separation culture probability of new species of microorganisms, and have completed the present invention.

The present invention can have the following configurations.
(1) A hydrogel solution, a microorganism-containing solution, a culture medium, and a surfactant for an aqueous phase are mixed to prepare a four-component mixed solution. The step of forming the emulsion, the step of cooling the solution containing the emulsion to 0 ° C. or higher and 4 ° C. or lower while stirring, and the step of preparing the hydrogel fine particles from the emulsion, and further stirring the solution containing the hydrogel fine particles A step of preparing an aggregate-containing solution containing in the oil an aggregate formed by agglomerating hydrogel fine particles containing a surfactant for microorganisms, a medium, and an aqueous phase; and the aggregate-containing solution at a predetermined temperature. And separating and culturing the microorganisms in the hydrogel microparticles for a predetermined period of time.

  (2) The method for separating and culturing microorganisms according to (1), wherein the diameter of the hydrogel fine particles is 1 μm or more and 50 μm or less.

  (3) The method for separating and culturing microorganisms according to (1) or (2), wherein the aggregate-containing solution is cultured under conditions of 4 ° C. or higher and 70 ° C. or lower and 500 hours or shorter.

  (4) In the step of forming a W / O emulsion and the step of creating hydrogel fine particles, the solution is stirred at 1000 rpm or more, and in the step of creating an aggregate-containing solution and in the separation culture step, the solution is stirred at 400 rpm or less. (1) The method for separating and culturing a microorganism according to any one of (3).

  (5) The method according to (1) to (4), which includes a step of preparing a microorganism-containing solution and a hydrogel solution so that the concentration of microorganisms is 1 or less with respect to one hydrogel fine particle formed from the hydrogel solution. The method for separating and culturing a microorganism according to any one of the above.

  (6) After adding water to the agglomerate-containing solution containing the hydrogel fine particles from which microorganisms have been separated and cultured, the mixture is allowed to stand, and a two-phase solution consisting of two phases, an aqueous phase and an oil phase, is prepared, and further centrifuged. The microgel recovery step of recovering the microgel, and the step of culturing the microorganism by containing a part of the aqueous phase of the two-phase solution in the agar medium, the microorganism of any one of (1) to (5) Separation culture method.

  (7) having an oil and an aggregate contained in the oil, wherein the aggregate is formed by aggregation of 50 or more hydrogel fine particles, and the hydrogel fine particles have a diameter of 1 μm or more and 50 μm or less; An aggregate-containing solution containing a medium and a surfactant for an aqueous phase.

  According to the method for separating and culturing microorganisms of the present invention, microorganisms can be isolated and stored in each hydrogel fine particle, and the interaction between cells can be strengthened while eliminating contact between adjacent microorganisms. The culture efficiency can be increased, and the movement of water-soluble substances is not limited, so the interaction between cells is strengthened, the cell density per unit volume is increased to a level comparable to the environment, and the environment in which microorganisms grow is increased. The structure that can be simulated and the aggregate is formed in the oil can stably hold the aggregate, has only the surfactant for the water phase, and does not contain the surfactant for the oil phase. Even in a state where the aggregate is stably held and isolated from the outside of the oil, the microorganisms in the hydrogel particles can be cultured, and each microorganism is isolated and stored in the hydrogel particles. Living organisms are separated and isolated in each hydrogel particle, and microorganisms cannot move between each hydrogel, so a single type of microorganism can be purely separated and cultured in each hydrogel particle, and high throughput is achieved. Therefore, it is possible to increase the culture efficiency of microorganisms, and even difficult-to-cultivate microorganisms, which were difficult to separate and culture by conventional methods, can be separated and cultured, and the probability of separating and culturing new types of microorganisms can be increased.

  According to the aggregate-containing solution of the present invention, the interaction between cells can be strengthened, and a single type of microorganism can be purely separated and cultured in each hydrogel fine particle. New species) can be separated and cultured.

It is process drawing and a photograph which show an example of the method of an agar culture method. It is a flowchart figure which shows an example of the isolation | separation culture method of the microorganisms which are embodiment of this invention. Schematic diagram (a) of the state immediately after putting four components (hydrogel, culture medium, microorganism sample, water-soluble surfactant) into oil, and enlarged view (b) of part A in FIG. 3 (a), emulsion It is the schematic diagram (c) of 133 and the AA part enlarged view (d) of FIG.3 (c).

It is the schematic diagram (a) of an aggregate containing solution, and the B section enlarged view (b) of Fig.4 (a). It is the C section enlarged view of FIG.4 (b). It is the schematic diagram (a) of the aggregate containing solution containing the hydrogel microparticles | fine-particles which isolate | separated microorganisms inside, and the D section enlarged view (b) of Fig.6 (a). It is the E section enlarged view of Drawing 6 (b). It is the elements on larger scale of Drawing 7, and is a figure showing two hydrogel particulates. It is the schematic diagram (a) of the aggregate containing solution containing the hydrogel fine particle which isolate | separated and culture | cultivated microorganisms inside, the schematic diagram (b) in the two-phase solution after centrifuging, and the F section enlarged view (c). It is a photograph which shows the external appearance immediately after micro gel particle formation. It is the external appearance of the aggregate containing solution at the time of 150-hour progress from the start of culture | cultivation, Comprising: It is a photograph which shows in culture (aggregate formation). It is the microscope picture of the aggregate containing solution at the time of 150-hour progress from the culture | cultivation start, and is a 50 micrometer bar scale (a) and a 20 micrometer bar scale (b). It is a fluorescence micrograph which shows the hydrogel microparticles | fine-particles disperse | distributed in water and the colony formed in the inside. It is a differential interference image which shows the hydrogel microparticles | fine-particles disperse | distributed in water and the colony formed in the inside. It is a graph which shows the result of the culture | cultivation efficiency at the time of 150-hour progress from the culture | cultivation start. It is a graph which shows the result of the culture time dependence of colony formation rate. It is a graph which shows the result of the ratio of a new kind. It is a photograph which shows the result of Subculture performed by Agar plating (agar plate method). It is a graph which shows the comparison result of the culture time dependence of the colony formation rate of a dispersion system and an aggregation system.

(Embodiment of the present invention)
Hereinafter, a method for separating and culturing microorganisms and an aggregate-containing solution, which are embodiments of the present invention, will be described with reference to the accompanying drawings.

<Method for separating and culturing microorganisms>
FIG. 2 is a flowchart showing an example of the method for separating and culturing microorganisms according to the embodiment of the present invention. As shown in FIG. 2, the method for separating and culturing microorganisms according to the embodiment of the present invention includes a W / O emulsion forming step S1, a hydrogel fine particle creating step S2, an aggregate-containing solution creating step S3, and a separating and culturing step. S4, and is schematically configured.

<W / O emulsion formation step S1>
In the W / O emulsion formation process, a hydrogel solution, a microorganism-containing solution, a medium, and a surfactant for the aqueous phase are mixed to prepare a four-component mixed solution at room temperature, and then the four-component mixed solution is put into oil. In this step, the W / O emulsion is formed by vigorous stirring.

  First, a hydrogel solution, a microorganism-containing solution, a medium, and a surfactant for an aqueous phase are mixed to prepare a four-component mixed solution. Next, the four-component mixed solution is put into oil and vigorously stirred to form a W / O emulsion. The W / O emulsion is formed by stirring the 5-component mixed solution at 40 ° C. By vigorously stirring the five-component mixed solution, a plurality of fine droplets can be formed from the hydrogel solution to easily form a W / O emulsion. A W / O emulsion is a state in which small particles of an aqueous phase are dispersed in an oil phase.

  In this step, it is preferable to vigorously stir. In this step, the solution is preferably stirred at 1000 rpm or more, and more preferably stirred at 2000 rpm or more.

  The hydrogel should just be a hydrogel which can embed microorganisms and can be propagated in the inside. For example, any one material selected from the group of agarose gel, alginate gel, gellan gum gel or polyvinyl alcohol can be mentioned. For example, there is a liquid low melting point agarose gel (hereinafter abbreviated as Cell Gel). These materials can stably hold microorganisms inside.

  The oil is a hydrophobic liquid as long as it can form hydrogel fine particles. Any one material selected from the group of mineral oil, silicone oil or fluorine oil can be mentioned. These materials can stably hold aggregates in the oil.

  The medium may be any medium that can be used as a microorganism medium. Examples thereof include peptones, yeast extracts or glucose. For example, the medium may be a standard NB medium diluted 1/20 so that the final concentration is 0.32 g / L. These materials can sufficiently cultivate microorganisms even in a small amount.

  The surfactant for the aqueous phase needs to be a nonionic water-soluble surfactant. There is a function to smooth the formation of the emulsion, and the particle size of the gel fine particles can be made uniform. From the viewpoint of cytotoxicity, it is preferably not an ionic surfactant. In addition, a water-insoluble surfactant cannot be applied because it does not dissolve in the water phase, and it is not preferable to add a water-insoluble surfactant to the oil phase because the agglomeration property of the aggregate is reduced.

  First, it is preferable to adjust the microorganism-containing solution and the hydrogel solution in this step so that the concentration of microorganisms is one or less for one hydrogel fine particle formed from the hydrogel solution in the subsequent step. Thereby, adjacent microorganisms can be isolated one by one in the hydrogel fine particles, and can be cultured efficiently.

  It is preferable to control the number of microbial cells to be inoculated to one hydrogel fine particle to 1 or less. For example, it is more preferable to control the concentration so that there is one cell for ten hydrogel particles to one cell for two hydrogel particles. That is, it is more preferable that the number of cells / the number of hydrogel particles = 1/10 to 1/2. As a result, two or more cells at the time of inoculation can be prevented from being included in one gel particle. Moreover, it is possible to prevent the overall inoculated cell density from becoming too low.

  The concentration condition is calculated from the capacity per gel particle produced from the average particle diameter of the gel particles. For example, in the preparation of a microorganism-containing solution, a predetermined amount of a sample medium containing microorganisms (microorganism-containing solution) is taken, the concentration of microorganisms in the sample medium is detected, and a microorganism-containing solution with an appropriate proportion of microorganisms is prepared. To do. Based on this, the hydrogel solution which adjusted the amount of hydrogel solutions to an appropriate ratio is adjusted.

  Samples are river water, soil, biofilm, and the like. The soil contains bottom mud. In the case of river water, the collected river water is the sample medium, and in the case of soil, the sample medium is obtained by putting soil and pure water in a tube.

  The sample medium is usually subjected to ultrasonic waves for 1 to 10 minutes to form a sample medium. Irradiation time is not limited to this.

  Pass the supernatant through a 5 μm filter, collect the filtrate as a microorganism sample, remove a portion of it, stain the microorganism in it, calculate the number of microorganism cells in the sample by microscopic observation, Calculate the microbial concentration in the inside.

Thereafter, for example, the sample cell concentration is adjusted to 1 to 5 × 10 ⁸ cells / ml to adjust the sample solution.
Based on this concentration, the hydrogel solution is adjusted so that the microorganisms are included so that it is 1 or less per one hydrogel fine particle.

  FIG. 3 is a schematic diagram (a) of a state immediately after mixing four components (hydrogel, medium, microorganism sample, water-soluble surfactant) and putting it in oil, and an enlarged view of part A in FIG. FIG. 4B is a schematic diagram (c) of the emulsion 133 and an enlarged view (d) of the AA portion of FIG. 3 (c).

  3 (a) and 3 (b), the four components (hydrogel, medium, microorganism sample, water-soluble surfactant) are mixed in the test tube 10 sealed with the cap 11 and then put into oil. An example of the state immediately after is shown. This state is not an emulsion state.

FIG. 3C is an example of a schematic diagram of the emulsion 133, and the emulsion 13 is filled inside the test tube 10 sealed with the cap 11.
As shown in FIG. 3 (d), the emulsion 13 is composed of a hydrogel solution 33 dispersed in an oil 31, and in the hydrogel solution 33, a microorganism group 20, a medium 32, and an aqueous phase surfactant 34 are contained. Is distributed. The microorganism group consists of microorganisms 21 to 24. It is dispersed in the oil phase, the microbial cells are not growing, and the gel is not yet solidified.

<Hydrogel fine particle preparation process S2>
The hydrogel fine production step S3 is a step of producing hydrogel fine particles from the emulsion solution by cooling the emulsion solution to 0 ° C. or higher and 4 ° C. or lower while stirring. The water phase side (hydrogel phase) of the emulsion is gelled to produce fine gel particles. Even in this step, vigorous stirring is preferred. In this step, it is preferable to stir the solution at 1000 rpm or more.

<Aggregate-containing solution preparation step S3>
The agglomerate-containing solution preparation step S3 is performed by reducing the stirring speed of the solution containing the hydrogel fine particles and aggregating the hydrogel fine particles containing the microbe, the medium and the surfactant for the aqueous phase. This is a step of preparing an aggregate-containing solution contained in oil.
In this step, stirring is preferable, but stirring is performed more slowly (at a low speed) than in the step of forming an emulsion and the step of forming hydrogel fine particles. In this step, it is preferable to stir the solution at 400 rpm or less.

    By cooling the emulsion to 0 ° C. or higher and 4 ° C. or lower, an aggregate-containing solution containing an aggregate in which hydrogel fine particles are aggregated in oil can be easily prepared. The cooling rate is preferably rapid cooling at a rate of −1 ° C./min or more. Thereby, hydrogel microparticles | fine-particles can be formed easily.

<Aggregate-containing solution>
FIG. 4 is a schematic diagram (a) of the aggregate-containing solution and an enlarged view (b) of part B of FIG. 4 (a). In FIG. 4A, the aggregate-containing solution 14 is filled in the test tube 10 sealed with the cap 11. As shown in FIG. 4B, the aggregate-containing solution 14 has an oil 31 and an aggregate 41 contained in the oil 31. The aggregate 41 is formed such that adjacent hydrogel fine particles 42 are in contact with each other.

  The aggregate 41 is preferably an aggregate of 50 or more hydrogel fine particles. Thereby, the aggregate can be stably held.

  FIG. 5 is an enlarged view of a portion C in FIG. As shown in FIG. 5, the hydrogel fine particles 42 include microorganisms 21 to 24, a medium 32, and a surfactant 34 for an aqueous phase. Each hydrogel fine particle 42 contains a single type of microorganisms 21 to 24 so as to be one or less.

  The hydrogel fine particles preferably have a diameter of 1 μm or more and 50 μm or less. Thereby, cohesion can be improved.

<Separation culture process S4>
The separation culture step S4 is a step of separating and culturing microorganisms in the hydrogel fine particles by holding the aggregate-containing solution at a predetermined temperature for a predetermined time.

  Also in this step, it is preferable to stir the solution. In this step, it is preferable to stir the solution at 400 rpm or less. Thereby, it can culture | cultivate, keeping the magnitude | size of an aggregate to the magnitude | size of about several hundred micrometers to 1 mm, and reducing the ratio of the mixing of the microorganisms between particles as a result. Thereby, the magnitude | size of an aggregate can be controlled appropriately and the isolation culture efficiency of microorganisms can be improved.

  The culture conditions for the aggregate-containing solution are not particularly limited. For example, it is preferable to perform the culture under conditions of 4 ° C. or higher and 70 ° C. or lower and 500 hours or shorter. Thereby, microorganisms suitable for this condition can be efficiently cultured.

  FIG. 6 is a schematic diagram (a) of an aggregate-containing solution containing hydrogel fine particles in which microorganisms are separated and cultured, and an enlarged view (D) of part D in FIG. 6 (a). In FIG. 6A, an agglomerate-containing solution 15 containing hydrogel fine particles in which microorganisms are separated and cultured is filled inside the test tube 10 sealed with the cap 11.

  As shown in FIG. 6 (b), an aggregate-containing solution 15 containing hydrogel fine particles in which microorganisms are separated and cultured is composed of aggregates 41 dispersed in oil 31, and the hydrogel fine particles are contained in the aggregate 41. 42 is agglomerated.

  FIG. 7 is an enlarged view of a portion E in FIG. Adjacent hydrogel particles 42 are formed so as to contact each other. Each hydrogel fine particle 42 contains microorganisms 21 to 24 grown by culture for each type.

  FIG. 8 is a partially enlarged view of FIG. 7 and shows two hydrogel particles. The dissolved substances 43 and ions 44 can pass between the hydrogel particles 42, but the microorganisms 22 and 23 cannot pass. Therefore, the microorganism does not move from one to the other hydrogel fine particles.

  As a subsequent process of the separation culture process S4, a microgel particle recovery process S5 and a microorganism culture process S6 on the surface of the agar medium may be provided. Thereby, the number of cells can be increased, and subsequent analysis and utilization are possible.

<Micro gel particle recovery step S5>
In the microgel particle recovery step S5, water is added to the agglomerate-containing solution containing the hydrogel fine particles in which microorganisms are separated and cultured, and stirred to create a two-phase solution composed of two phases, an aqueous phase and an oil phase. Then, it is a step of recovering the microgel particles in the aqueous phase by subsequent centrifugation. By adding water to the agglomerate-containing solution containing the hydrogel fine particles in which microorganisms are separated and cultured, stirring, and then centrifuging, the hydrogel fine particles can be moved from the oil phase to the water phase. It is possible to collect and secure hydrogel fine particles obtained by separating and culturing microorganisms in the aqueous phase after centrifugation. Centrifugation conditions are 120G and 5 minutes. After adding water, the gel particles do not move to the aqueous phase unless they are centrifuged.

First, water is added to an agglomerate-containing solution obtained by separating and culturing each microorganism in each hydrogel fine particle and stirred to prepare a solution (two-phase solution) composed of two phases, an aqueous phase and an oil phase.
Next, the aqueous phase is recovered.

FIG. 9 is a schematic diagram (a) of an aggregate-containing solution containing hydrogel fine particles in which microorganisms are separated and cultured, a schematic diagram (b) in a two-phase solution after centrifugation, and an enlarged view of F part (c). It is. In FIG. 9A, an agglomerate-containing solution 15 containing hydrogel fine particles in which microorganisms are separated and cultured is filled inside the test tube 10 sealed with the cap 11. In FIG. 9 (b), the inside of the test tube 10 sealed with the cap 11 is filled with a two-phase solution consisting of the oil phase 31 and the aqueous phase 16 after being centrifuged inside. These are F section enlarged views of FIG.9 (b). As shown in FIG. 9 (c), the aggregate-containing solution 15 containing the hydrogel fine particles in which the microorganisms are separated and cultured is the aqueous phase 16, and the hydrogel fine particles 42 are dispersed in the water 51. The hydrogel fine particles 42 each contain a single type of microorganisms 21 to 24.

<Microbial culture step S6 on the surface of the agar medium>
The microorganism culturing step S6 on the surface of the agar medium is a process of culturing the microorganisms by allowing a part of the aqueous phase of the two-phase solution to be contained in the agar medium.

First, the hydrogel fine particles and the cells existing outside the hydrogel fine particles are separated by percoll density gradient centrifugation, and the washed hydrogel fine particles are collected. Thereafter, water is added and centrifugation (120 G, 5 minutes) is performed to remove Percoll particles.
Next, the washed hydrogel fine particles are put into the gel solution 5 to prepare the solution.
Next, the solution is poured into an agar medium and then cooled in a refrigerator to harden the solution.
Next, after removing the plate from the refrigerator, it is incubated at room temperature.
Thereby, more microorganisms can be obtained from microorganisms that have already been separated and cultured.

  In the method for separating and culturing microorganisms according to the embodiment of the present invention, a hydrogel solution, a microorganism-containing solution, a medium, and a surfactant for an aqueous phase are mixed to prepare a four-component mixed solution, Step S1 in which the mixture is put into oil and vigorously stirred to form a W / O emulsion, and a solution containing the emulsion is cooled to 0 ° C. or higher and 4 ° C. or lower while stirring to prepare hydrogel fine particles from the emulsion solution. In step S2, the agitation of the hydrogel fine particles containing the surfactant for the microorganism, the medium, and the aqueous phase is included in the oil at a low stirring speed of the solution containing the hydrogel fine particles. Step S3 for producing an aggregate-containing solution, and separation culture step S4 for separating and culturing microorganisms in the hydrogel fine particles by holding the aggregate-containing solution at a predetermined temperature for a predetermined time. Because it is configured to isolate and store microorganisms in each hydrogel fine particle, while eliminating the contact between adjacent microorganisms, it is possible to strengthen the interaction between cells, thereby increasing the culture efficiency, Since the movement of water-soluble substances is not restricted, the interaction between cells can be strengthened, the cell density per unit volume can be increased to a level comparable to that in the environment, and the environment in which microorganisms grow can be simulated. In the state where the aggregates can be stably held by the structure formed in the above, having only the surfactant for the water phase and not having the surfactant for the oil phase, the aggregates are stably held in the oil. Even in a state of being isolated from the outside of the oil, the microorganisms in the hydrogel particles can be cultured, and the microorganisms are stored in the hydrogel particles so that each microorganism has its own hydrogen. Since each microgel is separated and isolated within each microgel, and microorganisms cannot move between each hydrogel, a single type of microbe can be purely separated and cultured within each hydrogel microparticle, and high throughput can be achieved. Thus, even difficult-to-cultivate microorganisms, which were difficult to separate and culture by conventional methods, can be separated and cultured, and the probability of separating and culturing new species of microorganisms can be increased.

  In the method for separating and culturing microorganisms according to the embodiment of the present invention, the diameter of the hydrogel fine particles 42 is 1 μm or more and 50 μm or less, so that it is possible to adjust gel fine particles corresponding to a large number of independent culture vessels. As a result, a large number of microorganism culture strains can be separated relatively easily and at low cost.

  Since the method for separating and culturing microorganisms according to an embodiment of the present invention is configured to culture the aggregate-containing solution 14 under conditions of 4 ° C. or higher and 70 ° C. or lower and 500 hours or shorter, various microorganisms can be grown. As a result, this method is excellent in generality and can be used for many samples and various purposes.

  In the method for separating and culturing microorganisms, which is an embodiment of the present invention, the solution is stirred at 1000 rpm or more in step S1 for forming a W / O emulsion and step S2 for creating hydrogel fine particles, thereby creating an aggregate-containing solution S3. Since the solution is stirred at 400 rpm or less in the step and in the separation culture step S4, gel fine particles having a particle size of 50 μm or less can be prepared with a relatively large particle size.

  In the method for separating and culturing microorganisms according to the embodiment of the present invention, the microorganism-containing solution 14 and the hydrogel solution 33 are adjusted so that the concentration of microorganisms is one or less with respect to one hydrogel fine particle 42 formed from the hydrogel solution 33. Therefore, the probability of obtaining a pure culture can be increased.

  The method for separating and culturing microorganisms according to an embodiment of the present invention is a two-phase solution comprising two phases of an aqueous phase and an oil phase by adding and stirring water to an aggregate-containing solution 14 containing hydrogel fine particles from which microorganisms are separated and cultured. And then centrifuging and collecting the microgel particles, and a step S6 for culturing the microorganism by containing a part of the aqueous phase of the two-phase solution in the agar medium. The number of cells can be increased. As a result, various types of analysis and use are possible.

  The aggregate-containing solution 14 according to the embodiment of the present invention includes an oil 31 and an aggregate 41 contained in the oil 31, and the aggregate 41 is formed by aggregating 50 or more hydrogel fine particles 42. Since the hydrogel fine particle 42 has a diameter of 1 μm or more and 50 μm or less and contains the microorganisms 21 to 24, the culture medium 32, and the aqueous phase surfactant 34, the interaction between cells is strengthened, Thus, a single type of microorganism can be purely separated and cultured, and the efficiency of separating and culturing microorganisms and the efficiency of separating and culturing uncultured microorganisms (new species) can be improved.

  The method for separating and culturing microorganisms and the aggregate-containing solution, which are embodiments of the present invention, are not limited to the above embodiments, and can be implemented with various modifications within the scope of the technical idea of the present invention. . Specific examples of this embodiment are shown in the following examples. However, the present invention is not limited to these examples.

Example 1
First, river water was collected as a sample medium.
Next, a predetermined amount of river water was taken into a 25 ml tube, and ultrasonic waves were applied for 3 minutes.
Next, the 25 ml tube was allowed to stand and the supernatant was collected.
Next, the supernatant was passed through a 5 μm filter, and the filtrate was collected as a microorganism (hereinafter referred to as microbial cell) sample.
Next, the cells were stained.
Next, the number of bacterial cells in the sample was calculated by microscopic observation.
As described above, the bacterial cell concentration in the river water was calculated.

Next, based on this, a sample solution (microorganism-containing solution) in which the bacterial cell concentration of the sample was 0.5 × 10 ⁸ cells / ml was prepared.

  As a hydrogel solution (gel state), a liquid low-melting point agarose gel (manufactured by One cell systems, hereinafter abbreviated as Cell Gel) was prepared. As the medium, a standard 1/20 diluted NB medium was prepared so that the final concentration was 0.32 g / L. PLORONIC F-68 was prepared as a surfactant for the aqueous phase.

  Next, 500 μl of the hydrogel solution was mixed with 100 μl of the sample solution, 50 μl of the medium, and 25 μl of the surfactant for the aqueous phase to prepare 675 μl of the four-component mixed solution.

  Next, 15 ml of Cell Mix oil (manufactured by One Cell Systems) was prepared as an oil in a 20 ml vial. Next, after mixing 4 component mixed solution in oil, it mixed on the conditions of 40 degreeC with the constant temperature water tank, and prepared 5 component mixed solution.

  Next, a stirrer was placed in a 20 ml vial, and the five-component mixed solution was stirred under conditions of room temperature and 2100 rpm for 1 min.

  Next, a 20 ml vial was placed in the crushed ice filled in the container, and the 5-component mixed solution was rapidly cooled to about 4 ° C. or lower and stirred under the condition of onice 2100 rpm for 1 min. Next, it agitated on the conditions of on ice 1100rpm 1min.

  As described above, a plurality of hydrogel fine particles were formed from the hydrogel solution, and an aggregate-containing solution containing an aggregate in which the hydrogel fine particles were aggregated in oil was prepared.

FIG. 10 is a photograph showing the appearance immediately after the formation of the fine gel particles. The average particle diameter of the hydrogel particles by microscopic observation was 25 μm, and the number of hydrogel particles formed from 675 μl of the four-component mixed solution was 10 ⁷ .

Next, the mixture was quickly brought to room temperature (about 25 ° C.) and stirred at 400 rpm for 1 min.
Thereafter, stirring was continued at room temperature for up to 310 hours under the condition of 400 rpm for 1 min.
Thereby, microorganisms were separated and cultured in the hydrogel fine particles.

  FIG. 11 is a photograph showing the appearance of the aggregate-containing solution at the time when 150 hours have elapsed from the start of the culture and showing that the culture is in progress (aggregate formation). Aggregates were formed.

  FIG. 12 is a photomicrograph of the aggregate-containing solution after 150 hours from the start of culture, which are 50 μm bar scale (a) and 20 μm bar scale (b). As shown in FIG. 12A, an aggregate of hydrogel fine particles having a diameter of about 1 mm was formed in the oil. Moreover, as shown in FIG.12 (b), the hydrogel microparticles | fine-particles about 5-30 micrometers in diameter were aggregated.

  In the course of the separation culture for 0 to 310 hours from the start of the culture, a part of the aggregate-containing solution was taken out every predetermined time and observed with a microscope to count the number of colonies.

  Specifically, first, a predetermined amount of the aggregate-containing solution was transferred from a 20 ml vial into a 1.5 μl tube. Next, a predetermined amount of water was added, followed by centrifugation. As a result, a solution composed of two phases of an oil phase (upper phase) and an aqueous phase (lower phase) was prepared. Aggregates were dispersed in the aqueous phase. Next, the oil phase was removed, and an aqueous phase solution in which aggregates were dispersed was recovered. Next, the microorganisms in the hydrogel fine particles dispersed in the aqueous phase were stained with a fluorescent dye. As the fluorescent dye, SYTOX Green was used. Next, the fluorescence microscope was observed using the fluorescence microscope apparatus (Olympus BX53). FIG. 13 is a fluorescence micrograph showing hydrogel fine particles dispersed in water and colonies formed inside. The number of fluorescent colonies in the hydrogel fine particles was counted.

  Next, the differential interference image was observed using a microscope. FIG. 14 is a differential interference image showing hydrogel fine particles dispersed in water and colonies formed inside. The number of microbial colonies in the hydrogel fine particles was counted. The number of colonies was counted by averaging the counts with the fluorescence microscope. Thereby, the count result of the number of colonies for every predetermined time was put together.

<Culture efficiency>
The culture efficiency was calculated based on the result of the number of colonies. The culture efficiency is a colony formation rate, that is, a ratio of colonies formed among all inoculated microbial cells, and is calculated by the number of colonies that appear / the total number of inoculated cells. As the total number of inoculated cells, the value obtained by counting the number of cells by a microscopic direct counting method was used. The number of colonies that appeared was the result of counting with the microscope described above.

  FIG. 15 is a graph showing the results of the culture efficiency when 150 hours have elapsed from the start of culture. The culture efficiency (colony formation rate) of this method (new method) was 20%. FIG. 15 also shows the results of the agar culture method (conventional method) of Comparative Example 1 for comparison. The culture efficiency of the conventional method at the point of 150 hours after the start of culture was 2.2%.

  As can be seen from FIG. 15, Example 1 showed superior results compared to Comparative Example 1. Generally, since the colony formation rate of microorganisms in the environment is said to be about 1%, it was extremely high compared to that.

<Culture time dependency of colony formation rate>
FIG. 16 is a graph showing the results of the culture time dependency of the colony formation rate. The data indicated by the squares are the results of Example 1. In addition, two experimental results (Examples 2 and 3) in which the experiment was performed under the same conditions are also described with data indicated by circles and triangles. The variation in the results of the culture time dependence of the colony formation rates in Examples 1 to 3 was small. In either case, the culture time showed a colony formation rate of 18-22% by 50 hours, then became saturated in 50-200 hours and reached a colony formation rate of 25-30%. Thereafter, almost the same characteristics were exhibited that the colony formation rate decreased to 10 to 20%.

<Ratio of new species>
First, a Percoll density gradient centrifugation was performed on the aqueous phase solution in which the aggregates were dispersed. Aggregates were precipitated in the lower part of the aqueous phase, and cells were precipitated in the lowermost layer. As a result, the hydrogel fine particles and the cells existing outside the hydrogel fine particles were separated. Next, the supernatant containing the precipitated aggregate was collected with a pipette. Thereby, the washed hydrogel fine particles were collected.

  Next, the washed hydrogel fine particles were placed in 5 ml of a low melting point agarose gel sea-place 1% solution heated to 40 ° C. to prepare a solution. Next, the solution was poured into an agar medium (containing DR2A 0.32 g / L) and then cooled in a refrigerator for 15 minutes. This solidified the sea-place 1% solution. Next, the plate was taken out from the refrigerator and cultured at room temperature. By culturing, several single species of colonies could be formed, and more than 50 strains were obtained.

  Next, with respect to the obtained strains (50 strains or more), the full length of 16S rRNA gene was amplified using PCR, and the DNA sequence was analyzed. Next, the homology of the obtained DNA sequence with the 16S rRNA gene of the existing culture strain registered in the database was compared. Next, those with less than 98% homology were defined as new species, and the proportion of new species was calculated.

  FIG. 17 is a graph showing the results of the ratio of new species. Table 1 shows the calculation results of the ratio of new species.

  As shown in FIG. 17 and Table 1, in this method (new method: GMD in Oil), 55% were not new species with homology of 100% or less and 98% or more. Since 38% of strains with a homology of less than 98% and 95% or more were obtained, and 7% of strains with a homology of less than 95% were obtained, the proportion of new species obtained by this method (new method) was 45%. It was.

  FIG. 17 and Table 1 also show the results of the agar culture method (conventional method) of Comparative Example 1 for comparison.

  FIG. 18 is a photograph showing the results of secondary culture performed by Agar plating (agar plate method). In the agar culture method (conventional method), 5% of strains with a homology of less than 97% and 95% or more were obtained, and strains with a homology of less than 95% were not obtained. there were. This method (new method) showed an excellent effect compared to the agar culture method (conventional method).

(Comparative Example 1)
The microorganisms were cultured in the same manner as in Example 1 except that an agar plate was used. Colonies on the agar plate were counted visually.

(Examples 2 and 3)
Culture was performed in the same manner as in Example 1, and the dependency of colony formation rate on the culture time was examined.

Example 4
Culture was performed in the same manner as in Example 1 except that soil was used instead of river water.

  Sample adjustment was performed as follows. First, about 5 g of soil and 5 ml of pure water were placed in a 25 ml tube, treated with ultrasonic waves for about 3 minutes, and then the 25 ml tube was left standing to collect the supernatant. Next, the supernatant liquid was passed through a 5 μm filter, and the filtrate was recovered as a microorganism (hereinafter also referred to as microbial cell) sample.

  The culture efficiency of this method (new method), that is, the rate of colony formation (colony formation rate) among all the inoculated microbial cells was 9.0% (at the time 150 hours passed from the start of culture).

(Comparative Example 2)
The microorganisms were cultured in the same manner as in Example 4 except that an agar plate was used. Colonies on the agar plate were counted visually. The colony formation rate of the agar culture method (conventional method) was 1.1% (at the time when 150 hours had elapsed from the start of the culture).

(Comparative Example 3)
A solution was prepared in the same manner as in Example 1 except that 0.1% of a surfactant (Span 80) was added to the oil phase. In the solution, since the surfactant was added to the oil phase, the emulsion was stable, and the microgel particles were dispersed in the oil phase and did not form aggregates.

  Culture was performed using this solution. In the culturing process, the count results of the number of colonies every predetermined time were summarized.

  The maximum colony formation rate was 1.6% (at the time when 150 hours had elapsed from the start of culture). This was the same level as the conventional method.

  FIG. 19 is a graph showing a comparison result of the culture time dependency of the colony formation rate of the aggregation system (Example 4) and the dispersion system (Comparative Example 3).

  Comparing the results of Example 4 and Comparative Example 3, in Example 4, aggregates were formed in the oil phase, but in Comparative Example 3, no aggregates were formed. It became clear that this difference has a great influence on the colony formation rate. That is, it has been proved that the effect of forming aggregates has been demonstrated.

  The method for separating and culturing microorganisms and the aggregate-containing solution of the present invention have the effect of increasing the efficiency of separating and culturing microorganisms and the difficulty of separating and culturing difficult-to-cultivate microorganisms (new species). New types of difficult-to-culture microorganisms that can create new drugs, and can be used in the medical, pharmaceutical, and biological industries.

10 test tubes, 11 ... cap, 13 ... emulsion, 14 ... aggregate-containing solution, 15 ... aggregate-containing solution containing hydrogel fine particles in which microorganisms are separated and cultured, 20 ... Microorganism group, 21, 22, 23, 24 ... Microorganism, 31 ... Oil, 32 ... Medium, 33 ... Hydrogel solution, 34 ... Surfactant for aqueous phase, 41 ... -Aggregates, 42 ... hydrogel fine particles, 43 ... dissolved substances, 44 ... ions, 16 ... aqueous phase, 51 ... water, 133 ... emulsion, 422 ... still solidified Gel not in the state.

Claims (7)

After preparing a four-component mixed solution by mixing a hydrogel solution, a microorganism-containing solution, a medium, and a surfactant for an aqueous phase, the four-component mixed solution is put into oil and stirred to form a W / O emulsion. A process of performing
Cooling the solution containing the emulsion to 0 ° C. or higher and 4 ° C. or lower while stirring to create hydrogel fine particles from the emulsion;
The solution containing the hydrogel fine particles is further stirred to prepare an aggregate-containing solution containing in the oil an aggregate formed by aggregating the hydrogel fine particles containing a surfactant for microorganisms, a medium and an aqueous phase. And a process of
A method for separating and culturing microorganisms, comprising: a step of separating and culturing microorganisms in the hydrogel fine particles by holding the aggregate-containing solution at a predetermined temperature for a predetermined time.   The method for separating and culturing microorganisms according to claim 1, wherein the diameter of the hydrogel fine particles is 1 µm or more and 50 µm or less.   The method for separating and culturing microorganisms according to claim 1 or 2, wherein the aggregate-containing solution is cultured under conditions of 4 ° C or higher and 70 ° C or lower and 500 hours or shorter. In the step of forming the W / O emulsion and the step of creating hydrogel fine particles, the solution is stirred at 1000 rpm or more,
The method for separating and culturing microorganisms according to any one of claims 1 to 3, wherein the solution is stirred at 400 rpm or less in the step of creating the aggregate-containing solution and the step of separating and culturing.   The method further comprises the step of preparing the microorganism-containing solution and the hydrogel solution so that the concentration of microorganisms is one or less with respect to one hydrogel fine particle formed from the hydrogel. The method for separating and culturing a microorganism according to any one of 1 to 4. Water is added to the agglomerate-containing solution containing microgel particles from which microorganisms are separated and cultured, and then left to stir to create a two-phase solution consisting of two phases, an aqueous phase and an oil phase, and further centrifuged to form microgel particles. A microgel particle recovery process for recovering
The method for separating and culturing a microorganism according to any one of claims 1 to 5, further comprising a step of culturing the microorganism by containing a part of the aqueous phase of the two-phase solution in an agar medium. . Oil and aggregates contained in the oil,
The aggregate is formed by aggregation of 50 or more hydrogel fine particles,
The agglomerate-containing solution, wherein the hydrogel fine particles have a diameter of 1 μm or more and 50 μm or less, and contain a microorganism, a medium, and a surfactant for an aqueous phase.