Methods for culturing environmental microorganisms

Abstract

The objective is to provide a method for agglomerating and culturing microorganisms derived from environmental microorganisms. [Solution] According to the present invention, a water-in-oil droplet for use in a method for agglutinating and culturing microorganisms or for use in the isolation and detection of microorganisms is provided, wherein the aqueous phase of the water-in-oil droplet comprises (1) a plurality of types and a plurality of gel microdroplets, and (2) microorganisms and / or activity measurement compounds, wherein each gel microdroplet independently comprises (1a) one or more types of microorganisms; (1b) a plurality of culture media of different compositions; and (1c) low-melting-point agarose.

Description

[Technical Field]

[0001] This invention relates to a water-in-oil droplet containing a gel microdroplet for screening and isolating microorganisms with useful functions from a library of microorganisms present in various environments or genetically modified microorganisms, a method for agglutinating and complex culturing microorganisms using the droplet, and a method for isolating and detecting microorganisms. In particular, the invention features a water-in-oil droplet containing a gel microdroplet having a structure in which microorganisms are encapsulated in gel microdroplets composed of multiple culture medium compositions, and multiple such droplets are encapsulated in water-in-oil droplets, and the invention relates to a technology that enables the exploration of microbial species that were previously unused due to the difficulty of culturing them. [Background technology]

[0002] The Earth is home to a vast array of microorganisms, and by isolating, culturing, and utilizing them, humanity has enjoyed numerous benefits in a wide range of fields, including medicine, agriculture, and environmental engineering. Conventional methods involved isolating actinomycete strains from the environment, culturing them as single microorganisms, and then screening them using the substances they produce as indicators. Alternatively, isolated microorganisms were co-culturized, and the interaction between them was used to screen for the desired microorganism. However, these methods of isolating microorganisms leave many microorganisms uncultivable, revealing that the microorganisms humanity has utilized to date represent only a small fraction of the total (Non-Patent Literature 1). If a screening technology were to be developed that could simultaneously screen a large number of unused microorganisms in the environment in diverse combinations, and further enable the growth of useful microorganisms through inter-microorganism interactions, it is expected that previously unusable microorganisms, as well as the compounds they produce, enzymes, genes, etc., could be utilized in the development of new pharmaceuticals, pesticides, and other products.

[0003] One reason why isolation and culture are difficult is the difficulty in finding optimal culture conditions for microorganisms in the environment. Individual microorganisms have different optimal temperature, pH, moisture content, salt concentration, essential nutrients, and gas phase conditions for growth, making it difficult to culture them together. Furthermore, it is believed that many environmental microorganisms can not only grow on their own, but also depend on or live in symbiotic relationships with other microorganisms or organisms. In other words, by culturing microorganisms in close proximity (hereinafter referred to as "complex microbial culture"), it is possible to induce microbial interactions, which can affect the metabolic systems and growth of each microorganism, and multiple cases have been reported to date in which they become able to grow together (Non-Patent Literature 2).

[0004] In the agricultural field, a technique has been proposed to search for antagonistic microorganisms against Ralstonia solanacearum, a plant pathogenic bacterium known to cause bacterial wilt, by encapsulating the bacterium and soil-derived bacteria in water-in-oil droplets (WODLs) and culturing them together. This technique has been successfully used to isolate antagonistic microorganisms (Patent Document 1).

[0005] In the field of ecology, it has been reported that when soil-derived environmental microorganisms are encapsulated in GMDs and then cultured in close proximity, substances move between the GMDs, resulting in a significant improvement in the colony formation rate of microorganisms compared to when they are cultured in a dispersed state (Patent Document 2).

[0006] Furthermore, there is a known example in which several microorganisms from the environment were isolated in millions of gel microdroplets (GMDs), combined with a single cell of the alga Chlorella sorokiniana, isolated and cultured in a WODL, and then Pseudomonas bacteria were isolated as microorganisms that promote algal growth (Non-Patent Literature 2).

[0007] Methods for encapsulating one or more gel microdroplets containing nucleic acids, proteins, or cells within a WODL have been reported (Patent Document 3). Furthermore, a method for encapsulating cells in the liquid phase of a WODL while simultaneously encapsulating GMDs containing other cells has also been reported, and this method makes it possible to sequence nucleic acids from various biological sources (Patent Document 4). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2023 / 210755 [Patent Document 2] Japanese Patent Publication No. 2017-63764 [Patent Document 3] U.S. Patent No. 10150963 [Patent Document 4] U.S. Patent Application Publication No. 2015 / 0376609 [Non-patent literature]

[0009] [Non-Patent Document 1] Lok C., 2015. Mining the microbial dark matter. Nature 522:270-273. doi: 10.1038 / 522270a [Non-Patent Document 2] Yan C. et al., 2023. Microbial Interaction is Among the Key Factors for Isolation of Previous Uncultured Microbes. Journal of Microbiology 61:655-662.doi: 10.1007 / s12275-023-00063-3 [Non-Patent Document 3] Ohan J. et al., 2019. High-Throughput Phenotyping of Cell-to-Cell Interactions in Gel Microdroplet Pico-Cultures. BioTechniques 66(5):218-224.doi:10.2144 / btn-2018-0124 [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention addresses the problem that, when encapsulating GMDs in a WODL (Wood-Owned Microorganism) as disclosed in the prior art, particularly Non-Patent Document 2, and simultaneously culturing microorganisms and cells within the WODL along with environmental microorganisms isolated within the GMDs, the culture rate of environmental microorganisms is low and the screening efficiency for environmental microorganisms with desirable superior characteristics is poor, as described above, because the GMD medium composition or the medium composition constituting the aqueous layer within the WODL is single. A similar problem also exists when screening and isolating strains with superior properties from a library of artificially and randomly genetically modified mutant microorganisms. [Means for solving the problem]

[0011] Therefore, in view of the above problems, the present inventors conducted diligent research and development and, as a result, created droplets by combining GMDs made with multiple culture medium compositions and encapsulating multiple GMDs in WODL, established a culture method that induces intermicrobial interactions using these droplets, and successfully developed a screening system for actually isolating and detecting microorganisms, thus completing the invention. In other words, the present invention is characterized in that, in the culture of microorganisms, microorganisms are cultured using a water-in-oil emulsion containing gel microdroplets (GMDs) composed of different culture medium compositions and an aggregated complex culture system using the same, and target microorganisms are isolated and detected from the environment.

[0012] In this method, small GMDs are created by suspending environmental microorganisms in water-soluble culture media of multiple different compositions. These GMDs are then combined and dispersed in oil within a WODL (Whole Oven Water) to produce tiny water-soluble droplets (hereinafter sometimes referred to as "droplets" or "DLs") with a diameter of several hundred micrometers (e.g., 100 μm to 200 μm). Because the space (volume) of such DLs is very narrow, it is expected that the concentration of substances reaching the DL space composed of the culture medium and between GMDs will increase rapidly. Furthermore, by culturing these droplets at high density, substances can move between DLs, which is expected to promote interactions between the same or different species of microorganisms separated in different DLs. Generally, it was thought that this method would not work because the culture medium within a GMD is equilibrated by diffusion. However, this method is characterized by demonstrating that the different culture medium compositions within the GMD are maintained for a long time, which is sufficient for the growth of environmentally derived microorganisms adapted to the culture medium composition that constitutes the GMD. While technologies such as GMDs, WODLs, and combinations thereof are known technologies, as shown in this disclosure, by encapsulating multiple GMDs containing different culture medium compositions and utilizing low-melting-point agarose to reduce thermal damage to the encapsulated microorganisms within a single WODL, and by inducing intermicrobial interactions that enable the exchange of substances between GMDs and between the aqueous phase of the WODL, it has become possible to cultivate microorganisms that could not be cultured conventionally.

[0013] Furthermore, the WODL does not necessarily have to contain microorganisms; it can also contain compounds for measuring microbial function. Specifically, by preparing a gel microdroplet (GMD) containing microorganisms and culturing the microorganisms in a WODL containing or without microorganisms while the GMD is introduced, it was possible to independently maintain and aggregate diverse microorganisms within the WODL, and to generate and measure the interactions between different microorganisms. In connection with this, high-throughput isolation and culture of functional microorganisms became possible by individually separating the target GMD from the WODL.

[0014] Figure 1 is a schematic diagram conceptually representing the technical configuration and features of the present invention. The ten large white circles visible in the figure, including parts of them, represent WODLs (W). Each WODL is separated and independent by the oxygen-permeable oil (O) surrounding it, but not only gases but also substances are exchanged between adjacent WODLs. The WODL in the center right shows that five small circles indicate the encapsulation of GMDs. Specifically, GMD (T) encapsulating tester cells, and four other GMDs encapsulating environmental microorganisms 1-4, are composed of different culture media (the intensity of the gray background indicates different culture media compositions). For example, if environmental microorganism 1 encapsulated in GMD1 is compatible with the culture medium it comprises, grows, and exhibits positive activity toward the survival and proliferation of tester cells, then by detecting the proliferation of tester cells, it is possible to screen for environmental microorganisms with the desired function. The bidirectional arrows indicate that an interaction occurred between environmental microorganism 1 and the tester cells.

[0015] Meanwhile, in another WODL shown in the center left, GMD(K) interacts with microorganisms and substrates (M) encapsulated in the aqueous phase of the WODL. For example, if M is a microorganism, and the microorganism K in the GMD is the desired microorganism, then it becomes possible to grow microorganism K through interaction with microorganism M. Furthermore, if M is a substrate, the solution in the WODL can be configured to exhibit luminescence, fluorescence, color reaction, etc. This makes it possible to detect environmentally derived microorganism K with a desired function. By changing the combination of culture medium composition and microorganisms constituting each GMD, and the solution constituting the WODL, it is possible to grow, propagate, and isolate diverse and unknown environmentally derived microorganisms with functions that previously had low culture rates and could not be screened.

[0016] The inventors of this invention have successfully completed the present invention by efficiently culturing microorganisms from the environment and isolating microorganisms with desired functions by culturing microorganisms in WODL containing multiple GMDs composed of different culture medium compositions.

[0017] That is, the present invention is as follows. [1] A water-in-oil droplet for use in a method for aggregate mixed culture of microorganisms, or for use in isolation and detection of microorganisms, wherein the aqueous phase of the water-in-oil droplet comprises (1) a plurality of types and a plurality of gel microdroplets, and (2) a microorganism and / or an activity measurement compound, wherein each of the gel microdroplets independently comprises the following: (1a) one or more microorganisms of one or more species; (1b) a plurality of culture media having different compositions; and (1c) low-melting point agarose The water-in-oil droplet as described above, comprising the above components. [2] The water-in-oil droplet according to [1], wherein the average diameter of the gel microdroplets is 10 µm to 50 µm. [3] The water-in-oil droplet according to [1], wherein the average diameter of the water-in-oil droplet is 50 µm to 200 µm. [4] The average diameter of the gel microdroplets is 10 µm to 50 µm, and The water-in-oil droplet according to any one of [1] to [3], wherein the average diameter of the water-in-oil droplet is 50 µm to 200 µm. [5] The water-in-oil droplet according to any one of [1] to [4], wherein the microorganism is a microorganism collected from the environment. [6] The gel microdroplet-containing water-in-oil droplet according to any one of [1] to [5], wherein the microorganism is one or more selected from the group consisting of bacteria, fungi, protists, and archaea. [7] A method for aggregate mixed culture of microorganisms, comprising (a) a step of encapsulating one or more microorganisms of one or more species in gel microdroplets, and (b) a step of introducing the gel microdroplets encapsulating the microorganisms obtained above into each water-in-oil droplet, and (c) A step of culturing microorganisms with gel microdroplets encapsulated in a water-in-oil droplet. A method for agglutinating and culturing microorganisms, including [specific element]. [8] In addition to steps (a) to (c) above, (d) A step comprising adding a microorganism and / or a compound for measuring activity to the aqueous phase of the water-in-oil droplet, A method for agglutinating and complex culturing of microorganisms as described in [7], including the above. [9] The method for agglutinating and complex culturing of microorganisms according to [7] or [8], wherein in step (a), the gel microdroplet comprises a plurality of different culture medium components.

[10] A method for agglutinating and culturing microorganisms according to any one of [7] to [9], wherein in step (a), the gel microdroplet comprises low-melting-point agarose.

[11] A method for agglutinating and culturing microorganisms according to any one of [7] to

[10] , wherein the average diameter of the gel microdroplets is 10 μm to 50 μm.

[12] A method for agglutinating and complex culturing of microorganisms according to any one of [7] to

[11] , wherein the average diameter of the water-in-oil droplets is 50 μm to 200 μm.

[13] The average diameter of the gel microdroplets is 10 μm to 50 μm, and A method for agglutinating and complex culturing of microorganisms according to any one of [7] to

[12] , wherein the average diameter of the water-in-oil droplets is 50 μm to 200 μm.

[14] The method for agglutinating and culturing microorganisms according to [7] to

[13] , wherein the microorganism is a microorganism collected from the environment.

[15] The method for agglutinating and culturing microorganisms according to [7] to

[14] , wherein the microorganism is selected from the group consisting of bacteria, fungi, protists, and archaea.

[16] A method for isolating and detecting microorganisms, (e) A step of preparing a water-in-oil droplet as described in any one of the above items [1] to [6], (f) A step of culturing microorganisms contained in a water-in-oil droplet containing multiple gel microdroplets using a microorganism aggregation complex culture method described in any one of the above items [7] to

[15] , (g) A step of detecting cells that have grown in a droplet by adding a fluorescent nucleic acid staining dye to the oil phase of the water-in-oil emulsion, (h) A step of separating the aqueous layer of a water-in-oil droplet from a gel microdroplet, (i) A step of separating gel microdroplets stained with a fluorescent nucleic acid staining dye from unstained gel microdroplets, (j) A step to recover microorganisms from the gel microdroplets separated in step (i) and (k) A step to extract nucleic acids from the microorganisms recovered in step (j), (l) A step of comparing the 16S rRNA base sequence contained in the nucleic acid with the sequence of a known microorganism, and determining that the isolated microorganism containing the nucleic acid is a novel microorganism if the identity is 97% or less, And, (m) Steps to measure the function of isolated and cultured microorganisms. A method for isolating and detecting microorganisms, comprising the above. [Effects of the Invention]

[0018] According to the microorganism culture method of this disclosure, by creating an aggregated complex culture system in which GMD is encapsulated within WODL, interactions between different microorganisms can be induced, providing a high-throughput isolation and culture technology for microorganisms with desired functions. [Brief explanation of the drawing]

[0019] [Figure 1] This diagram illustrates a conceptual system for a microbial GMD-WODL aggregated culture system, in which microorganisms are individually encapsulated in gel microdroplets (GMDs), and these GMDs are cultured in water-in-oil droplets (WODLs). [Figure 2] This is a microscopic image showing E. coli growing within a GMD enclosed in WODL. [Figure 3] These are microscopic images showing the growth of different bacterial strains after culturing GMDs prepared in three different culture media, enclosed in WODL. [Figure 4] These are microscopic images showing the independent growth of different strains of microorganisms in the liquid phase of GMD and WODL enclosed within WODL in a GMD-WODL combined culture system. [Figure 5] This is a microscopic image showing the culture of WODL containing actinomycetes that produce antimicrobial substances and E. coli that emit fluorescence. [Figure 6] This is a histogram plotting the number of WODLs according to the intensity of green fluorescence in WODLs where two different types of GMDs exist. [Figure 7] This document shows an example of actinomycete clones isolated by the composite culture method described herein, colonized on a 96-well agar plate (after 14 days of culture). [Figure 8] This graph shows the proportion of green-fluorescent droplets in WODLs containing GMDs encapsulating environmental microorganisms and GMDs encapsulating green-fluorescent E. coli. [Figure 9] This figure shows the antibacterial activity of bacteria isolated by the composite culture method described herein. [Modes for carrying out the invention]

[0020] According to one aspect of the present invention, a water-in-oil droplet is provided for use in a method for agglomerating and culturing microorganisms or for use in the separation and detection of microorganisms, wherein the aqueous phase of the water-in-oil droplet is (1) Multiple types and multiple gel microdroplets, (2) Microorganisms and / or activity measurement compounds, Here, each of the gel microdroplets is independently defined as follows: (1a) One or more microorganisms of one or more species; (1b) Multiple culture media with different compositions; and (1c) Low melting point agarose It can include...

[0021] According to another aspect of the present invention, a method for agglutinating and culturing microorganisms is provided, and this method is (a) A step of encapsulating one or more microorganisms of one or more species in a gel microdroplet, (b) A step of introducing the gel microdroplets containing the microorganisms obtained above into each water-in-oil droplet, (c) A step of culturing microorganisms with gel microdroplets encapsulated in a water-in-oil droplet. It can include...

[0022] According to yet another aspect of the present invention, a method for the isolation and detection of microorganisms is provided, the method is (e) A step of producing a water-in-oil droplet as described in any one of claims 1 to 6, (f) A step of culturing microorganisms contained in a water-in-oil droplet containing a plurality of gel microdroplets using the microorganism aggregation complex culture method described in any one of claims 7 to 15, (g) A step of detecting cells that have grown in a droplet by adding a fluorescent nucleic acid staining dye to the oil phase of the water-in-oil emulsion, (h) A step of separating the aqueous layer of a water-in-oil droplet from a gel microdroplet, (i) A step of separating gel microdroplets stained with a fluorescent nucleic acid staining dye from unstained gel microdroplets, (j) A step to recover microorganisms from the gel microdroplets separated in step (i) and (k) A step to extract nucleic acids from the microorganisms recovered in step (j), (l) A step of comparing the 16S rRNA base sequence contained in the nucleic acid with the sequence of a known microorganism, and determining that the isolated microorganism containing the nucleic acid is a novel microorganism if the identity is 97% or less, And, (m) Steps to measure the function of isolated and cultured microorganisms. It can include...

[0023] As used herein, the term “environmental microorganisms” (also simply referred to as “environmental microorganisms”) refers to a general term for microorganisms that inhabit the natural environment, and these microorganisms are known to exist in a variety of places, including the human body, soil, water, air, the surfaces and interiors of plants and animals, and extreme environments (e.g., hydrothermal vents, glaciers). Environmental microorganisms include bacteria, archaea, fungi, and algae. The microorganisms that can be cultured by the culture method of the present invention are not limited to those mentioned above, but the above-mentioned environmental microorganisms may be microorganisms that cannot be cultured in commonly used liquid culture media and nutrient agar media (such as Tryptic soy or R2A, and their agar media).

[0024] As used herein, the terms "water-in-oil emulsion" or "W / O emulsion" refer to a state in which particulate water droplets (DLs) exist as a dispersed phase (aqueous phase) in a continuous oil phase. Furthermore, "droplet" refers to a compartmentalized water droplet within an emulsion. In this specification, droplets in a water-in-oil emulsion are sometimes specifically referred to as "water-in-oil droplets (WODL)." Individual DLs exist independently of each other by the oil phase, and microorganisms encapsulated within DLs are isolated from microorganisms in other DLs; however, some compounds secreted by microorganisms can move between DLs.

[0025] The aqueous phase of a water-in-oil emulsion can be any hydrophilic liquid that is immiscible with the oil phase and on which microorganisms can grow. Suitable solutions for this aqueous phase are not limited to those mentioned above, but examples include culture media such as LB and R2A. Lake water or seawater can also be used directly as the aqueous phase. On the other hand, the oil phase of a water-in-oil emulsion can be any hydrophobic liquid that is immiscible with the aqueous phase. Such oil phases are well known and not limited to those mentioned above, but examples include FC40, Novec 7500, mineral oil, or combinations thereof.

[0026] To stabilize the water-in-oil emulsion, surfactants may be added to the aqueous phase, the oil phase, or both. Examples of usable surfactants are not limited to those mentioned above, but include Span80 and Tween20 for the aqueous phase, and Pico-surf1 and Krytox for the oil phase, or a combination thereof. The concentration of the surfactant added to the aqueous and / or oil phase can be appropriately adjusted depending on the type of surfactant used and the desired droplet size.

[0027] Methods for preparing water-in-oil emulsions consisting of the aqueous and oil phases described above are well known. For example, water-in-oil emulsions can be prepared using an On-chip Droplet Generator (On-chip, hereinafter referred to as "Generator"). This Generator is generally a device that produces droplets using microchannels, but by defining and selecting the channel size of the microchannels, droplets with a desired average diameter can be produced. Details regarding the Generator and related equipment used in the examples described later are shown below: (1) Types, model numbers, and applications of the chips used in the generator. • 2D Chip-800DG (On-chip Corporation) used for fabricating WODL and GMD with an average diameter of 30 μm (Part No. 1003002) • 2D Chip-1060DG (On-chip Co.) is used for fabricating WODLs with a diameter of 60-80 μm (currently with an average diameter of 80 μm) (product number 1003003). • DG8 cartridge (BIO RAD) used for the preparation of WODL with an average diameter of 140 μm (Part number 1864008) (2) Types, model numbers, and applications of chips used for dispensing multiple droplets • 2D Chip-Z1000-w150 (On-chip): Used for fluorescence detection of WODL containing GMDs and for preparative sampling of multiple WODLs (Catalog No. 1002005) (3) Type, model number, and application of the chip used for single droplet dispensing. • 2D Chip-SD1000 (On-chip): Used for fluorescence detection of WODL with an average diameter of 30 μm and for preparative extraction of single droplets (Catalog No. 1004001) • 2D Chip-SD1000-w150 (On-chip): Used for fluorescence detection of WODL containing GMD and for preparative extraction of single droplets (Catalog No. 1004002)

[0028] As used herein, the term "gel microdroplet (GMD)" refers to a fine gel-like droplet, typically formed on a gel substrate mainly composed of a water-soluble or hydrophilic polymer material. GMDs can typically be designed with an average diameter in the range of approximately 1 to 500 μm, with the 100 to 200 μm range being used for the isolation and cultivation of microorganisms. However, in this disclosure, considering the encapsulation of multiple GMDs in a WODL, the average diameter only needs to be smaller than that of the WODL, and from the viewpoint of encapsulating multiple GMDs, average diameters of 1 to 50 μm, 5 to 50 μm, 10 to 50 μm, 20 to 50 μm, 30 to 50 μm, and 40 to 50 μm are preferred.

[0029] Because GMDs are small enough to encapsulate individual microorganisms or cells, they are suitable for single-cell culture and analysis. Specifically, they can be applied to the isolation and culture of microorganisms from the environment, analysis of intestinal bacteria, screening of antibiotic-producing microorganisms, and evaluation of cellular metabolic activity, as well as, for example, isolating clones with superior characteristics from any microbial library, although this is not limited to these applications. This includes microbial libraries created by randomly modifying organisms through genetic recombination. Unlike conventional agar and liquid media, GMDs can maintain microorganisms in a closed environment on a nanoliter (nL) scale, thus contributing to the promotion of growth of non-cultured microorganisms and the control of intermicrobial interactions.

[0030] GMDs can be fabricated using methods such as emulsification, microfluidic devices, and inkjet printing. Microfluidic technology is particularly effective for producing fine, uniformly sized GMDs, allowing for precise particle size control. Hydrophilic polymers such as alginic acid, agar, agarose, polyethylene glycol (PEG), and polyvinyl alcohol (PVA) can generally be used as the gel substrate for GMDs. This provides a culture environment suitable for the growth conditions of the target microorganism. Unlike liquid culture media, GMDs, being gel-like solids, have the advantage of serving as a scaffold for microorganisms like solid culture media and retaining components such as culture medium around the cells. In particular, the use of low-melting-point agarose is preferred to minimize damage to microorganisms due to temperature changes such as heating and cooling. In this disclosure, considering that GMDs are encapsulated in WODL, the size, material, and culture conditions of the GMDs are appropriately selected to enable highly efficient growth and isolation of microorganisms.

[0031] According to this disclosure, high-throughput isolation of functional microorganisms from environmental microorganisms is possible using GMD-containing WODL. For example, by combining it with fluorescence assays or flow cytometry (FACS), rapid and highly accurate selection of microorganisms is possible. Specifically, by designing microorganisms grown in GMD to emit fluorescence in response to substrate conversion activity or metabolite secretion, only target microorganisms can be identified and isolated by flow cytometry.

[0032] Furthermore, by using microfluidic devices, it becomes possible to process millions to hundreds of millions of GMDs simultaneously and perform large-scale microbial screening. By applying the method of the present invention, diverse applications can be expected, such as the selection of antibiotic-producing bacteria, enzyme-producing bacteria, biofuel-producing microorganisms, and intestinal bacteria. In particular, it contributes to the identification and cultivation of non-culturable microorganisms that were difficult to isolate with conventional culture methods, and can accelerate the search for novel functional microorganisms. As described above, the technology for culturing and separating microorganisms using GMDs dramatically improves the efficiency of functional analysis and industrial utilization of microorganisms, and will be an extremely useful method in the field of biotechnology.

[0033] As used in this specification, microbial "diversity" refers to differences in species, genetic characteristics, functions, and interactions within a microbial community. High diversity means a rich variety of microorganisms and their interactions within a particular environment or ecosystem. Microbial diversity primarily includes species diversity, genetic diversity, functional diversity, ecological diversity, interaction diversity, and microbial diversity.

[0034] In one embodiment, environmental microorganisms are encapsulated in GMDs composed of different culture media, and multiple species and multiple GMDs are encapsulated in WOLD. This allows for cultivation according to the properties of each microorganism contained in each GMD, and enables isolation and sorting of GMDs exhibiting different properties (e.g., growth ability, differentiation ability). One or more microorganisms can be encapsulated in a GMD. This allows for verification of the effects of interactions between microorganisms encapsulated in a single GMD, or between microorganisms in individual GMDs and microorganisms encapsulated in the aqueous phase of WOLD. Furthermore, by encapsulating reagents capable of detecting desired functions of environmental microorganisms in the aqueous phase of WOLD, the effects of microorganism interactions between GMDs can also be verified. Not only can interactions between multiple microorganisms encapsulated in a single GMD be verified, but the effects of interactions between multiple GMDs, each containing one type of microorganism, can also be verified. From this perspective, it is preferable that the GMDs contain microorganisms at the single-cell level.

[0035] As long as the microorganisms are contained within the GMD at the single-cell level, the manufacturing method is not restricted. For example, by adjusting the microbial concentration used in GMD production and the number of GMDs produced, it is possible to produce GMDs containing single cells. The microorganisms contained within such GMDs are derived from a single cell. Therefore, when a microbial community containing multiple microbial species is used as a sample, selectively separating each GMD exhibiting different properties can contribute to the analysis and scaling up of each microorganism.

[0036] The present invention's method for culturing microorganisms includes the step of culturing microorganisms in WODL containing the GMD prepared above, but those skilled in the art can determine the culturing conditions by appropriately selecting the type of culture medium, temperature, and culturing period for general microorganisms.

[0037] Any method known to those skilled in the art can be used to detect microorganisms that have grown in the GMD and the aqueous phase of the WODL. In particular, as shown in this disclosure, when detecting living cells, it is desirable to use a fluorescent stain with low cytotoxicity in combination with a separation device equipped with a fluorescence detector. Also, as shown in this disclosure, it is possible to use stains other than cell stains. Although not limited to these, for example, SYBR green I is a nucleic acid fluorescent staining reagent characterized by fluorescence when it intercepts double-stranded DNA. Because it is amphiphilic, it can be applied to emulsions of fluorine-based oil and an aqueous layer, making it possible to detect microbial growth in real time while avoiding adverse effects on microbial cells by the stain. Since the number of living cells and the number of genomic DNA are proportional, staining with this dye indicates the presence of cultured cells. It also has the advantage that the number of cultured cells can be relatively quantified by fluorescence intensity. It also has the advantage of being more sensitive than conventional OD (Optical Density)-based evaluation of microbial growth, and can detect even slight growth. In addition to the above, methods using SYBR Green I (registered trademark), GelGreen (registered trademark), GelRed (registered trademark), etc. are also preferred.

[0038] The step of culturing microorganisms within the GMD described above may also be a step of culturing the GMD in a "dense state". The term "dense state" refers to a state in which multiple GMDs are arranged in close proximity to each other and in a microspace within the WODL, where the boundaries of individual GMDs are maintained and the contents (e.g., microorganisms and culture medium) do not mix with each other or with the aqueous phase of the WODL. In a preferred embodiment, the average number of GMDs contained in the WODL may be 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3, as long as it is a number that can be contained in the WODL. Alternatively, it may be any number within the above range, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0039] Other technical terms used herein have the common meanings in the art in which they are used, as exemplified by various technical dictionaries. Specific values ​​and configurations discussed in these non-limiting embodiments are variable and are cited merely to illustrate at least one embodiment and are not intended to limit its scope. [Examples]

[0040] [Experimental Example 1: Preparation of GMDs containing E. coli, removal of the surfactant layer from the GMDs, encapsulation of the GMDs in WODL, and microscopic observation] A genetically modified Escherichia coli strain (DH5α strain) was used as the bacterium for the experiment. This strain constitutively emits green fluorescence because it has the plasmid pIJ8668, which encodes the GFP biosynthesis gene, introduced into it (hereinafter simply referred to as "E. coli"). This strain was spread onto an agar plate (9 cm in diameter) containing LB agar medium (Nacalai Tesque Co., Ltd.) and cultured at 37°C for 1 day. Colonies that grew on the plate were scraped off with a sterile disposable stick (AS ONE, Japan) and suspended in a 1.5 mL Eppendorf tube containing 1 mL of LB liquid medium concentrated to twice the normal concentration (Tryptone (BD, USA), sodium chloride (Fujifilm Wako Pure Chemical Industries, Japan), Yeast extract (BD)). Subsequently, the suspension was diluted to 7.0 × 10⁶. 8The cells were prepared in LB liquid medium concentrated to twice its original concentration to achieve a cell count of cells / mL. SeaPlaque® agarose (LONZA, Switzerland) was suspended in distilled water to a concentration of 3% (w / w), sterilized and dissolved by autoclaving at 121°C for 20 minutes. Additionally, a fluorine oil was prepared by diluting fluorine oil containing 5% surfactant 008-FluoroSurfactant (On-Chip, Japan) with fluorine oil HFE-7500 (NT Science, Japan) to a surfactant concentration of 2%. The bacterial suspension, agarose solution, and prepared fluorine oil were each transferred to 2 mL Eppendorf tubes (1 mL each), and then incubated at 40°C in a constant temperature bath (TAITEC, Japan). The bacterial suspension and agarose solution were then mixed in a 1:1 ratio in the tubes (Agarose 1.5% (w / w)). Using an on-chip droplet generator (On-Chip, Japan), a chip heated to 45°C on a hot plate (Thermo Fisher Scientific, USA) was placed in a mixed agarose solution and fluorine oil, respectively, to create gel microdroplets (GMDs) with an average diameter of 30 μm. The GMDs dispersed in oil were transferred to a 2 mL tube and cooled in a refrigerator at 4°C for 10 minutes to solidify. Subsequently, 10 mL each of sterile water and fluorine oil (On-Chip, Inc.) with 0.1% surfactant 008-FluoroSurfactant added were placed in 25 mL Falcon tubes and vortexed for 1 minute to create WODLs with non-uniform sizes. The solidified GMDs and the prepared WODLs were placed in a 1:5 ratio in a 2 mL tube, and then a plasma ball was applied at 12 V, 500 mA for 10-20 seconds to separate them into two layers: an oil layer and a water layer. During this process, the GMDs migrate to the aqueous layer, so only the aqueous layer was extracted into a 1.5 mL tube. Sterile water containing GMDs with an average diameter of 30 μm was then mixed into a 1.5 × 10⁻¹⁶ tube. 7The GMD was diluted with sterile water to a concentration of 1 / mL, i.e., a concentration that would allow multiple GMDs to be encapsulated during WODL preparation. WODLs with an average diameter of 140 μm were prepared using an on-chip droplet generator with a GMD suspension prepared with fluorine oil containing 2% surfactant. To prevent clogging of the flow path during WODL preparation and to disperse the GMDs in water, the tip was gently tapped and vibrated during the process. The prepared WODLs were incubated statically in 2 mL tubes at 30°C for 7 days. After incubation, the WODLs were placed on a microslide 6(VI) flat (ibidi, Germany) and observed under a microscope. Specifically, using an inverted fluorescence microscope (ZEISS, Germany), 10x and 40x objective lenses, a fluorescence filter set (eGFP), and a microscope camera (ZEISS), fluorescence images and bright-field and phase-contrast field images of the same field of view were taken using a digital camera (ZEISS) attached to the microscope. These images captured WODLs contained within GMDs formed by Escherichia coli colonies emitting green fluorescence.

[0041] The results are shown in Figure 2. Microscopic observation of the WODL revealed that multiple GMDs were encapsulated within the WODL on day 0 of culture. Furthermore, on day 7 of culture, colony formation of E. coli was observed in many GMDs, and the green fluorescence also increased accordingly.

[0042] This fabrication method was made possible by using low-melting-point agarose and minimizing the use of surfactants and plasma.

[0043] [Experimental Example 2: Mounting of three or more GMDs composed of different culture medium components into a WODL, and observation of bacteria growing independently in three different GMDs within the same WODL] As in Experimental Example 1, the bacteria used in the experiment were E. coli DH5α strain, which emits green fluorescence; Streptomyces tanashiensis, a bacterium that forms hyphae; and Rhizobium skierniewicense, a bacterium that does not fluoresce, all of which were used. E. coli was spread under the conditions of Experimental Example 1, S. tanashiensis on agar plates containing ISP2 agar (BD), and R. skierniewicense on agar plates containing R2A agar (BD), and incubated at 25°C or 30°C for 1 to 3 days. As in Experimental Example 1, the colonies of each strain grown on the plates were suspended in 1.5 mL Eppendorf tubes containing 1 mL of liquid medium. For E. coli, the same conditions as in Experimental Example 1 were used. For S. tanashiensis, ISP2 liquid medium concentrated to twice the normal concentration (Yeast extract (BD), Malt extract (Thermo Fisher Scientific), glucose (Fujifilm Wako Pure Chemical Industries)) was used. For R. skierniewicense, R2A liquid medium concentrated to twice the normal concentration (Peptone (BD), Yeast extract (BD), Casamino acid (BD), glucose (Fujifilm Wako Pure Chemical Industries), soluble starch (Fujifilm Wako Pure Chemical Industries), monopotassium phosphate (Fujifilm Wako Pure Chemical Industries), magnesium sulfate heptahydrate (Fujifilm Wako Pure Chemical Industries), sodium pyruvate (Fujifilm Wako Pure Chemical Industries)) was used. Subsequently, each suspension was measured using the same method as in Experimental Example 1, yielding 7.0 × 10⁶ of each suspension. 8 After adjusting the cell count to a specific number of cells / mL, GMDs with an average diameter of 30 μm were prepared from each bacterial suspension. The prepared GMDs were then processed to remove the surfactant layer as in Experimental Example 1, and each was measured to 1.5 × 10⁶. 7The GMDs were diluted with sterile water to a concentration of 1 / mL. 200 μL each of the three prepared GMD suspensions were added to a 1.5 mL tube and mixed. Then, using this mixture, WODLs with an average diameter of 140 μm were prepared using an on-chip droplet generator, similar to Experimental Example 1, to create WODLs containing the three types of GMDs. The prepared WODLs were cultured statically in 2 mL tubes at 30°C for 7 days. After culturing, the presence of GMDs with three different types of bacteria growing within each WODL was observed using an inverted fluorescence microscope, similar to Experimental Example 1, and photographs were taken.

[0044] The results are shown in Figure 3. Microscopic observation of the cultured WODLs revealed that after 7 days of culture, each microorganism—namely, the autofluorescent E. coli colonies, the hyphae-forming S. tanashiensis, and the non-fluorescent R. skierniewicense colonies—proliferated independently within the same WODL's GMD. Furthermore, although the GMDs containing each bacterium were composed of three different culture medium components, the independent growth of each bacterium in its respective GMD demonstrated that even when two or three different GMDs composed of different culture medium components are enclosed in the same WODL, microorganisms can grow independently within the GMD.

[0045] [Experimental Example 3: Analysis of bacterial community structure in WODL containing three or more types of GMD composed of different culture medium components] The bacterial community structure in WODL containing three different GMDs prepared in Experimental Example 2 was calculated as follows. Specifically, 100 μL of the cultured WODL was placed in the sample reservoir of the analysis chip 2D Chip-Z1000-w150 (On-Chip Co., Ltd.), then fluorine oil with 0.1% 008-FluoroSurfactant added was placed in the reservoir for the sheath-slip solution, and the WODL was dispersed in the oil by the air pressure of the On-chip sort (On-Chip Co., Ltd.) and passed through the flow channel (150 μm × 150 μm) of the analysis chip. At that time, the intensity of green fluorescence from each droplet of the numerous WODLs flowing through the flow channel was observed using an excitation laser (488 nm) and a photomultiplier tube (543 ± 22 nm) for green fluorescence detection attached to the On-chip sort. Subsequently, only WODLs with a fluorescence intensity of 100 a.u. or higher, indicating the growth of at least one E. coli that emitted green fluorescence (i.e., GFP biosynthesis), were isolated, and a total of 128,000 WODLs were recovered. The recovered WODLs were destroyed along with the cells by bead beating using the Extra Soil DNA Kit Plus ver.2 (BDL, Japan), separated into aqueous and oil layers by centrifugation, and then the cells in the aqueous layer were collected and DNA was extracted according to the prescribed protocol. Using this extracted DNA, a library was prepared according to the prescribed protocol, and next-generation sequencing analysis targeting the 16S rRNA gene was performed (Illumina, iSeq, 92087 reads). The obtained read data were clustered after removing low-quality sequences and chimeric sequences using 16S-based MTP in EzBiCloud (https: / / www.ezbiocloud.net / ), and the bacterial community structure and the relative abundance of each bacterial species were calculated based on the PKSSU database (ver4.0).

[0046] The results are shown in Table 1. Analysis of the bacterial community structure revealed that the relative abundance of each bacterial species in the recovered WODL was 86.9% for E. coli, 4.3% for S. tanashiensis, and 6.8% for R. skierniewicense, confirming that the three bacterial species grew independently within the same WODL's GMD. This result demonstrates that even when multiple GMDs containing different culture media compositions or different bacterial species are present in a WODL, each bacterium can grow independently within its own GMD.

[0047] [Table 1]

[0048] Surprisingly, when encapsulating and culturing multiple microorganisms within a WODL, it was expected that only some bacteria would proliferate due to differences in their growth rate and compatibility with the culture medium components of the liquid phase. Therefore, in this experiment, it was anticipated that only E. coli, which has the fastest growth rate, would be detected. However, contrary to this expectation, it was shown that by preparing separate culture environments called GMDs and selecting a culture medium suitable for the microorganism being grown, different microorganisms could grow independently without inhibiting each other's growth.

[0049] [Experimental Example 4: Culture of different microbial species in GMD enclosed in WODL and in the external liquid phase] As with Experimental Example 1, the microorganisms used in the experiment were genetically modified Escherichia coli DH5α strain that emits green fluorescence and the fungus Lipomyces starkeyi (hereinafter referred to as "L. starkeyi"). E. coli was cultured under the conditions of Experimental Example 1, while L. starkeyi was cultured on agar plates solidified with 1.5% agar (Fujifilm Wako Pure Chemical Industries) in Potato Dextrose liquid medium (potato dextrose broth (BD)) and incubated at 30°C for 1-3 days. As with Experimental Example 1, the L. starkeyi colonies grown on the plates were suspended in 1.5 mL Eppendorf tubes containing 1 mL of liquid medium. The liquid medium used was Potato Dextrose liquid medium concentrated to 2:1. Subsequently, the suspension was cultured to 7.0 × 10⁶ using the same method as in Experimental Example 1. 8 After adjusting the cell count to the specified number of cells / mL, GMDs with an average diameter of 30 μm were prepared from the prepared suspension. The surfactant layer of the prepared GMDs was removed as in Experimental Example 1, but for WODLs of non-uniform size prepared using the vortex method, sterile water was replaced with LB liquid medium. Of the two layers separated into an oil layer and an aqueous layer, the GMDs migrated to the aqueous phase, i.e., into the LB liquid medium, so only the aqueous layer was extracted into a 1.5 mL tube. Subsequently, the LB liquid medium containing the suspended GMDs with an average diameter of 30 μm was divided into 1.5 × 10⁻⁶ tubes. 7 LB liquid medium was diluted to achieve a GMD concentration of cells / mL. E. coli cultured on agar plates was then added to this LB liquid medium, with a cell count of 9.6 × 10⁶. 5 The cells were suspended to a concentration of cells / mL. Using this suspension, WODLs with an average diameter of 140 μm were prepared using an on-chip droplet generator, similar to Experimental Example 1, in which E. coli was independently present in the liquid phase and L. starkeyi in the GMD. The prepared WODLs were cultured statically in 2 mL tubes at 30°C for 3 days. After culturing, they were observed using an inverted fluorescence microscope and photographs were taken, similar to Experimental Example 1.

[0050] The results are shown in Figure 4. As a result of observing the cultured WODL with a microscope, it was confirmed that on the 3rd day of culture, E. coli grew independently in the liquid phase and L. starkeyi grew independently in GMD, respectively. These results indicated that microorganisms of different lineages can grow independently in GMD and the liquid phase.

[0051] [Experimental Example 5: Method for inducing intermicrobial interactions between different GMDs within the same WODL and preparation of control test groups] As microorganisms used for the experiment, in the same manner as in Experimental Example 1, E. coli strain DH5α that emits green fluorescence and the actinomycete Streptomyces ghanaensis (hereinafter referred to as "S. ghanaensis") were used. This actinomycete has been confirmed to produce moenomycin, an antibiotic that inhibits the growth of E. coli. S. ghanaensis was spread on an agar plate containing LB agar medium (Nacalai Tesque) and cultured at 30°C for 7 days. In the same manner as in Experimental Example 1, colonies of S. ghanaensis grown on the plate were suspended in a 1.5 mL Eppendorf tube containing 1 mL of liquid medium. Note that LB liquid medium concentrated to twice the specified concentration was used as the liquid medium. Thereafter, each suspension was adjusted to 7.0 × 10 9 After adjusting the cell count to cells / mL, GMD with an average diameter of 30 µm was prepared from the bacterial suspension. The prepared GMD was transferred to a 2 mL tube, and statically cultured at 30°C for 7 days while dispersed in oil. After 7 days of culture, GMD with an average diameter of 30 µm was prepared using colonies of E. coli that had been cultured on an LB agar plate for 1 day by the same method as in Experimental Example 1, cooled at 4°C for 10 minutes, and solidified. After removing the surfactant layer from GMD encapsulating E. coli and GMD that had been cultured for 7 days with S. ghanaensis encapsulated by the same method as in Experimental Example 1, each was adjusted to 1.5 × 10 7The GMDs were diluted with sterile water to a concentration of 1 / mL. 300 μL each of the two prepared GMD suspensions were added to 1.5 mL tubes and mixed in a 1:1 ratio. Then, using this mixture, WODLs with an average diameter of 140 μm were prepared using an on-chip droplet generator, similar to Experimental Example 1, to create WODLs containing the two types of GMDs. The prepared WODLs were cultured statically in 2 mL tubes at 30°C for several days. After culturing, the presence of GMDs with two different types of bacteria growing within each WODL, as well as fluorescent colonies of E. coli, were observed and photographed using an inverted fluorescence microscope, similar to Experimental Example 1.

[0052] As a control group, SeaPlaque® agarose was suspended in distilled water to a concentration of 1.5% (w / w), sterilized, and dissolved. Using this agarose solution, GMDs with an average diameter of 30 μm were prepared using the same method as in Experimental Example 1. After removing the surfactant layer, 1.5 × 10⁻⁶ GMDs were prepared. 7 The GMD was diluted with sterile water to a concentration of 1 / mL. 300 μL each of the sterile water-only GMD and the GMD suspension containing the prepared E. coli were added to 1.5 mL tubes and mixed in a 1:1 ratio. Then, using this mixture, WODLs with an average diameter of 140 μm were prepared using an on-chip droplet generator, similar to Experimental Example 1, to create WODLs containing two types of GMDs. The prepared WODLs were incubated statically in 2 mL tubes at 30°C for several days.

[0053] The results are shown in Figure 5. Microscopic observation of the cultured WODL revealed that multiple GMDs were encapsulated. In WODL containing only GMDs in which E. coli grew, numerous colonies exhibiting green fluorescence were observed. On the other hand, in WODL containing GMDs with elongated hyphae of the actinomycete S. ghanaensis, no fluorescent colonies were observed in the other GMDs (Figure 5). This result indicates that the antibiotic moenomycin, derived from the actinomycete encapsulated in the GMD within the same WODL, inhibits the growth of E. coli. Specifically, this refers to Escherichia coli and S. ghanaensis encapsulated in different GMDs using the agglutination complex culture method using GMD-containing WODL described herein.

[0054] [Experimental Example 6: Separation of WODLs in which intermicrobial interactions occurred between different GMDs and secondary culture on agar plates] The fluorescence intensity of WODL prepared in Experimental Example 5 was measured. Specifically, using the same method as in Experimental Example 3, the green fluorescence intensity of approximately 5000 droplets of cultured WODL was observed using On-chip sort. Subsequently, 100 μL of the cultured WODL was placed in the sample reservoir of the analysis chip 2D Chip-SD1000-w150 (On-Chip Co., Ltd.), and then fluorine oil with 0.1% 008-FluoroSurfactant added was placed in the sheath liquid reservoir. The WODL was dispersed in the oil by the air pressure of the On-chip selector (On-Chip Co., Ltd.) and passed through the flow channel (150 μm × 150 μm) of the analysis chip. At that time, the green fluorescence intensity of each droplet of the numerous WODL flowing through the flow channel was measured again using an excitation laser (488 nm) and a photomultiplier tube (543 ± 22 nm) for green fluorescence detection attached to the On-chip selector. Subsequently, WODL (Whole Oxide Volume) (20 a.u. or less) where green fluorescence was attenuated, i.e., where E. coli growth was being inhibited by S. ghanaensis, was gated, and WODL within that region was separated into one droplet each. The separated WODL were dropped onto a 96-well flat-bottom plate (Corning, USA) filled with 180 μL of LB agar medium (Nacalai Tesque) containing aztreonam to a final concentration of 50 μg / mL, at a rate of one droplet per well. The plates were then incubated at 30°C for two weeks.

[0055] The results are shown in Figure 6. When the number of WODLs with attenuated green fluorescence was calculated (below 70 au), 16.4% of WODLs containing actinomycetes showed this (845 droplets (number of droplets with attenuated fluorescence) / 5154 droplets (total number of droplets detected)). On the other hand, in the control experiment, where the actinomycetes-containing GMDs were replaced with GMDs composed only of sterile water, the percentage of WODLs with attenuated green fluorescence (below 50 au) was 2.4% (124 droplets / 5199 droplets), indicating that green-fluorescent E. coli proliferated sufficiently in most WODLs without inhibiting growth. From these results, it was shown that, similar to Experimental Example 5, it is possible to induce microbial interactions between GMDs within the same WODL. Furthermore, when one droplet was isolated from the fluorescence-attenuated region, placed on a 96-well flat-bottom plate, and cultured, colony formation of actinomycetes was confirmed on the LB agar plate (Figure 7). This indicates that the actinomycete S. ghanaensis can grow in the GMD within the WODL region where fluorescence decay occurred, and that these can be secondary cultured on agar plates.

[0056] It has been suggested that actinomycetes often produce antibiotics vigorously under nutrient-rich conditions (Non-patent document 1). Although it was expected that GMDs, being small in size, would have insufficient culture medium components and therefore inadequate for actinomycetes to grow and produce antimicrobial substances, contrary to this expectation, it was shown that sufficient antimicrobial substances were produced and that the growth of E. coli growing in different GMDs was inhibited.

[0057] [Experimental Example 7: A method for screening microorganisms that produce useful substances such as antimicrobial agents using intermicrobial interactions] For the experiment, 100g of soil was collected from the National Institute of Advanced Industrial Science and Technology (AIST) in Tsukuba City, Ibaraki Prefecture. The collected soil sample was sieved to remove large particles using a 5mm mesh sieve. Then, 1g of the sieved soil was added to a 15mL Falcon tube containing 10mL of sterile water and vortexed at maximum intensity for 5 minutes. After that, the sample was filtered stepwise using a Miracross (Merck, Germany) with a pore size of 22-25μm, a Cell Strainer (PLS, Germany) with a pore size of 20μm, and a filter (Sartorius, Germany) with a pore size of 5μm, so as not to stress the microbial cells and to remove minute soil particles that would cause noise when measuring fluorescence intensity. Furthermore, the filtrate was filtered through a 0.2μm filter (Merck) to trap the cells on the filter. The filter containing the trapped microorganisms was placed in a 15mL Falcon tube, 5mL of sterile water was added, and the cells were detached by vortexing at maximum intensity for 5 minutes. The filter was then removed to prepare a cell suspension. Cell suspensions were prepared in R2A liquid medium and ISP2 liquid medium concentrated to twice the specified concentration so that an average of 1 cell was inscribed in each GMD with an average diameter of 30 μm. Subsequently, GMDs with an average diameter of 30 μm were prepared using the same method as in Experimental Example 5. The prepared GMDs were transferred to 2 mL tubes and cultured statically at 30°C for 5-7 days while dispersed in oil. Then, as in Experimental Example 5, GMDs with an average diameter of 30 μm were prepared using E. coli colonies cultured on LB agar plates for 1 day. After removing the surfactant layer from each GMD, each was measured to 1.5 × 10⁶ 7The GMD was diluted with sterile water to a concentration of 1 / mL. The GMD suspensions composed of the two prepared media and the GMD suspension containing E. coli were mixed in a 1:1 ratio in 1.5 mL tubes. Using this mixture, WODLs with an average diameter of 140 μm were prepared using an On-chip droplet generator, similar to Experimental Example 1, containing both GMDs containing soil-derived microorganisms and GMDs containing E. coli. The prepared WODLs were cultured statically in 2 mL tubes at 30°C for several days. After culturing, 100 μL of the WODLs were measured for green fluorescence intensity per droplet using an On-chip selector (On-Chip Inc.) in the same manner as in Experimental Example 6. Subsequently, WODLs with reduced green fluorescence, i.e., those where E. coli growth was inhibited by soil-derived microorganisms (less than 10 a.u.), were gated, and WODLs within that region were separated one droplet at a time. The separated WODL was dropped onto a 96-well flat-bottom plate (Corning) filled with 180 μL each of R2A agar (BD) or ISP2 agar (BD) containing aztreonam to a final concentration of 50 μg / mL, at a rate of 1 droplet per well. The cells were then cultured at 30°C for 2 weeks. After culturing, the formed colonies were isolated, and PCR targeting the 16S rRNA gene was performed. The amplified nucleic acids were then sequenced by Sanger sequencing (3730xl DNA analyzer, Applied Biosystems). The determined gene sequences were then subjected to phylogenetic analysis using the same method as in Experimental Example 3.

[0058] The results are shown in Figure 8. Using an on-chip selector, the number of droplets with attenuated green fluorescence was calculated for WODLs containing GMDs encapsulating soil-derived microorganisms. In WODLs where soil microorganisms were encapsulated in ISP2 liquid medium, the percentage was 16.9% (694 droplets (number of fluorescence-attenuated droplets) / 4108 droplets (total number of droplets detected)). In WODLs where soil microorganisms were encapsulated in R2A liquid medium, the percentage was 3.4% (141 droplets / 4155 droplets) (5a.u. or less). These results indicate that the growth of E. coli was inhibited within the WODL by substances derived from environmental microorganisms encapsulated in the GMDs. Furthermore, when the fluorescence-attenuated regions were isolated into individual droplets, divided into 96-well flat-bottom plates, and cultured, colony formation of multiple microorganisms was confirmed on R2A and ISP2 agar plates. This indicates that environmental microorganisms can grow in the GMD within WODL in the fluorescence-attenuated region, and that they can be secondary cultured on agar plates. Furthermore, analysis of the 16S rRNA genes of the isolated bacteria allowed for the identification of 10 different bacterial species (Table 2). These results demonstrate that this method allows for the independent culture and isolation of diverse environmental microorganisms within the GMD in WODL.

[0059] [Table 2]

[0060] [Experimental Example 8: Measurement of antibacterial activity of isolated bacteria] In Experimental Example 7, we performed antimicrobial activity tests on soil-derived bacterial strains that showed potential to inhibit E. coli growth between GMDs within the WODL. The isolated bacterial strains were spread onto R2A agar and ISP2 agar plates and cultured at 30°C for 7 days. Three species of bacteria were used as targets for antimicrobial activity testing: Escherichia coli, Micrococcus luteus, and Bacillus subtilis. The three strains were cultured on LB agar plates at 37°C for 1 day, then suspended in a Falcon tube containing 30 mL of LB liquid medium using a disposable stick (AS ONE), and cultured at 37°C for 1 day with shaking at 200 rpm. After sterilizing and dissolving the LB agar medium prepared at the specified concentration in an autoclave, it was cooled to 40°C in a water bath (THERMAL ROBO, AS ONE), and each bacterial culture solution was dropped into it in a volume of 1 / 20 of the agar medium. The mixture was then uniformly mixed by shaking, and the bacterial suspension LB agar medium was placed in a rectangular petri dish (14 cm x 10 cm) and allowed to solidify. Subsequently, the agar medium from the petri dish in which the isolated bacterial strain had grown was scooped out with a straw, along with the bacterial colonies, and placed at equal intervals on three different bacterial suspension LB agar petri dishes. The agar dishes were cultured at 37°C for 3 days, and the antibacterial activity was evaluated by the formation of an inhibition zone, where the growth of the target bacteria was inhibited by substances leached from the agar in which the isolated strain had grown.

[0061] The results are shown in Figure 9. Antimicrobial activity tests were performed on the bacterial isolates shown in Table 2 against the three types of bacteria in a rectangular petri dish. Bacillus wiedmannii I strain formed an inhibition zone against all three types of bacteria, demonstrating antimicrobial activity. These results demonstrate that functional microorganisms with antimicrobial activity can be detected and isolated using the method described herein. [Industrial applicability]

[0062] The high-density culture method for microorganisms using gel microdroplets of the present invention makes it possible to achieve a high culture rate of environmental microorganisms while maintaining high microbial diversity. Furthermore, it becomes possible to improve the efficiency of systematically isolating and culturing novel bacterial species and difficult-to-culture bacterial groups, which have been difficult to do until now. Such a microbial culture method is expected to make a great contribution not only to microbiology but also to the field of drug discovery.

[0063] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for any purpose. However, no reference to any references, articles, publications, patents, patent publications, or patent applications cited herein constitutes, and should not be construed as, an endorsement or any suggestion that they constitute valid prior art or form part of common technical knowledge in any country of the world.

Claims

1. A water-in-oil droplet for use in a method of agglomerating and culturing microorganisms or for use in the isolation and detection of microorganisms, wherein the aqueous phase of the water-in-oil droplet is (1) Multiple types and multiple gel microdroplets, (2) comprising microorganisms and / or activity measurement compounds, Here, each of the gel microdroplets is independent of the following: (1a) One or more microorganisms of one or more species; (1b) Multiple culture media of different compositions; and (1c) Low melting point agarose The above-mentioned water-in-oil droplets, including the above-mentioned water-in-oil type.

2. The water-in-oil droplet according to claim 1, wherein the average diameter of the gel microdroplets is 10 μm to 50 μm.

3. The water-in-oil droplet according to claim 1, wherein the average diameter of the water-in-oil droplet is 50 μm to 200 μm.

4. The average diameter of the gel microdroplets is 10 μm to 50 μm, and The water-in-oil droplet according to claim 1, wherein the average diameter of the water-in-oil droplet is 50 μm to 200 μm.

5. The water-in-oil droplet according to claim 1, wherein the microorganism is a microorganism collected from the environment.

6. The water-in-oil droplet containing a gel microdroplet according to claim 1, wherein the microorganism is selected from the group consisting of bacteria, fungi, protists, and archaea.

7. A method for agglutinating and complex culturing of microorganisms, (a) A step of encapsulating one or more microorganisms of one or more species in a gel microdroplet, (b) A step of introducing the gel microdroplets containing the microorganisms obtained above into each water-in-oil droplet, (c) A step of culturing microorganisms with gel microdroplets encapsulated in a water-in-oil droplet. A method for agglutinating and culturing microorganisms, including [specific element].

8. In addition to the above steps (a) to (c), (d) A step comprising adding a microorganism and / or an activity measurement compound to the aqueous phase of the water-in-oil droplet, A method for agglutinating and complex culturing of microorganisms according to claim 7, comprising the above.

9. The method for agglutinating and complex culturing of microorganisms according to claim 7, wherein in step (a), the gel microdroplet comprises a plurality of different culture medium components.

10. The method for agglutinating and culturing microorganisms according to claim 7, wherein in step (a), the gel microdroplet comprises low-melting-point agarose.

11. The method for agglomerating and complex culturing of microorganisms according to claim 7, wherein the average diameter of the gel microdroplets is 10 μm to 50 μm.

12. The method for agglomerating and complex culturing of microorganisms according to claim 7, wherein the average diameter of the water-in-oil droplets is 50 μm to 200 μm.

13. The average diameter of the gel microdroplets is 10 μm to 50 μm, and The method for agglomerating and complex culturing of microorganisms according to claim 7, wherein the average diameter of the water-in-oil droplets is 50 μm to 200 μm.

14. The method for agglomerating and complex culturing of microorganisms according to claim 7, wherein the microorganism is a microorganism collected from the environment.

15. The method for agglutinating and complex culturing of microorganisms according to claim 7, wherein the microorganism is selected from the group consisting of bacteria, fungi, protists, and archaea.

16. A method for isolating and detecting microorganisms, (e) A step of producing a water-in-oil droplet as described in any one of claims 1 to 6, (f) A step of culturing microorganisms contained in a water-in-oil droplet containing a plurality of gel microdroplets using the microorganism aggregation complex culture method described in any one of claims 7 to 15, (g) A step of detecting cells that have grown in a droplet by adding a fluorescent nucleic acid staining dye to the oil phase of the water-in-oil emulsion, (h) A step of separating the aqueous layer of a water-in-oil droplet from a gel microdroplet, (i) A step of separating gel microdroplets stained with a fluorescent nucleic acid staining dye from unstained gel microdroplets, (j) A step of recovering microorganisms from the gel microdroplets separated in step (i) and (k) A step of extracting nucleic acids from the microorganisms recovered in step (j), (l) A step of comparing the 16S rRNA base sequence contained in the nucleic acid with the sequence of a known microorganism, and determining that the isolated microorganism containing the nucleic acid is a novel microorganism if the identity is 97% or less, And, (m) A step to measure the function of isolated and cultured microorganisms. A method for isolating and detecting microorganisms, comprising the above.

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