Methods for culturing environmental microorganisms

Abstract

The objective is to provide a high-density culture method that achieves a high culture rate for environmental microorganisms. [Solution] According to the present invention, a method for culturing microorganisms to improve the cultureability rate of environmental microorganisms is provided, comprising the steps of (a) preparing droplets having an average diameter of 50 μm or less in an oil-in-water emulsion containing microorganisms, and (b) culturing microorganisms in the droplets.

Description

[Technical Field]

[0001] The present invention relates to a high-density isolation culture method that achieves a higher culturability rate for microorganisms collected from various environments (hereinafter referred to as "environmental microorganisms") compared to conventional culture methods. In particular, the present invention relates to a high-density isolation culture method for microorganisms that significantly improves the culturability rate of environmental microorganisms while maintaining high diversity by performing high-density culture using a water-in-oil emulsion. The present invention further relates to a method for detecting unknown microorganisms using this isolation culture method. [Background Art]

[0002] A wide variety of microorganisms exist on Earth, and by isolating, culturing, and utilizing these microorganisms, humankind has obtained various benefits in a wide range of fields such as medicine, agriculture, and environmental engineering. Conventional methods involve isolating microorganisms from the environment, culturing them as single strains, and then screening for the type and presence of substances produced by the microorganisms. However, it is becoming increasingly clear through conventional methods that there are a large number of microorganisms that cannot be isolated and cultured, that only a very small fraction of microorganisms have been utilized by humans, and that a large number of unutilized microorganisms remain on Earth (Non-Patent Document 1). If the culturability rate of uncultured microorganisms in the environment can be improved, it is expected to drive technological developments such as the development of new pharmaceuticals and agricultural chemicals.

[0003] One of the reasons why isolation culture is difficult is that it is hard to find optimal culture conditions for microorganisms in the environment. Furthermore, among environmental microorganisms, it is thought that there are many species that not only can grow alone, but can only grow through dependence or symbiotic relationships with different microorganisms or other organisms. That is, it is known that when microorganisms are cultured in close proximity to each other (hereinafter referred to as "mixed culture") to induce intermicrobial interactions, the metabolic system and growth of each microorganism are affected, allowing the microorganisms to grow in association with each other (Non-Patent Document 2).

[0004] In the pharmaceutical field, the following example is known: When Tsukamurella pulmonis, a Gram-positive aerobic bacterium of the genus Tsukamurella, was co-cultured with various actinomycetes to promote intermicrobial interactions, increased production of secondary metabolites and the production of substances not observed in individual cultures were confirmed in several actinomycetes. Among the substances whose production was confirmed in the combined culture were compounds exhibiting antibacterial activity, and these were novel compounds with structures different from known substances (Non-Patent Literature 3).

[0005] In the health field, there are known examples of compounds contributing to human health being commercialized through the complex culture of intestinal bacteria. Uroritin A, a functional polyphenol, is a substance attracting attention as a novel anti-aging functional food ingredient. Uroritin A is known to be an intestinal metabolite produced when ellagic acid, a type of polyphenol found in pomegranates and other fruits, is metabolized by intestinal bacteria. However, even if one ingests foods containing ellagic acid, it cannot be metabolized into uroritin A unless one's own intestinal microbiota is compatible. Therefore, intestinal bacteria that can convert ellagic acid into uroritin A and make this uroritin A ingestible have been searched for. As a result, it was revealed that this metabolism is carried out by two different types of microorganisms, and large-scale cultivation has actually been achieved, leading to commercialization (Non-Patent Literature 4). Furthermore, it has been reported that equol and its related substances, which are intestinal metabolites used in health foods, can also be industrially produced through the complex culture of microorganisms (Non-Patent Literature 5).

[0006] In the agricultural sector, there are known examples where intermicrobial interactions have a significant impact. Currently, the environmental burden and impact on human health of chemical fertilizers and pesticides used in agriculture are major concerns, and efforts to reduce their use are underway globally, particularly in the United States and EU countries where legal regulations are progressing. In Japan, the Ministry of Agriculture, Forestry and Fisheries has formulated the "Green Food System Strategy," which aims to achieve both improved productivity and sustainability in food, agriculture, forestry, and fisheries through innovation, and is aiming to reduce the use of chemical fertilizers by 30% by 2050. However, in agriculture, the use of fertilizers in the crop production process will continue to be indispensable for maintaining food supply, so technologies to improve plant productivity and disease resistance using symbiotic microorganisms are also being considered. Arbuscular mycorrhizal fungi (AM fungi), which parasitize plant roots, are fungi that inhabit the soil and help plant growth in agricultural land and natural ecosystems by absorbing phosphorus and nitrogen, which are essential nutrients for plants, from the soil and supplying them to their symbiotic host plants. AM fungi, in particular, are known to form symbiotic relationships with many plants, including grasses, legumes, and nightshades, and therefore have the potential to contribute to improving the productivity of a wide range of crops. On the other hand, because AM fungi depend on the carbon source supplied by the host plant when in symbiosis, they are difficult to grow on their own, and there has been a challenge in that AM fungi must always be cultured in co-existence with plants in order to proliferate them. Recently, a research group in Germany reported that AM fungi grow vigorously when co-cultured with the bacterium Paenibacillus validus (Non-Patent Literature 6). It is thought that compounds released by Paenibacillus validus activate the metabolism of AM fungi, and it has become clear that intermicrobial interactions are involved.

[0007] However, conventional methods have limitations in finding the optimal culture conditions for microorganisms that can be isolated and cultured from environmental microorganisms, resulting in extremely low culture success rates. Furthermore, even with complex cultures, the combinations of microorganisms are limited, throughput is low, and culture success rates remain low. Therefore, a method called gel microdroplets (GMDs) has been proposed, which involves creating numerous droplets in which microorganisms are embedded in a carrier such as agarose, and then culturing them (Non-Patent Literature 7). In addition, WODL( W ater-in- O il D rop l Although methods such as et al. were also developed, the culturability rate was limited, and it was not practical for industrial use (Patent Document 1).

[0008] While WODL itself is a known technology, as shown in this disclosure, culture methods using droplets of normal size still suffer from the problem of low cultureability. By making the average diameter of the droplets extremely small compared to what is typically done by those skilled in the art, a remarkable effect is achieved, and as a result, the cultureability of environmental microorganisms can be significantly improved compared to conventional methods. Furthermore, this method makes it possible to isolate and culture microorganisms individually as single cell species in a single DL, without having to culture environmental microorganisms together in a liquid medium, which was previously difficult due to their mixed composition. As a result, diverse environmental microorganisms can be individually preserved and maintained.

[0009] Generally, the smaller the size or average diameter of GMDs and WODLs, the smaller the volume, and the less nutrients necessary for microorganisms in the culture medium, making cell proliferation more difficult. For this reason, until now, there has been no active effort to produce small GMDs or WODLs, nor has it been considered desirable. Furthermore, even in examples where the average size of WODL diameter has been examined, the focus has not been on the cultureability rate, but on the proliferation rate (Non-Patent Literature 8). [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2017-63764 [Non-patent literature]

[0011] [Non-Patent Document 1] Lok C. 2015. Mining the microbial dark matter. Nature 522:270-273. [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. [Non-Patent Document 3] Onaka H. 2017. Novel antibiotic screening methods to awaken silent or cryptic secondary metabolic pathways in actinomycetes. The Journal of Antibiotics 70:865-870. [Non-Patent Document 4] World's first manufacturing method for pomegranate extract intestinal metabolites, "URORICH (trademark)," launched ~Supplied to supplement manufacturers as a functional ingredient that reactivates cells~ https: / / www.daicel.com / news / assets / pdf / 20200525.pdf [Non-Patent Document 5] Co-culturing multiple beneficial bacteria enables diverse variations! Establishment of an efficient and safe method for producing functional intestinal metabolites such as equol ~Topics from the 2022 Annual Meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry https: / / www.daicel.com / news / assets / pdf / 20220316.pdf [Non-Patent Document 6] Abdellatif L, Lokuruge P, and Hamel C. 2019. Axenic growth of the arbuscular mycorrhizal fungus Rhizophagus irregularis and growth stimulation by coculture with plant growth-promoting rhizobacteria. Mycorrhiza 29:591-598. [Non-Patent Document 7] Zengler K et al.,2002. Cultivating the uncultured. Proc Natl Acad Sci 99:15681-15686. [Non-Patent Document 8] Tan Y et al.,2022. The effect of droplet size on syntrophic dynamics in droplet-enabled microbial co-cultivation. PLOS one 17(3): e0266282. [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] This invention relates to a high-density isolation and culture method that achieves a high cultureability rate for environmental microorganisms, which was difficult with conventional techniques. Furthermore, it relates to a systematically novel method for the isolation and detection of microorganisms. [Means for solving the problem]

[0013] In view of the above-mentioned problems, the inventors have diligently conducted research and development and have succeeded in developing a microbial isolation and culture method, a microbial complex culture method, and a system that increase the culturability rate, which is expected to lead to the utilization of industrially important microorganisms that have not been utilized until now, and have completed the invention. The present invention is characterized by using a water-in-oil emulsion and a water-in-oil droplet (WODL) method for culturing microorganisms. In this method, an aqueous culture solution containing suspended environmental microorganisms is dispersed in oil to create "droplets" (hereinafter sometimes referred to as "DL") with a diameter of several tens of micrometers (for example, 1 to 50 μm). Since the space (volume) of such DLs is very narrow, it is expected that the concentration of substances secreted by microorganisms into the DL space composed of the culture medium 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 microorganisms separated in different DLs. While WODL itself is a known technology, the method described in this disclosure, which makes the average diameter of droplets extremely small compared to what is typically done by those skilled in the art, exhibits remarkable effects, and as a result, the cultureability rate of environmental microorganisms can be significantly improved compared to conventional methods. Furthermore, this method makes it possible to isolate and culture microorganisms individually as single cell species in a single DL, without having to culture environmental microorganisms together in a liquid medium, which was previously difficult due to their mixed composition. As a result, diverse environmental microorganisms can be individually preserved and maintained.

[0014] As described above, the inventors have succeeded in efficiently culturing environmental microorganisms by culturing them in droplets with a diameter of several tens of micrometers in a water-in-oil emulsion, thereby completing the present invention.

[0015] In other words, the present invention is as follows: [1] A method for culturing microorganisms to improve the cultureability rate, (a) A step of preparing droplets containing microorganisms with an average diameter of 1 to 50 μm in an oil-water emulsion, (b) culturing the microorganism in said droplet; and A method for culturing a microorganism, comprising: [2] The method for culturing a microorganism according to [1], further comprising a step of adjusting, in said step (a), such that microorganisms are contained in droplets on a single-cell basis. [3] The method for culturing a microorganism according to [1] or [2], wherein said microorganism is a microorganism collected from the environment. [4] The method for culturing a microorganism according to any one of [1] to [3], wherein said microorganism includes a microorganism that cannot be cultured on a nutrient agar medium. [5] The method for culturing a microorganism according to any one of [1] to [4], wherein the average diameter of said droplets is 1 to 30 µm. [6] The method for culturing a microorganism according to any one of [1] to [5], wherein said step (b) is a step of culturing droplets in a dense state. [7] A method for isolating and detecting a novel microorganism, comprising: (a) producing droplets having an average diameter of 1 to 50 µm in a water-in-oil emulsion, wherein the droplets contain a microorganism; and (b) culturing the microorganism in said droplet; and (c) detecting cells proliferated in the droplets by adding a fluorescent nucleic acid staining dye to the oil phase of said water-in-oil emulsion; and (d) separating droplets stained with a fluorescent nucleic acid staining dye from unstained droplets; and (e) isolating a microorganism from the stained droplets obtained in step (d), and extracting a nucleic acid from the microorganism; and (f) comparing the 16S rRNA nucleotide sequence contained in said nucleic acid with the sequence of a known microorganism, and determining that the isolated microorganism comprising the nucleic acid is a novel microorganism if the identity is 97.0% or less; and An isolation and detection method, comprising: Effects of the Invention

[0016] The microbial culture method described herein enables high-density culture by using WODL with an average diameter of 1 to 50 μm, thereby improving the culturability rate of environmental microorganisms. Furthermore, the method for detecting unknown microorganisms opens the way to utilizing unknown microorganisms that were previously unavailable to humankind. [Brief explanation of the drawing]

[0017] [Figure 1] These are microscopic images visualizing microorganisms growing in WODLs of different sizes. Average diameters of WODLs: (A) 140 μm, (B) 130 μm, (C) 75 μm, (D) 60 μm, (E) 30 μm. [Figure 2] These are microscopic images showing microorganisms grown in WODL stained with different fluorescent nucleic acid stains. (A) SYBR® Green I, (B) SYBR® Gold. [Figure 3] This graph shows the culture rate of environmentally derived microorganisms in WODLs (Wood of Occlusion Limbs) prepared by encapsulating cells at a single-cell level and ensuring different average diameters. [Figure 4] This graph compares the diversity of microbial species cultured in WODLs of different average diameters using environmentally derived microorganisms, with the number (types) of amplicon sequence variants (ASVs) as an indicator. [Figure 5] This graph shows the distribution rate of novel bacteria cultured in WODLs of different average diameters, using microorganisms originating from the environment. [Figure 6] This graph shows the distribution rate of difficult-to-culture bacteria (acI) when environmentally derived microorganisms are grown in WODLs of different average diameters. [Figure 7] This image shows the appearance of microorganisms that have formed colonies on a 96-well agar plate (cultured for 14 days). [Modes for carrying out the invention]

[0018] The present invention relates to a high-density culture method for achieving a high cultureability rate of environmental microorganisms. In one embodiment, the microbial culture method of the present invention is a method for culturing microorganisms to improve the cultureability rate, (a) A step of preparing droplets containing microorganisms with an average diameter of 1 to 50 μm in an oil-water emulsion, (b) A step of culturing microorganisms in the droplet; It can include...

[0019] 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).

[0020] "Cultureability rate" refers to the proportion of environmental microorganisms that can be cultured. Cultureability means that microorganisms can grow in the DL to a cell density detectable by known cell detection methods or detection methods using fluorescent nucleic acid staining dyes used in this disclosure, although this definition is not limited to these.

[0021] 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.

[0022] 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 listed 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 listed above, but examples include FC40, Novec® 7500, mineral oil, or combinations thereof.

[0023] 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 Tween® 20 for the aqueous phase, and Pico-surf® 1 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.

[0024] The present invention relates to a microbial culture method that aims to significantly improve the cultureability rate of environmental microorganisms while maintaining high diversity, and is particularly characterized by the size (average diameter) of droplets contained in a water-in-oil emulsion. The size of the droplets is not limited as long as the above objective can be achieved.

[0025] In this disclosure, when expressed as "average diameter X μm," X represents the size of the droplet in the WODL (Whole Oven Flow Line) that is the target size, i.e., a set value, defined by the width X of the flow channel in the microfluidic device. This may also be referred to as the average diameter (set value). The measured average diameter of droplets in a WODL fabricated according to the embodiments shown in this disclosure will be substantially close to X. Furthermore, it will include droplets with diameters within the tolerance of the device, for example, ±1% to ±10% of the value X expressed herein. The variation in droplet size can be shown by the standard deviation based on the measured droplet diameter.

[0026] Typically, droplets with a diameter of 120-150 μm have been used for the isolation and culture of environmental microorganisms using WODL. Larger droplet sizes offer the advantage of easier creation of uniformly sized droplets, and it was generally accepted that the increased volume of aqueous layer (culture medium) in each droplet facilitates cell proliferation. When reducing droplet size, uniformly encapsulating microorganisms within each droplet requires a significantly higher cell density in the microbial suspension compared to droplets of 120-150 μm diameter, necessitating a concentration process. However, this concentration process also concentrates particles other than microorganisms, leading to the problem of channel blockage, which rarely occurs in the production of 120-150 μm droplets. Therefore, small droplets, or droplets with extremely small diameters like those of the present invention, have rarely been produced. In this disclosure, WODLs with a diameter of 120 or more and less than 150 μm may be referred to as conventional droplets, WODLs with an average diameter of 50 or more and less than 120 μm may be referred to as small droplets, and WODLs with a diameter of 1 or more and less than 50 μm may be referred to as droplets of the present invention.

[0027] According to the present invention, the average diameter X of WODL is preferably a positive number less than 50 μm. For example, X may be 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1. Less than 40 μm is more preferable, and less than 30 μm is particularly preferable. Furthermore, the average diameter may be a range Y to Z rather than a specific numerical value. For example, Y to Z is preferably 1 or more and less than 50 μm (sometimes simply expressed as 1 to 50 μm to avoid complexity), more preferably 1 to 40 μm, and even more preferably 1 to 30 μm. Z is a number greater than Y. In addition, as stated above, ±10% of any of the numerical values ​​is also acceptable as the average diameter. More specifically, for example, an average diameter of 30 μm means a WODL that includes droplets with a diameter of 27-33 μm in the widest range, and an average diameter of 1-50 μm means a WODL that includes droplets with a diameter of 0.9-55 μm in the widest range.

[0028] 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 Co.) used for fabricating WODL and GMD with an average diameter of 30 μm (product code 1003002) • 2D Chip-1060DG (On-chip) 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 preparing WODL with an average diameter of 130-140 μm (product 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)

[0029] This disclosure provides a method for agglomerating and complex culturing of microorganisms to improve the cultureability rate of environmental microorganisms, and can significantly improve the proportion of cultureable microorganisms among environmental microorganisms compared to conventional microorganism culture methods. According to the present invention, the cultureability rate of environmental microorganisms can be increased to, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more compared to conventional methods.

[0030] As used herein, microbial "diversity" generally 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.

[0031] In one embodiment, one or more microorganisms can be encapsulated in a droplet. This allows for the verification of the effects of interactions between microorganisms encapsulated in a single droplet. Alternatively, microorganisms can be encapsulated in droplets at the single-cell level, enabling the isolation of microorganisms exhibiting different growth capabilities from each droplet. In particular, when the goal is to isolate a specific microbial species from a sample containing multiple microorganisms, such as a novel microorganism that has never been isolated before, it is preferable to include microorganisms at the single-cell level within the droplet.

[0032] As long as the droplet contains microorganisms at the single-cell level, the manufacturing method is not restricted. For example, by adjusting the number of microorganisms present in the aqueous phase and the number of droplets, droplets containing single cells can be produced. The microorganisms contained in such droplets are derived from a single cell. Therefore, when a microbial community containing multiple microbial species is used as a sample, selectively separating droplets exhibiting different properties can contribute to the analysis and scaling up of each microorganism.

[0033] The present invention's method for culturing microorganisms includes the step of culturing microorganisms in the droplet prepared as described above. However, those skilled in the art can determine the culturing conditions by appropriately selecting the culture medium, temperature, and culturing period used for culturing general microorganisms.

[0034] Any method known to those skilled in the art can be used to detect microorganisms that have grown in droplets. In particular, as shown in this disclosure, when distinguishing and separating droplets containing grown microorganisms from those that do not by detecting living cells, it is desirable to use a fluorescent cell stain in conjunction with a separation device equipped with a fluorescence detector. As shown in this disclosure, stains other than cell stains can also be used. For example, SYBR® green I is a fluorescent nucleic acid stain that emits fluorescence when it intercepts double-stranded DNA. Because it is amphiphilic, it can be applied to emulsions of fluorine-based oil and aqueous layers, 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 microbial growth evaluation and can detect even slight growth. In addition to the above, methods using SYBR® Gold, GelGreen®, GelRed®, etc. are also preferred.

[0035] The step of culturing microorganisms in the droplets described above may also be a step of culturing the droplets in a "dense state". The term "dense state" refers to a state in which multiple droplets are arranged in close proximity to each other, where the boundaries of each droplet are maintained and the contents (e.g., microorganisms and culture medium) do not mix. In a preferred embodiment, the distance between the centers of the droplets is in the range of 1.0 to 1.5 times the droplet diameter, preferably 1.0 to 1.2 times, and more preferably 1.0. Within this range, the droplets can be densely packed together, but their contents will not mix, and the individual culture environments can be maintained.

[0036] By using the microorganism culture method of the present invention, the isolation and culture efficiency of novel bacteria and difficult-to-culture bacterial groups can be improved, as shown in Example 4 below, and novel microorganisms from the environment that could not be obtained before can also be obtained, as shown in Example 5 below.

[0037] In another embodiment, the present invention relates to a method for isolating and detecting novel microorganisms. In one embodiment, a method for isolating and detecting novel microorganisms, (a) A step of preparing droplets having an average diameter of 1 to 50 μm in a water-in-oil emulsion containing microorganisms, (b) A step of culturing microorganisms in the droplet, (c) A step of detecting microorganisms that have grown in a droplet by adding a fluorescent nucleic acid staining dye to the oil phase of the water-in-oil emulsion, (d) A step of separating droplets stained with a fluorescent nucleic acid stain from droplets that are not stained, (e) A step of isolating microorganisms from the stained droplets obtained in step (d) and extracting nucleic acids from the microorganisms, (f) 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 97.0% or less. It can include...

[0038] For steps (a) and (b) described above, please refer to steps (a) and (b) in the method for culturing the microorganism.

[0039] In step (c), any method known to those skilled in the art can be used to detect the proliferated microorganisms. In particular, when using a separation apparatus equipped with a fluorescence detector, as shown in this disclosure, a method using a fluorescent nucleic acid staining dye (such as SYBR® Green I, SYBR® Gold, GelGreen®, GelRed®, etc.) is preferred. For example, SYBR green I is a nucleic acid fluorescence staining reagent characterized by emitting fluorescence when it intercepts double-stranded DNA. Because it is amphiphilic, it can be applied to emulsions of fluorine-based oil and aqueous layers, making it possible to detect microbial growth in real time while avoiding adverse effects on microbial cells by the staining agent. Since the number of viable 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 is more sensitive than conventional OD (Optical Density)-based microbial growth evaluation and has the advantage of being able to detect even slight growth.

[0040] In the droplet preparation or separation step in step (d), although not particularly limited, methods such as dilution and centrifugation, cell sorters, microfluidic devices, and optical tweezers can be used to separate the droplets while maintaining them as much as possible. The use of microfluidic devices is particularly preferred. At the same time, using a separation device equipped with a detector capable of detecting the staining reagent used in step (c), the droplets can be distinguished and prepared or separated from droplets containing stained microorganisms with high sensitivity by irradiating them with an excitation wavelength specific to the staining dye used and detecting the fluorescence wavelength specific to the fluorescent dye. This can be done using a fluorescence microscope, flow cytometer, or microplate reader.

[0041] Methods for extracting nucleic acids in step (e) are well known to those skilled in the art. When the nucleic acid to be extracted is DNA, the phenol-chloroform method, spin column method, salting-out method, etc., are commonly used. When the nucleic acid to be extracted is RNA, the guanidine thiocyanate method or the phenol-chloroform method can be used.

[0042] Step (f) is a step to determine that the isolated microorganism is a novel microorganism, and includes comparing the base sequence of the nucleic acid of the isolated microorganism with the base sequence of a known species. Generally, it is preferable to use the 16S rRNA gene as the base sequence to be compared. If the base sequence of the 16S rRNA gene of the isolated microorganism has an identity of 97.0% or less, 96.0% or less, 95.0% or less, 94.0% or less, 93.0% or less, 92.0% or less, 91.0% or less, 90.0% or less, 85.0% or less, 80.0% or less, 75.0% or less, 70.0% or less, 65.0% or less, 60.0% or less, 55.0% or less, or 50.0% or less with the base sequence of the 16S rRNA gene of a known species, it can be determined that the isolated microorganism is a novel microorganism.

[0043] Furthermore, the nucleotide sequence information of the 16S rRNA gene derived from the cultured microorganisms obtained in this step (f) can also be used as an indicator of microbial diversity in the culture. Specifically, although not particularly limited, by querying the clustered data obtained by ampicolon sequence variant (ASV) analysis with, for example, the SILVA database (High Quality Ribosomal RNA databases, ver. 138, https: / / www.arb-silva.de / news / view / 2024 / 07 / 11 / silva-release-1382 / ), taxonomic groups can be identified, the bacterial community structure cultured by aggregate complex culture can be determined, and the relative abundance ratio of microorganisms in each sample can be calculated.

[0044] Furthermore, the nucleotide sequence information of the 16S rRNA gene derived from the cultured microorganisms obtained in this step (f) can be used as an indicator of the culturability rate of difficult-to-culture microorganisms present in the environment. While not particularly limited, for example, there is a group of difficult-to-culture bacteria in the environment called acI. The acI-classified phylogenetic groups refer to microorganisms that are difficult to grow on ordinary liquid or agar media. In laboratory settings, they have been reported to grow only in media supplemented with catalase or hemin (Kim S et al., 2020. Journal of Microbiology 58:893-905.; Kim S et al., 2021. Proc Natl Acad Sci 118: e2102750118). Therefore, the detection of a large number of acI-class microorganisms as a result of agglutination complex culture can be considered one indicator of the high culturability rate of that culture method.

[0045] 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]

[0046] [Experimental Example 1: Collection of environmental microorganisms, preparation of WODL, and control experiment] For the experimental example, 300 mL of freshwater was collected from the Sakuragawa River in Tsuchiura City, Ibaraki Prefecture, as a cell sample. The collected water sample was filtered through a 22-25 μm pore size Miracross (Merck, Germany) to remove particles that could clog the flow path during water-in-oil droplet (WODL) preparation, and then filtered again using a 5 μm pore size syringe filter (Sartorius, Germany). The resulting filtrate was further filtered through a 0.2 μm filter (Sartorius) to trap the cells on the filter. The filter containing the trapped microorganisms was removed and placed in a tube containing sterile water. 5 mL of sterile water was added, and the cells were detached by vortexing for 5 minutes. Finally, the filter was removed to prepare a cell suspension.

[0047] Cell suspensions were diluted with Tryptic soy broth liquid medium (BD, USA), diluted 100 times to the specified concentration, so that theoretically one cell would be encapsulated in each WODL, i.e., the average number of encapsulated cells would be one. Subsequently, using an On-chip droplet generator (On-Chip, Japan), WODLs were prepared with the diluted cell suspension and fluorine oil (On-Chip, Japan) to which 5% surfactant 008-FluoroSurfactant had been added, and then adjusted to a surfactant concentration of 2% (W / W) with fluorine oil HFE-7500 (NT Science, Japan), to produce WODLs with an average diameter of 30 μm. The prepared WODLs with an average diameter of 30 μm were transferred to 2 mL Eppendorf tubes and incubated statically at 20°C for 14 days. Similarly, WODLs were prepared with average diameters of 60 μm, 75 μm, 130 μm, and 140 μm, respectively, and were cultured statically at 20°C for 14 days as described above. Furthermore, as a comparative example, cell suspensions were spread onto agar plates (9 cm in diameter) solidified with 1.5% (w / w) agar (Fujifilm Wako Pure Chemical Industries, Japan) using Tryptic soy broth liquid medium diluted 100 times to the specified concentration, and cultured at 20°C for 14 days as described above. In addition, as one of the control groups, cell suspensions were inoculated at 1 / 400th the volume into the diluted Tryptic soy broth liquid medium described above, and cultured statically as described above.

[0048] The measured values ​​of each droplet in WODL, as measured from microscope images, were as follows: for an average diameter of 30 μm: 32.5 μm (26.3~44.2 μm, n=1844, standard deviation=2.0); for an average diameter of 60 μm: 64.8 μm (52.0~70.1 μm, n=683, standard deviation=2.4); for 75 μm: 77.7 μm (71.0~108.6 μm, n=1846, standard deviation=3.2); for 130 μm: 134.8 μm (110.5~150.3 μm, n=669, standard deviation=4.1); and for 140 μm: 141.2 μm (125.9~183.9 μm, n=718, standard deviation=5.4). The percentage of droplets with an error of 10% or more was highest at 13% when the average diameter was 30 μm (Table 1).

[0049] [Table 1]

[0050] [Experimental Example 2: Method for Evaluating Cultureability] The cultureability rate was calculated as follows: After stirring, 20 μL of the cultured WODL sample was transferred to a new 1.5 mL tube, and 1 μL of SYBR® Green I Nucleic Acid Gel Stain (TAKARA, Japan) or SYBR® Gold (Thermo Fisher Scientific) was added and stained at room temperature for 30 minutes. After staining, cultured WODL with an average diameter of 30 μm were observed on slides prepared using 12-well 5 mmφ highly water-repellent printed slides (Matsunami Glass Industry Co., Ltd., Japan) and 18 mm × 18 mm square cover slips (Matsunami Glass Industry Co., Ltd.). WODL with an average diameter of 60 μm or more were observed on microslide 6(VI) flat (ibidi, Germany).

[0051] For the culture experiments using WODLs with different average diameters, fluorescence images of cells stained with SYBR® Green I were randomly taken using an inverted fluorescence microscope (ZEISS, Germany), a 10x objective lens, and a fluorescence filter set (for SYBR Green I) (Figure 1, with average diameters of (A) 140 μm, (B) 130 μm, (C) 75 μm, (D) 60 μm, and (E) 30 μm, respectively). As a result, droplets (DLs) with a large number of growing microorganisms were confirmed, particularly in the experimental group cultured using WODLs with an average diameter of 30 μm.

[0052] Next, to demonstrate that staining can be performed similarly with different nucleic acid fluorescent stains, Figure 2 shows the results of staining microorganisms grown in WODL with the following two nucleic acid fluorescent stains. (A) was stained with SYBR® Green I, and (B) was stained with SYBR® Gold. When microbial cells in WODL were observed under a microscope using fluorescence, droplets in which microorganisms were growing could be confirmed in both (A) SYBR® Green I staining and (B) SYBR® Gold staining.

[0053] After measuring WODLs (Whole Oxide Droplets) exhibiting green fluorescence from SYBR Green I, i.e., WODLs in which microorganisms had grown, the cultureability rate was calculated using a method also used for microwell plates (Journal of Microbiology 58:893-905). Specifically, it was calculated by dividing the number of droplets in which microorganisms had grown by the number of droplets in which microorganisms were detected (%). On the other hand, for the control group cultured on agar medium, the cultureability rate was calculated by dividing the number of colonies by the number of inoculated cells (%). As shown in Figure 3, the average number of WODLs stained with SYBR Green I was approximately 5% (54 stained cells / 1027 inoculated cells) for WODLs with an average diameter of 140 μm, approximately 8% (76 stained cells / 1013 inoculated cells) for WODLs with an average diameter of 130 μm, approximately 14% (313 stained cells / 2242 inoculated cells) for WODLs with an average diameter of 75 μm, approximately 28% (127 stained cells / 458 inoculated cells) for WODLs with an average diameter of 60 μm, and approximately 54% (1408 stained cells / 2616 inoculated cells) for WODLs with an average diameter of 30 μm. Furthermore, when cultured on agar plates, the percentage of bacteria that showed colony formation, i.e., the percentage of cultureable bacteria, was only 0.3% (7.6 × 10⁶). 5 Growing cells / 3.0×10 8 (Inoculated cells)

[0054] Thus, it was revealed that up to 54% of microorganisms could be cultured when cultured in WODL with an average diameter of 30 μm. This figure is approximately 1.9 times (compared to an average diameter of 60 μm) to approximately 10.8 times (compared to an average diameter of 140 μm) higher than when environmental microorganisms were cultured under the same conditions in WODL of 140 μm, 130 μm, 75 μm, and 60 μm. Furthermore, the culture rate in the conventional method of spreading environmental microorganisms on agar plates was only 0.3%, making this figure approximately 180 times higher. From these results, it was shown that it is possible to significantly increase the culture rate by culturing microorganisms in smaller WODL, i.e., in high-density WODL (Figure 3).

[0055] Furthermore, surprisingly, although WODL cultures were thought to have a lower culture success rate due to the small amount of culture medium components, reducing the average diameter unexpectedly improved the culture success rate of environmentally derived microorganisms, demonstrating that a wider variety of microorganisms could be isolated by WODL. In particular, WODLs with an average diameter of 30 μm resulted in approximately 45% more cultured WODLs compared to commonly used WODs (average diameter of 120-140 μm).

[0056] [Experimental Example 3: Analysis of Microbial Community Structure] Similar to Experimental Example 1, cell suspensions derived from freshwater environmental samples were prepared. Also, similar to Experimental Example 1, WODLs were prepared using an on-chip droplet generator, transferred to 2 mL tubes, and incubated statically at 20°C for 28 days. As a control experiment, 9.6 × 10⁶ cells were cultured in a 1 / 100-fold diluted Tryptic soy broth. 5One mL of a suspension adjusted to cells / mL was transferred to a two mL tube and cultured statically at 20°C for 28 days. After culturing, 500 μL of cells from each WODL and liquid medium with different average diameters were destroyed along with the WODL by bead beating using the Extra Soil DNA Kit Plus ver.2 (BDL, Japan). After separating the aqueous and oil layers by centrifugation, 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, MiSeq, 63,000-85,000 reads). From the obtained read data, low-quality sequences and chimeric sequences were removed using the QIIME2 pipeline (ver.2022.8), and the data was clustered into ampiclon sequence variants (ASVs). These ASVs are used as one of the indicators of species diversity. The diversity of the microbial community in each sample was calculated, and each clustered ASV was queried against the SILVA database (ver. 138) to identify the taxonomic group, thereby determining the bacterial community structure and calculating the relative abundance of microorganisms in each sample.

[0057] Figure 4 shows the results of the bacterial community structure analysis. The number of ASV species in each sample was 192-300 for WODLs with an average diameter of 140 μm and 392-444 for WODLs with an average diameter of 30 μm. Thus, the diversity of microorganisms grown in WODLs was best maintained when cultured in WODLs with an average diameter of 30 μm, with the number of species being up to 2.2 times that of WODLs with an average diameter of 140 μm. In contrast, the liquid culture prepared as a control experiment showed only 24-32 species, and the number of species in this culture was up to 18 times higher. These results indicate that culturing microorganisms in smaller WODLs, i.e., in high-density WODLs, allows for the growth of microorganisms while maintaining higher diversity than in normal WODL cultures.

[0058] Compared to conventional liquid culture, the nutrient sources in droplets, especially small droplets, are limited and are likely to be depleted as cells proliferate during culture. In fact, a previously reported study showed that when Candida pseudotropicalis, a fungus, was cultured in WODL, protein depletion occurred within 0.5 to 1 day (Pfammatter N. et al., 1992, Biotechnology and Bioengineering 40:167-172). Therefore, in this study, it was expected that only a small number of microorganisms capable of growing even with low nutrition levels would proliferate in WODL, resulting in a low ASV count, i.e., low diversity, in the microbial community. However, contrary to this expectation, the WODL with the smallest volume and lowest nutrient content, with an average diameter of 30 μm, had the highest ASV count. At 28 days of culture, this value was approximately 15 times that of liquid culture and approximately 2.2 times that of WODL with an average diameter of 140 μm, indicating that reducing the average diameter allows for the cultivation and maintenance of diverse microorganisms from the environment.

[0059] [Experimental Example 4: Detection of systematically novel bacterial species and bacterial groups belonging to the difficult-to-culture bacterial community] Among the clustered ASVs, those showing a relative abundance of 0.1% or more in each sample were re-analyzed using Ezbiocloud (https: / / www.ezbiocloud.net / ) to compare their nucleotide sequence identity with known bacterial species. According to a general definition, a novel bacterium was defined as one in which the nucleotide sequence of the 16S rRNA gene of a strain isolated from the environment showed 97.0% or less identity with the species having the closest nucleotide sequence (Stackebrandt E, Goebel BM. 1994. Microbiology 44:846-849). Furthermore, the results of bacterial community structure analysis suggested the distribution of the acI lineage (Candidatus Nanopelagicales) of the Actinobacteriota phylum, known as a difficult-to-culture bacterial group. Furthermore, in general microbial community structure analysis, the Silva database, which is used for phylogenetic estimation, sometimes failed to assign ASVs belonging to this microbial lineage to the appropriate lineage. Therefore, for ASVs classified under the phylum Actinobacteriota, we re-analyzed them using Ezbiocloud, which allows for more detailed phylogenetic analysis, to identify the lineages and calculate their relative abundance.

[0060] Figure 5 shows the relative abundance of novel bacteria among the bacterial species that could be isolated. The relative abundance of novel bacteria in each sample with different average WODL diameters was approximately 6% for WODLs with an average diameter of 140 μm and approximately 26% for WODLs with an average diameter of 30 μm. This was approximately 4.3 times higher than that of WODLs of commonly used sizes. On the other hand, in the control experiment, no novel bacteria were obtained (0%) when cultured in liquid medium. These results indicate that culturing environmental microorganisms at high density in small-sized WODLs not only improves the cultureability of environmental microorganisms but also significantly improves the cultureability of novel bacteria.

[0061] Next, Figure 6 shows the relative abundance of difficult-to-culture bacteria (acI) among the bacterial species that could be isolated. The relative abundance of difficult-to-culture bacteria in each sample with different average WODL diameters was 5% for WODL with an average diameter of 140 μm and 22% for WODL with an average diameter of 30 μm. This was approximately 4.4 times higher than that of WODL of commonly used sizes. On the other hand, in the control experiment, no difficult-to-culture bacteria (acI) were obtained when cultured in liquid medium (0%). These results indicate that culturing environmental microorganisms in high-density WODLs of smaller size can significantly improve the culture rate of difficult-to-culture bacteria (acI).

[0062] [Example 5: Isolation and identification of novel environmental microorganisms] Similar to Experimental Example 1, a cell suspension derived from a freshwater environmental sample was prepared. Also, similar to Experimental Example 1, WODL with an average diameter of 30 μm was prepared using an On-chip droplet generator and transferred to a 2 mL tube for static incubation at 20°C for 28 days. After incubation, the microorganisms that had grown in the WODL were stained with SYBR Green I Nucleic Acid Gel Stain, similar to Experimental Example 2. After staining, 50 μL of the WODL was placed in the sample reservoir of the analysis chip 2D Chip-SD1000 (On-Chip Co., Ltd.), then fluorine oil with 0.1% 008-FluoroSurfactant added was placed in the sheath fluid reservoir, and the WODL was dispersed in the oil by air pressure from the On-chip selector (On-Chip Co., Ltd.) and passed through the flow path (80 μm × 80 μm) of the analysis chip. During this process, the intensity of green fluorescence from each droplet of numerous WODLs flowing through the channel was measured using an excitation laser (488 nm) attached to an on-chip selector and a photomultiplier tube (543 ± 22 nm) for green fluorescence detection. Subsequently, regions with high green fluorescence, i.e., WODLs where environmental microorganisms were proliferating (500 a.u. or higher), were gated, and the WODLs within these regions were separated one droplet at a time. The separated WODLs were then dropped onto a 96-well flat-bottom plate (Corning) filled with 180 μL of Tryptic soy broth liquid medium, diluted 100 times to the specified concentration and solidified with 1.5% (w / w) agar, at a rate of one droplet per well. The plates were then incubated at 20-25°C for 14 days. After culturing, the formed colonies were isolated, and PCR targeting the 16S rRNA gene was performed. The nucleic acids amplified according to standard procedures were sequenced by Sanger sequencing (3730xl DNA analyzer, Applied Biosystems). Subsequently, the determined gene sequences were subjected to phylogenetic analysis using the same method as in Experimental Example 4.

[0063] As a result, microbial colony formation was confirmed on the well plate (Figure 7, showing two different examples). Furthermore, the proportion of WODL containing microorganisms that proliferated in the above experiment was 58% (167 droplets / 288 droplets) of the separated WODL. Analysis of the 16S rRNA gene of some of the bacteria derived from individually separated droplets revealed nine novel microorganisms with a nucleotide sequence identity of 97.0% or less with the 16S rRNA gene of known microorganisms (Table 2). These results demonstrate that novel microorganisms in the environment can be easily isolated and detected using the method based on this disclosure.

[0064] [Table 2] [Industrial applicability]

[0065] The present invention's high-density culture method for microorganisms using droplets makes it possible to maintain high microbial diversity while achieving a high culture rate of environmental microorganisms. Furthermore, it makes it possible to improve the efficiency of systematically isolating and culturing novel bacterial species and difficult-to-culture bacterial groups, which have been challenging until now. Such a microbial culture method is expected to make a significant contribution not only to microbiology but also to the field of drug discovery.

[0066] 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 method for culturing microorganisms to improve the cultureability rate, (a) A step of preparing droplets containing microorganisms having an average diameter of 1 to 50 μm in an oil-water emulsion, (b) A step of culturing microorganisms in the droplet and A method for culturing microorganisms, including [the specified term].

2. The method for culturing microorganisms according to claim 1, further comprising the step of adjusting the droplet so that it contains microorganisms on a single-cell basis in step (a).

3. The method for culturing microorganisms according to claim 1 or 2, wherein the microorganism is a microorganism collected from the environment.

4. The method for culturing microorganisms according to claim 1 or 2, wherein the microorganisms include microorganisms that cannot be cultured on nutrient agar medium.

5. The method for culturing microorganisms according to claim 1 or 2, wherein the average diameter of the droplets is 1 to 30 μm.

6. The method for culturing microorganisms according to claim 1 or 2, wherein step (b) is a step of culturing droplets in a dense state.

7. A method for isolating and detecting novel microorganisms, (a) A step of preparing droplets having an average diameter of 1 to 50 μm in a water-in-oil emulsion containing microorganisms, (b) A step of culturing microorganisms in the droplet, (c) 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, (d) A step of separating droplets stained with a fluorescent nucleic acid stain from droplets that are not stained, (e) A step of isolating microorganisms from the stained droplets obtained in step (d) and extracting nucleic acids from the microorganisms, (f) 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 97.0% or less. A separation and detection method, including

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