JP4822689B2 - Microorganism isolation apparatus, microorganism isolation method, and microorganism-containing microparticles obtained by the method - Google Patents

Description

  The present invention relates to a microorganism isolation apparatus, a microorganism isolation method, and a microorganism-containing microparticle obtained by the method. More specifically, the present invention relates to a microorganism isolation device that can isolate microorganisms that exist in nature, particularly microorganisms that survive but are difficult to cultivate; microorganisms that can isolate microorganisms in order to individually evaluate many types of microorganisms. And a microbe isolation method; and microparticles containing any number, preferably substantially one, of such microorganisms.

  Conventionally, a plate culture method is widely known as a method for separating and culturing microorganisms from nature. In the plate culture method, a single colony or single cell of a microorganism is obtained by seeding a sample mixed with a wide variety of microorganisms on a plate medium (plate) and isolating colonies formed on the plate after culturing for a certain period of time. It is a method of separation. According to the plate culture method, a microorganism that meets the purpose is usually searched from isolated microorganisms (colony). Based on this plate culture method, various methods have been developed, and many industrially useful microorganisms have been separated and used.

  In the late 1970s, a method for counting the total number of bacteria in a separation source using a fluorescence microscope was established. As a result, techniques for counting a wide variety of microorganisms have been developed. On the other hand, a technique for detecting a biological activity caused by a microorganism in a natural separation source by a fluorescent dye staining method has been developed. Through these technological developments, it has been elucidated that there are many microorganisms in nature that cannot be cultured by the above plate culture method. That is, it has been clarified in nature that there are a group of microorganisms that can be cultured in a normal nutrient medium and a group of microorganisms that exhibit biological activity but are difficult to culture. A group of microorganisms that exhibit such biological activity but are difficult to culture has become known as VBNC-like microorganisms (living microorganisms that are difficult to culture).

  Among the above-mentioned VBNC-like microorganisms, the existence of unknown microbial species (new microbial species) can be assumed. Furthermore, the presence of unknown microbial species can be assumed in extreme environments such as the deep sea, or in living organisms such as higher animals such as humans, insects, soil protozoa or marine organisms, or in artificial environments such as activated sludge. .

  In addition, since a VBNC-like microorganism is a living cell, a DNA gene is present therein. Therefore, it can be assumed that an unknown gene having a function not yet known exists in the DNA gene of the VBNC-like microorganism. Such unknown genes may be useful in the food industry, pharmaceutical industry, environmental conservation such as water quality, medical care, and the like.

  However, as described above, since VBNC-like microorganisms cannot be cultured by the plate culture method, such microorganisms (in some cases, their genes) cannot be isolated.

  In order to solve such a problem, a method called a gel microdrop method has been proposed as a method for separating only VBNC-like microorganisms from a microbial resource in which normal microorganisms and VBNC-like microorganisms are mixed (see Patent Document 1). ). In this method, a sample solution containing microorganisms is passed through a porous membrane having a pore diameter of about 15 μm, and W / O emulsion is formed to form gel microparticles (gel microdrops) having a diameter of about 40 μm. This gel microdrop population is placed in a growth environment (according to Patent Document 1, normal microorganisms grow in the gel and the gel contains a plurality of cells, whereas VBNC-like microorganisms do not grow, so the cells in the gel It is supposed to remain one). Finally, gel microparticles (VBNC-modified microorganisms) having one microbial cell are identified and separated by a cell sorter by flow cytometry. In this way, it is said that VBNC-like microorganisms can be separated.

However, since this method uses a porous film (a film having a so-called pumice-like pore structure) for the generation of gel fine particles, the particle size of the obtained fine particles is very uneven. Furthermore, the average diameter of the pores is as large as about 15 μm, and the average diameter of the resulting fine particles is as large as about 40 μm. As a result, the number of microorganisms (cells) contained in one fine particle becomes uneven, and the separation efficiency is extremely insufficient.
JP 2000-197479 A

  The present invention has been made to solve the above-described conventional problems, and the object of the present invention is to provide a microorganism isolation apparatus and microorganism capable of isolating VBNC-like microorganisms and / or known microorganisms with very high efficiency. It is in providing the isolation method of this.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have applied a pulsed pressure to the microchannel through which the raw material liquid containing a predetermined microorganism flows, and pulsated the raw material liquid to join the confluence portion of the microchannel It was found that the above-mentioned object can be achieved by generating a fine particle by instantaneously causing a physical process and / or a chemical process at the joining portion to complete the present invention.

  The microorganism isolation apparatus of the present invention includes at least one microorganism supply unit that contains a microorganism supply solution containing a predetermined microorganism, and a contact solution that contains a contact solution that contacts the microorganism supply solution to generate fine particles. A liquid supply section; at least one microorganism supply flow path extending from the at least one microorganism supply section and through which the microorganism supply liquid passes; and a contact liquid supply flow extending from the contact liquid supply section and through which the contact liquid passes. A microparticle generator that forms the microparticles by bringing the microbe supply liquid and the contact liquid into contact with each other, the path; and at least one microbe supply path and the contact liquid supply path The generated microparticles include a predetermined number of the predetermined microorganisms in the microparticles.

  In a preferred embodiment, the apparatus includes one microorganism supply unit containing a microorganism supply liquid containing a predetermined microorganism; and a contact liquid supply containing a contact liquid that contacts the microorganism supply liquid to generate fine particles. A microorganism supply passage extending from the microorganism supply section and through which the microorganism supply liquid passes; a contact liquid supply passage extending from the contact liquid supply section and through which the contact liquid passes; and the microorganism supply flow path and the contact A liquid supply flow path is formed to join the microorganism supply liquid and the contact liquid to generate fine particles, and the generated fine particles are contained in the predetermined fine particles in the fine particles. A predetermined number of each of the microorganisms.

  In another preferred embodiment, the apparatus contains a first microorganism supply section containing a first microorganism supply liquid containing a first microorganism; and a second microorganism supply liquid containing a second microorganism. A second microorganism supply section; a contact liquid supply section containing a contact liquid that is brought into contact with the first and second microorganism supply liquids to produce fine particles; and extends from the first microorganism supply section; A first microorganism supply flow path through which the first microorganism supply liquid passes; a second microorganism supply flow path extending from the second microorganism supply section and through which the second microorganism supply liquid passes; and the contact liquid supply A contact liquid supply channel extending from the portion through which the contact liquid passes; and the first and second microorganism supply channels and the contact liquid supply channel are joined together to form the first and second microorganisms. A fine particle generation unit that generates fine particles by bringing the supply liquid and the contact liquid into contact with each other. That fine particles, each containing a predetermined number of said predetermined microorganism in the microparticles.

  In a preferred embodiment, the produced microparticles contain substantially only one microorganism each in the microparticles.

  In a preferred embodiment, the contact liquid supply channel has a cross-sectional area larger than that of the microorganism supply channel.

  According to another aspect of the present invention, a method for isolating a microorganism is provided. The method comprises the steps of: preparing a microorganism supply liquid containing a predetermined microorganism; preparing a contact liquid that is brought into contact with the microorganism supply liquid to generate fine particles; and contacting the microorganism supply liquid and the contact liquid. Producing fine particles, and the produced fine particles contain a predetermined number of the predetermined microorganisms in the fine particles.

  In a preferred embodiment, the volume of the fine particles produced is 0.5 to 500 picoliters.

  In a preferred embodiment, the microorganism feed solution includes a gelling material.

In a preferred embodiment, the concentration of the predetermined microorganism in the microorganism supply solution is 2 × 10 ⁶ to 2 × 10 ⁹ cells / ml.

  In a preferred embodiment, the contact liquid includes a gelation initiating component.

  In a preferred embodiment, the predetermined microorganism is at least one selected from the group consisting of eubacteria, archaea, yeasts and genetic recombinants.

  According to still another aspect of the present invention, microorganism-containing microparticles are provided. The microorganism-containing fine particles are obtained by the above method.

  According to the present invention, a very small size of microparticles can be generated by contacting a micro amount of a microbe supply liquid in a microscopic area with a contact liquid that can generate microparticles by contacting the microbe supply liquid. . By adjusting the concentration of the microorganism supply solution, substantially one microorganism can be contained in the minute amount of the supply solution. Therefore, substantially one microorganism can be contained in the generated fine particles.

  Preferred embodiments of the present invention will be described with reference to the drawings, but the present invention is not limited to these embodiments.

A. Microorganism Isolation Device FIG. 1 is a schematic perspective view illustrating the overall configuration of a microorganism isolation device according to a preferred embodiment of the present invention. For the sake of simplicity, the illustrated example will be described in the case of having a single microorganism supply unit, but it goes without saying that the present invention can be applied to a case of having two or more microorganism supply units (two or more microorganisms). In the case of having a supply unit, only the characteristic part will be briefly described later). A microorganism isolation device 100 in FIG. 1 extends from a microorganism supply unit 1a formed on a substrate 10, a contact liquid supply unit 1b, a microorganism supply channel 2a extending from the microorganism supply unit 1a, and a contact liquid supply unit 1b. The contact liquid supply flow path 2b, and the microparticle production | generation part 5 formed by the microorganisms supply flow path 2a and the contact liquid supply flow path 2b joining are provided. The microorganism supply liquid contained in the microorganism supply unit 1a is supplied to the fine particle generation unit 5 through the microorganism supply channel 2a. Similarly, the contact liquid contained in the contact liquid supply unit 1b is supplied to the fine particle generation unit 5 through the contact liquid supply channel 2b. In the fine particle production | generation part 5, a microparticle is produced | generated by producing a physical process and / or a chemical process between microorganisms supply liquid and a contact liquid. A typical example of a physical process is phase separation, and a typical example of a chemical process is a chemical reaction (eg, oxidation, reduction, salt formation). Note that the liquid supply mechanism in this apparatus is an application of the liquid mixing mechanism described in Japanese Patent Application Laid-Open No. 2003-220322, and the disclosure of the publication is incorporated herein by reference.

  The external dimensions of such an apparatus 100 are typically about 25 mm × 30 mm × 0.5 mm. Preferably, the contact liquid supply channel 2b has a cross-sectional area larger than that of the microorganism supply channel 2a. By adopting such a configuration, the supply amount of each microorganism supply liquid can be made extremely small, and as a result, the probability that fine particles containing only one microorganism (cell) can be generated is remarkable. Become bigger. Specifically, the width of the microorganism supply channel 2a is typically 50 to 200 μm, and the depth is typically 30 to 120 μm. The width of the contact liquid supply channel 2b is typically 100 to 500 μm, and the depth is typically 50 to 500 μm. The fine particle generation unit 5 substantially extends continuously from the contact liquid supply flow channel 2b, and its width is typically 100 to 500 μm and the depth is typically 50 to 500 μm. The ratio 2b / 2a of the cross-sectional area of the contact liquid supply channel 2b to the microorganism supply channel 2a is preferably 2 to 50. Preferably, the width of the microorganism supply channel 2a is narrow in the portion immediately before joining the contact liquid supply channel 2b. The width of the microorganism supply channel 2a immediately before the merge is typically 10 to 50 μm. The microorganism supply channel 2a may be narrowed in a tapered shape or may be narrowed in stages. It is one of the features of the present invention that fine particles are produced by contacting a minute amount of a microorganism supply solution with a contact solution in such a minute region. By contacting a minute amount of the microorganism supply solution, it is possible to produce particles of extremely small size, which has been difficult in the past. As a result, the number of microorganisms (cells) taken up by the fine particles can be made very small (typically, only one is taken up). In the present invention, the substrate 10 is not particularly limited, and any appropriate substrate can be adopted. Typically, a silicon wafer is used. Formation of the flow path and the like on the substrate 10 is performed by any appropriate fine processing technique (for example, photolithography).

The apparatus 100 includes pressure generation mechanisms 3a and 3b in the microorganism supply channel 2a and the contact liquid supply channel 2b, respectively. Any appropriate device can be adopted as the pressure generating mechanism, and is typically a micropump (preferably a diffuser type micropump). FIG. 2 is a schematic cross-sectional view of the diffuser type micro pump in the flow path direction. The micropump 3 a is formed by attaching PZT [Pb (Zr, Ti) O ₃ ] 33, which is a ceramic piezoelectric material, to a diaphragm 32 that faces the pump chamber 31. When a drive voltage is applied by the drive unit 34, the PZT 33 is bent toward the pump chamber 31, the volume of the pump chamber 31 varies, and pressure is generated. By applying a pulsed drive voltage, a pulsed pressure corresponding to the drive voltage is generated. When pressure is generated, the raw material liquid is fed due to the difference in flow path impedance between the front and rear diffusers 35 and 36. A constant driving voltage is applied to the micropump 3b, and a constant pressure corresponding to the driving voltage is generated. As a result, a certain amount of contact liquid can be continuously supplied. As means for continuously flowing a certain amount of contact liquid into the contact liquid supply channel 2b, a syringe pump or the like may be connected to the contact liquid supply unit 1b instead of the micropump 3b.

  The pulse shape of the pressure can be appropriately set according to the purpose. Typically, the pulse shape can be set independently for each channel. Specifically, as shown in FIG. 3A, a predetermined pulse pressure is applied to the microorganism supply flow path 2a. As a result, the microorganism supply liquid is intermittently supplied to the fine particle generation unit. As described above and as shown in FIG. 3A, a constant pressure is applied to the contact liquid supply channel 2b. As a result, a certain amount of contact liquid is continuously supplied to the fine particle generation unit. Needless to say, any appropriate pulsed pressure may be applied to the contact liquid supply channel 2b in accordance with the purpose to intermittently supply the contact liquid to the fine particle generation unit.

Particularly preferably, the pulse shape of the pressure corresponds to a large pressure portion (droplet forming pulse) corresponding to the case where the microorganism supply liquid is supplied to the particle generation unit and the case where the microorganism supply liquid is not supplied to the particle generation unit. With a small pressure portion (backflow prevention pulse). That is, as shown in FIG. 3B, a small pressure (backflow prevention pulse) is generated when the microorganism supply liquid is not supplied to the fine particle generation unit. By generating such a small pressure, it is possible to prevent a back flow from the fine particle generation unit 5 to the microorganism supply channel 2a. The small pressure can be appropriately set according to the type of the microorganism supply liquid to be used. For example, the ratio P _L / P _S between the pressure P _L of the large pressure portion and the pressure P _S of the small pressure portion is in the range of ½ to 1/100. The applied voltage for generating a large pressure portion (droplet formation pulse) is typically 5 to 40 V, and the applied voltage for generating a small pressure portion (backflow prevention pulse) is typically 1 to 10 V. . According to the present invention, the switching cycle of the supply of the microorganism supply liquid can be very short as will be described later. However, if the switching cycle is shortened, backflow is likely to occur, and the accuracy of fine particle generation is greatly adversely affected. Therefore, the realization of such a backflow prevention means is extremely important for the production of ultrafine particles, and is one of the great achievements of the present invention. Of course, in addition to the shape as shown in FIG. 3, it is possible to change the supply amount and supply interval of the microorganism supply liquid in the fine particle generation unit by generating a pulse of any appropriate shape according to the purpose. .

  Referring to FIG. 3 (a), the frequency of the droplet forming pulse S in the pulse is preferably 1 to 50 kHz, more preferably 8 to 13 kHz. The pulse switching period is preferably 10 Hz to 10 kHz, and more preferably 100 Hz to 5 kHz. It is one of the features of the present invention that such a very short switching cycle can be achieved (as a result, the microorganism supply liquid can be sent to the fine particle generation unit with a very small supply amount). This is because the drive element is very short, so that the resonance frequency can be greatly increased. By supplying the fine particle supply liquid to the fine particle generation unit with a very short switching cycle (with a very small supply amount), the scale of the contact space or the reaction space becomes very small. As a result, fine particles having a very small and very sharp particle size distribution can be produced. As a result, the statistical accuracy of including a predetermined number of microorganisms in the fine particles is remarkably increased. In addition, since the chance of contact between the fine particle supply liquid and the contact liquid can be greatly increased, productivity is extremely high. In addition, since the apparatus scale (reaction scale) is extremely small, it is possible to produce fine particles with extremely small power consumption.

FIG. 4 is a schematic diagram for explaining the mechanism of the fine particle formation process in the merging portion of the microorganism supply flow channel 2a and the contact liquid supply flow channel 2b (the most upstream portion of the fine particle generation unit 5). In accordance with the pulse shape of the pressure, a mass (layer) 41 of the microorganism supply liquid is supplied to the flow 42 of the contact liquid. By appropriately selecting the solvent (dispersion medium) of the microorganism supply liquid and the contact liquid, the mass (layer) 41 of the microorganism supply liquid forms gel-like fine particles 43 at the moment of contact with the flow 42 of the contact liquid (note that Details of the microorganism supply liquid and the contact liquid will be described in Section B below). The gel fine particles 43 move downstream along the flow of the contact liquid. For example, if the microorganism concentration in the microorganism supply liquid is set to 1 × 10 ⁸ to 2 × 10 ⁹ cells / ml and the supply amount of the microorganism supply liquid per pulse is set to 0.5 to 10 picoliters, the microorganism supply One microorganism is substantially contained per one pulse of the liquid. In the present specification, “substantially including one microorganism” means that the microorganism can proliferate and grow substantially without being affected by other microorganisms of the same type and / or other types of microorganisms. It means that it is present in the supply liquid or generated fine particles in one pulse. When the supply liquid for one pulse comes into contact with the contact liquid, gel-like fine particles having an average particle diameter of about 10 to 27 μm are obtained. Since the concentration distribution and degree of contact of the microorganism supply liquid are extremely uniform at the liquid-liquid interface between the microorganism supply liquid and the contact liquid, fine particles having a very sharp particle size distribution can be obtained. The CV value of the obtained fine particles (which is an index of particle size distribution and is represented by standard deviation / average particle size × 100 (%)) is typically 2% to 20%, preferably 5% to 15%. is there. By forming microparticles having a very sharp particle size distribution, the statistical probability that substantially one microbe exists in each microparticle is greatly increased.

Preferably, the fine particle generation unit 5 is provided with a temperature control unit (not shown). The temperature control unit is configured by any appropriate temperature control means. A typical temperature control means is formed by forming TaSiO ₂ or Ta ₂ N as a thin film resistor on the back side of the flow path substrate 10. In addition, when Si is used for the flow path substrate, an impurity-doped Si resistor can be used. The method of temperature control can be appropriately set according to the purpose. For example, the entire fine particle generation unit 5 may be controlled to a constant temperature, a positive (or negative) temperature gradient may be formed from upstream to downstream, and only an arbitrary part of the fine particle generation unit 5 is heated to a high temperature. (Or low temperature) may be controlled. In this way, the fine particle production process can be controlled precisely and satisfactorily.

  Preferably, a particulate collection unit 6 is provided downstream of the particulate generation unit 5. The fine particle collection unit 6 collects (collects) fine particles using the size and / or physical characteristics of the fine particles. Examples of means for utilizing the size of the fine particles include filter means (for example, holes and pillars) previously processed in the flow path, and a centrifugal separation mechanism. Examples of means for utilizing the physical characteristics of the fine particles include means for utilizing a dielectric constant or electric charge. Specifically, collection is performed by dielectrophoresis, electrophoresis, or the like using an electrode pattern, a water repellent / hydrophilic pattern, or the like previously processed in the flow path.

FIG. 5 is a schematic plan view illustrating the channel shape of a microorganism isolation device according to another embodiment of the present invention. This embodiment is a case where two microorganism supply channels 2a and 2c are formed. When the flow path as shown in FIG. 5 is used, a first microorganism supply liquid containing microorganisms α is placed in the first microorganism supply unit 1a, and a second microorganism containing β is contained in the second microorganism supply unit 1c. By adding the microorganism supply solution, microparticles containing substantially one microorganism α and one microorganism β can be formed. FIG. 6 is a schematic diagram for explaining the mechanism of the fine particle formation process at the junction of the microorganism supply channel and the contact liquid supply channel in such an embodiment. In accordance with the pulse shape of the pressure applied to the pressure generating mechanism, the first microorganism supply liquid mass (layer) 61 is supplied to the microorganism supply flow path 2a. By adjusting the concentration of the first microorganism supply liquid and the supply amount per pulse, the mass (layer) 61 substantially contains one microorganism α. Similarly, a mass (layer) 64 of the second microorganism supply liquid is supplied to the microorganism supply channel 2c. By adjusting the concentration of the second microorganism supply liquid and the supply amount per pulse, the mass (layer) 64 substantially contains one microorganism β. The lump (layer) 61 and the lump (layer) 64 merge at the junction of the microorganism supply channels 2a and 2c to form a lump (layer) 65. By adjusting the timing of the pulses, the lump (layer) 61 and the lump (layer) 64 can be appropriately merged. The lump (layer) 65 forms gel-like fine particles 63 at the moment of contact with the flow 62 of the contact liquid. The gel-like fine particles 63 move downstream along the flow of the contact liquid. For example, the concentration of the microorganism α in the first microorganism supply liquid is set to 1 × 10 ⁸ to 2 × 10 ⁹ cells / ml, and the supply amount of the microorganism supply liquid per pulse is set to 0.5 to 10 picoliters. Then, one microorganism α is substantially included in one lump (layer) 61. Similarly, substantially one microorganism β can be included per lump (layer) 64. As a result, a lump (layer) 65 containing substantially one microorganism α and one microorganism β is formed. Finally, gel-like fine particles 63 containing one microorganism α and one microorganism β are finally formed. Can be generated. In this case, the volume of the lump (layer) 65 is 1.0 to 20 picoliters, and the average particle size of the obtained fine particles is about 12 to 34 μm. By controlling the concentration of the microorganism supply solution and the supply amount per pulse, the number of microorganisms contained in the fine particles on the gel can be appropriately set according to the purpose. For example, any combination such as (α1, β2), (α2, β1), (α2, β2), (α3, β2), (α2, β3) is adopted. obtain. Needless to say, an embodiment having three or more microorganism supply units may be employed depending on the purpose. Also, any suitable form may be adopted as the joining form of the two or more microorganism supply channels depending on the purpose.

B. Next, a preferable example of the microorganism isolation method of the present invention will be described. For simplicity, only the case of isolating one type of microorganism will be described.

First, a microorganism supply solution containing predetermined microorganisms is prepared. The microorganism supply liquid is prepared by dispersing microorganisms in a predetermined liquid. A dispersant (for example, a surfactant) or the like can be added to the liquid as necessary. This process is carried out according to the same procedure as before. An example is as follows. Collect a source (eg, soil). It is said that 10 ⁵ to 10 ⁹ microorganisms are present in 1 g of soil. This microorganism concentration is the basis for determining the concentration of the microorganism supply solution and the supply amount per pulse. The collected soil is suspended in water, and impurities are removed by physical means (typically, a filter). Next, the suspension from which impurities have been removed is diluted in multiple stages to a predetermined concentration using an appropriate liquid according to the purpose to prepare a microorganism supply solution. Specific examples of the liquid for dispersing the microorganism include alginic acid. When alginic acid is used, the microorganism concentration in the microorganism supply solution can be preferably set to 2 × 10 ⁶ to 2 × 10 ⁹ cells / ml. In this case, the viscosity of the microorganism supply solution is typically 1 to 3 cps. Preferably, the microorganism supply liquid contains a gelling material. In the present specification, the gelling material refers to a material that can form a gel by physical and / or chemical stimulation from the outside. Specific examples of the gelling material include alginic acid, carrageenan, pectin, cellulose, cyanoacrylate, polysaccharide amino acid carbamate derivative, polyvinyl alcohol, sodium silicate, alkyl group-containing silica matrix and the like.

  Next, a contact liquid is prepared. As the contact liquid, any appropriate liquid capable of generating fine particles (typically, gel-like fine particles) in contact with the microorganism supply liquid can be adopted. Typically, the contact liquid includes a gelation initiating component. In the present specification, the gelation initiating component refers to a substance that causes gelation of the gelled material by physical and / or chemical stimulation. A specific example of the gelation initiation component is an aqueous calcium chloride solution. When using an aqueous calcium chloride solution, the viscosity is typically 1 to 3 cps.

  Next, an apparatus as shown in FIG. 1 is used. The microorganism supply liquid is put into the microorganism supply part 1a, and the contact liquid is put into the contact liquid supply part 1b. A predetermined voltage (10 KHz, 10 V) is applied to the microorganism supply channel 2a by a micropump to generate a pulsed pressure (droplet formation pulse). By generating such a pressure, the supply amount of the microorganism supply liquid per pulse can be set to 0.5 to 10 picoliters. In this way, substantially one microorganism can be contained in the microorganism supply liquid for each pulse. On the other hand, rectification is generated in the contact liquid supply channel 2b at a flow rate of, for example, 50 nl / s, and the contact liquid is allowed to flow continuously at a constant amount.

  Next, the microorganism supply liquid and the contact liquid are brought into contact with each other at the junction of the microorganism supply flow path 2a and the contact liquid supply flow path 2b. Then, gel-like fine particles are generated by the mechanism shown in FIG. As described above, since one microorganism is substantially contained in the microorganism supply solution for each pulse, substantially one microorganism is also contained in the obtained fine particles. By controlling the concentration of the microorganism supply solution and the supply amount per pulse, any appropriate number of microorganisms can be included in the microparticles.

  The microparticles obtained in this way are collected and propagated and / or grown in a predetermined environment. By doing in this way, the possibility that VBNC-like microorganisms can be isolated can be dramatically increased. Further, with respect to known microorganisms, the culture efficiency can be remarkably increased by eliminating the influence of other microorganisms.

Specific examples of known microorganisms that can be used in the present invention include natural environments such as soil and rivers, living space environments, eubacteria, archaea, yeasts, and recombinants present in the digestive tract. Specific examples of eubacteria include Rhodococcus, Norcardia, Alcaligenes, Lactococcus, Acetobacter, Pseudomonas, Sphingomonas, Corynebacterium, Synechococcus, Botryococcus, Chlamydomonas, Zymomonus, Methanosarcina, Methanosarcina,
Examples include genus, Bachillus genus, Gordonia genus, Actinobacteria genus and actinomycetes. Specific examples of yeast include Candida genus and Cryptococcus genus. As a specific example of the gene recombinant, Escherichia
coli and Bachillus subtilis. Fine particles containing a predetermined number (typically, substantially one) of these can be used very suitably for various bioreactors. This is because according to such fine particles, the diffusion in the particles is fast, so that the production efficiency of the reactor can be remarkably improved. More specifically, Rhodococcus genus is suitably used for, for example, oil decomposition, nitrile decomposition, and cyano group hydrolysis. The genus Candida is suitably used for, for example, hydrocarbon oxidation, lipase reaction, and optical isomerization. The genus Norcardia is suitably used, for example, for cyano group hydrolysis. The genus Alcaligenes is preferably used for, for example, retroaldol reaction and PHB generation. The Lactococcus genus is suitably used for producing a raw material for polylactic acid, for example. The genus Acetobacter is suitably used for cellulose production, for example. Escherichia
Escherichia coli and Bachillus subtilis are suitably used, for example, as genetically modified strains. The genus Pseudomonas and the genus Sphingomonas are preferably used, for example, for the decomposition of contaminating organic compounds. Corynebacterium is preferably used for, for example, wastewater treatment and carbon dioxide recovery. The genus Synechococcus and the genus Botryococcus are preferably used for photosynthesis (oil synthesis), for example. The genus Chlamydomonas is preferably used for hydrogen production, for example. The genus Cryptococcus is preferably used for biodiesel production, for example. The genus Zymomonus is preferably used for ethanol production, for example. The Methanosarcina genus is preferably used for methane production, for example. The genus Methylosinus is suitably used for, for example, methanol production and chlorine compound production.

  The microorganisms that can be used in the present invention are not limited to the above, and any appropriate microorganism can be adopted depending on the purpose. Microorganisms isolated by the present invention are used for production of various substances (for example, pharmaceuticals, diagnostics, fine chemicals, optically active substances, general-purpose resins, ethanol, methane, fats and oils); wastewater / sewage / garbage treatment, It can be suitably used for environmental conservation, environmental purification, and hygiene management such as soil treatment by aerobic organic waste treatment and anaerobic organic waste treatment.

  The microorganism isolation device of the present invention can be suitably used for searching and evaluating useful microorganisms such as materials for drug discovery, materials for foods and chemical products, and environmental purification / environmental sensing materials. The separated microorganism can be suitably used for a microbial reactor.

It is a schematic perspective view explaining the whole structure of the microorganism isolation apparatus by preferable embodiment of this invention. It is a schematic sectional drawing of the flow-path direction of the diffuser type | mold micropump used for the microorganisms isolation apparatus by preferable embodiment of this invention. (A) And (b) is a schematic diagram explaining the pulse shape of the pulse-like pressure applied to a flow path. It is a schematic diagram explaining the mechanism of the fine particle formation process in the confluence | merging part of a flow path. It is a schematic plan view explaining the flow-path shape of the microorganisms isolation apparatus by another embodiment of this invention. It is a schematic diagram explaining the mechanism of the fine particle formation process in the confluence | merging part of the flow path in embodiment of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Microorganism isolation apparatus 1a Microbe supply part 1b Contact liquid supply part 2a Microbe supply flow path 2b Contact liquid supply flow path 5 Fine particle production | generation part

Claims (8)

At least one microbial supply, each containing a microbial supply comprising gelling material and a predetermined microorganism;
A contact liquid supply unit comprising a contact liquid that contains a gelling initiation component and that generates fine particles upon contact with the microorganism supply liquid;
At least one microorganism supply channel extending from each of the at least one microorganism supply section and through which the microorganism supply liquid passes;
A contact liquid supply channel extending from the contact liquid supply section and through which the contact liquid passes;
A fine particle generation unit that is formed by joining the at least one microorganism supply channel and the contact liquid supply channel, and that generates fine particles by bringing the microorganism supply liquid and the contact liquid into contact with each other;
The ratio of the cross-sectional area of the contact liquid supply channel to the cross-sectional area of the microorganism supply channel is 2-50.
Microbe-containing microparticle production equipment. One microorganism supply part containing a microorganism supply liquid containing a gelling material and a predetermined microorganism;
A contact liquid supply unit comprising a contact liquid that contains a gelling initiation component and that generates fine particles upon contact with the microorganism supply liquid;
A microorganism supply channel extending from the microorganism supply section and through which the microorganism supply liquid passes;
A contact liquid supply channel extending from the contact liquid supply section and through which the contact liquid passes;
The microbe supply channel and the contact liquid supply flow channel are joined together to form microparticles by bringing the microbe supply liquid and the contact liquid into contact with each other,
The ratio of the cross-sectional area of the contact liquid supply channel to the cross-sectional area of the microorganism supply channel is 2-50.
The microorganism-containing microparticle production apparatus according to claim 1.   The microbe-containing microparticle production apparatus according to claim 1 or 2, wherein the generated microparticles include substantially only one microbe in each microparticle. A method for producing microbe-containing microparticles using the microbe-containing microparticle production apparatus according to claim 1 or 2,
Preparing a microorganism supply solution containing a gelling material and a predetermined microorganism;
Preparing a contact liquid containing a gelation initiating component and generating fine particles upon contact with the microorganism supply liquid;
A step of bringing the microorganism supply liquid into contact with the contact liquid to produce fine particles,
The ratio of the cross-sectional area of the contact liquid supply passage through which the contact liquid passes to the cross-sectional area of the microorganism supply flow path through which the microorganism supply liquid passes is 2-50.
A method for producing microparticles containing microorganisms.   The method for producing microbe-containing microparticles according to claim 4, wherein the volume of the microparticles generated is 0.5 to 500 picoliters.   6. The method for producing microbe-containing microparticles according to claim 5, wherein the generated microparticles contain substantially only one microbe in each microparticle. The method for producing microbe-containing microparticles according to any one of claims 4 to 6 , wherein the concentration of the predetermined microbe in the microbe supply liquid is 2 × 10 ⁶ to 2 × 10 ⁹ cells / ml. The method for producing microbe-containing microparticles according to any one of claims 4 to 7 , wherein the predetermined microorganism is at least one selected from the group consisting of eubacteria, archaea, yeast, and a gene recombinant.

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