JP6579544B2 - Substance conversion method, bioreactor manufacturing method, bioreactor - Google Patents

Description

  The present invention relates to a technique for performing substance conversion using microorganisms, particularly microorganisms other than filamentous fungi.

  Fermentation methods and substance conversion methods that use various enzymes of microorganisms for substance conversion include production of useful compounds such as pharmaceutical raw materials, cosmetic raw materials, chemical products and electronic materials, energy such as biodiesel and biogas, or tetrachlorethylene and Applications to a wide range of industrial fields such as decomposition and removal of harmful and hardly decomposable substances such as benzo [a] pyrene are expected.

  Among these application fields, catabolite suppression (Non-Patent Document 1) and feedback inhibition (Non-Patent Document 2) are cited as phenomena that become a major problem when microorganisms are used for fermentation. The former is a phenomenon in which the production of target fermentation products is suppressed at the gene level when a large amount of carbon sources such as glucose that can be easily metabolized are present in the medium, improving the productivity of the target substance. It becomes a very big barrier in making it. As a measure to avoid this catabolite suppression, a fed-batch culture method (Non-patent Document 3) or a continuous addition method (Non-patent Document 4) that maintains the addition of a readily metabolizable carbon source at a low level, induces catabolite suppression. Examples include various methods such as gene disruption of a repressor protein (Non-Patent Document 5) and application of a solid culture method (Non-Patent Document 6). In particular, the former two methods for keeping the addition level of a readily metabolizable carbon source low are often used in actual industrial production.

  However, among these measures, solid culture methods that are difficult to scale up are unsuitable for industrial production oriented to mass production, and culture management is complicated for fed-batch culture methods and continuous addition methods. is there. In addition, the creation of a repressor protein gene-disrupted strain is troublesome, and the stability of the obtained disrupted strain is also difficult.

  In addition to these methods, the present inventors layered a hydrophobic organic solvent on a mold-hollow microparticle composite mat formed on the liquid medium surface using hollow microparticles, and produced fat-soluble secondary metabolism. In the extraction liquid surface immobilization system (Non-patent Document 7) for accumulating substances in the organic layer, it was reported that the above-mentioned catabolite suppression did not appear (Non-patent Document 8). Furthermore, in this system, since the hydrophobic organic solvent layered on the mold-hollow particle composite mat functions as an extraction solvent, the fat-soluble secondary metabolite produced by fermentation is continuously accumulated without being accumulated in the cells. Extracted from the fungus body into the organic layer. As a result, since these secondary metabolites do not accumulate excessively in the microbial cells, feedback inhibition is not a problem at all (Non-patent Document 7).

  On the other hand, there is a liquid-liquid interface bioreactor as a bioreactor in which hollow fine particles are applied to microbial conversion by mold (Non-patent Document 7, Patent Document 1). In the bioreactor, a hydrophobic organic solvent is layered on top of a mold-hollow particle composite mat formed by entraining hollow fine particles, as in the extraction liquid surface immobilization system. In this organic layer, the fat-soluble substrate used for the microbial conversion reaction can be dissolved, and the fat-soluble product produced by the reaction can be accumulated in the organic layer at a high concentration. Liquid-liquid interface bioreactors have so far been hydrolyzed with fungi (Non-patent Document 9), asymmetric reduction (Non-patent Document 10), position / stereoselective hydroxylation (Non-patent Document 11), It has been applied to various reactions such as stereoselective epoxidation (Non-patent Document 12), and has achieved product concentrations and optical purity far surpassing conventional liquid culture methods.

  Since reaction substrates used in microbial conversion generally exhibit strong toxicity to microorganisms, in a normal liquid culture method, microorganisms as biocatalysts are killed immediately after addition of a high concentration of substrate. Therefore, the reaction does not proceed significantly. In contrast, in a liquid-liquid interface bioreactor, the layered solvent functions as a toxic substrate or product reservoir, so that the conversion reaction proceeds efficiently even when a high concentration of toxic substrate is converted. Soluble products can be accumulated in the organic layer. For example, when β-caryophyllene, which is a toxic substrate, is regio- and stereoselectively epoxidized with Nemania aenea using a liquid-liquid interface bioreactor, (−)-β-caryophorene oxide is a highly toxic product. The accumulated concentration reaches as high as 150 times its lethal concentration (Non-patent Document 12).

  Furthermore, a liquid surface immobilization system (Non-patent Document 7, Patent Document 1) for producing and accumulating water-soluble fermentation products and enzymes produced on mold-hollow fine particle composite mats on the liquid medium side is particularly suitable for enzyme production. The enzyme productivity is more than twice that of the liquid culture method (Non-patent Documents 13 and 14).

Shinobu Oda, Hiroyuki Kumagai, JP 2008-29251

de Vargas Andradem S. , Et al. , Process Biochem. 39: 931 (2004). Robert-Gero, M.M. , Et al. , J. et al. Bacteriol. 112: 251 (1972) Szabo, I.D. J. et al. , Et al. , J. et al. Biotechnol. 48: 221 (1996). Aharonowitz, Y.A. , Demain, A .; L. , Antimicrob. Agents Chemother. , 14: 159 (1978) Bagga, P.A. S. , Et al. , Acta Biotechnol. 11: 395 (1991) Wali, A .; S. , Et al. , Arch. Microbio1. 118: 49 (1978). Oda, S .; , Isshiki, K .; , Process Biochem. 42: 1553 (2007) Oda, S .; , Et a1. , J .; Biosci. Bioeng. 113: 742 (2012) Oda, S .; , Et al. , J. et al. Biosci. Bioeng. , 112: 151 (2011) Oda, S .; , Isshiki, K .; Biosci. Biotchnol. Biochem. 72: 1364 (2008) Oda, S .; , Et al. Bull. Chem. Soc. Jpn. , 82: 105 (2009) Oda, S .; , Et al. , J .; Biosci. Bioeng. , 112, 561 (2011) Ozeki, K .; , Et al. , J .; Biosci. Bioeng. 109: 224 (2010) Ozeki, K .; , Et al. , J .; Biol. Macromol. 11:23 (2011) Kozo Sato, Adhesion, 41: 492 (1997)

  As described above, the extraction liquid surface immobilization system can overcome two major obstacles that are always problems in actual industrial production such as catabolite suppression and feedback inhibition. A liquid-liquid interface bioreactor is an effective device that can overcome both the problems of water insolubility of substrates and products and the problem of microbial toxicity.

  However, the application of both these systems is limited only to filamentous fungi (molds) that can form vegetative mycelium and take in hollow microparticles to form a cell-hollow microparticle composite mat. Therefore, there is a problem that it cannot be applied to microorganisms other than filamentous fungi such as bacteria, yeast, and algae.

  Specifically, even if it is microorganisms other than mold, it is possible to form a biofilm of microorganisms on the surface of the hollow fine particle layer on the liquid medium liquid surface under static conditions. However, the mold mycelium network can entangle hollow microparticles to form a stable mold-hollow microparticle composite mat, whereas microorganisms such as bacteria and yeast cannot form such a network structure. . Therefore, in a microorganism that does not form hyphae, the biofilm and the hollow fine particle layer are collapsed by slight vibration. Also, in the case of microorganisms that do not form mycelia, when an organic solvent is layered on the biofilm, the biofilm and the hollow can be quickly removed even under stationary conditions, as indicated by arrows in FIG. The fine particle layer will collapse.

  Further, the hollow fine particles used in the extraction liquid surface immobilization system are made of polyacrylonitrile, and have an average particle size of 10 to 200 μm and a specific gravity of 0.06 to 0.50, which are lightweight and minute. These hollow fine particles showed solvent resistance to organic solvents such as styrene, while being very stable to water. That is, the hollow microparticles thrown into the water are stably formed without any change and are densely formed while maintaining the form and surface properties to form a layer. Therefore, no adhesive force or adhesive force acts between the fine particles, and the hollow fine particle layer is physically extremely unstable, and the layered structure collapses even with a slight impact. It was.

  In particular, in the extraction liquid surface immobilization system and the liquid-liquid interface bioreactor described above, a hydrophobic organic solvent is layered on the cell-hollow microparticle composite mat. However, in the case of bacteria, actinomycetes, yeasts and microalgae whose microorganisms do not form hyphae, a large number of vegetative mycelia like molds can take in hollow microparticles and form a physically strong composite mat, And the hollow microparticles do not adhere or stick. As a result, as shown in FIG. 1, the biofilm / hollow particle layer easily collapses. In such a state, there is a problem that it is practically impossible to cause fermentation production or microbial conversion.

  Bacteria and yeasts containing actinomycetes, or microalgae and plant cells are extremely important as pharmaceutical raw materials such as antibiotics, anticancer agents and anti-inflammatory agents, and as raw materials for producing biofuels. Therefore, if these microorganisms can be applied to the extraction liquid surface immobilization system and the liquid-liquid interface bioreactor, it can be expected to have a great impact on a wide range of industrial fields such as the pharmaceutical industry, the cosmetics industry, and the chemical industry.

  The present invention has been made in view of such a situation, and the object of the present invention is a technique for efficiently converting a substance into a substance, as well as bioactive secondary metabolism, even when a biocatalyst is a microorganism that does not form hyphae. The purpose is to provide technology for fermenting products and biofuels.

  In order to solve the above problems, a substance conversion method according to an aspect of the present invention includes an aqueous solution containing a hydrophilic substrate, a plurality of synthetic polymer fine particles having a specific gravity smaller than that of the aqueous solution, and a plurality of the synthetic polymer fine particles in the aqueous solution. And a plurality of synthetic polymer fine particles that retain the microorganisms from below by buoyancy on the liquid surface of the aqueous layer formed by adding a binder material that collects the synthetic polymer fine particles and microorganisms, and Forming a composite layer containing a binder material; and reacting a hydrophilic substrate with a microorganism in the composite layer as a biocatalyst. In the present specification, substance conversion means conversion of a substrate into another substance using a microorganism as a biocatalyst. Specifically, for example, conversion to citric acid by metabolism using glucose, which is also a nutrient source, as a substrate.

  According to this aspect, even when a microorganism that does not form mycelia is used as a biocatalyst, the substrate can be efficiently converted into a substance. Moreover, even when a microorganism that does not form mycelia is used in the extraction liquid surface immobilization system, the problems of catabolite suppression and feedback inhibition can be effectively avoided. It is envisaged that the hydrophilic / hydrophobic balance and charge state optimum for substance conversion vary greatly depending on the microorganism used, fermentation, and type of microbial conversion reaction. However, for example, if the combination and blending amount of the synthetic polymer fine particles and the binder material are arbitrarily set and mixed at an arbitrary ratio, the charged state of the polymer fine particle layer formed on the liquid surface of the liquid medium is arbitrarily changed. be able to. For this reason, by using the substance conversion method of the present embodiment, custom-made substance conversion according to the type of microorganism, fermentation, and microorganism conversion reaction can be easily performed.

  In the substance conversion method, the binder material may be carboxylated cellulose, or a polyvinyl alcohol derivative having a butyral group, a hydroxy group, and an acetyl group. According to this aspect, the binder material efficiently holds the plurality of synthetic polymer fine particles, whereby microorganisms can be retained for a long period of time.

  In the substance conversion method, the binder material may be cellulose having a degree of etherification of 20 to 30 mol% or a polyvinyl alcohol derivative having a degree of etherification of 60 to 70 mol%. According to this aspect, the balance between the insolubility of the binder material in the aqueous solution and the binding properties of the plurality of synthetic polymer fine particles is optimized, so that microorganisms can be stably retained for a long period of time.

  In the substance conversion method, the microorganism may be a microorganism other than the filamentous fungus. According to this aspect, by using various microorganisms, it is possible to perform substance conversion that could not be performed by conventional filamentous fungi.

  The substance conversion method may further include a step of overlaying an organic layer containing a hydrophobic organic solvent on the liquid surface of the composite layer. According to this embodiment, the hydrophobic substrate can be converted into a substance.

  In the substance conversion method, the hydrophilic substrate may be converted into a hydrophilic compound by a microorganism, and the compound may be accumulated in the aqueous layer. According to this embodiment, a hydrophilic product can be obtained by substance conversion using a hydrophilic substrate.

  In the substance conversion method, the hydrophilic substrate may be converted into a hydrophobic compound by a microorganism, and the compound may be accumulated in the organic layer. According to this embodiment, a hydrophobic product can be obtained by substance conversion using a hydrophilic substrate.

  Another embodiment of the present invention is also a substance conversion method. In this substance conversion method, an aqueous solution containing a hydrophilic substrate, a plurality of synthetic polymer fine particles having a specific gravity smaller than that of the aqueous solution, a binder material for collecting the plurality of synthetic polymer fine particles in the aqueous solution, a microorganism, , And on the liquid surface of the aqueous layer formed by the aqueous solution, microorganisms, a plurality of synthetic polymer fine particles that retain the microorganisms from below by buoyancy, and a binder that collects the plurality of synthetic polymer fine particles in the aqueous solution Forming a composite layer containing a material, overlaying an organic layer containing a hydrophobic substrate and a hydrophobic organic solvent on the top surface of the composite layer, and using a microorganism in the composite layer as a biocatalyst to form a hydrophobic layer Reacting the substrate.

  Another embodiment of the present invention is a method for producing a bioreactor. In this bioreactor manufacturing method, an aqueous solution, microorganisms, a plurality of synthetic polymer fine particles having a specific gravity smaller than that of the aqueous solution, and a binder material that collects the plurality of synthetic polymer fine particles in the aqueous solution are added to the culture tank. Mixed on the surface of the aqueous layer formed by the aqueous solution, microorganisms, a plurality of synthetic polymer fine particles that retain the microorganisms from below by buoyancy, and a binder material that combines the plurality of synthetic polymer fine particles in the aqueous solution Forming a composite layer comprising:

  Another aspect of the invention is a bioreactor. The bioreactor includes a culture tank, an aqueous solution contained in the culture tank, and a composite layer that includes microorganisms and is formed on a liquid surface of the aqueous solution. The composite layer has a specific gravity smaller than that of the aqueous solution and lowers the microorganisms. A plurality of synthetic polymer fine particles retained by buoyancy and a binder material that collects the plurality of synthetic polymer fine particles in an aqueous solution.

  A combination of the above-described elements as appropriate can also be included in the scope of the invention for which patent protection is sought by this patent application.

  According to the present invention, a substrate can be efficiently converted into a substance using a microorganism.

FIG. 1 (A) is a reference diagram showing the results over time of the results of layering organic solvents on a composite layer containing hollow microparticles and microbial cells and not containing a binder material in microorganisms that do not form mycelia. FIG. 1 (B) is a graph showing the results of aging organic solvents on a composite layer containing hollow microparticles / binder material / microbial cells in microorganisms that do not form mycelia over time. It is a figure which shows typically the substance conversion method which concerns on embodiment. FIG. 2A is a diagram schematically showing the substance conversion method according to the first embodiment. FIG. 2B is a diagram schematically illustrating the substance conversion method according to the second embodiment. FIG. 2C is a diagram schematically illustrating the substance conversion method according to the third embodiment. FIG. 2D is a diagram schematically showing the substance conversion method according to the fourth embodiment. It is a graph which shows the production | generation density | concentration of the target substance 2-octanone in Example 4 and Comparative Examples 4-1 and 4-2.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. In addition, the description which overlaps in description of drawing is abbreviate | omitted suitably.

[First Embodiment]
In the substance conversion method of the present embodiment, an aqueous solution containing a hydrophilic substrate, a plurality of synthetic polymer fine particles having a specific gravity smaller than that of the aqueous solution, and a binder for collecting the plurality of synthetic polymer fine particles in the aqueous solution in the culture tank 12 Adding a material and microorganisms to form a composite layer 30 containing low-specific gravity synthetic polymer fine particles / binder material / microbial cells on the liquid surface of the aqueous layer 20 formed of an aqueous solution; and composite layer And reacting the hydrophilic substrate X with the microorganism in 30 as a biocatalyst.

  FIG. 2A is a diagram schematically showing the substance conversion method according to the first embodiment.

  In the material conversion method of the present embodiment, the bioreactor 10 is used. The bioreactor 10 includes a culture tank 12 and a composite layer 30 containing low-specific gravity synthetic polymer particles / binder material / microbial cells. The culture tank 12 is a culture reactor for culturing filamentous fungi to perform substance conversion. The aqueous layer 20 is formed from a liquid medium that is an aqueous solution containing water and a nutrient source supplied to microorganisms. In the present embodiment, the aqueous layer 20 also includes a hydrophilic substrate X that is converted into a substance using a microorganism as a biocatalyst. The hydrophilic substrate X may be the nutrient source itself. The composite layer 30 includes a plurality of synthetic polymer fine particles and a binder material that collects the plurality of synthetic polymer fine particles in an aqueous solution. The plurality of synthetic polymer fine particles have a specific gravity smaller than that of the aqueous solution, and the microorganisms can be floated on the liquid surface of the aqueous solution in a state of being collected by the binder material. In the present specification, the specific gravity refers to a value that takes into account the gas contained in the case of fine particles having a hollow structure. On the other hand, the true specific gravity refers to a specific gravity when fine particles are powdered, that is, a value that does not consider the gas contained therein.

(Synthetic polymer fine particles)
Examples of the synthetic polymer fine particles used in the present embodiment include hollow fine particles described in Patent Document 1 (Japanese Patent Laid-Open No. 2008-29251) described above. The synthetic polymer fine particles are preferably about 10 to about 200 μm and specific gravity of about 0.06 to about 0.50 and lightweight and fine. As a material, it is preferable that a synthetic polymer such as polyacrylonitrile or polystyrene is emulsion-polymerized or suspension-polymerized and then voids are provided inside the fine particles by a process such as thermal expansion. Moreover, what coated fine powder of inorganic materials, such as a talc and a calcium carbonate, as a specific gravity preparation material may be used. As synthetic polymer fine particles satisfying these properties, for example, Advancell HB-2051 (manufactured by Sekisui Chemical Co., Ltd., average particle size 20 μm, true specific gravity 0.5), MFL-80GCA (manufactured by Matsumoto Yushi Seiyaku Co., Ltd., calcium carbonate coat) Mold, average particle size 20 μm, true specific gravity 0.2), MFL-80GTA (manufactured by Matsumoto Yushi Seiyaku Co., Ltd., calcium carbonate coat type, average particle size 20 μm, true specific gravity 0.2).

(Binder material)
As shown in FIG. 1A, when the microorganism is held using only the synthetic polymer fine particles that are hollow fine particles as the composite layer 30, the composite layer 30 formed on the liquid surface is physically extremely It is fragile and easily collapses even if the composite layer 30 is formed on the surface and the layer. In particular, when the hydrophobic organic layer 40 is overlaid on the composite layer 30, the composite layer 30 rapidly collapses.

  When material conversion is performed using microorganisms that do not form mycelia as microorganisms, it is effective to adhere or closely adhere the fine particles to improve the brittleness of the synthetic polymer fine particles and add physical strength. is there. Therefore, it is effective to add an adhesive material or a pressure-sensitive adhesive material (hereinafter referred to as a binder material) to the composite layer 30.

  However, since the synthetic polymer fine particles are always in contact with water and the hydrophobic organic solvent, the binder material (1) keeps the adhesive force without dissolving in both the water and the hydrophobic organic solvent. (2) The particle size is equal to or smaller than that of the synthetic polymer fine particles (not excessively large), and (3) the composite layer is formed on the liquid surface as the synthetic polymer fine particles float. Some restrictions are imposed, such as being able to be incorporated into 30 and (4) not being toxic to the microorganisms used, such as bacteria, actinomycetes, yeasts, microalgae and the like.

  The constraint of (1) is physically very strict. Adhesives such as tackifiers exhibit adhesion and tackiness at the molecular level, more strictly, by the presence of polar functional groups such as carboxyl groups and hydroxy groups (Non-patent Document 15). Adhesion and tackiness at the level involves dissolution and diffusion of a single tackifier molecule in water or in an organic layer, so it is not possible to adhere and adhere synthetic polymer particles to each other over a long period of time. Is possible. In fact, it has been confirmed that a water-soluble polymer such as polyvinyl alcohol is completely dissolved in water, so that the composite layer 30 cannot be kept physically stable. Also, ordinary solid tackifiers such as terpene resins and low molecular weight styrene resins are too cohesive, so they aggregate strongly even when pulverized into fine powders, making classification impossible even in water. It is. Accordingly, it has become indispensable to search for a material that not only dissolves in water, but also hydrates only on the surface of the fine particles and exhibits adhesive strength, as well as stability in organic solvents.

  The condition (2) is that the particle diameter can be easily set to a desired range, for example, a range of 10 μm to 200 μm, by pulverization and classification. Since it is impossible to float on the liquid surface together with the synthetic polymer fine particles with a particle size of 200 μm or more, the restriction (3), that is, the intended purpose of adhering the fine particles by being taken into the composite layer 30 Can't reach. Furthermore, if the condition (4) cannot be satisfied, it cannot be subjected to fermentation or microbial conversion reaction.

  The binder materials, which are fine particles capable of adhering the synthetic polymer fine particles to each other in water for a long time as described above, have been intensively searched and examined. Water-soluble natural polymers such as agar and starch, alginic acid and gelatin, water-soluble synthetic polymers such as polyvinyl alcohol and polyacrylate, and water-soluble semi-synthetic polymers such as modified starch and carboxymethyl cellulose Although physical stabilization of the composite layer 30 is brought about, it was confirmed that it is difficult to stabilize the composite layer 30 for a long time because their water solubility is too high. In particular, when a hydrophobic organic solvent is overlaid on the composite layer 30 in the embodiment of the extraction liquid surface immobilization system or the liquid-liquid interface bioreactor, the composite layer 30 easily collapses even under stationary conditions.

  Compared with these water-soluble polymers, the present inventors have found that (1) lightly carboxylated cellulose or (2) polyvinyl alcohol derivatives having a butyral group, a hydroxy group and an acetyl group have been used for a long time. It has been found that the adhesiveness is maintained in water and the composite layer 30 is given physical strength. In the present embodiment, “lightly carboxylated” of cellulose means that the degree of etherification (unit) is about 0.20 to about 0.30 (about 20 to about 30 mol%).

  The former (1) is a semi-synthetic polymer, which is a polymer in which the C-6 hydroxy group of cellulose is partially carboxymethylated and insoluble in water. It hydrates and swells, and exhibits adhesive strength. Further, since the material is cellulose, it is harmless to human bodies and microorganisms as it is used as an additive for foods and samples and a cosmetic raw material. An example of such a product is the Sunrose SLD series manufactured by Nippon Paper Industries Co., Ltd. In the present specification, the semi-synthetic polymer is also a kind of synthetic polymer.

  The latter (2) is a completely synthetic polymer, a material based on a polyvinyl acetal resin synthesized by reacting polyvinyl alcohol and an aldehyde, and having various physical properties depending on the chain length of the aldehyde. In addition to the cyclic acetal structure, small amounts of acetyl and hydroxy groups are also formed, and many products are insoluble in both the polar solvent water and the nonpolar solvent hexane. On the other hand, the surface of many products can be hydrated in water and can exhibit adhesive strength by containing 5-8% of water. Examples of such products include SLECK B and K series of Sekisui Chemical Co., Ltd.

In addition, the binder material preferably has the following characteristics.
Average molecular weight: about 10,000 to about 50,000 (Dalton), preferably about 20,000 to about 40,000. Degree of etherification (mol%): about 20 to about 30 mol% in the case of lightly carboxylated cellulose. In the case of a polyvinyl alcohol derivative having a butyral group, a hydroxy group and an acetyl group, about 50 to about 80 mol%, preferably about 60 to about 70 mol%
Viscosity (mPa · s): about 5 to about 70 mPa · s, preferably about 10 to about 60 mPa · s
Glass transition temperature (° C.): about 50 to about 80 ° C., preferably about 60 to about 70 ° C. The glass transition temperature is a temperature at which a phenomenon occurs in which the polymer changes from a glassy hard state to a rubbery soft state with heating.
Softening point (° C.): about 100 to about 160 ° C., preferably about 125 to about 150 ° C. The softening point refers to a temperature at which the softening occurs due to temperature rise and deformation starts.
Adhesive strength (gf / cm ² ): Assuming that the binder material was once dissolved in isopropanol and formed into a thin film by desolvation and measured, about 50 to about 300 gf / cm ² at 180 ° C. peeling, preferably about 100 ~ About 200 gf / cm ²
-Solubility (insoluble) and water absorption (%): About 1 to about 20% (w / w), preferably about 2 to about 10% for SREC B and K series (Sekisui Chemical Co., Ltd.). About 5 to about 30%, preferably about 10 to about 20% in the Sunrose SLD series (Nippon Paper Industries Co., Ltd.)
・ Insoluble in nonpolar organic solvents (hexane, styrene, etc.)

  The above-mentioned semi-synthetic or synthetic polymer material is suitable as a binder material that promotes adhesion between a plurality of synthetic polymer fine particles, but it is necessary to pay attention to the amount of these blended with respect to the synthetic polymer fine particles. The stability of the composite layer 30 is improved as the blending amount of the binder material is increased. However, when the amount is excessively added, the incorporation rate into the composite layer 30 and the preparation cost are increased. Since the specific gravity of the binder material is often around 1.0, the liquid material itself has no liquid surface floating property or is very small. Therefore, when an excessive amount of the binder material is blended, the amount of the binder material that is levitated by the synthetic polymer fine particles is limited, and the remaining material settles to the bottom of the incubator.

  The blending ratio of the binder material to the synthetic polymer fine particles varies depending on the specific gravity and particle size of the synthetic polymer fine particles. For example, hollow fine particles having a specific gravity of 0.23 and an average particle size of 20 mm (Matsumoto Microsphere MFL-81GTA, Matsumoto Yushi Seiyaku) Industrial) 50 mg of binder is sufficient for 1 g, and 25 mg of binder is sufficient for 1 g of hollow microparticles (Advancel HB-2051, Sekisui Chemical) having a specific gravity of 0.50 and an average particle size of 20 mm. . The ratio of the synthetic polymer fine particles to the binder material is preferably about 5: about 1, and more preferably about 40: about 1. Accordingly, the binder material can be effectively used by reducing the binder material that does not contribute to the adhesion of the synthetic polymer fine particles while maintaining the floating property of the plurality of synthetic polymer fine particles to some extent.

(Aqueous solution)
There is no restriction | limiting in particular in the liquid culture medium which can be used as the aqueous solution which forms the water layer 20, What is necessary is just to use the thing of suitable composition and pH for production of a target substance. For example, monosaccharides such as glucose and sucrose, polysaccharides such as soluble starch, and polyols such as glycerol can be used as the carbon / energy source. When microalgae are used as microorganisms, they can be subjected to culture without adding a carbon / energy source. There are no particular restrictions on the nitrogen source, and inorganic nitrogen sources such as ammonium nitrate and ammonium chloride, organic nitrogen sources such as peptone and soyton, and yeast additives, amino acids, vitamins and phosphates as other additives are used as appropriate. Use it. In addition, when using microalgae as microorganisms, it is necessary to perform light irradiation. However, there is no particular limitation on the light irradiation condition, and for example, 1500 Lux light may be irradiated for 12 hours / 12 hours in a dark place.

(Microorganism)
There are no particular restrictions on applicable microorganisms, including Bacillus genus and Escherichia genus for bacteria, Streptomyces genus and Rhodococcus genus for actinomycetes, Saccharomyces genus and Candida genus for yeast, and Chlorella genus for microalgae. A representative example is Chlorococcum genus. By using such various microorganisms, it is possible to carry out substance conversion that could not be performed by conventional filamentous fungi. Note that the material conversion method of the present embodiment is sufficiently applicable to molds. For example, it can be applied to almost all genus species such as Aspergillus genus and Penicillium genus.

(Production method)
Next, a method for manufacturing the bioreactor 10 will be described. In the present embodiment, first, a plurality of synthetic polymer fine particles and a binder material are mixed in advance. To this mixture, the medium and the cell suspension of the strain used are added and mixed so as to have a predetermined mixing ratio, and a predetermined amount is dispensed into the culture tank 12 and left to stand. As a result, when a plurality of synthetic polymer fine particles are densely floated on the liquid surface, both the microorganisms and the binder material fine particles also float, and the liquid medium liquid surface contains low specific gravity synthetic polymer fine particles / binder material / microbial cells. A composite layer 30 is formed. FIG. 1B shows an example of a bioreactor 10 manufactured using a binder material for the composite layer 30. As shown with time from left to right, the composite layer 30 is stably formed as compared with the case where no binder material is used (FIG. 1A).

Regarding the ratio of the culture medium to be added and the inoculum suspension, this culture system differs from normal flask culture and jar fermenter culture, and the amount of inoculum to be added, the amount of synthetic polymer fine particles, and the amount of binder material are It is determined not depending on the hit setting but depending on the liquid medium liquid surface area. This is because the microorganism growth rate, substance production rate, and substance treatment rate in the main culture system do not depend on the liquid volume of the liquid medium but all on the liquid area. Accordingly, the amount of inoculation that can be set in terms of the liquid area is about 5 to about 100 μl, preferably about 10 to about 50 μl per 1 cm ² , and the amount of synthetic polymer fine particles varies depending on the specific gravity. For example, hollow fine particles (MMF with a specific gravity of 0.06) -DE-1: Matsumoto Yushi Pharmaceutical Co., Ltd.) per ² cm ² wave area is about 2 to about 50 mg, preferably about 5 to about 20 mg, and the amount of binder material depends on the amount of synthetic polymer fine particles. Is generally in the range of about 0.5 to about 10 mg, preferably about 1 to about 5 mg per cm ^{2 of} liquid area.

  As long as the bioreactor 10 shown in FIG. 2A can be manufactured, the order of adding each substance is not limited to that shown here. For example, a plurality of synthetic polymer fine particles and a binder material may be mixed later. Moreover, you may sterilize suitably using a well-known sterilization method.

(Substance conversion)
Next, substance conversion using the bioreactor 10 will be described. Due to the action of microorganisms in the composite layer 30 formed as a biofilm on the upper surface of the composite layer 30, the hydrophilic substrate X is converted into a product X ′, which is a target product, using filamentous fungi as a biocatalyst. In addition, at the time of substance conversion, that is, at the time of culturing microorganisms, the bioreactor 10 may be left stationary or rotated at a low speed.

  Various products X ′ can be obtained by appropriately combining the microorganism and the hydrophilic substrate X. By using a nutrient source in a liquid medium as the hydrophilic substrate X, for example, useful secondary metabolites such as antibiotics, primary metabolites such as organic acids and amino acids, and useful enzymes such as glucosidase and lipase can be used efficiently. Can be produced and accumulated in the water layer 20 at a high concentration.

  As an example of substance conversion using the substance conversion method of the present embodiment, for example, a reaction of generating citric acid using glucose, which is also a nutrient source, as a substrate can be mentioned.

  As described above, according to the substance conversion method of the present embodiment, even when a microorganism that does not form mycelia is used as a biocatalyst, the substrate can be efficiently converted into a substance. Moreover, even when a microorganism that does not form mycelia is used in the extraction liquid surface immobilization system, the problems of catabolite suppression and feedback inhibition can be effectively avoided. It is envisaged that the hydrophilic / hydrophobic balance and charge state optimum for substance conversion vary greatly depending on the microorganism used, fermentation, and type of microbial conversion reaction. However, for example, if the combination and blending amount of the synthetic polymer fine particles and the binder material are arbitrarily set and mixed at an arbitrary ratio, the charged state of the polymer fine particle layer formed on the liquid surface of the liquid medium is arbitrarily changed. be able to. For this reason, by using the substance conversion method of the present embodiment, custom-made substance conversion according to the type of microorganism, fermentation, and microorganism conversion reaction can be easily performed.

(Second Embodiment)
FIG. 2B is a diagram schematically illustrating the substance conversion method according to the second embodiment.

  In the present embodiment, the bioreactor 10 of the first embodiment has a structure in which an organic layer 40 is stacked on the upper surface of the composite layer 30. The organic layer 40 includes a hydrophobic organic solvent that is not substantially toxic to microorganisms.

  Microorganisms in the composite layer 30 located at the liquid / liquid interface between the organic layer 40 and the aqueous layer 20 are actively supplied with nutrients and water from the aqueous layer 20 and oxygen supplied from the atmosphere in contact with the organic layer 40. The metabolic activity can continue and the targeted product X ′ can be produced and accumulated at high concentrations. In the present embodiment, a product X ′, which is a hydrophobic secondary metabolite, is accumulated in the organic layer 40 from the hydrophilic substrate X.

  As an example of substance conversion using the substance conversion method of the present embodiment, for example, fermentation in which γ-decalactone is produced using glucose as a substrate can be mentioned.

  As the organic solvent layered on the composite layer 30, it is preferable to use a solvent that is not substantially toxic to microorganisms and does not inhibit growth. Such a hydrophobic organic solvent is not particularly limited as long as it is a hydrophobic organic solvent having a Log P value of 3 or more, preferably 4 or more. However, it is preferable to satisfy the conditions such as low toxicity to humans, excellent solubility of the reaction substrate and target lipophilic product, moderate boiling point, poor volatility, and low cost. . As an organic solvent, for example, medium chain normal paraffins such as decane and dodecane, medium chain aliphatic ethers such as hexyl ether and isoamyl ether, and low viscosity dimethyl silicone oil such as KF-96L-1CS are preferably used. can do.

  As described above, the same effect as that of the first embodiment can also be obtained by the material conversion method of the present embodiment. In addition, since the organic layer 40 functions as an extraction solvent layer and a reservoir layer for hydrophobic secondary metabolites, product inhibition or feedback inhibition can be effectively avoided.

(Third embodiment)
FIG. 2C is a diagram schematically illustrating the substance conversion method according to the third embodiment.

  This embodiment is different from the bioreactor 10 of the second embodiment in that the hydrophobic substrate X is added to the organic layer 40 instead of the aqueous layer 20.

  According to the bioreactor 10 of the present embodiment, the hydrophobic product X ′ is generated from the hydrophobic substrate X and accumulated in the organic layer 40.

  Examples of substance conversion using the substance conversion method of the present embodiment include, for example, Pichia kluyveri NBRC 1165 as a microorganism, citronellol acetate as a substrate (Example 2), and Candida viswanathii NBRC 10321 as a microorganism. Examples include a reaction for producing citronellic acid using citronellol as a substrate (Example 3), a reaction for producing Rhodococcus equi NBRC3730 as a microorganism and 2-octanone using 2-octanol as a substrate (Example 4).

  As described above, according to the substance conversion method of the present embodiment, as in the second embodiment, the organic layer 40 not only functions as a reaction solvent but also functions as a reservoir for the substrate X and the product X ′. That is, the hydrophobic substrate X is often toxic to microorganisms, but the concentration of the substrate X in contact with the microorganisms is diluted by the organic layer 40. Therefore, various substances can be selected as the substrate X that does not affect the survival of microorganisms. Further, the concentration of the substrate X that can be added to the organic layer 40 and the concentration of the product X ′ accumulated in the organic layer 40 are dramatically improved as compared with the conventional liquid culture method. According to the substance conversion method of the present embodiment, microorganisms located at the interface between the composite layer 30 and the organic layer 40 can actively perform metabolic activities without being inhibited from growth. Therefore, a coenzyme-requiring reaction such as an oxidation-reduction reaction or an induced enzyme reaction can be catalyzed without any problem. According to the material conversion method of the present embodiment, a highly difficult microbial conversion reaction such as a hydroxylation reaction of inert carbon can be efficiently advanced. Further, according to the material conversion method of the present embodiment, the product can be recovered only by taking it out from the organic layer 40. Therefore, a complicated solvent extraction step can be omitted.

(Fourth embodiment)
FIG. 2D is a diagram schematically showing the substance conversion method according to the fourth embodiment.

  In the present embodiment, a hydrophobic substrate X is added to the organic layer 40 as in the third embodiment. On the other hand, the bioreactor 10 of the present embodiment is different from the third embodiment in that the hydrophilic product X ′ is accumulated in the aqueous layer 20 from the hydrophobic substrate X.

  As an example of substance conversion using the substance conversion method of the present embodiment, for example, a reaction of generating fluorocatechol using fluorobenzene as a substrate can be mentioned.

  As described above, the same effect as that of the third embodiment can also be obtained by the material conversion method of the present embodiment.

  In the first to fourth embodiments, the composite layer 30 may be formed by previously forming a hollow fine particle / binder material layer on the liquid surface of the water layer 20 and placing microorganisms thereon. This formation method is particularly effective as a screening method for microorganisms or enzymes targeting a large number of microorganisms. In each embodiment mentioned above, it has high substance productivity compared with the liquid culture method. Further, no aeration or stirring operation is required, and the solvent extraction step can be omitted. Therefore, this formation method is effective for large-scale screening for a large number of microorganisms.

[Usage]
The substance conversion method described above is assumed to be applied to production of organic acids such as lactic acid and butyric acid, fragrance raw materials such as γ-decalactone, or pharmaceutical raw materials such as ursodeoxycholic acid and vitamin D3. By appropriately selecting microorganisms, their production can be easily made efficient.

  Microorganisms are useful as high productivity microorganisms of various enzymes. Microorganisms are suitable for high production of proteases, lipases and the like, in addition to saccharide-degrading enzymes such as amylase and cellulase. By using these microorganisms in the above-described material conversion method, it is possible to achieve a high yield of a product that surpasses not only the liquid culture method but also the solid culture method.

  On the other hand, application to the field of microbial conversion is not limited to general reactive species such as hydrolysis of esters, oxidation of alcohols and aldehydes, reduction of ketones and olefins, and the like. For example, it can be used for various reactive species such as hydroxylation of inert carbon and epoxidation reaction.

  Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to these examples.

  Hereinafter, in Examples 1-1 to 1-5 and Comparative Examples 1-1 to 1-4, screening of the binder material was performed. Moreover, in Examples 2-4, the experimental result which performed substance conversion using microorganisms as a biocatalyst is shown, respectively using citronellol, citronellol, and 2-octanol as a substrate. Moreover, as Comparative Examples 2 to 4, experimental results obtained by performing substance conversion in the emulsion culture system and the water-organic solvent two-phase system using the same substances as in Examples 1 to 4 as substrates are shown. Description of operations that are the same as those in Embodiment 2 is omitted as appropriate.

(Examples 1-1 to 1-5 and Comparative Examples 1-1 to 1-4)
On the other hand, fine particles (binder material) whose surface is hydrated and develops stickiness are not extensively dissolved in water but are extensively screened.

  In a glass vial having an inner diameter of 30 mm and a volume of 50 ml, 300 mg of polyacrylonitrile hollow microparticles (HB-2051, Sekisui Chemical Co., Ltd.) as synthetic polymer microparticles and binder materials for test (0, 5, 10, 15, 20, 25, 30 mg) 7) and 0.005% Rose Bengal aqueous solution (15 ml) were mixed by rotating and shaking at 120 rpm for 60 minutes, and allowed to stand overnight to form a composite layer containing hollow fine particles and a binder material on the liquid surface. As a test binder material, Sunrose SLD-F1 (Nippon Paper Industries) (Example 1-1), Sunrose SLD-FM (Nippon Paper Industries) (Example 1-2), ESREC BL-S'DA-C51 (Sekisui) Chemical Industry) (Example 1-3) ESREC BL-1BC-H47 (Sekisui Chemical) (Example 1-4), ESREC BL-1HCJ-740 (Sekisui Chemical) (Example 1-5), agar ( Nine types of Comparative Example 1-1), sodium carboxymethylcellulose (Comparative Example 1-2), sodium alginate (Comparative Example 1-3), and polyvinyl alcohol (Comparative Example 1-4) were used (all 100 μm mesh pass). . 2 ml of decane was layered on the composite layer formed on the liquid surface of the rose bengal aqueous solution, and the mixture was shaken and rotated at 60 rpm for 24 hours, and the state of the composite layer was visually observed.

  In the blending systems of Examples 1-1 to 1-5, the composite layer containing the hollow fine particles and the binder material did not collapse if the blending amount was 5 mg or more. Therefore, it was shown that these binder materials can also be used to retain microorganisms that do not form mycelia in water. On the other hand, in the compounding system of the visor material of Comparative Example (Comparative Examples 1-1 to 1-4), the composite layer collapsed even when the compounding amount was 30 mg.

(Example 2)
In a glass vial with an inner diameter of 30 mm and a volume of 50 ml, 300 mg of polyacrylonitrile hollow microparticles (HB-2051, Sekisui Chemical Co., Ltd.) as synthetic polymer microparticles and 20 mg of Sunrose SLD-F1 or SLD-FM (Nippon Paper Industries) as binder materials Then, 200 μl of a daily culture solution of Pichia kluyveri NBRC 1165 as a microorganism was blended, and allowed to stand for 1 day to form a composite layer containing hollow microparticles / binder material / microbial cells. Thereafter, 3 ml of a 10% citronellol / decane solution as a substrate and an organic layer was layered on this composite layer, and reacted at 30 ° C. for 10 days under a rotary shaking condition of 60 rpm.

  As a result of quantifying the concentration of citronellyl acetate as the target substance in the organic layer produced by culturing for 10 days by gas chromatography, an accumulated concentration of 5.8 g / L was confirmed.

(Comparative Example 2)
As a comparison object, P.I. An emulsion culture system (Comparative Example 2-1) in which citronellol is directly added to the daily culture of kluyveri NBRC 1165 at a 10% level, and a 10% citronellol / decane solution are added to the daily culture of the same strain in an Erlenmeyer flask. Then, a water-organic solvent two-phase system (Comparative Example 2-2) for shaking culture was employed.

  As a result, citronellol strong toxicity was expressed in the emulsion culture system, and citronellyl acetate was not produced at all. Even in the water-organic solvent two-phase system, the strong toxicity of citronellol could not be sufficiently avoided, and the accumulated concentration of citronellyl acetate produced in the decane layer was only 0.1 g / L.

(Example 3)
200 mg of polyacrylonitrile hollow microparticles (MFL-80GTA, Matsumoto Yushi Seiyaku Kogyo Co., Ltd.) as synthetic polymer microparticles in a glass vial with an inner diameter of 30 mm and a volume of 50 ml, and 10 mg of ESREC BL-1BC-H47 (Sekisui Chemical Co., Ltd.) as a binder material, 100 μl of a daily culture solution of Candida viswanathii NBRC 10321 as a microorganism was blended and allowed to stand for 1 day to form a composite layer containing hollow microparticles / binder material / microbial cells. Thereafter, 3 ml of a 10% citronellol / decane solution as a substrate / organic layer was layered on this composite layer, and the mixture was reacted at 30 ° C. for 14 days under a rotary shaking condition of 60 rpm.

As a result of quantifying the concentration of citronellic acid as the target substance in the organic layer produced by culturing for 14 days by gas chromatography, an accumulated concentration of 38.7 g / L was confirmed.
(Comparative Example 3)
As a comparison object, C.I. An emulsion culture system (Comparative Example 3-1) in which citronellol is directly added to the daily culture of Viswanathii NBRC 110321 at a 10% level and a 10% citronellol / decane solution are added to the daily culture of the same strain in an Erlenmeyer flask. A water-organic solvent two-phase system (Comparative Example 3-2) for shaking culture was employed.

  As a result, strong toxicity of citronellol was expressed in the emulsion culture system, and no generation of citronellic acid was observed. Even in the water-organic solvent two-phase system, the strong toxicity of citronellol could not be sufficiently avoided, and the accumulated concentration of the generated silonic acid in the decane layer was only 0.3 g / L.

Example 4
200 mg of polyacrylonitrile hollow microparticles (MFL-80GCA, Matsumoto Yushi Seiyaku Kogyo Co., Ltd.) as synthetic polymer microparticles in a glass vial having an inner diameter of 30 mm and a volume of 50 ml, and 20 mg of ESREC BL-1HCJ-740 (Sekisui Chemical Co., Ltd.) as a binder material, 100 μl of a daily culture solution of Rhodococcus equi NBRC3730 as a microorganism was blended and left to stand for 1 day to form a composite layer containing hollow microparticles / binder material / microbial cells. Thereafter, 3 ml of a 5% 2-octanol / KF-96L-1CS (dimethylsilicone oil, Shin-Etsu Chemical Co., Ltd.) solution as a substrate / organic layer was layered on this composite layer, and the conditions of rotational shaking at 60 ° C. for 10 days at 30 ° C. Reacted below.

  As a result of quantifying the concentration of the target substance 2-octanone in the organic layer produced by culturing for 10 days by gas chromatography, an accumulation concentration of 33.7 g / L was confirmed. FIG. 3 shows the production concentration of the target substance 2-octanone in Example 4 and Comparative Examples 4-1 and 4-2.

(Comparative Example 4)
For comparison, an emulsion culture system (Comparative Example 4-1) in which 2-octanol was directly added to the daily culture of Rhodococcus equi NBRC3730 at a 5% level and a 5% 2-octanol / KF-96L-1CS solution were added. A water-organic solvent two-phase system (Comparative Example 4-2) that was added to the daily culture of the same strain in an Erlenmeyer flask and shake-cultured was used.

  As a result, 2-octanol was highly toxic in the emulsion culture system, and 2-octanone was not produced at all (FIG. 3). Even in the water-organic solvent two-phase system, the strong toxicity of 2-octanol could not be sufficiently avoided, and the accumulated concentration of the produced 2-octanone in the organic layer was only 0.9 g / L (FIG. 3).

  If the synthetic polymer fine particles and the binder material in this example are used in an appropriate blending ratio, the composite layer can be stabilized over a long period of time, and if the material conversion method of this example is used, It was confirmed that it was possible to produce substances that far exceeded the liquid medium method and the water-organic solvent two-phase reaction method.

  As described above, the present invention has been described with reference to the above-described embodiment. However, the present invention is not limited to the above-described embodiment, and the present invention can be appropriately combined or replaced with the configuration of the embodiment. It is included in the present invention. In addition, it is possible to appropriately change the combinations and the order of steps in the embodiments based on the knowledge of those skilled in the art and to add various modifications such as various design changes to the embodiments. The described embodiments can also be included in the scope of the present invention.

Claims (11)

An aqueous solution containing a hydrophilic substrate, a plurality of synthetic polymer particles having a specific gravity smaller than that of the aqueous solution, a binder material for collecting the plurality of synthetic polymer particles in the aqueous solution, and a microorganism are added to the culture tank. And forming a composite layer containing the microorganism, the plurality of synthetic polymer fine particles that hold the microorganism from below by buoyancy, and the binder material on the liquid surface of the aqueous layer formed by the aqueous solution;
The microorganisms of the composite layer as biocatalysts, look including the steps of: reacting the hydrophilic substrate,
The binder material is a carboxylated cellulose having an etherification degree of 20 to 30 mol%, or a polyvinyl alcohol derivative having a butyral group, a hydroxy group, and an acetyl group having an etherification degree of 60 to 70 mol%. Substance conversion method. 2. The substance conversion method according to claim 1 , wherein the microorganism is a microorganism other than a filamentous fungus. Material conversion method according to claim 1 or 2 an organic layer containing a hydrophobic organic solvent, and further comprising the step of overlaying the liquid surface of the composite layer. The substance conversion method according to claim 1 or 2 , wherein the hydrophilic substrate is converted into a hydrophilic compound by the microorganism and the compound is accumulated in the water layer. 4. The substance conversion method according to claim 3 , wherein the hydrophilic substrate is converted into a hydrophobic compound by the microorganism, and the compound is accumulated in the organic layer. The culture tank were added a liquid medium, wherein a small plurality of synthetic polymer microparticles having a specific gravity than the liquid medium, the binder material to combine the plurality of synthetic polymer particles in the liquid medium, and the microorganism, the said Forming a composite layer containing the microorganism, the plurality of synthetic polymer fine particles that retain the microorganism by buoyancy from below, and the binder material on the liquid surface of the aqueous layer formed by the liquid medium ;
Overlaying an organic layer comprising a hydrophobic substrate and a hydrophobic organic solvent on top of the composite layer;
The microorganisms of the composite layer as biocatalysts, look including the steps of: reacting the hydrophobic substrate,
The binder material is a carboxylated cellulose having an etherification degree of 20 to 30 mol%, or a polyvinyl alcohol derivative having a butyral group, a hydroxy group, and an acetyl group having an etherification degree of 60 to 70 mol%. Substance conversion method. The substance conversion method according to claim 6 , wherein the hydrophobic substrate is converted into a hydrophilic compound by the microorganism, and the compound is accumulated in the aqueous layer. The substance conversion method according to claim 6 , wherein the hydrophobic substrate is converted into a hydrophobic compound by the microorganism, and the compound is accumulated in the organic layer. In a culture tank, an aqueous solution, a microorganism, a plurality of synthetic polymer fine particles having a specific gravity smaller than that of the aqueous solution, and a binder material for collecting the plurality of synthetic polymer fine particles in the aqueous solution are added and mixed, the liquid surface of the aqueous layer formed by an aqueous solution, viewed including the steps of forming said microorganism, said plurality of synthetic polymer particles that holds the buoyancy the microorganisms from the lower side, and a composite layer containing the binder material, the The biomaterial characterized in that the binder material is a carboxylated cellulose having an etherification degree of 20 to 30 mol%, or a polyvinyl alcohol derivative having a butyral group, a hydroxy group and an acetyl group having an etherification degree of 60 to 70 mol%. Reactor manufacturing method. A culture tank;
An aqueous solution contained in the culture vessel;
A composite layer containing microorganisms and formed on the liquid surface of the aqueous solution,
The composite layer is seen containing a plurality of synthetic polymer fine particles held by the buoyancy of the microorganism specific gravity smaller than the aqueous solution from the lower side, and a binder material to combine the plurality of synthetic polymer particles in the aqueous solution ,
The binder material is a carboxylated cellulose having an etherification degree of 20 to 30 mol%, or a polyvinyl alcohol derivative having a butyral group, a hydroxy group, and an acetyl group having an etherification degree of 60 to 70 mol%. Bioreactor. The bioreactor according to claim 10 , further comprising an organic layer including a hydrophobic organic solvent layered on an upper surface of the composite layer.