Method for producing phenolic compound

Abstract

To provide a method for producing a phenolic compound, comprising a step of separating the compound from a microbial culture system.SOLUTION: Provided is a production method comprising a step of separating a phenolic compound contained in a culture medium from the culture medium, by contacting an organic solvent with the culture medium in which microorganisms have been cultured, through a hollow fiber membrane. Specifically, a phenolic compound is produced by a step for utilizing a bioprocess system that cultures phenolic compound-producing microorganisms to separate the phenolic compound into an organic solvent through a hollow fiber membrane.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to a method for producing a phenol compound using a microorganism. [Background technology]

[0002] In the field of chemical manufacturing, efforts are being made to switch from petroleum-derived raw materials to biomass-derived raw materials. Patent Document 1 discloses a technology for fermentatively producing phenol from glucose using a microorganism with an altered sugar metabolic pathway.

[0003] Non-Patent Document 1 also discloses a technology for producing phenol by a bioprocess using phenol-producing bacteria. The technology disclosed in Non-Patent Document 1 includes a step of culturing phenol-producing bacteria in the presence of a phenol extractant, but the phenol extractant is limited to an organic solvent that does not inhibit the growth of microorganisms.

[0004] Low molecular weight compounds such as fermentation products produced using microorganisms, enzymes for biopharmaceuticals, and various alkaloids are generally water-soluble and accumulate in microbial culture systems without adversely affecting the growth of the microorganisms. However, when attempting to expand the use of bioprocesses to the general production of chemicals, such as general-purpose chemicals, the accumulation of such hydrophobic substances (aliphatic hydrocarbon compounds such as fatty acids and their esters) can have an adverse effect on the growth of the microorganisms and their ability to produce substances, particularly in the bioprocess production of hydrophobic substances, and therefore the separation and recovery of hydrophobic substances from the bioreactor has been a challenge.

[0005] As a solution to this problem, a two-phase culture system as described in Non-Patent Document 2 has attracted attention. A two-phase culture system is a culture system formed of two phases, an aqueous phase in which microorganisms are cultured and an organic phase consisting of an organic solvent, and hydrophobic substances produced by microorganisms are extracted from the aqueous phase to the organic phase. The advantage of a two-phase system is that it is possible to lower the concentration of hydrophobic substances in the aqueous phase by distributing the products unevenly in the organic phase, preventing unnecessary contact with the microorganisms in the aqueous phase, and making it possible to suppress the inhibition of growth and production of microorganisms caused by the influence of hydrophobic substances. In fact, it has been reported that a two-phase culture system can produce hydrophobic substances more effectively than a culture system using only an aqueous phase. However, since the two-phase culture system is a batch-type liquid-liquid extraction, it is not a practical process because extraction cannot be performed continuously. In addition, the liquid-liquid interfacial area is limited, which causes problems with the low extraction (production) amount of hydrophobic substances. As an alternative bioprocessing system that does not rely on such a two-phase culture system, a bioprocess that uses a hollow fiber membrane to extract and separate hydrophobic substances from the culture system through the hollow fiber membrane and transport them out of the system is being investigated.

[0006] Furthermore, there are particular difficulties in the production of phenols by microorganisms, which is the object of the present invention. One of these difficulties is that phenols have a low solubility in water and an extremely high cytotoxicity. That is, phenols are difficult to accumulate in the body of a microorganism, and are secreted from the microorganism and accumulated in the medium. However, phenols, which are more hydrophobic than fatty acids, have a low solubility in the medium and undergo phase separation, making them difficult to recover. Furthermore, since the phenols accumulated in the medium have an extremely high cytotoxicity, they have a negative effect on the growth and productivity of the microorganisms, and therefore it has been extremely difficult to efficiently produce phenols by bioprocesses. Therefore, there has been a demand for a process for efficiently producing phenols by efficiently separating and extracting phenols from a medium containing microorganisms without adversely affecting the growth and productivity of the microorganisms. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication Pamphlet WO2012 / 033112 [Non-patent literature]

[0008] [Non-Patent Document 1] B. Kim et al., Metabolic engineering of Escherichia coli for the production of phenol from glucose, 9(5), 621-629(2014). [Non-Patent Document 2] Takiguchi, Noboru. "Two-phase culture system as a field for hydrophobic substance production," Journal of Bioengineering, 90(7) 438 (2012). Summary of the Invention [Problem to be solved by the invention]

[0009] In two-phase culture systems, there are issues such as toxicity of the organic phase to the bacteria and a decrease in yield due to emulsification of the aqueous and organic phases. Furthermore, in the case of a batch-type two-phase culture system, it is not a practical process because extraction cannot be performed continuously, and the extraction amount (production amount) of hydrophobic substances is low due to the limited interfacial area between the liquid and liquid. In view of the above circumstances, the present invention aims to provide a method for producing phenols by a bioprocess using microorganisms, which can efficiently produce phenols without adversely affecting the growth and productivity of the microorganisms by efficiently separating and extracting phenols from a culture medium containing the microorganisms. [Means for solving the problem]

[0010] In order to solve the above problems, the present disclosure provides the following [1]-[5]. [1] A method for producing phenols by culturing a microorganism that produces phenols, comprising a step of separating the phenols contained in the culture solution from the culture solution by contacting an organic solvent with a culture solution in which the microorganism is cultured via a hollow fiber membrane. [2] The method according to [1] above, wherein the phenol is phenol. [3] The method according to [1] or [2] above, wherein the organic solvent is tributyrin. [4] The method according to the above [1] or [2], wherein the hollow fiber membrane is a polyvinylidene fluoride membrane prepared by adding a hydrophilizing agent. [5] The production method described in [1] or [2] above, wherein the microorganism is any one of yeast, coryneform bacteria, and Escherichia coli. Effect of the Invention

[0011] The present disclosure provides a technique that can be used to produce phenols from biomass. The production method of the present invention has the effect of efficiently separating and extracting phenols from a culture medium containing a microorganism, thereby enabling efficient production of phenols without adversely affecting the growth and productivity of the microorganism. [Brief description of the drawings]

[0012] [Figure 1] Cross-sectional photograph of PVDF / PVP hollow fiber membrane observed by SEM [Diagram 2] SEM image of the outer membrane surface of a PVDF / PVP hollow fiber membrane [Diagram 3] SEM image of the inner membrane surface of a PVDF / PVP hollow fiber membrane [Figure 4] Diagram of hollow fiber membrane module [Diagram 5] Schematic diagram of extraction flow using hollow fiber membrane module [Figure 6] Schematic diagram of the experimental system including a hollow fiber membrane module, an organic solvent circulating inside the hollow fiber membrane in the module, and a culture medium containing microorganisms circulating outside the hollow fiber membrane in the module. [Figure 7]A graph showing the time course of phenol concentration accumulated in the medium during yeast growth. [Figure 8] A graph showing the growth curves of yeast in media containing various concentrations of phenol. [Figure 9] A graph plotting the amount of phenol extracted from the culture medium using the hollow fiber membrane module and the phenol concentration in the aqueous phase (culture medium) versus the culture time. BEST MODE FOR CARRYING OUT THEINVENTION

[0013] Hereinafter, preferred embodiments for carrying out the present disclosure will be described. Note that the scope of the present invention is not limited to the following examples and illustrated examples, and many modifications and variations are possible.

[0014] (1) Microorganisms that produce phenols Examples of microorganisms that can be preferably used in the present invention include the following microorganisms: Escherichia bacteria such as Escherichia coli; Pseudomonas bacteria such as Pseudomonas putida; Corynebacterium bacteria such as Corynebacterium glutamicum; Rhodococcus bacteria such as Rhodococcus opacus; Brevibacterium bacteria such as Brevibacterium linens; Bacillus bacteria such as Bacillus subtilis; Pichia yeast such as Pichia pastoris; Saccharomyces yeast such as Saccharomyces cerevisiae; Aspergillus filamentous fungi such as Aspergillus oryzae, Aspergillus nidulans, and Aspergillus niger. The microorganisms can be used alone or in combination of two or more types selected from these. Furthermore, the microorganism is not limited to a wild-type strain, but may be a mutant strain obtained by subjecting the wild-type strain to a mutation treatment such as UV irradiation or chemical treatment, or a genetic recombinant induced by a genetic technique such as cell fusion or gene recombination.

[0015] As a microorganism that can be preferably used in the present invention, a budding yeast is Pichia yeast, as shown in the Examples described below. Specifically, P. pastoris CBS7435 Δdnl4 Δhis4 strain in which genes of the phenol production pathway are introduced into Pichia yeast is used. Pichia yeast is used for substance production, and it is known that genes involved in the biosynthesis of various phenol derivatives are introduced into Pichia yeast to efficiently produce various phenols such as phenol, resveratrol, and narigenin. Another example of yeast that produces phenols is a system in which yeast (genus Brettanomyces) decarboxylates and reduces ferulic acid and coumaric acid contained in grape juice to produce 4-ethylphenol. As described above, by using various yeasts, it is possible to produce various useful substances such as phenol, various stilbenoids, flavonoids, and benzylisoquinoline alkaloids by bioprocessing. These accumulate in the medium and are soluble in various organic solvents, so that by using them in the present invention, it is possible to efficiently extract and separate phenols from the medium to produce them.

[0016] As a medium for culturing yeast, a medium containing yeast extract, peptone, and glycerol is preferably used, with the yeast extract concentration being preferably in the range of 5 to 100 g / L, the peptone concentration being preferably in the range of 10 to 200 g / L, and the glycerol concentration being preferably in the range of 1 to 100 g / L. It is preferable to add a solution containing each of these components to the medium during the culture in accordance with the growth rate of the yeast, and in this case, it is preferable to add a concentrated solution with the concentration of each component in the solution added exceeding the above concentration range, and it is possible to keep the increase in the volume of the entire medium within an appropriate range. In addition to these components, the medium may contain various amino acids such as leucine, tryptophan, uracil, and histidine, nucleic acids, and various inorganic salts such as ammonium sulfate, magnesium sulfate, calcium chloride, and sodium chloride.

[0017] Examples of microorganisms other than yeast that can be preferably used in the present invention include coryneform bacteria. Determinant Bacteriology, Vol. 8, 599 (1974)), and is not particularly limited as long as it grows under normal aerobic conditions. Specific examples include bacteria of the genus Corynebacterium, Brevibacterium, and Arthrobacter, and among coryneform bacteria, bacteria of the genus Corynebacterium are particularly preferred. Among them, Corynebacterium glutamicum is particularly used for the production of various amino acids and nucleic acids including glutamic acid, and phenols such as 4-hydroxybenzoic acid (4-HBA), proprotocatechuic acid, shikimic acid, and phenol. All of these are released from the bacterial cells into the medium and are soluble in organic solvents, and therefore can be extracted with various organic solvents that can be used in the present invention. Therefore, the present invention can be preferably applied to bioprocesses that utilize bacteria of the genus Corynebacterium.

[0018] As a medium for culturing Corynebacterium bacteria, medium A (Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions, J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)). The culture temperature may be about 15 to 45°C, and the culture time may be about 1 to 7 days. Furthermore, it is possible to promote the production of various target phenols by suppressing the growth of bacteria of the genus Corynebacterium, and in this case, it is also preferable to reduce the concentration of biotin in the above-mentioned medium A.

[0019] Furthermore, Escherichia coli can be mentioned as a microorganism other than yeast that can be preferably used in the present invention. It has been shown that it is possible to produce phenols in a bioprocess using genetically modified Escherichia coli. In addition, a method is known in which a gene encoding tyrosine-phenol lyase is incorporated into Escherichia coli with an enhanced tyrosine biosynthesis system to produce phenol from glucose as a raw material (Non-Patent Document 1 mentioned above). Non-Patent Document 1 reports that growth inhibition of Escherichia coli is reduced and phenol production is improved by 2.2 times compared to monophase culture by efficiently transferring phenol to the organic phase in a two-phase culture system in which tributyrin is added to the medium.

[0020] (2) Organic solvent used as the organic phase In the present invention, examples of organic solvents that can be preferably used when extracting and separating phenols from a medium include glycerin triesters to which a carboxylic acid having 3 to 12 carbon atoms is bonded, and phosphoric acid triesters to which an alcohol having 3 to 12 carbon atoms is bonded. As glycerin triesters, lauric acid, undecenoic acid (having 12 carbon atoms), capric acid (having 10 carbon atoms), caprylic acid (having 8 carbon atoms), tributyrin (glycerol tributyrate), tripropionin (glycerol tripropionate), and the like can be preferably used. Alternatively, as phosphoric acid esters, phosphoric acid triesters to which an alcohol having 3 to 12 carbon atoms, such as trioctylphosphine oxide, is bonded can be preferably used. All of these can be preferably used as organic solvents that are substantially insoluble in water at room temperature, and therefore do not exhibit growth toxicity to microorganisms that produce phenols. In addition to the above glycerol triesters and phosphate triesters, other organic solvents may be included. In this case, examples of organic solvents that can be used include tertiary amines to which an aliphatic hydrocarbon having 8 or more carbon atoms may be substituted. Specific examples of such organic solvents include trioctylamine and tridecylamine. In this case, the extraction rate and recovery rate of phenols from the medium may be further increased. Alternatively, examples of other organic solvents that can be used in combination include aliphatic alcohols having 6 to 12 carbon atoms. The recovery rate of phenols from the medium can be increased by adding these aliphatic alcohols.

[0021] (3) Membrane used in extracting phenols in the present invention In the present invention, a hollow fiber membrane is used. The function of the hollow fiber membrane in the present invention is to physically stabilize the interface between the aqueous phase and the organic phase of the medium by forming the interface inside the pores of the membrane in order to extract and separate phenols from the medium, and to significantly increase the surface area of ​​the interface by forming the interface over the entire membrane surface, thereby dramatically increasing the speed at which phenols move from the aqueous phase to the organic phase, and to isolate yeast and other fungi contained in the medium from the organic phase, thereby avoiding the biotoxicity of the organic phase.

[0022] The hollow fiber membrane that can be used in the present invention can be any membrane, regardless of its physical shape or chemical composition, as long as it can be used for the purpose of separating phenols from a culture solution containing phenols. For example, it is necessary for phenols produced by microorganisms in the aqueous phase to migrate to the organic phase, so that the pores must be present. The size and number of the pores can be appropriately selected depending on the type of solvent and the type of microorganism used. For example, the pore size can be set to 0.01 to 2 μm, preferably 0.02 to 1 μm, and more preferably 0.03 to 0.1 μm.

[0023] The hollow fiber membrane of the present invention may be surface-treated to prevent deterioration during use. When the hollow fiber membrane comes into contact with an organic solvent, the organic solvent may penetrate into the material constituting the hollow fiber membrane, causing the hollow fiber membrane to swell, or the hollow fiber membrane may be partially dissolved, resulting in a decrease in strength of the hollow fiber membrane and making it difficult to use it continuously for a long period of time. Alternatively, the organic solvent may leak into the culture medium, adversely affecting the growth of microorganisms in the culture medium. As a method for improving the surface properties of such a hollow fiber membrane, it is preferable to subject the membrane surface to a hydrophilic treatment. The hydrophilic treatment improves the resistance of the hollow fiber membrane to organic solvents and prevents the organic solvent from passing through the membrane and leaking into the culture medium.

[0024] As a method for hydrophilizing the interface between the hollow fiber membrane and the organic solvent, various hydrophilic materials can be blended with the membrane forming material to form the hollow fiber membrane when manufacturing the hollow fiber membrane. As a hydrophilizing material that can be used for such a purpose, hydrophilic polymers such as polyvinylpyrrolidone and hydrophilic inorganic pigments such as colloidal silica are preferable. Alternatively, after forming the hollow fiber membrane, the hydrophilic material can be exposed on the membrane surface by coating. Alternatively, when the material forming the hollow fiber membrane is a polymer material, it is also possible to manufacture a copolymer using various known methods and use this. As the hydrophilizing material in this case, a random copolymer, a block copolymer, a graft copolymer, etc. can be formed using vinylpyrrolidone, acrylic acid, acrylic acid-2-hydroxyethyl ester, acrylamide and its derivatives, vinyl acetate, other hydrophilic monomers, or copolymer monomers that are hydrophilized by hydrolysis or the like, and can be used.

[0025] As other hydrophilization treatments, various plasma treatments are also preferably used. In this case, the plasma treatment method is preferably a low-temperature plasma treatment method, and a method in which plasma generated between high-voltage electrodes under atmospheric pressure is brought into contact with the membrane surface to perform hydrophilization treatment is particularly preferred. In addition, it is also preferable to perform hydrophilization treatment on the membrane surface by vacuum plasma or low-temperature spraying of various ceramics, metals, etc.

[0026] Examples of materials constituting the hollow fiber membrane in the present invention include at least one selected from various polymers, ceramics, and metals, and are preferably used, which have good water resistance and chemical resistance, excellent mechanical strength, and excellent pressure resistance and heat resistance. In particular, it is preferable to use an organic polymer-based material that is easy to mold. More specifically, the membrane material is preferably made mainly of at least one selected from polyvinylidene fluoride, cellulose, polyolefin, polysulfone, polyethersulfone, polyvinyl chloride, polytetrafluoroethylene (PTFE), polyetherimide, and ceramic. Among them, it is particularly preferable to include polyvinylidene fluoride, since it has excellent organic solvent resistance, is easy to mold, and is relatively easy to control the pore size and porosity of the hollow fiber membrane. Furthermore, it is preferable that the surface of the hollow fiber membrane made of polyvinylidene fluoride is hydrophilized with polyvinylpyrrolidone, and in this case, it is particularly preferable to use a membrane made by blending polyvinylidene fluoride with polyvinylpyrrolidone during membrane production.

[0027] The shape of the hollow fiber membrane used in the present invention is a cylindrical or fibrous separation membrane with a hollow center, the longitudinal length is preferably in the range of 50 to 2000 mm, the average outer diameter is preferably in the range of 0.4 to 10 mm, and the inner diameter of the hollow fiber membrane is preferably in the range of 0.3 to 9.5 mm.

[0028] Furthermore, the solid layer sandwiched between the outer surface and inner diameter of the hollow fiber membrane forms a porous structure having a porosity preferably in the range of 5 to 90%, more preferably 30 to 90%, and the pore size on the outer surface of the porous structure exposed to the outer surface of the hollow fiber membrane is preferably in the range of 0.025 to 2 microns, and the pore size on the inner surface exposed to the inner diameter surface of the inner surface is preferably in the range of 0.025 to 2 microns. The sizes of the pores exposed on the surface and the inner surface may be the same or different, the pore size on the outer surface may be smaller than the pore size on the inner surface, and the mesh structure forming the separation layer may be formed with a mesh density that is inclined from dense to sparse or vice versa from the outer surface to the inner surface. Here, the culture medium and the organic solvent may be placed either outside or inside the hollow fiber membrane. That is, by placing a culture medium containing a microorganism that produces phenols in the internal flow path of the hollow fiber membrane and placing an organic solvent on the outer surface of the hollow fiber membrane, it is possible to efficiently extract the produced phenols. Alternatively, it is also possible to place an organic solvent in the internal flow path of the hollow fiber membrane and extract and separate phenols from the culture medium outside the hollow fiber membrane into the organic solvent inside the hollow fiber membrane.

[0029] The hollow fiber membrane that can be used in the present invention has a substance layer with a pore size and porosity in the above-mentioned range, but it is preferable to optimize the pore size and porosity in order to prevent leakage of organic solvent into the medium and to increase the extraction efficiency of phenols. For this purpose, additives can be added to the material used to manufacture the hollow fiber membrane to control the pore size and porosity of the substance layer, and soluble pore formers that are known as various pore formers can be used as additives. For example, various inorganic salts such as table salt, various sugars such as sucrose, polymer particles such as acrylic or styrene-based polymer particles, and silicone-based particles such as colloidal silica, whose size and shape can be controlled in advance, can be used. Alternatively, when producing hollow fiber membranes, non-solvent induced phase separation (NIPS) or thermally induced phase separation (TIPS) can be used to induce a phase separation structure by combining two or more incompatible polymers as materials, and one of the polymer components can be selectively dissolved and removed to produce hollow fiber membranes with various pore sizes and porosities. There are many immiscible, dissolvable polymers available for this purpose, of which polyvinylpyrrolidone is preferred.

[0030] A single hollow fiber membrane or a plurality of hollow fiber membranes may be used. When a plurality of hollow fiber membranes are used, they may be arranged in parallel to each other and bundled together, and the hollow fiber membranes may be arranged in contact with each other or at appropriate intervals.

[0031] In the process of separating phenols from a medium using a hollow fiber membrane, an organic solvent that dissolves phenols and is not miscible with water is used, and the water in the medium and the organic solvent are separated through the hollow fiber membrane, so that the phenols can be extracted and separated from the medium into the organic solvent. The organic solvent used must be substantially insoluble in water at room temperature so that the medium and the organic solvent do not come into contact with each other through the hollow fiber membrane and do not mix or emulsify. The medium and the organic solvent may be placed either outside or inside the hollow fiber membrane, and it is possible to efficiently extract the phenols produced by placing a medium containing a microorganism that produces phenols in the internal flow path of the hollow fiber membrane and placing an organic solvent on the outer surface of the hollow fiber membrane. Alternatively, it is also possible to place an organic solvent in the internal flow path of the hollow fiber membrane and extract and separate phenols from the medium outside the hollow fiber membrane into the organic solvent inside the hollow fiber membrane. In any of these cases, the extraction efficiency of phenols can be increased by adjusting the flow rate of the fluid flowing inside the hollow fiber membrane or adjusting the pressure inside or outside the hollow fiber membrane. The hollow fiber membrane may be used in a form in which both ends of the internal liquid supply side and permeation side are open, or in a form in which one side of the permeation side is sealed with potting. The hollow fiber membrane can be used in various ways together with a bioreactor used for culture as a separation device integrated as a module.

[0032] For example, as shown in the examples described below, a method may be used in which a hollow fiber membrane module prepared by bundling a plurality of hollow fiber membranes is used, an organic solvent is introduced into the hollow fiber membrane, and a culture medium is placed on the outside of the hollow fiber membrane inside the module, or a method in which the hollow fiber membrane module is directly immersed inside the bioreactor. For example, as shown in JP-A-2020-532414, a method may be used in which a plurality of hollow fiber membranes sealed on one side by potting are arranged inside a cylindrical space, and a device incorporating a cylindrical hollow fiber membrane is immersed in the culture medium inside the bioreactor as a filter probe with each of them arranged at an appropriate interval, and useful components are filtered and aspirated from the culture medium.

[0033] In the present invention, the function of the hollow fiber membrane is to remove, by means of a filter function, yeast, other fungi and insoluble components contained in the culture medium so as not to mix with the organic phase in order to extract and separate phenols from the culture medium. In addition, the function of the hollow fiber membrane is to physically stabilize the separation interface between the aqueous phase (i.e. the culture medium) and the organic phase by forming the interface inside the pores of the hollow fiber membrane, and to significantly increase the surface area of ​​the interface by forming such a separation interface over the entire surface of the hollow fiber membrane, thereby dramatically increasing the speed at which phenols transfer from the aqueous phase to the organic phase. That is, in the conventional two-phase culture system, the organic phase exists as a phase-separated layer on the upper surface of the medium, but the interface can be disturbed by stirring or an emulsified state can occur, causing contact between the phenol-producing microorganisms and the organic phase, which can have a detrimental effect on the growth of the microorganisms. In contrast, when a hollow fiber membrane is used, the interface between the aqueous phase and the organic phase is formed inside the countless minute pores formed on the outer surface of the hollow fiber membrane, so the stability of the liquid-liquid interface inside the pores is not lost even when disturbed from the outside, such as by stirring. When water and an organic solvent face each other on both sides of a membrane with fine pores, it is thought that each liquid penetrates into the pores by capillary action. In this case, the driving force that causes each phase to penetrate into the pores can be explained by the capillary pressure (Δp) expressed by the Laplace equation in the following formula 1.

[0034]

number

[0035] In the above formula 1, γ lv is the surface tension of the liquid, θ is the contact angle of the liquid, and r p represents the radius of the pore. Therefore, the smaller the pore diameter, the greater the capillary force that acts on the liquid, but on the other hand, if the liquid has poor wettability with the hollow fiber membrane surface, it will be expelled from the pores. Therefore, the wettability of each liquid with the hollow fiber membrane surface is very important.

[0036] When a liquid penetrates into a pore, the change in the penetration distance h(t) from the surface over time t is expressed by the Lucas-Washburn equation, Equation 2 below.

[0037]

number

[0038] When the aqueous phase and the organic phase confront each other inside the pores, the two are in equilibrium at a point where their respective permeation distances depend on the surface tension, wettability, and viscosity (η) included in the above formula. Therefore, when the pore size is large or the wettability is excessively good, the liquid passes through the pores and flows out to the opposite side, so in order to stably maintain a liquid-liquid interface in the hollow fiber membrane, it is necessary to adjust the wettability of each phase and the pore size.

[0039] In the present invention, as shown in the examples described later, pores are formed by adding hydrophilic polyvinylpyrrolindone to polyvinylidene fluoride during the formation of the hollow fiber membrane. By appropriately hydrophilizing the highly hydrophobic polyvinylidene fluoride, the membrane exhibits appropriate wettability with respect to both the aqueous and organic phases, and the pore size on the surface of the hollow fiber membrane is adjusted to keep the penetration of both phases within an appropriate range.

[0040] A preferred embodiment of the present invention is a method for separating and recovering phenols in which the organic solvent introduced into the inside of the hollow fiber is circulated by a pump, and the culture system and the organic solvent are continuously in contact with each other through the hollow fiber membrane. When the culture system is placed inside the hollow fiber membrane, the surface of the hollow fiber membrane may be covered with various insoluble components generated during the culture, causing the pores to become clogged, which may hinder the diffusion of the generated phenols. In contrast, a system in which an organic solvent is placed inside the hollow fiber membrane is preferable because the hollow fiber membrane is less likely to become clogged and can be used for long-term culture. In this case, in order to separate and recover phenols more efficiently, it is more preferable to circulate the organic solvent in the hollow fiber membrane with a pump or the like. The phenols extracted into the organic solvent can be preferably separated and recovered from the organic solvent by contacting it with a solution containing an alkali such as sodium hydroxide.

[0041] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the examples, percentages are by weight unless otherwise specified. Example 1

[0042] (Preparation of hollow fiber membrane) N,N-dimethylacetamide (DMAC) was used as a solvent, and 18 parts of polyvinylidene fluoride (PVDF; Arkema (registered trademark) Kynar) and 2 parts of polyvinylpyrrolidone (PVP; K-30; Fujifilm Wako Pure Chemical Industries) were added to 80 parts of DMAC, and the mixture was left overnight at room temperature to obtain a uniformly dissolved solution. In order to produce a hollow fiber membrane, a non-solvent induced phase separation method (NIPS method) was used, and a PVDF solution containing PVP was introduced into the outer periphery of a double-tube nozzle, and at the same time, ion-exchanged water was introduced as a coagulation liquid into the central outlet of the double-tube nozzle. The hollow fiber membrane consisting of a PVDF / PVP blend membrane precipitated by introducing the PVDF solution and the coagulation liquid (ion-exchanged water) into a coagulation bath filled with water adjusted to 40 ° C. while simultaneously discharging the PVDF solution and the coagulation liquid (ion-exchanged water) from the double-tube nozzle was wound up, and the hollow fiber membrane was wound up from the coagulation bath using a roller and wound up on a winder drum. The air gap between the tip of the double-tube nozzle and the water surface of the coagulation bath was set to 15 cm, and the winding speed was set to 5.5 m / min.

[0043] The wound hollow fiber membrane sample was immersed in tap water for 2 days, and then immersed in a 50% glycerin aqueous solution at room temperature for 3 days to remove soluble components from the membrane, and then the glycerin was removed and the membrane was thoroughly dried at room temperature. The cross section, surface and inner membrane surface of the sample were observed using a scanning electron microscope (FE-SEM). The results are shown in Figures 1 to 3. Figure 1 shows a cross section photograph of the PVDF / PVP hollow fiber membrane produced in this example by SEM observation, Figure 2 shows an SEM observation image of the outer surface of the hollow fiber membrane, and Figure 3 shows an SEM observation image of the inner membrane surface of the hollow fiber membrane. The outer diameter of the obtained hollow fiber membrane was 2.2 mm, the inner diameter was 2.0 mm, and the thickness of the solid layer was 0.2 mm.

[0044] From the cross-sectional observation in Figure 1, it was observed that the average thickness of the substantive layer of the hollow fiber membrane was 0.2 mm, and that the inside of the substantive layer formed a mesh structure with pores of around 50 μm on average. From the cross-sectional photograph, it was confirmed that the outer surface was covered with a dense skin layer. It was confirmed that voids were formed below the skin layer, which were connected to a large mesh structure inside the substantive layer, and further connected to voids formed on the inner surface of the hollow fiber membrane and communicated with the porous membrane on the inner surface. The porosity of the substantive layer was calculated to be 87% from the ratio of the mass and volume of the substantive layer.

[0045] The average diameters of the pores formed on the outer and inner surfaces of the hollow fiber membrane were found to be 0.037 μm and 0.063 μm, respectively, from the enlarged images in Figures 2 and 3. The outer surface of the hollow fiber membrane forms a dense overall surface with minute pores, but the inner surface showed the appearance of relatively large voids, as confirmed by the cross-sectional images. Example 2

[0046] (Preparation of hollow fiber membrane module) The hollow fiber membrane prepared in Example 1 was used and cut to a length of 31 cm. 20 hollow fiber membranes were bundled together and fixed in a tube with both ends open using an isocyanate adhesive. Two openings were provided in the tube perpendicular to the longitudinal direction of the hollow fiber membrane, providing a structure that allows the culture medium containing microorganisms to circulate. Figures 4 and 5 show the configuration of the hollow fiber membrane module prepared in this example and a schematic diagram of the extraction flow, respectively.

[0047] As shown in Fig. 4, the hollow fiber membrane module 10 is configured such that bundled hollow fiber membranes 1 (PVDF / PVP membranes) are connected at both ends to separate tube piping connected to the outside via connectors (15a, 15b), and organic solvents (7a, 7b) in containers provided on the outside are circulated inside the hollow fiber membranes 1. An inlet opening 19a and an outlet opening 19b are provided on the outside of the hollow fiber membranes 1 in the hollow fiber membrane module 10 so that a culture medium 12 containing microorganisms can be circulated. Phenols 14 produced outside the body by fine particles enter the organic solvent inside the hollow fiber membranes 1 from the culture medium 12 containing microorganisms outside the hollow fiber membranes 1 in the hollow fiber membrane module 10. The volume fraction of the hollow fiber membrane module that was fabricated was 44%, and the surface area of ​​the hollow fiber membrane was 213 cm. 3 It was.

[0048] The extraction flow in the hollow fiber membrane 1 in the hollow fiber membrane module will be described with reference to Fig. 5. Fig. 5(1) shows a plurality of cylindrical hollow fiber membranes 1 in the hollow fiber membrane module 10 shown in Fig. 4 and the medium (aqueous phase) 12 containing microorganisms around them. Fig. 5(2) is an enlarged image of the dashed line A in Fig. 5(1), and shows one cylindrical hollow fiber membrane 1, the medium (aqueous phase) 12 containing microorganisms around it, and the organic phase 11 of the organic solvent flowing inside the hollow fiber membrane 1. The diagram shows the state in which the microorganism 16 takes in sugars 17 and extracts the phenols 18 produced outside the body from the aqueous phase 12 into the organic phase 11 via the hollow fiber membrane 1.

[0049] The appearance of the experimental system is shown in a model diagram in FIG. 6. FIG. 6 is a schematic diagram including a hollow fiber membrane module 10 used in this example, an organic solvent 7 circulating inside the hollow fiber membrane in the module, and a culture medium 6 containing microorganisms circulating outside the hollow fiber membrane in the module. Each liquid is stirred in its respective container (8a, 8b, 9a, 9b) and pumped by pumps (2a, 2b), and the flow rate and pressure of the liquid are measured by flow meters (3a, 3b) and pressure gauges (4a, 4b), respectively. Note that the arrangement of the pumps is not limited to the arrangement in FIG. 6. Example 3

[0050] (Yeast culture and phenol tolerance evaluation) Prior to the test using the hollow fiber membrane module of Example 2, the phenol production curve due to yeast growth and the tolerance of the yeast to the phenol concentration in the medium were evaluated. The yeast used was P. pastoris CBS7435 Δdnl4 Δhis4 strain (47m-Cf_tpl strain), in which one gene of the phenol production pathway was introduced into Pichia yeast. The medium was a mixture of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glycerol, and the rest of the medium had the same composition as the standard, and the culture was performed with shaking at 30°C. The phenol-producing bacteria was pre-cultured to ensure a sufficient number of cells required for the following experiments.

[0051] 5 mL of medium containing pre-cultured yeast was introduced into an L-type tube so that the initial OD660 was 0.05. The concentration of phenol accumulated in the medium due to yeast growth was determined by directly sampling the medium and measuring the concentration of phenol contained in the medium by HPLC. The results are shown in Figure 7. Figure 7 shows the time course of the phenol concentration accumulated in the medium due to yeast growth. It can be seen from this figure that the rate of increase in phenol concentration slows down once the phenol concentration in the medium exceeds 500 mg / L.

[0052] 5 ml of the medium obtained from the preculture containing yeast was introduced into an L-shaped tube, and phenol was added to each tube at five levels between 100 and 2000 mg / L. The tubes were incubated at 30°C and 40 rpm with shaking, together with the tubes that did not contain phenol. The OD660 was measured with a biophoto recorder (TVS062CA, Advantec) to monitor the cell growth every 20 minutes. The results are shown in Figure 8. Figure 8 shows the growth curves of yeast in media containing phenol at various concentrations. From the results shown in Figure 8, it was confirmed that the growth of phenol-producing bacteria was inhibited when the phenol concentration in the medium was 500 mg / L or higher. Example 4

[0053] (Phenol extraction test using yeast culture in a jar fermenter and a hollow fiber membrane module, and a comparative example in which phenol extraction is not performed) Using the hollow fiber membrane module prepared in Example 2, a 2 L jar fermenter containing a medium (volume 1 L) was connected to a container containing an organic solvent by a pipe as shown in FIG. 6. The yeast of Example 3 was placed in the 2 L jar fermenter together with the medium. The culture conditions in the jar fermenter were 30° C., 100 ml / min air flow, and stirring at a stirring speed of 400 rpm while maintaining the pH at 6.0 (ammonia water was added appropriately). From 30 hours to 96 hours after the start of culture, a solution containing yeast extract at a concentration of 50 g / L, peptone at a concentration of 100 g / L, and glycerol at a concentration of 200 g / L was added to the medium at a flow rate of 5 ml / h at 15-minute intervals.

[0054] Tributyrin was used as the organic solvent for phenol extraction. The flow rates of the aqueous and organic phases fed to the hollow fiber membrane module were set at 210 ml / min and 250 ml / min, respectively. Circulation of the organic phase began 30 hours after the start of cultivation, and phenol was extracted from the medium continuously for 18 hours, from the start of cultivation until 48 hours later. During this time, 1 ml samples were taken from the aqueous and organic phases every 6 hours, and the concentration of phenol dissolved in each phase was measured using HPLC. Similarly, phenol was extracted by circulating the organic phase between 54 and 72 hours after the start of cultivation and between 78 and 96 hours after the start of cultivation, and phenol was quantified by sampling each phase every 6 hours.

[0055] For comparison, cultivation was carried out under the same conditions, and the phenol concentration in a liquid sampled from the aqueous phase (culture medium) was measured without extracting phenol using the hollow fiber membrane module. This was set as Comparative Example 1. In addition, a culture experiment was carried out in a two-phase culture system in which tributyrin was added directly to the medium without using a hollow fiber membrane module as Comparative Example 2. Tributyrin was added to the medium in an amount of 30 parts by volume per 100 parts by volume of the medium, and culture was carried out with stirring in the same manner, but the organic phase was emulsified and mixed, inhibiting yeast growth and preventing phenol production.

[0056] The results of Example 4 and Comparative Example 1 are shown together in Figure 9. Figure 9 is a diagram plotting the amount of phenol extracted from the medium using the hollow fiber membrane module in Example 4 and the phenol concentration contained in the aqueous phase (medium) against the culture time, and also shows the amount of phenol contained in the medium when culture was carried out in the same manner as in Comparative Example 1 without using the hollow fiber membrane module.

[0057] 9, each bar indicates the amount of phenol contained in the aqueous phase measured at each time point at the bottom, and the amount of phenol contained in the organic phase at the top. The marker ◇ shown in the graph indicates the amount of phenol contained in the medium determined in Comparative Example 1. 9, it was confirmed that in Comparative Example 1, the phenol concentration in the medium was almost constant throughout the culture period, whereas in Example 4, when phenol was extracted into the organic phase using the hollow fiber module, the amount of extracted phenol increased with the culture time. In addition, since the amount of phenol contained in the aqueous phase remained at a level that did not adversely affect the growth of yeast, this example demonstrated that efficient production of phenols and efficient extraction and separation of phenol into an organic solvent through the hollow fiber membrane could be performed efficiently without affecting the growth of phenol-producing microorganisms. [Industrial Applicability]

[0058] The hollow fiber membrane produced by the present invention can efficiently extract and separate various phenols from a culture system, and therefore can be used for the production of phenols for various industrial and food applications. It can also be used in bioprocesses involving microorganisms, as well as in bioreactors using various enzymes, and for water treatment and other applications. [Explanation of symbols]

[0059] 1 Hollow fiber membrane (PVDF / PVP membrane) 2a,2b Pump 3a, 3b Flow meter 4a, 4b Pressure gauge 5a,5b Valve 6. Culture medium 7,7a,7b Organic Solvents 8a, 8b Motor 9a,9b Stirring blade 10 Hollow fiber membrane module 11 Organic phase 12 Culture medium containing microorganisms (aqueous phase) 14,18 Phenols 15a,15b Connector 16 microorganisms 17 saccharides 19a outlet side opening 19b inlet side opening

Claims

1. A method for producing phenols by culturing a microorganism that produces phenols, the method comprising a step of separating the phenols contained in the culture solution from the culture solution by contacting an organic solvent with a culture solution in which the microorganism is cultured via a hollow fiber membrane.

2. The method according to claim 1 , wherein the phenol is phenol.

3. The method according to claim 1 or 2, wherein the organic solvent is tributyrin.

4. 3. The method according to claim 1, wherein the hollow fiber membrane is a polyvinylidene fluoride membrane prepared by adding a hydrophilizing agent.

5. The method according to claim 1 or 2, wherein the microorganism is any one of yeast, coryneform bacteria, and Escherichia coli.

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