WO2013105651A1 - 化学品の製造方法 - Google Patents
化学品の製造方法 Download PDFInfo
- Publication number
- WO2013105651A1 WO2013105651A1 PCT/JP2013/050435 JP2013050435W WO2013105651A1 WO 2013105651 A1 WO2013105651 A1 WO 2013105651A1 JP 2013050435 W JP2013050435 W JP 2013050435W WO 2013105651 A1 WO2013105651 A1 WO 2013105651A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fermentation
- culture
- medium
- membrane
- xylose
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/54—Acetic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
- C12P7/60—2-Ketogulonic acid
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to a method for producing a chemical product by continuous fermentation using a fermentation raw material containing hexose and pentose.
- Biomass-derived chemicals such as biodegradable polymer raw materials such as lactic acid and biofuels such as ethanol have become sustainable (sustainable) as well as carbon dioxide emissions into the atmosphere and the emergence of energy problems. It is attracting a great deal of attention as a life cycle assessment (LCA) compatible product.
- LCA life cycle assessment
- As a method for producing these biodegradable polymer materials and biofuels glucose, which is a hexose refined from edible biomass such as corn, is generally used as a fermentation material for microorganisms, and is obtained as a fermentation product. If edible biomass is used, competition with food causes price increases, which may prevent stable procurement of raw materials. Therefore, attempts have been made to use sugar derived from non-edible biomass such as rice straw as a fermentation raw material for microorganisms (see Patent Document 1).
- sugars derived from non-edible biomass are used as fermentation raw materials
- cellulose, hemicellulose, etc. contained in non-edible biomass are decomposed into sugars by saccharifying enzymes.
- saccharifying enzymes not only hexose sugars such as glucose are decomposed.
- Pentoses such as xylose can be obtained at the same time.
- sugars derived from non-edible biomass are used as fermentation raw materials for microorganisms, mixed sugars of hexose and pentose sugars are used as fermentation raw materials (patents) Reference 1).
- Continuous fermentation can be adopted as a fermentation method using sugar derived from non-edible biomass, which is a mixed sugar of hexose sugar and pentose sugar, as a fermentation raw material for microorganisms. It has not been confirmed (see Patent Document 1).
- sugar derived from non-edible biomass which is a mixed sugar of hexose sugar and pentose sugar
- the batch is obtained by continuously receiving catabolite suppression compared to batch fermentation. It is also known that the fermentation yield is significantly lower than that of fermentation (see Non-Patent Document 1).
- microorganisms that undergo catabolite suppression are known as microorganisms that fermentatively produce biodegradable polymer raw materials and biofuels.
- microorganisms that fermentatively produce biodegradable polymer raw materials and biofuels.
- this invention makes it a subject to improve the fermentation yield in the case of continuously fermenting a mixed sugar of hexose and pentose as a fermentation raw material for microorganisms subject to catabolite suppression.
- the present inventor used a separation membrane for microorganisms having catabolite suppression in a method for producing a chemical that continuously fermented a mixed sugar of hexose and pentose as a fermentation raw material for microorganisms.
- the present inventors have found that the above-mentioned problems can be solved by performing continuous fermentation, which has led to the present invention.
- the present invention is as follows (1) to (5).
- a method for producing a chemical product by continuous fermentation wherein the microorganism is a microorganism that is subject to catabolite suppression, and the fermentation material contains hexose and pentose.
- a chemical product can be produced in a high yield despite the fact that a mixed sugar of hexose and pentose is used as a fermentation raw material for microorganisms that are subject to catabolite suppression.
- the present invention relates to a method for producing a chemical product in which a microorganism is cultured with a fermentation raw material to produce a chemical product by fermentation.
- the culture solution is filtered through a separation membrane, and the unfiltered solution is retained or refluxed in the culture solution.
- a method for producing a continuously fermented chemical that recovers a product in a filtrate in addition to a culture solution, wherein the microorganism that is subject to catabolite suppression is used, and the fermentation raw material is hexose and pentose. It is the manufacturing method of the chemical characterized by including.
- the carbon source of the fermentation raw material includes a mixed sugar containing pentose and hexose.
- the pentose sugar has five carbons constituting the sugar, and is also called pentose.
- the pentose used in the present invention may be any pentose as long as it can assimilate microorganisms, but is preferably xylose or arabinose, more preferably from the viewpoint of the natural ratio or availability. Is xylose.
- the hexose has 6 carbons constituting the sugar, and is also called hexose.
- aldose include glucose, mannose, galactose, allose, growth, and talose.
- ketose include fructose, psicose, and sorbose.
- the hexose used in the present invention may be any as long as it can assimilate microorganisms, but from the viewpoint of the natural ratio, availability, etc., preferably glucose, mannose, galactose, More preferred is glucose.
- the mixed sugar used in the present invention is not particularly limited, but a cellulose-containing biomass-derived sugar solution known to contain both hexose and pentose is preferably used.
- the cellulose-containing biomass include plant biomass such as bagasse, switchgrass, corn stover, rice straw, and straw, and woody biomass such as trees and waste building materials.
- Cellulose-containing biomass contains cellulose or hemicellulose, which is a polysaccharide obtained by dehydrating and condensing sugar, and a sugar solution that can be used as a fermentation raw material is produced by hydrolyzing such a polysaccharide. Any method may be used for preparing the cellulose-containing biomass-derived sugar solution.
- a method for producing a sugar solution by acid hydrolysis of biomass using concentrated sulfuric acid discloses a method for producing a sugar solution by hydrolyzing biomass with dilute sulfuric acid and further treating with an enzyme such as cellulase (A).
- Aden et al. “Lignocellulosic Biomass to Ethanol Process Design and Economics Optimized Co-Current Dirty Acid Prehydrology and Enzymatic Physiology Hydrology. ver "NREL Technical Report (2002)).
- a method not using an acid a method of hydrolyzing biomass using subcritical water at about 250 to 500 ° C.
- the weight ratio of the pentose and hexose contained in the mixed sugar is not particularly limited, but the weight ratio of the pentose and hexose in the mixed sugar is expressed as (pentose): (hexose). And 1: 9 to 9: 1 are preferred. This is a sugar ratio when a cellulose-containing biomass-derived sugar solution is assumed as a mixed sugar.
- the total sugar concentration contained in the fermentation raw material used in the present invention is not particularly limited and is preferably as high as possible as long as it does not inhibit the production of microbial chemicals.
- the concentration of the carbon source in the medium is preferably 15 to 500 g / L, more preferably 20 to 300 g / L. If the total concentration is 15 g / L or less, the effect of improving the yield from pentose may be reduced. In addition, when the total sugar concentration is low, the production efficiency of chemicals also decreases.
- the concentration of hexose contained in the fermentation raw material used in the present invention is not particularly limited in the range of the total sugar concentration and the ratio of pentose and hexose, but if the method for producing a chemical product of the present invention is used. A good yield can be obtained even in a mixed sugar solution containing hexose at a concentration of 5 g / L or more.
- the fermentation raw material used in the present invention is preferably a normal liquid medium appropriately containing a carbon source, a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins.
- nitrogen source used in the present invention examples include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysate, casein decomposition products, and others.
- inorganic salts phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and the like can be appropriately added and used.
- the nutrient is added as a preparation or a natural product containing it.
- An antifoaming agent is also used as necessary.
- the culture solution refers to a solution obtained as a result of the growth of microorganisms on the fermentation raw material. You may change suitably the composition of the fermentation raw material to add from the fermentation raw material composition at the time of a culture
- the porous membrane used in the present invention is not particularly limited as long as it has a function of separating and filtering a culture solution obtained by culturing using a microbial stirring type incubator or a stirring type bioreactor from microorganisms.
- Ceramic ceramic membranes, porous glass membranes, porous organic polymer membranes, metal fiber woven fabrics, nonwoven fabrics, and the like can be used. Among these, a porous organic polymer film or a ceramic film is particularly preferable.
- the configuration of a porous membrane used as a separation membrane in the present invention will be described.
- the porous membrane used in the present invention preferably has separation performance and water permeability according to the quality of water to be treated and the application.
- the porous membrane is preferably a porous membrane including a porous resin layer from the viewpoint of blocking performance, water permeability performance and separation performance, for example, stain resistance.
- the porous membrane including the porous resin layer preferably has a porous resin layer that acts as a separation functional layer on the surface of the porous substrate.
- the porous substrate supports the porous resin layer and gives strength to the separation membrane.
- the porous substrate used in the present invention has a porous resin layer on the surface of the porous substrate, the porous substrate is porous even if the porous resin layer penetrates the porous substrate. It does not matter if the porous resin layer does not penetrate, and it is selected according to the application.
- the average thickness of the porous substrate is preferably 50 ⁇ m or more and 3000 ⁇ m or less.
- the material of the porous substrate is made of an organic material and / or an inorganic material, and an organic fiber is preferably used.
- the preferred porous substrate is a woven or non-woven fabric made of organic fibers such as cellulose fiber, cellulose triacetate fiber, polyester fiber, polypropylene fiber and polyethylene fiber. More preferably, the density control is relatively easy and the production is easy. Inexpensive nonwoven fabric is used.
- An organic polymer film can be suitably used for the porous resin layer.
- the material of the organic polymer film include polyethylene resin, polypropylene resin, polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, polyacrylonitrile resin, cellulose resin, and the like. Examples thereof include cellulose triacetate resins.
- the organic polymer film may be a mixture of resins mainly composed of these resins.
- the main component means that the component is contained in an amount of 50% by weight or more, preferably 60% by weight or more.
- the organic polymer film is made of a polyvinyl chloride resin, a polyvinylidene fluoride resin, a polysulfone resin, a polyethersulfone resin, which is easy to form a film with a solution and has excellent physical durability and chemical resistance.
- Polyacrylonitrile-based resins are preferable, and polyvinylidene fluoride-based resins or resins containing them as the main components are most preferably used.
- polyvinylidene fluoride-based resin a homopolymer of vinylidene fluoride is preferably used.
- polyvinylidene fluoride resin a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride is also preferably used.
- vinyl monomers copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene, and ethylene trichloride fluoride.
- the porous membrane used as a separation membrane in the present invention is not particularly limited, and may be any microorganism as long as it does not pass through microorganisms used for fermentation. It is desirable that the clogging is less likely to occur and the filtration performance is within a range that continues stably for a long period of time. Therefore, the average pore diameter of the porous separation membrane is preferably 0.01 ⁇ m or more and less than 5 ⁇ m. More preferably, when the average pore diameter of the porous membrane is 0.01 ⁇ m or more and less than 1 ⁇ m, it is possible to achieve both a high exclusion rate at which microorganisms do not leak and a high water permeability. Holding for a long time can be carried out with higher accuracy and reproducibility.
- the average pore diameter of the porous membrane is preferably less than 1 ⁇ m.
- the average pore diameter of the porous membrane is preferably not too large compared to the size of the microorganism in order to prevent the occurrence of a problem that the microorganism leaks out, that is, the rejection rate decreases.
- the average pore diameter is preferably 0.4 ⁇ m or less, and more preferably less than 0.2 ⁇ m.
- the average pore size of the porous membrane in the present invention is preferably 0.01 ⁇ m. It is above, More preferably, it is 0.02 micrometer or more, More preferably, it is 0.04 micrometer or more.
- the average pore diameter can be obtained by measuring and averaging the diameters of all pores that can be observed within a range of 9.2 ⁇ m ⁇ 10.4 ⁇ m in a scanning electron microscope observation at a magnification of 10,000 times. it can.
- the average pore diameter can be obtained by taking a photograph of the surface of the membrane at a magnification of 10,000 using a scanning electron microscope and randomly selecting 10 or more, preferably 20 or more pores. It can also be obtained by measuring and number average.
- a circle having an area equal to the area of the pores (equivalent circle) can be obtained by an image processing apparatus or the like, and the equivalent circle diameter can be obtained by the method of setting the diameter of the pores.
- the standard deviation ⁇ of the average pore diameter of the porous membrane used in the present invention is preferably 0.1 ⁇ m or less.
- the standard deviation ⁇ of the average pore diameter is N as the number of pores that can be observed within the above-mentioned range of 9.2 ⁇ m ⁇ 10.4 ⁇ m, the measured diameter is X k, and the average pore diameter is X (ave ) And the following (Equation 1).
- the permeability of the fermentation broth is one of the important performances.
- the pure water permeability coefficient of the porous membrane before use can be used as an index of the permeability of the porous membrane.
- the pure water permeability coefficient of the porous membrane is 5.6 ⁇ 10 ⁇ 10 m when calculated by measuring the water permeability at a head height of 1 m using purified water at a temperature of 25 ° C. by a reverse osmosis membrane.
- a pure water permeability coefficient is 5.6 ⁇ 10 ⁇ 10 m 3 / m 2 / s / pa or more 6 ⁇ 10 ⁇ 7 m 3 / m 2 / s. If it is / pa or less, a practically sufficient amount of permeated water can be obtained.
- the surface roughness is the average value of the height in the direction perpendicular to the surface.
- the membrane surface roughness is one of the factors for facilitating separation of microorganisms adhering to the separation membrane surface by the membrane surface cleaning effect by the liquid flow by stirring or a circulation pump.
- the surface roughness of the porous membrane is not particularly limited as long as it is within a range in which microorganisms attached to the membrane and other solid substances are peeled off, but is preferably 0.1 ⁇ m or less. When the surface roughness is 0.1 ⁇ m or less, microorganisms adhering to the film and other solid substances are easily peeled off.
- the membrane surface roughness of the porous membrane is 0.1 ⁇ m or less, the average pore diameter is 0.01 ⁇ m or more and less than 1 ⁇ m, and the pure water permeability coefficient of the porous membrane is 2 ⁇ 10 ⁇ 9 m 3 / It has been found that by using a porous membrane of m 2 / s / pa or more, an operation that does not require excessive power required for membrane surface cleaning can be performed more easily.
- the surface roughness of the porous membrane to 0.1 ⁇ m or less, in the filtration of microorganisms, the shearing force generated on the membrane surface can be reduced, the destruction of microorganisms is suppressed, and the porous membrane is clogged.
- the surface roughness of the porous membrane is preferably as small as possible.
- the film surface roughness of the porous film is measured under the following conditions using the following atomic force microscope (AFM).
- FAM atomic force microscope
- Device Atomic force microscope device (“Nanoscope IIIa” manufactured by Digital Instruments)
- Condition probe SiN cantilever manufactured by Digital Instruments
- Scan mode Contact mode in-air measurement
- Underwater tapping mode underwater measurement
- Scanning range 10 ⁇ m, 25 ⁇ m square (measurement in air) 5 ⁇ m
- 10 ⁇ m square underwater measurement
- Scanning resolution 512 ⁇ 512 -Sample preparation The membrane sample was immersed in ethanol at room temperature for 15 minutes, then immersed in RO water for 24 hours, washed, and then air-dried.
- the RO water refers to water that has been filtered using a reverse osmosis membrane (RO membrane), which is a type of filtration membrane, to exclude impurities such as ions and salts.
- RO membrane reverse osmosis membrane
- the pore size of the RO membrane is approximately 2 nm or less.
- the film surface roughness d rough is calculated by the following (Equation 2) from the height of each point in the Z-axis direction by the above-described atomic force microscope (AFM).
- the shape of the porous membrane used in the present invention is preferably a flat membrane.
- the average thickness is selected according to the application.
- the average thickness when the shape of the porous membrane is a flat membrane is preferably 20 ⁇ m or more and 5000 ⁇ m or less, and more preferably 50 ⁇ m or more and 2000 ⁇ m or less.
- the shape of the porous membrane used in the present invention is preferably a hollow fiber membrane.
- the inner diameter of the hollow fiber is preferably 200 ⁇ m or more and 5000 ⁇ m or less
- the film thickness is preferably 20 ⁇ m or more and 2000 ⁇ m or less.
- a woven fabric or a knitted fabric in which organic fibers or inorganic fibers are formed in a cylindrical shape may be included in the hollow fiber.
- the above-mentioned porous membrane can be manufactured by the manufacturing method described in WO2007 / 097260, for example.
- the separation membrane in the present invention can be a membrane containing at least ceramics.
- the ceramics in the present invention refers to those containing a metal oxide and baked and hardened by heat treatment at a high temperature.
- the metal oxide include alumina, magnesia, titania, zirconia and the like.
- the separation membrane may be formed of only a metal oxide, and may include silica, silicon carbide, mullite, cordierite, or the like, which is a compound of silica and a metal oxide.
- Components other than the ceramics forming the separation membrane are not particularly limited as long as they can form a porous body as a separation membrane.
- the shape is not particularly limited, and any of monolithic membrane, flat membrane, tubular membrane and the like can be used. From the viewpoint of filling efficiency into the container, a columnar structure and a structure having a through hole in the longitudinal direction is preferable. A monolith film is preferred from the viewpoint of increasing the filling efficiency.
- the separation membrane When a separation membrane having a columnar structure is housed in a module container and used as a separation membrane module, the separation membrane can be modularized and filtered by selecting a suitable mode from an external pressure type and an internal pressure type.
- the side where the separation membrane is in contact with the fermentation broth is referred to as the primary side
- the side where the filtrate containing the chemical product is obtained by filtration is referred to as the secondary side.
- the output of the circulation pump can be saved when cross-flow filtration is performed because the flow path on the primary side is narrow. Furthermore, the action of discharging turbidity deposited on the surface of the separation membrane to the outside of the module becomes stronger, which is preferable because the surface of the separation membrane is easily kept clean.
- the internal pressure type separation membrane has an inlet and an outlet for the fermentation broth. In this case, it is preferable that the inlet and the outlet are in a state of through holes arranged in a straight line, since the liquid flow resistance becomes small. Further, since the separation membrane has a columnar structure and the through-holes are vacant in the longitudinal direction, the container for accommodating the separation membrane can be made thinner. When the separation membrane module is thin, it can be preferably used from the viewpoint of production and handling.
- the porosity of the separation membrane is not particularly limited, but if it is too low, the filtration efficiency will deteriorate, and if it is too high, the strength will decrease. In order to achieve both the filtration efficiency and the strength of the separation membrane and to have resistance to repeated steam sterilization, it is preferably 20% or more and 60% or less.
- the average pore diameter of the separation membrane is preferably 0.01 ⁇ m or more and 1 ⁇ m or less. If the membrane has an average pore diameter in this range, the separation membrane is less likely to block and the filtration efficiency is excellent. In addition, if the average pore size is in the range of 0.02 ⁇ m or more and 0.2 ⁇ m or less, fermentation by-products by microorganisms and cultured cells exemplified by proteins and polysaccharides, and microorganisms / cultured cells in the culture solution This is particularly preferable because clogging of a substance that easily clogs the separation membrane, such as a crushed cell produced by death, is less likely to occur.
- the separation membrane having a columnar structure having a through hole has a secondary surface on the secondary side, it is preferable to provide a module container for collecting filtrate and use the module as a module in which the separation container is filled.
- One or more separation membranes are filled in one module.
- the module container is preferably made of a material that can withstand repeated steam sterilization.
- raw materials that can withstand steam sterilization include stainless steel and ceramics having a low average porosity.
- Such a ceramic membrane module can be manufactured by, for example, a manufacturing method described in WO2012 / 086763, but a commercially available one can also be used. Specific examples of commercially available products include MEMBLAROX microfiltration membranes (Pall), ceramic membrane filter cefilt MF membranes (NGK), and the like.
- the transmembrane pressure difference during filtration is not particularly limited as long as the fermentation broth can be filtered.
- the organic polymer membrane is filtered at a transmembrane differential pressure higher than 150 kPa to filter the culture solution, the structure of the organic polymer membrane is likely to be destroyed, and the ability to produce chemicals is increased. May decrease.
- the transmembrane pressure is lower than 0.1 kPa, the permeated water amount of the fermentation broth may not be obtained sufficiently, and the productivity when producing a chemical product tends to decrease.
- the permeated water amount of the fermentation broth is increased by setting the transmembrane differential pressure, which is the filtration pressure, preferably in the range of 0.1 kPa to 150 kPa. Further, since there is no decrease in chemical production capacity due to destruction of the film structure, it is possible to maintain a high ability to produce chemical products.
- the transmembrane pressure difference is preferably in the range of 0.1 kPa to 50 kPa, and more preferably in the range of 0.1 kPa to 20 kPa.
- the transmembrane pressure difference during filtration is not particularly limited as long as the fermentation broth can be filtered, but is preferably 500 kPa or less.
- the operation is performed at 500 kPa or more, clogging of the membrane occurs, which may cause a problem in the continuous fermentation operation.
- a transmembrane differential pressure can be generated in the separation membrane by a siphon using a liquid level difference (water head difference) of the fermented liquid and the porous membrane treated water or a cross flow circulation pump.
- a suction pump may be installed on the secondary side of the separation membrane as a driving force for filtration.
- the transmembrane pressure difference can be controlled by the suction pressure.
- the transmembrane pressure difference can be controlled also by the pressure of the gas or liquid that introduces the pressure on the fermentation broth side.
- the concentration of the pentose in the total amount of filtrate that has passed through the separation membrane is maintained at 5 g / l or less.
- the medium is continuously used for fermentation.
- pentose remains in a large amount in the culture medium, resulting in a decrease in the production yield.
- the use of a separation membrane for continuous fermentation allows for catabolite.
- the pentose concentration in the total amount of filtrate can be maintained at 5 g / l or less, and as a result, the production yield of chemicals is improved compared to continuous fermentation without using a separation membrane. be able to. If 5 g / l or more of pentose remains in the total amount of filtrate obtained from the separation membrane, the yield improvement effect from pentose may be reduced, resulting in a decrease in production yield. May end up.
- the concentration of pentose in the total amount of filtrate can be adjusted according to the culture conditions. For example, it is possible to reduce the concentration of pentose in the total amount of filtrate by changing the concentration of sugar contained in the fermentation raw material, the supply rate of sugar, and the dilution rate. Alternatively, by increasing the nutrient sources contained in the fermentation raw material, it is possible to improve the consumption of microbial sugars and reduce the concentration of pentose in the total amount of filtrate.
- the pH and temperature are not particularly limited as long as the microorganisms grow, but the pH is preferably 4 to 8 and the temperature is preferably 20 to 75 ° C.
- the pH of the culture solution is usually adjusted to a predetermined value within a pH range of 4 to 8 with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, or the like. If it is necessary to increase the oxygen supply rate, means such as adding oxygen to the air to maintain the oxygen concentration at 21% or higher, pressurizing the culture solution, increasing the agitation rate, or increasing the aeration rate can be used. .
- continuous fermentation filtration of the culture solution
- continuous fermentation may be started after batch culture or fed-batch culture is performed at the initial stage of culture to increase the microorganism concentration.
- the start timing of the medium supply and the filtration of the culture solution is not necessarily the same. Further, the medium supply and the filtration of the culture solution may be continuous or intermittent.
- a nutrient necessary for cell growth may be added to the raw material culture solution so that the cell growth can be continuously performed.
- the concentration of microorganisms in the culture solution is preferably maintained in a state where the productivity of chemicals is high in order to obtain efficient productivity. For example, good production efficiency can be obtained by maintaining the concentration of microorganisms in the culture solution as a dry weight of 5 g / L or more.
- microorganisms in the culture tank are obtained by removing a part of the culture solution containing the microorganisms from the fermenter and diluting with a medium as necessary during the continuous fermentation.
- the density may be adjusted. For example, if the concentration of microorganisms in the fermenter becomes too high, clogging of the separation membrane is likely to occur, so avoiding clogging by removing a part of the culture solution containing microorganisms and diluting with a medium. There are things you can do.
- the production performance of chemicals may change depending on the concentration of microorganisms in the fermenter. Using the production performance as an indicator, remove a part of the culture solution containing microorganisms and dilute it in the medium to maintain the production performance. It is also possible.
- the number of fermenters is not limited as long as it is a continuous fermentation culture method for producing a product while growing cells.
- the production rate in continuous fermentation culture is calculated by the following equation (4).
- Fermentation production rate (g / L / hr) product concentration in the filtrate (g / L) ⁇ fermentation culture liquid withdrawal rate (L / hr) ⁇ operating fluid amount (L) of the apparatus (Formula 4).
- the fermentation production rate in batch culture is determined by the amount of product (g) at the time when all of the raw carbon source is consumed by the time (hr) required for consumption of the carbon source and the amount of fermentation broth (L) at that time. It is obtained by dividing.
- the yield in continuous culture is calculated by the following equation (5), and the amount of chemical product (g) produced by consuming the raw material carbon source within a certain period is calculated as the amount of input carbon source (g ) Divided by the value obtained by subtracting the amount of unused carbon source (g) from which microorganisms could not be used.
- the yield mentioned in the present specification refers to this unless otherwise specified.
- Yield (g / g) product amount (g) ⁇ ⁇ input carbon source amount (g) ⁇ unused carbon source amount (g) ⁇ (Formula 5).
- the continuous fermentation apparatus used in the present invention filters the fermentation broth of microorganisms through a separation membrane, collects the product from the filtrate, holds or refluxs the unfiltrated liquid in the fermentation broth, and supplies the fermentation raw material.
- a separation membrane collects the product from the filtrate in addition to the fermentation broth, but when using an organic polymer membrane, a specific example is given.
- the apparatus described in WO2007 / 097260 can be used.
- the apparatus described in WO2012 / 086763 can be used.
- the microorganism subjected to catabolite suppression used in the present invention generally means that when fermenting a microorganism that assimilate pentose, and using a fermentation raw material containing a mixed sugar containing hexose and pentose, pentose
- the consumption rate of xylose in a medium containing xylose alone in batch culture using a microorganism capable of assimilating glucose and xylose More than that, a microorganism with a slow consumption rate of xylose in a mixed sugar medium containing glucose and xylose is said to be “a microorganism subject to catabolite suppression”.
- the consumption rate of xylose in a medium containing xylose alone is calculated by the following (formula 6).
- Xylose consumption rate (g / L / hr) total amount of xylose contained in the fermentation raw material at the start of cultivation (g) ⁇ time spent until the xylose contained in the fermentation raw material is completely consumed (hr) ) / Fermentation liquid amount (L) (Formula 6).
- the consumption rate of xylose in a mixed sugar medium containing glucose and xylose is the consumption rate of xylose in the presence of glucose in the mixed sugar medium containing glucose and xylose. ).
- Xylose consumption rate (g / L / hr) xylose amount consumed from the start of culture to time T (g) ⁇ time from start of culture to time T (hr) ⁇ fermented liquid amount (L) (Formula 7).
- the weight ratio of glucose and xylose in the mixed sugar is 1: 1.
- the sugar concentration is not particularly limited as long as the microorganism can be completely consumed without residual sugar.
- the sugar concentration of xylose in a medium containing xylose is the same as the total sugar concentration in a mixed sugar medium containing glucose and xylose.
- time T is the time when glucose is completely consumed.
- the time T can be obtained by measuring the glucose concentration of the sampled culture solution using HPLC, a kit, or a sensor.
- the time T is the time when xylose is completely consumed.
- the time T can be obtained by measuring the xylose concentration in the same manner as the glucose concentration measuring method.
- the calculation is performed in consideration of the amount of liquid added to the culture liquid or the amount of liquid reduced.
- microorganisms that are subject to catabolite suppression are selected from yeasts such as baker's yeast often used in the fermentation industry, bacteria such as Escherichia coli and coryneform bacteria, filamentous fungi, actinomycetes, and the like.
- Pichia genus candy The genus Candida, the genus Pachisolen, the genus Kluyveromyces, the genus Hansenula, the genus Torulopsis, the genus Debaryomyes, the genus Isachenkia, the Isachenchia genus Yeasts such as the genus (Lindnera) and Wickerhamomyces (Clostridial) um), Enterobacter genus, Escherichia genus, Klebsiella genus bacteria, Lactobacillus genus, Lactococcus genus Lactococcus Actinoplanes, Arthrobacter, Streptomyces, and other actinomycetes, Bacillus, Paenibacillus, Aerobacter, Aerobacter, Aerobacter , Staphylococcus genus, Thermoanae Citrobacter (Thermoanaerobacter) genus, may be selected from Thermoan
- microorganisms that are catabolite-suppressed are isolated from the natural environment, they were originally not assimilated pentose, but were modified to assimilate pentose by mutation or genetic recombination It can also be selected from microorganisms.
- microorganisms modified so as to assimilate pentose by gene recombination include microorganisms in which a pentose metabolic gene is introduced or enhanced by gene recombination.
- xylose metabolic genes include enzymes such as xylose isomerase, xylose reductase, xylitol dehydrogenase, xylulose kinase, and microorganisms to which xylose is assimilated by such genetic recombination techniques. Examples thereof include microorganisms described in JP-T-2006-525029, JP-A-2009-112289, JP-T 2010-504756, and the like.
- the chemical product produced by the present invention is not limited as long as it is a substance produced by the microorganism in the fermentation broth, and includes substances that are mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, and nucleic acids. Can do. For example, as alcohol, ethanol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, glycerol, butanol, isobutanol, 2-butanol, isopropanol, etc.
- organic acids include acetic acid, lactic acid, adipic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, citric acid, and nucleic acids, nucleosides such as inosine and guanosine, nucleotides such as inosinic acid and guanylic acid, Examples thereof include diamine compounds such as cataverine.
- the present invention can also be applied to the production of substances such as enzymes, antibiotics, and recombinant proteins. These chemicals can be recovered from the filtrate by known methods (membrane separation, concentration, distillation, crystallization, extraction, etc.).
- Reference Example 3 Calculation of Xylose Consumption Rate in Bacillus coagulans
- As the medium a lactic acid fermentation xylose medium having the composition shown in Table 1 and a lactic acid fermentation mixed sugar medium 1 shown in Table 2 were used. Sampling was performed as appropriate, and the concentrations of glucose and xylose in the culture solution were measured by the method of Reference Example 1, and the concentration of lactic acid as a product was measured by the method of Reference Example 2.
- Bacillus coagulans NBRC12714 strain was cultured with calcium carbonate in a flask in 50 mL of a preculture medium (polypeptone 10 g / L, yeast extract 2 g / L, magnesium sulfate heptahydrate 1 g / L) for 24 hours (preculture).
- the preculture was inoculated into 1 L of lactic acid fermentation xylose medium and lactic acid fermentation mixed sugar medium 1 purged with nitrogen gas, and batch fermentation was performed under the following conditions.
- Fermentation reactor capacity 2 (L) Temperature adjustment: 50 (°C) Reaction tank ventilation rate (nitrogen gas): 100 (mL / min) Reaction tank stirring speed: 200 (rpm) pH adjustment: adjusted to pH 7 with 5N Ca (OH) 2 Sterilization: All culture tanks and culture media are autoclaved at 121 ° C. for 20 minutes and autoclaved for 20 minutes.
- the xylose consumption rates in the lactic acid fermentation xylose medium and the lactic acid fermentation mixed sugar medium 1 were calculated according to the above-mentioned (formula 6) and (formula 7), and are shown in Table 7. From this result, it was determined that the Bacillus coagulans NBRC12714 strain was a microorganism subject to catabolite suppression.
- Reference Example 4 Calculation of Xylose Consumption Rate in Candida tropicalis
- As the medium an ethanol fermentation xylose medium having the composition shown in Table 3 and an ethanol fermentation mixed sugar medium 1 shown in Table 4 were used. Sampling was performed as appropriate, and the concentrations of glucose and xylose in the culture solution and the concentration of ethanol as a product were measured by the method of Reference Example 1.
- Candida tropicalis NBRC0199 strain was cultured with shaking in 2 mL of YPD medium in a test tube at 30 ° C. overnight (pre-culture).
- the obtained culture solution was inoculated into 50 mL of a fresh YPD medium, and cultured with shaking overnight in a 500 mL pleated Erlenmeyer flask (preculture).
- the preculture liquid was inoculated into 2 L of ethanol fermentation xylose medium and ethanol fermentation mixed sugar medium, and batch culture was performed under the following conditions.
- Fermentation reactor capacity 2 (L) Temperature adjustment: 30 (° C) Aeration rate of reaction tank: 100 (mL / min) Reaction tank stirring speed: 800 (rpm) pH adjustment: None Sterilization: All culture tanks and culture media are autoclaved at 121 ° C for 20 min and autoclaved.
- the xylose consumption rates in the ethanol-fermented xylose medium and ethanol-fermented mixed sugar medium 1 were calculated according to the above-described (formula 6) and (formula 7), and are shown in Table 7.
- Candida tropicalis NBRC0199 strain was determined to be a microorganism that is subject to catabolite suppression.
- Reference Example 5 Calculation of Xylose Consumption Rate in Paenibacillus polymixer
- As the medium 2,3-butanediol-fermented xylose medium having the composition shown in Table 5 and 2,3-butanediol-fermented mixed sugar medium 1 shown in Table 6 were used. Sampling was performed as appropriate, and the concentrations of glucose and xylose in the culture solution and the concentration of 2,3-butanediol as a product were measured by the method of Reference Example 1.
- Paenibacillus polymixer ATCC12321 strain was cultured in a test tube with shaking in a 50 mL preculture medium (glucose 5 g / L, peptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L) for 24 hours (preculture).
- the preculture was inoculated into 1 L of 2,3-butanediol-fermented xylose medium and 2,3-butanediol-fermented mixed sugar medium, respectively, and batch culture was performed under the following conditions.
- Fermentation reactor capacity 2 (L) Temperature adjustment: 30 (° C) Aeration rate of reaction tank: 100 (mL / min) Reaction tank stirring speed: 800 (rpm) pH adjustment: pH 6.5 adjusted with 5N NaOH Sterilization: All culture tanks and culture media are autoclaved at 121 ° C for 20 minutes and autoclaved for 20 minutes.
- the xylose consumption rates in the 2,3-butanediol-fermented xylose medium and the 2,3-butanediol-fermented mixed sugar medium 1 were calculated according to the above (formula 6) and (formula 7), and are shown in Table 7. As a result, it was determined that Paenibacillus polymixer ATCC 12321 strain is a microorganism that is subject to catabolite suppression.
- Comparative Example 2 Production of L-lactic acid by batch culture of Bacillus coagulans using mixed sugar (glucose, xylose) as a fermentation raw material Comparative Example 1 using Bacillus coagulans NBRC12714 strain with lactic acid fermentation mixed sugar medium shown in Table 2 Batch culture was performed for 128 hours under the same conditions as in Table 11 (Table 11).
- Fermentation reactor capacity 2 (L) Temperature adjustment: 50 (°C) Reaction tank ventilation rate (nitrogen gas): 100 (mL / min) Reaction tank stirring speed: 200 (rpm) pH adjustment: adjusted to pH 7 with 5N Ca (OH) 2 Extracted amount of fermentation broth: 3 (L / Day) Sterilization: All culture tanks and culture media are autoclaved at 121 ° C for 20 min and autoclaved.
- Example 1 Production of L-lactic acid by continuous culture using a separation membrane of Bacillus coagulans using a mixed sugar (glucose, xylose) as a fermentation raw material 1
- Continuous culture was performed using a separation membrane using a lactic acid fermentation mixed sugar medium having the composition shown in Table 2 using mixed sugar (glucose, xylose) as a fermentation raw material.
- a hollow fiber form was adopted as the separation membrane element.
- the Bacillus coagulans NBRC12714 strain was cultured with shaking under the same conditions as the preculture in Comparative Example 1 (pre-culture).
- Pre-culture medium was inoculated into 1.5 L lactic acid fermentation mixed sugar medium purged with nitrogen gas, and the fermentation reaction tank was stirred at 200 rpm with the attached stirrer to fill the fermentation reaction tank with nitrogen. The temperature was adjusted, and batch culture was performed until the sugar in the culture solution was completely consumed (preculture). Immediately after completion of the pre-culture, the fermenter circulation pump is operated, the medium is continuously supplied, and the filtration amount of the culture solution is controlled so that the amount of the fermentation solution in the continuous fermentation apparatus is 1.5 L under the following conditions. Lactic acid was produced by continuous culture for 290 hours (Table 11).
- Fermentation reactor capacity 2 (L) Separation membrane used: Polyvinylidene fluoride filtration membrane membrane separation element Effective filtration area: 473 (cm 2 ) Temperature adjustment: 50 (°C) Fermentation reactor aeration volume (nitrogen gas): 100 (mL / min) Fermentation reactor stirring speed: 200 (rpm) Extracted amount of fermentation broth: 3 (L / Day) Sterilization: All the culture tank and separation medium containing the separation membrane element are autoclaved at 121 ° C for 20 min.
- the membrane used was a membrane having the following properties, and the transmembrane pressure difference during filtration was changed between 0.1 and 20 kPa.
- Average pore diameter 0.1 ⁇ m Standard deviation of average pore diameter: 0.035 ⁇ m
- Film surface roughness 0.06 ⁇ m
- Pure water permeability coefficient 50 ⁇ 10 ⁇ 9 m 3 / m 2 / s / pa.
- Example 2 Continuous culture 2 using a separation membrane of Bacillus coagulans using a mixed sugar (glucose, xylose) as a fermentation raw material 2 L-lactic acid was produced by using the lactic acid fermentation mixed sugar medium 2 shown in Table 9 for 305 hours using the separation membrane under the same conditions as in Example 1 (Table 11).
- Example 3 Continuous culture 3 using Bacillus coagulans using a mixed sugar (glucose, xylose) as a fermentation raw material Using the lactic acid fermentation mixed sugar medium 3 shown in Table 10, continuous culture using a separation membrane for 300 hours was performed under the same conditions as in Example 1 to produce L-lactic acid (Table 11).
- Candida tropicalis NBRC0199 strain was used as the ethanol fermentation microorganism, and the composition shown in Table 12 was used as the medium. An ethanol fermentation medium was used.
- Candida tropicalis NBRC0199 strain was cultured with shaking in 2 mL of YPD medium in a test tube at 30 ° C. overnight (pre-culture). The obtained culture solution was inoculated into 50 mL of a fresh YPD medium, and cultured with shaking overnight in a 500 mL pleated Erlenmeyer flask (preculture).
- the preculture was inoculated into 1.5 L of ethanol fermentation medium, and batch culture was performed for 16 hours under the following conditions to produce ethanol (Table 14).
- Fermentation reactor capacity 2 (L) Temperature adjustment: 30 (° C) Aeration rate of reaction tank: 100 (mL / min) Reaction tank stirring speed: 800 (rpm) pH adjustment: None Sterilization: All culture tanks and culture media are autoclaved at 121 ° C for 20 min and autoclaved.
- Fermentation reactor capacity 2 (L) Temperature adjustment: 30 (° C) Aeration volume of fermentation reaction tank: 100 (mL / min) Fermentation reactor stirring speed: 800 (rpm) pH adjustment: None Fermentation liquid withdrawal: 1 (L / Day) Sterilization: All culture tanks and culture media are autoclaved at 121 ° C for 20 min and autoclaved.
- Example 4 Production of ethanol by continuous culture using a separation membrane of Candida tropicalis using mixed sugar (glucose, xylose) as a fermentation raw material
- ethanol-fermented mixed sugar medium 2 having the composition shown in Table 13 is used.
- the continuous fermentation using the separated membrane was performed.
- a flat membrane was adopted as the separation membrane element.
- Candida tropicalis NBRC0199 strain was cultured in a test tube with shaking in 2 mL of YPD medium at 30 ° C. overnight (pre-culture).
- the obtained culture broth was inoculated into 50 mL of fresh YPD medium, and cultured overnight in a 500 mL capacity fluted Erlenmeyer flask (pre-culture).
- Pre-culture medium is inoculated into 1.5L ethanol fermentation mixed sugar medium 2 of continuous fermentation apparatus, the fermentation reaction tank is stirred at 800 rpm with the attached stirrer, adjustment of aeration volume of fermentation reaction tank, temperature adjustment And cultured for 36 hours (pre-culture).
- the fermenter circulation pump is operated, the medium is continuously supplied, and the filtration amount of the culture solution is controlled so that the amount of the fermentation solution in the continuous fermentation apparatus is 1.5 L under the following conditions. Ethanol was produced by continuous culture for 300 hours (Table 14).
- Fermentation reactor capacity 2 (L) Separation membrane used: Polyvinylidene fluoride filtration membrane membrane separation element Effective filtration area: 120 (cm 2 ) Temperature adjustment: 30 (° C) Aeration volume of fermentation reaction tank: 100 (mL / min) Fermentation reactor stirring speed: 800 (rpm) pH adjustment: None Fermentation liquid withdrawal: 1 (L / Day) Sterilization: The culture tank containing the separation membrane element and the medium used are all autoclaved at 121 ° C. for 20 minutes and autoclaved.
- the membrane used was a membrane having the same properties as in Example 1, and the transmembrane pressure difference during filtration was changed between 0.1 and 19.8 kPa.
- Paenibacillus polymixer ATCC12321 strain was cultured in a test tube with shaking in 50 mL of a preculture medium (glucose 5 g / L, peptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L) for 24 hours (preculture).
- the preculture was inoculated into 1 L of 2,3-butanediol fermentation medium, and batch culture was performed for 27 hours under the following conditions to produce 2,3-butanediol (Table 17).
- Fermentation reactor capacity 2 (L) Temperature adjustment: 30 (° C) Aeration rate of reaction tank: 100 (mL / min) Reaction tank stirring speed: 800 (rpm) pH adjustment: pH 6.5 adjusted with 5N NaOH Sterilization: All culture tanks and culture media are autoclaved at 121 ° C for 20 minutes and autoclaved for 20 minutes.
- Fermentation reactor capacity 2 (L) Temperature adjustment: 30 (° C) Aeration rate of reaction tank: 100 (mL / min) Reaction tank stirring speed: 800 (rpm) pH adjustment: adjusted to pH 6.5 with 5N NaOH Extraction amount of fermentation broth: 0.6 (L / Day) Sterilization: All culture tanks and culture media are autoclaved at 121 ° C for 20 min and autoclaved.
- Example 5 Production of 2,3-butanediol by continuous culture using a separation membrane of Paenibacillus polymixer using mixed sugar (glucose, xylose) as fermentation raw material Separation using mixed sugar (glucose, xylose) as fermentation raw material Continuous culture using a membrane was performed. A flat membrane was adopted as the separation membrane element.
- Paenibacillus polymixer ATCC12321 strain was cultured with shaking under the same conditions as the preculture of Comparative Example 7 (pre-culture). Pre-culture solution is inoculated into 1.2 L of mixed sugar 2,3-butanediol fermentation medium 2 and pre-culture solution is stirred at 200 rpm with the attached stirrer to adjust the temperature and culture.
- Batch culture was performed until the sugar in the liquid was completely consumed (pre-culture). Immediately after completion of the pre-culture, the fermenter circulation pump is operated, the medium is continuously supplied, and the filtration amount of the culture solution is controlled so that the amount of the fermentation solution in the continuous fermentation apparatus is 1.2 L under the following conditions. Continuous culture for 310 hours was performed to produce 2,3-butanediol (Table 17).
- Fermentation reactor capacity 2 (L) Separation membrane used: Polyvinylidene fluoride filtration membrane membrane separation element Effective filtration area: 120 (cm 2 ) Temperature adjustment: 30 (° C) Aeration volume of fermentation reaction tank: 100 (mL / min) Fermentation reactor stirring speed: 800 (rpm) Extracted amount of fermentation broth: 0.6 L / Day Sterilization: All the culture tank and separation medium containing the separation membrane element are autoclaved at 121 ° C for 20 min.
- the membrane used was a membrane having the same properties as in Example 1, and the transmembrane pressure difference during filtration was changed between 0.1 and 20 kPa.
- Reference Example 7 Calculation of Xylose Consumption Rate in Candida utilis Candida utilis CuLpLDH strain prepared by the method disclosed in WO2010 / 140602 was used as a microorganism.
- a D-lactic acid fermentation xylose medium having the composition shown in Table 18 and a D-lactic acid fermentation mixed sugar medium 1 shown in Table 19 were used as the medium.
- Batch culture was performed for 40 hours under the same conditions as in Comparative Example 1 except that the pH was adjusted to pH 6.0 with 1N calcium hydroxide, and the xylose consumption rate during D-lactic acid fermentation was calculated.
- the xylose consumption rates in the D-lactic acid fermented xylose medium and the D-lactic acid fermented mixed sugar medium 1 were calculated according to the above (formula 6) and (formula 7), and are shown in Table 22.
- the Candida utilis CuLpLDH strain was determined to be a microorganism that is subject to catabolite suppression.
- Reference Example 8 Calculation of Xylose Consumption Rate in Escherichia coli KO11 Strain
- the xylose consumption rate during ethanol fermentation of Escherichia coli KO11 strain which is an ethanol fermentation microorganism, was calculated.
- As the medium ethanol fermentation xylose medium 2 having the composition shown in Table 20 and ethanol fermentation mixed sugar medium 3 shown in Table 21 were used. Sampling was performed as appropriate, and the concentrations of glucose and xylose in the culture solution and the concentration of ethanol as a product were measured by the method of Reference Example 1.
- Escherichia coli KO11 strain was cultured overnight in a test tube at 30 ° C. in a 2 mL preculture medium (glucose 20 g / L yeast extract 10 g / L, tryptone 5 g / L, NaCl 5 g / L).
- the obtained culture solution was inoculated into a 50 mL preculture medium in a 500 mL pleated Erlenmeyer flask and cultured overnight (preculture).
- the preculture was inoculated into 1.5 L of ethanol-fermented xylose medium 2 and ethanol-fermented mixed sugar medium 3, and batch fermentation was performed for 16 hours under the following operating conditions while adjusting the temperature and pH.
- Culture tank capacity 2 (L) Temperature adjustment: 30 (° C) Aeration volume of fermentation reaction tank: 100 (mL / min) Fermentation reactor stirring speed: 800 (rpm) pH adjustment: adjusted to pH 6 with 5N Ca (OH) 2 Sterilization: All fermenters and culture media are autoclaved at 121 ° C. for 20 min under high pressure steam sterilization.
- the xylose consumption rates in the ethanol-fermented xylose medium 2 and the ethanol-fermented mixed sugar medium 3 were calculated according to the above (formula 6) and (formula 7), and are shown in Table 22. As a result, it was determined that Escherichia coli KO11 strain is a microorganism that is subject to catabolite suppression.
- Example 6 Production of Ethanol by Continuous Fermentation Using Pichia Stippitis Separation Membrane Using Mixed Sugar as Fermentation Raw Material 1 As a microorganism, Pichia stipitis NBRC1687 strain was used, and continuous fermentation using a separation membrane was performed. Ethanol was produced by continuous fermentation for 305 hours under the same conditions as in Example 4 except that the preculture was performed for 48 hours and the transmembrane pressure difference was adjusted to 0.1 to 19.8 kPa.
- Comparative Example 15 Production of D-lactic acid by continuous fermentation of Candida utilis using mixed sugar as a fermentation raw material
- Candida utilis CuLpLDH strain was used as a microorganism, and continuous fermentation was performed without using a separation membrane.
- the pre-culture was performed for 40 hours, the pH was adjusted to 6.0 with 1N calcium hydroxide, and the medium was the same as in Comparative Example 6 except that the D-lactic acid fermentation mixed sugar medium 2 shown in Table 24 was used.
- D-lactic acid was produced by continuous fermentation for 290 hours (Table 25).
- Example 7 Production of D-lactic acid by continuous fermentation using a separation membrane of Candida utilis using mixed sugar as a fermentation raw material 1 Using Candida utilis CuLpLDH strain as a microorganism, continuous fermentation using a separation membrane was performed. The pre-culture was performed for 50 hours, the pH was adjusted to 6.0 with 1N calcium hydroxide, and the medium was used under the same conditions as in Example 4 except that the D-lactic acid fermentation mixed sugar medium 2 shown in Table 24 was used. D-lactic acid was produced by continuous fermentation for 310 hours.
- the preculture was inoculated into 1 L of ethanol fermentation medium 2 having the composition shown in Table 26, and subjected to batch fermentation for 16 hours under the following operating conditions while adjusting the temperature and pH to produce ethanol (Table 28). ).
- Culture tank capacity 2 (L) Temperature adjustment: 30 (° C) Kla: 30 (h -1 ) pH adjustment: adjusted to pH 6 with 5N Ca (OH) 2 Sterilization: All fermenters and culture media are autoclaved at 121 ° C. for 20 min under high pressure steam sterilization.
- Comparative Example 17 Production of Ethanol by Batch Fermentation of Escherichia coli Using Mixed Sugar as Fermentation Raw Material 24 Using ethanol-fermented mixed sugar medium 4 having the composition shown in Table 27 as the fermentation medium under the same conditions as in Comparative Example 16 Time batch fermentation was performed to produce ethanol (Table 28).
- Ethanol was produced for 290 hours while controlling the medium supply rate so that the drawing rate of the culture solution containing microorganisms was constant and the amount of the culture solution in the culture tank was 1.5 L (Table 28).
- Culture tank capacity 2 (L) Temperature adjustment: 30 (° C) Kla: 30 (h -1 ) pH adjustment: adjusted to pH 6 with 5N Ca (OH) 2 Fermentation liquid withdrawal rate: 2 L / day Sterilization: All fermenters and culture media are autoclaved at 121 ° C for 20 min under high pressure steam sterilization.
- Example 8 Production of ethanol by continuous fermentation using Escherichia coli separation membrane using mixed sugar as a fermentation raw material
- Escherichia coli KO11 strain was cultured overnight at 30 ° C. in 2 mL of a preculture medium (glucose 20 g / L, yeast extract 10 g / L, tryptone 5 g / L, NaCl 5 g / L) in a test tube. ).
- the obtained culture solution was inoculated into a 50 mL preculture medium in a 500 mL pleated Erlenmeyer flask and cultured overnight (pre-culture).
- the transmembrane pressure difference during filtration ranges from 0.1 to 19.8 kPa.
- the ethanol was produced by continuous fermentation for 310 hours (Table 28).
- Fermenter capacity 2 (L) Separation membrane used: Polyvinylidene fluoride filtration membrane separation element Effective filtration area: 473 cm 2 Pure water permeability coefficient of separation membrane: 50 ⁇ 10 ⁇ 9 m 3 / m 2 / s / Pa Separation membrane average pore size: 0.1 ⁇ m Standard deviation of average pore diameter: ⁇ 0.035 ⁇ m Separation membrane surface roughness: 0.06 ⁇ m Temperature adjustment: 30 (° C) pH adjustment: adjusted to pH 6 with 5N Ca (OH) 2 Fermentation liquid withdrawal rate: 2 L / day Sterilization: All fermenters and separation media including separation membrane elements are autoclaved at 121 ° C. for 20 min under high pressure steam sterilization.
- Example 9 Production of L-lactic acid by continuous culture using Bacillus coagulans separation membrane using biomass-derived sugar liquid (glucose, xylose) as fermentation raw material Biomass-derived sugar liquid was used as a fermentation raw material.
- biomass-derived sugar liquid glucose, xylose
- the medium shown in Table 29 was used as the lactic acid fermentation sugar solution medium. Lactic acid was produced by continuous culture using a separation membrane for 260 hours under the same conditions as in Example 1 except that the medium used and the neutralizing agent were changed to 4N KOH.
- Example 10 Production of Ethanol by Continuous Culture Using Escherichia coli Separation Membrane Using Biomass-derived Sugar (Glucose, Xylose) as Fermentation Raw Material As in Example 8 using the ethanol fermentation sugar solution medium shown in Table 27 Under these conditions, continuous culture using a separation membrane was performed for 295 hours to produce ethanol.
- Biomass-derived Sugar Glucose, Xylose
- Comparative Example 24 Production of Ethanol by Continuous Culture of Candida tropicalis Using Mixed Sugar as Fermentation Raw Material Continuous culture for 350 hours under the same conditions as in Comparative Example 6 using ethanol fermentation mixed sugar medium 5 shown in Table 33 To produce ethanol (Table 34).
- Example 11 Production of ethanol by continuous culture using a separation membrane of Candida tropicalis using mixed sugar (glucose, xylose) as fermentation raw material 2 Using ethanol-fermented mixed sugar medium 5 shown in Table 33, continuous culture was performed for 360 hours under the same conditions as in Example 4 to produce ethanol (Table 34).
- Example 12 Production of ethanol by continuous fermentation using a ceramic separation membrane of Candida tropicalis using mixed sugar as a fermentation raw material 3
- Candida tropicalis NBRC0199 strain was used for continuous fermentation using a ceramic separation membrane.
- the medium shown in Table 33 was used as the fermentation medium.
- Candida tropicalis NBRC0199 strain was cultured in a test tube with shaking in 2 mL of YPD medium at 30 ° C. overnight (pre-culture).
- the obtained culture broth was inoculated into a 500 mL pleated Erlenmeyer flask containing 50 mL of YPD medium and cultured overnight with shaking (pre-culture).
- the culture solution was inoculated into a membrane-separated continuous fermentation apparatus (apparatus shown in FIG.
- Culture tank capacity 2L Separation membrane used: Celfit microfiltration membrane monolith ⁇ 4-19 (NGK) Membrane separation element length: 500mm Separation membrane average pore size: 0.1 ⁇ m Temperature adjustment: 30 ° C Aeration volume of fermentation reaction tank: 100 (mL / min) Fermentation reactor stirring speed: 800 (rpm) pH adjustment: None.
- Example 13 Production of 2,3-butanediol by continuous culture using a separation membrane made of Paenibacillus polymixer ceramic using a mixed sugar (glucose, xylose) as a fermentation raw material 2
- Paenibacillus polymixer ATCC12321 strain continuous fermentation using a ceramic separation membrane was performed.
- Paenibacillus polymixer ATCC12321 strain was cultured overnight at 30 ° C. in 2 mL of a preculture medium (glucose 5 g / L, peptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L) in a test tube. culture).
- the obtained culture solution was inoculated into a 50 mL preculture medium in a 500 mL pleated Erlenmeyer flask and cultured overnight (pre-culture).
- the culture solution Prior to inoculation, the culture solution was inoculated into a continuous culture apparatus (apparatus shown in FIG. 2 of WO2007 / 097260) containing a 2,3-butanediol fermentation mixed sugar medium having the composition shown in Table 16 to adjust the temperature and pH.
- batch fermentation was performed for 30 hours under the following operating conditions (preculture). After completion of the preculture, continuous culture was started immediately using a 2,3-butanediol fermentation mixed sugar medium having the composition shown in Table 16 to produce 2,3-butanediol.
- 2,3-butanediol was produced by continuous fermentation for 300 hours while adjusting the transmembrane pressure difference during filtration to 500 kPa or less (Table 35).
- Fermenter capacity 2 (L) Separation membrane used: Celfit microfiltration membrane monolith ⁇ 4-19 (NGK) Membrane separation element length: 500mm Separation membrane average pore size: 0.1 ⁇ m Temperature adjustment: 30 (° C) Aeration volume of fermentation reaction tank: 100 (mL / min) Fermentation reactor stirring speed: 800 (rpm) pH adjustment: adjusted to pH 6.5 with 5N Ca (OH) 2
- the efficiency of fermentation production of various chemicals using fermentation raw materials containing pentose and hexose can be greatly improved.
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
(1)微生物の培養液を分離膜で濾過すること、未濾過液を培養液に保持または還流すること、発酵原料を培養液に追加すること、および濾過液中の生産物を回収することを含む、連続発酵による化学品の製造方法であって、前記微生物がカタボライト抑制を受ける微生物であり、前記発酵原料が六炭糖および五炭糖を含む、化学品の製造方法。
(2)前記濾過液総量の五炭糖の濃度が5g/L以下である、(1)に記載の化学品の製造方法。
(3)前記発酵原料中に含まれる六炭糖と五炭糖の重量比率が1:9~9:1である、(1)または(2)に記載の化学品の製造方法。
(4)前記発酵原料がバイオマス由来の糖液を含む、(1)または(2)に記載の化学品の製造方法。
(5)前記五炭糖がキシロースである、(1)から(4)のいずれかに記載の化学品の製造方法。
・装置 原子間力顕微鏡装置(Digital Instruments(株)製“Nanoscope IIIa”)
・条件 探針 SiNカンチレバー(Digital Instruments(株)製)
走査モード コンタクトモード(気中測定)
水中タッピングモード(水中測定)
走査範囲 10μm、25μm四方(気中測定)
5μm、10μm四方(水中測定)
走査解像度 512×512
・試料調製 測定に際し膜サンプルは、常温でエタノールに15分浸漬後、RO水中に24時間浸漬し洗浄した後、風乾し用いた。RO水とは、濾過膜の一種である逆浸透膜(RO膜)を用いて濾過し、イオンや塩類などの不純物を排除した水を指す。RO膜の孔の大きさは、概ね2nm以下である。
気孔率[%]=100×(湿潤膜重量[g]-乾燥膜重量[g])/水比重[g/cm3]/(膜体積[cm3])。
生産収率(g/g)=生産物量(g)÷投入炭素源量(g)・・・(式3)。
発酵生産速度(g/L/hr)=濾液中の生産物濃度(g/L)×発酵培養液抜き取り速度(L/hr)÷装置の運転液量(L)・・・(式4)。
収率(g/g)=生産物量(g)÷{投入炭素源量(g)-未利用炭素源量(g)}・・・・(式5)。
キシロースの消費速度(g/L/hr)=培養開始時の発酵原料に含まれる総キシロース量(g)÷培養開始から発酵原料に含まれるキシロースを完全に消費し尽くすまでに費やした時間(hr)÷発酵液量(L)・・・(式6)。
キシロースの消費速度(g/L/hr)=培養開始時から時間Tまでに消費されたキシロース量(g)÷培養開始から時間Tまでの時間(hr)÷発酵液量(L)・・・(式7)。
発酵液中のグルコース、キシロース、エタノールおよび2,3-ブタンジオールの濃度は、下記に示すHPLC条件で、標品との比較により定量した。
カラム:Shodex SH1011(昭和電工株式会社製)
移動相:5mM 硫酸(流速0.6mL/分)
反応液:なし
検出方法:RI(示差屈折率)
温度:65℃。
発酵液中の乳酸を下記に示すHPLC条件で標品との比較により定量した。
カラム:Shim-Pack SPR-H(株式会社島津製作所製)
移動相:5mM p-トルエンスルホン酸(流速0.8mL/min)
反応液:5mM p-トルエンスルホン酸、20mM ビストリス、0.1mM EDTA・2Na(流速0.8mL/min)
検出方法:電気伝導度
温度:45℃。
乳酸発酵微生物であるバチルス・コアギュランスNBRC12714株の乳酸発酵時のキシロース消費速度を算出した。培地として表1に示す組成の乳酸発酵キシロース培地と表2に示す乳酸発酵混合糖培地1を用いた。適宜、サンプリングを行い、培養液中のグルコースおよびキシロースの濃度は参考例1の方法により、生産物である乳酸の濃度は参考例2の方法により測定した。
発酵反応槽容量:2(L)
温度調整:50(℃)
反応槽通気量(窒素ガス):100(mL/分)
反応槽攪拌速度:200(rpm)
pH調整:5N Ca(OH)2によりpH7に調整
滅菌:培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
エタノール発酵微生物であるキャンディダ・トロピカリスNBRC0199株のエタノール発酵時のキシロース消費速度を算出した。培地として表3に示す組成のエタノール発酵キシロース培地と表4に示すエタノール発酵混合糖培地1を用いた。適宜、サンプリングを行い、培養液中のグルコースおよびキシロースの濃度と、生産物であるエタノールの濃度は参考例1の方法により測定した。
発酵反応槽容量:2(L)
温度調整:30(℃)
反応槽通気量:100(mL/分)
反応槽攪拌速度:800(rpm)
pH調整:なし
滅菌:培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
2,3-ブタンジオール発酵微生物であるパエニバチラス・ポリミキサATCC12321株の2,3-ブタンジオール発酵時のキシロース消費速度を算出した。培地として表5に示す組成の2,3-ブタンジオール発酵キシロース培地と表6に示す2,3-ブタンジオール発酵混合糖培地1を用いた。適宜、サンプリングを行い、培養液中のグルコースおよびキシロースの濃度と、生産物である2,3-ブタンジオールの濃度は参考例1の方法により測定した。
発酵反応槽容量:2(L)
温度調整:30(℃)
反応槽通気量:100(mL/分)
反応槽攪拌速度:800(rpm)
pH調整:5N NaOHによりpH6.5に調整
滅菌:培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
L-乳酸発酵微生物としてバチルス・コアギュランスNBRC12714株を用い、培地として表8に示す組成の乳酸発酵培地を用いた。バチルス・コアギュランスNBRC12714株を炭酸カルシウムと共にフラスコで50mLの前培養培地(ポリペプトン10g/L、酵母エキス2g/L、硫酸マグネシウム7水和物1g/L)で24時間振とう培養した(前培養)。前培養液を窒素ガスでパージした1Lの乳酸発酵培地に植菌し、参考例3の条件で96時間のバッチ培養を行った(表11)。
バチルス・コアギュランスNBRC12714株を表2に示す乳酸発酵混合糖培地を用いて比較例1と同様の条件で128時間のバッチ培養を行った(表11)。
混合糖(グルコース、キシロース)を発酵原料にした表2に示す組成の乳酸発酵混合糖培地1を用いた分離膜を利用しない連続培養を行なった。バチルス・コアギュランスNBRC12714株を上記比較例1の前培養と同じ条件で振とう培養した(前々培養)。前々培養液を、前培養液を窒素ガスでパージした1.5Lの乳酸発酵混合糖培地に植菌し、発酵反応槽を付属の撹拌機によって200rpmで撹拌し、発酵反応槽を窒素で満たし、温度調整を行い、培養液中の糖が完全に消費されるまでバッチ培養を行った(前培養)。前培養完了後直ちに、前培養完了後、直ちに発酵液抜きポンプを稼動させ、さらに培地の連続供給を行い、連続発酵装置の発酵液量を1.5Lとなるよう微生物を含む培養液の引き抜き量の制御を行いながら以下の条件で300時間の連続培養を行い、乳酸を製造した(表11)。
発酵反応槽容量::2(L)
温度調整:50(℃)
反応槽通気量(窒素ガス):100(mL/分)
反応槽攪拌速度:200(rpm)
pH調整:5N Ca(OH)2によりpH7に調整
発酵液の抜き量:3(L/Day)
滅菌:培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
混合糖(グルコース、キシロース)を発酵原料にした表2に示す組成の乳酸発酵混合糖培地を用いた分離膜を利用した連続培養を行なった。分離膜エレメントとしては中空糸の形態を採用した。バチルス・コアギュランスNBRC12714株を上記比較例1の前培養と同じ条件で振とう培養した(前々培養)。前々培養液を、前培養液を窒素ガスでパージした1.5Lの乳酸発酵混合糖培地に植菌し、発酵反応槽を付属の撹拌機によって200rpmで撹拌し、発酵反応槽を窒素で満たし、温度調整を行い、培養液中の糖が完全に消費されるまでバッチ培養を行った(前培養)。前培養完了後、直ちに発酵液循環ポンプを稼動させ、さらに培地の連続供給を行い、連続発酵装置の発酵液量を1.5Lとなるよう培養液の濾過量の制御を行いながら以下の条件で290時間の連続培養を行い、乳酸を製造した(表11)。
発酵反応槽容量:2(L)
使用分離膜:ポリフッ化ビニリデン濾過膜
膜分離エレメント有効濾過面積:473(cm2)
温度調整:50(℃)
発酵反応槽通気量(窒素ガス):100(mL/分)
発酵反応槽攪拌速度:200(rpm)
発酵液の抜き量:3(L/Day)
滅菌:分離膜エレメントを含む培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
平均細孔径:0.1μm
平均細孔径の標準偏差:0.035μm
膜表面粗さ:0.06μm
純水透過係数:50×10-9m3/m2/s/pa。
表9に示す乳酸発酵混合糖培地2を用い、実施例1と同様の条件で305時間の分離膜利用連続培養を行い、L-乳酸を製造した(表11)。
表10に示す乳酸発酵混合糖培地3を用い、実施例1と同様の条件で300時間の分離膜利用連続培養を行い、L-乳酸を製造した(表11)。
エタノール発酵微生物としてキャンディダ・トロピカリスNBRC0199株を用い、培地として表12に示す組成のエタノール発酵培地を用いた。キャンディダ・トロピカリスNBRC0199株を試験管で2mLのYPD培地にて30℃で一晩振とう培養した(前々培養)。得られた培養液を新鮮なYPD培地50mLに植菌し、500mL容量のひだ付三角フラスコで一晩振とう培養した(前培養)。前培養液を、1.5Lのエタノール発酵培地に植菌し、以下の条件で16時間のバッチ培養を行い、エタノールを製造した(表14)。
発酵反応槽容量:2(L)
温度調整:30(℃)
反応槽通気量:100(mL/分)
反応槽攪拌速度:800(rpm)
pH調整:なし
滅菌:培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
表13に示すエタノール発酵混合糖培地2を用い、比較例4と同様の条件で23時間のバッチ培養を行い、エタノールを製造した(表14)。
培地として表13に示すエタノール発酵混合糖培地2を用いた分離膜を利用しない連続発酵を行った。キャンディダ・トロピカリスNBRC0199株を試験管で2mLのYPD培地にて30℃で一晩振とう培養した(前々々培養)。得られた培養液を新鮮なYPD培地50mLに植菌し、500mL容量のひだ付三角フラスコで一晩振とう培養した(前々培養)。1.5Lのエタノール発酵混合糖培地2の入った連続発酵装置に前々培養液を植菌し、発酵反応槽を付属の撹拌機によって800rpmで撹拌して、発酵反応槽の通気量の調整、温度調整を行い、16時間培養を行った(前培養)。前培養完了後、直ちに発酵液抜きポンプを稼動させ、さらに培地の連続供給を行い、連続発酵装置の発酵液量を1.5Lとなるよう微生物を含む培養液の引き抜き量の制御を行いながら以下の条件で295時間の連続培養を行い、エタノールを製造した(表14)。
発酵反応槽容量:2(L)
温度調整:30(℃)
発酵反応槽通気量:100(mL/分)
発酵反応槽撹拌速度:800(rpm)
pH調整:なし
発酵液の抜き量:1(L/Day)
滅菌:培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
培地として表13に示す組成のエタノール発酵混合糖培地2を用いた分離膜を利用した連続発酵を行った。分離膜エレメントとしては平膜の形態を採用した。キャンディダ・トロピカリスNBRC0199株を試験管で2mLのYPD培地にて30℃で一晩振とう培養した(前々々培養)。得られた培養液を新鮮なYPD培地50mLに植菌し、500mL容量のひだ付三角フラスコで一晩振とう培養した(前々培養)。前々培養液を、連続発酵装置の1.5Lのエタノール発酵混合糖培地2に植菌し、発酵反応槽を付属の撹拌機によって800rpmで撹拌し、発酵反応槽の通気量の調整、温度調整を行い、36時間培養を行った(前培養)。前培養完了後、直ちに発酵液循環ポンプを稼動させ、さらに培地の連続供給を行い、連続発酵装置の発酵液量を1.5Lとなるよう培養液の濾過量の制御を行いながら以下の条件で300時間の連続培養を行い、エタノールを製造した(表14)。
発酵反応槽容量:2(L)
使用分離膜:ポリフッ化ビニリデン濾過膜
膜分離エレメント有効濾過面積:120(cm2)
温度調整:30(℃)
発酵反応槽通気量:100(mL/分)
発酵反応槽撹拌速度:800(rpm)
pH調整:なし
発酵液の抜き量:1(L/Day)
滅菌:分離膜エレメントを含む培養槽、および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
2,3-ブタンジオール微生物であるパエニバチラス・ポリミキサATCC12321株を用い、培地として表15に示す組成の2,3-ブタンジオール発酵培地を用いた。
発酵反応槽容量:2(L)
温度調整:30(℃)
反応槽通気量:100(mL/分)
反応槽攪拌速度:800(rpm)
pH調整:5N NaOHによりpH6.5に調整
滅菌:培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
表16に示す混合糖2,3-ブタンジオール発酵培地2を用い、比較例7と同様の条件で50時間のバッチ培養を行い、2,3-ブタンジオールを製造した(表17)。
混合糖(グルコース、キシロース)を発酵原料にした分離膜を利用しない連続培養を行なった。パエニバチラス・ポリミキサATCC12321株を上記比較例7の前培養と同じ条件で振とう培養した(前々培養)。前々培養液を、1.2Lの表17に示す混合糖2,3-ブタンジオール発酵培地2に植菌し、発酵反応槽を付属の撹拌機によって200rpmで撹拌し、温度調整を行い、培養液中の糖が完全に消費されるまでバッチ培養を行った(前培養)。前培養完了後直ちに、前培養完了後、直ちに発酵液抜きポンプを稼動させ、さらに培地の連続供給を行い、連続発酵装置の発酵液量を1.2Lとなるよう微生物を含む培養液の引き抜き量の制御を行いながら以下の条件で280時間の連続培養を行い、2,3-ブタンジオールを製造した(表17)。
発酵反応槽容量:2(L)
温度調整:30(℃)
反応槽通気量:100(mL/分)
反応槽攪拌速度:800(rpm)
pH調整:5N NaOHによりpH6.5に調整
発酵液の抜き量:0.6(L/Day)
滅菌:培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
混合糖(グルコース、キシロース)を発酵原料にした分離膜を利用した連続培養を行なった。分離膜エレメントとしては平膜の形態を採用した。パエニバチラス・ポリミキサ ATCC12321株を上記比較例7の前培養と同じ条件で振とう培養した(前々培養)。前々培養液を、前培養液を1.2Lの混合糖2,3-ブタンジオール発酵培地2に植菌し、発酵反応槽を付属の撹拌機によって200rpmで撹拌し、温度調整を行い、培養液中の糖が完全に消費されるまでバッチ培養を行った(前培養)。前培養完了後、直ちに発酵液循環ポンプを稼動させ、さらに培地の連続供給を行い、連続発酵装置の発酵液量を1.2Lとなるよう培養液の濾過量の制御を行いながら以下の条件で310時間の連続培養を行い、2,3-ブタンジオールを製造した(表17)。
発酵反応槽容量:2(L)
使用分離膜:ポリフッ化ビニリデン濾過膜
膜分離エレメント有効濾過面積:120(cm2)
温度調整:30(℃)
発酵反応槽通気量:100(mL/分)
発酵反応槽攪拌速度:800(rpm)
発酵液の抜き量:0.6L/Day
滅菌:分離膜エレメントを含む培養槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
参考例4と同様の条件でバッチ培養を行い、培地として表3に示す組成のエタノール発酵キシロース培地と表4に示すエタノール発酵混合糖培地1を用い、エタノール発酵微生物であるピキア・スティピティスNBRC1687株のエタノール発酵時のキシロース消費速度を算出した。
微生物として、WO2010/140602に開示された方法によって作製したキャンディダ・ユーティリスCuLpLDH株を用いた。培地として表18に示す組成のD-乳酸発酵キシロース培地と表19に示すD-乳酸発酵混合糖培地1を用いた。pHを1N 水酸化カルシウムでpH6.0に調整する以外は、比較例1と同様の条件で40時間バッチ培養を行い、D-乳酸発酵時のキシロース消費速度を算出した。
エタノール発酵微生物であるエシェリシア・コリKO11株のエタノール発酵時のキシロース消費速度を算出した。培地として表20に示す組成のエタノール発酵キシロース培地2と表21に示すエタノール発酵混合糖培地3を用いた。適宜、サンプリングを行い、培養液中のグルコースおよびキシロースの濃度と、生産物であるエタノールの濃度は参考例1の方法により測定した。
培養槽容量:2(L)
温度調整:30(℃)
発酵反応槽通気量:100(mL/分)
発酵反応槽攪拌速度:800(rpm)
pH調整:5N Ca(OH)2によりpH6に調整
滅菌:発酵槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
微生物として、ピキア・スティピティスNBRC1687株を用い、比較例4と同様の条件で23時間バッチ培養を行い、エタノールを製造した。(表23)
微生物として、ピキア・スティピティスNBRC1687株を用い、比較例5と同様の条件で40時間バッチ培養を行い、エタノールを製造した(表23)。
微生物として、ピキア・スティピティスNBRC1687株を用い、混合糖原料にて分離膜を利用しない連続発酵を行った。前培養時間を40時間とする以外は比較例6と同様の条件で行い、298時間の連続発酵によりエタノールを製造した(表23)。
微生物として、ピキア・スティピティスNBRC1687株を用い、分離膜を利用した連続発酵を行った。前培養を48時間とし、膜間差圧を0.1~19.8kPaとした以外は実施例4と同様の条件で行い、305時間の連続発酵によりエタノールを製造した。
微生物として、WO2010/140602に開示された方法によって作製したキャンディダ・ユーティリスCuLpLDH株を用いた。pHを1N 水酸化カルシウムでpH6.0に調整し、培地は表24に示すD-乳酸発酵混合糖培地2を使用する以外は、比較例4と同様の条件で23時間バッチ培養を行い、D-乳酸を製造した(表25)。
微生物としてキャンディダ・ユーティリスCuLpLDH株を用い、pHを1N 水酸化カルシウムでpH6.0に調整し、培地は表24に示すD-乳酸発酵混合糖培地2を使用する以外は、比較例5と同様の条件で40時間バッチ培養を行い、D-乳酸を製造した(表25)。
微生物としてキャンディダ・ユーティリスCuLpLDH株を用い、分離膜を利用しない連続発酵を行った。前培養を40時間とし、pHを1N 水酸化カルシウムでpH6.0に調整し、培地は表24に示すD-乳酸発酵混合糖培地2を使用する以外は比較例6と同様の条件で行い、290時間の連続発酵によりD-乳酸を製造した(表25)。
微生物としてキャンディダ・ユーティリスCuLpLDH株を用い、分離膜を利用した連続発酵を行った。前培養を50時間とし、pHを1N 水酸化カルシウムでpH6.0に調整し、培地は表24に示すD-乳酸発酵混合糖培地2を使用する以外は実施例4と同様の条件で行い、310時間の連続発酵によりD-乳酸を製造した。
エシェリシア・コリKO11株を試験管で2mLの前培養培地(グルコース20g/L酵母エキス10g/L、トリプトン5g/L、NaCl5g/L)にて30℃で一晩培養した(前々培養)。得られた培養液を500mL容量のひだ付三角フラスコに入った50mLの前培養培地に植菌し、一晩培養した(前培養)。前培養液を表26に示す組成の1Lのエタノール発酵培地2に植菌し、温度調整およびpH調整を行いながら以下に示す運転条件にて16時間バッチ発酵を行い、エタノールを製造した(表28)。
培養槽容量:2(L)
温度調整:30(℃)
Kla:30(h-1)
pH調整:5N Ca(OH)2によりpH6に調整
滅菌:発酵槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
発酵培地として表27に示す組成のエタノール発酵混合糖培地4を用いて、比較例16と同様の条件で24時間バッチ発酵を行い、エタノールを製造した(表28)。
混合糖を発酵原料にした、分離膜を利用しない連続発酵を行った。エシェリシア・コリKO11株を試験管で2mLの前培養培地(グルコース 20g/L、酵母エキス 10g/L、トリプトン 5g/L、NaCl 5g/L)にて30℃で一晩培養した(前々々培養)。得られた培養液を500mL容量のひだ付三角フラスコに入った50mLの前培養培地に植菌し、一晩培養した(前々培養)。前々培養液を表27に示す組成のエタノール発酵混合糖培地4の入った連続培養装置(WO2007/097260の図2に示す装置より分離膜エレメントを除いたもの)に植菌し、温度調整およびpH調整を行いながら以下に示す運転条件にて24時間バッチ発酵を行った(前培養)。前培養完了後、直ちに連続培養を開始し、エタノールの製造を行った。表27に示す組成のエタノール発酵混合糖培地3の供給と微生物を含む培養液の引き抜きにはペリスタ・バイオミニポンプAC-2120型(ATTO社)を用いて、培養槽内に直接培地供給を行い、培養槽内から直接微生物を含む培養液の引き抜きを行った。微生物を含む培養液の引き抜き速度を一定にして、培養槽内の培養液量を1.5Lとなるように培地供給速度を制御しながら290時間エタノール製造を行った(表28)。
培養槽容量:2(L)
温度調整:30(℃)
Kla:30(h-1)
pH調整:5N Ca(OH)2によりpH6に調整
発酵液の抜き速度:2L/day
滅菌:発酵槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
混合糖を発酵原料にした、分離膜を利用した連続発酵を行った。エシェリシア・コリKO11株を試験管で2mLの前培養培地(グルコース 20g/L、酵母エキス 10g/L、トリプトン 5g/L、NaCl 5g/L)にて30℃で一晩培養した(前々々培養)。得られた培養液を500mL容量のひだ付三角フラスコに入った50mLの前培養培地に植菌し、一晩培養した(前々培養)。前々培養液を、表27に示す組成のエタノール発酵混合糖培地4の入った膜一体型の以下の条件を具備した連続発酵装置(WO2007/097260の図2に示す装置)に植菌し、温度調整およびpH調整を行いながら以下に示す運転条件にて24時間バッチ発酵を行った(前培養)。前培養完了後、直ちに連続培養を開始し、エタノールの製造を行った。表27に示す組成のエタノール発酵混合糖培地3の供給と培養液の濾過にはペリスタ・バイオミニポンプAC-2120型(ATTO社)を用いた。培地供給は培養槽内に直接行い、培養液の濾過は分離膜を固定したエレメントを通して行った。培養液の濾過速度を一定にして、培養槽内の培養液量を1.5Lとなるように培地供給速度を制御しつつ、濾過時の膜間差圧は0.1~19.8kPaの範囲を推移させることで、310時間の連続発酵によりエタノールを製造した(表28)。
発酵槽容量:2(L)
使用分離膜:ポリフッ化ビニリデンろ過膜
膜分離エレメント有効濾過面積:473cm2
分離膜の純水透過係数:50×10-9m3/m2/s/Pa
分離膜の平均細孔径:0.1μm
平均細孔径の標準偏差:±0.035μm
分離膜の表面粗さ:0.06μm
温度調整:30(℃)
pH調整:5N Ca(OH)2によりpH6に調整
発酵液の抜き速度:2L/day
滅菌:分離膜エレメントを含む発酵槽および使用培地は全て121℃、20minのオートクレーブにより高圧蒸気滅菌。
発酵原料として、バイオマス由来の糖液を使用した。乳酸発酵糖液培地は、WO2010/067785の実施例2に記載のナノ濾過膜による調製方法にて調製したセルロース糖化液を使用し、適宜試薬により表29に示すとおり調製した。使用した培地と、中和剤を4N KOHにする以外は、比較例2と同様の条件で70時間バッチ培養を行い、L-乳酸を製造した(表30)。
発酵原料として、バイオマス由来の糖液を使用した。乳酸発酵糖液培地は、比較例19と同様に表29記載の培地を用いた。使用した培地と、中和剤を4N KOHにする以外は、比較例3と同様の条件で250時間の連続培養を行い、乳酸を製造した(表30)。
発酵原料として、バイオマス由来の糖液を使用した。乳酸発酵糖液培地は、比較例19と同様に表29記載の培地を用いた。使用した培地と、中和剤を4N KOHにする以外は、実施例1と同様の条件で260時間の分離膜利用連続培養を行い、乳酸を製造した。
発酵原料として、バイオマス由来の糖液を使用した。エタノール発酵糖液培地は、WO2010/067785の実施例2に記載のナノ濾過膜による調製方法にて調製したセルロース糖化液を使用し、適宜試薬により表31に示すとおり調製した。使用した培地以外は、比較例17と同様の条件で23時間バッチ培養を行い、エタノールを製造した(表32)。
エタノール発酵糖液培地は、比較例21と同様に表31記載の培地を用い、比較例18と同様の条件で285時間の連続培養を行い、エタノールを製造した(表32)。
表27に示すエタノール発酵糖液培地を用い、実施例8と同様の条件で295時間の分離膜利用連続培養を行い、エタノールを製造した。
表33に示すエタノール発酵混合糖培地5を用い、比較例4と同様の条件で45時間のバッチ培養を行い、エタノールを製造した(表34)。
表33に示すエタノール発酵混合糖培地5を用い、比較例6と同様の条件で350時間の連続培養を行い、エタノールを製造した(表34)。
表33に示すエタノール発酵混合糖培地5を用い、実施例4と同様の条件で360時間の連続培養を行い、エタノールを製造した(表34)。
キャンディダ・トロピカリスNBRC0199株を用い、セラミック製分離膜を利用した連続発酵を行った。発酵培地は表33に示す培地を使用した。キャンディダ・トロピカリスNBRC0199株を試験管で2mLのYPD培地にて30℃で一晩振とう培養した(前々々培養)。得られた培養液をYPD培地50mLの入った500mL容量のひだ付三角フラスコに植菌し、一晩振とう培養した(前々培養)。前々培養液を、1.5LのYPDX培地の入った膜分離型の連続発酵装置(WO2012/086763の図12に示す装置)に植菌し、培養槽を付属の撹拌機によって800rpmで撹拌し、培養槽の通気速度の調整、温度調整を行い、36時間培養を行った(前培養)。前培養完了後、直ちに連続発酵を開始した。濾過時の膜間差圧は500kPa以下となるよう調整しながら、400時間の連続発酵によりエタノールを製造した(表34)。
培養槽容量:2L
使用分離膜:Celfit精密ろ過膜 モノリス φ4-19(日本ガイシ)
膜分離エレメント長さ:500mm
分離膜の平均細孔径:0.1μm
温度調整:30℃
発酵反応槽通気量:100(mL/分)
発酵反応槽撹拌速度:800(rpm)
pH調整:なし。
パエニバチラス・ポリミキサATCC12321株を用い、セラミック製分離膜を利用した連続発酵を行った。パエニバチラス・ポリミキサATCC12321株を試験管で2mLの前培養培地(グルコース5g/L、ペプトン5g/L、酵母エキス3g/L、麦芽エキス3g/L)にて30℃で一晩培養した(前々々培養)。得られた培養液を500mL容量のひだ付三角フラスコに入った50mLの前培養培地に植菌し、一晩培養した(前々培養)。前々培養液を表16に示す組成の2,3-ブタンジオール発酵混合糖培地の入った連続培養装置(WO2007/097260の図2に示す装置)に植菌し、温度調整およびpH調整を行いながら以下に示す運転条件にて30時間バッチ発酵を行った(前培養)。前培養完了後、表16に示す組成の2,3-ブタンジオール発酵混合糖培地を用いて直ちに連続培養を開始し、2,3-ブタンジオールの製造を行った。濾過時の膜間差圧は500kPa以下となるよう調整しながら、300時間の連続発酵により2,3-ブタンジオールを製造した(表35)。
発酵槽容量:2(L)
使用分離膜:Celfit精密ろ過膜 モノリス φ4-19(日本ガイシ)
膜分離エレメント長さ:500mm
分離膜の平均細孔径:0.1μm
温度調整:30(℃)
発酵反応槽通気量:100(mL/分)
発酵反応槽攪拌速度:800(rpm)
pH調整:5N Ca(OH)2によりpH6.5に調整
Claims (5)
- 微生物の培養液を分離膜で濾過すること、未濾過液を培養液に保持または還流すること、発酵原料を培養液に追加すること、および濾過液中の生産物を回収することを含む、連続発酵による化学品の製造方法であって、前記微生物がカタボライト抑制を受ける微生物であり、前記発酵原料が六炭糖および五炭糖を含む、化学品の製造方法。
- 前記濾過液総量の五炭糖の濃度が5g/L以下である、請求項1に記載の化学品の製造方法。
- 前記発酵原料中に含まれる六炭糖と五炭糖の重量比率が1:9~9:1である、請求項1または2に記載の化学品の製造方法。
- 前記発酵原料がバイオマス由来の糖液を含む、請求項1または2に記載の化学品の製造方法。
- 前記五炭糖がキシロースである、請求項1から4のいずれかに記載の化学品の製造方法。
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES13735747T ES2837800T3 (es) | 2012-01-13 | 2013-01-11 | Procedimiento para producir una sustancia química mediante fermentación continua |
US14/371,860 US10378029B2 (en) | 2012-01-13 | 2013-01-11 | Method of producing chemical substance |
KR1020147014765A KR20140116844A (ko) | 2012-01-13 | 2013-01-11 | 화학품의 제조방법 |
BR112014017215A BR112014017215B8 (pt) | 2012-01-13 | 2013-01-11 | Método para produzir um produto químico |
CN201380005122.3A CN104039971A (zh) | 2012-01-13 | 2013-01-11 | 化学品的制造方法 |
EP13735747.1A EP2803732B1 (en) | 2012-01-13 | 2013-01-11 | Method for producing chemical substance by continuous fermentation |
AU2013208439A AU2013208439B2 (en) | 2012-01-13 | 2013-01-11 | Method for producing chemical substance |
CA2860756A CA2860756A1 (en) | 2012-01-13 | 2013-01-11 | Method for producing chemical substance |
RU2014133168/10A RU2595387C2 (ru) | 2012-01-13 | 2013-01-11 | Способ получения химического вещества |
PH12014501607A PH12014501607B1 (en) | 2012-01-13 | 2014-07-11 | Method for producing chemical substance |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2012005255 | 2012-01-13 | ||
JP2012-005255 | 2012-01-13 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013105651A1 true WO2013105651A1 (ja) | 2013-07-18 |
Family
ID=48781592
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2013/050435 WO2013105651A1 (ja) | 2012-01-13 | 2013-01-11 | 化学品の製造方法 |
Country Status (13)
Country | Link |
---|---|
US (1) | US10378029B2 (ja) |
EP (1) | EP2803732B1 (ja) |
JP (2) | JPWO2013105651A1 (ja) |
KR (1) | KR20140116844A (ja) |
CN (2) | CN107043791A (ja) |
AU (1) | AU2013208439B2 (ja) |
BR (1) | BR112014017215B8 (ja) |
CA (1) | CA2860756A1 (ja) |
ES (1) | ES2837800T3 (ja) |
MY (1) | MY173716A (ja) |
PH (1) | PH12014501607B1 (ja) |
RU (1) | RU2595387C2 (ja) |
WO (1) | WO2013105651A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2016086707A (ja) * | 2014-10-31 | 2016-05-23 | トヨタ自動車株式会社 | 連続培養によるエタノールの製造方法及び連続培養装置 |
CN107043791A (zh) * | 2012-01-13 | 2017-08-15 | 东丽株式会社 | 化学品的制造方法 |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3647428A4 (en) * | 2017-06-30 | 2020-07-29 | Toray Industries, Inc. | PRODUCTION PROCESS AND DEVICE FOR CHEMICAL PRODUCTION BY CONTINUOUS FERMENTATION |
CN109207652A (zh) * | 2018-09-14 | 2019-01-15 | 许昌鑫瑞德化工科技有限公司 | 一种皮革化学品生产工艺 |
CN109207653A (zh) * | 2018-09-17 | 2019-01-15 | 许昌鑫瑞德化工科技有限公司 | 一种皮革化学品制备方法 |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH11506934A (ja) | 1995-06-07 | 1999-06-22 | アーケノール,インコーポレイテッド | 強酸加水分解法 |
JP3041380B2 (ja) | 1997-06-02 | 2000-05-15 | 工業技術院長 | 水溶性オリゴ糖類及び単糖類の製造方法 |
JP2001095597A (ja) | 1999-07-27 | 2001-04-10 | Shiseido Co Ltd | ステロイド5α−リダクターゼの阻害活性の検出方法 |
JP2003212888A (ja) | 2002-01-18 | 2003-07-30 | Asahi Kasei Corp | グルコース及び/又は水溶性セロオリゴ糖の製造方法 |
JP2005229821A (ja) | 2004-02-17 | 2005-09-02 | Jgc Corp | バイオマスから単糖を製造する方法及び単糖製造装置 |
JP2006525029A (ja) | 2003-05-02 | 2006-11-09 | ネイチャーワークス・エル・エル・シー | 遺伝子組換え酵母及び遺伝子組換え酵母を用いた発酵方法 |
WO2007097260A1 (ja) | 2006-02-24 | 2007-08-30 | Toray Industries, Inc. | 化学品の製造方法、および、連続発酵装置 |
JP2009112289A (ja) | 2007-09-14 | 2009-05-28 | National Institute Of Advanced Industrial & Technology | キシロース発酵酵母およびそれを用いたエタノールの生産方法 |
JP2010504756A (ja) | 2006-09-28 | 2010-02-18 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | キシロース資化性ザイモモナス(Zymomonas)のキシリトール合成変異体を使用するエタノール産生 |
WO2010067785A1 (ja) | 2008-12-09 | 2010-06-17 | 東レ株式会社 | 糖液の製造方法 |
WO2010140602A1 (ja) | 2009-06-03 | 2010-12-09 | 東レ株式会社 | D-乳酸脱水素酵素活性を有するポリペプチド、該ポリペプチドをコードするポリヌクレオチドおよびd-乳酸の製造方法 |
WO2012086763A1 (ja) | 2010-12-24 | 2012-06-28 | 東レ株式会社 | 分離膜モジュールの滅菌方法、滅菌用装置および化学品製造用装置 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NO884986D0 (no) * | 1987-03-10 | 1988-11-09 | Effenberger Helmut | Fremgangsmÿte ved kontinuerlig fermentering av medier inneholdende karbohydrat ved hjelp av bakterier. |
CN101553572A (zh) * | 2006-02-24 | 2009-10-07 | 东丽株式会社 | 化学品的制备方法和连续发酵装置 |
CN101255446B (zh) * | 2007-12-18 | 2010-09-29 | 大连理工大学 | 一种利用固定化酵母细胞与渗透蒸发膜耦合连续发酵葡萄糖木糖的方法 |
AU2009299152B2 (en) | 2008-09-30 | 2015-07-16 | Toray Industries, Inc. | Method for producing chemical and continuous culture system |
AU2013208439B2 (en) * | 2012-01-13 | 2016-11-24 | Toray Industries, Inc. | Method for producing chemical substance |
-
2013
- 2013-01-11 AU AU2013208439A patent/AU2013208439B2/en not_active Ceased
- 2013-01-11 RU RU2014133168/10A patent/RU2595387C2/ru not_active IP Right Cessation
- 2013-01-11 KR KR1020147014765A patent/KR20140116844A/ko not_active Application Discontinuation
- 2013-01-11 EP EP13735747.1A patent/EP2803732B1/en active Active
- 2013-01-11 BR BR112014017215A patent/BR112014017215B8/pt not_active IP Right Cessation
- 2013-01-11 CN CN201710099848.8A patent/CN107043791A/zh active Pending
- 2013-01-11 WO PCT/JP2013/050435 patent/WO2013105651A1/ja active Application Filing
- 2013-01-11 US US14/371,860 patent/US10378029B2/en active Active
- 2013-01-11 CA CA2860756A patent/CA2860756A1/en not_active Abandoned
- 2013-01-11 JP JP2013501473A patent/JPWO2013105651A1/ja active Pending
- 2013-01-11 MY MYPI2014701897A patent/MY173716A/en unknown
- 2013-01-11 CN CN201380005122.3A patent/CN104039971A/zh active Pending
- 2013-01-11 ES ES13735747T patent/ES2837800T3/es active Active
-
2014
- 2014-07-11 PH PH12014501607A patent/PH12014501607B1/en unknown
-
2017
- 2017-08-15 JP JP2017156747A patent/JP6447682B2/ja active Active
Patent Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH11506934A (ja) | 1995-06-07 | 1999-06-22 | アーケノール,インコーポレイテッド | 強酸加水分解法 |
JP3041380B2 (ja) | 1997-06-02 | 2000-05-15 | 工業技術院長 | 水溶性オリゴ糖類及び単糖類の製造方法 |
JP2001095597A (ja) | 1999-07-27 | 2001-04-10 | Shiseido Co Ltd | ステロイド5α−リダクターゼの阻害活性の検出方法 |
JP2003212888A (ja) | 2002-01-18 | 2003-07-30 | Asahi Kasei Corp | グルコース及び/又は水溶性セロオリゴ糖の製造方法 |
JP2006525029A (ja) | 2003-05-02 | 2006-11-09 | ネイチャーワークス・エル・エル・シー | 遺伝子組換え酵母及び遺伝子組換え酵母を用いた発酵方法 |
JP2005229821A (ja) | 2004-02-17 | 2005-09-02 | Jgc Corp | バイオマスから単糖を製造する方法及び単糖製造装置 |
WO2007097260A1 (ja) | 2006-02-24 | 2007-08-30 | Toray Industries, Inc. | 化学品の製造方法、および、連続発酵装置 |
JP2010504756A (ja) | 2006-09-28 | 2010-02-18 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | キシロース資化性ザイモモナス(Zymomonas)のキシリトール合成変異体を使用するエタノール産生 |
JP2009112289A (ja) | 2007-09-14 | 2009-05-28 | National Institute Of Advanced Industrial & Technology | キシロース発酵酵母およびそれを用いたエタノールの生産方法 |
WO2010067785A1 (ja) | 2008-12-09 | 2010-06-17 | 東レ株式会社 | 糖液の製造方法 |
JP4770987B2 (ja) * | 2008-12-09 | 2011-09-14 | 東レ株式会社 | 糖液の製造方法 |
WO2010140602A1 (ja) | 2009-06-03 | 2010-12-09 | 東レ株式会社 | D-乳酸脱水素酵素活性を有するポリペプチド、該ポリペプチドをコードするポリヌクレオチドおよびd-乳酸の製造方法 |
WO2012086763A1 (ja) | 2010-12-24 | 2012-06-28 | 東レ株式会社 | 分離膜モジュールの滅菌方法、滅菌用装置および化学品製造用装置 |
Non-Patent Citations (7)
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107043791A (zh) * | 2012-01-13 | 2017-08-15 | 东丽株式会社 | 化学品的制造方法 |
JP2016086707A (ja) * | 2014-10-31 | 2016-05-23 | トヨタ自動車株式会社 | 連続培養によるエタノールの製造方法及び連続培養装置 |
Also Published As
Publication number | Publication date |
---|---|
EP2803732A1 (en) | 2014-11-19 |
EP2803732A4 (en) | 2015-08-26 |
JP6447682B2 (ja) | 2019-01-09 |
JPWO2013105651A1 (ja) | 2015-05-11 |
ES2837800T3 (es) | 2021-07-01 |
BR112014017215B1 (pt) | 2021-10-13 |
BR112014017215B8 (pt) | 2021-11-03 |
AU2013208439A1 (en) | 2014-08-21 |
BR112014017215A8 (pt) | 2017-07-04 |
JP2017195909A (ja) | 2017-11-02 |
RU2014133168A (ru) | 2016-03-10 |
PH12014501607A1 (en) | 2014-10-13 |
CA2860756A1 (en) | 2013-07-18 |
PH12014501607B1 (en) | 2014-10-13 |
MY173716A (en) | 2020-02-18 |
CN104039971A (zh) | 2014-09-10 |
AU2013208439B2 (en) | 2016-11-24 |
EP2803732B1 (en) | 2020-12-09 |
RU2595387C2 (ru) | 2016-08-27 |
BR112014017215A2 (pt) | 2017-06-13 |
KR20140116844A (ko) | 2014-10-06 |
US10378029B2 (en) | 2019-08-13 |
CN107043791A (zh) | 2017-08-15 |
US20140349354A1 (en) | 2014-11-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6447682B2 (ja) | 化学品の製造方法 | |
AU2013208440B2 (en) | Method for producing chemical substance | |
JPWO2017159764A1 (ja) | 化学品の製造方法および微生物の培養方法 | |
JP5061639B2 (ja) | 連続発酵装置 | |
JP6540515B2 (ja) | 連続発酵による化学品の製造方法 | |
JPWO2019054469A1 (ja) | エタノールの製造方法およびエタノール発酵液 | |
AU2013208441B2 (en) | Method for producing chemical substance |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
ENP | Entry into the national phase |
Ref document number: 2013501473 Country of ref document: JP Kind code of ref document: A |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13735747 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 20147014765 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2860756 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14371860 Country of ref document: US Ref document number: 12014501607 Country of ref document: PH |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2013735747 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2014133168 Country of ref document: RU Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2013208439 Country of ref document: AU Date of ref document: 20130111 Kind code of ref document: A |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112014017215 Country of ref document: BR |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 112014017215 Country of ref document: BR Kind code of ref document: A2 Effective date: 20140711 |