WO2021117849A1

WO2021117849A1 - Chemical product production method with sequential fermentation that improves membrane filtration

Info

Publication number: WO2021117849A1
Application number: PCT/JP2020/046216
Authority: WO
Inventors: 耳塚　孝; 拓也野口; 和美須田; 日笠　雅史
Original assignee: 東レ株式会社
Priority date: 2019-12-13
Filing date: 2020-12-11
Publication date: 2021-06-17
Also published as: JPWO2021117849A1

Abstract

Disclosed is a method in which a separation membrane that contains cane molasses as a main ingredient and that uses an unsterilized fermentation starting material is utilized to conduct sequential fermentation, wherein high membrane filtration can be maintained and desired chemical product can be produced without any problems. In this chemical product production method: yeast is cultured with an unsterilized fermentation starting material that contains cane molasses as a main ingredient; the obtained culture solution is filtered through a separation membrane, and a filtered liquid that contains a chemical product from which yeast has been separated is collected; an unfiltered solution that includes yeast is retained in or circulated into the culture solution; and more fermentation starting material is added to the culture solution to perform sequential fermentation. In this method, the pH of the fermentation starting material and of the culture solution is controlled to be 4.0 or below.

Description

Method for manufacturing chemicals by continuous fermentation to improve membrane filterability

The present invention relates to a method for producing a chemical product by continuous fermentation using a separation membrane using a fermentation raw material containing cane molasses as a main component.

Biomass-derived chemicals represented by biodegradable polymer raw materials such as lactic acid and biofuels such as ethanol have sustainability and sustainability as well as the manifestation of carbon dioxide emission problems and energy problems into the atmosphere. It is receiving a lot of attention as a life cycle assessment (LCA) compatible product. As a method for producing these biodegradable polymer raw materials and biofuels, glucose, which is a six-carbon sugar refined from edible biomass such as corn, and molasses produced in the process of refining sugar from sugar cane are used as fermentation raw materials. , It is generally obtained as a fermentation product by microorganisms. Molasses is consumed in large quantities as an ethanol fermentation raw material in sugar-producing countries such as Brazil and Thailand, and is an important fermentation raw material.

When culturing, it is common to sterilize the fermentation raw material under high temperature and high pressure conditions or add antibiotics to the fermentation raw material in order to prevent the growth of germs contained in the fermentation raw material. As a method for producing a chemical product by fermentation of microorganisms, there are a batch fermentation method, a fed-batch fermentation method, a continuous fermentation method and the like. Patent Document 1 discloses a method for producing a target chemical product by continuous fermentation using a separation membrane using a fermentation raw material containing cane molasses as a main component.

On the other hand, in order to sterilize a large amount of fermentation raw materials, a great deal of labor and cost are issues, and a large amount of antibiotics is problematic not only from the viewpoint of cost but also from the viewpoint of environmental problems.

Therefore, a method of culturing while preventing the growth of various germs existing in the fermentation raw material using a fermentation raw material that has not been sterilized is also being studied.

In Patent Document 2, in the continuous fermentation method using a separation membrane, two or more fermentation raw material supply lines are installed and used alternately, so that germs can grow even if the fermentation raw material that has not been sterilized is used. A method that enables culturing while preventing the above is described.

Further, Patent Document 3 describes a method of suppressing the growth of various germs by culturing under low pH conditions using acid-resistant yeast.

International Publication No. 2017/159764 International Publication No. 2018/147289 Japanese Unexamined Patent Publication No. 2004-344584

In continuous fermentation using a separation membrane, the culture solution is continuously filtered through the separation membrane for a long period of time, so it is desirable to perform it by a method that does not reduce the membrane filterability of the separation membrane. Therefore, in the present invention, high membrane filterability can be maintained and the target chemical product can be produced without any problem in continuous fermentation using a separation membrane using a fermentation raw material containing cane molasses as a main component and not sterilized. The purpose is to provide a method that can be produced.

As a result of diligent studies, the present inventor used the fermentation raw material for fermentation without sterilization in continuous fermentation using a separation membrane using a fermentation raw material containing cane molasses as a main component, and used the fermentation raw material and the culture solution at pH 4. We have found that the above problems can be solved by controlling the content to 0 or less, and have completed the present invention.

That is, the present invention provides the following.

(1) Yeast is cultivated with a fermentation raw material containing cane moraces as a main component and has not been sterilized, and the obtained culture solution is filtered through a separation membrane to obtain a filtrate containing the chemical product from which the yeast has been separated. A method for producing a chemical product, which is collected, holds or recirculates an unfiltered solution containing the yeast in the culture solution, adds the fermentation raw material to the culture solution, and continuously ferments, wherein the fermentation raw material and the culture solution are continuously fermented. A method for producing a chemical product, which controls the pH to 4.0 or less.
(2) The method according to (1), wherein the intermembrane pressure of the separation membrane is 0.1 kPa to 150 kPa.
(3) The method according to (1) or (2), wherein the yeast belongs to the genus Schizosaccharomyces.
(4) The method according to any one of (1) to (3), wherein the membrane filtration rate when the culture solution is filtered through the separation membrane is accelerated within 150 hours ± 50 hours from the start of fermentation.
(5) The method according to (4), wherein the membrane filtration rate is increased 1.5 to 2.0 times that before acceleration in 150 hours ± 50 hours from the start of fermentation.
(6) The method according to any one of (1) to (5), wherein the chemical product is an organic acid or an alcohol.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to maintain high membrane filterability and produce a desired chemical product in continuous fermentation using a separation membrane using a fermentation raw material containing cane molasses as a main component and not sterilized. Become.

In the present invention, yeast is cultured in a fermentation raw material that contains cane molaces as a main component and has not been sterilized, and the obtained culture solution is filtered through a separation membrane to contain a filtrate containing the chemical product from which the yeast has been separated. In a method for producing a chemical product in which an unfiltered solution containing yeast is retained or recirculated in the culture solution, and the fermentation raw material is added to the culture solution for continuous fermentation, the fermentation raw material and the culture solution are used. The present invention relates to a method for producing a chemical product, which controls the pH to 4.0 or less.

The yeast used in the present invention is not particularly limited as long as it is a yeast having an ability to produce a chemical product from a fermentation raw material containing cane molasses as a main component. The yeast used may be one isolated from the natural environment, or one whose properties have been partially modified by mutation or genetic recombination. For example, the genus Saccharomyces, the genus Kluyveromyces, and the genus Saccharomyces can be used. Among these, yeast belonging to the genus Schizosaccharomyces is preferable, and specifically, Schizosaccharomyces pombe, Schizosaccharomyces japonicus, and Schizosaccharomyces schizosaccharomyces can be preferably used.

Khene molasses is a by-product produced in the process of sugar production from sugar cane juice or raw sugar. That is, it refers to a solution containing a sugar component remaining after crystallization in the crystallization step in the sugar manufacturing process. In general, the crystallization step is usually performed a plurality of times, and the crystallization of the first sugar, which is the crystal component obtained by the first crystallization, and the residual liquid of the first molasses (molasses). The crystallization was repeated repeatedly to obtain the remaining liquid as the remaining liquid of the second sugar, which is the crystal component obtained by the above, and the third sugar, which was obtained by crystallization of the remaining liquid of the second sugar (molasses No. 2). The final stage of molasses is called cane crystallization. As the number of crystallizations increases, inorganic salts other than the sugar component are concentrated in the cane molasses. The cane molasses used in the present invention is preferably cane molasses after a large number of crystallizations, and is preferably cane molasses remaining after crystallization at least 2 times, more preferably 3 times or more. preferable.

Since the cane molasses prepared as described above contains a high concentration of sugar, the growth of various germs is suppressed. In the present invention, a fermentation raw material containing cane molasses as a main component can be prepared by diluting cane molasses to a extent that yeast can be fermented. In addition, in this specification, the cane molasses before being diluted to the extent that fermentation is possible may be described as the stock solution of cane molasses. The sugar component contained in cane molasses contains sucrose, glucose, and fructose as main components, and may also contain some other sugar components such as xylose and galactose. The sugar concentration in the stock solution of cane molasses is generally about 200 to 800 g / L. Water or the like may be used as the liquid for diluting the stock solution of cane molasses, and if necessary, a liquid containing a nutrient source necessary for the growth of yeast, which will be described later, may be used. Specifically, a sugar solution obtained by saccharifying cellulose-containing biomass may be used. The cane molasses and the sugar concentration in the sugar solution can be quantified by a known measuring method such as HPLC.

The fermentation raw material used in the present invention needs to contain a nutrient source necessary for yeast to grow. The fermentation raw material used in the present invention may be prepared so as to contain cane molasses as a main component, but other organic micronutrients such as carbon source, nitrogen source, inorganic salts and, if necessary, amino acids and vitamins may be added. It may be added as appropriate. In the present invention, the fermentation raw material containing cane molasses as a main component means that 50% by weight or more of the substances (excluding water) contained in the fermentation raw material is cane molasses.

As carbon sources, sugars such as glucose, sucrose, fructose, galactose, and lactose, starch saccharified liquid containing these sugars, molasses, sugar beet molasses, high test molasses, organic acids such as acetic acid, and alcohols such as ethanol. In addition to glycerin and the like, a cellulose-containing biomass-derived sugar solution is preferably used.

Examples of cellulose-containing biomass include plant-based biomass such as bagasse, switchgrass, corn stover, rice straw, and straw, and wood-based biomass such as trees and waste building materials. Cellulose-containing biomass contains cellulose or hemicellulose, which are polysaccharides obtained by dehydration condensation of sugars, and by hydrolyzing these polysaccharides, a sugar solution that can be used as a fermentation raw material is produced.

The method for preparing the cellulose-containing biomass-derived sugar solution is not particularly limited, and as a method for producing such sugar, a method for acid-hydrolyzing biomass using concentrated sulfuric acid to produce a sugar solution (Special Table No. 11-506934). Japanese Patent Application Laid-Open No. 2005-229821) discloses a method for producing a sugar solution by hydrolyzing biomass with dilute sulfuric acid and then further treating it with an enzyme such as cellulase (A. Aden et al., “Lignocellulosic”. Biomass to Ethanol Process Design and Economics Hydrolysis Co-Curent Dilute Acid Prehydrorysis and Enzymatic Hydrolysis forrtric Further, as a method without using an acid, a method of hydrolyzing biomass to produce a sugar solution using sub-critical water at about 250 to 500 ° C. (Japanese Patent Laid-Open No. 2003-212888), or treating the biomass with sub-critical water. Later, a method for producing a sugar solution by further enzymatic treatment (Japanese Patent Laid-Open No. 2001-95597), a method of hydrolyzing biomass with pressurized hot water at 240 to 280 ° C., and then further enzymatically treating the sugar solution. (Patent No. 3041380) is disclosed. After the above treatment, the obtained sugar solution and cane molasses may be mixed and purified. The method is disclosed, for example, in WO2012 / 118171.

Ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil lees, soybean hydrolyzate, casein hydrolyzate, other amino acids, vitamins, corn Steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and their hydrolysates are used.

As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be appropriately added.

Further, when the yeast used in the present invention requires a specific nutrient for growth, the nutrient can be added as a standard product or a natural product containing the nutrient and used.

The method of fermenting cane molasses with yeast is well known, and fermentation can be carried out by a well-known method except that the pH of the fermentation raw material and the culture solution is controlled to 4.0 or less.

Further, as the fermentation raw material of the present invention, it is necessary to use a fermentation raw material that has not been sterilized. As used herein, "sterilization" refers to the complete sterilization or removal of proliferative microorganisms. Specific examples of the sterilization treatment of the fermentation raw material include a method of keeping the fermentation raw material under high temperature conditions of 100 ° C. or higher or high temperature and high pressure conditions to kill microorganisms (heat sterilization treatment), and a method of filtering the fermentation raw materials with a filter to remove microorganisms. (Filter sterilization treatment), a method of irradiating a fermentation raw material with UV or γ-rays to lose the ability of microorganisms to grow (ultraviolet sterilization treatment, radiation sterilization treatment), a method of administering an antibiotic, and the like. To confirm whether the fermentation raw material is sterilized by the above sterilization treatment, for example, if the sterilized fermentation raw material is applied to the LB medium or YPD medium by a sterile operation and then the microorganisms do not grow in the main medium, the fermentation raw material is used. It can be confirmed that it is sterilized. The non-sterilized fermentation raw material used in the present invention is a fermentation raw material in a state in which the above-mentioned treatment has not been performed and the proliferative microorganisms have not been completely sterilized or removed.

In the present invention, the fermentation raw material and the culture solution are controlled to have a pH of 4.0 or less. When preparing a fermentation raw material, the stock solution of cane molasses has a high viscosity and it is difficult to control the pH. Therefore, after diluting the stock solution of cane molasses, an acid may be added so that the pH becomes 4.0 or less. The pH when cane molasses is diluted with water is usually about pH 5.0. The lower limit of the pH of the fermentation raw material and the culture solution is not particularly limited as long as the yeast can grow and the desired chemical product can be produced, and can be appropriately set, but is more than 3.0. Is preferable.

In the method of the present invention, it is important that the pH of the fermentation raw material (in the stage before inoculation of yeast) containing cane molasses as a main component is 4.0 or less, preferably more than 3.0 and 4.0 or less. is there. Adjusting the pH of the fermentation raw material to this range prior to yeast inoculation has the significant effect of suppressing the increase in membrane filtration resistance during continuous culture. It is considered that this effect is not caused by the suppression of the growth of various germs, but by some mechanism brought about when the fermentation raw material containing cane molasses as a main component is fermented with yeast. This is because, in Comparative Example 2 described later, although the fermentation raw material was sterilized by heating and the culture was performed by controlling the pH at the time of culturing to 4.0, the pH was 4.0 without sterilizing the fermentation raw material. This is because the membrane filtration resistance is much higher than that of Example 1 which is only controlled to. Further, in Comparative Example 4, the fermentation raw material was filtered and sterilized to remove not only germs but also other substances that may cause clogging, but the membrane filtration resistance was higher than that of the examples of the present invention. I grew up. As described above, although the "sterilization" defined in the present invention cannot be performed at pH 4.0, the membrane filtration resistance is smaller than that in the case of heat sterilization or filtration sterilization, and the target chemical product can be produced in a satisfactory production amount. , It was found that an unexpected remarkable effect was produced.

On the other hand, if the fermentation raw material that has not been sterilized is used and the culture is performed at a pH exceeding 4.0, various germs will grow. The growth of various germs can be confirmed by whether or not microorganisms other than yeast inoculated in the culture solution are detected. In addition, the concentration of chemical substances in the culture solution can be measured to indirectly confirm the growth of various germs when the amount of chemical substances not produced by the inoculated yeast is increasing. As the chemical substance to be measured, the concentration of a chemical substance other than the main product of yeast inoculated into the fermentation raw material may be measured, and examples thereof include lactic acid and succinic acid.

When controlling the pH of the culture solution to pH 4.0 or less, an acid or alkali may be added so that the pH during culturing becomes pH 4.0 or less. In the present specification, "controlling the pH" means not only adjusting the pH to a predetermined pH by adding an acid or an alkali, but also adjusting the pH of the culture solution to pH 4 by the organic acid or the organic alkali produced by the microorganism during fermentation. The state of pH 4.0 or less of the present invention is also included in the state controlled to pH 4.0 or less of the present invention.

There are no particular restrictions on the acid or alkali used to control the pH, and industrially available general-purpose products are preferable. For example, acids include hydrochloric acid, sulfuric acid, acetic acid, nitric acid, calcium carbonate and the like, and alkalis include aqueous ammonia, sodium hydroxide and potassium hydroxide.

The separation membrane used in the present invention is not particularly limited as long as it has a function of separating and filtering the culture solution obtained by fermentation with a stirring type fermenter or a stirring type bioreactor of microorganisms from microorganisms, and the material may be used. For example, a porous ceramic film, a porous glass film, a porous organic polymer film, a metal fiber braid, a non-woven fabric, or the like can be used, and among these, a porous organic polymer film or a ceramic film is particularly preferable. is there.

In the present invention, the separation membrane is preferably a separation membrane containing a porous resin layer as a functional layer, for example, from the viewpoint of stain resistance.

The separation membrane containing the porous resin layer preferably has a porous resin layer acting as a separation functional layer on the surface of the porous base material. The porous substrate supports the porous resin layer and imparts strength to the separation membrane. Further, when the porous resin layer is provided on the surface of the porous base material, even if the porous resin layer permeates the porous base material, the porous resin layer does not permeate the porous base material. Either of them may be used, and it is selected according to the application.

The average thickness of the porous substrate is preferably 50 to 3000 μm.

The material of the porous base material is composed of an organic material and / or an inorganic material, and organic fibers are preferably used. Preferred porous substrates are woven fabrics and non-woven fabrics made of organic fibers such as cellulose fibers, cellulose triacetate fibers, polyester fibers, polypropylene fibers and polyethylene fibers, and more preferably, the density is relatively easy to control and easy to manufacture. Inexpensive non-woven fabric is used.

As the porous resin layer, an organic polymer film can be preferably used. Examples of 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 resin. The organic polymer film may be a mixture of resins containing these resins as main components. Here, 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 material of the organic polymer film is polyvinylidene chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, etc., which are easy to form with a solution and have excellent physical durability and chemical resistance. A polyacrylonitrile-based resin is preferable, and a polyvinylidene fluoride-based resin or a resin containing the same as a main component is most preferably used.

Here, as the polyvinylidene fluoride-based resin, a homopolymer of vinylidene fluoride is preferably used. Further, as the polyvinylidene fluoride-based resin, a copolymer of vinylidene fluoride and a copolymerizable vinyl-based monomer is also preferably used. Examples of the vinyl-based monomer copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene and ethylene trichloride.

The separation membrane used in the present invention may not allow the microorganisms used for fermentation to pass through, but it is less likely to be clogged by the secretions of the microorganisms used for fermentation and fine particles in the fermentation raw material, and has filtration performance. It is desirable that the range is stable for a long period of time. Therefore, the average pore diameter of the porous separation membrane is preferably 0.01 to 5 μm. Further, more preferably, when the average pore diameter of the separation membrane is 0.01 to 1 μm, it is possible to achieve both a high exclusion rate without leakage of microorganisms and a high water permeability, and the water permeability can be maintained for a long time. Holding can be carried out with higher accuracy and reproducibility.

If the size is close to that of microorganisms, they may directly block the pores, so the average pore diameter of the separation membrane is preferably 1 μm or less. The average pore size of the separation membrane is preferably not too large compared to the size of the microorganisms in order to prevent the leakage of microorganisms, that is, the occurrence of defects that reduce the exclusion rate. In addition, microorganisms may produce substances other than chemicals, which are desired products, such as substances that easily aggregate, such as proteins and polysaccharides, and cells are further killed by the death of some of the microorganisms in the culture medium. In order to avoid clogging of the separation membrane by these substances, it is more preferable that the average pore diameter is 0.1 μm or less. If the average pore diameter is too small, the water permeability of the separation membrane deteriorates and efficient operation cannot be performed even if the membrane is not dirty. Therefore, the average pore diameter of the separation membrane in the present invention is preferably 0.01 μm or more. Yes, more preferably 0.02 μm or more, still more preferably 0.04 μm or more.

Here, the average pore diameter can be obtained by measuring and averaging the diameters of all the pores that can be observed within the range of 9.2 μm × 10.4 μm in the scanning electron microscope observation at a magnification of 10,000 times. it can. For the average pore diameter, or the membrane surface was photographed at a magnification of 10,000 times using a scanning electron microscope, and 10 or more, preferably 20 or more pores were randomly selected, and the pores of these pores were selected. It is also possible to measure the diameter and calculate by averaging the numbers. When the pores are not circular, a circle (equivalent circle) having an area equal to the area of the pores is obtained by an image processing device or the like, and the equivalent circle diameter is used as the diameter of the pores.

The standard deviation σ of the average pore diameter of the separation membrane used in the present invention is preferably 0.1 μm or less. The smaller the standard deviation σ of the average pore diameter, the smaller it is desirable. For the standard deviation σ of the average pore diameter, the number of pores that can be observed within the above range of 9.2 μm × 10.4 μm is N, each measured diameter is Xk, and the average pore diameter is X (ave). It is calculated by the following (Equation 1).

In the separation membrane used in the present invention, the permeability of the culture solution is one of the important performances. As an index of the permeability of the separation membrane, the pure water permeability coefficient of the separation membrane before use can be used. ^{In the present invention, the pure water permeability coefficient of the separation membrane is 5.6 × 10 -10} m ³ when the water permeability is measured and calculated at a head height of 1 m using purified water having a temperature of 25 ° C. by a reverse osmosis membrane. It is preferably / m ² / s / pa or more, and the pure water permeability coefficient is 5.6 × 10 ^-10 m ³ / m ² / s / pa or more and 6 × 10 ^-7 m ³ / m ² / s /. If it is pa or less, a practically sufficient amount of permeated water can be obtained.

In the separation membrane used in the present invention, the surface roughness is the average value of the heights in the direction perpendicular to the surface. Membrane surface roughness is one of the factors that make it easier for microorganisms adhering to the separation membrane surface to peel off due to the effect of cleaning the membrane surface by stirring or liquid flow by a circulation pump. The surface roughness of the separation membrane is not particularly limited as long as it is within the range where microorganisms and other solids adhering to the membrane can be peeled off, but it is preferably 0.1 μm or less. When the surface roughness is 0.1 μm or less, microorganisms attached to the film and other solid substances are easily peeled off.

More preferably, the membrane surface roughness of the separation membrane is 0.1 μm or less, the average pore diameter is 0.01 to 1 μm, and the pure water permeability coefficient of the separation membrane is 2 × ^10-9 m ³ / m ² /. It was found that by using a separation membrane of s / pa or more, it is easier to perform an operation that does not excessively require the power required for cleaning the membrane surface. By setting the surface roughness of the separation membrane to 0.1 μm or less, the shearing force generated on the membrane surface can be reduced in the filtration of microorganisms, the destruction of microorganisms is suppressed, and the clogging of the separation membrane is also suppressed. By doing so, long-term stable filtration becomes easier. Further, by setting the surface roughness of the separation membrane to 0.1 μm or less, continuous fermentation can be carried out with a lower intermembrane differential pressure, and even when the separation membrane is clogged, the operation is performed with a high intermembrane differential pressure. Compared with the case, the washing recovery is good. The smaller the surface roughness of the separation membrane, the more preferable it is, because stable continuous fermentation is possible by suppressing the clogging of the separation membrane.

Here, the membrane surface roughness of the separation film is measured under the following conditions using the following atomic force microscope (AFM).
-Device Atomic force microscope device ("Nanoscape IIIa" manufactured by Digital Instruments Co., Ltd.)
・ Condition probe SiN cantilever (manufactured by Digital Instruments Co., Ltd.)
Scanning mode Contact mode (air measurement)
Underwater tapping mode (underwater measurement)
Scanning range 10 μm, 25 μm square (measurement in air)
5 μm, 10 μm square (measured in water)
Scanning resolution 512 x 512

-Sample preparation For the measurement, the membrane sample was immersed in ethanol at room temperature for 15 minutes, immersed in RO water for 24 hours, washed, and then air-dried. RO water refers to water that has been filtered using a reverse osmosis membrane (RO membrane), which is a type of filtration membrane, to remove impurities such as ions and salts. The size of the pores of the RO membrane is approximately 2 nm or less.

The film surface roughness device is calculated by the following (Equation 2) from the height of each point in the Z-axis direction by the above-mentioned atomic force microscope (AFM).

The shape of the separation membrane used in the present invention is preferably a flat membrane or a hollow fiber membrane. When the shape of the separation membrane is a flat membrane, the average thickness thereof is selected according to the application. When the shape of the separation membrane is a flat membrane, the average thickness is preferably 20 to 5000 μm, more preferably 50 to 2000 μm. When the separation membrane is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably 200 to 5000 μm, and the film thickness is preferably 20 to 2000 μm. Further, a woven fabric or knitted fabric in which organic fibers or inorganic fibers are formed into a tubular shape may be contained inside the hollow fiber.

The above-mentioned separation membrane can be produced by a known method (for example, the production method described in WO2007 / 097260).

In the continuous fermentation in the present invention, the culture solution of the microorganism is filtered through a separation membrane, the unfiltered solution is retained or refluxed in the culture solution, and the fermentation raw material is added to the culture solution to recover the product from the filtrate. It is characterized by being fermented.

In the method for producing a chemical product of the present invention, the differential pressure between membranes during filtration is not particularly limited, and it is sufficient that the culture solution can be filtered. However, if the organic polymer membrane is filtered at a differential pressure higher than 150 kPa in order to filter the culture solution, the structure of the organic polymer membrane is more likely to be destroyed, and the ability to produce chemicals is increased. May decrease. Further, if the intermembrane pressure is lower than 0.1 kPa, the amount of permeated water in the culture solution is often insufficient, and the productivity when producing a chemical product tends to decrease. Therefore, in the method for producing a chemical product of the present invention, in the organic polymer membrane, the differential pressure between the membranes, which is the filtration pressure, is preferably in the range of 0.1 to 150 kPa, so that the amount of permeated water in the culture solution is large. Since there is no decrease in the chemical product production capacity due to the destruction of the membrane structure, it is possible to maintain a high chemical product production capacity. The differential pressure between the membranes is preferably in the range of 0.1 to 50 kPa, more preferably in the range of 0.1 to 30 kPa, and particularly preferably in the range of 0.1 to 20 kPa in the organic polymer film. ..

It is determined by calculating the membrane filtration resistance that the membrane filterability is improved by the method of the present invention.

The membrane filtration resistance is a value obtained by dividing the differential pressure between membranes by the flux and is calculated by the following (Equation 3). The membrane filtration resistance is the value of the differential pressure between the membranes required to obtain the unit flux, and the smaller the value of the membrane filtration resistance, the higher the filterability. Flux represents the membrane filtration rate. Specifically, a membrane filtration water per unit membrane area and unit time, units commonly used, per membrane area 1 m ^2, as membrane filtration water per day ^{(m 3 / day), m} 3 Expressed as / (m ² · day) or m / day.

Membrane filtration resistance (kPa / (m / day)) = Intermembrane differential pressure (kPa) / flux (m / day) ... (Equation 3).

Here, in the method for producing a chemical product of the present invention, if the culture solution can be filtered at a higher membrane filtration rate and continuous fermentation can be performed for a longer period of time, the amount of membrane-filtered water will increase, which is an object of the present invention. It is possible to improve the production volume of chemical products. In general, acceleration of the membrane filtration rate causes an increase in the differential pressure between the membranes and causes an increase in the membrane filtration resistance value. However, if the method of the present invention is used, the culture solution is filtered at a high membrane filtration rate. However, since the membrane filterability is improved and the membrane filtration resistance value is lowered, continuous fermentation for a long period of time becomes possible. Further, if the culture solution is filtered at a low membrane filtration rate in the initial stage of continuous fermentation and then accelerated to a high membrane filtration rate, the membrane filtration resistance value can be maintained low for a longer period of time, and the membrane filtration resistance value can be maintained for a longer period of time. Can be continuously fermented. Specifically, it is preferable to accelerate the membrane filtration rate when the culture solution is filtered through the separation membrane at 150 hours ± 50 hours from the start of fermentation, and it is possible to accelerate the acceleration within 150 hours ± 24 hours from the start of fermentation. More preferred. Further, it is preferable that the membrane filtration rate is 1.5 to 2.0 times that before acceleration.

In the method for producing a chemical product of the present invention, filtration of the culture solution may be started after batch fermentation or fed batch fermentation is performed at the initial stage of fermentation to increase the microbial concentration. Alternatively, high-concentration bacterial cells may be seeded and the culture solution may be filtered at the start of fermentation. Here, the start of fermentation of the present invention refers to the time when the microorganism is inoculated into the culture tank for continuous culture. In the method for producing a chemical product of the present invention, it is possible to supply a fermentation raw material and filter a culture solution from an appropriate time. The start time of the fermentation raw material supply and the filtration of the culture solution does not necessarily have to be the same. Further, the supply of the fermentation raw material and the filtration of the culture solution may be continuous or intermittent. In addition, when the membrane filtration rate of the culture solution is changed, the supply rate of fermentation raw materials is also adjusted according to the change in the membrane filtration rate so that the amount of the culture solution in the fermenter for continuous fermentation is within a certain range. There is a need to.

As for the concentration of microorganisms in the culture solution, it is preferable to maintain the productivity of chemicals in a high state in order to obtain efficient productivity. As an example, good production efficiency can be obtained by maintaining the concentration of microorganisms in the culture solution at 5 g / L or more as a dry weight.

In the present invention, the concentration of microorganisms in the fermenter is adjusted by removing a part of the culture solution containing microorganisms from the fermenter and diluting with the fermentation raw material as necessary during the continuous fermentation. May be good. In addition, the production performance of chemicals may change depending on the concentration of microorganisms in the fermenter, and the production performance is maintained by removing a part of the culture solution containing microorganisms and diluting with fermentation raw materials using the production performance as an index. It is also possible to let it.

The temperature in yeast fermentation may be set to a temperature suitable for the yeast to be used, and is not particularly limited as long as the microorganism grows, but the temperature is in the range of 20 to 75 ° C.

In the present invention, the number of fermenters used in continuous fermentation using a separation membrane does not matter.

In the continuous fermentation apparatus used in the present invention, the yeast culture solution is filtered through a separation membrane, the product is recovered from the filtered solution, the unfiltered solution is retained or refluxed in the culture solution, and the fermentation raw material is used as described above. There is no particular limitation as long as it is an apparatus for producing a chemical product by continuous fermentation in which the product in the filtrate is recovered in addition to the culture solution of Specific examples of known devices include the devices described in WO2007 / 097260 and WO2010 / 038613.

Examples of the chemical products produced by the present invention include substances mass-produced in the fermentation industry, such as alcohols, organic acids, and amino acids. For example, examples of alcohol include ethanol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, glycerol, butanol, isobutanol, 2-butanol, isopropanol and the like. Organic acids include acetic acid, lactic acid, adipic acid, pyruvate, succinic acid, malic acid, itaconic acid, citric acid, etc. Amino acids include valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid. , Proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, aspartic acid, glycine, serine and the like.

For example, when the target chemical is ethanol, it is preferable to use, for example, the genus Saccharomyces, the genus Kluyveromyces, and the genus Schizosaccharomyces. Among these, yeast belonging to the genus Schizosaccharomyces is preferable, and specifically, Schizosaccharomyces pombe, Schizosaccharomyces japonicus, and Schizosaccharomyces schizosaccharomyces schizosaccharomyces can be used as schizosaccharomyces or schizosaccharomyces.

The present invention can also be applied to the production of substances such as enzymes, antibiotics and recombinant proteins. These chemicals are recovered from the filtrate by well-known methods (membrane separation, concentration, distillation, crystallization, extraction, etc.).

Further, the present invention is not limited to the above-mentioned method for producing a chemical product, and may be a fermentation method for the purpose of growing microorganisms by the above-mentioned method. Specific examples of this include fermentation using microorganisms as a production target.

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited thereto.

Reference Example 1 Analytical method of saccharides, ethanol and lactic acid The concentrations of saccharides, ethanol and lactic acid in the raw materials were quantified by comparison with the standard under the HPLC conditions shown below.
Column: Shodex SH1011 (manufactured by Showa Denko KK)
Mobile phase: 5 mM sulfuric acid (flow rate 0.6 mL / min)
Reaction solution: None Detection method: RI (differential refractive index)
Temperature: 65 ° C.

Reference Example 2 Preparation of fermentation raw material containing cane molasses as the main component Preparation of fermentation raw material containing cane molasses as the main component by diluting the undiluted solution of cane molasses purchased from Seiwa Sugar Industry Co., Ltd. with a bagasse saccharification treatment solution. Was done. Specifically, the solid content (C6 fraction) obtained by hydrothermally treating bagasse purchased from Seiwa Sugar Industry Co., Ltd. and water are mixed in the same manner as in International Publication No. 2017/159764, and the final solid is obtained. 20 mg / g of dry bagasse of saccharifying enzyme was added to a mixed solution having a concentration of 10%, and a saccharification reaction was carried out for 48 hours to obtain a bagasse saccharified solution. The saccharification reaction was carried out at 50 ° C., and the pH was not controlled. After 48 hours, the bagasse saccharification treatment solution and water were added to the stock solution of cane molasses so as to finally have the ratio shown in Table 1, and then the saccharification residue and the fermentation raw material were solid-liquid separated by a filter press. The main fermentation raw material had a pH of 4.6. Table 2 shows the analysis results of the sugar concentration of the fermentation raw material containing cane molasses as the main component by the method shown in Reference Example 1.

Example 1
Using the ethanol-producing yeast Schizosaccharomyces pombe NBRC1628 strain as a fermenting microorganism and the fermentation raw material prepared by the method described in Reference Example 2 as a medium, continuous cell fermentation using a separation membrane was performed. A hollow fiber form was adopted as the separation membrane element.

The Saccharomyces ponbe NBRC1628 strain was inoculated into 5 ml of heat-sterilized test tubes containing the raw materials shown in Table 2 and cultured overnight with shaking (culture before and after). The obtained culture broth was inoculated into 45 ml of fresh heat-sterilized Erlenmeyer flask containing the raw materials shown in Table 2, and cultured at 30 ° C. and 120 rpm with shaking for 8 hours (pre-pre-culture). 35 mL of 50 mL of the culture solution before the previous one was separated and inoculated into a continuous fermentation device (5% inoculation) containing 700 mL of heat-sterilized raw materials shown in Table 2, and a fermentation reaction tank was attached to the agitator. The mixture was stirred at 300 rpm and fermented for 24 hours (preculture). Immediately after inoculation, the culture solution circulation pump was operated to circulate the solution between the separation membrane module and the fermenter. After the completion of the pre-culture, the filtration pump was operated and the extraction of the culture solution from the separation membrane module was started.

[Common conditions for continuous fermentation]
Fermentation reaction tank capacity: 2 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane membrane separation element Effective filtration area: 218 (cm ² )
Temperature adjustment: 30 (° C)
Fermentation reaction tank Aeration rate: No aeration Fermentation reaction tank Stirring speed: 300 (rpm)
Flux set value: 0.18 (m ³ / m ² / day)
Cross flow filtration flow rate 100 cm / s
Sterilization: The fermenter containing the separation membrane element is autoclaved at 121 ° C for 20 minutes with high-pressure steam sterilization. Average pore diameter: 0.1 μm
Standard deviation of average pore size: 0.035 μm
Membrane surface roughness: 0.06 μm
Pure water permeability coefficient: 50 × ^10-9 m ³ / m ² / s / pa.

[Presence / absence of sterilization of fermentation raw materials and pH control]
The fermentation raw material prepared by the method described in Reference Example 2 was controlled to pH 4.0 with 2N hydrochloric acid and used without sterilization. This fermentation raw material was continuously fermented for 200 hours while controlling the supply of the fermentation raw material so that the amount of the culture solution of the continuous fermentation apparatus became 700 mL after the start of filtration.

[PH control of culture solution]
During the culture, the culture solution was controlled to pH 4.0 with 2N hydrochloric acid and 2N potassium hydroxide.

[Analysis of filtration resistance]
The amount of the filtrate and the differential pressure between the membranes at 200 hours after the start of fermentation were measured, and the flux was calculated from the amount of the filtrate at 200 hours after the start of fermentation, and calculated according to the above formula 3. The results are shown in Table 3. The differential pressure between the membranes was 11 kPa and the membrane filtration resistance was 71.0 kPa / (m / day), indicating extremely high membrane filtration performance. In Example 1, the ethanol concentration in the culture solution was measured by the method described in Reference Example 1 and showed 70 g / L. In addition, as a result of measuring the lactic acid concentration contained in the raw material before and after use by the method of Reference Example 1, the lactic acid concentration did not change at 0.6 g / L before and after use, and no increase in lactic acid concentration was observed. It was judged that continuous culture could be carried out without the proliferation of lactic acid.

Further, when the amount of the membrane filtrate and the differential pressure between the membranes at 350 hours after the start of fermentation were measured in the same manner, the membrane filtration resistance was 400 kPa / (m / day).

Example 2
Except for the conditions and operations described below, the test was carried out in the same manner as in Example 1 to calculate the membrane filtration resistance and the ethanol concentration.

[Presence / absence of sterilization of fermentation raw materials and pH control]
The fermentation raw material prepared by the method described in Reference Example 2 was controlled to pH 3.5 with 2N hydrochloric acid and used without sterilization.

[PH control of culture solution]
During the culture, the culture solution was controlled to pH 3.5 with 2N hydrochloric acid and 2N potassium hydroxide. Table 3 shows the results of calculating the filtration resistance by measuring the amount of the filtrate and the differential pressure between the membranes 200 hours after the start of fermentation and calculating the flux from the amount of the filtrate 200 hours after the start of fermentation. The differential pressure between the membranes was 12.1 kPa and the membrane filtration resistance was 70.0 kPa / (m / day), indicating extremely high membrane filtration performance. Further, in Example 2, the ethanol concentration in the culture solution was 70 g / L. In addition, as a result of measuring the lactic acid concentration contained in the raw material before and after use by the method of Reference Example 1, the lactic acid concentration did not change at 0.6 g / L before and after use, and no increase in lactic acid concentration was observed. It was judged that continuous culture could be carried out without the proliferation of lactic acid.

Comparative Example 1
Except for the conditions and operations described below, a filtration test was conducted in the same manner as in Example 1, and the membrane filtration resistance and ethanol concentration were calculated.

[Presence / absence of sterilization of fermentation raw materials and pH control]
The fermentation raw material prepared by the method described in Reference Example 2 was used after high-pressure steam sterilization in an autoclave at 121 ° C. for 20 minutes. The fermentation raw material at this time was pH 4.6.

[PH control of culture solution]
During the culture, the pH of the culture solution was not controlled, and the pH of the culture solution in this test was in the range of 4.6 to 4.3.

Table 3 shows the results of calculating the filtration resistance by measuring the amount of filtrate and the differential pressure between membranes at 200 hours, calculating the flux from the amount of filtrate 200 hours after the start of fermentation. The differential pressure between the membranes was 60.5 kPa, and the membrane filtration resistance was 387.0 kPa / (m / day), and the membrane filterability was significantly reduced as compared with Examples 1 and 2. Further, in Comparative Example 1, the ethanol concentration in the culture solution was 70 g / L. In addition, as a result of analyzing the concentration of lactic acid contained in the raw materials before and after use, no increase in lactic acid was observed before and after use (0.6 g / L), so it was judged that continuous culture could be carried out without the growth of germs.

Comparative Example 2
Except for the conditions and operations described below, the test was carried out in the same manner as in Example 1 to calculate the membrane filtration resistance and the ethanol concentration.

[PH control of culture solution]
The culture broth was controlled to pH 4.0 with 2N hydrochloric acid and 2N potassium hydroxide.

Table 3 shows the results of calculating the filtration resistance by measuring the amount of filtrate and the differential pressure between membranes at 200 hours, calculating the flux from the amount of filtrate 200 hours after the start of fermentation. The differential pressure between the membranes was 31.4 kPa and the membrane filtration resistance was 185.5 kPa / (m / day), and the membrane filterability was significantly reduced as compared with Examples 1 and 2. Further, in Comparative Example 2, the ethanol concentration in the culture solution was 70 g / L. In addition, as a result of analyzing the concentration of lactic acid contained in the raw materials before and after use, no increase in lactic acid was observed before and after use (0.6 g / L), so it was judged that continuous culture could be carried out without the growth of germs.

Comparative Example 3
Except for the conditions and operations described below, the test was carried out in the same manner as in Example 1 to calculate the membrane filtration resistance and the ethanol concentration.

[Presence / absence of sterilization of fermentation raw materials and pH control]
The fermentation raw material prepared by the method described in Reference Example 2 was used as it was without sterilization. The fermentation raw material had a pH of 4.6.

In Comparative Example 3, the ethanol concentration was significantly lower than that in Example 1 and showed 44.4 g / L. In addition, as a result of analyzing the lactic acid concentration contained in the raw material before and after use, as a result of analyzing the lactic acid concentration in the used raw material tank, the result was significantly increased (5.8 g / L) from before use (0.6 g / L). Therefore, it was judged that various germs had grown, and the culture was discontinued.

Comparative Example 4
Except for the conditions and operations described below, the test was carried out in the same manner as in Example 1 to calculate the membrane filtration resistance and the ethanol concentration.

[Presence / absence of sterilization of fermentation raw materials and pH control]
The fermentation raw material prepared by the method described in Reference Example 2 was used after filter sterilization through a microfiltration membrane and an ultrafiltration membrane. At this time, the fermentation raw material had a pH of 4.6.

[PH control of culture solution]
The pH of the culture solution was not controlled, and the pH of the culture solution in this test was in the range of 4.6 to 4.3.

Table 3 shows the results of calculating the filtration resistance by measuring the amount of filtrate and the differential pressure between membranes at 200 hours, calculating the flux from the amount of filtrate 200 hours after the start of fermentation. The differential pressure between the membranes was 22 kPa and the membrane filtration resistance was 89.1 kPa / (m / day), and the membrane filterability was significantly reduced as compared with Examples 1 and 2. Further, in Comparative Example 4, the ethanol concentration in the culture solution was 70 g / L. In addition, as a result of analyzing the concentration of lactic acid contained in the raw materials before and after use, no increase in lactic acid was observed before and after use (0.6 g / L), so it was judged that continuous culture could be carried out without the growth of germs.

Example 3
Except for the conditions and operations described below, the test was carried out in the same manner as in Example 1 to calculate the membrane filtration resistance and the ethanol concentration.

[Membrane filtration rate setting]
The membrane filtration rate was 0.1 (m ³ / m ² / day) after the start of fermentation, and was accelerated 1.8 times to 0.18 (m ³ / m ² / day) at 150 hours.

The amount of filtrate and the differential pressure between membranes at 350 hours after the start of fermentation were measured, the flux was calculated from the amount of filtrate at 350 hours after the start of fermentation, and calculated according to the above formula 3. The differential pressure between membranes was 18 kPa. The membrane filtration resistance is 100 kPa / (m / day), and the membrane filtration resistance at 350 hours from the start of fermentation in Example 3 is about 4 minutes as compared with the membrane filtration resistance at the same points in Examples 1 and 2. It was found that it can be reduced to 1. In addition, the ethanol concentration in the culture solution was 70 g / L, and ethanol production was possible without any problem.

Summary From the results shown in Table 3, it was confirmed that the membrane filtration resistance of Examples 1 and 2 was smaller than that of Comparative Examples 1 to 4 and the membrane filtration resistance was excellent. In particular, since the fermentation raw material of Comparative Example 4 is subjected to filtration sterilization treatment using a microfiltration membrane and an ultrafiltration membrane, a substance that causes clogging of the separation membrane as compared with the fermentation raw material of Example 1 or 2. Is a fermentation raw material from which is removed, and it is expected that the value of membrane filtration resistance in continuous fermentation using a separation membrane will be low. However, from the above test results, Examples 1 and 2 showed lower membrane filtration resistance values than Comparative Example 4. From the above, the improvement of the membrane filterability according to the present invention is an unexpected and remarkable effect.

Further, in Example 3, the membrane filtration rate is slowed down in the initial stage of fermentation, and the membrane filtration rate is accelerated 1.8 times at 150 hours from the start of fermentation to maintain the membrane filtration rate lower for a longer period of time. I confirmed that I could do it.

Claims

Yeast was cultivated in a fermentation raw material containing cane moraces as a main component and not sterilized, and the obtained culture solution was filtered through a separation membrane to recover the filtrate containing the chemical product from which the yeast was separated. A method for producing a chemical product in which an unfiltered solution containing yeast is retained or recirculated in the culture solution, and the fermentation raw material is added to the culture solution for continuous fermentation. The pH of the fermentation raw material and the culture solution is 4. A method for producing a chemical product, which is controlled to be 0 or less.
The method according to claim 1, wherein the intermembrane pressure of the separation membrane is 0.1 kPa to 150 kPa.
The method according to claim 1 or 2, wherein the yeast belongs to the genus Schizosaccharomyces.
The method according to any one of claims 1 to 3, which accelerates the membrane filtration rate when the culture solution is filtered through the separation membrane within 150 hours ± 50 hours from the start of fermentation.
The method according to claim 4, wherein the membrane filtration rate is increased 1.5 to 2.0 times that before acceleration in 150 hours ± 50 hours from the start of fermentation.
The method according to any one of claims 1 to 5, wherein the chemical product is an organic acid or an alcohol.