JP2008245537A - Method for producing chemicals by continuous fermentation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing chemicals by continuous fermentation in which microorganisms and cultured cells are filtered by a separation membrane, a product is collected from a filtrate, and the microorganisms and the cultured cells are recycled to the culture liquid to improve the concentration of the microorganisms and the cells in the fermentation culture liquid, by maintaining the concentration to be high and high material productivity in the continuous fermentation method. <P>SOLUTION: The method for producing the chemicals by the continuous fermentation, by filtering the fermentation culture liquid of the microorganisms or the cultured cells by the separation membrane, collecting the product from the filtrate, holding or returning the unfiltered liquid in or to the fermentation culture liquid, and adding a fermentation raw material to the fermentation culture liquid includes carrying out the filtering treatment, by using a porous separation membrane having ≥1 μm and ≤5 μm average pore diameter as the separation membrane. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a chemical product by a continuous fermentation method.

  Fermentation methods, which are substance production methods involving the cultivation of microorganisms and cultured cells, are roughly classified into (1) batch fermentation methods (Batch fermentation methods), fed-batch fermentation methods (Fed-Batch fermentation methods), and (2) continuous fermentation methods. can do.

  The batch fermentation method and fed-batch fermentation method of the above (1) are simple in terms of equipment, and have the advantage that the culture is completed in a short time and damage caused by contamination with bacteria is small. However, the product concentration in the fermentation broth increases with the passage of time, and productivity and yield decrease due to the influence of osmotic pressure or product inhibition. Therefore, it is difficult to stably maintain a high yield and high productivity over a long period of time.

  The continuous fermentation method (2) is characterized in that a high yield and high productivity can be maintained over a long period of time by avoiding accumulation of the target substance at a high concentration in the fermentation reaction tank.

  Conventionally, a continuous culture method has been proposed for fermentation of L-glutamic acid and L-lysine (see Non-Patent Document 1). However, in these examples, in order to continuously supply raw materials to the fermentation broth and extract the fermentation broth containing microorganisms and cultured cells, the microorganisms and cultured cells in the fermentation broth are diluted. Therefore, the improvement of production efficiency was limited.

  In the continuous fermentation method, microorganisms and cultured cells are filtered through a separation membrane, and the product is recovered from the filtrate, and at the same time, the filtered microorganisms and cultured cells are retained or refluxed in the fermentation broth, A method for maintaining a high cell concentration has been proposed.

  For example, a technique for continuous fermentation in a continuous fermentation apparatus using a ceramic membrane is disclosed (see Patent Document 1, Patent Document 2, and Patent Document 3). However, the disclosed technique has a problem in reduction of filtration flow rate and filtration efficiency due to clogging of the ceramic film, and back cleaning or the like is performed to prevent clogging.

  Separately, a method for producing succinic acid using a separation membrane has been proposed (see Patent Document 4). In this technique, a high filtration pressure (about 200 kPa) is employed in membrane separation. The high filtration pressure is not only disadvantageous in terms of cost, but the microorganisms and cultured cells are physically damaged by the pressure during the filtration process, so the continuous fermentation method returns the microorganisms and cultured cells back to the fermentation broth continuously. Is not appropriate.

  In conventional continuous culture, the fresh medium is supplied to the fermentation reactor at a constant rate, and the same amount of fermentation broth is discharged outside the fermentation reactor so that the amount of liquid in the fermentation reactor is always kept constant. It is a culture method. In the conventional batch culture method, the culture is terminated when the initial substrate concentration is consumed, but in the continuous culture, the culture can theoretically be continued indefinitely. That is, in theory, it can be fermented infinitely.

  On the other hand, in the conventional continuous culture described above, the microorganisms are discharged together with the fermentation broth out of the fermentation reaction tank, and it is difficult to maintain a high microorganism concentration in the fermentation reaction tank. Therefore, when performing fermentation production, fermentation production efficiency per fermentation volume can be improved if the microorganisms to be fermented can be kept at a high concentration. For this purpose, it is necessary to hold or reflux the microorganisms in the fermenter. As a method for retaining or refluxing microorganisms in the fermentation reaction tank, the discharged fermentation broth is subjected to solid-liquid separation by gravity, for example, centrifugation, and the microorganisms as precipitates are returned to the fermentation reaction tank, or filtered. The method of separating the microorganisms which are solid content by this, and discharging only a fermentation culture-medium supernatant outside a fermentation reaction tank is mentioned. However, the above-described method using centrifugation is expensive because of high power cost. Moreover, since the method by filtration requires high pressure to filter as described above, most of the studies have been conducted at the laboratory level.

As described above, the conventional continuous fermentation method has various problems, and industrial application is difficult. That is, in a continuous fermentation method, microorganisms and cultured cells are filtered through a separation membrane, and the product is recovered from the filtrate. At the same time, the filtered microorganisms and cultured cells are refluxed to the fermentation broth, and the microorganisms and cell concentration in the fermentation broth It is still difficult to obtain high material productivity by improving and maintaining high, and technological innovation has been desired.
JP-A-5-95778 Japanese Patent Laid-Open No. 62-138184 JP-A-10-174594 JP 2005-333886 A Toshihiko Hirao et. Al. (Hirano Toshihiko et al.), Appl. Microbiol. Biotechnol. (Applied Microvials and Microbiology), 32, 269-273 (1989)

  Therefore, an object of the present invention is to provide a method for producing a chemical product by a continuous fermentation method that maintains high productivity stably over a long period of time by a simple operation method. Specifically, the object of the present invention is to filter microorganisms and cultured cells through a separation membrane in a continuous fermentation method, collect the product from the filtrate, and at the same time reflux the filtered microorganisms and cultured cells to the culture solution to perform fermentation culture. An object of the present invention is to provide a method for producing a chemical product by a continuous fermentation method in which the concentration of microorganisms and cells in a liquid is improved and maintained at a high level by maintaining a high substance productivity.

  In the present invention, the fermentation broth of microorganisms or cultured cells is filtered through a separation membrane, the product is recovered from the filtrate, the unfiltered liquid is retained or refluxed in the fermentation broth, and the fermentation raw material is In continuous fermentation to be added to the fermentation broth, a porous separation membrane having pores with an average pore diameter of 1 μm or more and 5 μm or less is used as a separation membrane, and filtration treatment is performed with a transmembrane differential pressure in the range of 0.1 kPa to 150 kPa. It is the manufacturing method of the chemical by the continuous fermentation method.

According to the method for producing a chemical product by the continuous fermentation method of the present invention, the pure water permeability coefficient of the porous separation membrane is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 2 × 10 ⁻⁶ m. ³ / m ² / s / pa or less.

  According to the method for producing a chemical product by the continuous fermentation method of the present invention, the average pore diameter of the porous separation membrane is 1 μm or more and 5 μm or less, and the standard deviation of the average pore diameter of the porous separation membrane is 0. 5 μm or less.

  According to the method for producing a chemical product by the continuous fermentation method of the present invention, the membrane surface roughness of the porous separation membrane is a porous separation membrane of 0.1 μm or less, and the porous separation membrane is a porous resin. A porous separation membrane comprising a layer.

  The present invention separates a fermentation broth into a filtrate and a non-filtrate by a separation membrane, collects a desired fermentation product from the filtrate, and retains or refluxs the non-filtrate in the fermentation broth. In the production method, a porous separation membrane having an average pore diameter in the range of 1 μm or more and 5 μm or less is used as a separation membrane, and filtration treatment is performed at a constant transmembrane pressure, so that the fermentation production efficiency is stable and low cost. The present invention provides a method for producing a chemical by a continuous fermentation method that significantly improves the above.

  According to the present invention, continuous fermentation that stably maintains high productivity over a long period of time can be performed under simple operation conditions, and chemical products that are fermentation products in a wide range of fermentation industries can be stably produced at low cost. Is possible.

  The present invention filters the fermentation broth of microorganisms or cultured cells through a separation membrane, collects the product from the filtrate, holds or refluxs the unfiltered liquid in the fermentation broth, and uses the fermentation raw material as the fermentation broth In a continuous fermentation to be added to the above, a porous separation membrane having pores with an average pore diameter of 1 μm or more and 5 μm or less is used as a separation membrane, and the chemical treatment is filtered with a transmembrane differential pressure in the range of 0.1 kPa to 150 kPa. It is a manufacturing method.

  The porous separation membrane used as the separation membrane in the present invention will be described. First, the structure of the porous separation membrane used as the separation membrane in the present invention will be described. The porous separation membrane 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 separation 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 separation 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 separation membrane used in the present invention preferably has a porous resin layer on the surface of the porous substrate, and even if the porous resin layer penetrates into the porous substrate, it is porous. It does not matter if the porous resin layer does not penetrate into the porous substrate, and it is selected according to the application.

  The average thickness of the porous substrate is preferably 50 μm or more and 3000 μm or less.

  The material of the porous substrate is made of an organic material and / or an inorganic material, and an organic fiber is desirably used. A preferred porous substrate is a woven fabric or a nonwoven fabric using organic fibers such as cellulose fiber, cellulose triacetate fiber, polyester fiber, polypropylene fiber and polyethylene fiber, and more preferably, the density control is relatively easy. A non-woven fabric that is easy to manufacture and inexpensive is used.

  As the porous resin layer, an organic polymer film made of an organic polymer compound 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 resins. The organic polymer film may be a mixture of resins mainly composed of these resins. 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 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.

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

  It is important that the porous separation membrane used in the present invention has an average pore diameter of 1 μm or more and 5 μm or less. When the average pore diameter of the porous separation membrane is less than 1 μm, clogging due to the secretions of microorganisms used for fermentation and the fine particles in the fermentation raw material tends to occur. The average pore diameter of the porous separation membrane is 1 μm or more and 5 μm or less, and more preferably, by utilizing a porous separation membrane having an average pore diameter of 1 μm or more and 2 μm or less, derived from microorganisms or raw materials used for fermentation Therefore, fermentation can be carried out while continuously filtering the fermentation broth with high water permeability.

  Here, the average pore diameter can be determined by measuring and averaging the diameters of all the pores that can be observed within a range of 46 μm × 52 μm in a scanning electron microscope observation at a magnification of 2,000 times. The average pore diameter is photographed with a scanning electron microscope at a magnification of 2,000 times, and 10 or more, preferably 20 or more pores are randomly selected and the diameters of these pores are measured. However, it can also be obtained by number averaging. When the pores are not circular, a circle having an area equal to the area of the pores (equivalent circle) is obtained by an image processing device or the like, and the equivalent circle diameter is obtained by the method of setting the diameter of the pores.

  The standard deviation σ of the average pore diameter of the porous separation membrane used in the present invention is preferably 0.5 μm or less. The smaller the standard deviation σ of the average pore diameter, the better. The standard deviation σ of the average pore diameter is the following, where the number of pores that can be observed within the above-mentioned range of 46 μm × 52 μm is N, each measured diameter is Xk, and the average pore diameter is X (ave) Calculated by (Equation 1).

In the porous separation membrane used in the present invention, the permeability of the fermentation broth is one of the important performances. As an indicator of the permeability of the porous separation membrane, the pure water permeability coefficient of the porous separation membrane before use can be used. In the present invention, the pure water permeability coefficient of the porous separation membrane is 2 × 10 ⁻⁹ m ³ 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. / M ² / s / pa or more, and the pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 2 × 10 ⁻⁶ m ³ / m ² / s / pa or less. If so, a sufficiently high amount of permeated water can be obtained practically.

  In the porous separation membrane used in the present invention, the surface roughness is an average value of heights in a direction perpendicular to the surface. Membrane surface roughness is one of the factors for facilitating separation of microorganisms or cultured cells adhering to the separation membrane surface due to the membrane surface cleaning effect by stirring and a liquid flow by a circulation pump. The surface roughness of the porous separation membrane is preferably 0.1 μm or less. When the surface roughness is 0.1 μm or less, microorganisms or cultured cells attached to the film are easily peeled off.

More preferably, the membrane surface roughness of the porous separation membrane is 0.1 μm or less, the average pore diameter is 1 μm or more and 5 μm or less, and the pure water permeability coefficient of the porous separation membrane is 2 × 10 ⁻⁹ m ³ / It has been found that by using a porous separation membrane of m ² / s / pa or more, an operation that does not require excessive power necessary for membrane surface cleaning can be performed more easily. By setting the surface roughness of the porous separation membrane to 0.1 μm or less, in the filtration of microorganisms or cultured cells, the shearing force generated on the membrane surface can be reduced, the destruction of microorganisms is suppressed, and the porosity is reduced. By suppressing clogging of the separation membrane, stable filtration over a long period of time becomes easier. In addition, by setting the surface roughness of the porous separation membrane to 0.1 μm or less, continuous fermentation can be performed at a lower transmembrane pressure difference, and even when the membrane is clogged, the operation is performed at a higher transmembrane pressure difference. Compared to the case, the cleaning recoverability is good, which is preferable. Since stable continuous fermentation is possible by suppressing clogging, the surface roughness of the porous separation membrane is preferably as small as possible.

Here, the membrane surface roughness of the porous separation membrane can be measured under the following conditions using an atomic force microscope (AFM) described below.
<Apparatus> Atomic force microscope apparatus (Nanoscope IIIa manufactured by Digital Instruments)
<Conditions>
-Probe: SiN cantilever (manufactured by Digital Instruments)
Scan mode Contact mode (in-air measurement)
Underwater tapping mode (underwater measurement)
・ Scanning range: 10μm, 25μm square (measurement in air)
5μm, 10μm square (underwater measurement)
Scanning resolution: 512 × 512.
Sample preparation: Membrane samples were 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, and impurities such as ions and salts are excluded. The pore size of the RO membrane is approximately 2 nm or less.

  The film surface roughness “dr” is calculated by the following (formula 2) from the height of each point in the Z-axis direction by the atomic force microscope (AFM).

  The shape of the porous separation membrane used in the present invention is preferably a flat membrane or a hollow fiber membrane. When the shape of the porous separation membrane is a flat membrane, the average thickness is selected according to the application. The average thickness when the shape of the porous separation membrane is a flat membrane is preferably 20 μm or more and 5000 μm or less, and more preferably 50 μm or more and 2000 μm or less.

  When the porous separation membrane used in the present invention is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably 200 μm or more and 5000 μm or less, and the film thickness is preferably 20 μm or more and 2000 μm or less. Further, 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.

  Next, the outline of the method for producing the porous separation membrane used in the present invention will be exemplified and described. First, an outline of a method for producing a flat membrane among the porous separation membranes will be described.

  A film of a stock solution containing a resin and a solvent is formed on the surface of the porous base material, and the porous base material is impregnated with the stock solution. Thereafter, only the coating-side surface of the porous substrate having a coating is brought into contact with a coagulation bath containing a non-solvent to solidify the resin and form a porous resin layer on the surface of the porous substrate.

  The stock solution is prepared by dissolving the resin in a solvent. In general, the temperature of the stock solution is preferably selected within a temperature range of 5 to 120 ° C. from the viewpoint of film forming properties.

  The solvent dissolves the resin and acts on the resin to encourage them to form a porous resin layer. As the solvent, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone, methyl ethyl ketone, tetrahydrofuran, Tetramethylurea, trimethyl phosphate, cyclohexanone, isophorone, γ-butyrolactone, methyl isoamyl ketone, dimethyl phthalate, propylene glycol methyl ether, propylene carbonate, diacetone alcohol, glycerol triacetate, acetone and methyl ethyl ketone can be used. Among these, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO), which have high resin solubility, are preferably used. These solvents may be used alone or in combination of two or more.

  For example, components other than solvents such as polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and glycerin may be added to the solvent. Non-solvents can also be added to the solvent. The non-solvent is a liquid that does not dissolve the resin. The non-solvent acts to control the pore size by controlling the rate of solidification of the resin. As the non-solvent, water, alcohols such as methanol and ethanol can be used. Of these, water and methanol are preferably used as the non-solvent from the viewpoint of cost. Components other than the solvent and the non-solvent may be a mixture.

  A pore opening agent may be added to the stock solution. The pore-opening agent is extracted when immersed in the coagulation bath, and has a function of making the resin layer porous. By adding a pore opening agent, the average pore size can be controlled. The pore-opening agent is preferably one having high solubility in the coagulation bath. As the pore opening agent, for example, inorganic salts such as calcium chloride, calcium carbonate and silica can be used. As the pore opening agent, polyoxyalkylenes such as polyethylene glycol and polypropylene glycol, water-soluble polymer compounds such as polyvinyl alcohol, polyvinyl butyral and polyacrylic acid, and glycerin can be used.

  Next, an outline of a method for producing a hollow fiber membrane among the porous separation membranes will be described.

  The hollow fiber membrane discharges a stock solution composed of a resin and a solvent from the outer tube of the double-tube base, and discharges a hollow portion forming fluid from the inner tube of the double-tube base in a cooling bath. It can be produced by cooling and solidifying.

  The above stock solution can be prepared by dissolving the resin described in the above-described method for producing a flat film in a solvent described in the above-described method for producing a flat film at a concentration of 20% by weight or more and 60% by weight or less. . Moreover, a gas or a liquid can be normally used for the hollow portion forming fluid. In addition, a new porous resin layer can be coated (laminated) on the outer surface of the obtained hollow fiber membrane. Lamination can be performed to change the properties of the hollow fiber membrane, for example, hydrophilicity / hydrophobicity, pore diameter, and the like to desired properties. A new porous resin layer to be laminated can be produced by bringing a stock solution obtained by dissolving a resin in a solvent into contact with a coagulation bath containing a non-solvent to coagulate the resin. As the material of the resin, for example, the same material as that of the organic polymer film can be preferably used. The method of lamination is not particularly limited, and the hollow fiber membrane may be immersed in the stock solution, or the stock solution may be applied to the surface of the hollow fiber membrane, and after lamination, a part of the deposited stock solution is scraped off. Or the amount of stacking can be adjusted by blowing off with an air knife.

  The porous separation membrane used in the present invention can be made into a separation membrane element by adhering / sealing the hollow portion of the hollow fiber membrane using a member such as a resin and setting it on a support.

  The porous separation membrane used in the present invention can be made into a separation membrane element by combining with a support. A separation membrane element having a porous separation membrane used in the present invention is suitable for a separation membrane element in which a porous separation membrane used in the present invention is disposed on at least one surface of the support plate as a support. One of the forms. In order to increase the amount of water permeation, it is also a preferred embodiment of the separation membrane element to dispose a porous separation membrane on both sides of the support plate.

  In the method for producing a chemical product by continuous fermentation according to the present invention, it is effective to perform filtration treatment with a transmembrane pressure difference in the range of 0.1 kPa to 150 kPa. When filtration is performed at a transmembrane pressure difference higher than 150 kPa in order to filter the fermentation broth, the power cost for applying the pressure increases, and the economic effect when producing chemicals tends to decrease. In addition, when a transmembrane pressure higher than 150 kPa is applied, microorganisms or cultured cells are often crushed, and the ability to produce chemicals decreases. In addition, when the transmembrane pressure is lower than 0.1 kPa, the permeated water amount of the fermentation broth is often not sufficiently obtained, and the productivity when producing a chemical product tends to decrease. In the method for producing a chemical product of the present invention, the transmembrane differential pressure, which is the filtration pressure, is preferably in the range of 0.1 kPa to 150 kPa, so that the amount of permeated water in the fermentation broth is large and the chemistry caused by disruption of microorganisms or cultured cells Since there is no decline in product manufacturing capacity, 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, more preferably in the range of 0.1 kPa to 20 kPa. When the transmembrane pressure difference is outside the range of 0.1 kPa or more and 150 kPa or less, clogging of microorganisms and medium components may occur rapidly, resulting in a decrease in the amount of permeate and a problem in continuous fermentation operation.

  The method for producing a chemical product by continuous fermentation of the present invention uses a fermentation raw material. The fermentation raw material used in the present invention may be any material that promotes the growth of microorganisms to be cultured and can favorably produce a chemical product that is a target fermentation product.

  As the fermentation raw material used in the present invention, a normal liquid medium suitably containing a carbon source, a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins is preferably used. As a carbon source, sugars such as glucose, sucrose, fructose, galactose and lactose, starch saccharified solution containing these sugars, sweet potato molasses, sugar beet molasses, high acid molasses, organic acids such as acetic acid, alcohols such as ethanol, And glycerin and the like are used. Nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein degradation products, other amino acids, vitamins, Corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and the like can be appropriately added.

  When the microorganism used in the present invention requires a specific nutrient for growth, the nutrient can be added as a preparation or a natural product containing it. Moreover, an antifoamer can be used as needed.

  In the present invention, the fermentation culture liquid refers to a liquid obtained as a result of growth of microorganisms or cultured cells as fermentation raw materials. 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 | cultivation start so that productivity of the target chemical product may become high.

  In the present invention, the saccharide concentration in the fermentation broth is preferably maintained at 5 g / l or less. In the present invention, the reason why it is preferable to keep the saccharide concentration in the fermentation broth at 5 g / l or less is to minimize the loss of saccharide due to withdrawal of the broth.

  The culture of microorganisms is usually performed in the range of pH 4-8 and temperature 20-40 ° C. The pH of the culture solution is usually adjusted to a predetermined value within the pH 4-8 range by an inorganic acid or an organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, or the like. If it is necessary to increase the oxygen supply rate, oxygen can be added to the air to maintain the oxygen concentration at, for example, 21% or higher, or the culture can be pressurized, the agitation rate can be increased, or the aeration rate can be increased. it can.

  In the method for producing a chemical product by continuous fermentation of the present invention, batch culture or fed-batch culture may be performed at the initial stage of culture to increase the microorganism concentration, and then continuous culture (pulling) may be started. In the method for producing a chemical product by continuous fermentation according to the present invention, after increasing the concentration of microorganisms, high-concentration cells may be seeded, and continuous culture may be performed at the start of culture. In the method for producing a chemical product by continuous fermentation according to the present invention, it is possible to supply a raw material culture solution and extract a culture from an appropriate time. The starting times of the supply of the raw material culture solution and the withdrawal of the culture are not necessarily the same. Further, the supply of the raw material culture solution and the withdrawal of the culture may be continuous or intermittent.

  Nutrients necessary for cell growth may be added to the raw material culture solution so that the cell growth is continuously performed. It is efficient to maintain the concentration of microorganisms or cultured cells in the fermentation broth at a high level as long as the environment of the fermentation broth is not appropriate for the growth of the microorganisms or cultured cells and does not increase the rate of death. It is preferable for obtaining productivity. For example, good production efficiency can be obtained by maintaining the concentration of microorganisms or cultured cells in the fermentation broth at 5 g / L or more as a dry weight.

  In the method for producing a chemical product by continuous fermentation of the present invention, microorganisms or cultured cells can be extracted from the fermentation reaction vessel as necessary. For example, if the concentration of microorganisms or cultured cells in the fermentation reaction tank becomes too high, the separation membrane is likely to be clogged, so that it can be avoided by drawing them out. In addition, the production performance of chemical products may change depending on the concentration of microorganisms or cultured cells in the fermentation reaction tank, and the production performance can be maintained by extracting microorganisms or cultured cells using the production performance as an index.

  In the method for producing a chemical product by continuous fermentation according to the present invention, the continuous culture operation performed while growing fresh cells having fermentation production ability is a continuous culture method for producing a product while growing cells, The number of fermentation reaction tanks does not matter. In the method for producing a chemical product by continuous fermentation according to the present invention, the continuous culture operation is usually preferably performed in a single fermentation reaction tank in terms of culture management. It is also possible to use a plurality of fermenters because the capacity of the fermentation reaction tank is small. In this case, high productivity of the fermentation product can be obtained even if continuous fermentation is performed by connecting a plurality of fermentation reaction tanks in parallel or in series by piping.

  Next, microorganisms or cultured cells that can be used in the method for producing a chemical product by continuous fermentation according to the present invention will be described. Examples of microorganisms and cultured cells used in the method for producing chemicals by continuous fermentation of the present invention include yeasts such as baker's yeast often used in the fermentation industry, bacteria such as Escherichia coli and coryneform bacteria, filamentous fungi, and actinomycetes. Examples include fungi, animal cells and insect cells. The microorganism or cultured cell used may be isolated from the natural environment, or may be partially modified in nature by mutation or genetic recombination.

  The chemical product produced by the production method by continuous fermentation of the chemical product of the present invention is not limited as long as it is a substance produced by the microorganism or cultured cell in the culture solution. Examples of the chemical product produced by the method for producing a chemical product by continuous fermentation of the present invention include substances that are mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, and nucleic acids. For example, the alcohol includes ethanol, 1,3-propanediol, 1,4-butanediol, and glycerol. The organic acid includes acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, and citric acid. Examples of the nucleic acid include nucleosides such as inosine and guanosine, nucleotides such as inosinic acid and guanylic acid, and diamine compounds such as cadaverine. The present invention can also be applied to the production of substances such as enzymes, antibiotics and recombinant proteins.

  Next, microorganisms or cultured cells that can be used in the method for producing a chemical product by continuous fermentation according to the present invention will be described with reference to specific chemical products.

  In the method for producing a chemical product by continuous fermentation according to the present invention, the microorganism or cultured cell that can be used for the production of L-lactic acid is not limited as long as it is a microorganism that can produce L-lactic acid. .

  In the method for producing a chemical product by continuous fermentation of the present invention, a lactic acid bacterium can be preferably used as the microorganism or cultured cell that can be used for the production of L-lactic acid. Here, lactic acid bacteria can be defined as prokaryotic microorganisms that produce 50% or more of lactic acid in terms of sugar yield with respect to consumed glucose. Preferred lactic acid bacteria include, for example, Lactobacillus genus, Pediococcus genus, Tetragenococcus genus, Carnobacterium genus, Genus Carnobacterium Vus Genus Leuconostoc, Genus Oenococcus, Genus Atopobium, Streptococcus, Genus Enterococcus L, Genus Enterococcus L, Genus Enterococcus L, Genus Enterococcus L ), And lactic acid bacteria belonging to the genus Bacillus (Genus Bacillus). Among them, a lactic acid bacterium having a high yield of lactic acid with respect to sugar can be selected and preferably used for the production of lactic acid.

  In the method for producing a chemical product by continuous fermentation according to the present invention, among lactic acid, a lactic acid bacterium having a high yield of L-lactic acid with respect to sugar can be selected and preferably used for the production of lactic acid. L-lactic acid is a kind of optical isomer of lactic acid and can be clearly distinguished from D-lactic acid which is an enantiomer thereof. Examples of lactic acid bacteria having a high yield of L-lactic acid to sugar include, for example, Lactobacillus yamanashiensis, Lactobacillus animalis, Lactobacillus agilis, Lactobacillus agilis, Lactobacillus aviaries, and Lactobacillus aviaries.・ Lactobacillus casei, Lactobacillus delbruekii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus ruminiacti, Lactobacillus ruminisacti (Lactobacillus sharpeae), Lactococcus dextrinicus, and Lactococcus lactis And the like can be selected and used for the production of L-lactic acid.

  When L-lactic acid is produced by the method for producing a chemical product by continuous fermentation of the present invention, microorganisms or cultured cells having artificially imparted or enhanced lactic acid production ability can be used. For example, it is possible to use microorganisms or cultured cells in which an L-lactic acid dehydrogenase gene (hereinafter sometimes referred to as L-LDH) has been introduced to impart or enhance L-lactic acid production ability. As a method for imparting or enhancing L-lactic acid production ability, a conventionally known method using drug mutation can also be used. More preferably, a recombinant microorganism whose L-lactic acid production ability is enhanced by incorporating L-LDH into the microorganism can be mentioned.

  When L-lactic acid is produced by the method for producing a chemical product by continuous fermentation according to the present invention, the host of the recombinant microorganism is preferably a prokaryotic cell such as Escherichia coli, a lactic acid bacterium, or a eukaryotic cell such as yeast. Yeast. Among the yeasts, a preferable yeast is a yeast belonging to the genus Saccharomyces, and a more preferable yeast is Saccharomyces cerevisiae.

  L-LDH used in the present invention encodes a protein having an activity of converting reduced nicotinamide adenine dinucleotide (NADH) and pyruvate into oxidized nicotinamide adenine dinucleotide (NAD +) and L-lactic acid. If it does, it is not limited. For example, L-LDH derived from lactic acid bacteria having a high yield of L-lactic acid with respect to sugar can be used. Preferably, mammal-derived L-LDH can be used. Among these, L-LDH derived from Homo sapiens and frog can be used. Among the frogs, L-LDH derived from frogs belonging to the family Pipidae is preferably used, and among frogs belonging to the family Frogidae, L-LDH derived from Xenopus laevis can be preferably used.

  The human or frog-derived L-LDH used in the present invention includes genetic polymorphisms and mutant genes caused by mutagenesis. Genetic polymorphism is a partial change in the base sequence of a gene due to a natural mutation on the gene. Mutagenesis refers to artificially introducing a mutation into a gene. Mutagenesis is, for example, a method using a site-directed mutagenesis kit (Mutan-K (Takara Bio) or a random mutagenesis kit (BD Diversify PCR Random Mutagenesis (CLONTECH)). There is. In addition, human- or frog-derived L-LDH used in the present invention lacks a part of the base sequence as long as it encodes a protein having an activity of converting NADH and pyruvate into NAD + and L-lactic acid. There may be lost or inserted.

  When L-lactic acid is produced by the method for producing a chemical product by continuous fermentation according to the present invention, the separation / purification of L-lactic acid contained in the produced filtered / separated fermentation broth is conventionally known concentration, distillation and crystallization. A combination of methods such as analysis can be performed. As a method, for example, a method of extracting the pH of a filtered / separated fermentation broth to 1 or less and then extracting with diethyl ether or ethyl acetate, a method of elution after adsorbing and washing to an ion exchange resin, and an alcohol in the presence of an acid catalyst Examples thereof include a method of reacting and distilling as an ester, and a method of crystallizing as a calcium salt and a lithium salt. Preferably, the concentrated L-lactic acid solution obtained by evaporating water from the filtered / separated fermentation broth can be subjected to a distillation operation. Here, when distilling, it is preferable to distill while supplying water so that the water concentration of the undistilled stock solution is constant. After distilling the L-lactic acid aqueous solution, the water can be concentrated by heating and evaporating to obtain purified L-lactic acid having a target concentration. When an L-lactic acid aqueous solution containing a low-boiling component such as ethanol or acetic acid is obtained as a distillate, it is a preferred embodiment that the low-boiling component is removed during the L-lactic acid concentration process. After the distillation operation, impurities can be removed from the distillate by ion exchange resin, activated carbon, chromatographic separation, or the like, if necessary, to obtain higher purity L-lactic acid.

  When D-lactic acid is produced by the method for producing a chemical product by continuous fermentation of the present invention, microorganisms that can be used for D-lactic acid production or microorganisms that can produce D-lactic acid are limited. There is no. Examples of microorganisms or cultured cells that can be used for D-lactic acid production include, for example, in the wild type strain, Lactobacillus, Bacillus, and Pediococcus having the ability to synthesize D-lactic acid. And microorganisms belonging to

  When D-lactic acid is produced by the method for producing a chemical product according to the present invention by continuous fermentation, the enzyme activity of wild-type D-lactate dehydrogenase (hereinafter sometimes referred to as D-LDH) is enhanced. Is preferred. As a method for enhancing the enzyme activity, a conventionally known method using drug mutation can also be used. More preferably, a recombinant microorganism in which the enzyme activity of D-lactate dehydrogenase is enhanced by incorporating a gene encoding D-lactate dehydrogenase.

  When D-lactic acid is produced by the method for producing a chemical product by continuous fermentation according to the present invention, the host of the recombinant microorganism is preferably a prokaryotic cell such as Escherichia coli, a lactic acid bacterium, or a eukaryotic cell such as yeast. It is.

  When D-lactic acid is produced by the method for producing a chemical product by continuous fermentation according to the present invention, the gene encoding D-lactate dehydrogenase is Lactobacillus plantarum (Lactobacillus plantarum) and Pediococcus acidilactici ( It is preferably a gene derived from Pediococcus acidilactici) and Bacillus laevolacticus, more preferably a gene derived from Bacillus laevolacticus.

  When D-lactic acid is produced by the method for producing a chemical product by continuous fermentation according to the present invention, separation / purification of D-lactic acid contained in the filtered / separated fermentation broth includes conventionally known concentration, distillation and crystallization. A combination of methods can be performed. As a method, for example, a method of extracting the pH of a filtered / separated fermentation broth to 1 or less and then extracting with diethyl ether or ethyl acetate, a method of elution after adsorbing and washing with an ion exchange resin, and alcohol in the presence of an acid catalyst Examples thereof include a method of reacting and distilling as an ester, and a method of crystallizing as a calcium salt and a lithium salt.

  When producing D-lactic acid by the method for producing a chemical product by continuous fermentation according to the present invention, preferably, a concentrated D-lactic acid solution obtained by evaporating water from the filtered / separated fermentation liquid can be subjected to a distillation operation. Here, when distilling, it is preferable to distill while supplying water so that the water concentration of the undistilled stock solution is constant. After distilling off the D-lactic acid aqueous solution, the water can be concentrated by heating to evaporate to obtain purified D-lactic acid having a target concentration. When a D-lactic acid aqueous solution containing a low-boiling component (ethanol, acetic acid, etc.) is obtained as a distillate, it is a preferred embodiment that the low-boiling component is removed during the D-lactic acid concentration process. After the distillation operation, impurities can be removed from the distillate by ion exchange resin, activated carbon, chromatographic separation, or the like, if necessary, to obtain higher purity D-lactic acid.

  When succinic acid is produced by the method for producing a chemical product by continuous fermentation according to the present invention, there is no particular limitation as long as it is a microorganism that can be used for producing succinic acid or a microorganism that can produce succinic acid as cultured cells. Absent. As microorganisms or cultured cells that can be used for the production of succinic acid, bacteria belonging to the genus Anaerobiospirillum or the genus Actinobacillus can be preferably used. Specifically, Anaerobiospirillum succiniciproducens and Actinobacillus succinogenes disclosed in US Pat. No. 5,143,833 are disclosed by Anaerobiospirillum succiniciproducens and James B. Mckinlay. (Actinobacillus succinogenes) (Appl. Microbiol. Biotechnol. (Applied Microvial and Microbiology), 71, 6651-6656 (2005). Moreover, Corynebacterium genus and Brevibacterium (Brevibacterium) Coryneform bacteria such as genus) and Escherichia are also available, including Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum and the like are preferable.

  Moreover, as the microorganism, a microorganism whose succinic acid production ability has been improved by genetic recombination can be used, thereby improving the productivity of succinic acid. Examples of such microorganisms include Brevibacterium flavum MJ233AB-41 (FERM BP-1498) lacking lactate dehydrogenase described in JP-A-2005-27533, and the above non-patents. Corynebacterium glutamicum described in Document 1 and pyruvate formate lyase and lactate dehydrogenase deficient strains described in US Pat. No. 5,770,435 Escherichia coli AFP111 strain and the like can be used.

  When succinic acid is produced by the method for producing a chemical product by continuous fermentation according to the present invention, an ordinary purification method of succinic acid can be applied to the separation and purification of succinic acid. For example, a purification method combining water-splitting electrodialysis treatment and vacuum concentration / crystallization described in JP-A-2005-333886 can be suitably used.

  When 1,3-propanediol is produced by the method for producing a chemical product by continuous fermentation of the present invention, 1,3-propanediol is used as a microorganism or cultured cell that can be used for production of 1,3-propanediol. If it is microorganisms which can be produced, there will be no restriction | limiting in particular. Examples of microorganisms or cultured cells that can be used for the production of 1,3-propanediol include, for example, the genus Klebsiella having the ability to synthesize 1,3-propanediol from glycerol, clostridium ( Examples include microorganisms belonging to the genus Clostridium and the genus Lactobacillus.

  When 1,3-propanediol is produced by the method for producing a chemical product by continuous fermentation according to the present invention, the microorganism comprises (a) at least one gene encoding a polypeptide having glycerol dehydratase activity; (b) glycerol dehydratase reactivation. Preferably comprises at least one gene encoding an activator; and (c) at least one gene encoding a non-specific catalytic activity that converts 3-hydroxypropionaldehyde to 1,3-propanediol. . In the present invention, more preferably, the microorganism is a recombinant microorganism capable of producing 1,3-propanediol.

  When 1,3-propanediol is produced by the method for producing a chemical by continuous fermentation according to the present invention, the microorganism having the ability to synthesize 1,3-propanediol from glycerol is preferably Klebsiella, Clostridium, Lactobacillus, Cytrobacter, Enterobacter (Aerobacter), Aerobacter (Aerobacter), Aspergillus (Aspergillus), Saccharomyces (Saccharomyces) Pichia, Kluyveromise (Kluyveromyces), Candida, Hansenula, Debaryomyces, Mucor, Toluropsis, Methylobacter, Salmonella, Ms. A recombinant microorganism selected from the group consisting of Streptomyces, Escherichia and Pseudomonas, and more preferably Escherichia.

  When 1,3-propanediol is produced by the method for producing a chemical product by continuous fermentation according to the present invention, the recombinant microorganism is improved so that 1,3-propanediol can be efficiently produced from glucose. Is preferable. The recombinant microorganism comprises, for example, (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; and (b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity. A recombinant microorganism containing a gene is preferred. Furthermore, the glycerol dehydratase reactivating factor is preferably a recombinant microorganism containing genes encoded by orfX and orfZ isolated from the dha regulon. Furthermore, the recombinant microorganism is preferably a recombinant microorganism that lacks glycerol kinase activity and / or glycerol dehydrogenase activity and / or triose phosphate isomerase activity.

  When 1,3-propanediol is produced by the method for producing a chemical product by continuous fermentation according to the present invention, separation and purification of 1,3-propanediol contained in the filtered / separated fermentation liquid is performed by concentration and crystallization. be able to. For example, a purification method using vacuum concentration / crystallization described in JP-A-35785 can be suitably used.

  When continuous fermentation is carried out according to the method for producing a chemical product by continuous fermentation of the present invention, a high volumetric production rate is obtained compared to conventional batch fermentation, and extremely efficient fermentation production is possible. Here, the production rate in continuous culture is calculated by the following equation (3).

  The fermentation production rate in batch culture is calculated by dividing the amount of product (g) at the time when all the raw carbon source is consumed by the time (h) required for consumption of the carbon source and the amount of culture solution (L) at that time. Is required.

  Next, the continuous fermentation apparatus used in the method for producing a chemical product of the present invention will be described with reference to the drawings.

  FIG. 1 is a schematic side view for explaining an example of a membrane separation type continuous fermentation apparatus used in the method for producing a chemical by continuous fermentation according to the present invention. FIG. 1 is a typical example in which the separation membrane element is installed outside the fermentation reaction tank.

  In FIG. 1, a membrane separation type continuous fermentation apparatus basically includes a fermentation reaction tank 1, a membrane separation layer 12, and a differential pressure control device 3. Here, the separation membrane element 2 incorporates a porous separation membrane. As the porous separation membrane, for example, a separation membrane and a separation membrane element disclosed in International Publication No. 2002/064240 are suitably used. The membrane separation tank 12 is connected to the fermentation reaction tank 1 via a fermentation culture medium circulation pump 11.

  In FIG. 1, a culture medium is put into the fermentation reaction tank 1 by a medium supply pump 7, the fermentation culture solution in the fermentation reaction tank 1 is stirred with a stirrer 5 as necessary, and a gas supply device 4 is used as necessary. Can supply the required gas. At this time, the supplied gas can be recovered and recycled and supplied again by the gas supply device 4. If necessary, the pH of the fermentation broth is adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8, and the temperature of the fermentation broth is adjusted by the temperature controller 10 as necessary. Fermentative production with high productivity can be performed. Furthermore, the fermentation liquid in the apparatus is circulated between the fermentation reaction tank 1 and the membrane separation tank 12 by the fermentation liquid circulation pump 11. The fermentation broth containing the fermentation product is filtered and separated into microorganisms and fermentation product by the separation membrane element 2 and can be taken out from the apparatus system.

  Moreover, the microorganisms filtered and separated remain in the apparatus system, so that the microorganism concentration in the apparatus system can be maintained high, and fermentation production with high productivity is possible. Here, the filtration / separation by the separation membrane element 2 can be performed without using any special power due to the water head differential pressure with respect to the water surface of the membrane separation tank 12. The pressure control device 3 can appropriately adjust the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation broth in the device system. If necessary, the required gas can be supplied into the membrane separation tank 12 by the gas supply device 4. At this time, the supplied gas can be recovered and recycled and supplied again by the gas supply device 4. The filtration / separation by the separation membrane element 2 can be performed by suction filtration using a pump or the like, or by pressurizing the inside of the apparatus system as necessary. In addition, microorganisms or cultured cells can be cultured for continuous fermentation in a culture tank, and supplied to the fermentation tank as necessary. By culturing microorganisms or cultured cells for continuous fermentation in a culture tank and supplying them into the fermentation tank as needed, continuous fermentation with microorganisms or cultured cells that are always fresh and have a high production capacity for chemicals is possible, resulting in high production. Continuous fermentation with long-term performance is possible.

  Next, FIG. 2 is a schematic side view for explaining an example of another membrane separation type continuous fermentation apparatus used in the present invention. Of the continuous fermentation apparatus used in the method for producing a chemical product of the present invention, a typical example in which a separation membrane element is installed inside a fermentation reaction tank is shown in the schematic diagram of FIG.

  In FIG. 2, the membrane separation type continuous fermentation apparatus basically includes a fermentation reaction tank 1 and a differential pressure control apparatus 3. A porous membrane is incorporated in the separation membrane element 2 in the fermentation reaction tank 1. As this porous separation membrane, for example, a separation membrane and a separation membrane element disclosed in WO2002 / 064240 can be used. The separation membrane element will be described in detail later.

  Next, the form of continuous fermentation by the membrane separation type continuous fermentation apparatus of FIG. 2 will be described.

  The culture medium is pumped continuously or intermittently into the fermentation reaction tank 1 by the culture medium supply pump 7. The medium can be sterilized by heat sterilization, heat sterilization, or sterilization using a filter as necessary before charging. At the time of fermentation production, the fermentation culture solution in the fermentation reaction tank 1 is agitated by the agitator 5 in the fermentation reaction tank 1 as necessary. At the time of fermentation production, the gas required by the gas supply device 4 can be supplied into the fermentation reaction tank 1 as necessary. During fermentation production, if necessary, the pH of the fermentation broth in the fermentation reaction tank 1 is adjusted by the pH sensor / control device 9 and the pH adjustment solution supply pump 8, and the fermentation is performed by the temperature controller 10 as necessary. By adjusting the temperature of the fermentation broth in the reaction tank 1, fermentation production with high productivity can be performed. Here, the pH and temperature are exemplified for the adjustment of the physicochemical conditions of the fermentation broth by the instrumentation / control device. However, if necessary, the dissolved oxygen and ORP are controlled by an analyzer such as an online chemical sensor, The concentration of the chemical in the fermentation broth can be measured, and the physicochemical conditions can be controlled using the concentration of the chemical in the fermentation broth as an index. In addition, the continuous or intermittent input of the medium is preferably performed by appropriately adjusting the amount and speed of the medium input using the measured value of the physicochemical environment of the fermentation broth by the instrumentation apparatus as an index.

  In FIG. 2, microorganisms and fermentation products are filtered and separated from the fermentation broth by the separation membrane element 2 installed in the fermentation reaction tank 1, and the fermentation products are taken out from the apparatus system. In addition, since the filtered and separated microorganisms remain in the apparatus system, the microorganism concentration in the apparatus system can be maintained high, and fermentation production with high productivity is possible. Here, the filtration / separation by the separation membrane element 2 is performed by the water head differential pressure with respect to the water surface of the fermentation reaction tank 1 and can be performed without using any special power. The pressure control device 3 can appropriately adjust the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation broth in the fermentation reaction tank 1. For the filtration / separation by the separation membrane element, filtration / separation can be performed by suction filtration using a pump or the like, or pressurizing the inside of the apparatus system, if necessary. In addition, microorganisms or cultured cells can be cultured for continuous fermentation in a separately prepared culture tank, and supplied to the fermentation tank as necessary. By culturing microorganisms or cultured cells for continuous fermentation in a culture tank and supplying them into the fermentation tank as needed, continuous fermentation with microorganisms or cultured cells that are always fresh and have a high production capacity for chemicals is possible, resulting in high production. Continuous fermentation with long-term performance is possible.

  The separation membrane element preferably used in the continuous fermentation apparatus used in the method for producing a chemical product of the present invention will be described based on the drawings. In the continuous fermentation apparatus used in the method for producing a chemical product of the present invention, preferably, a separation membrane and a separation membrane element disclosed in WO2002 / 064240 can be used.

  The form of the separation membrane element used in the present invention will be outlined below with reference to the drawings of the separation membrane and the separation membrane element disclosed in WO 2002/064240, which is an example of a preferred embodiment. To do. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention. As shown in FIG. 3, the separation membrane element is configured by arranging the flow path material 14 and the separation membrane 15 in this order on both surfaces of a rigid support plate 13. The support plate 13 has recesses 16 on both sides. The separation membrane 15 filters the fermentation broth. The flow path member 14 is for efficiently flowing the permeated water filtered by the separation membrane 15 to the support plate 13. The permeated water that has flowed to the support plate 13 passes through the recess 16 of the support plate 13 and is taken out of the continuous fermentation apparatus via a water collecting pipe 17 that is a discharge means.

  Next, another embodiment of the separation membrane element shown in FIG. 4 will be described. FIG. 4 is a cross-sectional explanatory diagram for explaining an example of another separation membrane element used in the present invention.

  In the separation membrane element, as shown in FIG. 4, a separation membrane bundle 18 formed of a hollow fiber membrane is bonded and fixed in a bundle by a partial resin sealing layer 19 and a lower resin sealing layer 20. Adhesion / fixation by the lower resin sealing layer 20 seals the hollow portion of the hollow fiber membrane and has a structure that prevents leakage of the fermentation broth. On the other hand, the upper resin sealing layer 19 does not seal the inner hole of the hollow fiber membrane, and has a structure in which permeate flows through the water collecting pipe 22. This separation membrane element can be installed in the continuous fermentation apparatus via the support frame 21. The permeated water filtered by the separation membrane bundle 18 passes through the hollow portion of the hollow fiber membrane and is taken out of the fermentation culture tank via the water collecting pipe 22. As power for taking out the permeated water, a method such as a water head differential pressure, a pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used.

  The member constituting the separation membrane element of the continuous fermentation apparatus used in the method for producing a chemical product by continuous fermentation according to the present invention is preferably a member resistant to high-pressure steam sterilization operation. If the inside of the fermentation apparatus can be sterilized, the risk of contamination by undesirable microorganisms during continuous fermentation can be avoided, and more stable continuous fermentation is possible. The member constituting the separation membrane element is preferably resistant to the treatment for 15 minutes at a temperature of 121 ° C., which is the condition of the high-pressure steam sterilization operation. Examples of separation membrane element members include metals such as stainless steel and aluminum, polyamide resins, fluorine resins, polycarbonate resins, polyacetal resins, polybutylene terephthalate resins, PVDF, modified polyphenylene ether resins, and polysulfone resins. These resins can be preferably selected.

  In the continuous fermentation apparatus used in the method for producing a chemical product by continuous fermentation according to the present invention, the separation membrane element may be installed outside the fermentation tank or in the fermentation reaction tank. When the separation membrane element is installed outside the fermentation reaction tank, a separate membrane separation tank can be provided and the separation membrane element can be installed inside it, and the fermentation broth is circulated between the fermentation reaction tank and the membrane separation tank. The fermentation broth can be continuously filtered by the separation membrane element.

  In the continuous fermentation apparatus used in the method for producing a chemical product by continuous fermentation according to the present invention, the membrane separation tank is preferably capable of high-pressure steam sterilization. By allowing the membrane separation tank to be autoclaved, it is easy to avoid contamination with germs.

  Hereinafter, in order to explain the present invention in more detail, L-lactic acid, D-lactic acid, succinic acid and 1,3-propanediol are selected as the above-mentioned chemical products, and microorganisms capable of producing each chemical product. Alternatively, specific embodiments of continuous fermentation using cultured cells using the continuous fermentation apparatus shown in FIGS. 1 and 2 will be described with reference to examples.

  In the examples, when the continuous fermentation apparatus shown in FIG. 1 is used, the fermentation reaction tank has a volume of 2 L, the membrane separation tank has a volume of 0.5 L, and in the continuous fermentation test, the fermentation in this apparatus. The culture medium volume was adjusted to 2L. When the continuous fermentation apparatus shown in FIG. 2 was used, the fermentation reactor volume was 2 L, and in the continuous fermentation test, the fermentation liquid volume in the apparatus was adjusted to 1.5 L.

(Reference Example 1) Production of yeast strain having L-lactic acid production ability A yeast strain having L-lactic acid production ability was constructed as follows. A yeast strain capable of producing L-lactic acid was constructed by linking a human-derived LDH gene downstream of the PDC1 promoter on the yeast genome. For polymerase chain reaction (PCR), KOD-Plus-polymerase (Toyobo) was used according to the attached instruction manual.

  After culturing and recovering human breast cancer cell line (MCF-7), total RNA was extracted using TRIZOL Reagent (Invitrogen), and SuperScript Choice System (Invitrogen) was used with the obtained total RNA as a template. CDNA was synthesized by reverse transcription reaction. Details of these operations followed the attached protocol. The obtained cDNA was used as an amplification template for subsequent PCR.

  The L-ldh gene was cloned by PCR using KOD-Plus-polymerase using the cDNA obtained by the above operation as an amplification template and the oligonucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 as a primer set. Each PCR amplified fragment was purified and the end was phosphorylated with T4 Polynucleotide Kinase (manufactured by TAKARA), and then ligated to a pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver.2 (manufactured by TAKARA). Escherichia coli DH5α was transformed with the ligation plasmid product, and plasmid DNA was collected to obtain a plasmid in which various L-ldh genes (SEQ ID NO: 3) were subcloned. The obtained pUC118 plasmid into which the L-ldh gene was inserted was digested with restriction enzymes XhoI and NotI, and the resulting DNA fragments were inserted into the XhoI / NotI cleavage sites of the yeast expression vector pTRS11 (FIG. 5). In this manner, a human-derived L-ldh gene expression plasmid pL-ldh5 (L-ldh gene) was obtained. The above-described pL-ldh5, which is a human-derived L-ldh gene expression vector, was deposited as a FERM AP-20421 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, alone as a plasmid (deposit date: February 2005). May 21).

  By PCR using the plasmid pL-ldh5 containing the human-derived LDH gene as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 5 as a primer set, a 1.3-kb human-derived LDH gene and Saccharomyces cerevisiae derived A DNA fragment containing the terminator sequence of the TDH3 gene was amplified. In addition, a DNA fragment containing the TRP1 gene derived from Saccharomyces cerevisiae of 1.2 kb was amplified by PCR using the plasmid pRS424 as an amplification template and the oligonucleotides represented by SEQ ID NO: 6 and SEQ ID NO: 7 as a primer set. Each DNA fragment was separated by 1.5% agarose gel electrophoresis and purified according to a conventional method. A product obtained by the PCR method using a mixture of the 1.3 kb fragment and the 1.2 kb fragment obtained here as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 7 as a primer set is 1 . 2.5% agarose gel electrophoresis was carried out to prepare a 2.5 kb DNA fragment linked with human-derived LDH gene and TRP1 gene according to a conventional method. Saccharomyces cerevisiae strain NBRC10505 was transformed with this 2.5 kb DNA fragment in a conventional manner so as not to require tryptophan.

Confirmation that the obtained transformed cells were cells in which the human-derived LDH gene was linked downstream of the PDC1 promoter on the yeast genome was performed as follows. First, genomic DNA of a transformed cell was prepared according to a conventional method, and a 0.7 kb amplified DNA fragment was obtained by PCR using the oligonucleotides represented by SEQ ID NO: 8 and SEQ ID NO: 9 as a primer set. Confirmed by being. In addition, whether or not the transformed cells have lactic acid production ability is determined by the fact that lactic acid is contained in the culture supernatant obtained by culturing the transformed cells in SC medium (METHODS IN YEAS GENETIC 2000 EDITION, CSHL PRESS). It confirmed by measuring the amount of lactic acid by HPLC method on the conditions shown.
・ Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
-Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
-Detection method: electrical conductivity-Temperature: 45 ° C.

The optical purity of L-lactic acid was measured by the HPLC method under the following conditions.
・ Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation)
-Mobile phase: 1 mM aqueous copper sulfate-Flow rate: 1.0 ml / min
・ Detection method: UV254nm
-Temperature: 30 degreeC.

Moreover, the optical purity of L-lactic acid is calculated by the following formula.
Optical purity (%) = 100 × (LD) / (L + D)
(Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid).

  As a result of HPLC analysis, 4 g / L of L-lactic acid was detected, and D-lactic acid was below the detection limit. From the above examination, it was confirmed that this transformant has L-lactic acid production ability. The obtained transformed cells were used in subsequent examples as yeast strain SW-1.

Reference Example 2 Production of Porous Separation Membrane (Part 1)
Of the porous separation membrane used in the present invention, the creation of a flat membrane will be described. Polyvinylidene fluoride (15.9 wt%), lithium chloride (0.9 wt%), water (3.7 wt%) and polyvinylpyrrolidone (2.3 wt%) were added to the solvent dimethylacetamide (DMAC) (77.2 wt%) and mixed. The resin stock solution was prepared by sufficiently dissolving. Next, after cooling the obtained resin undiluted solution to a temperature of 25 ° C., it was applied to a non-woven fabric made of polyester fiber, which was previously stuck on a glass plate, and left in air with a relative humidity of 80% for 2 minutes, Immediately immersed in a warm water bath at a temperature of 50 ° C. for 5 minutes, a porous substrate on which a porous resin layer was formed was obtained. The surface of the porous resin layer of the obtained separation membrane was observed with a scanning electron microscope at a magnification of 1,500 times. The average diameter of all the pores that can be observed was 2.0 μm.

Next, as a result of evaluating the pure water permeability coefficient of the separation membrane, it was 3.0 × 10 ⁻⁶ m ³ / m ² · s · Pa. The pure water permeability coefficient was measured using purified water having a temperature of 25 ° C. prepared with a reverse osmosis membrane at a head height of 1 m. The porous separation membrane thus produced could be suitably used in the present invention.

Reference Example 3 Production of Porous Separation Membrane (Part 2)
Of the porous separation membrane used in the present invention, the production of a hollow fiber membrane will be described. 14.8% by volume of hydrophobic silica (Aerogel R-972), 48.5% by volume of dioctyl phthalate and 4.4% by volume of dibutyl phthalate were mixed in a mixer, and 32.3% by volume of polyvinylidene fluoride was added thereto. And mixed well. This was formed into a hollow fiber shape using a hollow fiber production apparatus in which a hollow fiber spinneret was attached to a twin screw extruder. The formed hollow fiber was immersed in 1,1,1-trichloroethane and then dried. Subsequently, it was immersed in 50% ethanol aqueous solution for 30 minutes and then in water for 30 minutes. Furthermore, after being immersed in a 20% NaOH aqueous solution at a temperature of 60 ° C. for 1 hour to elute the silica particles, they were thoroughly washed with water and dried.

As a result of observing the pore diameter on the outer surface of the obtained polyvinylidene fluoride porous separation membrane with a scanning electron microscope, the average pore diameter was 1.6 μm. As a result of evaluating the pure water permeability coefficient of this porous separation membrane, it was 6 × 10 ⁻⁸ m ³ / m ² / s / pa. The pure water permeability coefficient was measured using purified water at 25 ° C. prepared with a reverse osmosis membrane at a head height of 1 m.

(Example 1) Production of L-lactic acid by continuous fermentation using yeast (part 1)
Using the yeast SW-1 strain prepared in Reference Example 1, L-lactic acid was produced using the continuous fermentation apparatus of FIG. 1 and the yeast lactic acid fermentation medium having the composition shown in Table 1. The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous membrane prepared in Reference Example 2 was used. The operating conditions in Example 1 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane-Membrane separation element effective filtration area: 60 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
-Aeration rate of membrane separation tank: 0.3 (L / min)
・ Fermentation reactor stirring speed: 100 (rpm)
-PH adjustment: adjusted to pH 5 with 1N NaOH-Lactic acid fermentation medium supply rate: 50-300 ml / hr. In the range of circulated fluid volume by variable control and fermentation fluid circulation device: 0.1 (L / min)
・ Membrane permeate flow control: Flow rate control by transmembrane pressure difference (50 hours after the start of continuous fermentation: Control at 0.1 kPa to 50 kPa or less)
50 hours to 100 hours: controlled at 0.1 kPa to 100 kPa or less 100 hours to 150 hours: controlled at 0.1 kPa to 150 kPa or less).

  The HPLC shown in Reference Example 1 was used for the evaluation of the concentration of lactic acid as a product, and “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used for the glucose concentration measurement. It was.

  First, the SW-1 strain was cultured with shaking in a 5 ml lactic acid fermentation medium overnight in a test tube to obtain a culture solution (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh lactic acid fermentation medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L lactic acid fermentation medium of the continuous fermentation apparatus shown in FIG. 1, the fermentation reaction tank 1 is stirred with the attached stirrer 5, the aeration rate of the fermentation reaction tank 1 is adjusted, and the temperature The pH was adjusted, and the culture was performed for 24 hours without operating the fermentation broth circulation pump 11 (pre-culture). Immediately after completion of the pre-culture, the fermentation broth circulation pump 11 is operated, in addition to the operating conditions during pre-culture, the membrane separation tank 12 is vented, and the lactic acid fermentation medium is continuously supplied. Continuous culture was performed while controlling the amount of permeated water so that the amount of the fermentation broth was 2 L, and L-lactic acid was produced by continuous fermentation. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the transmembrane pressure difference so that it was 0.1 kPa or more and 150 kPa or less by the differential pressure control device 3. The produced L-lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately.

  Table 2 shows the results of a 150-hour continuous fermentation test. By the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIG. 1, stable production of L-lactic acid by continuous fermentation was possible.

(Example 2) Production of L-lactic acid by continuous fermentation using yeast (part 2)
Using the continuous fermentation apparatus shown in FIG. 2 and a lactic acid fermentation medium having the composition shown in Table 1, L-lactic acid was produced. The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 2 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Separation membrane: Polyvinylidene fluoride filtration membrane ・ Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
Lactic acid fermentation medium supply rate: 50 to 300 ml / hr. Variable control within the range of-Stirring speed of fermentation reaction tank: 800 (rpm)
-PH adjustment: adjusted to pH 5 with 1N NaOH-Membrane permeate flow rate control: Flow rate control by transmembrane differential pressure (100 hours after starting continuous fermentation: control from 0.1 kPa to 50 kPa or less 100 hours to 140 hours: 0.1 kPa to Control at 100 kPa or less 140 hours to 180 hours: Control at 0.1 kPa to 150 kPa or less).

-Bacteria: All the culture tanks containing the separation membrane element and the working medium were autoclaved at 121 ° C for 20 minutes and autoclaved at high pressure.

  The yeast SW-1 strain prepared in the above Reference Example 1 is used as the microorganism, the lactic acid fermentation medium having the composition shown in Table 1 is used as the medium, and the above Reference Example is used for evaluating the concentration of L-lactic acid as a product. 1 was used, and “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measuring the glucose concentration.

  First, the SW-1 strain was cultured with shaking in a 5 ml lactic acid fermentation medium overnight in a test tube to obtain a culture solution (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh lactic acid fermentation medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture broth was inoculated in a 1.5 L lactic acid fermentation medium of the membrane separation type continuous fermentation apparatus shown in FIG. 2, and the fermentation reaction tank 1 was stirred at 400 rpm by the attached stirrer 5. The amount of aeration was adjusted, the temperature was adjusted, and the pH was adjusted, followed by culturing for 24 hours (preculture). Immediately after the completion of the pre-culture, the lactic acid fermentation medium is continuously supplied and continuously cultured while controlling the amount of permeate through the membrane so that the amount of fermentation liquid in the membrane separation type continuous fermentation apparatus is 1.5 L. -Production of lactic acid was carried out. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the transmembrane pressure difference so that it was 0.1 kPa or more and 150 kPa or less by the differential pressure control device 3. The produced L-lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. In addition, Table 2 shows L-lactic acid versus sugar yield and L-lactic acid production rate calculated from input glucose calculated from L-lactic acid and glucose concentration.

  By the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIG. 2, which was subjected to a fermentation test for 80 hours, stable production of L-lactic acid by continuous fermentation was possible.

(Comparative example 1) Production of L-lactic acid by batch fermentation The most typical batch fermentation was performed as a fermentation form using microorganisms, and the L-lactic acid productivity was evaluated. Using the lactic acid fermentation medium shown in Table 1, a batch fermentation test was conducted using only the fermentation reaction tank 1 of the membrane-separated continuous fermentation apparatus of FIG. The medium was used after autoclaving at 121 ° C. for 15 minutes. Also in this Comparative Example 1, the yeast SW-1 strain constructed in the above Reference Example 1 was used as a microorganism, and the HPLC shown in the above Reference Example 1 was used for evaluation of the concentration of L-lactic acid as a product. For evaluation and measurement of the glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The operating conditions of Comparative Example 1 are shown below.
・ Fermentation reactor capacity: 2 (L)
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
・ Fermentation reactor stirring speed: 100 (rpm)
-PH adjustment: Adjust to pH 5 with 1N NaOH.

  First, the SW-1 strain was cultured with shaking in a 5 ml lactic acid fermentation medium overnight in a test tube (pre-culture). The culture solution was inoculated into 100 ml of fresh lactic acid fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). The preculture liquid was inoculated into 1 L of a lactic acid fermentation medium of a membrane separation type continuous fermentation apparatus, the fermentation reaction tank 1 was stirred at 100 rpm by the attached stirrer 5, and the fermentation reaction tank 1 was aerated. Temperature adjustment and pH adjustment were performed, and batch fermentation culture was performed without operating the fermentation culture medium circulation pump 11. The amount of bacterial cell growth at this time was 14 in terms of absorbance at 600 nm. The results of batch fermentation are shown in Table 2.

  From the above results, it can be seen that the production rate of L-lactic acid was greatly improved by the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIGS.

(Example 3) Continuous production of succinic acid by continuous fermentation (part 1)
Succinic acid was produced using the continuous fermentation apparatus shown in FIG.

Unless otherwise specified, succinic acid and glucose in the production of succinic acid were measured by the following methods. Succinic acid was analyzed by HPLC (Shimadzu LC10A, RI monitor: RID-10A, column: Aminex HPX-87H) for the supernatant of the fermentation broth. The column temperature was 50 ° C., and the column was equilibrated with 0.01N H ₂ SO ₄ , and then the sample was injected and eluted with 0.01N H ₂ SO ₄ for analysis. Glucose was measured using a glucose sensor (BF-4, manufactured by Oji Scientific Instruments).

The medium used was autoclaved at a temperature of 121 ° C. for 15 minutes. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 3 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane-Membrane separation element effective filtration area: 60 square cm
・ Temperature adjustment: 39 (℃)
Fermentation reaction tank CO ₂ aeration amount: 10 (mL / min)
-Membrane separation tank CO ₂ aeration rate: 100 (mL / min)
・ Fermentation reactor stirring speed: 100 (rpm)
· PH Adjustment: 2M _Na 2 adjusted to CO ₃ at pH 6.4 · succinic acid fermentation medium feed rate: 50 to 300 / hr. Circulated fluid volume by variable control / fermentation culture fluid circulation device in the range: 0.1 (L / min)
・ Membrane permeate flow control: Flow control by transmembrane pressure difference.

(After start of continuous fermentation to 100 hours: controlled at 0.1 kPa to 50 kPa or less 100 hours to 130 hours: controlled at 0.1 kPa to 100 kPa or less 130 hours to 150 hours: controlled at 0.1 kPa to 150 kPa or less).

In this Example 3, succinic acid was continuously produced by Anaerobiospirillum succiniciproducens ATCC53488 as a microorganism capable of producing succinic acid. 20 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 3 g / L K ₂ HPO ₄ , 1 g / L NaCl, 1 g / L (NH ₄ ) ₂ SO ₄ , 0.2 g / L MgCl ₂ , and 0 100 mL of a seed culture medium consisting of .2 g / L CaCl ₂ .2H ₂ O was placed in a 125 mL Erlenmeyer flask and sterilized by heating. In an anaerobic glove box, 1 mL of 30 mM Na ₂ CO ₃ and 0.15 mL of 180 mM H ₂ SO ₄ were added, and 0.5 mL of a reducing solution consisting of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S was added. Then, ATCC53488 strain was inoculated and left to stand overnight at a temperature of 39 ° C. (pre-culture). To a 1.5 L succinic acid fermentation medium (Table 3) of the continuous fermentation apparatus shown in FIG. 1 is added 5 mL of a reducing solution composed of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S · 9H ₂ O. After that, 50 mL of the culture solution was inoculated in advance, the fermentation reaction tank 1 was stirred at 200 rpm with the attached stirrer 5, the CO ₂ aeration amount of the fermentation reaction tank 1 was adjusted, the temperature was adjusted, and the pH was adjusted. Culture was performed for 24 hours (pre-culture).

  Immediately after completion of the pre-culture, succinic acid fermentation medium is continuously supplied, and continuous culture is performed while controlling the amount of membrane permeate so that the amount of fermentation liquid in the membrane separation type continuous fermentation apparatus becomes 2 L. Succinic acid by continuous fermentation Was manufactured. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the transmembrane pressure difference so that it was 0.1 kPa or more and 150 kPa or less by the differential pressure control device 3. The produced succinic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. Table 4 shows the production rate of succinic acid and the yield of succinic acid calculated from the succinic acid and glucose concentrations.

(Example 4) Continuous production of succinic acid by continuous fermentation (part 2)
Succinic acid was produced using the continuous fermentation apparatus shown in FIG. The medium used was autoclaved at a temperature of 121 ° C. for 15 minutes. The operating conditions in Example 4 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Separation membrane: Polyvinylidene fluoride filtration membrane ・ Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 39 (℃)
Fermentation reaction tank CO ₂ aeration amount: 10 (mL / min)
-Succinic acid fermentation medium supply speed: Variable control in the range of 50-300 ml / hr-Fermentation reactor agitation speed: 600 (rpm)
- pH Adjustment: 2M _Na 2 CO ₃ at pH6.4 to adjust and membrane permeate flow control: flow rate control by the transmembrane pressure.

(After continuous fermentation starts ~ 120 hours: controlled at 0.1 kPa to 50 kPa or less 120 hours to 160 hours: controlled at 0.1 kPa to 100 kPa or less 160 hours to 210 hours: controlled at 0.1 kPa to 150 kPa or less).
Sterilization: All of the culture tank containing the separation membrane element and the used medium were autoclaved at 121 ° C. for 20 minutes under high pressure steam sterilization.

In Example 4, succinic acid was continuously produced by Anaerobiospirillum succiniciproducens ATCC53488 as a microorganism capable of producing succinic acid. 20 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 3 g / L K ₂ HPO ₄ , 1 g / L NaCl, 1 g / L (NH ₄ ) ₂ SO ₄ , 0.2 g / L MgCl ₂ , and 0 100 mL of a seed culture medium consisting of .2 g / L CaCl ₂ .2H ₂ O was placed in a 125 mL Erlenmeyer flask and sterilized by heating. In an anaerobic glove box, add 1 mL of 30 mM Na ₂ CO ₃ and 0.15 mL of 180 mM H ₂ SO _4, and then add 0.5 mL of a reducing solution consisting of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S. After that, the ATCC53488 strain was inoculated and left to stand overnight at a temperature of 39 ° C. (pre-culture).

To a 1.5 L succinic acid fermentation medium (Table 3) of the continuous fermentation apparatus shown in FIG. 2 is added 5 mL of a reducing solution composed of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S · 9H ₂ O. After that, 50 mL of the culture solution was inoculated before, the fermentation reaction tank 1 was stirred at 600 rpm with the attached stirrer 5, the CO ₂ aeration amount of the fermentation reaction tank 1 was adjusted, the temperature was adjusted, and the pH was adjusted. Culture was performed for 24 hours (pre-culture).

  Immediately after completion of the pre-culture, the succinic acid fermentation medium is continuously supplied, and the continuous culture is carried out while controlling the amount of permeated water in the membrane separation type continuous fermentation apparatus so that the amount of the fermentation liquid becomes 1.5 L. Succinic acid was produced. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the transmembrane pressure difference so that it was 0.1 kPa or more and 150 kPa or less by the differential pressure control device 3. The produced succinic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. Table 4 shows the production rate of succinic acid and the yield of succinic acid calculated from the succinic acid and glucose concentrations.

(Comparative Example 2) Production of succinic acid by batch culture Succinic acid production by batch fermentation of Anaerobiospirillum succiniciproducens was performed as follows.

20 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 3 g / L K ₂ HPO ₄ , 1 g / L NaCl, 1 g / L (NH ₄ ) ₂ SO ₄ , 0.2 g / L MgCl ₂ , 0.00 100 mL of a seed culture medium consisting of ₂ g / L CaCl ₂ · 2H ₂ O was placed in a 125 mL Erlenmeyer flask and sterilized by heating. In an anaerobic glove box, add 1 mL of 30 mM Na ₂ CO ₃ and 0.15 mL of 180 mM H ₂ SO _4, and then add 0.5 mL of a reducing solution consisting of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S. Thereafter, Anaerobiospirillum succiniciproducens ATCC53488 was inoculated and cultured at 39 ° C. overnight. 1 L of fermentation medium shown in Table 3 was added to a mini jar fermenter (manufactured by ABLE, BMJ type, 2 L), and sterilized by heating at a temperature of 120 ° C. for 20 minutes.

CO ₂ gas was passed through the sparger at 10 mL / min, 10 mL of 3M Na ₂ CO ₃ solution was added, and then the pH was adjusted to 6.8 with a sulfuric acid solution. After adding 5 mL of a reducing solution consisting of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S · 9H ₂ O, inoculate 50 mL of the above seed culture solution, stirring speed 200 rpm, temperature is 39 ° C., 2 M Na Cultivation was performed while adjusting the pH to 6.4 with ₂ CO ₃ solution. The results are shown in Table 4.

  The production method of the chemical product of the present invention using the fermentation apparatus shown in FIGS. 1 and 2 significantly improved the production rate of succinic acid.

(Example 5) Continuous production of succinic acid by continuous fermentation (part 3)
Succinic acid was produced using the continuous fermentation apparatus shown in FIG. The succinic acid and glucose concentrations were measured in the same manner as in Example 3. The medium used was autoclaved at a temperature of 121 ° C. for 15 minutes. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 5 are the same as those in Example 3 except for the following membrane permeation water amount control.
・ Membrane permeate flow control: Flow control by transmembrane pressure difference.

(After continuous fermentation starts ~ 120 hours: controlled at 0.1 kPa to 50 kPa or less 120 hours to 160 hours: controlled at 0.1 kPa to 100 kPa or less 160 hours to 185 hours: controlled at 0.1 kPa to 150 kPa or less).

In Example 5, succinic acid was continuously produced using Actinobacillus succinogenes ATCC 55618 as a microorganism capable of producing succinic acid. 75 mL of succinic acid fermentation medium for Actinobacillus and 4.0 g of MgCO ₃ shown in Table 5 were added to a 100 mL sea ram test tube, and the gas was replaced with CO ₂ , followed by heat sterilization. In advance, 7.5 mL of a cell suspension of ATCC 55618 strain was inoculated and cultured at a temperature of 37 ° C. for 24 hours to prepare a seed culture (pre-culture). The continuous fermentation apparatus shown in FIG. 1 was charged with 1.5 L of succinic acid fermentation medium (Table 5), and 75 mL of the culture solution was inoculated in advance. The CO ₂ aeration rate in the fermentation reaction tank 1 was 75 mL / min, the separation membrane tank CO ₂ aeration rate was 150 mL / min, and the temperature was adjusted to 6.8 at 39 ° C. and 5.5 M Na ₂ CO ₃ while adjusting the pH to 6.8. Continuous culture was performed in the same manner as in Example 3 except that culture was performed. The produced succinic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately, and the succinic acid production rate and the succinic acid production yield calculated from the succinic acid and glucose concentrations are shown in Table 6.

(Example 6) Continuous production of succinic acid by continuous fermentation (part 4)
Succinic acid was produced using the continuous fermentation apparatus shown in FIG. The medium used was autoclaved at a temperature of 121 ° C. for 15 minutes. The succinic acid and glucose concentrations were measured in the same manner as in Example 3. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 6 are the same as those in Example 4 except for the following membrane permeation water amount control.
・ Membrane permeate flow control: Flow control by transmembrane pressure difference.

(After continuous fermentation starts ~ 120 hours: controlled at 0.1 kPa to 50 kPa or less 120 hours to 165 hours: controlled at 0.1 kPa to 100 kPa or less 165 hours to 190 hours: controlled at 0.1 kPa to 150 kPa or less).

In Example 6, succinic acid was continuously produced using Actinobacillus succinogenes ATCC 55618 as a microorganism capable of producing succinic acid. In the continuous production of succinic acid by Actinobacillus succinogenes, the fermentation reactor CO ₂ aeration rate is 75 mL / min, and the pH is adjusted to 5.5 with Na ₂ CO _3. Was performed in the same manner as the continuous culture in Example 4.

First, 75 mL of succinic acid fermentation medium for Actinobacillus and 4.0 g of MgCO ₃ shown in Table 5 were added to a 100 mL sea ram test tube, gas-substituted with CO ₂ , and then heat sterilized. A seed suspension was prepared by inoculating 7.5 mL of a cell suspension of ATCC 55618 strain that had been cryopreserved in advance and culturing at 37 ° C. for 24 hours (pre-culture). A continuous fermentation apparatus shown in FIG. 2 was charged with 1.5 L of a succinic acid fermentation medium (Table 5), inoculated with 75 mL of a culture solution in advance, and cultured for 24 hours while adjusting the pH to 6.8 ( Pre-culture). After completion of the pre-culture, the succinic acid fermentation medium shown in Table 5 is continuously supplied, and the amount of the permeated water in the membrane-separated continuous fermentation apparatus shown in FIG. Succinic acid was produced by fermentation. The produced succinic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. Table 6 shows the production rate of succinic acid and the yield of succinic acid calculated from the succinic acid and glucose concentrations.

(Comparative Example 3) Production of succinic acid by batch culture (Part 2)
Succinic acid production by batch fermentation using Actinobacillus succinogenes was performed as follows. 50 mL of succinic acid fermentation medium for Actinobacillus and 4.0 g of MgCO ₃ shown in Table 5 were added to a 100 mL sea ram test tube, and the gas was replaced with CO ₂ , followed by heat sterilization. A seed culture was prepared by inoculating 5 mL of a cell suspension of Actinobacillus succinofenes ATCC 55618, which had been cryopreserved in advance, and culturing at 37 ° C. for 24 hours. After adjusting 1 L of the fermentation medium shown in Table 5 to pH 6.8, it was sterilized by heating at 120 ° C. for 20 minutes in addition to a mini jar fermenter (manufactured by ABLE, BMJ type, 2 L). CO ₂ gas was bubbled through the sparger at 50 mL / min and the temperature was adjusted to 39 ° C. 50 mL of the above seed culture was inoculated and cultured while adjusting the pH to 6.8 with 5.5 M Na ₂ CO ₃ while stirring at 600 rpm using the attached stirring blade. The results are shown in Table 6.

  The production method of the chemical product of the present invention using the continuous fermentation apparatus shown in FIGS. 1 and 2 significantly improved the production rate of succinic acid.

(Example 7) Continuous production of succinic acid by continuous fermentation (part 5)
Succinic acid was produced using the continuous fermentation apparatus shown in FIG. The succinic acid and glucose concentrations were measured in the same manner as in Example 3. The medium used was autoclaved at a temperature of 121 ° C. for 15 minutes. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in this Example 7 are the same as those in Example 3 except for the following membrane permeation water amount control.
-Membrane permeated water amount control: Flow rate control by transmembrane pressure (After continuous fermentation up to 140 hours: Control at 0.1 kPa to 50 kPa or less 140 hours to 180 hours: Control at 0.1 kPa to 100 kPa or less 180 hours to 200 hours: Control at 0.1 kPa to 150 kPa or less).

In Example 7, succinic acid was continuously produced using Escherichia coli B strain ATCC 11303 as a microorganism capable of producing succinic acid. In the production of succinic acid by E. coli, a seed culture medium consisting of 12 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 1 g / L K ₂ HPO ₄ , 1 g / L NaCl, and 0.2 g / L MgCl ₂ 150 mL was placed in a 200 mL Erlenmeyer flask and the pH was adjusted to 6.8. After 7.5 g of MgCO ₃ was added, the mixture was sterilized by heating, cooled to room temperature, inoculated with ATCC11303 strain in an anaerobic glove box, and allowed to stand overnight at a temperature of 37 ° C. (pre-culture). Succinic acid comprising 12 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 1 g / L K ₂ HPO ₄ , 1 g / L NaCl, and 0.2 g / L MgCl _{2 is} added to the continuous fermentation apparatus shown in FIG. The fermentation medium 1.5L was prepared and 150 mL of culture solution was inoculated beforehand. Continuous fermentation of succinic acid was performed under the same conditions as in Example 3 except that the culture temperature was 37 ° C.

  After the 24-hour culture, the succinic acid fermentation medium shown in Table 7 was continuously supplied, and the continuous culture was performed while controlling the amount of membrane permeate so that the amount of the fermentation broth in the continuous fermentation apparatus was 2L.

  The produced succinic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. Table 8 shows the production rate of succinic acid and the yield of succinic acid calculated from the succinic acid and glucose concentrations.

(Example 8) Continuous production of succinic acid by continuous fermentation (part 6)
Succinic acid was produced using the continuous fermentation apparatus shown in FIG. The medium used was autoclaved at a temperature of 121 ° C. for 15 minutes. The succinic acid and glucose concentrations were measured in the same manner as in Example 3. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 8 are the same as those in Example 4 except for the following membrane permeation water amount control.
・ Membrane permeate flow control: Flow control by transmembrane pressure difference (70 hours after the start of continuous fermentation: control at 0.1 kPa to 50 kPa or less)
70 hours to 90 hours: controlled at 0.1 kPa to 100 kPa or less 90 hours to 110 hours: controlled at 0.1 kPa to 150 kPa or less).

In Example 8, succinic acid was continuously produced using Escherichia coli B strain ATCC 11303 as a microorganism capable of producing succinic acid. 150 mL seed culture medium consisting of 12 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 1 g / L K ₂ HPO ₄ , 1 g / L NaCl, and 0.2 g / L MgCl ₂ in a 200 mL Erlenmeyer flask The pH was adjusted to 6.8. After addition of MgCO ₃ 7.5 g, heat sterilized, cooled to room temperature, inoculated with ATCC11303 strain was overnight static culture at a temperature of 37 ° C. (pre-preculture). Succinic acid comprising 12 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 1 g / L K ₂ HPO ₄ , 1 g / L NaCl, and 0.2 g / L MgCl _{2 is} added to the continuous fermentation apparatus shown in FIG. The fermentation medium 1.5L was prepared and 150 mL of culture solution was inoculated beforehand. The temperature was 37 ° C., and the culture was performed for 24 hours while adjusting the pH to 6.8 with 5.5 M Na ₂ CO ₃ (pre-culture). After completion of the preculture, the succinic acid fermentation medium shown in Table 7 was continuously supplied, and the culture was continuously performed while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus was 1.5L. Table 8 shows the production rate of succinic acid and the yield of succinic acid calculated from the succinic acid and glucose concentrations.

(Comparative Example 4) Production of succinic acid by batch culture (part 3)
Succinic acid production by batch fermentation using Escherichia coli was performed as follows.

100 mL of seed culture medium consisting of 12 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 1 g / L K ₂ HPO ₄ , 1 g / L NaCl, and 0.2 g / L MgCl ₂ in a 1250 mL Erlenmeyer flask The pH was adjusted to 6.8. After addition of MgCO ₃ 5 g, heat sterilized, cooled to room temperature, in an anaerobic glove box was inoculated with E. coli B strain (Escherichia coli B) ATCC11303 strain was overnight static culture at a temperature of 37 ° C.. After adjusting 1 L of fermentation medium consisting of 12 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 1 g / L K ₂ HPO ₄ , 1 g / L NaCl, 0.2 g / L MgCl ₂ to pH 6.8, mini In addition to a jar fermenter (made by ABLE, BMJ type, 2 L), it was heat sterilized (120 ° C., 20 minutes). CO ₂ gas was passed through the sparger at 50 mL / min, and the temperature was adjusted to 37 ° C. 100 mL of the above seed culture was inoculated, stirred at 600 rpm using the attached stirring blade, and cultured while adjusting the pH to 6.8 with 5.5 M Na ₂ CO ₃ . Cultivation was performed while adding 200 mL of a 100 g / L glucose solution little by little so that the glucose concentration in the fermentation broth did not exceed 20 g / L. The results are shown in Table 8.

  The production method of the chemical product of the present invention using the fermentation apparatus shown in FIGS. 1 and 2 significantly improved the production rate of succinic acid.

(Example 9) Production of 1,3-propanediol by continuous fermentation (part 1)
1,3-propanediol was produced using the continuous fermentation apparatus of FIG. 1 and a 1,3-propanediol production medium having the composition shown in Table 9.

  First, the isolation, identification and measurement methods of the product 1,3-propanediol will be described.

Conversion of glycerol to 1,3-propanediol was confirmed by HPLC. Analyzes were performed using standard methods and materials available to those skilled in the chromatographic arts. As one suitable method, detection by UV (210 nm) and RI was performed using a Waters Maxima 820 HPLC system connected to a Shodex SH-1011P precolumn (6 mm × 50 mm). Samples at a flow rate of 0.5 mL / min on a Shodex SH-1011 column (8 mm x 300 mm, purchased from Waters, Milford, MA) temperature controlled at 50 ° C. using 0.01N H ₂ SO ₄ as the mobile phase. Injected. For quantitative analysis, samples were prepared using a known amount of trimethylacetic acid as an external standard. The retention times for glucose (RI detection), glycerol, 1,3-propanediol (RI detection) and trimethylacetic acid (UV and RI detection) were approximately 15 minutes, 20 minutes, 26 minutes and 35 minutes, respectively.

  The production of 1,3-propanediol was confirmed by GC / MS. GC / MS was performed using standard methods. For example, using a Hewlett Packard 5890 Series II gas chromatograph coupled to a Hewlett Packard 5971 Series mass selective detector (EI) and HP-INNOWax column (length 30 m, inner diameter 0.25 mm, film thickness 0.25 microns). It was. The retention time and mass spectrum of the 1,3-propanediol produced were compared with those of the 1,3-propanediol standard (m / e: 57,58).

  Sample derivatization was performed as follows. 30 μL of concentrated (70% v / v) perchloric acid was added to a 1.0 mL sample (eg, culture supernatant), mixed, and then the sample was lyophilized. A 1: 1 mixture of bis (trimethylsilyl) trifluoroacetamide: pyridine (300 μL) was added to the lyophilized sample, mixed vigorously and placed at a temperature of 65 ° C. for 1 hour. The insoluble material was removed by centrifugation, and the supernatant was collected. The resulting liquid was divided into two phases, and the upper phase was used for analysis. The resulting trimethylsilylated derivative sample of 1,3-propanediol was chromatographed on a DB-5 column (48 m, ID 0.25 mm, film thickness 0.25 μm; from J & W Scientific) to give 1,3-propanediol. The retention time and mass spectrum of the trimethylsilylated derivative were compared to the retention time and mass spectrum obtained from a reference standard sample. The mass spectrum of the trimethylsilylated derivative of 1,3-propanediol contains 205, 177, 130 and 115 AMU characteristic ions.

The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 9 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane-Membrane separation element effective filtration area: 60 square cm
・ Temperature adjustment: 37 (℃)
Fermentation reaction tank CO ₂ aeration amount: 10 (mL / min)
-Membrane separation tank CO ₂ aeration rate: 100 (mL / min)
-Reaction vessel stirring speed: 800 (rpm)
-PH adjustment: adjusted to pH 7.0 with 5N NaOH (after continuous fermentation starts to 100 hours: controlled at 0.1 kPa to 50 kPa or less 100 hours to 130 hours: controlled at 0.1 kPa to 100 kPa or less 130 hours to 170 hours: 0 .Control at 1 kPa to 150 kPa or less)
Sterilization: Fermentation culture tank and separation medium including separation membrane element are all autoclaved for 20 minutes at a temperature of 121 ° C. High pressure steam sterilization. Membrane permeate flow rate control: Flow rate control by transmembrane pressure difference.

  Klebsiella pneumoniae ATCC 25955 strain is used as the microorganism, 1,3-propanediol production medium having the composition shown in Table 9 is used as the medium, and evaluation of the concentration of 1,3-propanediol as the product is performed by the HPLC method described above. It was measured by. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  First, Klebsiella pneumoniae ATCC 25955 strain was cultured overnight in a test tube with 5 ml of 1,3-propanediol production medium (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh 1,3-propanediol production medium, and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 24 hours (pre-culture). The culture solution was inoculated in a 1.5 L 1,3-propanediol production medium of the membrane separation type continuous fermentation apparatus shown in FIG. 1, and the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5; The aeration amount of the fermentation reaction tank 1, the temperature adjustment, and the pH adjustment were performed, and the culture was performed for 24 hours (pre-culture). Immediately after completion of the pre-culture, the fermentation liquid circulation pump is operated, in addition to the operating conditions during pre-culture, the membrane separation tank 12 is vented, and 1,3-propanediol production medium (glycerol concentration is 100 g / L) is continuously supplied. Then, continuous culture was performed while controlling the amount of permeated water in the continuous fermentation apparatus so that the amount of the fermentation broth became 2 L, and 1,3-propanediol was produced by continuous fermentation. The control of the amount of permeated water through the continuous fermentation test was performed by the differential pressure control device 3. The produced 1,3-propanediol concentration and residual glucose concentration in the membrane permeation fermentation broth were appropriately measured. Table 1 shows the production rate of 1,3-propanediol calculated from 1,3-propanediol and input glycerol.

  As a result of a 170-hour fermentation test, stable production of 1,3-propanediol by continuous fermentation was possible by the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIG.

(Example 10) Production of 1,3-propanediol by continuous fermentation (part 2)
1,3-propanediol was produced using the continuous fermentation apparatus of FIG. 2 and a 1,3-propanediol production medium having the composition shown in Table 9.

The medium was used after autoclaving at 121 ° C. for 15 minutes. As the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 10 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Separation membrane: Polyvinylidene fluoride filtration membrane ・ Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 37 (℃)
Fermentation reaction tank aeration: 0.6 (L / min) CO 2 gas fermentation reaction tank stirrer speed: 800 (rpm)
・ PH adjustment: pH 7.0 adjusted with 5N NaOH ・ Sterilization: All culture tanks containing separation membrane elements and culture media used are autoclaved at 121 ° C. for 20 min. ・ High pressure steam sterilization ・ Membrane permeate control: by transmembrane pressure difference Flow control.

(After start of continuous fermentation to 100 hours: controlled at 0.1 kPa to 50 kPa or less 100 hours to 130 hours: controlled at 0.1 kPa to 100 kPa or less 130 hours to 180 hours: controlled at 0.1 kPa to 150 kPa or less).

  Klebsiella pneumoniae ATCC 25955 strain is used as the microorganism, 1,3-propanediol production medium having the composition shown in Table 9 is used as the medium, and the concentration of the product 1,3-propanediol is evaluated by the HPLC method. did. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  First, Klebsiella pneumoniae ATCC 25955 strain was cultured in a test tube with shaking overnight in 5 ml of 1,3-propanediol production medium (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh 1,3-propanediol production medium, and cultured with shaking in a 500 ml Sakaguchi flask at a temperature of 30 ° C. for 24 hours (pre-culture). The culture solution was inoculated in a 1.5 L 1,3-propanediol production medium of the membrane-separated continuous fermentation apparatus shown in FIG. 2, and the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5; The aeration amount of the fermentation reaction tank 1, the temperature, and the pH were adjusted and cultured for 24 hours (pre-culture). Immediately after completion of the pre-culture, 1,3-propanediol production medium (glycerol concentration is 100 g / L) is continuously supplied, and the amount of membrane permeate is controlled so that the amount of fermentation liquid in the membrane-integrated continuous fermentation apparatus becomes 1.5 L. The culturing was continued, and 1,3-propanediol was produced by continuous fermentation. The control of the amount of permeated water through the continuous fermentation test was performed by the differential pressure control device 3. The produced 1,3-propanediol concentration and residual glucose concentration in the membrane permeation fermentation broth were appropriately measured according to the method shown in Example 9. Table 10 shows the production rate of 1,3-propanediol calculated from the 1,3-propanediol and input glycerol. As a result of the 180-hour fermentation test, stable production of 1,3-propanediol by continuous fermentation was possible by the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIG.

(Comparative Example 5) Production of 1,3-propanediol by fed-batch fermentation Fed-batch fermentation, which is often performed as a fermentation form using microorganisms, is carried out using a 2 L jar fermenter, and the 1,3-propanediol productivity is increased. evaluated. The medium was used after autoclaving at 121 ° C. for 15 minutes. In Comparative Example 5, Klebsiella pneumoniae ATCC 25955 strain was used as a microorganism, and the concentration of 1,3-propanediol as a product was evaluated using HPLC according to the method shown in Example 9, and the glucose concentration For the measurement, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The operating conditions of Comparative Example 5 are shown below.
・ Fermentation reactor capacity: 2.0 (L)
・ Temperature adjustment: 37 (℃)
Fermentation reaction tank aeration rate: 0.4 (L / min) CO ₂ gas Fermentation reaction tank stirring speed: 300 (rpm)
-PH adjustment: Adjust to pH 7.0 with 5N NaOH.

  First, Klebsiella pneumoniae ATCC 25955 strain was cultured overnight in a test tube with 5 ml of 1,3-propanediol production medium (pre-culture). The culture solution was inoculated into 50 ml of a fresh 1,3-propanediol production medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (preculture). The preculture was inoculated into 1 L of 1,3-propanediol production medium of a jar fermenter. 1,3-propanediol production medium (glycerol concentration is 500 g / L) was continuously supplied so that the glycerol concentration was changed from 0 g / L to 10 g / L, and fed-batch fermentation was performed. The results are shown in Table 10.

  The production rate of 1,3-propanediol was greatly improved by the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIGS. 1 and 2.

(Example 11) Production of L-lactic acid by continuous fermentation using lactic acid bacteria (part 1)
L-lactic acid was produced using the continuous fermentation apparatus shown in FIG. L-lactic acid bacteria lactic acid fermentation medium shown in Table 11 was used as the medium, and the mixture was used after autoclaving at 121 ° C. for 15 minutes. As the separation membrane, the porous membrane prepared in Reference Example 2 was used. The operating conditions in Example 11 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane-Membrane separation element effective filtration area: 60 square cm
・ Temperature adjustment: 37 (℃)
Fermentation reaction tank N ₂ aeration rate: 10 (mL / min)
Membrane separation tank _{N 2} aeration: 100 (mL / min)
・ Fermentation reactor stirring speed: 600 (rpm)
-PH adjustment: adjusted to pH 6.5 with 8N aqueous ammonia solution (70 hours after the start of continuous fermentation: controlled at 0.1 kPa to 50 kPa or less)
70 hours to 120 hours: controlled at 0.1 kPa to 100 kPa or less 120 hours to 165 hours: controlled at 0.1 kPa to 150 kPa or less)
・ Sterilization: The culture tank containing the separation membrane element and the medium to be used are all autoclaved at a temperature of 121 ° C. for 20 minutes by high-pressure steam sterilization. Membrane permeate flow rate control: flow rate control by transmembrane pressure difference.

  Lactococcus lactis JCM7638 strain was used as a prokaryotic microorganism, and a lactic acid bacteria lactic acid fermentation medium having the composition shown in Table 11 was used as the medium. L-lactic acid contained in the fermentation broth was evaluated by the same method as in Reference Example 1. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  First, the Lactococcus lactis JCM7638 strain was statically cultured at a temperature of 37 ° C. for 24 hours in a lactic acid fermentation medium purged with 5 ml of nitrogen gas shown in Table 11 in a test tube (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 37 ° C. for 48 hours (pre-culture). The culture solution was inoculated in a 1.5 L lactic acid fermentation medium purged with nitrogen gas from the continuous fermentation apparatus shown in FIG. 1, and the fermentation reaction tank 1 was stirred at 600 rpm by the attached stirrer 5. The amount of aeration was adjusted and the temperature was adjusted to a temperature of 37 ° C., followed by culturing for 24 hours (pre-culture). Immediately after completion of the pre-culture, the lactic acid fermentation medium is continuously supplied, and the continuous fermentation apparatus is controlled to control the amount of permeated water so that the amount of the fermentation liquor is 2 L, and L-lactic acid is produced by continuous fermentation. It was. At this time, nitrogen gas was supplied from the gas supply device into the fermentation reaction tank, and the discharged gas was recovered and supplied again to the fermentation reaction tank. That is, recycling supply of gas containing nitrogen gas was performed. The control of the amount of permeated water through the continuous fermentation test was performed by the differential pressure control device 3. The produced L-lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. Table 12 shows the L-lactic acid versus sugar yield and the L-lactic acid production rate calculated from the input glucose calculated from the L-lactic acid and glucose concentrations.

  As a result of the 165-hour fermentation test, stable production of L-lactic acid by continuous fermentation was possible by the method for producing a chemical product of the present invention using a continuous fermentation apparatus.

(Example 12) Production of L-lactic acid by continuous fermentation using lactic acid bacteria (part 2)
L-lactic acid was produced using the continuous fermentation apparatus shown in FIG. A lactic acid bacteria lactic acid fermentation medium shown in Table 11 was used as the medium, and the mixture was autoclaved at 121 ° C. for 15 minutes. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 12 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Separation membrane: Polyvinylidene fluoride filtration membrane ・ Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 37 (℃)
-Aeration volume of fermentation reaction tank: 50 (mL-nitrogen / min)
・ Fermentation reactor stirring speed: 600 (rpm)
・ Sterilization: Culture tank containing separation membrane element and all used media are autoclaved for 20 min at a temperature of 121 ° C. ・ High pressure steam sterilization ・ pH adjustment: adjusted to pH 6.5 with 8N ammonia aqueous solution ・ Permeate control: transmembrane difference Flow control by pressure (up to 85 hours after the start of continuous fermentation: controlled at 0.1 kPa to 50 kPa or less
85 hours to 120 hours: controlled at 0.1 kPa to 100 kPa or less 120 hours to 190 hours: controlled at 0.1 kPa to 150 kPa or less).

  Lactococcus lactis JCM7638 strain was used as a prokaryotic microorganism, and the same procedures as in Example 11 were performed until preculture. Immediately after completion of the pre-culture, the medium is continuously supplied, and the continuous fermentation is performed while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus is 1.5 L, and L-lactic acid is produced by continuous fermentation. It was. At this time, nitrogen gas was supplied from the gas supply device into the fermentation reaction tank, and the discharged gas was recovered and supplied again to the fermentation reaction tank. That is, recycling supply of gas containing nitrogen gas was performed. The control of the amount of permeated water through the continuous fermentation test was performed by the differential pressure control device 3. The L-lactic acid concentration produced and the residual glucose concentration in the membrane permeation fermentation broth were measured as appropriate. Table 12 shows the L-lactic acid versus sugar yield and the L-lactic acid production rate calculated from the input glucose calculated from the L-lactic acid and glucose concentrations.

  As a result of the 190-hour fermentation test, stable production of L-lactic acid by continuous fermentation was possible by the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIG.

(Comparative Example 6) Production of L-lactic acid by batch fermentation The most typical batch fermentation as a fermentation form using microorganisms was performed using a 2 L jar fermenter, and the L-lactic acid productivity was evaluated. The medium shown in Table 11 was used as the medium, and autoclaved at 121 ° C. for 15 minutes. In Comparative Example 6, Lactococcus lactis JCM7638 strain was used as a prokaryotic microorganism, and the concentration of L-lactic acid as a product was evaluated using the method shown in Reference Example 1 above. Used was “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.). The operating conditions of Comparative Example 6 are shown below.
・ Fermentation reactor capacity: 2 (L)
・ Temperature adjustment: 37 (℃)
-Aeration volume of fermentation reaction tank: 50 (mL-nitrogen / min)
・ Fermentation reactor stirring speed: 200 (rpm)
-PH adjustment: pH adjusted to 6.5 with 8N aqueous ammonia solution.

  First, the Lactococcus lactis JCM7638 strain was statically cultured at a temperature of 37 ° C. for 24 hours in a lactic acid fermentation medium purged with 5 ml of nitrogen gas shown in Table 11 in a test tube (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 37 ° C. for 48 hours (preculture). The preculture was inoculated into 1 L of a jar fermenter in a continuous / batch fermentation medium shown in Table 11, and batch fermentation was performed. The results of batch fermentation are shown in Table 12.

  It was clarified that the production rate of L-lactic acid was greatly improved by the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIG. 1 and FIG.

(Reference Example 4) Preparation of chromosomal DNA of Bacillus laevolacticus JCM2513 Bacillus laevolacticus JCM2513 was inoculated into 100 ml of GYP medium (GYP medium described in JP-A-2003-088392) at 30 ° C. The culture was obtained by culturing at temperature for 24 hours. The obtained culture was centrifuged at 3000 rpm for 15 minutes to obtain 0.5 g of wet cells, and from the wet cells, the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963 )) To obtain chromosomal DNA. Next, 60 μg of this chromosomal DNA and 3 units of restriction enzyme Sau3AI were mixed with 10 mM Tris-HCl buffer (containing 50 mM NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol (pH 7.4)), and reacted at a temperature of 37 ° C. for 30 minutes. I let you. The reaction-terminated solution was subjected to phenol extraction treatment by an ordinary method, ethanol precipitation treatment, and 50 μg of a chromosomal DNA fragment of Bacillus laevolacticus JCM2513 digested with Sau3AI.

(Reference Example 5) Preparation of gene library of Bacillus laevolacticus JCM2513 using plasmid vector DNA Plasmid vector DNA (pUC19) capable of autonomous replication in Escherichia coli (pUC19) and a restriction enzyme BamHI200 unit, It was mixed with 50 mM Tris-HCl buffer solution (containing 100 mM NaCl and 10 mM magnesium sulfate (pH 7.4)) and reacted at a temperature of 37 ° C. for 2 hours to obtain a digested solution. A precipitation treatment was performed. Thereafter, in order to prevent the DNA fragment derived from the plasmid vector from recombining, the DNA fragment was dephosphorylated by a bacterial alkaline phosphatase treatment, followed by phenol extraction treatment by an ordinary method, and further ethanol precipitation treatment.

  1 μg of this BamHI-digested pUC19, 1 μg of the chromosomal DNA fragment of Bacillus laevolacticus JCM2513 digested with Sau3AI obtained in Reference Example 4 above, and 2 units of T4 DNA ligase (Takara Shuzo Co., Ltd.) Was added to 66 mM Tris-HCl buffer (pH 7.5) containing 66 mM magnesium chloride, 10 mM dithiothreitol and 10 mM ATP, and reacted at a temperature of 16 ° C. for 16 hours to ligate DNA. Next, Escherichia coli JM109 was transformed with the obtained DNA mixture by a conventional method, and this was spread on an LB agar medium containing 50 μg / ml ampicillin sodium to obtain about 20,000 colonies. It was rally. Recombinant DNA was recovered from about 20,000 colonies. The method of recovery was according to the method of Saito and Miura shown above.

Reference Example 6 Preparation of a Host for Screening for D-Lactate Dehydrogenase Gene Screening of the D-LDH gene of Bacillus laevolacticus JCM2513 strain was performed by functional complementation. Details of the principle are described in “(Dominique, G., Appl. Environ. Microbiol., United States, (1995), 61, 266-272.)”. That is, it is a method of screening a gene that restores growth under anaerobic conditions of a strain lacking Escherichia coli D-lactate dehydrogenase enzyme activity and pyruvate formate lyase enzyme activity. By the method of Kirill et al. (Kirill, A., Proc. Natl. Acad. Sci., United States, (2000), 97, 6640-6645), Escherichia coli D-lactate dehydrogenase gene (ldhA) and pyruvate formate Strains from which the lyase genes (pflB and pflD) were deleted by deletion were prepared. The strain thus prepared was named Escherichia coli TM33 strain (E. coli ΔldhA ΔpflB :: Km ^r ΔpflD :: Cm ^r ) and used as a screening host for the D-lactate dehydrogenase gene.

(Reference Example 7) Screening of D-lactate dehydrogenase gene Escherichia coli TM33 strain was inoculated into 100 ml of LB medium containing 50 μg / ml kanamycin sulfate and 15 μg / ml chloramphenicol, and the temperature was 37 ° C. for 24 hours. The culture was obtained. The culture thus obtained was centrifuged at 3,000 rpm for 15 minutes to obtain 0.8 g of wet cells. The obtained wet cells were washed 3 times with 10 ml of 10% glycerol solution and then suspended in 0.1 ml of 10% glycerol solution to obtain a competent cell. To this competent cell, 1 μl of the gene library of Bacillus laevolacticus JCM2513 obtained in Reference Example 5 was added, and the strain introduced according to the conventional method of electroporation was added to M9GP agar containing 50 μg / ml ampicillin sodium. Several strains were obtained which were seeded on a medium (M9 medium + 0.4% glucose + 0.2% peptone) and were able to grow under anaerobic conditions.

(Reference Example 8) Analysis of base sequence of DNA containing D-lactate dehydrogenase gene Plasmids were prepared from Escherichia coli TM33 / pBL2 containing the recombinant DNA obtained above according to a conventional method, and the obtained set The nucleotide sequence was determined using the replacement DNA. The base sequence was determined using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biochemical) according to the method of Sanger. The base sequence of the DNA containing the obtained D-lactate dehydrogenase gene was 2,995 base pairs. This sequence was subjected to open reading frame search using Genetyx (Software Development Co., Ltd.) to tentatively determine the DNA sequence (SEQ ID NO: 10) of the 1,011 base pair D-lactate dehydrogenase gene.

(Reference Example 9) Preparation of D-lactate dehydrogenase gene expression vector The D-LDH gene was cloned from Bacillus laevolacticus. All of the D-LDH genes are cloned by the PCR method and introduced into the expression vector by the same method. The cloning method is as follows.

  After Bacillus laevoralticus was cultured and collected by centrifugation, genomic DNA was extracted using UltraClean Microbial DNA Isolation Kit (manufactured by MO BIO). The detailed operation method followed the attached protocol. The obtained genomic DNA was used as a template for subsequent PCR. The D-LDH gene was cloned by PCR using the DNA obtained above as a template. For the PCR amplification reaction, KOD-Plus-polymerase (manufactured by Toyobo Co., Ltd.), which is said to be 50 times more accurate than Taq, was used. The attached reaction buffer and dNTPmix were used. D-LDH gene amplification primers (SEQ ID NOs: 11 and 12) were prepared such that an XhoI recognition sequence was added to the 5 terminal side and a NotI recognition sequence was added to the 3 terminal side, respectively.

  Each PCR amplified fragment was purified, and the end was phosphorylated with T4 Polynucleotide Kinase (manufactured by TAKARA), and then ligated to pUC118 vector (cleaved with restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver. 2 (manufactured by TAKARA). By transforming into Escherichia coli DH5α and recovering the plasmid DNA, a plasmid in which the D-LDH gene was subcloned was obtained. The pUC118 vector into which each D-LDH gene was inserted was cleaved with restriction enzymes XhoI and NotI, and the resulting DNA fragments were introduced into the XhoI / NotI cleavage sites of the yeast expression vector pTRS11 (FIG. 5). The D-LDH gene expression vector thus prepared is designated as pTM63.

Reference Example 10 Introduction of D-LDH Gene Expression Vector into Yeast pTM63 obtained in Reference Example 9 was transformed into the yeast Saccharomyces cerevisiae NBRC10505 strain. Transformation was performed by the lithium acetate method using YEASTMAKER Yeast Transformation System (CLONTECH). For details, the attached protocol was followed. The Saccharomyces cerevisiae NBRC10505 strain used as a host is a strain lacking the ability to synthesize uracil. By the action of the URA3 gene of pTM63, it is possible to select a transformant introduced with pTM63 on a uracil-free medium.

  Confirmation of D-LDH gene expression vector introduction into the transformant thus obtained was performed using a genomic DNA extraction kit Gen Toru-kun (manufactured by TAKARA) from a transformant cultured in a liquid medium without uracil. Genomic DNA including plasmid DNA was extracted, and this was performed by PCR using PreMix Taq (manufactured by TAKARA) as a template. The primer used when cloning the D-LDH gene was used. As a result, it was confirmed that the D-LDH gene was introduced into all the transformants. The Saccharomyces cerevisiae NBRC10505 strain into which pTM63 has been introduced is hereinafter referred to as NBRC10505 / pTM63 strain.

(Example 13) Production of D-lactic acid by continuous fermentation (part 1)
Using the continuous fermentation apparatus shown in FIG. 1 and the D-lactic acid production medium having the composition shown in Table 13, D-lactic acid was produced. The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 13 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane-Membrane separation element effective filtration area: 60 square cm
・ Temperature adjustment: 30 (℃)
-Air flow rate of fermentation reaction tank: 10 (mL / min)
-Membrane separation tank air flow rate: 100 (mL / min)
・ Fermentation reactor stirring speed: 800 (rpm)
・ PH adjustment: pH 5.0 adjusted with 5N NaOH ・ Sterilization: Culture tanks containing separation membrane elements and used media are all sterilized by autoclaving at a temperature of 121 ° C. for 20 minutes ・ Membrane permeation control: transmembrane difference Flow control by pressure (100 to 100 hours after start of continuous fermentation: control at 0.1 kPa to 50 kPa or less 100 hours to 125 hours: control at 0.1 kPa to 100 kPa or less 125 hours to 175 hours: control at 0.1 kPa to 150 kPa or less) .

  The NBRC10505 / pTM63 strain was used as the microorganism, and the concentration of the product D-lactic acid was evaluated by the same HPLC method as in Reference Example 1. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  First, NBRC10505 / pTM63 strain was shaken overnight in a test tube with 5 ml of D-lactic acid production medium to obtain a culture solution (pre-culture). The obtained culture broth was inoculated into 50 ml of a fresh D-lactic acid production medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L D-lactic acid production medium of the continuous fermentation apparatus shown in FIG. 1, the fermentation reaction tank 1 is stirred at 800 rpm by the attached stirrer 5, and the aeration amount of the fermentation reaction tank 1 Adjustment, temperature adjustment, and pH adjustment were performed for 24 hours (pre-culture).

  Immediately after completion of the pre-culture, the fermentation broth circulating pump 11 is operated, in addition to the operating conditions during pre-culture, the membrane separation tank 12 is vented, the D-lactic acid production medium is continuously supplied, and the fermentation culture of the continuous fermentation apparatus Continuous culture was performed while controlling the amount of membrane permeate so that the liquid volume was 2 L, and D-lactic acid was produced by continuous fermentation. The control of the amount of permeated water through the continuous fermentation test was performed by the differential pressure control device 3. The produced lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. The results of a 175 hour continuous fermentation test are shown in Table 14. Stable production of D-lactic acid by continuous fermentation was possible by the method for producing a chemical product of the present invention using a membrane separation type continuous fermentation apparatus.

(Example 14) Production of D-lactic acid by continuous fermentation (part 2)
D-lactic acid was produced using the fermentation apparatus shown in FIG. 2 and the D-lactic acid production medium having the composition shown in Table 13. The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous separation membrane prepared in Reference Example 2 was used. The operating conditions in Example 14 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 2 (L)
・ Separation membrane: Polyvinylidene fluoride filtration membrane ・ Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 30 (℃)
-Fermentation reactor aeration rate: 0.2 (L / min) air-Fermentation reactor agitation speed: 800 (rpm)
・ PH adjustment: pH 5.0 adjusted with 5N NaOH ・ Sterilization: Culture tanks containing separation membrane elements and used media are all sterilized by autoclaving at a temperature of 121 ° C. for 20 minutes ・ Membrane permeation control: transmembrane difference Flow control by pressure (starting continuous fermentation up to 120 hours: controlled at 0.1 kPa to 50 kPa or less 120 hours to 145 hours: controlled at 0.1 kPa to 100 kPa or less 145 hours to 170 hours: controlled at 0.1 kPa to 150 kPa or less) .

  As the microorganism, NBRC10505 / pTM63 strain was used, and a D-lactic acid production medium having the composition shown in Table 13 was used as the medium. The concentration of the product D-lactic acid was evaluated by the same HPLC method as in Reference Example 1 above. did. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  First, NBRC10505 / pTM63 strain was shaken overnight in a test tube with 5 ml of D-lactic acid production medium to obtain a culture solution (pre-culture). The obtained culture broth was inoculated into 50 ml of a fresh D-lactic acid production medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture).

  The culture solution is inoculated in a 1.5 L D-lactic acid production medium of the continuous fermentation apparatus shown in FIG. 2, the fermentation reaction tank 1 is stirred at 800 rpm by the attached stirrer 5, and the aeration amount of the fermentation reaction tank 1 , Temperature and pH were adjusted and cultured for 24 hours (pre-culture). Immediately after completion of the pre-culture, D-lactic acid production medium is continuously supplied and continuously cultured while controlling the amount of permeated water so that the fermentation culture volume of the continuous fermentation apparatus is 1.5 L. D- by continuous fermentation Lactic acid was produced. The control of the amount of permeated water through the continuous fermentation test was performed by the differential pressure control device 3. The produced D-lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. Table 14 shows the D-lactic acid production rate calculated from D-lactic acid and input glucose.

  As a result of a 170-hour fermentation test, stable production of D-lactic acid by continuous fermentation was possible by the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIG.

(Comparative Example 7) Production of D-lactic acid by batch fermentation The most typical batch fermentation as a fermentation form using microorganisms was performed using a 2 L jar fermenter, and the D-lactic acid productivity was evaluated. The medium was used after autoclaving at 121 ° C. for 15 minutes. In Comparative Example 7, the NBRC10505 / pTM63 strain was used as the microorganism, the concentration of the product D-lactic acid was evaluated using HPLC, and the glucose concentration was measured by “Glucose Test Wako C” (registered trademark). ) (Wako Pure Chemical Industries, Ltd.) was used. The operating conditions of Comparative Example 7 are as follows.
・ Fermentation reactor capacity: 2.0 (L)
・ Temperature adjustment: 30 (℃)
-Fermentation reactor aeration rate: 0.2 (L / min) air-Fermentation reactor agitation speed: 300 (rpm)
-PH adjustment: Adjust to pH 5.0 with 5N NaOH.

  First, NBRC10505 / pTM63 strain was cultured with shaking in a 5 ml D-lactic acid production medium overnight in a test tube (pre-culture). The culture solution was inoculated into 50 ml of a fresh D-lactic acid production medium and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 24 hours (preculture). The preculture was inoculated into a 1.0 L D-lactic acid production medium of a jar fermenter. Batch fermentation was performed using a D-lactic acid production medium. The results are shown in Table 14.

  It has been clarified that the production rate of D-lactic acid is greatly improved by the method for producing a chemical product of the present invention using the continuous fermentation apparatus shown in FIG. 1 and FIG.

(Example 15) Production of L-lactic acid by continuous fermentation using a hollow fiber membrane (part 1)
The separation membrane element shown in FIG. 4 having an effective filtration area of 120 square cm produced using the hollow fiber-like porous separation membrane produced in Reference Example 3 was used as the separation membrane. A lactic acid continuous fermentation test was conducted. The results are shown in Table 15 together with the results of Comparative Example 1. Production of stable L-lactic acid by continuous fermentation was possible. The transmembrane pressure difference during the entire period of continuous fermentation was maintained at 2 kPa or less.

(Example 16) Production of L-lactic acid by continuous fermentation using a hollow fiber membrane (part 2)
The separation membrane element shown in FIG. 4 having an effective filtration area of 120 square centimeters produced using the hollow fiber porous separation membrane produced in Reference Example 3 above was used as the separation membrane. A lactic acid continuous fermentation test was conducted. The results are shown in Table 15 together with Comparative Example 1. It was confirmed that production of stable L-lactic acid by continuous fermentation was possible.

  The method for producing a chemical product by the continuous fermentation method of the present invention is capable of continuous fermentation that maintains high productivity stably over a long period of time under simple operating conditions, and is a chemical product that is a fermentation product widely in the fermentation industry. Can be produced stably at low cost and is useful.

FIG. 1 is a schematic side view for explaining an example of a membrane separation type continuous fermentation apparatus used in the present invention. FIG. 2 is a schematic side view for explaining an example of another membrane separation type continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining an example of the separation membrane element used in the present invention. FIG. 4 is a cross-sectional explanatory diagram for explaining an example of another separation membrane element used in the present invention. FIG. 5 is a diagram showing a physical map of the expression vector pTRS11 for yeast.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Separation membrane element 3 Differential pressure control device 4 Gas supply device 5 Stirrer 6 Level sensor 7 Medium supply pump 8 pH adjustment solution supply pump 9 pH sensor / control device 10 Temperature controller 11 Fermentation culture medium circulation pump 12 Membrane Separation tank 13 Support plate 14 Channel material 15 Separation membrane 16 Recessed portion 17 Water collecting pipe 18 Separation membrane bundle 19 Upper resin sealing layer 20 Lower resin sealing layer 21 Support frame 22 Water collection pipe

Claims (12)

  The fermentation broth of microorganisms or cultured cells is filtered through a separation membrane, the product is recovered from the filtrate, and the unfiltered liquid is retained or refluxed in the fermentation broth, and the fermentation raw material is used as the fermentation broth. In the continuous fermentation added to the above, a method for producing a chemical product by continuous fermentation, wherein the separation membrane is filtered using a porous separation membrane having pores having an average pore diameter of 1 μm or more and 5 μm or less.   The method for producing a chemical product by continuous fermentation according to claim 1, wherein the transmembrane pressure difference of the porous separation membrane is set in the range of 0.1 kPa to 150 kPa. The continuous water according to claim 1 or 2, wherein a pure water permeability coefficient of the porous separation membrane is 2 x 10 ^-9 m ³ / m ² / s / pa or more and 2 x 10 ^-6 m ³ / m ² / s / pa or less. A method for producing chemicals by fermentation.   The method for producing a chemical product by continuous fermentation according to claim 3, wherein the average pore size of the porous separation membrane is 1 µm or more and 2 µm or less, and the standard deviation of the average pore size of the porous separation membrane is 0.5 µm or less.   The method for producing a chemical product by continuous fermentation according to claim 3 or 4, wherein the membrane surface roughness of the porous separation membrane is a porous separation membrane of 0.1 µm or less.   The method for producing a chemical product by continuous fermentation according to any one of claims 3 to 5, wherein the porous separation membrane is a porous separation membrane comprising a porous resin layer.   The method for producing a chemical product by continuous fermentation according to claim 6, wherein the porous resin layer is a porous resin layer made of an organic polymer compound.   The method for producing a chemical product by continuous fermentation according to claim 7, wherein the material of the porous resin layer is polyvinylidene fluoride.   The method for producing a chemical product by continuous fermentation according to any one of claims 1 to 8, wherein the fermentation broth of the microorganism or cultured cell and the fermentation raw material contain saccharides.   The method for producing a chemical product by continuous fermentation according to claim 1, wherein the chemical product is an organic acid or an alcohol.   The method for producing a chemical product by continuous fermentation according to claim 10, wherein the organic acid is L-lactic acid, D-lactic acid or succinic acid.   The method for producing a chemical by continuous fermentation according to claim 10, wherein the alcohol is 1,3-propanediol.

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