JP2008104451A - Method for producing d-lactic acid by continuous fermentation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing D-lactic acid by a continuous fermentation method that stably maintains high productivity over a long time, under simple operating conditions. <P>SOLUTION: This method for producing D-lactic acid by a continuous fermentation method comprises separating a culture liquid into a filtrate and a non-filtrated liquid with a separation membrane, recovering the D-lactic acid of a desired fermentation product from the filtrate, and then holding or returning the non-filtrated liquid in the culture liquid. By using a porous membrane, having high permeability and high cell-stopping rate and hardly occluded as the separation membrane and by subjecting the culture liquid to filtration treatment at a low inter-membrane difference pressure, the fermentation production efficiency of the D-lactic acid can be improved markedly and stably at low cost. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention efficiently filters and collects a liquid containing a product through a porous membrane that is a separation membrane that is less likely to be clogged than a culture solution of microorganisms or cultured cells while culturing, and an unfiltered solution. It is related with the manufacturing method of D-lactic acid by continuous fermentation characterized by obtaining high productivity by improving the microbe density | concentration in connection with fermentation by returning to a culture solution.

Polylactic acid, a biodegradable polymer, has attracted strong attention as a sustainability and life cycle assessment (LCA) compatible product with the emergence of CO ₂ and energy problems. There is a demand for an efficient and inexpensive production method for lactic acid.

  Currently produced polylactic acid is an L-lactic acid polymer, but lactic acid has two optical isomers, L-lactic acid and D-lactic acid, and D-lactic acid is also used for polymer raw materials, agricultural chemicals and pharmaceuticals. In recent years, it has attracted attention as an intermediate. However, in any application, high optical purity is required for L-lactic acid and D-lactic acid as raw materials.

  In nature, there are bacteria that efficiently produce lactic acid such as lactic acid bacteria, and some lactic acid production methods using these bacteria have already been put into practical use. For example, Lactobacillus delbrueckii (Lactobacillus delbrueckii) is a bacterium that efficiently produces L-lactic acid, and a Bacillus laevolacticus microorganism is known as a bacterium that efficiently produces D-lactic acid. (See Patent Document 1).

In addition, in recombinant microorganisms, attempts have been made to produce D-lactic acid in yeast and Escherichia coli that have abundant genome information and have a sufficient track record as genetically modified hosts. For example, an attempt to produce D-lactic acid using E. coli as a recombinant host has been proposed (see Patent Document 2). There is also an attempt to produce D-lactic acid by yeast by introducing a D-lactic acid dehydrogenase gene derived from a known Lactobacillus plantarum (see Patent Document 3). However, the production of these D-lactic acids by fermentation is only related to batch culture, and the production speed of any D-lactic acid is not sufficient, and a more efficient production method has been desired. .
JP 2003-088392 A JP 2005-102625 A JP 2002-136293 A

  Therefore, an object of the present invention is to provide a method for producing D-lactic acid by a continuous fermentation method that maintains high productivity stably over a long period of time with a simple operation method.

  As a result of diligent research, the present inventors have found that clogging of the membrane is remarkably suppressed when the microorganisms and cells enter the separation membrane with little penetration and the microorganisms and cells are filtered under a low transmembrane pressure. As a result, it was possible to maintain the stable filtration of high-concentration microorganisms, which was a problem, and completed the present invention.

  That is, the present invention separates a culture solution into a filtrate and an unfiltered solution by a separation membrane, collects a desired fermentation product from the filtrate, and retains or refluxs the unfiltered solution in the culture solution. A method for producing lactic acid. Specifically, in the method for producing D-lactic acid by the continuous fermentation method of the present invention, the culture solution of microorganisms or cultured cells having the ability to produce D-lactic acid is filtered through a separation membrane, and the product is recovered from the filtrate. A method for producing D-lactic acid by continuous fermentation in which an unfiltered solution is retained or refluxed in a culture solution, and a fermentation raw material is added to the culture solution, wherein the separation membrane has an average pore diameter of 0.01 μm or more and less than 1 μm A method for producing D-lactic acid, which uses a porous membrane and has a transmembrane differential pressure in the range of 0.1 to 20 kPa and filtration at a low transmembrane differential pressure, thereby significantly improving fermentation production efficiency stably at low cost. Is to provide.

According to a preferred embodiment of the method for producing D-lactic acid by the continuous fermentation method of the present invention, the porous water has a pure water permeability coefficient of 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ^{−. 7} m ³ / m ² / s / pa or less.

  According to a preferred embodiment of the method for producing D-lactic acid by the continuous fermentation method of the present invention, the porous membrane has an average pore diameter of 0.01 μm or more and less than 0.2 μm, and a standard deviation of the average pore diameter. Is 0.1 μm or less, and the film surface roughness is 0.1 μm or less.

  According to the preferable aspect of the manufacturing method of D-lactic acid by the continuous fermentation method of this invention, the said porous membrane is a porous membrane containing a porous resin layer.

  According to a preferred embodiment of the method for producing D-lactic acid by the continuous fermentation method of the present invention, a polyvinylidene fluoride resin is used as the membrane material of the porous membrane.

  According to the preferable aspect of the manufacturing method of D-lactic acid by the continuous fermentation method of this invention, it is that the culture solution and fermentation raw material of the said microorganism contain saccharides.

  According to a preferred embodiment of the method for producing D-lactic acid by the continuous fermentation method of the present invention, the microorganism is a microorganism that enhances the enzyme activity of D-lactic acid dehydrogenase.

  According to a preferred embodiment of the method for producing D-lactic acid by the continuous fermentation method of the present invention, the microorganism is a recombinant microorganism into which a gene encoding D-lactic acid dehydrogenase is incorporated, and the recombinant microorganism is It is a eukaryotic cell and the eukaryotic cell is yeast.

  According to a preferred embodiment of the method for producing D-lactic acid by the continuous fermentation method of the present invention, the gene encoding D-lactic acid dehydrogenase is Lactobacillus plantarum, and Pediococcus acidilacti. It is a gene derived from pheasant (Pediococcus acidilacticis) and Bacillus laevolacticus.

  ADVANTAGE OF THE INVENTION According to this invention, the continuous fermentation which maintains the high productivity of a desired fermentation product stably over a long period of time by simple operation conditions is attained, and D-lactic acid which is a fermentation product is widely used in fermentation industry. It becomes possible to produce stably at low cost. D-lactic acid obtained by the method for producing D-lactic acid of the present invention is useful as a raw material for synthetic fibers, for example.

  The present invention is a continuous fermentation in which a culture solution of microorganisms or cultured cells is filtered through a separation membrane, a product is recovered from the filtrate, an unfiltered solution is retained or refluxed in the culture solution, and a fermentation raw material is added to the culture solution. A method for producing D-lactic acid by using a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm as a separation membrane, and performing filtration treatment with a transmembrane differential pressure in the range of 0.1 to 20 kPa. It is a manufacturing method of D-lactic acid by continuous fermentation.

  It is desirable that the porous membrane used in the present invention is not easily clogged by microorganisms and cultured cells used for fermentation, and the filtration performance continues stably for a long period of time.

  The porous membrane used in the present invention is a separation membrane having an average pore diameter of 0.01 μm or more and less than 1 μm. As will be described later, a porous membrane having a porous resin layer on at least one surface of a porous substrate is preferably used. When the porous resin layer exists on both surfaces of the porous separation membrane, it is sufficient that at least one of the porous resin layers satisfies the above average pore diameter condition. If the average pore size is within this range, it is possible to achieve both a high exclusion rate without leaking bacterial cells and sludge and high water permeability, and it is difficult to clog and keep water permeability for a long time. However, it can be implemented with higher accuracy and reproducibility. If the average pore diameter is within this range, when animal cells, yeast, filamentous fungi, or the like are used, clogging is small, and continuous fermentation can be performed stably without leakage of the cells into the filtrate. When bacteria smaller than animal cells, yeast, and filamentous fungi are used, the average pore size is preferably 0.4 μm or less, and the average pore size is less than 0.2 μm, and can be suitably implemented. is there. If the average pore diameter is too small, the water permeation rate may decrease. Therefore, it is usually preferably 0.01 μm or more, more preferably 0.02 μm or more, and further preferably 0.04 μm or more.

  Here, the average pore diameter can be obtained by measuring and averaging the diameters of all pores that can be observed within a range of 9.2 μm × 10.4 μm in a scanning electron microscope observation at a magnification of 10,000 times. it can. The standard deviation σ of the average pore diameter is preferably 0.1 μm or less. The standard deviation σ of the pore diameter is Nk, which is the number of pores that can be observed within the above-mentioned range of 9.2 μm × 10.4 μm, and the measured diameter is Xk, and the average pore diameter is X (ave). The following (Formula 1) was calculated.

In the porous membrane used in the present invention, the permeability of the culture solution is one of the important points, and the pure water permeability coefficient of the porous membrane before use can be used as the permeability index. When the pure water permeability coefficient of the porous membrane was 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, 2 × 10 ⁻⁹ m ³ / m ² / s. / Pa or more is preferable, and if it is 2 × 10 ⁻⁹ or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less, a practically sufficient amount of permeated water can be obtained.

  The surface roughness of the porous membrane used in the present invention is a factor that affects the clogging of the separation membrane. Preferably, when the surface roughness is 0.1 μm or less, the separation coefficient or membrane resistance of the separation membrane. The continuous fermentation can be carried out with a lower transmembrane pressure difference. In addition, the low surface roughness can be expected to reduce the shearing force generated on the membrane surface during filtration of microorganisms and cultured cells, the destruction of microorganisms or cultured cells is suppressed, and the porous membrane is clogged. It is considered that stable filtration is possible for a long time by being suppressed.

Here, the surface roughness can be measured under the following conditions using the following atomic force microscope (AFM).
・ Atomic force microscope (Nanoscope IIIa manufactured by Digital Instruments)
・ Condition probe SiN cantilever (manufactured by Digital Instruments)
Scan mode Contact mode (in-air measurement)
Underwater tapping mode (underwater measurement)
Scanning range 10μm, 25μm square (measurement in air)
5μm, 10μm square (underwater measurement)
Scanning resolution 512 × 512
Sample preparation For measurement, the membrane sample was immersed in ethanol at room temperature for 15 minutes, immersed in RO water for 24 hours, washed, and then air-dried. The RO water refers to water that has been filtered using a reverse osmosis membrane (RO membrane), which is a type of filtration membrane, to exclude impurities such as ions and salts. The pore size of the RO membrane is approximately 2 nm or less.

The surface roughness d _rough of the film was calculated from the following (Equation 2) from the height of each point in the Z-axis direction by AFM.

  The porous membrane in the present invention has separation performance and water permeability according to the quality of water to be treated and applications, and in terms of separation performance such as blocking performance, water permeability and dirt resistance, the porous resin layer It is preferable that the film contains a porous film. That is, a preferred porous membrane 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.

  When the porous membrane used in the present invention has a porous resin layer on the surface of the porous substrate, the porous substrate is porous even if the porous resin layer penetrates the porous substrate. It does not matter if the porous resin layer does not penetrate, and it is selected according to the application.

  The material of the porous substrate is not particularly limited, and is preferably an organic fiber, such as an organic material and an inorganic material. 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 among them, the density control is relatively easy, Nonwoven fabrics that are easy to manufacture and inexpensive are preferably used. The average thickness of the porous substrate is preferably 50 μm or more and 3000 μm or less.

  As described above, the porous resin layer functions as a separation functional layer, and an organic polymer membrane can be preferably used. The organic polymer membrane is a separation membrane basically composed of an organic polymer material. For example, a pore size smaller than the pore size of the organic fiber nonwoven fabric or macropore structure porous organic substrate and the porous organic substrate. In many cases, the porous resin layer has a composite structure.

The material of the porous resin layer includes polyethylene resin, polypropylene resin, polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, polyacrylonitrile resin, cellulose resin, and cellulose triacetate. And a mixture of resins mainly composed of these resins may be used. Among them, polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethersulfone resins, and polyacrylonitrile resins that are easy to form in solution and have excellent physical durability and chemical resistance. Is preferably used. In particular, a polyvinylidene fluoride resin or a resin containing the same as the main component is most preferably used.
Here, as the polyvinylidene fluoride-based resin, a homopolymer of vinylidene fluoride is preferably used. In addition to a homopolymer of vinylidene fluoride, a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride is used. A polymer is also preferably used. Examples of such vinyl monomers include tetrafluoroethylene, hexafluoropropylene, and trichlorofluoroethylene.

  The porous membrane used in the present invention may be a flat membrane or a hollow fiber membrane.

  When the porous membrane is a flat membrane, its thickness is selected according to the application, but is preferably in the range of 20 μm to 5000 μm, more preferably in the range of 50 μm to 2000 μm. As described above, the porous film may be formed of a porous substrate and a porous resin layer. The thickness of the porous substrate is preferably selected in the range of 50 μm or more and 3000 μm or less.

  When the porous membrane is a hollow fiber membrane, the inner diameter is preferably selected in the range of 200 μm to 5000 μm, and the film thickness is preferably selected in the range of 20 μm to 2000 μm. Moreover, the woven fabric and knitting which made the organic fiber or the inorganic fiber the cylinder shape may be included.

  First, an outline of a method for producing a flat film among the porous films 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 a porous resin layer is formed on the surface of the porous substrate.

  The stock solution is prepared by dissolving the resin in a solvent. The temperature of the stock solution is usually preferably selected within the 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. Examples of the solvent include N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethylsulfoxide (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. Of these, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO), which have high resin solubility, are preferably used. These solvents may be used alone or in combination of two or more. The stock solution can be prepared by dissolving the above-mentioned resin in the above-mentioned solvent at a concentration of preferably 5 wt% or more and 60 wt% or less.

  Further, for example, components other than the solvent 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 and alcohols such as methanol and ethanol can be used. Of these, water or methanol is 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, an inorganic salt such as calcium chloride or calcium carbonate 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 example of the outline of a method for producing a hollow fiber membrane among the porous membranes will be described as an example.

  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 stock solution can be prepared by dissolving the resin described in the above-described method for producing a flat film, preferably in a concentration of 20% by weight or more and 60% by weight or less, in the solvent described in the above-described method for forming a flat film. . 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 in order to change the properties of the hollow fiber membrane, for example, hydrophilicity / hydrophobicity or pore diameter, 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 is preferably used. As a lamination method, 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. After the lamination, a part of the attached stock solution is scraped off or an air knife is used. It is also possible to adjust the stacking amount by blowing it away.

  The porous membrane used in the present invention can be used as a membrane separation element by combining with a support. A membrane separation element having a porous membrane in which a support plate is used as a support and the porous membrane used in the present invention is disposed on at least one side of the support plate is one of the preferred forms of the membrane element used in the present invention. It is. In this embodiment, since it is difficult to increase the membrane area, it is also a preferable aspect to dispose a porous membrane on both surfaces of the support plate in order to increase the water permeability.

  In the present invention, the transmembrane pressure difference during filtration of microorganisms and cells may be any condition as long as the microorganisms, cells and medium components are not easily clogged, but the transmembrane pressure difference is 0.1 kPa or more and 20 kPa or less. It is important to have a range. As a driving force for filtration, it is possible to generate a transmembrane differential pressure in the porous membrane by siphon using the liquid level difference (water head difference) of the culture solution and the porous membrane treated water. As a force, a suction pump may be installed on the porous membrane treated water side, or a pressure pump may be installed on the culture solution side of the porous membrane. The transmembrane pressure can be controlled by changing the liquid level difference between the culture solution and the porous membrane treated water. In addition, when using a pump, it can be controlled by the suction pressure. Can be controlled by the pressure of the gas or liquid introduced. When performing these pressure controls, the pressure difference between the culture solution side pressure and the porous membrane treated water side can be used as the transmembrane pressure difference and used to control the transmembrane pressure difference.

Moreover, it is preferable that the porous membrane used in the present invention can be subjected to filtration treatment in a range of a transmembrane differential pressure of 0.1 kPa to 20 kPa. In addition, when the pure water permeability coefficient before use was 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, 2 × 10 ⁻⁹ m ³ / m ² / It is preferably in the range of s / pa or more, and more preferably in the range of 2 × 10 ⁻⁹ m ³ / m ² / s / pa to 6 × 10 ⁻⁷ m ³ / m ² / s / pa. .

  The fermentation raw material used in the present invention is not limited as long as it promotes the growth of microorganisms to be cultured and can produce the desired fermentation product D-lactic acid satisfactorily. A normal liquid medium containing salts and organic micronutrients such as amino acids and vitamins as needed is preferably used.

  Examples of the carbon source include glucose, sucrose, fructose, saccharides such as galactose and lactose, starch saccharified solution containing these saccharides, sweet potato molasses, sugar beet molasses, high test molasses, and organic acids such as acetic acid, ethanol, etc. Other alcohols and glycerin are also used. Here, sugar is the first oxidation product of polyhydric alcohol, which is a carbohydrate that has one aldehyde group or ketone group, categorized as aldose, saccharide with aldehyde group, and ketose as sugar with ketone group. Point to.

  As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysate, casein decomposition 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.

  Moreover, as said inorganic salt, phosphate, magnesium salt, calcium salt, iron salt, manganese salt, etc. can be added suitably.

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

  In the present invention, the culture solution refers to a solution obtained as a result of growth of microorganisms or cultured cells on the fermentation raw material, and the composition of the fermentation raw material to be added is such that the productivity of the target D-lactic acid is increased. The fermentation raw material composition at the start of culture may be changed as appropriate.

  In the present invention, the saccharide concentration in the culture solution is preferably maintained at 5 g / L or less. The reason is to minimize the loss of saccharide due to withdrawal of the culture solution. 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 adjusted to a predetermined value within the above 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 in culturing microorganisms or cultured cells, add oxygen to the air to maintain the oxygen concentration at 21% or higher, pressurize the culture, increase the agitation rate, increase the aeration rate, etc. The following means can be used. Conversely, if it is necessary to reduce the oxygen supply rate, it is also possible to supply a gas containing no oxygen such as carbon dioxide, nitrogen and argon mixed with air.

  In 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 (pullout) may be started. Culture may be performed. It is possible to supply the raw material culture solution and extract the 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 as described above may be added to the raw material culture solution so that the cell growth is continuously performed. The concentration of microorganisms or cultured cells in the culture solution should be maintained at a high level so that the environment of the culture solution is not suitable for the growth of microorganisms or cultured cells and the rate of death does not increase. As an example, good production efficiency can be obtained by maintaining the dry weight at 5 g / L or more. Moreover, the upper limit of the density | concentration of microorganisms will not be limited if the malfunction on the operation | movement of a continuous fermentation apparatus and the fall of production efficiency are not caused.

  The continuous culture operation performed while growing fresh cells having fermentation production ability is usually preferably performed in a single fermentation reaction tank in terms of culture management. However, the number of fermentation reaction tanks is not limited as long as it is a continuous culture method for producing a product while growing cells. A plurality of fermentation reaction tanks may be used 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.

  The microorganism or cultured cell used in the present invention is a microorganism or cultured cell capable of producing D-lactic acid. For example, in a wild type strain, Lactobacillus having an ability to synthesize D-lactic acid is used. And microorganisms belonging to the genus Bacillus and Pediococcus.

  In the present invention, it is preferable that the enzyme activity of these wild-type strains of D-lactate dehydrogenase (hereinafter also referred to as D-LDH) is enhanced. 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.

  In the present invention, the host of the recombinant microorganism is preferably prokaryotic cells such as Escherichia coli, lactic acid bacteria, and eukaryotic cells such as yeast.

  In the present invention, the gene encoding D-lactate dehydrogenase is derived from Lactobacillus plantarum, Pediococcus acidilactici, and Bacillus lavolacticus (Bacillus lavolactus). Preferably, it is a gene derived from Bacillus laevolacticus.

  The separation / purification of D-lactic acid contained in the filtered / separated fermentation liquid produced by the method for producing D-lactic acid by continuous fermentation according to the present invention is performed by combining conventionally known methods such as concentration, distillation and crystallization. It can be carried out.

  Examples of methods for separating and purifying D-lactic acid include, for example, a method in which the pH of the filtered / separated fermentation broth is 1 or less and extraction with diethyl ether, ethyl acetate, or the like, a method in which elution is performed after adsorption washing on an ion exchange resin, an acid Examples thereof include a method of reacting with an alcohol in the presence of a catalyst to distill as an ester, and a method of crystallizing as a calcium salt or a lithium salt. Preferably, the concentrated D-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 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.

  One example of a continuous fermentation apparatus used in the method for producing D-lactic acid by continuous fermentation of the present invention is mainly a fermentation reaction tank for holding microorganisms or cultured cells and producing D-lactic acid, and microorganisms or cultured cells. And a membrane separation element including a porous membrane for separating the culture solution by filtration. The membrane separation element may be installed either inside or outside the fermentation reaction tank.

  In the method for producing D-lactic acid by continuous fermentation according to the present invention, a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm is used, and filtration is performed in a range of a transmembrane differential pressure of 0.1 to 20 kPa. It is characterized by processing. Therefore, there is no need to keep the inside of the fermentation reactor in a pressurized state, so there is no need for a power means to circulate the culture solution between the filtration separator and the fermentation reactor, and a membrane separation element is installed inside the fermentation reactor. Thus, the fermentation apparatus can be made compact.

  Among the continuous fermentation apparatuses used in the method for producing D-lactic acid by continuous fermentation of the present invention, a typical example in which a membrane separation element is installed inside the fermentation reaction tank is shown in the schematic diagram of FIG.

  FIG. 1 is a schematic side view showing one embodiment of a continuous fermentation apparatus used in the method for producing D-lactic acid by continuous fermentation of the present invention. In FIG. 1, the continuous fermentation apparatus basically includes a fermentation reaction tank 1 having a membrane separation element 2 therein and a water head difference control device 3. A porous membrane is incorporated in the membrane separation element 2 in the fermentation reaction tank 1. As this porous membrane, for example, a membrane and a membrane element disclosed in International Publication No. 2002/064240 pamphlet can be used. The membrane separation element will be described in detail later.

  Next, the form of continuous fermentation by the continuous fermentation apparatus of FIG. 1 will be described. In FIG. 1, the medium is continuously or intermittently charged into the fermentation reaction tank 1 by the medium supply pump 7. The medium can be subjected to heat sterilization, heat sterilization, or sterilization using a filter as necessary before addition. During fermentation production, the culture solution in the fermentation reaction tank 1 is agitated by the agitator 5 as necessary, and the necessary gas is supplied by the gas supply device 4 as necessary, and the pH sensor / control is performed as necessary. Fermentative production with high productivity can be performed by adjusting the pH of the culture solution with the apparatus 9 and the pH adjusting solution supply pump 8 and adjusting the temperature of the culture solution with the temperature controller 10 as necessary. Here, the pH and temperature are exemplified for the adjustment of the physicochemical conditions of the culture solution by the instrumentation / control device. However, the dissolved oxygen and ORP are controlled as necessary, and the culture is performed by an analytical device such as an online chemical sensor. The concentration of D-lactic acid in the liquid can be measured, and physicochemical conditions can be controlled using the concentration as an index. In addition, regarding the form of continuous or intermittent addition of the medium, the amount and speed of the medium input can be appropriately adjusted using the measured value of the physicochemical environment of the culture solution by the instrumentation device as an index.

  The culture solution is filtered and separated into microorganisms or cultured cells and a fermentation product by a membrane separation element 2 installed in the fermentation reaction tank 1, and the fermentation product is taken out from the apparatus system. Moreover, the microorganisms or cultured cells filtered and separated remain in the apparatus system, so that the concentration of the microorganisms or cultured cells in the apparatus system can be maintained high, and fermentation production with high productivity is possible. Here, the filtration / separation by the membrane separation element 2 is performed by the water head differential pressure with the water surface of the fermentation reaction tank 1, and no special power is required. Further, the filtration / separation speed of the membrane separation element 2 and the amount of the culture solution in the fermentation reaction tank can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3 as necessary. The filtration / separation by the membrane separation element is exemplified by the water head differential pressure. However, if necessary, the filtration / separation can be performed by suction filtration using a pump or the like, or by pressurizing the inside of the apparatus system.

  FIG. 2 is a schematic side view for explaining an example of another continuous fermentation apparatus used in the method for producing D-lactic acid according to the present invention. FIG. 2 is a typical example of a continuous fermentation apparatus in which the membrane separation element is installed outside the fermentation reaction tank.

  In FIG. 2, the continuous fermentation apparatus basically includes a fermentation reaction tank 1, a membrane separation tank 12 having a membrane separation element 2, and a water head difference control device 3. Here, the membrane separation element 2 incorporates a porous membrane. As the membrane separation element having porosity, it is preferable to use, for example, the separation membrane and the membrane separation element disclosed in WO2002 / 064240. Further, the membrane separation tank 12 is connected to the fermentation reaction tank 1 via the culture solution circulation pump 11.

  In FIG. 2, the medium is introduced into the fermentation reaction tank 1 by the medium supply pump 7, the culture solution in the fermentation reaction tank 1 is stirred by the stirrer 5 as necessary, and the gas supply device 4 is used as necessary. Necessary gas can be supplied. 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 culture solution is adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8, and the temperature of the culture solution is adjusted by the temperature controller 10 as necessary. Fermentative production with high productivity can be performed. Furthermore, the culture solution in the apparatus is circulated between the fermentation reaction tank 1 and the membrane separation tank 12 by the culture solution circulation pump 11. The culture solution containing the fermentation product is filtered and separated into the microorganism and the fermentation product by the membrane separation element 2, and the fermentation product 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 membrane separation element 2 can be performed by the water head differential pressure with the water surface of the membrane separation tank 12, and no special power is required. If necessary, the filtration / separation speed of the membrane separation element 2 and the amount of culture solution in the apparatus system can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3. 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 into the membrane separation tank 12 by the gas supply device 4.

  The filtration / separation by the membrane separation element 2 may be performed by suction filtration using a pump or the like, or by pressurizing the inside of the apparatus system, if necessary. In addition, microorganisms or cultured cells can be cultured for continuous fermentation in a separate culture tank (not shown), and supplied to the fermentation reaction tank as necessary. By culturing microorganisms or cultured cells for continuous fermentation in a separate culture tank and supplying the obtained culture solution into the fermentor as necessary, the microorganisms or cultured cells that are always fresh and have a high production capacity for D-lactic acid Enables continuous fermentation with high production performance maintained for a long period of time.

  Next, the membrane separation element preferably used in the continuous fermentation apparatus used in the method for producing D-lactic acid of the present invention will be described.

  The membrane separation element shown in FIG. 3 will be described. FIG. 3 is a schematic perspective view for illustrating the membrane separation element used in the present invention. In the continuous fermentation apparatus used in the method for producing D-lactic acid according to the present invention, preferably, a separation membrane and a membrane separation element disclosed in WO 2002/064240 can be used. As shown in FIG. 3, the membrane separation element is configured by arranging a flow path material 14 and the separation membrane 15 (porous membrane) 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 culture solution. The flow path member 14 is for efficiently flowing the filtrate filtered by the separation membrane 15 to the support plate 13. The filtrate 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 the water collecting pipe 17. As a power for taking out the filtrate, a method such as a differential pressure at the water head, a pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used.

  The membrane separation element shown in FIG. 4 will be described. FIG. 4 is a schematic perspective view for illustrating another membrane separation element used in the present invention. As shown in FIG. 4, the membrane separation element is mainly constituted by a separation membrane bundle 18 constituted by a hollow fiber membrane (porous membrane), an upper resin sealing layer 19 and a lower resin sealing layer 20. The separation membrane bundle 18 is bonded and fixed in a bundle by an upper resin sealing layer 19 and a lower resin sealing layer 20. Adhesion / fixation by the lower resin sealing layer 20 has a structure in which the hollow portion of the hollow fiber membrane (porous membrane) of the separation membrane bundle 18 is sealed to prevent leakage of the culture solution. On the other hand, the upper resin sealing layer 19 does not seal the inner hole of the hollow fiber membrane (porous membrane) of the separation membrane bundle 18 and has a structure in which the filtrate flows through the water collecting pipe 22. This membrane separation element can be installed in the continuous fermentation apparatus via the support frame 21. The filtrate filtered by the separation membrane bundle 18 passes through the hollow portion of the hollow fiber membrane and is taken out to the outside of the continuous fermentation apparatus via the water collecting pipe 22. As a power for taking out the filtrate, a method such as a differential pressure at the water head, a pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used.

  The member constituting the membrane separation element of the continuous fermentation apparatus used in the method for producing D-lactic acid of the present invention is preferably a member resistant to high-pressure steam sterilization operation. If the inside of the continuous fermentation apparatus can be sterilized, it is possible to avoid the risk of contamination by undesirable microorganisms during continuous fermentation, and more stable continuous fermentation is possible. The members constituting the membrane separation element are preferably resistant to 15 minutes at 121 ° C., which is the condition of the high-pressure steam sterilization operation. Membrane separation element members include, for example, 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. The resin can be preferably selected.

  In the continuous fermentation apparatus used in the method for producing D-lactic acid of the present invention, the membrane separation element may be installed in the fermentation reaction tank as shown in FIG. 1 or installed outside the fermentation reaction tank as shown in FIG. You may do it. When the membrane separation element is installed outside the fermentation reaction tank, a separate membrane separation tank can be provided and the membrane separation element can be installed in the inside, and the culture fluid is circulated between the fermentation reaction tank and the membrane separation tank. The culture solution can be continuously filtered through the membrane separation element.

  In the continuous fermentation apparatus used in the method for producing D-lactic acid according to the present invention, the membrane separation tank is preferably capable of high-pressure steam sterilization. If the membrane separation tank can be autoclaved, it is easy to avoid contamination with germs.

When continuous fermentation is performed according to the method for producing D-lactic acid by continuous fermentation of the present invention, a high volumetric production rate is obtained as compared with conventional batch fermentation, and extremely efficient fermentation production is possible. Here, the production rate in continuous culture is calculated by the following (Equation 3).
Fermentation production rate (g / L / hr) = product concentration in the drawn solution (g / L) × culture solution drawing rate (L / hr) ÷ operating solution volume (L) of the apparatus (Formula 3)
In addition, the fermentation production rate in batch culture is calculated by dividing the amount of product (g) at the time when all of 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.

  The D-lactic acid obtained by the method for producing D-lactic acid by continuous fermentation of the present invention is used as a raw material for polyester resins, which are injection molding, extrusion molding, blow molding, vacuum molding, melt spinning, and film. It can be formed into a desired shape by any molding method such as molding, and can be used as a resin molded product such as a machine part, a fiber such as clothing or industrial material, and a film such as packaging or magnetic recording.

  Hereinafter, in order to more specifically describe the method for producing D-lactic acid according to the present invention, continuous fermentation production of D-lactic acid by using the continuous fermentation apparatus shown in the schematic diagrams of FIGS. An example will be described.

The method for analyzing the D-lactic acid concentration by HPLC is as follows.
[Analysis method of D-lactic acid concentration by HPLC]
The culture solution was centrifuged, and the resulting supernatant was subjected to membrane filtration, and then the amount of lactic acid was measured by the HPLC method under the conditions shown below.
Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5M 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 D-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 ℃
Moreover, the optical purity of D-lactic acid is calculated by the following formula.
Optical purity (%) = 100 × (DL) / (L + D)
Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.

(Reference Example 1) Production of porous membrane (part 1)
Polyvinylidene fluoride (PVDF) resin was used as the resin and N, N-dimethylacetamide (DMAc) was used as the solvent, and these were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
[Undiluted solution]
・ PVDF: 13.0% by weight
DMAc: 87.0% by weight
Next, after cooling the above stock solution to a temperature of 25 ° C., a polyester fiber non-woven fabric (porous substrate) having a density of 0.48 g / cm ³ and a thickness of 220 μm, which was previously pasted on a glass plate. And immediately immersed in a coagulation bath at 25 ° C. having the following composition for 5 minutes to obtain a porous film having a porous resin layer formed on a porous substrate.
[Coagulation bath]
-Water: 30.0% by weight
DMAc: 70.0% by weight
After peeling this porous membrane from the glass plate, it was immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc to obtain a separation membrane. When the surface of the porous resin layer was observed with a scanning electron microscope at a magnification of 10,000 within the range of 9.2 μm × 10.4 μm, the average diameter of all observable pores was 0.1 μm. . Next, when the pure water permeability coefficient was evaluated about the said separation membrane, it was 50 * 10 < ^-9 > m < ³ > / m < ² > / s / Pa. The pure water permeation amount was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.035 μm, and the membrane surface roughness was 0.06 μm.

(Reference Example 2) Production of porous membrane (part 2)
A vinylidene fluoride homopolymer having a weight average molecular weight of 41,000 and γ-butyrolactone were dissolved at a temperature of 170 ° C. at a rate of 38% by weight and 62% by weight, respectively, to prepare a stock solution. This stock solution was discharged from the base while stirring γ-butyrolactone as a hollow portion forming liquid, and solidified in a cooling bath composed of an 80% by weight aqueous solution of γ-butyrolactone at a temperature of 20 ° C. to produce a hollow fiber membrane.

Next, 14% by weight of vinylidene fluoride homopolymer having a weight average molecular weight of 284,000, 1% by weight of cellulose acetate propionate (Eastman Chemical Co., CAP482-0.5), and N-methyl-2-pyrrolidone 77% by weight, polyoxyethylene coconut oil fatty acid sorbitan (manufactured by Sanyo Chemical Co., Ltd., trade name “IONET T-20C” (registered trademark)) 5% by weight, and water 3% by weight at a temperature of 95 ° C. The stock solution was prepared by mixing and dissolving. This undiluted solution was uniformly applied to the surface of the hollow fiber membrane obtained above, and a hollow fiber membrane (porous membrane) used in the present invention was immediately solidified in a water bath. The average pore diameter of the treated water side surface of the obtained hollow fiber membrane (separation membrane) was 0.05 μm. Next, the pure water permeation rate of the hollow fiber membrane as the separation membrane was evaluated and found to be 5.5 × 10 ⁻⁹ m ³ / m ² · s · Pa. The amount of water permeation was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.006 μm.

Reference Example 3 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 a temperature of 30 ° C. The culture was obtained by culturing at a temperature of 24 hours 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 solution (containing 50 mM NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol (pH 7.4)), respectively, at a temperature of 37 ° C. for 30 minutes. Reacted. 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 4) Preparation of a gene library of Bacillus laevolacticus JCM2513 using plasmid vector DNA Plasmid vector DNA (pUC19) 20 μg capable of autonomous replication in Escherichia coli and a restriction enzyme BamHI200 unit were used. , Mixed with 50 mM Tris-HCl buffer (containing 100 mM NaCl and 10 mM magnesium sulfate (pH 7.4)), reacted at a temperature of 37 ° C. for 2 hours to obtain a digested solution, and the digested solution was phenol-extracted by a conventional method, Further, ethanol 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 pUC19 digested with BamHI, 1 μg of chromosomal DNA fragment of Bacillus laevolacticus JCM2513 digested with Sau3AI obtained in Reference Example 3, and 2 units of T4 DNA ligase (Takara Shuzo Co., Ltd.) It 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 5 Preparation of a Host for Screening for D-Lactate Dehydrogenase Gene Screening for 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 necessary to produce a strain that lacks the D-lactate dehydrogenase enzyme activity and pyruvate formate lyase enzyme activity of Escherichia coli. Destruction of Escherichia coli D-lactate dehydrogenase gene (ldhA) and pyruvate formate lyase gene (pflB and pflD) by the method of Kirill et al. (Kirill, A., PNAS, United States (2000) 97 6640-6645) The lost strain was made. 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 6 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 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 4 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 7) Analysis of base sequence of DNA containing D-lactate dehydrogenase gene Plasmid was prepared from Escherichia coli TM33 / pBL2 containing the recombinant DNA obtained above according to a conventional method, and the resulting 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: 1) of the 1,011 base pair D-lactate dehydrogenase gene.

(Reference Example 8) Production 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 shown below.

  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: 2 and 3) 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 (which was cleaved with restriction enzyme HincII and the cleaved surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver. 2 (manufactured by TAKARA) was used. 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 9 Introduction of D-LDH Gene Expression Vector into Yeast pTM63 obtained as in Reference Example 8 was transformed into the yeast Saccharomyces cerevisiae NBRC10505 strain. Transformation was performed by the lithium acetate method using YEASTMAKER Yeast Transformation System (manufactured by 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” (registered trademark) (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 1) Production of D-lactic acid by continuous fermentation (part 1)
In order to investigate whether or not a D-lactic acid continuous fermentation system can be obtained by operating the continuous fermentation apparatus of FIG. 1, a continuous fermentation test using this apparatus was performed using a D-lactic acid production medium having the composition shown in Table 1. . The medium was used after autoclaving at 121 ° C. for 15 minutes. As the membrane separation element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 1 was used. The average pore diameter of the porous membrane is 0.1 μm as described above, and the pure water permeability coefficient is 50 × 10 ⁻⁹ m ³ / m ² / s / pa. The operating conditions in this example are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF 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: All culture tanks containing membrane separation elements and used media are autoclaved at 121 ° C. for 20 min. High pressure steam sterilization ・ Membrane permeate flow control: By transmembrane pressure difference Flow control.

(After continuous fermentation up to 90 hours: controlled at 0.1 kPa to 5 kPa
90 to 180 hours: Control at 2 kPa or less
180 to 264 hours: controlled at 0.1 kPa or more and 20 kPa or less)
The NBRC10505 / pTM63 strain was used as the microorganism, the D-lactic acid production medium having the composition shown in Table 1 was used as the medium, and the concentration of the product D-lactic acid was evaluated by the HPLC method. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  First, the NBRC10505 / pTM63 strain was cultured with shaking in a 5 ml D-lactic acid production medium overnight in a test tube (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 fermentation reaction tank 1 is aerated. The amount was adjusted, the temperature was adjusted, and the pH was adjusted, and cultured for 24 hours (preculture). Immediately after completion of the pre-culture, the D-lactic acid production medium is continuously supplied, and the continuous fermentation apparatus is continuously cultured while controlling the amount of permeated water so that the amount of the culture solution becomes 1.5 L. Manufactured. Control of the amount of permeated water through the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m at maximum, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 6. went. The produced D-lactic acid concentration and residual glucose concentration in the membrane permeated filtrate were measured as appropriate. In addition, Table 2 shows the D-lactic acid production rate calculated from the D-lactic acid and input glucose. As a result of conducting a fermentation test for 264 hours, it was confirmed that stable production of D-lactic acid by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG.

(Example 2) Production of D-lactic acid by continuous fermentation (part 2)
A NBRC10505 / pTM63 strain was used as a microorganism or cultured cell, a D-lactic acid production medium having the composition shown in Table 1 was used as the fermentation medium, and a continuous fermentation test was performed using the continuous fermentation apparatus shown in FIG. The D-lactic acid production medium was used after being autoclaved at 121 ° C. for 15 minutes. As the membrane separation element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2 was used. The operating conditions in Example 2 are as follows unless otherwise specified.
[Operating conditions]
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 4000 square cm
・ Temperature adjustment: 30 (℃)
-Fermentation reactor aeration rate: 0.2 (L / min) air-Fermentation reactor agitation speed: 800 (rpm)
-PH adjustment: pH 7.0 adjusted with 5N NaOH-Culture medium circulation rate: 100 ml / min
Sterilization: All the culture tank and membrane used including the membrane separation element were autoclaved by autoclaving at a temperature of 121 ° C. for 20 minutes.
-Membrane permeate control: Flow control by transmembrane pressure (starting continuous fermentation up to 90 hours: 0.1 kPa to 5 kPa, 90 to 180 hours: 2 kPa or less, 180 to 264 hours: 0.1 kPa to 20 kPa Controlled by)
The D-lactic acid and glucose concentrations were evaluated using the same method as in Example 1.

  First, the NBRC10505 / pTM63 strain was cultured with shaking in a 5 ml D-lactic acid production medium overnight in a test tube (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 was inoculated in the 2.0 L D-lactic acid production medium of the continuous fermentation apparatus shown in FIG. 2, the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5, and the fermentation reaction tank 1 was aerated. The amount was adjusted, the temperature was adjusted, the pH was adjusted, and the culture medium circulation rate was adjusted, and the culture was performed for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the D-lactic acid production medium is continuously supplied, and the continuous fermentation apparatus is continuously cultured while controlling the amount of permeated water so that the culture liquid volume becomes 2.0 L. Manufactured. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the water head difference by the water head difference control device 3 so that the transmembrane pressure difference was 0.1 kPa to 20 kPa. The produced D-lactic acid concentration and residual glucose concentration in the membrane permeated filtrate were measured as appropriate. In addition, Table 2 shows the D-lactic acid fermentation productivity calculated from the D-lactic acid and input glucose.

Comparative Example 1 Production of D-lactic acid by batch fermentation (part 1)
The most typical batch fermentation as a fermentation form using microorganisms was performed using a 2 L jar fermenter, and its D-lactic acid productivity was evaluated. The medium was used after autoclaving at 121 ° C. for 15 minutes. In Comparative Example 1, the strain NBRC10505 / pTM63 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 1 are as follows.
-Fermentation reactor capacity (D-lactic acid production medium amount): 1.0 (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 2 comparing the D-lactic acid fermentation productivity obtained in the continuous fermentation test of Example 1 and Example 2.

  As a result of these comparisons, it was clarified that the production rate of D-lactic acid was significantly improved by using the continuous fermentation apparatus shown in FIGS. 1 and 2. By using a continuous fermentation apparatus incorporating a porous membrane, which is a separation membrane disclosed by the present invention, by controlling the transmembrane pressure difference, the culture solution is separated into a filtrate and an unfiltered solution through the porous membrane, and the filtrate is separated from the filtrate. It becomes clear that it is possible to produce a D-lactic acid by continuous fermentation while recovering a desired fermentation product and allowing a continuous fermentation method in which an unfiltered solution is returned to a culture solution while maintaining a high amount of microorganisms. It was.

(Example 3) Production of D-lactic acid by continuous fermentation (part 3)
In order to examine whether or not a D-lactic acid continuous fermentation system can be obtained by operating the continuous fermentation apparatus of FIG. 1, a continuous fermentation test using this apparatus was performed using a D-lactic acid fermentation medium having the composition shown in Table 3. . The medium was used after autoclaving at 121 ° C. for 15 minutes. As the membrane separation element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 1 was used. The average pore diameter of the porous membrane is 0.1 μm as described above, and the pure water permeability coefficient is 50 × 10 ⁻⁹ m ³ / m ² / s / pa. The operating conditions in Example 3 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 37 (℃)
-Fermentation reactor aeration rate: 0.2 (L / min) nitrogen gas-Fermentation reactor agitation speed: 800 (rpm)
・ PH adjustment: pH 6.0 adjusted with 5N NaOH ・ Sterilization: All culture tanks containing membrane separation elements and used media are autoclaved at 121 ° C. for 20 min. Flow control.

(After continuous fermentation up to 90 hours: controlled at 0.1 kPa to 5 kPa
90 to 180 hours: Control at 2 kPa or less
180 to 245 hours: controlled at 0.1 kPa or more and 20 kPa or less)
The Bacillus laevolacticus ATCC23492 strain was used as the microorganism, the D-lactic acid fermentation medium having the composition shown in Table 3 was used as the medium, and the concentration of the product D-lactic acid was evaluated by the HPLC method. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  First, Bacillus laevolacticus ATCC23492 strain was cultured overnight in a test tube with 5 ml of D-lactic acid fermentation medium overnight (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh D-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 D-lactic acid fermentation 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 fermentation reaction tank 1 is aerated. The amount was adjusted, the temperature was adjusted, and the pH was adjusted, and cultured for 24 hours (preculture). Immediately after completion of the pre-culture, the D-lactic acid fermentation medium is continuously supplied, and the continuous fermentation apparatus is continuously cultured while controlling the amount of permeated water so that the amount of the culture solution is 1.5 L. Manufactured. Control of the amount of permeated water through the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m at maximum, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 6. went. The produced D-lactic acid concentration and residual glucose concentration in the membrane permeated filtrate were measured as appropriate. In addition, Table 4 shows the D-lactic acid production rate calculated from the D-lactic acid and input glucose. As a result of conducting the fermentation test for 245 hours, it was confirmed that stable D-lactic acid production by continuous fermentation was possible by using this continuous fermentation apparatus.

(Example 4) Production of D-lactic acid by continuous fermentation (part 4)
A continuous fermentation test was carried out using a continuous fermentation apparatus shown in FIG. 2 using a Bacillus laevolacticus ATCC23492 strain as a microorganism or cultured cell and a D-lactic acid fermentation medium having the composition shown in Table 3 as the fermentation medium. The D-lactic acid fermentation medium was used after autoclaving at a temperature of 121 ° C. for 15 minutes. As the membrane separation element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2 was used. The operating conditions in Example 4 are as follows unless otherwise specified.
[Operating conditions]
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 4000 square cm
・ Temperature adjustment: 37 (℃)
Fermentation reactor aeration rate: 0.2 (L / min) Nitrogen Fermentation reactor agitation speed: 800 (rpm)
-PH adjustment: pH 6.0 adjusted with 5N NaOH-Culture medium circulation rate: 100 ml / min
Sterilization: All the culture tank and membrane used including the membrane separation element were autoclaved by autoclaving at a temperature of 121 ° C. for 20 minutes.
・ Membrane permeate flow control: Flow control by transmembrane pressure difference (90 hours after the start of continuous fermentation: control at 0.1 kPa to 5 kPa)
90 to 180 hours: controlled at 2 kPa or less 180 to 250 hours: controlled at 0.1 kPa or more and 20 kPa or less)
-D-lactic acid and glucose concentration were evaluated using the same method as in Example 1.

  First, Bacillus laevolacticus ATCC23492 strain was cultured in a test tube with shaking overnight in 5 ml of D-lactic acid fermentation medium (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh D-lactic acid fermentation 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 is inoculated in the 2.0 L D-lactic acid fermentation 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 fermentation reaction tank 1 is aerated. The amount was adjusted, the temperature was adjusted, the pH was adjusted, and the culture medium circulation rate was adjusted, and the culture was performed for 24 hours (preculture). Immediately after the completion of the pre-culture, the D-lactic acid fermentation medium is continuously supplied and continuously cultured while controlling the amount of permeated water so that the amount of the culture solution in the continuous fermentation apparatus becomes 2.0 L. Manufactured. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the water head difference by the water head difference control device 3 so that the transmembrane pressure difference was 0.1 kPa to 20 kPa. The produced D-lactic acid concentration and residual glucose concentration in the membrane permeated filtrate were measured as appropriate. In addition, Table 4 shows D-lactic acid fermentation productivity calculated from the D-lactic acid and input glucose.

Comparative Example 2 Production of D-lactic acid by batch fermentation (part 2)
The most typical batch fermentation as a fermentation form using microorganisms was performed using a 2 L jar fermenter, and its D-lactic acid productivity was evaluated. The medium was used after autoclaving at 121 ° C. for 15 minutes. In Comparative Example 2, Bacillus laevolacticus ATCC23492 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” ( (Registered trademark C) (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The operating conditions of Comparative Example 2 are as follows.
-Fermentation reactor capacity (D-lactic acid production medium amount): 1.0 (L)
・ Temperature adjustment: 37 (℃)
Fermentation reactor aeration rate: 0.2 (L / min) Nitrogen Fermentation reactor agitation speed: 300 (rpm)
-PH adjustment: Adjust to pH 6.0 with 5N NaOH.

  First, Bacillus laevolacticus ATCC23492 strain was cultured overnight in a test tube with 5 ml of D-lactic acid fermentation medium (pre-culture). The culture solution was inoculated into 50 ml of a fresh D-lactic acid fermentation medium, and cultured with shaking in a 500 ml Sakaguchi flask at a temperature of 30 ° C. for 24 hours (preculture). The preculture was inoculated into a 1.5 L D-lactic acid fermentation medium of a jar fermenter. Batch fermentation was performed using a D-lactic acid fermentation medium. The results are shown in Table 4 comparing the D-lactic acid fermentation productivity obtained in the continuous fermentation test of Example 3 and Example 4.

  As a result of comparing these, even if the microorganisms having the ability to produce D-lactic acid are different, the production rate of D-lactic acid is greatly improved by using the continuous fermentation apparatus shown in FIGS. I was able to clarify. By using a continuous fermentation apparatus incorporating a porous membrane, which is a separation membrane disclosed by the present invention, by controlling the transmembrane pressure difference, the culture solution is separated into a filtrate and an unfiltered solution through the porous membrane, and the filtrate is separated from the filtrate. It becomes clear that it is possible to produce a D-lactic acid by continuous fermentation while recovering a desired fermentation product and allowing a continuous fermentation method in which an unfiltered solution is returned to a culture solution while maintaining a high amount of microorganisms. It was.

FIG. 1 is a schematic side view for explaining an example of a continuous fermentation apparatus used in the present invention. FIG. 2 is a schematic side view for explaining an example of another continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining an example of the membrane separation element used in the present invention. FIG. 4 is a schematic perspective view for explaining an example of another membrane separation element used in the present invention. FIG. 5 is a construction diagram showing a physical map of the expression vector pTRS11 for yeast used in the method for producing D-lactic acid of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Membrane separation element 3 Water head 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 Culture fluid circulation pump 12 Membrane Separation tank 13 Support plate 14 Channel material 15 Separation membrane 16 Recess 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 culture solution of microorganisms or cultured cells having the ability to produce D-lactic acid is filtered through a separation membrane, the product is recovered from the filtrate, the unfiltered solution is retained or refluxed, and the fermentation raw material is used as the culture solution. A method for producing D-lactic acid by continuous fermentation added to the above, wherein a porous membrane having an average pore size of 0.01 μm or more and less than 1 μm is used as the separation membrane, and the transmembrane pressure difference is in the range of 0.1 to 20 kPa. A method for producing D-lactic acid by continuous fermentation, characterized by performing a filtration treatment. The pure water permeability coefficient of the porous membrane is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less. The manufacturing method of D-lactic acid by continuous fermentation of description.   3. The continuous fermentation according to claim 1, wherein the porous membrane has an average pore diameter of 0.01 μm or more and less than 0.2 μm, and a standard deviation of the average pore diameter of 0.1 μm or less. A method for producing D-lactic acid.   The method for producing D-lactic acid by continuous fermentation according to any one of claims 1 to 3, wherein the membrane surface roughness of the porous membrane is 0.1 µm or less.   The method for producing D-lactic acid according to any one of claims 1 to 4, wherein the porous membrane is a porous membrane comprising a porous resin layer.   The method for producing D-lactic acid by continuous fermentation according to any one of claims 1 to 5, wherein a polyvinylidene fluoride resin is used as a membrane material of the porous membrane.   The method for producing D-lactic acid by continuous fermentation according to any one of claims 1 to 6, wherein the culture solution of the microorganism and the fermentation raw material contain saccharides.   The method for producing D-lactic acid by continuous fermentation according to any one of claims 1 to 7, wherein the microorganism is a microorganism having enhanced enzyme activity of D-lactic acid dehydrogenase.   The method for producing D-lactic acid by continuous fermentation according to claim 8, wherein the microorganism is a recombinant microorganism into which a gene encoding D-lactic acid dehydrogenase is incorporated.   The method for producing D-lactic acid by continuous fermentation according to claim 9, wherein the recombinant microorganism is a eukaryotic cell.   The method for producing D-lactic acid by continuous fermentation according to claim 10, wherein the eukaryotic cell is yeast.   Genes encoding D-lactate dehydrogenase are Lactobacillus plantarum, Pediococcus acidilactici, and Bacillus laevolactic genes The method for producing D-lactic acid by continuous fermentation according to any one of claims 9 to 11.

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