JP2008212138A - Method for producing l-amino acid by continuous fermentation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing L-amino acid by a continuous fermentation method holding stable high productivity for a long time under a simple operating condition. <P>SOLUTION: The continuous fermentation method includes separation of fermentation culture medium of a microorganism having L-amino acid producing activity into a filtrate and a non-filtrate by a separating membrane, collection of the L-amino acid which is the fermentation product from the filtrate and retention or circulation of the non-filtrate to the fermentation culture medium. In the method, the production efficiency of the L-amino acid is remarkably improved stably with reduced cost by using a specific nonocclusive porous membrane with a high permeability, a high cell rejection and filtering in a specific reduced pressure difference between membranes. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention efficiently filters and collects a liquid containing L-amino acid from a fermentation broth of a microorganism capable of producing L-amino acid through a porous separation membrane that is less likely to be clogged, and an unfiltered liquid. The present invention relates to a method for producing an L-amino acid by continuous fermentation, which can improve the concentration of microorganisms involved in fermentation by returning to a fermentation broth and obtain high productivity.

  Fermentation methods, which are substance production methods that involve the cultivation of microorganisms and cultured cells, are largely divided into (1) batch fermentation methods (Batch fermentation methods) and fed-batch fermentation methods (Fed-Batch fermentation methods), and (2) continuous fermentation methods. Can be classified.

  The batch fermentation method and fed-batch fermentation method (1) are simple in terms of equipment, have an advantage that the culture is completed in a short time, and there is little damage due to contamination with bacteria. However, the product concentration in the fermentation broth increases with the passage of time, and the 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. On the other hand, the continuous fermentation method of (2) can maintain high yield and high productivity over a long period of time by avoiding accumulation of the target substance at a high concentration in the fermenter. There are advantages.

  Next, the background art regarding the manufacturing method of the amino acid containing L-threonine etc. useful as a feed additive etc. is demonstrated. In recent years, amino acids are important substances that are increasing in demand as pharmaceuticals, pharmaceutical intermediates, feed additives, food additives, etc., and traditionally fermentative production in batch culture using bacteria such as Escherichia coli and Corynebacterium (See Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2.) On the other hand, for the purpose of further improving production efficiency, a continuous culture method is disclosed for fermentation of L-glutamic acid (see Patent Document 3) and L-lysine (see Non-Patent Document 3). However, in these examples, since the raw material is continuously supplied to the fermentation broth and the microorganism in the fermentation broth is diluted in order to extract the fermentation broth containing the microorganisms, the production efficiency is improved. Was limited. From this, if these amino acids can be produced efficiently, a reduction in production cost can be expected, and it can be expected that the supply of amino acids to the above-mentioned various uses becomes easier.

  On the other hand, studies on continuous fermentation in which bacteria are cultured using a ceramic filter are being conducted. For example, Kobayashi et al. Are investigating continuous lactic acid fermentation by lactic acid bacteria using a ceramic filter. In order to ensure sufficient permeation performance in filtration of bacterial cells, the filtration pressure is about 90 kPa. Report that high transmembrane pressure and backwashing are necessary. Further, it is disclosed that the permeability is improved by increasing the circulation speed of the membrane surface, but at the same time, the shearing force on the membrane surface causes a malfunction such as a decrease in the activity or rupture of microorganisms (Non-Patent Document 4). reference.).

  Similarly, an apparatus for performing continuous fermentation using a ceramic filter is disclosed. In this apparatus, a gas such as air or nitrogen, water, or filtration is used for back cleaning of the ceramic filter. Since liquid such as membrane permeated water is added to the fermentation broth, it is difficult to maintain the optimal state of the culture due to changes in the production capacity of bacterial substances, and filtration for cell separation. There has been a problem that the membrane device becomes complicated (see Patent Document 4). Furthermore, although a technique for culturing bacteria using a filtration membrane has also been disclosed, there are problems associated with back-washing operations, as with the above-mentioned device, and suction by a pump is necessary during filtration, which complicates the filtration membrane device. There was a problem (see Patent Document 5).

In other words, bacteria represented by amino acid-producing bacteria are filtered through a separation membrane, and the product is recovered from the filtrate. At the same time, the filtered bacteria are refluxed to the culture solution to improve the bacteria concentration in the culture solution and maintain it at a high level. It is still difficult to obtain a high material productivity by making it, and technological innovation has been desired.
Japanese Patent Laid-Open No. 1-148194 JP-A-2-219582 JP-A-10-150996 Japanese Patent Laid-Open No. 62-138184 Japanese Patent Laid-Open No. 2-174673 Ishida, Nakamori et al., “Bioscience. Biotechnology. Biochemical”, 57 (10), 1755-6 (1993). Okamoto, Ikeda, “Journal of Bioscience and Bioengineering (J. Biosci. Bioeng), 89 (1), 87-9 (2000) Toshihiko Hirano et al., "Applied Microvials and Biotechnology", 32, 269-273 (1989). Takeshi Kobayashi, Chemical Engineer, 12, 49 (1988)

  Therefore, an object of the present invention is to provide a method for producing an L-amino acid by a continuous fermentation method that can maintain high productivity stably for a long time with a simple operation method.

  As a result of diligent research, the inventors of the present invention significantly reduced clogging of the separation membrane when the fermentation broth was filtered under the condition that the microorganism has less intrusion into the separation membrane and the microorganism has a low transmembrane pressure difference. As a result, it was possible to maintain the filtration of high-concentration microorganisms, which was a problem, stably for a long period of time, and the present invention was completed.

  The method for producing an L-amino acid according to the present invention comprises filtering a fermentation broth of microorganisms having the ability to produce L-amino acid through a separation membrane, recovering the product from the filtrate, and turning the unfiltered liquid into the fermentation broth. A method for producing an amino acid by continuous fermentation in which a microorganism fermentation raw material is added to the culture solution, wherein the average pore diameter is 0.01 μm or more and less than 1 μm as the separation membrane. A method for producing an L-amino acid by continuous fermentation, characterized by using a porous membrane having a pH of 0.2 to 20 kPa and performing filtration treatment.

  Specifically, the present invention separates the fermentation broth into a filtrate and an unfiltered liquid using a separation membrane, collects L-amino acid as a desired fermentation product from the filtrate, and converts the unfiltered liquid into the fermented broth. In the method for producing L-amino acid by the continuous fermentation method for holding or refluxing, the above-mentioned specific porous separation membrane is used which has high permeability and high cell blocking rate and is difficult to block, and the above-mentioned specific low transmembrane pressure difference Is a method for producing an L-amino acid, which can improve the fermentation production efficiency remarkably at low cost stably.

According to a preferred embodiment of the method for producing L-amino acid by continuous fermentation of the present invention, the porous membrane 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 an L-amino acid by continuous fermentation of the present invention, the average pore diameter of the porous membrane is 0.01 μm or more and less than 0.2 μm, and the standard deviation of the average pore diameter is 0.3. It is 1 μm or less, and its film surface roughness is 0.1 μm or less.

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

  According to a preferred embodiment of the method for producing L-amino acid by continuous fermentation of the present invention, the microorganism is a microorganism capable of producing L-amino acid, and the microorganism capable of producing L-amino acid is a bacterium. .

  According to a preferred embodiment of the method for producing an L-amino acid by continuous fermentation of the present invention, the L-amino acid is L-threonine, L-lysine, L-glutamic acid, L-tryptophan, L-isoleucine, L-glutamine, L-arginine, L-alanine, L-histidine, L-proline, L-phenylalanine, L-aspartic acid, L-tyrosine, L-methionine, L-serine, L-valine or L-leucine, more preferably L-threonine, L-lysine and L-glutamic acid.

  According to a preferred embodiment of the method for producing an L-amino acid by continuous fermentation of the present invention, the bacterium capable of producing the L-amino acid is selected from the group consisting of Genus Escherichia, Genus Providencia, and Corynebacterium. A bacterium belonging to any of the genera Genus Corynebacterium, Genus Brevibacterium or Genus Serratia, and particularly preferred bacteria are Escherichia coli, Providencia regenti (Prov) , Corynebacterium glutamicum, It is Brevibacterium flavum, Brevibacterium lactofermentum or Serratia marcescens.

  According to the present invention, it is possible to perform continuous fermentation that maintains high productivity of a desired fermentation product stably over a long period of time under simple operation conditions, and the L-amino acid that is a fermentation product can be stably produced at low cost. It becomes possible to produce. The L-amino acid obtained by the method for producing L-amino acid by continuous fermentation of the present invention is useful, for example, as a feed additive, a pharmaceutical product, a pharmaceutical intermediate or a food additive.

  The present invention is a method of filtering a fermentation broth of microorganisms capable of producing L-amino acids through a separation membrane to collect a product from the filtrate and holding or refluxing the unfiltrated liquid in the fermentation broth, and In the continuous fermentation in which the fermentation raw material of the microorganism is added to the fermentation broth, a porous membrane having pores with an average pore diameter of 0.01 μm or more and less than 1 μm is used as the separation membrane, and the difference between the membranes A method for producing an L-amino acid by continuous fermentation, wherein the filtration is performed at a pressure of 0.1 or more and less than 20 kPa.

  The porous membrane used as the separation membrane in the present invention is desirably one that is less likely to be clogged by microorganisms used for fermentation and has a performance that allows filtration performance to continue stably for a long period of time. Therefore, it is important that the porous membrane used in the present invention has an average pore diameter of 0.01 μm or more and less than 1 μm.

  The structure of the porous membrane used as a separation membrane in the present invention will be described. The porous membrane in the present invention has separation performance and water permeation performance according to the quality of water to be treated and its use, and in terms of separation performance such as blocking performance, water permeation performance and dirt resistance, the porous resin layer It is preferable that the film contains a porous film. Such a 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.

  Organic materials and inorganic materials can be used as the material of the porous substrate, but organic fibers are preferably used. Preferred porous substrates are woven and non-woven fabrics using organic fibers such as cellulose fibers, cellulose triacetate fibers, polyester fibers, polypropylene fibers and polyethylene fibers. Among them, the density control is relatively easy and the production is also easy. An easy and inexpensive nonwoven fabric is preferably used.

  Further, as described above, the porous resin layer functions as a separation functional layer, and an organic polymer membrane can be suitably 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. Among them, the materials constituting the porous resin layer include polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polysulfone resins, which are easy to form a film with a solution and have excellent physical durability and chemical resistance. Ether sulfone resins and polyacrylonitrile resins are preferable, and polyvinylidene fluoride resins or resins containing them as the main component are most preferably used.

  Here, as the polyvinylidene fluoride resin, a homopolymer of vinylidene fluoride is preferably used, but 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.

  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, the average thickness is selected according to the use, but is preferably 20 μm or more and 5000 μm or less, more preferably 50 μm or more and 2000 μm or less.

  As described above, the separation membrane used in the present invention is desirably a porous membrane formed from a porous substrate and a porous resin layer. At that time, either the porous resin layer permeates the porous base material or the porous resin layer does not permeate the porous base material, and it is selected according to the application. The average thickness of the porous substrate is preferably selected in the range of 50 μm to 3000 μm.

  When the porous membrane is a hollow fiber membrane, the inner diameter of the hollow fiber 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. 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.

  First, an outline of a method for producing a flat film of the porous film will be described.

  A film of a stock solution containing the resin and the solvent is formed on the surface of the porous substrate, and the porous substrate 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. The stock solution may further contain a non-solvent. The temperature of the stock solution is usually preferably selected within the range of 15 to 120 ° C. from the viewpoint of film forming properties.

    Here, 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 extracted when immersed in the coagulation bath, and has a function of making the resin layer porous. 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.

  The solvent dissolves the resin. The solvent acts on the resin and the pore-opening agent to encourage them to form a porous resin layer. Examples of such a solvent include 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 do. Among these, NMP, DMAc, DMF and 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. 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. Among these, water and methanol are preferable from the viewpoint of price. The non-solvent may be a mixture thereof.

  Next, an outline of a method for producing a hollow fiber membrane among the porous 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 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 made into a separation membrane element by combining with a support. A separation membrane element 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 a preferred embodiment of the separation membrane element having a porous membrane used in the present invention. One. In this form, when it is difficult to increase the membrane area, it is preferable to dispose a porous membrane on both sides of the support plate in order to increase the water permeability.

  When the average pore diameter of the porous membrane as the separation membrane is in the range of 0.01 μm or more and less than 1 μm as described above, both a high exclusion rate that prevents leakage of bacterial cells and sludge and a high water permeability are compatible. Further, it is possible to carry out with higher accuracy and reproducibility to prevent clogging and to maintain water permeability for a long time. When bacteria are used, the average pore diameter of the porous membrane is preferably 0.4 μm or less, and the average pore diameter is preferably less than 0.2 μm. If the average pore diameter is too small, the water permeation rate may decrease. Therefore, in the present invention, the average pore diameter is 0.01 μm or more, preferably 0.02 μm or more, more preferably 0.04 μm or more. is there.

  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. Further, a uniform permeate can be obtained when the standard deviation of the average pore diameter is smaller, that is, when the pore diameters are uniform. Since fermentation operation management becomes easy, the smaller the standard deviation of the average pore diameter, the better.

  The standard deviation σ of the average pore diameter is N (the number of pores that can be observed within the above-mentioned range of 9.2 μm × 10.4 μm), each measured diameter is Xk, and the average pore diameter is X (ave) It is calculated by the following (Formula 1).

In the porous membrane used in the present invention, the permeability of the fermentation broth 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. In the present invention, the pure water permeation coefficient of the porous membrane is 2 × 10 ⁻⁹ m ³ / when the water permeability is measured at a head height of 1 m using purified water at a temperature of 25 ° C. by a reverse osmosis membrane. It is preferably m ² / s / pa or more. If the pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less, a practically sufficient amount of permeated water can be obtained. A more preferable pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 1 × 10 ⁻⁷ m ³ / m ² / s / pa or less.

  The membrane surface roughness of the porous membrane used in the present invention is a factor that affects the clogging of the separation membrane. When the membrane surface roughness is preferably 0.1 μm or less, the separation coefficient and membrane resistance of the separation membrane can be suitably reduced, and continuous fermentation can be carried out with a lower transmembrane pressure difference. Therefore, since stable continuous fermentation becomes possible by suppressing clogging, the smaller the surface roughness, the better.

  In addition, by reducing the membrane surface roughness of the porous membrane, it can be expected that the shear force generated on the membrane surface will be reduced in the filtration of microorganisms, the destruction of microorganisms is suppressed, and the porous membrane is clogged. It is considered that stable filtration is possible for a long time by being suppressed.

Here, film | membrane surface roughness can be measured on the following apparatus and conditions using the following atomic force microscope apparatus (AFM).
・ Device: Atomic force microscope device (Nanoscope IIIa manufactured by Digital Instruments)
・ Conditions: Probe SiN cantilever (manufactured by Digital Instruments)
: Scanning 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, to exclude impurities such as ions and salts. The pore size of the RO membrane is approximately 2 nm or less.

Membrane surface roughness _{d rough} from the Z-axis direction of the height of each point by the above AFM, is calculated by the following equation (2).

  In the present invention, the transmembrane pressure difference when the microorganism is filtered with a separation membrane may be any condition as long as the microorganism and medium components are not easily clogged, but the transmembrane pressure difference is in the range of 0.1 kPa to 20 kPa. Thus, it is important to perform filtration. The transmembrane pressure difference is preferably in the range of 0.1 kPa to 10 kPa, and more preferably in the range of 0.1 kPa to 5 kPa. When the range of the above transmembrane pressure difference is exceeded, clogging of microorganisms and medium components may occur rapidly, leading to a decrease in the amount of permeated water, which may cause problems in continuous fermentation operation.

  As a driving force for filtration, it is possible to generate a transmembrane pressure difference in the porous membrane by a siphon utilizing a liquid level difference (water head difference) of the fermentation broth and the porous membrane treated water. Moreover, a suction pump may be installed on the porous membrane treated water side as a driving force for filtration, or a pressure pump may be installed on the fermentation culture solution side of the porous membrane. The transmembrane pressure can be controlled by changing the liquid level difference between the fermentation broth and the porous membrane treated water, and if a pump is used to generate the transmembrane pressure, suction The transmembrane pressure can be controlled by the pressure, and the transmembrane pressure can also be controlled by the pressure of the gas or liquid that introduces the pressure on the fermentation broth side. When these pressure controls are performed, the pressure difference between the pressure on the fermentation broth side and the pressure on the porous membrane treated water side can be used as the transmembrane pressure difference, which can be used to control the transmembrane pressure difference.

Moreover, it is preferable that the porous membrane used in this invention has the performance which can be filtered in the range of 0.1 kPa or more and 20 kPa or less as a transmembrane differential pressure to filter. In addition, as described above, the porous membrane used in the present invention has a pure water permeability coefficient before use of purified water having a temperature of 25 ° C. by a reverse osmosis membrane, and measures the water permeability at a head height of 1 m. when ^{calculated, 2 × 10 -9 m 3 /} m 2 / s / is preferably pa is above the range, further ^{2 × 10 -9 m 3 / m} 2 / s / pa or 6 × ^{10 -7} m It is preferably in the range of ³ / m ² / s / pa or less.

  The microorganism fermentation raw material used in the present invention may be any material that can promote the growth of microorganisms to be fermented and cultivate and can favorably produce an L-amino acid that is a target fermentation product. As a fermentation raw material, for example, a liquid medium or the like appropriately containing a carbon source, a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins are preferably used.

  Examples of the carbon source include saccharides such as glucose, sucrose, fructose, galactose, and lactose, starch saccharified solution containing these saccharides, sweet potato molasses, sugar beet molasses, high test molasses, sugar cane juice, and acetic acid Organic acids such as fumaric acid, alcohols such as ethanol and glycerin are used. Sugars are the first oxidation products of polyhydric alcohols, and are carbohydrates that have one aldehyde group or ketone group, sugars with aldehyde groups are classified as aldoses, and sugars with ketone groups are classified as ketoses. Point to.

  Examples of the nitrogen source include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysates, 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, a phosphate, magnesium salt, calcium salt, iron salt, manganese salt etc. can be suitably added and used, for example.

  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. An antifoaming agent can be added and used as necessary.

  In the present invention, the fermentation culture liquid refers to a liquid obtained as a result of growth of microorganisms on the fermentation raw material, and the composition of the fermentation raw material to be added may be appropriately changed from the fermentation raw material composition at the start of the culture. When changing from a fermentation raw material composition to the composition of a fermentation raw material to be added, a change that increases the productivity of the target L-amino acid is preferred. For example, by reducing the weight ratio of organic micronutrients such as nitrogen sources, inorganic salts, amino acids and vitamins to the above carbon source, the production cost of L-amino acids is reduced, that is, the productivity of L-amino acids in a broad sense is improved. May be realized. On the other hand, by increasing the weight ratio of organic micronutrients such as nitrogen sources, inorganic salts, amino acids and vitamins to the carbon source, the productivity of L-amino acids may be improved.

  In the present invention, the concentration of fermentation raw materials such as sugars in the fermentation broth is preferably maintained at 5 g / l or less. The reason is to minimize the loss of the fermentation raw material due to withdrawal of the fermentation broth. Therefore, it is desirable that the concentration of the fermentation raw material is as small as possible.

  In many cases, the fermentation culture of microorganisms is usually performed at a pH of 4 to 8 and a temperature of 20 to 40 ° C. The pH of the fermentation broth is adjusted to a predetermined value within the above range with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, and the like.

  If it is necessary to increase the oxygen supply rate in the fermentation culture of microorganisms, add oxygen to the air to maintain the oxygen concentration at 21% or higher, pressurize the fermentation broth, increase the stirring speed, or increase the aeration rate. The following means can be used. On the other hand, when 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 fermentation raw material liquid and extract the culture from an appropriate time. The start time of the fermentation raw material supply and the withdrawal of the culture are not necessarily the same. Further, the supply of the fermentation raw material liquid and the withdrawal of the culture may be continuous or intermittent. What is necessary is just to add a nutrient required for microbial cell growth as shown above to a fermentation raw material liquid so that microbial cell growth may be performed continuously.

  The concentration of microorganisms in the fermentation broth must be maintained at a high level in order to obtain efficient productivity, so long as the environment of the fermentation broth is inappropriate for the growth of microorganisms and the rate of death does not increase. preferable. As an example, better production efficiency can be obtained by maintaining the concentration at 5 g / L or more as the dry weight. Moreover, the upper limit of the concentration of microorganisms is not particularly limited as long as it does not cause problems in the operation of the continuous fermentation apparatus or decrease in production efficiency.

  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.

  In the present invention, it is possible to continuously and efficiently extract the fermentation broth from the fermentation reaction tank while maintaining the microorganism in the fermentation reaction tank. It is also possible to change the composition of the fermentation raw material liquid after securing the desired chemical product efficiently.

  The amino acids suitably fermentatively produced in the present invention include L-threonine, L-lysine, L-glutamic acid, L-tryptophan, L-isoleucine, L-glutamine, L-arginine, L-alanine, L-histidine, L -Proline, L-phenylalanine, L-aspartic acid, L-tyrosine, L-methionine, L-serine, L-valine, L-leucine and the like, in particular, L-threonine, L-lysine and L-glutamic acid Is preferred.

  The microorganism used in the present invention has the ability to produce L-amino acids because the essence of the present invention is the efficiency of fermentation culture, and for example, can conventionally produce L-amino acids efficiently. Can be suitably used. Examples of such microorganisms include bacteria such as Escherichia coli and coryneform bacteria often used in the fermentation industry.

  An L-amino acid producing bacterium is a bacterium capable of producing a corresponding L-amino acid.

  Examples of L-threonine-producing bacteria include Genus Escherichia, Genus Providencia, Genus Corynebacterium, Genus Brevibacterium, and Serratia genus. Is mentioned. Particularly preferred bacteria are Escherichia coli, Providencia rettgeri, Corynebacterium glutamicum, Brevibacterium brebium, Brevibactrum. Brevibacterium lactofermentum) and Serratia marcescens.

  Examples of L-lysine producing bacteria include bacteria belonging to the genus Escherichia, Corynebacterium, or Brevibacterium. Particularly preferred bacteria are Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum.

  As L-glutamic acid-producing bacteria, Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum are preferable.

  Examples of L-tryptophan-producing bacteria include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus amyloliquefaciens. Etc.

  Examples of L-isoleucine-producing bacteria include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Serratia marcescens, and the like.

  Examples of L-glutamine-producing bacteria include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Flavobacterium ligens and the like.

  Examples of L-arginine-producing bacteria include Corynebacterium glutamicum, Brevibacterium flavum, Serratia marcescens, Escherichia coli and Bacillus subtilis.

  Examples of L-alanine producing bacteria include Brevibacterium flavum and Arthrobacter oxydans.

  Examples of L-histidine-producing bacteria include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium amoniagenes, Serratia marcescens, Escherichia coli, Bacillus subtilis and Streptomyces coelicolor ( Streptomyces coelicolor) and the like.

  Examples of L-proline-producing bacteria include Corynebacterium glutamicum, Kurthia catenaforma, Serratia marcescens and Escherichia coli.

  Examples of L-phenylalanine producing bacteria include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum or Escherichia coli.

  Examples of L-aspartic acid producing bacteria include Brevibacterium flavum, Bacillus megaterium, Escherichia coli, Pseudomonas fluorescens, and the like.

  Examples of L-tyrosine-producing bacteria include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum and Escherichia coli.

  As L-methionine-producing bacteria, Corynebacterium glutamicum is preferable.

  Examples of serine-producing bacteria include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Arthrobacter oxydans, and the like.

  Examples of L-serine producing bacteria include Corynebacterium acetoacidophilum and Brevibacterium lactofermentum.

  Examples of L-valine-producing bacteria include Brevibacterium lactofermentum, Serratia marcescens and Klebsiella pneumoniae.

  Examples of L-leucine-producing bacteria include Corynebacterium glutamicum, Brevibacterium lactofermentum, Serratia marcescens, and the like.

  Microorganisms having the ability to produce L-amino acids such as L-threonine may be isolated from the natural environment, or may have been partially modified by mutation or genetic recombination. Good. As an example, Providencia retgeri with improved L-threonine productivity described in JP-A-2-219582 and coryne with improved L-alanine productivity described in JP-T-3-5000048 pamphlet. Examples include bacteria and glutamicum.

  Separation / purification of L-amino acid contained in the filtered / separated fermentation broth produced by the method for producing L-amino acid by continuous fermentation of the present invention is performed by combining conventionally known methods such as concentration, distillation and crystallization. It can be carried out.

  One example of the continuous fermentation apparatus used in the method for producing L-amino acids by continuous fermentation of the present invention is mainly a fermentation reactor for holding L-amino acid-producing bacteria and producing L-amino acids, and L- It is composed of a separation membrane element including a porous membrane for separating the amino acid-producing bacteria and the culture solution by filtration. The separation membrane element may be installed either inside or outside the fermentation reaction tank.

  In the method for producing L-amino 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 as a separation membrane, and a transmembrane differential pressure as a filtration pressure is 0.1 to 20 kPa. It is characterized by filtering in a range. Therefore, it is not necessary to keep the inside of the fermentation reaction tank in a pressurized state, so that no power means for circulating the fermentation broth between the filtration separation device and the fermentation reaction tank is required, and the separation membrane element is placed inside the fermentation reaction tank. It can also be installed to make the fermentation culture apparatus compact.

  FIG. 1 shows a typical example in which a separation membrane element is installed inside a fermentation reaction tank among continuous fermentation apparatuses used in the method for producing L-amino acids by continuous fermentation of the present invention. FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus used in the present invention. In FIG. 1, a membrane separation type continuous fermentation apparatus is basically composed of a fermentation reaction tank 1 for fermentation culture of microorganisms and a water head difference control apparatus 3 for controlling the amount of fermentation culture solution in the fermentation reaction tank 1. It is configured. A separation membrane element 2 is disposed in the fermentation reaction tank 1, and a porous membrane is incorporated in the separation membrane element 2. As the porous membrane, for example, a separation membrane and a separation membrane element disclosed in International Publication No. 2002/064240 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. 1 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 heating, sterilization, or sterilization using a filter as necessary before being put into the fermentation reaction tank 1. During fermentation production, the fermentation liquor in the fermentation reaction tank 1 can be stirred with the agitator 5 in the fermentation reaction tank 1 as necessary. Moreover, the gas required by the gas supply apparatus 4 can be supplied in the fermentation reaction tank 1 as needed. At this time, the supplied gas can be recovered and recycled and supplied to the gas supply device 4 again. If necessary, the pH of the fermentation broth in the fermentation reaction tank 1 can be adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8. Moreover, if necessary, highly productive fermentation production can be performed by adjusting the temperature of the fermentation broth in the fermentation reaction tank 1 with the temperature controller 10.

  Here, the adjustment of the pH and temperature was exemplified for the adjustment of the physicochemical conditions of the fermentation broth by the instrumentation / control device, but if necessary, the dissolved oxygen and ORP can be controlled, The concentration of L-amino acid in the fermentation broth can be measured by an analytical device such as an online chemical sensor, and the physicochemical conditions can be controlled using the concentration as an index. Further, the form of continuous or intermittent input of the medium is not particularly limited, but the input amount and the rate of the medium are measured using the measured value of the physicochemical environment of the fermentation broth by the instrumentation apparatus as an index. Can be adjusted as appropriate.

  The fermentation broth is filtered and separated into microorganisms and fermentation products by the separation membrane element 2 installed in the fermentation reaction tank 1 and 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 is performed by the water head differential pressure with respect to the water surface of the fermentation reaction tank 1, and no special power is required. Moreover, the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation liquid in the fermentation reaction tank 1 can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3 as necessary. Although the filtration / separation by the separation membrane element 2 is exemplified by the water head differential pressure, the filtration / separation is performed by suction filtration with a pump, liquid or gas, or pressurization in the apparatus system as necessary. You can also

  Next, among the continuous fermentation apparatuses used in the method for producing L-amino acids by continuous fermentation of the present invention, a typical example in which the separation membrane element is installed outside the fermentation reaction tank is shown in the schematic diagram of FIG. . FIG. 2 is a schematic side view for explaining one embodiment of another membrane separation type continuous fermentation apparatus used in the present invention.

  In FIG. 2, a membrane separation type continuous fermentation apparatus includes a fermentation reaction tank 1 for culturing and culturing microorganisms, and a fermentation culture solution circulation pump 11 connected to the fermentation reaction tank 1 and a separation membrane element 2 inside. The membrane separation tank 12 and the water head difference control device 3 for controlling the amount of the fermentation broth in the fermentation reaction tank 1 are basically configured. Here, a porous membrane is incorporated in the separation membrane element 2. As the porous membrane, for example, a separation membrane and a separation membrane element disclosed in International Publication No. 2002/064240 are suitably used.

  In FIG. 2, the medium can be introduced into the fermentation reaction tank 1 by the medium supply pump 7, and the fermentation broth in the fermentation reaction tank 1 can be stirred by the stirrer 5 as necessary. Moreover, the gas required by the gas supply apparatus 4 can be supplied as needed. 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 can be adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8. Moreover, highly productive fermentation production can be performed by adjusting the temperature of a fermentation broth with the temperature controller 10 as needed. Further, the fermentation broth in the apparatus is circulated between the fermentation reaction tank 1 and the membrane separation tank 12 by the fermentation broth circulation pump 11. The fermentation broth containing the fermentation product is filtered and separated into the microorganism and the fermentation product by the separation membrane element 2, and the fermentation product can be taken out from the apparatus system.

  In addition, the filtered and separated microorganisms can remain in the apparatus system to maintain a high concentration of microorganisms in the apparatus system, enabling highly productive fermentation production. Here, the filtration / separation by the separation membrane 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 separation membrane element 2 and the amount of fermentation broth in the apparatus system can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3. Moreover, the gas required by the gas supply apparatus 4 can be supplied in the membrane separation tank 12 as needed.

  As described above, the filtration / separation by the separation membrane element 2 can be performed by the water head differential pressure. However, if necessary, the filtration / separation can be performed by suction filtration using a pump, liquid or gas, or by pressurizing the inside of the apparatus system. It can also be separated. By such means, the transmembrane pressure difference can be adjusted and controlled.

  Next, the outline of the separation membrane and the separation membrane element disclosed in International Publication No. 2002/064240, which is an example of a preferred embodiment of the separation membrane element used in the present invention, will be described below with reference to the drawings. 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 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.

  FIG. 4 is a schematic perspective view for explaining another embodiment of another separation membrane element used in the present invention. As shown in FIG. 4, the separation membrane element is mainly composed of a separation membrane bundle 18 composed of 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 separation membrane element can be installed in the continuous fermentation apparatus via the support frame 21. The filtrate containing the fermentation product filtered by the separation membrane bundle 18 passes through the hollow portion of the hollow fiber membrane and is taken out 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 separation membrane element of the continuous fermentation apparatus used in the L-amino acid production method 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 separation membrane element are preferably resistant to 15 minutes at 121 ° C., which is the condition of the high-pressure steam sterilization operation. Examples of the separation membrane element member 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 L-amino acid production method of the present invention, the separation membrane 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 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 culture fluid is circulated between the fermentation reaction tank and the membrane separation tank. The culture solution can be continuously filtered by the separation membrane element.

  In the continuous fermentation apparatus used in the L-amino acid production method of 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 carried out according to the method for producing L-amino acids 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 fermentation production rate in continuous culture is calculated by the following (Equation 3).
・ Fermentation production rate (g / L / hr) = Product concentration in extraction liquid (g / L) × Fermentation culture liquid extraction speed (L / hr) ÷ Operation liquid volume of apparatus (L) 3)
In addition, the fermentation production rate by batch culture is the amount of product (g) at the time when all the raw carbon source is consumed, the time (h) required for consumption of the carbon source and the amount of fermentation broth (L) at that time. It is obtained by dividing.

  The L-amino acid obtained by the method for producing L-amino acid by continuous fermentation of the present invention is mainly used as a feed additive, a pharmaceutical product, a pharmaceutical intermediate, a food additive, and the like. L-amino acids can be provided.

  Hereinafter, in order to describe the method for producing L-amino acid by continuous fermentation of the present invention in more detail, continuous fermentation production of L-threonine by using the continuous fermentation apparatus shown in the schematic diagrams of FIGS. 1 and 2. Will be described with reference to examples. The present invention is not limited to these examples.

  In the following examples relating to the method for producing L-threonine, L-lysine production using L-threonine, Providencia lettelli SGR588-77 strain (FERM P-10528) among Providencia lettelli, is used. In the Example concerning, the strain | stump | stock which carried out the gene modification of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 among Corynebacterium glutamicum was used as microorganisms which produce L-lysine.

  Moreover, glucose was used for the carbon source among the fermentation raw materials, and the raw materials described in the following examples were used for the nitrogen source and inorganic salts.

Reference Example 1 Quantitative Method for L-Threonine, L-Lysine and Glucose The amount of L-threonine contained in the culture solution was measured by the following method. Take 25 μL of the fermentation broth containing L-threonine to be measured, and add 150 μl of NaHCO 3 (75 mM) and 25 μl of L-methionine (2 g / L) as an internal standard. Add 900 μl of ethanol and 150 μl of 0.2 M dinitrofluorobenzene (DNFB) to the above solution and mix. The solution was allowed to stand at 37 ° C. for 1 hour, and then subjected to HPLC analysis under the following conditions.
Column: CAPCELLPAK C18 TYPE SG120 (Shiseido)
Mobile phase: 0.1% (w / v) H3PO4: Acetonitrile = 7: 3 (flow rate 1.2 mL / min)
-Detection method: UV (360 nm)
・ Temperature: 23 ℃
A calibration curve is prepared by analyzing L-threonine with a known concentration as a standard, and plotting the L-threonine concentration on the horizontal axis and the area ratio of L-threonine area / L-methionine (internal standard) area on the vertical axis. did.

The amount of L-lysine contained in the culture solution was measured by the following method. Take 25 μL of the fermentation broth containing L-lysine to be measured, and add 400 μL of NaHCO 3 (75 mM) and 25 μL of 1,4-butanediol (2 g / L) as an internal standard. After adding 150 μl of 0.2MDNFB to the above solution, the mixture was reacted at 37 ° C. for 1 hour.
50 μl of the solution was dissolved in 1 mL of acetonitrile, and 10 μl of the supernatant centrifuged at 10,000 rpm for 5 minutes was analyzed by HPLC under the following conditions.
-Column: CAPCELLPAK C18 TYPE SG120 (Shiseido)
Mobile phase: 0.1% (w / w) phosphoric acid aqueous solution: acetonitrile = 45: 55 (flow rate 1 mL / min)
-Detection method: UV (360 nm)
・ Temperature: 23 ℃
The calibration curve is analyzed using L-lysine with a known concentration as a standard, and the horizontal axis plots the L-lysine concentration and the vertical axis plots the area ratio of L-lysine area / 1,4-butanediol (internal standard) area. And produced.

  For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

(Reference Example 2) 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.
・ 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 having a temperature of 25 ° C. having the following composition for 5 minutes to obtain a porous film having a porous resin layer formed on a porous substrate.
-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 on the side of the separation membrane in contact with the coagulation bath is observed with a scanning electron microscope at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm, the pores that can be observed The average of all diameters was 2.0 μm. Moreover, when the scanning electron microscope observation was performed at a magnification of 10,000 times in the range of 9.2 μm × 10.4 μm on the surface of the porous resin layer on the opposite side, the average diameter of all the observable pores was It was 0.1 μm. Next, when the permeation amount of pure water was evaluated for the separation membrane, it was 50 × 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 0.035 μm, and the membrane surface roughness was 18 μm.

(Reference Example 3) Production of porous membrane (part 2)
Polyvinylidene fluoride (PVDF) resin as a resin, polyethylene glycol (PEG) having a molecular weight of about 20,000 as a pore-opening agent, N, N-dimethylacetamide (DMAc) as a solvent, and pure water as a non-solvent. Each was used and stirred sufficiently at a temperature of 90 ° C. to obtain a stock solution having the following composition.
・ PVDF: 13.0% by weight
・ PEG: 5.5% by weight
DMAc: 78.0% by weight
・ Pure water: 3.5% by weight
Next, after cooling the stock solution to a temperature of 25 ° C., it was applied to a polyester fiber nonwoven fabric (porous substrate) having a density of 0.48 g / cm ³ and a thickness of 220 μm. It was immersed in pure water at a temperature for 5 minutes and further immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc and PEG, thereby obtaining a porous membrane (separation membrane) having a porous resin layer. When the surface of the porous resin layer on the side of the separation membrane on which the stock solution was applied was observed with a scanning electron microscope at a magnification of 10,000, all pores that could be observed were observed. The average diameter was 0.02 μm. When the water permeability of this separation membrane was evaluated, it was 2 × 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 0.0055 μm, and the membrane surface roughness was 0.1 μm.

(Reference Example 4) Production of porous membrane (part 3)
A porous membrane (separation membrane) having a porous resin layer was obtained in the same manner as in Reference Example 2 except that a stock solution having the following composition was used.
・ PVDF: 13.0% by weight
・ PEG: 5.5% by weight
DMAc: 81.5% by weight
When the surface of the porous resin layer on the side of the separation membrane on which the stock solution was applied was observed in a scanning electron microscope at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm, all the pores that could be observed The average diameter was 0.2 μm, and the pure water permeation rate of this separation membrane was evaluated to be 100 × 10 ⁻⁹ m ³ / m ² · s · Pa. The amount of water permeation was measured using purified water at 25 ° C. with a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.060 μm, and the membrane surface roughness was 0.08 μm.

Reference Example 5 Production of porous membrane (Part 4)
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 ratio 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 stock solution was uniformly applied to the surface of the hollow fiber membrane obtained above, and immediately solidified in a water bath to produce a hollow fiber membrane (porous membrane) used in the present invention. 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.

(Example 1) Production of L-threonine by continuous fermentation (part 1)
In order to investigate whether L-threonine can be obtained by continuous fermentation by operating the continuous fermentation apparatus shown in FIG. 1, a continuous fermentation test using the same apparatus was performed. The medium shown in Table 1 was used as the medium. This medium was used after being subjected to high-pressure (2 atm) steam sterilization at a temperature of 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 membrane produced in the above-mentioned Reference Example 2 containing polyvinylidene fluoride (PVDF) as a main component was used. When the characteristics of the porous membrane were examined, the average pore diameter was 0.1 μm, and the pure water permeability coefficient was 50 × 10 ⁻⁹ m ³ / m ² / s / pa. The operating conditions in Example 1 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 (℃)
-Aeration volume of fermentation reaction tank: 1.5 (L / min)
・ Fermentation reactor stirring speed: 800 (rpm)
・ Sterilization: Fermentation reaction tank containing separation membrane element and all used media were autoclaved at 121 ° C. for 20 min under high pressure (2 atm) steam sterilization.
・ PH adjustment: Adjusted to pH 7 with 28% ammonia solution ・ Membrane permeate flow control: Flow control by transmembrane pressure difference
(100 hours after the start of continuous fermentation: controlled at 0.1 kPa to 5 kPa
100 hours to 200 hours: controlled at 0.1 kPa to 2 kPa)
Providencia rettgeri strain SGR588-77 (FERM P-10528) was used as the microorganism, and an L-threonine fermentation medium having the composition shown in Table 1 was used as the medium. The concentrations of L-threonine and glucose contained in the fermentation broth were measured by the method shown in Reference Example 1.

    First, the Providencia reggeri SGR588-77 strain scraped from the agar medium was inoculated into a 500 ml Erlenmeyer flask charged with 100 ml of glucose broth medium (1% glucose, 3% broth (Nissui)). This was cultured with stirring at a temperature of 37 ° C. and a rotation speed of 140 rpm (pre-culture). The obtained pre-culture solution was inoculated into the continuous fermentation apparatus shown in FIG. 1 charged with 1 L of L-threonine preculture medium (Table 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 was adjusted and the temperature was adjusted to a temperature of 37 ° C., followed by culturing for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the L-threonine fermentation medium shown in Table 1 is continuously supplied and continuously cultured while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus is 1.5 L. L-threonine was produced by fermentation. The control of the amount of permeated water when performing the continuous fermentation test is controlled by the water head difference control device 3 at a rate of 0.1 kPa to 5 kPa for 100 hours after the start of continuous fermentation, and 0.1 kPa to 2 kPa for 100 hours to 200 hours. Control was as follows. Where appropriate, the produced L-threonine concentration and residual glucose concentration in the membrane permeate filtrate were measured. Table 4 shows the L-threonine production rate calculated from L-threonine and input glucose. As a result of conducting a fermentation test for 200 hours, it was confirmed that stable production of L-threonine by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG. The transmembrane pressure difference during the period of continuous fermentation changed from 0.1 kPa to 2 kPa.

(Example 2) Production of L-threonine by continuous fermentation (part 2)
Using the porous membrane prepared in Reference Example 3 as the separation membrane, the same L-threonine continuous fermentation test as in Example 1 was performed. The results are shown in Table 4. As a result, it was confirmed that stable production of L-threonine by continuous fermentation was possible. Moreover, the transmembrane pressure difference during the period of continuous fermentation changed at 2 kPa or less.

(Example 3) Production of L-threonine by continuous fermentation (part 3)
Using the porous membrane produced in Reference Example 4 as the separation membrane, the same L-threonine continuous fermentation test as in Example 1 was performed. The results are shown in Table 4. As a result, it was confirmed that stable production of L-threonine by continuous fermentation was possible. Moreover, the transmembrane pressure difference during the period of continuous fermentation was changed from 0.1 kPa to 2 kPa.

(Example 4) Production of L-threonine by continuous fermentation (Part 4) Separation type flat membrane 3
In order to examine whether or not L-threonine can be obtained by operating the continuous fermentation apparatus shown in FIG. 2, a continuous fermentation test using the same apparatus was performed. The medium shown in Table 1 was used as the medium, and it was used after being subjected to high-pressure steam sterilization 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, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 4 was used. The porous membrane has an average pore diameter of 0.1 μm and a pure water permeability coefficient of 50 × 10 ⁻⁹ m ³ / m ² / s / pa. The operating conditions in Example 4 are as follows unless otherwise specified.
・ 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: 120 square cm
・ Temperature adjustment: 37 (℃)
-Aeration volume of fermentation reaction tank: 1.5 (L / min)
・ Fermentation reactor stirring speed: 800 (rpm)
-Sterilization: All the culture tanks containing the separation membrane element and the medium used were autoclaved at 121 ° C for 20 min under high pressure steam sterilization.
-PH adjustment: adjusted to pH 7 with 28% aqueous ammonia solution-Membrane permeated water amount control: Flow rate was controlled by the difference in the water head of the membrane separation tank (the water head difference was controlled within 2 m).

  Providencia rettgeri strain SGR588-77 (FERM P-10528) was used as the microorganism, and an L-threonine fermentation medium having the composition shown in Table 1 was used as the medium. The concentrations of L-threonine and glucose contained in the fermentation broth were measured by the method shown in Reference Example 1.

  First, the Providencia reggeri SGR588-77 strain scraped from the agar medium was inoculated into a 500 ml Erlenmeyer flask charged with 100 ml of glucose broth medium (1% glucose, 3% broth (Nissui)). This was cultured with stirring at a temperature of 37 ° C. and a rotation speed of 140 rpm (pre-culture). The obtained pre-culture solution was inoculated into the continuous fermentation apparatus shown in FIG. 2 charged with 1 L of L-threonine preculture medium (Table 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 was adjusted and the temperature was adjusted to a temperature of 37 ° C., followed by culturing for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the L-threonine fermentation medium shown in Table 1 is continuously supplied and continuously cultured while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus is 1.5 L. L-threonine was produced by fermentation. Control of the amount of permeated water when performing the continuous fermentation test was performed by appropriately changing the water head difference so that the head of the fermentation reaction tank was within 2 m at the maximum, that is, the transmembrane pressure difference was within 20 kPa. Where appropriate, the produced L-threonine concentration and residual glucose concentration in the membrane permeate filtrate were measured. Table 4 shows the L-threonine production rate calculated from L-threonine and input glucose. As a result of conducting a fermentation test for 200 hours, it was confirmed that stable production of L-threonine by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG.

(Example 5) Production of L-threonine by continuous fermentation (part 5)
In order to investigate whether L-amino acid is obtained by continuous fermentation by operating the continuous fermentation apparatus shown in FIG. 1, the continuous fermentation test using the same apparatus was conducted. The medium shown in Table 1 was used as the medium, and it was used after being subjected to high-pressure steam sterilization 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, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 5 was used. The operating conditions in Example 5 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 (℃)
-Aeration volume of fermentation reaction tank: 1.5 (L / min)
・ Fermentation reactor stirring speed: 800 (rpm)
-Sterilization: All the culture tanks containing the separation membrane element and the medium used were autoclaved at 121 ° C for 20 min under high pressure steam sterilization.
-PH adjustment: adjusted to pH 7 with 28% aqueous ammonia solution-Membrane permeate flow rate control: Flow rate is controlled by membrane head difference (water head difference was controlled within 2 m).

  Providencia rettgeri strain SGR588-77 (FERM P-10528) was used as the microorganism, and an L-threonine fermentation medium having the composition shown in Table 1 was used as the medium. The concentrations of L-threonine and glucose contained in the fermentation broth were measured by the method shown in Reference Example 1.

  First, the Providencia reggeri SGR588-77 strain scraped from the agar medium was inoculated into a 500 ml Erlenmeyer flask charged with 100 ml of glucose broth medium (1% glucose, 3% broth (manufactured by Nissui)). This was cultured with stirring at a temperature of 37 ° C. and a rotation speed of 140 rpm (pre-culture). The obtained pre-culture solution was inoculated into the membrane-separated continuous fermentation apparatus shown in FIG. 1 charged with 1 L of L-threonine preculture medium (Table 2). The mixture was stirred at 800 rpm, the aeration amount of the fermentation reaction tank 1 was adjusted, and the temperature was adjusted to a temperature of 37 ° C., and cultured for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the L-threonine fermentation medium shown in Table 1 is continuously supplied and continuously cultured while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus is 1.5 L. L-threonine was produced by fermentation. Control of the amount of permeated water when performing the continuous fermentation test was performed by appropriately changing the water head difference so that the head of the fermentation reaction tank was within 2 m at the maximum, that is, the transmembrane pressure difference was within 20 kPa. Where appropriate, the produced L-threonine concentration and residual glucose concentration in the membrane permeate filtrate were measured. Table 4 shows the L-threonine production rate calculated from L-threonine and input glucose. As a result of conducting a fermentation test for 200 hours, it was confirmed that stable production of L-threonine by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG.

(Comparative Example 1) Production of L-threonine by batch fermentation The most typical batch fermentation as a fermentation form using microorganisms was performed using a 2 L jar fermenter, and its L-threonine productivity was evaluated. The medium shown in Table 2 was used as the medium at the start of batch culture. This medium was used after being subjected to high-pressure (2 atm) steam sterilization at a temperature of 121 ° C. for 15 minutes. In Comparative Example 1, the same Providencia retgeri SGR588-77 strain as in Example 1 was used as the microorganism, and the concentrations of L-threonine and glucose contained in the fermentation broth were measured by the method shown in Reference Example 1. The operating conditions of Comparative Example 1 are as follows.
Fermentation reactor capacity (L-threonine fermentation medium amount): 1 (L)
・ Temperature adjustment: 37 (℃)
-Fermentation reactor aeration rate: 1 (L / min)
・ Fermentation reactor stirring speed: 800 (rpm)
-PH adjustment: adjusted to pH 7 with 28% aqueous ammonia solution First, Providencia scraped from an agar medium into a 500 ml Erlenmeyer flask containing 90 ml of glucose broth medium (1% glucose, 3% broth (Nissui)) -Leggery SGR588-77 strain was inoculated. This was cultured while stirring at a temperature of 37 ° C. and a rotation speed of 140 rpm (pre-culture). The obtained preculture was inoculated into a mini jar fermenter charged with 810 ml of the medium shown in Table 2 and subjected to batch fermentation. Table 3 shows the composition of the medium added during the culture. The medium was added at 50 mL each 24 hours, 32 hours, 40 hours, and 48 hours after the start of culture. The results of batch fermentation are shown in Table 4 in comparison with the L-threonine fermentation productivity obtained in the continuous fermentation test of Examples 1 to 5 above.

  From the results in Table 4, it was confirmed that the production rate of L-threonine was significantly improved by using the continuous fermentation apparatus shown in FIGS. 1 and 2 used in the present invention.

(Example 6) Production of L-lysine by continuous fermentation (production of recombinant strain)
A homoserine dehydrogenase (HOM) gene disruption strain of Corynebacterium glutamicum ATCC13032 (hereinafter abbreviated as ATCC13032 strain) was produced as a microorganism having L-lysine production ability.

(1) Cloning of levansucrase (SacB) gene The SacB gene derived from Bacillus subtilis was cloned.

  Oligonucleotide primers (SEQ ID NOs: 1 and 2) were synthesized with reference to the base sequence of the SacB gene (Accession No. X02730) registered in the database (GenBank). Take 0.2 μl each of a genomic DNA solution prepared from Bacillus subtilis IFO13719 strain according to a conventional method as a template in a 0.2 ml microcentrifuge tube, 20 pmol of each primer, Tris-HCl buffer pH 8.0 (20 mM), chloride Reagents were added to give potassium (2.5 mM), gelatin (100 μg / ml), each dNTP (50 μM), LATaq DNA polymerase (2 units) (manufactured by Takara Shuzo) to a total volume of 50 μl. 30 cycles of polymerase chaining using a BioRad thermal cycler under conditions of DNA denaturation at 94 ° C for 30 seconds, primer annealing at 55 ° C for 30 seconds, DNA primer extension at 72 ° C for 3 minutes Reaction was performed (hereinafter abbreviated as PCR method). The PCR method in Example 6 was performed under these conditions unless otherwise specified. The product obtained by this PCR method was electrophoresed with 1% agarose, and a DNA fragment of about 1.4 kb containing the SacB gene was excised from the gel and purified with Gene Clean Kit (manufactured by BIO101). This fragment was digested with the restriction enzyme SacI, and the resulting 1.4 kb SacI fragment was ligated to the EcoRI / BamHI gap of pHSG298 (Takara Shuzo) previously digested with SacI. 1 (Takara Shuzo Co., Ltd.) was inserted, and the resulting plasmid was named pTM38.

(2) Cloning of homoserine dehydrogenase (HOM) gene In order to delete the HOM activity, a gene corresponding to the 300 amino acid region from the N-terminal was cloned.

  An oligonucleotide primer (SEQ ID NO: 3 and 4) was synthesized with reference to the base sequence of the HOM gene (Accession No. BA00000036) registered in the database (GenBank). PCR was performed using a genomic DNA solution prepared from the ATCC 13032 strain according to a conventional method as an amplification template and the oligonucleotides of SEQ ID NOs: 3 and 4 as primer sets, and the product obtained by this PCR was electrophoresed in 1% agarose. A DNA fragment of about 0.9 kb containing the HOM gene was excised from the gel and purified by Gene Clean Kit (manufactured by BIO101). This fragment was digested with restriction enzymes SphI and BamHI, and the obtained 0.9 kb SphI-BamHI fragment was ligated into the SphI / BamHI gap of pTM38 previously digested with SphI and BamHI. 1 (Takara Shuzo Co., Ltd.) was inserted, and the resulting plasmid was designated as pTM44.

(3) Cloning of chloramphenicol resistance gene (Cm gene) Oligonucleotide primers (SEQ ID NOs: 5 and 6) were synthesized with reference to the base sequence of pHSG399 (Accession No. M19087) registered in the database (GenBank). .

  PCR was performed using pHSG399 as an amplification template and the oligonucleotides of SEQ ID NOs: 5 and 6 as a primer set, and the product obtained by this PCR was electrophoresed with 1% agarose, and about 1.0 kb containing Cm gene was obtained. The DNA fragment was excised from the gel and purified with Gene Clean Kit (BIO101). This fragment was digested with the restriction enzyme Aor51HI, and the resulting 1 kb Aor51HI fragment was ligated into the Aor51HI gap of pTM44 previously digested with Aor51HI. 1 (Takara Shuzo Co., Ltd.) was inserted, and the resulting plasmid was named pTM62.

(4) Disruption of homoserine dehydrogenase gene Plasmid pTM62 was transformed into ATCC13032 strain by electroporation [FEMS Microbiology Letters, 65, p. 299 (1989)] and added with kanamycin (25 μg / ml) LB (tryptone (10 g / l) (Bacto), yeast extract (5 g / l) (Bacto), sodium chloride ( 10 g / l)) Selected on agar medium.

  A genomic DNA solution was prepared from the thus selected transformant according to a conventional method. PCR was carried out using this genomic DNA as a template and oligonucleotides (SEQ ID NOs: 3 and 4) as primer sets, and the resulting product was electrophoresed on a 1.0% agarose gel. A band was observed. From this, it was confirmed that the selected transformant had the Cm gene inserted into the HOM locus and destroyed. This transformant was named Corynebacterium glutamicum delta-HOM strain (hereinafter abbreviated as delta-HOM strain).

(Example 7) Production of L-lysine by continuous fermentation (part 1)
In order to investigate whether L-lysine can be obtained by continuous fermentation by operating the continuous fermentation apparatus shown in FIG. 1, a continuous fermentation test using the same apparatus was performed. The medium shown in Table 5 was used as the medium. This medium was used after being subjected to high-pressure (2 atm) steam sterilization at a temperature of 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 membrane produced in the above-mentioned Reference Example 4 containing polyvinylidene fluoride (PVDF) as a main component was used. The average pore diameter of this 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 7 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 (℃)
-Aeration volume of fermentation reaction tank: 2 (L / min)
・ Fermentation reactor stirring speed: 650 (rpm)
・ Sterilization: Fermentation reaction tank containing separation membrane element and all used media were autoclaved at 121 ° C. for 20 min under high pressure (2 atm) steam sterilization.
・ PH adjustment: pH adjusted to 7.3 with 4N NH ₄ OH ・ Membrane permeate flow rate control: Flow rate is controlled by difference in membrane separation tank water head (water head difference was controlled within 2 m).

  The delta-HOM strain produced in Example 6 was used as the microorganism, and the L-lysine fermentation medium having the composition shown in Table 5 was used as the medium. The concentration of L-lysine and glucose contained in the fermentation broth was measured by the method shown in Reference Example 1.

  First, delta-HOM scraped from an agar medium into a test tube charged with 5 ml of BY medium (0.5% yeast extract, 0.7% meat extract, 1% peptone, 0.3% sodium chloride). The strain was inoculated and cultured with shaking at a temperature of 30 ° C. for 24 hours (pre-culture). The whole culture solution obtained was inoculated in a 500 mL Erlenmeyer flask containing 50 mL of the medium shown in Table 5 and cultured with shaking at 30 ° C. for 24 hours (pre-culture). The obtained pre-culture solution was inoculated into the continuous fermentation apparatus shown in FIG. 1 in which 1 L of the medium shown in Table 5 was added, and the fermentation reaction tank 1 was stirred at 650 rpm by the attached stirrer 5 to give the fermentation reaction tank 1 The aeration amount was adjusted and the temperature was adjusted to 30 ° C., followed by culturing for 24 hours (preculture). Immediately after completion of the pre-culture, the medium shown in Table 5 is continuously supplied, and the continuous culture 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. -Preparation of lysine. Control of the amount of permeated water when performing the continuous fermentation test was performed by appropriately changing the water head difference so that the head of the fermentation reaction tank was within 2 m at the maximum, that is, the transmembrane pressure difference was within 20 kPa. The produced L-lysine concentration and residual glucose concentration in the membrane permeate filtrate were measured as appropriate. In addition, Table 6 shows L-lysine production rates calculated from L-lysine and input glucose. As a result of performing the fermentation test for 200 hours, it was confirmed that stable production of L-lysine by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG.

(Example 8) Production of L-lysine by continuous fermentation (part 2)
In order to investigate whether L-lysine can be obtained by continuous fermentation by operating the continuous fermentation apparatus shown in FIG. 1, a continuous fermentation test using the same apparatus was performed. The medium shown in Table 5 was used as the medium. This medium was used after being subjected to high-pressure (2 atm) steam sterilization at a temperature of 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 membrane produced in the above-mentioned Reference Example 5 containing polyvinylidene fluoride (PVDF) as a main component was used. The average pore diameter of this 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 9 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 (℃)
-Aeration volume of fermentation reaction tank: 2 (L / min)
・ Fermentation reactor stirring speed: 650 (rpm)
・ Sterilization: Fermentation reaction tank containing separation membrane element and all used media were autoclaved at 121 ° C. for 20 min under high pressure (2 atm) steam sterilization.
・ PH adjustment: pH adjusted to 7.3 with 4N NH ₄ OH ・ Membrane permeate flow rate control: Flow rate is controlled by difference in membrane separation tank water head (water head difference was controlled within 2 m).

  The delta-HOM strain produced in Example 6 was used as the microorganism, and the L-lysine fermentation medium having the composition shown in Table 5 was used as the medium. The concentration of L-lysine and glucose contained in the fermentation broth was measured by the method shown in Reference Example 1.

  First, delta-HOM scraped from an agar medium into a test tube charged with 5 ml of BY medium (0.5% yeast extract, 0.7% meat extract, 1% peptone, 0.3% sodium chloride). The strain was inoculated and cultured with shaking at a temperature of 30 ° C. for 24 hours (pre-culture). The whole culture solution obtained was inoculated in a 500 mL Erlenmeyer flask containing 50 mL of the medium shown in Table 5 and cultured with shaking at 30 ° C. for 24 hours (pre-culture). The obtained pre-culture solution was inoculated into the continuous fermentation apparatus shown in FIG. 1 in which 1 L of the medium shown in Table 5 was added, and the fermentation reaction tank 1 was stirred at 650 rpm by the attached stirrer 5 to give the fermentation reaction tank 1 The amount of aeration was adjusted and the temperature was adjusted to 30 ° C., followed by culturing for 24 hours (pre-culture). Immediately after completion of the pre-culture, the medium shown in Table 5 is continuously supplied, and the continuous culture 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. -Preparation of lysine. Control of the amount of permeated water when performing the continuous fermentation test was performed by appropriately changing the water head difference so that the head of the fermentation reaction tank was within 2 m at the maximum, that is, the transmembrane pressure difference was within 20 kPa. The produced L-lysine concentration and residual glucose concentration in the membrane permeate filtrate were measured as appropriate. In addition, Table 6 shows L-lysine production rates calculated from L-lysine and input glucose. As a result of performing the fermentation test for 200 hours, it was confirmed that stable production of L-lysine by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG.

(Example 9) Production of L-lysine by continuous fermentation (part 3)
In order to investigate whether L-lysine can be obtained by continuous fermentation by operating the continuous fermentation apparatus shown in FIG. 2, a continuous fermentation test using the apparatus was performed. The medium shown in Table 5 was used as the medium. This medium was used after being subjected to high-pressure (2 atm) steam sterilization at a temperature of 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 membrane produced in the above-mentioned Reference Example 4 containing polyvinylidene fluoride (PVDF) as a main component was used. The average pore diameter of this 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 9 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 (℃)
-Aeration volume of fermentation reaction tank: 2 (L / min)
・ Fermentation reactor stirring speed: 650 (rpm)
・ Sterilization: Fermentation reaction tank containing separation membrane element and all used media were autoclaved at 121 ° C. for 20 min under high pressure (2 atm) steam sterilization.
・ PH adjustment: pH adjusted to 7.3 with 4N NH ₄ OH ・ Membrane permeate flow rate control: Flow rate is controlled by difference in membrane separation tank water head (water head difference was controlled within 2 m).

  The delta-HOM strain produced in Example 6 was used as the microorganism, and the L-lysine fermentation medium having the composition shown in Table 5 was used as the medium. The concentration of L-lysine and glucose contained in the fermentation broth was measured by the method shown in Reference Example 1.

  First, delta-HOM scraped from an agar medium into a test tube charged with 5 ml of BY medium (0.5% yeast extract, 0.7% meat extract, 1% peptone, 0.3% sodium chloride). The strain was inoculated and cultured with shaking at a temperature of 30 ° C. for 24 hours (pre-culture). The whole culture solution obtained was inoculated in a 500 mL Erlenmeyer flask containing 50 mL of the medium shown in Table 5 and cultured with shaking at 30 ° C. for 24 hours (pre-culture). The obtained pre-culture solution was inoculated into the continuous fermentation apparatus shown in FIG. 2 in which 1 L of the medium shown in Table 5 was added, and the fermentation reaction tank 1 was stirred at 650 rpm by the attached stirrer 5. The amount of aeration was adjusted and the temperature was adjusted to 30 ° C., followed by culturing for 24 hours (pre-culture). Immediately after completion of the pre-culture, the medium shown in Table 5 is continuously supplied, and the continuous culture 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. -Preparation of lysine. Control of the amount of permeated water when performing the continuous fermentation test was performed by appropriately changing the water head difference so that the head of the fermentation reaction tank was within 2 m at the maximum, that is, the transmembrane pressure difference was within 20 kPa. The produced L-lysine concentration and residual glucose concentration in the membrane permeate filtrate were measured as appropriate. In addition, Table 6 shows L-lysine production rates calculated from L-lysine and input glucose. As a result of conducting a fermentation test for 200 hours, it was confirmed that stable L-lysine production by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG.

(Example 10) Production of L-lysine by continuous fermentation (part 4)
In order to investigate whether L-lysine can be obtained by continuous fermentation by operating the continuous fermentation apparatus shown in FIG. 2, a continuous fermentation test using the apparatus was performed. The medium shown in Table 5 was used as the medium. This medium was used after being subjected to high-pressure (2 atm) steam sterilization at a temperature of 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 membrane produced in the above-mentioned Reference Example 5 containing polyvinylidene fluoride (PVDF) as a main component was used. The average pore diameter of this 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 10 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 (℃)
-Aeration volume of fermentation reaction tank: 2 (L / min)
・ Fermentation reactor stirring speed: 650 (rpm)
・ Sterilization: Fermentation reaction tank containing separation membrane element and all used media were autoclaved at 121 ° C. for 20 min under high pressure (2 atm) steam sterilization.
・ PH adjustment: pH adjusted to 7.3 with 4N NH ₄ OH ・ Membrane permeate flow rate control: Flow rate is controlled by difference in membrane separation tank water head (water head difference was controlled within 2 m).

  The delta-HOM strain produced in Example 6 was used as the microorganism, and the L-lysine fermentation medium having the composition shown in Table 5 was used as the medium. The concentration of L-lysine and glucose contained in the fermentation broth was measured by the method shown in Reference Example 1.

  First, delta-HOM scraped from an agar medium into a test tube charged with 5 ml of BY medium (0.5% yeast extract, 0.7% meat extract, 1% peptone, 0.3% sodium chloride). The strain was inoculated and cultured with shaking at a temperature of 30 ° C. for 24 hours (pre-culture). The whole culture solution obtained was inoculated in a 500 mL Erlenmeyer flask containing 50 mL of the medium shown in Table 5 and cultured with shaking at 30 ° C. for 24 hours (pre-culture). The obtained pre-culture solution was inoculated into the continuous fermentation apparatus shown in FIG. 2 in which 1 L of the medium shown in Table 5 was added, and the fermentation reaction tank 1 was stirred at 650 rpm by the attached stirrer 5. The amount of aeration was adjusted and the temperature was adjusted to 30 ° C., followed by culturing for 24 hours (pre-culture). Immediately after completion of the pre-culture, the medium shown in Table 5 is continuously supplied, and the continuous culture 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. -Preparation of lysine. Control of the amount of permeated water when performing the continuous fermentation test was performed by appropriately changing the water head difference so that the head of the fermentation reaction tank was within 2 m at the maximum, that is, the transmembrane pressure difference was within 20 kPa. The produced L-lysine concentration and residual glucose concentration in the membrane permeate filtrate were measured as appropriate. In addition, Table 6 shows L-lysine production rates calculated from L-lysine and input glucose. As a result of conducting a fermentation test for 200 hours, it was confirmed that stable L-lysine production by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG.

(Comparative Example 2) Production of L-lysine by batch fermentation The most typical batch fermentation as a fermentation form using microorganisms was performed using a 2L jar fermenter, and the L-lysine productivity was evaluated. The media shown in Table 5 were used as the media at the start of batch culture. This medium was used after being subjected to high-pressure (2 atm) steam sterilization at a temperature of 121 ° C. for 15 minutes. In Comparative Example 1, the delta-HOM strain prepared in Example 6 was used as the microorganism, and the concentrations of L-lysine and glucose contained in the fermentation broth were measured by the method shown in Reference Example 1. The operating conditions of Comparative Example 1 are as follows.
-Fermentation reaction tank capacity (L-lysine fermentation medium amount): 1.5 (L)
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 2 (L / min)
・ Fermentation reactor stirring speed: 650 (rpm)
・ PH adjustment: Adjust to pH 7.3 with 4N NH ₄ OH First, add 5 ml of BY medium (0.5% yeast extract, 0.7% meat extract, 1% peptone, 0.3% sodium chloride). The delta-HOM strain scraped from the agar medium was inoculated into the test tube, and cultured with shaking at a temperature of 30 ° C. for 24 hours (pre-culture). The entire amount of the obtained pre-cultured solution was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of the medium shown in Table 5, and cultured with shaking at 30 ° C. for 24 hours (pre-culture). The obtained culture solution was inoculated into a mini jar fermenter charged with 1450 mL of the medium shown in Table 5, and batch culture was performed. The results of batch fermentation are shown in Table 6 in comparison with the L-lysine fermentation productivity obtained in the continuous fermentation test of Examples 7 to 10 above.

  From the results in Table 6, it was confirmed that the production rate of L-lysine was greatly improved by using the continuous fermentation apparatus shown in FIGS. 1 and 2 used in the present invention.

FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus used in the present invention. FIG. 2 is a schematic side view for explaining one embodiment of another membrane separation type continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention. FIG. 4 is a schematic perspective view for explaining one embodiment of another separation membrane element used in the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Separation membrane element 3 Water head difference 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 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 (15)

  Filtration of a fermentation broth of a microorganism having the ability to produce L-amino acids through a separation membrane, recovering the product from the filtrate, holding or refluxing the unfiltered liquid in the fermentation broth, and fermentation of the microorganism A method for producing an L-amino acid by continuous fermentation in which a raw material is added to the fermentation broth, wherein a porous membrane having pores with an average pore diameter of 0.01 μm or more and less than 1 μm is used as the separation membrane, A process for producing an L-amino acid by continuous fermentation, wherein the transmembrane pressure difference is in the range of 0.1 to 20 kPa. The continuous water fermentation according to claim 1, wherein the porous membrane has a pure water permeability coefficient of 2 × 10 ⁻⁹ m ³ / m ² / s / pa to 6 × 10 ⁻⁷ m ³ / m ² / s / pa. A method for producing an L-amino acid.   3. The production of L-amino acid by continuous fermentation according to claim 1 or 2, 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 is 0.1 μm or less. Method.   The method for producing an L-amino 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 an L-amino acid by continuous fermentation 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 an L-amino acid by continuous fermentation according to any one of claims 1 to 4, wherein the material of the porous membrane is a polyvinylidene fluoride resin.   The method for producing an L-amino acid by continuous fermentation according to any one of claims 1 to 5, wherein the fermentation raw material of the microorganism contains a saccharide.   The method for producing an L-amino acid by continuous fermentation according to any one of claims 1 to 7, wherein the microorganism is a bacterium.   L-amino acid is L-threonine, L-lysine, L-glutamic acid, L-tryptophan, L-isoleucine, L-glutamine, L-arginine, L-alanine, L-histidine, L-proline, L-phenylalanine, L The method for producing an L-amino acid by continuous fermentation according to any one of claims 1 to 8, which is aspartic acid, L-tyrosine, L-methionine, L-serine, L-valine or L-leucine.   The method for producing an L-amino acid by continuous fermentation according to any one of claims 1 to 8, wherein the L-amino acid is L-threonine.   The bacterium is a genus belonging to the genus Escherichia, Genus Providencia, Genus Corynebacterium, Genus Brevibacterium, or Genus Serratia. Item 11. A method for producing an L-amino acid by continuous fermentation according to Item 10.   The bacteria are Escherichia coli, Providencia rettgeri, Corynebacterium glutamicum, Brevibacterium brebactrum. It is either Serratia marcescens (Serratia marcescens), The manufacturing method of L-amino acid by continuous fermentation of Claim 10 or 11.   The method for producing an L-amino acid by continuous fermentation according to any one of claims 1 to 8, wherein the L-amino acid is L-lysine.   The method for producing an L-amino acid by continuous fermentation according to claim 13, wherein the bacterium is a bacterium belonging to any of the genus Escherichia, Genus Corynebacterium, or Genus Brevibacterium.   The bacterium is Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, or Brevibacterium lactofermentum. Or the manufacturing method of L-amino acid by continuous fermentation of 14.

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