JP5659466B2 - Method and apparatus for producing chemicals by continuous culture - Google Patents

Description

  The present invention relates to a chemical production method and production apparatus by continuous culture of microorganisms. More specifically, the present invention produces a chemical product with high productivity by efficiently filtering and recovering a liquid containing a chemical product produced by culturing from a microorganism culture solution through a separation membrane while performing continuous culture. The present invention relates to a method for manufacturing a chemical product and a manufacturing apparatus.

  Fermentation methods, which are one of the methods for producing substances that involve the cultivation of microorganisms and cultured cells, are largely (1) batch fermentation methods (Batch fermentation methods), fed-batch fermentation methods (Fed-Batch fermentation methods), and (2) continuous culture methods. Can be classified.

  The batch and fed-batch fermentation methods have a merit that they are simple in terms of equipment, cultivate in a short time, and have less damage due to contamination with bacteria. However, the product concentration in the culture solution increases with time, and the productivity and yield decrease due to the influence of osmotic pressure or product inhibition. For this reason, it is difficult to stably maintain a high yield and high productivity over a long period of time.

  The continuous culture method is generally characterized in that high yield and high productivity can be maintained over a long period of time by avoiding accumulation of the target substance at a high concentration in the culture apparatus. For example, Non-Patent Document 1 discloses a continuous culture method for fermentation of L-glutamic acid or L-lysine. However, in the method described in this document, the microorganisms and the cultured cells in the culture solution are diluted to continuously supply the culture raw material to the culture solution and to extract the culture solution containing the microorganisms and the cultured cells. . Therefore, in reality, improvement in production efficiency has been limited.

  Further, in Patent Document 1, in a continuous culture method, microorganisms and cultured cells are filtered through a separation membrane, and a product is recovered from the filtrate, and at the same time, the filtered microorganisms and cultured cells are held or refluxed in the culture solution. A method for maintaining a high concentration of microorganisms and cells in the culture solution has been proposed. For example, a continuous culture technique capable of stably maintaining high productivity over a long period of time under simple operation conditions is disclosed. However, if continuous culture is performed while circulating the culture medium in the membrane separation tank, the membrane is clogged, and thus the membrane filtration flow rate needs to be controlled. On the other hand, if the circulation amount (the amount of liquid fed from the culture reaction tank) is changed in order to control the membrane filtration flow rate, the culture results (sugar yield) will change. Therefore, there is a possibility that continuous culture cannot be stably performed for a long time without lowering the culture results.

Toshihiko Hirao et. Al., Appl. Microbiol. Biotechnol., Applied Microvials and Microbiology, 32, 269-273 (1989)

JP 2007-252367 A

  As described in the background art, in order to provide a method for producing a chemical by a continuous culture method that can be stably performed over a long period of time, the present inventors have conducted intensive research, and as a result, it is important to control the membrane filtration flow rate. However, in order to control the membrane filtration flow rate, it was necessary to change the circulation amount, and the problem that the continuous culture results changed and became unstable by changing the circulation amount was newly generated.

  Accordingly, an object of the present invention is to provide a method and an apparatus capable of stably producing a chemical product by a continuous culture method over a long period of time, in which continuous culture results are hardly changed even when the membrane filtration flow rate is changed. To do.

  As a result of diligent research, the present inventors have conducted continuous culture stably for a long period of time without reducing the culture performance, which was a problem by directly refluxing the unfiltered culture solution into the culture solution stored in the culture reaction tank. The present invention has been completed. Furthermore, analysis of the effect of refluxing unfiltered culture directly into the culture revealed that changes in the oxygen transfer capacity coefficient, one of the factors affecting fermentation performance, could be significantly suppressed. .

That is, the present invention is characterized by any of the following configurations.
(1) A culture solution of microorganisms or cultured cells stored in a culture reaction tank is continuously supplied to the separation membrane while changing the circulation amount, and the filtrate containing chemicals produced by the culture is separated into the separation membrane. When the unfiltered culture solution that has not been filtered through the separation membrane is refluxed to the culture reaction tank and continuously cultured , the oxygen transfer capacity coefficient kLa in the culture reaction tank is determined from the set value. Production of chemicals by continuous culture, wherein the unfiltered culture solution is directly refluxed into the culture solution stored in the culture reaction tank in order to suppress the decrease rate within 30% of the set value. Method.
(2) The method for producing a chemical product by continuous culture according to (1), wherein L-lactic acid or D-lactic acid is recovered as the chemical product.

  According to the present invention, it is possible to perform stable continuous culture that maintains a high productivity of a desired fermentation product over a longer time than before with a simple device, and stably produce chemicals at low cost by continuous culture. It becomes possible.

FIG. 1 is a schematic diagram of a chemical production apparatus using continuous culture according to an embodiment of the present invention. FIG. 2 is a schematic diagram of an apparatus for producing a chemical by continuous culture according to another embodiment of the present invention. FIG. 3 is a schematic perspective view showing an embodiment of a separation membrane element used in the present invention. FIG. 4 is a schematic perspective view showing another embodiment of the separation membrane element used in the present invention. FIG. 5 is a schematic diagram of a conventional chemical production apparatus using continuous culture. FIG. 6 is a diagram showing a physical map of the expression vector pTRS11 for yeast.

  The present invention continuously feeds and circulates a culture solution of microorganisms stored in a culture reaction tank to a separation membrane, collects a filtrate containing chemicals produced by culture on the permeate side of the separation membrane, and When continuous culture is performed by refluxing the unfiltered culture solution that has not been filtered through the membrane to the culture reaction tank, the rate of decrease from the set value of the oxygen transfer capacity coefficient (1 / h) in the culture reaction tank is the set value. The chemical production method is characterized by being controlled within 30% of the above, and this production method can be carried out, for example, by the apparatus shown in FIG.

  The apparatus shown in FIG. 1 basically includes a culture reaction tank 1, a membrane separation tank 12, and a differential pressure control device 3. One or more separation membrane elements 2 are accommodated in a membrane separation tank 12 installed outside the culture reaction tank 1, and the culture reaction tank 1 and the membrane separation tank 12 intervene with a culture solution circulation pump 11. The culture medium circulation pipe 110 is connected so that the culture medium can be circulated. The culture medium circulation pipe 110 includes a liquid feeding port 111 for supplying the culture liquid from the culture reaction tank 1 to the membrane separation tank 12, and a recirculation port 112 for returning the unfiltered culture liquid to the culture reaction tank 1. However, it opens to the position immersed in the culture solution stored by the culture reaction tank.

  In the culture reaction tank 1, the stirrer 5, the temperature controller 10, the pH sensor / control are provided so that the agitation, temperature, pH, dissolved oxygen concentration, liquid volume, etc. of the culture solution can be adjusted to conditions suitable for the culture. A device 9, a pH adjusting solution supply pump 8, a gas supply device 4, a medium supply pump 7, and the like are provided.

  As described above, the separation membrane element 12 is provided inside the membrane separation tank 12 provided outside the culture reaction tank.

  Between the culture reaction tank 1 and the membrane separation tank 12 installed outside the culture reaction tank, a means for circulating the culture solution, for example, a culture solution circulation pump 11 is provided. There are various types of the pump 11 such as a centrifugal pump, a tube pump, and a diaphragm pump. In the present invention, a pump that can calculate the amount of circulating fluid by the output setting of the pump is preferable, and a diaphragm pump is preferable. .

  In the apparatus of FIG. 1, a culture medium containing microorganisms, cultured cells, and nutrients (culture raw materials) used for culture is stored in the culture reaction tank 1. And culture | cultivation and fermentation progress by controlling to predetermined temperature etc., and a chemical product is produced | generated.

  At this time, the culture medium is fed into the culture reaction tank 1 by the culture medium supply pump 7, and the fermentation culture solution in the culture reaction tank 1 is stirred with the stirrer 5 as necessary, or the gas supply device 4 is placed in the culture reaction tank. Supply gas. If necessary, the pH of the culture solution is adjusted by the pH sensor / control device 9 and the pH adjustment solution supply pump 8, and the temperature of the culture solution is adjusted by the temperature controller 10. By doing in this way, highly productive fermentation production can be performed.

  The culture medium in the apparatus is circulated between the culture reaction tank 1 and the membrane separation tank 12 by the culture medium circulation pump 11, and the fermentation product (chemical product) is removed from the system by, for example, periodically operating the membrane separation. Take out. That is, since the culture solution sent to the membrane separation tank is filtered and separated by the separation membrane element 2 into an unfiltered culture solution containing microorganisms and a filtrate containing fermentation products, the filtrate containing the fermentation products on the membrane permeation side By collecting the chemical, the chemical product can be taken out from the apparatus system. On the other hand, the filtered and separated microorganisms remain in the culture reaction system by being refluxed to the culture reaction tank 1 through the culture solution circulation pipe 110, so that the microorganism concentration in the culture reaction system can be maintained high, and the productivity is improved. High fermentation production is possible.

  The filtration / separation by the separation membrane element 2 can be carried out without using any special power by the water head differential pressure between the water surface of the culture reaction tank 1 and the water surface of the membrane separation tank 12, but if necessary, the level The sensor 6 and the differential pressure control device 3 can appropriately adjust the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation broth in the apparatus system. Filtration / separation by the separation membrane element 2 can also be performed by suction filtration with a pump or the like or pressurizing the inside of the apparatus system.

  FIG. 2 shows a schematic perspective view of another apparatus used for carrying out the method for producing a chemical product of the present invention. FIG. 2 is the same as the apparatus of FIG. 1 except that the gas supply device 4 is also installed in the culture solution circulation pipe 110. By providing the gas supply device 4 also in the culture solution circulation pipe 110, the gas can be efficiently circulated through the culture solution circulation pipe 110 and the membrane separation tank 12, and the same is applied to the entire place where the culture and fermentation of the device are performed. It is possible to supply gas at a volume ratio.

  Next, the separation membrane element 2 that is preferably used in the apparatus shown in FIGS. 1 and 2 will be described.

  As the separation membrane element 2, for example, a separation membrane and a separation membrane element disclosed in International Publication No. 2002/064240 pamphlet can be used.

  Specifically, for example, as shown in FIG. 3, the separation membrane element 2 is mainly composed of a separation membrane bundle 13 made of a hollow fiber membrane, an upper resin sealing layer 14, and a lower resin sealing layer 15. The separation membrane bundle 13 is bonded and fixed in a bundle by the upper resin sealing layer 14 and the lower resin sealing layer 15. Adhesion / fixation by the lower resin sealing layer 15 seals the hollow portion of the hollow fiber membrane and has a structure that prevents leakage of the culture solution. On the other hand, the upper resin sealing layer 14 does not seal the hollow portion of the hollow fiber membrane, and has a structure in which a filtrate (that is, a liquid containing a chemical produced by culture) flows through the water collecting pipe 17. The separation membrane element can be installed in the membrane separation tank via the support frame 16. The filtrate that has permeated the outer surface of the hollow fiber membrane constituting the separation membrane bundle 13 and entered the hollow portion passes through the hollow portion and is taken out of the membrane separation tank through 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.

  For example, the separation membrane element 2 may be configured as shown in FIG. The separation membrane element shown in FIG. 4 is configured by arranging a flow path material 19 and a separation membrane 20 in this order on both surfaces of a rigid support plate 18. The support plate 18 has recesses 21 on both sides. The separation membrane 20 filters the culture solution. The flow path member 19 is for efficiently flowing the filtrate that has passed through the separation membrane 20 to the support plate 18. The filtrate that has flowed to the support plate 18 passes through the recess 21 of the support plate 18 and is taken out of the membrane separation tank through 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 members constituting these separation membrane elements are preferably members having resistance to high-pressure steam sterilization operation. If the inside of the membrane separation tank can be sterilized in addition to the culture reaction tank, it is possible to avoid the risk of contamination by undesirable microorganisms during continuous fermentation, and more stable continuous fermentation is possible. Specifically, the member constituting the separation membrane element is preferably one having resistance to the conditions of high-pressure steam sterilization at 121 ° C. for 15 minutes. For example, metals such as stainless steel and aluminum, polyamide resins, fluorine resins, polycarbonate resins, polyacetal resins, polybutylene terephthalate resins, PVDF, modified polyphenylene ether resins, and polysulfone resins can be preferably selected.

  Next, the separation membrane that can be used in the present invention will be described.

  The separation membrane is preferably a porous membrane having separation performance and permeation performance according to the properties of the liquid to be treated and the application. As the porous membrane, it is possible to use a porous membrane made of an inorganic material such as ceramics or an organic material such as a resin, but preferably a porous separation membrane including a porous resin layer. preferable. 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 resin layer may or may not permeate the porous base material, but a membrane permeated into the porous base material is preferably employed in terms of strength.

  The porous substrate supports the porous resin layer and gives strength to the separation membrane, and the material is made of an organic material and / or an inorganic material, among which organic fibers are preferably used. Preferred porous substrates are woven fabrics and nonwoven fabrics made of organic fibers such as cellulose fibers, cellulose triacetate fibers, polyester fibers, polypropylene fibers and polyethylene fibers. Among them, a nonwoven fabric that is relatively easy to control the density, easy to manufacture, and inexpensive is preferably used. The average thickness of the porous substrate is preferably 50 μm or more and 3000 μm or less.

  As described above, the porous resin layer functions as a separation functional layer, and an organic polymer membrane can be preferably used. 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, polyolefin resin, Cellulosic resins and cellulose triacetate resins are exemplified. The organic polymer film may be made of 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, as a membrane material constituting the porous resin layer, a polyvinyl chloride resin, a polyvinylidene fluoride resin, a polysulfone resin, which is easy to form a film with a solution and excellent in physical durability and chemical resistance, A polyethersulfone resin, a polyacrylonitrile resin or a polyolefin resin is preferable, a polyvinylidene fluoride resin or a polyolefin resin is more preferable, and a polyvinylidene fluoride resin or a resin containing the same as the main component is most preferably used.

  As the polyvinylidene fluoride resin, a homopolymer of vinylidene fluoride is preferable, 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. Examples of the polyolefin resin include polyethylene, polypropylene, chlorinated polyethylene, and chlorinated polypropylene, and chlorinated polyethylene is preferably used.

  The shape of the separation membrane used in the present invention is not particularly limited, and specific examples include a flat membrane and a hollow fiber membrane. When the shape of the separation membrane is a flat membrane, the average thickness is preferably 20 μm or more and 5000 μm or less, and more preferably 50 μm or more and 2000 μm or less. When the shape of the separation membrane is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably 200 μm or more and 5000 μm or less, and the film thickness is preferably 20 μm or more and 2000 μm or less. Further, a woven fabric or a knitted fabric in which organic fibers or inorganic fibers are formed in a cylindrical shape may be included in the hollow fiber.

  An outline of a method for producing a porous membrane preferably used as a separation membrane in the present invention will be described as an example.

  First, an outline of a method for producing a flat film among the porous films will be described. The flat membrane is formed on the surface of the porous substrate with a film of a film-forming stock solution containing a resin and a solvent constituting the porous resin layer, and impregnated with the film-forming stock solution in the porous substrate, It is obtained by forming the porous resin layer on the surface of the porous substrate by bringing only the coating-side surface of the porous substrate having the coating into contact with a coagulation bath containing a non-solvent to coagulate the resin.

  Next, an outline of a method for producing a hollow fiber membrane will be described. The hollow fiber membrane discharges a membrane-forming stock solution composed of a resin and a solvent constituting the porous resin layer from the outer tube of the double tube die, and the hollow portion forming fluid is supplied to the inner tube of the double tube die. It is possible to produce by cooling from a cooling bath and solidifying by cooling in a cooling bath.

  Another porous resin layer can be coated (laminated) on the outer surface of the obtained hollow fiber membrane. Such lamination of the porous resin layer can be performed in order to change the properties of the hollow fiber membrane, for example, hydrophilicity / hydrophobicity, pore diameter, and the like into desired properties.

  The porous resin layer laminated on the surface can be prepared 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. The method of lamination is not particularly limited, and the hollow fiber membrane may be immersed in the membrane-forming stock solution, or the membrane-forming stock solution may be applied to the surface of the hollow fiber membrane. The amount of lamination can also be adjusted by scraping off the part or blowing it off using an air knife.

  The hollow fiber membrane produced as described above is formed by adhering and sealing the hollow portion with a member such as a resin, and the flat membrane is installed on a support, so that the separation membrane element shown in FIGS. can do. In the present invention, it is preferable to use a hollow fiber membrane from the viewpoint that it is advantageous in increasing the membrane area per volume.

  In the method for producing a chemical product of the present invention, microorganisms or cultured cells are cultured using the apparatus as described above, and the culture solution is continuously supplied and circulated from the culture reaction tank to the separation membrane, for example, periodically separated. A chemical product contained in the culture solution is extracted by generating a differential pressure between the stock solution side and the permeate side of the membrane and collecting the filtrate containing the chemical product on the permeate side of the separation membrane. During the membrane separation, the unfiltered culture solution that has not been filtered through the separation membrane is refluxed to the culture reaction tank. Moreover, the number of culture apparatuses is not limited as long as continuous culture for producing chemicals can be performed while growing microorganisms or cultured cells. The continuous culture operation is usually preferably performed in a single culture apparatus for management purposes, but a plurality of culture apparatuses can be used because the capacity of the culture apparatus is small. In this case, a fermentation product can be obtained with high productivity by connecting a plurality of culture apparatuses in parallel or in series by piping and performing continuous culture.

  Examples of microorganisms and cultured cells used in the present invention include bacteria, such as yeast, Escherichia coli, and coryneform bacteria, industrially frequently used, filamentous fungi, actinomycetes, animal cells, and insect cells. The microorganisms and cells used may be those isolated from the natural environment, or may be those whose properties have been partially modified by mutation or genetic recombination. Among these microorganisms and cultured cells, it is preferable to select and use those having a high production capacity of the target chemical product. In the present invention, the culture of microorganisms is sometimes referred to as “fermentation” or “fermentation culture”.

  In order to promote the growth of microorganisms or cultured cells for cultivation, microorganisms or cultured cells are continuously grown, and the desired chemicals are produced well. .

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

  When the microorganisms or cultured cells used in the present invention require specific nutrients for growth, the nutrients are added as preparations or natural products containing them.

  Culture raw materials such as nutrients are added as appropriate so as to increase the productivity of the target chemical product. At this time, the raw material composition at the start of the culture may be changed as appropriate.

  In culture, an antifoaming agent is used as necessary.

  In the present invention, the saccharide concentration in the culture solution in the culture reaction tank is preferably maintained at 5 g / l or less. The reason why it is preferable to keep the saccharide concentration at 5 g / l or less is to minimize the loss of saccharide due to withdrawal of the culture solution. On the other hand, the concentration of microorganisms or cultured cells in the culture solution in the culture reaction tank is maintained at a high level as long as the environment of the culture solution is not suitable for the growth of microorganisms or cultured cells and does not increase the rate of death. It is preferable for improving productivity. Good production efficiency can be obtained by maintaining the concentration of the microorganism or cultured cell at, for example, 5 g / l or more as a dry weight.

  In the present invention, the culture solution refers to a solution obtained as a result of growth of microorganisms or cultured cells from the culture raw material.

  Moreover, culture | cultivation of microorganisms or a cultured cell is normally performed in the range of pH4-8, temperature 20-40 degreeC. The pH of the culture solution is usually adjusted to a predetermined value within the pH 4-8 range with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, or the like. If it is necessary to increase the oxygen supply rate, oxygen can be added to the air to keep the oxygen concentration at 21% or higher, or the culture reaction tank is pressurized and cultured, the stirring speed is increased, the aeration rate is increased, etc. Can be used.

  In the method for producing a chemical product of the present invention, batch culture or fed-batch culture may be performed at the initial stage of culture to increase the microorganism concentration, and then continuous culture and withdrawal of the culture solution (membrane separation) may be started. In the method for producing a chemical product of the present invention, after increasing the concentration of microorganisms, high concentration cells may be seeded, and continuous culture and withdrawal of the culture solution may be performed at the start of culture. That is, in the method for producing a chemical product of the present invention, it is possible to supply a culture raw material and extract a culture solution from an appropriate time. The supply timing of the culture raw material and the start time of drawing out the culture solution are not necessarily the same. Moreover, the supply of the culture raw material and the extraction of the culture solution may be continuous or intermittent.

  The extracted culture solution, that is, a solution containing chemicals recovered on the permeation side of the separation membrane is preferably recovered by further concentration, distillation, crystallization and the like to increase the concentration.

  Moreover, in the method for producing a chemical product of the present invention, microorganisms or cultured cells can be extracted from the culture apparatus as necessary. For example, if the microorganism concentration or the cultured cell concentration in the culture apparatus becomes too high, the separation membrane is easily clogged. Therefore, the separation membrane can be prevented from being clogged by pulling out the microorganisms and the cultured cells. In addition, since the production performance of chemicals may change depending on the concentration of microorganisms or cultured cells in the culture apparatus, it is possible to maintain production performance by extracting microorganisms or cultured cells using the production performance as an index. .

  It should be noted that the extracted microorganisms and cultured cells are preferably discarded after sterilization by high-pressure steam sterilization or the like.

  In the present invention, the culture solution is continuously supplied from the culture reaction tank to the separation membrane and circulated. However, in order to improve the production rate of the target chemical product, the extraction rate of the culture solution as shown in formula (1) That is, the filtrate flow rate may be increased. However, if the filtrate flow rate is increased, clogging by microorganisms is likely to occur, and it becomes difficult to stably control the filtration performance for a long period of time. Therefore, in the present invention, by increasing the circulation amount of the culture solution circulating between the culture reaction vessel and the membrane separation vessel, the linear velocity of the culture solution flowing on the surface of the separation membrane in the membrane separation vessel is increased, and the membrane It is preferable to prevent clogging. As a result, it becomes possible to improve the production rate of the target chemical product. The circulation amount means the flow rate of the culture solution fed from the culture reaction tank to the membrane separation tank through the culture solution circulation pipe. The amount of the fermentation broth refers to the sum of the amount of the culture solution in the culture reaction tank 1 and the amount of the culture solution in the culture solution circulation pipe 110 and the membrane separation tank 12.

  In the present invention, when performing the continuous culture as described above, for example, the culture solution circulation pipe 110, the liquid feeding port 111 for supplying the culture solution from the culture reaction tank 1 to the membrane separation tank 12, and the unfiltered culture. The circulation port 112 for refluxing the liquid from the membrane separation tank 12 to the culture reaction tank 1 is opened at a position where it is immersed in the culture solution stored in the culture reaction tank 1, so that the inside of the culture reaction tank 1 The rate of decrease of the oxygen transfer capacity coefficient kLa from the set value is suppressed to a range within 30% of the set value.

  Here, the oxygen transfer capacity coefficient kLa will be described. The oxygen transfer capacity coefficient kLa (hereinafter simply abbreviated as kLa) indicates the ability to transfer dissolved oxygen from the gas phase to the liquid phase per unit time during aeration and agitation culture. 2). In addition, kLa is used in the balance equation of oxygen in the culture medium represented by oxygen supplied from the gas phase and oxygen consumed by microorganisms during the aeration and agitation culture, Equation (3) (Biotechnology Experiment) Book, edited by Japan Society for Biotechnology, Baifukan, p.310 (1992)).

  KLa in the culture reactor can be measured by sulfite oxidation method (batch method / continuous method), gas displacement method (Gassing out method (static method, dynamic method)), exhaust gas analysis method, etc. etc). Below, an example of the measurement of kLa by the gas substitution method (dynamic method) is shown.

  First, water or a medium to be used is placed in the culture reaction tank, and the oxygen in these liquids is replaced with nitrogen gas or deoxygenated by adding sodium sulfite to a nearly saturated concentration. Reduce the oxygen concentration of the liquid. Next, a dissolved oxygen concentration electrode is inserted into the liquid, the aeration speed, the stirring speed, and the temperature are set to arbitrary conditions, and the rising process of the dissolved oxygen under the conditions is measured. Here, since the following formula (4) is derived from the above formula (2), kLa can be obtained by plotting the logarithm of C * -C against the aerated time.

  In the present invention, this measurement is repeated in advance to find kLa (setting value) at which the fermentation titer is maintained at a high level. When a chemical product is produced by continuous culture, the reduction rate corresponds to the setting value. Set the value within 30% of the set value.

  kLa varies depending on various factors. Examples of the element include the amount of the culture solution, the agitation of the culture reaction tank, the aeration amount, and the temperature, and these are relatively easy to control even during continuous culture. In addition, kLa changes due to vibration of the liquid level. For example, when the unfiltered culture medium is circulated to the culture reaction tank 1, if the unfiltered culture liquid is dropped from an upper position away from the culture liquid level in the culture reaction tank 1, the liquid level of the culture liquid is reduced. It vibrates greatly and kLa changes greatly. However, the vibration of the liquid level is difficult to control. Therefore, in the present invention, the culture solution circulation pipe 110 is used to supply the culture solution 111 from the culture reaction tank 1 to the membrane separation tank 12 and to return the unfiltered culture solution to the culture reaction tank 1. When the unfiltered culture liquid is returned to the culture reaction tank 1 through the membrane separation tank 12, the culture reaction is performed. It is preferable to directly reflux into the culture solution stored in the tank 1. By doing so, the liquid level is not greatly disturbed, and as a result, the rate of decrease from the set value of kLa can be suppressed to a range within 30%.

  By culturing while keeping the rate of decrease from the set value of the oxygen transfer capacity coefficient kLa in the culture reaction tank within 30% of the set value, it is possible to culture while maintaining a high fermentation titer. For example, if kLa is reduced by 30% or more from the set value, the anaerobic culture becomes too much under the optimum culture condition, the sugar consumption rate is lowered, the carbon source is excessive, and the production rate of the target chemical is lowered. On the other hand, according to the present invention that suppresses the decrease rate of kLa to within 30%, continuous culture results such as sugar yield are unlikely to change and stable continuous fermentation can be performed. Compared with batch fermentation, chemical products can be obtained at a high volumetric production rate, and extremely efficient chemical products can be produced.

  Here, the production rate of the chemical in the continuous culture is calculated by the following equation (5). Moreover, the sugar yield in continuous culture is calculated by the following formula (6), and the amount of chemical product (g) produced by consuming the raw material carbon source within a certain period is calculated as the amount of input carbon source during that period. It is obtained by dividing by (g).

  Note that the chemical production rate in batch culture is determined by the amount of product (g) at the time when all the raw carbon source is consumed by the time (h) required for consumption of the carbon source and the amount of culture solution (L) at that time. It is obtained by dividing.

  The chemical product in the present invention is not limited as long as it is a substance produced by microorganisms or cultured cells in the culture solution, and can be appropriately selected depending on the microorganisms or cultured cells to be cultured. Specific examples of the chemical products include substances that are desired to be mass-produced in the fermentation industry, such as alcohols and organic acids. For example, alcohol, ethanol, 1,3-propanediol, 1,4-butanediol, glycerol, etc., and organic acid, acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, citric acid, etc. It is. In addition, 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 And amino acids such as L-tyrosine, methionine, serine, valine and leucine, nucleic acids such as inosine and guanosine, diamine compounds such as cadaverine, enzymes, antibiotics and recombinant proteins.

  Next, microorganisms or cultured cells that can be used for the production of the above-described chemical products will be described with reference to specific chemical products.

  The method for producing a chemical product of the present invention can be used for production of L-lactic acid, for example. L-lactic acid is a kind of optical isomer of lactic acid and can be clearly distinguished from D-lactic acid which is an enantiomer thereof. The microorganism or cultured cell used for the production of L-lactic acid is not particularly limited as long as it is a microorganism capable of producing L-lactic acid. For example, lactic acid bacteria can be used. Here, lactic acid bacteria can be defined as prokaryotic microorganisms that produce 50% or more of lactic acid in terms of sugar yield with respect to consumed glucose.

  Preferred lactic acid bacteria include, for example, Lactobacillus genus, Pediococcus genus, Tetragenococcus genus, Carnobacterium genus, Genus Carnobacterium Vus Genus Leuconostoc, Genus Oenococcus, Genus Atopobium, Streptococcus, Genus Enterococcus L, Genus Enterococcus L, Genus Enterococcus L, Genus Enterococcus L ), And lactic acid bacteria belonging to the genus Bacillus (Genus Bacillus). Among them, a lactic acid bacterium having a high yield of lactic acid to saccharide can be selected and preferably used for the production of lactic acid, and among them, a lactic acid bacterium having a high yield of L-lactic acid to saccharide can be selected to produce lactic acid. It can be preferably used.

  Examples of lactic acid bacteria having a high yield of L-lactic acid to sugar include, for example, Lactobacillus yamanashiensis, Lactobacillus animalis, Lactobacillus agilis, Lactobacillus agilis, Lactobacillus aviaries, and Lactobacillus aviaries.・ Lactobacillus casei, Lactobacillus delbruekii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus ruminiacti, Lactobacillus ruminisacti (Lactobacillus sharpeae), Lactococcus dextrinicus, and Lactococcus lactis And the like can be selected and used for the production of L-lactic acid.

  Moreover, when manufacturing L-lactic acid by this invention, the microorganisms or cultured cell which artificially provided or enhanced the lactic acid production ability can be used. For example, it is possible to use microorganisms or cultured cells in which an L-lactic acid dehydrogenase gene (hereinafter sometimes referred to as L-LDH) has been introduced to impart or enhance L-lactic acid production ability. As a method for imparting or enhancing L-lactic acid production ability, a conventionally known method using drug mutation can also be used. More preferably, a recombinant microorganism whose L-lactic acid production ability is enhanced by incorporating L-LDH into the microorganism can be mentioned.

  In the case of producing L-lactic acid, the host of the recombinant microorganism is preferably prokaryotic cells such as Escherichia coli, lactic acid bacteria, or eukaryotic cells such as yeast, and particularly preferably yeast. Among the yeasts, yeast belonging to the genus Saccharomyces is preferable, and Saccharomyces cerevisiae is more preferable.

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

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

  When producing L-lactic acid according to the present invention, a liquid containing chemicals recovered on the permeate side of the separation membrane (hereinafter sometimes referred to as “filtered / separated fermentation liquid”) is further concentrated conventionally. It is preferable to separate and purify by a method such as distillation and crystallization. For example, a method of extracting the collected filtered / separated fermentation broth to 1 or less and then extracting with diethyl ether or ethyl acetate, a method of elution after adsorption washing with an ion exchange resin, or a reaction with alcohol in the presence of an acid catalyst. And a method of distilling as an ester and a method of crystallizing as a calcium salt or a lithium salt. Among these, a method of subjecting the concentrated L-lactic acid solution obtained by evaporating the water of the filtered / separated fermentation broth to a distillation operation is preferable. Here, when distilling the concentrated L-lactic acid solution, it is preferable to distill while supplying water so that the water concentration of the distillation stock solution is constant. After distilling the L-lactic acid aqueous solution, the water can be concentrated by heating and evaporating to obtain purified L-lactic acid having a target concentration. When an L-lactic acid aqueous solution containing a low-boiling component such as ethanol or acetic acid is obtained as the distillate, it is preferable to remove the low-boiling component in the L-lactic acid concentration process. After the distillation operation, impurities can be removed from the distillate by ion exchange resin, activated carbon, chromatographic separation, or the like, if necessary, to obtain higher purity L-lactic acid.

  The method for producing a chemical product of the present invention can also be used for producing D-lactic acid, for example.

  The microorganism or cultured cell that can be used for D-lactic acid production is not limited as long as it is a microorganism that can produce D-lactic acid. For example, a wild type strain has the ability to synthesize D-lactic acid. Examples include microorganisms belonging to the genus Lactobacillus, Bacillus, and Pediococcus.

  Moreover, when manufacturing D-lactic acid by this invention, it is also preferable to use the microorganism of the wild type strain | stump | stock which has enhanced the enzyme activity of D-lactic acid dehydrogenase (it may also be called D-LDH below). As a method for enhancing the enzyme activity, a conventionally known method using drug mutation can also be used. More preferably, a recombinant microorganism in which the enzyme activity of D-lactate dehydrogenase is enhanced by incorporating a gene encoding D-lactate dehydrogenase.

  In the case of producing D-lactic acid, the host of the recombinant microorganism is preferably prokaryotic cells such as Escherichia coli, lactic acid bacteria, or eukaryotic cells such as yeast.

  Genes encoding D-lactate dehydrogenase include Lactobacillus plantarum, Pediococcus acidilactici, and Bacillus lavolactus gene from Bacillus lavolactus It is preferably a gene, and more preferably a gene derived from Bacillus laevolacticus.

  In addition, also when manufacturing D-lactic acid by this invention, it is preferable to isolate | separate and refine | purify D-lactic acid contained in a filtration and isolation | separation fermentation liquid similarly to the case where L-lactic acid described previously is manufactured.

  In the case of producing ethanol according to the present invention, there is no particular limitation as long as it is a microorganism or cultured cell capable of producing pyruvic acid. For example, Genus Saccharomyces, Genus Kluyveromyces, Yeast belonging to the genus Schizosaccharomyces can be used. Of these, Saccharomyces cerevisiae, Kluyveromyces lactis, and Schizosaccharomyces pombe can be preferably used. Further, bacteria belonging to the genus Lactobacillus and Genus Zymomonas can also be preferably used. Among these, Lactobacillus brevis (Lactobacillus brevis) and Zymomonas mobilis (Zymomonas mobilis) can be used conveniently.

  The microorganisms or cultured cells that can be used for ethanol production may be microorganisms or cultured cells that have artificially increased ethanol production ability. Specifically, some of the properties may be modified by mutation or gene recombination. As an example of a partially modified nature, yeast that has acquired the ability to assimilate raw starch by incorporating a fungal glucoamylase gene belonging to the genus Rhizopus can be cited (microorganism, 3: 555-564 (1987)). .

  In the case of producing ethanol according to the present invention, the separation / purification of ethanol contained in the filtered / separated fermentation broth is, for example, a purification method using a distillation method, or concentration using a separation membrane made of NF, RO membrane, or zeolite. -It can carry out suitably by a purification method.

  In the case of producing pyruvic acid according to the present invention, there is no particular limitation as long as it is a microorganism or cultured cell capable of producing pyruvic acid. For example, Genus Pseudomonas, Genus Corynebacterium Bacteria belonging to the genus Escherichia and Genus Acinetobacter can be preferably used. More preferably, bacteria such as Pseudomonas fluorescens, Pseudomonas aeruginosa and Escherichia coli can be used. Those bacteria whose properties are partially modified by mutation or gene recombination may be used. For example, a bacterium in which an ATPase gene directly involved in ATP production by oxidative phosphorylation is mutated or deleted is also preferably used. Molds, yeasts and the like can also be preferably used. For example, molds and yeasts belonging to the genus Saccharomyces, the genus Toluropsis, the genus Candida, the genus Schizophyllum can be used. Further preferably, Saccharomyces Serebise (Saccharomyces cerevisiae), Saccharomyces Kopushisu (Saccharomyces copsis), Candida glabrata (Candida glabrata), Candida lipolytica (Candida lipolytica), Torulopsis glabrata (Toluropusis glabrata), Shizofiriumu-Komune (Schizophyllum commune Pyruvic acid can be produced using fungi such as) and yeast.

  When producing pyruvic acid according to the present invention, separation and purification of pyruvic acid contained in the filtered / separated fermentation broth can be performed by a method using an anion exchange column. For example, a purification method using a weak salt ion exchanger disclosed in JP-A-6-345683 can be suitably used.

  Next, when producing succinic acid according to the present invention, there is no particular limitation as long as it is a microorganism or cultured cell capable of producing succinic acid. For example, the genus Anaerobiospirillum or Actinobacillus ( Bacteria belonging to the genus Actinobacillus can be preferably used. Specifically, Anaerobiospirillum succiniciproducens and Actinobacillus succinogenes disclosed in US Pat. No. 5,143,833 are disclosed by Anaerobiospirillum succiniciproducens and James B. Mckinlay. (Actinobacillus succinogenes) (Appl. Microbiol. Biotechnol. (Applied Microvial and Microbiology), 71, 6651-6656 (2005). Moreover, Corynebacterium genus and Brevibacterium (Brevibacterium) Coryneform bacteria such as genus), Escherichia, etc. are also available, such as Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum and the like are preferable.

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

  In the case of producing succinic acid according to the present invention, an ordinary purification method of succinic acid can be applied to separation / purification of succinic acid contained in the filtered / separated fermentation broth. For example, a purification method combining water-splitting electrodialysis treatment and vacuum concentration / crystallization disclosed in JP-A-2005-333886 can be suitably used.

  When itaconic acid is produced according to the present invention, it is not particularly limited as long as it is a microorganism capable of producing itaconic acid. For example, mold or yeast can be preferably used. More preferably, production of itaconic acid using fungi belonging to the genus Aspergillus or Genus Ustilago, and yeast belonging to the genus Candida and Genus Rhodotorula can be mentioned. Among them, Aspergillus terreus (Aspergillus terreus), Aspergillus Itakonikusu (Aspergillus itaconicus), Usutirago Meidisu (Ustilago maydis), Usutirago synodontis (Ustilago cynodontis), and Usutirago Raven mold Horus Tina (Ustilago rabenhorstina) or Candida Ann talc Tikka (Candia antarctica, ) Can be preferably used for the production of itaconic acid.

  When itaconic acid is produced according to the present invention, itaconic acid contained in the filtered / separated fermentation broth can be preferably separated and purified by ultrafiltration or electrodialysis. For example, an ultrafiltration method disclosed in Japanese Patent Publication No. Sho-50958 and a purification method by electrodialysis using a salt-type cation exchange resin membrane can be suitably used.

  The production of 1,3-propanediol according to the present invention is not particularly limited as long as it is a microorganism or cultured cell capable of producing 1,3-propanediol. And microorganisms belonging to the genus Klebsiella, Clostridium, and Lactobacillus having the ability to synthesize 3-propanediol.

  Then, when producing 1,3-propanediol, the microorganism is (a) at least one gene encoding a polypeptide having glycerol dehydratase activity; (b) at least one gene encoding a glycerol dehydratase reactivation factor. And (c) preferably comprises at least one gene encoding a non-specific catalytic activity that converts 3-hydroxypropionaldehyde to 1,3-propanediol. In the present invention, more preferably, the microorganism is a recombinant microorganism capable of producing 1,3-propanediol.

  As such a recombinant microorganism, when producing 1,3-propanediol, a microorganism having the ability to synthesize 1,3-propanediol from glycerol is preferably Klebsiella, Clostridium, Lactobacillus, Cytrobacter, Enterobacter (Aerobacter), Aerobacter (Aerobacter), Aspergillus (Aspergillus), Saccharomyces (Saccharomyces), Sescharomyces (Saccharomyces) Kluyveromyces (Kluy eromyces, Candida, Hansenula, Debaryomyces, Mucor, Toluropsis, Methylobacter, Salumella (A), Salmonella (A) A recombinant microorganism selected from the group consisting of (Streptomyces), Escherichia and Pseudomonas is preferred, and Escherichia coli is more preferred.

  In addition, it is preferable that the recombinant microorganism be improved so that 1,3-propanediol can be efficiently produced from glucose. The recombinant microorganism comprises, for example, (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; and (b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity. A recombinant microorganism containing a gene is preferred.

 More preferably, the glycerol dehydratase reactivating factor is a recombinant microorganism containing genes encoded by orfX and orfZ isolated from dha regulon. Furthermore, the recombinant microorganism is preferably a recombinant microorganism that lacks glycerol kinase activity and / or glycerol dehydrogenase activity and / or triose phosphate isomerase activity.

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

  When producing cadaverine according to the present invention, there is no particular limitation as long as it is a microorganism or cultured cell capable of producing cadaverine. For example, the enzyme activity of lysine decarboxylase and / or lysine cadaverine antiporter is enhanced. Microorganisms are preferred. More preferably, a recombinant microorganism into which a gene encoding lysine decarboxylase and / or lysine cadaverine antiporter is incorporated. More preferably, it is a recombinant microorganism into which one or more genes encoding lysine decarboxylase are integrated.

  When producing cadaverine, the recombinant microorganism is preferably Escherichia coli or coryneform bacterium, more preferably lysine decarboxylase activity and homoserine auxotrophy or S- (2-aminoethyl) -L-cysteine. It is a coryneform bacterium having at least one characteristic of resistance. Furthermore, it is preferable to lack homoserine dehydrogenase activity. More preferably, it lacks homoserine dehydrogenase activity due to gene insertion mutagenesis. Moreover, it is preferable that the genus of coryneform bacteria is at least one genus selected from the group consisting of the genus Corynebacterium and Brevibacterium. More preferably, it is Corynebacterium glutamicum.

  When producing cadaverine according to the present invention, the separation / purification of cadaverine contained in the filtered / separated fermentation broth can be performed by combining conventionally known methods such as concentration, distillation and crystallization. For example, a purification method using crystallization disclosed in Japanese Patent Application Laid-Open No. 2004-222569 can be suitably used. The cadaverine obtained by the present invention can be made into various polymer raw materials depending on the acid used in the continuous culture. However, a purification method by crystallization is preferably used for polymer raw materials that require good purity. If the pH of the culture solution is maintained with hydrochloric acid, cadaverine dihydrochloride can be recovered from the filtered / separated fermentation broth by a crystallization step. Furthermore, it is also preferred to maintain the pH of the culture solution with dicarboxylic acid during continuous culture and recover cadaverine dicarboxylate from the filtered / separated fermentation solution by a crystallization step. The dicarboxylic acid is an aliphatic and / or aromatic dicarboxylic acid having only two carboxyl groups as functional groups, and further adipic acid, sebacic acid, 1,12-dodecanedicarboxylic acid, succinic acid, It is preferably either isophthalic acid or terephthalic acid.

  Hereinafter, in order to describe the present invention in more detail, L-lactic acid, D-lactic acid, ethanol, pyruvic acid, succinic acid, 1,3-propanediol, itaconic acid, and cadaverine are selected as the chemicals, respectively. A specific embodiment of continuous fermentation using the apparatus shown in FIG. 1 and FIG. 2 using microorganisms or cultured cells capable of producing a chemical product will be described with reference to examples.

Reference Example 1 Production of porous membrane (Part 1)
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 film forming 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 coating stock solution was prepared by mixing and dissolving. This coating 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 following Examples and Comparative Examples.

The average pore diameter of the surface to be treated (outer surface) of the obtained porous membrane was 0.05 μm. Moreover, when the pure water permeability coefficient of this porous membrane was evaluated, it was 5.5 × 10 ⁻⁹ m ³ / m ² · s · Pa. The pure water permeability coefficient was measured at a head height of 1 m using purified water having a temperature of 25 ° C. prepared with a reverse osmosis membrane. The standard deviation of the average pore diameter was 0.006 μm.

(Reference Example 2) Production of porous membrane (part 2)
Polyvinylidene fluoride (15.9 wt%), lithium chloride (0.9 wt%), water (3.7 wt%) and polyvinylpyrrolidone (2.3 wt%) were added to the solvent dimethylacetamide (DMAC) (77.2 wt%) and mixed. A film-forming stock solution was prepared by sufficiently dissolving. Next, after the obtained film-forming stock solution was cooled to a temperature of 25 ° C., it was applied to a non-woven fabric made of polyester fiber (porous substrate) that had been previously pasted on a glass plate, and the relative humidity was 80%. The porous membrane was left in air for 2 minutes and immediately immersed in a warm water bath at a temperature of 50 ° C. for 5 minutes to obtain a porous membrane having a porous resin layer formed on the porous substrate.

The surface of the porous resin layer of the obtained porous membrane was observed with a scanning electron microscope at a magnification of 1,500 times. The average diameter of all the pores that can be observed was 2.0 μm. Further, as a result of evaluating the pure water permeability coefficient of this porous membrane, it was 3.0 × 10 ⁻⁶ m ³ / m ² · s · Pa. The pure water permeability coefficient was measured at a head height of 1 m using purified water having a temperature of 25 ° C. prepared with a reverse osmosis membrane.

Reference Example 3 Production of Yeast Strain Having L-Lactic Acid Production Ability As a yeast having a lactic acid production ability, the Xenopus-derived ldh gene having the base sequence described in SEQ ID NO: 1 is the PDC1 gene, SED1 gene, and TDH3 gene Yeast introduced into the locus was made. The ldh gene derived from Xenopus laevis was cloned by PCR. For PCR, phagemid DNA prepared from Xenopus kidney cDNA library (manufactured by STRATAGENE) according to the attached protocol was used as a template.

  For the PCR amplification reaction, KOD-Plus polymerase (manufactured by Toyobo Co., Ltd.) was used, and the attached reaction buffer, dNTPmix, etc. were used. A 50 μl reaction system was prepared so that the phagemid DNA prepared as described above was 50 ng / sample, the primer was 50 pmol / sample, and the KOD-Plus polymerase was 1 unit / sample. The reaction solution was thermally denatured at 94 ° C. for 5 minutes with a PCR amplification apparatus iCycler (manufactured by BIO-RAD), then 94 ° C. (thermal denature): 30 seconds, 55 ° C. (primer annealing): 30 seconds, 68 C. (Complementary strand extension): One cycle was performed for 30 cycles, and then cooled to a temperature of 4.degree. The gene amplification primer (SEQ ID NO: 2 and 3) was prepared such that a SalI recognition sequence was added to the 5 terminal side and a NotI recognition sequence was added to the 3 terminal side.

  The PCR amplified fragment was purified, the end was phosphorylated with T4 polynucleotide Kinase (manufactured by Takara Bio Inc.), and then ligated to the pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver.2 (Takara Bio Inc.). The ligation solution was transformed into competent cells of Escherichia coli DH5α (manufactured by Takara Bio Inc.) and plated on an LB plate containing 50 μg / mL of the antibiotic ampicillin and cultured overnight. For the grown colonies, plasmid DNA was recovered with a miniprep, cut with restriction enzymes SalI and NotI, and a plasmid into which the Xenopus-derived ldh gene was inserted was selected. All of these series of operations were performed according to the attached protocol.

  The pUC118 vector into which the Xenopus laevis ldh gene was inserted was cleaved with restriction enzymes SalI and NotI, the DNA fragments were separated by 1% agarose gel electrophoresis, and the fragment containing the Xenopus laevis ldh gene was purified according to a conventional method. The fragment containing the obtained ldh gene was ligated to the XhoI / NotI cleavage site of the expression vector pTRS11 shown in FIG. 6, the plasmid DNA was recovered in the same manner as described above, and cleaved with the restriction enzymes XhoI and NotI. An expression vector into which the Xenopus laevis ldh gene was inserted was selected. Hereinafter, an expression vector incorporating the Xenopus-derived ldh gene prepared in this manner is referred to as pTRS102.

  A 1.3 kb PCR fragment containing the Xenopus laevis ldh gene and the TDH3 terminator sequence was amplified by PCR using pTRS102 as an amplification template and oligonucleotides (SEQ ID NOs: 4 and 5) as primer sets. Here, SEQ ID NO: 4 was designed such that a sequence corresponding to 60 bp upstream from the start codon of the PDC1 gene was added.

  Next, a 1.2 kb PCR fragment containing the TRP1 gene, a yeast selection marker, was amplified by PCR using plasmid pRS424 as an amplification template and oligonucleotides (SEQ ID NOs: 6 and 7) as primer sets. Here, SEQ ID NO: 7 was designed such that a sequence corresponding to 60 bp downstream from the stop codon of the PDC1 gene was added.

  Each DNA fragment was separated by 1% agarose gel electrophoresis and purified according to a conventional method. A mixture of each 1.3 kb fragment and 1.2 kb fragment obtained as described above was used as an amplification template, and PCR was performed using oligonucleotides (SEQ ID NOs: 4 and 7) as primer sets. A PCR fragment of about 2.5 kb to which a sequence corresponding to 60 bp upstream and downstream of the gene was added and to which the Xenopus laevis ldh gene, TDH3 terminator and TRP1 gene were linked was amplified.

  The above PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, transformed into yeast Saccharomyces cerevisiae NBRC10505, and cultured in a medium lacking tryptophan. A transformant introduced downstream of the PDC1 gene promoter was selected.

  The transformant obtained as described above was confirmed to be a yeast in which the Xenopus-derived ldh gene was introduced downstream of the PDC1 gene promoter on the chromosome as follows. First, the genomic DNA of the transformant was prepared using a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.), and this was used as an amplification template, and by PCR using oligonucleotides (SEQ ID NOs: 7 and 8) as primer sets, This was confirmed by obtaining an amplified DNA fragment of 2.8 kb. For non-transformed strains, an amplified DNA fragment of about 2.1 kb can be obtained by the PCR. Hereinafter, a transformant in which the above-mentioned Xenopus-derived ldh gene is introduced downstream of the PDC1 gene promoter on the chromosome is referred to as B2 strain. The upstream and downstream sequences of the PDC1 gene can be obtained from Saccharomyces Genome Database (URL: http://www.yeastgenome.org/).

  Subsequently, the ldh gene described in SEQ ID NO: 1 was introduced into the BED strain at the SED1 locus. The introduction into the SED1 gene locus includes the ldh gene derived from Xenopus and the TDH3 terminator sequence by PCR using the pTRS102 prepared in Reference Example 1 as an amplification template and the oligonucleotide (SEQ ID NOs: 5 and 9) as a primer set. A .3 kb PCR fragment was amplified. Here, SEQ ID NO: 9 was designed such that a sequence corresponding to 60 bp upstream from the start codon of the SED1 gene was added.

  Next, a PCR fragment of about 1.3 kb containing the HIS3 gene, which is a yeast selection marker, was amplified by PCR using plasmid pRS423 as an amplification template and oligonucleotides (SEQ ID NOs: 6, 10) as primer sets. Here, SEQ ID NO: 10 was designed so that a sequence corresponding to 60 bp downstream from the stop codon of the SED1 gene was added.

  Each DNA fragment was separated by 1% agarose gel electrophoresis and purified according to a conventional method. A mixture of the two kinds of about 1.3 kb fragments obtained here was used as an amplification template, and PCR was performed using oligonucleotides (SEQ ID NOs: 9 and 10) as primer sets. An about 2.6 kb PCR fragment to which sequences corresponding to upstream and downstream 60 bp were added and to which Xenopus laevis ldh gene, TDH3 terminator and HIS3 gene were linked was amplified.

  The above PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, transformed into the B2 strain, and cultured in a medium not added with histidine, whereby the Xenopus-derived ldh gene was transformed into the SED1 gene promoter on the chromosome. The transformant introduced downstream of was selected.

  Confirmation that the transformant obtained as described above was a yeast in which the Xenopus laevis ldh gene was introduced downstream of the SED1 gene promoter on the chromosome was performed as follows. First, genomic DNA of the transformed strain was prepared by a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.), and this was used as an amplification template, and by PCR using oligonucleotides (SEQ ID NOs: 11 and 12) as primer sets, This was confirmed by obtaining an amplified DNA fragment of 2.9 kb. For non-transformed strains, an amplified DNA fragment of about 1.4 kb can be obtained by the PCR. Hereinafter, a transformant in which the above-mentioned Xenopus-derived ldh gene is introduced downstream of the SED1 gene promoter on the chromosome is referred to as SU014-I strain.

  Subsequently, the ldh gene described in SEQ ID NO: 1 was introduced into SU014-I at the TDH3 locus. For introduction into the TDH3 locus, a plasmid in which the TDH3 terminator of pTRS102 was changed to an ADH1 terminator was prepared.

  First, genomic DNA was extracted from the NBRC10505 strain using a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.), by using the extracted genomic DNA as a template and PCR using oligonucleotides (SEQ ID NOs: 13 and 14) as primer sets, A PCR fragment containing the ADH1 promoter was amplified. Here, SEQ ID NO: 13 was prepared such that a NotI recognition sequence was added to the 5 terminal side, and a HindIII recognition sequence was added to SEQ ID NO: 14 on the 3 terminal side.

  The PCR amplified fragment was purified, the end was phosphorylated with T4 polynucleotide Kinase (manufactured by Takara Bio Inc.), and then ligated to the pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). The ligation solution was transformed into competent cells of Escherichia coli DH5α (manufactured by Takara Bio Inc.) and plated on an LB plate containing 50 μg / mL of the antibiotic ampicillin and cultured overnight. For the grown colonies, plasmid DNA was collected with a miniprep, cut with restriction enzymes NotI and HindIII, and a plasmid in which the ADH1 terminator was inserted was selected. The prepared plasmid is designated as pUC118-ADH1t.

  Next, pUC118-ADH1t was cleaved with restriction enzymes NotI and HindIII, DNA fragments were separated by 1% agarose gel electrophoresis, and a fragment containing ADH1 terminator was purified according to a routine method. The obtained fragment containing the ADH1 terminator was ligated to the NotI / HindIII cleavage site of pTRS102, and plasmid DNA was recovered by the same method as described above, and then cleaved with the restriction enzymes NotI and HindIII. Modified plasmids were selected. Hereinafter, the plasmid thus prepared is designated as pTRS150.

  A 1.3 kb PCR fragment containing the frog-derived L-ldh gene and the ADH1 terminator sequence was amplified by PCR using pTRS150 as a template and oligonucleotides (SEQ ID NOs: 15 and 16) as primer sets. Here, the primer of SEQ ID NO: 16 was designed such that a sequence corresponding to 60 bp upstream from the start codon of the TDH3 gene was added.

  Next, a 1.2 kb PCR fragment containing the URA3 gene, a yeast selection marker, was amplified by PCR using plasmid pRS426 as an amplification template and oligonucleotides (SEQ ID NOs: 17 and 18) as primer sets. Here, the primer of SEQ ID NO: 18 was designed such that a sequence corresponding to 60 bp downstream from the stop codon of the TDH3 gene was added.

  Each PCR fragment was separated by 1% agarose gel electrophoresis and purified according to a conventional method. A mixture of each 1.3 kb fragment and 1.2 kb fragment obtained here was used as an amplification template, and by PCR using oligonucleotides (SEQ ID NOs: 16 and 18) as primer sets, a frog-derived L-ldh gene, An approximately 2.5 kb PCR fragment to which the ADH1 terminator and URA3 gene were linked was amplified.

  The above PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, transformed into SU014-I strain, and cultured in a uracil-free medium, so that the frog-derived L-ldh gene was located on the chromosome. A transformant introduced downstream of the TDH3 gene promoter was selected.

  Confirmation that the transformant obtained as described above was a yeast in which the frog-derived L-ldh gene was introduced downstream of the TDH3 gene promoter on the chromosome was performed as follows. First, the genomic DNA of the transformant was prepared using a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.), and this was used as an amplification template, and by PCR using oligonucleotides (SEQ ID NOs: 19 and 20) as primer sets, This was confirmed by obtaining an amplified DNA fragment of 2.8 kb. For non-transformed strains, an amplified DNA fragment of about 2.1 kb can be obtained by the PCR. Hereinafter, a transformant in which the frog-derived L-ldh gene is introduced downstream of the TDH3 gene promoter on the chromosome is referred to as SU014-II strain.

  Next, the yeast SW015 strain having a temperature-sensitive mutation in the pdc5 gene and the obtained SU014-II strain described in International Publication WO2007 / 097260 were joined to obtain diploid cells. The diploid cells were allowed to form ascending with an ascending medium. The ascending sac was dissected with a micromanipulator to obtain each haploid cell, and the auxotrophy of each haploid cell was examined. From the obtained haploid cells, a strain having an xenopus-derived ldh gene inserted into the pdc1, sed1 and tdh3 loci and having a temperature-sensitive mutation in the pdc5 gene (not viable at 34 ° C.) is designated as MATa. The mating type of MATα and MATα was selected. Among the obtained yeast strains, a strain having a mating type of MATa was designated as SU014-8A strain, and a strain having a mating type of MATα was designated as SU014-4B strain.

(Reference Example 4) Preparation of lysine auxotrophic return strain The lysine auxotrophy of the SU014-8A strain obtained in Reference Example 3 was restored. A PCR fragment of about 2 kb in the first half of the LYS2 gene was amplified by PCR using BY4741 genomic DNA manufactured by Funakoshi as a template and oligonucleotides (SEQ ID NOS: 21 and 22) as primer sets. The PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, transformed into SU014-8A strain, and the amber mutation of lys2 gene was released. By culturing in a lysine-free medium, a transformant having restored lysine synthesis ability was selected.

  Confirmation that the transformant obtained as described above was a yeast from which the amber mutation of the lys2 gene was released was performed as follows. First, the obtained transformant and 20GY77 strain having a wild-type LYS2 gene were joined to obtain diploid cells. The diploid cells were allowed to form ascending with an ascending medium. The ascending sac was dissected with a micromanipulator to obtain each haploid cell, and the auxotrophy of each haploid cell was examined. All of the obtained haploid cells were confirmed to have lysine synthesis ability. In addition, when the lys2 mutation is not released and lysine synthesis ability is restored, cells having no lysine synthesis ability among the haploid cells obtained above are obtained. Hereinafter, the strain in which the lysine synthesis ability of the SU014-8A strain has been restored is referred to as HI001.

(Reference example 5) Acquisition of lysine auxotrophic return strain The leucine requirement of the SU014-3B strain obtained in Reference Example 3 was recovered. About 2 kb of the PCR fragment of the LEU2 gene was amplified by PCR using PRS425 as a template and oligonucleotides (SEQ ID NOs: 23, 24) as primer sets. The above PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, transformed into SU014-3B strain, and the mutation of leu2 gene was released. By culturing in a leucine-free medium, a transformant in which the ability to synthesize leucine was restored was selected.

  Confirmation that the transformant obtained as described above was a yeast in which the mutation of the leu2 gene was canceled was performed as follows. First, the obtained transformant and 20GY7 strain having a wild type LEU2 gene were joined to obtain diploid cells. The diploid cells were allowed to form ascending with an ascending medium. The ascending sac was dissected with a micromanipulator to obtain each haploid cell, and the auxotrophy of each haploid cell was examined. All of the obtained haploid cells were confirmed to have the ability to synthesize leucine. When the leucine gene mutation is not released and leucine synthesis ability is restored, cells that do not have leucine synthesis ability are obtained from the haploid cells obtained above. Hereinafter, a strain in which the leucine synthesis ability of the SU014-3B strain has been restored is referred to as HI002.

(Reference Example 6) Acquisition of a diploid prototrophic strain The HI001 strain and the HI002 strain obtained in Reference Examples 4 and 5 were joined to obtain a diploid prototrophic strain having no auxotrophy. The obtained strain is hereinafter referred to as HI003.

(Reference Example 7) Acquisition of a diploid auxotrophic strain The SU014-8A strain and the SU014-3B strain obtained in Reference Example 3 were joined to obtain a diploid auxotrophic strain having auxotrophy. . The obtained strain is designated SU014.

Reference Example 8 Quantification of Medium Components such as Lactic Acid Lactic acid was confirmed by measuring the amount of lactic acid by the HPLC method under the following conditions for the centrifugal supernatant of the filtrate.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: electrical conductivity Temperature: 45 ° C.

  The concentration of L-lactic acid and D-lactic acid was measured by the HPLC method under the following conditions.

Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation)
Mobile phase: 1 mM aqueous copper sulfate flow rate: 1.0 ml / min
Detection method: UV254 nm
Temperature: 30 ° C.

  Moreover, the optical purity of L-lactic acid was calculated by the following formula.

Optical purity (%) = 100 × (LD) / (L + D)
Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.

  Moreover, the measurement of the sucrose, glucose, and fructose concentrations was confirmed by measuring the centrifugal supernatant of the filtrate by the HPLC method under the following conditions.

Column: Asahipak NH2P-50 4E (manufactured by SHODEX)
Mobile phase: 75% acetonitrile solution Detection method: Differential refractive index Temperature: 30 ° C
Flow rate: 0.6 ml / min.

(Comparative example 1) Batch culture using a diploid prototrophic strain The most typical batch fermentation was performed as a fermentation form using microorganisms, and L-lactic acid productivity was evaluated. That is, a batch fermentation test using only the culture reaction tank 1 of the continuous culture apparatus of FIG. As shown in Table 1, raw sugar (crude sugar) and the like were used for the yeast lactic acid fermentation medium. As the raw sugar, eucalyptus (trade name) from Muso Co., Ltd. was used. The medium was used after autoclaving at 121 ° C. for 15 minutes. As the microorganism, the yeast HI003 strain prepared in the above Reference Example 6 was used, and the HPLC method shown in the above Reference Example 8 was used for the concentration of the product (lactic acid) in the filtrate and the quantification of the filter sugar.

  The culture conditions are shown below.

・ Culture reactor capacity: 1.5 (L)
・ Temperature adjustment: 32 (℃)
-Aeration volume of culture reaction tank: 0.1 (L / min)
-Stirring speed of culture reaction tank: 400 (rpm)
・ PH adjustment: pH adjusted to 5 with 5N Ca (OH) ₂・ Culture reaction tank is autoclaved at 121 ° C. for 20 min under high pressure steam sterilization.

  Specifically, first, the HI003 strain was cultured with shaking in a 5 ml lactic acid fermentation medium overnight in a test tube (pre-culture). The culture solution was inoculated into 100 ml of fresh lactic acid fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). The preculture was inoculated into 1.5 L of lactic acid fermentation medium in the continuous culture apparatus of FIG. 1, and the culture reaction tank 1 was stirred with the attached stirrer 5 and aerated. Temperature adjustment and pH adjustment were performed, and batch fermentation culture was performed without operating the culture medium circulation pump 11. The results of batch fermentation are shown in Table 2. The lactic acid to sugar yield was 80%. Moreover, kLa at the time of this culture was 7.0.

(Comparative example 2) Production of L-lactic acid by continuous fermentation using an external circulation device Yeast HI003 strain prepared in the above Reference Example 6 was used as a microorganism for producing L-lactic acid, and yeast having the composition shown in Table 3 was used as a medium. Using a lactic acid fermentation medium, L-lactic acid was produced by the continuous culture apparatus of FIG. In the apparatus shown in FIG. 5, the separation membrane element 2 is installed outside the culture reaction tank 1, and the culture reaction liquid is transferred from the culture reaction tank 1 to the membrane separation tank 12 of the culture liquid circulation pipe 110. And a reflux port 112 for refluxing the unfiltered culture solution from the membrane separation tank 12 to the culture reaction tank 1, and the liquid feed port 111 is connected to the culture reaction tank 1. It is opened at a position where it is immersed in the stored culture solution, and the reflux port 112 is opened at a position where it is not immersed in the culture solution stored in the culture reaction tank 1, and the reflux port 112 is immersed in the culture solution. The continuous culture apparatus of FIG. 1 is the same as that of FIG. As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  The medium was used after autoclaving (121 ° C., 15 minutes). The HPLC shown in Reference Example 8 was used for the concentration of the product (lactic acid) in the filtrate and the quantification of saccharides.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 60 square cm
Temperature adjustment: 32 (℃)
Aeration volume of culture reaction tank: 0.1 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 5 with 5N Ca (OH) ₂ Fermentation medium supply rate: 50-300 ml / hr. Variable control within the range of circulating fluid volume of culture fluid: 0.1 (L / min)
Filtrate flow rate control: Flow rate control by transmembrane differential pressure (transmembrane differential pressure: controlled at 0.1 kPa to less than 20 kPa)
Specifically, first, the HI003 strain was shaken and cultured overnight in a 5 ml lactic acid fermentation medium in a test tube to obtain a culture solution (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh lactic acid fermentation medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L lactic acid fermentation medium of the continuous culture apparatus shown in FIG. 5 and the culture reaction tank 1 is stirred by the attached stirrer 5 to adjust the aeration amount of the culture reaction tank 1 and the temperature. The culture medium circulation pump 11 was operated (0.1 L / min) and the culture was performed for 24 hours (preculture). Immediately after the completion of the pre-culture, the lactic acid fermentation medium is continuously supplied, and the fermentation culture volume of the continuous culture apparatus is 2 L (1.5 L for the culture reaction tank, 0.5 L for the culture circulation pipe and the membrane separation tank) Thus, continuous culture was performed while controlling the filtrate flow rate, and L-lactic acid was produced by continuous fermentation. Control of the amount of filtrate when performing the continuous fermentation test was performed by appropriately changing the pressure difference control device 3. The L-lactic acid concentration and residual sugar concentration in the filtrate were measured as appropriate.

  Table 4 shows the results of the 800-hour continuous fermentation test. By performing fermentation using the continuous culture apparatus shown in FIG. 5 while performing external circulation, the yield of lactic acid versus sugar was slightly lower than in Comparative Example 1 of batch fermentation. The lactic acid to sugar yield was 73%. Moreover, kLa at the time of this culture was 9.1.

(Comparative Example 3) Change in filtration flow rate when the amount of circulation is changed To improve the production rate, increase the extraction rate of the culture solution, that is, the filtrate flow rate as shown in the above formulas (1) and (5). Improve. However, if the filtrate flow rate is increased, clogging by microorganisms is likely to occur, and it becomes difficult to stably control the filtration performance for a long period of time.

  Therefore, by increasing the circulation amount of the culture solution between the culture reaction tank and the membrane separation tank, a crossflow flow rate is generated, and the membrane surface is washed, so that clogging by microorganisms is less likely to occur, and the filtrate flow rate is increased. Whether or not the filtration performance can be maintained stably was confirmed under the following operating conditions.

  Production of L-lactic acid using the yeast lactic acid fermentation medium having the composition shown in Table 3 as a culture medium using the yeast HI003 strain prepared in Reference Example 6 as a microorganism for producing L-lactic acid, and the continuous culture apparatus of FIG. Went. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 60 square cm
Temperature adjustment: 32 (℃)
Aeration volume of culture reaction tank: 0.1 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 5 with 5N Ca (OH) ₂ Circulating fluid volume in culture medium circulation: variable control within the range of 0.25 to 1 (L / min) (200 hours after the start of continuous fermentation: circulating fluid volume 0. 25L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: circulating fluid volume 1 L / min).

  Specifically, first, the HI003 strain was shaken and cultured overnight in a 5 ml lactic acid fermentation medium in a test tube to obtain a culture solution (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh lactic acid fermentation medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L lactic acid fermentation medium of the continuous culture apparatus shown in FIG. 5 and the culture reaction tank 1 is stirred by the attached stirrer 5 to adjust the aeration amount of the culture reaction tank 1 and the temperature. The culture medium circulation pump 11 was operated (0.1 L / min) and the culture was performed for 24 hours (preculture). Immediately after the completion of the pre-culture, the lactic acid fermentation medium is continuously supplied, and the continuous culture apparatus is continuously cultivated while controlling the flow rate of the filtrate so that the amount of the fermentation broth is 2 L, and L-lactic acid is produced by continuous fermentation. It was. The circulation rate was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. Table 5 shows the results of a 600 hour continuous fermentation test. It became clear that the filtrate flow rate was improved by increasing the circulation rate. Moreover, as a result of measuring kLa for every circulation amount, kLa became 7.0 same as the time of batch culture at the time of operation of 0.25 L / min. Further, kLa was 3.1 at the time of operation of 0.5 L / min, and kLa was 1.9 at the time of operation of 1.0 L / min.

(Example 1) Production of L-lactic acid by continuous fermentation using yeast (part 1)
Production of L-lactic acid using the yeast lactic acid fermentation medium having the composition shown in Table 3 as a medium using the yeast HI003 strain prepared in Reference Example 6 as a microorganism producing L-lactic acid, and the continuous culture apparatus of FIG. Went. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The HPLC shown in Reference Example 8 was used for the concentration of lactic acid as a product and the quantification of residual saccharides.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 60 square cm
Temperature adjustment: 32 (℃)
Aeration volume of culture reaction tank: 0.1 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: Adjusted to pH 5 with 5N NaOH Circulating fluid volume: Variable control in the range of 0.25 to 1 (L / min) (200 hours after starting continuous fermentation: Circulating fluid volume 0.25 L / min)
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the HI003 strain was cultured with shaking in a test tube overnight in 5 ml of yeast lactic acid fermentation medium (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh yeast lactic acid fermentation medium, and cultured with shaking at 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L yeast fermentation medium of the continuous culture apparatus shown in FIG. 1, the culture reaction tank 1 is stirred by the attached stirrer 5, the aeration rate of the culture reaction tank 1 is adjusted, and the temperature Adjustment and pH adjustment were performed, the culture fluid circulation pump 11 was operated (circulating fluid amount: 0.25 L / min), and culture was performed for 24 hours (preculture). Immediately after completion of the pre-culture, the yeast lactic acid fermentation medium is continuously supplied, and the continuous culture apparatus is continuously cultivated while controlling the flow rate of the filtrate so that the amount of the fermentation broth is 2 L, thereby producing L-lactic acid by continuous fermentation. went. The circulation rate was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. The L-lactic acid concentration and residual glucose concentration in the filtrate were measured as appropriate.

  Table 6 shows the results of a 600 hour continuous fermentation test. kLa started when the culture was started under the condition of 7.0 (set value), where the yield to sugar was high in batch culture, but decreased when the circulation rate was increased, but the decrease rate was within 30% of the set value. It was. By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 1, it was possible to produce L-lactic acid by continuous fermentation in a stable yield.

  The reduction rate of kLa is calculated by dividing the difference between the set value of kLa and the kLa value at a certain point by the set value and multiplying that value by 100.

(Example 2) Production of L-lactic acid by continuous fermentation using yeast (part 2)
An L-lactic acid continuous fermentation test was conducted under the same conditions as in Example 1 except that the porous membrane prepared in Reference Example 2 was used as the separation membrane, and the separation membrane element shown in FIG. The results are shown in Table 6. kLa started when the culture was started under the condition of 7.0 (set value), where the yield to sugar was high in batch culture, but decreased when the circulation rate was increased, but the decrease rate was within 30% of the set value. It was. By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 1, it was possible to produce L-lactic acid by continuous fermentation in a stable yield.
(Example 3) Production of L-lactic acid by continuous fermentation using yeast (part 3)
Production of L-lactic acid using the yeast lactic acid fermentation medium having the composition shown in Table 3 as a culture medium using the yeast HI003 strain prepared in Reference Example 6 as a microorganism producing L-lactic acid, and the continuous culture apparatus of FIG. Went. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The HPLC shown in Reference Example 8 was used for the concentration of lactic acid as a product and the quantification of residual saccharides.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 60 square cm
Temperature adjustment: 32 (℃)
Aeration volume of culture reaction tank: 0.1 (L / min)
Aeration volume of culture medium circulation pipe: 0.03 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: Adjusted to pH 5 with 5N NaOH Circulating fluid volume: Variable control in the range of 0.25 to 1 (L / min) (200 hours after starting continuous fermentation: Circulating fluid volume 0.25 L / min)
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the HI003 strain was cultured with shaking in a test tube overnight in 5 ml of yeast lactic acid fermentation medium (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh yeast lactic acid fermentation medium, and cultured with shaking at 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L yeast fermentation medium of the continuous culture apparatus shown in FIG. 2 and the culture reaction tank 1 is stirred with the attached stirrer 5 to adjust the aeration amount of the culture reaction tank 1 and the temperature. Adjustment and pH adjustment are performed, and the culture solution circulation piping part is aerated so as to have the same aeration amount (volume ratio) as the culture reaction tank, and the culture solution circulation pump 11 is operated (circulation fluid amount: 0.25 L / min). And cultured for 24 hours (pre-culture). Immediately after completion of the pre-culture, the yeast lactic acid fermentation medium is continuously supplied, and the continuous culture apparatus is continuously cultivated while controlling the flow rate of the filtrate so that the amount of the fermentation broth is 2 L, thereby producing L-lactic acid by continuous fermentation. went. The circulation rate was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. The produced L-lactic acid concentration and residual glucose concentration in the filtrate were measured as appropriate.

  Table 6 shows the results of a 600 hour continuous fermentation test. kLa was cultured under the condition of 7.0 (set value), where the yield to sugar was high in batch culture, and was almost constant even when the circulation rate was increased, and the rate of decrease was 30% of the set value. Was within. According to the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 2, stable production of L-lactic acid at a high yield was possible.

Comparative Example 4 Production of L-lactic acid by continuous fermentation using yeast (part 4)
An L-lactic acid continuous fermentation test was conducted in the same manner as in Example 1 except that the continuous culture apparatus of FIG. 5 in which the circulation port of the culture solution circulation pipe was opened at a position not immersed in the culture solution in the culture reaction tank was used. . The L-lactic acid concentration and residual glucose concentration in the filtrate were measured as appropriate. Further, Table 6 shows the yield of L-lactic acid versus sugar calculated from the L-lactic acid and glucose concentrations. kLa decreased when the amount of circulation was increased when the culture was started under the condition of 7.0 (set value), which yielded high sugar yield in batch culture, and the decrease rate was 30% or more of the set value. .

(Comparative Example 5) Production of L-lactic acid by continuous fermentation using yeast (part 5)
It was carried out in the same manner as in Example 2 except that the continuous culture apparatus of FIG. 5 in which the circulation port of the culture solution circulation pipe was opened at a position not immersed in the culture solution in the culture reaction tank. The results are shown in Table 6. kLa decreased when the amount of circulation was increased when the culture was started under the condition of 7.0 (set value), which yielded high sugar yield in batch culture, and the decrease rate was 30% or more of the set value. .

  Looking at the results shown in Table 6 (Comparative Example 4 and Comparative Example 5), the continuous culture apparatus (FIG. 5) in which the circulation port of the culture solution circulation pipe is opened at a position not immersed in the culture solution in the culture reaction tank. When culturing was carried out while changing the amount of circulation, the yield to sugar, which is one of the continuous culture results, changed and decreased as the amount of circulation increased. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG. On the other hand, in the continuous culture apparatus (FIGS. 1 and 2) in which the circulation port of the culture solution circulation pipe is opened at a position where it is immersed in the culture solution in the culture reaction tank, the continuous culture results can be obtained even if the circulation rate is changed. It was possible to operate stably for a long time without changing the yield to sugar. Furthermore, by using the continuous culture apparatus of FIG. 2 to ventilate the culture solution circulation piping portion at the same volume ratio as in the culture reaction tank, it was possible to perform culture for a long period of time with a higher yield.

(Example 4) Production of ethanol by continuous fermentation (part 1)
The NBRC10505 strain was used as the microorganism producing ethanol, and the ethanol fermentation medium having the composition shown in Table 7 was used as the medium, and ethanol was produced using the continuous culture apparatus shown in FIG. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The concentration of ethanol as a product was evaluated by gas chromatography. Shimadzu GC-2010 Capillary GC TC-1 (GL science) 15 meter L. * 0.53 mm ID, df1.5 μm was used to detect and calculate with a flame ionization detector and evaluated. The HPLC shown in Reference Example 8 was used for quantification of the residual saccharide.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 32 (℃)
Aeration volume of culture reaction tank: 0.1 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 5 with 5N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the NBRC10505 strain was cultured with shaking in a 5 ml ethanol fermentation medium overnight in a test tube (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh ethanol fermentation medium, and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 24 hours (pre-culture). The culture solution is inoculated in a 1.5 L ethanol fermentation medium of the continuous culture apparatus shown in FIG. 1 and the culture reaction tank 1 is stirred by the attached stirrer 5 to adjust the aeration amount of the culture reaction tank 1 and the temperature. Adjustment and pH adjustment were performed, the culture fluid circulation pump 11 was operated (circulating fluid amount: 0.25 L / min), and culture was performed for 24 hours (preculture). Immediately after completion of the preculture, the ethanol fermentation medium was continuously supplied, and the culture was continuously performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus was 2 L, and ethanol was produced by continuous fermentation. The reflux amount was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 8 shows the results of measuring the ethanol concentration and residual glucose concentration produced in the filtrate as appropriate.

  By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 1, stable production of ethanol by continuous fermentation was possible.

(Example 5) Production of ethanol by continuous fermentation (part 2)
NBRC10505 strain was used as a microorganism producing ethanol, and an ethanol fermentation medium having the composition shown in Table 7 was used as a medium, and ethanol was produced using the continuous culture apparatus shown in FIG. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The concentration of ethanol as a product was evaluated by gas chromatography. The HPLC shown in Reference Example 8 was used for quantification of the residual saccharide.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 32 (℃)
Aeration volume of culture reaction tank: 0.1 (L / min)
Aeration volume of culture medium circulation pipe: 0.03 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 5 with 5N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the NBRC10505 strain was cultured with shaking in a 5 ml ethanol fermentation medium overnight in a test tube (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh ethanol fermentation medium, and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 24 hours (pre-culture). The culture solution is inoculated in a 1.5 L ethanol fermentation medium of the continuous culture apparatus shown in FIG. 2 and the culture reaction tank 1 is stirred by the attached stirrer 5 to adjust the aeration amount of the culture reaction tank 1 and the temperature. Adjustment and pH adjustment are performed, and the culture solution circulation piping part is aerated so as to have the same aeration amount (volume ratio) as the culture reaction tank, and the culture solution circulation pump 11 is operated (circulation fluid amount: 0.25 L / min). And cultured for 24 hours (pre-culture). Immediately after completion of the preculture, the ethanol fermentation medium was continuously supplied, and the culture was continuously performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus was 2 L, and ethanol was produced by continuous fermentation. The reflux amount was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. The results are shown in Table 8.

  By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 2, stable production of ethanol by continuous fermentation was possible.

(Comparative Example 6) Production of ethanol by continuous fermentation (part 3)
The same procedure as in Example 4 was performed except that the continuous culture apparatus of FIG. 5 was used in which the reflux port of the culture solution circulation pipe was opened at a position where it was not immersed in the culture solution in the culture reaction tank.

  The results are shown in Table 8, and the yield to sugar, which is one of the continuous culture results, was changed and decreased by changing the circulation amount. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG.

  By the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIG. 1 and FIG. 2, the continuous culture results did not change even if the circulation amount was changed, and it was possible to operate stably for a long time.

(Example 6) Production of pyruvic acid by continuous fermentation (part 1)
The production of pyruvic acid is performed using the continuous culture apparatus of FIG. 1, using Toluropsis glabrata strain P120-5a (FERM P-16745) as the microorganism producing pyruvic acid, and using the pyruvic acid fermentation medium having the composition shown in Table 9 as the medium. went. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  The concentration of pyruvic acid as a product was measured by the HPLC method under the following conditions. Further, the HPLC shown in Reference Example 8 was used for quantification of the residual saccharide.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: electrical conductivity Temperature: 45 ° C.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 30 (° C)
Aeration volume of culture reaction tank: 1.5 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 5.5 with 4N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the P120-5a strain was cultured with shaking in a 5 ml pyruvic acid fermentation medium overnight in a test tube (pre-culture). The obtained culture broth was inoculated into 100 ml of a fresh pyruvate fermentation medium, and cultured with shaking at 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). Before inoculation of the culture solution into 1.5 L pyruvic acid fermentation medium of the continuous culture apparatus shown in FIG. 1, the culture reaction tank 1 is stirred by the attached stirrer 5, and the aeration amount of the culture reaction tank 1 is adjusted. Temperature adjustment and pH adjustment were performed, the culture solution circulation pump 11 was operated (circulation solution amount: 0.25 L / min), and culture was performed for 24 hours (pre-culture). Immediately after completion of the pre-culture, the pyruvic acid fermentation medium is continuously supplied, and the continuous culture is performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture device is 2 L. It was. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 10 shows the results of appropriately measuring the concentration of pyruvic acid produced and the residual glucose concentration in the filtrate.

By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 1, stable production of pyruvic acid by continuous fermentation was possible.
(Example 7) Production of pyruvic acid by continuous fermentation (part 2)
The production of pyruvic acid is performed using the continuous culture apparatus of FIG. 2, using Toluropsis glabrata strain P120-5a (FERM P-16745) as the microorganism producing pyruvic acid, and using the pyruvic acid fermentation medium having the composition shown in Table 9 as the medium. went. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  The concentration of pyruvic acid as a product was measured by the HPLC method under the following conditions. Further, the HPLC shown in Reference Example 8 was used for quantification of the residual saccharide.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: electrical conductivity Temperature: 45 ° C.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 30 (° C)
Aeration volume of culture reaction tank: 1.5 (L / min)
Aeration volume of culture solution circulation pipe: 0.5 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 5.5 with 4N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the P120-5a strain was cultured with shaking in a 5 ml pyruvic acid fermentation medium overnight in a test tube (pre-culture). The obtained culture broth was inoculated into 100 ml of a fresh pyruvate fermentation medium, and cultured with shaking at 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The incubating medium is inoculated into a 1.5 L pyruvic acid fermentation medium of the continuous culture apparatus shown in FIG. 2, the culture reaction tank 1 is stirred with the attached stirrer 5, and the aeration rate of the culture reaction tank 1 is adjusted. Temperature adjustment and pH adjustment are performed, and the culture solution circulation piping part is aerated so as to have the same aeration amount (volume ratio) as the culture reaction tank, and the culture solution circulation pump 11 is operated (circulation fluid amount: 0.25 L / min) for 24 hours (pre-culture). Immediately after completion of the pre-culture, the pyruvic acid fermentation medium is continuously supplied, and the continuous culture is performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture device is 2 L. It was. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 10 shows the results of appropriately measuring the concentration of pyruvic acid produced and the residual glucose concentration in the filtrate.

  By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 2, stable production of pyruvic acid by continuous fermentation was possible.

(Comparative Example 7) Production of pyruvic acid by continuous fermentation (part 3)
The same procedure as in Example 6 was performed except that the continuous culture apparatus of FIG. 5 in which the circulation port of the culture solution circulation pipe was opened at a position where it was not immersed in the culture solution in the culture reaction tank.

  The results are shown in Table 10. The yield to sugar, which is one of the continuous culture results, was changed and decreased by changing the circulation amount. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG.

  By the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIG. 1 and FIG. 2, the continuous culture results did not change even if the circulation amount was changed, and it was possible to operate stably for a long time.

(Example 8) Continuous production of succinic acid by continuous fermentation (part 1)
As a microorganism for producing succinic acid, Anaerobiospirillum succiniciproducens ATCC53488 strain is used, and a succinic acid fermentation medium having the composition shown in Table 11 is used as a medium, and succinic acid is produced by a continuous culture apparatus shown in FIG. Was manufactured. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  Unless otherwise specified, succinic acid and glucose in the production of succinic acid were measured by the following methods.

Succinic acid was analyzed by HPLC (Shimadzu LC10A, RI monitor: RID-10A, column: Aminex HPX-87H) for the supernatant of the culture broth. The column temperature was 50 ° C. and the column was equilibrated with 0.01N H ₂ SO ₄ , and then the sample was injected and analyzed by eluting with 0.01N H ₂ SO ₄ . Glucose was measured using a glucose sensor (BF-4, Oji Scientific Instruments).

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 39 (℃)
Culture reactor CO ₂ aeration rate: 10 (mL / min)
O ₂ ventilation rate: 1 (mL / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 6.4 with 2M Na ₂ CO ₃ Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

Specifically, first, the fermentation medium is put in a 125 mL Erlenmeyer flask and sterilized by heating. Then, 1 mL of 30 mM Na ₂ CO ₃ and 0.15 mL of 180 mM H ₂ SO ₄ are added in an anaerobic glove box, and further 0.1. After adding 0.5 mL of a reducing solution consisting of 25 g / L cysteine · HCl and 0.25 g / L Na ₂ S, the strain ATCC53488 was inoculated and left to stand overnight at 39 ° C. (pre-culture). Thereafter, after adding 5 mL of a reducing solution composed of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S · 9H ₂ O to 1.5 L of succinic acid fermentation medium in the continuous culture apparatus shown in FIG. Before inoculation of 50 mL of the culture solution, the culture reaction tank 1 is agitated by the attached agitator 5, the aeration amount of the culture reaction tank 1 is adjusted, the temperature is adjusted, and the pH is adjusted, and the culture solution circulation pump 11 is operated ( Circulating fluid amount: 0.25 L / min), and cultured for 24 hours (pre-culture).

  Immediately after completion of the pre-culture, the succinic acid fermentation medium was continuously supplied, and the continuous culture was performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus was 2 L, and succinic acid was produced by continuous fermentation. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 12 shows the results of measuring the succinic acid concentration and residual glucose concentration produced in the filtrate as appropriate.

  Stable production of succinic acid by continuous fermentation was possible by the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG.

(Example 9) Continuous production of succinic acid by continuous fermentation (part 2)
As a microorganism for producing succinic acid, Anaerobiospirillum succiniciproducens ATCC53488 strain is used, and a succinic acid fermentation medium having the composition shown in Table 12 is used as a medium, and succinic acid is produced by a continuous culture apparatus shown in FIG. Was manufactured. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  Unless otherwise specified, succinic acid and glucose in the production of succinic acid were measured by the following methods.

Succinic acid was analyzed by HPLC (Shimadzu LC10A, RI monitor: RID-10A, column: Aminex HPX-87H) for the supernatant of the culture broth. The column temperature was 50 ° C. and the column was equilibrated with 0.01N H ₂ SO ₄ , and then the sample was injected and analyzed by eluting with 0.01N H ₂ SO ₄ . Glucose was measured using a glucose sensor (BF-4, Oji Scientific Instruments).

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 39 (℃)
Culture reactor CO ₂ aeration rate: 10 (mL / min)
O ₂ ventilation rate: 1 (mL / min)
Aeration volume of culture solution circulation pipe: 0.03 (mL / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 6.4 with 2M Na ₂ CO ₃ Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

Specifically, first, the fermentation medium is put in a 125 mL Erlenmeyer flask and sterilized by heating. Then, 1 mL of 30 mM Na ₂ CO ₃ and 0.15 mL of 180 mM H ₂ SO ₄ are added in an anaerobic glove box, and further 0.1. After adding 0.5 mL of a reducing solution consisting of 25 g / L cysteine · HCl and 0.25 g / L Na ₂ S, the strain ATCC53488 was inoculated and left to stand overnight at 39 ° C. (pre-culture).

Then, after adding 5 mL of a reducing solution consisting of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S · 9H ₂ O to 1.5 L of succinic acid fermentation medium in the continuous culture apparatus shown in FIG. Before inoculation of 50 mL of the culture solution, the culture reaction tank 1 is agitated by the attached agitator 5, the aeration amount of the culture reaction tank 1 is adjusted, the temperature is adjusted, and the pH is adjusted. Aeration was performed so that the aeration volume (volume ratio) was the same as that in the culture reaction tank, the culture medium circulation pump 11 was operated (circulation volume: 0.25 L / min), and culture was performed for 24 hours (preculture).

  Immediately after completion of the pre-culture, the succinic acid fermentation medium was continuously supplied, and the continuous culture was performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus was 2 L, and succinic acid was produced by continuous fermentation. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 12 shows the results of measuring the succinic acid concentration and residual glucose concentration produced in the filtrate as appropriate.

  By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 2, stable production of succinic acid by continuous fermentation was possible.

(Comparative Example 8) Production of succinic acid by continuous fermentation (part 3)
The same procedure as in Example 8 was performed, except that the continuous culture apparatus of FIG. 5 was used in which the circulation port of the culture solution circulation pipe was opened in a position where the culture reaction vessel was not immersed in the culture solution.

  The results are shown in Table 12, and the yield to sugar, which is one of the continuous culture results, was changed and decreased by changing the circulation amount. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG.

  By the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIG. 1 and FIG. 2, the continuous culture results did not change even if the circulation amount was changed, and it was possible to operate stably for a long time.

(Example 10) Production of 1,3-propanediol by continuous fermentation (part 1)
As a microorganism producing 1,3-propanediol, Klebsiella pneumoniae ATCC 25955 strain is used, and a 1,3-propanediol production medium having the composition shown in Table 13 is used as a medium. Propanediol was produced. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  The measurement of 1,3-propanediol as a product was performed as follows. 30 μL of concentrated (70% v / v) perchloric acid was added to 1.0 mL of the culture supernatant, and after mixing, the sample was lyophilized. A 1: 1 mixture of bis (trimethylsilyl) trifluoroacetamide: pyridine (300 μL) was added to the lyophilized material, mixed vigorously and placed at 65 ° C. for 1 hour. The sample was clarified by removing insoluble material by centrifugation. The resulting liquid was divided into two phases, and the upper phase was used for analysis. Samples were analyzed by chromatography on a DB-5 column (48 m, ID 0.25 mm, film thickness 0.25 μm; from J & W Scientific). In addition, glucose test Wako C (Wako Pure Chemical Industries) was used for measuring the glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 37 (℃)
Culture reactor N ₂ aeration rate: 0.6 (L / min)
O ₂ ventilation rate: 0.05 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: pH 7.0 adjusted with 5N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, Klebsiella pneumoniae ATCC 25955 strain was cultured overnight in a test tube with 5 ml of 1,3-propanediol production medium (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh 1,3-propanediol production medium, and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 24 hours (pre-culture). The culture solution was inoculated in a 1.5 L 1,3-propanediol production medium of the continuous culture apparatus shown in FIG. 1 and the culture reaction tank 1 was stirred with the attached stirrer 5. Aeration volume adjustment, temperature adjustment and pH adjustment were performed, the culture fluid circulation pump 11 was operated (circulation fluid volume: 0.25 L / min), and culture was performed for 24 hours (pre-culture). Immediately after completion of the preculture, 1,3-propanediol production medium (glycerol concentration is 100 g / L) is continuously supplied, and continuous culture is performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus becomes 2 L. Then, 1,3-propanediol was produced by continuous fermentation. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 14 shows the results of measurement of the produced 1,3-propanediol concentration and residual glucose concentration in the filtrate as appropriate.

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

(Example 11) Production of 1,3-propanediol by continuous fermentation (part 2)
As a microorganism producing 1,3-propanediol, Klebsiella pneumoniae ATCC 25955 strain is used, and a 1,3-propanediol production medium having the composition shown in Table 13 is used as a medium. Propanediol was produced. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  The measurement of 1,3-propanediol as a product was performed as follows. 30 μL of concentrated (70% v / v) perchloric acid was added to 1.0 mL of the culture supernatant, and after mixing, the sample was lyophilized. A 1: 1 mixture of bis (trimethylsilyl) trifluoroacetamide: pyridine (300 μL) was added to the lyophilized material, mixed vigorously and placed at 65 ° C. for 1 hour. The sample was clarified by removing insoluble material by centrifugation. The resulting liquid was divided into two phases, and the upper phase was used for analysis. Samples were analyzed by chromatography on a DB-5 column (48 m, ID 0.25 mm, film thickness 0.25 μm; from J & W Scientific). In addition, glucose test Wako C (Wako Pure Chemical Industries) was used for measuring the glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 37 (℃)
Culture reactor N ₂ aeration rate: 0.6 (L / min)
O ₂ ventilation rate: 0.05 (L / min)
Culture solution circulation pipe aeration rate: 0.017 L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: pH 7.0 adjusted with 5N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, Klebsiella pneumoniae ATCC 25955 strain was cultured overnight in a test tube with 5 ml of 1,3-propanediol production medium (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh 1,3-propanediol production medium, and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 24 hours (pre-culture). The culture solution was inoculated in a 1.5 L 1,3-propanediol production medium of the continuous culture apparatus shown in FIG. 1 and the culture reaction tank 1 was stirred with the attached stirrer 5. Adjust the aeration volume, adjust the temperature, adjust the pH, and ventilate the culture fluid circulation piping to the same aeration volume (volume ratio) as the culture reaction tank, and operate the culture fluid circulation pump 11 (circulation fluid volume) : 0.25 L / min) and cultured for 24 hours (pre-culture). Immediately after completion of the preculture, 1,3-propanediol production medium (glycerol concentration is 100 g / L) is continuously supplied, and continuous culture is performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus becomes 2 L. Then, 1,3-propanediol was produced by continuous fermentation. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 14 shows the results of measurement of the produced 1,3-propanediol concentration and residual glucose concentration in the filtrate as appropriate.

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

(Comparative Example 9) Production of 1,3-propanediol by continuous fermentation (part 3)
The same procedure as in Example 10 was performed except that the continuous culture apparatus of FIG. 5 was used in which the reflux port of the culture solution circulation pipe was opened at a position where it was not immersed in the culture solution in the culture reaction tank.

  The results are shown in Table 14, and the sugar yield, which is one of the continuous culture results, was changed and decreased by changing the circulating amount. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG.

  By the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIG. 1 and FIG. 2, the continuous culture results did not change even if the circulation amount was changed, and it was possible to operate stably for a long time.

(Example 12) Production of itaconic acid by continuous fermentation (part 1)
Itaconic acid was produced by the continuous culture apparatus shown in FIG. 1 using Aspergillus terreus ATCC 10020 strain as a microorganism producing itaconic acid and using an itaconic acid fermentation medium having the composition shown in Table 15 as a medium. The medium was used after high-pressure steam sterilization (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The concentration of the product itaconic acid was measured by the method of Koppeshaar (Kyoritsu Shuppan Co., Ltd., Microbiological Optics Course Vol. 5, “Must Utilization Industry” p72-73, published in 1951). Moreover, glucose test Wako C (Wako Pure Chemical Industries) was used for the measurement of glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 35 (℃)
Aeration volume of culture reaction tank: 1.5 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 5.0 with 4N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the Aspergillus terreus ATCC 10020 strain was cultured in a test tube with shaking overnight in 5 ml of the preculture medium shown in Table 15 (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh preculture medium, and cultured with shaking in a 500 ml Sakaguchi flask at a temperature of 35 ° C. for 48 hours (pre-culture). Pre-culture medium is inoculated into a 1.5 L continuous / batch fermentation medium of the continuous culture apparatus shown in FIG. 1, the culture reaction tank 1 is stirred with the attached stirrer 5, and the aeration volume of the culture reaction tank 1 is adjusted. Then, temperature adjustment and pH adjustment were performed, and the culture solution circulation pump 11 was operated (circulation solution amount: 0.25 L / min), and cultured for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the itaconic acid fermentation medium was continuously supplied, and the continuous flow was controlled while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus was 2 L, and itaconic acid was produced by continuous fermentation. . The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 16 shows the results of appropriately measuring the concentration of itaconic acid produced and the residual glucose concentration in the filtrate.

  By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 1, stable production of itaconic acid by continuous fermentation was possible.

(Example 13) Production of itaconic acid by continuous fermentation (part 2)
Itaconic acid was produced by the continuous culture apparatus shown in FIG. 2 using Aspergillus terreus ATCC 10020 strain as a microorganism producing itaconic acid and using an itaconic acid fermentation medium having the composition shown in Table 15 as a medium. The medium was used after high-pressure steam sterilization (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  The concentration of the product itaconic acid was measured by the method of Koppeshaar (Kyoritsu Shuppan Co., Ltd., Microbiological Optics Course Vol. 5, “Must Utilization Industry” p72-73, published in 1951). Moreover, glucose test Wako C (Wako Pure Chemical Industries) was used for the measurement of glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 35 (℃)
Aeration volume of culture reaction tank: 1.5 (L / min)
Culture solution circulation pipe aeration rate: 0.5L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 5.0 with 4N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the Aspergillus terreus ATCC 10020 strain was cultured in a test tube with shaking overnight in 5 ml of the preculture medium shown in Table 15 (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh preculture medium, and cultured with shaking in a 500 ml Sakaguchi flask at a temperature of 35 ° C. for 48 hours (pre-culture). The culture solution is inoculated in a 1.5 L continuous / batch fermentation medium of the continuous culture apparatus shown in FIG. 2, the culture reaction tank 1 is stirred by the attached stirrer 5, and the aeration amount of the culture reaction tank 1 is adjusted. Then, temperature adjustment and pH adjustment are performed, and the culture solution circulation piping part is aerated so as to have the same aeration amount (volume ratio) as the culture reaction tank, and the culture solution circulation pump 11 is operated (circulation fluid amount: 0.25 L). / Min) and cultured for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the itaconic acid fermentation medium was continuously supplied, and the continuous flow was controlled while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus was 2 L, and itaconic acid was produced by continuous fermentation. . The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 16 shows the results of appropriately measuring the concentration of itaconic acid produced and the residual glucose concentration in the filtrate.

Comparative Example 10 Production of Itaconic Acid by Continuous Fermentation (Part 3)
The continuous culture apparatus of FIG. 5 in which the reflux port of the culture solution circulation pipe was opened at a position where it was not immersed in the culture solution in the culture reaction tank, was used in the same manner as in Example 12.

  The results are shown in Table 16, and the yield to sugar, which is one of the continuous culture results, was changed and decreased by changing the circulation amount. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG.

  By the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIG. 1 and FIG. 2, the continuous culture results did not change even if the circulation amount was changed, and it was possible to operate stably for a long time.

(Example 14) Production of cadaverine by continuous fermentation (part 1)
As a microorganism for producing cadaverine, the Corynebacterium glutamicum TR-CAD1 strain described in JP-A No. 2004-22269 is used, and a cadaverine production medium having the composition shown in Table 17 is used as the medium, and the continuous culture apparatus shown in FIG. Cadaverine was produced. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  Cadaverine was evaluated by the HPLC method shown below.

Column used: CAPCELL PAK C18 (Shiseido)
Mobile phase: 0.1% (w / w) phosphoric acid aqueous solution: acetonitrile = 4.5: 5.5
Detection: UV360nm
Sample pretreatment: 25 μl of analytical sample was 25 μl of 1,4-diaminobutane (0.03 M), 150 μl of sodium hydrogen carbonate (0.075 M) and 2,4-dinitrofluorobenzene (0.2 M) as internal standards. The ethanol solution is added and mixed, and kept at a temperature of 37 ° C. for 1 hour. After dissolving 50 μl of the above reaction solution in 1 ml acetonitrile, 10 μl of the supernatant after centrifugation at 10,000 rpm for 5 minutes was analyzed by HPLC. In addition, glucose test Wako C (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measuring the glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 30 (° C)
Aeration volume of culture reaction tank: 1.5 (L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: pH 7.0 adjusted with 3M HCl and 3M aqueous ammonia Fermentation medium feed rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, Corynebacterium glutamicum TR-CAD1 strain was cultured with shaking overnight in a test tube with addition of cadaverine fermentation medium supplemented with 5 ml of kanamycin (25 μg / ml) (pre-culture). The obtained culture broth was inoculated into 50 ml of cadaverine production medium supplemented with fresh kanamycin (25 μg / ml), and placed in a 500 ml Sakaguchi flask for 24 hours at a temperature of 30 ° C. with an amplitude of 30 cm and 180 rpm. Culture was performed (pre-culture). The culture solution is inoculated in a 1.5 L cadaverine fermentation medium of the continuous culture apparatus shown in FIG. 1 and the culture reaction tank 1 is stirred by the attached stirrer 5 to adjust the aeration amount of the culture reaction tank 1 and the temperature. Adjustment and pH adjustment were performed, the culture fluid circulation pump 11 was operated (circulating fluid amount: 0.25 L / min), and culture was performed for 24 hours (preculture).

  Immediately after the completion of the pre-culture, the cadaverine fermentation medium was continuously supplied, and the culture was continuously performed while controlling the filtrate flow rate so that the amount of the fermentation broth in the continuous culture apparatus was 2 L, and cadaverine was produced by continuous fermentation. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 18 shows the results of measuring the cadaverine concentration and residual glucose concentration produced in the filtrate as appropriate.

  By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 1, stable production of cadaverine by continuous fermentation was possible.

(Example 15) Production of cadaverine by continuous fermentation (part 2)
As a microorganism for producing cadaverine, the Corynebacterium glutamicum TR-CAD1 strain described in JP-A No. 2004-22269 is used, and a cadaverine production medium having the composition shown in Table 17 is used as the medium, and the continuous culture apparatus shown in FIG. Cadaverine was produced. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG.

  Cadaverine was evaluated by the HPLC method shown below.

Column used: CAPCELL PAK C18 (Shiseido)
Mobile phase: 0.1% (w / w) phosphoric acid aqueous solution: acetonitrile = 4.5: 5.5
Detection: UV360nm
Sample pretreatment: 25 μl of analytical sample was 25 μl of 1,4-diaminobutane (0.03 M), 150 μl of sodium hydrogen carbonate (0.075 M) and 2,4-dinitrofluorobenzene (0.2 M) as internal standards. The ethanol solution is added and mixed, and kept at a temperature of 37 ° C. for 1 hour. After dissolving 50 μl of the above reaction solution in 1 ml acetonitrile, 10 μl of the supernatant after centrifugation at 10,000 rpm for 5 minutes was analyzed by HPLC. In addition, glucose test Wako C (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measuring the glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 30 (° C)
Aeration volume of culture reaction tank: 1.5 (L / min)
Culture solution circulation pipe aeration rate: 0.5L / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: pH 7.0 adjusted with 3M HCl and 3M aqueous ammonia Fermentation medium feed rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, Corynebacterium glutamicum TR-CAD1 strain was cultured with shaking overnight in a test tube with addition of cadaverine fermentation medium supplemented with 5 ml of kanamycin (25 μg / ml) (pre-culture). The obtained culture broth was inoculated into 50 ml of cadaverine production medium supplemented with fresh kanamycin (25 μg / ml), and placed in a 500 ml Sakaguchi flask for 24 hours at a temperature of 30 ° C. with an amplitude of 30 cm and 180 rpm. Culture was performed (pre-culture). The culture solution is inoculated in a 1.5 L cadaverine fermentation medium in the continuous culture apparatus shown in FIG. 2, the culture reaction tank 1 is stirred by the attached stirrer 5, the aeration amount of the culture reaction tank 1 is adjusted, and the temperature Adjustment and pH adjustment are performed, and the culture solution circulation piping part is aerated so as to have the same aeration amount (volume ratio) as the culture reaction tank, and the culture solution circulation pump 11 is operated (circulation fluid amount: 0.25 L / min). And cultured for 24 hours (pre-culture).

  Immediately after the completion of the pre-culture, the cadaverine fermentation medium was continuously supplied, and the culture was continuously performed while controlling the filtrate flow rate so that the amount of the fermentation broth in the continuous culture apparatus was 2 L, and cadaverine was produced by continuous fermentation. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 18 shows the results of measuring the cadaverine concentration and residual glucose concentration produced in the filtrate as appropriate.

(Comparative Example 11) Production of cadaverine by continuous fermentation (part 3)
The same procedure as in Example 14 was performed except that the continuous culture apparatus of FIG.

  The results are shown in Table 18, and the yield to sugar, which is one of the continuous culture results, was changed and decreased by changing the circulating amount. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG.

  By the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIG. 1 and FIG. 2, the continuous culture results did not change even if the circulation amount was changed, and it was possible to operate stably for a long time.

(Example 16) Production of L-lactic acid by continuous fermentation using lactic acid bacteria (part 1)
As a microorganism producing L-lactic acid, Lactococcus lactis JCM7638 strain is used, and a lactic acid bacteria lactic acid fermentation medium having the composition shown in Table 19 is used as a medium, and the production of L-lactic acid is performed by the continuous culture apparatus shown in FIG. went. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The concentration of L-lactic acid as a product was evaluated by the HPLC method shown in Reference Example 8. Moreover, glucose test Wako C (Wako Pure Chemical Industries) was used for the measurement of glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 37 (℃)
Culture reactor N ₂ aeration rate: 50 (mL / min)
O ₂ ventilation rate: 5 (mL / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 6.5 with 8N aqueous ammonia fermentation medium feed rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the Lactococcus lactis JCM7638 strain was statically cultured at a temperature of 37 ° C. for 24 hours in a lactic acid fermentation medium purged with 5 ml of nitrogen gas shown in Table 20 in a test tube (pre-culture). . The obtained culture solution was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 37 ° C. for 48 hours (pre-culture). The culture solution is inoculated in a 1.5 L lactic acid fermentation medium purged with nitrogen gas from the continuous culture apparatus shown in FIG. 1, the culture reaction tank 1 is stirred by the attached stirrer 5, and the culture reaction tank 1 is vented. After adjusting the amount, adjusting the temperature, and adjusting the pH, the culture fluid circulation pump 11 was operated (circulation fluid amount: 0.25 L / min) and cultured for 24 hours (preculture). Immediately after completion of the pre-culture, the lactic acid fermentation medium was continuously supplied, and the culture was continuously cultivated while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture device was 2 L, and L-lactic acid was produced by continuous fermentation. . The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 20 shows the results of measuring the L-lactic acid concentration and residual glucose concentration produced in the filtrate as appropriate.

  With the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 1, stable production of L-lactic acid by continuous fermentation was possible.

(Example 17) Production of L-lactic acid by continuous fermentation using lactic acid bacteria (part 2)
As a microorganism producing L-lactic acid, Lactococcus lactis JCM7638 strain is used, and a lactic acid bacteria lactic acid fermentation medium having the composition shown in Table 19 is used as a medium, and the production of L-lactic acid is performed by the continuous culture apparatus shown in FIG. went. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The concentration of L-lactic acid as a product was evaluated by the HPLC method shown in Reference Example 8. Moreover, glucose test Wako C (Wako Pure Chemical Industries) was used for the measurement of glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 37 (℃)
Culture reactor N ₂ aeration rate: 50 (mL / min)
O ₂ ventilation rate: 5 (mL / min)
Aeration volume of culture solution circulation pipe: 1.7 (mL / min)
Cultivation speed of culture reaction tank: 400 (rpm)
pH adjustment: adjusted to pH 6.5 with 8N aqueous ammonia fermentation medium feed rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, the Lactococcus lactis JCM7638 strain was statically cultured at a temperature of 37 ° C. for 24 hours in a lactic acid fermentation medium purged with 5 ml of nitrogen gas shown in Table 20 in a test tube (pre-culture). . The obtained culture solution was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 37 ° C. for 48 hours (pre-culture). The culture solution is inoculated in a 1.5 L lactic acid fermentation medium purged with nitrogen gas from the continuous culture apparatus shown in FIG. 1, the culture reaction tank 1 is stirred by the attached stirrer 5, and the culture reaction tank 1 is vented. Adjust the amount, adjust the temperature, adjust the pH, and ventilate the culture fluid circulation piping to the same aeration volume (volume ratio) as the culture reaction tank, and operate the culture fluid circulation pump 11 (circulation fluid volume: 0.25 L / min) and cultured for 24 hours (pre-culture). Immediately after completion of the pre-culture, the lactic acid fermentation medium was continuously supplied, and the culture was continuously cultivated while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture device was 2 L, and L-lactic acid was produced by continuous fermentation. . The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 20 shows the results of measuring the L-lactic acid concentration and residual glucose concentration produced in the filtrate as appropriate.

Comparative Example 12 Production of L-lactic acid by continuous fermentation using lactic acid bacteria (part 3)
The continuous culture apparatus of FIG. 5 in which the reflux port of the culture solution circulation pipe was opened at a position where it was not immersed in the culture solution in the culture reaction tank, was used in the same manner as in Example 16.

  The results are shown in Table 20, and the yield to sugar, which is one of the continuous culture results, was changed and decreased by changing the circulation amount. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG.

  By the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIG. 1 and FIG. 2, the continuous culture results did not change even if the circulation amount was changed, and it was possible to operate stably for a long time.

(Example 18) Production of D-lactic acid by continuous fermentation using lactic acid bacteria (part 1)
As a microorganism that produces D-lactic acid, Sporactobacillus laevolacticus ATCC23492 strain is used, and a D-lactic acid production medium having the composition shown in Table 21 is used as a medium. Was manufactured. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The concentration of the product D-lactic acid was measured by the HPLC method shown in Reference Example 8. In addition, glucose test Wako C (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measuring the glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 37 (℃)
Culture reactor N ₂ aeration rate: 0.2 (L / min)
O ₂ ventilation rate: 2 (mL / min)
Membrane separation tank aeration rate: none Culture reaction tank stirring speed: 400 (rpm)
pH adjustment: adjusted to pH 6.0 with 8N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variably controlled (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, Sporolactobacillus laevolacticus ATCC23492 strain was cultured overnight under anaerobic conditions in a test tube using 10 ml of lactic acid fermentation medium (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh D-lactic acid production medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask under anaerobic conditions (pre-culture). The culture solution is inoculated in a 1.5 L D-lactic acid production medium of the continuous culture apparatus shown in FIG. 1 and the culture reaction tank 1 is stirred by the attached stirrer 5 to adjust the aeration amount of the culture reaction tank 1. Then, temperature adjustment and pH adjustment were performed, and the culture solution circulation pump 11 was operated (circulation solution amount: 0.25 L / min), and cultured for 24 hours (pre-culture).

  Immediately after the completion of the pre-culture, the D-lactic acid production medium is continuously supplied, and the continuous culture is performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus becomes 2 L. went. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 22 shows the results of measuring the concentration of lactic acid produced and the concentration of residual glucose as appropriate in the filtrate.

  By the method for producing a chemical product of the present invention using the continuous culture apparatus shown in FIG. 1, stable production of D-lactic acid by continuous fermentation was possible.

(Example 19) Production of D-lactic acid by continuous fermentation using lactic acid bacteria (part 2)
As a microorganism that produces D-lactic acid, Sporactobacillus laevolacticus ATCC23492 strain is used, and a D-lactic acid production medium having the composition shown in Table 21 is used as the medium. Was manufactured. The medium was used after autoclaving (121 ° C., 15 minutes). As the separation membrane, the porous membrane produced in Reference Example 1 was used to constitute the separation membrane element shown in FIG. The concentration of the product D-lactic acid was measured by the HPLC method shown in Reference Example 8. In addition, glucose test Wako C (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measuring the glucose concentration.

  The culture conditions are shown below.

Culture reactor capacity: 1.5 (L)
Membrane separation tank capacity: 0.5 (L)
Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 1)
Membrane separation element effective filtration area: 200 square cm
Temperature adjustment: 37 (℃)
Culture reactor N ₂ aeration rate: 0.2 (L / min)
O ₂ ventilation rate: 2 (mL / min)
Aeration volume of culture medium circulation piping: 0.7 (mL / min)
Membrane separation tank aeration rate: none Culture reaction tank stirring speed: 400 (rpm)
pH adjustment: adjusted to pH 6.0 with 8N NaOH Fermentation medium supply rate: 50-300 ml / hr. Variable control in the range of circulated fluid volume: 0.25 to 1 (L / min) variable control (after continuous fermentation up to 200 hours: circulating fluid volume 0.25 L / min
200 hours to 400 hours: Circulating fluid amount 0.5 L / min
400 hours to 600 hours: Circulating fluid volume 1 L / min)
Filtration amount: 50 mL to 300 mL / hr. Variable control within the range (200 hours after the start of continuous fermentation: filtration rate 400 mL / h
200 hours to 400 hours: filtration amount 600 mL / h
400 hours to 600 hours: filtration amount 800 mL / h).

  Specifically, first, Sporolactobacillus laevolacticus ATCC23492 strain was cultured overnight under anaerobic conditions in a test tube using 10 ml of lactic acid fermentation medium (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh D-lactic acid production medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask under anaerobic conditions (pre-culture). The culture solution is inoculated in a 1.5 L D-lactic acid production medium of the continuous culture apparatus shown in FIG. 2 and the culture reaction tank 1 is stirred by the attached stirrer 5 to adjust the aeration amount of the culture reaction tank 1. Then, temperature adjustment and pH adjustment are performed, and the culture solution circulation piping part is aerated so as to have the same aeration amount (volume ratio) as the culture reaction tank, and the culture solution circulation pump 11 is operated (circulation fluid amount: 0.25 L). / Min) and cultured for 24 hours (pre-culture).

  Immediately after the completion of the pre-culture, the D-lactic acid production medium is continuously supplied, and the continuous culture is performed while controlling the flow rate of the filtrate so that the amount of the fermentation broth in the continuous culture apparatus becomes 2 L. went. The amount of circulating fluid was variably controlled at 0.25, 0.5, and 1.0 L / min every 200 hours. The filtrate flow rate was variably controlled at 400, 600, and 800 mL / h every 200 hours. Table 22 shows the results of measuring the concentration of lactic acid produced and the concentration of residual glucose as appropriate in the filtrate.

Comparative Example 13 Production of D-lactic acid by continuous fermentation using lactic acid bacteria (part 3)
The continuous culture apparatus of FIG. 5 in which the reflux port of the culture solution circulation pipe was opened at a position where it was not immersed in the culture solution in the culture reaction tank, was used in the same manner as in Example 18.

  The results are shown in Table 22, and the yield to sugar, which is one of the continuous culture results, was changed and decreased by changing the circulating amount. From this, it has been clarified that there is a problem in that the culture results are not stable in performing continuous culture using the continuous culture apparatus of FIG.

  By the method for producing a chemical product of the present invention using the fermentation apparatus shown in FIG. 1 and FIG. 2, the continuous culture results did not change even if the circulation amount was changed, and it was possible to operate stably for a long time.

DESCRIPTION OF SYMBOLS 1 Culture 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 Culture fluid circulation pump 12 Membrane separation Tank 13 Separation membrane bundle 14 Upper resin sealing layer 15 Lower resin sealing layer 16 Support frame 17 Water collecting pipe 18 Support plate 19 Channel material 20 Separation membrane 21 Recess 22 Water collecting pipe 110 Culture fluid circulation piping 111 Liquid feeding port 112 Recirculation opening

Claims (2)

Supply and circulate the culture solution of microorganisms or cultured cells stored in the culture reaction tank continuously to the separation membrane while changing the circulation rate, and pass the filtrate containing chemicals produced by the culture on the permeate side of the separation membrane When the unfiltered culture solution that has not been filtered through the separation membrane is refluxed to the culture reaction tank and continuously cultured, the rate of decrease from the set value of the oxygen transfer capacity coefficient kLa in the culture reaction tank is A method for producing a chemical product by continuous culture, characterized in that the unfiltered culture solution is directly refluxed into the culture solution stored in the culture reaction tank in order to suppress it within 30% of the set value .   The method for producing a chemical product by continuous culture according to claim 1, wherein L-lactic acid or D-lactic acid is recovered as the chemical product.

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