JP5061639B2 - Continuous fermentation equipment - Google Patents

Description

  The present invention relates to an improvement of a microorganism or cell culture method and a culture apparatus used therefor. More specifically, the present invention efficiently filters and collects a liquid containing a product from a fermentation broth of microorganisms or cultured cells through a porous separation membrane that is less likely to be clogged, while performing culture, and unfiltered. The present invention relates to a continuous fermentation apparatus for producing a chemical product that improves the concentration of microorganisms involved in fermentation by returning the liquid to a fermentation broth and obtains high productivity.

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

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

  On the other hand, the continuous fermentation method (2) is characterized in that a high yield and high productivity can be maintained over a long period of time by avoiding accumulation of the target substance at a high concentration in the fermenter. A continuous culture method is disclosed for fermentation of L-glutamic acid (see Patent Document 1) and L-lysine (see Non-Patent Document 1). However, in these examples, since the raw material is continuously supplied to the culture solution and the culture solution containing microorganisms and cells is extracted, the microorganisms and cells in the culture solution are diluted. The improvement was limited.

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

  For example, in a continuous fermentation apparatus using a ceramic membrane, techniques for continuous fermentation have been proposed (see Patent Document 2, Patent Document 3 and Patent Document 4). In these proposals, filtration by clogging of the separation membrane is performed. There is a problem in the flow rate and the reduction in filtration efficiency, and back washing or the like is performed to prevent clogging. Separately, a method for producing succinic acid using a separation membrane has been proposed (see Patent Document 5). However, this proposal employs a high filtration pressure (about 200 kPa) in membrane separation. The high filtration pressure is not only disadvantageous in terms of cost, but also in the continuous fermentation method in which microorganisms and cells are physically damaged by pressure during filtration, so that microorganisms and cells are continuously returned to the culture medium. Not appropriate.

As described above, the conventional continuous fermentation method has various problems, and industrial application is difficult. That is, in a continuous fermentation method, microorganisms and cells are filtered through a separation membrane, and the product is recovered from the filtrate, and at the same time, the filtered microorganisms and cells are refluxed to the culture solution to improve the microorganisms and cell concentration in the culture solution, In addition, it is still difficult to obtain high material productivity by maintaining a high level, and technological innovation has been desired.
JP-A-10-150996 JP-A-5-95778 Japanese Patent Laid-Open No. 62-138184 JP-A-10-174594 JP 2005-333886 A Toshihiko Hirao et. Al. (Hirano Toshihiko et al.), Appl. Microbiol. Biotechnol. (Applied Microvials and Microbiology), 32, 269-273 (1989)

  An object of the present invention is to provide a continuous fermentation apparatus for producing a chemical product by a continuous fermentation method that stably maintains high productivity over a long period of time by a simple operation method.

  As a result of diligent research, the inventors of the present application have significantly reduced clogging of the separation membrane when the microorganisms and cells have entered the separation membrane less and the microorganisms and cells are filtered under a low transmembrane pressure. It was found that the filtration of high-concentration microorganisms, which was a problem, can be stably maintained for a long time, and the present invention has been completed.

  The present invention separates a fermentation broth into a filtrate and a non-filtrate by a separation membrane, collects a desired fermentation product from the filtrate, and retains or refluxs the non-filtrate in the fermentation broth. By using a porous membrane having an average pore diameter in the range of 0.01 μm or more and less than 1 μm as a separation membrane, and performing filtration treatment with a low transmembrane pressure difference, fermentation can be performed stably at low cost. The present invention provides a chemical manufacturing apparatus that significantly improves production efficiency.

  That is, the continuous fermentation apparatus of the present invention filters the fermentation broth of microorganisms or cultured cells with a separation membrane, collects the product from the filtrate and holds or refluxs the unfiltrated liquid in the fermentation broth, and An apparatus for producing a chemical product by continuous fermentation in which fermentation raw materials are added to the fermentation broth, comprising a fermentation reaction tank for fermenting microorganisms or cultured cells, and a separation membrane disposed inside the fermentation reaction tank A separation membrane element for filtering the fermented culture broth, means for discharging the permeate containing the filtered fermentation product connected to the separation membrane element, and the transmembrane pressure difference of the separation membrane to 0. The continuous fermentation apparatus is characterized in that it comprises means for controlling in the range of 1 to 20 kPa, and the separation membrane is a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm.

According to a preferred aspect of the continuous fermentation apparatus of the present invention, the porous membrane has a pure water permeability coefficient of 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ² /. It is below s / pa.

  According to a preferred embodiment of the continuous fermentation apparatus of the present invention, the average pore diameter of the porous membrane is in the range of 0.01 μm or more and less than 0.2 μm, and the standard deviation of the pore diameter is 0.1 μm or less. It is.

  According to a preferred aspect of the continuous fermentation apparatus of the present invention, the membrane surface roughness of the porous membrane is a porous membrane of 0.1 μm or less.

  According to a preferred aspect of the continuous fermentation apparatus of the present invention, the porous membrane is a porous membrane including a porous resin layer, and the material of the porous resin layer includes polyvinylidene fluoride.

  According to a preferred aspect of the continuous fermentation apparatus of the present invention, the means for controlling the transmembrane pressure difference is a water head difference control apparatus for controlling the liquid level difference between the fermentation culture solution and the porous membrane treated water.

  According to a preferred aspect of the continuous fermentation apparatus of the present invention, the means for controlling the transmembrane pressure difference is a pressurization pump or / and a suction pump.

  According to a preferred aspect of the continuous fermentation apparatus of the present invention, the member constituting the membrane separation element including the porous membrane is resistant to high-pressure steam sterilization operation.

  According to a preferred embodiment of the continuous fermentation apparatus of the present invention, the chemical is L-lactic acid.

  According to the continuous fermentation apparatus of the present invention, continuous fermentation capable of maintaining high productivity of a desired fermentation product stably over a long period of time under simple operation conditions is possible, and is a fermentation product widely in the fermentation industry. Various chemical products can be stably produced at low cost.

  The continuous fermentation apparatus of the present invention filters the fermentation broth of microorganisms or cultured cells through a separation membrane, collects the product from the filtrate, holds or refluxs the unfiltrated liquid in the fermentation broth, and provides a fermentation raw material. Is an apparatus for producing a chemical product by continuous fermentation, wherein a fermentation reaction vessel for fermenting microorganisms and a fermentation culture solution provided inside the fermentation reaction vessel and provided with a separation membrane are provided. Separation membrane element for filtration, means for discharging filtered microorganisms and fermentation products connected to the separation membrane element, and controlling the transmembrane pressure difference of the separation membrane within the range of 0.1 to 20 kPa The continuous fermentation apparatus is characterized in that the separation membrane is a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm.

  A porous membrane used as a separation membrane in the present invention will be described. The porous membrane used in the present invention has separation performance and water permeability according to the quality of water to be treated and the application. The porous membrane is preferably a porous membrane including a porous resin layer from the viewpoint of separation performance such as blocking performance, water permeability performance, and dirt resistance. Such a porous membrane has a porous resin layer that acts as a separation functional layer on the surface of the porous substrate. The porous substrate supports the porous resin layer and gives strength to the separation membrane.

  The material of the porous substrate is made of an organic material and / or an inorganic material, and organic fiber is preferably used among them. 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.

  Further, as described above, the porous resin layer functions as a separation functional layer, and an organic polymer membrane can be suitably used. Examples of the material of the organic polymer film include polyethylene resin, polypropylene resin, polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, polyacrylonitrile resin, cellulose resin, and the like. Examples thereof include cellulose triacetate resins. The organic polymer film may be 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, the materials constituting the porous resin layer include polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polysulfone resins, which are easy to form a film with a solution and have excellent physical durability and chemical resistance. Ethersulfone resins and polyacrylonitrile resins are preferably used. The material constituting the porous resin layer is most preferably a polyvinylidene fluoride resin or a resin containing it as a main component.

  Here, 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.

  The porous membrane used in the present invention is desirably one that is not easily clogged by microorganisms and cultured cells used for fermentation and has a performance that allows filtration performance to continue stably for a long period of time. Therefore, it is important that the porous membrane used in the present invention is a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm.

  When the average pore diameter is in the range of 0.01 μm or more and less than 1 μm, it is possible to achieve both a high exclusion rate that does not cause leakage of bacterial cells and sludge, and high water permeability. Can be carried out with higher accuracy and reproducibility. If the average pore size is in the range of 0.01 μm or more and less than 1 μm, when animal cells, yeasts, filamentous fungi, etc. are used, there is little clogging and stable continuous fermentation without leakage of cells into the filtrate. It can be implemented. When bacteria smaller than animal cells, yeast, and filamentous fungi are used, the average pore diameter is preferably 0.4 μm or less, and the average pore diameter is preferably 0.2 μm or less. is there. If the average pore diameter is too small, the water permeation rate may be lowered. Therefore, it is usually preferably 0.02 μm or more, and more preferably 0.04 μm or more.

  Here, the average pore diameter can be obtained by measuring and averaging the diameters of all pores that can be observed within a range of 9.2 μm × 10.4 μm in a scanning electron microscope observation at a magnification of 10,000 times. it can.

  The standard deviation σ of the pore diameter is preferably 0.1 μm or less. The standard deviation σ of the pore diameter is defined as N, where X is the number of pores that can be observed within the above-mentioned range of 9.2 μm × 10.4 μm, and X (ave) is the average pore diameter. Is calculated by the following equation (1).

In the porous membrane used in the present invention, the permeability of the fermentation broth is one of the important points, and the pure water permeability coefficient of the porous membrane before use can be used as the permeability index. When the pure water permeability coefficient of the porous membrane was calculated by measuring the water permeability at a head height of 1 m using purified water at a temperature of 25 ° C. by a reverse osmosis membrane, 2 × 10 ⁻⁹ m ³ / m ² / s. / Pa or more is preferable, and if it is 2 × 10 ⁻⁹ or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less, a practically sufficient amount of permeated water can be obtained. A more preferable pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 1 × 10 ⁻⁷ m ³ / m ² / s / pa or less.

  The surface roughness of the porous membrane used in the present invention is a factor that affects the clogging of the separation membrane. The surface roughness of the porous membrane is preferably 0.1 μm or less. When the surface roughness is 0.1 μm or less, the separation coefficient and membrane resistance of the separation membrane can be further reduced, and continuous fermentation can be carried out with a lower transmembrane pressure difference. In addition, by reducing the surface roughness, it can be expected that the shearing force generated on the membrane surface will be reduced in filtration of microorganisms and cultured cells, the destruction of microorganisms or cultured cells is suppressed, and the porous membrane is clogged. It is considered that stable filtration is possible for a long period of time by suppressing the above.

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

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

  The porous membrane used in the present invention may be a flat membrane or a hollow fiber membrane. In the case of a flat membrane, the thickness is selected according to the application. For example, the thickness is preferably 20 μm or more and 5000 μm or less, more preferably 50 μm or more and 2000 μm or less. As described above, the porous film may be formed of a porous substrate and a porous resin layer. At that time, either the porous resin layer permeates the porous base material or the porous resin layer does not permeate the porous base material, and it is selected according to the application. The thickness of the porous substrate is preferably selected in the range of 50 μm or more and 3000 μm or less. In the case of a hollow fiber membrane, the inner diameter is preferably selected in the range of 200 μm to 5000 μm, and the film thickness is preferably selected in the range of 20 μm to 2000 μm. Moreover, the woven fabric and knitting which made the organic fiber or the inorganic fiber the cylinder shape may be included.

  The porous membrane used in the present invention can be made into a separation membrane element by combining with a support. The form of the separation membrane element is not particularly limited, but a separation membrane element in which a support plate is used as a support and the porous membrane of the present invention is disposed on at least one surface of the support plate is suitable for the separation membrane element of the present invention. One of the forms. In this embodiment, since it is difficult to increase the membrane area, it is also a preferable aspect to dispose a porous membrane on both surfaces of the support plate in order to increase the water permeability.

  In the present invention, the transmembrane pressure difference during filtration of microorganisms and cultured cells may be any condition as long as the microorganisms, cultured cells and medium components are not easily clogged, and the transmembrane pressure difference is 0.1 kPa or more and 20 kPa or less. The range. As the driving force of filtration, which is a means for controlling the transmembrane pressure difference, the transmembrane pressure difference is applied to the porous membrane by siphon using the liquid level difference (water head difference) of the fermentation broth and the porous membrane treated water. Can be generated. Moreover, a suction pump may be installed on the porous membrane treated water side as a driving force for filtration, or a pressure pump may be installed on the fermentation culture solution side of the porous membrane. Thus, the transmembrane pressure difference can be controlled by changing the liquid level difference between the fermentation broth and the porous membrane treated water. Moreover, when using a pump, it can control by a suction pressure, Furthermore, it can control by the pressure of the gas or liquid which introduces the pressure by the side of a fermentation culture solution. When these pressure controls are performed, the pressure difference between the pressure on the fermentation broth side and the pressure on the porous membrane treated water side can be used as the transmembrane pressure difference, and can be used to control the transmembrane pressure difference.

  In addition, the porous membrane used in the present invention is preferably one that can be filtered in the range of 0.1 kPa to 20 kPa.

  The fermentation raw material used in the present invention is not limited as long as it promotes the growth of the microorganisms to be cultured and can produce a desired chemical product, particularly pyruvic acid. A normal liquid medium suitably containing a source, inorganic salts, and, if necessary, organic micronutrients such as amino acids and vitamins is preferably used.

  Carbon sources include sugars such as glucose, sucrose, fructose, galactose and lactose, starch saccharified liquids containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, and organic acids such as acetic acid, alcohols such as ethanol And glycerin are also used.

  Nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein degradation products, other amino acids, vitamins, Corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used.

  As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and the like can be appropriately added. When the microorganism used in the present invention requires a specific nutrient for growth, the nutrient can be added as a preparation or a natural product containing it. Moreover, an antifoamer can also be used as needed.

  In the present invention, the fermentation broth refers to a liquid obtained as a result of growth of microorganisms or cultured cells on the fermentation raw material, and the composition of the fermentation raw material to be added has the productivity of the target chemical product, particularly pyruvic acid. You may change suitably from the fermentation raw material composition at the time of a culture | cultivation start so that it may become high.

  In the present invention, the sugar concentration in the fermentation broth is preferably maintained at 5 g / l or less. The reason is to minimize the loss of sugar due to withdrawal of the fermentation broth. The culture of microorganisms is usually carried out at a pH of 4 to 8 and a temperature of 20 to 40 ° C. The pH of the fermentation broth can be adjusted to a predetermined value within the above range with an inorganic acid or an organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, or the like. If it is necessary to increase the oxygen supply rate, means such as adding oxygen to the air to keep the oxygen concentration at 21% or higher, pressurizing the culture, increasing the stirring rate, or increasing the aeration rate can be used.

  In the present invention, batch culture or fed-batch culture may be performed at the initial stage of culture to increase the microorganism concentration, and then continuous culture (pullout) may be started. May be performed. It is possible to supply the raw material culture solution and extract the culture from an appropriate time. The starting times of the supply of the raw material culture solution and the withdrawal of the culture are not necessarily the same. Further, the supply of the raw material culture solution and the withdrawal of the culture may be continuous or intermittent. What is necessary is just to add a nutrient required for microbial cell growth as shown above to a raw material culture solution so that microbial cell growth may be performed continuously. Efficient productivity to maintain the concentration of microorganisms or cultured cells in the culture solution at a high level so long as the environment of the fermentation broth is not suitable for the growth of microorganisms or cultured cells and does not increase the rate of death. It is preferable to obtain As an example, good production efficiency can be obtained by maintaining the concentration at 5 g / L or more as the dry weight.

  Moreover, microorganisms or cultured cells can be extracted from the fermentation reaction tank as necessary. For example, if the concentration of microorganisms or cultured cells in the fermentation reaction tank becomes too high, the separation membrane may be easily clogged. In addition, the production performance of chemical products may change depending on the concentration of microorganisms or cultured cells in the fermentation reaction tank, and the production performance can be maintained by extracting microorganisms or cultured cells using the production performance as an index.

  The continuous culturing operation performed while growing fresh microbial cells capable of fermentation production is usually preferably performed in a single fermentation reaction tank in terms of culture management. However, the number of fermentation reaction tanks is not limited as long as it is a continuous culture method for producing a product while growing cells. A plurality of fermentation reaction tanks may be used because the capacity of the fermentation reaction tank is small. In this case, high productivity of the fermentation product can be obtained even if continuous fermentation is performed by connecting a plurality of fermentation reaction tanks in parallel or in series by piping.

  Examples of microorganisms and cultured cells used in the present invention include yeasts such as baker's yeast often used in the fermentation industry, bacteria such as Escherichia coli and coryneform bacteria, filamentous fungi, actinomycetes, animal cells and insect cells. Can be mentioned. 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.

  The chemical product produced by the continuous fermentation apparatus of the present invention is not particularly limited as long as it is a substance produced by the microorganisms or cells in the culture solution, but is mass-produced in the fermentation industry such as alcohol, organic acid, amino acid and nucleic acid. Can be mentioned. The present invention can also be applied to the production of substances such as enzymes, antibiotics and recombinant proteins. For example, alcohols include ethanol, 1,3-propanediol, 1,4-butanediol, cadaverine and glycerol, and organic acids include acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid and citric acid. An acid etc. can be mentioned. By using the continuous fermentation apparatus of the present invention, high productivity of the chemical product can be obtained.

  Next, the form of continuous fermentation performed using the continuous fermentation apparatus of this invention is demonstrated.

  In conventional continuous fermentation culture, a fresh medium is supplied to the fermentation reaction tank at a constant rate, and the same amount of fermentation broth is discharged out of the tank so that the amount of liquid in the fermentation reaction tank is always kept constant. Is the law. In batch culture, fermentation culture is terminated when the initial substrate concentration is consumed. However, in continuous fermentation culture, fermentation culture can theoretically be maintained indefinitely. That is, theoretically, it can be fermented indefinitely.

  In batch culture, the environmental conditions of microorganisms such as microbial concentration, substrate concentration, product concentration, pH and dissolved oxygen, etc., in the course of fermentation culture time depend on microbial growth, substrate consumption and microbial product. It changes with it. On the other hand, in continuous fermentation culture, a state (steady state) in which these environmental conditions are kept constant appears and persists.

  On the other hand, in the conventional continuous culture described above, the microorganisms are also discharged out of the tank together with the fermentation broth, and it is difficult to maintain a high microorganism concentration in the fermentation reaction tank. Here, when performing fermentation production, the fermentation production efficiency per fermentation volume can be improved if the microorganisms for fermentation can be maintained at a high concentration. For that purpose, it is necessary to hold or reflux the microorganisms in the fermentation reaction tank. As a method for retaining or refluxing microorganisms in the fermentation reaction tank, the discharged fermentation broth is subjected to solid-liquid separation by gravity, for example, centrifugation, and the microorganisms as precipitates are returned to the fermentation reaction tank, or filtered. For example, a method may be used in which microorganisms that are solids are separated, and only the supernatant of the fermentation broth is discharged out of the fermentation reaction tank. However, the method using centrifugation is expensive because of the high power cost. Since the filtration method requires a high pressure for filtration as described above, most of the studies are performed at the laboratory level.

  Next, the configuration and characteristics of the continuous fermentation apparatus of the present invention will be described here. The main constitution of the continuous fermentation apparatus of the present invention is a membrane comprising a fermentation reaction vessel for holding microorganisms or cultured cells and producing chemicals, and a porous membrane for separating the microorganisms or cultured cells and the fermentation broth by filtration The separation element is characterized in that the porous membrane is installed inside the fermentation reaction tank. The porous membrane used here has an average pore diameter of 0.01 μm or more and less than 1 μm. A high filtration pressure is required when separating and filtering a fermentation broth using a conventional continuous fermentation apparatus, for example, a conventional filtration apparatus such as a ceramic filter, but it is difficult to keep the inside of the fermentation apparatus in a pressurized state. The filtration / separation apparatus was installed outside the fermentation reaction tank. However, in the continuous fermentation apparatus of the present invention, the transmembrane differential pressure, which is the filtration pressure, is in the range of 0.1 to 20 kPa. Since it is not necessary to keep the inside in a pressurized state, it is possible to install a porous membrane inside the fermenter. As described above, the continuous fermentation apparatus of the present invention does not have a filtration / separation apparatus, and it is not necessary to install an apparatus such as a pump for circulating the fermentation broth between the continuous fermentation apparatus and the filtration / separation tank. The apparatus can be made compact and the structure of the apparatus can be simplified. For this reason, it is possible to reduce the frequency of microbial culture problems such as contamination of microorganisms and the frequency of mechanical troubles in the movable parts, which facilitates maintenance of the apparatus. Moreover, since the fermentation culture fluid circulation power between the filtration separation apparatus and the fermentation reaction tank is not required, the operation cost can be reduced.

  As the separation membrane and the support member constituting the separation membrane element of the continuous fermentation apparatus of the present invention, a member resistant to high-pressure steam sterilization operation is preferably used. Thereby, the inside of the fermentation reaction tank containing the separation membrane element can be sterilized. If the inside of the fermentation reaction tank can be sterilized, the risk of contamination by undesirable microorganisms during continuous fermentation culture can be avoided, so that stable continuous fermentation culture is possible. The type of the separation membrane and the support member is not particularly limited as long as the separation membrane and the support member constituting the separation membrane element are resistant to the condition of 121 minutes at a temperature of 121 ° C., which is a high-pressure steam sterilization operation condition. As the material of the separation membrane, the material of the porous membrane used as the above-described separation membrane can be used. Examples of the support member include metals such as stainless steel and aluminum, or polyamide resins, fluorine resins, polycarbonate resins, polyacetal resins, polybutylene terephthalate resins, PVDF, modified polyphenylene ether resins, and polysulfone resins. A resin or the like can be preferably selected.

  Next, a typical example of the continuous fermentation apparatus of the present invention will be described with reference to the drawings.

  FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus of the present invention.

  In FIG. 1, a membrane-separated continuous fermentation apparatus is used for filtering a fermentation reaction tank 1 for fermenting microorganisms or cultured cells, and a fermentation broth having a separation membrane disposed in the fermentation reaction tank 1. Separation membrane element 2, means for discharging the permeate containing the filtered fermentation product connected to the separation membrane element 2, and the transmembrane differential pressure of the separation membrane in the range of 0.1 to 20 kPa It consists of means for controlling. A separation membrane element 2 is disposed in the fermentation reaction tank 1, and a porous membrane having pores with an average pore diameter of 0.01 μm or more and less than 1 μm is incorporated in the separation membrane element 2. As the porous membrane, for example, a separation membrane and a separation membrane element disclosed in International Publication No. 2002/064240 can be used. The separation membrane element will be described in detail later.

  Next, the form of continuous fermentation by the membrane separation type continuous fermentation apparatus of FIG. 1 will be described. The culture medium is pumped into the fermentation reaction tank 1 continuously or intermittently by the culture medium supply pump 7. The medium can be sterilized by heating, sterilization, or sterilization using a filter as necessary before being put into the fermentation reaction tank 1. At the time of fermentation production, the fermentation culture solution in the fermentation reaction tank 1 can be agitated with the agitator 5 in the fermentation reaction tank 1 as necessary. Moreover, the gas required by the gas supply apparatus 4 can be supplied in the fermentation reaction tank 1 as needed. At this time, the supplied gas can be recovered and recycled and supplied to the gas supply device 4 again. If necessary, the pH of the fermentation broth in the fermentation reaction tank 1 can be adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8. Moreover, if necessary, highly productive fermentation production can be performed by adjusting the temperature of the fermentation broth in the fermentation reaction tank 1 with the temperature controller 10.

  Here, the adjustment of the pH and temperature was exemplified for the adjustment of the physicochemical conditions of the fermentation broth by the instrumentation / control device, but if necessary, the dissolved oxygen and ORP can be controlled, The concentration of microorganisms in the fermentation broth can be measured by an analytical device such as an on-line chemical sensor, and the physicochemical conditions can be controlled using the concentration as an index. Further, the form of continuous or intermittent input of the medium is not particularly limited, but the amount of medium input is determined using the measured value of the physicochemical environment of the fermentation broth by the instrumentation / control apparatus as an index. And the speed can be adjusted accordingly.

  The fermentation broth is filtered and separated into microorganisms and fermentation products by the separation membrane element 2 installed in the fermentation reaction tank 1 and discharged from the continuous fermentation apparatus system and taken out. This discharge method will be described in detail below.

  In addition, the filtered and separated microorganisms can remain in the apparatus system to maintain a high concentration of microorganisms in the apparatus system, enabling highly productive fermentation production. Here, the filtration / separation by the separation membrane element 2 is performed by the water head differential pressure with respect to the water surface of the fermentation reaction tank 1, and no special power is required. Moreover, the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation culture solution in the fermentation reaction tank 1 can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3 as necessary. The filtration / separation by the separation membrane element 2 is exemplified by the water head differential pressure. However, if necessary, the filtration / separation can be performed by pumping, suction filtration with a liquid, gas, or the like, or pressurizing the apparatus system. It can also be separated.

  That is, as a means for controlling the transmembrane pressure difference, the water head difference control device 3 for controlling the liquid level difference of the fermentation broth and the porous membrane treated water, the pressure pump and / or the suction pump can be used. In addition, since the transmembrane pressure difference can be controlled by the pressure of gas or liquid, stable fermentation production for a long period of time becomes possible.

  Although the form of the separation membrane element 2 used in the present invention is not particularly limited, the separation membrane and the separation membrane element disclosed in International Publication No. 2002/064240, which is an example of a preferred embodiment, are described below with reference to the drawings. The outline will be described. FIG. 2 is a schematic perspective view for explaining one embodiment of the separation membrane element 2 used in the present invention.

  As shown in FIG. 2, the separation membrane element is configured by arranging a flow path material 12 and the separation membrane 13 in this order on both surfaces of a rigid support plate 11. The support plate 11 has recesses 14 on both sides. The separation membrane 13 filters the fermentation broth. The flow path member 12 is for efficiently flowing the permeated water filtered by the separation membrane 13 to the support plate 11. The permeate containing the fermentation product that has flowed to the support plate 11 passes through the recess 14 of the support plate 13 and is taken out of the continuous fermentation apparatus via the water collecting pipe 15 that is a discharging means. Here, the above-mentioned water head differential pressure, pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used as power for extracting permeated water.

As described above, when continuous fermentation is performed using the continuous fermentation apparatus of the present invention, a high volumetric production rate is obtained compared to conventional batch fermentation, and extremely efficient fermentation production is possible. Here, the production rate in continuous culture is calculated by the following equation (3).
・ Fermentation production rate (g / L / hr) = Product concentration in extraction liquid (g / L) × Fermentation culture liquid extraction speed (L / hr) ÷ Operation liquid volume of apparatus (L) 3)
In addition, the fermentation production rate in batch culture is calculated by dividing the amount of product (g) at the time when all of the raw carbon source is consumed by the time (h) required for consumption of the carbon source and the amount of culture solution (L) at that time. Is required.

  Hereinafter, in order to describe the continuous fermentation apparatus of the present invention in more detail, L-lactic acid is selected as the above-mentioned chemical product, and the continuous fermentation apparatus shown in FIG. This will be described with reference to examples. The present invention is not limited to these examples.

  Here, yeast Saccharomyces cerevisae was used as a microorganism for producing L-lactic acid. Yeast Saccharomyces cerevisiae originally did not have L-lactic acid fermentation, but by introducing a gene encoding L-lactic acid dehydrogenase, a Saccharomyces cerevisiae strain having L-lactic acid fermentation ability was constructed and carried out. Specifically, a yeast strain having L-lactic acid fermentation ability was constructed and used by linking the human-derived LDH gene downstream of the PDC1 promoter on the yeast genome.

Reference Example 1 Production of Yeast Strain Having Lactic Acid Production Capacity A yeast strain having lactic acid production capacity was constructed as follows. Specifically, a yeast strain capable of producing L-lactic acid is constructed by linking a human-derived LDH gene downstream of the PDC1 promoter on the yeast genome. For polymerase chain reaction (PCR), La-Taq (manufactured by Takara Shuzo) or KOD-Plus-polymerase (manufactured by Toyobo Co., Ltd.) was used according to the attached instruction manual.

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

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

  By PCR using the plasmid pL-ldh5 containing the human-derived LDH gene as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 5 as primer sets, the human-derived LDH gene of 1.3 kb and Saccharomyces cerevisiae are derived. A DNA fragment containing the terminator sequence of the TDH3 gene was amplified. In addition, a DNA fragment containing the TRP1 gene derived from Saccharomyces cerevisiae of 1.2 kb was amplified by PCR using the plasmid pRS424 as an amplification template and the oligonucleotides represented by SEQ ID NO: 6 and SEQ ID NO: 7 as a primer set. Each DNA fragment was separated by 1.5% agarose gel electrophoresis and purified according to a conventional method. A product obtained by PCR using a mixture of the 1.3 kb fragment obtained here and the 1.2 kb fragment as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 7 as a primer set, 1.5% agarose gel electrophoresis was performed, and a 2.5 kb DNA fragment to which the human-derived LDH gene and TRP1 gene were linked was prepared according to a conventional method. Saccharomyces cerevisiae NBRC10505 strain was transformed with this 2.5 kb DNA fragment into tryptophan non-requirement according to a conventional method.

Confirmation that the obtained transformed cells were cells in which the human-derived LDH gene was linked downstream of the PDC1 promoter on the yeast genome was performed as follows. First, genomic DNA of a transformed cell was prepared according to a conventional method, and a 0.7 kb amplified DNA fragment was obtained by PCR using the oligonucleotides represented by SEQ ID NO: 8 and SEQ ID NO: 9 as a primer set. It was confirmed by obtaining. In addition, whether or not the transformed cells have lactic acid production ability is determined by the fact that lactic acid is contained in the culture supernatant obtained by culturing the transformed cells in SC medium (METHODS IN YEAS GENETIC 2000 EDITION, CSHL PRESS). It confirmed by measuring the amount of lactic acid by HPLC method on the conditions shown.
・ Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
-Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
-Detection method: electrical conductivity-Temperature: 45 ° C.
-The optical purity of L-lactic acid was measured by the HPLC method under the following conditions.
・ Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation)
-Mobile phase: 1 mM aqueous copper sulfate-Flow rate: 1.0 ml / min
・ Detection method: UV254nm
・ Temperature: 30 ℃
Moreover, the optical purity of L-lactic acid is calculated by the following formula.
Optical purity (%) = 100 × (LD) / (L + D)
(Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.)
As a result of HPLC analysis, 4 g / L of L-lactic acid was detected, and D-lactic acid was below the detection limit. From the above examination, it was confirmed that this transformant has L-lactic acid production ability. The obtained transformed cells were used in subsequent examples as yeast strain SW-1.

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

(Reference Example 3) Production of porous membrane (part 2)
Polyvinylidene fluoride (PVDF) resin as a resin, polyethylene glycol (PEG) having a molecular weight of about 20,000 as a pore-opening agent, N, N-dimethylacetamide (DMAc) as a solvent, and pure water as a non-solvent. These were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
・ PVDF: 13.0% by weight
・ PEG: 5.5% by weight
DMAc: 78.0% by weight
・ Pure water: 3.5% by weight
Next, after cooling the stock solution to a temperature of 25 ° C., it was applied to a polyester fiber nonwoven fabric (porous substrate) having a density of 0.48 g / cm ³ and a thickness of 220 μm. It was immersed in pure water at a temperature for 5 minutes, and further immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc and PEG to obtain a separation membrane. When the surface of the porous resin layer on the side of the separation membrane on which the stock solution was applied was observed in a scanning electron microscope at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm, all the pores that could be observed The average diameter was 0.02 μm. When this separation membrane was evaluated for its pure water permeability coefficient, it was 2 × 10 ⁻⁹ m ³ / m ² · s · Pa. The amount of water permeation was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane with a water head difference of 1 m. The standard deviation of the average pore diameter was 0.0055 μm, and the membrane surface roughness was 0.1 μm. Thus, the porous membrane produced in Reference Example 3 can be suitably used in the present invention.

(Reference Example 4) Production of porous membrane (part 3)
A separation membrane was obtained in the same manner as in Reference Example 3 except that the stock solution having the following composition was used.
・ PVDF: 13.0% by weight
・ PEG: 5.5% by weight
DMAc: 81.5% by weight
When the surface of the porous resin layer on the side of the separation membrane on which the stock solution was applied was observed in a scanning electron microscope at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm, all the pores that could be observed The average diameter was 0.19 μm, and the pure water permeability coefficient of this separation membrane was evaluated to be 100 × 10 ⁻⁹ m ³ / m ² · s · Pa. The amount of water permeation was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane with a water head difference of 1 m. The standard deviation of the average pore diameter was 0.060 μm, and the membrane surface roughness was 0.08 μm. Thus, the porous film produced in Reference Example 4 can be suitably used in the present invention.

(Example 1) Production of L-lactic acid by continuous fermentation (part 1)
In order to examine whether or not an L-lactic acid continuous fermentation system can be obtained by operating the membrane-separated continuous fermentation apparatus of FIG. 1, a continuous fermentation test of this apparatus was conducted using a lactic acid fermentation medium having the composition shown in Table 1. went. The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. Moreover, the porous membrane produced in Reference Example 2 was used as the separation membrane. The operating conditions in Example 1 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.1 (L / min)
・ Fermentation reactor stirring speed: 400 (rpm)
・ PH adjustment: adjusted to pH 5 with 1N NaOH ・ Sterilization: Fermentation reaction tank 1 including separation membrane element 2 and the used culture medium are all autoclaved at 121 ° C. for 20 minutes. The flow rate was controlled by the difference (transmembrane pressure difference was controlled to 2 kPa or less).
-Membrane permeate flow rate control: The flow rate was controlled by the water separation difference in the membrane separation tank (the water difference was controlled within 2 m).

  The yeast SW-1 strain prepared in Reference Example 1 was used as the microorganism, the lactic acid fermentation medium having the composition shown in Table 1 was used as the medium, and the HPLC shown in Reference Example 1 was used for evaluation of the concentration of lactic acid as the product. The glucose concentration was measured using “Glucose Test Wako C” (registered trademark) (Wako Pure Chemical Industries, Ltd.).

  First, the SW-1 strain was cultured with shaking in a 5 ml lactic acid fermentation medium in a test tube overnight (pre-culture). The obtained fermentation broth 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 incubating medium is inoculated in a 1.5 L lactic acid fermentation medium of the continuous fermentation apparatus shown in FIG. 1 and the fermentation reaction tank 1 is stirred at 400 rpm by the attached stirrer 5. Adjustment, temperature adjustment, and pH adjustment were performed, and culture was performed for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the lactic acid fermentation medium is continuously supplied, and the continuous fermentation culture is performed while controlling the amount of permeated water in the continuous fermentation apparatus so that the amount of the fermentation broth in the continuous fermentation apparatus becomes 1.5 L. Production of lactic acid by continuous fermentation culture Went. The control of the amount of permeated water when performing the continuous fermentation test is performed by appropriately changing the water head difference by the water head difference control device 3 so that the water head difference of the membrane separation tank is within 2 m at the maximum, that is, the transmembrane pressure difference is within 20 kPa. I went there.

  The produced lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. In addition, Table 2 shows the lactic acid-to-sugar yield calculated from the lactic acid and the input glucose calculated from the glucose concentration, and the lactic acid production rate. As a result of conducting a fermentation test for 280 hours, it was confirmed that stable production of L-lactic acid by continuous fermentation was possible by using the continuous fermentation apparatus shown in FIG. The transmembrane pressure difference during the continuous fermentation period was 2 kPa or less and 0.1 or more.

(Example 2) Production of L-lactic acid by continuous fermentation (part 2)
In order to examine whether or not an L-lactic acid continuous fermentation system can be obtained by operating the continuous fermentation apparatus of FIG. 1, a continuous fermentation test of this apparatus was performed using a lactic acid fermentation medium having the composition shown in Table 1. The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member 2, a molded product of stainless steel and polysulfone resin was used. Further, the porous membrane prepared in Reference Example 1 was used as the separation membrane. The operating conditions in Example 2 are as follows unless otherwise specified.
・ Reaction tank capacity: 1.5 (L)
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
Lactic acid fermentation medium supply rate: 50 to 300 ml / hr. Variable control within the range of-Stirring speed of fermentation reaction tank: 800 (rpm)
・ PH adjustment: pH adjusted to 5 with 1N NaOH ・ Membrane permeate flow rate control: Flow rate control by transmembrane pressure difference.

(80 hours after the start of continuous fermentation: controlled at 0.1 kPa to 5 kPa
80 hours to 160 hours: Control between 0.1 kPa and 2 kPa
160 to 240 hours: controlled at 0.1 kPa to 20 kPa)
・ Sterilization: Culture tank containing separation membrane element and all used media are autoclaved at 121 ° C for 20 min. High pressure steam sterilization. Membrane permeate flow control: Flow rate is controlled by membrane separation tank water head difference (water head difference is controlled within 2 m. did.).

  The yeast SW-1 strain prepared in Reference Example 1 was used as the microorganism, and the lactic acid fermentation medium having the composition shown in Table 1 was used as the medium. The glucose concentration was measured using “Glucose Test Wako C” (registered trademark) (Wako Pure Chemical Industries, Ltd.).

  First, the SW-1 strain was cultured with shaking in a 5 ml lactic acid fermentation medium in a test tube overnight (pre-culture). The obtained fermentation broth 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 incubating medium is inoculated in a 1.5 L lactic acid fermentation medium of the continuous fermentation apparatus shown in FIG. 1 and the fermentation reaction tank 1 is stirred at 400 rpm by the attached stirrer 5. Adjustment, temperature adjustment, and pH adjustment were performed, and culture was performed for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the lactic acid fermentation medium is continuously supplied, and the continuous fermentation culture is performed while controlling the amount of permeated water in the continuous fermentation apparatus so that the amount of the fermentation broth in the continuous fermentation apparatus becomes 1.5 L. Production of lactic acid by continuous fermentation culture Went. Control of the amount of water permeated through the continuous fermentation test was carried out by measuring the water head difference as a transmembrane pressure difference with the water head difference control device 3 and changing it under the above-mentioned membrane permeated water amount control conditions. The produced lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. In addition, Table 2 shows the lactic acid-to-sugar yield calculated from the input glucose calculated from the lactic acid and glucose concentrations, and the lactic acid production rate. As a result of conducting a fermentation test for 240 hours, it was confirmed that stable production of L-lactic acid by continuous fermentation was possible by using this membrane separation type continuous fermentation apparatus. In addition, the transmembrane differential pressure during the period of continuous fermentation changed at 2 kPa or less and 0.1 or more.

(Example 3) Production of L-lactic acid by continuous fermentation (part 3)
Using the porous membrane produced in Reference Example 3 as the separation membrane, the same L-lactic acid continuous fermentation test as in Example 2 was performed. The results are shown in Table 2. As a result, it was confirmed that stable production of L-lactic acid by continuous fermentation was possible. The transmembrane pressure difference during the continuous fermentation period was 2 kPa or less and 0.1 or more.

(Example 4) Production of L-lactic acid by continuous fermentation (part 4)
Using the porous membrane produced in Reference Example 4 as the separation membrane, the same L-lactic acid continuous fermentation test as in Example 2 was performed. The results are shown in Table 2. As a result, it was confirmed that stable production of L-lactic acid by continuous fermentation was possible. The transmembrane pressure difference during the continuous fermentation period was 2 kPa or less and 0.1 or more.

(Comparative example 1) Production of L-lactic acid by batch fermentation In order to examine whether L-lactic acid fermentation productivity is improved by using the membrane-separated continuous fermentation apparatus shown in Fig. 1, fermentation form using microorganisms As the most typical batch fermentation, the lactic acid productivity was evaluated. Using a lactic acid fermentation medium shown in Table 1, a batch fermentation test was conducted without performing filtration with a separation membrane element in the continuous fermentation apparatus of FIG. The medium was used after autoclaving at 121 ° C. for 15 minutes. Also in Comparative Example 1, the yeast SW-1 strain prepared in Reference Example 1 was used as a microorganism, and the concentration of lactic acid as a product was evaluated using the HPLC shown in Reference Example 1, and the glucose concentration For the measurement, “Glucose Test Wako C” (registered trademark) (Wako Pure Chemical Industries, Ltd.) was used. The operating conditions of Comparative Example 1 are shown below.
-Fermentation reactor capacity (lactic acid fermentation medium amount): 1 (L)
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.1 (L / min)
・ Fermentation reactor stirring speed: 400 (rpm)
-PH adjustment: Adjusted to pH 5 with 1N NaOH First, the SW-1 strain was cultured with shaking in a 5 ml lactic acid fermentation medium overnight in a test tube (pre-culture). The culture solution was inoculated into 100 ml of fresh lactic acid fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). The preculture was inoculated into a 1.5 L lactic acid fermentation medium of a membrane separation type continuous fermentation apparatus, the fermentation reaction tank 1 was stirred at 400 rpm with the attached stirrer 5, and the fermentation reaction tank 1 was aerated. Temperature adjustment and pH adjustment were performed, and batch fermentation culture was performed without operating the fermentation broth circulation pump 10. The amount of bacterial cell growth at this time was 15 in terms of absorbance at 600 nm. The results of batch fermentation are shown in Table 2 comparing L-lactic acid fermentation productivity obtained in the continuous fermentation test of Example 1.

  As a result of these comparisons, it was clarified that the production rate of L-lactic acid was significantly improved by using the continuous fermentation apparatus of FIG. That is, using a membrane separation type continuous fermentation apparatus incorporating a porous membrane disclosed by the present invention, by controlling the transmembrane pressure difference, the fermentation broth is separated into a filtrate and an unfiltered liquid by the separation membrane, The desired fermentation product can be recovered from the filtrate, and a continuous fermentation method can be used to return the unfiltered liquid to the fermentation broth, allowing the production of chemicals such as L-lactic acid by continuous fermentation while maintaining a high microbial content. It became clear that.

  The continuous fermentation apparatus of the present invention is capable of continuous fermentation that maintains high productivity of a desired fermentation product stably over a long period of time under simple operating conditions, and is widely used as a fermentation product in the fermentation industry. It is useful because it can produce chemicals stably at low cost.

FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus of the present invention. FIG. 2 is a schematic perspective view for explaining one embodiment of a separation membrane element used in the present invention. It is a figure which shows the physical map of the expression vector pTRS11 for yeast used in the Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Separation membrane element 3 Water head difference control device 4 Gas supply device 5 Stirrer 6 Level sensor 7 Medium supply pump 8 pH adjustment solution supply pump 9 pH sensor / control device 10 Temperature controller 11 Support plate 12 Channel material 13 Separation membrane 14 Recess 15 Water collecting pipe

Claims (10)

  The fermentation broth of microorganisms or cultured cells is filtered through a separation membrane, the product is recovered from the filtrate, the unfiltered liquid is retained or refluxed in the fermentation broth, and the fermentation raw material is added to the fermentation broth An apparatus for producing a chemical by continuous fermentation, a fermentation reaction tank for fermenting microorganisms or cultured cells, and a separation for filtering a fermentation broth provided in the fermentation reaction tank and provided with a separation membrane A membrane element, means for discharging the permeate containing the filtered fermentation product connected to the separation membrane element, and for controlling the transmembrane differential pressure of the separation membrane in the range of 0.1 to 20 kPa A continuous fermentation apparatus characterized in that the separation membrane is a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm. 2. The continuous fermentation apparatus according to claim 1, wherein the porous membrane has a pure water permeability coefficient of 2 × 10 ⁻⁹ m ³ / m ² / s / pa to 6 × 10 ⁻⁷ m ³ / m ² / s / pa. .   The continuous fermentation apparatus according to claim 1 or 2, wherein the porous membrane has an average pore diameter in the range of 0.01 µm or more and less than 0.2 µm, and a standard deviation of the average pore diameter is 0.1 µm or less.   The continuous fermentation apparatus according to any one of claims 1 to 3, wherein the porous membrane has a membrane surface roughness of 0.1 µm or less.   The continuous fermentation apparatus according to any one of claims 1 to 4, wherein the porous membrane is a porous membrane including a porous resin layer.   The continuous fermentation apparatus according to any one of claims 1 to 5, wherein the material of the porous resin layer is polyvinylidene fluoride.   The continuous fermentation apparatus according to any one of claims 1 to 6, wherein the means for controlling the transmembrane pressure difference is a water head difference control apparatus that controls a liquid level difference between the fermentation broth and the porous membrane treated water.   The continuous fermentation apparatus according to any one of claims 1 to 7, wherein the means for controlling the transmembrane pressure difference is a pressure pump or / and a suction pump. The continuous fermentation apparatus according to any one of claims 1 to 8 , wherein a member constituting the separation membrane element including the porous membrane is resistant to high-pressure steam sterilization operation.   The fermentation broth of microorganisms or cultured cells is filtered through a separation membrane, the product is recovered from the filtrate, the unfiltered liquid is retained or refluxed in the fermentation broth, and the fermentation raw material is added to the fermentation broth An apparatus for producing a chemical by continuous fermentation, a fermentation reaction tank for fermenting microorganisms or cultured cells, and a separation for filtering a fermentation broth provided in the fermentation reaction tank and provided with a separation membrane A membrane element, means for discharging the permeate containing the filtered fermentation product connected to the separation membrane element, and for controlling the transmembrane differential pressure of the separation membrane in the range of 0.1 to 20 kPa Characterized in that the separation membrane is a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm, and continuously fermenting microorganisms or cultured cells using a continuous fermentation apparatus Method of manufacturing the chemicals to be.

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