JP2009296921A - Continuous culture device and method for producing chemical - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compact continuous culture device for producing chemicals by a culture method, stably maintaining the high productivity over a long time. <P>SOLUTION: The continuous culture device includes a culture reaction vessel for culturing microorganisms or culture cells, a membrane separation vessel in which a separation membrane for filtering a culture liquid continuously fed from the culture reaction vessel is arranged, and a culture liquid-circulating means for feeding the culture liquid from the culture reaction vessel to the membrane separation vessel, and returning the unfiltered culture liquid which is not filtered to the culture liquid at the upstream side of the membrane separation vessel. The membrane separation vessel and the culture reaction vessel have volumes regulated so that the culture liquid volume ratio of the culture liquid in the culture reaction vessel to the culture liquid in the membrane separation vessel may be ≥5 and ≤100. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a culture apparatus used for culturing microorganisms or cultured cells. More specifically, the present invention manufactures chemicals and the like with high productivity by efficiently filtering and recovering a liquid containing substances produced by culturing from a culture liquid of microorganisms or cultured cells through a separation membrane while culturing. The present invention relates to a continuous culture apparatus that can be used.

  Substance production methods involving cultivation of microorganisms and cultured cells can be broadly classified into (1) batch culture methods (Batch culture methods) and fed-batch culture methods (Fed-Batch culture methods), and (2) continuous culture methods. it can.

  The batch culture method and fed-batch culture method described in (1) above are simple in terms of equipment, have the advantage that the culture is completed in a short time, and there is little damage caused by contamination with bacteria. Conventionally, microorganisms and cultured cells have been used. Has been used as a material production method. However, these methods increase the concentration of the product in the culture solution over time, and the productivity and yield decrease due to the influence of osmotic pressure or product inhibition. Therefore, it is difficult for these culture methods to stably maintain a high yield and high productivity over a long period of time.

  On the other hand, the continuous culture method (2) is characterized in that high yield and high productivity can be maintained over a long period of time by avoiding the accumulation of the product at a high concentration in the culture reaction tank.

  For example, 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, although raw materials such as nutrients are continuously supplied to the culture solution, the microorganisms and culture cells in the culture solution are diluted to extract the culture solution containing microorganisms and culture cells. The improvement in production efficiency was limited.

  Therefore, in a continuous culture 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, a technique for continuous culture using a continuous culture apparatus using a separation membrane has been proposed (see Patent Document 2). In this proposal, various chemical products can be obtained by using a continuous culture device with a tank for culturing microorganisms and cultured cells and a tank for performing membrane separation of the target product and microorganisms and cultured cells from the culture solution. Compared to batch culture and fed-batch culture, it produces at a higher production rate. However, in this example, the volume of the tank for membrane separation is large compared to the tank for culturing, so the power cost required for the culture medium circulation between the culture tank and the membrane separation tank is high and the chemistry of the continuous culture apparatus as a whole is high. Product production rate is low. In addition, the culture conditions are adjusted to suit the production of chemicals in the culture tank. However, if the volume of the membrane separation tank is large, the residence time of the culture solution in the membrane separation tank becomes longer. Therefore, there is a concern that the productivity may be further lowered by deviating from the culture conditions suitable for chemical products. As described above, the conventional continuous culture apparatus contains problems such as waste of driving power, low production speed, and complicated operation management of the apparatus, and improvement of continuous culture technology has been desired.
JP-A-10-150996 International Publication No. 07/097260 Pamphlet Toshihiko Hirao et. Al., Appl. Microbiol. Biotechnol., Applied Microvials and Microbiology, 32, 269-273 (1989)

  Therefore, an object of the present invention is to provide a compact continuous culture apparatus that can solve problems such as waste of driving power, low production speed, and complexity of apparatus operation management.

The inventors of the present invention have conducted extensive research for the purpose of reducing the scale of a continuous culture apparatus using a separation membrane, and as a result, the apparatus can be made compact by any of the following configurations (1) to (3). At the same time, it was found that the production efficiency of chemicals was improved, and the present invention was completed.
(1) A culture reaction tank for culturing microorganisms or cultured cells, a membrane separation tank in which a separation membrane for filtering the culture solution continuously supplied from the culture reaction tank is disposed, and the culture solution is cultured in the culture medium A culture medium circulating means for supplying an unfiltered culture solution supplied from the reaction tank to the membrane separation tank and refluxed to the culture medium upstream of the membrane separation tank; and the membrane separation tank and the The continuous culture apparatus, wherein the culture reaction tank has a volume in which the culture liquid volume ratio of the culture liquid in the culture reaction tank to the culture liquid in the membrane separation tank is 5 or more and 100 or less.
(2) The continuous culture apparatus according to (1), wherein the membrane separation tank and the culture reaction tank have a volume in which the culture liquid volume ratio is 15 or more and 100 or less.
(3) Incubating microorganisms or cultured cells in a culture reaction tank, continuously supplying the culture solution from the culture reaction tank to the membrane separation tank, and filtering through the separation membrane to recover the product. In refluxing the filtered culture solution to the culture solution upstream of the membrane separation tank, the culture solution volume ratio of the culture solution in the culture reaction tank to the culture solution in the membrane separation tank is set to 5 or more and 100 or less. A method for producing a characteristic chemical product.

  According to the continuous culture apparatus of the present invention, downsizing of the continuous culture apparatus is realized, and continuous culture that maintains high productivity of a desired product stably over a long period of time is realized under simple operation conditions. In addition, it is possible to stably produce various chemical products as products at low cost.

  The continuous culture apparatus of the present invention is an apparatus for producing chemicals and the like by continuous culture. As a basic structure, the culture solution of microorganisms or cultured cells is filtered through a separation membrane, and the product is recovered from the filtrate. Means for holding or refluxing the unfiltered liquid in the culture medium. Specifically, a culture reaction tank for culturing microorganisms or cultured cells, a membrane separation tank in which a separation membrane for filtering the culture solution continuously supplied from the culture reaction tank is disposed, and the culture solution A culture medium circulation means is provided for supplying an unfiltered culture solution that has been supplied from the culture reaction tank to the membrane separation tank and that has not been filtered, to the culture medium on the upstream side of the membrane separation tank. In the present invention, the membrane separation tank and the culture reaction tank have a volume in which the culture liquid volume ratio of the culture liquid in the culture reaction tank to the culture liquid in the membrane separation tank is 5 or more and 100 or less. By reducing the volume ratio of the culture solution to 5 or more and 100 or less, the apparatus can be made compact, and the culture reaction tank retention time of the culture solution can be lengthened and adjustment to appropriate culture conditions can be realized. It is possible to improve the production rate of chemical products and to easily manage the operation of the equipment.

  Hereinafter, the basic configuration and characteristics of the continuous culture apparatus of the present invention will be specifically illustrated and described. In FIG. 1, the schematic side view of the continuous culture apparatus which shows one Embodiment of this invention is shown. The continuous culture apparatus shown in FIG. 1 is connected to a culture reaction tank 1 for continuously culturing microorganisms or cultured cells, and connected to the culture reaction tank 1 via a culture medium circulation means (pipe and culture medium circulation pump 5). It is comprised with the membrane separation tank 12 provided with the separation membrane 3 for filtering a culture solution.

  The culture reaction tank 1 has a function of continuously culturing microorganisms or cultured cells, and may be connected to a culture medium circulation means. A jar fermenter that has been conventionally used for production of chemicals can be used. The shape of the membrane separation tank 12 is not limited as long as the separation membrane 3 can be installed inside and the culture medium circulating means can be connected like the culture reaction tank 1. In addition, as a material for the separation membrane used in the present invention, any inorganic or organic material can be used. A preferable separation membrane will be described in detail later.

  In the present invention, the culture reaction tank 1 and the membrane separation tank 12 have a volume in which the culture liquid volume ratio of the culture liquid in the culture reaction tank to the culture liquid in the membrane separation tank is 5 or more and 100 or less. In the present invention, the culture medium volume ratio is defined as the ratio of the amount of each culture solution that is filled in the membrane separation tank and the culture reaction tank during the culture operation, such as a head space that is not filled with the culture solution in each tank. The volume of is not taken into account.

  In the apparatus shown in FIG. 1, the culture reaction tank 1 is connected to a medium supply pump 8 and is equipped with a stirrer 2. The medium is supplied to the culture reaction tank 1 by the medium supply pump 8, and if necessary. Thus, the stirrer 2 is configured to stir the culture solution in the culture reaction tank 1. Further, a gas supply device 11 is also connected, and necessary gas is supplied by the gas supply device 11 as necessary. At this time, for example, a pipe is connected between the head space of the culture reaction tank 1 and the gas supply device 11 so that the supplied gas can be recovered and recycled and supplied again by the gas supply device 11. It is also preferable to collect and recycle by supplying the supply gas in the order of the pipe and the gas supply device 11.

  The culture reaction tank 1 is provided with a pH sensor / control device 10 and a pH adjusting solution supply pump 9 so that the pH of the culture solution can be adjusted as necessary. Furthermore, a temperature controller 7 is also provided so that a chemical product with high productivity can be produced by adjusting the temperature of the culture solution as required. Here, the adjustment of the pH and temperature was exemplified for the adjustment of the physicochemical conditions of the culture solution by the instrumentation / control device, but the dissolved oxygen and ORP can be controlled as necessary, and further online The concentration of microorganisms in the culture solution can be measured by an analyzer such as a chemical sensor, and the physicochemical conditions can be controlled using the measured concentration as an index. Further, the form of continuous or intermittent addition of the medium is not particularly limited, but the measured amount of the culture medium by the instrumentation / control apparatus as described above is used as an index, and the amount of medium input and The speed can be adjusted accordingly.

  In the apparatus shown in FIG. 1, a level sensor 6 is provided in the culture reaction tank 1 in order to adjust the filtration speed in the separation membrane 3 and the amount of the culture solution in the culture reaction tank, and the water head differential pressure control. A device 4 is also provided.

  In the continuous culture apparatus configured as described above, the culture is performed, for example, as follows. That is, culturing microorganisms or cultured cells in the culture reaction tank 1, continuously supplying the culture solution from the culture reaction tank 1 to the membrane separation tank 12, and filtering through the separation membrane 3 to collect products such as lactic acid, In refluxing the unfiltered culture solution that has not been filtered to the culture solution upstream of the membrane separation tank 12, the culture solution volume ratio of the culture solution in the culture reaction tank to the culture solution in the membrane separation tank 12 is 5 or more and 100 or less. To do.

  More specifically, first, microorganisms and a culture raw material (medium) are stored in the culture reaction tank 1, and the inside of the culture reaction tank 1 is maintained within a pH range of 4 to 8 by appropriately adding a neutralizing agent. And the temperature is maintained in the range of 20 to 50 ° C. In this way, microorganisms are cultured, and desired substances (products such as chemicals) such as alcohol, organic acid, amino acid and nucleic acid are produced.

  During this time, a medium containing nutrients used for the culture is continuously or intermittently supplied to the culture reaction tank 1 via the medium supply pump 8 so that the desired product can be obtained by continuous culture. . In addition, the culture solution in the culture reaction tank 1 is continuously circulated between the culture reaction tank 1 and the membrane separation tank 12 by the culture solution circulation pump 5, and the culture solution containing the product in the membrane separation tank 12. Is separated and separated into a filtrate containing microorganisms and products by a separation membrane. As a result, the filtrate containing the product is removed from the apparatus system, while the filtered and separated microorganisms and cultured cells remain in the culture reaction tank 1, so that the microorganism concentration can be maintained high, Highly productive culture is possible.

  The concentration of the microorganisms or cultured cells in the culture solution is preferably maintained at a high level within a range where the growth rate of the microorganisms or cultured cells is inappropriate and does not increase, so that efficient productivity can be obtained. As an example, good production efficiency can be obtained by maintaining the concentration at 5 g / L or more as the dry weight.

  In order to maintain the culture solution at such an appropriate concentration, it is also preferable to pull out microorganisms or cultured cells from the culture reaction tank as necessary. If the concentration of microorganisms or cultured cells in the culture reaction tank becomes too high, clogging of the separation membrane may occur easily. However, the clogging of the separation membrane is avoided by pulling out and maintaining the concentration. You can also. In addition, the production performance of chemicals may change depending on the concentration of microorganisms or cultured cells in the culture reaction tank, but it is also possible to maintain the production performance by extracting microorganisms or cultured cells using the production performance as an index.

  Further, in the present invention, continuous culture may be started after batch culture or fed-batch culture is performed at an early stage of culture to increase the concentration of microorganisms or cultured cells. In addition, continuous culture may be performed.

  The supply of the culture raw material and the extraction of the culture solution may be performed from an appropriate time. That is, the start timing of supplying the culture raw material and drawing out the culture solution are not necessarily the same. Further, the supply of the culture raw material and the extraction of the culture solution may be continuous or intermittent. Filtration / separation of the culture solution using the separation membrane can be performed by the water head differential pressure between the culture solution liquid surface of the membrane separation tank 12 and the filtrate, and no special power is required. Alternatively, filtration and separation can be performed by pressurizing the inside of the apparatus system with gas, liquid, or the like. That is, as a means for controlling the transmembrane pressure difference, the head differential pressure control device 4 for controlling the liquid level difference between the culture solution and the filtrate, a pressurization pump and / or a suction pump can be used. Alternatively, the transmembrane pressure can be controlled by the liquid pressure. By appropriately using these means together, it becomes possible to culture stably for a long time.

  Moreover, it is also preferable to appropriately adjust the filtration rate of the separation membrane 3 and the amount of the culture solution in the culture reaction tank by the level sensor 6 and the head differential pressure control device 4 as necessary. For adjusting the amount of the culture solution in the culture reaction tank, it is also possible to measure and adjust the weight of the culture solution instead of the culture solution level in the culture reaction vessel.

  Here, the transmembrane pressure difference and filtration rate when the culture solution of microorganisms or cultured cells is filtered through a separation membrane will be described. There is a correlation between the transmembrane pressure difference and the filtration rate, and the higher the filtration rate, the higher the transmembrane pressure difference. That is, by controlling the transmembrane pressure during continuous culture operation, the filtration rate can be controlled, and the transmembrane pressure is an important operating parameter. In continuous culture operation, if it is operated under high transmembrane pressure and filtration rate conditions, the production rate of the target chemical product can be improved, while microorganisms or cultured cells into the pores of the separation membrane and Clogging of medium components is likely to occur, resulting in problems in continuous culture operation. Therefore, the transmembrane pressure difference is preferably such that the filtration rate is such that microorganisms or cultured cells and medium components are not easily clogged. Specifically, it is preferable to perform filtration treatment with a transmembrane pressure difference in the range of 0.1 kPa to 20 kPa, and more preferably in the range of 0.1 kPa to 10 kPa. When the range of the transmembrane pressure is out of the range, clogging of microorganisms or cultured cells and medium components may occur rapidly, resulting in a decrease in the permeate amount and a problem in continuous culture operation.

  In the present invention, the culture liquid refers to a liquid obtained as a result of the growth of microorganisms or cultured cells on the culture raw material, and the culture raw material refers to the target production that promotes the growth of the microorganisms or cultured cells to be cultured. It can be used to produce good chemicals. The composition of the culture raw material may be appropriately changed from the culture raw material composition at the start of the culture so that the productivity of the target chemical product is increased.

  Examples of microorganisms and cultured cells used in the present invention include yeasts such as baker's yeast, which are often used industrially, 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. 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”.

  The culture raw material may be any material as long as it can achieve the above-mentioned purpose, but a normal liquid medium appropriately containing a carbon source, a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins. Preferably used.

  Carbon sources include sugars such as glucose, sucrose, fructose, galactose and lactose, starch saccharified liquid 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 cultured 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 microorganisms require specific nutrients for growth, the nutrients can be added as preparations or natural products containing them. Moreover, an antifoamer can also be used as needed.

  The culture solution is preferably maintained at a saccharide concentration of 5 g / l or less. The reason is to minimize the loss of saccharide due to withdrawal of the culture solution. Culture of microorganisms or cultured cells is usually performed at a pH of 4 to 8 and a temperature of 20 to 50 ° C. The pH of the culture solution 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.

  And it is preferable to perform a continuous culture operation normally with a single culture reaction tank on culture management. However, the number of culture reaction tanks is not limited as long as it is a continuous culture method in which microorganisms and cultured cells are cultured to produce a product. It is also preferable to use a plurality of culture reaction tanks because the capacity of the culture reaction tank is small. In this case, high productivity of the product can be obtained even if continuous culture is performed by connecting a plurality of culture reaction tanks in parallel or in series by piping.

    In the present invention, it is also preferable to culture microorganisms or cultured cells in a separate culture tank in advance, supply a culture solution containing the cultured fresh microorganisms or cultured cells to the culture reaction tank, and continuously culture. That is, by supplying a culture solution containing microorganisms or cultured cells with a high culture capacity prepared separately to the culture reaction tank, it is possible to always maintain high production performance of chemical products.

  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, a porous membrane made of an inorganic material such as ceramics or an organic material such as resin can be used, 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 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.

  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.

  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. Examples of the polyolefin resin include polyethylene, polypropylene, chlorinated polyethylene, and chlorinated polypropylene, and chlorinated polyethylene is preferably used.

  An outline of a method for producing a porous membrane used in the present invention will be described. First, a film of a film-forming stock solution containing the resin and solvent is formed on the surface of the porous base material, and the film-forming stock solution is impregnated into the porous base material. Thereafter, only the coating-side surface of the porous substrate having a coating is brought into contact with a coagulation bath containing a non-solvent to solidify the resin and form a porous resin layer on the surface of the porous substrate. The temperature of the film-forming stock solution is usually preferably selected within a temperature range of 15 to 120 ° C. from the viewpoint of film-forming properties.

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

  The solvent dissolves the resin. The solvent acts on the resin and the pore-opening agent to encourage them to form a porous resin layer. As such a solvent, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone, and the like can be used. . Among these, NMP, DMAc, DMF and DMSO, which have high resin solubility, are preferably used.

  Furthermore, a non-solvent can also be added to the film forming stock solution. The non-solvent is a liquid that does not dissolve the resin. The non-solvent acts to control the pore size by controlling the rate of solidification of the resin. As the non-solvent, water, alcohols such as methanol and ethanol can be used. Among these, water and methanol are preferable from the viewpoint of price. The non-solvent may be a mixture thereof.

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

  As described above, the porous film used in the present invention is desirably a porous film 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 average thickness of the porous substrate is preferably selected in the range of 50 μm to 3000 μm.

  When the porous membrane is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably selected in the range of 200 μm to 5000 μm, and the thickness of the porous resin layer is preferably selected in the range of 20 μm to 2000 μm. . Further, a woven fabric or a knitted fabric in which organic fibers or inorganic fibers are formed in a cylindrical shape may be included in the hollow fiber.

  If the separation membrane has an average pore diameter in the range of 0.01 μm or more and 2 μm or less, it is possible to achieve both a high exclusion rate that prevents the microbial catalyst from leaking and a high permeability, and further, it is difficult to clog and the permeability. Can be carried out with higher accuracy and reproducibility. When bacteria are used as the microorganism, the average pore diameter of the separation membrane is preferably 0.4 μm or less, and can be suitably implemented as long as it is less than 0.2 μm. If the average pore diameter is too small, the amount of permeate may decrease. Therefore, in the present invention, it is 0.01 μm or more, 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 separation membrane that can be used in the present invention is preferably as the standard deviation σ of the pore diameter is smaller, that is, the distribution of the pore diameter is narrower. It is preferable that the pore size distribution is narrowed and the standard deviation is 0.1 μm or less. When the standard deviation of the pore diameter is small, that is, the pore diameters are uniform, a permeate having uniform characteristics can be obtained, and the operation management of the apparatus is facilitated.

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

In the separation membrane used in the present invention, the permeability of the reaction solution containing the chemical is one of the important points, and the pure water permeability coefficient of the separation membrane before use can be used as the permeability index. In the present invention, the pure water permeability coefficient of the separation membrane is 1 × 10 ⁻¹⁰ m ³ / m when calculated by measuring the water permeability at a head height of 1 m using purified water at a temperature of 25 ° C. by a reverse osmosis membrane. It is preferable that it is ² / s / pa or more. If the pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less, a practically sufficient amount of permeate can be obtained. . A more preferable pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 2 × 10 ⁻⁷ m ³ / m ² / s / pa or less.

  The membrane surface roughness of the separation membrane used in the present invention is a factor that affects the clogging of the separation membrane. When the membrane surface roughness of the separation membrane is preferably 0.1 μm or less, the separation coefficient and membrane resistance of the separation membrane can be suitably reduced, and the production of chemicals and the like can be performed with a lower transmembrane pressure difference. It is. Therefore, by suppressing clogging, it becomes possible to produce a stable chemical product or the like, so that the smaller the surface roughness is, the better.

  In addition, by reducing the membrane surface roughness of the separation membrane, it can be expected that the shearing force generated on the membrane surface will be reduced in the filtration of the microbial catalyst, the destruction of microorganisms is suppressed, and the clogging of the separation membrane is also suppressed. By doing so, it is considered that stable filtration is possible for a long period of time.

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

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

  The separation membrane as described above can be used by appropriately processing the shape according to the shape of the membrane separation tank. For example, a separation membrane element can be obtained by combining a separation membrane in the form of a flat membrane with a support prepared separately. This separation membrane element in which a separation membrane is disposed on at least one side of the support plate (on both sides of the support plate in order to increase the membrane area is difficult or to increase permeability) is suitable for the separation membrane in the present invention. Such a separation membrane element is installed in a membrane separation tank. Hereinafter, the outline will be described with reference to the drawings.

FIG. 2 is a schematic perspective view for explaining one embodiment of a separation membrane element used in the present invention. As shown in FIG. 2, the separation membrane element is configured by arranging a flow path material 14 and a separation membrane 15 in this order on both surfaces of a rigid support plate 13. The support plate 13 has recesses 16 on both sides. The separation membrane 15 filters the culture solution. The flow path member 14 is for efficiently flowing the permeate filtered through the separation membrane 15 to the support plate 13. The permeate containing the product that has flowed to the support plate 13 passes through the recess 16 of the support plate 13 and is taken out of the continuous culture apparatus via the liquid collection pipe 17 that is a discharge means. Here, a method such as pumping, suction filtration with a liquid or gas, or pressurizing the inside of the system can be used as power for extracting the permeated liquid, including the differential pressure of the head.
When the separation membrane is a hollow fiber membrane, it is processed into a separation membrane element of another form as shown in FIG. FIG. 3 is a schematic perspective view of the separation membrane element, which is mainly composed of a support plate 13, a hollow fiber-shaped separation membrane 15, an upper resin sealing layer 18 and a lower resin sealing layer 19. The separation membrane 15 is bonded and fixed to the support plate 13 in a bundle by an upper resin sealing layer 18 and a lower resin sealing layer 19. Adhesion / fixation by the lower resin sealing layer 19 has a structure in which the hollow portion of the separation membrane 15 in the form of a hollow fiber is sealed to prevent leakage of the culture solution. On the other hand, the upper resin sealing layer 18 does not seal the hollow portion of the hollow fiber form separation membrane 15, and the hollow portion and the liquid collection pipe 17 communicate with each other. This separation membrane element can be installed in the continuous culture apparatus via the support plate 13. The permeate filtered by the separation membrane 15 passes through the hollow portion of the hollow fiber membrane and is taken out to the outside of the continuous culture apparatus via the liquid collection pipe 17. As the power for taking out the permeated liquid, a method such as a water head differential pressure, a pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used.

  The membrane separation tank 12 provided with the separation membrane is desirably capable of high-pressure steam sterilization, and in this way, contamination from various germs can be avoided. The high-pressure steam sterilization of the present invention is to sterilize microorganisms or cultured cells present in the tank by heating and pressurizing the membrane separation tank with steam. As heating and pressurizing conditions, for example, it is preferable to pressurize and warm for 20 minutes or more under conditions of 121.1 ° C. and a vapor pressure of 1 atm. Therefore, the membrane separation tank 12 of the continuous culture apparatus of the present invention, the separation membrane disposed in the membrane separation tank 12, and the element constituent members should be members resistant to high-pressure steam sterilization operation under such conditions. Is preferred. Thereby, the inside of the culture reaction tank containing the separation membrane element can be sterilized. If the inside of the culture reaction vessel can be sterilized, it is possible to avoid the risk of contamination by undesirable microorganisms during continuous culture, and stable continuous culture is possible.

  If the separation membrane element and the support plate constituting the separation membrane element are resistant to the conditions of high-pressure steam sterilization operation, such as 121.1 ° C. and a vapor pressure of 1 atm for 20 minutes or more, the separation membrane In addition, the type of element constituent member is not particularly limited. As the material of the separation membrane having such resistance, the above-mentioned porous membrane material can be used. In addition, as an element constituent member such as a support plate, for example, a metal such as stainless steel or aluminum, or polyamide resin, fluorine resin, polycarbonate resin, polyacetal resin, polybutylene terephthalate resin, PVDF, modified polyphenylene ether Resins and polysulfone resins can be preferably selected.

  The chemical product produced by the continuous culture apparatus of the present invention is not particularly limited as long as it is a substance produced by the microorganism or cultured cell in the culture solution. For example, industrial products such as alcohol, organic acid, amino acid, and nucleic acid are used. Can be mentioned in mass production. Examples of the alcohol include ethanol, 1,3-propanediol, 1,4-butanediol, and glycerol. Examples of organic acids include acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, and citric acid, and examples of nucleic acids include nucleosides such as inosine and guanosine, and nucleotides such as inosinic acid and guanylic acid. Can be mentioned. Other examples of chemicals include diamine compounds such as cadaverine, antibiotics, enzymes, and recombinant proteins. By using the continuous culture apparatus of the present invention, high productivity of the chemical product can be obtained.

  Here, the production rate in continuous culture is calculated by the following equation (3).

  In addition, the production rate of chemicals in batch culture is determined by determining the amount of product (g) at the time of consumption of the raw carbon source, 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 by.

  Hereinafter, the continuous culture apparatus of the present invention will be described in detail with reference to examples. Specifically, L-lactic acid as a chemical, yeast having a L-lactic acid production ability in microorganisms or cultured cells (Reference Example 1), and a porous membrane as a separation membrane (Reference Example 2: flat membrane, Reference Example 3: hollow) 1 is selected, and the continuous culture apparatus shown in FIG. 1 is used to produce a continuous chemical product in which the culture liquid volume ratio of the culture reaction tank to the membrane separation tank of the continuous culture apparatus is changed in the range of 5 to 100 Will be described with reference to examples.

(Reference Example 1) Production of Yeast Strain with Lactic Acid Production Capacity (SW-1 Strain) A yeast strain with 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) 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 the plasmid DNA was recovered 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 fragment was inserted into the XhoI / NotI cleavage site of the yeast expression vector pTRS11 (FIG. 4). Thus, a human-derived L-ldh gene expression plasmid pL-ldh5 was obtained. The above-mentioned pL-ldh5, which is a human-derived L-ldh gene expression vector, is FERM AP- in the independent biological corporation National Institute of Advanced Industrial Science and Technology Patent Biology Depositary Center (1-1-1, Chuo 6-1-1, Tsukuba, Ibaraki). Deposited as 20421 (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 and the 1.2 kb fragment obtained here as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 7 as a primer set was obtained as 1 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 by electrophoresis on 5% agarose gel. With this 2.5 kb DNA fragment, the budding yeast strain NBRC10505 was transformed to 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. Confirmed by 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 copper sulfate aqueous solution ・ Flow rate: 1.0 ml / min
・ Detection method: UV254nm
-Temperature: 30 degreeC.

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

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

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

(Reference Example 2) Production of porous flat membrane (Part 1)
Polyvinylidene fluoride (PVDF) resin was used as a resin and N, N-dimethylacetamide (DMAc) was used as a 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.

The porous membrane was peeled off from the glass plate and then immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc to obtain 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 pure water permeation amount was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the pore diameter was 0.035 μm, and the membrane surface roughness was 0.06 μm.

Reference Example 3 Production of Porous Hollow Fiber Membrane A vinylidene fluoride homopolymer having a weight average molecular weight of 417,000 and γ-butyrolactone were dissolved at a temperature of 170 ° C. at a ratio of 38% by weight and 62% by weight, respectively. Stock solutions were made. 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 (Sanyo Kasei Co., Ltd., trade name Ionette T-20C) was mixed and dissolved at a temperature of 95 ° C. at a rate of 5% by weight and 3% by weight of water to prepare a stock solution. . This stock solution was uniformly applied to the surface of the hollow fiber membrane obtained above, and a porous hollow fiber membrane used in the present invention was immediately solidified in a water bath. The average pore diameter of the surface of the treated water side of the obtained porous hollow fiber membrane was 0.05 μm. Next, the pure water permeation rate of the porous hollow fiber membrane as the separation membrane was evaluated and found to be 5.5 × 10 ⁻⁹ m ³ / m ² · s · Pa. The amount of permeated liquid was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the pore diameter was 0.006 μm.

Example 1 Production of L-lactic acid by continuous culture (Part 1)
Using the yeast SW-1 strain, the continuous culture apparatus shown in the schematic diagram of FIG. 1, and the lactic acid production medium having the composition shown in Table 1, L-lactic acid was produced by continuous culture. In Example 1, a 2 L culture reaction tank and a 150 ml membrane separation tank were used, and the separation membrane element shown in FIG. 2 equipped with the porous flat membrane prepared in Reference Example 2 was used as the separation membrane element.

  Note that the amount of the culture solution filled in the culture reaction tank and the membrane separation tank during the culture operation is 1.5 L and 100 mL (culture solution volume ratio 15), respectively, and the amount of the culture solution in the continuous culture apparatus (excluding the inside of the pipe) is 1. Continuous culture was performed to obtain 6 L. The lactic acid production medium was used after autoclaving at 121 ° C. for 15 minutes. For the base member of the separation membrane element, a molded product of stainless steel and polysulfone resin was used.

The operating conditions in Example 1 were as follows.
-Culture reactor capacity: 2 (L) (1.5 L as the culture volume during operation)
・ Membrane separation tank capacity: 150 (mL) (100 mL as culture volume during operation)
-Separation membrane used: flat membrane (see Reference Example 2)
・ Membrane separation element effective filtration area: 120 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of culture reaction tank: 0.05 (L / min)
-Stirring speed of culture reaction tank: 100 (rpm)
-PH adjustment: adjusted to pH 5 with 1N NaOH-Lactic acid production medium supply rate: 50-300 ml / hr. Circulated fluid volume with variable control and culture fluid circulation pump in the range: 0.1 (L / min)
-Membrane permeate volume control: flow rate control by transmembrane differential pressure (after continuous culture up to 100 hours: controlled at transmembrane differential pressure of 0.1 kPa to 5 kPa 100 hours to 200 hours: transmembrane differential pressure of 0.1 kPa to 2 kPa Control at 200 hours to 300 hours: Control at a transmembrane pressure difference of 0.1 kPa to 20 kPa)
More specifically, first, the SW-1 strain was cultured with shaking overnight in a test tube using 5 ml of lactic acid production medium (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh lactic acid production medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution was inoculated in a 1.5 L lactic acid production medium of the membrane separation type continuous culture apparatus shown in FIG. 1 and the culture reaction tank 1 was stirred at 400 rpm by the attached stirrer 2. While adjusting the aeration amount, adjusting the temperature, and adjusting the pH, the culture medium circulation pump 5 was operated and the culture was performed for 24 hours (pre-culture). Immediately after completion of the preculture, the culture medium circulation pump 5 was operated. In addition to the operating conditions at the time of pre-culture, the medium is continuously supplied, and the continuous culture is performed while controlling the amount of the membrane permeate so that the total culture volume in the continuous culture apparatus is 2 L. Manufactured.

  Control of the amount of membrane permeate when performing continuous culture was performed by measuring the head differential as the transmembrane differential pressure with the head differential pressure controller 4 and changing it under the above-mentioned membrane permeate control conditions. As a result, the transmembrane pressure difference during the continuous culture was maintained at 2 kPa or less. The produced L-lactic acid concentration and residual glucose concentration in the membrane permeation culture solution were measured as appropriate. The HPLC shown in Reference Example 1 was used for evaluation of the concentration of L-lactic acid, and “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measurement of the glucose concentration.

  Table 2 shows the results of continuous culture for 300 hours under the above conditions. As a result, it was confirmed that stable L-lactic acid production by continuous culture was possible by using the continuous culture apparatus of FIG.

(Example 2) Production of L-lactic acid by continuous culture (part 2)
Using the porous membrane produced in Reference Example 2 as the separation membrane, L-lactic acid was produced by continuous culture in the same manner as in Example 1. However, in Example 2, a 2 L culture reaction tank and a 400 ml volume membrane separation tank were used, and the amount of the culture solution filled in the culture reaction tank and the membrane separation tank during the culture operation was 1.5 L and 300 mL, respectively. 5), and continuous culture was performed so that the amount of the culture solution in the continuous culture apparatus (excluding the inside of the pipe) was 1.8 L. Also in Example 2, the transmembrane pressure difference during the continuous culture period changed as a result of 2 kPa or less. The results are shown in Table 2. As a result, it was confirmed that stable L-lactic acid production by continuous culture of stable L-lactic acid was possible.

(Example 3) Production of L-lactic acid by continuous culture (part 3)
Using the porous membrane produced in Reference Example 2 as the separation membrane, L-lactic acid was produced by continuous culture in the same manner as in Example 1. However, in Example 3, a 2 L culture reaction tank and a 100 ml membrane separation tank were used, and the volume of the culture solution filled in the culture reaction tank and the membrane separation tank during continuous culture operation was 1.5 L and 50 mL, respectively. The ratio was 30), and the continuous culture was performed so that the amount of the culture solution in the continuous culture apparatus (excluding the pipe) was 1.55 L. Also in Example 3, the transmembrane pressure difference during the period of continuous culture changed as a result of 2 kPa or less. The results are shown in Table 2. As a result, it was confirmed that stable L-lactic acid production by continuous culture was possible.

Comparative Example 1 Production of L-lactic acid by continuous culture (Part 1)
Using the porous membrane produced in Reference Example 2 as the separation membrane, continuous culture similar to Example 1 was performed. However, in Comparative Example 1, a 2 L culture reaction tank and a 500 ml membrane separation tank were used, and the amount of the culture solution filled in the culture reaction tank and the membrane separation tank during the culture operation was 1.5 L and 500 mL, respectively. 3), and the continuous culture was performed so that the amount of the culture solution in the continuous culture apparatus (excluding the inside of the pipe) was 2L. Also in Comparative Example 1, the transmembrane pressure difference during the continuous culture period changed as a result of 2 kPa or less. The results are shown in Table 2.

(Example 4) Production of L-lactic acid by continuous culture (part 4)
The yeast SW-1 strain, the continuous culture apparatus shown in the schematic diagram of FIG. 1, and the yeast lactic acid production medium having the composition shown in Table 2 were used for continuous culture. In Example 4, a 2 L culture reaction tank and 150 ml membrane separation tank were used, and the separation membrane element shown in FIG. 3 equipped with the porous hollow fiber membrane prepared in Reference Example 3 was used as the separation membrane element. It was.

  The volume of the culture solution filled in the culture reaction tank and the membrane separation tank during continuous culture operation is 1.5 L and 100 mL (culture solution volume ratio of 15), respectively. A continuous culture test was conducted so as to obtain 1.6 L. The yeast lactic acid production medium was used after autoclaving at 121 ° C. for 15 minutes. For the base member of the separation membrane element, a molded product of stainless steel and polysulfone resin was used.

Moreover, the operating conditions in Example 4 were as follows.
-Culture reactor capacity: 2 (L) (1.5 L as the culture volume during operation)
・ Membrane separation tank capacity: 150 (mL) (100 mL as culture volume during operation)
-Separation membrane used: hollow fiber membrane (see Reference Example 3)
・ Membrane separation element effective filtration area: 120 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of culture reaction tank: 0.05 (L / min)
-Stirring speed of culture reaction tank: 100 (rpm)
-PH adjustment: adjusted to pH 5 with 1N NaOH-Lactic acid production medium supply rate: 50-300 ml / hr. Circulated fluid volume by variable control and culture fluid circulation pump: 0.1 (L / min)
-Membrane permeate volume control: flow rate control by transmembrane pressure difference (after continuous culture up to 100 hours: control at transmembrane pressure difference 0.1 kPa to 5 kPa 100 hours to 200 hours: transmembrane pressure difference 0.1 kPa to 2 kPa Control at 200 hours to 300 hours: Control at a transmembrane pressure difference of 0.1 kPa to 20 kPa)
More specifically, first, the SW-1 strain was cultured with shaking overnight in a test tube using 5 ml of lactic acid production medium (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh lactic acid production medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L lactic acid production medium of the continuous culture apparatus shown in FIG. 1 and the culture reaction tank 1 is stirred at 400 rpm by the attached stirrer 2 so that the aeration volume of the culture reaction tank 1 is adjusted. While performing the adjustment, temperature adjustment and pH adjustment, the culture medium circulation pump 5 was operated for 24 hours (preculture). Immediately after completion of the preculture, the culture medium circulation pump 5 was operated. In addition to the operating conditions at the time of pre-culture, the lactic acid production medium is continuously supplied, and the continuous culture is performed while controlling the amount of the membrane permeate so that the total culture volume in the continuous culture apparatus is 2 L. Lactic acid was produced.

  Control of the amount of membrane permeate when performing continuous culture was performed by measuring the head differential as the transmembrane differential pressure with the head differential pressure controller 4 and changing it under the above-mentioned membrane permeate control conditions. As a result, the transmembrane pressure difference during the continuous culture was maintained at 2 kPa or less. The produced L-lactic acid concentration and residual glucose concentration in the membrane permeation culture solution were measured as appropriate. The HPLC shown in Reference Example 1 was used to evaluate the concentration of L-lactic acid, which is a chemical product, and “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used to measure the glucose concentration. It was.

  Table 3 shows the results of continuous culture for 300 hours under the above conditions. As a result, it was confirmed that stable L-lactic acid production by continuous culture was possible by using the continuous culture apparatus of FIG.

(Example 5) Production of L-lactic acid by continuous culture (5)
Using the porous hollow fiber membrane prepared in Reference Example 3 as a separation membrane, continuous culture similar to Example 4 was performed. However, in Example 5, a 2 L culture reaction tank and a 100 ml membrane separation tank were used, and the amount of the culture solution filled in the culture reaction tank and the membrane separation tank during the culture operation was 1.5 L and 50 mL, respectively. 30), and continuous culture was performed so that the amount of the culture solution in the continuous culture apparatus (excluding the pipe) was 1.55 L. Also in Example 5, the transmembrane pressure difference during the period of continuous culture changed as a result of 2 kPa or less. The results are shown in Table 3. As a result, it was confirmed that stable L-lactic acid production by continuous culture was possible.

(Example 6) Production of L-lactic acid by continuous culture (Part 6)
Using the porous hollow fiber membrane prepared in Reference Example 3 as a separation membrane, continuous culture similar to Example 4 was performed. However, in Example 6, a 2 L culture reaction tank and a 50 ml membrane separation tank were used, and the volume of the culture solution filled in the culture reaction tank and the membrane separation tank during continuous culture operation was 1.5 L and 15 mL, respectively. The ratio was 100), and the continuous culture was performed so that the amount of the culture solution in the continuous culture apparatus (excluding the pipe) was 1.515 L. Also in Example 6, the transmembrane pressure difference during the period of continuous culture changed as a result of 2 kPa or less. The results are shown in Table 3. As a result, it was confirmed that stable L-lactic acid production by continuous culture was possible.

Comparative Example 2 Production of L-lactic acid by continuous culture (part 2)
Using the porous hollow fiber membrane prepared in Reference Example 3 as a separation membrane, continuous culture similar to Example 4 was performed. However, in Comparative Example 2, a 2 L culture reaction tank and a 500 ml membrane separation tank were used, and the volume of the culture solution filled in the culture reaction tank and the membrane separation tank during continuous culture operation was 1.5 L and 500 mL, respectively. The ratio was 3), and the continuous culture was performed so that the amount of the culture solution in the continuous culture apparatus (excluding the inside of the pipe) was 2L. Also in Comparative Example 2, the transmembrane pressure difference during the period of continuous culture changed as a result of 2 kPa or less. The results are shown in Table 3.

From the continuous culture results of Examples 1 to 6 and Comparative Examples 1 and 2, the culture solution volume ratio of the culture reaction tank to the membrane separation tank is in the range of 5 to 100 as compared with the conventional continuous culture apparatus. It has been clarified that, while realizing a more compact size, the residence time of the culture solution in the culture reaction tank becomes longer and adjustment to appropriate culture conditions is realized, thereby improving the production rate of chemical products.

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

Explanation of symbols

1 Culture Reaction Tank 2 Stirrer 3 Separation Membrane (Element)
4 Hydrohead differential pressure control device 5 Culture fluid circulation pump 6 Level sensor 7 Temperature controller 8 Medium supply pump 9 pH adjustment solution supply pump 10 pH sensor / control device 11 Gas supply device 12 Membrane separation tank 13 Support plate 14 Channel material 15 Separation membrane 16 Concave portion 17 Liquid collecting pipe 18 Upper resin sealing layer 19 Lower resin sealing layer

Claims (3)

  A culture reaction tank for culturing microorganisms or cultured cells, a membrane separation tank in which a separation membrane for filtering the culture solution continuously supplied from the culture reaction tank is disposed, and the culture solution from the culture reaction tank A culture medium circulation means for supplying unfiltered culture medium that has been supplied to the membrane separation tank and refluxed to the culture medium upstream of the membrane separation tank; and the membrane separation tank and the culture reaction tank Is a continuous culture apparatus characterized in that the culture solution volume ratio of the culture solution in the culture reaction vessel to the culture solution in the membrane separation vessel has a volume of 5 or more and 100 or less.   The continuous culture apparatus according to claim 1, wherein the membrane separation tank and the culture reaction tank have a volume in which the culture liquid volume ratio is 15 or more and 100 or less.   A microorganism or cultured cell is cultured in a culture reaction tank, and the culture solution is continuously supplied from the culture reaction tank to the membrane separation tank and filtered through the separation membrane to recover the product. Is refluxed to the culture medium upstream of the membrane separation tank, the culture liquid volume ratio of the culture liquid in the culture reaction tank to the culture liquid in the membrane separation tank is 5 or more and 100 or less. Manufacturing method of chemical products.

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