JP5130811B2 - Process for producing 1,3-propanediol by continuous fermentation - Google Patents

Description

  The present invention efficiently filters and collects a liquid containing a product through a porous separation membrane that is less likely to be clogged than a culture liquid of microorganisms or cultured cells and returns an unfiltered liquid to the culture liquid while culturing. It is related with the manufacturing method of 1, 3- propanediol characterized by obtaining high productivity by improving the microbe density | concentration in connection with fermentation.

  The technical background regarding the method for producing 1,3-propanediol will be described. 1,3-propanediol is a chemical used in the field of synthetic fibers and is extremely useful. If this 1,3-propanediol can be produced efficiently, it can be expected that the supply of the fibers will be easier. On the other hand, as for 1,3-propanediol, a chemical synthesis method using a catalyst from 3-hydroxypropanol (Patent Document 1) is known. However, the price of oil has risen, it has escaped from an oil-dependent society, Due to social needs such as conversion to carbon-neutral raw materials, production from oil-independent raw materials is being studied. There is a resting cell method (Patent Document 2) in which 1,3-propanediol is converted from glycerol by Klebsiella cells. Since glucose cannot be produced by this method, which is an inexpensive fermentation raw material, a study of culture (Patent Document 3) in which glycerol is fermented with yeast and glycerol to 1,3-propanediol is produced with Klebsiella (Patent Document 3) is also conducted. It has been broken.

  In addition, a fermentation method using one kind of microbial cells, which is the simplest method, has been studied, and there are fermentation methods using recombinant E. coli (Non-patent Documents 1 and 4).

However, the production of 1,3-propanediol by fermentation is only related to culture and fed-batch (fed-batch), and any 1,3-propanediol production method is not sufficient in production rate. Therefore, a more efficient production method has been desired.
Japanese Patent Laid-Open No. 11-515021 JP 2005-304362 A Japanese National Patent Publication No. 10-507082 Japanese Patent Publication No. 2003-507022 Charles E Nakamura et.al. (Current Opinion in Biotechnology), 14, 454-459 (2003)

  The subject in this invention is providing the manufacturing method of 1, 3- propanediol by the continuous fermentation method which maintains high productivity stably over a long time with a simple operation method.

  As a result of diligent research, the inventors of the present application are able to significantly suppress clogging of membranes when microorganisms and cells are less likely to enter the separation membrane and the microorganisms and cells are filtered under low transmembrane pressure. As a result, it was possible to maintain the stable filtration of high-concentration microorganisms, which was a problem, and completed the present invention.

That is, the present invention separates a culture solution of microorganisms or cultured cells having the ability to produce 1,3-propanediol into a filtrate and an unfiltered solution by a separation membrane, and collects a desired fermentation product from the filtrate. In a method for producing 1,3-propanediol by a continuous fermentation method in which an unfiltered liquid is retained or refluxed, a porous membrane having an average pore diameter in the range of 0.01 μm or more and less than 1 μm is used as a separation membrane. Thus, the present invention provides a method for producing 1,3-propanediol, which can stably improve the fermentation production efficiency at a low cost by performing filtration with a low transmembrane pressure.

  According to the present invention, continuous fermentation that stably maintains a high productivity of a desired fermentation product over a long period of time can be performed under simple operation conditions. Propanediol can be stably produced at low cost.

  1,3-propanediol obtained by the method for producing 1,3-propanediol of the present invention is useful as a raw material for synthetic fibers, for example.

In the present invention, a culture solution of microorganisms or cultured cells having the ability to produce 1,3-propanediol is filtered through a separation membrane, and the product is recovered from the filtrate and the unfiltered solution is retained or refluxed in the culture solution. In addition, continuous fermentation in which a fermentation raw material is added to a culture solution, and a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm is used as a separation membrane, and a transmembrane pressure difference is in a range of 0.1 to 20 kPa. It is a manufacturing method of 1, 3- propanediol by continuous fermentation characterized by performing a filtration process.

  It is desirable that the porous membrane used in the present invention is less likely to be clogged by microorganisms and cultured cells used for fermentation, and the filtration performance continues stably for a long period of time.

  The porous membrane used in the present invention has an average pore diameter of 0.01 μm or more and less than 1 μm. When the porous resin layer is present on both surfaces of the porous membrane, it is sufficient that at least one of the porous resin layers satisfies this condition. If the average pore size is within this range, it is possible to achieve both a high exclusion rate that does not cause leakage of bacterial cells and sludge, and high water permeability, and it is more difficult to clog and maintain water permeability for a long time. It can be carried out with high accuracy and reproducibility. If the average pore diameter is within this range, when animal cells, yeast, filamentous fungi, or the like are used, clogging is small, and continuous fermentation can be performed stably without leakage of the cells into the filtrate. When bacteria smaller than animal cells, yeasts, and filamentous fungi are used, the average pore size is preferably 0.4 μm or less, and the average pore size is less than 0.2 μm, and can be suitably implemented. is there. If the average pore diameter is too small, the water permeation rate may decrease. Therefore, it is usually preferably 0.01 μm or more, more preferably 0.02 μm or more, and further preferably 0.04 μm or more. Here, the average pore diameter can be obtained by measuring and averaging the diameters of all pores that can be observed within a range of 9.2 μm × 10.4 μm in a scanning electron microscope observation at a magnification of 10,000 times. it can. The standard deviation σ of the pore diameter is preferably 0.1 μm or less. The standard deviation σ of the pore diameter is Nk, which is the number of pores that can be observed within the above-mentioned range of 9.2 μm × 10.4 μm, and the measured diameter is Xk, and the average pore diameter is X (ave). It computed from Formula (1) below.

In the porous membrane used in the present invention, the permeability of the culture solution is one of the important points, and the pure water permeability coefficient of the porous membrane before use can be used as the permeability index. When the pure water permeability coefficient of the porous membrane was calculated by measuring the water permeability at a head height of 1 m using purified water at 25 ° C. by a reverse osmosis membrane, 2 × 10 ⁻⁹ m ³ / m ² / s / pa Preferably, the permeated water amount is practically sufficient if it is 2 × 10 ⁻⁹ or more and 6 × 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. Preferably, when the surface roughness is 0.1 μm or less, the separation coefficient and membrane resistance of the separation membrane are reduced. And continuous fermentation can be carried out with a lower transmembrane pressure difference. In addition, the low surface roughness can be expected to reduce the shearing force generated on the membrane surface during filtration of microorganisms and cultured cells, the destruction of microorganisms or cultured cells is suppressed, and the porous membrane is clogged. It is considered that stable filtration is possible for a long time by being suppressed. Here, the surface roughness can be measured under the following conditions using the following atomic force microscope (AFM).

Equipment Atomic Force Microscope (Nanoscope IIIa from Digital Instruments)
Conditions Probe SiN cantilever (Digital Instruments)
Scan mode Contact mode (in-air measurement)
Underwater tapping mode (underwater measurement)
Scanning range 10μm, 25μm square (measurement in air)
5μm, 10μm square (underwater measurement)
Scanning resolution 512 × 512
Sample preparation In the measurement, the membrane sample was immersed in ethanol at room temperature for 15 minutes, then immersed in RO water for 24 hours, washed, and then air-dried.

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

  The material of the porous membrane in the present invention is not particularly limited as long as separation performance and water permeation performance corresponding to the quality of water to be treated and the use can be obtained, but from the viewpoint of separation performance such as blocking performance, water permeation performance and dirt resistance. Is preferably a porous film including a porous resin layer.

  The porous membrane including the porous resin layer preferably has a porous resin layer that acts as a separation functional layer on the surface of the porous substrate. The porous substrate supports the porous resin layer and gives strength to the separation membrane.

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

  The average thickness of the porous substrate is preferably 50 μm or more and 3000 μm or less.

  The material of the porous membrane in the present invention is a woven fabric or nonwoven fabric using organic fibers such as cellulose fiber, cellulose triacetate fiber, polyester fiber, polypropylene fiber, polyethylene fiber, etc., a porous substrate made of an inorganic material, and porous It may be formed from a resin layer. As a separation membrane for realizing the present invention, an organic polymer membrane can be used. The organic polymer membrane is a separation membrane basically composed of an organic polymer material, for example, an organic fiber. In many cases, a non-woven fabric or a macroporous structure porous organic base material and a porous resin layer having a pore size smaller than the pore size of the porous organic base material are combined.

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

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

  In the case where the porous membrane is a flat membrane, the thickness is selected according to the application, but is selected in the range of, for example, 20 μm to 5000 μm, preferably 50 μm to 2000 μm. As described above, it 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 selected in the range of 50 μm to 3000 μm.

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

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

  A film of a stock solution containing a resin and a solvent is formed on the surface of the porous base material, and the porous base material is impregnated with the stock solution. Thereafter, only the coating-side surface of the porous substrate having a coating is brought into contact with a coagulation bath containing a non-solvent to solidify the resin, and a porous resin layer is formed on the surface of the porous substrate.

  The stock solution is prepared by dissolving the resin in a solvent. The temperature of the stock solution is usually preferably selected within the range of 5 to 120 ° C. from the viewpoint of film forming properties. The solvent dissolves the resin and acts on the resin to encourage them to form a porous resin layer. Examples of the solvent include N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), N-methyl-2-pyrrolidone, methyl ethyl ketone, tetrahydrofuran, Tetramethylurea, trimethyl phosphate, cyclohexanone, isophorone, γ-butyrolactone, methyl isoamyl ketone, dimethyl phthalate, propylene glycol methyl ether, propylene carbonate, diacetone alcohol, glycerol triacetate, acetone, and methyl ethyl ketone can be used. Of these, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO), which have high resin solubility, are preferably used. These solvents may be used alone or in combination of two or more.

  Further, for example, components other than the solvent such as polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone and glycerin may be added to the solvent. Non-solvents can also be added to the solvent. The non-solvent is a liquid that does not dissolve the resin. The non-solvent acts to control the pore size by controlling the rate of solidification of the resin. As the non-solvent, water and alcohols such as methanol and ethanol can be used. Of these, water or methanol is preferably used as the non-solvent from the viewpoint of cost. Components other than the solvent and the non-solvent may be a mixture.

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

  Next, an example of the outline of a method for producing a hollow fiber membrane among the porous membranes will be described as an example.

  The hollow fiber membrane discharges a stock solution composed of a resin and a solvent from the outer tube of the double-tube base, and discharges a hollow portion forming fluid from the inner tube of the double-tube base in a cooling bath. It can be produced by cooling and solidifying.

  The stock solution can be prepared by dissolving the resin described in the above-described method for producing a flat film at a concentration of 20% by weight or more and 60% by weight or less in the solvent described in the above-described method for forming a flat film. Moreover, a gas or a liquid can be normally used for the hollow portion forming fluid. In addition, a new porous resin layer can be coated (laminated) on the outer surface of the obtained hollow fiber membrane. Lamination can be performed in order to change the properties of the hollow fiber membrane, such as hydrophilicity / hydrophobicity or pore diameter, to desired properties. A new porous resin layer to be laminated can be produced by bringing a stock solution obtained by dissolving a resin in a solvent into contact with a coagulation bath containing a non-solvent to coagulate the resin. As the material of the resin, for example, the same material as that of the organic polymer film is preferably used. As a lamination method, the hollow fiber membrane may be immersed in the stock solution, or the stock solution may be applied to the surface of the hollow fiber membrane. After the lamination, a part of the attached stock solution is scraped off or an air knife is used. It is also possible to adjust the stacking amount by blowing it away.

  The porous membrane of the present invention can be made into a membrane separation element by combining with a support. A porous 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 one of the preferred forms of the membrane element of the present invention. In this embodiment, since it is difficult to increase the membrane area, it is also preferable to dispose a porous membrane on both sides of the support plate in order to increase the water permeability.

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

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. Moreover, when the pure water permeation coefficient before use was calculated by measuring the amount of water permeation at a head height of 1 m using purified water at 25 ° C. by a reverse osmosis membrane, 2 × 10 ⁻⁹ m ³ / m ² / s / It is preferably at least pa, and more preferably within a range of 2 × 10 ⁻⁹ m ³ / m ² / s / pa to 6 × 10 ⁻⁷ m ³ / m ² / s / pa.

  The fermentation raw material used in the present invention is not limited as long as it promotes the growth of microorganisms to be cultured and can successfully produce 1,3-propanediol, which is the target fermentation product. Ordinary liquid media containing inorganic salts and organic micronutrients such as amino acids and vitamins as appropriate are suitable. Carbon sources include sugars such as glucose, sucrose, fructose, galactose, lactose, starch saccharified solution 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 is added as a preparation or a natural product containing it. An antifoaming agent is also used as necessary. In the present invention, the culture liquid 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 high productivity of the desired 1,3-propanediol. Thus, the fermentation raw material composition at the start of culture may be appropriately changed.

  In the present invention, the saccharide concentration in the culture solution is preferably maintained at 5 g / l or less. The reason is to minimize the loss of saccharide due to withdrawal of the culture solution. Microorganisms are usually cultured in the range of pH 4-8 and temperature 20-40 ° C. The pH of the culture solution is adjusted to a predetermined value within the above range with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, or the like. If it is necessary to increase the oxygen supply rate, 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 initial stage of the culture of the present invention, batch culture or fed-batch culture may be performed to increase the microbial concentration, and then continuous culture (pulling) may be started. You can go. 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. The concentration of microorganisms or cultured cells in the culture solution should be maintained at a high level so that the environment of the culture solution is not suitable for the growth of microorganisms or cultured cells and the rate of death does not increase. As an example, good production efficiency can be obtained by maintaining the dry weight at 5 g / L or more.

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

  The microorganisms and cultured cells used in the present invention are not particularly limited as long as they can produce 1,3-propanediol. For example, wild-type strains have the ability to synthesize 1,3-propanediol from glycerol. Examples include microorganisms belonging to the genus Klebsiella, the genus Clostridium, and the genus Lactobacillus.

  In the present invention, the microorganism comprises (a) at least one gene encoding a polypeptide having glycerol dehydratase activity; (b) at least one gene encoding a glycerol dehydratase reactivating factor; and (c) 3-hydroxypropion It preferably contains at least one gene encoding a non-specific catalytic activity that converts an aldehyde to 1,3-propanediol. In the present invention, more preferably, the microorganism is a recombinant microorganism capable of producing 1,3-propanediol.

In the present invention, a microorganism having the ability to synthesize 1,3-propanediol from glycerol is good Mashiku is Klebsiella (Klebsiella), cross tree indium (Clostridium), Lactobacillus (Lactobacillus), Shitorobakuteru (Cytrobacter), Enterobaku Enterobacter, Aerobacter, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Zygosaccharomyces Hanse Hansenula, Debaryomyces, Mucor, Toluropsis, Methylobacter, Salmonella, Bacillus, Aerobacter, S And a recombinant microorganism selected from the group consisting of Pseudomonas, more preferably Escherichia coli.

  In addition, it is preferable that the recombinant microorganism be improved so that 1,3-propanediol can be efficiently produced from glucose. The recombinant microorganism comprises, for example, (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; and (b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity. A recombinant microorganism containing a gene is preferred. More preferably, the glycerol dehydratase reactivating factor is a recombinant microorganism containing genes encoded by orfX and orfZ isolated from dha regulon. Furthermore, the recombinant microorganism is more preferably a recombinant microorganism that lacks glycerol kinase activity and / or glycerol dehydrogenase activity and / or triose phosphate isomerase activity.

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

  One example of a continuous fermentation apparatus used in the production method of the present invention is mainly a fermentation reaction vessel for holding microorganisms or cultured cells and producing 1,3-propanediol, and filtering the microorganisms or cultured cells and the fermentation liquor. It is composed of a membrane separation element including a porous membrane for separation. The membrane separation element may be installed either inside or outside the fermentation reaction tank. The production method of the present invention is characterized in that a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm is used, and filtration treatment is performed in a range of a transmembrane differential pressure as a filtration pressure of 0.1 to 20 kPa. . Therefore, there is no need to keep the inside of the fermenter in a pressurized state, so there is no need for a power means for circulating the fermented liquid between the filtration separator and the fermenter, and a membrane separation element is installed inside the fermenter for fermentation. It is possible to make the apparatus compact.

  Of the continuous fermentation apparatus used in the production method of the present invention, the separation element is shown in the schematic diagram of FIG. 1 as a representative example installed in the fermentation reaction tank. In FIG. 1, 1 is a fermentation reaction tank, and 2 is a membrane separation element. Here, a porous membrane is incorporated in the membrane separation element 2. As this porous membrane, for example, the membrane and membrane element disclosed in International Publication No. 2002/064240 pamphlet can be used.

  Next, the form of continuous fermentation by the continuous fermentation apparatus of FIG. 1 will be described.

  The medium is continuously or intermittently charged into the fermentation reaction tank 1 by the medium supply pump 7. The medium can be subjected to heat sterilization, heat sterilization, or sterilization using a filter as necessary before addition. During fermentation production, the fermented liquid in the fermentation reaction tank 1 is agitated by the agitator 5 as necessary, and the necessary gas is supplied by the gas supply device 4 as necessary, and the pH sensor / control is performed as necessary. Fermentative production with high productivity can be performed by adjusting the pH of the fermentation broth with the apparatus 9 and the pH adjusting solution supply pump 8, and adjusting the temperature of the fermented liquor with the temperature controller 10 as necessary. Here, the adjustment of the physicochemical conditions of the fermented liquor by the instrumentation / control device is exemplified by the pH and temperature, but if necessary, the dissolved oxygen and ORP are controlled, and further by an analytical device such as an online chemical sensor The concentration of 1,3-propanediol in the fermentation broth can be measured, and the physicochemical conditions can be controlled using it as an index. Further, the form of continuous or intermittent addition of the medium is not particularly limited, but the medium input amount and speed are appropriately determined using the measured value of the physicochemical environment of the fermentation broth by the instrumentation device as an index. Can be adjusted.

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

  FIG. 2 is a schematic side view for explaining an example of another continuous fermentation apparatus used in the method for producing 1,3-propanediol of the present invention. FIG. 2 is a typical example of a continuous fermentation apparatus in which the membrane separation element is installed outside the fermentation reaction tank.

  In FIG. 2, the continuous fermentation apparatus basically includes a fermentation reaction tank 1, a membrane separation tank 12 including a membrane separation membrane element 2, and a water head difference control device 3. Here, a porous membrane is incorporated in the separation membrane element 2. As the porosity and the membrane separation element, it is preferable to use, for example, a separation membrane and a membrane separation element disclosed in International Publication No. 2002/064240 pamphlet. Further, the membrane separation tank 12 is connected to the fermentation reaction tank 1 via the culture solution circulation pump 11.

  In FIG. 2, the medium is introduced into the fermentation reaction tank 1 by the medium supply pump 7, the culture solution in the fermentation reaction tank 1 is stirred by the stirrer 5 as necessary, and the gas supply device 4 is used as necessary. Necessary gas can be supplied. At this time, the supplied gas can be recovered and recycled and supplied again by the gas supply device 4. If necessary, the pH of the fermentation broth is adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8, and the temperature of the fermentation broth is adjusted by the temperature controller 10 as necessary. Fermentative production with high productivity can be performed. Furthermore, the culture solution in the apparatus is circulated between the fermentation reaction tank 1 and the membrane separation tank 12 by the culture solution circulation pump 11. The culture solution containing the fermentation product is filtered and separated into the microorganism and the fermentation product by the membrane separation element 2, and the fermentation product can be taken out from the apparatus system. Moreover, the microorganisms filtered and separated remain in the apparatus system, so that the microorganism concentration in the apparatus system can be maintained high, and fermentation production with high productivity is possible.

  Here, the filtration / separation by the membrane separation element 2 can be performed by the water head differential pressure with the water surface of the membrane separation tank 12, and no special power is required. If necessary, the filtration / separation speed of the membrane separation element 2 and the amount of culture solution in the apparatus system can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3. If necessary, the required gas can be supplied into the membrane separation tank 12 by the gas supply device 4. At this time, the supplied gas can be recovered and recycled and supplied again into the membrane separation tank 12 by the gas supply device 4.

  The filtration / separation by the membrane separation element 2 may be performed by suction filtration using a pump or the like, or by pressurizing the inside of the apparatus system, if necessary. In addition, microorganisms or cultured cells can be cultured for continuous fermentation in a separate culture tank (not shown), and supplied to the fermentation reaction tank as necessary. By culturing microorganisms or cultured cells for continuous fermentation in a separate culture tank and supplying the obtained culture solution into the fermentation tank as necessary, it is always fresh and highly capable of producing 1,3-propanediol. Alternatively, continuous fermentation using cultured cells is possible, and continuous fermentation with high production performance maintained for a long period of time is possible.

  Next, the membrane separation element preferably used in the continuous fermentation apparatus used in the method for producing 1,3-propanediol of the present invention will be described.

  The membrane separation element shown in FIG. 3 will be described. FIG. 3 is a schematic perspective view for illustrating the membrane separation element used in the present invention. In the continuous fermentation apparatus used in the method for producing 1,3-propanediol of the present invention, preferably, a separation membrane and a membrane separation element disclosed in International Publication No. 2002/064240 can be used. As shown in FIG. 3, the membrane separation element is configured by arranging a flow path material 14 and the separation membrane 15 (porous membrane) in this order on both surfaces of a rigid support plate 13. The support plate 13 has recesses 16 on both sides. The separation membrane 15 filters the culture solution. The flow path member 14 is for efficiently flowing the permeated water filtered by the separation membrane 15 to the support plate 13. The permeated water that has flowed to the support plate 13 passes through the recess 16 of the support plate 13 and is taken out of the continuous fermentation apparatus through the water collecting pipe 17. As power for extracting the permeated water, 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 element shown in FIG. 4 will be described. FIG. 4 is a schematic perspective view for illustrating another separation membrane element used in the present invention. As shown in FIG. 4, the membrane separation element is mainly constituted by a separation membrane bundle 18 constituted by a hollow fiber membrane (porous membrane), an upper resin sealing layer 19 and a lower resin sealing layer 20. The separation membrane bundle 18 is bonded and fixed in a bundle by an upper resin sealing layer 19 and a lower resin sealing layer 20. Adhesion / fixation by the lower resin sealing layer 20 has a structure in which the hollow portion of the hollow fiber membrane (porous membrane) of the separation membrane bundle 18 is sealed to prevent leakage of the culture solution. On the other hand, the upper resin sealing layer 19 does not seal the inner hole of the hollow fiber membrane (porous membrane) of the separation membrane bundle 18 and has a structure in which permeate flows through the water collecting pipe 22. This separation membrane element can be installed in the continuous fermentation apparatus via the support frame 21. The permeated water filtered by the separation membrane bundle 18 passes through the hollow portion of the hollow fiber membrane and is taken out of the continuous fermentation apparatus via the water collecting pipe 22. As power for extracting the permeated water, 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 member constituting the membrane separation element of the continuous fermentation apparatus used in the method for producing 1,3-propanediol of the present invention is preferably a member resistant to high-pressure steam sterilization operation. If the inside of the continuous fermentation apparatus can be sterilized, it is possible to avoid the risk of contamination by undesirable microorganisms during continuous fermentation, and more stable continuous fermentation is possible. The members constituting the membrane separation element are preferably resistant to 15 minutes at 121 ° C., which is the condition of the high-pressure steam sterilization operation. Membrane separation element members include, for example, metals such as stainless steel and aluminum, polyamide resins, fluorine resins, polycarbonate resins, polyacetal resins, polybutylene terephthalate resins, PVDF, modified polyphenylene ether resins, and polysulfone resins. A resin can be preferably selected.

  In the continuous fermentation apparatus used in the method for producing 1,3-propanediol of the present invention, the membrane separation element may be installed in the fermentation reaction tank as shown in FIG. 1, or the fermentation reaction tank as shown in FIG. It may be installed outside. When the membrane separation element is installed outside the fermentation reaction tank, a separate membrane separation tank can be provided and the membrane separation element can be installed in the inside, and the culture fluid is circulated between the fermentation reaction tank and the membrane separation tank. The culture solution can be continuously filtered through the membrane separation element.

  In the continuous fermentation apparatus used in the method for producing 1,3-propanediol of the present invention, it is desirable that the membrane separation tank is capable of high-pressure steam sterilization. If the membrane separation tank can be autoclaved, it is easy to avoid contamination with germs.

  When continuous fermentation is performed according to the present invention described above, a higher 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).

  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 more specifically explain the method for producing 1,3-propanediol of the present invention, continuous 1,3-propanediol by using the continuous fermentation apparatus shown in the schematic diagrams of FIGS. An example is given and demonstrated about fermentative production of. In the examples relating to the production of 1,3-propanediol shown below, Klebsiella pneumoniae ATCC 25955 strain was used as a microorganism for producing 1,3-propanediol.

(Reference Example 1) Production of porous membrane (part 1)
Polyvinylidene fluoride (PVDF) resin was used as a resin and N, N-dimethylacetamide (DMAc) was used as a solvent, and these were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
[Undiluted solution]
・ PVDF: 13.0% by weight
DMAc: 87.0% by weight
Next, after cooling the above stock solution to a temperature of 25 ° C., a polyester fiber non-woven fabric (porous substrate) having a density of 0.48 g / cm ³ and a thickness of 220 μm, which was previously pasted on a glass plate. And immediately immersed in a coagulation bath at 25 ° C. having the following composition for 5 minutes to obtain a porous film having a porous resin layer formed on a porous substrate.
[Coagulation bath]
-Water: 30.0% by weight
DMAc: 70.0% by weight.

After peeling this porous membrane from the glass plate, it was immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc to obtain a separation membrane. When the surface of the porous resin layer was observed with a scanning electron microscope at a magnification of 10,000 within the range of 9.2 μm × 10.4 μm, the average diameter of all observable pores was 0.1 μm. . Next, when the pure water permeability coefficient was evaluated about the said separation membrane, it was 50 * 10 < ^-9 > m < ³ > / m < ² > / s / Pa. The pure water permeation amount was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.035 μm, and the membrane surface roughness was 0.06 μm.

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

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

Isolation and identification of 1,3-propanediol The conversion of glycerol to 1,3-propanediol was confirmed by HPLC. The analysis was performed using standard methods and materials available to those skilled in the chromatographic arts. One suitable method used a Waters Maxima 820 HPLC system with UV (210 nm) and RI detection. A Shodex SH-1011P pre-column (6 mm × 50 mm) is equipped on a Shodex SH-1011 column (8 mm × 300 mm, purchased from Waters, Milford, Mass.), Temperature controlled at 50 ° C., using 0.01 N H ₂ SO ₄ as the mobile phase. The sample was injected at a flow rate of 0.5 mL / min. Samples were prepared using known amounts of trimethylacetic acid as an external standard when quantitative analysis was desired. The retention times of glucose (RI detection), glycerol, 1,3-propanediol (RI detection) and trimethylacetic acid (UV and RI detection) were approximately 15 minutes, 20 minutes, 26 minutes and 35 minutes, respectively.

  The production of 1,3-propanediol was confirmed by GC / MS. The analysis was performed using standard methods and materials available to those skilled in the GC / MS art. For example, using a Hewlett Packard 5890 Series II gas chromatograph coupled to a Hewlett Packard 5971 Series mass selective detector (EI) and HP-INNOWax column (length 30 m, inner diameter 0.25 mm, film thickness 0.25 microns). It was. The retention time and mass spectrum of the 1,3-propanediol produced were compared to that of the reference 1,3-propanediol (m / e: 57,58).

  The sample was also derivatized. 30 μL of concentrated (70% v / v) perchloric acid was added to a 1.0 mL sample (eg, culture supernatant). After mixing, the sample was lyophilized. A 1: 1 mixture of bis (trimethylsilyl) trifluoroacetamide: pyridine (300 μL) was added to the lyophilized material, mixed vigorously and placed at 65 ° C. for 1 hour. The sample was clarified by removing insoluble material by centrifugation. The resulting liquid was divided into two phases, and the upper phase was used for analysis. Samples were chromatographed on a DB-5 column (48 m, ID 0.25 mm, film thickness 0.25 μm; from J & W Scientific) and retention times and mass spectra of 1,3-propanediol derivatives obtained from culture supernatants. Was compared to that obtained from the reference standard. The mass spectrum of TMS-derivatized 1,3-propanediol contains 205, 177, 130 and 115 AMU characteristic ions.

Example 1 Production of 1,3-propanediol by continuous fermentation (part 1)
In order to examine whether or not a 1,3-propanediol continuous fermentation system can be obtained by operating the membrane-integrated continuous fermentation apparatus of FIG. 1, a 1,3-propanediol production medium having the composition shown in Table 1 is used. A continuous fermentation test of this apparatus was conducted. 1 is a schematic side view showing one embodiment of the present invention, and the present invention is not limited to this embodiment. The medium was used after autoclaving (121 ° C., 15 minutes). As the membrane separation element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) was used. When the characteristics of the porous membrane were examined, the average pore diameter was 0.1 μm, and the pure water permeability coefficient was 50 × 10 ⁻⁹ m ³ / m ² / s / pa. Unless otherwise specified, the operating conditions in this example are as follows.

Fermentation reactor capacity: 1.5 (L)
Separation membrane used: PVDF filtration membrane Effective membrane filtration area: 120 square cm
Temperature adjustment: 37 (℃)
Fermentation reactor aeration rate: 0.6 (L / min) Nitrogen gas Fermentation reactor agitation speed: 800 (rpm)
pH adjustment: Adjustment to pH 7.0 with 5N NaOH Sterilization: All culture tanks containing separation membrane elements and used media are autoclaved at 121 ° C for 20 min. High pressure steam sterilization Control of membrane permeate flow rate: Flow rate control by transmembrane pressure difference ( Control was performed at 0.1 kPa or more and 20 kPa or less (transmembrane pressure difference of 20 kPa or less corresponds to 2 m or less in the head differential control).

  The Klebsiella pneumoniae ATCC 25955 strain was used as the microorganism, the 1,3-propanediol production medium having the composition shown in Table 1 was used as the medium, and the concentration of the product 1,3-propanediol was evaluated by the HPLC method. .

  In addition, glucose test Wako C (Wako Pure Chemical Industries) was used for measuring the glucose concentration.

  First, Klebsiella pneumoniae ATCC 25955 strain was cultured in a test tube with shaking overnight in 5 ml of 1,3-propanediol production medium (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh 1,3-propanediol production medium, and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 24 hours (pre-culture). The incubating medium was inoculated in a 1.5 L 1,3-propanediol production medium of the membrane integrated continuous fermentation apparatus shown in FIG. 1, and the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5; The aeration amount of the fermentation reaction tank 1, the temperature adjustment, and the pH adjustment were performed, and the culture was performed for 24 hours (pre-culture). Immediately after completion of the pre-culture, 1,3-propanediol production medium (glycerol concentration is 100 g / L) is continuously supplied, and the amount of membrane permeate is controlled so that the amount of fermentation liquid in the membrane-integrated continuous fermentation apparatus becomes 1.5 L. The culturing was continued, and 1,3-propanediol was produced by continuous fermentation. Control of the amount of permeated water when performing the continuous fermentation test is performed by adjusting the water head difference appropriately by the head differential control device 6 so that the head of the fermentation reaction tank is within 2 m at the maximum, that is, the transmembrane pressure difference is 0.1 kPa to 20 kPa. It was done by changing. The produced 1,3-propanediol concentration and residual glucose concentration in the membrane permeation fermentation broth were appropriately measured. The production rate of 1,3-propanediol calculated from the 1,3-propanediol and input glycerol is shown in Table 4. As a result of conducting a fermentation test for 264 hours, it was confirmed that stable production of 1,3-propanediol by continuous fermentation was possible by using this membrane integrated continuous fermentation apparatus.

(Example 2) Production of 1,3-propanediol by continuous fermentation (part 2)
The ATCC25955 strain was used as a microorganism or cultured cell, a 1,3-propanediol fermentation medium having the composition shown in Table 1 was used as the fermentation medium, and a continuous fermentation test was conducted using the continuous fermentation apparatus shown in FIG. The 1,3-propanediol fermentation 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. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2 was used. The operating conditions in Example 2 are as follows unless otherwise specified.

[Operating conditions]
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: PVDF filtration membrane-Separation membrane element effective filtration area: 4000 square cm
・ Temperature adjustment: 37 (℃)
-Fermentation reactor aeration rate: 0.6 (L / min) nitrogen gas-Fermentation reactor agitation speed: 800 (rpm)
-PH adjustment: pH 7.0 adjusted with 5N NaOH-Culture medium circulation rate: 100 ml / min
Sterilization: The culture tank containing the separation membrane element and the used medium were all autoclaved at a temperature of 121 ° C. for 20 minutes by autoclaving.
-Membrane permeated water amount control: Flow rate control by transmembrane pressure difference (controlled at 0.1 kPa to 20 kPa).

  The 1,3-propanediol and glucose concentrations were evaluated using the same method as in Example 1.

  First, Klebsiella pneumoniae ATCC 25955 strain was cultured overnight in a test tube with 5 ml of 1,3-propanediol fermentation medium (pre-culture). The obtained culture broth was inoculated into 50 ml of a fresh 1,3-propanediol fermentation medium and cultured with shaking at 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution was inoculated in the 2.0 L 1,3-propanediol fermentation medium of the continuous fermentation apparatus shown in FIG. 2 and the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5 to give a fermentation reaction tank. The aeration amount, temperature adjustment, pH adjustment, and culture medium circulation rate adjustment of No. 1 were performed, and culture was performed for 24 hours (pre-culture). Immediately after completion of the pre-culture, 1,3-propanediol fermentation medium (glycerol concentration is 100 g / L) is continuously supplied, and the amount of permeated water in the continuous fermentation apparatus is controlled to 2.0 L. Continuous culture was performed, and 1,3-propanediol was produced by continuous fermentation. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the water head difference by the water head difference control device 3 so that the transmembrane pressure difference was 0.1 kPa to 20 kPa. The produced 1,3-propanediol concentration and residual glucose concentration in the membrane permeation fermentation broth were appropriately measured. In addition, Table 1 shows 1,3-propanediol fermentation productivity calculated from the 1,3-propanediol and input glycerol.

Comparative Example 1 Production of 1,3-propanediol by fed-batch fermentation The most typical fed-batch fermentation as a fermentation form using microorganisms was performed using a 2L jar fermenter, and its 1,3-propanediol productivity was evaluated. did. The medium was used after autoclaving (121 ° C., 15 minutes). In this comparative example, Klebsiella pneumoniae ATCC 25955 strain was used as a microorganism, the concentration of 1,3-propanediol as a product was evaluated using HPLC, and glucose test Wako C Photopure drug) was used. The operating conditions of Comparative Example 1 are shown below.

Fermentation reactor capacity (1,3-propanediol production medium amount): 1.0 (L)
Temperature adjustment: 37 (℃)
Fermentation reactor aeration rate: 0.4 (L / min) Nitrogen gas Fermentation reactor agitation speed: 300 (rpm)
pH adjustment: Adjusted to pH 7.0 with 5N NaOH.

  First, Klebsiella pneumoniae ATCC 25955 strain was cultured overnight in a test tube with 5 ml of 1,3-propanediol production medium (pre-culture). The culture solution was inoculated into 50 ml of a fresh 1,3-propanediol production medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (preculture). The preculture was inoculated into 1.5 L of 1,3-propanediol production medium of a jar fermenter. 1,3-propanediol production medium (glycerol concentration is 500 g / L) was continuously supplied so that the glycerol concentration was changed from 0 g / L to 10 g / L, and fed-batch fermentation was performed. The results are shown in Table 2, comparing the 1,3-propanediol fermentation productivity obtained in the continuous fermentation test of Example 1 and Example 2.

  As a result of these comparisons, it was clarified that the production rate of 1,3-propanediol was greatly improved by using the continuous fermentation apparatus shown in FIG. 1 and FIG. That is, by using a continuous fermentation apparatus incorporating a porous membrane disclosed by the present invention and controlling the transmembrane pressure difference, the culture solution is separated into a filtrate and an unfiltered solution by a separation membrane, and the desired fermentation is performed from the filtrate. It becomes clear that the production of 1,3-propanediol by continuous fermentation is possible while recovering the product and allowing a continuous fermentation method in which the unfiltered liquid is returned to the culture solution while maintaining a high amount of microorganisms. It was.

FIG. 1 is a schematic side view for explaining an example of a membrane-integrated continuous fermentation apparatus used in the present invention. FIG. 2 is a schematic side view for explaining an example of another continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining an example of the membrane separation element used in the present invention. FIG. 4 is a cross-sectional explanatory diagram for explaining an example of another membrane separation element used in the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Membrane separation element 3 Water head difference control device 4 Gas supply device 5 Stirrer 6 Level sensor 7 Medium supply pump 8 NaOH solution supply pump 9 pH sensor / control device 10 Temperature controller 11 Culture fluid circulation pump 12 Membrane separation tank 13 Support plate 14 Channel material 15 Separation membrane 16 Concave portion 17 Water collecting pipe 18 Separation membrane bundle 19 Upper resin sealing layer 20 Lower resin sealing layer 21 Support frame 22 Water collection pipe

Claims (11)

The culture solution of microorganisms or cultured cells having the ability to produce 1,3-propanediol is filtered through a separation membrane, the product is recovered from the filtrate, and the unfiltered solution is retained or refluxed in the culture solution, and fermentation is performed. Continuous fermentation in which the raw material is added to the culture solution, and a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm is used as a separation membrane, and the intermembrane differential pressure is filtered within the range of 0.1 to 20 kPa. A process for producing 1,3-propanediol by continuous fermentation. The pure water permeability coefficient of the porous membrane is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less. A method for producing 1,3-propanediol by continuous fermentation as described in 1.   The porous membrane is an organic polymer membrane, the average pore diameter 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. A method for producing 1,3-propanediol by continuous fermentation according to claim 1 or 2.   The porous membrane is an organic polymer membrane, and the membrane surface roughness is a porous membrane having a surface roughness of 0.1 μm or less. -A method for producing propanediol.   The method for producing 1,3-propanediol according to any one of claims 1 to 4, wherein the porous membrane is a porous membrane including a porous resin layer. The method for producing 1,3-propanediol by continuous fermentation according to any one of claims 1 to 5, wherein a polyvinylidene fluoride resin is used as a membrane material of the porous membrane. A microorganism capable of producing 1,3-propanediol is (a) at least one gene encoding a polypeptide having glycerol dehydratase activity; (b) at least one gene encoding a glycerol dehydratase reactivation factor; and (C) contains at least one gene encoding non-specific catalytic activity for converting 3-hydroxypropionaldehyde to 1,3-propanediol, and has the ability to synthesize 1,3-propanediol from glycerol. The method for producing 1,3-propanediol by continuous fermentation according to any one of claims 1 to 6 . Microorganisms capable of synthesizing 1,3-propanediol from glycerol are Klebsiella, Clostridium, Lactobacillus, Cytrobacter, Enterobacter, Aerobacter (Aer) , Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Hlu, Cluberomise , Debaromyces, Mucor, Toluropsis, Methylobacter, Salmonella, Bacillus, S, Aerobacter, S The method for producing 1,3-propanediol according to claim 7 , wherein the microorganism is selected from the group consisting of: The recombinant microorganism further comprises: (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; and (b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity. The method for producing 1,3-propanediol by continuous fermentation according to claim 8 , which is a recombinant microorganism containing a gene. The method for producing 1,3-propanediol by continuous fermentation according to claim 9 , wherein the glycerol dehydratase reactivating factor is a recombinant microorganism encoded by orfX and orfZ isolated from dha regulon. The 1,3-propane by continuous fermentation according to claim 10 , wherein the recombinant microorganism is a recombinant microorganism further lacking glycerol kinase activity and / or glycerol dehydrogenase activity and / or triosephosphate isomerase activity. A method for producing a diol.

Images