JP2011188791A - Method for operating continuous fermentation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for supplying a washing agent which enables the holding of filterability and the control of a microorganism concentration for the high concentration culture of a microorganism-mixed liquid, when the product is produced and recovered by fermentation using a separation membrane. <P>SOLUTION: The method for operating a continuous fermentation apparatus, includes supplying a washing agent containing an aqueous hypochlorite solution from the penetrated liquid side of a membrane module to wash the membrane, on a continuous fermentation operation for introducing an undiluted solution containing an unconverted substance into a fermentation tank, converting the unconverted substance with a microorganism-containing solution, filtering the conversion solution with the membrane module, and continuously holding a non-penetrated solution in the fermentation tank, and simultaneously taking out a penetrated solution containing the converted substance. The method includes controlling conditions for supplying the washing agent based on the concentration of the microorganisms in the fermentation tank. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for operating a continuous fermentation apparatus that continuously produces a chemical product by fermentation using a separation membrane.

  Separation membranes are used in various fields including water treatment fields such as drinking water production, water purification, and wastewater treatment, pharmaceutical production, and food industry, including application to the field of fermentation. In water treatment fields such as drinking water production, water purification treatment, and wastewater treatment, separation membranes are used to remove impurities in water as an alternative to conventional sand filtration and coagulation sedimentation processes. Furthermore, in the wastewater treatment and food industry fields, high-concentration wastewater or raw materials are introduced into a treatment tank in large quantities, and the treatment is performed while maintaining a high concentration of microorganisms, resulting in a high removal rate and high-purity treatment liquid. Therefore, separation membrane technology is widely used.

  In the water treatment field, food industry field, etc. using such separation membranes, improvement in water permeability performance is required from the viewpoint of cost reduction, and the membrane area is reduced by a separation membrane with excellent water permeability performance, and the apparatus is made compact. We are trying to reduce facility costs, membrane replacement costs and installation area. From such a viewpoint of cost, a hollow fiber membrane having a large filtration area with respect to volume has attracted attention.

  However, such separation membranes, including hollow fiber membranes, are subject to SS (Suspended Solid) and adsorbate adhering to the filtration surface through the filtration operation, resulting in a decrease in filtration capacity and the inability to obtain the required amount of filtrate. There is. For this reason, by regularly or irregularly washing the filtration surface, the SS content and the like adhering to the filtration surface is removed to maintain the filtration capacity. For example, in Patent Document 1, filtration performance is maintained by reversely permeating filtered water from the filtration side periodically. In Patent Document 2, it is proposed to perform filtration while washing the filtration surface by supplying air from below the filter body. Furthermore, Patent Document 3 proposes a method of chemically cleaning the filter body in the case where the filtration surface cannot be cleaned even by the methods disclosed in Patent Document 1 and Patent Document 2.

  On the other hand, among the methods of chemically washing the filter body, hypochlorite treatment is widely used for washing clogged filter membranes generated by organic substances. For example, in patent document 4, the hypochlorite solution whose effective chlorine concentration is 70-200 mg / L about the fall of the membrane filtration performance which generate | occur | produced when purifying the hydroponic culture nutrient solution mainly on tomato etc. by membrane filtration. The filter membrane is washed by using a solution to eliminate clogging of the filtration membrane, and sterilization of slime-forming bacteria by sterilization in the vicinity of the filtration membrane is expected to prevent the pore from being clogged again.

  However, from this method, in the hypochlorite treatment, the amount of hypochlorite solution to be washed while contacting the upstream side of the membrane without passing through the membrane, The ratio to the amount of the chlorite solution (cleaning solution) is 5: 5 to 9: 1, and the cleaning is performed while contacting the upstream side of the membrane. In this method, the flow of the liquid to be filtered that has flowed upstream of the membrane is blocked, and after washing with a hypochlorite solution, the flow of the hypochlorite solution is blocked, and the liquid to be filtered is removed. A complicated process of reflowing and resuming filtration must be performed. In addition, cleaning was performed with clean water so that the hypochlorite solution did not remain on the surface of the filtration membrane after cleaning, and it was generated by cleaning with a hypochlorite solution and cleaning with clean water. Wastewater needs to be treated.

  Regarding this wastewater treatment problem, for example, in Patent Document 5, after performing washing twice, the waste liquid generated from each washing step is mixed and neutralized, thereby enabling the discharge to the public water area. is suggesting. From this method, membrane cleaning is performed using a mixed solution of sodium hydroxide and sodium hypochlorite in the first cleaning step, and sodium bisulfite solution, sodium bisulfite solution, sodium sulfite solution, thiosulfuric acid in the second step. Membrane cleaning is performed using a reducing acid solution such as a sodium solution. After washing, it is proposed that the washing waste liquid generated from each of the two steps can be mixed and neutralized, and discharged directly into the public water area. However, this method does not consider filtration membranes that do not require cleaning until the second step, and membrane contamination that does not provide a cleaning effect in the second step, and further increases the cost by cleaning two steps. There was a problem.

  On the other hand, in recent years, a proposal of a culture technique using a membrane has been actively made. Among these, in the continuous production method involving cultivation of microorganisms and cultured cells, the microorganisms and cultured cells are filtered through a separation membrane, and the product is recovered from the filtrate. At the same time, the filtered microorganisms and cultured cells are retained in the culture solution or refluxed. By doing so, it is possible to maintain a high concentration of microorganisms and cells in the culture solution. For example, Patent Document 6 proposes that high substance productivity can be obtained by performing continuous fermentation using a separation membrane, improving the microorganism and cell concentration in the culture solution, and maintaining the concentration high. However, from this method, it is necessary to maintain a high microbial concentration in order to obtain high substance productivity, and in order to recover the product from the culture solution of high-concentration microorganisms, cleaning such as chemical cleaning is performed and the filterability is reduced. Need to hold. Furthermore, if the medium supply rate is increased in order to obtain a high microorganism concentration, a high production rate can be obtained, but microorganisms grow rapidly due to the abundant supply of the medium, and microorganisms with a concentration higher than the filterable microorganism concentration are cultured. There was a problem that the filtration membrane was clogged. When microorganisms with a concentration higher than the concentration of microorganisms that can be filtered are grown, in order to reduce the microorganism concentration, a part of the culture solution must be extracted and disposed of, which raises the production cost. .

  As described above, in the conventional technology, problems such as a complicated washing process, treatment of waste liquid generated during washing, maintenance of filterability for high concentration culture of microorganisms, and control method for excessive increase of microorganism concentration have not been studied. Therefore, development of a membrane filtration operation method and a microorganism concentration control method for maintaining the filterability is required.

JP-A-8-141375 JP 2001-38177 A JP 2002-126470 A Japanese Patent No. 3538385 JP 2005-193119 A International Publication WO2007 / 097260 Pamphlet

  The present invention provides a method for supplying a cleaning agent capable of simultaneously maintaining filterability and controlling the concentration of microorganisms in a high concentration culture of a microorganism mixed solution when producing and recovering a product by fermentation using a separation membrane. The task is to do.

  In order to solve the above problems, the present invention is achieved by the following configurations (1) to (2).

  (1) The raw solution containing the pre-conversion substance is introduced into the fermenter, the pre-conversion substance is converted using the microorganism-containing liquid, and then filtered using a membrane module, and the non-permeate is continuously held in the fermenter. In the continuous fermentation operation for removing the permeate containing the substance after conversion, the operation method of the continuous fermentation apparatus for supplying the cleaning agent containing the hypochlorite aqueous solution from the permeate side of the membrane module to perform the membrane cleaning A method for operating a continuous fermentation apparatus, characterized in that the supply condition of the cleaning agent is controlled by the microorganism concentration in the fermenter.

  (2) The continuous fermentation apparatus according to (1), wherein the post-conversion substance is a fluid product containing at least one of chemical products, dairy products, pharmaceuticals, foods or brewed products, or waste water. Driving method.

  According to the present invention, in a continuous fermentation operation in which a fermentation broth is filtered using a filtration membrane and a permeate containing a post-conversion substance is taken out while continuously retaining the non-permeate in the fermentation tank, membrane filtration is used. It is possible to effectively clean the generated membrane dirt, control the concentration of microorganisms in the fermenter, significantly improve the fermentation production efficiency at low cost stably, and wash waste liquid and extraction culture liquid. By not generating, it is possible to reduce the processing cost and further reduce the cost, and it is possible to stably produce a fermentation product at a low cost widely in the fermentation industry.

It is a schematic side view for demonstrating the example of the membrane separation type | mold continuous fermentation apparatus used by this invention. It is a figure which shows the back liquid washing | cleaning method and procedure of the membrane filtration module in this invention. It is a figure which shows the time-dependent change of the microorganism concentration by the continuous fermentation filtration experiment which concerns on Example 1 and Comparative Example 1. FIG. It is a figure which shows the time-dependent change of the membrane filtration differential pressure by the continuous fermentation filtration experiment which concerns on Example 1 and Comparative Example 1. FIG. It is a figure which shows the time-dependent change of the microorganisms concentration by the continuous fermentation filtration experiment which concerns on Example 2 and Comparative Example 2. FIG. It is a figure which shows the time-dependent change of the membrane filtration differential pressure by the continuous fermentation filtration experiment which concerns on Example 2 and Comparative Example 2. FIG. It is a figure which shows the time-dependent change of the microorganisms concentration by the continuous fermentation filtration experiment which concerns on Example 3 and Comparative Example 3. FIG. It is a figure which shows the time-dependent change of the membrane filtration differential pressure by the continuous fermentation filtration experiment which concerns on Example 3 and Comparative Example 3. FIG.

  In the continuous fermentation operation in which the permeate containing the substance after conversion is taken out while being filtered using the membrane module and continuously retaining the non-permeate in the fermenter, the hypothesis is added from the permeate side of the membrane module. A method for operating a continuous fermentation apparatus is characterized in that a cleaning agent containing an aqueous chlorate solution is supplied to perform membrane cleaning, and the supply condition of the cleaning agent is controlled by the microorganism concentration in the fermenter.

  The separation membrane used in the membrane module of the present invention is chemically resistant, such as polyvinylidene fluoride, polysulfone, polyethersulfone, polytetrafluoroethylene, polyethylene, polypropylene, and ceramic membranes, regardless of whether they are organic or inorganic. Any separation membrane that has

  The separation membrane used in the present invention is preferably a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm. In addition, the shape of the separation membrane may be any shape such as a flat membrane and a hollow fiber membrane.

  The average pore diameter of the surface of the three-dimensional network structure is obtained by taking a photograph of the surface of the three-dimensional network structure at 60000 times using a scanning electron microscope, measuring the diameters of 20 arbitrary pores, and averaging the numbers. . When the pores are not circular, a circle having an area equal to the area of the pores (equivalent circle) is obtained by an image processing device or the like, and the equivalent circle diameter is obtained by the method of setting the diameter of the pores.

  According to a preferred embodiment of the present invention, filtration can be performed using the separation membrane with a transmembrane pressure difference in the range of 0.1 to 20 kPa.

  The membrane module in the present invention may be made of a material having excellent chemical resistance and may have a shape capable of injecting a cleaning liquid from the secondary side to the primary side of the module.

  The fermentation raw material for microorganisms and cultured cells used in the present invention, that is, the substance before conversion, can promote the growth of the microorganisms and cultured cells to be fermented and cultured, and can produce a chemical product that is the desired fermentation product. I just need it. As a fermentation raw material, for example, a normal liquid medium that appropriately contains a carbon source, a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins is preferably used. If it is a liquid that partially promotes the growth of microorganisms and cultured cells to be fermented and can successfully produce a chemical product that is the target fermentation product, for example, waste water or sewage is used as it is or as a fermentation raw material. May be used.

  Examples of the carbon source include sugars such as glucose, sucrose, fructose, galactose and lactose, starch containing these sugars, starch hydrolysates, sugar cane molasses, sugar beet molasses, cane juice, sugar beet molasses or cane juice. Extracts or concentrates, sugar beet molasses or cane juice filtrate, syrup (high test molasses), sugar beet molasses or cane juice purified or crystallized raw sugar, vegetable molasses or cane juice purified or crystallized Purified saccharides, organic acids such as acetic acid and fumaric acid, alcohols such as ethanol, glycerin and the like are used. Sugars are the first oxidation products of polyhydric alcohols, and are carbohydrates that have one aldehyde group or ketone group, sugars with aldehyde groups are classified as aldoses, and sugars with ketone groups are classified as ketoses. Point to.

  Examples of the nitrogen source include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein decomposition products, Other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used.

  Moreover, as said inorganic salt, a phosphate, magnesium salt, calcium salt, iron salt, manganese salt etc. can be used suitably, for example.

  In the present invention, fermentation culture of microorganisms can usually be performed at a pH of 4 to 8 and a temperature of 20 to 65 ° C. The pH of the fermentation broth is adjusted to a predetermined value within the above range with an inorganic acid or an organic acid, an alkaline substance, urea, calcium hydroxide, calcium carbonate, ammonia gas, and the like.

  If it is necessary to increase the oxygen supply rate in the culture, oxygen is added to the air to keep the oxygen concentration preferably at 21% or higher, the fermentation broth is pressurized, the stirring speed is increased, or the aeration rate is increased. The following means can be used. Conversely, if it is necessary to reduce the oxygen supply rate, it is also possible to supply a gas containing no oxygen such as carbon dioxide, nitrogen and argon mixed with air.

  In the present invention, when extracting the fermentation broth containing microorganisms or cultured cells from the fermentation broth from the fermentation broth, the fermentation culture is performed so that the concentration of the microorganisms or cultured cells does not decrease and the productivity of the fermented product does not decrease. It is preferable to adjust appropriately based on the turbidity of liquid or MLSS (Mixed Liquor Suspended Solid).

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

  As the microorganisms and cultured cells used in the present invention, eukaryotic cells or prokaryotic cells are used, for example, yeasts such as baker's yeast often used in the fermentation industry, bacteria such as E. coli, lactic acid bacteria, coryneform bacteria, filamentous forms, etc. Examples include fungi, actinomycetes, animal cells and insect cells. The microorganisms and cells used may be those isolated from the natural environment, or may be those whose properties have been partially modified by mutation or genetic recombination.

  The most distinguishing feature of eukaryotic cells used in the present invention is clearly distinguished from prokaryotes having a structure called a cell nucleus (nucleus) in the cell and not having a cell nucleus (nucleus). In the present invention, yeast is more preferably used among the eukaryotic cells. Suitable yeasts in the present invention include, for example, yeasts belonging to the genus Saccharomyces and yeasts belonging to Saccharomyces cerevisiae.

  The most prominent feature of the prokaryotic cell used in the present invention is that it does not have a structure called a cell nucleus (nucleus) in the cell, and is clearly distinguished from a eukaryote having a cell nucleus (nucleus). In the present invention, lactic acid bacteria can be preferably used among the eukaryotic cells.

  The chemical product obtained by the production method of the present invention, that is, the post-conversion substance, is a substance produced by the above-mentioned microorganisms or cultured cells in the fermentation broth. Examples of the chemicals include substances that are mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, and nucleic acids. The present invention can also be applied to the production of substances such as enzymes, antibiotics and recombinant proteins. For example, the alcohol includes ethanol, 1,3-butanediol, 1,4-butanediol, cadaverine, glycerol and the like. 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 inosine, guanosine, and cytidine.

  Further, the post-conversion substance obtained by the production method of the present invention is preferably a fluid containing at least one of chemical products, dairy products, pharmaceuticals, foods or brewed products, or waste water. Here, as the chemical product, for example, organic acids, amino acids and nucleic acids, substances applicable to making chemical products by the process after membrane separation filtration, and dairy products, for example, low-fat milk, membranes, etc. As substances and pharmaceuticals that can be applied as dairy products by the process after separation and filtration, for example, as substances and foods that can be applied to make pharmaceuticals by the process after membrane separation and filtration, such as enzymes, antibiotics, and recombinant proteins. Is a substance that can be applied as a food by a process after membrane separation filtration, such as a lactic acid beverage, and as a brewed product, for example, a substance that can be applied as a beverage containing alcohol by a process after membrane separation filtration, such as beer or shochu Examples of the wastewater include wastewater after washing of product such as food washing wastewater and dairy product washing wastewater, and domestic wastewater containing abundant organic substances.

  In the case of producing lactic acid according to the present invention, it is preferable to use yeast for eukaryotic cells and lactic acid bacteria for prokaryotic cells. Among these, yeast in which a gene encoding lactate dehydrogenase is introduced into cells is preferable. Of these, lactic acid bacteria are preferably lactic acid bacteria that produce 50% or more of lactic acid as a yield of sugar relative to consumed glucose, and more preferably 80% or more of lactic acid bacteria as a comparable yield. .

  Examples of lactic acid bacteria that are preferably used in producing lactic acid in the present invention include, for example, in the wild type strain, Lactobacillus, Bacillus, Pediococcus having the ability to synthesize lactic acid, Genus Carnobacterium, Genus Carnobacterium, Genus Carnobacterium, Genus Carnobacterium, Genus Carnobacterium, Genus Carnobacteria ), Genus Leuconostoc Genus Oenococcus, Genus Atopobium, Streptococcus, Genus Enterococcus, Genus Lactococcus, Genus Lactococcus Can be mentioned.

  In addition, lactic acid bacteria having high yield and optical purity of lactic acid can be selected and used. For example, as lactic acid bacteria having the ability to select and produce D-lactic acid, D-lactic acid belonging to the genus Sporolactocillus Production microorganisms can be mentioned, and preferred specific examples include Sporolactobacillus laevolacticus or Sporolactobacillus inulinus. More preferably, Sporolactobacillus laevolacticus ATCC 23492, ATCC 23493, ATCC 23494, ATCC 23495, ATCC 23396, ATCC 223549, IAM 12326, IAM 12327, IAM 12328, IAM 12329, IAM 12330, IAM 12331, IAM 12379 , DSM 2315, DSM 6477, DSM 6510, DSM 6511, DSM 6763, DSM 6764, DSM 6771, and Sporolactocillus inulinas JCM 6014.

  Examples of lactic acid bacteria having a high yield of L-lactic acid to saccharide include, for example, Lactobacillus yamanasiensis, Lactobacillus animalis, Lactobacillus bilis Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Casei (Lactobacillus casei), Lactobacillus delbruecki, Lactobacillus paracasei, Lactobacillus rhamnosus rhactobaslus Lactobacillus ruminis, Lactobacillus salivarius, Lactobacillus sharpeae, Lactobacillus sharpeae, Lactobacillus sharvarie, Lactobacillus sharvarie It can be selected and used for the production of L-lactic acid.

  In the present invention, the transmembrane pressure difference during filtration of the fermentation broth of microorganisms and cultured cells with the separation membrane in the membrane module may be any condition as long as the microorganisms, cultured cells and medium components are not easily clogged. It is important to carry out the filtration treatment with the transmembrane pressure difference in the range of 0.1 kPa to 20 kPa. The transmembrane pressure difference is preferably in the range of 0.1 kPa to 10 kPa, and more preferably in the range of 0.1 kPa to 5 kPa. When the range of the transmembrane pressure difference is exceeded, clogging of prokaryotic microorganisms and medium components occurs rapidly, leading to a decrease in the amount of permeated water, and may cause problems in continuous fermentation operation.

  As a driving force for filtration, a transmembrane differential pressure can be generated in the separation membrane by a siphon utilizing a liquid level difference (water head difference) of the fermentation culture solution and the porous membrane treated water or a cross flow circulation pump. Moreover, you may install a suction pump in the separation membrane process water side as a driving force of filtration. In addition, when a cross flow circulation pump is used, the transmembrane pressure difference can be controlled by the suction pressure. Furthermore, the transmembrane pressure difference can be controlled by the pressure of the gas or liquid that introduces the pressure on the fermentation broth side. When these pressure controls are performed, the pressure difference between the pressure on the fermentation broth side and the pressure on the porous membrane treated water side can be used as the transmembrane pressure difference, which can be used to control the transmembrane pressure difference.

  In the present invention, a hypochlorite aqueous solution that has a membrane cleaning effect and can reduce the concentration of microorganisms is used as the cleaning liquid, and among them, an aqueous solution of sodium hypochlorite and potassium hypochlorite. In view of cost, a sodium hypochlorite aqueous solution is desirable.

  The hypochlorite aqueous solution of the present invention may contain an alkaline solution such as sodium hydroxide or calcium hydroxide as long as the effects of the invention are not impaired.

  The concentration of the hypochlorite aqueous solution used as the cleaning liquid is such that the free chlorine concentration is in the range of 10 to 5,000 ppm, more preferably 100 to 3,000 ppm. If the free chlorine concentration is higher than this range, the filtration membrane may be damaged, and if it is lower than this range, the cleaning effect and the microorganism control effect may not be sufficiently obtained. Moreover, the back pressure washing | cleaning rate of the hypochlorite aqueous solution used as a washing | cleaning liquid is the range of 0.5-5 times of the membrane filtration rate, More preferably, it is 1-3 times. When the back pressure washing rate is higher than this range, the filtration membrane module may be damaged, and when it is lower than this range, the washing effect and the microorganism control effect may not be sufficiently obtained.

  Here, the back pressure washing is a method of removing the fouling substance on the membrane surface by sending a liquid from the porous membrane treated water side to the fermentation culture solution side.

  The counter pressure cleaning cycle of the hypochlorite aqueous solution used as the cleaning liquid can be determined by the film differential pressure and the change in the film differential pressure. The counter pressure washing cycle is in the range of 0.1 to 12 times / hour, more preferably 4 to 8 times / hour. If the counter pressure cleaning cycle is larger than this range, the filtration membrane may be damaged, and if it is smaller than this range, the cleaning effect and the microorganism control effect may not be sufficiently obtained.

  The back pressure cleaning time of the hypochlorite aqueous solution used as the cleaning liquid can be determined by the back pressure cleaning cycle, the membrane differential pressure, and the changes in the membrane differential pressure. The back pressure washing time is in the range of 5 to 300 seconds / time, more preferably 30 to 120 seconds / time. If the back pressure washing time is longer than this range, the filtration membrane may be damaged, and if it is shorter than this range, the washing effect and the microorganism control effect may not be sufficiently obtained.

  The cleaning agent storage tank, the cleaning agent supply pump, the piping and valves from the cleaning agent storage tank to the module may be those having excellent chemical resistance. The cleaning agent can be injected manually, but a filtration / backwash control device is provided, and the filtration pump and filtration side valve, cleaning agent supply pump and cleaning agent supply valve can be automatically controlled by a timer, etc. desirable.

  In the present invention, it is necessary to monitor the microorganism concentration in order to control the microorganism concentration in the microorganism culture tank. Microbial concentration can be measured by taking a sample and measuring it, but it is desirable to install a microorganism concentration sensor such as an MLSS measuring device in the microorganism culture tank and continuously monitor the change in the microorganism concentration.

  Next, the continuous fermentation apparatus used by this invention is demonstrated using figures.

  FIG. 1 is a schematic side view for illustrating and explaining a continuous fermentation apparatus used in the method for supplying a cleaning agent of the present invention. FIG. 1 is an example of a typical continuous fermentation apparatus in which a separation membrane module is installed outside a fermentation culture tank. In FIG. 1, the continuous fermentation apparatus is basically composed of a fermentation culture tank 1, a separation membrane module 2, and a cleaning agent supply unit. Here, a large number of hollow fiber membranes are incorporated in the separation membrane module 2. The cleaning agent supply unit includes a filtration valve 13, a cleaning agent supply pump 12, and a cleaning agent supply valve 14. The separation membrane module and the cleaning agent supply unit will be described in detail later. The separation membrane module 2 is connected to the fermentation culture tank 1 via a circulation pump 8.

  In FIG. 1, the medium is fed into the fermentation culture tank 1 by the medium supply pump 9, the fermentation culture liquid in the fermentation culture tank 1 is stirred by the stirring device 4 as necessary, and the gas is supplied as necessary. The device 15 can supply the required gas. At this time, the supplied gas can be recovered and recycled and supplied again by the gas supply device 15. Further, if necessary, by adjusting the pH of the fermentation broth with the pH sensor / control device 5 and the neutralizing agent supply pump 10, fermentation production with high productivity can be performed.

  Further, the fermentation broth in the apparatus is circulated between the fermentation culture tank 1 and the separation membrane module 2 by the circulation pump 8. The fermentation broth containing the fermentation product is filtered and separated into microorganisms and fermentation product by the separation membrane module 2 and 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 separation membrane module 2 can be carried out by using the pressure of the circulation pump 8 without using any special power. However, if necessary, a filtration pump 11 is provided to provide a differential pressure sensor / control. The amount of the fermented liquid can be appropriately adjusted by the device 7. If necessary, the temperature controller 3 can maintain the temperature of the fermentation culture tank 1 constant, and can maintain a high microorganism concentration.

  The cleaning agent supply unit used in the cleaning agent supply method of the present invention includes a filtration valve 13, a cleaning agent supply pump 12, and a cleaning agent supply valve 14. The cleaning agent can be input after monitoring the microbial concentration.

  The control procedure used in the operation method of the present invention is performed according to the procedure shown in FIG.

  First, membrane filtration of the product is started, and changes in microbial concentration and membrane pressure are monitored. When the membrane is fouled by membrane filtration and the membrane differential pressure rises, the microorganism concentration is confirmed. If the concentration is higher than the assumed microorganism concentration, back pressure washing is performed using a hypochlorite aqueous solution.

  When the membrane differential pressure increases, the microorganism concentration is confirmed. If the concentration is lower than the assumed microorganism concentration, backwashing is performed using another cleaning solution other than the hypochlorite aqueous solution. As the other washing liquid, since it does not affect the fermentation, the liquid agent used in the fermentation is desirable. The medium, neutralizing agent, filtrate, water, or one or more of these components to be put into the fermentation culture tank is used. A liquid containing is desirable.

  After performing membrane cleaning with a hypochlorite aqueous solution or other cleaning solution, the membrane differential pressure is confirmed to confirm the cleaning effect. When the confirmed differential pressure is higher than the assumed reference differential pressure, the microorganism concentration is confirmed again, and when the confirmed differential pressure is lower than the assumed reference differential pressure, the back pressure washing may be stopped. The assumed reference differential pressure may be determined by the characteristics of the filtration membrane and the characteristics of the filtration membrane module.

  By performing the reverse pressure cleaning using the above hypochlorite aqueous solution, it is possible to clean the filtration membrane without generating waste liquid and to control the excessively proliferating microorganism concentration. Microorganisms to maintain the filtration performance by increasing the filtration performance of the membrane filtration module in the filtration of the microorganism culture solution by performing the cleaning agent supply method capable of maintaining filterability and controlling the microorganism concentration Concentration control can be performed, and even when a cleaning agent is used, no waste liquid is generated, and even when the membrane is clogged, a favorable long-term stable filtration is possible compared to when operating at a high transmembrane pressure difference, Easier and possible.

Hereinafter, in order to describe the present invention in more detail, specific embodiments of continuous fermentation using the apparatus shown in the schematic diagram of FIG. 1 will be described with reference to examples.

Example 1
First, a membrane filtration module was manufactured. The hollow fiber membrane used for the production of the membrane module was dismantled from the pressure-type PVDF hollow fiber membrane module “HFS1020” manufactured by Toray Industries, Inc. used. A polycarbonate resin molded product was used as the separation membrane module member. The capacity of the produced membrane filtration module was 0.06 L, and the effective filtration area of the membrane filtration module was 200 cm 2. Example 1 was performed using the produced porous hollow fiber membrane and membrane filtration module. The operating conditions in Example 1 are as follows unless otherwise specified.
Fermentation tank capacity: 2 (L)
Fermentation culture tank effective volume: 1.5 (L)
Separation membrane used: 60 polyvinylidene fluoride hollow fiber membranes Temperature control: 37 (° C)
Fermentation tank aeration volume: 0.2 (L / min)
Fermentation tank agitation speed: 600 (rpm)
pH adjustment: adjusted to pH 6 with 3N NaOH Lactic acid fermentation medium supply rate: Variable in the range of 15 to 300 mL / hr
Membrane filtration flow rate control: flow rate control by suction pump The medium was used after autoclaving (121 ° C, 15 minutes). Sporolactobacillus laevolacticus JCM2513 (SL strain) is used as the microorganism, the lactic acid fermentation medium having the composition shown in Table 1 is used as the medium, and the concentration of the product lactic acid is evaluated using the HPLC shown below under the following conditions: I went there.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5 mM p-toluenesulfonic acid (0.8 mL / min)
Reaction phase: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (0.8 mL / min)
Detection method: Electrical conductivity Column temperature: 45 ° C
The optical purity of lactic acid was analyzed under the following conditions.
Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation)
Mobile phase: 1 mM aqueous copper sulfate flow rate: 1.0 mL / min Detection method: UV 254 nm
Temperature: 30 ° C
The optical purity of L-lactic acid is calculated by the following formula (i).
Optical purity (%) = 100 × (LD) / (D + L) (i)
Further, the optical purity of D-lactic acid is calculated by the following formula (ii).
Optical purity (%) = 100 × (DL) / (D + L) (ii)
Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.

  For the culture, the SL strain was first cultured overnight in a test tube with 5 mL of lactic acid fermentation medium (pre-culture). The obtained culture solution was inoculated into 100 mL of a fresh lactic acid fermentation medium, and cultured with shaking in a 500 mL Sakaguchi flask at 30 ° C. for 24 hours (pre-culture). Pre-culture medium is inoculated by placing the medium in the 1.5 L fermentation culture tank of the continuous fermentation apparatus shown in Fig. 1 and stirred with the attached stirring apparatus 4, adjusting the aeration volume of the fermentation culture tank 1 and adjusting the temperature Then, the pH was adjusted, and the culture was performed for 50 hours without operating the circulation pump 8 (pre-culture). Immediately after completion of the pre-culture, the circulating pump 8 is operated, and in addition to the operating conditions during pre-culture, the lactic acid fermentation medium is continuously supplied, and the amount of permeated water in the membrane separation type continuous fermentation apparatus is 1.5 L Then, continuous culture was performed, and D-lactic acid was produced by continuous fermentation. Control of the amount of permeated water through the continuous fermentation test was carried out by measuring the amount of filtration coming out of the filtration pump 11 and changing it under membrane filtration rate control conditions. The produced D-lactic acid concentration and optical purity in the membrane filtration fermentation broth were appropriately measured.

  Continuous fermentation filtration operation was performed for 250 hours, and backwashing was performed from 80 hours to 180 hours for continuous fermentation and from 230 hours to 250 hours, respectively. The membrane filtration operation is performed using the filtration pump 11, and the filtration flow rate is 0 to 50 hours without filtration, and the filtration is performed at a flow rate of 100 mL / h for 50 to 200 hours and 135 mL / h for 200 to 250 hours. Went. Filtration was repeated for 9 minutes and then repeatedly stopped for 1 minute. Back pressure washing was performed for a filtration time of 80 to 250 hours, followed by filtration for 9 minutes, followed by 1 minute of back pressure washing at a flow rate of a back pressure washing rate of 170 mL / h. As for the microbial concentration in the fermenter, since the filterability tends to decrease as the microbial concentration increases, the microbial concentration is considered to be able to maintain the filtration rate properly and maintain productivity. After that, the operation was performed so that the microorganism concentration was reached. The chemical solution used for back pressure washing was diluted with distilled water using commercially available sodium hypochlorite having an effective chlorine concentration of 10% so that the free chlorine concentration was 1,000 ppm or 500 ppm. The chemical solution used for back pressure washing was a chemical solution with a free chlorine concentration of 1,000 ppm from 80 hours to 180 hours of continuous fermentation, and a chemical solution with a free chlorine concentration of 500 ppm from 230 hours to 250 hours. The filtration differential pressure was measured once / day using a differential pressure gauge, and the microorganism concentration was measured once / day using OD600. To measure OD600, first collect a sample from the fermenter, dilute it with distilled water so that the OD600 of the sample is around 1, then measure the absorbance at a wavelength of 600 nm using an absorptiometer (Shimadzu UV-2450). And measured. The obtained measured value was multiplied by the magnification diluted with distilled water, and calculated as the OD600 of the sample. The experimental results obtained are shown in FIG. 3 and FIG. As a result, it was possible to stably maintain the membrane filtration differential pressure while maintaining the microorganism concentration at about 30 turbidity, and it was possible to perform effective membrane cleaning while controlling the microorganism concentration.

Example 2
A commercially available sodium hypochlorite having an effective chlorine concentration of 10% was used as a chemical solution for back pressure washing, diluted with distilled water to a free chlorine concentration of 3,000 ppm, and used from 180 to 250 hours for continuous fermentation. The membrane filtration operation is performed using the filtration pump 11, and the filtration flow rate is 0 to 50 hours without filtration. Went. Except for the above method, the same D-lactic acid continuous fermentation test as in Example 1 was performed. The results are shown in FIG. 5 and FIG. As a result, it was possible to stably maintain the membrane filtration differential pressure while maintaining the microbial concentration at about 40 turbidity, and it was possible to perform effective membrane cleaning while controlling the microbial concentration.

Example 3
A commercially available sodium hypochlorite with an effective chlorine concentration of 10% was used as a chemical solution for back pressure washing, diluted with distilled water to a free chlorine concentration of 100 ppm, and used for 100 to 250 hours of continuous fermentation. The membrane filtration operation is performed using the filtration pump 11, and the filtration flow rate is 0 to 50 hours without filtration. Went. Except for the above method, the same D-lactic acid continuous fermentation test as in Example 1 was performed. The results are shown in FIG. 7 and FIG. As a result, it was possible to stably maintain the membrane filtration differential pressure while maintaining the microbial concentration at about 20 turbidity, and it was possible to perform effective membrane cleaning while controlling the microbial concentration.

Comparative Example 1
The same D-lactic acid continuous fermentation test as in Example 1 was performed without performing back pressure washing with the back pressure washing chemical. The results are shown in FIG. 3 and FIG. The turbidity of continuous fermentation for 250 hours increased to 75.1, the microbial concentration could not be controlled, the membrane filtration differential pressure also increased, and stable continuous fermentation filtration operation could not be performed.

Comparative Example 2
The same D-lactic acid continuous fermentation test as in Example 2 was performed without performing back pressure cleaning with a back pressure cleaning chemical. The results are shown in FIG. 5 and FIG. The turbidity of continuous fermentation for 250 hours increased to 79.7, the microbial concentration could not be controlled, the membrane filtration differential pressure also increased, and stable continuous fermentation filtration operation could not be performed.

Comparative Example 3
The same D-lactic acid continuous fermentation test as in Example 3 was performed without performing back pressure washing with the back pressure washing chemical. The results are shown in FIG. 7 and FIG. The turbidity of continuous fermentation for 250 hours increased to 58.8, the microbial concentration could not be controlled, the membrane filtration differential pressure also increased, and stable continuous fermentation filtration operation could not be performed.

  The present invention effectively cleans membrane dirt generated by membrane filtration with a simple operation method, and can control the concentration of microorganisms in the fermenter, significantly improving fermentation production efficiency stably at low cost. In addition, it is possible to reduce the processing costs generated from the washing waste liquid and the drawn culture liquid, and further to reduce the cost, and in the fermentation industry, it is possible to stably produce fermentation products at low cost. It becomes possible.

DESCRIPTION OF SYMBOLS 1 Fermentation culture tank 2 Separation membrane module 3 Temperature control device 4 Stirring device 5 pH sensor / control device 6 Level sensor / control device 7 Differential pressure sensor / control device 8 Circulation pump 9 Medium supply pump 10 Neutralizing agent supply pump 11 Filtration pump 12 Cleaning agent supply pump 13 Filtration valve 14 Cleaning agent valve 15 Gas supply device

Claims (2)

  The raw solution containing the pre-conversion substance is introduced into the fermentor, the pre-conversion substance is converted using the microorganism-containing liquid, and then filtered using a membrane module, and the non-permeate is continuously retained in the fermentor for conversion. In a continuous fermentation operation for taking out a permeate containing a post-substance, an operation method of a continuous fermentation apparatus for performing membrane cleaning by supplying a cleaning agent containing a hypochlorite aqueous solution from the permeate side of the membrane module. A method for operating a continuous fermentation apparatus, characterized in that the supply condition of the detergent is controlled by the microorganism concentration in the fermenter.   The method for operating a continuous fermentation apparatus according to claim 1, wherein the substance after conversion is a fluid containing at least one of chemical products, dairy products, pharmaceuticals, foods or brewed products, or waste water. .

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