JP5082496B2 - Process for producing chemicals by continuous fermentation and continuous fermentation apparatus - Google Patents

Process for producing chemicals by continuous fermentation and continuous fermentation apparatus Download PDF

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JP5082496B2
JP5082496B2 JP2007040518A JP2007040518A JP5082496B2 JP 5082496 B2 JP5082496 B2 JP 5082496B2 JP 2007040518 A JP2007040518 A JP 2007040518A JP 2007040518 A JP2007040518 A JP 2007040518A JP 5082496 B2 JP5082496 B2 JP 5082496B2
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秀樹 澤井
勝成 山田
孝 耳塚
健司 澤井
徹 米原
世人 伊藤
昌弘 辺見
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Description

本発明は、微生物または細胞の培養方法の改良およびそれに用いられる培養装置に関するものである。さらに詳しくは、本発明は、培養を行いながら、微生物または培養細胞の発酵培養液から、目詰まりが生じにくい多孔性分離膜を通して生産物を含む液を効率よく濾過・回収することおよび未濾過液を発酵培養液に戻すことにより、発酵に関与する微生物濃度を向上させて高い生産性を得ることができる連続発酵による化学品の製造方法およびそれに用いられる連続発酵装置に関するものである。   The present invention relates to an improvement of a microorganism or cell culture method and a culture apparatus used therefor. More specifically, the present invention efficiently filters and collects a liquid containing a product from a fermentation broth of microorganisms or cultured cells through a porous separation membrane that is less likely to be clogged while culturing, and an unfiltered liquid. It is related with the manufacturing method of the chemical product by continuous fermentation which can improve the microbe density | concentration in connection with fermentation, and can obtain high productivity by returning this to a fermentation culture solution, and the continuous fermentation apparatus used for it.

微生物や培養細胞の培養を伴う物質生産方法である発酵法は、大きく（１）バッチ発酵法（Ｂａｔｃｈ発酵法）および流加発酵法（Ｆｅｄ−Ｂａｔｃh発酵法）と（２）連続発酵法に分類することができる。   Fermentation methods, which are substance production methods involving the cultivation of microorganisms and cultured cells, are roughly classified into (1) batch fermentation methods (Batch fermentation methods), fed-batch fermentation methods (Fed-Batch fermentation methods), and (2) continuous fermentation methods. can do.

上記（１）のバッチ発酵法および流加発酵法は、設備的には簡素であり、短時間で培養が終了し、雑菌汚染による被害が少ないという利点がある。しかしながら、時間経過とともに培養液中の生産物濃度が高くなり、浸透圧あるいは生産物阻害等の影響により生産性および収率が低下してくる。そのため、長時間にわたり安定して高収率かつ高生産性を維持するのが困難である。   The batch fermentation method and fed-batch fermentation method of the above (1) are simple in terms of equipment, and have an advantage that culture is completed in a short time and damage caused by contamination with bacteria is small. However, as the time elapses, the product concentration in the culture solution increases, and the productivity and yield decrease due to the influence of osmotic pressure or product inhibition. Therefore, it is difficult to maintain high yield and high productivity stably over a long period of time.

一方、上記（２）の連続発酵法は、発酵槽内で目的物質が高濃度に蓄積するのを回避することによって、長時間にわたって高収率かつ高生産性を維持できるという特徴がある。Ｌ−グルタミン酸やＬ−リジンの発酵について、このような連続培養法が開示されている（非特許文献１参照。）。しかしながら、これらの例では、培養液へ原料の連続的な供給を行うと共に、微生物や細胞を含んだ培養液を抜き出すために、培養液中の微生物や細胞が希釈されることから、生産効率の向上は限定されたものであった。   On the other hand, the continuous fermentation method (2) is characterized in that a high yield and high productivity can be maintained over a long period of time by avoiding accumulation of the target substance at a high concentration in the fermenter. Such a continuous culture method is disclosed about fermentation of L-glutamic acid or L-lysine (refer nonpatent literature 1). However, in these examples, since the raw material is continuously supplied to the culture solution and the culture solution containing microorganisms and cells is extracted, the microorganisms and cells in the culture solution are diluted. The improvement was limited.

このことから、連続発酵法において、微生物や培養細胞を分離膜で濾過し、濾液から生産物を回収すると同時に濾過された微生物や培養細胞を培養液に保持または還流させることで、培養液中の微生物や細胞濃度を高く維持する方法が提案されている。   Therefore, in the continuous fermentation method, microorganisms and cultured cells are filtered through a separation membrane, and the product is recovered from the filtrate, and at the same time, the filtered microorganisms and cultured cells are held or refluxed in the culture solution, A method of maintaining a high microorganism or cell concentration has been proposed.

例えば、セラミックス膜を用いた連続発酵装置において、連続発酵する技術が提案されている（特許文献１、特許文献２および特許文献３参照。）が、これらの提案では、分離膜の目詰りによる濾過流量や濾過効率の低下に問題があり、目詰まり防止のために、逆洗浄等を行っている。また、分離膜を用いたコハク酸の製造方法が提案されている（特許文献４参照。）。しかしながら、この提案では、膜分離において高い濾過圧（約２００ｋＰａ）が採用されている。高い濾過圧は、コスト的にも不利であるばかりでなく、濾過処理において微生物や細胞が圧力によって物理的なダメージをうけることから、微生物や細胞を連続的に発酵培養液に戻す連続発酵法においては適切ではない。   For example, in a continuous fermentation apparatus using a ceramic membrane, techniques for continuous fermentation have been proposed (see Patent Document 1, Patent Document 2 and Patent Document 3). In these proposals, filtration by clogging of the separation membrane is performed. There is a problem in the flow rate and the reduction in filtration efficiency, and back washing or the like is performed to prevent clogging. Further, a method for producing succinic acid using a separation membrane has been proposed (see Patent Document 4). However, in this proposal, a high filtration pressure (about 200 kPa) is employed in membrane separation. High filtration pressure is not only disadvantageous in terms of cost, but also in the continuous fermentation method in which microorganisms and cells are physically damaged by pressure during filtration, so that microorganisms and cells are continuously returned to the fermentation broth. Is not appropriate.

このように、従来の連続発酵法には様々な問題があり、産業的応用が難しかった。   As described above, the conventional continuous fermentation method has various problems, and industrial application is difficult.

すなわち、連続発酵法において、微生物や細胞を分離膜で濾過し、濾液から生産物を回収すると同時に濾過された微生物や細胞を発酵培養液に還流させ、発酵培養液中の微生物や細胞濃度を向上させ、かつ、高く維持させることで高い物質生産性を得ることは、依然として困難であり、技術の革新が望まれていた。
特開平５−９５７７８号公報 特開昭６２−１３８１８４号公報 特開平１０−１７４５９４号公報 特開２００５−３３３８８６号公報 Toshihiko Hirao et. al.（ヒラノ・トシヒコ ら）、 Appl. Microbiol. Biotechnol.（アプライド マイクロバイアル アンド マイクロバイオロジー），３２，２６９−２７３（１９８９） That is, in a continuous fermentation method, microorganisms and cells are filtered through a separation membrane, and the product is recovered from the filtrate, and at the same time, the filtered microorganisms and cells are refluxed to the fermentation broth to improve the microorganisms and cell concentration in the fermentation broth. However, it is still difficult to obtain high material productivity by maintaining it at a high level, and technological innovation has been desired.
JP-A-5-95778 Japanese Patent Laid-Open No. 62-138184 JP-A-10-174594 JP 2005-333886 A Toshihiko Hirao et. Al. (Hirano Toshihiko et al.), Appl. Microbiol. Biotechnol. (Applied Microvials and Microbiology), 32, 269-273 (1989)

本発明の目的は、簡便な操作方法で、長時間にわたり安定して高生産性を維持することができる連続発酵による化学品の製造方法と、その製造方法に用いられる連続発酵装置を提供することにある。   An object of the present invention is to provide a method for producing a chemical product by continuous fermentation capable of maintaining high productivity stably over a long period of time with a simple operation method, and a continuous fermentation apparatus used in the production method. It is in.

本発明者らは、微生物や培養細胞の分離膜内への侵入が少なく、微生物や培養細胞を膜間差圧が低い条件で濾過した場合に、分離膜の目詰まりが著しく抑制されることを見出し、課題であった高濃度の微生物や培養細胞の濾過が長期間安定に維持できることを可能とし、本発明に到達したのである。   The present inventors show that clogging of the separation membrane is remarkably suppressed when the microorganisms and the cultured cells are less likely to enter the separation membrane and the microorganisms and the cultured cells are filtered under a condition where the transmembrane pressure is low. The present invention has reached the present invention by enabling the filtration of high-concentration microorganisms and cultured cells, which were found and problematic, to be stably maintained for a long period of time.

すなわち、本発明は、微生物もしくは培養細胞の発酵培養液を分離膜で濾過し、濾液から生産物を回収すると共に未濾過液を前記の発酵培養液に保持または還流し、かつ、発酵原料を前記の発酵培養液に追加する連続発酵による化学品の製造方法であって、前記の分離膜として平均細孔径が０.０１μｍ以上１μｍ未満の細孔を有する多孔性膜を用い、その膜間差圧を０.１から２０ｋＰａの範囲にして濾過処理することを特徴とする連続発酵による化学品の製造方法である。   That is, the present invention filters the fermentation broth of microorganisms or cultured cells with a separation membrane, collects the product from the filtrate, holds or refluxs the unfiltrated liquid in the fermentation broth, A method for producing a chemical product by continuous fermentation added to the fermentation broth, wherein a porous membrane having pores with an average pore diameter of 0.01 μm or more and less than 1 μm is used as the separation membrane, and the transmembrane pressure difference In a range of 0.1 to 20 kPa, and a chemical production method by continuous fermentation.

本発明の好ましい態様によれば、前記の多孔性膜の純水透過係数は、２×１０-9ｍ３／ｍ２／ｓ／ｐａ以上６×１０-7ｍ３／ｍ２／ｓ／ｐａ以下である。 According to a preferred embodiment of the present invention, 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. It is as follows.

本発明の好ましい態様によれば、前記の多孔性膜の平均細孔径は０.０１μｍ以上０.２μｍ未満の範囲内にあり、その平均細孔径の標準偏差は０.１μｍ以下であり、そして、その膜表面粗さは０.１μｍ以下である。   According to a preferred embodiment of the present invention, the average pore size of the porous membrane is in the range of 0.01 μm or more and less than 0.2 μm, the standard deviation of the average pore size is 0.1 μm or less, and The film surface roughness is 0.1 μm or less.

本発明の好ましい態様によれば、前記の多孔性膜は多孔質樹脂層を含む多孔性膜であり、その多孔質樹脂層は好ましくはポリフッ化ビニリデン等の有機高分子化合物からなるものである。   According to a preferred embodiment of the present invention, the porous membrane is a porous membrane including a porous resin layer, and the porous resin layer is preferably composed of an organic polymer compound such as polyvinylidene fluoride.

本発明の好ましい態様によれば、前記の微生物または培養細胞の発酵培養液および発酵原料が、糖類を含むことである。   According to a preferred embodiment of the present invention, the fermentation broth and fermentation raw material of the microorganism or cultured cell contain saccharides.

本発明の好ましい態様によれば、前記の化学品は、乳酸等の有機酸またはエタノール等のアルコールである。   According to a preferred embodiment of the present invention, the chemical product is an organic acid such as lactic acid or an alcohol such as ethanol.

本発明の好ましい態様によれば、前記の微生物は酵母等の真核細胞であり、その好適な酵母は、サッカロミセス属（Ｇｅｎｕｓ Ｓａｃｃｈａｒｏｍｙｃｅｓ）に属する酵母とサッカロミセス・セレビセ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）に属する酵母である。   According to a preferred embodiment of the present invention, the microorganism is a eukaryotic cell such as a yeast, and the preferred yeast is a yeast belonging to the genus Saccharomyces and a yeast belonging to Saccharomyces cerevisiae. .

また、本発明の連続発酵装置は、微生物もしくは培養細胞の発酵培養液を分離膜で濾過し、濾液から生産物を回収すると共に未濾過液を前記の発酵培養液に保持または還流し、かつ、発酵原料を前記の発酵培養液に追加する連続発酵による化学品の製造装置であって、微生物もしくは培養細胞を発酵培養させるための発酵反応槽と、該発酵反応槽に発酵培養液循環手段を介して接続され内部に分離膜を備えた発酵培養液を濾過するための膜分離槽と、分離膜の膜間差圧を０.１から２０ｋＰａの範囲に制御する手段からなり、該分離膜が平均細孔径０.０１μｍ以上１μｍ未満の多孔性膜であることを特徴とする連続発酵装置である。   In addition, the continuous fermentation apparatus of the present invention filters the fermentation broth of microorganisms or cultured cells through a separation membrane, collects the product from the filtrate and holds or refluxs the unfiltrated liquid in the fermentation broth, and An apparatus for producing a chemical product by continuous fermentation in which fermentation raw materials are added to the fermentation broth, a fermentation reaction tank for fermenting and culturing microorganisms or cultured cells, and a fermentation broth circulation means in the fermentation reaction tank And a means for controlling the transmembrane differential pressure of the separation membrane within the range of 0.1 to 20 kPa, and the separation membrane is an average. It is a continuous fermentation apparatus characterized by being a porous membrane having a pore diameter of 0.01 μm or more and less than 1 μm.

本発明の連続発酵装置の好ましい態様によれば、前記の分離膜の膜間差圧は、水頭差制御装置による発酵培養液と多孔性膜処理水の液位差で制御することができる。   According to the preferable aspect of the continuous fermentation apparatus of this invention, the transmembrane differential pressure | voltage of the said separation membrane can be controlled by the liquid level difference of the fermentation culture solution by a water head difference control apparatus, and porous membrane treated water.

本発明の連続発酵装置の好ましい態様によれば、前記の分離膜の膜間差圧は、加圧ポンプまたは／および吸引ポンプで制御することができる。   According to a preferred aspect of the continuous fermentation apparatus of the present invention, the transmembrane pressure difference of the separation membrane can be controlled by a pressurizing pump and / or a suction pump.

本発明の連続発酵装置の好ましい態様によれば、前記の分離膜の膜間差圧は、気体または液体の圧力で制御することができる。   According to a preferred aspect of the continuous fermentation apparatus of the present invention, the transmembrane pressure difference of the separation membrane can be controlled by the pressure of gas or liquid.

本発明の連続発酵装置の好ましい態様によれば、前記の発酵培養液循環手段は、循環ポンプである。   According to a preferred aspect of the continuous fermentation apparatus of the present invention, the fermentation culture medium circulation means is a circulation pump.

本発明によれば、分離膜として高い透過性と高い細胞阻止率を持ち閉塞しにくい多孔性膜を用い、低い膜間差圧で濾過処理することにより、安定に低コストで発酵生産効率を著しく向上させることができる。また、本発明によれば、簡便な操作条件で、長時間にわたり安定して高生産性を維持する連続発酵が可能となり、広く発酵工業において、発酵生産物である化学品を低コストで安定に生産することが可能となる。   According to the present invention, by using a porous membrane having high permeability and high cell blocking rate as a separation membrane and hardly clogging, and performing filtration treatment with a low transmembrane pressure difference, fermentation production efficiency is remarkably reduced at low cost. Can be improved. In addition, according to the present invention, continuous fermentation that stably maintains high productivity over a long period of time can be performed under simple operation conditions, and chemical products that are fermentation products can be stably stabilized at low cost in the fermentation industry. It becomes possible to produce.

本発明は、微生物もしくは培養細胞の発酵培養液を分離膜で濾過し、濾液から生産物を回収すると共に未濾過液を発酵培養液に保持または還流し、かつ、発酵原料を発酵培養液に追加する連続発酵において、分離膜として平均細孔径が０.０１μｍ以上１μｍ未満の細孔を有する多孔性膜を用い、その膜間差圧を０.１から２０ｋＰａの範囲にして濾過処理することを特徴とするものである。   The present invention filters the fermentation broth of microorganisms or cultured cells through a separation membrane, collects the product from the filtrate, holds or refluxs the unfiltered liquid in the fermentation broth, and adds fermentation raw materials to the fermentation broth In the continuous fermentation, a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm is used as a separation membrane, and the filtration is performed with a transmembrane differential pressure in the range of 0.1 to 20 kPa. It is what.

本発明において分離膜として用いられる多孔性膜は、発酵に使用される微生物や培養細胞による目詰まりが起こりにくく、そして、濾過性能が長期間安定に継続する性能を有するものであることが望ましい。そのため、本発明で使用される多孔性膜は、平均細孔径が、０.０１μｍ以上１μｍ未満であることが重要である。   The porous membrane used as a separation membrane in the present invention is desirably one that is not easily clogged by microorganisms and cultured cells used for fermentation, and has a performance that allows filtration performance to continue stably for a long period of time. Therefore, it is important that the porous membrane used in the present invention has an average pore diameter of 0.01 μm or more and less than 1 μm.

本発明で分離膜として用いられる多孔性膜の構成について説明する。本発明における多孔性膜は、被処理水の水質や用途に応じた分離性能と透水性能を有するものである。多孔性膜は、阻止性能および透水性能や耐汚れ性という分離性能の点からは、多孔質樹脂層を含む多孔性膜であることが好ましい。このような多孔性膜は、多孔質基材の表面に、分離機能層として作用とする多孔質樹脂層を有している。多孔質基材は、多孔質樹脂層を支持して分離膜に強度を与えるものである。   The structure of the porous membrane used as a separation membrane in the present invention will be described. The porous membrane in the present invention has separation performance and water permeability according to the quality of water to be treated and the application. The porous membrane is preferably a porous membrane including a porous resin layer from the viewpoint of separation performance such as blocking performance, water permeability performance, and dirt resistance. Such a porous membrane has a porous resin layer that acts as a separation functional layer on the surface of the porous substrate. The porous substrate supports the porous resin layer and gives strength to the separation membrane.

多孔質基材の材質は、有機材料および／または無機材料等からなり、中でも有機繊維が望ましく用いられる。好ましい多孔質基材は、セルロース繊維、セルローストリアセテート繊維、ポリエステル繊維、ポリプロピレン繊維およびポリエチレン繊維などの有機繊維を用いてなる織布や不織布等である。中でも、密度の制御が比較的容易であり製造も容易で安価な不織布が好ましく用いられる。   The material of the porous substrate is made of an organic material and / or an inorganic material, and organic fiber is preferably used among them. Preferred porous substrates are woven fabrics and nonwoven fabrics made of organic fibers such as cellulose fibers, cellulose triacetate fibers, polyester fibers, polypropylene fibers and polyethylene fibers. Among them, a nonwoven fabric that is relatively easy to control the density, easy to manufacture, and inexpensive is preferably used.

また、多孔質樹脂層は、上述したように分離機能層として作用するものであり、有機高分子膜を好適に使用することができる。有機高分子膜の材質としては、例えば、ポリエチレン系樹脂、ポリプロピレン系樹脂、ポリ塩化ビニル系樹脂、ポリフッ化ビニリデン系樹脂、ポリスルホン系樹脂、ポリエーテルスルホン系樹脂、ポリアクリロニトリル系樹脂、セルロース系樹脂およびセルローストリアセテート系樹脂等が挙げられる。有機高分子膜は、これらの樹脂を主成分とする樹脂の混合物からなるものであってもよい。ここで主成分とは、その成分が５０重量％以上、好ましくは６０重量％以上含有することをいう。中でも、多孔質樹脂層を構成する膜素材としては、溶液による製膜が容易で物理的耐久性や耐薬品性にも優れているポリ塩化ビニル系樹脂、ポリフッ化ビニリデン系樹脂、ポリスルホン系樹脂、ポリエーテルスルホン系樹脂およびポリアクリロニトリル系樹脂が好ましく用いられる。膜素材には、ポリフッ化ビニリデン系樹脂またはそれを主成分とする樹脂が最も好ましく用いられる。   Further, as described above, the porous resin layer functions as a separation functional layer, and an organic polymer membrane can be suitably used. Examples of the material of the organic polymer film include polyethylene resin, polypropylene resin, polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, polyacrylonitrile resin, cellulose resin, and the like. Examples thereof include cellulose triacetate resins. The organic polymer film may be made of a mixture of resins mainly composed of these resins. Here, the main component means that the component is contained in an amount of 50% by weight or more, preferably 60% by weight or more. Among them, as a membrane material constituting the porous resin layer, a polyvinyl chloride resin, a polyvinylidene fluoride resin, a polysulfone resin, which is easy to form a film with a solution and excellent in physical durability and chemical resistance, Polyether sulfone resins and polyacrylonitrile resins are preferably used. For the film material, a polyvinylidene fluoride resin or a resin containing it as the main component is most preferably used.

ここで、ポリフッ化ビニリデン系樹脂としては、フッ化ビニリデンの単独重合体が好ましいが、フッ化ビニリデンと共重合可能なビニル系単量体との共重合体も好ましく用いられる。フッ化ビニリデンと共重合可能なビニル系単量体としては、テトラフルオロエチレン、ヘキサフルオロプロピレンおよび三塩化フッ化エチレンなどが例示される。   Here, as the polyvinylidene fluoride resin, a homopolymer of vinylidene fluoride is preferable, but a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride is also preferably used. Examples of vinyl monomers copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene, and ethylene trichloride fluoride.

本発明で用いられる多孔性膜の作成法の概要を説明する。まず、前記の多孔質基材の表面に、前記の樹脂と溶媒を含む原液の被膜を形成するとともに、その原液を多孔質基材に含浸させる。その後、被膜を有する多孔質基材の被膜側表面のみを、非溶媒を含む凝固浴と接触させて樹脂を凝固させると共に多孔質基材の表面に多孔質樹脂層を形成する。原液に、さらに非溶媒を含ませることもできる。原液の温度は、製膜性の観点から、通常、１５〜１２０℃の範囲内で選定することが好ましい。   An outline of a method for producing a porous membrane used in the present invention will be described. First, a film of the stock solution containing the resin and the solvent is formed on the surface of the porous substrate, and the porous substrate 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 form a porous resin layer on the surface of the porous substrate. The stock solution may further contain a non-solvent. The temperature of the stock solution is usually preferably selected within the range of 15 to 120 ° C. from the viewpoint of film forming properties.

ここで、原液には、開孔剤を添加することもできる。開孔剤は、凝固浴に浸漬された際に抽出されて、樹脂層を多孔質にする作用を持つものである。開孔剤を添加することにより、平均細孔径の大きさの制御することができる。開孔剤は、凝固浴に浸漬された際に抽出されて、樹脂層を多孔質にする作用を持つものである。開孔剤は、凝固浴への溶解性の高いものであることが好ましい。開孔剤としては、例えば、塩化カルシウムや炭酸カルシウムなどの無機塩を用いることができる。また、開孔剤として、ポリエチレングリコールやポリプロピレングリコールなどのポリオキシアルキレン類や、ポリビニルアルコール、ポリビニルブチラールおよびポリアクリル酸などの水溶性高分子化合物や、グリセリンを用いることができる。   Here, 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 extracted when immersed in the coagulation bath, and has a function of making the resin layer porous. The pore-opening agent is preferably one having high solubility in the coagulation bath. As the pore opening agent, for example, an inorganic salt such as calcium chloride or calcium carbonate can be used. As the pore opening agent, polyoxyalkylenes such as polyethylene glycol and polypropylene glycol, water-soluble polymer compounds such as polyvinyl alcohol, polyvinyl butyral and polyacrylic acid, and glycerin can be used.

また、溶媒は、樹脂を溶解するものである。溶媒は、樹脂および開孔剤に作用してそれらが多孔質樹脂層を形成するのを促す。このような溶媒としては、Ｎ−メチルピロリジノン（ＮＭＰ）、Ｎ，Ｎ−ジメチルアセトアミド（ＤＭＡｃ）、Ｎ，Ｎ−ジメチルホルムアミド（ＤＭＦ）、ジメチルスルホキシド（ＤＭＳＯ）、アセトンおよびメチルエチルケトンなどを用いることができる。中でも、樹脂の溶解性の高いＮＭＰ、ＤＭＡｃ、ＤＭＦおよびＤＭＳＯが好ましく用いられる。   The solvent dissolves the resin. The solvent acts on the resin and the pore-opening agent to encourage them to form a porous resin layer. As such a solvent, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone, and the like can be used. . Among these, NMP, DMAc, DMF and DMSO, which have high resin solubility, are preferably used.

さらに、原液には、非溶媒を添加することもできる。非溶媒は、樹脂を溶解しない液体である。非溶媒は、樹脂の凝固の速度を制御して細孔の大きさを制御するように作用する。非溶媒としては、水やメタノールおよびエタノールなどのアルコール類を用いることができる。中でも、価格の点から水やメタノールが好ましい。非溶媒は、これらの混合物であってもよい。   Furthermore, a non-solvent can be added to the stock solution. The non-solvent is a liquid that does not dissolve the resin. The non-solvent acts to control the pore size by controlling the rate of solidification of the resin. As the non-solvent, water, alcohols such as methanol and ethanol can be used. Among these, water and methanol are preferable from the viewpoint of price. The non-solvent may be a mixture thereof.

本発明で用いられる多孔性膜は、平膜であっても中空糸膜であっても良い。平膜の場合、その平均厚みは用途に応じて選択されるが、好ましくは２０μｍ以上５０００μｍ以下であり、より好ましくは５０μｍ以上２０００μｍ以下の範囲で選択される。   The porous membrane used in the present invention may be a flat membrane or a hollow fiber membrane. In the case of a flat membrane, the average thickness is selected according to the application, but is preferably 20 μm or more and 5000 μm or less, and more preferably 50 μm or more and 2000 μm or less.

上述のように、本発明で用いられる分離膜は、多孔質基材と多孔質樹脂層とから形成されている多孔性膜であることが望ましい。その際、多孔質基材に多孔質樹脂層が浸透していても、多孔質基材に多孔質樹脂層が浸透していなくてもどちらでも良く、用途に応じて選択される。多孔質基材の平均厚みは、好ましくは５０μｍ以上３０００μｍ以下の範囲で選択される。また、多孔性膜が中空糸膜の場合、中空糸の内径は好ましくは２００μｍ以上５０００μｍ以下の範囲で選択され、膜厚は好ましくは２０μｍ以上２０００μｍ以下の範囲で選択される。また、有機繊維または無機繊維を筒状にした織物や編物を中空糸の内部に含んでいても良い。   As described above, the separation membrane used in the present invention is desirably a porous membrane formed from a porous substrate and a porous resin layer. At that time, either the porous resin layer permeates the porous base material or the porous resin layer does not permeate the porous base material, and it is selected according to the application. The average thickness of the porous substrate is preferably selected in the range of 50 μm to 3000 μm. When the porous membrane is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably selected in the range of 200 μm to 5000 μm, and the film thickness is preferably selected in the range of 20 μm to 2000 μm. Further, a woven fabric or a knitted fabric in which organic fibers or inorganic fibers are formed in a cylindrical shape may be included in the hollow fiber.

本発明で用いられる多孔性膜は、支持体と組み合わせることによって分離膜エレメントとすることができる。支持体として支持板を用い、その支持板の少なくとも片面に、本発明で用いられる多孔性膜を配した分離膜エレメントは、本発明で用いられる多孔性膜を有する分離膜エレメントの好適な形態の一つである。この形態で、膜面積を大きくすることが困難な場合には、透水量を大きくするために、支持板の両面に多孔性膜を配することが好ましい。   The porous membrane used in the present invention can be made into a separation membrane element by combining with a support. A separation membrane element in which a support plate is used as a support and the porous membrane used in the present invention is disposed on at least one side of the support plate is a preferred embodiment of the separation membrane element having a porous membrane used in the present invention. One. In this form, when it is difficult to increase the membrane area, it is preferable to dispose a porous membrane on both sides of the support plate in order to increase the water permeability.

分離膜の平均細孔径が上記のように０．０１μｍ以上１μｍ未満の範囲内にあると、菌体や汚泥などがリークすることのない高い排除率と、高い透水性を両立させることができ、さらに目詰まりをしにくく、透水性を長時間保持することが、より高い精度と再現性を持って実施することができる。微生物として細菌類を用いた場合、多孔性膜の平均細孔径は好ましくは０.４μｍ以下であり、平均細孔径は０.２μｍ未満であればなお好適に実施することが可能である。平均細孔径は、小さすぎると透水量が低下することがあるので、本発明では、平均細孔径は０.０１μｍ以上であり、好ましくは０．０２μｍ以上であり、さらに好ましくは０．０４μｍ以上である。   When the average pore diameter of the separation membrane is within the range of 0.01 μm or more and less than 1 μm as described above, it is possible to achieve both a high exclusion rate without leaking bacterial cells or sludge and high water permeability, Further, clogging is less likely to occur and the water permeability can be maintained for a long time with higher accuracy and reproducibility. When bacteria are used as the microorganism, the average pore diameter of the porous membrane is preferably 0.4 μm or less, and the average pore diameter is preferably less than 0.2 μm. If the average pore diameter is too small, the water permeation rate may decrease. Therefore, in the present invention, the average pore diameter is 0.01 μm or more, preferably 0.02 μm or more, more preferably 0.04 μm or more. is there.

ここで、平均細孔径は、倍率１０，０００倍の走査型電子顕微鏡観察における、９．２μｍ×１０．４μｍの範囲内で観察できる細孔すべての直径を測定し、平均することにより求めることができる。   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.

上記の平均細孔径の標準偏差σは、０．１μｍ以下であることが好ましい。更に、平均細孔径の標準偏差が小さい、すなわち細孔径の大きさが揃っている方が均一な透過液を得ることができる。発酵運転管理が容易になることから、平均細孔径の標準偏差は小さければ小さい方が望ましい。   The standard deviation σ of the average pore diameter is preferably 0.1 μm or less. Further, a uniform permeate can be obtained when the standard deviation of the average pore diameter is smaller, that is, when the pore diameters are uniform. Since fermentation operation management becomes easy, the smaller the standard deviation of the average pore diameter, the better.

平均細孔径の標準偏差σは、上述の９．２μｍ×１０．４μｍの範囲内で観察できる細孔数をＮとして、測定した各々の直径をＸｋとし、細孔直径の平均をＸ（ａｖｅ）とした下記の（式１）により算出される。   The standard deviation σ of the average pore diameter is N (the number of pores that can be observed within the above-mentioned range of 9.2 μm × 10.4 μm), each measured diameter is Xk, and the average pore diameter is X (ave) It is calculated by the following (Formula 1).

Figure 0005082496
Figure 0005082496

本発明で用いられる多孔性膜においては、発酵培養液の透過性が重要点の一つであり、透過性の指標として、使用前の多孔性膜の純水透過係数を用いることができる。本発明において、多孔性膜の純水透過係数は、逆浸透膜による２５℃の温度の精製水を用い、ヘッド高さ１ｍで透水量を測定し算出したとき、２×１０−９ｍ３／ｍ２／ｓ／ｐａ以上であることが好ましい。純水透過係数が２×１０−９ｍ３／ｍ２／ｓ／ｐａ以上６×１０−７ｍ３／ｍ２／ｓ／ｐａ以下であれば、実用的に十分な透過水量が得られる。より好ましい純水透過係数は、２×１０−９ｍ３／ｍ２／ｓ／ｐａ以上１×１０−７ｍ３／ｍ２／ｓ／ｐａ以下である。 In the porous membrane used in the present invention, the permeability of the fermentation broth is one of the important points, and the pure water permeability coefficient of the porous membrane before use can be used as the permeability index. In the present invention, the pure water permeation coefficient of the porous membrane is 2 × 10 −9 m 3 / when the water permeability is measured at a head height of 1 m using purified water at a temperature of 25 ° C. by a reverse osmosis membrane. It is preferably m 2 / s / pa or more. If the pure water permeability coefficient is 2 × 10 −9 m 3 / m 2 / s / pa or more and 6 × 10 −7 m 3 / m 2 / s / pa or less, a practically sufficient amount of permeated water can be obtained. A more preferable pure water permeability coefficient is 2 × 10 −9 m 3 / m 2 / s / pa or more and 1 × 10 −7 m 3 / m 2 / s / pa or less.

本発明で用いられる多孔性膜の膜表面粗さは、分離膜の目詰まりに影響を与える因子である。膜表面粗さが好ましくは０.１μｍ以下のときに、分離膜の剥離係数や膜抵抗を好適に低下させることができ、より低い膜間差圧で連続発酵が実施可能である。従って、目詰まりを抑えることにより、安定した連続発酵が可能になることから、表面粗さは小さければ小さいほど好ましい。   The membrane surface roughness of the porous membrane used in the present invention is a factor that affects the clogging of the separation membrane. When the membrane surface roughness is preferably 0.1 μm or less, the separation coefficient and membrane resistance of the separation membrane can be suitably reduced, and continuous fermentation can be carried out with a lower transmembrane pressure difference. Therefore, since stable continuous fermentation becomes possible by suppressing clogging, the smaller the surface roughness, the better.

また、多孔性膜の膜表面粗さを低くすることにより、微生物や培養細胞の濾過において、膜表面で発生する剪断力を低下させることが期待でき、微生物や培養細胞の破壊が抑制され、多孔性膜の目詰まりも抑制されることにより、長期間安定な濾過が可能になると考えられる。   In addition, by reducing the membrane surface roughness of the porous membrane, it can be expected that the shearing force generated on the membrane surface will be reduced during filtration of microorganisms and cultured cells. It is considered that stable filtration is possible for a long time by suppressing clogging of the conductive film.

ここで、膜表面粗さは、下記の原子間力顕微鏡装置（ＡＦＭ）を使用して、下記の装置と条件で測定することができる。
・装置：原子間力顕微鏡装置(Digital Instruments（株）製Nanoscope IIIa)
・条件：探針 SiNカンチレバー(Digital Instruments（株）製)
：走査モード コンタクトモード（気中測定）
水中タッピングモード（水中測定）
：走査範囲 １０μm、２５μm 四方（気中測定）
５μm、１０μm 四方（水中測定）
：走査解像度 ５１２×５１２
・試料調製 測定に際し膜サンプルは、常温でエタノールに１５分浸漬後、ＲＯ水中に２４時間浸漬し洗浄した後、風乾し用いた。ＲＯ水とは、ろ過膜の一種である逆浸透膜（ＲＯ膜）を用いてろ過し、イオンや塩類などの不純物を排除した水を指す。ＲＯ膜の孔の大きさは、概ね２ｎｍ以下である。 Here, film | membrane surface roughness can be measured on the following apparatus and conditions using the following atomic force microscope apparatus (AFM).
・ Device: Atomic force microscope device (Nanoscope IIIa manufactured by Digital Instruments)
・ Conditions: Probe SiN cantilever (manufactured by Digital Instruments)
: Scanning mode Contact mode (in-air measurement)
Underwater tapping mode (underwater measurement)
: Scanning range 10μm, 25μm square (measurement in air)
5μm, 10μm square (underwater measurement)
: Scanning resolution 512 × 512
-Sample preparation 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 RO water refers to water that has been filtered using a reverse osmosis membrane (RO membrane), which is a type of filtration membrane, and impurities such as ions and salts are excluded. The pore size of the RO membrane is approximately 2 nm or less.

膜表面粗さｄｒｏｕｇｈは、上記ＡＦＭにより各ポイントのＺ軸方向の高さから、下記の（式２）により算出する。 Membrane surface roughness d rough from the Z-axis direction of the height of each point by the AFM, is calculated by the following equation (2).

Figure 0005082496
Figure 0005082496

本発明で使用される微生物や培養細胞の発酵原料は、発酵培養する微生物や培養細胞の生育を促し、目的とする発酵生産物である化学品を良好に生産させ得るものであればよい。発酵原料としては、例えば、炭素源、窒素源、無機塩類、および必要に応じてアミノ酸、およびビタミンなどの有機微量栄養素を適宜含有する通常の液体培地等が好ましく用いられる。   The fermentation raw material for microorganisms and cultured cells used in the present invention may be any material that promotes the growth of microorganisms and cultured cells for fermentation and can produce a desired chemical product that is a desired fermentation product. 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.

上記の炭素源としては、例えば、グルコース、シュークロース、フラクトース、ガラクトースおよびラクトース等の糖類、これら糖類を含有する澱粉糖化液、甘藷糖蜜、甜菜糖蜜、ハイテストモラセス、サトウキビ搾汁、更には酢酸やフマル酸等の有機酸、エタノールなどのアルコール類、およびグリセリンなどが使用される。ここで糖類とは、多価アルコールの最初の酸化生成物であり、アルデヒド基またはケトン基をひとつ持ち、アルデヒド基を持つ糖をアルドース、ケトン基を持つ糖をケトースと分類される炭水化物のことを指す。   Examples of the carbon source include saccharides such as glucose, sucrose, fructose, galactose and lactose, starch saccharified solution containing these saccharides, sweet potato molasses, sugar beet molasses, high test molasses, sugarcane juice, acetic acid and Organic acids such as fumaric acid, alcohols such as ethanol, and glycerin 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.

また、上記の窒素源としては、例えば、アンモニアガス、アンモニア水、アンモニウム塩類、尿素、硝酸塩類、その他補助的に使用される有機窒素源、例えば、油粕類、大豆加水分解液、カゼイン分解物、その他のアミノ酸、ビタミン類、コーンスティープリカー、酵母または酵母エキス、肉エキス、ペプトン等のペプチド類、各種発酵菌体およびその加水分解物などが使用される。   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.

また、上記の無機塩類としては、例えば、リン酸塩、マグネシウム塩、カルシウム塩、鉄塩およびマンガン塩等を適宜添加使用することができる。   Moreover, as said inorganic salt, a phosphate, magnesium salt, calcium salt, iron salt, manganese salt etc. can be added and used suitably, for example.

本発明で使用される微生物や培養細胞が生育のために特定の栄養素を必要とする場合には、その栄養物を標品もしくはそれを含有する天然物として添加することができる。また、消泡剤も必要に応じて添加使用することができる。   When the microorganisms or cultured cells used in the present invention require specific nutrients for growth, the nutrients can be added as preparations or natural products containing them. An antifoaming agent can be added and used as necessary.

本発明において、発酵培養液とは、発酵原料に微生物または培養細胞が増殖した結果得られる液のことを言う。追加する発酵原料の組成は、目的とする化学品の生産性が高くなるように、培養開始時の発酵原料組成から適宜変更することができる。   In the present invention, the fermentation culture liquid refers to a liquid obtained as a result of growth of microorganisms or cultured cells as fermentation raw materials. The composition of the fermented raw material to be added can be appropriately changed from the fermented raw material composition at the start of the culture so that the productivity of the target chemical product is increased.

本発明では、発酵培養液中の糖類濃度は５ｇ／ｌ以下に保持されるようにすることが好ましい。その理由は、発酵培養液の引き抜きによる糖類の流失を最小限にするためである。そのため、糖類の濃度は可能な限り小さいことが望ましい。   In the present invention, the saccharide concentration in the fermentation broth is preferably maintained at 5 g / l or less. The reason is to minimize the loss of sugar due to withdrawal of the fermentation broth. Therefore, it is desirable that the saccharide concentration be as small as possible.

微生物の発酵培養は、通常、ｐＨが４〜８で温度が２０〜４０℃の範囲で行われることが多い。発酵培養液のｐＨは、無機の酸あるいは有機の酸、アルカリ性物質、さらには尿素、炭酸カルシウムおよびアンモニアガスなどによって、上記範囲内のあらかじめ定められた値に調節される。   In many cases, the fermentation culture of microorganisms is usually performed at a pH of 4 to 8 and a temperature of 20 to 40 ° 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 carbonate, ammonia gas, or the like.

培養において、酸素の供給速度を上げる必要があれば、空気に酸素を加えて酸素濃度を好適には２１％以上に保つ、発酵培養液を加圧する、攪拌速度を上げる、あるいは通気量を上げるなどの手段を用いることができる。逆に、酸素の供給速度を下げる必要があれば、炭酸ガス、窒素およびアルゴンなど酸素を含まないガスを空気に混合して供給することも可能である。   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.

本発明においては、培養初期にＢａｔｃｈ培養またはＦｅｄ−Ｂａｔｃｈ培養を行って微生物濃度を高くした後に連続培養（引き抜き）を開始しても良いし、高濃度の菌体をシードし、培養開始とともに連続培養を行っても良い。適当な時期から、原料培養液の供給および培養物の引き抜きを行うことが可能である。原料培養液供給と培養物の引き抜きの開始時期は、必ずしも同じである必要はない。また、原料培養液の供給と培養物の引き抜きは連続的であってもよいし、間欠的であってもよい。原料培養液には、上記に示したような菌体増殖に必要な栄養素を添加し、菌体増殖が連続的に行われるようにすればよい。   In the present invention, batch culture or fed-batch culture may be performed at the initial stage of culture to increase the microorganism concentration, and then continuous culture (pullout) may be started. Culture may be performed. From an appropriate time, it is possible to supply the raw material culture solution and extract the culture. 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. Nutrients necessary for cell growth as described above may be added to the raw material culture solution so that the cell growth is continuously performed.

発酵培養液中の微生物または培養細胞の濃度は、発酵培養液の環境が微生物または培養細胞の増殖にとって不適切となって死滅する比率が高くならない範囲で、高い状態で維持することが効率的でよい生産性を得る上で好ましい態様である。一例として、濃度を、乾燥重量として５ｇ／Ｌ以上に維持することにより良好な生産効率が得られる。連続発酵装置の運転上の不具合や生産効率の低下を招かなければ、発酵培養液中の微生物または培養細胞の濃度の上限は特に限定されない。   The concentration of microorganisms or cultured cells in the fermentation broth is efficient to maintain at a high level as long as the environment of the fermentation broth is not suitable for the growth of microorganisms or cultured cells and does not increase the rate of death. This is a preferred embodiment for obtaining good productivity. As an example, good production efficiency can be obtained by maintaining the concentration at 5 g / L or more as the dry weight. The upper limit of the concentration of microorganisms or cultured cells in the fermentation broth is not particularly limited as long as it does not cause problems in the operation of the continuous fermentation apparatus or decrease in production efficiency.

発酵生産能力のあるフレッシュな菌体を増殖させつつ行う連続培養操作は、培養管理上、通常、単一の発酵反応槽で行うことが好ましい。しかしながら、菌体を増殖しつつ生産物を生成する連続発酵培養法であれば、発酵反応槽の数は問わない。発酵反応槽の容量が小さい等の理由から、複数の発酵反応槽を用いることもあり得る。その場合、複数の発酵反応槽を配管で並列または直列に接続して連続培養を行っても、発酵生産物の高生産性は得られる。   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.

本発明においては、微生物や培養細胞を発酵反応槽に維持したままで、発酵反応槽からの発酵培養液の連続的かつ効率的な抜き出しが可能である。そのため、微生物や細胞を連続的に発酵培養し、十分な増殖を確保した後に発酵原料液組成を変更し、目的とする化学品を効率よく製造することも可能である。   In the present invention, it is possible to continuously and efficiently extract the fermentation culture solution from the fermentation reaction tank while maintaining the microorganisms and cultured cells in the fermentation reaction tank. For this reason, it is possible to produce a desired chemical product efficiently by continuously fermenting and culturing microorganisms and cells and changing the composition of the fermentation raw material solution after ensuring sufficient growth.

本発明で使用される微生物や培養細胞としては、例えば、発酵工業においてよく使用されるパン酵母などの酵母、大腸菌、コリネ型細菌などのバクテリア、糸状菌、放線菌、動物細胞および昆虫細胞などが挙げられる。使用する微生物や細胞は、自然環境から単離されたものでもよく、また、突然変異や遺伝子組換えによって一部性質が改変されたものであってもよい。   Examples of microorganisms and cultured cells used in the present invention include yeasts such as baker's yeast often used in the fermentation industry, bacteria such as Escherichia coli and coryneform bacteria, filamentous fungi, actinomycetes, animal cells and insect cells. Can be mentioned. The microorganisms and cells used may be those isolated from the natural environment, or may be those whose properties have been partially modified by mutation or genetic recombination.

本発明において好適な酵母としては、例えば、サッカロミセス属（Ｇｅｎｕｓ Ｓａｃｃｈａｒｏｍｙｃｅｓ）に属する酵母とサッカロミセス・セレビセ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）に属する酵母が挙げられる。   Suitable yeasts in the present invention include, for example, yeasts belonging to the genus Saccharomyces and yeasts belonging to Saccharomyces cerevisiae.

本発明における化学品は、上記の微生物や細胞が発酵培養液中に生産する物質である。化学品としては、例えば、アルコール、有機酸、アミノ酸、核酸など発酵工業において大量生産されている物質を挙げることができる。また、本発明は、酵素、抗生物質、組換えタンパク質のような物質の生産に適用することも可能である。例えば、アルコールとしては、エタノール、１，３−プロパンジオール、１,４−ブタンジオール、カダベリン、グリセロール等が挙げられる、また、有機酸としては、酢酸、乳酸、ピルビン酸、コハク酸、リンゴ酸、イタコン酸およびクエン酸等を挙げることができ、核酸であればイノシン、グアノシンおよびシチジン等を挙げることができる。   The chemical product in the present invention is a substance produced in the fermentation broth by the microorganisms and cells described above. Examples of chemical products 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-propanediol, 1,4-butanediol, cadaverine, glycerol and the like, and the organic acid includes acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, Examples thereof include itaconic acid and citric acid, and examples of nucleic acids include inosine, guanosine and cytidine.

本発明において、微生物や培養細胞の発酵培養液を分離膜で濾過処理する際の膜間差圧は、微生物や培養細胞および培地成分が容易に目詰まりしない条件であればよいが、膜間差圧を０.１ｋＰａ以上２０ｋＰａ以下の範囲にして濾過処理することが重要である。膜間差圧は、好ましくは０.１ｋＰａ以上１０ｋＰａ以下の範囲であり、さらに好ましくは０.１ｋＰａ以上５ｋＰａの範囲である。上記膜間差圧の範囲を外れた場合、原核微生物および培地成分の目詰まりが急速に発生し、透過水量の低下を招き、連続発酵運転に不具合を生じることがある。   In the present invention, the transmembrane pressure difference when the fermentation broth of microorganisms and cultured cells is filtered with a separation membrane may be any condition as long as the microorganisms, cultured cells and medium components are not easily clogged. It is important to perform the filtration treatment at a pressure 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.

濾過の駆動力としては、発酵培養液と多孔性膜処理水の液位差（水頭差）を利用したサイホンにより多孔性膜に膜間差圧を発生させることができる。また、濾過の駆動力として多孔性膜処理水側に吸引ポンプを設置してもよいし、多孔性膜の発酵培養液側に加圧ポンプを設置することも可能である。上記の範囲に膜間差圧を制御する手段としては、発酵培養液と多孔性膜処理水の液位差を変化させることにより制御することができる、また、膜間差圧を発生させるためにポンプを使用する場合には、吸引圧力により膜間差圧を制御することができる。更に、発酵培養液側の圧力を導入する気体または液体の圧力によっても膜間差圧を制御することができる。これら圧力制御を行う場合には、発酵培養液側の圧力と多孔性膜処理水側の圧力差をもって膜間差圧とし、膜間差圧の制御に用いることができる。   As a driving force for filtration, a transmembrane pressure difference can be generated in the porous membrane by a siphon utilizing the liquid level difference (water head difference) of the fermentation broth and the porous membrane treated water. Moreover, a suction pump may be installed on the porous membrane treated water side as a driving force for filtration, or a pressure pump may be installed on the fermentation culture solution side of the porous membrane. As a means for controlling the transmembrane pressure difference within the above range, it can be controlled by changing the liquid level difference of the fermentation broth and the porous membrane treated water, and in order to generate the transmembrane pressure difference When a 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.

また、本発明において使用される多孔性膜は、濾過処理する膜間差圧として、０．１ｋＰａ以上２０ｋＰａ以下の範囲で濾過処理することができる性能を有することが好ましい。   Moreover, it is preferable that the porous membrane used in this invention has the performance which can be filtered in the range of 0.1 kPa or more and 20 kPa or less as a transmembrane differential pressure to filter.

次に、本発明の連続発酵による化学品の製造方法を具体的に実施し、本発明を完成させる手段として使用される、発酵反応層の外部に分離膜エレメントが設置された連続発酵装置の代表的な一例を
1172031386281_0
１に示す。図１は、本発明の膜分離型の連続発酵装置の一つの実施の形態を説明するための概略側面図である。 Next, a representative example of a continuous fermentation apparatus in which a separation membrane element is installed outside the fermentation reaction layer, which is used as a means for completing the present invention by specifically carrying out the method for producing a chemical product by continuous fermentation of the present invention. Example
1172031386281_0
It is shown in 1. FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus of the present invention.

図１において、膜分離型の連続発酵装置は、微生物もしくは培養細胞を発酵培養させる発酵反応槽１と、その発酵反応槽１に発酵培養液循環ポンプ１１を介して接続され内部に分離膜エレメント２を備えた膜分離槽１２と、発酵反応槽１内の発酵培養液の量を制御するための水頭差制御装置３で基本的に構成されている。ここで、分離膜エレメント２には分離膜である多孔性膜が組み込まれている。この多孔性膜としては、例えば、国際公開第２００２／０６４２４０号パンフレットに開示されている膜を使用することが好適である。   In FIG. 1, a membrane separation type continuous fermentation apparatus includes a fermentation reaction tank 1 for fermenting and culturing microorganisms or cultured cells, and a fermentation culture solution circulation pump 11 connected to the fermentation reaction tank 1 and a separation membrane element 2 inside. And a water head difference control device 3 for controlling the amount of the fermentation broth in the fermentation reaction tank 1. Here, the separation membrane element 2 incorporates a porous membrane as a separation membrane. As this porous membrane, for example, a membrane disclosed in International Publication No. 2002/064240 is preferably used.

図１において、培地供給ポンプ７によって培地を発酵反応槽１に投入し、必要に応じて、攪拌機５で発酵反応槽１内の発酵培養液を攪拌することができる。また必要に応じて、気体供給装置４によって必要とする気体を供給することができる。このとき、供給した気体を回収リサイクルして再び気体供給装置４によって供給することができる。また、必要に応じて、ｐＨセンサ・制御装置９およびｐＨ調整溶液供給ポンプ８によって発酵培養液のｐＨを調整することができる。また必要に応じて、温度調節器１０によって発酵培養液の温度を調節することにより、生産性の高い発酵生産を行うことができる。   In FIG. 1, the culture medium can be charged into the fermentation reaction tank 1 by the medium supply pump 7, and the fermentation broth in the fermentation reaction tank 1 can be stirred by the stirrer 5 as necessary. Moreover, the gas required by the gas supply apparatus 4 can be supplied as needed. At this time, the supplied gas can be recovered and recycled and supplied again by the gas supply device 4. Further, the pH of the fermentation broth can be adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8 as necessary. Moreover, highly productive fermentation production can be performed by adjusting the temperature of a fermentation broth with the temperature controller 10 as needed.

さらに、装置内の発酵培養液は、発酵培養液循環ポンプ１１によって発酵反応槽１と膜分離槽１２の間を循環する。発酵生産物を含む発酵培養液は、分離膜エレメント２によって微生物と発酵生産物に濾過・分離され、装置系から取り出すことができる。   Further, the fermentation broth in the apparatus is circulated between the fermentation reaction tank 1 and the membrane separation tank 12 by the fermentation broth circulation pump 11. The fermentation broth containing the fermentation product is filtered and separated into microorganisms and fermentation product by the separation membrane element 2 and can be taken out from the apparatus system.

また、濾過・分離された微生物は、装置系内に留まることにより装置系内の微生物濃度を高く維持することができ、生産性の高い発酵生産を可能としている。ここで、分離膜を備えた分離膜エレメント２による発酵培養液の濾過・分離は、膜分離槽１２の水面との水頭差圧によって行うことができ、特別な動力は必要ない。また、必要に応じて、レベルセンサ６および水頭差圧制御装置３によって、分離膜エレメント２の濾過・分離速度および装置系内の発酵培養液量を適当に調節することができる。   In addition, the filtered and separated microorganisms can remain in the apparatus system to maintain a high concentration of microorganisms in the apparatus system, enabling highly productive fermentation production. Here, the filtration / separation of the fermentation broth by the separation membrane element 2 provided with the separation membrane 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 separation membrane element 2 and the amount of fermentation broth in the apparatus system can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3.

上記のように、図１では分離膜エレメント２を膜分離槽１２外に設置する形態を例示したが、分離膜エレメント２を発酵反応槽１内に設置することができる。分離膜エレメント２を発酵反応槽１内に設置した場合は、膜分離槽１２、発酵培養液循環ポンプ１１およびそれに付帯する設備を省略することができる。また、分離膜エレメント２を発酵反応槽１内に設置した場合における分離膜エレメント２による濾過・分離は、発酵反応槽１との水頭差圧によって行うことができ、特別な動力は必要ない。   As described above, FIG. 1 illustrates a mode in which the separation membrane element 2 is installed outside the membrane separation tank 12, but the separation membrane element 2 can be installed in the fermentation reaction tank 1. When the separation membrane element 2 is installed in the fermentation reaction tank 1, the membrane separation tank 12, the fermentation culture medium circulation pump 11 and the equipment attached thereto can be omitted. Further, when the separation membrane element 2 is installed in the fermentation reaction tank 1, the filtration / separation by the separation membrane element 2 can be performed by the water head differential pressure with respect to the fermentation reaction tank 1, and no special power is required.

上記のように、分離膜エレメント２による濾過・分離は、水頭差圧によって行うことができるが、必要に応じて、ポンプや気体・液体等による吸引濾過あるいは装置系内を加圧することにより濾過・分離することもできる。このような手段により、膜間差圧を制御することができる。   As described above, the filtration / separation by the separation membrane element 2 can be performed by the water head differential pressure, but if necessary, the filtration / It can also be separated. The transmembrane pressure difference can be controlled by such means.

本発明で用いられる分離膜エレメント２の好適な形態の例である国際公開第２００２／０６４２４０号パンフレットに開示されている分離膜および分離膜エレメントを、以下、図面を用いてその概略を説明する。図２は、本発明で用いられる分離膜エレメントの一つの実施の形態を説明するための概略斜視図である。   The outline of the separation membrane and the separation membrane element disclosed in International Publication No. 2002/064240 pamphlet, which is an example of a preferred embodiment of the separation membrane element 2 used in the present invention, will be described below with reference to the drawings. FIG. 2 is a schematic perspective view for explaining one embodiment of a separation membrane element used in the present invention.

分離膜エレメントは、図２に示すように、剛性を有する支持板１３の両面に、流路材１４と分離膜１５とをこの順序で配し構成されている。支持板１３は、両面に凹部１６を有している。分離膜１５は発酵培養液を濾過する。流路材１４は、分離膜１５で濾過された透過水を効率よく支持板１３に流すためのものである。支持板１３に流れた発酵生成物を含む透過液は、支持板１３の凹部１６を通り、排出手段である集水パイプ１７を介して連続発酵装置外部に取り出される。ここで、上述の水頭差圧、ポンプ、液体や気体等による吸引濾過、あるいは装置系内を加圧するなどの方法を透過水を取り出すための動力として用いることができる。   As shown in FIG. 2, the separation membrane element is configured by arranging a flow path material 14 and a separation membrane 15 in this order on both surfaces of a rigid support plate 13. The support plate 13 has recesses 16 on both sides. The separation membrane 15 filters the fermentation broth. The flow path member 14 is for efficiently flowing the permeated water filtered by the separation membrane 15 to the support plate 13. The permeate containing the fermentation product that has flowed to the support plate 13 passes through the recess 16 of the support plate 13 and is taken out of the continuous fermentation apparatus via the water collecting pipe 17 serving as a discharge means. Here, the above-mentioned water head differential pressure, pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used as power for extracting permeated water.

本発明に従って連続発酵を行った場合、従来のバッチ発酵と比較して、高い体積生産速度が得られ、極めて効率のよい発酵生産が可能となる。ここで、連続発酵培養における生産速度は、次の式（３）で計算される。
・発酵生産速度（ｇ／Ｌ／ｈｒ）＝抜き取り液中の生産物濃度（ｇ／Ｌ）×発酵培養液抜き取り速度（Ｌ／ｈｒ）÷装置の運転液量（Ｌ） ・・・・（式３）
また、バッチ培養での発酵生産速度は、原料炭素源をすべて消費した時点の生産物量（ｇ）を、炭素源の消費に要した時間（ｈ）とその時点の発酵培養液量（Ｌ）で除して求められる。 When continuous fermentation is performed according to the present invention, a high volumetric production rate is obtained compared to conventional batch fermentation, and extremely efficient fermentation production is possible. Here, the production rate in continuous fermentation culture is calculated by the following equation (3).
・ Fermentation production rate (g / L / hr) = Product concentration in extraction liquid (g / L) × Fermentation culture liquid extraction speed (L / hr) ÷ Operation liquid volume of apparatus (L) 3)
In addition, the fermentation production rate in batch culture is calculated based on the amount of product (g) at the time when all the raw carbon source is consumed by the time (h) required for consumption of the carbon source and the amount of fermentation broth (L) at that time. It is obtained by dividing.

以下、本発明の連続発酵による化学品の製造方法をさらに詳細に説明するために、上記の発酵生産物としてＬ−乳酸を選定し、図１に示す連続発酵装置を用いて連続的なＬ−乳酸の発酵生産について、実施例を挙げて説明する。本発明は、これらの実施例に限定されない。   Hereinafter, in order to describe the method for producing a chemical product by continuous fermentation of the present invention in more detail, L-lactic acid is selected as the fermentation product, and continuous L- using the continuous fermentation apparatus shown in FIG. The fermentation production of lactic acid will be described with reference to examples. The present invention is not limited to these examples.

ここで、Ｌ−乳酸を生産させる微生物には、酵母サッカロミセス・セレビセ（Saccharomyces cerevisae）を用いた。サッカロミセス・セレビセは、本来Ｌ−乳酸発酵を持たないが、Ｌ−乳酸脱水素酵素をコードする遺伝子をサッカロミセス・セレビセに導入することによりＬ−乳酸発酵能力をもつサッカロミセス・セレビセ株を造成し実施した。具体的には、ヒト由来ＬＤＨ遺伝子を酵母ゲノム上のＰＤＣ１プロモーターの下流に連結することにより、Ｌ−乳酸発酵能力を持つ酵母株を造成して使用した。   Here, yeast Saccharomyces cerevisae was used as a microorganism for producing L-lactic acid. Saccharomyces cerevisiae originally did not have L-lactic acid fermentation, but by introducing a gene encoding L-lactate dehydrogenase into Saccharomyces cerevisiae, a Saccharomyces cerevisiae strain having L-lactic acid fermentation ability was constructed and implemented. . Specifically, a yeast strain having L-lactic acid fermentation ability was constructed and used by linking the human-derived LDH gene downstream of the PDC1 promoter on the yeast genome.

［参考例１］乳酸生産能力を持つ酵母株の作製
乳酸生産能力を持つ酵母株を、下記のように造成した。具体的には、ヒト由来ＬＤＨ遺伝子を酵母ゲノム上のＰＤＣ１プロモーターの下流に連結することにより、Ｌ−乳酸生産能力を持つ酵母株を造成する。ポリメラーゼ・チェーン・リアクション（ＰＣＲ）には、Ｌａ−Ｔａｑ（宝酒造社製）あるいはKOD-Plus-polymerase（東洋紡社製）を用い、付属の取扱説明に従って行った。 [Reference Example 1] Preparation of yeast strain having lactic acid production ability A yeast strain having lactic acid production ability was constructed as follows. Specifically, a yeast strain capable of producing L-lactic acid is constructed by linking a human-derived LDH gene downstream of the PDC1 promoter on the yeast genome. For polymerase chain reaction (PCR), La-Taq (manufactured by Takara Shuzo) or KOD-Plus-polymerase (manufactured by Toyobo) was used according to the attached instruction manual.

ヒト乳ガン株化細胞（ＭＣＦ−７）を培養回収後、TRIZOL Reagent（Invitrogen）を用いてtotal ＲＮＡを抽出し、得られたtotal ＲＮＡを鋳型としてSuperScript Choice System（Invitrogen）を用いた逆転写反応によりｃＤＮＡの合成を行った。これらの操作の詳細は、それぞれ付属のプロトコールに従った。得られたｃＤＮＡを、続くＰＣＲの増幅鋳型とした。   After culturing and recovering human breast cancer cell lines (MCF-7), total RNA was extracted using TRIZOL Reagent (Invitrogen), and reverse transcription using SuperScript Choice System (Invitrogen) was performed using the obtained total RNA as a template. cDNA synthesis was performed. Details of these operations followed the attached protocol. The obtained cDNA was used as an amplification template for subsequent PCR.

上記の操作で得られたｃＤＮＡを増幅鋳型とし、配列番号１および配列番号２で表されるオリゴヌクレオチドをプライマーセットとしたKOD-Plus-polymeraseによるＰＣＲにより、Ｌ−ｌｄｈ遺伝子のクローニングを行った。各ＰＣＲ増幅断片を精製し末端をT4 Polynucleotide Kinase（TAKARA社製）によりリン酸化後、ｐＵＣ１１８ベクター（制限酵素HincIIで切断し、切断面を脱リン酸化処理したもの）にライゲーションした。ライゲーションは、DNA Ligation Kit Ver.2（TAKARA社製）を用いて行った。   The L-ldh gene was cloned by PCR with KOD-Plus-polymerase using the cDNA obtained by the above operation as an amplification template and the oligonucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 as a primer set. Each PCR amplified fragment was purified and the end was phosphorylated with T4 Polynucleotide Kinase (manufactured by TAKARA), and then ligated to a pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver.2 (manufactured by TAKARA).

ライゲーションプラスミド産物で大腸菌DH５αを形質転換し、プラスミドＤＮＡを回収することにより、各種Ｌ−ｌｄｈ遺伝子（配列番号３）がサブクローニングされたプラスミドを得た。得られたＬ−ｌｄｈ遺伝子が挿入されたｐＵＣ１１８プラスミドを制限酵素ＸｈｏＩおよびＮｏｔＩで消化し、得られた各ＤＮＡ断片を酵母発現用ベクターｐＴＲＳ１１（図３）のＸｈｏＩ／ＮｏｔＩ切断部位に挿入した。このようにして、ヒト由来Ｌ−ｌｄｈ遺伝子発現プラスミドｐＬ−ｌｄｈ５（Ｌ−ｌｄｈ遺伝子）を得た。ヒト由来のＬ−ｌｄｈ遺伝子発現ベクターである上記ｐＬ−ｌｄｈ５は、プラスミド単独で独立行政法人産業技術総合研究所 特許生物寄託センター（茨城県つくば市東１−１−１中央第６）にＦＥＲＭＡＰ−２０４２１として寄託した（寄託日：平成１７年２月２１日）。   Escherichia coli DH5α was transformed with the ligation plasmid product, and the plasmid DNA was recovered to obtain a plasmid in which various L-ldh genes (SEQ ID NO: 3) were subcloned. The obtained pUC118 plasmid into which the L-ldh gene was inserted was digested with restriction enzymes XhoI and NotI, and the resulting DNA fragments were inserted into the XhoI / NotI cleavage sites of the yeast expression vector pTRS11 (FIG. 3). In this manner, a human-derived L-ldh gene expression plasmid pL-ldh5 (L-ldh gene) was obtained. The above-mentioned pL-ldh5, which is a human-derived L-ldh gene expression vector, is FERMAP-20421 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki Prefecture) as a plasmid alone. (Deposit date: February 21, 2005).

ヒト由来ＬＤＨ遺伝子を含むプラスミドｐＬ−ｌｄｈ５を増幅鋳型とし、配列番号４および配列番号５で表されるオリゴヌクレオチドをプライマーセットとしたＰＣＲにより、１．３ｋｂのヒト由来ＬＤＨ遺伝子およびサッカロミセス・セレビセ由来のＴＤＨ３遺伝子のターミネーター配列含むＤＮＡ断片を増幅した。また、プラスミドｐＲＳ４２４を増幅鋳型として、配列番号６および配列番号７で表されるオリゴヌクレオチドをプライマーセットとしたＰＣＲにより、１．２ｋｂのサッカロミセス・セレビセ由来のＴＲＰ１遺伝子を含むＤＮＡ断片を増幅した。それぞれのＤＮＡ断片を、１．５％アガロースゲル電気泳動により分離し、常法に従い精製した。   By PCR using the plasmid pL-ldh5 containing the human-derived LDH gene as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 5 as a primer set, a 1.3-kb human-derived LDH gene and Saccharomyces cerevisiae derived A DNA fragment containing the terminator sequence of the TDH3 gene was amplified. In addition, a DNA fragment containing the TRP1 gene derived from Saccharomyces cerevisiae of 1.2 kb was amplified by PCR using the plasmid pRS424 as an amplification template and the oligonucleotides represented by SEQ ID NO: 6 and SEQ ID NO: 7 as a primer set. Each DNA fragment was separated by 1.5% agarose gel electrophoresis and purified according to a conventional method.

ここで得られた１．３ｋｂ断片と１．２ｋｂ断片を混合したものを増幅鋳型とし、配列番号４および配列番号７で表されるオリゴヌクレオチドをプライマーセットとしたＰＣＲ法によって得られた産物を１．５％アガロースゲル電気泳動して、ヒト由来ＬＤＨ遺伝子およびＴＲＰ１遺伝子が連結された２．５ｋｂのＤＮＡ断片を、常法に従い調整した。この２．５ｋｂのＤＮＡ断片で、出芽酵母ＮＢＲＣ１０５０５株を常法に従いトリプトファン非要求性に形質転換した。   A product obtained by PCR using a mixture of the 1.3 kb fragment and the 1.2 kb fragment obtained here as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 7 as a primer set was obtained as 1 A 2.5 kb DNA fragment to which the human-derived LDH gene and TRP1 gene were linked was prepared according to a conventional method by electrophoresis on 5% agarose gel. With this 2.5 kb DNA fragment, the budding yeast strain NBRC10505 was transformed to tryptophan non-requirement according to a conventional method.

得られた形質転換細胞が、ヒト由来ＬＤＨ遺伝子を酵母ゲノム上のＰＤＣ１プロモーターの下流に連結されている細胞であることの確認は、下記のようにして行った。まず、形質転換細胞のゲノムＤＮＡを常法に従って調製し、これを増幅鋳型とした配列番号８および配列番号９で表されるオリゴヌクレオチドをプライマーセットとしたＰＣＲにより、０．７ｋｂの増幅ＤＮＡ断片が得られることにより確認した。   Confirmation that the obtained transformed cells were cells in which the human-derived LDH gene was linked downstream of the PDC1 promoter on the yeast genome was performed as follows. First, genomic DNA of a transformed cell was prepared according to a conventional method, and a 0.7 kb amplified DNA fragment was obtained by PCR using the oligonucleotides represented by SEQ ID NO: 8 and SEQ ID NO: 9 as a primer set. This was confirmed by obtaining.

また、形質転換細胞が乳酸生産能力を持つかどうかは、ＳＣ培地（ＭＥＴＨＯＤＳ ＩＮ ＹＥＡＳＴ ＧＥＮＥＴＩＣＳ ２０００ ＥＤＩＴＩＯＮ、 ＣＳＨＬ ＰＲＥＳＳ）で形質転換細胞を培養した培養上澄に乳酸が含まれていることを、下記に示す条件でＨＰＬＣ法により乳酸量を測定することにより確認した。
・カラム：Shim-Pack SPR-H（島津社製）
・移動相：５mM ｐ−トルエンスルホン酸（流速０．８ｍＬ／ｍｉｎ）
・反応液：５mM ｐ−トルエンスルホン酸、２０mM ビストリス、０．１mM EDTA・２Na（流速０．８ｍＬ／ｍｉｎ）
・検出方法：電気伝導度
・温度：４５℃。 In addition, whether or not the transformed cells have lactic acid production ability is determined by the fact that lactic acid is contained in the culture supernatant obtained by culturing the transformed cells in SC medium (METHODS IN YEAS GENETIC 2000 EDITION, CSHL PRESS). It confirmed by measuring the amount of lactic acid by HPLC method on the conditions shown.
・ Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
-Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
-Detection method: electrical conductivity-Temperature: 45 ° C.

また、Ｌ−乳酸の光学純度測定は、下記の条件でＨＰＬＣ法により測定した。
・カラム：TSK-gel Enantio L1（東ソー社製）
・移動相 ：１ｍＭ 硫酸銅水溶液
・流速：１．０ｍｌ／ｍｉｎ
・検出方法 ：ＵＶ２５４ｎｍ
・温度 ：３０℃。 The optical purity of L-lactic acid was measured by the HPLC method under the following conditions.
・ Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation)
-Mobile phase: 1 mM aqueous copper sulfate-Flow rate: 1.0 ml / min
・ Detection method: UV254nm
-Temperature: 30 degreeC.

また、Ｌ−乳酸の光学純度は、次式で計算される。
・光学純度（％）＝１００×（Ｌ−Ｄ）／（Ｌ＋Ｄ）。 Moreover, the optical purity of L-lactic acid is calculated by the following formula.
Optical purity (%) = 100 × (LD) / (L + D).

ここで、ＬはＬ−乳酸の濃度であり、ＤはＤ−乳酸の濃度を表す。   Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.

ＨＰＬＣ分析の結果、４ｇ／ＬのＬ−乳酸が検出され、Ｄ−乳酸は検出限界以下であった。以上の検討により、この形質転換体がＬ−乳酸生産能力を持つことが確認された。得られた形質転換細胞を、酵母ＳＷ−１株として、後の実施例で用いた。   As a result of HPLC analysis, 4 g / L of L-lactic acid was detected, and D-lactic acid was below the detection limit. From the above examination, it was confirmed that this transformant has L-lactic acid production ability. The obtained transformed cell was used in a later example as a yeast SW-1 strain.

［参考例２］多孔性膜の作製（その１）
樹脂としてポリフッ化ビニリデン（ＰＶＤＦ）樹脂を、また溶媒としてＮ，Ｎ−ジメチルアセトアミド（ＤＭＡｃ）をそれぞれ用い、これらを９０℃の温度下に十分に攪拌し、次の組成を有する原液を得た。
・ＰＶＤＦ：１３．０重量％
・ＤＭＡｃ：８７．０重量％
次に、上記原液を２５℃の温度に冷却した後、あらかじめガラス板上に貼り付けて置いた、密度が０．４８ｇ／ｃｍ3で、厚みが２２０μｍのポリエステル繊維製不織布（多孔質基材）に塗布し、直ちに次の組成を有する２５℃の温度の凝固浴中に５分間浸漬して、多孔質基材に多孔質樹脂層が形成された多孔性膜を得た。
・水 ：３０．０重量％
・ＤＭＡｃ：７０．０重量％
この多孔性膜をガラス板から剥がした後、８０℃の温度の熱水に３回浸漬してＤＭＡｃを洗い出し、分離膜（多孔性膜）を得た。多孔質樹脂層表面の９．２μｍ×１０．４μｍの範囲内を、倍率１０，０００倍で走査型電子顕微鏡観察を行ったところ、観察できる細孔すべての直径の平均は０．１μｍであった。次に、上記分離膜について純水透水透過係数を評価したところ、５０×１０-9ｍ3／ｍ2／ｓ／Ｐａであった。純水透水量の測定は、逆浸透膜による２５℃の温度の精製水を用い、ヘッド高さ１ｍで行った。また、平均細孔径の標準偏差は０．０３５μｍで、膜表面粗さは０．０６μｍであった。 [Reference Example 2] Production of porous membrane (Part 1)
Polyvinylidene fluoride (PVDF) resin was used as the resin and N, N-dimethylacetamide (DMAc) was used as the solvent, and these were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
・ PVDF: 13.0% by weight
DMAc: 87.0% by weight
Next, after the stock solution is cooled to a temperature of 25 ° C., a non-woven fabric made of polyester fiber (porous substrate) having a density of 0.48 g / cm 3 and a thickness of 220 μm, which is previously pasted on a glass plate. And immediately immersed in a coagulation bath at 25 ° C. having the following composition for 5 minutes to obtain a porous film having a porous resin layer formed on a porous substrate.
-Water: 30.0% by weight
DMAc: 70.0% by weight
After peeling this porous membrane from the glass plate, it was immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc, thereby obtaining a separation membrane (porous membrane). When the surface of the porous resin layer was observed with a scanning electron microscope at a magnification of 10,000 within the range of 9.2 μm × 10.4 μm, the average diameter of all observable pores was 0.1 μm. . Next, when the pure water permeability coefficient 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.

［参考例３］多孔性膜の作製（その２）
樹脂としてポリフッ化ビニリデン（ＰＶＤＦ）樹脂を、開孔剤として分子量が約２０，０００のポリエチレングリコール（ＰＥＧ）を、溶媒としてＮ，Ｎ−ジメチルアセトアミド（ＤＭＡｃ）を、そして非溶媒として純水をそれぞれ用い、これらを９０℃の温度下に十分に攪拌し、次の組成を有する原液を得た。
・ＰＶＤＦ：１３．０重量％
・ＰＥＧ ： ５．５重量％
・ＤＭＡｃ：７８．０重量％
・純水 ： ３．５重量％
次に、上記原液を２５℃の温度に冷却した後、密度が０．４８ｇ／ｃｍ3 で、厚みが２２０μｍのポリエステル繊維製不織布に塗布し、塗布後、直ちに２５℃の温度の純水中に５分間浸漬し、さらに８０℃の温度の熱水に３回浸漬してＤＭＡｃおよびＰＥＧを洗い出し、分離膜（多孔性膜）を得た。この分離膜の原液を塗布した側における、多孔質樹脂層表面の９．２μｍ×１０．４μｍの範囲内を、倍率１０，０００倍で走査型電子顕微鏡観察を行ったところ、観察できる細孔すべての直径の平均は０．０２μｍであった。この分離膜について純水透過係数を評価したところ、２×１０-9ｍ3／ｍ2／ｓ／Ｐａであった。透水量の測定は、逆浸透膜による２５℃の温度の精製水を用い、ヘッド高さ１ｍで行った。また、平均細孔径の標準偏差は０．００５５μｍで、膜表面粗さは０．１μｍであった。 [Reference Example 3] Production of porous membrane (part 2)
Polyvinylidene fluoride (PVDF) resin as a resin, polyethylene glycol (PEG) having a molecular weight of about 20,000 as a pore-opening agent, N, N-dimethylacetamide (DMAc) as a solvent, and pure water as a non-solvent. These were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
・ PVDF: 13.0% by weight
・ PEG: 5.5% by weight
DMAc: 78.0% by weight
・ Pure water: 3.5% by weight
Next, after cooling the stock solution to a temperature of 25 ° C., it was applied to a non-woven fabric made of polyester fiber having a density of 0.48 g / cm 3 and a thickness of 220 μm. It was immersed for 5 minutes and further immersed three times in hot water at a temperature of 80 ° C. to wash out DMAc and PEG, thereby obtaining a separation membrane (porous membrane). When the surface of the porous resin layer on the side of the separation membrane on which the stock solution was applied was observed in a scanning electron microscope at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm, all the pores that could be observed The average diameter was 0.02 μm. When this separation membrane was evaluated for its pure water permeability coefficient, it was 2 × 10 −9 m 3 / m 2 / s / Pa. The amount of water permeation was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.0055 μm, and the membrane surface roughness was 0.1 μm.

［参考例４］多孔性膜の作製（その３）
次に示す組成の原液を用いた他は、参考例２と同様にして分離膜を得た。
・ＰＶＤＦ：１３．０重量％
・ＰＥＧ ： ５．５重量％
・ＤＭＡｃ：８１．５重量％
この分離膜の原液を塗布した側における、多孔質樹脂層表面の９．２μｍ×１０．４μｍの範囲内を、倍率１０，０００倍で走査型電子顕微鏡観察を行ったところ、観察できる細孔すべての直径の平均は０．１９μｍであり、この分離膜について純水透過係数を評価したところ、１００×１０-9ｍ3／ｍ2／ｓ／Ｐａであった。透水量の測定は、逆浸透膜による２５℃の温度の精製水を用い、ヘッド高さ１ｍで行った。また、平均細孔径の標準偏差 は０．０６０μｍで、膜表面粗さは０．０８μｍであった。 [Reference Example 4] Production of porous membrane (part 3)
A separation membrane was obtained in the same manner as in Reference Example 2, except that the stock solution having the following composition was used.
・ PVDF: 13.0% by weight
・ PEG: 5.5% by weight
DMAc: 81.5% by weight
When the surface of the porous resin layer on the side of the separation membrane on which the stock solution was applied was observed in a scanning electron microscope at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm, all the pores that could be observed The average diameter was 0.19 μm, and the pure water permeability coefficient of this separation membrane was evaluated to be 100 × 10 −9 m 3 / m 2 / s / Pa. The amount of water permeation was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.060 μm, and the membrane surface roughness was 0.08 μm.

［参考例５］ 多孔性膜の作製（その４）
樹脂としてポリフッ化ビニリデン（ＰＶＤＦ）樹脂を、また溶媒としてＮ，Ｎ−ジメチルアセトアミド（ＤＭＡｃ）をそれぞれ用い、これらを９０℃の温度下に十分に攪拌し、次の組成を有する原液を得た。
・ＰＶＤＦ：１５．０重量％
・ＤＭＡｃ：８５．０重量％
次に、上記原液を２５℃の温度に冷却した後、あらかじめガラス板上に貼り付けて置いた、密度が０．４８ｇ／ｃｍ3、厚みが２２０μｍのポリエステル繊維製不織布（多孔質基材）に塗布し、直ちに次の組成を有する２５℃の温度の凝固浴中に５分間浸漬して、多孔質基材に多孔質樹脂層が形成された多孔性膜を得た。
・水 ：１００．０重量％
この多孔性膜をガラス板から剥がした後、８０℃の温度の熱水に３回浸漬してＤＭＡｃを洗い出し、分離膜を得た。多孔質樹脂層表面の９．２μｍ×１０．４μｍの範囲内を、倍率１０，０００倍で走査型電子顕微鏡観察を行ったところ、観察できる細孔すべての直径の平均は０．００８μｍであった。次に、上記分離膜について純水透過係数を評価したところ、０．３×１０-9ｍ3／ｍ2・ｓ・Ｐａであった。透水量の測定は、逆浸透膜による２５℃の精製水を用い、ヘッド高さ１ｍで行った。また、平均細孔径の標準偏差 は０．００２μｍで、膜表面粗さは０．０６μｍであった。 [Reference Example 5] Production of porous membrane (part 4)
Polyvinylidene fluoride (PVDF) resin was used as the resin and N, N-dimethylacetamide (DMAc) was used as the solvent, and these were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
・ PVDF: 15.0% by weight
DMAc: 85.0% by weight
Next, after cooling the undiluted solution to a temperature of 25 ° C., a polyester fiber nonwoven 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, was placed. It was applied and immediately immersed in a coagulation bath having a temperature of 25 ° C. having the following composition for 5 minutes to obtain a porous film having a porous resin layer formed on a porous substrate.
-Water: 100.0% 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 the observable pores was 0.008 μm. . Next, when the pure water permeability coefficient of the separation membrane was evaluated, it was 0.3 × 10 −9 m 3 / m 2 · s · Pa. The amount of water permeation was measured using purified water at 25 ° C. with a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.002 μm, and the membrane surface roughness was 0.06 μm.

［実施例１］連続発酵によるＬ−乳酸の製造（その１）
図１の膜分離型の連続発酵装置を稼働させることにより、Ｌ−乳酸連続発酵系が得られるかどうかを調べるため、表１に示す組成の乳酸発酵培地を用い、この装置の連続発酵試験を行った。図１の膜分離型の連続発酵装置は、本発明の一実施の形態を示すものであり、本発明はその形態になんら限定されるものではない。該乳酸発酵培地は、１２１℃の温度で１５分間、高圧蒸気滅菌して用いた。分離膜には、前記の参考例２で作製した多孔性膜を用いた。実施例１における運転条件は、特に断らない限り、下記のとおりである。
・発酵反応槽容量：１．５（Ｌ）
・膜分離槽容量：０．５（Ｌ）
・使用分離膜：ＰＶＤＦ濾過膜
・膜分離エレメント有効濾過面積：６０平方ｃｍ
・温度調整：３０（℃）
・発酵反応槽通気量：０．２（Ｌ／ｍｉｎ）
・膜分離槽通気量：０．３（Ｌ／ｍｉｎ）
・発酵反応槽攪拌速度：４００（ｒｐｍ）
・ｐＨ調整：０．５Ｎ ＮａＯＨによりｐＨ５に調整
・ 発酵培養液循環装置による循環液量：０．１（Ｌ／ｍｉｎ）
・ 膜透過水量制御：膜分離槽水頭差により流量を制御（膜間差圧として２ ｋＰａ以下に制御した。）。 [Example 1] Production of L-lactic acid by continuous fermentation (part 1)
In order to examine whether or not an L-lactic acid continuous fermentation system can be obtained by operating the membrane-separated continuous fermentation apparatus of FIG. 1, a continuous fermentation test of this apparatus was conducted using a lactic acid fermentation medium having the composition shown in Table 1. went. The membrane-separated continuous fermentation apparatus of FIG. 1 shows an embodiment of the present invention, and the present invention is not limited to that form. The lactic acid fermentation medium was used after being autoclaved at 121 ° C. for 15 minutes. As the separation membrane, the porous membrane prepared in Reference Example 2 was used. The operating conditions in Example 1 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
・ Use separation membrane: PVDF filtration membrane ・ Membrane separation element Effective filtration area: 60 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.2 (L / min)
-Aeration rate of membrane separation tank: 0.3 (L / min)
・ Fermentation reactor stirring speed: 400 (rpm)
-PH adjustment: pH 5 adjusted with 0.5N NaOH-Circulating fluid volume by fermentation culture fluid circulation device: 0.1 (L / min)
-Membrane permeate flow rate control: The flow rate was controlled by the difference in the water head of the membrane separation tank (the transmembrane pressure difference was controlled to 2 kPa or less).

微生物として前記の参考例１で造成した酵母ＳＷ−１株を用い、培地として表１に示す組成の乳酸発酵培地を用いた。生産物であるＬ−乳酸の濃度の評価には、前記の参考例１に示したＨＰＬＣを用い、グルコース濃度の測定には“グルコーステストワコーＣ”（登録商標）（和光純薬社製）を用いた。   The yeast SW-1 strain constructed in Reference Example 1 was used as the microorganism, and a lactic acid fermentation medium having the composition shown in Table 1 was used as the medium. For the evaluation of the concentration of L-lactic acid as a product, the HPLC shown in Reference Example 1 was used, and for measuring the glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used. Using.

Figure 0005082496
Figure 0005082496

まず、試験管中で５ｍｌの乳酸発酵培地を用いＳＷ−１株を一晩振とう培養した（前々々培養）。得られた培養液を新鮮な乳酸発酵培地１００ｍｌに植菌し、５００ｍｌ容坂口フラスコ中で２４時間、３０℃の温度で振とう培養した（前々培養）。前々培養液を、図１に示した膜分離型の連続発酵装置の１．５Ｌの乳酸発酵培地に植菌し、発酵反応槽１を付属の攪拌機５によって４００ｒｐｍで攪拌し、発酵反応槽１の通気量の調整、温度調整およびｐＨ調整を行った。発酵培養液循環ポンプ１１を稼働させることなく、２４時間培養を行った（前培養）。前培養完了後、直ちに、発酵培養液循環ポンプ１１を稼働させた。前培養時の運転条件に加え、膜分離槽２を通気し、乳酸発酵培地の連続供給を行い、膜分離型の連続発酵装置の発酵培養液量を２Ｌとなるよう膜透過水量の制御を行いながら連続培養し、連続発酵によるＬ−乳酸の製造を行った。連続発酵試験を行うときの膜透過水量の制御は、水頭差制御装置３により、膜分離槽水頭を最大２ｍ以内、すなわち膜間差圧が２０ｋＰａ以内となるように適宜水頭差を変化させることによって行った。適宜、膜透過発酵液中の生産されたＬ−乳酸濃度および残存グルコース濃度を測定した。また、該Ｌ−乳酸およびグルコース濃度から算出された投入グルコースから算出された該Ｌ−乳酸対糖収率と乳酸生産速度を、表３に示した。２６４時間の発酵試験を行った結果、図１の膜分離型の連続発酵装置を用いることにより、安定したＬ−乳酸の連続発酵による製造が可能であることが確認できた。   First, the SW-1 strain was cultured with shaking overnight in a test tube using 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 at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution was inoculated in a 1.5 L lactic acid fermentation medium of the membrane separation type continuous fermentation apparatus shown in FIG. 1 and the fermentation reaction tank 1 was stirred at 400 rpm by the attached stirrer 5. The air flow rate was adjusted, the temperature was adjusted, and the pH was adjusted. The culture was performed for 24 hours without operating the fermentation culture medium circulation pump 11 (pre-culture). Immediately after completion of the preculture, the fermentation broth circulation pump 11 was operated. In addition to the operating conditions at the time of pre-culture, the membrane separation tank 2 is vented, the lactic acid fermentation medium is continuously supplied, and the amount of fermentation permeate in the membrane separation type continuous fermentation apparatus is controlled to 2 L. Then, L-lactic acid was produced by continuous fermentation and continuous fermentation. The control of the amount of permeated water when performing the continuous fermentation test is performed by appropriately changing the water head difference so that the water head of the membrane separation tank is within 2 m at the maximum, that is, the transmembrane pressure difference is within 20 kPa. went. The L-lactic acid concentration produced and the residual glucose concentration in the membrane permeation fermentation broth were measured as appropriate. In addition, Table 3 shows the L-lactic acid-to-sugar yield and lactic acid production rate calculated from the input glucose calculated from the L-lactic acid and glucose concentrations. As a result of performing a fermentation test for 264 hours, it was confirmed that stable production of L-lactic acid by continuous fermentation was possible by using the membrane-separated continuous fermentation apparatus of FIG.

［実施例２］連続発酵によるＬ−乳酸の製造（その２）
図１の膜分離型の連続発酵装置を稼働させることにより、Ｌ−乳酸連続発酵系が得られるかどうかを調べるため、表１に示す組成の乳酸発酵培地を用い、この装置の連続発酵試験を行った。該乳酸発酵培地は、１２１℃の温度で１５分間、高圧蒸気滅菌して用いた。分離膜には、前記の参考例２で作製した多孔性膜を用いた。実施例２における運転条件は、特に断らない限り、次のとおりである。
・発酵反応槽容量：１．５（Ｌ）
・膜分離槽容量：０．５（Ｌ）
・使用分離膜：ＰＶＤＦ濾過膜
・膜分離エレメント有効濾過面積：６０平方ｃｍ
・温度調整：３０（℃）
・発酵反応槽通気量：０．０５（Ｌ／ｍｉｎ）
・膜分離槽通気量：０．３（Ｌ／ｍｉｎ）
・発酵反応槽攪拌速度：１００（ｒｐｍ）
・ｐＨ調整：１Ｎ ＮａＯＨによりｐＨ５に調整
・乳酸発酵培地供給速度：５０〜３００ｍｌ／ｈｒ．の範囲で可変制御
・発酵培養液循環装置による循環液量：０．１（Ｌ／ｍｉｎ）
・膜透過水量制御：膜間差圧による流量制御。 [Example 2] Production of L-lactic acid by continuous fermentation (part 2)
In order to examine whether or not an L-lactic acid continuous fermentation system can be obtained by operating the membrane-separated continuous fermentation apparatus of FIG. 1, a continuous fermentation test of this apparatus was conducted using a lactic acid fermentation medium having the composition shown in Table 1. went. The lactic acid fermentation medium was used after being autoclaved at 121 ° C. for 15 minutes. As the separation membrane, the porous membrane prepared in Reference Example 2 was used. The operating conditions in Example 2 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
・ Use separation membrane: PVDF filtration membrane ・ Membrane separation element Effective filtration area: 60 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
-Aeration rate of membrane separation tank: 0.3 (L / min)
・ Fermentation reactor stirring speed: 100 (rpm)
-PH adjustment: adjusted to pH 5 with 1N NaOH-Lactic acid fermentation medium supply rate: 50-300 ml / hr. Circulated fluid volume by variable control / fermentation culture fluid circulation device in the range: 0.1 (L / min)
・ Membrane permeate flow control: Flow control by transmembrane pressure difference.

（連続発酵開始後〜１００時間：膜間差圧０．１ｋＰａ以上５ｋＰａ以下で制御
１００時間〜２００時間：膜間差圧０．１ｋＰａ以上２ｋＰａ以下で制御
２００時間〜３００時間：膜間差圧０．１ｋＰａ以上２０ｋＰａ以下で制御）。 (After the start of continuous fermentation up to 100 hours: controlled at a transmembrane differential pressure of 0.1 kPa to 5 kPa
100 hours to 200 hours: Control at a transmembrane differential pressure of 0.1 kPa to 2 kPa
200 hours to 300 hours: controlled at a transmembrane differential pressure of 0.1 kPa to 20 kPa).

微生物として前記の参考例１で造成した酵母ＳＷ−１株を用い、培地として表１に示す組成の乳酸発酵培地を用いた。生産物であるＬ−乳酸の濃度の評価には、参考例１に示したＨＰＬＣを用い、グルコース濃度の測定には“グルコーステストワコーＣ”（登録商標）（和光純薬社製）を用いた。   The yeast SW-1 strain constructed in Reference Example 1 was used as the microorganism, and a lactic acid fermentation medium having the composition shown in Table 1 was used as the medium. The HPLC shown in Reference Example 1 was used to evaluate the concentration of L-lactic acid as a product, and “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used to measure the glucose concentration. .

まず、試験管中で５ｍｌの乳酸発酵培地を用いＳＷ−１株を一晩振とう培養した（前々々培養）。得られた培養液を新鮮な乳酸発酵培地１００ｍｌに植菌し、５００ｍｌ容坂口フラスコ中で２４時間、３０℃の温度で振とう培養した（前々培養）。前々培養液を、図１に示した膜分離型の連続発酵装置の１．５Ｌの乳酸発酵培地に植菌し、発酵反応槽１を付属の攪拌機５によって攪拌し、発酵反応槽１の通気量の調整、温度調整およびｐＨ調整を行った。発酵液循環ポンプ１１を稼働させることなく、２４時間培養を行った（前培養）。前培養完了後、直ちに、発酵液循環ポンプ１１を稼働させた。前培養時の運転条件に加え、膜分離槽１２を通気し、乳酸発酵培地の連続供給を行い、膜分離型の連続発酵装置の発酵培養液量を２Ｌとなるよう膜透過水量の制御を行いながら連続培養し、連続発酵によるＬ−乳酸の製造を行った。連続発酵試験を行うときの膜透過水量の制御は、水頭差制御装置３により水頭差を膜間差圧として測定し、上記膜透過水量制御条件で変化させることにより行った。適宜、膜透過発酵液中の生産された乳酸濃度および残存グルコース濃度を測定した。３００時間の連続発酵試験を行った結果、を表３に示す。その結果、この膜分離型の連続発酵装置を用いることにより、安定したＬ−乳酸の連続発酵による製造が可能であることを確認することができた。連続発酵の期間中の膜間差圧は、２ｋＰａ以下０．１以上で推移した。   First, the SW-1 strain was cultured with shaking overnight in a test tube using 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 at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L lactic acid fermentation medium of the membrane separation type continuous fermentation apparatus shown in FIG. 1, the fermentation reaction tank 1 is stirred by the attached stirrer 5, and the fermentation reaction tank 1 is vented. Adjustment of amount, temperature adjustment and pH adjustment were performed. The culture was performed for 24 hours without operating the fermented liquid circulation pump 11 (pre-culture). Immediately after completion of the pre-culture, the fermentation liquid circulation pump 11 was operated. In addition to the operating conditions at the time of pre-culture, the membrane separation tank 12 is aerated, the lactic acid fermentation medium is continuously supplied, and the amount of fermentation permeate in the membrane separation type continuous fermentation apparatus is controlled to 2 L. Then, L-lactic acid was produced by continuous fermentation and continuous fermentation. Control of the amount of water permeated through the continuous fermentation test was performed by measuring the water head difference as a transmembrane pressure difference with the water head difference control device 3 and changing it under the above-mentioned membrane permeated water amount control conditions. The produced lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. The results of 300 hours continuous fermentation test are shown in Table 3. As a result, it was confirmed that stable production of L-lactic acid by continuous fermentation was possible by using this membrane separation type continuous fermentation apparatus. The transmembrane pressure difference during the continuous fermentation period was 2 kPa or less and 0.1 or more.

［実施例３］連続発酵によるＬ−乳酸の製造（その３）
分離膜として前記の参考例３で作製した多孔性膜を用い、実施例２と同様のＬ−乳酸連続発酵試験を行った。その結果を、表３に示す。その結果、安定したＬ−乳酸の連続発酵による製造が可能であることを確認することができた。 [Example 3] Production of L-lactic acid by continuous fermentation (part 3)
The same L-lactic acid continuous fermentation test as in Example 2 was performed using the porous membrane prepared in Reference Example 3 as a separation membrane. The results are shown in Table 3. As a result, it was confirmed that stable production of L-lactic acid by continuous fermentation was possible.

［実施例４］連続発酵によるＬ−乳酸の製造（その４）
分離膜として前記の参考例４で作製した多孔性膜を用い、実施例２と同様のＬ−乳酸連続発酵試験を行った。その結果を、表３に示す。その結果、安定したＬ−乳酸の連続発酵による製造が可能であることを確認することができた。 [Example 4] Production of L-lactic acid by continuous fermentation (part 4)
Using the porous membrane prepared in Reference Example 4 as a separation membrane, the same L-lactic acid continuous fermentation test as in Example 2 was performed. The results are shown in Table 3. As a result, it was confirmed that stable production of L-lactic acid by continuous fermentation was possible.

［実施例５］連続発酵によるエタノールの製造（その１）
図１の膜分離型の連続発酵装置を稼働させることにより、エタノール連続発酵系が得られるかどうかを調べるため、表２に示す組成のエタノール発酵培地を用い、この装置の連続発酵試験を行った。該エタノール発酵培地は、１２１℃の温度で１５分間、高圧蒸気滅菌して用いた。分離膜には、前記の参考例２で作製した多孔性膜を用いた。実施例５における運転条件は、特に断らない限り、次のとおりである。
・発酵反応槽容量：１．５（Ｌ）
・膜分離槽容量：０．５（Ｌ）
・使用分離膜：ＰＶＤＦ濾過膜
・膜分離エレメント有効濾過面積：６０平方ｃｍ
・温度調整：３０（℃）
・発酵反応槽通気量：０．０５（Ｌ／ｍｉｎ）
・膜分離槽通気量：０．３（Ｌ／ｍｉｎ）
・発酵反応槽攪拌速度：１００（ｒｐｍ）
・ｐＨ調整：１Ｎ ＮａＯＨによりｐＨ５に調整
・エタノール発酵培地供給速度：５０〜３００ｍｌ／ｈｒ．の範囲で可変制御
・発酵培養液循環装置による循環液量：０．１（Ｌ／ｍｉｎ）
・膜透過水量制御：膜間差圧による流量制御
（連続発酵開始後〜１００時間：膜間差圧０．１ｋＰａ以上５ｋＰａ以下で制御
１００時間〜２００時間：膜間差圧０．１ｋＰａ以上２ｋＰａ以下で制御
２００時間〜３００時間：膜間差圧０．１ｋＰａ以上２０ｋＰａ以下で制御）
微生物としてＮＢＲＣ１０５０５株を用い、培地として表２に示す組成のエタノール発酵培地を用いた。生産物であるエタノール濃度は、ガスクロマトグラフ法により定量した。Shimadzu GC-2010キャピラリーGC TC-1(GL science) 15 meter L.＊0.53 mm I.D., df=1.5 μmを用いて、水素炎イオン化検出器により検出・算出して、エタノール生産量を評価した。また、グルコース濃度の測定には“グルコーステストワコーＣ”（登録商標）（和光純薬社製）を用いた。 [Example 5] Production of ethanol by continuous fermentation (part 1)
In order to examine whether or not an ethanol continuous fermentation system can be obtained by operating the membrane separation type continuous fermentation apparatus of FIG. 1, a continuous fermentation test of this apparatus was performed using an ethanol fermentation medium having the composition shown in Table 2. . The ethanol fermentation medium was used after autoclaving at 121 ° C. for 15 minutes. As the separation membrane, the porous membrane prepared in Reference Example 2 was used. The operating conditions in Example 5 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
・ Use separation membrane: PVDF filtration membrane ・ Membrane separation element Effective filtration area: 60 square cm
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
-Aeration rate of membrane separation tank: 0.3 (L / min)
・ Fermentation reactor stirring speed: 100 (rpm)
-PH adjustment: adjusted to pH 5 with 1N NaOH-Ethanol fermentation medium supply rate: 50-300 ml / hr. Circulated fluid volume by variable control / fermentation culture fluid circulation device in the range: 0.1 (L / min)
・ Membrane permeate flow control: Flow control by transmembrane pressure difference (After continuous fermentation up to 100 hours: Control at transmembrane pressure difference of 0.1 kPa to 5 kPa
100 hours to 200 hours: Control at a transmembrane differential pressure of 0.1 kPa to 2 kPa
200 hours to 300 hours: Controlled between transmembrane pressures 0.1 kPa and 20 kPa)
NBRC10505 strain was used as the microorganism, and an ethanol fermentation medium having the composition shown in Table 2 was used as the medium. The ethanol concentration of the product was quantified by gas chromatography. Shimadzu GC-2010 Capillary GC TC-1 (GL science) 15 meter L. * 0.53 mm ID, df = 1.5 μm was used for detection and calculation by a flame ionization detector to evaluate ethanol production. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

Figure 0005082496
Figure 0005082496

まず、試験管中で５ｍｌのエタノール発酵培地を用いＮＢＲＣ１０５０５株を一晩振とう培養した（前々々培養）。得られた培養液を新鮮なエタノール発酵培地１００ｍｌに植菌し、５００ｍｌ容坂口フラスコ中で２４時間、３０℃の温度で振とう培養した（前々培養）。前々培養液を、図１に示した膜分離型の連続発酵装置の１．５Ｌのエタノール発酵培地に植菌し、発酵反応槽１を付属の攪拌機５によって１００ｒｐｍで攪拌し、発酵反応槽１の通気量の調整、温度調整およびｐＨ調整を行い、発酵培養液循環ポンプ１１を稼働させることなく、２４時間培養を行った（前培養）。前培養完了後、直ちに、発酵培養液循環ポンプ１１を稼働させた。前培養時の運転条件に加え、膜分離槽１２を通気し、エタノール発酵培地の連続供給を行い、膜分離型の連続発酵装置の発酵液量を２Ｌとなるように膜透過水量の制御を行いながら連続培養し、連続発酵によるエタノールの製造を行った。連続発酵試験を行うときの膜透過水量の制御は、水頭差制御装置３により水頭差を膜間差圧として測定し、上記膜透過水量制御条件で変化させることで行った。適宜、膜透過発酵液中の生産されたエタノール濃度および残存グルコース濃度を測定した。３００時間の発酵試験を行った結果を表４に示す。その結果、この膜分離型の連続発酵装置を用いることにより、安定したエタノールの連続発酵による製造が可能であることを確認することができた。連続発酵の期間中の膜間差圧は、２ｋＰａ以下０．１ｋＰａ以上で推移した。   First, NBRC10505 strain was cultured with shaking overnight in a test tube using 5 ml of ethanol fermentation medium (pre-culture). The obtained culture solution was inoculated into 100 ml of a fresh ethanol fermentation medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture medium was inoculated in a 1.5 L ethanol fermentation medium of the membrane-separated continuous fermentation apparatus shown in FIG. 1 and the fermentation reaction tank 1 was stirred at 100 rpm by the attached stirrer 5 to give the fermentation reaction tank 1 The aeration amount was adjusted, the temperature was adjusted, and the pH was adjusted, and the culture was performed for 24 hours without operating the fermentation broth circulation pump 11 (pre-culture). Immediately after completion of the preculture, the fermentation broth circulation pump 11 was operated. In addition to the operating conditions at the time of pre-culture, the membrane separation tank 12 is vented, the ethanol fermentation medium is continuously supplied, and the amount of fermentation water in the membrane separation type continuous fermentation apparatus is controlled to 2 L. Then, the cells were continuously cultured, and ethanol was produced by continuous fermentation. Control of the amount of water permeated through the continuous fermentation test was carried out by measuring the water head difference as a transmembrane pressure difference with the water head difference control device 3 and changing it under the above-mentioned membrane permeated water amount control conditions. Where appropriate, the ethanol concentration produced and the residual glucose concentration in the membrane permeation fermentation broth were measured. Table 4 shows the results of 300 hours fermentation test. As a result, it was confirmed that stable production of ethanol by continuous fermentation was possible by using this membrane separation type continuous fermentation apparatus. The transmembrane pressure difference during the period of continuous fermentation changed at 2 kPa or less and 0.1 kPa or more.

［実施例６］連続発酵によるエタノールの製造（その２）
分離膜には前記の参考例３で作製した多孔性膜を用い、実施例２と同様のエタノール連続発酵試験を行った。その結果を、表４に示す。その結果、安定したエタノールの連続発酵による製造が可能であることを確認することができた。 [Example 6] Production of ethanol by continuous fermentation (part 2)
The same continuous ethanol fermentation test as in Example 2 was performed using the porous membrane prepared in Reference Example 3 described above as the separation membrane. The results are shown in Table 4. As a result, it was confirmed that stable ethanol production by continuous fermentation was possible.

［実施例７］連続発酵によるエタノールの製造（その３）
分離膜には前記の参考例４で作製した多孔性膜を用い、実施例２と同様のエタノール連続発酵試験を行った。その結果を、表４に示す。その結果、安定したエタノールの連続発酵による製造が可能であることを確認することができた。 [Example 7] Production of ethanol by continuous fermentation (part 3)
The same continuous ethanol fermentation test as in Example 2 was performed using the porous membrane prepared in Reference Example 4 described above as the separation membrane. The results are shown in Table 4. As a result, it was confirmed that stable ethanol production by continuous fermentation was possible.

［比較例１］回分発酵によるＬ−乳酸の製造（その１）
図１に示す膜分離型の連続発酵装置を用いることによりＬ−乳酸発酵生産性が向上するかどうかを調べるため、微生物を用いた発酵形態として最も典型的な回分発酵を行い、その乳酸生産性を評価した。表１に示す乳酸発酵培地を用い、図１の膜分離型の連続発酵装置の発酵反応槽１のみを用いた回分発酵試験を行った。該乳酸発酵培地は、１２１℃の温度で１５分間、高圧蒸気滅菌して用いた。比較例１でも、微生物として前記の参考例１で造成した酵母ＳＷ−１株を用いた。生産物であるＬ−乳酸の濃度は、前記の参考例１に示したＨＰＬＣを用いて評価し、グルコース濃度の測定には“グルコーステストワコーＣ”（登録商標）（和光純薬社製）を用いた。比較例１の運転条件を次に示す。
・発酵反応槽容量（乳酸発酵培地量）：１（Ｌ）
・温度調整：３０（℃）
・発酵反応槽通気量：０．２（Ｌ／ｍｉｎ）
・発酵反応槽攪拌速度：４００（ｒｐｍ）
・ｐＨ調整：０．５Ｎ ＮａＯＨによりｐＨ５に調整。 [Comparative Example 1] Production of L-lactic acid by batch fermentation (part 1)
In order to investigate whether or not L-lactic acid fermentation productivity is improved by using the membrane-separated continuous fermentation apparatus shown in FIG. 1, the most typical batch fermentation is performed as a fermentation form using microorganisms, and the lactic acid productivity is determined. Evaluated. Using the lactic acid fermentation medium shown in Table 1, a batch fermentation test was conducted using only the fermentation reaction tank 1 of the membrane-separated continuous fermentation apparatus of FIG. The lactic acid fermentation medium was used after being autoclaved at 121 ° C. for 15 minutes. Also in Comparative Example 1, the yeast SW-1 strain constructed in Reference Example 1 was used as a microorganism. The concentration of L-lactic acid as a product is evaluated using the HPLC shown in Reference Example 1 above, and “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring the glucose concentration. Using. The operating conditions of Comparative Example 1 are shown below.
-Fermentation reactor capacity (lactic acid fermentation medium amount): 1 (L)
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.2 (L / min)
・ Fermentation reactor stirring speed: 400 (rpm)
-PH adjustment: Adjust to pH 5 with 0.5N NaOH.

まず、試験管中で５ｍｌの乳酸発酵培地を用いＳＷ−１株を一晩振とう培養した（前々培養）。前々培養液を新鮮な乳酸発酵培地１００ｍｌに植菌し、５００ｍｌ容坂口フラスコ中で２４時間振とう培養した（前培養）。前培養液を膜分離型連続発酵装置の１．５Ｌの乳酸発酵培地に植菌し、発酵反応槽１を付属の攪拌機５で４００ｒｐｍで攪拌し、発酵反応槽１を通気した。温度調整とｐＨ調整を行い、発酵培養液循環ポンプ１１を稼働させることなく、回分発酵培養を行った。このときの菌体増殖量は、６００ｎｍでの吸光度で１５であった。回分発酵の結果を、実施例１の連続発酵試験で得られたＬ―乳酸発酵生産性と比較して表３に示す。   First, the SW-1 strain was cultured with shaking overnight in a test tube using 5 ml of lactic acid fermentation medium (pre-culture). The culture solution was inoculated into 100 ml of fresh lactic acid fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). The preculture was inoculated into a 1.5 L lactic acid fermentation medium of a membrane separation type continuous fermentation apparatus, the fermentation reaction tank 1 was stirred at 400 rpm with the attached stirrer 5, and the fermentation reaction tank 1 was aerated. Temperature adjustment and pH adjustment were performed, and batch fermentation culture was performed without operating the fermentation broth circulation pump 11. The amount of bacterial cell growth at this time was 15 in terms of absorbance at 600 nm. The results of batch fermentation are shown in Table 3 in comparison with the L-lactic acid fermentation productivity obtained in the continuous fermentation test of Example 1.

これら比較の結果、図１の膜分離型の連続発酵装置を用いることにより、Ｌ−乳酸の生産速度が大幅に向上することを明らかにすることができた。すなわち、本発明によって開示された多孔性膜を組み込んだ膜分離型の連続発酵装置を用い、膜間差圧を制御することにより、発酵培養液を分離膜によって濾液と未濾過液に分離し、濾液から所望の発酵生産物を回収すると共に、未濾過液を発酵培養液に戻す連続発酵方法を可能とし、微生物量を高く維持しながら、連続発酵によるＬ―乳酸等の化学品の製造が可能であることが明らかとなった。   As a result of these comparisons, it was clarified that the production rate of L-lactic acid was greatly improved by using the membrane separation type continuous fermentation apparatus of FIG. That is, using a membrane separation type continuous fermentation apparatus incorporating a porous membrane disclosed by the present invention, by controlling the transmembrane pressure difference, the fermentation broth is separated into a filtrate and an unfiltered liquid by the separation membrane, A desired fermentation product is recovered from the filtrate and a continuous fermentation method is possible in which the unfiltered liquid is returned to the fermentation broth, allowing the production of chemicals such as L-lactic acid by continuous fermentation while maintaining a high amount of microorganisms. It became clear that.

［比較例２］回分発酵によるＬ−乳酸の製造（その２）
図１に示す膜分離型の連続発酵装置を用いることによりＬ−乳酸発酵生産性が向上するかどうかを調べるため、微生物を用いた発酵形態として最も典型的な回分発酵を行い、その乳酸生産性を評価した。表１に示す乳酸発酵培地を用い、図１の膜分離型の連続発酵装置の発酵反応槽１のみを用いた回分発酵試験を行った。該乳酸発酵培地は、１２１℃の温度で１５分間、高圧蒸気滅菌して用いた。比較例２でも、微生物として前記の参考例１で造成した酵母ＳＷ−１株を用いた。生産物であるＬ−乳酸の濃度は、参考例１に示したＨＰＬＣを用いて評価し、グルコース濃度の測定には“グルコーステストワコーＣ”（登録商標）（和光純薬）を用いた。比較例２の運転条件を以下に示す。
・発酵反応槽容量（乳酸発酵培地量）：１（Ｌ）
・温度調整：３０（℃）
・発酵反応槽通気量：０．０５（Ｌ／ｍｉｎ）
・発酵反応槽攪拌速度：１００（ｒｐｍ）
・ｐＨ調整：１Ｎ ＮａＯＨによりｐＨ５に調整。 [Comparative Example 2] Production of L-lactic acid by batch fermentation (part 2)
In order to investigate whether or not L-lactic acid fermentation productivity is improved by using the membrane-separated continuous fermentation apparatus shown in FIG. 1, the most typical batch fermentation is performed as a fermentation form using microorganisms, and the lactic acid productivity is determined. Evaluated. Using the lactic acid fermentation medium shown in Table 1, a batch fermentation test was conducted using only the fermentation reaction tank 1 of the membrane-separated continuous fermentation apparatus of FIG. The lactic acid fermentation medium was used after being autoclaved at 121 ° C. for 15 minutes. Also in Comparative Example 2, the yeast SW-1 strain constructed in Reference Example 1 was used as the microorganism. The concentration of L-lactic acid as a product was evaluated using the HPLC shown in Reference Example 1, and “glucose test Wako C” (registered trademark) (Wako Pure Chemical Industries) was used for measuring the glucose concentration. The operating conditions of Comparative Example 2 are shown below.
-Fermentation reactor capacity (lactic acid fermentation medium amount): 1 (L)
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
・ Fermentation reactor stirring speed: 100 (rpm)
-PH adjustment: Adjust to pH 5 with 1N NaOH.

まず、試験管中で５ｍｌの乳酸発酵培地を用いＳＷ−１株を一晩振とう培養した（前々培養）。前々培養液を新鮮な乳酸発酵培地１００ｍｌに植菌し、５００ｍｌ容坂口フラスコ中で２４時間振とう培養した（前培養）。前培養液を膜分離型の連続発酵装置の１．５Ｌの乳酸発酵培地に植菌し、発酵反応槽１を付属の攪拌機５で１００ｒｐｍで攪拌し、発酵反応槽１を通気した。温度調整とｐＨ調整を行い、発酵培養液循環ポンプ１１を稼働させることなく、回分発酵培養を行った。このときの菌体増殖量は、６００ｎｍでの吸光度で１４であった。回分発酵の結果を、実施例２、３および４の連続発酵試験で得られたＬ―乳酸発酵生産性と比較して表３に示す。これら比較の結果、図１の膜分離型の連続発酵装置を用いることにより、Ｌ−乳酸の生産速度が大幅に向上することを明らかにすることができた。すなわち、本発明によって開示された多孔性膜を組み込んだ膜分離型の連続発酵装置を用い、膜間差圧を制御することにより、発酵培養液を分離膜によって濾液と未濾過液に分離し、濾液から所望の発酵生産物を回収すると共に、未濾過液を発酵培養液に戻す連続発酵方法を可能とし、微生物量を高く維持しながら、連続発酵によるＬ―乳酸等の化学品の製造が可能であることが明らかとなった。   First, the SW-1 strain was cultured with shaking overnight in a test tube using 5 ml of lactic acid fermentation medium (pre-culture). The culture solution was inoculated into 100 ml of fresh lactic acid fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). The preculture was inoculated into a 1.5 L lactic acid fermentation medium of a membrane separation type continuous fermentation apparatus, the fermentation reaction tank 1 was stirred at 100 rpm with the attached stirrer 5, and the fermentation reaction tank 1 was aerated. Temperature adjustment and pH adjustment were performed, and batch fermentation culture was performed without operating the fermentation broth circulation pump 11. The amount of bacterial cell growth at this time was 14 in terms of absorbance at 600 nm. The results of batch fermentation are shown in Table 3 in comparison with L-lactic acid fermentation productivity obtained in the continuous fermentation tests of Examples 2, 3 and 4. As a result of these comparisons, it was clarified that the production rate of L-lactic acid was greatly improved by using the membrane separation type continuous fermentation apparatus of FIG. That is, using a membrane separation type continuous fermentation apparatus incorporating a porous membrane disclosed by the present invention, by controlling the transmembrane pressure difference, the fermentation broth is separated into a filtrate and an unfiltered liquid by the separation membrane, A desired fermentation product is recovered from the filtrate and a continuous fermentation method is possible in which the unfiltered liquid is returned to the fermentation broth, allowing the production of chemicals such as L-lactic acid by continuous fermentation while maintaining a high amount of microorganisms. It became clear that.

［比較例３］回分発酵によるエタノールの製造
図１に示す膜分離型の連続発酵装置を用いることによりエタノール発酵生産性が向上するかどうかを調べるため、微生物を用いた発酵形態として最も典型的な回分発酵を行い、そのエタノール生産性を評価した。表２に示すエタノール発酵培地を用い、図１の膜分離型の連続発酵装置の発酵反応槽１のみを用いた回分発酵試験を行った。該エタノール発酵培地は、１２１℃の温度で１５分間、高圧蒸気滅菌して用いた。比較例３でも、微生物としてＮＢＲＣ１０５０５株を用いた。生産物であるエタノールの濃度は、実施例５に示したＨＰＬＣを用いて評価し、グルコース濃度の測定には“グルコーステストワコーＣ”（登録商標）を用いた。比較例３の運転条件を次に示す。
・反応槽容量（エタノール発酵培地量）：１（Ｌ）
・温度調整：３０（℃）
・反応槽通気量：０．０５（Ｌ／ｍｉｎ）
・反応槽攪拌速度：１００（ｒｐｍ）
・ｐＨ調整：１Ｎ ＮａＯＨによりｐＨ５に調整。 [Comparative Example 3] Production of ethanol by batch fermentation In order to examine whether ethanol fermentation productivity is improved by using the membrane separation type continuous fermentation apparatus shown in Fig. 1, the most typical fermentation form using microorganisms is used. Batch fermentation was performed and the ethanol productivity was evaluated. Using the ethanol fermentation medium shown in Table 2, a batch fermentation test using only the fermentation reaction tank 1 of the membrane separation type continuous fermentation apparatus of FIG. 1 was conducted. The ethanol fermentation medium was used after autoclaving at 121 ° C. for 15 minutes. Also in Comparative Example 3, NBRC10505 strain was used as a microorganism. The concentration of ethanol as a product was evaluated using HPLC shown in Example 5, and “glucose test Wako C” (registered trademark) was used for measurement of glucose concentration. The operating conditions of Comparative Example 3 are shown below.
-Reaction tank capacity (ethanol fermentation medium amount): 1 (L)
・ Temperature adjustment: 30 (℃)
-Reaction tank aeration: 0.05 (L / min)
-Reaction vessel stirring speed: 100 (rpm)
-PH adjustment: Adjust to pH 5 with 1N NaOH.

まず、試験管中で５ｍｌのエタノール発酵培地を用いＮＢＲＣ１０５０５株を一晩振とう培養した（前々培養）。前々培養液を新鮮なエタノール発酵培地１００ｍｌに植菌し５００ｍｌ容坂口フラスコ中で２４時間振とう培養した（前培養）。前培養液を膜分離型の連続発酵装置の１．５Ｌのエタノール発酵培地に植菌し、発酵反応槽１を付属の攪拌機５で１００ｒｐｍで攪拌し、発酵反応槽１を通気した。温度調整とｐＨ調整を行い、発酵液循環ポンプ１１を稼働させることなく、回分発酵培養を行った。このときの菌体増殖量は、６００ｎｍでの吸光度で１８であった。回分発酵の結果を、実施例５の連続発酵試験で得られたＬ―エタノール発酵生産性と比較して表４示す。これら比較の結果、図１の膜分離型の連続発酵装置を用いることにより、エタノールの生産速度が大幅に向上することを明らかにすることができた。すなわち、本発明によって開示された多孔性膜を組み込んだ膜分離型連続発酵装置を用い、膜間差圧を制御することで、発酵培養液を分離膜によって濾液と未濾過液に分離し、濾液から所望の発酵生産物を回収するとともに、未濾過液を発酵培養液に戻す連続発酵方法を可能とし、微生物量を高く維持しながら、連続発酵によるエタノール等の化学品の製造が可能であることが明らかとなった。   First, the NBRC10505 strain was cultured overnight in a test tube using 5 ml of ethanol fermentation medium (pre-culture). The culture solution was inoculated into 100 ml of fresh ethanol fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). The preculture was inoculated into a 1.5 L ethanol fermentation medium of a membrane separation type continuous fermentation apparatus, the fermentation reaction tank 1 was stirred at 100 rpm with the attached stirrer 5, and the fermentation reaction tank 1 was aerated. Temperature adjustment and pH adjustment were performed, and batch fermentation culture was performed without operating the fermentation liquid circulation pump 11. The amount of bacterial cell growth at this time was 18 in terms of absorbance at 600 nm. Table 4 shows the results of batch fermentation in comparison with the L-ethanol fermentation productivity obtained in the continuous fermentation test of Example 5. As a result of these comparisons, it was clarified that the production rate of ethanol was significantly improved by using the membrane separation type continuous fermentation apparatus of FIG. That is, by using a membrane separation type continuous fermentation apparatus incorporating a porous membrane disclosed by the present invention, and controlling the transmembrane pressure difference, the fermentation broth is separated into a filtrate and an unfiltered liquid by the separation membrane, and the filtrate It is possible to produce a chemical product such as ethanol by continuous fermentation while allowing a continuous fermentation method to recover the desired fermentation product from the fermentation and returning the unfiltered liquid to the fermentation broth, while maintaining a high amount of microorganisms. Became clear.

［比較例４］連続発酵によるＬ−乳酸の製造
細孔径が小さく、純水透過係数が小さい多孔性膜を用い、図１の膜分離型連続発酵装置を稼働させることにより、Ｌ−乳酸連続発酵系が得られるかどうかを調べるため、表１に示す組成の酵母乳酸発酵培地を用い、この装置による連続発酵試験を行った。比較例４は、分離膜としては参考例５で作製した細孔径が小さく、純水透過係数が小さい多孔性膜を用い、膜透過水量制御方法を膜間差圧による流量制御（連続発酵全期間０．１ｋＰａ以上２０ｋＰａ以下で制御）し、その他試験条件は実施例２と同様に行った。その結果、培養開始後８０時間で、膜間差圧が２０ｋＰａを超え膜の閉塞が発生したため、連続発酵を停止した。このことから、本発明の連続発酵による化学品の製造方法と連続発酵装置には、参考例５で作製した多孔性膜は不適であることが明らかになった。 [Comparative Example 4] Production of L-lactic acid by continuous fermentation Using a porous membrane having a small pore diameter and a small pure water permeability coefficient, the membrane-separated continuous fermentation apparatus of Fig. 1 was operated, whereby L-lactic acid continuous fermentation. In order to examine whether or not a system was obtained, a yeast fermentation medium having the composition shown in Table 1 was used, and a continuous fermentation test using this apparatus was performed. Comparative Example 4 uses a porous membrane with a small pore diameter and a small pure water permeability coefficient produced in Reference Example 5 as a separation membrane, and controls the flow rate of membrane permeate through a transmembrane differential pressure (all continuous fermentation period). The other test conditions were the same as in Example 2. As a result, 80 hours after the start of the culture, the transmembrane pressure exceeded 20 kPa and the membrane was blocked, so the continuous fermentation was stopped. From this, it became clear that the porous membrane produced in Reference Example 5 is unsuitable for the chemical production method and continuous fermentation apparatus according to the present invention.

Figure 0005082496
Figure 0005082496

Figure 0005082496
Figure 0005082496

本発明の連続発酵による化学品の製造方法は、分離膜として高い透過性と高い細胞阻止率を持ち閉塞しにくい多孔性膜を用い、低い膜間差圧で濾過処理することにより、安定に低コストで発酵生産効率を著しく向上させることができる。また、簡便な操作条件で、長時間にわたり安定して高生産性を維持する連続発酵が可能となり、広く発酵工業において、発酵生産物である化学品を低コストで安定に生産することが可能である。   The method for producing a chemical product by continuous fermentation of the present invention uses a porous membrane that has high permeability and high cell blocking rate as a separation membrane and is difficult to block, and is stably filtered by performing filtration with a low transmembrane pressure difference. Fermentation production efficiency can be remarkably improved at a cost. In addition, continuous fermentation that stably maintains high productivity over a long period of time under simple operating conditions is possible, and in the fermentation industry, chemical products that are fermentation products can be stably produced at low cost. is there.

図１は、本発明の膜分離型の連続発酵装置の一つの実施の形態を説明するための概略側面図である。FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus of the present invention. 図２は、本発明で用いられる分離膜エレメントの一つの実施の形態を説明するための概略斜視図である。FIG. 2 is a schematic perspective view for explaining one embodiment of a separation membrane element used in the present invention. 図３は、実施例で用いた酵母用発現ベクターｐＴＲＳ１１のフィジカルマップを示す図である。FIG. 3 is a diagram showing a physical map of the expression vector for yeast pTRS11 used in the examples.

符号の説明Explanation of symbols

１ 発酵反応槽
２ 分離膜エレメント
３ 水頭差制御装置
４ 気体供給装置
５ 攪拌機
６ レベルセンサ
７ 培地供給ポンプ
８ ｐＨ調整溶液供給ポンプ
９ ｐＨセンサ・制御装置
１０ 温度調節器
１１ 発酵培養液循環ポンプ
１２ 膜分離槽
１３ 支持板
１４ 流路材
１５ 分離膜
１６ 凹部
１７ 集水パイプ DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Separation membrane element 3 Water head difference control device 4 Gas supply device 5 Stirrer 6 Level sensor 7 Medium supply pump 8 pH adjustment solution supply pump 9 pH sensor / control device 10 Temperature controller 11 Fermentation culture medium circulation pump 12 Membrane Separation tank 13 Support plate 14 Channel material 15 Separation membrane 16 Recess 17 Water collecting pipe

Claims (13)

微生物もしくは培養細胞の発酵培養液を分離膜で濾過し、濾液から生産物を回収すると共に未濾過液を前記の発酵培養液に保持または還流し、かつ、発酵原料を前記の発酵培養液に追加する連続発酵による化学品の製造方法であって、前記の分離膜として平均細孔径が０.０１μｍ以上１μｍ未満の細孔を有する多孔性膜を用い、その膜間差圧を０.１から２０ｋＰａの範囲にして濾過処理することを特徴とする連続発酵による化学品の製造方法。   The fermentation broth of microorganisms or cultured cells is filtered through a separation membrane, the product is recovered from the filtrate, the unfiltered liquid is retained or refluxed in the fermentation broth, and the fermentation raw material is added to the fermentation broth A method for producing a chemical by continuous fermentation, wherein a porous membrane having pores with an average pore diameter of 0.01 μm or more and less than 1 μm is used as the separation membrane, and the transmembrane pressure difference is 0.1 to 20 kPa. A method for producing a chemical product by continuous fermentation, wherein the filtration treatment is performed in the range described above. 多孔性膜の純水透過係数が、２×１０−９ｍ３／ｍ２／ｓ／ｐａ以上６×１０−７ｍ３／ｍ２／ｓ／ｐａ以下であることを特徴とする請求項１記載の連続発酵による化学品の製造方法。 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. The manufacturing method of the chemical product by continuous fermentation of description. 多孔性膜の平均細孔径が、０.０１μｍ以上０.２μｍ未満の範囲内にあり、かつ、該平均細孔径の標準偏差が０.１μｍ以下であることを特徴とする請求項１または２記載の連続発酵による化学品の製造方法。   The average pore size of the porous membrane is in the range of 0.01 µm or more and less than 0.2 µm, and the standard deviation of the average pore size is 0.1 µm or less. A method for producing chemicals by continuous fermentation. 多孔性膜の膜表面粗さが０．１μｍ以下の多孔性膜であることを特徴とする請求項１から３のいずれかに記載の連続発酵による化学品の製造方法。   The method for producing a chemical product by continuous fermentation according to any one of claims 1 to 3, wherein the porous membrane has a membrane surface roughness of 0.1 µm or less. 多孔性膜が多孔質樹脂層を含む多孔性膜である請求項１から４のいずれかに記載の連続発酵による化学品の製造方法。   The method for producing a chemical product by continuous fermentation according to any one of claims 1 to 4, wherein the porous membrane is a porous membrane including a porous resin layer. 多孔性膜の膜素材がポリフッ化ビニリデンを含むことを特徴とする請求項１から５のいずれかに記載の連続発酵による化学品の製造方法。   The method for producing a chemical product by continuous fermentation according to any one of claims 1 to 5, wherein the membrane material of the porous membrane contains polyvinylidene fluoride. 微生物または培養細胞の発酵培養液および発酵原料が、糖類を含むことを特徴とする請求項１から６のいずれかに記載の連続発酵による化学品の製造方法。   The method for producing a chemical product by continuous fermentation according to any one of claims 1 to 6, wherein the fermentation broth and fermentation raw material of microorganisms or cultured cells contain saccharides. 化学品が有機酸またはアルコールであることを特徴とする請求項１から７のいずれかに記載の連続発酵による化学品の製造方法。   The method for producing a chemical product by continuous fermentation according to any one of claims 1 to 7, wherein the chemical product is an organic acid or an alcohol. 微生物もしくは培養細胞の発酵培養液を分離膜で濾過し、濾液から生産物を回収すると共に未濾過液を前記の発酵培養液に保持または還流し、かつ、発酵原料を前記の発酵培養液に追加する連続発酵による化学品の製造装置であって、微生物もしくは培養細胞を発酵培養させるための発酵反応槽と、該発酵反応槽に発酵培養液循環手段を介して接続され内部に分離膜を備えた発酵培養液を濾過するための膜分離槽と、分離膜の膜間差圧を０.１から２０ｋＰａの範囲に制御する手段からなり、該分離膜が平均細孔径０.０１μｍ以上１μｍ未満の多孔性膜であることを特徴とする連続発酵装置。   The fermentation broth of microorganisms or cultured cells is filtered through a separation membrane, the product is recovered from the filtrate, the unfiltered liquid is retained or refluxed in the fermentation broth, and the fermentation raw material is added to the fermentation broth An apparatus for producing a chemical product by continuous fermentation, comprising a fermentation reaction vessel for fermenting and culturing microorganisms or cultured cells, and a separation membrane inside which is connected to the fermentation reaction vessel via a fermentation culture medium circulation means A membrane separation tank for filtering the fermentation broth and means for controlling the transmembrane pressure difference of the separation membrane in the range of 0.1 to 20 kPa, and the separation membrane is porous with an average pore diameter of 0.01 μm or more and less than 1 μm. A continuous fermentation apparatus characterized by being a membrane. 分離膜の膜間差圧を、水頭差制御装置による発酵培養液と多孔性膜処理水の液位差で制
御することを特徴とする請求項９記載の連続発酵装置。 10. The continuous fermentation apparatus according to claim 9 , wherein the transmembrane pressure difference of the separation membrane is controlled by the difference in liquid level between the fermentation broth and the porous membrane treated water by the head differential control device. 分離膜の膜間差圧を、加圧ポンプまたは／および吸引ポンプで制御することを特徴とす
る請求項９または１０記載の連続発酵装置。 The continuous fermentation apparatus according to claim 9 or 10 , wherein the transmembrane pressure difference of the separation membrane is controlled by a pressurizing pump or / and a suction pump. 分離膜の膜間差圧を、気体または液体の圧力で制御することを特徴とする請求項９から１１のいずれかに記載の連続発酵装置。 The continuous fermentation apparatus according to any one of claims 9 to 11 , wherein the transmembrane pressure difference of the separation membrane is controlled by gas or liquid pressure. 発酵培養液循環手段が、循環ポンプであることを特徴とする請求項９〜１２のいずれかに記載の連続発酵装置。 The continuous fermentation apparatus according to any one of claims 9 to 12 , wherein the fermentation culture medium circulation means is a circulation pump.
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Publication number Priority date Publication date Assignee Title
JP5130811B2 (en) * 2006-07-19 2013-01-30 東レ株式会社 Process for producing 1,3-propanediol by continuous fermentation
JP5130816B2 (en) * 2006-08-02 2013-01-30 東レ株式会社 Method for producing succinic acid by continuous fermentation
JP5141126B2 (en) * 2006-09-26 2013-02-13 東レ株式会社 Method for producing D-lactic acid by continuous fermentation
JP5092487B2 (en) * 2007-03-27 2012-12-05 東レ株式会社 Production method of chemicals by continuous fermentation
JP5130826B2 (en) * 2007-03-28 2013-01-30 東レ株式会社 Process for producing lactic acid by continuous fermentation
JP5141133B2 (en) * 2007-08-10 2013-02-13 東レ株式会社 Protein production method by continuous fermentation
JP2009142210A (en) * 2007-12-14 2009-07-02 Toray Ind Inc Method for producing lactic acid by continuous fermentation
JP5992135B2 (en) 2008-02-04 2016-09-14 東レ株式会社 Process for producing lactic acid by continuous fermentation
JP2009296921A (en) * 2008-06-12 2009-12-24 Toray Ind Inc Continuous culture device and method for producing chemical
JP5593594B2 (en) * 2008-07-23 2014-09-24 東レ株式会社 Process for producing chemicals by continuous culture
BR112012010949A2 (en) 2009-11-10 2017-11-07 Toray Industries hollow membrane module for use in producing a chemical and process for producing a chemical using the hollow membrane module for use in producing a chemical
BRPI1004963A2 (en) * 2010-08-19 2015-03-24 Companhia Refinadora Da Amazônia Constant volume acidity correction system and process of fermentative media for the production of organic acids
WO2012077742A1 (en) 2010-12-09 2012-06-14 東レ株式会社 Method for producing chemical by continuous fermentation
CN103261427B (en) 2010-12-22 2015-04-08 东丽株式会社 Method for producing chemical by continuous fermentation
WO2012090556A1 (en) * 2010-12-27 2012-07-05 東レ株式会社 Method for producing chemicals by continuous fermentation
JP2013048583A (en) * 2011-08-31 2013-03-14 Panasonic Corp Method and apparatus for producing bioethanol
WO2014156998A1 (en) * 2013-03-28 2014-10-02 旭硝子株式会社 Process and device for producing chemical product
AU2014258258B2 (en) 2013-04-25 2018-07-05 Toray Industries, Inc. Cartridge type hollow fiber membrane module
JP6467920B2 (en) 2013-09-30 2019-02-13 東レ株式会社 Manufacturing method of cartridge type hollow fiber membrane module
CN108779478A (en) * 2016-03-17 2018-11-09 东丽株式会社 The manufacturing method and method for culturing microbes of chemicals
JP6142042B1 (en) 2016-03-18 2017-06-07 株式会社村田製作所 Filtration filter for nucleated cells and filtration method using the same
JP6249124B1 (en) 2017-04-26 2017-12-20 株式会社村田製作所 Filtration filter for nucleated cells and filtration method using the same
JP6815307B2 (en) * 2017-11-09 2021-01-20 株式会社村田製作所 Filtration filter for nucleated cells and filtration method using it
WO2020170228A1 (en) 2019-02-24 2020-08-27 Oncosimis Biotech Private Limited Method for continous production of recombinant glp-1 peptide by bacteria

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63248396A (en) * 1987-04-06 1988-10-14 Nok Corp Production of dihydrooxoisophorone
JPH10118461A (en) * 1996-10-16 1998-05-12 Kao Corp Recovery of fermentation product
JPH10323543A (en) * 1997-05-22 1998-12-08 Arakawa Chem Ind Co Ltd Method for separating product in microbial reaction
JPH11113587A (en) * 1997-10-14 1999-04-27 Ube Ind Ltd Production of alcohol by alcoholic fermentation and apparatus therefor
KR100874079B1 (en) * 2001-02-16 2008-12-12 도레이 카부시키가이샤 Separating Film, Separating Film Element, Separating Film Module, Sewage and Waste Water Treatment Device, and Separating Film Manufacturing Method
JP2003053164A (en) * 2001-08-20 2003-02-25 Kuraray Co Ltd Removing method of pathogenic protozoa and separating membrane used therein
JP3896101B2 (en) * 2003-07-11 2007-03-22 ダイセル化学工業株式会社 Car wash wastewater treatment equipment
JP2005111326A (en) * 2003-10-06 2005-04-28 Toray Ind Inc Liquid separation membrane and its use

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