JPH0530962A - Method for culturing microorganism - Google Patents
Method for culturing microorganismInfo
- Publication number
- JPH0530962A JPH0530962A JP3214598A JP21459891A JPH0530962A JP H0530962 A JPH0530962 A JP H0530962A JP 3214598 A JP3214598 A JP 3214598A JP 21459891 A JP21459891 A JP 21459891A JP H0530962 A JPH0530962 A JP H0530962A
- Authority
- JP
- Japan
- Prior art keywords
- microorganism
- shear stress
- culturing
- stirrer
- controlled
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/20—Degassing; Venting; Bubble traps
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/42—Means for regulation, monitoring, measurement or control, e.g. flow regulation of agitation speed
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は攪拌機を備えた培養槽内
での微生物の培養方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing microorganisms in a culture tank equipped with a stirrer.
【０００２】[0002]
【従来の技術】微生物に対し形質転換、遺伝子組替え或
いは細胞融合を施すことで、有用物質を大量に生産し得
る技術の研究が盛んである。特に微生物は動物や植物に
比べ増殖速度が速く、死滅しにくいので有利である。2. Description of the Related Art Research on a technique capable of producing a large amount of a useful substance by subjecting a microorganism to transformation, gene recombination or cell fusion has been actively conducted. In particular, microorganisms are advantageous because they have a higher growth rate and are less likely to die than animals and plants.
【０００３】斯かる微生物の培養法としては、栄養源と
なる基質を含んだ培養液を槽内に入れ、更にこの培養液
に微生物を投入した懸濁液を攪拌することで懸濁状態
（微生物濃度）の均一化を図るとともに微生物と溶存酸
素とを接触させて増殖を促進するようにしている。As a method for culturing such microorganisms, a culture solution containing a substrate serving as a nutrient source is placed in a tank, and the suspension obtained by adding microorganisms to this culture solution is stirred to obtain a suspended state (microorganisms). The concentration is made uniform, and the microorganisms are brought into contact with dissolved oxygen to promote the growth.
【０００４】[0004]
【発明が解決しようとする課題】ところで、微生物懸濁
液を攪拌すると剪断応力が発生し、この剪断応力が大き
いと微生物の細胞が破壊されてしまう。逆に攪拌しない
と前記したように溶存酸素との接触が図れない等の不利
が生じる。このため、微生物懸濁液に作用する剪断応力
を所定範囲にして攪拌を継続することが必要となるが、
個々の微生物によって剪断応力耐性値が異なり、そのコ
ントロールが困難である。By the way, when the microbial suspension is stirred, shear stress is generated, and if the shear stress is large, the cells of the microorganism are destroyed. On the contrary, if stirring is not carried out, disadvantages such as contact with dissolved oxygen cannot be achieved as described above. Therefore, it is necessary to keep the stirring within a predetermined range of shear stress acting on the microbial suspension,
It is difficult to control the shear stress resistance value depending on individual microorganisms.
【０００５】[0005]
【課題を解決するための手段】上記課題を解決すべく本
願の第１発明は、微生物懸濁液に加えられる剪断応力を
微生物懸濁液の粘度等の流動特性から算出し、この剪断
応力が個々の微生物に応じて設定した所定値を超えない
ように攪拌機の回転数を制御するようにした。In order to solve the above-mentioned problems, the first invention of the present application is to calculate the shear stress applied to a microbial suspension from flow characteristics such as viscosity of the microbial suspension. The rotation speed of the stirrer was controlled so as not to exceed a predetermined value set according to each microorganism.
【０００６】また本願の第２発明は培養槽内の微生物の
剪断破壊量を直接測定し、この剪断破壊量が所定値を超
えないように攪拌機の回転数を制御するようにした。In the second invention of the present application, the shear fracture amount of the microorganism in the culture tank is directly measured, and the rotation speed of the stirrer is controlled so that the shear fracture amount does not exceed a predetermined value.
【０００７】[0007]
【作用】培養槽中の微生物懸濁液に作用する剪断応力
は、槽の大きさ、攪拌機の形式及び攪拌機の回転速度に
よって決定される。そして、一旦培養槽を製作した後に
剪断応力を制御できるパラメータは攪拌機の回転速度の
みとなる。そこで、剪断応力が所定値を超えないように
するには攪拌機の回転数を制御すればよいことになる。The shear stress acting on the microbial suspension in the culture tank is determined by the size of the tank, the type of stirrer and the rotation speed of the stirrer. The parameter that can control the shear stress after the culture tank is once manufactured is only the rotation speed of the stirrer. Therefore, in order to prevent the shear stress from exceeding a predetermined value, the rotation speed of the stirrer may be controlled.
【０００８】[0008]
【実施例】以下に本発明の実施例を添付図面に基づいて
説明する。ここで、図１は本願の第１発明に係る培養方
法の実施に用いる培養装置の概略図であり、培養装置は
槽１内に微生物を混入した培養液すなわち微生物懸濁液
２を満たし、この微生物懸濁液２をモータ３にて回転せ
しめられる攪拌機４にて攪拌するようにしている。Embodiments of the present invention will be described below with reference to the accompanying drawings. Here, FIG. 1 is a schematic view of a culturing apparatus used for carrying out the culturing method according to the first invention of the present application. The culturing apparatus fills a tank 1 with a culture solution containing a microorganism, that is, a microbial suspension 2. The microbial suspension 2 is agitated by an agitator 4 rotated by a motor 3.
【０００９】また、槽１の底部には多孔質なガス供給プ
レート５を配置し、フィルタ６及びガス供給プレート５
を介して外部から酸素ガス等を微生物懸濁液２に供給す
るようにし、槽１の上部にはフィルタを備えたガス抜き
パイプ７を設け、更に微生物懸濁液２の粘度をレオメー
タ８で測定し、このレオメータ８の測定値を制御装置９
に入力し、制御装置９では測定粘度に基づき、モータ３
にオン・オフ信号或いは回転数の制御信号を出力するよ
うにしている。A porous gas supply plate 5 is arranged at the bottom of the tank 1, and the filter 6 and the gas supply plate 5 are arranged.
Oxygen gas or the like is supplied to the microbial suspension 2 from the outside via a gas outlet pipe 7 provided with a filter at the upper part of the tank 1, and the viscosity of the microbial suspension 2 is measured by a rheometer 8. Then, the measured value of this rheometer 8 is set to the control device 9
To the motor 3 based on the measured viscosity.
An on / off signal or a control signal for the number of revolutions is output to.
【００１０】図２はモータの回転数を粘度に応じて変化
せしめた本発明方法と一定の回転数とした従来の培養法
とを菌体濃度と生成物濃度において比較したものであ
る。菌体としてはMicromonospora olivasterospora（KY
-11587）を用い、この菌体からアミノグロシド系抗生物
質であるフォーチミン−Ａを生成するものとする。FIG. 2 shows a comparison between the method of the present invention in which the rotation speed of the motor is changed according to the viscosity and the conventional culture method in which the rotation speed is constant, in terms of bacterial cell concentration and product concentration. As a microbial cell, Micromonospora olivasterospora (KY
-11587) is used to produce the aminogloside antibiotic Fortimine-A.
【００１１】上記のフォーチミン−Ａの生成の条件は、
槽１を１００リットル、攪拌翼は１段式６枚タービン羽
根とし、攪拌翼の長さを２８０ｍｍ、高さを６０ｍｍ、
攪拌翼の長さ／槽径＝０．５１とし、運転液量は８０リ
ットルで運転量当りの通気量は０．７ｖｖｍとした。The conditions for the production of Fortimine-A are:
The tank 1 is 100 liters, the stirring blade is a single-stage 6-blade turbine blade, and the stirring blade has a length of 280 mm and a height of 60 mm.
The length of the stirring blade / tank diameter = 0.51, the operating liquid amount was 80 liters, and the aeration amount per operating amount was 0.7 vvm.
【００１２】そして、本発明方法の実施においては培養
開始とともに菌体濃度は上昇し、それにともなって粘度
も上昇した。特に粘度は培養開始の１ｃｐから培養開始
後１０時間で６００ｃｐと大巾に増加したので、剪断応
力による微生物の破壊を防ぐために攪拌機４の回転数を
当初の６００ｒｐｍから２時間後に５００ｒｐｍ、６時
間後に４００ｒｐｍ、８時間後に２００ｒｐｍに変更し
た。In the practice of the method of the present invention, the bacterial cell concentration increased with the start of culture, and the viscosity also increased accordingly. In particular, the viscosity greatly increased from 1 cp at the start of the culture to 600 cp at 10 hours after the start of the culture. Therefore, in order to prevent the destruction of microorganisms due to shear stress, the rotation speed of the stirrer 4 was changed from the initial 600 rpm to 500 rpm after 2 hours and after 6 hours It was changed to 400 rpm and 200 rpm after 8 hours.
【００１３】培養結果は本発明方法による場合は培養開
始後１０時間で菌体濃度が最大値の７．４×１０7cells
/mlとなり、成生物であるフォーチミン−Ａの培養液中
での濃度は３５４mg/lであった。一方、従来法による場
合は菌体濃度の最大値は６．０×１０7cells/mlで、フ
ォーチミン−Ａの培養液中での濃度は２９０mg/lで収率
は本発明方法の８２％でしかなかった。According to the method of the present invention, the culturing result shows that the cell concentration reached a maximum value of 7.4 × 10 7 cells 10 hours after the start of culturing.
/ ml, and the concentration of adult fortimin-A in the culture solution was 354 mg / l. On the other hand, according to the conventional method, the maximum cell concentration was 6.0 × 10 7 cells / ml, the concentration of Fortimin-A in the culture solution was 290 mg / l, and the yield was 82% of that of the method of the present invention. It was only.
【００１４】図３〜図５はそれぞれ酵母、大腸菌及び枯
草菌に対する剪断応力と破壊率との関係についての実験
結果を示すグラフである。これらのグラフから微生物の
剪断応力に対する耐性値は個々の微生物毎に異なり、酵
母は１．３ｋＮ／ｍ2、大腸菌は２．２ｋＮ／ｍ2、枯草
菌は３．０ｋＮ／ｍ2が剪断応力耐性値であることが判
明した。FIGS. 3 to 5 are graphs showing the results of experiments on the relationship between shear stress and destruction rate for yeast, Escherichia coli and Bacillus subtilis, respectively. Resistance values for the shear stress of microorganisms from these graphs differ for each individual microorganism, yeast 1.3kN / m 2, E. coli 2.2kN / m 2, Bacillus subtilis 3.0 kN / m 2 is the shear stress resistance It turned out to be a value.
【００１５】また、微生物の培養においては少なくとも
剪断力は破壊率が５０％以下となるものでなければなら
ない。このことを考慮すれば、酵母については攪拌によ
って加えられる剪断応力が８，０００Ｎ／ｍ2を超えな
いように、大腸菌については攪拌によって加えられる剪
断応力が９，０００Ｎ／ｍ2を超えないように、また枯
草菌については攪拌によって加えられる剪断応力が１
０，０００Ｎ／ｍ2を超えないように攪拌機の回転数を
制御すべきである。Further, in culturing microorganisms, at least the shearing force must have a breaking rate of 50% or less. Taking this into consideration, for yeast, the shear stress applied by stirring should not exceed 8,000 N / m 2 , and for E. coli, the shear stress applied by stirring should not exceed 9,000 N / m 2. For Bacillus subtilis, the shear stress applied by stirring is 1
The rotation speed of the stirrer should be controlled so as not to exceed 10,000 N / m 2 .
【００１６】図６は第２発明に係る培養方法の実施に用
いる培養装置の概略図であり、この培養装置にあっては
前記第１発明ではレオメータ８を用いて微生物懸濁液の
粘度を測定し、この粘度から剪断応力を算出し、剪断応
力と破壊率との関係から攪拌機の回転数を制御するよう
にしていたが、この第２発明にあっては酵素等を組込ん
だ検出器１０で直接微生物の破壊量を測定し、この測定
値を制御装置９に入力してモータ３を制御するようにし
ている。このようにした場合、第１発明の方法よりも対
応に遅れが多少生じるが正確な制御が可能になる。FIG. 6 is a schematic diagram of a culturing apparatus used for carrying out the culturing method according to the second invention. In this culturing apparatus, the viscosity of the microbial suspension is measured using the rheometer 8 in the first invention. Then, the shear stress was calculated from this viscosity, and the rotation speed of the stirrer was controlled from the relationship between the shear stress and the fracture rate. However, in the second invention, the detector 10 incorporating an enzyme or the like is used. The amount of destruction of the microorganisms is directly measured with and the measured value is input to the control device 9 to control the motor 3. In such a case, accurate control can be performed although a slight delay occurs in correspondence with the method of the first aspect of the invention.
【００１７】[0017]
【発明の効果】以上に説明した如く本発明によれば、培
養槽内に設けられる攪拌機の回転数を、微生物に対する
剪断応力が所定値を超えないようにしたので、微生物の
増殖を効果的に行なうことができ、特に当該剪断応力を
微生物懸濁液の粘度から算出するようにすれば、制御遅
れもなく、微生物増殖の自動運転が可能となる。As described above, according to the present invention, the rotation speed of the agitator provided in the culture tank is set so that the shear stress against microorganisms does not exceed a predetermined value. If the shear stress is calculated from the viscosity of the microbial suspension, the microbial growth can be automatically operated without control delay.
【図１】本願の第１発明に係る培養方法の実施に用いる
培養装置の概略図FIG. 1 is a schematic view of a culture device used for carrying out a culture method according to a first invention of the present application.
【図２】本発明方法と従来法によって菌株を培養した場
合の培養時間と菌体濃度、微生物懸濁液の粘度、攪拌機
の回転数、生成物濃度との関係を示すグラフFIG. 2 is a graph showing the relationship between the culture time, the bacterial cell concentration, the viscosity of the microbial suspension, the rotation speed of the stirrer, and the product concentration when the strains are cultured by the method of the present invention and the conventional method.
【図３】酵母に対する剪断応力と破壊率との関係を示す
グラフFIG. 3 is a graph showing the relationship between shear stress and fracture rate for yeast.
【図４】大腸菌に対する剪断応力と破壊率との関係を示
すグラフFIG. 4 is a graph showing the relationship between shear stress and fracture rate for E. coli.
【図５】枯草菌に対する剪断応力と破壊率との関係を示
すグラフFIG. 5 is a graph showing the relationship between shear stress and fracture rate against Bacillus subtilis.
【図６】本願の第２発明に係る培養方法の実施に用いる
培養装置の概略図FIG. 6 is a schematic view of a culture device used for carrying out the culture method according to the second invention of the present application.
１…槽、２…微生物懸濁液、３…モータ、４…攪拌機、
８…レオメータ、９…制御装置。1 ... Tank, 2 ... Microbial suspension, 3 ... Motor, 4 ... Stirrer,
8 ... Rheometer, 9 ... Control device.
フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 (Ｃ１２Ｎ 1/00 Ｃ１２Ｒ 1:125） Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI technical display location (C12N 1/00 C12R 1: 125)
Claims (5)
て攪拌しつつ微生物を培養する方法において、前記微生
物懸濁液に加えられる剪断応力を微生物懸濁液の粘度等
の流動特性から算出し、この剪断応力が所定値を超えな
いように攪拌機の回転数を制御するようにしたことを特
徴とする微生物の培養方法。1. A method for culturing a microorganism while stirring the microorganism suspension in a culture tank with a stirrer, wherein the shear stress applied to the microorganism suspension is calculated from the flow characteristics such as viscosity of the microorganism suspension. A method for culturing a microorganism, characterized in that the rotational speed of the stirrer is controlled so that the shear stress does not exceed a predetermined value.
加えられる剪断応力は８，０００Ｎ／ｍ2を超えないよ
うに攪拌機の回転数を制御するようにした請求項１に記
載の微生物の培養方法。2. The method for culturing a microorganism according to claim 1, wherein the microorganism is yeast, and the rotation speed of the agitator is controlled so that the shear stress applied by stirring does not exceed 8,000 N / m 2. .
て加えられる剪断応力は９，０００Ｎ／ｍ2を超えない
ように攪拌機の回転数を制御するようにした請求項１に
記載の微生物の培養方法。3. The method for culturing a microorganism according to claim 1, wherein the microorganism is Escherichia coli, and the rotation speed of the agitator is controlled so that the shear stress applied by the agitation does not exceed 9,000 N / m 2. .
て加えられる剪断応力は１０，０００Ｎ／ｍ2を超えな
いように攪拌機の回転数を制御するようにした請求項１
に記載の微生物の培養方法。4. The rotation speed of the stirrer is controlled so that the microorganism is Bacillus subtilis and the shear stress applied by stirring does not exceed 10,000 N / m 2.
The method for culturing a microorganism according to 1.
て攪拌しつつ微生物を培養する方法において、前記微生
物の剪断破壊量を測定し、この剪断破壊量が所定値を超
えないように攪拌機の回転数を制御するようにしたこと
を特徴とする微生物の培養方法。5. A method for culturing a microorganism while stirring a microbial suspension in a culture tank with a stirrer, wherein the shear fracture amount of the microorganism is measured, and the shear fracture amount of the stirrer is controlled so as not to exceed a predetermined value. A method for culturing a microorganism, characterized in that the number of rotations is controlled.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3214598A JPH0530962A (en) | 1991-07-31 | 1991-07-31 | Method for culturing microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3214598A JPH0530962A (en) | 1991-07-31 | 1991-07-31 | Method for culturing microorganism |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0530962A true JPH0530962A (en) | 1993-02-09 |
Family
ID=16658372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3214598A Withdrawn JPH0530962A (en) | 1991-07-31 | 1991-07-31 | Method for culturing microorganism |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0530962A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000055295A1 (en) * | 1999-03-12 | 2000-09-21 | Shinko Pantec Co., Ltd. | Agitation tank for storing yeast solution, method of producing fermented foods such as beer using the agitation tank, and agitating vanes provided in the agitation tank |
| JP2014124139A (en) * | 2012-12-26 | 2014-07-07 | Hitachi Ltd | Culture control method, cell culture apparatus, and apparatus for evaluation of cellular characteristics |
| CN112265150A (en) * | 2020-10-21 | 2021-01-26 | 湖南机电职业技术学院 | Mixing drum rotating speed self-adaptive control device, method, equipment and storage medium |
| FR3112147A1 (en) * | 2020-07-02 | 2022-01-07 | Universite De Paris | Method of calibrating a fluidic system for the production of extracellular vesicles and associated production fluidic system |
-
1991
- 1991-07-31 JP JP3214598A patent/JPH0530962A/en not_active Withdrawn
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000055295A1 (en) * | 1999-03-12 | 2000-09-21 | Shinko Pantec Co., Ltd. | Agitation tank for storing yeast solution, method of producing fermented foods such as beer using the agitation tank, and agitating vanes provided in the agitation tank |
| US7368283B1 (en) | 1999-03-12 | 2008-05-06 | Kobelco Eco-Solutions Co. Ltd. | Agitation tank for storing beer yeast slurry |
| JP2014124139A (en) * | 2012-12-26 | 2014-07-07 | Hitachi Ltd | Culture control method, cell culture apparatus, and apparatus for evaluation of cellular characteristics |
| EP2749635A3 (en) * | 2012-12-26 | 2016-07-27 | Hitachi, Ltd. | Culture control method, cell culture apparatus, and apparatus for evaluation of cellular characteristics |
| US10011812B2 (en) | 2012-12-26 | 2018-07-03 | Hitachi, Ltd. | Culture control method, cell culture apparatus, and apparatus for evaluation of cellular characteristics |
| FR3112147A1 (en) * | 2020-07-02 | 2022-01-07 | Universite De Paris | Method of calibrating a fluidic system for the production of extracellular vesicles and associated production fluidic system |
| CN112265150A (en) * | 2020-10-21 | 2021-01-26 | 湖南机电职业技术学院 | Mixing drum rotating speed self-adaptive control device, method, equipment and storage medium |
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