JPH02286071A - Method and device for culture - Google Patents
Method and device for cultureInfo
- Publication number
- JPH02286071A JPH02286071A JP11035489A JP11035489A JPH02286071A JP H02286071 A JPH02286071 A JP H02286071A JP 11035489 A JP11035489 A JP 11035489A JP 11035489 A JP11035489 A JP 11035489A JP H02286071 A JPH02286071 A JP H02286071A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- cylindrical container
- tank
- medium
- main
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title description 7
- 238000012258 culturing Methods 0.000 claims abstract description 14
- 238000003756 stirring Methods 0.000 claims description 29
- 238000012136 culture method Methods 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 abstract description 6
- 238000011109 contamination Methods 0.000 abstract description 5
- 238000011081 inoculation Methods 0.000 abstract description 5
- 238000012546 transfer Methods 0.000 abstract description 3
- 238000011218 seed culture Methods 0.000 description 14
- 239000002609 medium Substances 0.000 description 13
- 239000001963 growth medium Substances 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 8
- 230000008878 coupling Effects 0.000 description 8
- 238000010168 coupling process Methods 0.000 description 8
- 238000005859 coupling reaction Methods 0.000 description 8
- 244000005700 microbiome Species 0.000 description 6
- 239000000498 cooling water Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000010025 steaming Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000005273 aeration Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/34—Internal compartments or partitions
-
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/58—Reaction vessels connected in series or in parallel
-
- 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
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Clinical Laboratory Science (AREA)
- Molecular Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
この発明は、微生物工業の培養工程における前培養から
主培養への移行を容易に行うことができる新規かつ改良
された培養方法、およびこの方法を実施するための培養
装置に関するものである。The present invention relates to a new and improved culture method that allows easy transition from preculture to main culture in the culture process of microorganism industry, and a culture device for carrying out this method.
一般に微生物を培養するに際しては、微生物濃度が低い
段階から大きな培￥を槽で培養すると時間がかかるだけ
でなく、場合によっては予定していた培養生産物が生成
されないこともある。
そのため典型的な微生物工業の培養工程においては、振
とうフラスコおよび種菌培養槽（種菌発酵槽）を用いて
二段にわたる前培養を行い、主培養槽（主発酵槽）を用
いる主培養の開始に必要な接種菌体量を増殖させること
か行われている。前培養から主培養へは通常約５〜６０
倍の規模で容積が拡大されていき、主培養の容積が大き
くなれば、それに応じて前培養の過程を多段階にしなけ
ればならないから、前培養槽の容積も大きくなる。
従って、従来の一般的な培養工程における主要な装置は
、種菌培養槽と主培養槽とからなっている。種菌培養槽
は主培養槽にて微生物を培養するに必要な初期菌体量を
培養することを目的とし、目的生産物は主培養槽で取得
することになる。これら各培養槽にはそれぞれ、培地の
調製、蒸煮殺菌、冷却、通気用空気の圧縮、除菌、撹拌
、消泡などのための装置や設備が付属している。
かような従来の培養装置によって微生物を培養するに際
しては、先ず使用菌株は、試験管による斜面培養から振
とうフラスコ培養へと拡大され、この菌体は次いで種菌
培養槽へ接種されて前培養される。種菌培養槽で前培養
された菌体は更に主培養槽へ接種され、この主培養槽で
目的とする培養生産物を生成させる。
種菌培養槽は、菌体の接種に先立って、加圧蒸気による
予備殺菌を行ったのち培地を仕込み、この培地を蒸煮殺
菌しておく。また主培養槽も、菌体の接種に先立って予
備殺菌および無菌培地の仕込みを行っておく必要がある
。Generally, when culturing microorganisms, culturing a large culture in a tank from a stage where the microorganism concentration is low not only takes time, but in some cases, the expected culture product may not be produced. Therefore, in a typical culture process in the microbial industry, a shake flask and a seed culture tank (seed fermenter) are used for two stages of pre-culture, and a main culture tank (main fermenter) is used to start the main culture. The necessary amount of inoculated bacteria is grown. From pre-culture to main culture usually about 5-60
If the volume is doubled and the volume of the main culture becomes larger, the preculture process must be performed in multiple stages accordingly, so the volume of the preculture tank also becomes larger. Therefore, the main equipment in a conventional general culture process consists of a seed culture tank and a main culture tank. The purpose of the seed culture tank is to cultivate the initial amount of microorganisms necessary for culturing microorganisms in the main culture tank, and the target product is obtained in the main culture tank. Each of these culture tanks is attached with devices and equipment for preparing the culture medium, steaming and sterilizing, cooling, compressing air for ventilation, sterilization, stirring, defoaming, and the like. When culturing microorganisms using such conventional culture equipment, the bacterial strain used is first expanded from a slant culture in a test tube to a shake flask culture, and then the bacterial cells are inoculated into a seed culture tank and precultured. Ru. The bacterial cells precultured in the seed culture tank are further inoculated into the main culture tank, and the desired culture product is produced in this main culture tank. Prior to inoculation of bacterial cells, the seed culture tank is pre-sterilized using pressurized steam, then a culture medium is charged therein, and this culture medium is sterilized by steaming. The main culture tank also needs to be pre-sterilized and filled with a sterile medium before inoculation of bacterial cells.
上記したような培養規模の拡大に伴い、振とうフラスコ
から種菌培養槽へ、さらに種菌培養槽から主培養槽へと
菌体を含む培養液を移し変えて順次接種しなくてはなら
ないが、かような接種作業は通常人手によるか、または
ポンプや配管等によって行われている。
しかしながら、培養液の移送接種時に、雑菌が混入する
いわゆるコンタミネーションが起こりやすいという問題
がある。
さらに、種菌培養槽や主培養槽は、菌体の接種に先立っ
て、それぞれ個々に予備殺菌や無菌培地の仕込み等を行
わなければならず、培養開始までの準備操作が煩雑とな
る。
そこでこの発明の目的は、より大容量の培養槽に菌体を
移送接種する際に生じやすいコンタミネーションの問題
のない新規かつ改良された培養方法を提供することであ
る。
この発明のもう１つの目的は、種菌培養に用いる前培養
槽や、より大容量の主培養槽に予備殺菌や無菌培地の仕
込み等の準備操作を６槽ごとに別個に行う煩雑さを低減
できる新規かつ改良された培養方法を提供することであ
る。
この発明のもう１つの目的は、上記の方法を実施するた
めに使用する培養装置を提供することである。With the expansion of the culture scale as described above, it is necessary to transfer the culture solution containing the bacteria from the shake flask to the seed culture tank, and then from the seed culture tank to the main culture tank and inoculate them one after another. Such inoculation work is usually carried out manually or by means of pumps, piping, etc. However, there is a problem in that so-called contamination, in which various bacteria are mixed in, is likely to occur when the culture solution is transferred and inoculated. Furthermore, the seed culture tank and the main culture tank must be individually pre-sterilized and filled with a sterile culture medium prior to inoculation of bacterial cells, making the preparation operations until the start of culture complicated. Therefore, an object of the present invention is to provide a new and improved culture method that does not cause the problem of contamination that tends to occur when transferring and inoculating bacterial cells into a larger capacity culture tank. Another object of the present invention is to reduce the complexity of performing pre-culture tanks used for seed culture and preparatory operations such as pre-sterilization and charging of sterile culture medium into the larger-capacity main culture tank separately for every six tanks. An object of the present invention is to provide a new and improved culture method. Another object of the invention is to provide a culture device for use in carrying out the above method.
すなわちこの発明の培養方法は、竪型円筒容器からなる
培養槽の下部のみに培地を仕込み前培養し、次いで該培
養槽に無菌培地をさらに追加供給して主培養することに
よって、前培養から主培養にいたるまでを同一の培養槽
内で行うことを特徴とするものである。
さらにこの発明の培養装置は、主培養用竪型円筒容器の
下部に該円筒容器の径より小径でかつ内部が該円筒容器
と連続する前培養用円筒容器を連設するとともに、各円
筒容器に内部撹拌手段および／または無菌空気供給手段
をそれぞれ配設したことを特徴とするものである。
またこの発明の培養装置は、必要に応じて前培養用円筒
容器を複数個設けることもできる。
この場合には、主培養用竪型円筒容器の下部に下方へい
くに従い該円筒容器の径より順次段階的に小径となりか
つ内部が該円筒容器と連続する複数の前培養用円筒容器
を多段に連設するとともに、各円筒容器に内部撹拌手段
および／または無菌空気供給手段をそれぞれ配設する。That is, in the culture method of the present invention, a culture medium is charged only to the lower part of a culture tank consisting of a vertical cylindrical container for pre-culture, and then a sterile medium is further supplied to the culture tank for main culture. It is characterized in that all steps up to culturing are carried out in the same culture tank. Furthermore, in the culture apparatus of the present invention, a pre-culture cylindrical container having a diameter smaller than that of the cylindrical container and whose inside is continuous with the cylindrical container is connected to the lower part of the vertical cylindrical container for main culture, and each cylindrical container is It is characterized in that an internal stirring means and/or a sterile air supply means are respectively provided. Further, the culture apparatus of the present invention may be provided with a plurality of cylindrical containers for pre-culture, if necessary. In this case, a plurality of cylindrical containers for pre-culture are arranged in multiple stages at the bottom of a vertical cylindrical container for main culture, the diameter of which is gradually smaller than the diameter of the cylindrical container as it goes downward, and whose inside is continuous with the cylindrical container. In addition, each cylindrical container is provided with an internal stirring means and/or a sterile air supply means.
上記のごときこの発明によれば、主培養用竪型円筒容器
の下部に連接した小径の円筒容器内で先ず比較的小容量
の種菌培養等の前培養を行ったのち、無菌培地をさらに
追加供給して主培養用円筒容器に至るまで満たし、引続
き主培養を行う。
主培養用円筒容器の下部に複数の小径円筒容器を多段に
連設した装置を用いる場合には、先ず最下部の最小径円
筒容器内で最初の種菌培養を行い、次いでその上の２番
目に小さい径の円筒容器内まで無菌培地を追加して引続
き種菌培養を行い、順次上方の円筒容器にいくに従って
培養液容量を拡大して、最終的に主培養用円筒容器まで
無菌培地を満たして主培養を行う。
かくして、培養液を前培養槽から主培養槽へ移し変える
ことなく、前培養から主培養に至るまでを同一の培養槽
内で行うことができる。According to the invention as described above, a relatively small volume of preculture such as seed culture is first performed in a small diameter cylindrical container connected to the lower part of the vertical cylindrical container for main culture, and then a sterile medium is further supplied. Fill up to the cylindrical container for main culture and continue with main culture. When using an apparatus in which multiple small-diameter cylindrical containers are arranged in a row at the bottom of a main culture cylindrical container, the initial inoculum culture is first carried out in the smallest diameter cylindrical container at the bottom, and then in the second cylindrical container above it. Continue culturing the seed culture by adding sterile medium to the inside of the small diameter cylindrical container, and gradually expand the culture solution volume as you go to the upper cylindrical container, and finally fill the sterile medium up to the main culture cylindrical container. Perform culture. In this way, everything from preculture to main culture can be carried out in the same culture tank without transferring the culture solution from the preculture tank to the main culture tank.
第１図は比較的大容量の培養に適したこの発明による培
養装置を示し、竪型円筒容器】１からなる主培養槽１０
と、その下部に連設された比較的小径の円筒容器３１か
らなる種菌培養用の前培養ｔ！３０とからなる。
主培養槽１０の中心には撹拌軸１２が配設され、この撹
拌軸の上部は無菌シール１３を介して主培養槽頂壁から
伸長し、軸受装置１４およびギアボックス１５を介して
駆動モーター１６と連結されている。撹拌軸１２が駆動
モータ１６により回転されると、撹拌軸に取付けられた
撹拌羽根１７と消泡用羽根１８が回転する。
撹拌軸下端は撹拌軸支持アーム１９により支持されてい
る。主培養槽１０の側壁には無菌空気送入口２０が設け
られ、主培養槽底部のスパージャ−２１と連結されてい
る。冷却水コイル２２は支持アーム２３に支持されて主
培養槽１０内の底部から頂部へ向かって配設されている
。番号２４および２５はそれぞれ主項Ｌ％ Ｗｊ頂壁に
設けられた培地仕込口およびガス排出口であり、番号２
６は主培養槽内壁から突設させた邪魔板である。かよう
な主培養槽の構造は、従来から慣用されている主培養槽
の構造と実質的に同じである。
この発明の特徴は、主培養槽１０の下部に、竪型円筒容
器１１の径より小径でかつ内部がこの円筒容器と連続す
る第２の円筒容器３１を連設し、これを前培養槽３０と
して使用する点である。主培養槽１０と同様に前培養槽
３０も、外部の駆動モーター３２により回転する撹拌軸
３３と撹拌羽根３４、無菌空気送入口３５とスパージャ
−３６、および邪魔板３７等が配設される。番号３８お
よび３９は、それぞれ熱伝対挿入口およびｐＨ電極／Ｄ
、０．電極挿入口であり、番号４０は培養液排出口であ
る。
上記したごとき構造を有する培養装置の操作手順は以下
の通りである。先ずこの培養装置の内部全体の予備殺菌
が加圧蒸気を用いて行われる。具体的には、例えばスパ
ージャ−２１３６から加圧蒸気を噴射したり、冷却水コ
イル２２に加圧蒸気を流すことにより行うことができる
。次いで、主培養槽１０頂部の培地仕込口２４から培地
を導入して、前培養ｔ４１３０のみに所定レベルまで培
地を仕込み、スパージャ−３６から加圧蒸気を噴射して
培地を蒸煮殺菌する。あるいは外部の殺菌装置で予め殺
菌した培地を仕込んでもよい。前培養槽３０に仕込んだ
培地を、スパージャ−３６から無菌空気を通気すること
により培養温度まで冷却した後、振とうフラスコ培養し
た菌体をこの培地に接種して培養し、菌体を増殖させる
。この前培養に際しては、前培養槽３０用の内部撹拌手
段３３゜３４および無菌空気供給手段３５．３６が使用
される。
所定時間前培養した後、外部の殺菌装置で殺菌し培養温
度まで冷却した培地を主培養槽１０頂部の培地仕込口２
４から追加供給して、主培養槽１０内の所定レベルまで
培地を満たし、この状態で通常の主培養を行う。主培養
中は、常法通り温度やｐＨを設定値に制御し、好気培養
の場合には通気撹拌する。主培養に際しては、主培養用
の内部撹拌手段１２．１７や無菌空気供給手段２０．２
１と共に、前培養用の内部撹拌手段３３．３４や無菌空
気供給手段３５３６も使用する。
目的とする培養生産物が所定濃度に達した時点で主培養
を終了する。培養終了後、生産物を含む培養液は、培養
装置最下部の培養液排出口４０から取出され、生産物回
収工程へと移される。その後この培養装置は、次の培養
開始のために洗浄、殺菌が行われる。
第２図は、比較的小容量の培養に適したこの発明の培養
装置の別な実施例を示すものであり、第１図の装置と同
じ部材には同じ番号を付すことにより説明を省略する。
第１図の装置と異なる構造は、前培養槽３０における撹
拌手段としてマグネットカップリング４１．４２を採用
した点である。すなわち、駆動モーター４４により回転
する駆動マグネットカップリング４１を前培養槽底部外
側に配置し、一方、前培養槽底部内側には従動マグネッ
トカップリング４２を配置し、この従動マグネットカッ
プリング４２から上方に撹拌軸３３を伸長させて撹拌羽
根３４を取り付ける。かような構造によって、駆動マグ
ネットカップリング４１を駆動モーター４４で回転させ
れば従動マグネットカップリング４２もそれに追従して
回転し、撹拌軸３３および撹拌羽根３４を回転させて、
前培養槽３゜内の培養液の撹拌がなされる。
駆動モーター４４と駆動マグネットカップリング４１と
は支持アーム４５を介して支持軸４６に対し回転可能に
支持されるようにしたから、駆動モーター４４および駆
動マグネットカップリング４１の位置を必要に応じて回
動させることができる。また培養液排出口４ｏは前培養
槽３０の最下部に設ける必要がら、マグネットカップリ
ング４１．４２は図示したように傾斜して取り付けてい
る。なお、スパージャ−や冷却水コイル等の培養に必要
なその他の部材は、図面を簡単にするため省略しである
。
第３図は、比較的小容量の培養に適したこの発明の培養
装置の更に別な実施例を示すものであり、第２図の装置
における前培養槽撹拌手段のマグネットカップリングに
代えて、マグネチック・スターラー５１．５２を採用し
たものである。前培養槽底部外側に配置した磁石５１を
回転させることによって、前培養槽底部内側に配置した
回転子５２を回転させ、これによって前培養槽３０内の
培養液の撹拌がなされる。なお第３図においても、スパ
ージャ−や冷却水コイル等の培養に必要なその他の部材
は、図面を簡単にするため省略しである。
上述した各実施例においては、主培養槽の下部に小径の
前培養槽を１段のみ連接し、振とうフラスコ培養した菌
体をこの前培養槽に接種して菌体を増殖させる、いわゆ
る種菌培養槽として使用した例を示したが、この発明は
かような実施例にのみ限定されるものではない。例えば
、種菌培養槽の下部に更に小径の円筒容器を多段に連接
し、振とうフラスコに相当する段階を含む更に小容量の
前培養段階から単一の培養槽で培養を行うようにしても
よい。
また内部撹拌手段は、撹拌羽根等の機械的手段を用いる
例を示したが、かような機械的手段を使用せずに、培養
槽底部に無菌空気を供給してスパージャ−から培地内に
放散させることによって撹拌を行うようにしてもよい。FIG. 1 shows a culture apparatus according to the present invention suitable for relatively large-capacity culture.
A preculture t! for inoculum culture consists of a cylindrical container 31 with a relatively small diameter and connected to the lower part of the preculture t! It consists of 30. A stirring shaft 12 is disposed at the center of the main culture tank 10 , and the upper part of the stirring shaft extends from the top wall of the main culture tank via a sterile seal 13 and a drive motor 16 via a bearing device 14 and a gearbox 15 . is connected to. When the stirring shaft 12 is rotated by the drive motor 16, the stirring blade 17 and defoaming blade 18 attached to the stirring shaft are rotated. The lower end of the stirring shaft is supported by a stirring shaft support arm 19. A sterile air inlet 20 is provided on the side wall of the main culture tank 10 and is connected to a sparger 21 at the bottom of the main culture tank. The cooling water coil 22 is supported by a support arm 23 and is disposed inside the main culture tank 10 from the bottom to the top. Numbers 24 and 25 are the culture medium inlet and gas outlet provided on the top wall of the main term L% Wj, respectively;
6 is a baffle plate protruding from the inner wall of the main culture tank. The structure of such a main culture tank is substantially the same as that of a conventional main culture tank. A feature of the present invention is that a second cylindrical container 31 having a diameter smaller than that of the vertical cylindrical container 11 and whose inside is continuous with this cylindrical container is provided in a row below the main culture tank 10, and this is connected to the pre-culture tank 30. The point is to use it as a. Similar to the main culture tank 10, the pre-culture tank 30 is also provided with a stirring shaft 33 and stirring blades 34 rotated by an external drive motor 32, a sterile air inlet 35, a sparger 36, a baffle plate 37, and the like. Numbers 38 and 39 are the thermocouple insertion port and pH electrode/D, respectively.
,0. It is an electrode insertion port, and number 40 is a culture solution discharge port. The operating procedure for the culture device having the above structure is as follows. First, the entire interior of this culture device is pre-sterilized using pressurized steam. Specifically, this can be done, for example, by injecting pressurized steam from the sparger 2136 or by flowing pressurized steam through the cooling water coil 22. Next, a medium is introduced from the medium inlet 24 at the top of the main culture tank 10, and the medium is charged only to the preculture t4130 to a predetermined level, and pressurized steam is injected from the sparger 36 to sterilize the medium by steaming. Alternatively, a culture medium previously sterilized using an external sterilizer may be used. After cooling the medium charged in the pre-culture tank 30 to the culture temperature by blowing sterile air through the sparger 36, the microbial cells cultured in the shake flask are inoculated into this medium and cultured to multiply the microbial cells. . During this preculture, internal stirring means 33, 34 and sterile air supply means 35, 36 for the preculture tank 30 are used. After pre-cultivation for a predetermined period of time, the medium is sterilized using an external sterilizer and cooled to the culture temperature.
4 is additionally supplied to fill the main culture tank 10 with the medium to a predetermined level, and normal main culture is performed in this state. During the main culture, the temperature and pH are controlled to set values as usual, and in the case of aerobic culture, aeration and stirring are performed. During main culture, internal stirring means 12.17 and sterile air supply means 20.2 for main culture are used.
1, internal stirring means 33, 34 for pre-culture and sterile air supply means 3536 are also used. The main culture is terminated when the desired culture product reaches a predetermined concentration. After the cultivation is completed, the culture solution containing the product is taken out from the culture solution outlet 40 at the bottom of the culture device and transferred to a product recovery step. Thereafter, this culture device is cleaned and sterilized in order to start the next culture. FIG. 2 shows another embodiment of the culture device of the present invention suitable for relatively small-volume culture, and the same members as those in the device in FIG. 1 are given the same numbers and their explanation will be omitted. . The structure differs from the apparatus shown in FIG. 1 in that magnetic couplings 41 and 42 are employed as stirring means in the preculture tank 30. That is, a driving magnetic coupling 41 rotated by a driving motor 44 is arranged on the outside of the bottom of the pre-culture tank, and a driven magnetic coupling 42 is arranged on the inside of the bottom of the pre-culture tank. The stirring shaft 33 is extended and the stirring blades 34 are attached. With such a structure, when the driving magnetic coupling 41 is rotated by the driving motor 44, the driven magnetic coupling 42 also follows and rotates, thereby rotating the stirring shaft 33 and the stirring blade 34.
The culture solution in the preculture tank 3° is stirred. Since the drive motor 44 and the drive magnetic coupling 41 are rotatably supported on the support shaft 46 via the support arm 45, the positions of the drive motor 44 and the drive magnetic coupling 41 can be rotated as necessary. can be moved. Further, since the culture solution outlet 4o needs to be provided at the lowest part of the pre-culture tank 30, the magnetic couplings 41 and 42 are attached at an angle as shown. Note that other members necessary for culturing, such as a sparger and a cooling water coil, are omitted to simplify the drawing. FIG. 3 shows yet another embodiment of the culture apparatus of the present invention, which is suitable for relatively small-volume culture. A magnetic stirrer 51.52 is used. By rotating the magnet 51 placed outside the bottom of the pre-culture tank, the rotor 52 placed inside the bottom of the pre-culture tank is rotated, thereby stirring the culture solution in the pre-culture tank 30. Also in FIG. 3, other members necessary for culturing, such as a sparger and a cooling water coil, are omitted to simplify the drawing. In each of the above-mentioned examples, only one small-diameter preculture tank is connected to the lower part of the main culture tank, and bacterial cells cultured in a shake flask are inoculated into this preculture tank to multiply the cells, so-called seed culture. Although an example of use as a culture tank has been shown, the present invention is not limited to such an example. For example, smaller diameter cylindrical containers may be connected in multiple stages at the bottom of the seed culture tank, so that culture can be carried out in a single culture tank starting from an even smaller volume pre-culture stage including a stage corresponding to a shake flask. . In addition, as for the internal stirring means, an example using mechanical means such as stirring blades was shown, but instead of using such mechanical means, sterile air is supplied to the bottom of the culture tank and diffused into the culture medium from a sparger. Stirring may be performed by
以上説明したようにこの発明によれば、同一の培養槽内
で前培養から主培養に至るまでを行うようにしたから、
培養液を前培養槽から主培養槽へ移送接種する必要がな
くなり、従って移送接種に際して生じやすいコンタミネ
ーションの問題を低減することができる。
さらに、従来のように前培養槽や主培養槽として別個の
槽を用いる場合には、使用に先立ってそれぞれ個々の槽
ごとに予備殺菌等の準備操作を行わなければならなかっ
たが、内部が連続した単一の円筒容器を用いるこの発明
においては、培養開始までの準備操作を極めて簡略化す
ることができる。As explained above, according to the present invention, everything from pre-culture to main culture is carried out in the same culture tank.
There is no need to transfer and inoculate the culture solution from the pre-culture tank to the main culture tank, and therefore it is possible to reduce the problem of contamination that tends to occur when transferring and inoculating. Furthermore, when using separate tanks as the pre-culture tank and main culture tank as in the past, preparatory operations such as preliminary sterilization had to be performed for each tank before use, but the internal In the present invention, which uses a single continuous cylindrical container, preparatory operations up to the start of culture can be extremely simplified.
第１図はこの発明の培養装置の実施例を示す説明図；第
２図はこの発明の培養装置の別な実施例を示す説明図；
第３図はこの発明の培養装置のさらに別な実施例を示す
説明図である。
１０・・・主培養槽、１１・・・竪型円筒容器、１２３
３・・・撹拌軸、１７．３４・・・撹拌羽根、２０゜５
・・
無菌空気送入口、
２１゜
６・・・スパージ
ャ−
Ｏ・・・前培養槽、
・・・第２の円筒容器。FIG. 1 is an explanatory diagram showing an embodiment of the culture device of the present invention; FIG. 2 is an explanatory diagram showing another embodiment of the culture device of the present invention;
FIG. 3 is an explanatory diagram showing still another embodiment of the culture apparatus of the present invention. 10... Main culture tank, 11... Vertical cylindrical container, 123
3... Stirring shaft, 17.34... Stirring blade, 20°5
... Sterile air inlet, 21°6... Sparger O... Preculture tank, ... Second cylindrical container.
Claims (1)
込み前培養し、次いで該培養槽に無菌培地をさらに追加
供給して主培養することによって、前培養から主培養に
いたるまでを同一の培養槽内で行うことを特徴とする培
養方法。 ２、竪型円筒容器の下部に該円筒容器の径より小径でか
つ内部が該円筒容器と連続する前培養用円筒容器を連設
するとともに、各円筒容器に内部撹拌手段および／また
は無菌空気供給手段をそれぞれ配設したことを特徴とす
る培養装置。 ３、竪型円筒容器の下部に下方へいくに従い該円筒容器
の径より順次段階的に小径となりかつ内部が該円筒容器
と連続する複数の前培養用円筒容器を多段に連設すると
ともに、各円筒容器に内部撹拌手段および／または無菌
空気供給手段をそれぞれ配設したことを特徴とする培養
装置。[Scope of Claims] 1. Pre-culture by charging a medium only to the lower part of a culture tank consisting of a vertical cylindrical container, and then additionally supplying a sterile medium to the culture tank and carrying out main culture. A culture method characterized by carrying out everything up to culturing in the same culture tank. 2. At the bottom of the vertical cylindrical container, a preculture cylindrical container having a diameter smaller than that of the cylindrical container and whose inside is continuous with the cylindrical container is installed in series, and each cylindrical container is provided with internal stirring means and/or sterile air supply. A culture device characterized in that means are respectively provided. 3. At the bottom of the vertical cylindrical container, a plurality of preculture cylindrical containers are arranged in a row in stages, each having a diameter that gradually becomes smaller than the diameter of the cylindrical container as it goes downward, and whose interior is continuous with the cylindrical container. A culture apparatus characterized in that a cylindrical container is provided with an internal stirring means and/or a sterile air supply means.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11035489A JPH02286071A (en) | 1989-04-28 | 1989-04-28 | Method and device for culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11035489A JPH02286071A (en) | 1989-04-28 | 1989-04-28 | Method and device for culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02286071A true JPH02286071A (en) | 1990-11-26 |
| JPH0435154B2 JPH0435154B2 (en) | 1992-06-10 |
Family
ID=14533643
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11035489A Granted JPH02286071A (en) | 1989-04-28 | 1989-04-28 | Method and device for culture |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02286071A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016073937A (en) * | 2014-10-07 | 2016-05-12 | 三菱化学株式会社 | Multitubular separation membrane module and liquid treatment method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5247977A (en) * | 1975-10-14 | 1977-04-16 | Res Inst For Prod Dev | Semi-batch process of fermentation wherein feed rate of substrate or i ts solution is increased exponentially with time |
-
1989
- 1989-04-28 JP JP11035489A patent/JPH02286071A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5247977A (en) * | 1975-10-14 | 1977-04-16 | Res Inst For Prod Dev | Semi-batch process of fermentation wherein feed rate of substrate or i ts solution is increased exponentially with time |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016073937A (en) * | 2014-10-07 | 2016-05-12 | 三菱化学株式会社 | Multitubular separation membrane module and liquid treatment method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0435154B2 (en) | 1992-06-10 |
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