JPH0361439B2 - - Google Patents
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- Publication number
- JPH0361439B2 JPH0361439B2 JP58157526A JP15752683A JPH0361439B2 JP H0361439 B2 JPH0361439 B2 JP H0361439B2 JP 58157526 A JP58157526 A JP 58157526A JP 15752683 A JP15752683 A JP 15752683A JP H0361439 B2 JPH0361439 B2 JP H0361439B2
- Authority
- JP
- Japan
- Prior art keywords
- rate
- amount
- dissolved oxygen
- fed
- solution
- 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.)
- Expired - Lifetime
Links
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 29
- 239000001301 oxygen Substances 0.000 claims description 29
- 229910052760 oxygen Inorganic materials 0.000 claims description 29
- 235000015097 nutrients Nutrition 0.000 claims description 20
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 claims description 17
- 244000005700 microbiome Species 0.000 claims description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 238000012136 culture method Methods 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 229920002472 Starch Polymers 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 claims 1
- 230000007423 decrease Effects 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 235000014633 carbohydrates Nutrition 0.000 description 4
- AANLCWYVVNBGEE-IDIVVRGQSA-L Disodium inosinate Chemical compound [Na+].[Na+].O[C@@H]1[C@H](O)[C@@H](COP([O-])([O-])=O)O[C@H]1N1C(NC=NC2=O)=C2N=C1 AANLCWYVVNBGEE-IDIVVRGQSA-L 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000013890 disodium inosinate Nutrition 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- DVNYTAVYBRSTGK-UHFFFAOYSA-N 5-aminoimidazole-4-carboxamide Chemical compound NC(=O)C=1N=CNC=1N DVNYTAVYBRSTGK-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241001467578 Microbacterium Species 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 2
- 229950006790 adenosine phosphate Drugs 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- -1 carbohydrate compounds Chemical class 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- SGFRUQZYZBPQCJ-UHFFFAOYSA-N 1h-pyrazolo[3,4-c]pyridin-7-amine Chemical compound NC1=NC=CC2=C1NN=C2 SGFRUQZYZBPQCJ-UHFFFAOYSA-N 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229910000361 cobalt sulfate Inorganic materials 0.000 description 1
- 229940044175 cobalt sulfate Drugs 0.000 description 1
- KTVIXTQDYHMGHF-UHFFFAOYSA-L cobalt(2+) sulfate Chemical compound [Co+2].[O-]S([O-])(=O)=O KTVIXTQDYHMGHF-UHFFFAOYSA-L 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- DSKLYHDHQJANOE-UHFFFAOYSA-N pyrazolo[4,3-d]pyrimidine Chemical compound C1=NC=N[C]2C=NN=C21 DSKLYHDHQJANOE-UHFFFAOYSA-N 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 150000008223 ribosides Chemical class 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
Description
【発明の詳細な説明】
本発明は微生物の培養液を貯溜する発酵槽中へ
糖などの栄養物を培養時間の経過とともに少量ず
つ添加するようにした微生物の流加培養法に関す
る。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for fed-batch culture of microorganisms, in which nutrients such as sugar are added little by little over the course of culture time into a fermenter in which a culture solution of microorganisms is stored.
従来から発酵法によつて目的生成物を製造する
にあたり、流加培養法を用いて収率を向上せしめ
る手法が採られている。 BACKGROUND ART Conventionally, when producing a target product by a fermentation method, a method has been adopted in which a fed-batch culture method is used to improve the yield.
斯る流加培養法は添加する栄養物の濃度を任意
に制御できる点にその特徴を有するのであるが、
その制御にあたつて種々の問題を有している。 The characteristic of this fed-batch culture method is that the concentration of added nutrients can be controlled arbitrarily.
There are various problems in controlling this.
例えば、3′，5′−サイクリツク・アデニル酸を
工業的に製造する場合、培地中に出来るだけ高濃
度に3′，5′−サイクリツク・アデニル酸を蓄積す
ることが要求され、そのため培地中に糖質原料又
は前駆体を予じめ高濃度に添加しておくことが前
提条件となる。 For example, when producing 3',5'-cyclic adenylic acid industrially, it is required to accumulate 3',5'-cyclic adenylic acid in the medium as high as possible; A prerequisite is to add the carbohydrate raw material or precursor at a high concentration in advance.
一方、3′，5′−サイクリツク・アデニル酸の収
率を高めるには、3′，5′−サイクリツク・アデニ
ル酸を生産する菌体の増殖と糖濃度との関係も考
慮しなければならない。 On the other hand, in order to increase the yield of 3',5'-cyclic adenylic acid, it is also necessary to consider the relationship between the growth of bacterial cells that produce 3',5'-cyclic adenylic acid and the sugar concentration.
即ち、培地中の糖濃度が15％程度以上の高濃度
となると、菌体の生育に変調をきたし、しかも添
加前駆体に対する3′，5′−サイクリツク・アデニ
ル酸の生成率が低濃度の場合に比較して著しく劣
ることとなる。 In other words, if the sugar concentration in the medium becomes high (approximately 15% or more), the growth of bacterial cells will be affected, and if the production rate of 3',5'-cyclic adenylic acid with respect to the added precursor is low. This is significantly inferior to .
一方、3′，5′−サイクリツク・アデニル酸の培
地中への蓄積は菌体の増殖が平衡期に入る時期か
ら顕著に認められ、特に低糖濃度培地において
は、菌体の増殖が平衡期に入る時期が短縮され、
早い時期に3′，5′−サイクリツク・アデニル酸の
生産に移行し、且つ添加前駆体に対する3′，5′−
サイクリツク・アデニル酸の生成率も高まるので
あるが、培地中への最終蓄積量が低いという欠点
がある。 On the other hand, the accumulation of 3',5'-cyclic adenylic acid in the medium is noticeable from the time when bacterial growth enters the equilibrium phase, especially in low sugar concentration medium. The time to enter is shortened,
The production of 3',5'-cyclic adenylic acid occurred at an early stage, and the 3',5'-
Although the production rate of cyclic adenylic acid is increased, the disadvantage is that the final amount accumulated in the medium is low.
したがつて、例えば3′，5′−サイクリツク・ア
デニル酸を効率よく生産するには、糖濃度が高過
ぎても、低過ぎてもよくなく、常に適正な糖濃度
を維持する必要がある。 Therefore, in order to efficiently produce, for example, 3',5'-cyclic adenylic acid, the sugar concentration must not be too high or too low, and it is necessary to maintain an appropriate sugar concentration at all times.
また、上記3′，5′−サイクリツク・アデニル酸
の生成に限らず、流加培養法を利用して、微生物
により栄養物（基質）から目的物を工業的に満足
し得る程度に効率よく生成するには、培地中に添
加される栄養物の濃度を常に一定範囲に制御する
ことが必要とされ、この制御を如何に簡単且つ確
実に行うかが課題とされている。 In addition to the above-mentioned production of 3',5'-cyclic adenylic acid, the fed-batch culture method can also be used to efficiently produce the target product from nutrients (substrates) by microorganisms to an industrially satisfactory level. To do this, it is necessary to always control the concentration of nutrients added to the culture medium within a certain range, and the challenge is how to easily and reliably perform this control.
本発明は上述した課題を解決すべく成したもの
であつて、その目的とする処は、3′，5′−サイク
リツク・アデニル酸の生産能を有する微生物を培
養するに際し、培養液中の栄養物濃度を常に最適
範囲内に簡単且つ確実に制御し、もつて工業的に
も十分利用し得る収率を得ることができる微生物
の流加培養法を提供するにある。 The present invention has been made to solve the above-mentioned problems, and its purpose is to provide nutrients in the culture solution when culturing microorganisms capable of producing 3',5'-cyclic adenylic acid. It is an object of the present invention to provide a fed-batch culture method for microorganisms that can easily and reliably control the concentration of a substance within an optimum range at all times and obtain a yield that can be used industrially.
斯る目的を達成するため、本発明は培養液中の
溶存酸素量を測定して溶存酸素量の上昇変化率を
算出し、この上昇変化率を指標として、該上昇変
化率曲線の変曲点と上昇変化率が零となる点との
間において、栄養物を培養液に流加せしめるよう
にしたことをその要旨とする。 In order to achieve such an objective, the present invention measures the amount of dissolved oxygen in the culture solution, calculates the rate of increase in the amount of dissolved oxygen, and uses this rate of increase as an index to determine the inflection point of the rate of increase curve. The gist is that nutrients are added to the culture solution between the point and the point where the rate of increase becomes zero.
以下に本発明の実施例を添付図面に基づいて説
明する。 Embodiments of the present invention will be described below based on the accompanying drawings.
第１図は本発明方法を実施する装置の制御系統
の概略図であり、密閉発酵槽１内には微生物の培
養液２が満され、また発酵槽１内には撹拌羽根３
が配設され、また、コンプレツサー１９より散気
管２０を介して、該槽１内に無菌空気が送入さ
れ、一方槽１内の空気は排気管２１を通つて排出
される。また発酵槽１の上部には栄養液４を満し
た栄養液槽５から栄養液４を発酵槽１に供給する
パイプ６が臨んでいる。 FIG. 1 is a schematic diagram of the control system of an apparatus for carrying out the method of the present invention, in which a closed fermenter 1 is filled with a culture solution 2 of microorganisms, and a stirring blade 3 is provided in the fermenter 1.
is provided, and sterile air is fed into the tank 1 from the compressor 19 via the diffuser pipe 20, while the air in the tank 1 is discharged through the exhaust pipe 21. Further, a pipe 6 for supplying the nutrient solution 4 to the fermenter 1 from a nutrient solution tank 5 filled with the nutrient solution 4 faces the upper part of the fermenter 1.
一方、発酵槽１には培養液２中の溶存酸素量を
測定するためのセンサー７を装着し、このセンサ
７で測定した溶存酸素量を信号として取り出し、
この信号を増巾器８で増巾し、これを記録針９に
入力するとともに、アナログ・デジタル変換器１
０にてデジタル信号に変換して、マイクロコンピ
ユータ１１に入力する。 On the other hand, the fermenter 1 is equipped with a sensor 7 for measuring the amount of dissolved oxygen in the culture solution 2, and the amount of dissolved oxygen measured by this sensor 7 is taken out as a signal.
This signal is amplified by an amplifier 8 and inputted to a recording needle 9, and an analog-to-digital converter 1
0, it is converted into a digital signal and input to the microcomputer 11.
このマイクロコンピユータ１１には記憶装置１
２から、予じめ設定された測定間隔時間及び基準
となる溶存酸素量の上昇変化率が入力インターフ
エース１３を介して入力され、マイクロコンピユ
ータ１１にて実測値に基づく溶存酸素量の上昇変
化率と基準となる上昇変化率とを比較し、実測の
上昇変化率が基準の上昇変化率よりも小さくなつ
たときに、出力インターフエース１４を介して出
力信号を出し、この出力信号はタイマー１５を介
して出力スイツチ１６に送られ、上記パイプ６に
設けた栄養液の流加ポンプ１７を所定時間だけ駆
動し、一定量の栄養液４を発酵槽１に供給するよ
うにしている。尚１８はプリンターである。 This microcomputer 11 has a storage device 1.
2, the preset measurement interval time and the reference rate of increase in the amount of dissolved oxygen are input via the input interface 13, and the microcomputer 11 calculates the rate of increase in the amount of dissolved oxygen based on the actual measured value. and a reference rate of increase, and when the actually measured rate of increase is smaller than the reference rate of increase, an output signal is outputted via the output interface 14, and this output signal triggers the timer 15. The nutrient solution is sent to the output switch 16 via the nutrient solution, and the nutrient solution fed-batch pump 17 provided in the pipe 6 is driven for a predetermined period of time to supply a fixed amount of nutrient solution 4 to the fermenter 1. Note that 18 is a printer.
次に上記装置を用いて3′，5′−サイクリツク・
アデニル酸を生産する場合の具体例を第２図及び
第３図に基づいて説明する。 Next, using the above device, 3', 5'-cyclic
A specific example of producing adenylic acid will be explained based on FIGS. 2 and 3.
ここで、初発培地に存在させる糖質原料及び培
養途中に添加する糖質原料としては、グルコー
ス、澱粉加水分解物或いはグリセリン等の糖質化
合物を使用し、微生物としては例えばミクロバク
テリウムNo.205（FERM−ＰNo.106ATCC21376）
を使用し、また使用菌株のＣ−AMP前駆体とし
て例えば、アデニン、ハイポキサンチン、サクシ
ニル・アデニン、５−アミノ−４−イミダゾー
ル・カルボキサマイド、７−アミノ−ピラゾロ
（４・３−ｄ）−ピリジン、ピラゾロ−（４・３−
ｄ）−ピリミジン、４−アミノ−ピロロ（２・
３・ｄ）−ピリミジン、ピロロ（２・３−ｄ）−ピ
リミジン又はこれらを塩基とするリボサイド若し
くはデオキシリボサイド、又はリボヌクレオチド
を使用し、更に無機塩類としては、例えばリン酸
１カリウム、リン酸１ソーダ、リン酸２カリウ
ム、リン酸２ソーダ、硫酸マグネシウム、塩化マ
グネシウム、硫酸鉄、塩化鉄、塩化マンガン、硫
酸亜鉛、硫酸コバルト等を使用する。 Here, carbohydrate compounds such as glucose, starch hydrolyzate, or glycerin are used as carbohydrate raw materials present in the initial medium and carbohydrate raw materials added during cultivation, and examples of microorganisms include Microbacterium No. 205. (FERM-PNo.106ATCC21376)
and the C-AMP precursor of the strain used, for example, adenine, hypoxanthine, succinyl adenine, 5-amino-4-imidazole carboxamide, 7-amino-pyrazolo(4.3-d)- Pyridine, pyrazolo-(4,3-
d)-pyrimidine, 4-amino-pyrrolo(2.
3・d)-pyrimidine, pyrrolo(2・3-d)-pyrimidine, riboside or deoxyriboside based on these, or ribonucleotide, and further inorganic salts such as monopotassium phosphate, monopotassium phosphate, Soda, dipotassium phosphate, disodic phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese chloride, zinc sulfate, cobalt sulfate, etc. are used.
先ず、培養液中の糖濃度を５％（ｗ／ｖ）に調
整して発酵を開始した。この時の菌株接種前の培
地中の溶存酸素量を100％飽和とする。 First, the sugar concentration in the culture solution was adjusted to 5% (w/v) and fermentation was started. At this time, the amount of dissolved oxygen in the medium before inoculating the bacterial strain is set to 100% saturation.
この状態から発酵が進むと、微生物（菌株）は
糖を消費するとともに溶存酵素を消費して3′，
5′−サイクリツク・アデニル酸を生成しつつ増殖
する。そのため培養液中の糖濃度は低下し、一方
培養液中の酸素はその消費量の方が空気中から培
養液中に溶け込む量よりも多いため急激に減少す
る。 As fermentation progresses from this state, the microorganism (strain) consumes sugar and dissolved enzymes, resulting in 3′,
Proliferates while producing 5'-cyclic adenylate. Therefore, the sugar concentration in the culture solution decreases, and on the other hand, the amount of oxygen consumed in the culture solution is greater than the amount dissolved into the culture solution from the air, so the concentration of oxygen in the culture solution decreases rapidly.
そして、糖濃度が０％（ｗ／ｖ）近くまで減少
すると、栄養源が少なくなつたことになり、それ
だけ菌株による溶存酸素の消費が少なくなり、し
たがつて散気管２０からの酸素供給量が消費量よ
りも多くなり、溶存酸素の変化率を表わす曲線ｌ
は上昇する。 When the sugar concentration decreases to nearly 0% (w/v), this means that the nutrient source has decreased, and the consumption of dissolved oxygen by the bacterial strain decreases accordingly, so that the amount of oxygen supplied from the aeration pipe 20 decreases. The curve l representing the rate of change in dissolved oxygen is greater than the amount consumed.
will rise.
このようにして約23時間経過すると、培養開始
時添加した糖が消費しつくされるので、新たに糖
を発酵槽中に添加し、その濃度が２％（ｗ／ｖ）
程度となるようにする。すると、培養液中の菌株
は糖を消費して、溶存酸素量及び糖濃度は再び低
下し、糖濃度が０％（ｗ／ｖ）に近くなると消費
酸素量が少くなるので溶存酸素量は多くなり、そ
の変化率を表わす曲線ｌは再び上昇する。 After about 23 hours have passed in this way, the sugar added at the start of the culture has been consumed, so new sugar is added to the fermenter and its concentration is 2% (w/v).
The degree of Then, the bacterial strain in the culture solution consumes sugar, and the amount of dissolved oxygen and sugar concentration decrease again. When the sugar concentration approaches 0% (w/v), the amount of consumed oxygen decreases, so the amount of dissolved oxygen increases. The curve l representing the rate of change rises again.
このような糖添加の操作を前記第１図に示した
装置によつて行うわけであるが、糖の添加時期の
制御は第３図の如くして行う。即ち、第３図は上
記変化率曲線ｌの一部を取り出して示したもので
あり、糖濃度が０％（ｗ／ｖ）近くになつた時点
P0から変化率曲線ｌは上昇し、変曲点P1を経過
した後、曲線ｌの二次微分値は負となり、最終的
には点P2において変化率は零となる。つまり糖
濃度が零となり微生物が酸素を消費しないため、
培養液中の溶存酸素量は100％飽和となる。 Such sugar addition operation is performed using the apparatus shown in FIG. 1, and the timing of sugar addition is controlled as shown in FIG. 3. That is, FIG. 3 shows a part of the above rate of change curve l, and shows the point at which the sugar concentration approaches 0% (w/v).
The rate of change curve l rises from P0 , and after passing through the inflection point P1 , the second-order differential value of the curve l becomes negative, and finally the rate of change becomes zero at point P2 . In other words, since the sugar concentration is zero and microorganisms do not consume oxygen,
The amount of dissolved oxygen in the culture solution becomes 100% saturated.
ここで、糖を添加する時点P3を変曲点P1に経
過する以前に設定すると、微生物の糖消費能が減
退し、3′，5′−サイクリツク・アデニル酸の収率
が低下し、また変化率が零となる点P2以降に添
加点P3を設定すると、培養液中に糖が完全に無
くなつてから糖を添加することとなり、前記同
様、収率が低下する。そこで本実施例にあつて
は、添加時点P3を点P1とP2の間に設定した。 Here, if the time point P3 at which sugar is added is set before the inflection point P1 , the sugar consuming ability of microorganisms will decrease, and the yield of 3',5'-cyclic adenylic acid will decrease. Furthermore, if the addition point P 3 is set after the point P 2 where the rate of change is zero, the sugar will be added after the sugar has completely disappeared from the culture solution, and the yield will decrease as described above. Therefore, in this example, the addition time point P3 was set between points P1 and P2 .
具体的には、点P1とP2との間の最も適切と思
われる変化率を設定しておき、前記記憶装置１２
から入力インターフエース１３を介して該設定値
を予じめマイクロコンピユータ１１に入力してお
き、実際の変化率Ｄ・Ｏを、Ｄ・Ｏ＝Do+1−
Do／to+1−to＝ｂ／ａによつて算出し、このＤ・
Ｏが該設定値よりも小さくなつたことをマイクロ
コンピユータ１１によつて判断し、所定時間ポン
プ１７を駆動して培養液中の糖濃度が２％（ｗ／
ｖ）となるように糖を添加する。 Specifically, the most appropriate rate of change between points P 1 and P 2 is set, and the rate of change between points P 1 and P 2 is set, and
The set value is input into the microcomputer 11 in advance via the input interface 13, and the actual rate of change D.O. is calculated as D.O=D o+1 −
Calculated by D o /t o+1 −t o = b/a, and this D・
The microcomputer 11 determines that O has become smaller than the set value, and drives the pump 17 for a predetermined period of time until the sugar concentration in the culture solution is 2% (w/
v) Add sugar so that
斯る操作を連続することにより、3′，5′−サイ
クリツク・アデニル酸の生成収率を高水準に維持
することができる。尚、培養中にあつては、塩基
を加えて培養液のPHを約6.5〜7.0に保持する。 By continuing such operations, the production yield of 3',5'-cyclic adenylic acid can be maintained at a high level. During cultivation, a base is added to maintain the pH of the culture solution at about 6.5 to 7.0.
次に具体的な実験結果を以下に挙げる。 Next, specific experimental results are listed below.
実験例 １
イノシン酸ソーダ４％（ｗ／ｖ）、ポリペプト
ン１％（ｗ／ｖ）、KH2PO42％（ｗ／ｖ）、硫安
0.5％（ｗ／ｖ）、ビオチン100ｇ／、ZnSO4・
7H2O0.01％（ｗ／ｖ）、FeSO4・7H2O10mg／、
消泡剤0.1％（ｗ／ｖ）よりなる培地1.6分を1.4
の水に溶解し、これを３の発酵槽に投入して
殺菌した。一方これとは別に殺菌したグルコース
及びMgSO4・7H2Oからなる混合液を最終濃度と
してグルコース５％（ｗ／ｖ）、MgSO4・7H2O1
％（ｗ／ｖ）となるように200mlの水に溶解した
ものを添加し、ミクロバクテリウムNo.205
（FERM−ＰNo.106ATCC21376）の種培養液を接
種して、30℃、430RPM、通気量1.6／分の条
件で培養した。Experimental example 1 Sodium inosinate 4% (w/v), polypeptone 1% (w/v), KH 2 PO 4 2% (w/v), ammonium sulfate
0.5% (w/v), biotin 100g/, ZnSO 4 .
7H 2 O 0.01% (w/v), FeSO 4・7H 2 O 10 mg/,
Medium 1.6 min with antifoaming agent 0.1% (w/v) 1.4
The mixture was dissolved in water and put into the fermenter (No. 3) for sterilization. On the other hand, a mixture of glucose and MgSO 4 7H 2 O that had been sterilized separately was prepared with a final concentration of 5% glucose (w/v) and MgSO 4 7H 2 O1.
% (w/v) in 200 ml of water, Microbacterium No. 205
(FERM-P No. 106ATCC21376) was inoculated and cultured at 30° C., 430 RPM, and air flow rate 1.6/min.
そして、この培養にあたり、オリエンタル電気
株式会社製の酸素分析計の検出端を培地中に挿入
し、溶存酸素量が90％飽和となつた際に、コンピ
ユータから糖添加信号を送り、グルコース（45％
グルコースとして使用）が初発液量に対して1.0
％（ｗ／ｖ）添加されるように設定して培養を続
けた。尚、培地中にグルコースが存在している状
態での溶存酸素量は約20％飽和であつた。 During this culture, the detection end of an oxygen analyzer manufactured by Oriental Electric Co., Ltd. is inserted into the medium, and when the amount of dissolved oxygen reaches 90% saturation, a sugar addition signal is sent from the computer, and glucose (45%
(used as glucose) is 1.0 relative to the initial fluid volume.
% (w/v) was added, and culture was continued. The amount of dissolved oxygen in the presence of glucose in the medium was approximately 20% saturated.
そして、PHが約6.5〜7.0となるように自動的に
アンモニアガスでPHを調整しつつ培養を続けたと
ころ、105時間で初発液量に対して21％のグルコ
ースを消費した。 Then, when culturing was continued while automatically adjusting the pH with ammonia gas to about 6.5 to 7.0, 21% of the initial volume of glucose was consumed in 105 hours.
また培養終了後の3′，5′−サイクリツク・アデ
ニル酸量は22.5mg／mlで対イノシン酸ソーダ収率
は90％であつた。 The amount of 3',5'-cyclic adenylic acid after the completion of the culture was 22.5 mg/ml, and the yield based on sodium inosinate was 90%.
実験例 ２
培地としては実験例１と同様とし、発酵槽の温
度を30℃、撹拌器を530RPM、通気量を1.6／
分とし、溶存酸素濃度が95％飽和となつたとき、
コンピユータから糖添加信号を送り、グルコース
（45％グルコースとして使用）が初発液量に対し
て0.5％添加されるように設定して培養を続けた。
尚、培養液中にグルコースが存在している状態で
の溶存酸素量は約60％飽和であつた。Experimental Example 2 The culture medium was the same as Experimental Example 1, the temperature of the fermenter was 30℃, the stirrer was 530RPM, and the aeration rate was 1.6/
minutes, and when the dissolved oxygen concentration reaches 95% saturation,
A sugar addition signal was sent from the computer, and the culture was continued by setting glucose (used as 45% glucose) to be added at 0.5% to the initial solution volume.
The amount of dissolved oxygen in the presence of glucose in the culture solution was approximately 60% saturated.
そして実験例１と同様にPHを調整しつつ培養を
続けたところ、96時間で初発液量に対し21％のグ
ルコースを消費した。 Then, when culturing was continued while adjusting the pH in the same manner as in Experimental Example 1, 21% of the initial amount of glucose was consumed in 96 hours.
また培養終了後の3′，5′−サイクリツク・アデ
ニル酸は23.5mg／mlで、対イノシン酸ソーダ収率
は94％であつた。 Further, after the completion of the culture, 3',5'-cyclic adenylic acid was 23.5 mg/ml, and the yield based on sodium inosinate was 94%.
上記の実験例からも分かるように本発明方法に
よれば3′，5′−サイクリツク・アデニル酸を高収
率で得ることができる。 As can be seen from the above experimental examples, 3',5'-cyclic adenylic acid can be obtained in high yield according to the method of the present invention.
以上に説明したように、本発明によれば、コン
ピユータにより、培養液中の栄養分濃度が常に最
適範囲内となるように制御したため、高収率な流
加培養を行え、工業的な利用価値が極めて大であ
る等多くの効果を発揮する。 As explained above, according to the present invention, the nutrient concentration in the culture solution is controlled by a computer so that it is always within the optimal range, so high-yield fed-batch culture can be performed, and the industrial value is high. It has many effects such as being extremely large.
第１図は本発明に係る流加培養法を実施する装
置の制御系を示す概略図、第２図は溶存酸素量或
いは糖濃度と培養時間との関係を示すグラフ、第
３図は溶存酸素量の変化曲線を示すグラフであ
る。
尚、図面中１は密閉発酵槽、２は培養液、４は
栄養液、１１はマイクロコンピユータ、ｌは溶存
酸素量の変化率曲線、P1は変曲点、P2は溶存酸
素量の変化率が零となる点、P3は栄養液を添加
する時点である。
Fig. 1 is a schematic diagram showing the control system of the apparatus for implementing the fed-batch culture method according to the present invention, Fig. 2 is a graph showing the relationship between dissolved oxygen amount or sugar concentration and culture time, and Fig. 3 is a graph showing the relationship between dissolved oxygen amount or sugar concentration and culture time. It is a graph showing a change curve of quantity. In the drawing, 1 is a closed fermenter, 2 is a culture solution, 4 is a nutrient solution, 11 is a microcomputer, l is a rate of change curve of dissolved oxygen amount, P 1 is an inflection point, and P 2 is a change in dissolved oxygen amount. The point at which the rate becomes zero, P 3 , is the point at which the nutrient solution is added.
Claims (1)
を有する微生物を培養するに際し、培養液中の溶
存酸素量の上昇変化率（溶存酸素量の変化率／測
定間隔時間）を算出し、該溶存酸素の上昇変化率
曲線の変曲点付近を経過した後で、且つ該上昇変
化率が零となる以前に、該培養液に栄養液を添加
するようにしたことを特徴とする微生物の流加培
養法。 ２ 前記栄養液はグルコース、澱粉加水分解物及
びグリセリンのうちの少なくとも一種からなる糖
を含有する液であることを特徴とする特許請求の
範囲第１項に記載の微生物の流加培養法。 ３ 前記培養液中に添加された栄養液の濃度は２
％（ｗ／ｖ）以内であることを特徴とする特許請
求の範囲第１項に記載の微生物の流加培養法。 ４ 前記微生物は細菌であることを特徴とする特
許請求の範囲第１項に記載の微生物の流加培養
法。[Scope of Claims] 1. When culturing microorganisms capable of producing 3',5'-cyclic adenylic acid, the rate of increase in the amount of dissolved oxygen in the culture solution (rate of change in amount of dissolved oxygen/measurement interval time) ), and the nutrient solution was added to the culture solution after passing around the inflection point of the upward change rate curve of dissolved oxygen and before the upward change rate became zero. Characteristic fed-batch culture method of microorganisms. 2. The fed-batch culture method for microorganisms according to claim 1, wherein the nutrient solution is a solution containing sugar consisting of at least one of glucose, starch hydrolyzate, and glycerin. 3 The concentration of the nutrient solution added to the culture solution is 2
% (w/v) or less, the fed-batch culture method for microorganisms according to claim 1. 4. The microorganism fed-batch culture method according to claim 1, wherein the microorganism is a bacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15752683A JPS6049792A (en) | 1983-08-29 | 1983-08-29 | Feeding culture of microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15752683A JPS6049792A (en) | 1983-08-29 | 1983-08-29 | Feeding culture of microorganism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6049792A JPS6049792A (en) | 1985-03-19 |
| JPH0361439B2 true JPH0361439B2 (en) | 1991-09-19 |
Family
ID=15651591
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15752683A Granted JPS6049792A (en) | 1983-08-29 | 1983-08-29 | Feeding culture of microorganism |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6049792A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0755149B2 (en) * | 1988-05-20 | 1995-06-14 | 鐘淵化学工業株式会社 | Culture method for increasing lipase activity in cells |
| JP2528727Y2 (en) * | 1990-12-18 | 1997-03-12 | 未来工業株式会社 | Support devices for pipes, cables, etc. |
| EP2226380B1 (en) * | 2009-03-05 | 2014-12-17 | BioSilta Oy | Enzyme-based fed-batch technique in liquid cultures |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS4950989A (en) * | 1972-05-10 | 1974-05-17 |
-
1983
- 1983-08-29 JP JP15752683A patent/JPS6049792A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS4950989A (en) * | 1972-05-10 | 1974-05-17 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6049792A (en) | 1985-03-19 |
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