WO2017017830A1 - Bioréacteur utilisant des micro/nano-bulles enrichies en oxygène et procédé de bioréaction utilisant le bioréacteur utilisant des micro/nano-bulles enrichies en oxygène - Google Patents

Bioréacteur utilisant des micro/nano-bulles enrichies en oxygène et procédé de bioréaction utilisant le bioréacteur utilisant des micro/nano-bulles enrichies en oxygène Download PDF

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Publication number
WO2017017830A1
WO2017017830A1 PCT/JP2015/071597 JP2015071597W WO2017017830A1 WO 2017017830 A1 WO2017017830 A1 WO 2017017830A1 JP 2015071597 W JP2015071597 W JP 2015071597W WO 2017017830 A1 WO2017017830 A1 WO 2017017830A1
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Prior art keywords
biological
culture
micro
culture solution
mnb
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Ceased
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PCT/JP2015/071597
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English (en)
Japanese (ja)
Inventor
正守 樋口
信秀 国友
小林　祐一
和矩 熊田
伸宏 田中
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Mitsubishi Chemical Engineering Corp
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Mitsubishi Chemical Engineering Corp
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Priority to PCT/JP2015/071597 priority Critical patent/WO2017017830A1/fr
Priority to PCT/JP2016/059728 priority patent/WO2017017998A1/fr
Priority to JP2016518206A priority patent/JP6087476B1/ja
Publication of WO2017017830A1 publication Critical patent/WO2017017830A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes

Definitions

  • diaphragm pumps, tube pumps, screw pumps with relatively little stress and damage to microorganisms, etc. as a pump for circulating the biological culture solution to the outside of the culture vessel as the amount of biological culture solution extracted from the culture vessel decreases. It has also been found that a positive displacement pump such as a rotary pump can be suitably used, and this can further reduce stress and damage to microorganisms.
  • the bioreactor of the present invention and the bioreaction method using the bioreactor include reaction products produced by microorganisms such as brewing, fermentation, etc. for the production of foods, chemicals, chemicals, etc., and the production of bioethanol using biomass. It is useful not only for the production of but also for the growth of microorganisms and the like.
  • the biological reaction of the present invention is to cause a reaction product to be produced by a microorganism or the like in a culture solution containing a microorganism or the like contained in a culture tank, and to cause the microorganism to grow.
  • a culture solution containing a saccharide and a nitrogen source is used.
  • saccharides saccharides such as maltose, sucrose, glucose, fructose, and mixtures thereof are usually used.
  • concentration of saccharides in the culture solution is not particularly limited, but is set to 0.1 to 10 w / v%. preferable.
  • nitrogen source ammonium chloride, ammonium sulfate, corn steep liquor, yeast extract, meat extract, peptone or the like is used, and it is preferably set to 0.1 to 10 w / v%.
  • vitamins, inorganic salts, and the like is added to the culture solution as needed in addition to the saccharides and the nitrogen source.
  • microorganism in the present invention examples include aerobic and facultative anaerobic microorganisms such as koji molds such as Aspergillus, Bacillus natto, acetic acid bacteria, yeasts, and lactic acid bacteria conventionally used in technical fields such as brewing and fermentation.
  • aerobic and facultative anaerobic microorganisms created by gene recombination technology can be used.
  • the cells include animal cells for producing physiologically active peptides or proteins used as antibody drugs, particularly genetically modified animal cells.
  • the concentration of the microorganism or cell added to the culture solution is not particularly limited, but is preferably 0.5 to 10 g / L, and more preferably 3.0 to 6.0 g / L.
  • the first feature of the present invention is that the biological culture liquid extracted from the culture tank contains oxygen-enriched MNB, and the biological culture liquid containing the oxygen-enriched MNB is added to the culture tank. To reflux.
  • the amount of the biological culture solution extracted from the culture tank it is possible to reduce stress and damage to microorganisms and the like, and to reduce the energy required for circulation of the biological culture solution. Furthermore, by reducing the amount of MNB contained in the culture solution, the energy required for driving the MNB generator can be reduced.
  • the second feature of the present invention is that, as described above, by using oxygen-enriched MNB, the amount of the biological culture solution extracted from the culture vessel can be reduced, and the biological culture solution is circulated outside the culture vessel.
  • a positive displacement pump such as a diaphragm pump, a tube pump, a screw pump, a rotary pump, or the like that causes relatively little stress and damage to microorganisms or the like is preferably used. The use of such a positive displacement pump can further reduce stress and damage to microorganisms.
  • Nano bubbles which are very small bubbles, are also called “ultra fine bubbles”.
  • ISO International Organization for Standardization
  • the creation of an international standard for fine bubble technology is being considered, and once the international standard is created, the name of “nanobubble”, which is currently commonly used, There is a possibility that it will be unified into “Ultra Fine Bubble”.
  • micro / nano bubble generating device As the micro / nano bubble generating device, a known or commercially available device can be used. For example, after a sufficient amount of gas is dissolved in water at a certain high pressure, the dissolved gas is released by releasing the pressure. “Pressure-dissolving microbubble generator” that creates supersaturation conditions, utilizing the phenomenon that bubbles are broken apart when large bubbles are entrained in the vortex by causing a water flow and large vortices are engulfed in the vortex The “gas-liquid two-phase flow swirl type microbubble generator” or the like can be used.
  • the oxygen concentration of the oxygen-enriched MNB is preferably 25 to 40%, more preferably 35 to 40%.
  • the oxygen concentration is 25% or more, the respiratory action of microorganisms and the like can be promoted, and the activity of microorganisms and the like can be increased.
  • the oxygen concentration is 40% or less, microorganisms and the like are not easily damaged by oxidation.
  • oxygen-enriched MNB is blown into the biological culture solution extracted from the culture tank containing microorganisms, so that the microorganisms are stress-damaged in the step of blowing oxygen-enriched MNB.
  • stress damage may be reduced in the filtration step.
  • the oxygen-enriched MNB is directly added to the biological culture solution extracted from the culture tank. Therefore, it is not necessary to increase the amount of the biological culture solution, and the operating cost of the apparatus is not increased, and the stress and damage to the microorganisms are not increased.
  • the biological reaction apparatus employing the method 1) will be described based on the first embodiment of the present invention shown in FIG. 1, and the biological reaction apparatus employing the method 2) will be illustrated in FIG. A description will be given based on the second embodiment of the invention.
  • the first embodiment is a biological reaction apparatus for causing a microorganism or the like to generate a reaction product, and includes oxygen-enriched MNB in a biological culture solution as follows.
  • the culture solution 1 is supplied to the culture tank 2.
  • the valve 12 is closed, the valve 13 and the valve 14 are opened, the culture tank pump 8 is driven, and the biological culture solution 3 containing the culture solution, microorganisms and the like is extracted from the culture vessel 2 and supplied to the filter 4.
  • the biological culture solution B that is, the biological culture solution in which microorganisms and the like are concentrated
  • the biological culture solution B that is, the biological culture solution in which microorganisms and the like are concentrated
  • the filter 4 includes a filtration membrane and a container that accommodates the filtration membrane.
  • the filtration membrane may be an organic membrane or an inorganic membrane.
  • the shape of the filtration membrane may be any shape such as a flat membrane, a hollow fiber membrane, and a spiral type. Among these, a hollow fiber membrane module is preferable. Any of the pressure type shapes can be employed.
  • a culture solution containing reaction products, microorganisms, and the like is filtered while being supplied to the inside of the hollow fiber membrane, and the filtrate is taken out from the outside. Microorganisms deposited inside the hollow fiber membrane And so on, so that a stable filtration state can be maintained over a long period of time.
  • the biological culture solution containing microorganisms and the like to be filtered contains oxygen-enriched MNB, even if it is flowed at a lower flow rate than usual, it is possible to scrape membrane dirt, Can significantly reduce the stress and damage to them.
  • the soot circulation flow rate is about 1 to 2 m / s when an organic membrane is used, and about 1 to 3 m / s when a ceramic membrane is used.
  • oxygen-enriched MNB oxygen-enriched MNB
  • the necessary circulation flow rate can be reduced to about 0.2 to 1.5 m / s.
  • the flux can be increased by about 1.2 to 2.0 times.
  • an organic polymer compound can be suitably used from the viewpoints of separation performance, water permeability, and dirt resistance.
  • examples include polyethylene resins, polypropylene resins, polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethersulfone resins, polyacrylonitrile resins, cellulose resins, and cellulose triacetate resins. A mixture of these resins as the main component may be used.
  • Polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethersulfone resins and polyacrylonitrile resins which are easy to form in solution and have excellent physical durability and chemical resistance, are preferred.
  • a vinylidene chloride resin or a resin containing the vinylidene fluoride resin as a main component is more preferably used because it has a characteristic of having both chemical strength (particularly chemical resistance) and physical strength.
  • the polyvinylidene fluoride-based resin a homopolymer of vinylidene fluoride is preferably used.
  • the polyvinylidene fluoride resin may be a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride.
  • vinyl monomers copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene, and ethylene trichloride fluoride.
  • the average pore diameter of the filtration membrane can be appropriately determined according to the purpose and situation of use, but it is preferably smaller to some extent, and is usually preferably 0.01 ⁇ m or more and 1 ⁇ m or less.
  • the average pore diameter of the hollow fiber membrane is less than 0.01 ⁇ m, components such as microorganisms, such as sugars and proteins, and membrane dirt components such as aggregates thereof block the pores, and stable operation cannot be performed.
  • it is preferably 0.02 ⁇ m or more, and more preferably 0.03 ⁇ m or more.
  • the average pore diameter approaches the size of a microorganism or the like, these may directly block the pores.
  • the average pore diameter is 0.4 ⁇ m. The following is preferable, and 0.2 ⁇ m or less is preferable.
  • the average pore diameter of the filtration membrane can be obtained by measuring and averaging the diameters of a plurality of pores observed by scanning electron microscope observation at a magnification of 10,000 times or more.
  • 10 or more, preferably 20 or more pores are randomly selected, the diameters of these pores are measured, and the number average is obtained.
  • an image processing device or the like it is also preferable to use an image processing device or the like to obtain a circle having an area equal to the area of the pores, that is, an equivalent circle, and obtain the equivalent circle diameter as the pore diameter. it can.
  • the filtrate A which is the liquid made to contain MNB in the MNB generator 7a is driven out of the MNB generation tank 6 by driving the liquid supply pump 10, and the MNB generator In addition to being supplied to 7a, air C having an increased air concentration obtained by the oxygen enrichment means is supplied to the MNB generator 7a.
  • the MNB generating apparatus 7a used in the first embodiment As the MNB generating apparatus 7a used in the first embodiment, as shown in FIG. 2, an apparatus that can generate a large amount of MNB economically and that is driven using a water flow (nozzle system) is used.
  • the filtrate A is supplied from the inlet 21 of the nozzle in a state where pressure is applied, and turbulence is generated in the throat 22 while reducing the diameter of the pipe and increasing the flow velocity.
  • air C with increased oxygen concentration is supplied from the gas inlet 24, mixed with the filtrate A in the suction portion 23, becomes MNB by the water flow, and contains MNB of air with increased oxygen concentration from the outlet portion 25.
  • the filtrate D to be discharged is discharged and supplied to the macro / nano bubble generating tank 6.
  • the amount and size of the MNB can be adjusted by adjusting the flow rate of the filtrate A and the air C with increased oxygen concentration supplied to the MNB generator 7a.
  • oxygen enrichment means using an oxygen enriched film as shown in FIG. 3 is used.
  • the container 31 in which the oxygen-enriched film 30 is disposed has an air introduction part 33 and a lead-out part for discharging air F having a low oxygen concentration at both ends.
  • the air pressurized by the intake fan 32 is vented from the air inlet 33 to the oxygen-enriched film 30, and the air C having an increased oxygen concentration is discharged from the outlet 35, and the oxygen concentration The low air F is discharged from the outlet 34.
  • the oxygen concentration of the gas constituting the MNB is made higher than the concentration in the air (about 21%), thereby reducing the amount of the biological culture solution extracted from the culture tank, and the biological culture solution contains Even if the amount of MNB to be reduced is decreased, the activity of the microorganisms and the like can be maintained by supplying the microorganisms with a high concentration of oxygen that is easily absorbed in the MNB state. Furthermore, by reducing the amount of the biological culture solution extracted from the culture tank, it is possible to reduce stress and damage to microorganisms and the like, and to reduce the energy required for circulation of the biological culture solution.
  • positive displacement pumps such as a diaphragm pump, a tube pump, a screw pump, and a rotary pump that have relatively little stress and damage to microorganisms and the like are preferably used. This also makes it possible to further reduce the stress and damage to microorganisms. Furthermore, by reducing the amount of MNB contained in the culture solution, the energy required for driving the MNB generator can be reduced.
  • the second embodiment is a biological reaction apparatus for causing a microorganism or the like to generate a reaction product, and contains the oxygen-enriched MNB in the biological culture as follows.
  • the culture solution 1 is supplied to the culture tank 2.
  • the valve 15 is closed, the valve 16 is opened, and the culture tank pump 8 is driven to extract the biological culture solution 3 containing microorganisms and the like from the culture tank 2 and supply it to the micro / nano bubble generation tank 6.
  • the biological culture solution 3 is stored in the micro / nano bubble generating tank 6 and oxygen enriched MNB is contained by the micro / nano bubble generating device 7a.
  • the return pump 9 is driven to return the biological culture solution G containing the oxygen-enriched MNB to the culture tank 2. e) In this way, the biological reaction is advanced while stirring the biological culture solution 3 in the culture tank 2 with the culture tank agitator 11. f) When the biological reaction has sufficiently progressed, the valve 16 is closed, the valve 15 is opened and the culture tank pump 8 is driven, and the reaction product generated in the culture tank 2 is collected together with the filtrate A to obtain the filtrate. Store in storage tank 5.
  • the method of 1) (first embodiment) and the method of 2) (second embodiment), which are methods for adding oxygen-enriched MNB to the biological culture solution extracted from the culture tank, are the types of microorganisms and the like. It is preferable to adopt a method in which the stress and damage to the microorganisms are generally reduced depending on the conditions of the biological reaction.
  • the biological culture solution extracted from the culture tank mentioned as the first feature point contains the oxygen-enriched MNB and is returned to the culture tank.
  • first means means (hereinafter referred to as “first means”) is used, other means may be used in combination.
  • the first means When the first means is used alone, it may take time to set the MNB content in the biological culture solution in the culture tank to an appropriate value.
  • the second means is preferable as a means used in combination with the first means because the microorganisms and the like are not subjected to stress or damage due to the blowing of MNB.
  • the third embodiment is a biological reaction apparatus for causing a microorganism or the like to generate a reaction product, and uses the second means in combination with the first embodiment (using the first means) of the present invention.
  • the oxygen-enriched MNB is contained in a culture solution such as a microorganism as follows. a) The culture solution 1 supplied to the culture tank 2 is caused to contain oxygen-enriched MNB (culture solution E containing oxygen-enriched MNB) by the micro / nano bubble generator 7b. b) In this way, the biological reaction is advanced while stirring the biological culture solution 3 in the culture tank 2 with the culture tank agitator 11.
  • the fourth embodiment is a biological reaction apparatus for causing a microorganism or the like to generate a reaction product, and the second means and the third means are used in combination with the first embodiment (using the first means) of the present invention. Is.
  • the oxygen-enriched MNB is contained in a culture solution such as a microorganism as follows. a) The culture solution 1 supplied to the culture tank 2 is caused to contain oxygen-enriched MNB (culture solution E containing oxygen-enriched MNB) by the micro / nano bubble generator 7b. b) In this way, the biological reaction is advanced while stirring the biological culture solution 3 in the culture tank 2 with the culture tank agitator 11.
  • the first embodiment (using the first means) of the present invention which is the combination of the second means, the second means, and the third means, respectively, has been described.
  • the second embodiment (using the first means) of the present invention can be used in combination with the second means, the second means, and the third means, respectively, and the same effects can be obtained. It can be easily understood by a contractor.
  • the oxygen concentration of the gas constituting the MNB is made higher than the concentration in air (about 21%).
  • a pump for circulating a biological culture solution such as a pump for extracting the biological culture solution from the culture vessel, a pump for refluxing the biological culture solution containing oxygen-enriched MNB to the culture vessel, It is possible to use positive displacement pumps such as diaphragm pumps, tube pumps, screw pumps, rotary pumps, etc. that cause relatively little stress and damage to the This can be further reduced.

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Abstract

Ce bioréacteur et le procédé de bioréaction qui utilise le bioréacteur aborde les problèmes visant à réduire le stress/l'endommagement subi par un micro-organisme ou similaire pendant une bioréaction et permettant de réaliser une bioréaction de manière efficace et économique qui utilise un micro-organisme ou similaire. Pour résoudre lesdits problèmes, ce bioréacteur et le procédé de bioréaction qui utilise le bioréacteur sont caractérisés en ce qu'un générateur de micro/nano-bulles est utilisé pour incorporer des micro/nano-bulles (MNB) enrichies en oxygène, qui sont formées à partir d'air présentant une concentration accrue en oxygène, dans une solution de culture biologique qui a été extraite d'un réservoir de culture et en ce que la solution de culture biologique dans laquelle les MNB enrichies en oxygène ont été incorporées est renvoyée vers le réservoir de culture. Soit 1) un procédé selon lequel la solution de culture biologique qui a été extraite du réservoir de culture est séparée au moyen d'un filtre en un filtrat et en une solution de culture biologique de laquelle le filtrat a été retiré et les MNB enrichies en oxygène sont incorporées dans le filtrat soit 2) un procédé selon lequel les MNB enrichies en oxygène sont directement incorporées dans la solution de culture biologique qui a été extraite du récipient de culture peut être utilisé comme procédé visant à incorporer les MNB enrichies en oxygène dans la solution de culture biologique qui a été extraite du récipient de culture.
PCT/JP2015/071597 2015-07-30 2015-07-30 Bioréacteur utilisant des micro/nano-bulles enrichies en oxygène et procédé de bioréaction utilisant le bioréacteur utilisant des micro/nano-bulles enrichies en oxygène Ceased WO2017017830A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/JP2015/071597 WO2017017830A1 (fr) 2015-07-30 2015-07-30 Bioréacteur utilisant des micro/nano-bulles enrichies en oxygène et procédé de bioréaction utilisant le bioréacteur utilisant des micro/nano-bulles enrichies en oxygène
PCT/JP2016/059728 WO2017017998A1 (fr) 2015-07-30 2016-03-25 Bioréacteur utilisant des micro/nano-bulles enrichies en oxygène et procédé de bioréaction utilisisant le bioréacteur utilisant des micro/nano-bulles enrichies en oxygène
JP2016518206A JP6087476B1 (ja) 2015-07-30 2016-03-25 酸素富化マイクロナノバブルを用いた生物反応装置及びこの生物反応装置を用いた生物反応方法

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PCT/JP2015/071597 WO2017017830A1 (fr) 2015-07-30 2015-07-30 Bioréacteur utilisant des micro/nano-bulles enrichies en oxygène et procédé de bioréaction utilisant le bioréacteur utilisant des micro/nano-bulles enrichies en oxygène

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PCT/JP2016/059728 Ceased WO2017017998A1 (fr) 2015-07-30 2016-03-25 Bioréacteur utilisant des micro/nano-bulles enrichies en oxygène et procédé de bioréaction utilisisant le bioréacteur utilisant des micro/nano-bulles enrichies en oxygène

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JP7651253B2 (ja) 2019-02-21 2025-03-26 三菱ケミカルエンジニアリング株式会社 窒素ガスを主成分とする気体のファインバブル・ウルトラファインバブルを培養液に含有させる機構を備えた嫌気性菌培養装置を用いた嫌気性菌培養方法

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DE102020103333B3 (de) * 2020-02-10 2021-05-06 Krinner Drucklufttechnik Gmbh Verfahren zum Vermehren von Hefezellen

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JP7651253B2 (ja) 2019-02-21 2025-03-26 三菱ケミカルエンジニアリング株式会社 窒素ガスを主成分とする気体のファインバブル・ウルトラファインバブルを培養液に含有させる機構を備えた嫌気性菌培養装置を用いた嫌気性菌培養方法

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