JP6499203B2 - Biological reaction device provided with device for supplying micro-nano bubbles of oxygen-containing gas, device for removing dissolved carbon dioxide, and biological reaction method using this biological reaction device - Google Patents

Description

  The present invention relates to a biological reaction apparatus for culturing microorganisms or cells (hereinafter referred to as “microorganisms”) and a biological reaction method using the biological reaction apparatus, and oxygen is contained in a culture solution containing microorganisms or the like at the time of biological reaction. In addition to supplying and containing gaseous micro / nano bubbles, the biological reaction is efficiently carried out by removing dissolved carbon dioxide from the culture solution containing microorganisms and the like.

  Biological reactions in which microorganisms and the like are cultured in a culture tank to produce chemicals such as organic acids and alcohols and useful chemical substances such as pharmaceuticals are performed.

  Unlike chemical reactions, biological reactions are slow, but they do not use a lot of energy or chemicals, so they are mild and meaningful for the environment. However, the biological reaction generally has a problem that the reaction is mild and slow. That is, for chemical reactions, a reaction within one hour is often sufficient, whereas in the case of biological reactions, a reaction time of several days or longer, several days or particularly several weeks or longer is required. There is also. For this reason, it is required to perform biological reactions efficiently and economically.

  As a technique for increasing the efficiency of biological reactions, Patent Document 1 discloses that cell growth is promoted by culturing cells in a culture solution containing microbubbles. In Patent Documents 2 to 5, in the cultivation of microorganisms, the presence of micro-nano bubbles or nano bubbles in the culture solution promotes the activation of microorganisms, improves the reaction efficiency of biological reactions, shortens the reaction time, etc. Is disclosed.

  Specifically, Patent Document 1 describes that microbubbles are contained in the filtrate extracted from the culture solution in the culture vessel and supplied to the culture vessel. Patent Document 2 describes that in a batch system, a culture solution is extracted from a culture tank, filtered through a bacterial cell filter to obtain a filtrate, and micronano bubbles are mixed with the filtrate and returned to the culture tank. ing. Patent Document 3 describes mixing micro-nano bubbles and nano bubbles in the culture solution before supplying the culture solution to the culture vessel. Patent Document 4 supplies the culture solution to the culture vessel. It is described that the micro / nano bubbles are mixed in the previous stage.

  In addition, in order to remove carbon dioxide dissolved in the culture solution in the culture tank in cell culture, Patent Document 5 supplies oxygen to the culture solution by the first air diffuser that generates bubbles having a relatively small diameter, It is described that a carbon dioxide-free gas is passed through a second air diffuser that generates relatively large-sized bubbles to diffuse dissolved carbon dioxide in the culture solution into the bubbles and remove it from the culture solution. In addition, in Patent Document 6, while supplying an oxygen-containing gas into the culture solution, an air mixed gas is passed through the gas exchange means 4 having carbon dioxide permeable membrane properties as shown in FIGS. Thus, it is described that dissolved carbon dioxide in the culture solution is removed from the culture solution.

  However, in the dissolved carbon dioxide removing means described in Patent Document 5, it is necessary to increase the aeration rate in order to efficiently remove the dissolved carbon dioxide with large-sized bubbles, which may cause cell damage. Increase.

  Moreover, the dissolved carbon dioxide removal means described in patent document 6 is a gaseous-phase part of the carbon dioxide permeable membrane 4 by the carbon dioxide dissolved in the culture solution M by a density | concentration difference, as FIG. 41, which is removed by being put on an air mixed gas vented in the gas phase portion 41, but the dissolved carbon dioxide is passed through the carbon dioxide permeable membrane by the weak force generated by the concentration difference of carbon dioxide. Therefore, it is difficult to sufficiently remove carbon dioxide dissolved in the culture solution.

JP 2011-120535 A Japanese Patent No. 4146476 Japanese Patent No. 4805120 Japanese Patent No. 4956552 JP 2008-182899 A JP 2014-018174 A

  The subject of the biological reaction device of the present invention and the biological reaction method using this biological reaction device is that a sufficient amount of oxygen necessary for the respiratory action of microorganisms and the like is supplied to the culture solution of microorganisms and the like. Bioreactor capable of promoting the respiration action of microorganisms by sufficiently removing the dissolved carbon dioxide produced by the respiration action of microorganisms, etc., and performing the biological reaction using microorganisms efficiently and economically And providing a biological reaction method using the biological reaction apparatus.

  In order to solve the above problems, the biological reaction apparatus of the present invention and the biological reaction method using the biological reaction apparatus remove dissolved carbon dioxide through a carbon dioxide separation membrane by vacuum decompression from the culture solution extracted from the culture tank, After the micro-nano bubble generator contains oxygen-containing gas micro-nano bubbles, the culture solution is refluxed to the culture tank.

  The “micro-nano bubbles” of the present invention means “micro bubbles” and / or “nano bubbles”. While “normal bubbles” rapidly rise in water and burst and disappear on the surface, microbubbles with a diameter of 50 μm or less called “microbubbles” shrink in water and disappear. Together with free radicals, “nanobubbles”, which are ultrafine bubbles having a diameter of 100 nm or less, are generated, and these “nanobubbles” remain in water for a certain amount of time. “Nano bubbles”, which are extremely fine bubbles, are also called “ultra fine bubbles”. Currently, in the 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”.

  The present invention contains micro-nano bubbles of oxygen-containing gas in a culture solution containing microorganisms by a micro-nano bubble generator, and supplies oxygen necessary for the respiration action of microorganisms, etc., and by a dissolved carbon dioxide removing device, Efficient and economical biological reaction using microorganisms by removing dissolved carbon dioxide from the culture solution containing microorganisms and reducing the concentration of dissolved carbon dioxide in the culture solution, which increases due to the respiratory action of microorganisms, etc. Can be done automatically.

It is a schematic diagram which shows 1st Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows 2nd Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows 3rd Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows 4th Embodiment of the biological reaction apparatus of this invention. It is FIG. 1 shown by patent document 6 (Unexamined-Japanese-Patent No. 2014-018174). It is FIG. 2A shown by patent document 6. FIG. It is FIG. 2B shown by patent document 6. FIG.

  First, general items of the biological reaction apparatus and biological reaction method of the present invention will be described.

  The biological reaction of the present invention is to cause a microorganism or the like to produce a reaction product using a culture solution as a nutrient source in a culture solution containing microorganisms or the like housed in a culture tank.

  As the culture solution in the present invention, a culture solution containing a saccharide and a nitrogen source is used. As saccharides, saccharides such as maltose, sucrose, glucose, fructose, and mixtures thereof are usually used, and the concentration of saccharides in the culture solution is not particularly limited, but is set to 0.1 to 10 w / v%. preferable. As the 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%. Furthermore, it is preferable to add vitamins, inorganic salts, and the like to the culture solution as needed in addition to the saccharides and the nitrogen source.

  Examples of the cell in the present invention include an animal cell for producing a physiologically active peptide or protein used as an antibody drug, particularly a genetically modified animal cell.

  In addition to microorganisms such as Aspergillus oryzae, natto, acetic acid, yeast, lactic acid, and other aerobic microorganisms that are conventionally used in technical fields such as brewing and fermentation, etc. Various aerobic microorganisms can be used. The reaction product generated in the culture tank is extracted from the culture tank together with a culture solution containing microorganisms and the like, separated into microorganisms and a filtrate by a filter, and the reaction product is recovered from the filtrate.

  The filter of the present invention comprises 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.

  As the filtration method of the present invention, cross flow filtration using a hollow fiber membrane module is preferred. In this filtration method, 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.

  When performing cross-flow filtration using a hollow fiber membrane module, it is necessary to flow the liquid to be filtered into the hollow fiber membrane at a flow rate of a certain level or more in order to scrape membrane dirt. However, in the present invention, since the culture solution containing microorganisms and the like to be filtered contains micro-nano bubbles of oxygen-containing gas, even if it is flowed at a lower flow rate than usual, it can scrape membrane dirt. Stress and damage to microorganisms can be greatly reduced.

  Specifically, in general cross-flow filtration, the circulation flow rate is about 1 to 2 m / s when using an organic membrane, and about 1 to 3 m / s when using a ceramic membrane. However, by containing micro-nano bubbles of oxygen-containing gas in the culture solution, the membrane flux is reduced and the filtration resistance can be kept small, so that the same flux (the amount of membrane filtered water per unit time and unit membrane area) can be obtained. Can be reduced to about 0.2 to 1.5 m / s. In addition, when operating at the same circulation flow rate, the flux can be increased by about 1.2 to 2.0 times.

  As the filtration membrane, an organic polymer compound can be preferably used from the viewpoints of separation performance and water permeation performance, and stain 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.

  Here, as the polyvinylidene fluoride resin, a homopolymer of vinylidene fluoride is preferably used. Furthermore, the polyvinylidene fluoride resin may be a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride. Examples of 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 preferable that it is small to some extent, and it is usually preferably 0.01 μm or more and 1 μm or less. When the average pore diameter of the hollow fiber membrane is less than 0.01 μm, 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. In consideration of the balance with water permeability, it is preferably 0.02 μm or more, and more preferably 0.03 μm or more. In addition, when it exceeds 1 μm, the film surface smoothness and the shearing force due to the flow of the film surface, and the peeling of dirt components from the pores by physical cleaning such as backwashing and air scrubbing are insufficient, and stable operation is possible. Disappear.

  Moreover, when an average pore diameter approaches the magnitude | size of microorganisms etc., these may block | close a pore directly. In addition, there may be cases where broken cells of microorganisms or cultured cells in the fermented liquid are killed to produce cell crushed materials. In order to avoid pore clogging by these crushed materials, the average pore diameter is 0.4 μm. The following is preferable, and 0.2 μm or less is preferable.

  Here, the average pore diameter of the filtration membrane can be determined 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. Preferably, 10 or more, preferably 20 or more pores are randomly selected, the diameters of these pores are measured, and the number average is obtained. When the pores are not circular, 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.

  In the bioreactor of the present invention and the bioreaction method using this bioreactor, the target product may be collected batchwise or continuously.

  In the case of performing the batch process, after the biological reaction in the culture tank is completed, the culture tank pump is driven to transfer the filtered filtrate to the filtrate storage tank.

  In the case of continuous operation, the filtered filtrate is balanced with the amount of the culture solution supplied to the culture vessel so that the water level of the culture solution containing microorganisms in the culture vessel is kept constant. Extract and transfer to the filtrate storage tank.

  The batch type or continuous type can be selected as appropriate in consideration of the efficiency of biological reaction, the required purity of the target product, the economic efficiency, and the like.

Next, the main features of the biological reaction apparatus of the present invention and the biological reaction method using this biological reaction apparatus are as described above, during the biological reaction,
O The micro-nano bubble generating device contains micro-nano bubbles of oxygen-containing gas in the culture solution containing microorganisms, and ○ The dissolved carbon dioxide removal device removes dissolved carbon dioxide from the culture solution containing microorganisms. Reducing the concentration of dissolved carbon dioxide in the culture medium, which increases due to the respiratory action of microorganisms,
These items will be described below.

<About adding micro-nano bubbles of oxygen-containing gas to a culture solution containing microorganisms or the like by a micro-nano bubble generator>
The first feature of the present invention is that the micro-nano bubble generator causes the culture solution containing microorganisms or the like to contain micro-nano bubbles of oxygen-containing gas, so that oxygen necessary for the respiratory action of the microorganisms is sufficiently contained in the culture solution. It is to promote the respiratory action by supplying to the body, and to perform biological reaction efficiently.

  By adding micro-nano bubbles of oxygen-containing gas to a culture solution containing microorganisms or the like during biological reaction, activation of microorganisms is promoted as described in Patent Documents 1 to 4, and It is possible to improve reaction efficiency and shorten reaction time.

  In the present invention, micro-nano bubbles of oxygen-containing gas are included in a culture solution containing microorganisms or the like during a biological reaction. From the viewpoint of efficiently supplying oxygen necessary for respiration to microorganisms or the like, oxygen of the oxygen-containing gas It is preferable to set the content rate high.

As a means for containing micro-nano bubbles in a culture solution or washing solution containing microorganisms,
a) means for releasing the micro / nano bubbles into the culture solution containing microorganisms in the culture tank by the micro / nano bubble generator provided outside the culture tank;
b) Means for containing micro / nano bubbles in the culture solution supplied to the culture tank by the micro / nano bubble generator provided in the conduit for supplying the culture solution to the culture tank,
c) Means for causing the filtrate collected from the culture solution containing microorganisms to contain micro / nano bubbles by a micro / nano bubble generator and refluxing the filtrate containing the micro / nano bubbles to the culture tank d) Pre-containing the micro / nano bubbles A means for supplying the culture solution to the culture tank through a conduit can be employed.

  These means can be adopted singly or in combination, but which means is selected as appropriate in consideration of resistance to shearing force of microorganisms used, efficiency of biological reaction, economy, etc. can do.

  The means with the least stress and damage to microorganisms are the means of b) and d). When using microorganisms that are extremely vulnerable to stress and damage, the means of b) or d) is used. preferable.

  In general, the means a) often gives stress or damage to microorganisms or the like due to the shearing force generated by the release of nanobubbles. Further, in the above c) means, stress and damage are given to microorganisms and the like due to the shearing force generated during filtration, but by selecting the filtration means, the stress and damage given to microorganisms and the like can be considerably reduced. .

  Further, as will be described later, if a technique of supporting microorganisms or the like with a porous structure is employed, such stress and damage can be greatly reduced.

  As the micro / nano bubble generating apparatus, a known or commercially available apparatus can be used.

  As a microbubble generator, for example, a sufficient amount of gas is dissolved in water at a certain high pressure, and then the pressure is released to create a supersaturated condition of the dissolved gas. "A gas-liquid two-phase flow swirl type micro-bubble generator that utilizes the phenomenon of generating a vortex by generating a water flow, entraining a large bubble in the vortex, and breaking the vortex into bubbles. Or the like.

  Examples of nanobubble generators include, for example, JP 2007-31690 A, JP 2006-289183 A, JP 2005-245817 A, JP 2007-136255 A, and JP 2009-39600 A. Those described can be used.

<About removing dissolved carbon dioxide from a culture solution containing microorganisms or the like with a dissolved carbon dioxide removal device to reduce the dissolved carbon dioxide concentration in the culture solution that increases due to the respiratory action of microorganisms or the like>
The second feature of the present invention is that the dissolved carbon dioxide is removed from the culture solution extracted from the culture tank and containing microorganisms through a carbon dioxide permeable membrane under reduced pressure, thereby causing the respiratory action of microorganisms and the like. It is to reduce the concentration of dissolved carbon dioxide in the culture solution that becomes high enough to promote the respiratory action, and to perform biological reactions efficiently.

  As described above, as a means for removing dissolved carbon dioxide from the cell culture solution, there is a means for supplying relatively large-sized bubbles of carbon dioxide-free gas into the culture solution as described in Patent Document 5. However, with this measure, increasing the aeration rate to sufficiently remove dissolved carbon dioxide increases the potential for cell damage. In addition, as described in Patent Document 6, there is a means for drawing in the gas phase part through a carbon dioxide permeable membrane membrane disposed in the culture solution using a difference in carbon dioxide concentration. Since dissolved carbon dioxide is drawn through the carbon dioxide permeable membrane by a small force generated by the concentration difference of carbon dioxide, it is difficult to sufficiently remove carbon dioxide dissolved in the culture solution.

  In contrast, in the present invention, a culture solution containing microorganisms and the like extracted from the culture tank is guided to a dissolved carbon dioxide removing device, and from this culture solution, a pressure difference caused by decompression is used as a driving force, and carbon dioxide permeability is obtained. The dissolved carbon dioxide is sufficiently removed through the membrane.

  In addition to carbon dioxide, oxygen, nitrogen, and the like are dissolved in the culture solution. The size of oxygen molecules and nitrogen molecules (3.64Å and 3.78 そ れ ぞ れ respectively) is the size of carbon dioxide molecules (4.6Å). ), The dissolved carbon dioxide removal device removes not only carbon dioxide dissolved in the culture solution but also oxygen, nitrogen, etc. In the present invention, first, The removal is sufficiently performed, and the dissolved oxygen removed together with this is supplemented with micro-nano bubbles of oxygen-containing gas generated by the micro-nano bubble generator.

Examples of the carbon dioxide permeable membrane of the dissolved carbon dioxide removal apparatus of the present invention include those known or commercially available, for example, polymer membranes such as polyimide membrane polymer membranes, zeolite membranes having nanometer order pores, silica An inorganic film such as a film or a carbon film can be used. In addition, an uncrosslinked vinyl alcohol-acrylate copolymer aqueous solution as disclosed in JP-B-7-102310 is applied in a film form on a carbon dioxide permeable support, and then heated. A carbon dioxide permeable membrane produced by cross-linking and water insolubilization, and gelling by absorbing an aqueous solution containing a carbon dioxide carrier (a substance having an affinity for carbon dioxide) into the water insolubilized product A gel layer obtained by adding an additive made of cesium carbonate, cesium bicarbonate or cesium hydroxide to a polyvinyl alcohol-polyacrylic acid copolymer gel film as disclosed in JP-A-195900 by supporting form a CO ₂ -facilitated transport membrane, the feed gas contains at least carbon dioxide and water vapor to a predetermined main component gas CO ₂ -facilitated transport membrane Charge supplied at 100 ° C. or more feed temperature on the side surface, the carbon dioxide permeable membrane such as to take out the carbon dioxide that has passed through the CO ₂ -facilitated transport membrane from the permeate side may also be used.

As the structure of the dissolved carbon dioxide removal device of the present invention, preferably,
1) A structure in which a culture solution containing microorganisms and the like extracted from a culture tank is flowed into a large number of carbon dioxide permeable hollow fiber membranes in parallel, and a reduced-pressure gas is caused to flow in a direction substantially orthogonal to these hollow fiber membranes,
2) A structure in which a reduced pressure gas is caused to flow in a large number of carbon dioxide permeable hollow fiber membranes in parallel, and a culture solution containing microorganisms and the like extracted from the culture tank is allowed to flow in a direction substantially orthogonal to these hollow fiber membranes.
3) A structure in which a culture solution containing microorganisms or the like extracted from a culture tank is allowed to flow in a large number of parallel carbon dioxide permeable hollow fiber membranes, and a reduced-pressure gas is caused to flow in a direction substantially parallel to these hollow fiber membranes; 4) A structure in which a reduced pressure gas is caused to flow through a large number of parallel carbon dioxide permeable hollow fiber membranes, and a culture solution containing microorganisms and the like extracted from the culture tank is allowed to flow in a direction substantially parallel to the hollow fiber membranes.
Can be adopted. When the dissolved carbon dioxide removing apparatus having the structure 1) is used, it is more preferable because the dissolved carbon dioxide can be removed efficiently. Various types of apparatuses having these structures are commercially available from Polypore Corporation under the trade name “Liqui-Cel”, for example.

  When using microorganisms that are resistant to the shear stress given by the dissolved carbon dioxide removal device and the micro / nano bubble generation device, the culture solution containing the microorganisms extracted from the culture tank may be supplied directly to these devices, When using microorganisms that are vulnerable to shear stress, supply the culture solution to the filter once, supply the filtrate separated by the filter to these devices, and then remove the filtrate separated by the filter. It is preferable to return the culture solution from which the microorganisms have been removed (that is, the culture solution in which microorganisms and the like are concentrated) to the culture tank through the pipeline.

  In order to further reduce the shear stress applied to microorganisms or the like, a porous structure (hereinafter referred to as “supporting structure”) in which microorganisms are supported in the pores can be used. A biological reaction apparatus and a biological reaction method using this carrying structure will be described below.

1) Support structure The support structure has a high support density in which microorganisms and the like are supported to the back of the pores, and the support structure contains a porous structure, microorganisms, and micro-nano bubbles. It can manufacture by making it contact with a culture solution.

  As the porous structure, it is preferable to use one having pores, particularly one having a three-dimensional solid network structure having continuous pores. If only a porous structure having such a structure is used, oxygen is not sufficiently supplied to the inside of the pores, so that microorganisms or the like can be supported only on the surface of the porous structure.

  On the other hand, by using a culture solution containing microorganisms and micro-nano bubbles as a culture solution to be brought into contact with the porous structure, oxygen can be supplied to the depths of the pores of the porous structure, and the microorganisms etc. to the depths of the pores. Can be carried, and the carrying density of microorganisms and the like can be increased.

  Further, when recovering a reaction product such as a metabolite such as a microorganism (hereinafter referred to as “target product”) obtained from a biological reaction from a culture tank, the microorganism is separated from the culture solution and the filtrate is recovered. Although it is necessary, if a carrying structure is used, separation of microorganisms and the like can be performed easily and economically.

  Next, a preferred example of the method for manufacturing the carrying structure will be described.

  As a method of supporting microorganisms or the like on the porous structure, a method of adding microorganisms or the like to the culture solution and culturing and growing the microorganisms and then bringing them into contact with the porous structure or supporting the microorganism in the culture solution And a porous structure, and a method of supporting microorganisms while culturing and growing them.

The former method is performed by the following procedure.
a) First, sterilize the porous structure, the culture solution, etc. so that microorganisms other than the necessary microorganisms, bacteria, etc. do not exist.
b) Next, microorganisms etc. are added to a culture solution, and microorganisms etc. are culture | cultivated and propagated, making a culture solution contain a micro nano bubble.
c) In a stage where microorganisms or the like have grown to a certain concentration or higher, the medium is brought into contact with the porous structure while containing the micro / nano bubbles in the culture solution.

The latter method is performed in the following procedure.
a) First, sterilize the porous structure, the culture solution, etc. so that microorganisms other than the necessary microorganisms, bacteria, etc. do not exist.
d) Next, the porous structure is added to the culture solution, and the culture solution contains micro-nano bubbles.
e) A microorganism or the like is added to the culture solution, and the microorganism or the like is cultured and grown while the culture solution contains micro-nano bubbles.

  The culture solution used in the above method mainly contains saccharides and a nitrogen source. As saccharides, saccharides such as maltose, sucrose, glucose, fructose, and mixtures thereof are usually used, and the concentration of saccharides in the culture solution is not particularly limited, but is set to 0.1 to 10 w / v%. preferable. As the 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%. Furthermore, it is preferable to add vitamins, inorganic salts, and the like to the culture solution as needed in addition to the saccharides and the nitrogen source.

  The concentration added to the culture solution of microorganisms and the like in step b) and step e) is not particularly limited, but is preferably 0.5 to 10 g / L, and is preferably 3.0 to 6.0 g / L. Is more preferable.

  In the above step b), micronano bubbles are contained in the treated water in order to promote the growth of microorganisms, etc., but when microorganisms and the like can be cultured and grown sufficiently quickly without containing micronano bubbles. The inclusion of micro-nano bubbles can be omitted.

  The contact between the culture solution containing the micro-nano bubbles and the porous structure in the step c) can be performed by, for example, placing the porous structure in the treated water and stirring with shaking.

  The pore diameter of the continuous pores of the porous structure having a three-dimensional three-dimensional network structure having continuous pores is preferably 1 to 30 μm, although it depends on the size of microorganisms to be supported, etc. More preferably.

  Examples of the material of the porous structure suitably used in the present invention include vinyl alcohol resins such as polyvinyl alcohol, ether resins such as polyethylene glycol, acrylic resins such as polymethacrylic acid, acrylamide resins such as polyacrylamide, polyethylene, and polypropylene. Olefin resins, styrene resins such as polystyrene, ester resins such as polyethylene terephthalate and polybutylene terephthalate, acrylonitrile resins such as polyacrylonitrile, urethane resins such as polyurethane sponge, calcium alginate, κ (kappa) carrageenan, agar, and cellulose derivatives Sugars, polyester acrylate, epoxy acrylate, urethane acrylate Tsu photocurable resin, and the like can be exemplified porous inorganic compounds such as activated carbon.

  More preferably, a polyvinyl alcohol-based porous gel is preferable in that it has a porous and network structure up to the inside and a large amount of water can be taken into the gel.

  In addition, the mechanical strength of the porous gel can be improved sufficiently, and it has the strength that it can withstand even with strong agitation during a biological reaction. Formalized polyvinyl alcohol porous gel and acetalization A polyvinyl alcohol-based porous gel is more preferable. Specific examples of the polyvinyl alcohol-based porous gel include, for example, Kuraray Co., Ltd. trade name.

  As described above, the support structure is one in which the support density of microorganisms or the like is increased by adding micro-nano bubbles to the culture solution.

  An element for efficiently performing a biological reaction is to increase the density of microorganisms in the culture solution. However, if the density is too high, nutrients and oxygen are not sufficiently provided to the microorganisms. The efficiency of biological reactions will be reduced.

  When culturing microorganisms etc. using only a culture solution, the cell density which can culture aerobic microorganisms appropriately is about 3-6 g / L.

  On the other hand, when microorganisms are supported on a porous structure, there is no problem in providing nutrients and oxygen to the microorganisms, but in the conventional supporting method, microorganisms are penetrated deep into the pores of the porous structure. It is difficult to carry microorganisms etc. at a high density in the porous structure. (There are no examples of supporting microorganisms or the like deep inside the pores of the porous structure in pure culture.)

  On the other hand, in the supporting structure of the present invention, by incorporating micro-nano bubbles in the culture solution, microorganisms and the like can be supported deep inside the pores of the porous structure. Depending on the material, etc., the density can be about 5 to 6 times the cell density of the culture solution.

  The shape of the support structure is not particularly limited, but for example, a granular shape such as a spherical shape, a rectangular parallelepiped shape, or a cubic shape is preferable. If the powder is used, the surface area for aerobic microorganism immobilization can be greatly increased, and the target product can be produced with higher efficiency. The particle size (diameter) at the time of drying of the porous gel particles is preferably 0.5 to 10 mm.

  The use structure of the support structure may be fixed in the microorganism culture solution by a column, a net, or the like, or may be present in a dispersed state in the microorganism culture solution.

2) Inclusion of micro-nano bubbles in the microorganism culture solution In the present invention, the support structure obtained in 1) above is housed in a microorganism culture tank together with the culture solution, and the microorganism culture solution contains micro-nano bubbles. Biological reactions take place.

  In this way, by using a porous structure having a high loading density of microorganisms and the like, in which microorganisms are supported to the back of the pores, and further containing micro-nano bubbles in the culture solution, the pores of the porous structure Since the oxygen necessary for respiration can be sufficiently supplied to the microorganisms and the like carried in the back of the body, the efficiency of the biological reaction can be improved.

  As described above, the biological reaction apparatus of the present invention and the biological reaction method using this biological reaction apparatus sufficiently supply oxygen necessary for the respiratory action of microorganisms to the culture solution of microorganisms and the like. By sufficiently removing the dissolved carbon dioxide produced by the respiratory action of microorganisms, etc. from the culture fluid such as microorganisms, the respiratory action of microorganisms is promoted, and biological reactions using microorganisms are performed efficiently and economically. It is a very good thing that can be.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.

  FIG. 1 schematically shows a first embodiment of the biological reaction apparatus of the present invention. This first embodiment can be suitably used in the case of using microorganisms and the like that are resistant to shear stress, and the culture solution 5 containing microorganisms and the like extracted from the culture tank 1 is directly added to the dissolved carbon dioxide removal device 2 and This is supplied to the micro / nano bubble generator 3. In the first embodiment, the culture tank 1 is provided with a culture tank agitator 10 for stirring the culture solution 5 containing microorganisms and the like.

In the first embodiment, removal of dissolved carbon dioxide from a culture solution such as microorganisms and inclusion of micro-nano bubbles of oxygen-containing gas in the culture solution such as microorganisms are performed as follows.
a) The culture solution 4 is supplied to the culture tank 1.
b) The valve 14 is closed, the valve 13 is opened, the culture tank pump 8 is driven, the culture solution 5 containing microorganisms and the like is extracted from the culture tank 1, and the dissolved carbon dioxide is removed by decompression through the carbon dioxide permeable membrane. The dissolved carbon dioxide removing device 2 is supplied.
c) The culture solution from which the dissolved carbon dioxide has been removed by the dissolved carbon dioxide removing apparatus 2 is supplied to the micro / nano bubble generating apparatus 3 to contain oxygen-containing gas micro / nano bubbles.
d) The culture solution containing the micro-nano bubbles of the oxygen-containing gas in the micro-nano bubble generator 3 is returned to the culture tank 1.
e) The reaction product produced in the culture tank 1 is recovered together with the filtrate through the filter 6 by driving the culture tank pump 8 with the valve 13 closed and the valve 14 opened at an appropriate time, and the filtrate storage tank. It is stored in 9.

  In the first embodiment, dissolved carbon dioxide is removed from a culture solution containing microorganisms and the like, and the dissolved carbon dioxide concentration in the culture solution that increases due to the respiratory action of microorganisms and the like is reduced. By supplying the necessary micro-nano bubbles and supplying oxygen necessary for the respiratory action of microorganisms or the like, a biological reaction using microorganisms or the like can be performed efficiently and economically.

  Further, in the first embodiment, the culture solution 4 extracted from the culture vessel 1 is returned to the culture vessel 1 again after removing dissolved carbon dioxide and containing micro-nano bubbles of oxygen-containing gas. It is possible to maintain a high concentration of microorganisms in the inside.

  FIG. 2 schematically shows a second embodiment of the biological reaction apparatus of the present invention. This second embodiment can be suitably used in the case of using a microorganism or the like that is weak against shear stress, and supplies the culture solution 5 containing the microorganism or the like extracted from the culture tank 1 to the filter 6. The filtrate separated in 6 is supplied to the dissolved carbon dioxide removing device 2 and the micro / nano bubble generating device 3. In the second embodiment, the culture tank 1 is provided with a culture tank agitator 10 for stirring the culture solution 5 containing microorganisms and the like.

  In the second embodiment, the removal of dissolved carbon dioxide from the culture solution of microorganisms and the inclusion of oxygen-containing gas micro-nano bubbles in the culture solution of microorganisms and the like are performed as follows.

a) The culture solution 4 is supplied to the culture tank 1.
b) The valve 14 is closed, the valve 13 and the valve 15 are opened, the culture tank pump 8 is driven, and the culture solution 5 containing microorganisms and the like is extracted from the culture tank 1 and supplied to the filter 6.
c) The culture solution separated from the filter 6 from which the filtrate is removed (that is, the culture solution in which microorganisms and the like are concentrated) is returned to the culture tank 1.
d) The filtrate separated by the filter 6 is supplied to the dissolved carbon dioxide removing apparatus 2 that removes the dissolved carbon dioxide by reducing the pressure through the carbon dioxide permeable membrane.
e) The filtrate from which the dissolved carbon dioxide has been removed by the dissolved carbon dioxide removing apparatus 2 is supplied to the micro / nano bubble generating apparatus 3 to contain oxygen-containing gas micro / nano bubbles.
f) The filtrate containing the micro-nano bubbles of the oxygen-containing gas in the micro-nano bubble generator 3 is returned to the culture tank 1.
g) The reaction product produced in the culture tank 1 is collected at a suitable time with the valve 13 closed and the valve 14 opened to drive the culture tank pump 8 and collected together with the filtrate and stored in the filtrate storage tank 9. .

  In the second embodiment, dissolved carbon dioxide is removed from the filtrate of a culture solution containing microorganisms, and the share stress applied to the microorganisms is reduced by adding micro-nano bubbles of oxygen-containing gas to the filtrate. However, a biological reaction using microorganisms or the like can be performed efficiently and economically.

FIG. 3 schematically shows a third embodiment of the biological reaction apparatus of the present invention.
The third embodiment is characterized in that a supporting structure 11 in which a microorganism or the like is supported on a porous structure such as a polyvinyl alcohol porous gel is used. In addition, by using the support structure 11, when filtering a culture solution containing microorganisms or the like, a flat membrane with a low filtration accuracy such as a porous membrane made of an organic polymer compound such as polyvinylidene fluoride or a metal wire mesh is used. A membrane 7 can be used.

  In the third embodiment, the removal of dissolved carbon dioxide from a culture solution such as microorganisms and the inclusion of micro-nano bubbles of oxygen-containing gas in the culture solution such as microorganisms are performed as follows.

a) The culture solution 4 is supplied to the culture tank 1, and the supporting structure 11 carrying microorganisms or the like is dispersed in the culture solution 4 in the culture tank 1.
b) The valve 14 is closed, the valve 13 is opened and the culture tank pump 8 is driven, the culture solution 5 containing microorganisms and the like is extracted from the culture vessel 1 through the flat membrane 7, and the filtrate is passed through the carbon dioxide permeable membrane. Then, it is supplied to the dissolved carbon dioxide removing device 2 that removes the dissolved carbon dioxide by reducing the pressure.
c) The filtrate from which the dissolved carbon dioxide has been removed by the dissolved carbon dioxide removing apparatus 2 is supplied to the micro / nano bubble generating apparatus 3 to contain oxygen-containing gas micro / nano bubbles.
d) The filtrate containing oxygen-containing gas micro-nano bubbles in the micro-nano bubble generator 3 is returned to the culture tank 1.
g) The reaction product produced in the culture tank 1 is collected at a suitable time with the valve 13 closed and the valve 14 opened to drive the culture tank pump 8 and collected together with the filtrate and stored in the filtrate storage tank 9. .

According to the third embodiment, when the dissolved carbon dioxide concentration in the culture solution is reduced and the micro-nano bubbles of the oxygen-containing gas are contained, a biological reaction using microorganisms or the like can be performed efficiently and economically. In addition to the features described above, the following features can be obtained by using the supporting structure 11 supporting microorganisms or the like.
O Since the density of microorganisms or the like in the culture tank 1 can be increased, the efficiency of biological reaction can be increased.
〇To separate microorganisms from culture solution 5 containing microorganisms, it is not necessary to adopt a microfilter usually used in the technical field of brewing or fermentation, and from organic polymer compounds such as polyvinylidene fluoride. Since a flat membrane 7 having a low filtration accuracy such as a porous membrane or a metal wire mesh can be employed, the filtration process can be carried out economically and efficiently.
〇 Biological reactions can be performed efficiently and economically without causing significant stress or damage to microorganisms.

FIG. 4 schematically shows a fourth embodiment of the biological reaction apparatus of the present invention.
In the fourth embodiment, the carrying structure 11 carrying the microorganisms or the like in the third embodiment is fixed in the culture tank 1 by a fixing member 12 such as a column or a net.

  In the fourth embodiment, the removal of dissolved carbon dioxide from the filtrate of the culture solution 5 containing microorganisms and the like, and the inclusion of oxygen-containing gas micro-nano bubbles in the filtrate are performed in the same manner as in the third embodiment. Is called.

  In the fourth embodiment, since the supporting structure 11 supporting microorganisms or the like is fixed in the culture tank 1, the supporting structure 11 is less damaged or destroyed. Features such as little desorption.

DESCRIPTION OF SYMBOLS 1 Culture tank 2 Dissolved carbon dioxide removal apparatus 3 Micro-nano bubble generation apparatus 4 Culture liquid 5 Culture liquid containing microorganisms 6 Filter 7 Flat membrane 8 Culture tank pump 9 Filtrate storage tank 10 Culture tank agitator 11 Porous structure carrying aerobic microorganisms)
12 Fixing member 13-15 Valve

Claims (10)

A culture vessel containing a culture solution containing microorganisms or cells;
A conduit for extracting the culture solution from the culture tank;
A dissolved carbon dioxide removing device that removes dissolved carbon dioxide by decompression through a carbon dioxide permeable membrane from the culture solution extracted from the culture tank;
A micro / nano bubble generator for containing micro / nano bubbles of oxygen-containing gas in the culture solution from which dissolved carbon dioxide has been removed,
A conduit for removing the dissolved carbon dioxide and returning the culture solution containing oxygen-containing gas micro-nano bubbles to the culture tank;
A biological reaction apparatus comprising:   In the dissolved carbon dioxide removing device, the culture solution extracted from the culture tank is caused to flow through a large number of carbon dioxide permeable hollow fiber membranes in parallel, and a reduced-pressure gas is caused to flow in a direction substantially orthogonal to these hollow fiber membranes. Or a decompressed gas is allowed to flow through the hollow fiber membrane, and the culture solution extracted from the culture vessel is caused to flow in a direction substantially orthogonal to the hollow fiber membranes, thereby being extracted from the culture vessel. The biological reaction apparatus according to claim 1, wherein dissolved carbon dioxide is removed from the culture broth. A filter is disposed in a conduit for extracting the culture solution from the culture tank, and the filtrate separated by the filter is supplied to the dissolved carbon dioxide removing device, and the filtrate separated by the filter is removed. the culture medium he is characterized in that it is returned to the culture tank through a conduit, the biological reaction apparatus of claim 1 or 2.   The filter is composed of a hollow fiber membrane and a container for containing the hollow fiber membrane, and the culture liquid extracted from the culture tank is flowed into the hollow fiber membrane, and the filtrate is taken out from the outside of the hollow fiber membrane. The biological reaction apparatus according to claim 3, wherein: The microorganism or cell is carried in the pores of the porous structure, and a porous film made of an organic polymer compound such as polyvinylidene fluoride is formed in a conduit for extracting the culture solution from the culture tank. The biological reaction apparatus according to claim 1 or 2, wherein a filtration membrane such as a wire mesh is provided, and the filtrate separated by the filtration membrane is supplied to the dissolved carbon dioxide removal device.   The biological reaction apparatus according to claim 5, wherein the porous structure has a three-dimensional three-dimensional network structure with continuous pores.   The biological reaction apparatus according to claim 6, wherein the porous structure is a polyvinyl alcohol-based porous gel.   The biological reaction apparatus according to any one of claims 5 to 7, wherein the porous structure carrying the microorganisms or cells is present in a dispersed state in a culture solution in the culture tank.   The biological reaction according to any one of claims 5 to 7, wherein the porous structure carrying the microorganisms or cells is fixed in the culture tank by a fixing member such as a column or a net. apparatus.   A biological reaction method, wherein a reaction product such as a metabolite of a microorganism or a cell aerobic microorganism is obtained by the biological reaction device according to any one of claims 1 to 9.

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