JP6850609B2 - Biological reaction device equipped with a device for supplying micro-nano bubbles containing oxygen and micro-nano bubbles containing a bactericidal gas such as ozone. - Google Patents

Description

The present invention relates to a biological reaction device for culturing a microorganism or a cell (hereinafter referred to as “microorganism or the like”) and a biological reaction method using the biological reaction device, and a culture solution containing the microorganism, the cell, or a medium after the completion of the biological reaction. Alternatively, the cleaning liquid contains micro-nanobubbles containing a bactericidal gas such as ozone to efficiently, economically and sufficiently sterilize and sterilize microorganisms and the like.

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

Unlike chemical reactions, biological reactions are slow, but they do not use a lot of energy or many chemical substances, so they are mild and meaningful reactions for the environment.

However, biological reactions generally have the problem of being mild and slow. That is, in many cases, a reaction within one hour is sufficient for a chemical reaction, whereas in the case of a biological reaction, a reaction time of several hours to several days if it is long, or several weeks or more if it is particularly long, is required. There is also. Therefore, it is required to carry out biological reactions efficiently and economically.

As a technique for improving the efficiency of biological reactions, Patent Document 1 discloses that cell proliferation is promoted by culturing cells in a culture medium containing microbubbles. Further, Patent Documents 2 to 5 describe that in culturing 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, and the like. Is disclosed.

Specifically, Patent Document 1 describes that the filtrate taken out from the culture solution in the culture tank contains microbubbles and is supplied to the culture tank. Patent Document 2 describes that, in a batch method, a culture solution is extracted from a culture tank, filtered with a cell filter to obtain a filtrate, and micro-nano 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 tank, and Patent Document 4 supplies the culture solution to the culture tank. It is described that the micro-nano bubbles are mixed in the pre-stage.

Furthermore, in order to carry out pure culture in a state where contamination is prevented in the biological reaction, it is necessary to sterilize the culture tank, peripheral equipment, filter membrane, etc. before the biological reaction, and the biological reaction is completed. After that, it is necessary to sufficiently sterilize the microorganisms used in the biological reaction and discharge them to the outside of the biological reaction device. As a method for sterilizing and sterilizing such microorganisms, a method using water vapor, a method using a chemical, and the like as disclosed in Patent Documents 5 to 7 have been conventionally performed.

However, the sterilization / sterilization method using steam requires equipment and labor for sterilization / sterilization, and the sterilization / sterilization method using chemicals makes it difficult to ensure sufficient worker safety. , A separate wastewater treatment means for neutralizing or decomposing the chemicals remaining after sterilization is required, and there is a risk of damage such as corrosion to the biological reaction apparatus. Especially for sterilization of filtration membranes
1) Having a complicated shape,
2) If the filtration membrane dries, the filtration function will deteriorate.
3) Since the filtration membrane is inferior in heat resistance to the culture tank and peripheral equipment, it is difficult to sufficiently sterilize the details without deteriorating the function of the filtration membrane.

Japanese Unexamined Patent Publication No. 2011-120535 Japanese Patent No. 4146476 Japanese Patent No. 4805120 Japanese Patent No. 4956052 Japanese Unexamined Patent Publication No. 61-242605 Special Fair 08-004726 Gazette Japanese Unexamined Patent Publication No. 2013-226486

The subject of the biological reaction apparatus of the present invention and the biological reaction method using this biological reaction apparatus is that after the completion of the biological reaction, the microorganisms used in the biological reaction can be sterilized and sterilized efficiently, economically and sufficiently. The purpose of the present invention is to provide a biological reaction device and a biological reaction method using the biological reaction device, and more specifically, to provide a biological reaction device.
1) At the time of biological reaction, it is possible to efficiently and economically carry out the biological reaction using microorganisms, etc., and at the time of cleaning / sterilization after the completion of the biological reaction, cleaning / sterilization of the biological reaction device, especially the filter membrane, etc. To provide a biological reaction device that can be efficiently, economically and sufficiently performed and a biological reaction method using this biological reaction device, and 2) to use the culture solution used for the biological reaction after the biological reaction is completed outside the biological reaction device. It is an object of the present invention to provide a biological reaction apparatus capable of efficiently, economically and sufficiently sterilizing and sterilizing microorganisms and the like, and a biological reaction method using the biological reaction apparatus.

In order to solve the above problems, the biological reaction device of the present invention and the biological reaction method using the biological reaction device are used to remove microorganisms and the like into micro-nano bubbles containing a bactericidal gas such as ozone (hereinafter referred to as “micro-nano bubbles”) after the biological reaction is completed. , "Micro-nano bubbles such as ozone") is used for sterilization and sterilization.

More specifically, in order to solve the above-mentioned problems, the biological reaction device of the present invention and the biological reaction method using the biological reaction device use the micro-nano bubble generator provided in the biological reaction device to generate microorganisms and the like during the biological reaction. The containing culture solution contains oxygen-containing micro-nano bubbles (hereinafter referred to as "oxygen micro-nano bubbles"), and during cleaning and sterilization after the completion of the biological reaction, the cleaning solution contains micro-nano bubbles such as ozone. It is characterized in that, after the completion of the biological reaction, the concentrated solution of microorganisms or the like contained in the inactivation tank contains micro-nano bubbles such as ozone.

The "micro-nano bubbles" of the present invention mean "micro-bubbles" and / or "nano-bubbles". While "ordinary bubbles" rapidly rise in the liquid and burst at the surface and disappear, microbubbles with a diameter of 50 μm or less, called "microbubbles," shrink and disappear in the liquid. At that time, "nano bubbles", which are microbubbles having a diameter of 100 nm or less, are generated together with free radicals, and these "nano bubbles" remain in the liquid for a certain period of time.

"Nano bubbles", which are micro bubbles, are also called "ultra fine bubbles". At present, the ISO (International Organization for Standardization) is considering the creation of an international standard for fine bubble technology, and if an international standard is created, the name "nano bubble" that is generally used at present will be used. There is a possibility that it will be unified into "ultra fine bubble".

As the "bactericidal gas such as ozone" contained in the micro-nanobubbles such as ozone of the present invention, ozone, carbon dioxide (CO ₂ ), chlorine dioxide (ClO ₂ ), or a mixture thereof can be used. However, it is preferable to use ozone alone from the viewpoint of sterilization, handleability and the like.

In particular, since ozone has self-decomposability, when sterilizing and sterilizing microorganisms using ozone-containing micro-nano bubbles, there is an advantage that the wastewater treatment facility can be simplified compared to the case where chemicals are used. There is also.

In the present invention, after completion of a biological reaction, sterilization / sterilization of microorganisms or the like is rapidly dissolved and diffused in a cleaning solution, a culture solution containing microorganisms or the like, and micro-nano bubbles such as ozone that remain for a long time are used. , Efficient, economical and adequate. That is, the method using steam requires equipment and labor, and the method using chemicals makes it difficult to sufficiently secure the safety of workers, and neutralizes or decomposes the chemicals remaining after sterilization. A separate wastewater treatment means is required for this purpose, and there is a risk of damage such as corrosion to the biological reaction device. On the other hand, in the sterilization / sterilization method using micro-nanobubbles such as ozone of the present invention, ozone or the like is used. Since the micro-nano bubbles quickly dissolve and diffuse in a cleaning solution, a culture solution containing microorganisms, etc. and remain for a long time, sterilization and sterilization of microorganisms, etc. can be performed efficiently, economically and sufficiently. Furthermore, even in places where liquids such as cleaning liquids and culture liquids containing microorganisms do not come into direct contact, such as the upper part of tanks such as culture tanks and inactivation tanks, sterilize with a bactericidal gas such as ozone. Can exert a bactericidal action.

Further, the present invention makes the biological reaction using microorganisms efficient by containing micro-nanobubbles of oxygen that rapidly dissolves and diffuses in the solution and remains for a long time in the culture solution containing microorganisms during the biological reaction. It can be carried out economically, economically and sufficiently, and at the time of cleaning and sterilization after the completion of the biological reaction, the cleaning liquid contains micro-nanobubbles such as ozone that rapidly dissolves and diffuses in the liquid and remains for a long time. It is possible to economically and sufficiently perform cleaning and sterilization of biological reaction devices, particularly filtration membranes. Further, as described above, a sterilizing / sterilizing action can be exerted by a bactericidal gas such as ozone even in a place where the cleaning liquid does not come into direct contact, such as the upper part of the culture tank.

Further, according to the present invention, when the culture solution used for the biological reaction is discharged to the outside of the biological reaction apparatus after the completion of the biological reaction, the culture solution is rapidly dissolved and diffused in the concentrated solution of microorganisms or the like contained in the inactivation tank. By containing micro-nano bubbles such as ozone that remain for a long time, microorganisms and the like can be efficiently, economically and sufficiently sterilized and discharged to the outside of the biological reaction apparatus. Further, as described above, a sterilizing / sterilizing action can be exerted by a bactericidal gas such as ozone even in a place where the culture solution containing microorganisms or the like does not come into direct contact, such as the upper part of the inactivated tank. ..

It is a schematic diagram which shows the 1st Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows the 2nd Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows the 3rd Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows the 4th Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows the 5th Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows the 6th Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows the 7th Embodiment of the biological reaction apparatus of this invention.

First, general matters of the biological reaction apparatus and the biological reaction method of the present invention will be described.
In the biological reaction of the present invention, a reaction product is produced by a microorganism or the like in a culture solution containing a microorganism or the like contained in a culture tank using the culture solution as a nutrient source.

As a biological reaction device that performs a biological reaction,
1) A type in which microorganisms generate reaction products in one culture tank and collect the reaction products, and 2) grow microorganisms in a culture tank and transfer them to another tank such as a fermenter. There is a type in which a reaction product is produced by a microorganism or the like in this separate tank and the reaction product is recovered. In the above-mentioned type 1), the "culture tank" in the present invention means "one culture tank", and in the above-mentioned type 2), "culture tank" and "another tank such as a fermenter". Means.

As the medium (nutrient source for microorganisms and the like) of the culture solution in the present invention, sugars, nitrogen-containing compounds and the like are used. As the saccharide, saccharides such as maltose, sucrose, glucose, fructose, and a mixture thereof are usually used, and the concentration of the saccharide in the culture solution is not particularly limited, but is set to 0.1 to 10 w / v%. preferable. As the nitrogen-containing compound, ammonium chloride, ammonium sulfate or corn steep liquor, yeast extract, meat extract, peptone and the like are used, and it is preferably set to 0.1 to 10 w / v%. Further, it is preferable to add vitamins, inorganic salts and the like to the culture broth, if necessary, in addition to the saccharides and nitrogen-containing compounds.

Examples of the cells in the present invention include animal cells for producing physiologically active peptides or proteins used as antibody drugs, particularly genetically modified animal cells.

In addition, as microorganisms, aerobic or facultative anaerobic microorganisms such as aspergillus bacteria and other aspergillus bacteria, natto bacteria, acetic acid bacteria, yeasts and lactic acid bacteria, which are conventionally used in technical fields such as brewing and fermentation, as well as genes Various aerobic microorganisms created by recombination technology can be used.

The reaction product produced in the culture tank is extracted from the culture tank together with the culture solution containing microorganisms and the like, separated into the microorganism and the filter solution by a filter, and the reaction product is recovered from the filter solution.

The filter of the present invention comprises a filtration membrane and a container for accommodating the filtration membrane. The filtration membrane may be an organic membrane or an inorganic membrane. As the shape of the filtration membrane, any shape such as a flat membrane, a hollow fiber membrane, and a spiral type can be adopted. Among them, the hollow fiber membrane module is preferable, and as long as it is a hollow fiber membrane module, either an external pressure type or an internal pressure type can be adopted.

As the filtration method of the present invention, cross-flow filtration using a hollow fiber membrane module is preferable. In this filtration method, a culture solution containing reaction products, microorganisms, etc. is filtered while being supplied to the inside of the hollow fiber membrane, and the filtered solution is taken out from the outside through the hollow fiber membrane. Since the film stains such as the accumulated microorganisms are scraped off by the shearing force of the parallel flow of the culture solution, a stable filtration state can be maintained for a long period of time.

When cross-flow filtration using the hollow fiber membrane module is performed, it is necessary to flow the liquid to be filtered through the hollow fiber membrane at a flow rate of a certain level or higher in order to scrape off the membrane stains. However, in the present invention, since the culture solution containing microorganisms and the like to be filtered contains micro-nano bubbles of oxygen, the membrane stains can be scraped off even if the culture solution is flowed at a flow velocity lower than usual, and the microorganisms and the like can be scraped off. It can greatly reduce the stress and damage given to.

Specifically, in general cross-flow filtration, the circulation flow velocity 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 for steady operation. However, by including micro-nano bubbles of oxygen in the culture solution, membrane contamination can be reduced and filtration resistance can be kept small, so it is necessary to obtain the same flux (the amount of membrane filtration water per unit time / unit membrane area). Circulation flow velocity can be reduced to about 0.2 to 1.5 m / s. Further, when operating at the same circulation flow velocity, 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 viewpoint of separation performance, water permeability, and stain resistance. Examples thereof include polyethylene-based resins, polypropylene-based resins, polyvinyl chloride-based resins, polyvinylidene fluoride-based resins, polysulfone-based resins, polyether sulfone-based resins, polyacrylonitrile-based resins, cellulose-based resins, and cellulose triacetate-based resins. It may be a mixture of resins containing these resins as main components.

Polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin and polyacrylonitrile resin, which are easy to form a film with a solution and have excellent physical durability and chemical resistance, are preferable. A vinylidene fluoride resin or a resin containing the same 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-based resin, a homopolymer of vinylidene fluoride is preferably used. Further, as the polyvinylidene fluoride-based resin, a copolymer of vinylidene fluoride and a copolymerizable vinyl-based monomer may be used. Examples of the vinyl-based monomer copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene and ethylene trichloride.

The average pore diameter of the filtration membrane can be appropriately determined depending on the purpose and situation of use, but it is preferably small to some extent, and usually 0.01 μm or more and 1 μm or less. If the average pore diameter of the hollow fiber membrane is less than 0.01 μm, components such as microorganisms, sugars and proteins, and film stain components such as aggregates thereof block the pores, and stable operation cannot be performed. Considering the balance with the water permeability, it is preferably 0.02 μm or more, and more preferably 0.03 μm or more. If it exceeds 1 μm, the smoothness of the film surface, 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 become insufficient, and stable operation is possible. It disappears.

Further, when the average pore diameter approaches the size of microorganisms or the like, these may directly block the pores. Furthermore, crushed cells may be produced due to the death of some microorganisms or cultured cells in the fermented broth, and in order to avoid blockage of pores by these crushed substances, 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 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. It is preferable that 10 or more, preferably 20 or more pores are randomly selected, the diameters of the pores are measured, and the number of the pores is averaged. 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 to obtain the equivalent circle diameter as the diameter of the pores. it can.

In the biological reaction apparatus of the present invention and the biological reaction method using this biological reaction apparatus, the target product may be recovered in a batch manner or in a continuous manner.

In the case of batch type, 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 the continuous method, the filtered solution is filtered while balancing the amount of the culture solution supplied to the culture tank so that the water level of the culture solution containing the microorganisms in the culture tank is kept constant. Extract and transfer to the filtrate storage tank.

Whether to select the batch type or the continuous type can be appropriately selected in consideration of the efficiency of the biological reaction, the required purity of the target product, the economic efficiency, and the like.

First, as described above, the first feature of the present invention is that after the completion of the biological reaction, sterilization and sterilization of microorganisms and the like are rapidly dissolved and diffused in the liquid, and micro-nano bubbles such as ozone that remain for a long time are used. Efficient, economical and adequate.

As a result, as described above, the conventional method using steam requires equipment and labor, and the method using conventional chemicals makes it difficult to sufficiently secure the safety of the operator and sterilizes. While a separate wastewater treatment means for neutralizing or decomposing the remaining chemicals is required and there is a risk of damage such as corrosion to the biological reaction device, the micro-nanobubbles such as ozone of the present invention are used. In the conventional sterilization / sterilization method, micro-nanobubbles such as ozone rapidly dissolve and diffuse in the culture solution or cleaning solution and remain for a long time, so that sterilization / sterilization of microorganisms can be performed efficiently, economically and sufficiently. Furthermore, as shown in Examples and Comparative Examples described later, it also has bactericidal properties such as ozone even in places where the washing liquid or the culture liquid does not come into direct contact, such as the upper part of the tank such as a culture tank and an inactivation tank. The gas can exert sterilization and sterilization effects.

Specific methods for sterilizing and sterilizing microorganisms and the like using micro-nano bubbles such as ozone will be described in the following first to seventh embodiments.

Next, the second feature of the present invention is that, as described above, the micro-nano bubble generator provided in the biological reaction device allows the culture solution containing microorganisms and the like to contain oxygen micro-nano bubbles at the time of the biological reaction. At the time of cleaning and sterilization after the completion of the biological reaction, the cleaning liquid contains micro-nano bubbles such as ozone.

As a result, as described above, the biological reaction using microorganisms and the like can be efficiently and economically carried out, and at the time of washing and sterilization after the completion of the biological reaction, the biological reaction is rapidly dissolved and diffused in the washing liquid and remains for a long time. By containing micro-nano bubbles such as ozone, it is possible to efficiently, economically and sufficiently perform cleaning and sterilization of a biological reaction device, particularly a filter membrane. Further, as shown in Examples and Comparative Examples described later, a sterilizing / sterilizing action can be exerted by a bactericidal gas such as ozone even in a place where the cleaning liquid does not come into direct contact, such as the upper part of the culture tank.

The second feature of the present invention will be described below with reference to the first to sixth embodiments.
As a means for containing micro-nano bubbles in a culture solution or a washing solution containing microorganisms, etc.
a) A means for releasing micro-nano bubbles into a culture solution containing microorganisms in the culture tank by a micro-nano bubble generator provided outside the culture tank.
b) A means for incorporating micro-nano bubbles into the culture solution supplied to the culture tank by a micro-nano bubble generator provided in a conduit for supplying the culture solution to the culture tank.
c) A means for allowing a micro-nano bubble generator to contain micro-nano bubbles in a filter solution recovered from a culture solution containing microorganisms and the like, and refluxing the filter solution containing the micro-nano bubbles to a culture tank.
d) A means for supplying a culture solution containing micro-nano bubbles in advance to a culture tank through a conduit.
Etc. can be adopted.

These means can be adopted alone or in combination, but which means is appropriately selected in consideration of resistance to shearing force of the microorganisms to be used, efficiency of biological reaction, economic efficiency, etc. can do.

The means with the least stress and damage to microorganisms and the like are the means b) and d) above. In general, the means of a) above cause stress and damage to microorganisms and the like due to the shearing force generated by the release of micro-nano bubbles, and the means of c) above cause stress and damage due to the shearing force generated during filtration. When a microorganism or the like that is vulnerable to damage is used, it is preferable to use the means b) or d) above.

As will be described later, if a method of supporting microorganisms or the like with a porous structure is adopted, such stress and damage can be significantly reduced.

As the micro-nano bubble generator, a known or commercially available device can be used. Specifically, for example, a "pressurized dissolution type microbubble generator" that creates a supersaturated condition of the dissolved gas by dissolving a sufficient amount of gas in the liquid at a certain high pressure and then releasing the pressure. , "Gas-liquid two-phase flow swirling type micro bubble generator" that utilizes the phenomenon that a water flow is generated to generate a vortex, large bubbles are entrained in the vortex, and the bubbles are subdivided into pieces when the vortex is destroyed. Etc. can be used.

Examples of the micro-nano bubble generator include JP-A-2007-31260, JP-A-2006-289183, JP-A-2005-245817, JP-A-2007-136255, and JP-A-2009-39600. The ones described in the above can be used.

The greatest feature of the present invention is that by utilizing this micro-nano bubble generator, a culture solution containing microorganisms and the like at the time of a biological reaction contains oxygen micro-nano bubbles, and at the time of washing and sterilization after the completion of the biological reaction. , The cleaning liquid contains micro-nano bubbles such as ozone, but the generation of oxygen micro-nano bubbles and the generation of micro-nano bubbles such as ozone may be performed by the same micro-nano bubble generator or separate micros. This may be done using a nanobubble generator. When the same micro-nano bubble generator is used, the supply of oxygen-containing gas and the supply of ozone-containing gas to this device are switched by a switching valve or the like.

It is preferable to generate micro-nano bubbles of oxygen and micro-nano bubbles of ozone or the like in the same micro-nano bubble generator because the manufacturing cost of the biological reaction device can be reduced and the device can be made compact.

At the time of biological reaction, by adding micro-nano bubbles of oxygen to the culture solution containing microorganisms, etc., activation of microorganisms, etc. is promoted as described in Patent Documents 1 to 4, and the reaction efficiency of the biological reaction is promoted. It is possible to improve the reaction time and shorten the reaction time.

In the present invention, oxygen micro-nano bubbles are contained in a culture solution containing microorganisms during a biological reaction, but from the viewpoint of efficiently supplying oxygen necessary for respiration to microorganisms, the oxygen content of the micro-nano bubbles is used. It is preferable to set it high.

Further, in the present invention, at the time of cleaning / sterilization after the completion of the biological reaction, the cleaning liquid contains micro-nano bubbles such as ozone to economically and sufficiently perform cleaning / sterilization to the details of the biological reaction device, particularly the filtration membrane. be able to.

In the present invention, ozone can be preferably used as a "bactericidal gas such as ozone" contained in micro-nano bubbles such as ozone, but ozone is used for short-wavelength ultraviolet irradiation by a commonly used mercury lamp. It can be generated from air or oxygen by low temperature discharge with high voltage or the like.

The amount of ozone supplied to the cleaning liquid varies depending on the type, temperature, pH, etc. of the bacteria to be sterilized, but it is preferable to supply ozone so that the ozone concentration in the cleaning liquid is around 1 ppm. If the ozone concentration in the cleaning solution is too low, the bactericidal activity is insufficient, and in particular, the details of the filtration membrane cannot be sufficiently sterilized. On the other hand, if the ozone concentration in the cleaning liquid is too high, it is uneconomical and may deteriorate the inside of the biological reaction device, the filtration membrane and the like.

As the cleaning liquid containing micro-nano bubbles such as ozone, for example, water such as ion-exchanged water, reverse osmosis membrane permeated water, distilled water, and alcohols are preferably used. Examples of alcohols include monohydric alcohols such as 1-butanol, 2-butanol and 1-heptanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, diethylene glycol, triethylene glycol and glycerin. In addition to polyhydric alcohols, butyl cellosolve, phenyl cellosolve and the like are exemplified. Further, silicone oil or water to which a surfactant is added can also be used.

The cleaning solution may be one in which an electrolyte is dissolved in water, or one in which an alkali, an acid, an oxidizing agent or a reducing agent is added. As the additive, one that does not adversely affect the filtration membrane, the module member, or the like due to the generation of a decomposed product or the like is preferably used. In particular, water containing no additives is preferable because the change in the properties of the filtrate is small even if it is mixed with the filtrate.

As the cleaning liquid, the higher the affinity with the filtration membrane, the easier it is to supply the liquid. Therefore, when the filtration membrane is hydrophilic, it is preferable to select a hydrophilic liquid, and for the hydrophobic filtration membrane, it is preferable to select a hydrophobic liquid. Alternatively, even when a hydrophobic filter membrane is used, for example, by immersing the hydrophobic filter membrane in glycerin compatible with water, or by once replacing glycerin with alcohol, glycerin Can be replaced with water, so that water can be selected as the liquid of the hydrophobic filter membrane.

When a solvent other than water is used as the cleaning liquid, it is preferable to supply water to the filtration membrane after cleaning to clean the remaining liquid.

Cleaning and sterilization are usually performed in the following steps.
1) Remove the microorganisms used in the biological reaction and the culture solution containing the microorganisms from the biological reaction apparatus.
2) A cleaning solution is contained in a culture tank, and the cleaning solution contains micro-nano bubbles such as ozone. As described in a) to d) above, the content of micro-nano bubbles such as ozone in the washing liquid passes through the washing liquid contained in the culture tank, the cleaning liquid at the stage of being supplied to the culture tank, and the filtration membrane into the culture tank. It is performed on the flushed wash solution and / or the wash solution before being supplied to the culture tank.
3) At the time of biological reaction, a cleaning solution containing micro-nano bubbles such as ozone is flowed in the same route as the culture solution or filtrate containing microorganisms is flowed to clean and sterilize the inside of the biological reaction device.

In this way, by simply changing the liquid contained in the culture tank from the culture solution to the washing solution, the inside of the biological reaction device can be washed and sterilized by using the device for carrying out the biological reaction as it is. In addition, it is possible to sufficiently clean and sterilize the corners inside the biological reaction apparatus, the pores of the filtration membrane, etc., which are difficult to clean and sterilize by a normal method.

In particular, the filtration membrane has a large number of pores, and it is difficult to sufficiently remove organic substances such as microorganisms clogged in these pores and sterilize them. Cleaning and sterilization of the filtration membrane is performed by supplying the cleaning liquid to the primary side of the filters 1) to 3) above and filtering to the secondary side of the filter (hereinafter referred to as "forward washing"). However, if you want to perform more sufficient cleaning and sterilization, or if you want to shorten the cleaning and sterilization time, you can use backwashing together.

In this backwash, a cleaning solution containing micro-nano bubbles such as ozone is supplied from a conduit connected to the secondary side of the filter, and the filtration membrane is cleaned and sterilized by filtering the primary side of the filter. Is to do. In order to sufficiently perform cleaning / sterilization or to shorten the cleaning / sterilization time, it is preferable to repeat normal washing and reverse line a plurality of times.

The cleaning liquid after cleaning and sterilizing the filtration membrane may be discharged to the outside of the biological reaction device, or may be refluxed to the culture tank to clean and sterilize the culture tank and the like. Since the cleaning liquid obtained by cleaning and sterilizing the filter membrane at the initial stage of cleaning contains a high concentration of organic substances such as microorganisms, the cleaning liquid is discharged to the outside of the biological reaction device at the initial stage of cleaning, and the filter membrane is sufficient to some extent. At the stage of cleaning and sterilization, it is preferable to return the cleaning solution to the culture tank to clean and sterilize the inside of the culture tank.

Further, the cleaning liquid after cleaning and sterilizing the filtration membrane may be discharged from the outlet of the filter in one cleaning and sterilization, or may be returned to the inlet of the filter to clean and sterilize the filtration membrane again. You can do it. The cleaning solution that has been cleaned and sterilized from the filtration membrane at the initial stage of cleaning contains organic substances such as microorganisms at a high concentration, so it is discharged from the outlet of the filter, and when the filtration membrane is sufficiently cleaned and sterilized, this cleaning solution is used. It is preferable to recirculate the filtration membrane to the inlet of the filter to clean and sterilize the filtration membrane again because the cleaning liquid can be saved.

Further, when cleaning and sterilizing the inside of the culture tank with the cleaning liquid, it is preferable to spray the cleaning liquid over a wide area like a shower because the inner wall of the culture tank can be sufficiently cleaned and sterilized.

Cleaning and sterilization should be performed in a time that allows them to be sufficiently cleaned and sterilized, depending on the condition of the filtration membrane, resistance to microorganisms, and the like.

As described above, the biological reaction apparatus of the present invention and the biological reaction method using the biological reaction apparatus can efficiently and economically carry out the biological reaction using microorganisms and the like at the time of the biological reaction. When the culture device is washed and sterilized after the completion of the biological reaction, it is extremely excellent in that the biological reaction device, particularly the filter membrane and the like, can be washed and sterilized efficiently, economically and sufficiently.

In the present invention, since the biological reaction is carried out in a culture solution containing microorganisms or the like containing micro-nano bubbles of oxygen, a porous structure having a high carrying density and carrying the microorganisms or the like deep into the pores is provided. Even when it is used, it is possible to sufficiently supply oxygen necessary for respiration to microorganisms and the like carried in the back of the pores of the porous structure, so that the efficiency of the biological reaction can be improved. In this case, filtration can be performed efficiently and economically, and stress and damage given to microorganisms and the like can be reduced.

As described above, the biological reaction device of the present invention and the biological reaction method using this biological reaction device can efficiently and economically carry out a biological reaction using microorganisms and the like by utilizing micro-nano bubbles, and can be used for fermentation and brewing. It is useful not only for the production of foods, chemicals, chemicals, etc. that utilize biological reactions such as fermentation, but also for biorefinery that produces bioethanol, etc. using biomass.

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.

<First Embodiment>
FIG. 1 schematically shows a first embodiment of the biological reaction device of the present invention. In the first embodiment, the culture tank 1 is provided with a culture tank agitator 17 for stirring the culture solution 6 containing microorganisms and the like.

In the first embodiment, at the time of biological reaction,
a) The culture solution 7 supplied to the culture tank 1 is contained with micro-nano bubbles of oxygen by the micro-nano bubble generator 3 provided in the conduit for supplying the culture solution 7 to the culture tank 1, and b) culture. The micro-nano bubble generator 2 provided outside the tank 1 allows the culture solution 6 containing the microorganisms and the like in the culture tank 1 to contain micro-nano bubbles of oxygen.

The reaction product produced in the culture tank 1 was extracted from the culture tank 1 together with the culture liquid 6 containing microorganisms and the like by the culture tank pump 11, and the filter liquid and the filter liquid were removed in the filter 10. The filtrate is separated into a culture solution containing microorganisms (hereinafter referred to as "concentrate of microorganisms"), and the filtrate is stored in the filtrate storage tank 16 with the valves 12 and 13 open, and the microorganisms and the like are stored. The concentrate is returned to the culture tank 1 through the conduit.

At the time of washing and sterilization after the completion of the biological reaction, the culture solution 6 and the filter solution containing microorganisms and the like are removed from the culture tank 1 and the filter solution storage tank 16, respectively.
a) The cleaning solution supplied to the culture tank 1 instead of the culture solution 7 by the micro-nano bubble generator 3 contains micro-nano bubbles such as ozone, and b) the micro-nano bubble generator 2 in the culture tank 1 The cleaning liquid contains micro-nano bubbles such as ozone.

First, the inside of the culture tank 1 is cleaned and sterilized with a cleaning solution containing a sufficient amount of micro-nano bubbles such as ozone.

After that, the cleaning solution containing a sufficient amount of micro-nano bubbles such as ozone is extracted from the culture tank 1 by the culture tank pump 11, the valve 12 is closed, the valve 13 is opened, and the filtration membrane of the filter 10 is cleaned and sterilized (in order). Wash), and clean and sterilize the inside of the conduit of the filter 10 → filtrate storage tank 16. Further, if the valve 12 is opened and the valve 13 is closed, the inside of the pipe line of the filter 10 → the culture tank 1 can be cleaned and sterilized.

In the first embodiment,
1) The gas supplied to the micro-nano bubble generators 2 and 3 is switched from "oxygen-containing gas" to "ozone-containing gas" at the time of biological reaction.
2) The liquid supplied to the culture tank 1 is switched from the "culture solution" at the time of the biological reaction to the "washing solution".
It is possible to perform cleaning and sterilization by the same operation as the biological reaction by such a simple operation.

Furthermore, since the cleaning solution containing micro-nanobubbles such as ozone spreads to the details of the filtration membrane of the filter in the same way as the culture solution and the filtration solution, it should be sufficiently cleaned and sterilized without imposing a burden on the filtration membrane. Can be done.

Further, in the first embodiment, since the concentrated solution of microorganisms or the like separated by the filter 10 is returned to the culture tank 1 at the time of the biological reaction, the concentration of the microorganisms or the like in the culture tank 1 can be maintained high. ..

<Second Embodiment>
FIG. 2 schematically shows a second embodiment of the biological reaction device of the present invention.
In the second embodiment, the micro-nano bubble generator 4 is used instead of the micro-nano bubble generators 2 and 3 in the first embodiment, and the filter solution of microorganisms or the like contains the micro-nano bubbles and is refluxed to the culture tank 1. It is something that I tried to do.

Further, in the second embodiment,
1) A function that allows you to select whether to discharge the cleaning solution that has been washed and sterilized from the filtration membrane to the outside of the biological reaction device or to return it to the culture tank, and 2) to wash and sterilize the filtration membrane once. A function that allows you to select whether to discharge from the outlet of the filter or to return to the inlet of the filter for cleaning and sterilization.
Is provided. The functions 1) and 2) can be appropriately provided in other embodiments.

In the second embodiment, as follows, the culture solution containing microorganisms and the like contains micro-nano bubbles of oxygen at the time of the biological reaction, and the cleaning solution at the time of washing / sterilization after the completion of the biological reaction is added. , Ozone and other micro-nano bubbles are included.

At the time of biological reaction :
a) The culture solution 7 is supplied to the culture tank 1.
b) The valve 13, valve 22, valve 23 and valve 24 are closed, the valve 12, valve 14 and valve 21 are opened to drive the culture tank pump 11, and the concentrated solution of microorganisms or the like separated by the filter 10 is piped. It is returned to the culture tank 1 through the passage, and the filtrate separated by the filter 10 is guided to the micro-nano bubble generator 4, contains micro-nano bubbles of oxygen, and then returned to the culture tank 1.
c) The reaction product produced in the culture tank 1 is collected together with the filtrate by opening the valve 13 at an appropriate time and stored in the filtrate storage tank 16.

During washing and sterilization : The culture solution 6 and the filter solution containing microorganisms and the like are removed from the culture tank 1 and the filter solution storage tank 16, respectively.
a) A cleaning solution is supplied to the culture tank 1 instead of the culture solution 7.
b) The valve 13, valve 14, valve 21, valve 23 and valve 24 are closed, the valve 12 and valve 22 are opened to drive the culture tank pump 11, and the filter membrane of the filter 10 is mainly cleaned, and the initial cleaning process is performed. A cleaning solution containing a high concentration of organic substances such as microorganisms is discharged to the outside of the biological reaction device.
c) After that, when the filter membrane of the filter 10 has been sufficiently cleaned, the valve 13, the valve 14, the valve 22, the valve 23 and the valve 24 are closed, the valve 12 and the valve 21 are opened, and the culture tank pump 11 is opened. By driving, the cleaning liquid is guided to the micro-nano bubble generator 4 through the conduit of the culture tank 1 → the filter 10 → the valve 12, and the cleaning liquid containing micro-nano bubbles such as ozone is returned to the culture tank 1 to become the cleaning liquid. While containing micro-nano bubbles such as ozone, clean the inside of the culture tank 1, the filter membrane of the filter 10, the culture tank 1 → the filter 10 → the valve 12 → the micro-nano bubble generator 4 → the inside of the conduit of the culture tank 1. Sterilize.
d) After that, the cleaning liquid containing a sufficient amount of micro-nano bubbles such as ozone is extracted from the culture tank 1 by the culture tank pump 11, the valve 12 and the valve 14 are closed, and the valve 13 is opened to filter the filter 10. The membrane can be washed and sterilized (sequential washing), and the inside of the conduit of the filter 10 → valve 13 → filter solution storage tank 16 can be washed and sterilized. Further, if the valve 12 and the valve 13 are closed and the valve 14 is opened, the inside of the conduit of the filter 10 → the valve 14 → the culture tank 1 can be cleaned and sterilized.
e) Further, when the filtration membrane of the filter 10 has been sufficiently washed and sterilized (sequential washing), the valve 12, the valve 13 and the valve 14 are closed, the valve 23 and the valve 24 are opened, and the filter 10 is opened. The cleaning liquid obtained by cleaning and sterilizing the filtration membrane is returned to the inlet of the filter 10 to clean and sterilize the filtration membrane of the filter 10 again. As a result, the cleaning liquid can be saved, and the filtration membrane of the filter 10 can be sufficiently cleaned and sterilized.

In the second embodiment, as in the first embodiment, cleaning and sterilization can be performed by the same operation as the biological reaction, and sufficient cleaning and sterilization can be performed without imposing a burden on the filtration membrane. it can.

Further, in the second embodiment, since the filtrate separated by the filter 10 contains micro-nano bubbles at the time of the biological reaction, the stress and damage given to the microorganisms and the like can be reduced, and the filter 10 can be reduced. Since the concentrated solution of the microorganisms and the like separated in 1 is returned to the culture tank 1, the concentration of the microorganisms and the like in the culture tank 1 can be maintained high.

<Third Embodiment>
FIG. 3 schematically shows a third embodiment of the biological reaction device of the present invention.
In the third embodiment, the means (micro-nano bubble generator 2) and the means for incorporating micro-nano bubbles in the culture solution 6 containing the microorganisms and the like in the culture tank 1 used in the first embodiment in the second embodiment. A means (micro-nano bubble generator 3) for containing micro-nano bubbles in the culture solution 7 supplied to the culture tank 1 is used in combination.

When it is desired to increase the content of micro-nano bubbles in the culture solution and the washing solution, it is effective to use a plurality of micro-nano bubble-containing means in combination in this way.

<Fourth Embodiment>
FIG. 4 schematically shows a fourth embodiment of the biological reaction device of the present invention.
The fourth embodiment is characterized in that a supported structure 8 in which a microorganism or the like is supported on a porous structure such as a polyvinyl alcohol-based porous gel is used.

In the fourth embodiment, the inclusion of oxygen micro-nano bubbles in the culture solution at the time of the biological reaction and the micro of ozone or the like in the washing solution at the time of washing / sterilization after the completion of the biological reaction are as follows. Nanobubbles are included.

At the time of biological reaction :
a) The culture solution 7 is supplied to the culture tank 1.
b) The culture tank pump 11 is driven with the valve 12 opened and the valve 13 closed, and the filtrate separated by the filter 10 provided in the culture tank 1 is guided to the micro-nano bubble generator 4 and contains oxygen micro-nano bubbles. After that, it is returned to the culture tank 1.
c) The reaction product produced in the culture tank 1 is collected together with the filtrate by opening the valve 13 at an appropriate time and stored in the filtrate storage tank 16.

During washing and sterilization : The culture solution 6 and the filter solution containing microorganisms and the like are removed from the culture tank 1 and the filter solution storage tank 16, respectively.
a) A cleaning solution is supplied to the culture tank 1 instead of the culture solution 7.
b) With the valve 12 open and the valve 13 closed, the culture tank pump 11 is driven to guide the cleaning liquid to the micro-nano bubble generator 4 through the culture tank 1 → filter 10 → valve 12 conduit, and micro-ozone or the like. By returning the cleaning liquid containing nanobubbles to the culture tank 1, the inside of the culture tank 1 and the culture tank 1 → filter 10 → valve 12 → micro-nano bubble generator 4 → culture while the cleaning liquid contains micro-nano bubbles such as ozone. The inside of the pipeline of the tank 1 is washed and sterilized, and the filter membrane of the filter 10 is washed and sterilized (sequential washing).
c) After that, the cleaning liquid containing a sufficient amount of micro-nano bubbles such as ozone is extracted from the culture tank 1 through the filter 10 by the culture tank pump 11, the valves 12 and 14 are closed, and the valve 13 is opened for filtration. The filter membrane of the vessel 10 can be washed and sterilized again (sequential washing), and the inside of the conduit of the filter 10 → valve 13 → filter solution storage tank 16 can be cleaned and sterilized.

The fourth embodiment has the following features by using the supported structure 8 on which microorganisms and the like are supported.
○ Since the density of microorganisms and the like in the culture tank 1 can be increased, the efficiency of biological reactions can be increased.
○ As the filter 10 for separating microorganisms and the like from the culture solution 6 containing microorganisms and the like, it is not necessary to adopt a microfiltration filter usually used in the technical fields of brewing and fermentation, and an organic polymer such as polyvinylidene fluoride is used. Since a flat membrane having low filtration accuracy such as a porous membrane made of a compound or a metal wire net can be adopted, the filtration step can be performed economically and efficiently.
○ Biological reactions can be carried out efficiently and economically without causing great stress or damage to microorganisms.

<Fifth Embodiment>
FIG. 5 schematically shows a fifth embodiment of the biological reaction device of the present invention.
In the fifth embodiment, the supported structure 8 on which the microorganisms and the like in the fourth embodiment are supported is fixed in the culture tank 1 with a fixing member 9 such as a column and a net body.

In the fifth embodiment, the inclusion of oxygen micro-nano bubbles in the culture solution 6 containing microorganisms and the like during the biological reaction, and ozone and the like in the cleaning solution during cleaning and sterilization after the completion of the biological reaction. The inclusion of micro-nano bubbles is carried out in the same manner as in the fourth embodiment.

In the fifth embodiment, since the carrier structure 8 on which the microorganisms and the like are supported is fixed in the culture tank 1, the carrier structure 8 is less likely to be damaged or destroyed. It has features such as less detachment.

<Sixth Embodiment>
FIG. 6 schematically shows a sixth embodiment of the biological reaction device of the present invention.
In the sixth embodiment, a cleaning liquid containing micro-nano bubbles such as ozone is supplied from a conduit connected to the secondary side of the filter 10, so that back-washing is performed together with normal washing. is there.

In the sixth embodiment, with the valve 15 and the valve 20 closed, the following operations 1) at the time of biological reaction and the operation 2 at the time of cleaning / sterilization are similar to those of the second embodiment. ) Is performed.

1) At the time of biological reaction :
a) The culture solution 7 is supplied to the culture tank 1.
b) The valve 13 is closed, the valve 12 and the valve 14 are opened, the culture tank pump 11 is driven, and the concentrated solution of microorganisms and the like separated by the filter 10 is returned to the culture tank 1 through the conduit, and the filter 10 is used. The filtrate separated in (1) is guided to the micro-nano bubble generator 4, contains micro-nano bubbles of oxygen, and then returned to the culture tank 1.
c) The reaction product produced in the culture tank 1 is collected together with the filtrate by opening the valve 13 at an appropriate time and stored in the filtrate storage tank 16.

2) During washing / sterilization (sequential washing) : The culture solution 6 and the filter solution containing microorganisms and the like are removed from the culture tank 1 and the filter solution storage tank 16, respectively.
a) A cleaning solution is supplied to the culture tank 1 instead of the culture solution 7.
b) The valve 12 is opened, the valve 13 and the valve 14 are closed, and the culture tank pump 11 is driven to guide the cleaning liquid to the micro-nano bubble generator 4 through the pipeline of the culture tank 1 → the filter 10 → the valve 12, and ozone. By returning the cleaning solution containing micro-nano bubbles such as, etc. to the culture tank 1, the inside of the culture tank 1 and the culture tank 1 → filter 10 → valve 12 → micro-nano bubble generator while the cleaning solution contains micro-nano bubbles such as ozone. 4 → Clean and sterilize the inside of the pipeline of the culture tank 1.
c) After that, the cleaning liquid containing a sufficient amount of micro-nano bubbles such as ozone is extracted from the culture tank 1 by the culture tank pump 11, the valve 12 and the valve 14 are closed, and the valve 13 is opened to filter the filter 10. The membrane can be washed and sterilized (sequential washing), and the inside of the conduit of the filter 10 → valve 13 → filter solution storage tank 16 can be washed and sterilized. Further, if the valve 12 and the valve 13 are closed and the valve 14 is opened, the inside of the conduit of the filter 10 → the valve 14 → the culture tank 1 can be cleaned and sterilized.

In the sixth embodiment, as in the other embodiments, washing and sterilization can be performed by the same operation as the biological reaction, and washing and sterilization can be sufficiently performed without imposing a burden on the filtration membrane. It is possible to reduce the stress and damage given to the above, and it is possible to maintain a high concentration of microorganisms and the like in the culture tank.

Further, in the sixth embodiment, at the time of washing and sterilization, in addition to the normal washing of 2) above, the backwashing of the filtration membrane of 3) below is also performed.
3) After interrupting the normal washing of 2) above, the backwash of the filtration membrane is performed by the following steps d) to f). d) With the valves 12, 13 and 14 closed and the valves 15 and 20 open, a pump (not shown) is driven to guide the cleaning liquid in the cleaning liquid tank 19 to the micro-nano bubble generator 5. Then, the cleaning liquid contains micro-nano bubbles such as ozone.
e) The cleaning liquid containing micro-nano bubbles such as ozone is supplied to the secondary side of the filter 10, filtered to the primary side of the filter 10, and the filtration membrane is backwashed for cleaning and sterilization. ..
f) The washing water after backwashing the filtration membrane is discharged to the outside via the valve 20.

In the sixth embodiment, the above-mentioned 2) forward washing and the above-mentioned 3) backwashing are repeated a plurality of times as necessary.

In the sixth embodiment, since cleaning / sterilization of the filtration membrane having a large number of pores and requiring labor and time for cleaning / sterilization is performed by using both normal washing and backwashing, cleaning / sterilization of the filtration membrane is performed. It has features such as sufficient performance and shortening of cleaning and sterilization time.

Next, as described above, the third feature of the present invention is that after the biological reaction is completed, the culture solution containing the medium such as microorganisms is extracted from the culture tank, and the microorganisms and the like are separated and concentrated by the filter. Is separated and stored in an inactivating tank, and the concentrated liquid contained in this inactivating tank contains micro-nanobubbles containing a bactericidal gas such as ozone to sterilize microorganisms and the like in the recovered concentrated liquid. It is to be.
The third feature of the present invention will be described below with reference to the seventh embodiment.

<7th Embodiment>
FIG. 7 schematically shows a seventh embodiment of the biological reaction device of the present invention.
In the seventh embodiment, at the time of the biological reaction, the reaction product produced in the culture tank 1 with the valves 25 and 27 closed is the culture solution 6 containing a medium such as microorganisms by the culture tank pump 11. It is taken out from the culture tank 1 together with, and separated into a filtrate containing a reaction product and a concentrate of microorganisms (hereinafter referred to as "recovery concentrate") in the filter 10, and the valve 12, the valve 13 and the valve 13 and the concentrate are separated from each other. With the valve 14 open, the filtrate is stored in the filtrate storage tank 16, and the recovered concentrate is returned to the culture tank 1 through the conduit.

After the biological reaction is completed, the microorganisms used in the biological reaction are sufficiently sterilized and discharged to the outside of the biological reaction apparatus. However, in the seventh embodiment, the microorganisms and the like are sterilized in the culture tank (hereinafter, "culture tank sterilization"). ”) And a mode in which microorganisms and the like are transferred from the culture tank to the inactivation tank and sterilized (hereinafter, referred to as“ inactivation tank sterilization ”) can be adopted.

First, the inactivated tank sterilization can be performed as follows.
a) With the valve 27 and the valve 14 closed, the culture medium 6 containing the medium such as microorganisms is extracted from the culture tank 1 by the culture tank pump 11, and the filter solution 10 contains the medium. , Separate into recovery concentrate.
b) With the valves 12, 13 and 25 opened and the valve 26 closed, the filtrate is stored in the filtrate storage tank 16 and the recovered concentrate is stored in the inactivated tank 30.
c) The recovered concentrate contained in the inactivating tank 30 is sterilized with micro-nano bubbles such as ozone by the micro-nano bubble generator 28.
d) When the sterilization of microorganisms and the like is completed, the valves 26 and 33 are opened, and the sterilized recovery concentrate is discharged to the outside of the biological reaction apparatus.

Next, the culture tank sterilization can be performed as follows.
a) With the valve 27 and the valve 25 closed, the culture medium 6 containing the medium such as microorganisms is extracted from the culture tank 1 by the culture tank pump 11, and the filter solution 10 contains the medium. , Separate into recovery concentrate.
b) With the valves 12, 13 and 14 open, the filtrate is stored in the filtrate storage tank 16 and the recovered concentrate is refluxed to the culture tank 1.
c) The recovered concentrate contained in the culture tank 1 contains micro-nano bubbles such as ozone by the micro-nano bubble generator 29 to sterilize microorganisms and the like.
d) When the sterilization of microorganisms and the like is completed, the valve 27 and the valve 33 are opened, the valve 26 is closed, and the wastewater pump 32 is driven to discharge the sterilized recovery concentrate to the outside of the biological reaction apparatus.

This seventh embodiment can basically be performed by selecting inactivated tank sterilization or culture tank sterilization by switching between the valve 14 and the valve 25, but either inactivated tank sterilization or culture tank sterilization. If only can be done, the equipment not used for the sterilization can be omitted. For example, if only culture tank sterilization can be performed, it is not necessary to install equipment on the downstream side of the valve 25 such as the inactivating tank 30 and the micro-nano bubble generator 28.

In this seventh embodiment, the recovered concentrate is separated by using the filter 10 used to recover the reaction product during the biological reaction, but the recovered concentrate is separated without using a filter medium. It can also be performed by centrifugation, sedimentation separation, etc. using the difference in specific gravity.

In the inactivated tank sterilization, the inactivated tank 30 takes into consideration that the sterilizing effect of micro-nano bubbles such as ozone is enhanced and that a bactericidal gas such as ozone is prevented from being discharged to the outside of the biological reaction device. Therefore, it is desirable to make it a closed type.

If the viscosity of the recovered concentrate is too high, micro-nano bubbles such as ozone will not spread evenly throughout the recovered concentrate, and microorganisms and the like cannot be sterilized sufficiently. Therefore, in the inactivated tank sterilization, the inactivated tank is used. It is preferable that the 30 is provided with a liquid feeder 31 capable of supplying a liquid, and the viscosity of the recovered concentrate is adjusted within an appropriate range. Further, also in the sterilization of the culture tank, it is preferable to supply the liquid to the culture tank 1 through a conduit or the like for supplying the culture liquid 7 to the culture tank 1 and adjust the viscosity of the recovered concentrate to an appropriate range.

The biological reaction apparatus of the seventh embodiment basically sterilizes the culture tank and / or inactivates the culture tank after the biological reaction is completed. However, if germs are mixed in the culture solution during the biological reaction, the culture tank is sterilized. When it becomes necessary to use the culture tank 1 for the next biological reaction during sterilization or inactivation tank sterilization, the valve 27 and the valve 26 are opened, the valve 33 is closed, and the waste water pump 32 is driven. As a result, the culture solution 6 or the concentrated solution in the culture tank 1 can be immediately stored in the inactivation tank 30.

In the present invention, 1) sterilization of microorganisms and the like using micro-nanobubbles such as ozone that rapidly dissolves and diffuses in a solution such as a culture medium and a washing solution and remains for a long time, and 2) separation and concentration of the microorganisms and the like. By sterilizing the collected concentrated solution, microorganisms and the like can be sterilized efficiently, economically and sufficiently. Further, the present inventors have prepared a culture medium containing a medium such as microorganisms. When separating the recovered concentrate using a separator such as a filter, the ratio of the medium contained in the culture medium entering the recovered concentrate should be 50% or less, preferably 25% or less, that is, the filtrate. It has been found that the sterilization / bactericidal action of micro-nanobubbles such as ozone can be remarkably enhanced by allowing 50% or more, preferably 75% or more of the medium contained in the culture solution to enter the liquid side of the culture medium. It is a thing.

As shown in Examples and Comparative Examples described later, the sterilization / bactericidal action is remarkably enhanced by setting the proportion of the medium that enters the recovered concentrate to 50% or less, preferably 25% or less by using a separator such as a filter. be able to.

The reason for this is that if the medium concentration in the recovered concentrate is too high, micro-nano bubbles such as ozone contained in the recovered concentrate are consumed in chemical reactions such as denaturation of saccharides and nitrogen-containing compounds, resulting in sterilization and sterilization. It is possible that the action is reduced.

Further, as shown in Examples and Comparative Examples described later, a sterilizing / sterilizing action is exerted by a bactericidal gas such as ozone even on a place where the culture solution does not come into direct contact, such as the upper part of an inactivated tank. Can be done.

The excellent sterilization / sterilization action of the present invention when micro-nano bubbles such as ozone are used will be further described below with reference to Examples and Comparative Examples.

First, the gist of the explanation using Examples and Comparative Examples will be briefly described. Using Examples 1 to 4 and Comparative Example 1, the form of bubbles of ozone-containing gas blown into the culture solution and the medium (sugar) of the culture solution. The effect of the content of (nutrient sources such as microorganisms such as nitrogen-containing compounds) on the sterilization / bactericidal action was explained, and using Examples 5 to 6, a place where the culture medium does not come into direct contact, such as the upper part of the culture tank. The sterilizing and bactericidal action of micro-nanobubbles such as ozone will be described.

<Examples 1 to 4 and Comparative Example 1>
A 1 L medium bottle (Shibata Scientific Technology model number: 017200-10002A) was used as a treatment tank, and a standard strain of a culture medium [Corynebacterium glutamicum as a microorganism, etc.] was placed in the culture medium (value of OD660). It contains 16 and contains 24 g / L of glucose as a medium, as well as peptone, yeast extract, etc.] in 500 mL.

Ozone generator (eco-design made by Model Number: ED-OG-RC12GC) using, ozone concentration 2.2% by weight, making the ozone-containing gas density 1.2kg / m ^3, the bubbles of the ozone-containing gas, large The culture solution at 20 ° C. was blown into the culture solution at 20 ° C. from the bottom of the treatment tank at an aeration rate of 0.5 L / min.

<Example 1>
Using a micro-nano bubble generator (trade name: OK nozzle, model number: OKE-MB200 ml), ozone-containing gas is blown into the culture solution from which all the medium has been removed in the form of micro-nano bubble bubbles, and 15 minutes at the start of blowing. After that, the viable cell counts in the culture medium after 30 minutes, 45 minutes and 60 minutes were measured by the plate culture method.

<Comparative example 1>
A silicon tube having an inner diameter of 4 mm was used as a ventilation device, and the viable cell count was measured in the same manner as in Example 1 except that the ozone-containing gas was blown in the form of normal bubbles.

<Example 2>
The viable cell count was measured in the same manner as in Example 1 except that the culture medium from which 87.5% of the medium had been removed was used as the culture medium.

<Example 3>
The viable cell count was measured in the same manner as in Example 1 except that the culture medium from which 75% of the medium had been removed was used as the culture medium.

<Example 4>
The viable cell count was measured in the same manner as in Example 1 except that the culture medium from which 50% of the medium had been removed was used as the culture medium.

The results of Examples 1 to 4 and Comparative Example 1 are shown in Table 1.
First, as can be seen from the comparison between Example 1 and Comparative Example 1 in Table 1, when ozone-containing gas is blown into a culture solution to sterilize and sterilize microorganisms, the form of bubbles is compared with the case where normal bubbles are used. , When micro-nano bubbles are used, the sterilization and sterilization effects can be significantly enhanced.

Further, as can be seen from Examples 1 to 4, when sterilizing / sterilizing microorganisms or the like in the culture solution, the more the medium is removed from the culture solution, the higher the sterilization / sterilization action can be. It can be seen that the removal rate of the medium is preferably 50% or more, and more preferably 75% or more.

<Examples 5 to 6>
In Examples 5 and 6, respectively, in Example 1 and Example 4, one round of the upper inner surface where the culture solution in the treatment tank does not come into direct contact is wiped with absorbent cotton at the start of blowing and 60 minutes later, and adhered to the absorbent cotton. The viable cell count was measured by the plate culture method. The results of Examples 5 to 6 are shown in Table 2.

As can be seen from Examples 5 to 6 in Table 2, when micro-nanobubbles of ozone-containing gas are blown into the culture solution to sterilize and sterilize the microorganisms, not only the microorganisms in the culture solution but also the treatment tank. It is possible to exert a sterilizing / sterilizing action on microorganisms and the like adhering to the upper part of the treatment tank which the culture solution does not come into direct contact with, and further, removing the medium from the culture solution can enhance the sterilizing / sterilizing action.

In this way, after the biological reaction is completed, micro-nano bubbles containing a bactericidal gas such as ozone are used to sterilize and sterilize the microorganisms, so that the upper part of the culture tank, inactivation tank, etc. It is possible to exert a sterilizing / sterilizing action even on a place where the washing liquid or the culture liquid does not come into direct contact, and it is possible to sterilize / sterilize every corner of the tank such as the culture tank and the inactivation tank.

1 Culture tank 2-5 Micro-nano bubble generator 6 Culture solution containing microorganisms, etc. 7 Culture solution 8 Support structure (porous structure carrying microorganisms, etc.)
9 Fixing member 10 Filter 11 Culture tank pump 12-15, 20-27 Valve 16 Filter liquid storage tank 17 Culture tank stirrer 18 Switching valve 19 Cleaning liquid tank 28-29 Micro-nano bubble generator 30 Inactivation tank 31 Liquid feeder 32 Waste water Pump 33 valve

Claims (19)

Microorganisms or cells are purely cultivated in a culture tank in a state where contamination with germs is prevented, and foods, chemicals, chemical substances such as organic acids and alcohols, useful chemical substances such as antibodies, or chemistry such as bioethanol from biomass. It is a biological reaction device that manufactures products.
A culture tank containing a culture solution containing the above-mentioned microorganisms, cells, and a medium is provided.
When cleaning and sterilizing the biological reaction device after the completion of the biological reaction, the culture solution in the culture tank or the cleaning solution supplied in the culture tank contains micro-nano bubbles containing a bactericidal gas such as ozone. A biological reaction apparatus comprising a micro-nano bubble generator to which a conduit for supplying a bactericidal gas such as ozone is connected. The biological reaction apparatus according to claim 1, further comprising a reflux conduit provided with a filter for refluxing the culture solution extracted from the culture tank to the culture tank. The biological reaction apparatus according to claim 2, wherein the reflux conduit is provided with a filter for separating the concentrated solution in which the microorganism or the cell is concentrated from the culture solution. The biological reaction apparatus according to claim 3, further comprising a liquid feeder for supplying a liquid to the concentrated liquid to adjust the viscosity. With the culture tank containing the culture solution or the washing solution,
A micro-nano bubble generator connected to a conduit for supplying an oxygen-containing gas, which contains oxygen-containing micro-nano bubbles in the culture solution during a biological reaction.
A pipeline for supplying a bactericidal gas such as ozone, which contains micro-nano bubbles containing a bactericidal gas such as ozone, in the cleaning liquid at the time of cleaning and sterilizing the biological reaction device after the completion of the biological reaction. With a micro-nano bubble generator connected to
The biological reaction apparatus according to any one of claims 1 to 4, wherein the biological reaction apparatus comprises. The micro-nano bubble generator in which the culture solution contains the micro-nano bubbles containing oxygen and the micro-nano bubble generator in which the cleaning solution contains micro-nano bubbles containing a bactericidal gas such as ozone are the same device. Therefore, the micro-nano bubble generator is provided with a switchable conduit for supplying the gas containing oxygen and a conduit for supplying a gas having bactericidal properties such as ozone. The biological reaction apparatus according to claim 5, wherein the biological reaction apparatus is characterized. The micro-nano bubble generator that contains the micro-nano bubbles containing oxygen in the culture solution and the micro-nano bubble generator that contains the micro-nano bubbles containing a bactericidal gas such as ozone in the cleaning solution are separate devices. The biological reaction apparatus according to claim 5, wherein there is. The biological reaction apparatus according to any one of claims 1 to 7, wherein the micro-nano bubble generator is provided outside the culture tank, and the micro-nano bubbles are blown into the culture solution or the washing solution. A claim, wherein a micro-nano bubble generator is provided in a conduit for supplying the culture solution or the cleaning solution to the culture tank, and the culture solution or the cleaning solution supplied to the culture tank contains micro-nano bubbles. Item 8. The biological reaction apparatus according to any one of Items 1 to 8. The filter comprises a hollow fiber membrane and a container for accommodating the hollow fiber membrane, and the culture solution is supplied to the inside of the hollow fiber membrane, and the filtrate is taken out from the outside through the hollow fiber membrane. The biological reaction apparatus according to any one of claims 3 to 9. The biological reaction apparatus according to any one of claims 3 to 10, further comprising a micro-nano bubble generator in which the filter solution or the cleaning solution that has passed through the filter contains micro-nano bubbles. A pipeline for supplying the cleaning liquid, which is connected to the secondary side of the filter, and
A micro-nano bubble generator to which a pipeline for supplying a bactericidal gas such as ozone is connected, in which the cleaning liquid contains micro-nano bubbles containing a bactericidal gas such as ozone.
The biological reaction apparatus according to any one of claims 3 to 11, wherein the biological reaction apparatus comprises. After completion of the biological reaction, a filter for separating the concentrated solution in which the microorganism or cells are concentrated from the above-mentioned culture solution contained in the above-mentioned culture tank, and a filter.
It is provided with an inactivating tank for accommodating the culture solution and / or the concentrate.
A tube for supplying a bactericidal gas such as ozone, which contains micro-nano bubbles containing a bactericidal gas such as ozone in the culture solution and / or the concentrated solution contained in the inactivation tank. The biological reaction apparatus according to claim 1, further comprising a micro-nano bubble generator to which a path is connected. The biological reaction apparatus according to claim 13, wherein the inactivating tank is provided with a liquid feeder for supplying a liquid to the concentrated liquid to adjust the viscosity. By the biological reaction apparatus according to any one of claims 5 to 7 above.
At the time of biological reaction, the culture solution contained in the culture tank contains the oxygen-containing micro-nano bubbles to produce a reactant such as a metabolite of the microorganism or cell.
At the time of cleaning and sterilization after the completion of the biological reaction, the cleaning liquid supplied to the culture tank contains micro-nano bubbles containing a bactericidal gas such as ozone, and the inside of the biological reaction device is cleaned and sterilized with the cleaning liquid. To do,
A biological reaction method characterized by. According to the biological reaction apparatus according to claim 11 or 12 above.
At the time of biological reaction, the culture solution contained in the culture tank contains the oxygen-containing micro-nano bubbles to produce a reactant such as a metabolite of the microorganism or cell.
At the time of cleaning and sterilization after the completion of the biological reaction, the cleaning solution supplied to the culture tank contains micro-nanobubbles containing a gas having bactericidal properties such as ozone, and the inside of the biological reaction device is cleaned and sterilized with the cleaning solution. Backwash to clean and sterilize the inside of the biological reactor with the normal washing and the washing liquid containing micro-nanobubbles containing a bactericidal gas such as ozone supplied to the secondary side of the filter. I do,
A biological reaction method characterized by the fact that. According to the biological reaction apparatus according to claim 13 or 14.
After the biological reaction is completed, the culture solution is withdrawn from the culture tank.
The concentrate is separated by the filter and stored in the inactivated tank.
A biological reaction method, which comprises sterilizing the microorganisms or cells by containing micro-nano bubbles containing a bactericidal gas such as ozone in the concentrated solution contained in the inactivating tank. The biological reaction according to claim 17, wherein the ratio of the medium contained in the culture solution entering the concentrate side is 50% or less, preferably 25% or less by separation by the filter. Method. The biological reaction method according to claim 17 or 18, wherein a liquid is supplied to the concentrated solution contained in the inactivating tank to adjust the viscosity.

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