JP2010046644A - Device for defoaming, culture system, and container with device for defoaming - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for defoaming, a culture system, and a container with the device for defoaming, which have high cleanability, are capable of easily carrying out disassembly cleaning, and excels in usability. <P>SOLUTION: The culture system 1 includes a culture vessel 2 filled with culture liquid, a stirrer 3, and a defoaming device 4 defoaming foam generated by stirring the culture liquid. The defoaming device 4 includes a vane wheel 41, a casing outer periphery part 42 and a casing rear face part 43 housing the vane wheel 41. The vane wheel 41 has a plurality of vane plates, and a suction plate provided at a suction side of the vane plates and formed with a suction hole. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an antifoaming device, a culture system, and a container with an antifoaming device, and includes, for example, foam generated when cultivating microorganisms (including foam generated when cultivating genetically modified bacteria), and the like. The present invention relates to a defoaming device, a culture system, and a container with a defoaming device that defoams bubbles generated by chemical synthesis by a physical action of an impeller.

Conventionally, culture apparatuses have been used in the manufacture of pharmaceuticals and the like. In particular, in the case of culturing genetically modified bacteria, etc., the following three items are required for the culture apparatus.
First, it must be completely sealed because a highly sterile environment is required.
The second is to have high detergency in order to perform pure culture. Here, having high detergency means that dirt is removed to a high level by washing.
The third is that the characteristics of the components that make up the device are clear and do not adversely affect the quality of the cells to be cultured or the product to be produced.
In addition, it is necessary to prove that the above three items can be realized in a culture apparatus for actually cultivating recombinant bacteria of genes.

According to the third demand, for example, the above-mentioned culture apparatus efficiently defoams bubbles generated when cultivating a recombinant bacterium without using an antifoaming agent such as silicone oil or a surfactant. There is a need. If an antifoaming agent such as silicone oil or surfactant is used, this antifoaming agent remains as an impurity in the product, so it must be removed or proved harmless. However, it is not desirable to introduce impurities into the bacteria.
For this reason, various techniques for defoaming without using an antifoaming agent have been studied.

For example, Patent Document 1 discloses an aeration tank or the like characterized in that a rotating shaft appropriately provided with a propeller-shaped fan having both a defoaming effect and a foam suction effect is mounted in a tubular body for preventing foam scattering. The technology of an antifoaming device is disclosed.
In this defoaming device, bubbles on the liquid surface are guided to the opening of the tube body, and the introduced bubbles are mechanically crushed by the fan and the inner surface of the tube body, and the liquid scattered on the inner surface of the tube body is discharged. Then, it travels down the inner surface of the tube and falls to the liquid level.

Further, in Patent Document 2, a disk that can be rotated via a rotation shaft and that has a plurality of bubble breaking blades radially attached to the lower surface thereof, and is installed so as to face the bubble breaking blades of the disk. In addition, there is disclosed a technique of a bubble breaking device characterized in that a portion corresponding to the rotation axis of the disk is composed of a counter plate in which a bubble suction port is formed.
In this foam breaking device, the disc and the foam breaking blade are housed in the opposing plate, the skirt-like stability frame and the liquid plate to be separated, and the bubbles on the liquid level are guided to the skirt-like stability frame, and the opposing plate The liquid that has been sucked into the bubble inlet and broken is transferred to the inner surface of the plate to be separated and falls to the liquid surface.

Patent Document 3 discloses a technology of a liquid tank that includes a tank that stores liquid and a foam suction device that sucks foam generated on the liquid level of the tank by the rotation of the impeller and can eliminate the foam. .
This foam suction device has a casing that houses the impeller, and has an inlet that opens toward the center of the impeller on the front side of the casing facing the impeller so that the foam is guided to the inlet. A cylindrical portion having a suction port at the lower end that opens on the liquid level of the tank through the inlet is provided in the casing, and the foam that has undergone centrifugal force due to the centrifugal action of the impeller is transferred from the inlet to the outer peripheral side of the impeller. It has a casing inner wall part that collides so as to be destroyed on the outer periphery of the impeller, and has a discharge port on the outer side of the impeller of the casing that discharges the liquid caused by the disappearance of bubbles together with air into the tank by collision with the casing inner wall part. .
Japanese Utility Model Publication No. 53-60365 Japanese Utility Model Publication No. 54-150176 Japanese Patent No. 3604873

However, since the defoaming device of Patent Document 1 discharges the defoamed gas from the inside of the tube to the outside of the defoaming device, it cannot be used for a culture device for culturing a recombinant bacterium or the like of a gene. There was a problem.
In addition, the foam breaking device of Patent Document 2 has a problem in that the structure of the counter plate, the skirt-like stability frame, the liquid plate to be separated, and the like is complicated, and the cleaning performance cannot be improved.
Furthermore, the liquid tank capable of defoaming in Patent Document 3 has a problem in that the structure of the cylindrical portion and the discharge port is complicated and the cleaning performance cannot be improved.
In addition, an antifoaming device used for a culture device is required to be easily disassembled and washed and to be easy to use. Furthermore, it is desired that it can be easily attached to an existing culture apparatus.

  In order to solve the above problems, the present invention provides a defoaming apparatus, a culture system, and a container with a defoaming apparatus that have high detergency, can be easily disassembled and washed, and are excellent in usability. With the goal.

  In order to achieve this object, an antifoaming device according to the present invention comprises an impeller and a casing for housing the impeller, wherein the impeller includes a plurality of blade plates and a suction plate for the blade plates. A suction plate formed integrally with the side and formed with a suction port for bubbles, and a casing having a rear surface portion located on the anti-suction side of the impeller, and an outer peripheral portion located on the outer peripheral side of the impeller It is set as the structure which has.

Further, the culture system of the present invention includes a culture tank containing a culture solution, a stirring device for stirring the culture solution, and a defoaming device for removing bubbles generated by stirring the culture solution. The configuration using the defoaming device of the present invention is used.

  Moreover, the container with a defoaming device of the present invention includes a container containing a liquid and a defoaming device for erasing bubbles generated from the liquid, and the defoaming device of the present invention is used as the defoaming device. . Here, examples of the container in the present invention include containers used for chemical synthesis of various liquids.

  According to the defoaming apparatus, the culture system, and the container with the defoaming apparatus in the present invention, it has high cleaning properties, can be easily disassembled and cleaned, and can improve usability.

[First embodiment of culture system and defoaming apparatus]
FIG. 1: has shown schematic front direction sectional drawing for demonstrating the culture system and antifoamer concerning 1st embodiment of this invention.
FIG. 2 is a schematic plan view for explaining the culture system and the defoaming apparatus according to the first embodiment of the present invention.
1 and 2, a culture system 1 includes a culture tank 2 containing a culture solution (not shown), a stirring device 3 for stirring the culture solution, and bubbles (not shown) generated by stirring the culture solution. ) Is removed.
In addition, the culture system 1 of the present embodiment is a culture apparatus that cultures recombinant bacteria of genes.

(Culture tank)
The culture tank 2 includes a flange 21, a cylindrical member 22, a bottom plate 23, a connecting rod 24, an upper lid 25, and the like.
Further, the flange 21 is usually a substantially annular flat plate made of stainless steel, and a bolt 211 that is rotatably disposed at six positions (equally spaced positions in the circumferential direction) of the peripheral edge, and the bolt 211. And a wing nut 212 that is tightened into the flange 21 and an O-ring 213 that seals the flange 21 and the upper lid 25. Thus, the upper lid 25 is easily attached to the flange 21 in a sealed state.
Furthermore, the cylindrical member 22 is made of transparent resin or tempered glass, and is sandwiched between the flange 21 and the bottom plate 23 via a gasket, a sealant, or the like. Here, the flange 21 and the bottom plate 23 sandwiching the cylindrical member 22 are connected by six connecting rods 24 so as to be disassembled.
Further, the bottom plate 23 is usually a resin disc, and a driven portion and a drive portion of the magnet coupling 34 are rotatably provided at substantially the center between the upper surface and the lower surface.

The upper lid 25 is usually a substantially annular flat plate made of stainless steel, and is provided with an air supply pipe 51, an exhaust pipe 52, three support columns 35, and the like at positions corresponding to the inside of the cylindrical member 22. Although not shown for easy understanding, a joint for detecting temperature, ph value, and the like is generally provided. Further, the air supply pipe 51, the exhaust pipe 52, the three support columns 35, and the like are attached to the upper lid 25 in a sealed state so that the culture tank 2 can maintain a sealed state.
Further, the casing 25 of the defoaming device 4 is welded and joined to the upper lid 25 substantially at the center, and a handle 251 is attached to the opposing outer edge.

The air supply pipe 51 has an upper end protruding above the upper lid 25, and an annular pipe 511 is connected to the lower end. This pipe 511 is provided in the lower part of the culture tank 2, and has a plurality of through holes (not shown). Thereby, the gas (for example, air) sent from the upper end part of the supply pipe | tube 51 is discharge | released in the culture tank 2 from a some through-hole.
Further, the lower end of the exhaust pipe 52 is at substantially the same height as the lower surface of the upper lid 25, and exhausts the gas above the culture tank 2.
In addition, the culture tank 2 is not limited to said structure, The thing of various structures can be used.

(Agitator)
The stirring device 3 includes stirring blades 31 and 32, a stirring shaft 33, a magnet coupling 34, a support 35, a shaft support plate 36, a bottom plate 37, and the like.
The three struts 35 are attached to the upper lid 25 so as to be substantially equally spaced in the circumferential direction, and a disc-shaped bottom plate 37 is provided at the lower end. Further, each support column 35 is provided with a shaft support plate 36 toward the center of the upper part of the culture tank 2.
The stirring shaft 33 has an upper end portion and a lower end portion that are pivotally supported by a shaft support plate 36 and a bottom plate 37, respectively, and a driven portion of a magnet coupling 34 is attached to the lower portion. A stirring blade 31 is attached. The drive unit of the magnet coupling 34 is connected to a drive shaft of a variable speed motor (not shown) and rotates at a predetermined rotation speed (usually several tens to several hundred rotations per minute).
In addition, the stirring apparatus 3 is not limited to said structure, A thing of various structures can be used.

(Defoaming device)
The defoaming device 4 of this embodiment includes an impeller 41, a casing outer peripheral portion 42, a casing back surface portion 43, a bearing / shaft housing 44, a joint housing 45, a motor 46, and the like.

FIG. 3: is the schematic for demonstrating the impeller of the defoaming apparatus of the culture system concerning 1st embodiment of this invention, (a) has shown the front view, (b) is A- A sectional view is shown.
In FIG. 3, the impeller 41 includes a base 411, a blade plate 412, and a suction plate 413. In addition, although stainless steel is used as a material of the impeller 41, it is not limited to this.
The base 411 has a columnar shape, and a through hole 414 into which the shaft 410 is inserted is formed along the central axis, and a female screw 415 is cut from the side surface direction toward the through hole 414. A hexagon socket set screw 419 is tightened on the female screw 415 and the impeller 41 is fixed to the shaft 410 by coming into contact with the shaft 410 inserted into the through hole 414.

The vane plate 412 is a substantially pentagonal flat plate, that is, has a pentagonal shape with inclined surfaces 416 formed on the base 411 side and the suction side of the rectangular plate. The six blade plates 412 are welded and joined to the side surfaces of the base portion 411 so that the end portions on the base portion 411 side are substantially equidistant in the circumferential direction.
Here, when the inclined surfaces 416 are formed, the rectangular plate is cut out, whereby a suction opening 418 is formed in the central portion on the suction side of the impeller 41. If it does in this way, when the impeller 41 rotates, this suction opening 418 becomes a negative pressure, so the impeller 41 can suck the bubbles effectively.

The suction plate 413 is an annular flat plate and is welded to the suction side edge of each vane plate 412. The suction plate 413 has an outer diameter corresponding to the position of the end portion on the outer peripheral side of the blade plate 412, and an inner diameter corresponding to the position of the end portion on the suction side of the inclined surface 416. Therefore, the inside of the inner diameter of the suction plate 413 is a suction hole 417.
Further, in the impeller 41 of this embodiment, the suction opening 418 is formed at a position corresponding to the suction hole 417. In this manner, the suction plate 413 and the suction hole 417 effectively exert the suction and discharge operations of the blade plate 412, so that the defoaming efficiency by the impeller 41 can be improved.

  Here, the suction plate 413 functions as, for example, a suction side portion of a fan casing (not shown), and the suction hole 417 functions as a suction port of a fan casing (not shown). That is, the impeller 41 has a suction plate 413 that functions as a suction side portion of the casing and a suction port. Thereby, a structure is simplified and the washing | cleaning property of the impeller 41, the casing outer peripheral part 42, and the casing back surface part 43 can be improved in total.

FIG. 4: is a schematic enlarged view for demonstrating the casing of the defoaming apparatus of the culture system concerning 1st embodiment of this invention, (a) has shown front direction sectional drawing, (b) is The BB arrow line view is shown.
In FIG. 4, the casing outer peripheral portion 42 has a cylindrical shape that accommodates the impeller 41 and is provided on the outer peripheral side of the impeller 41. The casing outer peripheral portion 42 is welded and joined to the upper lid 25 in a state where the lower end portion protrudes downward from the upper lid 25 and is at the same height as the lower surface of the flange 21. Further, the suction plate 413 of the impeller 41 is substantially at the same height as the lower surface of the upper lid 25. If it does in this way, even if it is a case where the liquid level is approaching the lower surface of the flange 21, the impeller 41 can suck | inhale the bubble which generate | occur | produced on the liquid level effectively, and can defoam.

  That is, when the impeller 41 rotates, the suction opening 418 has a negative pressure, and bubbles generated on the liquid surface can be effectively sucked from the suction holes 417 of the suction plate 413. The bubbles sucked into the impeller 41 are crushed by colliding with the blade plate 412, and are blown from the blade plate 412 in the outer peripheral direction, and are crushed by colliding with the inner surface of the casing outer peripheral portion 42, It is divided into a liquid and a gas and moves downward along the inner surface of the casing outer peripheral portion 42.

  Here, although not shown in the figure, at a position slightly below the suction plate 413 with respect to the wind generated in the outer peripheral direction by the impeller 41 and moving downward along the inner surface of the casing outer peripheral portion 42. A direction plate for changing the direction of the wind may be provided, and the plate may be sent in a substantially horizontal direction along the outer circumferential direction and along the lower surface of the upper lid 25. In this way, the wind sent out in the outer peripheral direction and substantially horizontally along the lower surface of the upper lid 25 descends along the inner surface of the cylindrical member 22 and circulates toward the approximate center of the liquid surface. Thereby, the foam around the inner surface of the cylindrical member 22 can be moved almost directly below the suction hole 417 by the circulating wind, and the defoaming performance can be improved.

The casing back surface portion 43 has a substantially disk shape that is fitted into the upper portion of the casing outer peripheral portion 42, and is provided on the anti-suction side of the impeller 41. The casing back surface portion 43 has a shaft 410 passing through the center, and a bearing / shaft seal housing 44 is screwed onto the top surface.
Further, the casing back surface portion 43 accommodates an O-ring 431 at the lower part of the side surface, and the O-ring 431 seals the inside of the culture tank 2 from the outside.

  Further, the casing back surface portion 43 has pin-attached screws 432 fastened at three locations on the upper side (positions at substantially equal intervals in the circumferential direction). The pin at the head of the pinned screw 432 is engaged in a hook-shaped locking groove 421 formed in the upper portion of the casing outer peripheral portion 42. That is, the casing back surface portion 43 is lowered, the pin at the head of the pin-attached screw 432 is engaged with the locking groove 421, and the casing back surface portion 43 is rotated in the clockwise direction, thereby the casing back surface portion 43. Can be easily attached to the outer peripheral portion 42 of the casing. By returning to this operation, the casing back surface portion 43 can be easily detached from the casing outer peripheral portion 42. If it does in this way, the defoaming apparatus 4 of this embodiment can perform a decomposition | disassembly washing | cleaning easily, and can further improve a washability.

  In this embodiment, when the casing back surface portion 43 is removed from the casing outer peripheral portion 42, the impeller 41 can come out of the casing outer peripheral portion 42. In this way, disassembly and cleaning can be performed more easily, and usability can be improved.

In the present embodiment, the casing back surface portion 43 is attached to the casing outer peripheral portion 42 using the pinned screw 432 and the locking groove 421, but the present invention is not limited to this configuration. For example, although not shown, a thumbscrew may be tightened into the screw hole instead of the pinned screw 432 and the locking groove 421.
In the present embodiment, the casing outer peripheral portion 42 is welded and joined to the upper lid 25, and the casing back surface portion 43 is detachably attached to the casing outer peripheral portion 42. However, the present invention is not limited to this configuration. For example, although not shown, the casing back surface portion 43 and the casing outer peripheral portion 42 may be integrally formed, and the casing outer peripheral portion 42 may be detachably attached to the upper lid 25.

The bearing / shaft housing 44 has a substantially cylindrical shape, and a lower portion is screwed to the casing back surface portion 43. Ball bearings 441 are attached to the bearing / shaft seal housing 44 at two locations in the middle part, and support the shaft 410. In addition, oil seals 442 are respectively attached to the upper end portion and the lower end portion to seal the liquid in the culture tank 2. Here, it is preferable that the pressure in the space sandwiched between the oil seals 442 be higher than the pressure in the culture tank 2, and in this way, the sealing performance can be improved.
The bearing / shaft housing 44 is configured to use a two-stage oil seal 442, but is not limited to this configuration. For example, although not shown, a mechanical seal or the like may be used instead of the oil seal 442.

The joint housing 45 has a substantially rectangular parallelepiped shape having a space for accommodating the coupling 451 therein, and is detachably attached to the upper surface of the bearing / shaft seal housing 44. The coupling 451 connects the upper end of the shaft 410 and the drive shaft of the motor 46.
The motor 46 is usually a variable speed motor, and rotates at a predetermined rotation speed (usually several hundred to several thousand rotations per minute).

As described above, the culture system 1 of the present embodiment has a simple structure because the impeller 41 of the defoaming device 4 includes the suction plate 413 that functions as a suction side portion and a suction port of the casing. Thus, the cleaning performance of the impeller 41, the casing outer peripheral portion 42, and the casing back surface portion 43 can be improved in total. Thus, for example, a high level of washing for pure culture can be performed.
Furthermore, since the casing back surface portion 43 and the impeller 41 can be easily detached from the casing outer peripheral portion 42, disassembly and cleaning can be easily performed, and usability can be improved.

Moreover, the culture system 1 of this embodiment has various application examples. For example, although not shown, a liquid feed pump and an aspirator connected to a discharge pipe of the liquid feed pump may be used instead of the stirring device 3 and the air supply pipe 51 (including an air supply fan). Good. The amount of air can be controlled to 1/10 to 1/50 by aeration using this aspirator.
In addition, an aspirator means the instrument for producing a pressure reduction state by the venturi effect using a fluid.

[Second embodiment of defoaming apparatus]
FIG. 5: has shown schematic front direction sectional drawing for demonstrating the defoaming apparatus concerning 2nd embodiment of this invention.
In FIG. 5, the defoaming device 4 a of this embodiment includes a foam introduction unit 425 for introducing foam and a casing collection unit 47 for collecting the defoamed liquid, as compared with the defoaming device 4 described above. And the point that the defoaming device 4a is attached to an existing jar fermenter (not shown). Other configurations are almost the same as those of the defoaming device 4.
Therefore, in FIG. 5, the same components as those in FIGS. 1, 2, 3, and 4 are denoted by the same reference numerals, and detailed description thereof is omitted.
The jar fermenter refers to an apparatus used for large-scale culture of microorganisms. Usually, conditions necessary for culture of microorganisms such as temperature, aeration volume, stirring speed, and ph can be kept constant.

  The defoaming device 4a of this embodiment includes an impeller 41, a casing outer peripheral portion 42a, a casing back surface portion 43a, a bearing / shaft seal housing 44, a joint housing 45, a motor 46, a casing recovery portion 47, and the like.

The casing outer peripheral part 42 a includes a pair of ferrules 422, a ferrule gasket 423, a sanitary pipe 424, and a foam introduction part 425.
The ferrule 422 and the sanitary pipe 424 have an inner diameter for accommodating the impeller 41, and the ferrule 422 is welded to the upper and lower parts of the sanitary pipe 424.
In addition, although not shown in figure, the casing outer peripheral part 42a may provide a sight glass (view window) in the front side, for example.

Further, the bubble introduction part 425 includes a ferrule 4251, a sanitary pipe 4252 and a sanitary T-shaped pipe 4253. The sanitary T-shaped pipe 4253 is provided so that the center line of the pipe having the opposed opening coincides with the central axis of the impeller 41, and the pipe having the remaining one opening faces leftward. ing.
Here, the pipe having the opposed opening has an upper end located between the upper surface of the suction plate 413 and the lower surface of the base 411 of the impeller 41, and a lower end located below the lower ferrule 422. ing. If it does in this way, a bubble will be reliably suck | inhaled by the impeller 41 from the upper end part of this piping, and a liquid can fall to the casing collection | recovery part 47 from a lower end part. In addition, the suction force of the bubble introduction part 425 can also be adjusted by making the opening area of a lower end part smaller than the opening area of an upper end part.

The pipe having the remaining one opening of the sanitary T-shaped pipe 4253 is welded and joined to the sanitary pipe 4252. The sanitary pipe 4252 is welded and joined in a state of penetrating the sanitary pipe 424. The other end of the sanitary pipe 4252 is welded to the ferrule 4251.
Although not shown, the ferrule 4251 is easily connected to the sanitary piping and ferrule from the jar fermenter and the ferrule gasket and clamp. That is, the foam in the jar fermenter is introduced into the impeller 41 through the foam introduction unit 425.

The casing back surface portion 43a has a substantially disc shape having a ferrule structure corresponding to the ferrule 422, and is detachably attached to the casing outer peripheral portion 42a via a ferrule gasket 423 and a clamp (not shown).
Further, the bearing / shaft seal housing 44, the joint housing 45, and the motor 46 described above are provided in this order on the upper surface of the casing back surface portion 43a.

The casing collection unit 47 includes a ferrule 471, a truncated cone member 472, and a ferrule 473. The ferrule 471 is detachably attached via a ferrule 422 below the casing outer peripheral portion 42a, a ferrule gasket 423, and a clamp (not shown).
The truncated cone member 472 is a member in which a thin stainless steel plate is formed into a truncated cone shape (funnel shape), and an end portion having a large diameter is welded to the ferrule 471 and an end portion having a small diameter is joined to the ferrule 473. It is welded. Although not shown, the ferrule 473 is easily connected to the sanitary piping and ferrule to the jar fermenter through a ferrule gasket and a clamp. That is, the defoamed liquid can be recovered smoothly and efficiently through the casing recovery unit 47 to the jar fermenter.

As described above, the defoaming device 4a of the present embodiment can obtain substantially the same effect as the defoaming device 4 of the first embodiment. Further, even when the defoaming device 4a is not attached to the jar / fermenter substantially integrally, the defoaming device 4a is detachably attached to the jar / fermentor via a pipe or a ferrule. Can do.
Furthermore, the defoaming device 4a draws the bubbles in the jar fermenter through the foam introduction part 425, defoams them by the impeller 41 and the casing outer peripheral part 42a, and further smoothly through the casing collection part 47. In addition, the defoamed liquid can be efficiently collected in the jar fermenter.

Moreover, the defoaming apparatus 4a of this embodiment has various application examples. For example, as indicated by a dotted line in FIG. 5, an exhaust part 426 including a sanitary pipe 4261 and a ferrule 4262 may be provided on the right side surface of the sanitary pipe 424. In this way, by providing the defoaming device 4a between the exhaust lines (not shown) of the jar fermenter, the bubbles in the exhaust line are drawn in via the foam introduction part 425, and the defoaming is performed. The liquid thus collected can be recovered via the casing recovery part 47, and the gas containing no bubbles can be discharged via the exhaust part 426.
Further, the exhaust unit 426 may exhaust air via a hepa filter. If it does in this way, the outflow of a spray-like liquid can be prevented effectively.
The hepa (HEPA) filter is a high-performance filter used in a clean room of a semiconductor factory.

By the way, the culture system (cultivation apparatus) for cultivating the recombinant bacteria of the gene, as described above, has excellent sealing properties, cleanability, and culture properties (without adversely affecting the cells to be cultured. , That can be cultured effectively).
Next, since culture was actually performed using the culture system 1 described above, an example will be described. In addition, this invention is not limited to this Example.

<Examples of culture system culture>
About the defoaming capability in the culture system 1, and the influence which it has on a culture microorganism, it verified as follows.
The culture conditions of the culture system 1 were as follows, and aerated and stirred culture.
Culture solution volume: 5000ml (milliliter)
Culture room temperature (starting temperature): 27 ° C
Air supply amount: 3000 ml (milliliter) / min
Stirring device: rotation speed of stirring blade = 200 rpm / min, outer diameter of stirring blade 31 = 104 mm, outer diameter of stirring blade 32 = 120 mm
Defoaming device: rotational speed of impeller 41 = 1400 rpm / min, outer diameter of impeller 41 = 100 mm, height of impeller 41 = 36 mm, diameter of suction hole 417 = 66 mm

<Culture reference example>
As a reference example for the culture system 1, a 1000 ml (milliliter) Erlenmeyer flask with baffle (hereinafter, Erlenmeyer flask) was used, and “culture volume: 100 ml (milliliter), stirring rotation speed: 145 rpm / min, culture chamber temperature ( Under the conditions of “starting temperature): 27 ° C.”, shaking culture was performed.

In the above culture examples and culture reference examples, Bacillus subtilis, which is an obligately aerobic Gram-positive rod, was prepared as a microorganism to be used. Furthermore, in order to verify the influence of the culture system 1 and the defoaming device 4 on the cells, the outer shell protein (His-JEC3) of Japanese encephalitis virus (JEV) with HisTag added to the N-terminus is added to the culture solution. A genetically engineered Bacillus subtilis secreted in was used.
Next, after inoculating inoculum corresponding to 1/100 of the culture volume into the culture system 1 and the Erlenmeyer flask, the culture is started under the above conditions, and the culture is performed for 48 hours while sampling over time. It was.

The results are shown below.
In the culture system 1, although the generation of bubbles by stirring was observed immediately after the start of the culture, the bubbles were colorless and transparent, the viscosity was low, and the diameter was often large. The bubbles could be extinguished by operating the defoaming device 4, and disappeared naturally by stopping the stirring.
As the culture time progressed (the growth of Bacillus subtilis), the foam became brownish yellow and became viscous and its diameter changed to a small one. As a result, when the defoaming device 4 was not movable, the bubbles rose to the top plate (the lower surface of the upper lid 25), and it was observed that the culture solution was discharged from the exhaust pipe 52. However, by operating the defoaming apparatus 4, the bubbles were crushed by suction and circulated into the culture solution, and even after 48 hours, the culture solution was hardly reduced.

FIG. 6 is a graph showing the transition of dissolved oxygen concentration (DO: ppm) in the culture solution up to 48 hours in the culture examples.
In FIG. 6, the dissolved oxygen concentration in the culture solution gradually decreased and consumed oxygen in the solution over about 15 hours from the start of the culture. This was correlated with the growth of Bacillus subtilis (FIG. 2), and it was found that oxygen was consumed with the growth. In addition, after 15 hours, the state of low oxygen concentration in the culture medium was observed, but the growth of Bacillus subtilis (Fig. 2) was slow and consumed oxygen for protein synthesis. It was inferred that

FIG. 7 shows O.D. up to 48 hours in the culture examples. D. It is a graph which shows transition of 600 (indicator of the number of microbial cells).
In FIG. 7, a sample was diluted 3 times with a culture solution, and a wavelength of 600 nm was measured with a spectrophotometer. It was observed that Bacillus subtilis grew rapidly from about 10 hours to about 24 hours from the start of the culture. After 24 hours, as shown by the variation in the graph, there was no large change in the numerical value, and it was observed that the growth rate was slow.

FIG. 8 is a graph showing changes in the temperature (° C.) of the culture solution up to 48 hours in the culture example.
In FIG. 8, an increase in temperature was observed over about 20 hours. This was correlated with the dissolved oxygen concentration (FIG. 1) and the growth of Bacillus subtilis (FIG. 2), and it was assumed that the temperature of the culture solution increased with the growth of Bacillus subtilis.
Moreover, after 20 hours, it fluctuates within a range of about 0.4 ° C., and the influence of the conduction heat from each drive motor of the stirring device 3 and the defoaming device 4 on the increase in the temperature of the culture solution Inferred to be low.

FIG. 9 shows the results of analyzing the protein secreted in the culture solution by SDS-PAGE (sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE)) in the culture examples and the culture reference examples. Yes.
As shown in FIG. 9, in both the culture examples and the culture reference examples, the protein bands are generally concentrated as the culture time elapses. It seems that the number of people is increasing.
The electrophoresis conditions were polyacrylamide gel concentration = 15%, sample amount = 6 μl (microliter), current = 20 mA, time = 60 min.

FIG. 10 shows the result of analyzing the sample used in FIG. 9 by Western blot.
As shown in FIG. 10, in both the culture examples and the culture reference examples, secretion of His-JEC3 was observed in the culture solution at 12 hours of culture. The expression level increased with the lapse of the culture time, but at the same time, the detected level of the degradation product of His-JEC3 was seen to increase.
Various conditions in the Western blot analysis were as follows.
Electrophoretic conditions: polyacrylamide gel concentration = 15%, sample amount = 3.2 μl (microliter), current = 20 mA, time = 60 min
Transfer conditions: Current = 100 mA, time = 60 min, PVDF (Polyvinylidene difluoride) membrane was used.
Primary antibody: anti-JE Rabbit was used at 1/1000 concentration.
Secondary antibody: anti-Rabbit IgG HRP labeling was used at 1/1000 concentration.
Staining: Eto WestBlue from Ato was used.

  As described above, in the culturing examples, by using the culturing system 1, it was possible to cultivate genetically modified Bacillus subtilis in a large amount and normally compared to the Erlenmeyer flask of the culturing reference example. That is, the effectiveness of the culture system 1 could be proved by an actual culture test.

[One Embodiment of Container with Defoaming Device]
The present invention is also effective as an invention of a container with a defoaming device.
That is, although the container with a defoaming device of this embodiment is not illustrated, it is provided with a container containing a liquid for chemical synthesis and a defoaming device that erases bubbles generated during chemical synthesis in the container. As the defoaming device, the defoaming device 4 or the defoaming device 4a is used.
Here, the shape, size, or structure of the container is not particularly limited.
Moreover, the liquid contained in the container is not particularly limited. For example, a liquid for culturing microorganisms or a liquid for performing predetermined chemical synthesis. Here, examples of the predetermined chemical synthesis include acetate synthesis, polymerization reaction of synthetic resin and latex, and emulsion polymerization reaction.

  According to this container with a defoaming device, the impeller 41 of the defoaming device 4 or the defoaming device 4a has the suction plate 413 functioning as a suction side portion and a suction port of the casing, as described above. In addition, the cleaning performance can be improved in total. Moreover, disassembly and cleaning can be easily performed, and usability can be improved.

As described above, the defoaming device, the culture system, and the container with the defoaming device according to the present invention have been described with reference to the preferred embodiments. Needless to say, the present invention is not limited to the embodiments, and various modifications can be made within the scope of the present invention.
For example, although the impeller 41 of the defoaming device 4 uses six flat blade plates 412, the number and shape of the blade plates 412 are not limited to the above. Various configurations can be made according to the above.
Moreover, although the defoaming apparatus 4a is attached to a jar fermenter, it is not limited to this, For example, it can attach to various containers etc.

FIG. 1: has shown schematic front direction sectional drawing for demonstrating the culture system and antifoamer concerning 1st embodiment of this invention. FIG. 2 shows a schematic plan view for explaining the culture system and the defoaming apparatus according to the first embodiment of the present invention. FIG. 3: is the schematic for demonstrating the impeller of the defoaming apparatus of the culture system concerning 1st embodiment of this invention, (a) has shown the front view, (b) is A- A sectional view is shown. FIG. 4: is a schematic enlarged view for demonstrating the casing of the defoaming apparatus of the culture system concerning 1st embodiment of this invention, (a) has shown front direction sectional drawing, (b) is The BB arrow line view is shown. FIG. 5: has shown schematic front direction sectional drawing for demonstrating the defoaming apparatus concerning 2nd embodiment of this invention. FIG. 6 is a graph showing the transition of dissolved oxygen concentration (DO: ppm) in the culture solution up to 48 hours in the culture examples. FIG. 7 shows O.D. up to 48 hours in the culture examples. D. It is a graph which shows transition of 600 (indicator of the number of microbial cells). FIG. 8 is a graph showing changes in the temperature (° C.) of the culture solution up to 48 hours in the culture example. FIG. 9 shows the results of analyzing the protein secreted in the culture solution by SDS-PAGE (sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE)) in the culture examples and the culture reference examples. Yes. FIG. 10 shows the result of analyzing the sample used in FIG. 9 by Western blot.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Culture system 2 Culture tank 3 Stirrer 4, 4a Defoamer 21 Flange 22 Cylindrical member 23 Bottom plate 24 Connecting rod 25 Top cover 31 Stirrer blade 32 Stirrer blade 33 Stirrer shaft 34 Magnet coupling 35 Strut 36 Shaft support plate 37 Bottom plate 41 Blade Cars 42, 42a Casing outer peripheral parts 43, 43a Casing rear part 44 Bearing / shaft housing 45 Joint housing 46 Motor 47 Casing recovery part 51 Air supply pipe 52 Exhaust pipe 211 Bolt 212 Wing nut 213 O-ring 251 Handle 410 Shaft 411 Base part 412 Blade Plate 413 Suction plate 414 Through hole 415 Female thread 416 Slope 417 Suction hole 418 Suction opening 419 Hexagon socket set screw 421 Locking groove 422 Ferrule 423 Ferrule gasket 424 Sanitary piping 425 Foam introduction part 426 Exhaust part 431 O phosphorus 432 Pinned screw 441 Ball bearing 442 Oil seal 451 Coupling 471 Ferrule 472 Ferrule 473 Ferrule 511 Pipe 4251 Ferrule 4252 Sanitary pipe 4253 Sanitary T-shaped pipe 4261 Sanitary pipe 4262 Ferrule

Claims (10)

In the defoaming apparatus provided with the impeller and a casing for accommodating the impeller,
The impeller has a plurality of blade plates, and a suction plate integrally provided on the suction side of the blade plates and formed with a bubble suction hole,
The defoaming apparatus, wherein the casing has a back surface portion located on the anti-suction side of the impeller and an outer peripheral portion located on the outer peripheral side of the impeller.   The defoaming device according to claim 1, wherein the suction hole is formed in a central portion of the suction plate, and is joined to a suction side edge of the blade plate on an outer peripheral side of the suction hole.   The defoaming device according to claim 1, wherein a suction opening is formed at a position corresponding to the suction hole of the blade.   The defoaming device according to any one of claims 1 to 3, wherein a back surface portion of the casing is detachably attached to the outer peripheral portion.   The defoaming device according to claim 4, wherein the impeller can be inserted into and removed from the casing when the rear surface of the casing is removed from the outer peripheral portion of the casing.   The defoaming apparatus according to any one of claims 1 to 5, wherein the casing includes a foam introduction unit that introduces foam and a collection unit that collects the defoamed liquid.   The defoaming apparatus according to claim 6, wherein the casing has an exhaust part that discharges gas in the casing.   The defoaming device according to claim 6 or 7, wherein the recovery part of the casing has a funnel shape. In a culture system comprising a culture tank containing a culture solution, a stirring device for stirring the culture solution, and a defoaming device for removing bubbles generated by stirring the culture solution,
A culture system using the defoaming device according to any one of claims 1 to 8 as the defoaming device. In a container with a defoaming device comprising a container containing a liquid and a defoaming device that eliminates bubbles generated from the liquid,
A container with a defoaming device, wherein the defoaming device according to any one of claims 1 to 8 is used as the defoaming device.