JP2005040061A - Fermentation tank (jar fermenter) - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fermentation tank (jar fermenter) that is made of a membrane and circulates a culture solution and with which a yield is increased and an inexpensive culture cost is maintained by forced dispersion fall. <P>SOLUTION: This energy-saving type fermentation tank (jar fermenter) having a low running cost is equipped with a pump from a lower stirrer to an upper forced dispersion apparatus of the membrane tank, various sensors and a by-pass furnished with an inoculation port. The fermentation tank (jar fermenter) is enfolded, sterilized, mounted on a suspension system, swollen with sterile air or a mixed gas of nitrogen and carbon dioxide gas, charged with a sterilized culture solution and inoculated with a bacterium. The culture solution is circulated and an aerobic bacterium or anaerobic bacterium is cultured while forced dispersion. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a small general-purpose fermentation tank (jar fermenter) for the purpose of microbial growth in the field of natural science.

  In the conventional fermentation tank (jar fermenter), the means for blocking from the outside world is a metal sealed tank, the inoculation port, the pH adjusting acid solution container and its mounting port, the alkaline solution container and its mounting port, The defoamer container and its attachment port, the bubble detection sensor attachment port, the pressure gauge attachment port and the exhaust port are located at the top of the tank, the pH detection sensor attachment port, the dissolved oxygen detection sensor attachment port, and the temperature detection sensor The mounting port and baffle plate were provided on the tank body, the sterile air supply unit, and the stirring bar rotated by the motor were provided on the bottom of the tank.

  Such a metal fermentation tank (jar fermenter) is sterilized by a high pressure steam method. The fermentation tank (jar fermenter) is fixed at a fixed position, as well as a tank that can withstand high pressures, as well as piping and valves, high-pressure steam from the high-pressure steam generator to the tank body, and the amount of high-pressure steam that exceeds the tank capacity Must be sent. As a result, a high-pressure steam generator is required as ancillary equipment, and the tank itself is heavy, and the pH adjusting acid solution container and its attachment port, the alkaline solution container and its attachment port, the antifoaming agent container and its Mounting port, foam detection sensor mounting port, inoculation port, pressure gauge mounting port, exhaust port, pH detection sensor mounting port, temperature detection sensor mounting port, dissolved oxygen detection sensor mounting port, sterile air input port, and stir bar mechanical It is not easy to attach the seal part to the tank body. In addition, it is difficult for the user to clean the tank, the number of valves is large, and the operation is complicated.

  On the other hand, the main method of aeration is to send sterilized air from the bottom of the tank, and although there is an effect of staying in the culture medium of sterilized air by stirring and an effect of a baffle, sufficient gas exchange cannot be obtained, causing foaming. Therefore, it is necessary to provide an antifoaming wing or to add an antifoaming agent.

  Also, in the current fermentation tank (jar fermenter), anaerobic bacteria are almost impossible to cultivate even if sterile nitrogen gas and carbon dioxide mixed gas is supplied instead of sterile air. It is.

Takashi Toganehara: Dokkyo Journal, 33, 198 (1975)

  The problem to be solved by the present invention is a fermentation tank (jar fermenter) that does not require any additional equipment such as a boiler, can be sterilized with a small amount of steam, has no valves, and is easy to operate.

  In order to increase the yield of microorganisms or metabolites, a fermentation tank that can maintain the dissolved oxygen concentration required by microorganisms in the culture solution and can be used with a smaller amount of aseptic air than before. Mentor).

  A fermentation tank (jar fermenter) that requires no or a small amount of antifoam.

  It must be a fermentation tank (jar fermenter) that can cultivate anaerobic bacteria.

  In order to solve these problems, the container of the fermentation tank (jar fermenter) itself is made into a cylindrical container with a flexible membrane. For example, it may be made of a vinyl film or a silicon rubber film, folded and then sterilized with an ethylene oxide gas (EOG) sterilizer in the former case and a high-pressure steam sterilizer (autoclave) in the latter case.

  In order to meet the dissolved oxygen concentration required by microorganisms even if the amount of sterile air to be supplied is less than that of conventional ones, a bypass is provided to circulate the culture solution and increase the surface area through a forced dispersion device attached to the top. Then, by dropping the inside of the tank, effective gas exchange is performed, and when falling and colliding with the culture liquid surface, the gas in the tank is involved in the liquid, and an increase in dissolved oxygen can be expected.

  In addition, the surface area of the culture solution increases through the forced dispersion device, and when it falls into the culture solution surface and collides, it has a defoaming effect to eliminate bubbles, so if the carbon source of the medium composition is about 2%, No antifoaming agent is required during the culture process.

  Furthermore, if the tank sterilized by inflating it is inflated with a mixed gas of nitrogen gas and carbon dioxide gas that has been sterilized by filtration with a filter, etc., anaerobic bacteria culture that was impossible with conventional metal fermentation tanks (jar fermenters) Is possible.

  By folding the tank itself, it can be sterilized with a small laboratory-level high-pressure steam sterilizer and EOG sterilizer or EOG gas cylinder, heater equipped pressure reducing valve, and EOG gas processing machine. ) Does not require the necessary boiler and piping. In other words, the economic effect of requiring less capital investment is great.

  The conventional medium sterilization method for metal fermentation tanks (jar fermenters) requires high-pressure steam sterilization to cool the whole to the optimum temperature for cultivation, whereas the fermentation tank (jar fermenters) of the present invention ), The organic medium components dissolved in as little purified water as possible are sterilized in a small laboratory-sized high-pressure steam sterilizer, and the purified water is sterilized by filtration through a membrane filter and sterilized in advance. Since the tank (jar fermenter) can be injected aseptically, not only energy can be saved, but also the destruction of the heat-sensitive nutrients of the medium components can be kept to a minimum. Can be inoculated immediately if it is poured into a fermentation tank (jar fermenter) while keeping the temperature at the optimum temperature for cultivation. Ri, the running cost of the culture requires less.

  After folding and sterilizing the tank, and assembling it on a suspension system, when cultivating aerobic bacteria, use sterile air, and when cultivating anaerobic bacteria, use a mixture of sterile nitrogen gas and carbon dioxide. What is necessary is just to inflate this fermentation tank (jar fermenter). Efficient and easy cultivation of anaerobes that were impossible with conventional metal fermentation tanks (jar fermenters) can contribute to academic progress in the field of anaerobes.

  The main body itself is lightweight and does not require piping to a drainage channel that is a source of contamination, and if it has a power source, it can be moved to a sealed aseptic room and cultured. As a result, there is an advantage that the risk of being contaminated with various germs is low, but the artificially produced germs by genetic manipulation are not released to the outside and can be easily contained.

  In the fermentation tank (jar fermenter) of the present invention, there are no pipes that are a source of contamination or valves that are currently used. Therefore, after removing the culture solution, the medium components sterilized by autoclaving and purified water sterilized by filtration are used. If the temperature is optimally maintained in advance and replenished in a sterile room, the second culture is possible, and continuous culture is possible without preparation of the inoculum.

  Iron ions from conventional metal fermentation tanks (jar fermenters), boilers and piping may have adverse effects. For example, when targeting diphtheria toxin preparation, the amount of toxin production depends on the iron ion concentration, but this fermentation tank (jar fermenter) that minimizes the contact of the culture medium with metal is optimal. is there.

  When culturing at a constant pH, it is desirable to use hydrochloric acid and sodium hydroxide as pH adjusters. In the case of leaning to alkalinity, the conventional stainless steel fermentation tank (jar fermenter) is not used because the stainless steel is corroded by hydrochloric acid, but the fermentation tank (jar fermenter) can use hydrochloric acid. It is convenient.

  By dispersing and dropping the culture solution from the forced dispersion device, the surface area of the culture solution is increased, the area in contact with the gas in the tank is increased, effective gas exchange is performed, and the culture is performed by being dispersed and dropped. Because of the gas entrainment effect when it falls to the liquid surface, a small amount of air flow is sufficient.

  When the culture solution is dispersed and dropped from the forced dispersion device of the fermentation tank (jar fermenter) of the present invention, the bubbles disappear due to physical collision. Therefore, if the carbon source component of the medium is within 2% of the usual amount, an antifoaming agent is unnecessary.

  The fermentation tank (jar fermenter) of the present invention can be transferred to a sealed aseptic room and cultured if there is a power source. As a result, if the inoculum is immersed in ice water for 5 minutes and inoculated in a small amount, the lag phase will be longer, but the slope of the logarithmic growth phase will be larger, and the OD value in the stationary phase will be the conventional fermentation tank (jar fermenter) The amount of bacteria per culture medium (metabolite) can be increased. In addition, as a synergistic effect of the large OD value, the autolysis of the cultured bacteria occurs efficiently in the stationary phase at a relatively early stage, and when the target substance is made in the cells by genetic manipulation, Since it comes out, there is an advantage that the work of breaking the culture can be omitted and the object can be easily taken out. In any case, the reason why the yield is high is largely due to a new aeration method using a circulation method in which a culture solution is dispersed and dropped from a forced dispersion device by providing a bypass.

  When two fermentation tanks (jar fermenters) of the present invention of the same scale are arranged side by side and cultured, the results of changing the conditions at one place can be experimented under the same conditions of the inoculum. Since it is not necessary to consider the inoculum, it is easy to analyze the experimental results.

  The main body of the fermentation tank (jar fermenter) of the present invention is a cylinder using a film such as a vinyl chloride film or a silicon rubber film 1 instead of a conventional stainless metal in order to reduce the volume as much as possible during sterilization. Make a shaped container. A part of this cylindrical container is cut into a circular shape from the upper and lower centers, and a flat ring 3 with a bolt 2 made of synthetic resin or stainless steel is attached to the inside from the inside, packing 4, upper circular plate 5, washer, nut Install with 6 and completely shut off from the outside world. The upper circular plate has a forced dispersion device (spray nozzle) 7 not present in a conventional fermentation tank (jar fermenter), a conventional antifoaming agent addition port 8, a foam detection sensor attachment port 9, and pressure. A pressure gauge mounting port 11 with a switch 10, an exhaust port 12 and a sterile air input port 14 with a filter 13 are attached. Similarly, a bottom circular plate 15 is attached to the bottom of the tank, and a conventional aseptic air diffusion device 16, a stirrer 17 having a method different from the conventional method, and its stirrer 18 are attached to the bottom circular plate. The aseptic air diffusion device at the bottom is connected by a silicone rubber tube 19 from the aseptic air inlet at the top. In addition, a bypass 20 is provided using a silicon rubber tube from the culture medium outlet of the stirring device to the upper forced dispersion device, and a mechanical seal-free magnet pump rotor portion 21, temperature detection sensor attachment port 22, pH is provided in this bypass. Detection sensor attachment port 23, dissolved oxygen detection sensor attachment port 24, pH adjustment acid solution inlet 25, pH adjustment alkali solution inlet 26, OD measurement sample outlet 27 and OD measurement sample return port 28, and culture solution Attach the injection / inoculation port 29.

  When cultivating microorganisms, the rotor part of the magnet pump is attached to the magnet pump drive motor part 30 and the rotor is rotated to flow the culture medium into the bypass. By dropping and colliding while increasing the surface area, there is an effect of eliminating the large foamed bubbles, and at the same time, the gas in the tank is entrained in the liquid, and an increase in dissolved oxygen can be expected, and the gas in the tank As a result of the increased surface area in contact and efficient gas exchange, a small amount of air flow is sufficient.

  Unlike the conventional method in which the stirrer is rotated by a motor, this stirrer rotates the stirrer by changing the rotor of the stirrer into a rotational motion by using the suction water flow of a pump that circulates the culture solution.

  In order to increase the amount of dissolved oxygen, it is possible to control by increasing the number of rotations of the pump for circulating the culture solution as well as increasing the aeration amount. If you want more, you can install another bypass and circulate and drop the culture fluid from the pump and forced dispersion device.

  When the pH is far from the set pH, the peristaltic pump 31 may be rotated so that the acid or alkali solution can be sent from the acid storage bottle 32 or the base storage bottle 33 to the inlet.

  When the culture solution is in contact with the tip of the flexible defoaming sensor 34 attached to the end of the tank, the peristaltic pump automatically rotates to remove the defoamer from the defoamer storage bottle 35 into the tank. What is necessary is just to control by the bubble measuring device 36 so that it may enter.

  Next, in order to make this flexible fermentation tank (jar fermenter) self-supporting, the upper and lower circular plates of the tank are attached to the suspension device 37 having a variable height, and then aseptic air is fed into the tank. . By sending sterile air, the tank begins to swell. At this time, if the upper part of the tank of the suspension system is assisted to gradually rise by the weight 38, the flexible tank can easily stand by itself.

  Aseptic air can be easily produced by passing the compressed air produced by the compressor 39 through the pre-filter 40 and the filter. In the case of anaerobic bacteria, the gas in the mixed gas cylinder 41 of nitrogen gas and carbon dioxide gas may be prepared by passing through a prefilter and a filter in the same manner.

  A protective cover 42 having a double structure for protecting the tank is provided so that the membrane is not broken when the culture solution is put into the tank from the culture solution inlet / inoculation port, and the temperature-controlled liquid is provided there. Circulating and controlling the temperature of the culture solution. In particular, when removal of reaction heat generated by fermentation must be performed quickly, control may be performed so that cold water flows.

  The bottom half of the protective cover with this double structure is fixed, the upper half is provided with a support 43, and the cover is opened to the left and right with a hinge-shaped metal fitting 44. Assembling this fermentation tank (jar fermenter) Make it easy to operate.

  Further, when the tank internal pressure becomes a positive pressure above a certain level, the pressure switch works to activate the pump 46 for circulating the sterilizing liquid in the exhaust treatment device 45, and the sterilizing liquid is sucked when it passes through the aspirator 47. In this way, the tank internal pressure is kept in equilibrium. If a filter is installed in the middle of the line, the microorganisms to be cultured are prevented from coming out to the outside world. Even if the filter breaks, take care to disperse it in the sterilizing solution and sterilize the microorganisms.

  A caster 48 is attached to the suspension device, and a pH measuring device 49, a dissolved oxygen measuring device 50, an OD measuring device 51, an exhaust treatment device, a compressor, and a hot water generating device 52 and a cold water generating device necessary for optimum temperature control of the culture solution. If it is possible to move by providing 53, it is possible to perform culture at any location as long as there is a high-pressure sterilizer and a gas sterilizer.

  Here, the culture process of the conventional metal fermentation tank (jar fermenter) is carried out by putting predetermined medium components and purified water into the tank, sending high-pressure steam into the outer pot, sterilizing the culture solution, and at the same time, acid solution for pH adjustment , The alkaline solution container, the defoamer solution container, etc. After sterilization, tap water is circulated in the outer pot, cooled to the optimum culture temperature, inoculated with the inoculum, and then cultivated and sterilized acid solution for pH adjustment, alkaline solution for pH adjustment, antifoam solution Is a conventional method, but the culture process of the flexible fermentation tank (jar fermenter) of the present invention is different and may be carried out as follows.

  This fermentation tank (jar fermenter) main body, bypass and various sensors are sterilized separately with a high-pressure sterilizer or an EOG gas sterilizer. These are assembled by aseptic operation. The upper and lower parts of the fermentation tank (jar fermenter) are fixed to the suspension system, the bypass is connected, the magnet pump rotor is fixed to the drive unit, and aseptic air is fed from the aseptic air input port. Inflate the fermentation tank (jar fermenter). When anaerobic bacteria are cultured, the fermentation tank (jar fermenter) is inflated with a mixture of aseptic nitrogen gas and carbon dioxide.

  Alternatively, assemble the tank in advance, such as a bubble detection sensor, a pressure gauge, a silicone tube connection from the sterile air inlet to the sterile air diffusion device, and a bypass connection equipped with various sensors, and operate the pump of the exhaust treatment device. After exhausting the air in the tank, inject EOG gas from the sterile air inlet / exhaust port and the culture solution inlet / inoculation port, inflate the tank, close it for several hours, Perform a leak test. Next, the pump of the exhaust treatment device is operated to make negative pressure, and EOG is extracted from the exhaust port, compressed air is sent from the sterile air input port, the tank is inflated, and then the sterile air in the tank is extracted from the exhaust port. This aseptic air flow is repeated several times to remove the remaining EOG as much as possible.

  Also, if the protective cover on the lower half of the suspension system is set, a cylindrical adapter with a diameter smaller than the tank diameter is attached, and the tank itself is recessed to perform gas sterilization, the amount of EOG or aseptic air can be reduced. .

  What is necessary is just to process the EOG gas discharged | emitted by the EOG gas processing apparatus 54 using a platinum catalyst under 300 degreeC.

  After sterilizing this tank (jar fermenter), dissolve the medium components in 1/10 to 1/5 volume of purified water, and after high-pressure sterilization, use a siphon or pump to inoculate and inoculate the culture medium. After putting into the tank from the mouth, 9/10 to 4/5 amount of purified water is put into the tank while sterilizing by filtration. Temperature-controlled hot water is circulated in the double-structured cover.

  Take the prepared inoculum in a sterilized syringe, wipe the rubber stopper of the culture solution inlet / inoculation port with alcohol cotton, inoculate the inoculum in the manner of injection, turn on the pump, circulate the culture solution and start culture .

  After completion of the culture, the rubber plug portion of the culture solution injection / inoculation port is removed, and the culture solution is taken out by changing the attachment to the silicon rubber tube and operating the pump. When continuous culture is desired, if the medium is injected, the remaining culture solution becomes a seed fungus and can be cultured, and preparation of the seed fungus is unnecessary.

  Pseudomonas aeruginosa N-10 strain was synthesized in a synthetic medium (sodium glutamate 2%, glucose 0.5%, magnesium sulfate heptahydrate 0.01%, calcium nitrate 0.0001% iron sulfate heptahydrate 0.000005%, Potassium dihydrogen phosphate 0.0252%, disodium hydrogen phosphate dodecahydrate 0.563%, pH 7.6) and the conventional metal fermentation tank (jar fermenter) and the fermentation tank of the present invention (jar fermenter) As a result of comparing the amount of bacteria when cultivated in) in terms of OD value, the tank (jar fermenter) of the present invention has an OD value of 2 in the stationary phase compared to the conventional metal tank (jar fermenter). .5, which is higher than 1.8-2.0 of the conventional metal fermentation tank (jar fermenter), the lag phase is long, but the rise of the logarithmic growth phase is large, and the final purified product is In about twice per land liter, protein content of the final purified product whereas was 70% for metallic fermentation tanks (jar fermentor) was superior to 90%. This is because the growth was significant during the logarithmic growth phase, so the medium was used effectively, and the subsequent autolysis was considered to have been successfully performed. The process is expected to be easy. In addition, the conventional metal tank (jar fermenter) needed an antifoaming agent while culturing the bacteria, whereas the tank (jar fermenter) of the present invention consumed the defoaming agent. Was zero.

  For the purpose of producing proteolytic enzyme from Pseudomonas aeruginosa IFO 3080 strain in semi-synthetic medium (glucose 7%, disodium hydrogen phosphate dodecahydrate 0.05%, diammonium hydrogen phosphate 1%, yeast extract 0.2%) They were cultured in a conventional metal fermentation tank (jar fermenter) and the fermentation tank of the present invention (jar fermenter). In the case of a conventional metal fermentation tank (jar fermenter), culture was carried out by adding aseptically as a pH adjuster so that the precipitated calcium carbonate was 2.5%. The fever started from 24 hours after the culture, and the foaming became more intense at 30 hours, and the culture had to be stopped. The enzyme titer at this time was 0.4 mPU. On the other hand, the fermentation tank (jar fermenter) of the present invention was cultured for 60 hours while automatically adjusting to pH 5.5 using a 1N sodium hydroxide solution. As a result, the culture solution did not overflow from the tank by the automatic addition of the antifoaming agent, as if there was a culture solution between the bubbles. The enzyme titer was 2.0 mPU.

  Although this fermentation tank (jar fermenter) must be assembled in the culture operation process, when compared with the current metal fermentation tank (jar fermenter) from a different perspective, this fermentation tank (jar)・ Fermenter's aeration method allows the target microorganisms to be easily cultivated at a high rate, and requires only a small operating cost. In a closed environment, not only aerobic bacteria but also anaerobes Considering that it can be easily cultured in large quantities, the availability of this fermentation tank (jar fermenter) is great, and it is considered that this fermentation tank (jar fermenter) is accepted by society.

It is an upper top view of this fermentation tank (jar fermenter). It is a perspective view of the tank body part constituted from the film (vinyl chloride film or silicon rubber film) of this fermentation tank (jar fermenter). It is the bottom part top view seen from the inside of this fermentation tank (jar fermenter). It is explanatory drawing which replaced various sensor attachment ports etc. with the rubber stopper by the cross-sectional view method during culture | cultivation with this fermentation tank (jar fermenter). It is explanatory drawing by the flow sheet of this fermentation tank (jar fermenter).

Explanation of symbols

1 Membrane 2 Bolt 3 Ring 4 Packing 5 Upper Circular Plate 6 Nut 7 Forced Dispersion Device (Spray Nozzle)
8 Defoamer addition port 9 Foam detection sensor mounting port 10 Pressure switch 11 Pressure gauge mounting port 12 Exhaust port 13 Filter 14 Aseptic air inlet 15 Bottom circular plate 16 Aseptic air diffusion device 17 Stirrer 18 Stirrer 19 Silicon rubber tube 20 Bypass 21 Magnut pump rotor portion 22 Temperature detection sensor mounting port 23 pH detection sensor mounting port 24 Dissolved oxygen detection sensor mounting port 25 Acid solution inlet 26 for pH adjustment Alkaline solution inlet 27 for pH adjustment Sample outlet 28 for OD measurement Sample return port for OD measurement 29 Culture solution inlet / inoculation port 30 Magnet pump drive motor portion 31 Peristal pump 32 Acid storage bottle 33 Base storage bottle 34 Antifoam sensor 35 Antifoam agent storage bottle 36 Foam measuring device 37 Suspension device 36 Foam measurement Device 37 Suspension device 38 Weight 39 Compressor 40 Pre Filter 41 Nitrogen gas and carbon dioxide mixed gas cylinder 42 Protective cover 43 Strut 44 Hinge fitting 45 Exhaust treatment device 46 Pump 47 Aspirator 48 Caster 49 pH measurement device 50 Dissolved oxygen measurement device 51 OD measurement device 52 Hot water generation device 53 Cold water generation device 54 EOG gas processing equipment

Claims (3)

  A small fermentation tank (jar fermenter) that can be folded and sterilized using a flexible membrane (for example, a vinyl chloride membrane or a silicone rubber membrane) as a means of completely blocking the outside.   A bypass is provided from the bottom to the top of the tank, and various sensor attachment ports, culture solution injection / inoculation ports and a magnet pump are attached to the bypass to circulate the culture solution and increase the surface area as much as possible into the tank. The fermentation tank (jar fermenter) according to claim 1, wherein a forcible dispersion device is attached to the upper part of the tank so as to be dropped. To control the temperature of the tank in a double-structured cover to protect the tank by attaching the upper layer and the base of the tank to a suspension system with a variable height so that the flexible tank can stand on its own. The fermentation tank (jar fermenter) according to claim 1, wherein a protective cover device capable of flowing a liquid is provided.

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