CN103525689B - High dissolved oxygen bioreactor for high-density culture of genetically engineered bacteria and culture control method - Google Patents

Abstract

The invention relates to a high dissolved oxygen bioreactor for high-density culture of genetically engineered bacteria and a culture control method. The reactor comprises a tank body, wherein the top of the tank body is provided with a stirring driving motor, and an output shaft of the stirring driving motor is in transmission connection with a stirring paddle; the bottom of the tank body is provided with a rotor driving motor, an output shaft of the rotor driving motor is in transmission connection with a gas-liquid dispersion rotor, the gas-liquid dispersion rotor is provided with a ventilation inner cavity, the top of the ventilation inner cavity is provided with a gas inlet, the lower part of the ventilation inner cavity is provided with a gas outlet, and the gas inlet is connected with a pressurized air source through a pipeline; the inner side of the bottom of the tank body is fixedly connected with a gas-liquid dispersion stator which is provided with a diversion trench; the gas-liquid dispersion stator is positioned in the circumferential direction of the gas-liquid dispersion rotor, and the gas-liquid dispersion stator and the gas-liquid dispersion rotor are coaxial and form a revolute pair. The method comprises a gas-liquid dispersion rotor ventilation control process, a gas-liquid dispersion rotor rotating speed control process and a stirring paddle stirring rotating speed control process. The invention has high dissolved oxygen level and is suitable for high-density culture of genetically engineered bacteria.

Description

High-dissolved oxygen bioreactor for high-density cultivation of genetically engineered bacteria and cultivation control method

technical field

The invention relates to a high-dissolved oxygen bioreactor for high-density cultivation of genetically engineered bacteria and a cultivation control method using the reactor, belonging to the technical field of biological fermentation engineering.

Background technique

With the development of genetic engineering technology, more and more biological products can be produced through genetic engineering process. Among them, Escherichia coli and Pichia pastoris have been widely used in the construction of genetically engineered bacteria to obtain exogenous gene products that are useful to people.

The use of genetically engineered bacteria for fermentation aims to obtain large quantities of high-quality exogenous gene products, and at the same time reduce the contamination of the host cell's own components as much as possible, so its fermentation process is different from that of traditional microorganisms.

The traditional batch culture adopts one-time feeding and putting into the tank. The initial concentration of the culture medium is high, which is easy to produce the inhibitory effect of substrates and metabolites, and the high concentration of the fermentation liquid is not conducive to the transfer of oxygen.

As far as the applicant knows, at present, the high-density fermentation process is becoming more and more mature and has become the main process of biotechnology pilot production. It has the advantages of stable process, short fermentation cycle, and high expression of exogenous genes. In some fermentation processes, its The fermentation period can be shortened by more than 50%, and the cell yield and exogenous gene expression are 10-50 times that of non-high-density culture. High-density culture technology has also become one of the important means for genetically engineered bacteria to increase the expression of exogenous genes.

However, there are many factors affecting the expression of exogenous genes in genetically engineered bacteria, such as: the characteristics of genetically engineered bacteria, the composition of the medium, the temperature, pH value, and concentration of dissolved oxygen during fermentation, the mixing degree of materials in the fermenter, and the induced Among them, the concentration of dissolved oxygen and the mixing degree of materials in the fermenter have a particularly significant impact on the expression of exogenous genes in high-density culture genetic engineering.

According to the existing method of high-density cultivation of genetically engineered bacteria, the dissolved oxygen concentration is generally controlled at about 30% in the growth period of the bacteria, and controlled at about 20% in the induction period. In the growth period of bacteria, because the number of bacteria in the bacteria is relatively small compared with the number of bacteria in the stable period, it is easy to meet the requirement of 30% dissolved oxygen concentration by adjusting the air intake and other conditions. In the cell induction period, it is difficult to control the dissolved oxygen concentration at about 20%. The reason is that the cell induction period belongs to the late stage of high-density culture, the number of cells expands exponentially, and the demand for dissolved oxygen increases sharply. However, the gas-liquid dispersion performance of ordinary bioreactors is limited, and it is difficult to meet the needs of bacteria for dissolved oxygen and mixed mass transfer only by increasing the ventilation rate and stirring speed. In response to this problem, many fermentation processes use pure oxygen instead of air to solve the problem. However, the use of pure oxygen will not only increase the production cost, but also bring inconvenience to the control of the entire fermentation process.

At present, there are three main types of bioreactors commonly used for high-density cultivation of genetically engineered bacteria: airlift reactors, ventilated stirring reactors, and self-priming reactors.

Airlift reactors are divided into airlift outer loop reactors and airlift inner loop reactors. Because of its simple structure and low shear stress during operation, it is widely used in the fermentation process of shear-sensitive microorganisms. The airlift reactor mainly uses the injection function of the gas and the density difference of the fluid medium to promote the circulation of the reaction system. In the airlift reactor, the effect of circulating flow and mixing mass transfer of the reaction system in the fermenter can be enhanced by installing a guide tube. The air-lift reactor can improve the uniformity of gas-liquid distribution and the rate of dissolved oxygen, but it requires higher aeration volume and aeration pressure, which leads to increased energy consumption to a certain extent, especially for fermentation systems with higher viscosity. The oxygen mass transfer coefficient is small and the mixing effect is poor. When used for high-density cultivation of genetically engineered bacteria, its mixed mass transfer effect is difficult to meet the requirements. When the ventilation volume is large, it is easy to produce a large amount of foam, which increases the probability of bacterial contamination.

Compared with the airlift reactor, the structure of the ventilated stirred reactor is more complicated. A ventilated stirred reactor usually consists of a tank, a stirring device, an aeration device and other auxiliary devices. Wherein, the stirring device is arranged in the middle of the tank body and sealed with the tank body aseptically, and the gas distributor of the ventilation device is arranged at the bottom of the tank body. The gas is ejected from the gas distributor through the ventilation device, and the mixing and mass transfer are realized by the high-speed rotating stirring blades. The ventilated stirred reactor is suitable for most aerobic fermentation processes, but there are also deficiencies. For example, in order to achieve a higher dissolved oxygen concentration and mixed mass transfer, the stirring speed and aeration volume must be increased, which will increase energy consumption and make the The overall shear stress in the tank increases, which is not conducive to the growth of shear-sensitive microorganisms, especially when the filamentous bacteria are cultured, the damage to the cells is greater.

The self-priming reactor is a reactor that uses the gas-liquid dispersion rotor to rotate at high speed and actively sucks the outside gas into the tank. It is widely used because of its excellent gas-liquid dispersion performance and low energy consumption, especially in food The application in the fermentation production of vinegar and yeast is more mature. However, the structure of the existing self-priming reactor also has unreasonable places, which are mainly reflected in: the gas-liquid dispersion rotor has a small suction capacity, the tank height-to-diameter ratio should not be too large, and the tank volume should not be too large. The fermentation capacity of the reactor is limited.

The gas-liquid dispersion rotor of the existing self-priming reactor is generally located at the bottom or the middle of the tank body.

When the gas-liquid dispersing rotor is installed at the bottom of the tank, due to the high liquid level difference, the critical speed of the gas-liquid dispersing rotor increases and the suction capacity decreases. This phenomenon is more serious with the increase of the height-to-diameter ratio of the tank. Since the gas-liquid dispersion rotor is small and placed at the bottom of the tank, during the fermentation process, the fermentation liquid in the middle and upper part of the tank is only driven by the air bubbles to carry out the overall macroscopic circulation, and its macroscopic mixing intensity is weak, which will lead to a decrease in mass transfer effect. The material is not mixed evenly. The mixing mass transfer effect will be worse in the reaction system with higher viscosity.

When the gas-liquid dispersion rotor is placed in the middle of the tank, compared with the reactor with the gas-liquid dispersion rotor placed at the bottom of the tank, its critical speed decreases, and the suction volume at the same speed increases, but there will be gas inclusions in the upper part of the tank. The phenomenon that the rate is greater than the gas holdup rate in the lower part of the tank, and this phenomenon is extremely unfavorable for the growth of microorganisms.

Contents of the invention

The technical problem to be solved by the present invention is: to overcome the problems existing in the prior art, to provide a high-dissolved oxygen bioreactor for high-density cultivation of genetically engineered bacteria, which can meet the needs of high dissolved oxygen concentration, and facilitate the implementation of high-density genetically engineered bacteria nourish. In addition, a culture control method using the reactor is also provided.

The technical scheme that the present invention solves its technical problem is as follows:

A high-dissolved oxygen bioreactor for high-density cultivation of genetically engineered bacteria, comprising a tank body with a feeding hole at the top and a discharge port at the bottom, characterized in that a stirring drive motor is provided on the outside of the top of the tank body, and the stirring The output shaft of the drive motor is connected to the stirring paddle extending into the tank; the bottom of the tank is provided with a rotor drive motor, and the output shaft of the rotor drive motor is connected to the gas-liquid dispersion rotor inside the tank. The liquid dispersing rotor has a ventilated inner cavity with an air inlet on the top and an air outlet on the lower part. The air inlet is connected to a pressurized air source through a pipeline; The gas-liquid dispersion stator has a diversion groove with a water inlet and a water outlet, the water inlet of the diversion groove is close to the gas-liquid dispersion rotor, and the water outlet is far away from the gas-liquid dispersion rotor; the gas-liquid dispersion stator is located around the gas-liquid dispersion rotor In the direction, the gas-liquid dispersion stator is coaxial with the gas-liquid dispersion rotor and constitutes a rotating pair.

When this structure is in use, on the one hand, the mixture in the tank is continuously stirred by the overhead stirring paddle, and on the other hand, the gas-liquid dispersion rotor continues to rotate, so that the gas input from the air source and the liquid in the tank are well mixed together, and the gas-liquid The mixture is sprayed into the tank through the gas-liquid dispersion stator, the dispersion effect is good, and the oxygen dissolution efficiency is high, which can meet the needs of high dissolved oxygen concentration.

The further perfect technical scheme of reactor of the present invention is as follows:

Preferably, the gas-liquid dispersing rotor is a cavity rotor with multiple curved blades, each curved blade is composed of an upper arc portion and a lower vertical portion, and the upper arc portion of each curved blade is twisted along the rotation direction of the rotor; the The top of the upper arc part of each curved leaf is fixedly connected with the upper port of the rotor, and the bottom end of the lower vertical part of each curved leaf is fixedly connected with the bottom surface of the rotor; the curved leaves, the bottom surface of the rotor and the upper port of the rotor together form a ventilating interior cavity, the inner side of each curved leaf forms an air diversion bend, and the outer side forms a liquid diversion bend; the air inlet of the ventilation inner cavity is located at the upper port of the rotor, and the air outlets of the ventilation inner cavity have multiple, respectively Located at the lower vertical part of each curved leaf.

The applicant found through in-depth practical research that the gas-liquid dispersion rotor adopts this structure to increase the space of its internal cavity, which is conducive to improving the air intake of the gas-liquid dispersion rotor, and the air in the gas-liquid dispersion rotor that is consistent with the rotation direction The diversion curve can make the gas inside the cavity get more power, the injection speed is higher, and the gas-liquid dispersion is more uniform; the liquid diversion curve outside the gas-liquid dispersion rotor is consistent with the rotation direction, which can Under the circumstances, the liquid around the upper part of the gas-liquid dispersion rotor is driven to move downward in a highly turbulent state. This can increase the liquid discharge volume and suction volume of the gas-liquid dispersing rotor, effectively mix the liquid with the gas ejected from the cavity, and increase the dissolved oxygen level.

More preferably, the arc diameter of the upper arc portion of each curved leaf is 2/5-3/5 of the diameter of the entire gas-liquid dispersion rotor; the height of the vertical portion of the lower portion of each curved leaf is the height of the entire gas-liquid dispersion rotor 1/3-1/2; the twist angle of the upper arc portion of each curved leaf is 20°-60°.

The applicant found through in-depth practical research that the above structure can further optimize the performance of the gas-liquid dispersion rotor and further increase the dissolved oxygen level.

Preferably, the gas-liquid dispersion stator is ring-shaped; the outer edge part of the top surface of the gas-liquid dispersion stator is inclined downward, and the middle part is parallel to the bottom surface and is provided with openings for arranging the gas-liquid dispersion rotor; A number of diversion grooves are arranged between the top surface and the bottom surface of the liquid dispersion stator, and the water inlets of each diversion groove are water inlet holes located on the top surface and evenly distributed around the opening; the outlets of each diversion groove The water port is located on the outer surface of the gas-liquid dispersion stator.

After in-depth practical research, the applicant found that after the gas-liquid dispersion stator adopts this structure, the opening area of the inner end of the diversion groove is larger than the opening area of the outer end, which can greatly reduce the resistance of the fluid passing through the gas-liquid dispersion stator, and can make the gas-liquid dispersion rotor swing The outflow fluid is dispersed more smoothly through the diversion groove; the position and arrangement of the water inlet holes can increase the liquid discharge volume of the gas-liquid dispersion rotor and improve the gas-liquid dispersion effect.

More preferably, the angle between the outer edge of the top surface of the gas-liquid dispersion stator and the bottom surface is 8°-20°; the radial angle between the diversion groove and the outer end of the diversion groove is 15°-45°.

The applicant found through in-depth practice and research that the above structure can effectively keep the direction of the diversion groove consistent with the direction of the fluid thrown out by the gas-liquid dispersion rotor, further reducing the resistance when the fluid passes.

In addition, the reactor of the present invention can also have the following preferred features:

Preferably, the air source includes an air compressor, an oil-water separator, an air storage tank, an air filter, and an air flow meter connected in sequence; the air outlet of the air flow meter communicates with the upper port of the intake pipe, and the air intake pipe The lower port communicates with the air inlet of the gas-liquid dispersing rotor ventilating inner cavity; the lower port of the air inlet pipe is connected with the gas-liquid dispersing rotor in a sealed and rotating manner.

Preferably, the stirring blade includes a drive shaft connected to the output shaft of the stirring drive motor, the drive shaft is provided with stirring paddles in the circumferential direction, and the upper part of the drive shaft is provided with a defoamer in the circumferential direction.

Preferably, the tank body includes a flat tank bottom, a cylindrical tank body and an oval-shaped tank top; the tank bottom is sealed and connected to the tank body through a transitional arc surface, and the diameter of the arc surface is 1/2 of the diameter of the tank body. 1/6-1/2; the tank top is fixedly connected with the tank body through the flange; the feeding hole is located on the tank top, and the discharge port is located at the bottom of the tank; the tank top is also provided with a manhole; the stirring The driving motor is sealed and connected to the top of the tank through a sterile mechanical seal, and the rotor driving motor is sealed and connected to the bottom of the tank through a sterile mechanical seal; there are several baffles on the inner side of the middle and lower part of the tank body, and a cooling water inlet and a cooling water inlet on the outer side. A jacket for the cooling water outlet; the tank body is also provided with a sensor interface.

The present invention also provides:

A high-density culture control method using the aforementioned high-density culture of genetically engineered bacteria using a high-dissolved oxygen bioreactor is characterized in that it includes a gas-liquid dispersion rotor ventilation control process, a gas-liquid dispersion rotor speed control process, and a stirring paddle Speed control process;

The air flow control process of the gas-liquid dispersing rotor is: control the air flow of the gas-liquid dispersing rotor at 70-95% of the critical air flow of the gas-liquid dispersing rotor; the determination process of the critical air flow of the gas-liquid dispersing rotor is: first control The gas-liquid dispersion rotor rotates at a predetermined speed, and then gradually increases the ventilation volume of the air inlet of the gas-liquid dispersion rotor ventilation cavity through the air source. When the air outlet of the inner cavity enters the tank, the air flow at this time is the critical air flow of the gas-liquid dispersion rotor;

The speed control process of the gas-liquid dispersion rotor is: controlling the speed of the gas-liquid dispersion rotor at 110-150% of the critical speed of the gas-liquid dispersion rotor; the determination process of the critical speed of the gas-liquid dispersion rotor is: firstly control the air source to preset The air flow is ventilated to the gas-liquid dispersion rotor, and then gradually increase the speed of the gas-liquid dispersion rotor, when the gas can move along the bottom surface of the tank with the liquid thrown out by the gas-liquid dispersion rotor to the farthest radial end of the tank in the form of gas-liquid dispersion , the speed at this time is the critical speed of the gas-liquid dispersion rotor;

The stirring speed control process of the stirring paddle is: the stirring speed of the stirring paddle is controlled at 60-90% of the maximum stirring speed of the stirring paddle; the determination process of the maximum stirring speed of the stirring paddle is: firstly control the gas-liquid dispersion rotor to rotate at a predetermined speed , and control the air source to ventilate the gas-liquid dispersing rotor with a predetermined amount of ventilation, and then gradually increase the stirring speed of the stirring paddle. In the process, the volumetric oxygen mass transfer coefficient K _L a increases with the increase of the stirring speed, and finally tends to be stable, when K _L a reaches 85% of the maximum K _L a, the corresponding stirring speed is the maximum stirring speed of the stirring paddle.

The applicant found through in-depth practical research on the basis of the aforementioned reactors that when the air flow of the gas-liquid dispersion rotor is greater than the critical air flow, the gas overflows in the form of bubbling, the dispersion effect is poor, the bubbles are larger and the residence time in the liquid phase is longer. Short, resulting in low oxygen mass transfer coefficient. When the speed of the gas-liquid dispersion rotor is lower than the critical speed, not only some bubbles will overflow in the form of bubbling, but also a dead zone will be formed on the outer edge of the bottom of the tank, which will easily lead to material accumulation here. Under the conditions of a certain ventilation rate and a certain speed of the gas-liquid dispersion rotor, when the stirring speed of the impeller is greater than the maximum speed, the increase in K _L a is small, and the improvement of the mixing and mass transfer effect is not obvious, and it will lead to the overall shear stress and Increased stirring power. The applicant made further in-depth research on this basis, and finally obtained the above-mentioned high-density cultivation control method. After adopting this control method, the aforementioned reactor is more suitable for the high-density culture and fermentation production of genetically engineered bacteria, especially suitable for the fermentation and production of high-viscosity, high-density and shear-sensitive microorganisms, and further effectively solves the problem of supply and demand in high-density culture of genetically engineered bacteria. The technical problem of insufficient oxygen can further improve the effect of mixing and mass transfer and reduce energy consumption.

Preferably, it also includes a stirring paddle selection process before culturing:

When the height-to-diameter ratio of the tank is less than 1.5, and the ratio of the filling height to the tank diameter is less than 1, use a layer of stirring paddle; when the tank height-diameter ratio is greater than or equal to 1.5, and the ratio of the filling height to the tank diameter is 1-2 In between, two or three layers of stirring paddles are used; the blades of the stirring paddles are radial paddles or downward-pressing axial flow paddles.

Compared with the prior art, the present invention has the following beneficial effects:

1. The gas-liquid dispersion performance is good, the oxygen mass transfer rate is high, the ventilation rate is smaller than that of the existing bioreactor, and the air utilization rate is high, which can effectively reduce the workload of the air compressor and reduce the energy consumption of production.

2. The stirring speed of the required stirring paddle can be lower than that of the existing mechanical stirring bioreactor, so that the reactor of the present invention has the characteristics of low shear stress, and can be applied to the culture of shear-sensitive bacteria.

3. The level of dissolved oxygen is high and the mixing effect is good. It is suitable for the cultivation of high-viscosity and high-density microorganisms, especially for the high-density cultivation of genetically engineered bacteria. It can meet the needs of high-density bacteria for dissolved oxygen and overcome the existing biological reactions. The device is used for the technical problem of insufficient dissolved oxygen in the high-density cultivation of genetically engineered bacteria, and improves the effect of mixed mass transfer.

4. The operation of the device is flexible, which can meet various fermentation conditions and has a wide range of applications.

5. With a complete set of device system, each link can be optimized, and it is convenient to carry out large-scale operation of the device under the condition that the effect of dissolved oxygen and mixed mass transfer remains unchanged.

Description of drawings

Fig. 1 is a structural schematic diagram of the reactor of the embodiment of the present invention.

Fig. 2 is a schematic structural view of the gas-liquid dispersion rotor of the embodiment in Fig. 1 .

FIG. 3 is a top view of FIG. 2 .

FIG. 4 is a cross-sectional view along line A-A of FIG. 3 .

Fig. 5 is a top view of the gas-liquid dispersion stator of the embodiment in Fig. 1 .

Fig. 6 is a B-B sectional view of Fig. 5 .

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and examples. However, the invention is not limited to the examples given.

Example

As shown in Figures 1 to 6, the high-dissolved oxygen bioreactor for high-density cultivation of genetically engineered bacteria in this embodiment includes a tank body with feeding holes 4 in the upper part and a discharge port 15 in the lower part. Stirring drive motor 6, the output shaft of stirring drive motor 6 is connected with the stirring paddle extending into the tank body; the outer side of the tank body bottom is provided with a rotor drive motor 16, and the output shaft of the rotor drive motor 16 is connected to the gas-liquid dispersion rotor 18 inside the tank body Transmission connection, the gas-liquid dispersion rotor 18 has a ventilated inner cavity with an air inlet at the top and an air outlet at the bottom, and the air inlet is connected to a compressed air source through a pipeline; a gas-liquid dispersion stator 19 is also fixedly connected to the inside of the bottom of the tank. The gas-liquid dispersing stator 19 has the diversion groove that establishes water inlet and water outlet; The gas-liquid dispersing stator 19 is positioned at 18 circumferential directions of the gas-liquid dispersing rotor, and the gas-liquid dispersing stator 19 is coaxial with the gas-liquid dispersing rotor 18 and forms a rotating pair; The water inlet of the launder is close to the gas-liquid dispersion rotor, and the water outlet is away from the gas-liquid dispersion rotor.

The tank body includes a flat tank bottom, a cylindrical tank body and an oval headed tank top. The bottom of the tank is flat, which can reduce the resistance of gas-liquid fluid movement at the bottom of the tank, effectively reduce the probability of material sedimentation and accumulation around the gas-liquid dispersion rotor at the bottom of the tank, and reduce the resistance of the gas-liquid dispersion rotor rotation and the number of equipment maintenance. The oval head can enhance the strength of the tank.

The bottom of the tank is sealed and connected with the tank body through the transition arc surface 14, and the diameter of the arc surface 14 is 1/6-1/2 of the diameter of the tank body. In this way, the dead zone on the outer edge of the bottom of the tank body can be reduced, and the strength of the tank body can also be enhanced, which is beneficial to the manufacture of large tank bodies.

The top of the tank is fixedly connected with the tank body through the flange 3; the feeding hole 4 is located on the top of the tank, and the discharge port 15 is located at the bottom of the tank; the top of the tank is also provided with a manhole 7; the stirring drive motor 6 is sealed and connected to the top of the tank through a sterile mechanical seal 5 , the rotor drive motor 16 is sealed and connected to the bottom of the tank through a sterile mechanical seal 17; the inner side of the middle and lower part of the tank body is provided with a number of baffles 11, and the outer side is provided with a jacket 10 with a cooling water inlet 13 and a cooling water outlet 21; the tank body is also A sensor interface 20 is provided. In the present embodiment, there are four baffle plates 11 whose width is 1/10 of the diameter of the can body.

The air source includes an air compressor 25, an oil-water separator 24, an air storage tank 23, an air filter 22, and an air flow meter 21 connected in sequence; The port communicates with the air inlet of the gas-liquid dispersing rotor 18 ventilating inner cavity; Using the air compressor to supply air can increase the intake volume, increase the dissolved oxygen level, reduce the influence of the liquid level difference in the tank on the intake volume, and facilitate the large-scale operation of the reactor.

As shown in Figures 2 to 4, the gas-liquid dispersion rotor 18 is a cavity rotor with multiple curved blades (specifically, a cavity rotor with nine curved blades can be used), and each curved blade is composed of an upper arc portion 27 and a lower vertical portion 26, each The upper arc portion 27 of the curved leaf is twisted along the direction of rotation of the rotor; the top end of the upper arc portion 27 of each curved leaf is fixedly connected with the upper port 28 of the rotor, and the bottom end of the vertical portion 26 of each curved leaf is fixedly connected with the bottom surface of the rotor; each curved leaf Together with the bottom surface of the rotor and the upper port 28 of the rotor, a ventilating inner cavity is formed, and the inside of each curved leaf forms an air diversion bend, and the outer side forms a liquid diversion bend; the air inlet of the ventilating inner cavity is located at the upper port 28 of the rotor, and the ventilating inner cavity There are a plurality of air outlets 29, which are respectively located in the lower vertical portion 26 of each curved leaf.

The diameter of the radian 30 of the curved part 27 on the upper part of each curved leaf is 2/5-3/5 of the diameter of the entire gas-liquid dispersion rotor 18; the height of the vertical part 26 at the bottom of each curved leaf is 1/3 of the height of the entire gas-liquid dispersion rotor 18 -1/2; the twist angle C of the upper arc portion 27 of each curved leaf is 20°-60°.

The gas-liquid dispersing stator 19 is annular; the outer edge part of the gas-liquid dispersing stator 19 top surface is inclined downward, and the middle part is parallel with the bottom surface and is provided with the opening for arranging the gas-liquid dispersing rotor 18; the top of the gas-liquid dispersing stator 19 There are several diversion grooves (12-18 diversion grooves can be used specifically) between the surface and the bottom surface, and the water inlets of each diversion groove are water inlet holes 31 located on the top surface and evenly distributed around the opening; The water outlet of the launder is located on the outer surface of the gas-liquid dispersion stator 19 .

The angle E between the outer edge part of the top surface of the gas-liquid dispersion stator 19 and the bottom surface is 8°-20°; the radial angle D between the diversion groove and the outer end point of the diversion groove is 15°-45°.

The stirring paddle includes a drive shaft 9 connected to the output shaft of the stirring drive motor 6 . The drive shaft 9 is provided with stirring paddles 12 circumferentially, and a defoamer 8 is provided circumferentially on the top of the drive shaft 9 .

Stirring the paddles can make the gas in the fermentation liquid circulate in the tank with the paddles, increase the gas-liquid contact time, and improve the oxygen mass transfer efficiency. At the same time, the rotation of the paddles can increase the turbulent intensity of the liquid and reduce the mixing time of the liquid phase. Improve mixing efficiency. Due to the action of the gas-liquid dispersion rotor, the gas-liquid dispersion is better and the bubble diameter is smaller. The stirring paddle does not need to rely on high speed to improve the gas-liquid dispersion, but only needs to maintain a low speed to strengthen the overall macroscopic mixing of the fermentation broth. That is, in this way, the shear stress of the whole reactor can be made smaller than that of the existing ventilated and stirred bioreactor. The defoamer can break and eliminate the bubbles generated during the fermentation process, reducing the chance of bacterial contamination.

The high-density cultivation control method of the high-density cultivation of genetically engineered bacteria using the high-dissolved oxygen bioreactor of this embodiment includes the gas-liquid dispersion rotor ventilation control process, the gas-liquid dispersion rotor speed control process, and the stirring speed control process of the stirring paddle.

The airflow control process of the gas-liquid dispersion rotor is: control the airflow of the gas-liquid dispersion rotor at 70-95% of the critical airflow volume of the gas-liquid dispersion rotor; the determination process of the critical airflow volume of the gas-liquid dispersion rotor is: first control the Rotate at a predetermined speed, and then gradually increase the ventilation volume of the air inlet of the gas-liquid dispersion rotor ventilation cavity through the air source, when the gas is no longer in the form of gas-liquid dispersion but in the form of bubbling from the gas-liquid dispersion rotor ventilation cavity outlet When entering the tank, the air volume at this time is the critical air volume of the gas-liquid dispersion rotor.

Since the kinetic energy of the air comes from the pressure of the air compressor and the rotation of the gas-liquid dispersion rotor, the liquid around the rotor is thrown out at a high speed and the negative pressure suction is formed around it. Therefore, the ventilation pressure required by the entire reactor is relatively low. , can effectively reduce the air compressor load and reduce energy consumption.

When the ventilation rate increases gradually, the liquid around the gas-liquid dispersion rotor is gradually discharged by the air, the liquid discharge volume of the gas-liquid dispersion rotor gradually decreases, the vacuum degree formed by the rotation also decreases, and the gas entrained and thrown out by the liquid gradually decreases. , and finally the gas will enter the tank from the gas-liquid dispersion rotor in the form of bubbling. The applicant defines the critical point of this process as the critical air flow of the gas-liquid dispersion rotor.

The applicant found that if the ventilation rate is too small, the oxygen supply of the system is insufficient, and the performance of the reactor cannot be fully exerted; if the ventilation rate is greater than the critical ventilation rate, the gas will enter the liquid in the form of bubbling, and the gas of the gas-liquid dispersion rotor cannot be exerted. Due to the effect of liquid dispersion, the diameter of the bubbles will increase significantly, and the oxygen mass transfer rate will decrease. At the same time, the liquid level in the tank will shake violently, which will affect the liquid filling capacity. After further in-depth research, and comprehensively considering factors such as the instability of the ventilation and the movement characteristics of the gas in the liquid, the applicant finally came up with the above gas-liquid dispersion rotor ventilation control process.

The speed control process of the gas-liquid dispersion rotor is as follows: the speed of the gas-liquid dispersion rotor is controlled at 110-150% of the critical speed of the gas-liquid dispersion rotor; Ventilate the liquid dispersion rotor, and then gradually increase the speed of the gas-liquid dispersion rotor. When the gas can move along the bottom surface of the tank with the liquid thrown out by the gas-liquid dispersion rotor to the farthest radial end of the tank in the form of gas-liquid dispersion, the The speed is the critical speed of the gas-liquid dispersion rotor.

Under a certain amount of ventilation, the gas-liquid dispersion rotor rotates at a certain speed, and the surrounding liquid violently turbulates and generates centrifugal motion to move toward the straight wall of the tank, forming a negative pressure around the gas-liquid dispersion rotor. Under the action of the pressure of the air compressor and the negative pressure suction formed by the rotation of the gas-liquid dispersion rotor, the air is thrown out with the liquid and moves along the bottom surface of the tank. Bubbles, the bubbles move evenly along the bottom of the tank to the tank wall, and the minimum speed required to reach the radially farthest end of the tank body is the critical speed of the gas-liquid dispersion rotor. The movement of gas and liquid along the bottom of the tank can avoid the accumulation of materials at the bottom of the tank and the gas-liquid dispersion rotor, improve the mixing uniformity of raw materials, and reduce the probability of equipment maintenance.

The applicant found that when the speed of the gas-liquid dispersion rotor is lower than the critical speed, the gas cannot reach the position of the tank wall, and it is easy to form a dissolved oxygen dead zone, which is not conducive to the growth of microorganisms; when the speed of the gas-liquid dispersion rotor is higher than the critical speed, the liquid phase spray intensity It is more intense, and the air is torn into smaller bubbles, which is beneficial to the mixing and mass transfer of oxygen, but the excessively high speed will increase the shear stress and energy consumption. After further in-depth research, the applicant finally obtained the above gas-liquid dispersion rotor speed control process.

The stirring speed control process of the stirring paddle is as follows: the stirring speed of the stirring paddle is controlled at 60-90% of the maximum stirring speed of the stirring paddle; The source is ventilated to the gas-liquid dispersion rotor with a predetermined amount of ventilation, and then the stirring speed of the stirring paddle is gradually increased. During this process, the volumetric oxygen mass transfer coefficient K _L a increases with the increase of the stirring speed, and finally tends to be stable. When K _L a reaches 85% of the maximum K _L a, the corresponding stirring speed is the maximum stirring speed of the stirring paddle.

The oxygen transfer efficiency of the reactor can be characterized by the volumetric oxygen mass transfer coefficient K _L a. When the air is fed with a certain amount of ventilation and the rotor rotates at a certain speed for gas-liquid dispersion, K _L a increases with the increase of the stirring speed, and finally tends to be stable. Because the gas-liquid dispersion rotor has good gas-liquid dispersion performance, it weakens the effect of high stirring speed on tearing bubbles and increasing the gas-liquid specific surface area to increase K _L a, thereby avoiding the increased shear and energy consumption caused by high stirring speed . The stirring paddle of the reactor in this embodiment only needs to enhance the effect of gas-liquid macroscopic mixing at a relatively low stirring speed. After further in-depth research, the applicant finally came to the conclusion that the above-mentioned stirring speed control process of the stirring paddle can not only ensure the overall macroscopic mixing of gas and liquid, but also reduce the energy consumption of the whole device.

In addition, there are many factors related to the maximum stirring speed, among which the type of paddle and the number of paddle layers have a greater impact on the maximum stirring speed.

For the fermentation liquid in the tank of this embodiment: the gas-liquid dispersion rotor rotates, and the surrounding liquid generates centrifugal motion to form a negative pressure, and the liquid on the upper part of the gas-liquid dispersion rotor is mixed with the gas introduced by the air inlet pipe, and the liquid is mixed along the bottom of the tank body. Shooting towards the wall of the tank, it moves upwards along the wall of the tank under the barrier of the wall. During the rising process, many small bubbles collide with each other, causing the thickness of the liquid film between the bubbles to become thinner, and finally the bubbles coalesce, and the diameter of the bubbles becomes larger; when reaching At the liquid surface, part of the gas overflows the liquid surface, and the remaining gas flows down along the stirring shaft to the gas-liquid dispersion rotor along with the liquid, forming a cycle.

When the height-to-diameter ratio of the tank is less than 1.5, and the ratio of the liquid filling height to the diameter of the tank is less than 1, a layer of stirring paddles can be installed. When the height-to-diameter ratio of the tank is greater than or equal to 1.5, and the ratio of the filling height to the diameter of the tank is between 1 and 2, two or three layers of stirring paddles can be used according to the characteristics of the fermentation broth system. At the same time, the type of stirring blade should be determined according to the viscosity of the fermentation broth system. When the viscosity is high, the mixing effect is poor, and the bubbles coalesce seriously, the stirring blade can use a radial paddle with a smaller power standard, such as six Parabolic turbine blades and six-half-tube turbine blades, but the use of these two blades is not limited. When the viscosity is not high, a down-pressing axial-flow paddle with a large displacement can be used, such as a three-blade paddle and a four-plum blade paddle, but the use of these two types of paddles is not limited. Selecting the paddle type and the number of paddle layers according to the actual situation can effectively reduce the maximum stirring speed, thereby reducing shear force and energy consumption.

Specific application cases are as follows:

Cold mold experiment 1: (cold mold experiment is a simulation experiment without chemical reaction, the same below)

The size of the reactor is: the diameter of the tank is 700mm, and the effective volume is 300L; the diameter of the gas-liquid dispersion rotor is 96mm; the gas-liquid dispersion stator is designed with 12 diversion grooves, and the outer diameter is 168mm; the width of the four baffles is 70mm. The filling height is 700mm, and there is a layer of stirring paddles, the paddles are four plum blossom axial flow paddles, and the paddle diameter is 200mm.

The measurement system is tap water, and the system temperature is 25°C.

Table 1 shows the critical air flow measurement results of the gas-liquid dispersion rotor at different speeds, and table 2 shows the critical air flow measurement results of the gas-liquid dispersion rotor at different air flow rates.

Table 1. The critical ventilation volume of the gas-liquid dispersion rotor at different speeds

Firstly, under the conditions of the gas-liquid dispersion rotor speed of 1500r/min and 85% of the critical air flow, the reactor control conditions are measured, that is, the critical speed of the gas-liquid dispersion rotor is measured under the condition of the air flow of 7.2m ³ /h. The result is 1320r/min, and the speed of the gas-liquid dispersion rotor is set to 120% of the critical speed, which is 1584r/min. Then measure the maximum stirring speed under the condition that the air flow is 7.2m ³ /h and the gas-liquid dispersion rotor speed is 1584r/min. The measurement result is 240r/min. The stirring speed is set as 75% of the maximum stirring speed, which is 180r/min. min.

The measured K _L a value under this condition is 0.065S ^-1 , which is more than 30% higher than the K _{L a} of the existing ventilated stirred reactor under the same condition.

Cold mold experiment 2:

The size of the reactor is: the diameter of the tank is 700mm, and the effective volume is 300L; the diameter of the gas-liquid dispersion rotor is 96mm; the gas-liquid dispersion stator is designed with 12 diversion grooves, and the outer diameter is 168mm; the width of the four baffles is 70mm. The filling height is 700mm, and there is a layer of stirring paddles, the paddles are four plum blossom axial flow paddles, and the paddle diameter is 200mm.

The measurement system is an aqueous solution of sodium carboxymethyl cellulose with a mass fraction of 0.5%, and the system temperature is 25°C.

Table 3 shows the critical ventilation rate measurement results of the gas-liquid dispersion rotor at different speeds, and Table 4 shows the critical speed measurement results of the gas-liquid dispersion rotor at different ventilation rates.

Table 3. The critical ventilation volume of the gas-liquid dispersion rotor at different speeds

Firstly, the reactor control conditions were measured under the conditions of the gas-liquid dispersion rotor speed of 1500r/min and 85% of the critical airflow, that is, the critical speed of the gas-liquid dispersion rotor was measured under the condition of the airflow of 6.9m ³ /h. The result is 1300r/min, and the speed of the gas-liquid dispersion rotor is set to 120% of the critical speed, which is 1560r/min. Then measure the maximum stirring speed under the condition that the air flow is 6.9m ³ /h and the gas-liquid dispersion rotor speed is 1560r/min. The measurement result is 260r/min, and the stirring speed is set as 75% of the maximum stirring speed, which is 195r/min. min.

The measured K _L a value under this condition is 0.055S ^-1 , which is more than 35% higher than the K _{L a} of the existing ventilated stirred reactor under the same condition.

Implementation case 1:

The size of the reactor is: the volume of the tank is 5m ³ , the height-to-diameter ratio of the tank is 2.2:1, the diameter of the gas-liquid dispersion rotor is 215mm, the gas-liquid dispersion stator is designed with 16 diversion grooves, the outer diameter is 405mm, and the width of four baffles Both are 140mm. There are two layers of stirring paddles with a diameter of 500 mm.

In this implementation case, under the condition of 2.8 tons of liquid, the critical air flow rate of the gas-liquid dispersion rotor at a speed of 2100r/min, the critical speed of the gas-liquid dispersion rotor at this air flow rate, and the maximum stirring speed were measured.

The measured critical air flow rate is 134.4m ³ /h, according to 85% of the critical air flow rate, which is 114.2m ³ /h air intake, the measured critical speed of the gas-liquid dispersion rotor is 1950r/min. The speed of the gas-liquid dispersion rotor is set to 120% of the critical speed, that is, 2340r/min, and the measured maximum stirring speed is 210r/min, and the stirring speed is set to 75% of the maximum stirring speed, that is, 158r/min.

Using the genetically engineered strain Pichia pastoris producing glutathione as the strain, after primary and secondary seed cultivation, it was inoculated into the reactor containing the fermentation medium according to the inoculation amount of 3% (v/v) for high Density culture.

The liquid volume is 2.8t. During the fermentation process, glycerin is added to meet the growth of the bacteria. The pH is controlled by adding ammonia water to 6.5, and the temperature is controlled at 31°C. L-cysteine, L-glycine and L- Glutamic acid ensures the growth of the thallus, cultured for 51 hours, and the content of glutamic acid in the fermentation broth was determined by the tetraoxo derivative method, and the content of glutamic acid was 6.5g/L.

Compared with this, under the same fermentation and operating conditions, with the existing general-purpose mechanically ventilated stirred reactor culture, the final determination of glutamic acid content is only 4.1g/L. The content of glutamic acid in the obtained fermented liquid of present embodiment case is its 1.59 times.

Implementation case 2:

The size of the reactor is: the volume of the tank is 5m ³ , the height-to-diameter ratio of the tank is 2.2:1, the diameter of the gas-liquid dispersion rotor is 215mm, the gas-liquid dispersion stator is designed with 16 diversion grooves, the outer diameter is 405mm, and the width of four baffles Both are 140mm. There are two layers of stirring paddles with a diameter of 500 mm.

In this implementation case, under the condition of 2.8 tons of liquid, the critical air flow rate of the gas-liquid dispersion rotor at a speed of 2100r/min, the critical speed of the gas-liquid dispersion rotor at this air flow rate, and the maximum stirring speed were measured.

The measured critical air volume is 134.4m ³ /h, according to 70% of the critical air volume, namely 94m ³ /h air intake, the measured critical speed of the gas-liquid dispersion rotor is 1780r/min. The speed of the gas-liquid dispersion rotor is set to 120% of the critical speed, that is, 2136r/min, and the measured maximum stirring speed is 240r/min, and the stirring speed is set to 75% of the maximum stirring speed, that is, 180r/min.

Using the genetically engineered strain Pichia pastoris producing glutathione as the strain, after primary and secondary seed cultivation, it was inoculated into the reactor containing the fermentation medium according to the inoculation amount of 3% (v/v) for high Density culture.

The liquid volume is 2.8t. During the fermentation process, glycerin is added to meet the growth of the bacteria. The pH is controlled by adding ammonia water to 6.5, and the temperature is controlled at 31°C. L-cysteine, L-glycine and L- Glutamic acid ensures the growth of the thallus, cultured for 51 hours, and the content of glutamic acid in the fermentation broth was determined by tetraoxo derivatives, and the content of glutamic acid was 5.2g/L.

Compared with this, under the same fermentation and operating conditions with the existing general-purpose mechanical ventilation stirring reactor culture, the final determination of the content of glutamic acid is only 3.3g/L. The content of glutamic acid in the obtained fermented liquid of present embodiment case is its 1.58 times.

Implementation case 3:

The size of the reactor is: the volume of the tank is 5m ³ , the height-to-diameter ratio of the tank is 2.2:1, the diameter of the gas-liquid dispersion rotor is 215mm, the gas-liquid dispersion stator is designed with 16 diversion grooves, the outer diameter is 405mm, and the width of four baffles Both are 140mm. There are two layers of stirring paddles with a diameter of 500 mm.

In this implementation case, under the condition of 2.8 tons of liquid, the critical air flow rate of the gas-liquid dispersion rotor at a speed of 2100r/min, the critical speed of the gas-liquid dispersion rotor at this air flow rate, and the maximum stirring speed were measured.

The measured critical air volume is 134.4m ³ /h, according to 95% of the critical air volume, that is, 127.7m ³ /h air intake, the measured critical speed of the gas-liquid dispersion rotor is 2020r/min. The speed of the gas-liquid dispersion rotor is set to 120% of the critical speed, that is, 2424r/min, and the measured maximum stirring speed is 195r/min, and the stirring speed is set to 75% of the maximum stirring speed, that is, 146r/min.

Using the genetically engineered strain Pichia pastoris producing glutathione as the strain, after primary and secondary seed cultivation, it was inoculated into the reactor containing the fermentation medium according to the inoculation amount of 3% (v/v) for high Density culture.

The liquid volume is 2.8t. During the fermentation process, glycerin is added to meet the growth of the bacteria. The pH is controlled by adding ammonia water to 6.5, and the temperature is controlled at 31°C. L-cysteine, L-glycine and L- Glutamic acid ensures the growth of the thallus, cultured for 51 hours, and the content of glutamic acid in the fermentation broth was determined by tetraoxo derivatives, and the content of glutamic acid was 5.4g/L.

Compared with this, under the same fermentation and operating conditions, with the existing general-purpose mechanically ventilated stirred reactor culture, the final determination of glutamic acid content is only 4.5g/L. The content of glutamic acid in the obtained fermented liquid of present embodiment case is its 1.2 times.

In addition to the above-mentioned embodiments, the present invention can also have other implementations. All technical solutions formed by equivalent replacement or equivalent transformation fall within the scope of protection required by the present invention.

Claims (9)

1. a high dissolved oxygen bio-reactor for genetic engineering bacterium high-density culture, comprise that top establishes the tank body that charge cavity, bottom are established drain hole, it is characterized in that, described tank body top outer is provided with stirring drive-motor, and described stirring drive-motor output shaft is in transmission connection with the stirring rake that stretches into tank body; Described tank base outside is provided with rotor drive-motor, described rotor drive-motor output shaft is in transmission connection with the Gas-Liquid Dispersion rotor that is positioned at tank interior, described Gas-Liquid Dispersion rotor has top and establishes the vent lumen that inlet mouth and bottom set out gas port, and described inlet mouth is connected with air with pressure source by the road; Described tank base inner side is also fixed with Gas-Liquid Dispersion stator, and described Gas-Liquid Dispersion stator has the diversion trench of establishing water-in and water outlet, the water-in of described diversion trench near Gas-Liquid Dispersion rotor, water outlet away from Gas-Liquid Dispersion rotor; Described Gas-Liquid Dispersion stator is positioned at Gas-Liquid Dispersion periphery of rotor, and described Gas-Liquid Dispersion stator and Gas-Liquid Dispersion rotor coaxial also form revolute pair;

Described Gas-Liquid Dispersion rotor is how curved leaf cavity rotor, and described each curved leaf partly consists of top arch section and lower vertical, and described each curved leaf top arch section is along the distortion of rotor turning direction; Top and the rotor upper port of described each curved leaf top arch section are connected, and the bottom of described each curved hypophyll vertical component and rotor bottom surface are connected; Described each curved leaf and rotor bottom surface and rotor upper port surround vent lumen jointly, and described each curved leaf inner side forms air conducting bend, outside forms liquid guide flow bend; The inlet mouth of described vent lumen is positioned at rotor upper port, and that the air outlet of described vent lumen has is a plurality of, lay respectively at each curved hypophyll vertical component.　

2. high dissolved oxygen bio-reactor for genetic engineering bacterium high-density culture according to claim 1, is characterized in that, the radian diameter of described each curved leaf top arch section is the 2/5-3/5 of whole Gas-Liquid Dispersion rotor diameter; The height of described each curved hypophyll vertical component is the 1/3-1/2 of whole Gas-Liquid Dispersion rotor height; The distortion angle of described each curved leaf top arch section is 20 °-60 °.　

3. high dissolved oxygen bio-reactor for genetic engineering bacterium high-density culture according to claim 1, is characterized in that, described Gas-Liquid Dispersion stator ringwise; The outer edge part of described Gas-Liquid Dispersion stator end face is downward-sloping and middle portion is parallel with bottom surface and be provided with to settle the perforate of Gas-Liquid Dispersion rotor; Between the end face of described Gas-Liquid Dispersion stator and bottom surface, be provided with some diversion trenchs, the water-in of described each diversion trench is to be positioned at end face and to be uniformly distributed in perforate prosopyle around; The water outlet of described each diversion trench is positioned at the outer side of Gas-Liquid Dispersion stator.　

4. high dissolved oxygen bio-reactor for genetic engineering bacterium high-density culture according to claim 3, is characterized in that, between described Gas-Liquid Dispersion stator end face outer edge part and bottom surface, angle is 8 °-20 °; Diversion trench is 15 °-45 ° with the radially angle of crossing the outer end points of diversion trench.　

5. according to high dissolved oxygen bio-reactor for genetic engineering bacterium high-density culture described in claim 1 to 4 any one, it is characterized in that, described air source comprises air compressor machine, water-and-oil separator, air accumulator, air filter and the air flowmeter connecting successively; The air outlet of described air flowmeter is communicated with inlet pipe upper port, and described inlet pipe lower port is communicated with the inlet mouth of Gas-Liquid Dispersion rotor vent lumen; Described inlet pipe lower port and Gas-Liquid Dispersion rotor seal are rotationally connected.　

6. according to high dissolved oxygen bio-reactor for genetic engineering bacterium high-density culture described in claim 1 to 4 any one, it is characterized in that, described stirring rake comprises the drive shaft being in transmission connection with stirring drive-motor output shaft, described drive shaft is circumferentially with agitating vane, and described drive shaft top is circumferentially with froth breaker.　

7. according to high dissolved oxygen bio-reactor for genetic engineering bacterium high-density culture described in claim 1 to 4 any one, it is characterized in that, at the bottom of described tank body comprises plane tank, cylindrical can body and ellipsoidal head tank deck; At the bottom of described tank, through transition arc surface and can body, be tightly connected, described arc surface diameter is the 1/6-1/2 of can body diameter; Described tank deck is fixedly connected with can body through flange; Described charge cavity is positioned at tank deck, at the bottom of described drain hole is positioned at tank; Described tank deck is also provided with manhole; Described stirring drive-motor is tightly connected through aseptic mechanical seal and tank deck, and described rotor drive-motor is tightly connected at the bottom of aseptic mechanical seal and tank; Inner side, described can body middle and lower part is provided with some baffle plates, outside is provided with the chuck with entrance of cooling water and cooling water outlet; Described can body is also provided with sensor interface.　

8. one kind adopts described in claim 1 to 7 any one the high-density culture control method of high dissolved oxygen bio-reactor for genetic engineering bacterium high-density culture, it is characterized in that, comprise Gas-Liquid Dispersion rotor air flow control process, Gas-Liquid Dispersion rotor speed control process and stirring rake mixing speed control process;

Described Gas-Liquid Dispersion rotor air flow control process is: the 70-95% that Gas-Liquid Dispersion rotor air flow is controlled to the critical air flow of Gas-Liquid Dispersion rotor; The deterministic process of the critical air flow of described Gas-Liquid Dispersion rotor is: first control Gas-Liquid Dispersion rotor and rotate with desired speed, by air source, strengthen gradually again the air flow of Gas-Liquid Dispersion rotor vent lumen inlet mouth, when gas no longer enters tank body with Gas-Liquid Dispersion form but with bubbling form from Gas-Liquid Dispersion rotor vent lumen air outlet, air flow is now the critical air flow of Gas-Liquid Dispersion rotor;

Described Gas-Liquid Dispersion rotor speed control process is: the 110-150% by Gas-Liquid Dispersion rotor speed control at Gas-Liquid Dispersion critical rotor speed; The deterministic process of described Gas-Liquid Dispersion critical rotor speed is: first control air source and ventilate to Gas-Liquid Dispersion rotor with predetermined air flow, increase gradually again the rotating speed of Gas-Liquid Dispersion rotor, the liquid that can throw away with Gas-Liquid Dispersion rotor with Gas-Liquid Dispersion form when gas moves to tank body radially during distal-most end along tank body bottom surface, rotating speed is now Gas-Liquid Dispersion critical rotor speed;

Described stirring rake mixing speed control process is: stirring rake mixing speed is controlled to the 60-90% that stirring rake stirs maximum speed of revolution; The deterministic process that described stirring rake stirs maximum speed of revolution is: first control Gas-Liquid Dispersion rotor and with desired speed, rotate and control air source and ventilate to Gas-Liquid Dispersion rotor with predetermined air flow, increase gradually again the mixing speed of stirring rake, volume oxygen mass transfer coefficients K in this process _la increases, also finally tends towards stability with the increase of mixing speed, works as K _la reaches maximum K _la 85% time corresponding mixing speed be stirring rake and stir maximum speed of revolution.　

9. high-density culture control method according to claim 8, is characterized in that, also comprise cultivate before stirring rake chosen process:

When tank body aspect ratio is less than 1.5 and dress liquid height while being less than 1 with tank diameter ratio, adopt one deck stirring rake; When tank body aspect ratio be more than or equal to 1.5 and dress liquid height and tank diameter than between 1-2 time, adopt two-layer or three layers of stirring rake; The blade of described stirring rake is radially oar or press-down type axial flow oar.