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

High dissolved oxygen bioreactor for high-density culture of genetically engineered bacteria and culture control method Download PDF

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Publication number
CN103525689B
CN103525689B CN201310502780.5A CN201310502780A CN103525689B CN 103525689 B CN103525689 B CN 103525689B CN 201310502780 A CN201310502780 A CN 201310502780A CN 103525689 B CN103525689 B CN 103525689B
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gas
liquid dispersion
rotor
dispersion rotor
tank
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CN103525689A (en
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洪厚胜
李军庆
蔡子金
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Nanjing Tech University
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Nanjing Tech University
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02Stirrer or mobile mixing elements
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06Nozzles; Sprayers; Spargers; Diffusers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/14Pressurized fluid
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation

Abstract

The invention relates to a high dissolved oxygen bioreactor for the 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 used for driving a stirring paddle and is connected with the stirring paddle; the bottom of the tank body is provided with a rotor driving motor, an output shaft of the rotor driving motor is used for driving a gas-liquid dispersion rotor and is connected with the gas-liquid dispersion rotor, the gas-liquid dispersion rotor is provided with an inner ventilation cavity which is provided with an air inlet at the top and an air outlet at the lower part, and the air inlet is connected with an under-pressure air source through a pipeline; the inner side at the bottom of the tank body is also fixedly connected with a gas-liquid dispersion stator provided with a diversion trench; the gas-liquid dispersion stator is located at the circumference of the gas-liquid dispersion rotor and is coaxial with the gas-liquid dispersion rotor to form a rotating 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 high dissolved oxygen bioreactor is high in dissolved oxygen level and suitable for the high-density culture of the genetically engineered bacteria.

Description

Genetic engineering bacterium high-density culture is with high dissolved oxygen bio-reactor and cultivate control method
Technical field
The present invention relates to high dissolved oxygen bio-reactor for a kind of genetic engineering bacterium high-density culture, and the cultivation control method that adopts this reactor, bio-fermentation engineering field belonged to.
Background technology
Along with the development of genetic engineering technique, increasing biological products can make by genetically engineered technique.Wherein, intestinal bacteria and pichia spp have been widely used in the structure of genetic engineering bacterium, in order to obtain the exogenous genes products that people is had to utility value.
Utilize genetic engineering bacterium fermentation to be intended to obtain large batch of high quality exogenous genes products, also will reduce as far as possible the pollution of host cell self moiety, thereby its zymotechnique is different from the zymotechnique of traditional microorganism simultaneously.
Traditional batch-type cultivation employing is disposable to feed intake and puts tank, and its nutrient solution starting point concentration is higher, easily produces the restraining effect of substrate and meta-bolites, and higher fermented liquid concentration is also unfavorable for the transmission of oxygen.
According to the knowledge of the applicant, at present, high cell density fermentation is day by day ripe, become the main technique of producing as a trial in biotechnology, there is process stabilizing, fermentation period is short, exogenous gene expression amount is high, in some fermenting process, its fermentation period can shorten more than 50%, and thalline output and exogenous gene expression amount are 10-50 times of non-high-density culture.High-density culture technique has also become genetic engineering bacterium and has improved one of important means of exogenous gene expression amount.
Yet, affect the many factors of genetic engineering bacterium exogenous gene expression amount, as: the characteristic of genetic engineering bacterium, medium component, the concentration of temperature during fermentation, pH value, dissolved oxygen, the degree of mixing of material in fermentor tank, the fed-batch mode of inductor and stream dosage etc., wherein, in the concentration of dissolved oxygen and fermentor tank, the degree of mixing of material is particularly remarkable on the impact of high-density culture gene engineering exogenous gene expression amount.
By the method for existing genetic engineering bacterium high-density culture, generally in thalli growth phase oxyty, be controlled at 30% left and right, inductive phase oxyty be controlled at 20% left and right.In the thalli growth phase, due to the thalline comparatively small amt of thalline quantity relatively stable period in thalline, by conditions such as regulation and control air inputs, be easy to reach the requirement of 30% oxyty.And in thalline inductive phase, oxyty is controlled at 20% left and right and is difficult for reaching, its reason is: thalline belongs to the high-density culture later stage inductive phase, thalline quantity is exponential type amplification, demand to dissolved oxygen sharply increases, and the Gas-Liquid Dispersion performance of common bio-reactor is limited, only by increasing the means of air flow and raising mixing speed, be difficult to meet the demand of thalline to dissolved oxygen and mixing mass transfer.For this difficult problem, many zymotechniques can be used pure oxygen to replace air to solve problem, yet use pure oxygen not only can increase production cost, and make troubles to the control of whole fermenting process.
At present, the conventional bio-reactor of genetic engineering bacterium high-density culture mainly contains three major types: airlift reactor, ventilation stirred reactor, self-priming reactor.
Airlift reactor is divided into external loop airlift reactor and gas lift type common loop reactor.Because it has simple in structure, when operation shearing stress feature such as less, be widely used in shear-sensitive type fermentation process.Airlift reactor mainly utilizes the ejection function of gas and the density difference of fluid medium to come driving a reaction system to circulate.In airlift reactor, can strengthen the effect that in fermentor tank, reaction system circulated and mixed mass transfer by guide shell is installed.Airlift reactor can improve homogeneity and the dissolved oxygen speed that gas-liquid distributes, but the air flow that it is had relatively high expectations and venting pressure, this causes energy consumption to increase to a certain extent, especially for the higher fermentation system of viscosity, its oxygen mass transfer coefficients is less, and mixed effect is poor.When for genetic engineering bacterium high-density culture, it mixes mass transfer effect and is difficult to reach requirement, easily produces a large amount of foams when air flow is larger, increases the probability of microbiological contamination.
Compare with airlift reactor, ventilation stirred reactor structure is comparatively complicated.Ventilation stirred reactor consists of tank body, whipping appts, breather and other auxiliary equipment conventionally.Wherein, whipping appts is located in the middle of tank body and with tank body and is adopted sterile seal, and the gas distributor of breather is located at tank base position.Gas ejects from gas distributor through breather, relies on the agitating vane of high speed rotating to realize mixing mass transfer.Ventilation stirred reactor is applicable to most of aerobic fermentation processes, but also Shortcomings, as, for realizing higher oxyty and mixing mass transfer, will increase mixing speed and air flow, this can increase energy consumption, and whole shearing stress in tank body is increased, and be unfavorable for shear-sensitive type microorganism growth, especially when thread yeast culture, the damage to cell is larger.
Self-priming reactor is a kind of reactor that utilizes Gas-Liquid Dispersion rotor high-speed to rotate, initiatively ambient atmos is sucked tank body, because having outstanding Gas-Liquid Dispersion performance and the characteristic of low power consuming, be widely used, especially in the fermentative production of vinegar and yeast, application is more ripe.Yet also there is unreasonable place in the structure of existing self-priming reactor, is mainly reflected in: Gas-Liquid Dispersion rotor inspiratory capacity is little, tank body aspect ratio can not be excessive, tank body volume also can not be too large, and this has just limited the fermentation capacity of reactor.
The Gas-Liquid Dispersion rotor of existing self-priming reactor is generally located at tank base or place, mid-way.
When being located at tank base, Gas-Liquid Dispersion rotor owing to there being higher liquid level difference, cause the increase of Gas-Liquid Dispersion critical rotor speed, inspiratory capacity to reduce, along with this phenomenon of increase of tank body aspect ratio is more serious.Because Gas-Liquid Dispersion rotor is less and be positioned over tank base, the fermented liquid of tank body middle and upper part just carries out integral macroscopic circulation under the drive of bubble during the fermentation, its micromixing intensity a little less than, can cause mass transfer effect to decline, mixing of materials is inhomogeneous.In the larger reaction system of viscosity, mixing mass transfer effect can be poorer.
When Gas-Liquid Dispersion rotor is located at tank body mid-way, the reactor that is placed in tank base with Gas-Liquid Dispersion rotor is compared the inspiratory capacity increase under its quick passage critical speed reduction, equal rotating speed, but there will be tank body top gas holdup to be greater than the phenomenon of tank body lower part gas holdup, and this phenomenon utmost point is unfavorable for microbial growth.
Summary of the invention
Technical problem to be solved by this invention is: overcome the problem that prior art exists, high dissolved oxygen bio-reactor for a kind of genetic engineering bacterium high-density culture is provided, can meet the demand of high oxyty, be beneficial to and implement genetic engineering bacterium high-density culture.In addition, also provide the cultivation control method that adopts this reactor.
The technical scheme that the present invention solves its technical problem is as follows:
High dissolved oxygen bio-reactor for a kind of 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.
This structure in use, with overhead type stirring rake, continue mixture in stirred pot on the one hand, Gas-Liquid Dispersion rotor continues to rotate on the other hand, gas and the pot liquid of air source input are mixed well, and make gas-liquid mixture spurt into tank body through Gas-Liquid Dispersion stator, dispersion effect is good, and dissolved oxygen efficiency is high, can meet the demand of high oxyty.
Reactor of the present invention further perfect technical scheme is as follows:
Preferably, 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.
Applicant finds in going deep into practical studies, Gas-Liquid Dispersion rotor adopts this structure can increase the space of its internal cavities, be conducive to improve the inspiratory capacity of Gas-Liquid Dispersion rotor, the air conducting bend that Gas-Liquid Dispersion internal rotor is consistent with turning direction, can make the gas of cavity inside obtain larger power, jet velocity is larger, Gas-Liquid Dispersion is more even; The liquid guide flow bend that Gas-Liquid Dispersion rotor exterior is consistent with turning direction can drive Gas-Liquid Dispersion rotor top liquid around to move downward with the state of height turbulence in the situation that of high speed rotating.Can increase like this lifting rate and the inspiratory capacity of Gas-Liquid Dispersion rotor, can make liquid effectively mix with the gas of cavity ejection, improve dissolved oxygen level.
More preferably, 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 °.
Applicant finds in going deep into practical studies, and above structure can further be optimized the performance of Gas-Liquid Dispersion rotor, further improves dissolved oxygen level.
Preferably, 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.
Applicant finds through going deep into practical studies, Gas-Liquid Dispersion stator adopts after this structure, the inner port area of diversion trench is greater than outer end opening area, can greatly reduce fluid by the resistance of Gas-Liquid Dispersion stator, and the fluid that can make Gas-Liquid Dispersion rotor throw away spreads out by diversion trench more swimmingly; The position of prosopyle and decoration form can increase the lifting rate of Gas-Liquid Dispersion rotor, improve Gas-Liquid Dispersion effect.
More preferably, 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.
Applicant finds through going deep into practical studies, and above structure can make diversion trench direction and Gas-Liquid Dispersion rotor throw away flow direction to be consistent effectively, further reduce fluid by time resistance.
In addition, reactor of the present invention also can have following preferred feature:
Preferably, 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.
Preferably, described stirring rake comprises the drive shaft being in transmission connection with stirring drive-motor output shaft, and described drive shaft is circumferentially with agitating vane, and described drive shaft top is circumferentially with froth breaker.
Preferably, 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.
The present invention also provides:
A kind ofly adopt the high-density culture control method of high dissolved oxygen bio-reactor for aforementioned 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.
Applicant finds through going deep into practical studies on previous reaction device basis, when Gas-Liquid Dispersion rotor air flow is greater than critical air flow, gas overflows with the form of bubbling, and dispersion effect is poor, bubble large and in liquid phase the residence time shorter, cause oxygen mass transfer coefficients step-down.Gas-Liquid Dispersion rotor speed during lower than quick passage critical speed, not only can make part bubble overflow with the form of bubbling, and can make the outer generation dead band, edge of tank base, easily causes solid accumulation herein.Under the condition of certain air flow and certain Gas-Liquid Dispersion rotor speed, when stirring rake mixing speed is greater than maximum speed of revolution, its K la increasing degree is less, and the raising that mixes mass transfer effect is also not obvious, and can cause the increase of whole shearing stress and power of agitator.Applicant is further further investigation on this basis, finally draws above-specified high density cultivation control method.Adopt after this control method, make previous reaction device more be applicable to genetic engineering bacterium high-density culture fermentative production, especially be applicable to the fermentative production of high viscosity, high-density and shear-sensitive type microorganism, effectively solve further the technical barrier that supplies hypoxgia in genetic engineering bacterium high-density culture, the effect that further improves mixing mass transfer also reduces energy consumption.
Preferably, also comprise and cultivate front 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.
Compared with prior art, the present invention has following beneficial effect:
1, Gas-Liquid Dispersion performance is good, oxygen transfer rate is high, and air flow is compared little with existing bio-reactor, and air utilization ratio is high, can effectively reduce air compressor machine working load, reduces production energy consumption.
2, the mixing speed of required stirring rake can, lower than existing machinery stirred bioreactor, make reactor of the present invention have the advantages that shearing stress is low, applicable to the yeast culture of shear-sensitive type.
3, dissolved oxygen level is high, good mixing effect, be suitable for the cultivation of high viscosity, high-density microorganism, be particularly suited for genetic engineering bacterium high-density culture, can meet the needs of high-density thalline to dissolved oxygen, overcome existing bio-reactor for the technical barrier of the highly dense cultivation dissolved oxygen of genetic engineering bacterium deficiency, improved the effect of mixing mass transfer.
4, device turndown ratio is large, can meet various fermentation condition, applied widely.
The apparatus system 5, with a whole set of, can be optimized links, is guaranteeing dissolved oxygen and is mixing the maximization operation of conveniently carrying out device under the condition that mass transfer effect is constant.
Accompanying drawing explanation
Fig. 1 is the structural representation of embodiment of the present invention reactor.
Fig. 2 is the structural representation of Fig. 1 embodiment Gas-Liquid Dispersion rotor.
Fig. 3 is the vertical view of Fig. 2.
Fig. 4 is the A-A sectional view of Fig. 3.
Fig. 5 is the vertical view of Fig. 1 embodiment Gas-Liquid Dispersion stator.
Fig. 6 is the B-B sectional view of Fig. 5.
Embodiment
With reference to the accompanying drawings and in conjunction with the embodiments the present invention is described in further detail.But the invention is not restricted to given example.
Embodiment
As shown in Figures 1 to 6, high dissolved oxygen bio-reactor for the genetic engineering bacterium high-density culture of the present embodiment, comprise that top establishes the tank body that charge cavity 4, bottom are established drain hole 15, tank body top outer is provided with and stirs drive-motor 6, stirs drive-motor 6 output shafts and is in transmission connection with the stirring rake that stretches into tank body; Tank base outside is provided with rotor drive-motor 16, rotor drive-motor 16 output shafts are in transmission connection with the Gas-Liquid Dispersion rotor 18 that is positioned at tank interior, Gas-Liquid Dispersion rotor 18 has top and establishes the vent lumen that inlet mouth and bottom set out gas port, and inlet mouth is connected with air with pressure source by the road; Tank base inner side is also fixed with Gas-Liquid Dispersion stator 19, and Gas-Liquid Dispersion stator 19 has the diversion trench of establishing water-in and water outlet; It is circumferential that Gas-Liquid Dispersion stator 19 is positioned at Gas-Liquid Dispersion rotor 18, and Gas-Liquid Dispersion stator 19 is coaxial and form revolute pair with Gas-Liquid Dispersion rotor 18; The water-in of diversion trench near Gas-Liquid Dispersion rotor, water outlet away from Gas-Liquid Dispersion rotor.
At the bottom of tank body comprises plane tank, cylindrical can body and ellipsoidal head tank deck.At the bottom of tank, be that plane can reduce gas-liquid fluid at the resistance of tank base motion, can effectively reduce material deposit buildup to tank spirit liquid dispersion rotor probability around, can reduce the resistance of Gas-Liquid Dispersion rotor rotation and the number of times of maintenance of equipment.Ellipsoidal head tank deck can strengthen the intensity of tank body.
At the bottom of tank, through transition arc surface 14 and can body, be tightly connected, arc surface 14 diameters are the 1/6-1/2 of can body diameter.Can reduce so the outer peripheral dead band of tank base, can also strengthen tank intensity, be conducive to the manufacture of large tank.
Tank deck is fixedly connected with can body through flange 3; Charge cavity 4 is positioned at tank deck, at the bottom of drain hole 15 is positioned at tank; Tank deck is also provided with manhole 7; Stir drive-motor 6 and be tightly connected with tank deck through aseptic mechanical seal 5, rotor drive-motor 16 is tightly connected with at the bottom of tank through aseptic mechanical seal 17; Inner side, can body middle and lower part is provided with some baffle plates 11, outside is provided with the chuck 10 with entrance of cooling water 13 and cooling water outlet 21; Can body is also provided with sensor interface 20.In the present embodiment, baffle plate 11 has four, and its width is 1/10 of can body diameter.
Air source comprises air compressor machine 25, water-and-oil separator 24, air accumulator 23, air filter 22 and the air flowmeter 21 connecting successively; The air outlet of air flowmeter 21 is communicated with inlet pipe 2 upper port, and inlet pipe 2 lower port are communicated with the inlet mouth of Gas-Liquid Dispersion rotor 18 vent lumen; Inlet pipe 2 lower port are connected with Gas-Liquid Dispersion rotor 18 sealed rotational.Utilize air compressor machine air feed, can increase air input, improve dissolved oxygen level, reduce the impact of liquid level difference on air input in tank body, can facilitate the maximization operation of reactor.
As shown in Figures 2 to 4, Gas-Liquid Dispersion rotor 18 is how curved leaf cavity rotor (specifically can adopt nine curved leaf cavity rotors), and each curved leaf consists of top arch section 27 and lower vertical part 26, and each curved leaf top arch section 27 is along the distortion of rotor turning direction; Top and the rotor upper port 28 of each curved leaf top arch section 27 are connected, and the bottom of each curved hypophyll vertical component 26 and rotor bottom surface are connected; Each curved leaf and rotor bottom surface and rotor upper port 28 surround vent lumen jointly, and each curved leaf inner side forms air conducting bend, outside forms liquid guide flow bend; The inlet mouth of vent lumen is positioned at rotor upper port 28, and that the air outlet 29 of vent lumen has is a plurality of, lay respectively at each curved hypophyll vertical component 26.
Radian 30 diameters of each curved leaf top arch section 27 are the 2/5-3/5 of whole Gas-Liquid Dispersion rotor 18 diameters; The height of each curved hypophyll vertical component 26 is the 1/3-1/2 of whole Gas-Liquid Dispersion rotor 18 height; The distortion angle C of each curved leaf top arch section 27 is 20 °-60 °.
Gas-Liquid Dispersion stator 19 ringwise; The outer edge part of Gas-Liquid Dispersion stator 19 end faces is downward-sloping and middle portion is parallel with bottom surface and be provided with to settle the perforate of Gas-Liquid Dispersion rotor 18; Between the end face of Gas-Liquid Dispersion stator 19 and bottom surface, be provided with some diversion trenchs (specifically can adopt 12-18 diversion trench), the water-in of each diversion trench is to be positioned at end face and to be uniformly distributed in perforate prosopyle 31 around; The water outlet of each diversion trench is positioned at the outer side of Gas-Liquid Dispersion stator 19.
Between Gas-Liquid Dispersion stator 19 end face outer edge part and bottom surface, angle E is 8 °-20 °; Diversion trench is 15 °-45 ° with the radially angle D that crosses the outer end points of diversion trench.
Stirring rake comprises the drive shaft 9 being in transmission connection with stirring drive-motor 6 output shafts, and drive shaft 9 is circumferentially with agitating vane 12, and drive shaft 9 tops are circumferentially with froth breaker 8.
Agitating vane can make the gas in fermented liquid circulate in tank body with blade, increases gas-liquid contact time, improves oxygen transfer efficiency, and the rotation of blade simultaneously can increase liquid turbulence intensity, reduces the mixing time of liquid phase, improves mixing efficiency.Under the effect at Gas-Liquid Dispersion rotor, Gas-Liquid Dispersion is better, bubble diameter is less, stirring rake does not need to improve Gas-Liquid Dispersion degree by high rotating speed, only need to keep the integral macroscopic of strengthening fermented liquid compared with the slow speed of revolution to mix, can make like this shearing stress of whole reactor little compared with the bio-reactor of existing ventilation stirring-type.Froth breaker can be eliminated the bubble breaking producing in fermenting process, reduces microbiological contamination probability.
Adopt the high-density culture control method of high dissolved oxygen bio-reactor for the present embodiment genetic engineering bacterium high-density culture, comprise Gas-Liquid Dispersion rotor air flow control process, Gas-Liquid Dispersion rotor speed control process and stirring rake mixing speed control process.
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 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.
Owing to passing into, the kinetic energy of air derives from the pressure of air compressor machine and Gas-Liquid Dispersion rotor rotates the negative-pressure sucking that causes peritrochanteric liquid to be thrown away at a high speed and form at it around, therefore, the required venting pressure of whole reactor is lower, can effectively reduce air compressor machine load, reduces energy consumption.
When air flow increases gradually, Gas-Liquid Dispersion peritrochanteric liquid is arranged by air gradually, the lifting rate of Gas-Liquid Dispersion rotor reduces gradually, rotate the vacuum tightness forming also reduces thereupon, the gas being thrown away by liquid entrainment reduces gradually, and final gas can enter tank body from Gas-Liquid Dispersion rotor in the mode of bubbling.Applicant is decided to be the critical air flow of Gas-Liquid Dispersion rotor by the stagnation point of this process.
Applicant's discovery, if air flow is too small, system is for hypoxgia, reactor performance cannot be not fully exerted; If air flow is greater than critical air flow, gas enters liquid by the form with bubbling, cannot bring into play the Gas-Liquid Dispersion effect of Gas-Liquid Dispersion rotor, the diameter of bubble will obviously increase, oxygen transfer rate declines, and in tank body, liquid level, by violent shake occurs, affects liquid amount simultaneously.Applicant is through further further investigation, and considers the unstable of ventilation and gas in factors such as Liquid characteristics, finally draws above-mentioned Gas-Liquid Dispersion rotor air flow control process.
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 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.
Under a certain amount of aeration condition, Gas-Liquid Dispersion rotor is with certain rotational speed, and the violent turbulence of its surrounding liquid also produces centrifugal motion and moves to described tank body straight flange wall, at Gas-Liquid Dispersion peritrochanteric, forms negative pressure.Air is at the pressure of air compressor machine and by Gas-Liquid Dispersion rotor, rotated under the negative-pressure sucking effect forming, with liquid, throw away and move along tank body bottom surface, in this process, spouting of liquid intensity is larger, air is divided into less bubble, bubble moves to tank skin face equably at the bottom of tank, reach tank body radially the required minimum speed of distal-most end be Gas-Liquid Dispersion critical rotor speed.Gas-liquid is moved and can be avoided material at the bottom of tank and the accumulation at gas-liquid dispersion rotor position along tank base, improve raw material mixture homogeneity, reduce the probability of maintenance of equipment.
Applicant's discovery, when Gas-Liquid Dispersion rotor speed is less than quick passage critical speed, gas can not arrive tank skin position, easily forms dissolved oxygen dead band, is unfavorable for microbial growth; When Gas-Liquid Dispersion rotor speed is greater than quick passage critical speed, liquid phase injection intensity is comparatively strong, and air is torn into less bubble, is conducive to the mixing mass transfer of oxygen, but too high rotating speed can make shearing stress and power consumption increase.Applicant, through further further investigation, draws above-mentioned Gas-Liquid Dispersion rotor speed control process finally.
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 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.
The height of reactor oxygen transfer efficiency can pass through volume oxygen mass transfer coefficients K lthe size of a characterizes.While rotating when the air flow air inlet with certain and with certain Gas-Liquid Dispersion rotor speed, K la increases along with the increase of mixing speed, finally tends towards stability.Because Gas-Liquid Dispersion rotor has good Gas-Liquid Dispersion performance, weakened high mixing speed for tearing bubble, increasing vapour-liquid ratio surface-area to improve K lthe effect of a, thereby shearing and the energy consumption of avoiding high mixing speed to increase.The stirring rake of the present embodiment reactor only need, under lower mixing speed, play the effect that strengthens gas-liquid micromixing.Applicant, through further further investigation, draws above-mentioned stirring rake mixing speed control process finally, both can guarantee that the integral macroscopic of gas-liquid mixes, and can reduce again the energy consumption of whole device.
In addition, the many factors relevant to maximum mixing speed, wherein, blade type and the blade number of plies are larger on maximum mixing speed impact.
For fermented liquid in the present embodiment tank body: Gas-Liquid Dispersion rotor rotates, its surrounding liquid produces centrifugal motion, form negative pressure, Gas-Liquid Dispersion rotor upper liquid and the gas that passed into by inlet pipe are mixed, along tank base directive tank body wall, under the stopping of wall, along tank wall face, move upward, in uphill process, a plurality of small bubbles mutually collide and cause thickness of liquid film attenuation between bubble, final bubble generation coalescence, it is large that bubble diameter becomes; When arriving liquid level place, portion gas overflows liquid level, and residual gas flows to Gas-Liquid Dispersion rotor with liquid downwards along stir shaft, forms circulation.
When tank body aspect ratio is less than 1.5 and liquid amount height while being less than 1 with tank diameter ratio, can establish 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, can adopt two-layer or three layers of stirring rake according to fermented liquid system characteristic.Simultaneously, to determine agitating vane type according to fermented liquid system viscosity, in the situation that viscosity is higher, mixed effect is poor, bubble coalescence phenomenon is serious, agitating vane can adopt the radially oar that power levels number is less, as six para-curve vane wheel oars and six semi-circular tube vane wheel oars, but do not limit, do not use these two kinds of blades.When viscosity is little, can adopt the press-down type axial flow oar that discharge capacity is larger, as three square blades and four plum blossom leaf oars, but do not limit, not use these two kinds of blades.According to practical situation, select blade type and the blade number of plies can effectively reduce maximum mixing speed, thereby reduce shearing force and energy consumption.
Concrete application case is as follows:
The cold mould experiment of cold mould experiment 1:(is without the simulated experiment of chemical reaction, lower same)
Reactor size is: tank diameter is 700mm, and useful volume is 300L; Gas-Liquid Dispersion rotor diameter is 96mm; Gas-Liquid Dispersion stator adopts 12 diversion trench designs, and external diameter is 168mm; Four barrier width are 70mm.Dress liquid height is 700mm, establishes one deck stirring rake, and blade is four plum blossom axial flow oars, and blade footpath is 200mm.
Mensuration system is tap water, and system temperature is 25 ℃.
Critical air flow measurement result under Gas-Liquid Dispersion rotor different rotating speeds is as shown in table 1, and under different air flows, Gas-Liquid Dispersion critical rotor speed measurement result is as shown in table 2.
Critical air flow under table 1, Gas-Liquid Dispersion rotor different rotating speeds
First under the condition of 1500r/min, 85% critical air flow, carry out reactor control condition mensuration take Gas-Liquid Dispersion rotor speed, take air flow as 7.2m 3under the condition of/h, measure Gas-Liquid Dispersion critical rotor speed, measurement result is 1320r/min, and Gas-Liquid Dispersion rotor speed is set as to 120% of quick passage critical speed, is 1584r/min.Take air flow as 7.2m again 3the condition of/h, Gas-Liquid Dispersion rotor speed 1584r/min is measured and is stirred maximum speed of revolution, and measurement result is 240r/min, and mixing speed is set as to 75% of maximum mixing speed, is 180r/min.
Record with this understanding K la value is 0.065S -1, than the K of existing ventilation stirred reactor under the same terms la improves more than 30%.
Cold mould experiment 2:
Reactor size is: tank diameter is 700mm, and useful volume is 300L; Gas-Liquid Dispersion rotor diameter is 96mm; Gas-Liquid Dispersion stator adopts 12 diversion trench designs, and external diameter is 168mm; Four barrier width are 70mm.Dress liquid height is 700mm, establishes one deck stirring rake, and blade is four plum blossom axial flow oars, and blade footpath is 200mm.
Mensuration system is that massfraction is 0.5% sodium carboxymethyl cellulose solution, and system temperature is 25 ℃.
Critical air flow measurement result under Gas-Liquid Dispersion rotor different rotating speeds is as shown in table 3, and under different air flows, Gas-Liquid Dispersion critical rotor speed measurement result is as shown in table 4.
Critical air flow under table 3, Gas-Liquid Dispersion rotor different rotating speeds
First under the condition of 1500r/min, 85% critical air flow, carry out reactor control condition mensuration take Gas-Liquid Dispersion rotor speed, take air flow as 6.9m 3under the condition of/h, measure Gas-Liquid Dispersion critical rotor speed, measurement result is 1300r/min, and Gas-Liquid Dispersion rotor speed is set as to 120% of quick passage critical speed, is 1560r/min.Take air flow as 6.9m again 3the condition of/h, Gas-Liquid Dispersion rotor speed 1560r/min is measured and is stirred maximum speed of revolution, and measurement result is 260r/min, and mixing speed is set as to 75% of maximum mixing speed, is 195r/min.
Record with this understanding K la value is 0.055S -1, than the K of existing ventilation stirred reactor under the same terms la improves more than 35%.
Case study on implementation 1:
Reactor size is: tank body volume is 5m 3, tank body aspect ratio is 2.2 ﹕ 1, and Gas-Liquid Dispersion rotor diameter is 215mm, and Gas-Liquid Dispersion stator adopts 16 diversion trench designs, and external diameter is 405mm, four barrier width are 140mm.If two-layer stirring rake, diameter of propeller blade is 500mm.
Under the condition that the implementation case is 2.8t at liquid amount, measure critical air flow, the Gas-Liquid Dispersion critical rotor speed under this air flow and maximum mixing speed that Gas-Liquid Dispersion rotor speed is 2100r/min.
Recording critical air flow is 134.4m 3/ h, the critical air flow according to 85% is 114.2m 3/ h air inlet, recording Gas-Liquid Dispersion critical rotor speed is 1950r/min.To set Gas-Liquid Dispersion rotor speed be quick passage critical speed 120% is 2340r/min, and recording maximum mixing speed is 210r/min, and it is 158r/min that mixing speed is set as to 75% of maximum mixing speed.
Using produce gsh genetic engineering bacterium pichia spp as bacterial classification, after one-level, secondary seed are cultivated, according to 3%(v/v) inoculum size be inoculated into containing carrying out high-density culture in the reactor of fermention medium.
Liquid amount is 2.8t, in fermenting process, flow glycerol adding and meet thalli growth, by Feeding ammonia water, controlling pH is 6.5, temperature is controlled at 31 ℃, in the fermentation later stage, add the growth that Cys, L-glycine and Pidolidone guarantee thalline, cultivate 51h, utilize four oxygen derivative methods to measure the content of fermented liquid two-story valley Gelucystine, the content of paddy Gelucystine is 6.5g/L.
In contrast to this, under identical fermentation and operational condition, with existing general form power ventilation stirred reactor, cultivate, the content of finally measuring paddy Gelucystine is only 4.1g/L.Paddy cystine in the implementation case gained fermented liquid is its 1.59 times.
Case study on implementation 2:
Reactor size is: tank body volume is 5m 3, tank body aspect ratio is 2.2 ﹕ 1, and Gas-Liquid Dispersion rotor diameter is 215mm, and Gas-Liquid Dispersion stator adopts 16 diversion trench designs, and external diameter is 405mm, four barrier width are 140mm.If two-layer stirring rake, diameter of propeller blade is 500mm.
Under the condition that the implementation case is 2.8t at liquid amount, measure critical air flow, the Gas-Liquid Dispersion critical rotor speed under this air flow and maximum mixing speed that Gas-Liquid Dispersion rotor speed is 2100r/min.
Recording critical air flow is 134.4m 3/ h, the critical air flow according to 70% is 94m 3/ h air inlet, recording Gas-Liquid Dispersion critical rotor speed is 1780r/min.To set Gas-Liquid Dispersion rotor speed be quick passage critical speed 120% is 2136r/min, and recording maximum mixing speed is 240r/min, and it is 180r/min that mixing speed is set as to 75% of maximum mixing speed.
Using produce gsh genetic engineering bacterium pichia spp as bacterial classification, after one-level, secondary seed are cultivated, according to 3%(v/v) inoculum size be inoculated into containing carrying out high-density culture in the reactor of fermention medium.
Liquid amount is 2.8t, in fermenting process, flow glycerol adding and meet thalli growth, by Feeding ammonia water, controlling pH is 6.5, temperature is controlled at 31 ℃, in the fermentation later stage, add the growth that Cys, L-glycine and Pidolidone guarantee thalline, cultivate 51h, utilize four oxygen derivatives to send out the content of measuring fermented liquid two-story valley Gelucystine, the content of paddy Gelucystine is 5.2g/L.
In contrast to this, in identical fermentation and operational condition, with existing general form power ventilation stirred reactor, cultivate, the content of finally measuring paddy Gelucystine is only 3.3g/L.Paddy cystine in the implementation case gained fermented liquid is its 1.58 times.
Case study on implementation 3:
Reactor size is: tank body volume is 5m 3, tank body aspect ratio is 2.2 ﹕ 1, and Gas-Liquid Dispersion rotor diameter is 215mm, and Gas-Liquid Dispersion stator adopts 16 diversion trench designs, and external diameter is 405mm, four barrier width are as 140mm.If two-layer stirring rake, diameter of propeller blade is 500mm.
Under the condition that the implementation case is 2.8t at liquid amount, measure critical air flow, the Gas-Liquid Dispersion critical rotor speed under this air flow and maximum mixing speed that Gas-Liquid Dispersion rotor speed is 2100r/min.
Measuring critical air flow is 134.4m 3/ h, the critical air flow according to 95% is 127.7m 3/ h air inlet, recording Gas-Liquid Dispersion critical rotor speed is 2020r/min.To set Gas-Liquid Dispersion rotor speed be quick passage critical speed 120% is 2424r/min, and recording maximum mixing speed is 195r/min, and it is 146r/min that mixing speed is set as to 75% of maximum mixing speed.
Using produce gsh genetic engineering bacterium pichia spp as bacterial classification, after one-level, secondary seed are cultivated, according to 3%(v/v) inoculum size be inoculated into containing carrying out high-density culture in the reactor of fermention medium.
Liquid amount is 2.8t, in fermenting process, flow glycerol adding and meet thalli growth, by Feeding ammonia water, controlling pH is 6.5, temperature is controlled at 31 ℃, in the fermentation later stage, add the growth that Cys, L-glycine and Pidolidone guarantee thalline, cultivate 51h, utilize four oxygen derivatives to send out the content of measuring fermented liquid two-story valley Gelucystine, the content of paddy Gelucystine is 5.4g/L.
In contrast to this, under identical fermentation and operational condition, with existing general form power ventilation stirred reactor, cultivate, the content of finally measuring paddy Gelucystine is only 4.5g/L.Paddy cystine in the implementation case gained fermented liquid is its 1.2 times.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of 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.
CN201310502780.5A 2013-10-23 2013-10-23 High dissolved oxygen bioreactor for high-density culture of genetically engineered bacteria and culture control method Expired - Fee Related CN103525689B (en)

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