CN109777721B - Continuous fermentation system and fermentation process for engineering bacteria - Google Patents
Continuous fermentation system and fermentation process for engineering bacteria Download PDFInfo
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- CN109777721B CN109777721B CN201910208725.2A CN201910208725A CN109777721B CN 109777721 B CN109777721 B CN 109777721B CN 201910208725 A CN201910208725 A CN 201910208725A CN 109777721 B CN109777721 B CN 109777721B
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Abstract
The invention relates to a continuous fermentation system and a fermentation process for engineering bacteria, wherein the continuous fermentation system comprises a first fermentation tank, a second fermentation tank, a butterfly centrifuge, a stirring tank and a peristaltic pump, wherein an outlet pipe of the first fermentation tank, an outlet pipe of the second fermentation tank and an inlet pipe of the butterfly centrifuge are mutually communicated through a first pipeline; the butterfly centrifuge is provided with a supernatant outlet and a fermentation thallus outlet, and the fermentation thallus outlet of the butterfly centrifuge is communicated with the inlet of the stirring tank through a second pipeline; the outlet pipe of the stirring tank, the inlet pipe of the first fermentation tank and the inlet pipe of the second fermentation tank are mutually communicated through a third pipeline; the peristaltic pump is arranged on the third pipeline; the outlet pipe of the first fermentation tank is provided with a first valve, the outlet pipe of the second fermentation tank is provided with a second valve, the inlet pipe of the butterfly centrifuge is provided with a third valve, the outlet pipe of the stirring tank is provided with a fourth valve, the inlet pipe of the first fermentation tank is provided with a fifth valve, and the inlet pipe of the second fermentation tank is provided with a sixth valve. The invention has high fermentation efficiency.
Description
Technical Field
The invention belongs to the technical field of strain fermentation, and particularly relates to a continuous fermentation system and a fermentation process for engineering bacteria.
Background
The fermentation engineering is to provide an environment condition which is most suitable for the growth of microorganisms, and utilize the metabolic function of the microorganisms to produce products required by human beings through modern technical means such as transgenic engineering and the like. The primary complete fermentation comprises the processes of sterilization, strain culture, inoculation, expansion, induction and the like of the whole fermentation system, meanwhile, the material supplementing control is needed in the culture induction process, and finally, the induced fermentation product is obtained.
The fermentation period is different according to different strain habits, but the culture of general engineering bacteria is time-consuming. At present, most of common fermentation is batch fermentation, batch feed fermentation, continuous fermentation and the like. The batch fermentation is a fermentation method in which a limited amount of nutrients are added into a closed system, and then a small amount of strains are inoculated for culture, so that the strains grow and reproduce, and only one growth cycle is completed under specific conditions. Fed-batch fermentation, also known as fed-batch fermentation, refers to a fermentation technique in which, during a microbial batch fermentation, a certain amount of material is fed into the fermentation system in some way, but the fermentation broth is not continuously discharged outwards. Continuous fermentation refers to a fermentation technique in which a certain amount of material is fed into a fermentation system in a certain manner while continuously discharging fermentation liquid outwards during a microbial batch fermentation process. During batch fermentation and batch feed fermentation, bacterial cells have a certain growth rule, and all have to undergo a delay period, a logarithmic growth phase, a stabilization phase and a decay phase, and each batch of fermentation needs to undergo the stages of strain expansion, equipment flushing, sterilization and the like, so that large manpower, material resources, power and low production efficiency are required to be consumed.
And a system that allows continuous batch fermentation harvesting solves the above problems. Therefore, how to design a continuous fermentation system with high fermentation efficiency is a technical point to be solved in the art.
Disclosure of Invention
Based on the defects in the prior art, the invention provides a continuous fermentation system and a fermentation process for engineering bacteria.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
the continuous fermentation system for engineering bacteria comprises a first fermentation tank, a second fermentation tank, a butterfly centrifuge, a stirring tank and a peristaltic pump, wherein an outlet pipe of the first fermentation tank, an outlet pipe of the second fermentation tank and an inlet pipe of the butterfly centrifuge are connected through a first pipeline so as to be communicated with each other; the butterfly centrifuge is provided with a supernatant outlet and a fermentation thallus outlet, and the fermentation thallus outlet of the butterfly centrifuge is communicated with the inlet of the stirring tank through a second pipeline; the outlet pipe of the stirring tank, the inlet pipe of the first fermentation tank and the inlet pipe of the second fermentation tank are connected through a third pipeline so as to be communicated with each other; the peristaltic pump is arranged on the third pipeline; the outlet pipe of the first fermentation tank is provided with a first valve, the outlet pipe of the second fermentation tank is provided with a second valve, the inlet pipe of the butterfly centrifuge is provided with a third valve, the outlet pipe of the stirring tank is provided with a fourth valve, the inlet pipe of the first fermentation tank is provided with a fifth valve, and the inlet pipe of the second fermentation tank is provided with a sixth valve.
As a preferable scheme, a seventh valve and an eighth valve are arranged on the first pipeline, the seventh valve and the eighth valve are respectively positioned at two sides of the communication position of the first pipeline and the outlet pipe of the first fermentation tank, and the seventh valve is used for controlling the input of steam; and a ninth valve and a tenth valve are further arranged on the first pipeline, the ninth valve and the tenth valve are respectively positioned at two sides of the communication position of the first pipeline and the outlet pipe of the second fermentation tank, and the eighth valve is adjacent to the ninth valve.
Preferably, the fifth valve and the sixth valve are three-way valves, and the three-way valves are used for controlling the third pipeline to be communicated with the corresponding fermentation tank or discharging the sterilizing steam.
Preferably, the first valve and the second valve are three-way valves, and the three-way valves are used for controlling the discharge of fermentation liquor or sterilizing steam in the fermentation tank.
As a preferable scheme, stirring devices are arranged in the first fermentation tank and the second fermentation tank, and the stirring speed of the stirring devices is 0-600 rpm.
As a preferable scheme, a temperature detection control joint, a pH value detection control joint and a dissolved oxygen detection control joint are arranged in the first fermentation tank and the second fermentation tank.
As a preferable scheme, the connecting fermentation system further comprises a control cabinet, and the temperature detection control joint, the pH value detection control joint and the dissolved oxygen detection control joint are all connected with the control cabinet; the control cabinet is used for controlling the opening or closing of each valve, and controlling the temperature, the pH value, the stirring speed and the dissolved oxygen in each fermentation tank.
The invention also provides a continuous fermentation process for engineering bacteria, which comprises the following steps:
step one: when the first fermentation tank carries out engineering bacteria fermentation, the second fermentation tank is added with a culture medium for engineering bacteria and sterilized;
before the first fermentation tank finishes fermentation, steam is adopted to sterilize each pipeline, a butterfly centrifuge and a stirring tank in sequence;
step two: when fermentation in the first fermentation tank is completed, conveying fermentation liquor to a butterfly centrifuge for centrifugation through a pipeline, collecting target protein through a supernatant outlet of the butterfly centrifuge, enabling concentrated thalli to enter a stirring tank, and pumping the stirred concentrated thalli into a second fermentation tank through a peristaltic pump for fermentation;
when the second fermentation tank carries out engineering bacteria fermentation, adding a culture medium for engineering bacteria after the first fermentation tank is cleaned, and sterilizing;
step three: when fermentation in the second fermentation tank is completed, the fermentation liquor is conveyed to a butterfly centrifuge for centrifugation through a pipeline, target proteins are collected through a supernatant outlet of the butterfly centrifuge, concentrated thalli enter a stirring tank, and are pumped into the first fermentation tank through a peristaltic pump after stirring for fermentation;
repeating the first to third steps until the activity of the engineering bacteria does not meet the standard requirement. The standard requirement of the activity of the engineering bacteria is that the bacterial activity is not lower than 90%, namely the bacterial activity is lower than 90%, and the continuous fermentation requirement is not met.
As a preferable scheme, after a preset amount of engineering bacteria culture medium is added into a first fermentation tank for the first time, twice the preset amount of engineering bacteria culture medium is added into both the first fermentation tank and a second fermentation tank in the continuous fermentation process; when one of the fermenters is completed, half of the twice predetermined amount of the culture medium for the engineering bacteria of the other fermenter is transferred into the fermenter completed by adjusting the pressure difference in the two fermenters to dilute the fermentation broth.
Preferably, after transferring half of the twice the predetermined amount of the culture medium for the engineering bacteria in the other fermenter to the fermenter after completion of fermentation to dilute the fermentation liquid, the method further comprises: and conveying part of the residual culture medium in the other fermentation tank into the stirring tank through a pipeline and a butterfly centrifuge, and conveying the diluted fermentation liquor in the fermentation tank after fermentation to the butterfly centrifuge.
Compared with the prior art, the invention has the beneficial effects that:
according to the continuous fermentation system and the fermentation process for engineering bacteria, only one-time fermentation culture of engineering bacteria is needed, after the engineering bacteria are cultured to a final concentration, the fermented engineering bacteria are recovered through the butterfly centrifugal machine to continue circulating fermentation, the density of the recovered engineering bacteria is not obviously reduced, the activity is high, the induction time is greatly shortened while the yield is ensured in the subsequent fermentation culture, and the production efficiency and quality are improved.
Drawings
FIG. 1 is a schematic diagram of a continuous fermentation system for engineering bacteria according to an embodiment of the present invention.
Detailed Description
The technical scheme of the invention is further described and illustrated by the following specific examples.
As shown in FIG. 1, the continuous fermentation system for engineering bacteria in the embodiment of the invention comprises a first fermentation tank 1, a second fermentation tank 2, a butterfly centrifuge 3, a stirring tank 4 and a peristaltic pump 5.
The first fermentation tank 1 and the second fermentation tank 2 are of vertical structures, and 150L of fermentation tank capacity is adopted; stirring devices are arranged in the first fermentation tank and the second fermentation tank, each stirring device comprises a stirring motor and a stirring rod, the stirring speed is 0-600 rpm, and the specific stirring speed is set according to a specific fermentation process. The outlet pipe 10 of the first fermentation tank, the outlet pipe 20 of the second fermentation tank and the inlet pipe 30 of the butterfly centrifuge are connected through a first pipeline 6 to realize mutual communication, namely, the outlet pipe 10 of the first fermentation tank is communicated with the outlet pipe 20 of the second fermentation tank through the first pipeline 6, the outlet pipe 10 of the first fermentation tank is communicated with the inlet pipe 30 of the butterfly centrifuge through the first pipeline 6, the outlet pipe 20 of the second fermentation tank is communicated with the inlet pipe 30 of the butterfly centrifuge through the first pipeline 6, and the first fermentation tank 1, the second fermentation tank 2 and the butterfly centrifuge are sequentially distributed along the extending direction (namely from left to right) of the first pipeline 6; the left end of the first pipeline 6 is a steam inlet for inputting steam to sterilize the pipelines and the equipment. Wherein, the outlet pipe 10 of the first fermentation tank is provided with a first valve a, the first valve a is a three-way valve, and two outlets of the three-way valve are respectively used for discharging fermentation liquor and sterilizing steam in the first fermentation tank 1; the outlet pipe 20 of the second fermentation tank is provided with a second valve b, the second valve b is a three-way valve, and two outlets of the three-way valve are respectively used for discharging fermentation liquor and sterilizing steam in the second fermentation tank 2; the inlet pipe 30 of the butterfly centrifuge is provided with a third valve c, and the third valve c is a common switch valve for controlling fermentation liquid or steam in the first pipeline 6 to enter the butterfly centrifuge so as to perform centrifugation or sterilization operation.
In addition, a seventh valve g and an eighth valve h are installed on the first pipeline 6, the seventh valve g and the eighth valve h are respectively positioned at the left side and the right side of the communication position of the first pipeline 6 and the outlet pipe 10 of the first fermentation tank, wherein the communication position of the first pipeline 6 and the outlet pipe 10 of the first fermentation tank is of a three-way structure, the seventh valve g positioned at the left side is used for controlling the input of steam, and the eighth valve h positioned at the right side is used for controlling the steam inlet of the first pipeline 6, the outlet pipe 10 of the first fermentation tank is communicated or not communicated with the outlet pipe 20 of the second fermentation tank and the inlet pipe 30 of the butterfly centrifuge. The first pipeline 6 is also provided with a ninth valve i and a tenth valve j, the ninth valve i and the tenth valve j are respectively positioned at the left side and the right side of the communication position of the first pipeline 6 and the outlet pipe 20 of the second fermentation tank, wherein the communication position of the first pipeline 6 and the outlet pipe 20 of the second fermentation tank is of a three-way structure, the ninth valve i positioned at the left side is used for controlling the steam inlet of the first pipeline 6, the outlet pipe 10 of the first fermentation tank and the outlet pipe 20 of the second fermentation tank and the inlet pipe 30 of the butterfly centrifuge to be communicated or not communicated, and the tenth valve j positioned at the right side is used for controlling the steam inlet of the first pipeline 6, the outlet pipe 10 of the first fermentation tank and the outlet pipe 20 of the second fermentation tank to be communicated or not communicated with the inlet pipe 30 of the butterfly centrifuge. The eighth valve h is adjacent to the ninth valve i, and the seventh valve g, the eighth valve h, the ninth valve i and the tenth valve j are all common switch valves.
The butterfly centrifuge 3 of the embodiment of the invention is used for separating solid and liquid of fermentation liquid, on one hand, collecting fermentation supernatant liquid, and on the other hand, recovering concentrated thalli for reuse. Specifically, the butterfly centrifuge 3 has a supernatant outlet 31 and a fermentation cell outlet, and the fermentation cell outlet of the butterfly centrifuge communicates with the inlet of the agitation tank 4 through the second pipe 7; the stirring tank 4 is used for stirring and uniformly mixing the concentrated thalli after the centrifugation of the butterfly centrifuge 3. The outlet pipe 400 of the stirring tank, the inlet pipe 100 of the first fermentation tank and the inlet 200 pipe of the second fermentation tank are connected through a third pipeline 8 to realize mutual communication, that is, the outlet pipe 400 of the stirring tank is communicated with the inlet pipe 100 of the first fermentation tank through the third pipeline 8, the outlet pipe 400 of the stirring tank is communicated with the inlet 200 of the second fermentation tank through the third pipeline 8, and the inlet pipe 100 of the first fermentation tank is communicated with the inlet 200 of the second fermentation tank through the third pipeline 8. Peristaltic pump 5 is mounted on third tube 8 adjacent to the junction of third tube 8 with outlet tube 400 of the agitation tank. The outlet pipe 400 of the stirring tank is provided with a fourth valve d for controlling the output of the fermentation thalli in the stirring tank; the inlet pipe 100 of the first fermenter is provided with a fifth valve e for controlling the fermentation cells in the agitator tank to be transferred to the first fermenter 1; the inlet pipe 200 of the second fermenter is provided with a sixth valve f for controlling the transfer of the fermentation cells in the stirrer to the second fermenter 2. The fifth valve e and the sixth valve f are three-way valves and are also used for controlling the discharge of sterilizing steam.
In order to realize intelligent control of the continuous fermentation system for engineering bacteria, which is provided by the embodiment of the invention, a temperature detection control joint, a pH value detection control joint and a dissolved oxygen detection control joint are arranged in the first fermentation tank 1 and the second fermentation tank 2; correspondingly, the connection fermentation system further comprises a control cabinet, the temperature detection control joint, the pH value detection control joint and the dissolved oxygen detection control joint are respectively connected with the control cabinet, a liquid crystal display screen is arranged on the control cabinet, the detection of the temperature, the pH value and the DO dissolved oxygen value in the two fermentation tanks is realized through the sensors, the data information of the pH value, the DO dissolved oxygen value, the temperature and the rotating speed of the stirring motor in the two fermentation tanks can be accurately displayed on the liquid crystal display screen of the control cabinet, the automatic control is realized, and the fermentation process can be regulated at any time through a computer. In addition, all valves are electromagnetic valves and are in communication connection with a control cabinet, and the control cabinet is also used for controlling the opening or closing or switching of the valves so as to realize intelligent control of the valves. According to the embodiment of the invention, the first fermentation tank 1 and the second fermentation tank 2 are both provided with the feeding channels, the material of the feeding channels is 304/316L stainless steel, and accordingly, the control cabinet can also perform feeding control. The specific structure and technology of the control cabinet in the embodiment of the present invention may refer to the prior art, and are not described herein.
The continuous fermentation system for engineering bacteria provided by the embodiment of the invention has reasonable structural layout, can be applied to continuous fermentation of engineering bacteria, can realize the heavy utilization of secretory yeast engineering bacteria to realize continuous fermentation until the activity of the engineering bacteria is lower than a set value, can continuously express target protein when the engineering bacteria have high bacterial density, and has few target protein impurities and high production efficiency. The butterfly type centrifugal machine is adopted to perform solid-liquid separation on line, the separated concentrated thalli are put into the fermentation tank again to induce and express target proteins, the time for culturing engineering bacteria again to high density is greatly reduced, and the waste of manpower, material resources and financial resources caused by batch fermentation is avoided.
Corresponding to the continuous fermentation system for engineering bacteria in the embodiment, the embodiment of the invention also provides a continuous fermentation process for engineering bacteria, which comprises the following steps:
step one: when the first fermentation tank carries out engineering bacteria fermentation, the second fermentation tank is added with a culture medium for engineering bacteria, sterilized according to a specified rule, and cooled to a specified temperature for later use;
before the fermentation of the first fermentation tank is completed, steam is adopted to sterilize each pipeline, the butterfly centrifugal machine and the stirring tank in sequence, so that the flow path of the whole fermentation liquid is in a sterilization state;
step two: when fermentation in the first fermentation tank is completed, conveying fermentation liquor to a butterfly centrifuge for centrifugation through a pipeline, collecting target protein through a supernatant outlet of the butterfly centrifuge, enabling concentrated thalli to enter a stirring tank, and pumping the stirred concentrated thalli into a second fermentation tank through a peristaltic pump for fermentation; the second fermentation tank has high initial bacterial density and engineering bacteria are in an induced state, so that the re-fermentation expression time is generally shortened greatly, and the re-induction expression time is generally about 48 hours;
when the second fermentation tank carries out engineering bacteria fermentation, after the first fermentation tank is cleaned, adding a culture medium for engineering bacteria, sterilizing according to a specified rule, and cooling to a specified temperature for standby;
step three: when fermentation in the second fermentation tank is completed, the fermentation liquor is conveyed to a butterfly centrifuge for centrifugation through a pipeline, target proteins are collected through a supernatant outlet of the butterfly centrifuge, concentrated thalli enter a stirring tank, and are pumped into the first fermentation tank through a peristaltic pump after stirring for fermentation;
repeating the first to third steps until the activity of the engineering bacteria does not meet the standard requirement. Typically yeast will support more than 7 cycles.
The embodiment of the invention adopts the following scheme to detect the activity of the yeast engineering bacteria.
Meran staining method: taking a clean glass slide, dripping a drop of the methylene blue dye solution in the center of the glass slide, diluting the methylene blue dye solution by using a small amount of water for injection, uniformly smearing the bacterial solution to be detected on a diluted methylene blue dye solution area, and covering a cover glass, wherein no bubbles are required; standing at room temperature for several minutes, and observing under a microscope; colorless yeast cells are living cells, blue cells are dead cells, and light blue cells are senescent cells; the percentages of various cells were counted.
The embodiment of the invention uses pichia to express human serum albumin-growth hormone fusion protein for detailed description, and a butterfly centrifuge adopts clara 20 250L/h.
(1) First-stage strain culture
One yeast engineering strain for production stored in a refrigerator at the temperature of minus 70 ℃ is taken, 400-700 mu L of bacterial liquid is taken in 50mL of YPD culture medium in a sterilized ultra-clean workbench, the culture medium is sealed by using sterile gauze and is put in a shaking table at the temperature of 30 ℃ for culturing for about 24 hours, and the revolution is 250rpm.
(2) Second-level strain culture
Taking a proper amount of cultured first-class strain to detect OD 600 OD is taken from the source of activity and purity 600 The primary strain with the value of 10-20 and good growth and no bacteria is used as the secondary culture strain. Ultra-clean work after sterilizationIn the bench, the primary strain was cultured in a shaking flask of 7.5mL, 250mL of YNB mother liquor, 3mL of biotin in 750mL of BMGY medium at a shaking speed of 250rpm at 30℃for about 12 hours.
(3) Fermentation culture
Primary fermentation: the BMGY medium, defoamer, etc. are sterilized in the first fermenter 1 and reduced to a suitable temperature before inoculation. 2.25L of the qualified second-level strain (OD) 600 Values 10-20 and well-grown and sterile) was inoculated into the first fermenter 1 and 0.1% biotin and PTM1 trace elements were added. Aeration-agitation culture, wherein preferred culture conditions are: the rotation speed is 200rpm, the ventilation amount is 1vvm, the tank pressure is 0.05Mpa, the pH is automatically adjusted to about 5.5, and the culture temperature is 30 ℃. Proliferation is carried out by feeding glycerol as a carbon source. When the wet weight reaches 260+/-5 g/L, the culture stage of the target protein induced by the methanol phase is carried out. Stopping feeding glycerol, starting feeding methanol for cooling culture, and performing induction culture for about 104h. Wherein the preferred culture conditions are: the rotation speed is 350rpm, the ventilation amount is 2.5vvm, the tank pressure is 0.07Mpa, the DO is controlled at about 20 percent, the pH is automatically regulated to about 5.5, and the culture temperature is 20 ℃.
At the end of the first fermentation, two times the amount of BMMY medium was added to the second fermenter 2, the valve g, h, i, j, c, d, e was opened, steam was allowed to enter the butterfly centrifuge, the stirring tank, the second fermenter 2 and the pipes through the valves to sterilize, and the steam was discharged from the valve e or the valve f after the completion of the sterilization.
And (3) secondary fermentation: after the first fermentation, the valve a, h, i, b is opened and half of the sterilized medium in the second fermenter 2 is transferred to the first fermenter 1 by adjusting the pressure difference between the two fermenters for dilution of the fermentation broth, the purpose of which is to increase the yield of supernatant. The valves b, j, c are opened and one fifth of the remaining medium of the second fermenter 2 is placed in the stirring tank 4 by manual setting of the butterfly centrifuge 3. And then sequentially opening the valve a, h, i, j, c to enable all fermentation liquor of the first fermentation tank 1 to enter a butterfly centrifuge, and collecting liquid containing target proteins from a supernatant outlet 31 of the centrifuge after centrifugation for further processing. The centrifuged fermentation thalli directly enter a stirring tank 4, and a magnetic stirrer is started to stir the concentrated thalli and the culture medium uniformly. And opening the valve d, switching the outlet of the three-way valve e, and transporting the thalli in the stirring tank to the second fermentation tank 2 through the peristaltic pump 5 for continuous cultivation. The culture methods such as feed supplement and the like are the same as those of the first fermentation. In contrast, the induction culture time was greatly shortened, and only 48 hours was required. The whole second fermentation requires only about 2 days to harvest in the tank.
And (3) carrying out washing, adding BMMY culture medium with twice the volume, sterilizing and cooling on the first fermentation tank 1 at the same time of secondary fermentation. At the end of fermentation in the second fermenter 2, the valve a, h, i, b is opened and half of the medium in the first fermenter 1 is pumped into the second fermenter 2 by means of the pressure difference between the two fermentors to dilute the fermentation broth. The lower valve a, h, i, j, c of the fermenter was then opened and one fifth of the remaining medium was placed in the stirrer by manual setting with a butterfly centrifuge.
And after the fermentation liquor after the second fermentation tank 2 is put into a butterfly centrifuge for solid-liquid separation through valves b, j and c, collecting fermentation supernatant, uniformly mixing the fermentation supernatant with concentrated thalli through a stirring tank, and pumping the fermentation liquor to the sterilized first fermentation tank 1 for continuous culture.
The cells recovered after the completion of the first fermentation may be fermented seven times in succession with the above-described cycle. The total protein content per harvest is greater than 250g. The continuous fermentation for many times saves time and material manpower compared with single-batch fermentation, and ensures the yield.
The activity and purity of the step (2) are as follows: and (3) dripping a drop of the methylene blue staining solution in the center of the glass slide by taking a clean glass slide, diluting the methylene blue staining solution by using a small amount of water for injection, uniformly smearing the taken fermentation broth on a diluted methylene blue staining solution area, and covering a cover glass, wherein no bubbles are required. Standing at room temperature for several minutes, and observing under a microscope. Colorless yeast cells are living cells, blue cells are dead cells, and light blue cells are senescent cells. The percentages of various cells were counted. And meanwhile, observing the shape and the size of the cells to check whether other bacteria pollute the cells.
The wet weight detection in the step (3) is as follows: four groups of 1mL fermentation broths were prepared, respectively, centrifuged at 10000rpm for 10min, and the centrifuged supernatant was removed and dried and weighed. The average value of the four groups of precipitation net values is taken as the wet weight of the fermentation liquor.
The next treatment of the liquid containing the target protein in the step (3) means that the protein concentration of the fermentation liquid is measured by a Bradford method after ultrafiltration of the fermentation supernatant, and the concentration is about 10 times, and the detection concentration is between 25.0 and 35.0 mg/mL.
The results of the related detection and protein content detection of seven continuous fermentations are shown in the following table:
| lot number | Culture medium | Induction time | Wet weight of | Supernatant volume | Protein content | Yeast vitality |
| A first part | BMGY | 104h | 402g/L | 108L | 302g | 99.5％ |
| Two (II) | BMMY | 48h | 413g/L | 112L | 278g | 99.4％ |
| Three kinds of | BMMY | 48h | 425g/L | 107L | 299g | 99.2％ |
| Fourth, fourth | BMMY | 48h | 430g/L | 115L | 305g | 98.1％ |
| Five kinds of | BMMY | 48h | 442g/L | 110L | 300g | 96.2％ |
| Six kinds of | BMMY | 48h | 456g/L | 108L | 286g | 92.3％ |
| Seven pieces of | BMMY | 48h | 478g/L | 113L | 295g | 90.1％ |
By continuously circulating seven times of fermentation, the fermentation time from the second fermentation is greatly shortened compared with the first fermentation time, the wet weight shows an increasing trend along with the increase of the fermentation times, the obtained fermentation broth supernatant and the protein content basically remain unchanged, the yeast activity shows a decreasing trend, and the fermentation is not continued when the yeast activity is lower than 90%.
According to the continuous fermentation process for engineering bacteria, disclosed by the embodiment of the invention, only one-time fermentation culture of engineering bacteria is needed, after the engineering bacteria are cultured to a final concentration, the fermented engineering bacteria are recovered through a butterfly centrifuge to continue circulating fermentation, the density of the recovered engineering bacteria is not obviously reduced, the activity is high, the induction time is greatly shortened while the yield is ensured in the subsequent fermentation culture, and the production efficiency and quality are improved.
It is to be understood that the foregoing is only illustrative of the preferred embodiments and principles of the present invention, and that modifications in detail will readily occur to those skilled in the art upon reading the teachings herein and are to be considered as within the scope and spirit of the invention.
Claims (3)
1. The continuous fermentation process for the engineering bacteria is characterized in that the continuous fermentation system for the engineering bacteria comprises a first fermentation tank, a second fermentation tank, a butterfly centrifuge, a stirring tank and a peristaltic pump, wherein an outlet pipe of the first fermentation tank, an outlet pipe of the second fermentation tank and an inlet pipe of the butterfly centrifuge are connected through a first pipeline so as to realize mutual communication; the butterfly centrifuge is provided with a supernatant outlet and a fermentation thallus outlet, and the fermentation thallus outlet of the butterfly centrifuge is communicated with the inlet of the stirring tank through a second pipeline; the outlet pipe of the stirring tank, the inlet pipe of the first fermentation tank and the inlet pipe of the second fermentation tank are connected through a third pipeline so as to be communicated with each other; the peristaltic pump is arranged on the third pipeline; the outlet pipe of the first fermentation tank is provided with a first valve, the outlet pipe of the second fermentation tank is provided with a second valve, the inlet pipe of the butterfly centrifuge is provided with a third valve, the outlet pipe of the stirring tank is provided with a fourth valve, the inlet pipe of the first fermentation tank is provided with a fifth valve, and the inlet pipe of the second fermentation tank is provided with a sixth valve;
a seventh valve and an eighth valve are arranged on the first pipeline, the seventh valve and the eighth valve are respectively positioned at two sides of the communication position of the first pipeline and the outlet pipe of the first fermentation tank, and the seventh valve is used for controlling the input of steam; a ninth valve and a tenth valve are further arranged on the first pipeline, the ninth valve and the tenth valve are respectively positioned at two sides of the communication position of the first pipeline and the outlet pipe of the second fermentation tank, and the eighth valve is adjacent to the ninth valve;
the method comprises the following steps:
step one: when the first fermentation tank carries out engineering bacteria fermentation, the second fermentation tank is added with a culture medium for engineering bacteria and sterilized; before the first fermentation tank finishes fermentation, steam is adopted to sterilize each pipeline, a butterfly centrifuge and a stirring tank in sequence;
step two: when fermentation in the first fermentation tank is completed, conveying fermentation liquor to a butterfly centrifuge for centrifugation through a pipeline, collecting target protein through a supernatant outlet of the butterfly centrifuge, enabling concentrated thalli to enter a stirring tank, and pumping the stirred concentrated thalli into a second fermentation tank through a peristaltic pump for fermentation; when the second fermentation tank carries out engineering bacteria fermentation, adding a culture medium for engineering bacteria after the first fermentation tank is cleaned, and sterilizing;
step three: when fermentation in the second fermentation tank is completed, the fermentation liquor is conveyed to a butterfly centrifuge for centrifugation through a pipeline, target proteins are collected through a supernatant outlet of the butterfly centrifuge, concentrated thalli enter a stirring tank, and are pumped into the first fermentation tank through a peristaltic pump after stirring for fermentation;
repeating the first to third steps until the activity of the engineering bacteria does not meet the standard requirement.
2. The continuous fermentation process for engineering bacteria according to claim 1, wherein after a predetermined amount of the culture medium for engineering bacteria is added to the first fermentation tank for the first time, twice the predetermined amount of the culture medium for engineering bacteria is added to both the first fermentation tank and the second fermentation tank during the continuous fermentation; when one of the fermenters is completed, half of the twice predetermined amount of the culture medium for the engineering bacteria of the other fermenter is transferred into the fermenter completed by adjusting the pressure difference in the two fermenters to dilute the fermentation broth.
3. The continuous fermentation process for an engineering bacterium according to claim 2, wherein after transferring half of twice the predetermined amount of the culture medium for an engineering bacterium of the other fermenter into the fermenter after completion of fermentation to dilute the fermentation liquid, further comprising: and conveying part of the residual culture medium in the other fermentation tank into the stirring tank through a pipeline and a butterfly centrifuge, and conveying the diluted fermentation liquor in the fermentation tank after fermentation to the butterfly centrifuge.
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| CN101280322A (en) * | 2008-04-21 | 2008-10-08 | 云浮市九方农业科技发展有限公司 | Anaerobic fermentation method and device of alcohol waste Liquid |
| CN105349308A (en) * | 2015-11-19 | 2016-02-24 | 湖北工业大学 | Method for producing Fen-flavor liquor through continuous liquid fermentation |
| WO2019030185A1 (en) * | 2017-08-07 | 2019-02-14 | Novozymes A/S | Ejector equipped fermenter |
| CN210215355U (en) * | 2019-03-19 | 2020-03-31 | 浙江优诺金生物工程有限公司 | Continuous fermentation system for engineering bacteria |
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| CN101280322A (en) * | 2008-04-21 | 2008-10-08 | 云浮市九方农业科技发展有限公司 | Anaerobic fermentation method and device of alcohol waste Liquid |
| CN105349308A (en) * | 2015-11-19 | 2016-02-24 | 湖北工业大学 | Method for producing Fen-flavor liquor through continuous liquid fermentation |
| WO2019030185A1 (en) * | 2017-08-07 | 2019-02-14 | Novozymes A/S | Ejector equipped fermenter |
| CN210215355U (en) * | 2019-03-19 | 2020-03-31 | 浙江优诺金生物工程有限公司 | Continuous fermentation system for engineering bacteria |
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