Automatic sampling system suitable for high-density fermentation process and control method

Abstract

The invention discloses an automatic sampling system suitable for a high-density fermentation process and a control method, the automatic sampling system comprises an assembly I: a fermentation reactor and a sampling tube, and the assembly I is used for extracting fermentation liquor from the fermentation reactor; the component II is provided with a switch valve, a pressure-resistant container, a separation membrane and the like, is used for intercepting solid components in fermentation liquor and cooperates with other components to complete liquid transfer; the assembly III comprises a switch valve, a fermentation liquor sample pool, a cleaning pump and the like, is used for accommodating filtered fermentation liquor clear liquid and can be used for cleaning operation; the fourth assembly is used for providing high-pressure gas or a vacuum environment so as to assist liquid in moving in the system; the fifth assembly is responsible for providing cleaning liquid to ensure that the system can be effectively cleaned in the using process; and the sixth assembly is used for collecting waste liquid and ensuring that the whole sampling process does not pollute the environment. Through the system, the application of an online monitoring technology can be effectively supported, the automation level and the product quality in the fermentation process are improved, and the development of a biological fermentation technology is promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemistry, and specifically relates to an automatic sampling system and a control method, which are suitable for sampling high-density fermentation broth in food production and biological processes. BACKGROUND

[0002] In the biofermentation industry, especially in high-density fermentation processes, real-time monitoring of fermentation status is crucial for optimizing process parameters, improving product quality and production efficiency. Traditional manual sampling methods not only consume time and effort, but also introduce external contaminants, affecting the stability of the fermentation process and the purity of the product. With the continuous development of biotechnology, automation and intelligentization of the fermentation process have become an inevitable trend in the industry. Online monitoring technology, as a key means to achieve this goal, can continuously obtain various parameters in the fermentation process in real time, greatly reducing the workload of fermentation engineers and improving production efficiency and product quality.

[0003] However, to achieve efficient online monitoring, reliable sampling technology is indispensable. In high-density fermentation processes, the fermentation broth usually contains a large number of microorganisms or cells, and traditional sampling methods often fail to meet the requirements. In particular, using membrane samplers in high-density fermentation conditions is prone to clogging, resulting in sampling failure, which seriously affects the accuracy and reliability of online monitoring. This not only increases production costs, but also may lead to instability of the fermentation process, thereby affecting the quality of the final product.

[0004] Therefore, developing an automatic sampling system suitable for high-density fermentation processes that can ensure sampling accuracy and efficiency while avoiding contamination and clogging problems is of great significance to the development of biofermentation technology. SUMMARY

[0005] The present application aims to solve the problem of sampling in high-density fermentation processes and provides an efficient, reliable and easy-to-operate automatic sampling system suitable for high-density fermentation processes to solve the problems of low sampling efficiency, easy contamination and easy clogging in the prior art, support the application of online monitoring technology and improve the automation level of the fermentation process and product quality.

[0006] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows: An automatic sampling system suitable for high-density fermentation processes, comprising: Component one, for containing and extracting fermentation broth; Component two, which is internally provided with a solid-liquid separation membrane for intercepting solid components in the fermentation liquor; the inlet end of component two is connected with component one to introduce the fermentation liquor into component two; the clear liquor outlet at the rear end of component two is respectively connected with component three, component five through pipelines; the middle part of component two is respectively connected with component four and component six through pipelines; Component three is used for containing fermentation liquor clear liquor, which is respectively connected with component two, component five and component six through pipelines; Component four is used for connecting high-pressure gas, which is connected with component two through a pipeline to introduce high-pressure gas into component two; Component five is used for containing cleaning liquid, which is connected with component two and component three through pipelines; Component six is used for containing fermentation liquor solid components and waste liquid, which is respectively connected with component two and component three through pipelines.

[0007] Specifically, component one comprises a fermentation reactor and a sampling tube; wherein, The sampling tube is fixed on the fermentation reactor by external threads, and the two are sealed by a rubber ring, and the lower end of the sampling tube is extended below the liquid level in the fermentation reactor; The upper end of the sampling tube is connected with component two through a pipeline for extracting fermentation liquor into component two; The fermentation reactor is used for containing fermentation liquor.

[0008] Specifically, component two comprises a switch valve one, a pressure-resistant container, a separation membrane and a tee one; wherein, The inlet end of the switch valve one is connected with component one through a pipeline, and the outlet end is connected with the pressure-resistant container through a pipeline; The upper end of the pressure-resistant container is connected with component four through a pipeline, the lower end is connected with component six through a pipeline, the left end is connected with the switch valve one through a pipeline, and the right end is connected with the separation membrane through threads; The separation membrane is fixed on the right interface of the pressure-resistant container by external threads, and the other end is connected with the tee one through a pipeline; The interface one of the tee one is connected with the separation membrane through a pipeline, the interface two is connected with component five through a pipeline, and the interface three is connected with component three through a pipeline; The switch valve one is used to isolate the connection between component one and component two, and only allows one-way flow from component one to component two; The pressure-resistant container is used for containing fermentation liquor, high-pressure gas or cleaning liquid, which can resist pressure ≥1.0 MPa; The separation membrane is used for intercepting solid components in the fermentation liquor.

[0009] Specifically, the third assembly comprises: the second switch valve, the fermentation broth sample pool, the cleaning pump, the first bubble sensor, the overflow pump, the liquid outlet pump and the three-way valve two; wherein, The inlet end of the second switch valve is connected with the second assembly through a pipeline, and the outlet end is connected with the fermentation broth sample pool through a pipeline; The fermentation broth sample pool is provided with four interfaces, which are respectively connected with the second switch valve, the cleaning pump, the first bubble sensor and the liquid outlet pump through pipelines, wherein the interface connected with the liquid outlet pump is located at the lowest point of the inner cavity of the fermentation broth sample pool, and the interface connected with the first bubble sensor is arranged in the middle of the fermentation broth sample pool; The inlet end of the cleaning pump is connected with the fifth assembly through a pipeline, and the outlet end is connected with the fermentation broth sample pool through a pipeline; The first bubble sensor is connected with the fermentation broth sample pool and the overflow pump through pipelines respectively; The inlet end of the overflow pump is connected with the first bubble sensor through a pipeline, and the outlet end is connected with the three-way valve two through a pipeline; The inlet end of the liquid outlet pump is connected with the fermentation broth sample pool through a pipeline, and the outlet end is connected with the three-way valve two through a pipeline; The first interface of the three-way valve two is connected with the overflow pump through a pipeline, the second interface is connected with the liquid outlet pump through a pipeline, and the third interface is connected with the sixth assembly through a pipeline; The second switch valve is used for isolating the connection between the second assembly and the third assembly, and only allows the flow path to flow from the second assembly to the third assembly in one direction; The fermentation broth sample pool is used for containing fermentation broth supernatant; The cleaning pump is used for pumping cleaning liquid into the fermentation broth sample pool; The first bubble sensor is used for judging whether there is liquid in the liquid transfer pipeline, so as to control the volume of liquid in the fermentation broth sample pool; The overflow pump is used for pumping excess liquid in the fermentation broth sample pool to the sixth assembly; The liquid outlet pump is used for pumping waste liquid in the fermentation broth sample pool to the sixth assembly.

[0010] Specifically, the fourth assembly comprises: the second bubble sensor, the three-way valve three, the third switch valve, the fourth switch valve, the high-pressure gas interface and the vacuum interface; wherein, The second bubble sensor is connected with the second assembly and the three-way valve three through pipelines respectively; The first interface of the three-way valve three is connected with the second bubble sensor through a pipeline, the second interface is connected with the third switch valve through a pipeline, and the third interface is connected with the fourth switch valve through a pipeline; The inlet end of the third switch valve is connected with the high-pressure gas interface through a pipeline, and the outlet end is connected with the three-way valve three through a pipeline; The inlet end of the fourth switch valve is connected with the three-way valve three through a pipeline, and the outlet end is connected with the vacuum interface through a pipeline; The bubble sensor two is used to determine whether there is liquid in the gas pipeline, so as to control the liquid volume in the pressure-resistant container in the second assembly; The switch valve three is used to isolate the connection between the second assembly and the high-pressure gas interface, and only allows the flow path to flow from the high-pressure gas interface to the second assembly in one direction; The switch valve four is used to isolate the connection between the second assembly and the vacuum interface, and only allows the flow path to flow from the second assembly to the vacuum interface in one direction; The high-pressure gas interface is used to connect the high-pressure gas to drive the fermentation liquid supernatant to pass through the separation membrane in the second assembly into the fermentation liquid sample pool in the third assembly; The vacuum interface is used to connect the vacuum pump to suck the liquid in the fermentation reactor in the first assembly or the cleaning liquid bottle in the fifth assembly into the pressure-resistant container in the second assembly.

[0011] Specifically, the fifth assembly comprises a switch valve five, a three-way four, a bubble sensor three, and a cleaning liquid bottle; wherein, The inlet end of the switch valve five is connected to the three-way four through a pipeline, and the outlet end is connected to the second assembly through a pipeline; The interface one of the three-way four is connected to the switch valve five through a pipeline, the interface two is connected to the bubble sensor four through a pipeline, and the interface three is connected to the third assembly through a pipeline; The bubble sensor three is connected to the three-way four and the cleaning liquid bottle through a pipeline, respectively; The cleaning liquid bottle is connected to the bubble sensor three through a pipeline; The switch valve five is used to isolate the connection between the second assembly and the cleaning liquid bottle, and only allows the flow path to flow from the cleaning liquid bottle to the second assembly in one direction; The bubble sensor three is used to determine whether there is liquid in the gas pipeline, so as to control the cleaning liquid volume in the cleaning liquid bottle; The cleaning liquid bottle is used to contain cleaning liquid.

[0012] Specifically, the sixth assembly comprises a switch valve six, a three-way five, and a waste liquid bottle; wherein, The inlet end of the switch valve six is connected to the second assembly through a pipeline, and the outlet end is connected to the three-way five through a pipeline; The interface one of the three-way five is connected to the switch valve six through a pipeline, the interface two is connected to the third assembly through a pipeline, and the interface three is connected to the waste liquid bottle through a pipeline; The waste liquid bottle is connected to the three-way five through a pipeline; The switch valve six is used to isolate the connection between the second assembly and the waste liquid bottle, and only allows the flow path to flow from the second assembly to the waste liquid bottle in one direction; The waste liquid bottle is used to contain waste liquid.

[0013] Specifically, the pipes in the system are independently selected from any one of stainless steel pipe, silica gel pipe, pressure-resistant PTFE pipe or pressure-resistant FEP pipe.

[0014] Preferably, the pipes in the second and fourth assemblies are stainless steel pipe, pressure-resistant PTFE pipe or pressure-resistant FEP pipe. In this way, the material of the pipes can make the system an automatic sampling system suitable for high-density fermentation process.

[0015] Preferably, the pipes in the first, third, fifth and sixth assemblies are silica gel pipe, pressure-resistant PTFE pipe or pressure-resistant FEP pipe. In this way, the material of the pipes can make the system an automatic sampling system convenient for sterilization and cleaning.

[0016] Preferably, the cleaning pump, overflow pump and liquid outlet pump are peristaltic pumps or diaphragm pumps. In this way, the setting of the pumps can make the system an automatic sampling system with sealed and rapid liquid discharge.

[0017] Preferably, the six switch valves are all automatic execution valves. In this way, the setting of the automatic execution valves can make the system a sterile and automatic sampling system.

[0018] Preferably, the three bubble sensors are capacitive sensors or ultrasonic sensors. In this way, the setting of the sensors can make the system an intelligent automatic sampling system.

[0019] Further, the application also claims the control method of the above-mentioned automatic sampling system suitable for high-density fermentation process, which comprises the following steps: Step S100: open the sixth switch valve and the third switch valve, and blow the residual liquid in the pressure-resistant container into the waste liquid bottle through the third switch valve with high-pressure gas, and then empty the pressure-resistant container; Step S200: close the sixth switch valve and the third switch valve, open the fourth switch valve, empty the high-pressure gas, open the first switch valve, and then draw the fermentation liquid in the fermentation reactor into the pressure-resistant container through the sampling pipe until the second bubble sensor senses the liquid signal, close the fourth switch valve and the first switch valve, open the sixth switch valve and the third switch valve, and then blow the fermentation liquid in the pressure-resistant container into the waste liquid bottle through the third switch valve with high-pressure gas, and then rinse; Step S300: close the sixth switch valve and the third switch valve, open the fourth switch valve, empty the high-pressure gas, open the first switch valve, and then draw the fermentation liquid in the fermentation reactor into the pressure-resistant container through the sampling pipe until the second bubble sensor senses the liquid signal, close the fourth switch valve and the first switch valve, and then complete the sampling; Step S400: Open switch valve two, switch valve three and overflow pump accordingly, high-pressure gas passes through switch valve three to blow the fermentation broth in the pressure-resistant container into the fermentation liquid sample pool through the separation membrane until the bubble sensor one senses the liquid signal, close switch valve two, switch valve three and overflow pump, filtration is completed; Step S500: Open switch valve six and switch valve three accordingly, high-pressure gas passes through switch valve three to blow the residual fermentation broth in the pressure-resistant container into the waste liquid bottle, close switch valve six and switch valve three, open switch valve four, empty the high-pressure gas, open switch valve five, extract the cleaning liquid in the cleaning liquid bottle to the pressure-resistant container through the separation membrane until the bubble sensor two senses the liquid signal, close switch valve four and switch valve three, open switch valve six and switch valve three accordingly, high-pressure gas passes through switch valve three to blow the cleaning waste liquid in the pressure-resistant container into the waste liquid bottle, this step is repeated three times, and the cleaning of component two is completed. Step S600: Open the waste liquid pump, pump the waste liquid in the fermentation liquid sample pool into the waste liquid bottle, close the waste liquid pump, open the cleaning pump and the overflow pump, pump the cleaning liquid in the cleaning liquid bottle into the fermentation liquid sample pool until the bubble sensor one senses the liquid signal, close the cleaning pump and the overflow pump, open the waste liquid pump, pump the waste liquid in the fermentation liquid sample pool into the waste liquid bottle, close the waste liquid pump, this step is repeated three times, and the cleaning of component three is completed.

[0020] Compared with the prior art, the present application has the following advantages: (1) High efficiency: through automatic control, the sampling time is greatly shortened, and the work efficiency is improved.

[0021] (2) Accuracy: ensures the accuracy and consistency of each sampling, which helps to more accurately monitor the fermentation process.

[0022] (3) Safety: reduces manual intervention, reduces the risk of pollution, and ensures product quality.

[0023] (4) Ease of use: the system is designed to be user-friendly, easy to operate and maintain.

[0024] (5) Compatibility: suitable for various high-density fermentation processes, can be seamlessly connected with existing online monitoring technology, and improve the overall production efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0025] The above and / or other aspects of the present application will become more apparent by describing in detail the following embodiments with reference to the accompanying drawings.

[0026] Figure 1 The above and / or other aspects of the present application will become more apparent by describing in detail the following embodiments with reference to the accompanying drawings.

[0027] Figure 2This is a flowchart of the automatic sampling control method applicable to high-density fermentation processes according to the present invention.

[0028] The reference numerals in the attached figures represent: 101 - Fermentation reactor; 102 - Sampling tube; 201-Switch valve one; 202-Pressure vessel; 203-Separation membrane; 204-Tee one; 301-Switch valve 2; 302-Fermentation broth sample cell; 303-Washing pump; 304-Bubble sensor 1; 305-Overflow pump; 306-Discharge pump; 307-T-connector 2; 401-Bubble Sensor II; 402-Tee III; 403-Switch Valve III; 404-Switch Valve IV; 405-High Pressure Gas Interface; 406-Vacuum Interface; 501-Switch valve five; 502-Three-way valve four; 503-Bubble sensor three; 504-Cleaning fluid bottle; 601-Switch valve six; 602-Three-way valve five; 603-Waste liquid bottle. Detailed Implementation

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings and other implementation methods can be obtained based on these drawings without any creative effort.

[0030] To keep the drawings concise, only the parts relevant to the invention are shown schematically in each figure, and they do not represent the actual structure of the product. Furthermore, for ease of understanding, in some figures, components with the same structure or function are shown only schematically, or only one is labeled. Example 1

[0031] like Figure 1 As shown, this embodiment discloses an automatic sampling system suitable for high-density fermentation processes, which includes component one, component two, component three, component four, component five and component six.

[0032] Component one is used to contain and extract fermentation broth; component one includes: fermentation reactor 101 and sampling tube 102; wherein, the sampling tube 102 is fixed to the fermentation reactor 101 with PG13.5 external thread, and the two are sealed by a rubber ring, and the lower end of the sampling tube 102 extends below the liquid surface of the fermentation reactor 101; the upper end of the sampling tube 102 is connected to component two through a pipeline; the sampling tube 102 is used to extract fermentation broth; the fermentation reactor 101 is used to contain fermentation broth.

[0033] The second component is used for intercepting solid components in the fermentation broth, and is connected with the first component, the third component, the fourth component, the fifth component and the sixth component through pipelines; the second component comprises a switch valve 201, a pressure-resistant container 202, a separation membrane 203 and a tee joint 204; the inlet end of the switch valve 201 is connected with the second component through a pipeline, and the outlet end is connected with the left side interface of the pressure-resistant container 202 through a pipeline; the upper end interface of the pressure-resistant container 202 is connected with the fourth component through a pipeline, the lower end interface is connected with the sixth component through a pipeline, the left end interface is connected with the switch valve 201 through a pipeline, and the right end interface is connected with the separation membrane 203 through a thread; the separation membrane 203 is fixed on the right side interface of the pressure-resistant container 202 through an external thread, and the other end is connected with the tee joint 204 through a pipeline; the interface one of the tee joint 204 is connected with the separation membrane 203 through a pipeline, the interface two is connected with the fifth component through a pipeline, and the interface three is connected with the third component through a pipeline; the switch valve 201 is used for isolating the connection between the first component and the second component, and only allows the flow path to pass from the first component to the second component, but not in the reverse direction; the pressure-resistant container 202 is used for containing fermentation broth, high-pressure gas or cleaning liquid, and can resist pressure ≥1.0MPa; the separation membrane 203 is used for intercepting solid components in the fermentation broth.

[0034] The component three is used for containing fermentation liquid supernatant, which is connected with the component two, the component five and the component six respectively by pipelines; the component three comprises: a switch valve two 301, a fermentation liquid sample pool 302, a cleaning pump 303, a bubble sensor one 304, an overflow pump 305, a liquid outlet pump 306 and a three-way pipe two 307; wherein the inlet end of the switch valve two 301 is connected with the component two by a pipeline, and the outlet end is connected with the fermentation liquid sample pool 302 by a pipeline; the fermentation liquid sample pool 302 has four interfaces, which are connected with the switch valve two 301, the cleaning pump 303, the bubble sensor one 304 and the liquid outlet pump 306 respectively by pipelines, wherein the interface connected with the liquid outlet pump 306 is located at the lowest point of the internal cavity of the fermentation liquid sample pool 302, and the interface connected with the bubble sensor one 304 is the highest interface among the four interfaces of the fermentation liquid sample pool 302; the inlet end of the cleaning pump 303 is connected with the component five by a pipeline, and the outlet end is connected with the fermentation liquid sample pool 302 by a pipeline; the bubble sensor one 304 is connected with the fermentation liquid sample pool 302 and the overflow pump 305 respectively by pipelines; the inlet end of the overflow pump 305 is connected with the bubble sensor one 304 by a pipeline, and the outlet end is connected with the three-way pipe two 307 by a pipeline; the inlet end of the liquid outlet pump 306 is connected with the fermentation liquid sample pool 302 by a pipeline, and the outlet end is connected with the three-way pipe two 307 by a pipeline; the interface one of the three-way pipe two 307 is connected with the overflow pump 305 by a pipeline, the interface two is connected with the liquid outlet pump 306 by a pipeline, and the interface three is connected with the component six by a pipeline; the switch valve two 301 is used for isolating the connection between the component two and the component three, and only allows the flow path to pass from the component two to the component three, but not in the opposite direction; the fermentation liquid sample pool 302 is used for containing fermentation liquid supernatant; the cleaning pump 303 is used for pumping cleaning liquid into the fermentation liquid sample pool 302; the bubble sensor one 304 is used for judging whether there is liquid in the liquid transfer pipeline, so as to control the liquid volume in the fermentation liquid sample pool 302; the overflow pump 305 is used for pumping the excess liquid in the fermentation liquid sample pool 302 to the component six; the liquid outlet pump 306 is used for pumping the waste liquid in the fermentation liquid sample pool 302 to the component six.

[0035] The fourth component is used for accessing high-pressure and low-pressure gas, and is connected to the second component through a pipeline; the fourth component comprises a bubble sensor 401, a three-way valve 402, a switch valve 403, a switch valve 404, a high-pressure gas interface 405, and a vacuum interface 405; wherein the bubble sensor 401 is connected to the second component and the three-way valve 402 through a pipeline respectively; the interface one of the three-way valve 402 is connected to the bubble sensor 401 through a pipeline, the interface two is connected to the switch valve 403 through a pipeline, and the interface three is connected to the switch valve 404 through a pipeline; the inlet end of the switch valve 403 is connected to the high-pressure gas interface 405 through a pipeline, and the outlet end is connected to the three-way valve 402 through a pipeline; the inlet end of the switch valve 404 is connected to the three-way valve 402 through a pipeline, and the outlet end is connected to the vacuum interface 405 through a pipeline; the bubble sensor 401 is used for judging whether there is liquid in the gas pipeline, so as to control the liquid volume in the pressure-resistant container 202 in the second component; the switch valve 403 is used for isolating the connection between the second component and the high-pressure gas interface 405, and only allows the flow path to pass from the high-pressure gas interface 405 to the second component, but not in the opposite direction; the switch valve 404 is used for isolating the connection between the second component and the vacuum interface 405, and only allows the flow path to pass from the second component to the vacuum interface 405, but not in the opposite direction; the high-pressure gas interface 405 is used for connecting high-pressure gas, so as to drive the fermentation liquid to pass through the separation membrane 203 in the second component into the fermentation liquid sample pool 302 in the third component; the vacuum interface 405 is used for connecting vacuum, so as to suck the liquid in the fermentation reactor 101 in the first component or the cleaning liquid bottle 504 in the fifth component into the pressure-resistant container 202 in the second component.

[0036] The fifth component is used for containing cleaning liquid, which is connected with the second component and the third component through pipelines; the fifth component comprises: a switch valve five 501, a three-way four 502, a bubble sensor three 503 and a cleaning liquid bottle 504; wherein, the inlet end of the switch valve five 501 is connected with the three-way four 502 through pipelines, and the outlet end is connected with the second component through pipelines; the interface one of the three-way four 502 is connected with the switch valve five 501 through pipelines, the interface two is connected with the bubble sensor four through pipelines, and the interface three is connected with the third component through pipelines; the bubble sensor three 503 is connected with the three-way four 502 and the cleaning liquid bottle 504 through pipelines respectively; the cleaning liquid bottle 504 is connected with the bubble sensor three 503 through pipelines; the switch valve five 501 is used for isolating the connection between the second component and the cleaning liquid bottle 504, and only allows the flow path from the cleaning liquid bottle 504 to the second component but not in the opposite direction; the bubble sensor three 503 is used for judging whether there is liquid in the gas pipeline, so as to control the volume of cleaning liquid in the cleaning liquid bottle 504; the cleaning liquid bottle 504 is used for containing cleaning liquid.

[0037] The sixth component is used for containing fermentation liquid solid components and waste liquid, which is connected with the second component and the third component through pipelines respectively; the sixth component comprises: a switch valve six 601, a three-way five 602 and a waste liquid bottle 603; wherein, the inlet end of the switch valve six 601 is connected with the second component through pipelines, and the outlet end is connected with the three-way five 602 through pipelines; the interface one of the three-way five 602 is connected with the switch valve six 601 through pipelines, the interface two is connected with the third component through pipelines, and the interface three is connected with the waste liquid bottle 603 through pipelines; the waste liquid bottle 603 is connected with the three-way five 602 through pipelines; the switch valve six 601 is used for isolating the connection between the second component and the waste liquid bottle 603, and only allows the flow path from the second component to the waste liquid bottle 603 but not in the opposite direction; the waste liquid bottle 603 is used for containing waste liquid.

[0038] In some embodiments, the switch valves one 201 to six are all automatic execution valves. The setting of such automatic execution valves can make the system become a sterile and automatic sampling system.

[0039] In some embodiments, the pipelines in the second component and the fourth component are stainless steel pipes, pressure-resistant PTFE pipes or pressure-resistant FEP pipes. The material of the pipelines can make the system become an automatic sampling system suitable for high-density fermentation process.

[0040] In some embodiments, the pipelines in the first component, the third component, the fifth component and the sixth component are silica gel pipes, pressure-resistant PTFE pipes or pressure-resistant FEP pipes. The material of the pipelines can make the system become an automatic sampling system which is convenient for sterilization and cleaning.

[0041] In some embodiments, the cleaning pump 303, the overflow pump 305 and the outlet pump 306 are peristaltic pumps or diaphragm pumps. The arrangement of such pumps can make the system a sealed and fast liquid discharge automatic sampling system.

[0042] In some embodiments, the bubble sensor one 304, the bubble sensor two 401 and the bubble sensor three 503 are capacitive sensors or ultrasonic sensors. The arrangement of such sensors can make the system an intelligent automatic sampling system.

[0043] The system is used for automatic sampling control method of high-density fermentation process, the fermentation reactor 101 is filled with fermentation broth, including the following sampling steps: Step S100: open the switch valve six 601 and the switch valve three 403, the high-pressure gas passes through the switch valve three 403 to blow the residual liquid in the pressure-resistant container 202 into the waste liquid bottle 603, and the emptying is completed; Step S200: close the switch valve six 601 and the switch valve three 403, open the switch valve four 404, empty the high-pressure gas, open the switch valve one 201, and draw the fermentation broth in the fermentation reactor 101 into the pressure-resistant container 202 through the sampling tube 102 until the bubble sensor two 401 senses the liquid signal, close the switch valve four 404 and the switch valve one 201, open the switch valve six 601 and the switch valve three 403, and the high-pressure gas passes through the switch valve three 403 to blow the fermentation broth in the pressure-resistant container 202 into the waste liquid bottle 603, and the rinsing is completed; Step S300: close the switch valve six 601 and the switch valve three 403, open the switch valve four 404, empty the high-pressure gas, open the switch valve one 201, and draw the fermentation broth in the fermentation reactor 101 into the pressure-resistant container 202 through the sampling tube 102 until the bubble sensor two 401 senses the liquid signal, close the switch valve four 404 and the switch valve one 201, and the sampling is completed; Step S400: open the switch valve two 301, the switch valve three 403 and the overflow pump 305, the high-pressure gas passes through the switch valve three 403 to blow the fermentation broth in the pressure-resistant container 202 into the fermentation broth sample pool 302 through the separation membrane 203 until the bubble sensor one 304 senses the liquid signal, close the switch valve two 301, the switch valve three 403 and the overflow pump 305, and the filtration is completed; Step S500: open the switch valve six 601 and the switch valve three 403, the high pressure gas blows the residual fermentation broth in the pressure-resistant container 202 into the waste liquid bottle 603 through the switch valve three 403, close the switch valve six 601 and the switch valve three 403, open the switch valve four 404, empty the high pressure gas, open the switch valve five 501, draw the cleaning liquid in the cleaning liquid bottle 504 into the pressure-resistant container 202 through the separation membrane 203, until the bubble sensor two 401 senses the liquid signal, close the switch valve four 404 and the switch valve three 403, open the switch valve six 601 and the switch valve three 403, the high pressure gas blows the cleaning waste liquid in the pressure-resistant container 202 into the waste liquid bottle 603 through the switch valve three 403, repeat this step three times, and the component two cleaning is completed. Step S600: open the waste liquid pump 306, pump the waste liquid in the fermentation broth sample pool 302 into the waste liquid bottle 603, close the waste liquid pump, open the cleaning pump 303 and the overflow pump 305, pump the cleaning liquid in the cleaning liquid bottle 504 into the fermentation broth sample pool 302, until the bubble sensor one 304 senses the liquid signal, close the cleaning pump 303 and the overflow pump 305, open the waste liquid pump, pump the waste liquid in the fermentation broth sample pool 302 into the waste liquid bottle 603, close the waste liquid pump, repeat this step three times, and the component three cleaning is completed.

[0044] The application case of the automatic sampling system is used for fermenting Escherichia coli BL21 (containing a recombinant plasmid) in a 15L mechanical stirring fermenter to produce alkaline protease, and a TB medium (12g / L of tryptone, 24g / L of yeast extract, 4g / L of glycerol, 2.31g / L of KH2PO4 and 12.54g / L of K2HPO4·3H2O) is used, the initial pH of which is 7.0, the temperature is kept at 37℃ during the fermentation process, the stirring speed is 500-800rpm (adjusted according to the dissolved oxygen), the aeration rate is 1.0vvm, the fermentation cycle is 48h, and glycerol is supplemented in the later stage to maintain the carbon source. The automatic sampling system is used for sampling. The sampling system is configured as follows: component one: a φ6mm stainless steel tube is used as a sampling tube, the lower end of which is 10cm away from the bottom of the fermenter, is connected with the side opening of the fermenter through a PG13.5 external thread, and is sealed by a rubber ring; component two: a pressure-resistant container with a volume of 500mL (pressure-resistant 1.0MPa) is used, a separation membrane with a length of 5cm and a filtering precision of 0.1μm is used, and the separation membrane is fixed through an external thread; component three: a fermentation broth sample pool with a volume of 100mL is used, and a peristaltic pump (flow range 0-50mL / min) is used as a cleaning pump, an overflow pump and a liquid outlet pump; component four: sterile compressed air (pressure 0.4MPa) is used as high-pressure gas, and a vacuum interface is connected with a vacuum pump (vacuum degree-0.08MPa); component five: sterile deionized water is used as cleaning liquid, and a cleaning liquid bottle with a volume of 2L is used; and pipeline materials: components two and four are made of pressure-resistant PTFE pipes, and the remaining components are made of silica gel pipes.

[0045] During sampling, the "S100-S200-S300-S400-S500-S600" process is operated. During the 48h fermentation cycle, the system completes 6 automatic samplings, and the time consumed from starting to collecting the clear liquid is controlled to be 10min each time, the valve switching, pump operation and gas driving process are not jammed, the pressure of the fermenter is stable (fluctuation≤0.02MPa), and no abnormal fermentation parameters caused by sampling occur.

[0046] The application provides a kind of suitable for high-density fermentation process automatic sampling system and control method's train of thought and method, the method and approach for specifically realizing this technical scheme are many, above-mentioned only is preferred implementation mode of the present application, it should be pointed out, for the ordinary skill in the art, without departing from the principle of the present application, can make a number of improvements and refinements, these improvements and refinements also should be considered as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by existing technology.

Claims

1. An automated sampling system suitable for use in high density fermentation processes, characterized in that, include: Component 1 is used to contain and extract the fermentation broth; Component 2 has a solid-liquid separation membrane inside to retain solid components in the fermentation broth; the inlet of Component 2 is connected to Component 1 to introduce fermentation broth into Component 2; the clear liquid outlet at the rear end of Component 2 is connected to Component 3 and Component 5 through pipelines; the two sides of the middle part of Component 2 are connected to Component 4 and Component 6 through pipelines. Component 3 is used to contain the fermentation broth and is connected to Component 2, Component 5 and Component 6 through pipelines. The fourth component is used to access high-pressure gas, and it is connected to the second component through a pipeline to introduce high-pressure gas into the second component. Component five is used to contain cleaning fluid and is connected to components two and three via pipelines; Component six is ​​used to contain the solid components of the fermentation broth and waste liquid, and it is connected to components two and three through pipelines.

2. The automatic sampling system for high-density fermentation processes according to claim 1, characterized in that, The first component includes: a fermentation reactor (101) and a sampling tube (102); wherein, The sampling tube (102) is fixed to the fermentation reactor (101) by external thread, and the two are sealed by a rubber ring. The lower end of the sampling tube (102) extends below the liquid surface inside the fermentation reactor (101). The upper end of the sampling tube (102) is connected to component two through a pipeline and is used to extract fermentation broth into component two; The fermentation reactor (101) is used to contain the fermentation broth.

3. The automatic sampling system for high-density fermentation processes according to claim 1, characterized in that, The second component includes: a switching valve (201), a pressure vessel (202), a separation membrane (203), and a three-way valve (204); wherein, The inlet end of the switching valve (201) is connected to component one via a pipeline, and the outlet end is connected to the interface of the pressure vessel (202) via a pipeline. The upper end interface of the pressure vessel (202) is connected to component four through a pipeline, the lower end interface is connected to component six through a pipeline, the left end interface is connected to switch valve one (201) through a pipeline, and the right end interface is connected to the separation membrane (203) through a thread. The separation membrane (203) is fixed to the right side interface of the pressure vessel (202) by external thread, and the other end is connected to the tee (204) by a pipeline; The first interface of the three-way connector (204) is connected to the separation membrane (203) through a pipeline, the second interface is connected to the fifth component through a pipeline, and the third interface is connected to the third component through a pipeline. The switching valve (201) is used to isolate the connection between component one and component two, and only allows unidirectional flow from component one to component two; The pressure-resistant container (203) is used to contain fermentation broth, high-pressure gas or cleaning liquid, and its pressure resistance is ≥1.0MPa; The separation membrane (203) is used to retain solid components in the fermentation broth.

4. The automatic sampling system for high-density fermentation processes according to claim 1, characterized in that, The third component includes: a second switching valve (301), a fermentation broth sample tank (302), a cleaning pump (303), a first bubble sensor (304), an overflow pump (305), a discharge pump (306), and a second three-way valve (307); wherein, The inlet end of the second switch valve (301) is connected to the second component via a pipeline, and the outlet end is connected to the fermentation broth sample tank (302) via a pipeline. The fermentation broth sample pool (302) is provided with four interfaces, which are respectively connected to the second switch valve (301), the cleaning pump (303), the first bubble sensor (304) and the outlet pump (306) through pipelines. The interface connected to the outlet pump (306) is located at the lowest point of the inner cavity of the fermentation broth sample pool (302), and the interface connected to the first bubble sensor (304) is located in the middle of the fermentation broth sample pool (302). The inlet of the cleaning pump (303) is connected to component five through a pipeline, and the outlet is connected to the fermentation broth sample tank (302) through a pipeline. The bubble sensor (304) is connected to the fermentation broth sample tank (302) and the overflow pump (305) via pipelines; The inlet of the overflow pump (305) is connected to the bubble sensor (304) via a pipeline, and the outlet is connected to the tee (307) via a pipeline. The inlet of the outlet pump (306) is connected to the fermentation broth sample tank (302) via a pipeline, and the outlet is connected to the three-way valve (307) via a pipeline. The first interface of the three-way connector (307) is connected to the overflow pump (305) through a pipeline, the second interface is connected to the outlet pump (306) through a pipeline, and the third interface is connected to component six through a pipeline. The second switching valve (301) is used to isolate the connection between component two and component three, and only allows unidirectional flow from component two to component three; The fermentation broth sample cell (302) is used to contain the fermentation broth clear liquid; The cleaning pump (303) is used to pump the cleaning solution into the fermentation broth sample tank (302). The bubble sensor (304) is used to determine whether there is liquid in the pipetting line in order to control the liquid volume in the fermentation broth sample cell (302); The overflow pump (305) is used to pump excess liquid from the fermentation broth sample tank (302) to component six; The discharge pump (306) is used to pump the waste liquid in the fermentation broth sample pool (302) to component six.

5. The automatic sampling system for high-density fermentation processes according to claim 1, characterized in that, The fourth component includes: a second bubble sensor (401), a third tee (402), a third switching valve (403), a fourth switching valve (404), a high-pressure gas interface (405), and a vacuum interface (406); wherein, The second bubble sensor (401) is connected to the second component and the third tee (402) via pipes; The first interface of the three-way valve (402) is connected to the second bubble sensor (401) through a pipeline, the second interface is connected to the third switch valve (403) through a pipeline, and the third interface is connected to the fourth switch valve (404) through a pipeline. The inlet end of the switch valve three (403) is connected to the high-pressure gas interface (405) through a pipeline, and the outlet end is connected to the three-way valve three (402) through a pipeline; The inlet end of the four-way valve (404) is connected to the three-way valve (402) via a pipeline, and the outlet end is connected to the vacuum port (406) via a pipeline; The second bubble sensor (401) is used to determine whether there is liquid in the gas pipeline in order to control the liquid volume in the pressure container (202) in component two; The third switching valve (403) is used to isolate the connection between component two and the high-pressure gas interface (405), and only allows unidirectional flow from the high-pressure gas interface (405) to component two; The fourth switching valve (404) is used to isolate the connection between component two and vacuum interface (406), and only allows unidirectional flow from component two to vacuum interface (406); The high-pressure gas interface (405) is used to connect high-pressure gas to drive the fermentation broth clear liquid through the separation membrane (203) in component two into the fermentation broth sample pool (303) in component three. The vacuum interface (406) is used to connect a vacuum pump to draw liquid from the fermentation reactor (101) in component one or the cleaning liquid bottle (504) in component five into the pressure container (202) in component two.

6. The automatic sampling system for high-density fermentation processes according to claim 1, characterized in that, The fifth component includes: a five-way valve (501), a three-way valve (502), a three-way bubble sensor (503), and a cleaning fluid bottle (504); wherein, The inlet end of the switch valve five (501) is connected to the three-way four (502) via a pipeline, and the outlet end is connected to component two via a pipeline; The first interface of the three-way four (502) is connected to the five-way valve five (501) through a pipeline, the second interface is connected to the four-way bubble sensor four (503) through a pipeline, and the third interface is connected to the three-way component three through a pipeline; The bubble sensor three (503) is connected to the three-way valve four (502) and the cleaning fluid bottle (504) through pipelines; The cleaning fluid bottle (504) and the bubble sensor three (503) are connected by a pipeline; The switch valve five (501) is used to isolate the connection between component two and the cleaning fluid bottle (504), and only allows unidirectional flow from the cleaning fluid bottle (504) to component two; The bubble sensor three (503) is used to determine whether there is liquid in the gas pipeline in order to control the volume of cleaning liquid in the cleaning liquid bottle (504); The cleaning fluid bottle (504) is used to contain the cleaning fluid.

7. The automatic sampling system for high-density fermentation processes according to claim 1, characterized in that, The sixth component includes: a six-way valve (601), a three-way valve (602), and a waste liquid bottle (603); wherein, The inlet end of the switching valve six (601) is connected to component two via a pipeline, and the outlet end is connected to tee five (602) via a pipeline; The first interface of the three-way valve (602) is connected to the sixth switch valve (601) via a pipeline, the second interface is connected to the third component via a pipeline, and the third interface is connected to the waste liquid bottle (603) via a pipeline. The waste liquid bottle (603) and the three-way valve (602) are connected by a pipeline; The switching valve six (601) is used to isolate the connection between component two and waste liquid bottle (603), and only allows unidirectional flow from component two to waste liquid bottle (603); The waste liquid bottle (603) is used to contain waste liquid.

8. The automatic sampling system suitable for high-density fermentation processes according to any one of claims 1 to 7, characterized in that, The pipelines are each independently selected from any one of stainless steel pipes, silicone pipes, pressure-resistant PTFE pipes, or pressure-resistant FEP pipes.

9. The automatic sampling system for high-density fermentation processes according to claim 4, characterized in that, The cleaning pump (303), overflow pump (305) and discharge pump (306) are peristaltic pumps or diaphragm pumps.

10. The control method for the automatic sampling system suitable for high-density fermentation processes according to claim 1, characterized in that, Includes the following steps: Step S100: Open valve six (601) and valve three (403) in turn. High pressure gas blows the residual liquid in the pressure container (202) into the waste liquid bottle (603) through valve three (403) and the container is emptied. Step S200: Close switch valve six (601) and switch valve three (403), open switch valve four (404) to vent the high-pressure gas, open switch valve one (201), and extract the fermentation liquid in the fermentation reactor (101) into the pressure container (202) through the sampling tube (102) until the bubble sensor two (401) senses the liquid signal. Close switch valve four (404) and switch valve one (201), and open switch valve six (601) and switch valve three (403) in turn. The high-pressure gas blows the fermentation liquid in the pressure container (202) into the waste liquid bottle (603) through switch valve three (403) to complete the rinsing. Step S300: Close switch valve six (601) and switch valve three (403), open switch valve four (404) to vent the high-pressure gas, open switch valve one (201), and extract the fermentation liquid in the fermentation reactor (101) into the pressure container (202) through the sampling tube (102) until the bubble sensor two (401) senses the liquid signal, close switch valve four (404) and switch valve one (201), and the sampling is completed; Step S400: Open valve 2 (301), valve 3 (403) and overflow pump (305) in sequence. High pressure gas blows the fermentation liquid in the pressure container (202) through the separation membrane (203) into the fermentation liquid sample pool (302) through valve 3 (403) until bubble sensor 1 (304) senses the liquid signal. Then close valve 2 (301), valve 3 (403) and overflow pump (305) to complete the filtration. Step S500: Open valve six (601) and valve three (403) in sequence. High-pressure gas blows the residual fermentation liquid in the pressure container (202) into the waste liquid bottle (603) through valve three (403). Close valve six (601) and valve three (403). Open valve four (404) to vent the high-pressure gas. Open valve five (501) to draw the cleaning liquid in the cleaning liquid bottle (504) into the pressure container (202) through the separation membrane (203) until the bubble sensor two (401) senses the liquid signal. Close valve four (404) and valve three (403). Open valve six (601) and valve three (403) in sequence. High-pressure gas blows the cleaning waste liquid in the pressure container (202) into the waste liquid bottle (603) through valve three (403). Repeat this step three times. Component two is cleaned. Step S600: Turn on the waste liquid pump (306) to pump the waste liquid in the fermentation broth sample pool (302) into the waste liquid bottle (603), turn off the waste liquid pump (306), turn on the cleaning pump (303) and the overflow pump (305) to pump the cleaning liquid in the cleaning liquid bottle (504) into the fermentation broth sample pool (302) until the bubble sensor (304) senses the liquid signal, turn off the cleaning pump (303) and the overflow pump (305), turn on the waste liquid pump (306) to pump the waste liquid in the fermentation broth sample pool (302) into the waste liquid bottle (603), turn off the waste liquid pump (306), repeat this step three times, and the cleaning of component three is completed.

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