Escherichia coli fermentor capable of stably measuring carbon dioxide concentration

Abstract

The present application relates to a kind of escherichia coli fermenter capable of stable measurement of carbon dioxide concentration, including tank body, stirring device and carbon dioxide electrode, stirring device and carbon dioxide electrode are all arranged at the top of tank body, stirring device is arranged at the center of the top of tank body, stirring device includes power device and stirring assembly, the power output end of power device is vertically downward overhanging into the inside of tank body, stirring assembly is drivingly connected with the power output end of power device, carbon dioxide electrode includes installation end and measurement end, installation end is fixedly installed at the top of tank body near edge, measurement end is vertically downward overhanging into the inside of tank body, still include measuring cylinder and liquid taking device, measuring cylinder is installed in tank body, the measurement end of carbon dioxide electrode can be inserted into measuring cylinder, liquid taking device ensures that the fermentation broth in measuring cylinder is over measurement end when measuring.Under the premise that normal stirring of fermentation broth in tank body is not adjusted, the damage rate of liquid wave flow to gas permeation membrane is reduced, and the sensitivity and accuracy of electrode detection are ensured.

Description

Technical Field

[0001] This invention relates to the field of bioengineering equipment, and more particularly to an Escherichia coli fermenter capable of stably measuring carbon dioxide concentration. Background Technology

[0002] The fermentation method for producing L-phenylalanine utilizes genetically engineered *E. coli* to convert glucose into L-phenylalanine. In this process, glucose serves as the primary carbon source, providing essential building blocks for bacterial growth, respiratory metabolism, and product synthesis. Respiratory metabolism generates a significant amount of carbon dioxide. When the carbon dioxide concentration in the fermentation broth reaches a certain level, it can negatively impact bacterial growth and metabolism, thereby affecting product synthesis. Therefore, controlling the carbon dioxide concentration in the fermentation broth is crucial for maintaining optimal bacterial growth and metabolism, maximizing product synthesis, and ensuring a high glucose conversion rate.

[0003] To control the carbon dioxide concentration in the fermentation broth, the primary step is to measure it. Currently, a well-established method is to install a carbon dioxide electrode in the fermenter. The principle of the carbon dioxide electrode is that it is separated from the fermentation broth through a chamber with a gas-permeable membrane, which is filled with electrolyte. Carbon dioxide gas diffuses through the membrane into the electrolyte inside the electrode, reaching equilibrium with bicarbonate ions and thus changing the pH value of the electrolyte. An internal pH electrode measures this pH change and converts it into a signal related to the carbon dioxide concentration.

[0004] Ideally, a carbon dioxide electrode can accurately measure the carbon dioxide concentration in the fermentation broth. However, the actual operating conditions in a bioreactor are complex and subject to numerous interferences. The biggest problem is that bioreactors typically involve high-frequency agitation during operation. The main function of agitation is to thoroughly mix the culture medium, microbial cells, oxygen, and other additives (such as nutrients and pH adjusters) within the fermenter, ensuring the homogeneity of the fermentation broth. This homogeneity is crucial for maintaining the stability and consistency of the fermentation process. Therefore, agitation is usually indispensable in bioreactors. However, agitation introduces several problems to the operation of the carbon dioxide electrode: First, agitation causes vigorous flow in the fermentation broth, and the impact of the liquid wave may cause the gas permeation membrane to rupture or deform, thus affecting the electrode's sensitivity and accuracy. Second, the impact of the liquid wave can destabilize the gas permeation membrane on the electrode surface, leading to fluctuations in the measurement signal. Additionally, the liquid wave may accelerate the flow of liquid around the electrode, causing the local carbon dioxide concentration to differ from the actual concentration in the fermentation broth. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is to provide an Escherichia coli fermenter that can stably measure carbon dioxide concentration, reduce the probability of damage to the gas permeation membrane by liquid ripples without adjusting the normal stirring of the fermentation liquid in the tank, and ensure the sensitivity and accuracy of electrode detection.

[0006] To solve the above-mentioned technical problems, the present invention proposes the following technical solution:

[0007] An E. coli fermenter capable of stably measuring carbon dioxide concentration includes a tank body, a stirring device, and a carbon dioxide electrode. Both the stirring device and the carbon dioxide electrode are located at the top of the tank body. The stirring device is positioned at the center of the top of the tank body and includes a power unit and a stirring assembly. The power output end of the power unit extends vertically downwards into the tank body, and the stirring assembly is connected to the power output end of the power unit. The carbon dioxide electrode includes an installation end and a measuring end. The installation end is fixedly installed at the top of the tank body near the edge, and the measuring end extends vertically downwards into the tank body. The fermenter also includes a measuring vessel and a liquid sampling device. The measuring vessel is installed inside the tank body, and the measuring end of the carbon dioxide electrode can extend into the measuring vessel. The liquid sampling device ensures that the fermentation liquid in the measuring vessel covers the measuring end during measurement.

[0008] Because the measuring vessel surrounds the measuring end of the carbon dioxide electrode, it can block the liquid flow caused by stirring of the fermentation broth, thereby protecting the gas permeation membrane of the carbon dioxide electrode. The liquid sampling device is used to keep the fermentation broth in the tank, ensuring stable and reliable measurement during the fermentation process.

[0009] Preferably, the liquid collection device includes a lifting assembly, which comprises a fixed component and a movable component. The fixed component is fixed to the top of the tank, and the movable component can move up and down relative to the fixed component. The measuring vessel is fixedly mounted on the movable component. The lifting assembly allows the position of the measuring vessel relative to the carbon dioxide electrode to change. During the change, the fermentation liquid around the carbon dioxide electrode can be relatively integrated or isolated from the fermentation liquid inside the tank, ensuring that the carbon dioxide concentration of the fermentation liquid around the carbon dioxide electrode changes with the fermentation liquid inside the tank.

[0010] Furthermore, the measuring end of the carbon dioxide electrode is located above the surface of the fermentation broth inside the tank, and the measuring vessel is a barrel-shaped vessel with a closed bottom and an open top. In this design, the measuring vessel extracts the test solution from the fermentation broth through a lifting assembly. When the measuring vessel is immersed in the fermentation broth and stirred by a stirring device, the fermentation broth exchanges with the test solution in the measuring vessel, ensuring the consistency between the concentration of the measured liquid in the measuring vessel and the concentration of the fermentation broth.

[0011] Preferably, the measuring end of the carbon dioxide electrode is located below the surface of the fermentation broth inside the tank, and the measuring vessel includes a wall that surrounds the carbon dioxide electrode, with openings at both the top and bottom. In this design, when carbon dioxide concentration needs to be measured, the measuring vessel sinks and surrounds the measuring end of the carbon dioxide electrode. At this time, liquid fluctuations around the carbon dioxide electrode are isolated by the wall of the measuring vessel. Measurement is performed after the liquid surface around the gas permeation membrane inside the measuring vessel has stabilized. This prevents the gas permeation membrane from becoming unstable due to liquid ripples during the detection process, thus preventing fluctuations in the measurement signal and effectively improving the measurement stability of the carbon dioxide electrode.

[0012] Preferably, the liquid sampling device includes an inlet pipe, a pump body, and an outlet pipe. One end of the inlet pipe is installed on the tank body and communicates with the inside of the tank body, while the other end of the inlet pipe is connected to the suction port of the pump body. One end of the outlet pipe is connected to the measuring vessel, and the other end of the outlet pipe is connected to the pump inlet of the pump body. A diaphragm pump is used to avoid contamination of the test liquid during transport. This design allows for real-time circulation of the fermentation broth in the measuring vessel. Firstly, the carbon dioxide electrode does not need to be inserted into the agitated fermentation broth, preventing damage to the gas permeability membrane from the agitated liquid. Secondly, the real-time circulation of the liquid ensures a consistent concentration of the measured liquid, further improving the accuracy of the measurement.

[0013] Furthermore, the outlet pipe includes a straight section and a reducing section. The straight section connects to the pump's suction port, and the reducing section connects the straight section to the measuring vessel. The diameter of the reducing section near the measuring vessel is larger than that near the straight section. This reducing section design gradually slows down the flow rate of the test liquid within the pipe, thereby reducing the flow rate of the liquid inside the measuring vessel. This further reduces the impact of the liquid on the gas permeation membrane and improves the measurement stability of the carbon dioxide electrode.

[0014] Preferably, the measuring vessel includes a vessel body and a slowing hopper. The vessel body has a liquid inlet at the bottom, the slowing hopper has a primary slowing port at the bottom, and a secondary slowing port at the top. The diameter of the secondary slowing port is larger than that of the primary slowing port. The primary slowing port is connected to a reducing tube, and the secondary slowing port is connected to the liquid inlet. This design further reduces the flow rate of the liquid inside the measuring vessel.

[0015] Furthermore, the axis of the carbon dioxide electrode is offset from the axis of the liquid inlet. This staggered design prevents the gushing test liquid from directly impacting the gas permeation membrane, thus avoiding damage to the membrane.

[0016] Specifically, the measuring vessel also includes a baffle plate, which is positioned between the liquid inlet and the carbon dioxide electrode, dividing the vessel into two chambers. The bottom of the baffle plate is connected to the bottom of the vessel, and the top of the baffle plate does not exceed the top of the vessel. The carbon dioxide electrode and the liquid inlet are located in the two chambers respectively, with the measuring end of the carbon dioxide electrode higher than the baffle plate. The baffle plate design blocks the test liquid flowing up from the slow-moving chamber, preventing the gushing liquid from impacting the gas permeation membrane. When the chamber on the liquid inlet side is filled, the test liquid will overflow the baffle plate and enter the chamber on the carbon dioxide electrode side. When the test liquid fills both sides of the baffle plate, the liquid level will tend to rise steadily and gradually submerge the measuring end of the carbon dioxide electrode. The stable liquid level can further improve the stability of data measurement.

[0017] Preferably, the measuring vessel also includes a drain outlet, which is located near the center of the tank and connects the measuring vessel to the tank. The horizontal height of the drain outlet is not lower than the horizontal height of the carbon dioxide electrode measuring end. The drain outlet being higher than the electrode measuring end ensures that the electrode is completely submerged in the test liquid, preventing premature loss and potential undetectable readings. This design allows for timely backflow of the test liquid, reducing the total circulating volume of the test liquid and minimizing the impact of the circulating liquid on the fermentation progress.

[0018] In summary, this E. coli fermenter, which can stably measure carbon dioxide concentration, reduces the probability of liquid ripples damaging the gas permeation membrane without adjusting the normal stirring of the fermentation broth inside the tank, thus ensuring the sensitivity and accuracy of electrode detection. Attached Figure Description

[0019] The invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Figure 1 This is a cross-sectional structural schematic diagram of the first embodiment of the technical solution;

[0021] Figure 2 This is a cross-sectional structural diagram of the second embodiment of the technical solution;

[0022] Figure 3 These are schematic diagrams of the lifting component structure in the first and second embodiments of the technical solution described above;

[0023] Figure 4 This is a structural schematic diagram of the third embodiment of the technical solution;

[0024] Figure 5 This is a cross-sectional structural diagram of the third embodiment of the technical solution;

[0025] Figure 6 It is the aforementioned Figure 5 An enlarged view of icon A in the middle;

[0026] Figure 7 This is a schematic diagram of the measuring vessel structure according to the third embodiment of the technical solution;

[0027] in,

[0028] Tank body-1, stirring device-2, power unit-21, stirring assembly-22, carbon dioxide electrode-3, mounting end-31, measuring end-32, measuring vessel-4, vessel body-41, liquid inlet-411, slowing hopper-42, primary slowing port-421, secondary slowing port-422, baffle plate-43, drain port-44, liquid taking device-5, lifting assembly-51, fixing component-52, moving component-53, liquid inlet pipe-54, pump body-55, liquid outlet pipe-56, straight pipe section-561, reducing pipe section-562. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0030] An E. coli fermenter capable of stably measuring carbon dioxide concentration includes a tank body 1, a stirring device 2, and a carbon dioxide electrode 3. Both the stirring device 2 and the carbon dioxide electrode 3 are located on the top of the tank body 1. The stirring device 2 is located at the center of the top of the tank body 1 and includes a power unit 21 and a stirring assembly 22. The power output end of the power unit 21 extends vertically downward into the tank body 1. The stirring assembly 22 is connected to the power output end of the power unit 21. The carbon dioxide electrode 3 includes an installation end 31 and a measuring end 32. The installation end 31 is fixedly installed on the top of the tank body 1 near the edge, and the measuring end 32 extends vertically downward into the tank body 1. The fermenter also includes a measuring vessel 4 and a liquid sampling device 5. The measuring vessel 4 is installed inside the tank body 1, and the measuring end 32 of the carbon dioxide electrode 3 can extend into the measuring vessel 4. The liquid sampling device 5 ensures that the fermentation liquid in the measuring vessel 4 does not exceed the measuring end 32 during measurement.

[0031] Since the measuring vessel 4 surrounds the measuring end 32 of the carbon dioxide electrode 3, the measuring vessel 4 can block the liquid flow caused by stirring of the fermentation liquid, thereby protecting the gas permeation membrane of the carbon dioxide electrode 3. The liquid sampling device 5 is used to keep the fermentation liquid in the tank 1 to ensure stable and reliable measurement during the fermentation process.

[0032] like Figure 1As shown, in the first embodiment of the technical solution, the liquid collection device 5 includes a lifting assembly 51, which includes a fixing member 52 and a moving member 53. The fixing member 52 is fixed to the top of the tank 1, and the moving member 53 can move up and down relative to the fixing member 52. The measuring vessel 4 is fixedly installed on the moving member 53. The lifting assembly 51 allows the position of the measuring vessel 4 relative to the carbon dioxide electrode 3 to change. During the change, the fermentation liquid around the carbon dioxide electrode 3 can be relatively fused or isolated from the fermentation liquid in the tank 1, ensuring that the carbon dioxide concentration of the fermentation liquid around the carbon dioxide electrode 3 changes with the fermentation liquid in the tank 1.

[0033] The shape of the measuring vessel 4 is further designed so that the measuring end 32 of the carbon dioxide electrode 3 is located above the surface of the fermentation liquid inside the tank 1. The measuring vessel 4 is a barrel shape with a closed bottom and an open top. In this design, the measuring vessel 4 extracts the test liquid from the fermentation liquid by being driven by the lifting component 51. When the measuring vessel 4 is immersed in the fermentation liquid and stirred by the stirring device 2, the fermentation liquid will exchange with the test liquid in the measuring vessel 4, ensuring the consistency between the concentration of the measured liquid in the measuring vessel 4 and the concentration of the fermentation liquid. Although the bottom-closed measuring vessel 4 has a simple structure, it is difficult for it to sink into the fermentation liquid to extract the test liquid when there is no liquid inside. Figure 3 As shown, in this scheme, the moving part 53 is a support part with a sliding screw seat, and the fixing part 52 is a motor set on the top of the tank 1. The power output end of the motor is provided with a sliding screw, which is installed in the sliding screw seat. The motor drives the sliding screw to rotate in the sliding screw seat, providing a force for the measuring pot 4 to move up and down, so that the empty measuring pot 4 can be completely immersed in the fermentation liquid.

[0034] like Figure 2 As shown, in the second embodiment of the technical solution, the measuring end 32 of the carbon dioxide electrode 3 is located below the surface of the fermentation broth inside the tank 1. The measuring vessel 4 includes a wall that surrounds the carbon dioxide electrode 3, and both the top and bottom ends of the measuring vessel 4 are open. In this solution, when it is necessary to detect the carbon dioxide concentration, the measuring vessel 4 sinks and surrounds the measuring end of the carbon dioxide electrode 3. At this time, the liquid fluctuations around the carbon dioxide electrode 3 are isolated by the wall of the measuring vessel 4. Measurement is performed after the liquid surface around the gas permeation membrane inside the measuring vessel 4 has stabilized. This prevents the gas permeation membrane from becoming unstable due to the impact of liquid waves during the detection process, thus preventing fluctuations in the measurement signal and effectively improving the measurement stability of the carbon dioxide electrode 3.

[0035] like Figure 4 as well as Figure 5As shown, in the third embodiment of the technical solution, the liquid sampling device 5 includes an inlet pipe 54, a pump body 55, and an outlet pipe 56. One end of the inlet pipe 54 is installed on the tank body 1 and communicates with the inside of the tank body 1. The other end of the inlet pipe 54 is connected to the suction port of the pump body 55. One end of the outlet pipe 56 is connected to the measuring vessel 4, and the other end of the outlet pipe 56 is connected to the pump port of the pump body 55. The pump body 55 uses a diaphragm pump to avoid contamination of the test liquid during transportation. This design allows the fermentation liquid to circulate in the measuring vessel 4 in real time. On the one hand, the carbon dioxide electrode 3 does not need to be inserted into the stirred fermentation liquid, avoiding damage to the gas permeability membrane by the stirred liquid. On the other hand, the real-time circulation of the liquid ensures the uniformity of the concentration of the measured liquid, further improving the accuracy of the measurement. However, both the first and second embodiments have the problem of not being able to measure the concentration in the fermentation liquid in real time. Compared with the third embodiment, the ease of use is poor and the amount of data read per unit time is too small, making it impossible to effectively form an effective and smooth concentration change curve for reference.

[0036] like Figure 6 As shown, to reduce the liquid flow rate inside the pipe, the outlet pipe 56 includes a straight pipe section 561 and a reducing pipe section 562. The straight pipe section 561 is connected to the suction port of the pump body 55, and the reducing pipe section 562 connects the straight pipe section 561 to the measuring vessel 4. The diameter of the reducing pipe section 562 near the measuring vessel 4 is larger than that near the straight pipe section 561. The design of the reducing pipe section 562 can gradually slow down the flow rate of the test liquid in the pipe, thereby reducing the flow rate of the liquid inside the measuring vessel 4, further reducing the impact of the liquid on the gas permeation membrane, and improving the measurement stability of the carbon dioxide electrode 3.

[0037] To reduce the flow rate of liquid entering the measuring vessel 4, the measuring vessel 4 includes a vessel body 41 and a retarder hopper 42. The vessel body 41 has a liquid inlet 411 at its bottom. The retarder hopper 42 has a primary retarder inlet 421 at its bottom and a secondary retarder inlet 422 at its top. The diameter of the secondary retarder inlet 422 is larger than that of the primary retarder inlet 421. The primary retarder inlet 421 is connected to a reducing pipe section 562, and the secondary retarder inlet 422 is connected to the liquid inlet 411. This design further reduces the flow rate of liquid inside the measuring vessel 4.

[0038] To prevent damage to the gas permeation membrane, the axis of the carbon dioxide electrode 3 is offset from the axis of the liquid inlet 411. This staggered design prevents the gushing test liquid from directly impacting the gas permeation membrane, thus avoiding damage to the membrane.

[0039] like Figure 7As shown, specifically, the measuring vessel 4 also includes a baffle 43. The baffle 43 is positioned between the liquid inlet 411 and the carbon dioxide electrode 3, dividing the vessel body 41 into two chambers. The bottom of the baffle 43 is connected to the bottom of the vessel body 41, and the top of the baffle 43 does not exceed the top of the vessel body 41. The carbon dioxide electrode 3 and the liquid inlet 411 are located in the two chambers respectively, and the measuring end 32 of the carbon dioxide electrode 3 is higher than the baffle 43. The design of the baffle can block the test liquid surging from the slow-moving container 42 and prevent the gushing liquid from impacting the gas permeation membrane. When the chamber on one side of the liquid inlet 411 is filled, the test liquid will overflow the baffle 43 and enter the chamber on the side of the carbon dioxide electrode 3. When the test liquid fills both sides of the baffle 43, the liquid level will tend to rise steadily and gradually submerge the measuring end 32 of the carbon dioxide electrode 3. The stable liquid level can also further improve the stability of data measurement.

[0040] The measuring vessel 4 also includes a drain port 44, which is located near the center of the tank 1 and connects the measuring vessel 4 to the tank 1. The horizontal height of the drain port 44 is not lower than the horizontal height of the measuring end 32 of the carbon dioxide electrode 3. The height of the drain port 44 being higher than the measuring end 32 of the electrode ensures that the electrode can be completely immersed in the test liquid, preventing premature loss that would render the electrode undetectable. This design allows for timely backflow of the test liquid, reducing the total circulation volume of the test liquid and minimizing the impact of the circulating liquid on the fermentation progress.

[0041] In summary, this E. coli fermenter, which can stably measure carbon dioxide concentration, reduces the probability of damage to the gas permeation membrane by liquid ripples without adjusting the normal stirring of the fermentation broth inside the tank, thus ensuring the sensitivity and accuracy of electrode detection.

[0042] In summary, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fermenter of E. coli capable of stably measuring carbon dioxide concentration, comprising a tank body, a stirring device and a carbon dioxide electrode, wherein the stirring device and the carbon dioxide electrode are arranged at the top of the tank body, the stirring device is arranged at the center of the top of the tank body, the stirring device comprises a power device and a stirring assembly, the power output end of the power device vertically downwardly overhangs into the inside of the tank body, the stirring assembly is in transmission connection with the power output end of the power device, the carbon dioxide electrode comprises a mounting end and a measuring end, the mounting end is fixedly mounted at the top of the tank body close to the edge, and the measuring end vertically downwardly overhangs into the inside of the tank body. The measuring end of the carbon dioxide electrode is above the liquid level of the fermentation liquid in the tank body, and the measuring cup is in the shape of a barrel with a closed bottom and an open top. The measuring end of the carbon dioxide electrode is below the liquid level of the fermentation liquid in the tank body, and the measuring cup comprises a wall part that can surround the carbon dioxide electrode, and both the top and bottom of the measuring cup are open.

2. The E. coli fermenter capable of stable measurement of carbon dioxide concentration according to claim 1, characterized in that: The liquid taking device comprises a liquid inlet pipe, a pump body and a liquid outlet pipe, one end of the liquid inlet pipe is installed on the tank body and communicates with the inside of the tank body, the other end of the liquid inlet pipe is connected with the water suction port of the pump body, one end of the liquid outlet pipe is connected with the measuring cup, and the other end of the liquid outlet pipe is connected with the water pumping port of the pump body.

3. The E. coli fermenter capable of stable measurement of carbon dioxide concentration according to claim 1, characterized in that: The liquid outlet pipe comprises a straight pipe part and a variable-diameter pipe part, the straight pipe part is connected with the water pumping port of the pump body, the variable-diameter pipe part connects the straight pipe part with the measuring cup, and the diameter of the variable-diameter pipe part near the measuring cup is larger than the diameter of the variable-diameter pipe part near the straight pipe part.

4. The E. coli fermenter capable of stable measurement of carbon dioxide concentration according to claim 1, wherein: The measuring cup comprises a cup body and a slow-speed bucket, the cup body is provided with a liquid inlet at the bottom, the slow-speed bucket is provided with a primary slow-speed opening at the bottom, and is provided with a secondary slow-speed opening at the top, the diameter of the secondary slow-speed opening is larger than the diameter of the primary slow-speed opening, the primary slow-speed opening is connected with the variable-diameter pipe, and the secondary slow-speed opening is connected with the liquid inlet.

5. The E. coli fermenter capable of stable measurement of carbon dioxide concentration according to claim 4, characterized in that: The axis of the carbon dioxide electrode is arranged to be offset from the axis of the liquid inlet.

6. The E. coli fermenter capable of stable measurement of carbon dioxide concentration according to claim 5, characterized in that: The measuring cup further comprises a partition plate, the partition plate is arranged between the liquid inlet and the carbon dioxide electrode to divide the cup body into two chambers, the bottom of the partition plate is connected with the bottom of the cup body, the top of the partition plate does not exceed the top of the cup body, the carbon dioxide electrode and the liquid inlet are respectively located in the two chambers, and the measuring end of the carbon dioxide electrode is higher than the partition plate.

7. The E. coli fermenter capable of stable measurement of carbon dioxide concentration according to claim 6, characterized in that: The measuring cup further comprises a liquid outlet, the liquid outlet is arranged on the side close to the center of the tank body and communicates the measuring cup with the tank body, and the horizontal height of the liquid outlet is not lower than the horizontal height of the measuring end of the carbon dioxide electrode.

8. The E. coli fermenter capable of stable measurement of carbon dioxide concentration according to claim 7, characterized in that: ​ 9. The E. coli fermenter capable of stable measurement of carbon dioxide concentration according to claim 4, characterized in that: ​

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