Enrichment culture device and method for on-line regulation and control of microorganisms in high-pressure environment

Abstract

The invention discloses an enrichment culture device and method for on-line regulation and control of microorganisms in a high-pressure environment, and relates to the technical field of on-line regulation and control culture of marine microorganisms, the enrichment culture device comprises a microorganism enrichment unit, an on-line detection unit, a filtering unit, a pressure control unit, a multi-sequence nutrient solution supply unit and a central control unit; the gas inlet end of the microorganism enrichment unit is connected with the gas outlet end of the pressure control unit, and the liquid inlet end of the microorganism enrichment unit is connected with the liquid outlet end of the multi-sequence nutrient solution supply unit; the liquid inlet end of the filter unit is connected with the liquid outlet end of the microorganism enrichment unit, the liquid outlet end of the filter unit is connected with the liquid inlet end of the online detection unit, and the gas inlet end of the online detection unit is connected with the gas outlet end of the pressure control unit. In the enrichment culture process and the detection process, the device is consistent with the ocean in-situ pressure environment, pressure release is not needed in the detection process, the detection result fits the actual situation, the microorganisms are in the optimal enrichment culture environment, and the enrichment culture efficiency of the microorganisms is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of online regulation and cultivation of marine microorganisms, and more specifically, to an enrichment and cultivation device and method for online regulation of microorganisms under a high-pressure environment. Background Art

[0002] Marine microorganisms are abundant in number, have extremely high metabolic diversity, and play a huge role in marine material and energy cycles, but there are still great difficulties in the cultivation of marine microorganisms. Enrichment culture, as an important means of studying marine microorganisms, is widely used in the cultivation of microorganisms such as bacteria and archaea. It is generally used for targeted enrichment, by continuously adding certain nutrients to the culture medium and performing regular subcultures to obtain the target bacterial community.

[0003] At present, microbial enrichment experiments have been carried out under high pressure and normal pressure environments. Under normal pressure, some marine microorganisms are regulated by pressure, causing them to enter a dormant state or die, and cannot fully reflect the growth state of the microbial community. At the same time, the in-situ simulated culture carried out in a high-pressure environment generally only monitors some environmental indicators such as temperature and pressure in the environment online. The test of other chemical indicators requires collecting samples and releasing the pressure before testing on the machine, which leads to large errors in the measurement results and cannot truly reflect the physiological and biochemical state of the microorganisms during the culture process. The existing technical solutions mostly use multi-stage dilution for enrichment culture, which provides sufficient nutrition for the microorganisms, but does not supply nutrition according to the culture state of the microorganisms, nor does it clarify the specific growth state of the microorganisms during the enrichment process, such as changes in the number of cells. Therefore, it is urgent to provide a technical solution that can accurately regulate the cultivation of microorganisms during high-pressure enrichment of microorganisms to improve the cultivation efficiency of microorganisms. Summary of the invention

[0004] One of the purposes of the present invention is to provide an enrichment and cultivation device for online regulation of microorganisms under a high-pressure environment, so as to overcome the defect that the above-mentioned prior art cannot accurately regulate the cultivation of microorganisms under a high-pressure environment; the second purpose is to provide an enrichment and cultivation method for online regulation of microorganisms under a high-pressure environment.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0006] The present invention provides an enrichment and cultivation device for online regulation of microorganisms under high pressure environment, comprising: a microorganism enrichment unit, an online detection unit, a filtration unit, a pressure control unit, a multi-sequence nutrient solution supply unit and a central control unit;

[0007] The air inlet of the microorganism enrichment unit is connected to the air outlet of the pressure control unit, and the liquid inlet of the microorganism enrichment unit is connected to the liquid outlet of the multi-sequence nutrient solution supply unit;

[0008] The liquid inlet end of the filtration unit is connected to the liquid outlet end of the microorganism enrichment unit, the liquid outlet end of the filtration unit is connected to the liquid inlet end of the online detection unit, and the air inlet end of the online detection unit is connected to the air outlet end of the pressure control unit;

[0009] The data output end of the microorganism enrichment unit is connected to the data input end of the central control unit, and the control ends of the microorganism enrichment unit, the online detection unit, the pressure control unit, and the multi-sequence nutrient solution supply unit are connected to the control end of the central control unit.

[0010] The present invention sets a microorganism enrichment unit for microorganism enrichment culture, uses a pressure control unit to provide the microorganism enrichment unit with culture gas required for microorganism culture, or fills in inert gas to keep the pressure environment in the microorganism enrichment unit consistent with the marine in-situ pressure environment, and uses a multi-sequence nutrient solution supply unit to provide the microorganism enrichment unit with the nutrient solution required for microorganism culture; after a period of enrichment culture, the microorganism liquid enters the online detection unit after filtering out large particle impurities through a filter component, and at the same time, the pressure control unit provides inert gas for the online detection unit, so that the pressure environment in the online detection unit is consistent with the marine in-situ pressure environment, and the cell data and environmental parameters of the enriched cultured microorganism liquid are detected under the marine in-situ pressure environment, and the supplementary amount of the nutrient solution is calculated according to the cell data and environmental parameters of the microorganism liquid, so that the microorganism is always in the best enrichment culture environment. The present invention is consistent with the marine in-situ pressure environment during enrichment culture and detection, and no pressure release is required during the detection process, the detection result fits the actual situation, the microorganism is always in the best enrichment culture environment, and the efficiency of microorganism enrichment culture is improved.

[0011] Preferably, the online detection unit comprises a measuring chamber, a measuring pool, a light source, a waste liquid pool, a viewing window, a handle, a cleaning liquid container, a diluent container and a first liquid discharge valve;

[0012] The upper surface of the measuring chamber is provided with a first liquid inlet, a second liquid inlet, a third liquid inlet and an air inlet, the liquid outlet of the filter unit is connected to the first liquid inlet, the liquid outlet of the cleaning liquid container is connected to the second liquid inlet, the liquid outlet of the diluent container is connected to the third liquid inlet, and the air outlet of the pressure control unit is connected to the air inlet;

[0013] The light source is arranged on the side surface of the measuring chamber, parallel to the measuring pool; the window is arranged on the upper surface of the measuring chamber, corresponding to the position of the measuring pool; the control end of the control end of the light source is connected to the control end of the central control unit;

[0014] The waste liquid pool is arranged in the measuring chamber, and the measuring chamber is arranged in the waste liquid pool; one end of the handle is connected to the side surface of the measuring chamber, and the other end of the handle passes through the waste liquid pool and the measuring chamber in sequence and extends out; one end of the first liquid discharge valve is connected to the side surface of the waste liquid pool, and the other end of the first liquid discharge valve passes through the measuring chamber and extends out;

[0015] The diluent container is provided with a reaction solution, and the cleaning liquid container is provided with a cleaning liquid.

[0016] The first liquid inlet, the second liquid inlet and the third liquid inlet arranged on the upper surface of the measuring chamber are connected to the filtering unit, the cleaning liquid container and the diluent container respectively; during the first detection, the sterilized substrate to be cultured is injected into the measuring pool as a blank sample, the light source is turned on, and the spectrophotometry is used to perform zero adjustment to eliminate background interference; after the zero adjustment is completed, the light source is turned off, the handle is rotated clockwise to pour the substrate to be cultured in the measuring pool into the waste liquid pool, and the handle is rotated counterclockwise to inject the filtered microbial bacterial liquid from the first liquid inlet; after reaching the preset volume, the diluent is injected from the diluent container through the second liquid inlet to obtain the detection solution; the light source is turned on again to detect the detection solution, and the transmittance value is obtained and sent to the central control unit; after the detection is completed, the handle is rotated clockwise again to pour the detection solution in the measuring pool into the waste liquid pool, and the handle is rotated counterclockwise; the cleaning liquid is injected from the cleaning liquid container through the third liquid inlet to clean the measuring pool several times. After cleaning, the above process is performed at regular intervals to perform long-term enrichment culture and detection on the substrate to be cultured. The substrate to be cultured can also be updated to achieve enrichment culture and detection of other substrates to be cultured.

[0017] Preferably, the online detection unit further comprises an annular wall sleeve;

[0018] The annular wall sleeve is clamped with the measuring pool, and one end of the handle is connected with the annular wall sleeve.

[0019] The annular wall sleeve is connected to the measuring cell by clamping, so that different types of measuring cells can be easily replaced during the detection process.

[0020] Preferably, the measuring pool is a rectangular parallelepiped structure, and an overflow port and an overflow groove are provided on the first side surface of the measuring pool, the overflow port and the overflow groove are connected, and the length of the overflow groove is not less than the distance between the first liquid inlet and the third liquid inlet on the upper surface of the measuring chamber;

[0021] The second side surface of the measuring cell is arranged parallel to the light source;

[0022] The first side surface and the second side surface of the measuring cell are adjacent to each other.

[0023] It can be understood that the measuring pool is a rectangular parallelepiped structure, with four rough surfaces on the upper and lower front and back sides and two smooth surfaces on the left and right sides; an overflow port and an overflow groove are arranged on the rough surface, the overflow port and the overflow groove are connected, and the opening of the overflow port is larger than the distance between the first liquid inlet and the third liquid inlet, so as to ensure that the microbial liquid, diluent or cleaning liquid can enter the measuring pool; the handle is connected to the annular wall sleeve, the annular wall sleeve is clamped with the measuring pool, the handle can be rotated in the vertical direction, and the rotation process drives the measuring pool to flip; when the handle is perpendicular to the horizontal plane, it indicates that the opening of the measuring pool is parallel to the horizontal plane; when the handle is parallel to the horizontal plane, it indicates that the opening of the measuring pool is perpendicular to the horizontal plane; the light source is parallel to the smooth surface and is located on the same horizontal plane, and the type and wavelength of the emitted light are set according to the absorption wavelength of the microbial liquid to be detected. The window is arranged directly above the measuring pool to facilitate the observation of the growth of microorganisms in the measuring pool.

[0024] Preferably, the filtration unit comprises a regulating valve, a filtration assembly and a first injection pump which are connected in sequence;

[0025] The liquid inlet end of the regulating valve is connected to the liquid outlet end of the microorganism enrichment unit, and the liquid outlet end of the first injection pump is connected to the first liquid inlet of the measuring chamber;

[0026] The control end of the first injection pump is connected to the control end of the central control unit.

[0027] The regulating valve is used to control the outflow of the microbial solution, the filtering component filters the large particle impurities in the microbial solution, and the first injection pump is used to control the flow rate of the microbial solution.

[0028] Preferably, the filter assembly comprises a filter frame and a filter net, and the filter net is covered on the surface of the filter frame.

[0029] Preferably, the microorganism enrichment unit comprises a microorganism enrichment kettle, a magnetic stirrer, a vent valve, a liquid inlet valve, a pressure sensor, a temperature sensor, a water bath, an air inlet valve and a second liquid discharge valve;

[0030] The side wall of the microorganism enrichment kettle is provided with a liquid outlet, and the liquid outlet serves as the liquid outlet of the microorganism enrichment unit and is connected to the liquid inlet of the regulating valve;

[0031] The microorganism enrichment kettle is arranged in a water bath, a magnetic stirrer is arranged on the lower surface of the microorganism enrichment kettle, and the control ends of the water bath and the magnetic stirrer are connected to the control end of the central control unit;

[0032] One end of the vent valve is connected to the upper surface of the microorganism enrichment kettle, the other end of the vent valve is suspended in the air, and the control end of the vent valve is connected to the control end of the central control unit;

[0033] One end of the air inlet valve is connected to the upper surface of the microorganism enrichment kettle, the other end of the air inlet valve is connected to the air outlet end of the pressure control unit as the air inlet end of the microorganism enrichment unit, and the control end of the air inlet valve is connected to the control end of the central control unit;

[0034] One end of the liquid inlet valve is connected to the side wall of the microorganism enrichment kettle, the other end of the liquid inlet valve is connected to the liquid outlet of the multi-sequence nutrient solution supply unit as the liquid inlet end of the microorganism enrichment unit, and the control end of the liquid inlet valve is connected to the control end of the central control unit;

[0035] One end of the second liquid discharge valve is connected to the side wall of the microorganism enrichment kettle, and the other end of the second liquid discharge valve is suspended in the air;

[0036] The temperature sensor and the pressure sensor are both arranged on the upper surface of the microorganism enrichment kettle, and the data output ends of the temperature sensor and the pressure sensor are both connected to the data input end of the central control unit.

[0037] The upper surface of the microbial enrichment kettle is detachable, which is convenient for sterilization and placement of substrates to be cultured; a magnetic stirrer is set on the lower surface to enhance the mass transfer process in the microbial enrichment kettle and increase the energy and nutrient utilization efficiency of the microbial culture. The pressure sensor and temperature sensor are used to monitor the pressure and temperature changes in the microbial enrichment kettle in real time, the vent valve is used to reduce the real-time pressure in the microbial enrichment kettle, and the water bath is used to maintain the constant temperature in the microbial enrichment kettle. The air inlet valve and liquid inlet valve are used to control the entry of culture gas or inert gas and nutrient solution respectively, so that the microbial culture is always in the best growth environment.

[0038] Preferably, the pressure control unit comprises a booster pump, an air compressor, an air storage tank and a pressure regulating valve connected in sequence;

[0039] The other end of the pressure regulating valve is connected to the other end of the air inlet valve as the air outlet of the pressure control unit, and the other end of the pressure regulating valve is also connected to the air inlet of the measuring chamber; the control end of the booster pump, the air compressor, and the pressure regulating valve is connected to the control end of the central control unit;

[0040] The gas storage tank is filled with culture gas or inert gas.

[0041] Preferably, the multi-sequence nutrient solution supply unit comprises a plurality of nutrient bottles, a plurality of liquid outlet valves and a second liquid injection pump;

[0042] The liquid outlet of each nutrient bottle is connected to one end of a liquid outlet valve, and the other end of each liquid outlet valve is connected to one end of a second liquid injection pump. The other end of the second liquid injection pump serves as the liquid outlet end of the multi-sequence nutrient solution supply unit and is connected to the other end of the liquid inlet valve; the control end of each liquid outlet valve and the control end of the second liquid injection pump are connected to the control end of the central control unit;

[0043] A nutrient solution is arranged in each nutrient bottle.

[0044] The present invention also provides an enrichment and cultivation method for microorganisms under high pressure environment with online regulation, based on the above-mentioned device, comprising:

[0045] S1: sterilizing the microorganism enrichment unit, the online detection unit, and the filtration unit; adding the substrate to be cultured into the microorganism enrichment kettle, adding the culture gas or inert gas into the gas storage tank, and adding the nutrient solution into the nutrient bottle;

[0046] S2: Control the real-time temperature of the water bath; control the opening of the air inlet valve, adjust the real-time pressure of the culture gas or inert gas output by the pressure control unit, and input it into the microorganism enrichment kettle; control the opening of the liquid inlet valve, select the nutrient solution, control the opening of the corresponding liquid outlet valve and the second liquid injection pump, input it into the microorganism enrichment kettle, and form a microbial bacterial liquid;

[0047] S3: inject the substrate to be cultured into the measuring pool as a blank sample, control the light source to turn on, generate light of the first preset wavelength, and use spectrophotometry to perform zero adjustment; after the zero adjustment is completed, turn off the light source, rotate the handle clockwise, pour the substrate to be cultured in the measuring pool into the waste liquid pool, and rotate the handle counterclockwise;

[0048] S4: controlling the magnetic stirrer to stir the microbial solution, and after a first preset time, controlling the regulating valve and the first injection pump to open, adjusting the flow rate of the first injection pump, filtering the microbial solution through the filter assembly to remove large particles of impurities, obtaining the filtered microbial solution, and injecting it into the measuring pool;

[0049] S5: After the second preset time has passed, the regulating valve and the first injection pump are closed, the diluent container is controlled to open, and the diluent enters the measuring cell to obtain a test solution;

[0050] S6: Control the light source to turn on, generate light of a second preset wavelength, detect the test solution, obtain the transmittance value and transmit it to the central control unit;

[0051] S7: Rotate the handle clockwise to pour the test solution in the measuring cell into the waste liquid tank, and rotate the handle counterclockwise;

[0052] S8: Control the cleaning liquid container to open, and the cleaning liquid enters the measuring tank until it flows out from the overflow port and flows into the waste liquid tank through the overflow tank;

[0053] S9: After the third preset time, rotate the handle clockwise to pour the cleaning liquid in the measuring tank into the waste liquid tank, and rotate the handle counterclockwise;

[0054] S10: Repeat steps S8-S9 until a preset number of times is reached;

[0055] S11: Repeat steps S4-S10 at intervals of a fourth preset time until a fifth preset time is reached;

[0056] S12: The central control unit calculates the cell data and environmental parameters of the microbial liquid at different times according to the permeability value, and calculates the replenishment amount of the nutrient solution; controls the second liquid discharge valve and the pressure control unit to open, and discharges the waste liquid in the microbial enrichment kettle; after the preset volume of waste liquid is discharged, controls the liquid inlet valve to open, and inputs the replenishment amount of nutrient solution into the microbial enrichment kettle.

[0057] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0058] The present invention sets a microorganism enrichment unit for microorganism enrichment culture, uses a pressure control unit to provide the microorganism enrichment unit with culture gas required for microorganism culture, or fills in inert gas to keep the pressure environment in the microorganism enrichment unit consistent with the marine in-situ pressure environment, and uses a multi-sequence nutrient solution supply unit to provide the microorganism enrichment unit with the nutrient solution required for microorganism culture; after a period of enrichment culture, the microorganism liquid enters the online detection unit after filtering out large particle impurities through a filter component, and at the same time, the pressure control unit provides inert gas to the online detection unit, so that the pressure environment in the online detection unit is consistent with the marine in-situ pressure environment, and the cell data and environmental parameters of the enriched cultured microorganism liquid are detected under the marine in-situ pressure environment, and the supplementary amount of the nutrient solution is calculated according to the cell data and environmental parameters of the microorganism liquid, so that the microorganism is always in the best enrichment culture environment. The present invention is consistent with the marine in-situ pressure environment during enrichment culture and detection, and no pressure release is required during the detection process, the detection result fits the actual situation, the microorganism is in the best enrichment culture environment, and the efficiency of the enrichment culture of the microorganism is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a schematic diagram of the structure of an enrichment and cultivation device for online regulation of microorganisms under a high pressure environment as described in Example 1;

[0060] Figure 2 This is a schematic diagram of the structure of an enrichment and cultivation device for online regulation of microorganisms under a high pressure environment as described in Example 2;

[0061] Figure 3 This is a schematic diagram of the structure of the measuring cell described in Example 2;

[0062] Figure 4 This is a flow chart of a method for enrichment and cultivation of microorganisms under online regulation in a high pressure environment as described in Example 2;

[0063] In the figure, 1-microorganism enrichment unit, 2-online detection unit, 3-filtration unit, 4-pressure control unit, 5-multi-sequence nutrient solution supply unit, 6-central control unit, 11-microorganism enrichment kettle, 12-magnetic stirrer, 13-vent valve, 14-liquid inlet valve, 15-pressure sensor, 16-temperature sensor, 17-water bath, 18-water bath, 19-second liquid discharge valve, 20-annular wall sleeve, 21-measuring chamber, 2 2-measuring pool, 23-light source, 24-waste liquid pool, 25-window, 26-handle, 27-cleaning liquid container, 28-dilution liquid container, 29-first liquid discharge valve, 31-regulating valve, 32-filtering assembly, 33-first liquid injection pump, 41-boosting pump, 42-air compressor, 43-gas storage tank, 44-pressure regulating valve, 51-nutrient bottle, 52-liquid outlet valve, 53-second liquid injection pump, 221-overflow port, 222-overflow trough. DETAILED DESCRIPTION

[0064] The drawings are for illustrative purposes only and should not be construed as limiting the present patent;

[0065] In order to better illustrate the present embodiment, some parts in the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product;

[0066] It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0067] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0068] Example 1

[0069] This embodiment provides an enrichment and cultivation device for microorganisms under high pressure environment and online regulation, such as Figure 1 As shown, it includes: a microorganism enrichment unit 1, an online detection unit 2, a filtration unit 3, a pressure control unit 4, a multi-sequence nutrient solution supply unit 5 and a central control unit 6;

[0070] The air inlet end of the microorganism enrichment unit 1 is connected to the air outlet end of the pressure control unit 4, and the liquid inlet end of the microorganism enrichment unit 1 is connected to the liquid outlet end of the multi-sequence nutrient solution supply unit 5;

[0071] The liquid inlet end of the filtration unit 3 is connected to the liquid outlet end of the microorganism enrichment unit 1, the liquid outlet end of the filtration unit 3 is connected to the liquid inlet end of the online detection unit 2, and the air inlet end of the online detection unit 2 is connected to the air outlet end of the pressure control unit 4;

[0072] The data output end of the microorganism enrichment unit 1 is connected to the data input end of the central control unit 6, and the control ends of the microorganism enrichment unit 1, the online detection unit 2, the pressure control unit 4, and the multi-sequence nutrient solution supply unit 5 are connected to the control end of the central control unit 6.

[0073] In the specific implementation process, this implementation sets a microbial enrichment unit for microbial enrichment culture, uses a pressure control unit to provide the microbial enrichment unit with the culture gas required for microbial culture, or fills in inert gas to keep the pressure environment in the microbial enrichment unit consistent with the marine in-situ pressure environment, and uses a multi-sequence nutrient solution supply unit to provide the microbial enrichment unit with the nutrient solution required for microbial culture; after a period of enrichment culture, the microbial liquid enters the online detection unit after filtering out large particle impurities through the filter component, and at the same time, the pressure control unit provides inert gas to the online detection unit, so that the pressure environment in the online detection unit is consistent with the marine in-situ pressure environment, and the cell data and environmental parameters of the enriched cultured microbial liquid are detected under the marine in-situ pressure environment, and the amount of nutrient solution supplemented is calculated based on the cell data and environmental parameters of the microbial liquid, so that the microorganisms are always in the best enrichment culture environment. This embodiment is consistent with the marine in-situ pressure environment during enrichment culture and detection, and there is no need to release pressure during the detection process. The detection results fit the actual situation, and the microorganisms are always in the best enrichment culture environment, which improves the efficiency of microbial enrichment culture.

[0074] Example 2

[0075] This embodiment provides an enrichment and cultivation device for microorganisms under high pressure environment and online regulation, such as Figure 2 As shown, it includes: a microorganism enrichment unit 1, an online detection unit 2, a filtration unit 3, a pressure control unit 4, a multi-sequence nutrient solution supply unit 5 and a central control unit 6;

[0076] The air inlet end of the microorganism enrichment unit 1 is connected to the air outlet end of the pressure control unit 4, and the liquid inlet end of the microorganism enrichment unit 1 is connected to the liquid outlet end of the multi-sequence nutrient solution supply unit 5;

[0077] The liquid inlet end of the filtration unit 3 is connected to the liquid outlet end of the microorganism enrichment unit 1, the liquid outlet end of the filtration unit 3 is connected to the liquid inlet end of the online detection unit 2, and the air inlet end of the online detection unit 2 is connected to the air outlet end of the pressure control unit 4;

[0078] The online detection unit 2 includes a measuring chamber 21, a measuring pool 22, a light source 23, a waste liquid pool 24, a viewing window 25, a handle 26, a cleaning liquid container 27, a diluent container 28, a first drain valve 29 and an annular wall sleeve 20;

[0079] The upper surface of the measuring chamber 21 is provided with a first liquid inlet, a second liquid inlet, a third liquid inlet and an air inlet, the liquid outlet of the filter unit 3 is connected to the first liquid inlet, the liquid outlet of the cleaning liquid container 27 is connected to the second liquid inlet, the liquid outlet of the diluent container 28 is connected to the third liquid inlet, and the air outlet of the pressure control unit 4 is connected to the air inlet;

[0080] The light source 23 is arranged on the side surface of the measuring chamber 21, parallel to the measuring pool 22; the window 25 is arranged on the upper surface of the measuring chamber 21, corresponding to the position of the measuring pool 22; the control end of the control end of the light source 23 is connected to the control end of the central control unit 6;

[0081] The waste liquid pool 24 is arranged in the measuring chamber 21, and the measuring chamber 22 is arranged in the waste liquid pool 24; one end of the handle 26 is connected to the annular wall sleeve 20, and the other end of the handle 26 sequentially passes through the waste liquid pool 24 and the measuring chamber 21 and extends out; one end of the first liquid discharge valve 29 is connected to the side surface of the waste liquid pool 24, and the other end of the first liquid discharge valve 29 passes through the measuring chamber 21 and extends out;

[0082] The diluent container 28 is provided with a reaction solution, and the cleaning liquid container 27 is provided with a cleaning liquid;

[0083] The annular wall sleeve 20 is clamped with the measuring cell 22;

[0084] like Figure 3 As shown, the measuring pool 22 is a rectangular parallelepiped structure, and an overflow port 221 and an overflow groove 222 are provided on the first side surface of the measuring pool 22, the overflow port 221 and the overflow groove 222 are connected, and the length of the overflow groove 222 is not less than the distance between the first liquid inlet and the third liquid inlet on the upper surface of the measuring chamber 21;

[0085] The second side surface of the measuring cell 22 is arranged parallel to the light source 23;

[0086] The first side surface and the second side surface of the measuring cell 22 are adjacent to each other.

[0087] It should be noted that the first liquid inlet, the second liquid inlet and the third liquid inlet provided on the upper surface of the measuring chamber 21 are connected to the filter unit, the cleaning liquid container 27 and the diluent container 28 respectively; during the first detection, the sterilized substrate to be cultured is injected into the measuring pool 22 as a blank sample, the light source 23 is turned on, and the spectrophotometry method is used to perform zero adjustment to eliminate background interference; after the zero adjustment is completed, the light source 23 is turned off, the handle 26 is rotated clockwise, the substrate to be cultured in the measuring pool 22 is poured into the waste liquid pool 24, and the handle 26 is rotated counterclockwise. , inject the filtered microbial liquid from the first liquid inlet; after reaching the preset volume, inject the diluent from the diluent container 28 through the second liquid inlet to obtain the test solution; turn on the light source 23 again, test the test solution, obtain the transmittance value and send it to the central control unit; after the test is completed, rotate the handle 26 clockwise again to pour the test solution in the measuring cell 22 into the waste liquid tank 24, and rotate the handle 26 counterclockwise; inject the cleaning liquid from the cleaning liquid container 27 through the third liquid inlet to clean the measuring cell 22 several times. After cleaning, perform the above process at intervals to perform long-term enrichment culture and detection on the substrate to be cultured, and also update the substrate to be cultured to achieve enrichment culture and detection of other substrates to be cultured;

[0088] It can be understood that the measuring pool 22 is a rectangular parallelepiped structure, with four rough surfaces on the upper and lower front and back sides and two smooth surfaces on the left and right sides; an overflow port 221 and an overflow groove 222 are arranged on the rough surface, the overflow port 221 and the overflow groove 222 are connected, and the opening of the overflow port 221 is larger than the distance between the first liquid inlet and the third liquid inlet, so as to ensure that the microbial liquid, diluent or cleaning liquid can enter the measuring pool 22; the handle 26 is connected to the annular wall sleeve 20, the annular wall sleeve 20 is clamped with the measuring pool 22, the handle 26 can rotate in the vertical direction, and the rotation process drives the measuring pool 22 to flip; when the handle 26 is perpendicular to the horizontal plane, it indicates that the opening of the measuring pool 22 is parallel to the horizontal plane; when the handle 26 is parallel to the horizontal plane, it indicates that the opening of the measuring pool 22 is perpendicular to the horizontal plane; the light source 23 is parallel to the smooth surface and is located on the same horizontal plane, and the type and wavelength of the emitted light are set according to the absorption wavelength of the microbial liquid to be detected. The window 25 is arranged directly above the measuring pool 22, so as to facilitate the observation of the growth of microorganisms in the measuring pool 22.

[0089] The filtering unit 3 comprises a regulating valve 31, a filtering assembly 32 and a first injection pump 33 which are connected in sequence;

[0090] The liquid inlet end of the regulating valve 31 is connected to the liquid outlet end of the microorganism enrichment unit 1, and the liquid outlet end of the first injection pump 33 is connected to the first liquid inlet of the measuring chamber 21;

[0091] The filter assembly 32 includes a filter frame and a filter net, and the filter frame is covered with the filter net;

[0092] The control end of the first injection pump 33 is connected to the control end of the central control unit 6;

[0093] It can be understood that the regulating valve 31 is used to control the outflow of the microbial liquid, the filtering component 32 filters the large particles of impurities in the microbial liquid, and the first injection pump 33 is used to control the flow rate of the microbial liquid.

[0094] The microorganism enrichment unit 1 includes a microorganism enrichment kettle 11, a magnetic stirrer 12, a vent valve 13, a liquid inlet valve 14, a pressure sensor 15, a temperature sensor 16, a water bath 17, an air inlet valve 18 and a second liquid discharge valve 19;

[0095] The side wall of the microorganism enrichment kettle 11 is provided with a liquid outlet, and the liquid outlet serves as the liquid outlet of the microorganism enrichment unit 1 and is connected to the liquid inlet of the regulating valve 31;

[0096] The microorganism enrichment kettle 11 is arranged in a water bath 17, a magnetic stirrer 12 is arranged on the lower surface of the microorganism enrichment kettle 11, and the control end of the water bath 17 and the magnetic stirrer 12 is connected to the control end of the central control unit 6;

[0097] One end of the vent valve 13 is connected to the upper surface of the microorganism enrichment kettle 11, and the other end of the vent valve 13 is suspended in the air. The control end of the vent valve 13 is connected to the control end of the central control unit 6;

[0098] One end of the air inlet valve 18 is connected to the upper surface of the microorganism enrichment kettle 11, and the other end of the air inlet valve 18 is connected to the air outlet end of the pressure control unit 4 as the air inlet end of the microorganism enrichment unit 1, and the control end of the air inlet valve 18 is connected to the control end of the central control unit 6;

[0099] One end of the liquid inlet valve 14 is connected to the side wall of the microorganism enrichment kettle 11, and the other end of the liquid inlet valve 14 is connected to the liquid outlet of the multi-sequence nutrient solution supply unit 5 as the liquid inlet end of the microorganism enrichment unit 1, and the control end of the liquid inlet valve 14 is connected to the control end of the central control unit 6;

[0100] One end of the second liquid discharge valve 19 is connected to the side wall of the microorganism enrichment kettle 11, and the other end of the second liquid discharge valve 19 is suspended in the air;

[0101] The temperature sensor 16 and the pressure sensor 15 are both arranged on the upper surface of the microorganism enrichment kettle 11 , and the data output ends of the temperature sensor 16 and the pressure sensor 15 are both connected to the data input end of the central control unit 6 .

[0102] It is understandable that the upper surface of the microbial enrichment kettle is detachable, which is convenient for sterilization and placement of substrates to be cultured; a magnetic stirrer is set on the lower surface to enhance the mass transfer process in the microbial enrichment kettle and increase the energy and nutrient utilization efficiency of the microbial culture. The pressure sensor and temperature sensor are used to monitor the pressure and temperature changes in the microbial enrichment kettle in real time, the vent valve is used to reduce the real-time pressure in the microbial enrichment kettle, and the water bath is used to maintain the constant temperature state in the microbial enrichment kettle. The air inlet valve and the liquid inlet valve are used to control the entry of culture gas or inert gas and nutrient solution respectively, so that the microbial culture is always in the best growth environment.

[0103] The pressure control unit 4 includes a booster pump 41, an air compressor 42, an air storage tank 43 and a pressure regulating valve 44 connected in sequence;

[0104] The other end of the pressure regulating valve 44 is connected to the other end of the air inlet valve 18 as the air outlet of the pressure control unit 4, and the other end of the pressure regulating valve 44 is also connected to the air inlet of the measuring chamber 21; the control ends of the booster pump 41, the air compressor 42, and the pressure regulating valve 44 are connected to the control end of the central control unit 6;

[0105] The gas storage tank 43 is filled with culture gas or inert gas.

[0106] The multi-sequence nutrient solution supply unit 5 includes a plurality of nutrient bottles 51, a plurality of liquid outlet valves 52 and a second liquid injection pump 53;

[0107] The liquid outlet of each nutrient bottle 51 is connected to one end of a liquid outlet valve 52, and the other end of each liquid outlet valve 52 is connected to one end of a second liquid injection pump 53. The other end of the second liquid injection pump 53 serves as the liquid outlet end of the multi-sequence nutrient solution supply unit 5 and is connected to the other end of the liquid inlet valve 14. The control end of each liquid outlet valve 52 and the control end of the second liquid injection pump 53 are connected to the control end of the central control unit 6.

[0108] Each of the nutrient bottles 51 is provided with a nutrient solution.

[0109] Example 3

[0110] This embodiment provides a method for online regulation of enrichment and cultivation of microorganisms under high pressure environment, based on the device described in embodiment 1 or 2, such as Figure 4 As shown, including:

[0111] S1: sterilizing the microorganism enrichment unit, the online detection unit, and the filtration unit; adding the substrate to be cultured into the microorganism enrichment kettle, adding the culture gas or inert gas into the gas storage tank, and adding the nutrient solution into the nutrient bottle;

[0112] S2: Control the real-time temperature of the water bath; control the opening of the air inlet valve, adjust the real-time pressure of the culture gas or inert gas output by the pressure control unit, and input it into the microorganism enrichment kettle; control the opening of the liquid inlet valve, select the nutrient solution, control the opening of the corresponding liquid outlet valve and the second liquid injection pump, input it into the microorganism enrichment kettle, and form a microbial bacterial liquid;

[0113] S3: inject the substrate to be cultured into the measuring pool as a blank sample, control the light source to turn on, generate light of the first preset wavelength, and use spectrophotometry to perform zero adjustment; after the zero adjustment is completed, turn off the light source, rotate the handle clockwise, pour the substrate to be cultured in the measuring pool into the waste liquid pool, and rotate the handle counterclockwise;

[0114] S4: controlling the magnetic stirrer to stir the microbial solution, and after a first preset time, controlling the regulating valve and the first injection pump to open, adjusting the flow rate of the first injection pump, filtering the microbial solution through the filter assembly to remove large particles of impurities, obtaining the filtered microbial solution, and injecting it into the measuring pool;

[0115] S5: After the second preset time has passed, the regulating valve and the first injection pump are closed, the diluent container is controlled to open, and the diluent enters the measuring cell to obtain a test solution;

[0116] S6: Control the light to turn on, generate light of a second preset wavelength, detect the test solution, obtain the transmittance value and transmit it to the central control unit;

[0117] S7: Rotate the handle clockwise to pour the test solution in the measuring cell into the waste liquid tank, and rotate the handle counterclockwise;

[0118] S8: Control the cleaning liquid container to open, and the cleaning liquid enters the measuring tank until it flows out from the overflow port and flows into the waste liquid tank through the overflow tank;

[0119] S9: After the third preset time, rotate the handle clockwise to pour the cleaning liquid in the measuring tank into the waste liquid tank, and rotate the handle counterclockwise;

[0120] S10: Repeat steps S8-S9 until a preset number of times is reached;

[0121] S11: Repeat steps S4-S10 at intervals of a fourth preset time until a fifth preset time is reached;

[0122] S12: The central control unit calculates the cell data and environmental parameters of the microbial liquid at different times according to the permeability value, and calculates the replenishment amount of the nutrient solution; controls the second liquid discharge valve and the pressure control unit to open, and discharges the waste liquid in the microbial enrichment kettle; after the preset volume of waste liquid is discharged, controls the liquid inlet valve to open, and inputs the replenishment amount of nutrient solution into the microbial enrichment kettle.

[0123] In this embodiment, the light source 23 is an infrared light source or an ultraviolet light source, which is determined according to the absorption wavelength of the measurement solution. In the detection process of determining the cell data of the microbial liquid, the light source 23 generates a wavelength of 600nm and is zeroed; then the magnetic stirrer is controlled to stir the microbial liquid for a period of time, and the regulating valve 31 and the first injection pump 33 are controlled to be turned on, and the flow rate v and the second preset time t of the first injection pump 33 are set, thereby controlling the volume V mL of the filtered microbial liquid entering the measuring pool 22; the diluent container 28 is controlled to be opened, and the volume of the diluent entering the measuring pool 22 is X times the volume of the microbial liquid, then the volume of the detection solution is V+XV mL, and the volume after the reaction reaches about 3 / 4 of the volume of the measuring pool 22; the light source 23 is turned on again to generate a wavelength of 600nm to detect the detection solution by spectrophotometry, and the transmittance value T is obtained, and the OD600 value A of the microbial liquid is converted according to the formula A=log101 / T, and the actual OD600 value of the detection solution is XA; the OD600 value is between 0.1 and 1.5, indicating that the test result is reliable. After the detection is completed, turn off the light source 23, rotate the handle 26 clockwise to pour all the solution in the measuring cell 22 into the waste liquid tank 24, and then rotate the handle 26 counterclockwise to make the opening of the measuring cell 22 parallel to the horizontal plane. Then inject cleaning liquid into the measuring cell 22 through the cleaning unit. When too much cleaning liquid is injected, the cleaning liquid flows out through the overflow port 221 and flows into the waste liquid tank through the overflow tank 222, and will not pollute the light surface of the measuring cell 22. Then, select the handle 26 clockwise again to pour all the solution in the measuring cell 22 into the waste liquid tank 24. Repeat the above cleaning process several times and start the next test.

[0124] Spectrophotometry can also be used to detect changes in metal ions during the culture process. 2+ , Fe 3+ According to the test principle, when the pH is in the range of 1.5 to 9.5, Fe 2+ It can react with o-phenanthroline to form a stable orange complex, which has a maximum absorption peak at a wavelength of 510nm. Based on this principle, the concentration of ferrous iron in the solution can be measured by colorimetry. 3+ All reduced to Fe 2 + , and then the concentration of total iron ions can be determined by reacting with o-phenanthroline. The operation steps are as follows:

[0125] a. Reagent preparation

[0126] Iron salt stock solution: Accurately weigh 0.863 g of NH4FeSO 4212H2O, placed in a 200mL beaker, add 100mL distilled water and 10mL sulfuric acid and stir thoroughly to dissolve, then transfer to a 1L volumetric flask, add distilled water to the mark to obtain a 100μg / L iron ion solution;

[0127] 0.2% o-phenanthroline solution: Store in a 4°C refrigerator away from light;

[0128] Acetic acid-sodium acetate buffer pH = 4.5: 9 g sodium acetate, 4.9 mL glacial acetic acid, add water to make up to 500 mL;

[0129] 2% ascorbic acid solution: Prepare and store in a 4°C refrigerator away from light. pH = 2.0 of distilled water is adjusted with dilute hydrochloric acid.

[0130] b. Establishment of standard curve

[0131] Dilute the iron salt stock solution by half to obtain a concentration of 50 μg / L Fe 3+ Standard solution, take 0, 50μL, 100μL, 150μL, 200μL, 250μL, 300μL, 350μL and 400μL of Fe 3+ The standard solution was placed in a 10 mL test tube, and 500 μL of acetic acid-sodium acetate buffer, 250 μL of ascorbic acid solution and 250 μL of o-phenanthroline solution were added. The solution was made up to 5 mL with water at pH 2.0, mixed thoroughly and allowed to stand for 10 min.

[0132] Remove the diluent bottle and clean it. Then take the mixed solution and add it to the measuring cell 22. At this time, the central control system needs to determine the flow rate v mL / min and the time tmin of the first injection pump 33, and then determine the specific amount of solution VmL added. The calculation formula is v×t=V. The final reaction solution reaches about 3 / 4 of the volume of the measuring cell 22. Finally, turn on the wavelength of 510nm, use spectrophotometry to detect the solution, and get the test value. After the test, turn off the light source 23, rotate the handle 26 clockwise to pour all the solution in the measuring cell 22 into the waste liquid tank 24, and then rotate the handle 26 counterclockwise to make the opening of the measuring cell 22 parallel to the horizontal plane. Then inject cleaning liquid into the measuring cell 22 through the cleaning unit. When too much cleaning liquid is injected, the cleaning liquid flows out through the overflow port 221 and flows into the waste liquid tank through the overflow tank 222, and will not pollute the light surface of the measuring cell 22. Then select the handle 26 clockwise again to pour all the solution in the measuring cell 22 into the waste liquid tank 24. Repeat the above method to measure other standard solutions. Finally, take the absorbance as the vertical coordinate, Fe 3+ The concentration is the horizontal axis, and Fe 3+ Standard curve, when the standardization coefficient R 2>0.9, you can start to measure the iron ion content of the reaction solution, otherwise re-establish the standard curve.

[0133] c. Total Fe and Fe in culture medium 2+ , Fe 3+ Determination

[0134] Total Fe concentration: First, open the regulating valve 31 to allow the bacterial solution in the microbial enrichment unit to automatically enter the filtration unit. The solution filtered by the filtration unit enters the first injection pump 33, and the filtered bacterial solution is injected into the measuring pool 22 through the injection pump. At this time, the flow rate v mL / min and the time t min of the first injection pump 33 need to be determined through the central control system, and then the specific amount of solution added V mL is determined, and the calculation formula is v×t=V. Then, the mixed solution 10VμL acetic acid-sodium acetate buffer, 50VμL ascorbic acid solution and 50VμL o-phenanthroline solution are injected into the measuring pool 22 through the dilution unit, and the pH=2.0 water is added to 0.5V mL, and it is left to stand for 10 minutes. The absorbance is measured by spectrophotometry and the total Fe concentration of the sample is obtained through the standard curve. After the detection is completed, turn off the light source 23, rotate the handle 26 clockwise to pour all the solution in the measuring cell 22 into the waste liquid tank 24, and then rotate the handle 26 counterclockwise to make the opening of the measuring cell 22 parallel to the horizontal plane, and inject cleaning liquid into the measuring cell 22 through the cleaning unit. When too much cleaning liquid is injected, the cleaning liquid flows out through the overflow port 221 and flows into the waste liquid tank through the overflow tank 222, and will not pollute the light surface of the measuring cell 22. Then, select the handle 26 clockwise again to pour all the solution in the measuring cell 22 into the waste liquid tank 24, repeat the above cleaning process in sequence, and then start the next solution test.

[0135] Fe 2+ Concentration: First, open the regulating valve 31 to allow the bacterial solution in the microbial enrichment unit to automatically enter the filtration unit. The solution filtered by the filtration module enters the first injection pump 33, and the filtered bacterial solution is injected into the measuring pool 22 through the injection pump. At this time, the flow rate v mL / min and the time t min of the first injection pump 33 need to be determined through the central control system, and then the specific amount of solution added V mL is determined, and the calculation formula is v×t=V. Then, 10VμL of the mixed solution (acetic acid-sodium acetate buffer, 50VμL o-phenanthroline solution) is injected into the measuring pool 22 through the dilution unit, and pH=2.0 water is added to 0.5V mL, and it is left to stand for 10 minutes. The absorbance is measured by spectrophotometry and the sample Fe is obtained through the standard curve. 2+Concentration. After the test is completed, turn off the light source 23, rotate the handle 26 clockwise to pour all the solution in the measuring cell 22 into the waste liquid tank 24, and then rotate the handle 26 counterclockwise to make the opening of the measuring cell 22 parallel to the horizontal plane, and inject the cleaning liquid into the measuring cell 22 through the cleaning unit. When too much cleaning liquid is injected, the cleaning liquid flows out through the overflow port 221 and flows into the waste liquid tank through the overflow tank 222, and will not pollute the light surface of the measuring cell 22. Then, select the handle 26 clockwise again to pour all the solution in the measuring cell 22 into the waste liquid tank 24, repeat the above cleaning process in sequence, and then start the next solution test.

[0136] Fe 3+ Concentration: Subtract the sample Fe from the total Fe concentration obtained. 2+ The concentration is Fe 3+ concentration.

[0137] Other metal ions or salts can be tested according to the above operation method. During the experiment, according to the difference of the test substances, appropriate standard substances and reaction substances are selected. After the test is completed, the switch of the waste liquid pool (24) is turned on to discharge the waste liquid through the drainage pipe. The pressure difference generated during the waste liquid discharge process is compensated by the pressure control unit.

[0138] The same or similar reference numerals correspond to the same or similar components;

[0139] The terms used in the drawings to describe positional relationships are only used for illustrative purposes and should not be construed as limiting this patent;

[0140] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A microbial enrichment and cultivation device for online regulation under high pressure environment, characterized in that: include: A microorganism enrichment unit (1), an online detection unit (2), a filtration unit (3), a pressure control unit (4), a multi-sequence nutrient solution supply unit (5) and a central control unit (6); The air inlet end of the microorganism enrichment unit (1) is connected to the air outlet end of the pressure control unit (4), and the liquid inlet end of the microorganism enrichment unit (1) is connected to the liquid outlet end of the multi-sequence nutrient solution supply unit (5); The liquid inlet end of the filtration unit (3) is connected to the liquid outlet end of the microorganism enrichment unit (1), the liquid outlet end of the filtration unit (3) is connected to the liquid inlet end of the online detection unit (2), and the air inlet end of the online detection unit (2) is connected to the air outlet end of the pressure control unit (4); The data output end of the microorganism enrichment unit (1) is connected to the data input end of the central control unit (6), and the control ends of the microorganism enrichment unit (1), the online detection unit (2), the pressure control unit (4), and the multi-sequence nutrient solution supply unit (5) are connected to the control end of the central control unit (6).

2. The enrichment and cultivation device for online regulation of microorganisms under high pressure environment according to claim 1, characterized in that: The online detection unit (2) comprises a measuring chamber (21), a measuring pool (22), a light source (23), a waste liquid pool (24), a viewing window (25), a handle (26), a cleaning liquid container (27), a diluent container (28) and a first liquid discharge valve (29); The upper surface of the measuring chamber (21) is provided with a first liquid inlet, a second liquid inlet, a third liquid inlet and an air inlet, the liquid outlet of the filter unit (3) is connected to the first liquid inlet, the liquid outlet of the cleaning liquid container (27) is connected to the second liquid inlet, the liquid outlet of the diluent container (28) is connected to the third liquid inlet, and the air outlet of the pressure control unit (4) is connected to the air inlet; The light source (23) is arranged on the side surface of the measuring chamber (21) and is parallel to the measuring pool (22); the window (25) is arranged on the upper surface of the measuring chamber (21) and corresponds to the position of the measuring pool (22); the control end of the control end of the light source (23) is connected to the control end of the central control unit (6); The waste liquid pool (24) is arranged in the measuring chamber (21), and the measuring chamber (22) is arranged in the waste liquid pool (24); one end of the handle (26) is connected to the side surface of the measuring chamber (22), and the other end of the handle (26) sequentially passes through the waste liquid pool (24) and the measuring chamber (21) to extend out; one end of the first liquid discharge valve (29) is connected to the side surface of the waste liquid pool (24), and the other end of the first liquid discharge valve (29) passes through the measuring chamber (21) to extend out; The diluent container (28) contains a reaction solution, and the cleaning liquid container (27) contains a cleaning liquid.

3. The enrichment and cultivation device for online regulation of microorganisms under high pressure environment according to claim 2, characterized in that: The online detection unit (2) further comprises an annular wall sleeve (20); The annular wall sleeve (20) is clamped with the measuring cell (22), and one end of the handle (26) is connected to the annular wall sleeve (20).

4. The enrichment and cultivation device for online regulation of microorganisms under high pressure environment according to claim 3, characterized in that: The measuring pool (22) is a rectangular parallelepiped structure, and an overflow port (221) and an overflow groove (222) are provided on a first side surface of the measuring pool (22), the overflow port (221) and the overflow groove (222) are connected, and the length of the overflow groove (222) is not less than the distance between the first liquid inlet and the third liquid inlet on the upper surface of the measuring chamber (21); The second side surface of the measuring cell (22) is arranged parallel to the light source (23); The first side surface and the second side surface of the measuring cell (22) are adjacent to each other.

5. The enrichment and cultivation device for online regulation of microorganisms under high pressure environment according to claim 2, characterized in that: The filter unit (3) comprises a regulating valve (31), a filter assembly (32) and a first injection pump (33) which are connected in sequence; The liquid inlet end of the regulating valve (31) is connected to the liquid outlet end of the microorganism enrichment unit (1), and the liquid outlet end of the first injection pump (33) is connected to the first liquid inlet of the measuring chamber (21); The control end of the first injection pump (33) is connected to the control end of the central control unit (6).

6. The enrichment and cultivation device for online regulation of microorganisms under high pressure environment according to claim 5, characterized in that: The filter assembly (32) comprises a filter frame and a filter net, and the filter net is covered on the surface of the filter frame.

7. The enrichment and cultivation device for online regulation of microorganisms under high pressure environment according to claim 5, characterized in that: The microorganism enrichment unit (1) comprises a microorganism enrichment kettle (11), a magnetic stirrer (12), a vent valve (13), a liquid inlet valve (14), a pressure sensor (15), a temperature sensor (16), a water bath (17), an air inlet valve (18) and a second liquid discharge valve (19); A liquid outlet is provided on the side wall of the microorganism enrichment kettle (11), and the liquid outlet serves as the liquid outlet of the microorganism enrichment unit (1) and is connected to the liquid inlet of the regulating valve (31); The microorganism enrichment kettle (11) is arranged in a water bath (17), a magnetic stirrer (12) is arranged on the lower surface of the microorganism enrichment kettle (11), and the control ends of the water bath (17) and the magnetic stirrer (12) are connected to the control end of the central control unit (6); One end of the vent valve (13) is connected to the upper surface of the microorganism enrichment kettle (11), the other end of the vent valve (13) is suspended in the air, and the control end of the vent valve (13) is connected to the control end of the central control unit (6); One end of the air inlet valve (18) is connected to the upper surface of the microorganism enrichment kettle (11), the other end of the air inlet valve (18) is connected to the air outlet of the pressure control unit (4) as the air inlet end of the microorganism enrichment unit (1), and the control end of the air inlet valve (18) is connected to the control end of the central control unit (6); One end of the liquid inlet valve (14) is connected to the side wall of the microorganism enrichment kettle (11), the other end of the liquid inlet valve (14) is connected to the liquid outlet of the multi-sequence nutrient solution supply unit (5) as the liquid inlet end of the microorganism enrichment unit (1), and the control end of the liquid inlet valve (14) is connected to the control end of the central control unit (6); One end of the second liquid discharge valve (19) is connected to the side wall of the microorganism enrichment kettle (11), and the other end of the second liquid discharge valve (19) is suspended in the air; The temperature sensor (16) and the pressure sensor (15) are both arranged on the upper surface of the microorganism enrichment kettle (11), and the data output ends of the temperature sensor (16) and the pressure sensor (15) are both connected to the data input end of the central control unit (6).

8. The enrichment and cultivation device for online regulation of microorganisms under high pressure environment according to claim 7, characterized in that: The pressure control unit (4) comprises a booster pump (41), an air compressor (42), an air storage tank (43) and a pressure regulating valve (44) which are connected in sequence; The other end of the pressure regulating valve (44) is connected to the other end of the air inlet valve (18) as the air outlet end of the pressure control unit (4), and the other end of the pressure regulating valve (44) is also connected to the air inlet of the measuring chamber (21); the control ends of the booster pump (41), the air compressor (42) and the pressure regulating valve (44) are connected to the control end of the central control unit (6); The gas storage tank (43) is filled with culture gas or inert gas.

9. The enrichment and cultivation device for online regulation of microorganisms under high pressure environment according to claim 7, characterized in that: The multi-sequence nutrient solution supply unit (5) comprises a plurality of nutrient bottles (51), a plurality of liquid outlet valves (52) and a second liquid injection pump (53); The liquid outlet of each of the nutrient bottles (51) is connected to one end of a corresponding liquid outlet valve (52); the other end of each of the liquid outlet valves (52) is connected to one end of a second liquid injection pump (53); the other end of the second liquid injection pump (53) serves as the liquid outlet of the multi-sequence nutrient solution supply unit (5) and is connected to the other end of the liquid inlet valve (14); the control end of each liquid outlet valve (52) and the control end of the second liquid injection pump (53) are connected to the control end of the central control unit (6); A nutrient solution is arranged in each of the nutrient bottles (51).

10. A method for enrichment and cultivation of microorganisms under high pressure environment with online regulation, characterized in that: The device according to any one of claims 1 to 9, comprising: S1: sterilizing the microorganism enrichment unit, the online detection unit, and the filtration unit; adding the substrate to be cultured into the microorganism enrichment kettle, adding the culture gas or inert gas into the gas storage tank, and adding the nutrient solution into the nutrient bottle; S2: Control the real-time temperature of the water bath; control the opening of the air inlet valve, adjust the real-time pressure of the culture gas or inert gas output by the pressure control unit, and input it into the microorganism enrichment kettle; control the opening of the liquid inlet valve, select the nutrient solution, control the opening of the corresponding liquid outlet valve and the second liquid injection pump, input it into the microorganism enrichment kettle, and form a microbial bacterial liquid; S3: inject the substrate to be cultured into the measuring pool as a blank sample, control the light source to turn on, generate light of the first preset wavelength, and use spectrophotometry to perform zero adjustment; after the zero adjustment is completed, turn off the light source, rotate the handle clockwise, pour the substrate to be cultured in the measuring pool into the waste liquid pool, and rotate the handle counterclockwise; S4: controlling the magnetic stirrer to stir the microbial solution, and after a first preset time, controlling the regulating valve and the first injection pump to open, adjusting the flow rate of the first injection pump, filtering the microbial solution through the filter assembly to remove large particles of impurities, obtaining the filtered microbial solution, and injecting it into the measuring pool; S5: After the second preset time has passed, the regulating valve and the first injection pump are closed, the diluent container is controlled to open, and the diluent enters the measuring cell to obtain a test solution; S6: Control the light source to turn on, generate light of a second preset wavelength, detect the test solution, obtain the transmittance value and transmit it to the central control unit; S7: Rotate the handle clockwise to pour the test solution in the measuring cell into the waste liquid tank, and rotate the handle counterclockwise; S8: Control the cleaning liquid container to open, and the cleaning liquid enters the measuring tank until it flows out from the overflow port and flows into the waste liquid tank through the overflow tank; S9: After the third preset time, rotate the handle clockwise to pour the cleaning liquid in the measuring tank into the waste liquid tank, and rotate the handle counterclockwise; S10: Repeat steps S8-S9 until a preset number of times is reached; S11: every fourth preset time interval, repeating steps S4-S10, the central control unit calculates the cell data and environmental parameters of the microbial solution at different times according to the transmittance value, and calculates the replenishment amount according to the cell data and environmental parameters of the microbial solution; S12: After reaching the fifth preset time, the second liquid discharge valve and the pressure control unit are controlled to open to discharge the waste liquid in the microorganism enrichment kettle; after the preset volume of waste liquid is discharged, the liquid inlet valve is controlled to open to input a supplementary amount of nutrient solution into the microorganism enrichment kettle.

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