A continuous separation device and method for marine microorganisms under high pressure environment

Abstract

This invention discloses a continuous separation device and method for marine microorganisms under high pressure, relating to the technical field of online separation of marine microorganisms. The method includes setting up a microbial enrichment unit for microbial enrichment, using a multi-sequence nutrient solution supply unit to provide the nutrient solution required for cultivation, and using a gas injection unit to provide the culture gas or inert gas required for cultivation. This ensures that the pressure within the microbial enrichment unit is consistent with the pressure value of the microorganisms in situ at sea, guaranteeing the effectiveness of the enrichment culture. After a period of enrichment culture, the microbial culture is pumped into a multi-stage separation unit to complete the multi-stage separation of cells of different sizes from the microbial culture, obtaining the target microbial cells. Finally, the target microbial cells are transferred to a monitoring culture unit for culture and fixation, and cell characteristic detection is performed using an external multi-omics unit. This invention enables continuous separation and characteristic detection of marine microorganisms of different sizes under in-situ high pressure at sea. It is simple to operate, improves separation efficiency, and provides accurate detection results that closely match the actual environment.

Description

Technical Field

[0001] This invention relates to the technical field of online separation of marine microorganisms, and more specifically, to a continuous separation device and method for marine microorganisms under high pressure. Background Technology

[0002] Marine microorganisms are small, single-celled, or multicellular, or even non-cellular, simple organisms that grow and reproduce in the marine environment. The ocean, on which these microorganisms depend, is the largest and oldest habitat on Earth, covering 71% of the planet's surface and averaging 3.8 km in depth. Due to the uneven distribution of land and sea on Earth, the marine habitat is actually composed of many different large habitats, each supporting different microbial populations.

[0003] Currently, enrichment and isolation experiments involving microorganisms have been conducted under both high-pressure and normal-pressure environments. Marine microorganisms are regulated by pressure factors; enrichment and isolation under normal-pressure environments can only yield some pressure-resistant microorganisms, and cannot obtain the unique indigenous microorganisms native to the deep sea. Furthermore, microorganisms enriched and cultured under high-pressure environments are generally isolated using methods such as streak plating and extreme dilution. In addition, to detect microbial characteristics, such as gene transcription, it is necessary to change the environmental pressure, resulting in results that deviate significantly from the actual environmental conditions. Summary of the Invention

[0004] To overcome the shortcomings of existing high-pressure enrichment culture processes for marine microorganisms, which require depressurization before the separation and detection of characteristics of microorganisms of different sizes, resulting in inaccurate detection results, this invention provides a continuous separation device and method for marine microorganisms under high pressure. This method enables continuous separation of marine microorganisms of different sizes under high pressure for characteristic detection. It is simple to operate, improves separation efficiency, and provides accurate detection results that are consistent with actual environments.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] This invention discloses a continuous separation device for marine microorganisms under high pressure, comprising: a microbial enrichment unit, a gas injection unit, a multi-sequence nutrient solution supply unit, a multi-stage separation unit, a monitoring and culture unit, and a central control unit;

[0007] The air inlet of the microbial enrichment unit is connected to the air outlet of the gas injection unit, and the liquid inlet of the microbial enrichment unit is connected to the liquid outlet of the multi-sequence nutrient solution supply unit.

[0008] The input end of the multi-stage separation unit is connected to the outlet end of the microbial enrichment unit, and the output end of the multi-stage separation unit is connected to the input end of the monitoring and culture unit.

[0009] The data output terminals of the microbial enrichment unit and the monitoring and culture unit are connected to the data input terminal of the central control unit, and the control terminals of the microbial enrichment unit, the gas injection unit, the multi-sequence nutrient solution supply unit, the multi-stage separation unit, and the monitoring and culture unit are connected to the control terminal of the central control unit.

[0010] This invention employs a microbial enrichment unit for microbial enrichment. A multi-sequence nutrient solution supply unit provides the necessary nutrient solution for microbial culture, while a gas injection unit provides the required culture gas or inert gas. The pressure within the enrichment unit is maintained consistent with the pressure at which the microorganisms are located in situ in the ocean, ensuring the effectiveness of the enrichment culture. After a period of enrichment culture, the microbial culture is pumped into a multi-stage separation unit to separate cells of different sizes, obtaining the target microbial cells. Finally, the target microbial cells are transferred to a monitoring and culture unit for immobilization, and cell characteristics are detected using an external multi-omics unit.

[0011] Preferably, the multi-stage separation unit includes a first filter, a microbial separator, a second filter, a first separation filter, a second separation filter, and a multi-channel single-cell sorter;

[0012] The first filter, the microbial separator, the second filter, and the first separation filter are connected in sequence; the first output end of the first separation filter is connected to the first input end of the multi-channel single-cell sorter, the second output end of the first separation filter is connected to the input end of the second separation filter, the first output end of the second separation filter is connected to the second input end of the multi-channel single-cell sorter, and the second output end of the second separation filter is connected to the third input end of the multi-channel single-cell sorter.

[0013] The input end of the first filter serves as the input end of the multi-stage separation unit and is connected to the outlet end of the microbial enrichment unit. The first, second, and third output ends of the multi-channel single-cell sorter all serve as the output ends of the multi-stage separation unit and are connected to the input end of the monitoring and culture unit.

[0014] The microbial culture is pumped into the first filter to remove large particulate impurities, obtaining a primary filtered microbial culture. This primary filtered culture then enters a microbial separator for dilution and separation, yielding a diluted microbial culture. The diluted microbial culture then enters the second filter to remove sediment, obtaining a secondary filtered microbial culture. The first filter has a larger diameter than the second filter. The secondary filtered microbial culture first enters the first filter for primary separation. Microbial culture that fails to pass through the first filter enters the first input of a multi-channel single-cell sorter via the first output of the first filter. Microbial culture that passes through the first filter enters the second input of the multi-channel single-cell sorter via the second output of the first filter. The filter performs two-stage separation. The microbial solution that fails to pass through the filter enters the second input of the multi-channel single-cell sorter through the first output of the second filter. The microbial solution that passes through the filter enters the third input of the multi-channel single-cell sorter through the second output of the filter, thus completing the multi-stage separation of cells of different sizes in the microbial solution. After entering the multi-channel single-cell sorter, the state and type of the single cells entering each input is observed through the window on the multi-channel single-cell sorter to determine whether the target microbial cells have been obtained. If no target microbial cells are obtained, the microbial solution filtered in the first stage of the microbial separator is refreshed, and the above steps are repeated until the target microbial cells are obtained.

[0015] Preferably, the first filter includes a first filter frame and a first filter membrane, wherein the surface of the first filter frame is covered with the first filter membrane;

[0016] The second filter includes a second filter frame and a second filter membrane, wherein the surface of the second filter frame is covered with the second filter membrane;

[0017] The filtration diameter of the second filter membrane is smaller than that of the first filter membrane.

[0018] The first filter is used to remove large particulate impurities, such as humus, during the enrichment and cultivation of microbial culture. The second filter is used to remove sediments larger than the diameter of microorganisms from the diluted microbial culture after separation.

[0019] Preferably, the first separation filter includes a first protective shell and a first filter screen, wherein the first filter screen is disposed inside the first protective shell;

[0020] The second separation filter includes a second protective shell and a second filter screen, wherein the second filter screen is disposed inside the second protective shell;

[0021] The filtration diameter of the second filter screen is smaller than that of the first filter screen.

[0022] The first and second separation filters are used to separate cells of different diameters. Since the filtration diameter of the second filter is smaller than that of the first filter, the microbial solution filtered in the second stage first enters the first separation filter for primary separation. The microbial solution that does not pass through the first filter has the largest cell size and enters the multi-channel single-cell sorter through the first output end of the first separation filter. The microbial solution that passes through the first filter enters the second separation filter for secondary separation through the second output end of the first separation filter. The microbial solution that does not pass through the second filter has the next smallest cell size and enters the multi-channel single-cell sorter through the first output end of the second separation filter. The microbial solution that passes through the second filter has the smallest cell size and enters the multi-channel single-cell sorter through the second output end of the second separation filter.

[0023] Preferably, the microbial separator includes a separation chamber, a diluent storage chamber, a filtration pump, a magnet, and magnetic beads;

[0024] The magnetic beads are installed inside the separation chamber, and the magnet is installed on the lower surface of the separation chamber, corresponding to the position of the magnetic beads.

[0025] The outlet end of the filtration pump is suspended in the air, the inlet end of the filtration pump extends into the interior of the separation chamber, and the control end of the filtration pump is connected to the control end of the central control unit.

[0026] The diluent storage chamber contains diluent, and the diluent storage chamber is connected to the upper surface of the separation chamber via a pipe.

[0027] After the primary filtered microbial culture enters the separation chamber of the microbial separator, the diluent storage chamber opens, and the diluent enters the separation chamber to dilute the primary filtered microbial culture according to a preset ratio, obtaining a diluted microbial culture. The diluent is mainly used to dilute the culture and protect the cells from damage, and also provides some nutrients. The magnet and magnetic beads rotate the diluted microbial culture in the separation chamber, separating the sediment and the bound cells to obtain the separated diluted microbial culture. The function of the vacuum pump is to extract and discharge the primary filtered microbial culture from the separation chamber when no target microbial cells are obtained in the multi-channel single-cell sorter, refreshing the primary filtered microbial culture in the separation chamber and repeating the separation process.

[0028] Preferably, the monitoring and culture unit includes a culture chamber, a support frame, several culture cups, a handle, an environmental indicator sensor, a first injection pump, and a fixed liquid container;

[0029] The environmental indicator sensor, support frame, and several culture cups are installed inside the culture chamber, and the data output terminal of the environmental indicator sensor is connected to the data input terminal of the central control unit.

[0030] The plurality of culture cups are evenly arranged on the upper surface of the support frame along the circumference; the handle is vertically arranged, with one end connected to the lower surface of the support frame and the other end extending from the bottom surface of the culture chamber;

[0031] The upper surface of the culture chamber is provided with a first through hole and a second through hole, the positions of which correspond to any two culture cups.

[0032] The first, second, and third outputs of the multi-channel single-cell sorter are connected to the first through hole; the output of the fixative container is connected to one end of the first injection pump, and the other end of the first injection pump is connected to the second through hole.

[0033] Preferably, the environmental indicator sensor includes an environmental pressure sensor, an environmental temperature sensor, and a gas concentration detector;

[0034] The culture chamber is cylindrical, and the support frame is circular. Several culture cups are evenly arranged along the circumference on the upper surface of the support frame. The handle is vertically connected to the lower surface of the support frame. The upper surface of the culture chamber is provided with a first through hole and a second through hole, which correspond to any two culture cups respectively. In use, the output end of the multi-channel single-cell sorter corresponding to the target microbial cell is turned on, and the cells enter the culture cup through the first through hole for culture. The handle is rotated to align the next culture cup with the first through hole. After a period of culture, the handle is rotated to align the culture cup containing the cultured target microbial cell with the second through hole, and the first injection pump is turned on to allow the fixative to enter the culture cup, thus completing the fixation of the cultured target microbial cell and obtaining a fixed sample.

[0035] Preferably, the microbial enrichment unit includes a microbial enrichment vessel, a magnetic stirrer, a vent valve, a liquid inlet valve, a pressure sensor, a temperature sensor, a water bath, and an air inlet valve;

[0036] The side wall of the microbial enrichment vessel is provided with a liquid outlet hole, which serves as the liquid outlet end of the microbial enrichment unit and is connected to the input end of the first filter.

[0037] The microbial enrichment vessel is set inside a water bath, and a magnetic stirrer is installed on the lower surface of the microbial enrichment vessel. The control terminals of the water bath and the magnetic stirrer are connected to the control terminal of the central control unit.

[0038] One end of the vent valve is connected to the upper surface of the microbial enrichment vessel, and the other end of the vent valve is suspended in the air. The control end of the vent valve is connected to the control end of the central control unit.

[0039] One end of the air inlet valve is connected to the upper surface of the microbial enrichment vessel, and the other end of the air inlet valve is connected to the air outlet of the gas injection unit as the air inlet of the microbial enrichment unit. The control end of the air inlet valve is connected to the control end of the central control unit.

[0040] One end of the inlet valve is connected to the side wall of the microbial enrichment vessel, and the other end of the inlet valve is connected to the outlet of the multi-sequence nutrient solution supply unit as the inlet end of the microbial enrichment unit. The control end of the inlet valve is connected to the control end of the central control unit.

[0041] Both the temperature sensor and the pressure sensor are mounted on the upper surface of the microbial enrichment vessel, and their data output terminals are connected to the data input terminal of the central control unit.

[0042] The upper surface of the microbial enrichment vessel is removable for easy sterilization and placement of substrates. A magnetic stirrer is installed on the lower surface to enhance mass transfer within the vessel, increasing the energy and nutrient utilization efficiency of the microbial solution. Pressure and temperature sensors monitor real-time pressure and temperature changes within the vessel. A vent valve reduces the real-time pressure, and a water bath maintains a constant temperature. Air and liquid inlet valves control the introduction of culture gases or inert gases and nutrient solutions, ensuring the microbial solution remains in an optimal growth environment.

[0043] Preferably, the gas injection unit includes a booster pump, an air compressor, a gas storage tank, and a pressure regulating valve connected in sequence;

[0044] The other end of the pressure regulating valve is connected to the other end of the gas injection unit as the gas outlet; the control terminals of the booster pump, air compressor, and pressure regulating valve are connected to the control terminal of the central control unit.

[0045] The gas storage tank is filled with either a culture gas or an inert gas.

[0046] Preferably, the multi-sequence nutrient solution supply unit includes several nutrient bottles, several dispensing valves, and a second injection pump;

[0047] Each of the nutrient bottles has its outlet connected to one end of an outlet valve, and the other end of each outlet valve is connected to one end of a second injection pump. The other end of the second injection pump serves as the outlet of the multi-sequence nutrient solution supply unit and is connected to the other end of the inlet valve. The control end of each outlet valve and the control end of the second injection pump are connected to the control end of the central control unit.

[0048] Each of the nutrient bottles contains a nutrient solution.

[0049] This invention also provides a method for the continuous separation of marine microorganisms under high pressure, applied to the aforementioned continuous separation apparatus, comprising:

[0050] S1: Sterilize the microbial enrichment unit, multi-stage separation unit and monitoring culture unit; add the substrate to be cultured to the microbial enrichment vessel, add culture gas or inert gas to the gas storage tank, and add nutrient solution to the nutrient bottle;

[0051] 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 gas injection unit, and input it into the microbial enrichment vessel; 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 microbial enrichment vessel, and form microbial liquid.

[0052] S3: Control the magnetic stirrer to stir the microbial liquid. After a first preset time, control the injection pump to pump the microbial liquid into the first filter to filter out large particulate impurities and obtain a first-stage filtered microbial liquid, which then enters the separation chamber of the microbial separator.

[0053] S4: Control the opening of the diluent storage chamber, the diluent enters the separation chamber, and dilutes the microbial solution filtered in the first stage according to the preset ratio to obtain the diluted microbial solution; control the opening of the magnet, the magnetic beads drive the diluted microbial solution in the separation chamber to rotate, and obtain the separated diluted microbial solution.

[0054] S5: The separated diluted microbial solution enters the second filter to remove sediment and obtain a secondary filtered microbial solution;

[0055] S6: The microbial culture from the secondary filtration enters the first separation filter. The microbial culture that does not pass through the first filter enters the first input of the multi-channel single-cell sorter through the first output of the first separation filter. The microbial culture that passes through the first filter enters the second filter through the second output of the first separation filter. The microbial culture that does not pass through the second filter enters the second input of the multi-channel single-cell sorter through the first output of the second separation filter. The microbial culture that passes through the second filter enters the third input of the multi-channel single-cell sorter through the second output of the second separation filter.

[0056] S7: Observe the state and type of the single cells entering from each input end through the window on the multi-channel single-cell sorter to determine whether the target microbial cells have been obtained; if not, proceed to step S8; otherwise, proceed to step S9.

[0057] S8: Control the filtration pump to start, extract and discharge the microbial liquid in the separation chamber, and repeat steps S3-S7.

[0058] S9: Control the output end of the multi-channel single-cell sorter corresponding to the target microbial cell to open, rotate the handle to make the culture cup correspond to the first through hole, and the target microbial cell enters the culture cup for culture; after the second preset time, rotate the handle to make the culture cup correspond to the second through hole, control the first injection pump to open, and make the fixation solution enter the culture cup to complete the fixation of the cultured target microbial cell and obtain a fixed sample;

[0059] S10: The fixed sample is transferred to an external multi-omics unit for cell characterization.

[0060] Preferably, the external multi-omics unit includes an automated DNA extraction instrument and a single-cell sequencer.

[0061] The automated DNA extraction instrument and single-cell sequencer can obtain the genetic characteristics of individual microorganisms, improving the detection efficiency and genome understanding of isolated individual microorganisms.

[0062] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0063] This invention employs a microbial enrichment unit for microbial enrichment. A multi-sequence nutrient solution supply unit provides the necessary nutrient solution for microbial culture, while a gas injection unit provides the required culture gas or inert gas. The pressure within the enrichment unit is maintained consistent with the pressure at which the microorganisms are located in situ in the ocean, ensuring the effectiveness of the enrichment culture. After a period of enrichment culture, the microbial culture is pumped into a multi-stage separation unit to separate cells of different sizes, obtaining the target microbial cells. Finally, the target microbial cells are transferred to a monitoring and culture unit for immobilization, and cell characteristic detection is performed using an external multi-omics unit. This invention enables continuous separation and characteristic detection of marine microorganisms of different sizes under in-situ high-pressure conditions in the ocean. It is simple to operate, improves separation efficiency, and provides accurate detection results that closely reflect the actual environment. Attached Figure Description

[0064] Figure 1 This is a schematic diagram of the structure of a continuous separation device for marine microorganisms under high pressure as described in Example 1;

[0065] Figure 2 This is a schematic diagram of the structure of a continuous separation device for marine microorganisms under high pressure as described in Example 2;

[0066] Figure 3 This is a schematic diagram of the microbial separator described in Example 2;

[0067] Figure 4This is a schematic diagram showing the distribution of the culture cups on the support frame as described in Example 2;

[0068] Figure 5 This is a flowchart of a continuous separation method for marine microorganisms under high pressure as described in Example 3;

[0069] In the diagram, 1-microbial enrichment unit, 2-multi-stage separation unit, 3-monitoring and culture unit, 4-gas injection unit, 5-multi-sequence nutrient solution supply unit, 6-central control unit, 11-microbial enrichment vessel, 12-magnetic stirrer, 13-vent valve, 14-liquid inlet valve, 15-pressure sensor, 16-temperature sensor, 17-water bath, 18-air inlet valve, 21-first filter, 22-microbial separator, 23-second filter, 24-first separation filter, 25-then... 26-Multi-channel single-cell sorter, 31-Cultivation chamber, 32-Support frame, 33-Cultivation cup, 34-Handle, 35-Environmental indicator sensor, 36-First injection pump, 37-Fixture container, 41-Boost pump, 42-Air compressor, 43-Air tank, 44-Pressure regulating valve, 51-Nutrient bottle, 52-Discharge valve, 53-Second injection pump, 221-Separation chamber, 222-Dilution solution storage chamber, 223-Suction filtration pump, 224-Magnet, 225-Magnetic bead. Detailed Implementation

[0070] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the scope of this patent.

[0071] To better illustrate this embodiment, some parts in the accompanying drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions;

[0072] It will be understood by those skilled in the art that certain well-known structures and their descriptions may be omitted in the accompanying drawings.

[0073] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0074] Example 1

[0075] This embodiment discloses a continuous separation device for marine microorganisms under high pressure, such as... Figure 1 As shown, it includes: a microbial enrichment unit 1, a gas injection unit 4, a multi-sequence nutrient solution supply unit 5, a multi-stage separation unit 2, a monitoring and culture unit 3, and a central control unit 6;

[0076] The air inlet of the microbial enrichment unit 1 is connected to the air outlet of the gas injection unit 4, and the liquid inlet of the microbial enrichment unit 1 is connected to the liquid outlet of the multi-sequence nutrient solution supply unit 5.

[0077] The input end of the multi-stage separation unit 2 is connected to the outlet end of the microbial enrichment unit 1, and the output end of the multi-stage separation unit 2 is connected to the input end of the monitoring and culture unit 3.

[0078] The data output terminals of the microbial enrichment unit 1 and the monitoring and culture unit 3 are connected to the data input terminal of the central control unit 6, and the control terminals of the microbial enrichment unit 1, the gas injection unit 4, the multi-sequence nutrient solution supply unit 5, the multi-stage separation unit 2, and the monitoring and culture unit 3 are connected to the control terminal of the central control unit 6.

[0079] In the specific implementation process, this embodiment sets up a microbial enrichment unit 1 for microbial enrichment. The multi-sequence nutrient solution supply unit 5 provides the nutrient solution required for microbial culture to the microbial enrichment unit 1, and the gas injection unit 4 provides the culture gas or inert gas required for microbial culture to the microbial enrichment unit 1. The pressure in the microbial enrichment unit 1 is made consistent with the pressure value of the microorganism in situ in the ocean, which ensures the effectiveness of enrichment culture. After enrichment culture for a period of time, the microbial culture is pumped into the multi-stage separation unit 2 to complete the multi-stage separation of cells of different sizes in the microbial culture and obtain the target microbial cells. Finally, the target microbial cells are transferred to the monitoring culture unit 3 for the culture and fixation of the target microorganism, and the cell characteristics are detected by the external multi-omics unit.

[0080] Example 2

[0081] This invention discloses a continuous separation device for marine microorganisms under high pressure, comprising: a microbial enrichment unit 1, a gas injection unit 4, a multi-sequence nutrient solution supply unit 5, a multi-stage separation unit 2, a monitoring and culture unit 3, and a central control unit 6.

[0082] The air inlet of the microbial enrichment unit 1 is connected to the air outlet of the gas injection unit 4, and the liquid inlet of the microbial enrichment unit 1 is connected to the liquid outlet of the multi-sequence nutrient solution supply unit 5.

[0083] The input end of the multi-stage separation unit 2 is connected to the outlet end of the microbial enrichment unit 1, and the output end of the multi-stage separation unit 2 is connected to the input end of the monitoring and culture unit 3.

[0084] The microbial enrichment unit 1 includes a microbial enrichment vessel 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, and an air inlet valve 18.

[0085] The side wall of the microbial enrichment vessel 11 is provided with a liquid outlet hole, which is connected to the input end of the first filter 21 as the liquid outlet end of the microbial enrichment unit 1.

[0086] The microbial enrichment vessel 11 is installed inside the water bath 17. A magnetic stirrer 12 is installed on the lower surface of the microbial enrichment vessel 11. The control terminals of the water bath 17 and the magnetic stirrer 12 are connected to the control terminal of the central control unit 6.

[0087] One end of the vent valve 13 is connected to the upper surface of the microbial enrichment vessel 11, and the other end of the vent valve 13 is suspended. The control end of the vent valve 13 is connected to the control end of the central control unit 6.

[0088] One end of the air inlet valve 18 is connected to the upper surface of the microbial enrichment vessel 11, and the other end of the air inlet valve 18 is connected to the air outlet of the gas injection unit 4 as the air inlet of the microbial enrichment unit 1. The control end of the air inlet valve 18 is connected to the control end of the central control unit 6.

[0089] One end of the inlet valve 14 is connected to the side wall of the microbial enrichment vessel 11, and the other end of the inlet valve 14 is connected to the outlet of the multi-sequence nutrient solution supply unit 5 as the inlet end of the microbial enrichment unit 1. The control end of the inlet valve 14 is connected to the control end of the central control unit 6.

[0090] The temperature sensor 16 and the pressure sensor 15 are both installed on the upper surface of the microbial enrichment vessel 11, and the data output terminals of the temperature sensor 16 and the pressure sensor 15 are both connected to the data input terminal of the central control unit 6.

[0091] The upper surface of the microbial enrichment vessel 11 is removable for easy sterilization and placement of substrates to be cultured. A magnetic stirrer 12 is installed on the lower surface to enhance mass transfer within the vessel, increasing the energy and nutrient utilization efficiency of the microbial culture. Pressure sensors 15 and 16 monitor pressure and temperature changes within the vessel in real time. A vent valve 13 reduces the real-time pressure within the vessel, and a water bath 17 maintains a constant temperature. An air inlet valve 18 and a liquid inlet valve 14 control the entry of culture gas or inert gas and nutrient solution, ensuring the microbial culture remains in an optimal growth environment.

[0092] The gas injection 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.

[0093] The other end of the pressure regulating valve 44 is connected to the other end of the gas injection unit 4 as the outlet end of the gas injection unit 4; the control ends of the booster pump 41, air compressor 42, and pressure regulating valve 44 are connected to the control end of the central control unit 6.

[0094] The gas storage tank 43 is filled with a culture gas or an inert gas.

[0095] The multi-sequence nutrient solution supply unit 5 includes several nutrient bottles 51, several dispensing valves 52, and a second injection pump 53.

[0096] Each of the nutrient bottles 51 has its outlet connected to one end of an outlet valve 52, and the other end of each outlet valve 52 is connected to one end of a second injection pump 53. The other end of the second injection pump 53 serves as the outlet of the multi-sequence nutrient solution supply unit 5 and is connected to the other end of the inlet valve 14. The control end of each outlet valve 52 and the control end of the second injection pump 53 are connected to the control end of the central control unit 6.

[0097] Each of the nutrient bottles 51 contains a nutrient solution.

[0098] The multi-stage separation unit 2 includes a first filter 21, a microbial separator 22, a second filter 23, a first separation filter 24, a second separation filter 25, and a multi-channel single-cell sorter 26;

[0099] The first filter 21, the microbial separator 22, the second filter 23, and the first separation filter 24 are connected in sequence; the first output end of the first separation filter 24 is connected to the first input end of the multi-channel single-cell sorter 26, the second output end of the first separation filter 24 is connected to the input end of the second separation filter 25, the first output end of the second separation filter 25 is connected to the second input end of the multi-channel single-cell sorter 26, and the second output end of the second separation filter 25 is connected to the third input end of the multi-channel single-cell sorter 26.

[0100] The input end of the first filter 21 serves as the input end of the multi-stage separation unit 2 and is connected to the liquid outlet of the microbial enrichment unit 1. The first output end, the second output end, and the third output end of the multi-channel single-cell sorter 26 all serve as the output ends of the multi-stage separation unit 2 and are connected to the input end of the monitoring and culture unit 3.

[0101] The first filter 21 includes a first filter frame and a first filter membrane, wherein the surface of the first filter frame is covered with the first filter membrane;

[0102] The second filter 23 includes a second filter frame and a second filter membrane, wherein the surface of the second filter frame is covered with the second filter membrane;

[0103] The filtration diameter of the second filter membrane is smaller than that of the first filter membrane;

[0104] The first separation filter 24 includes a first protective shell and a first filter screen, wherein the first filter screen is disposed inside the first protective shell;

[0105] The second separation filter 25 includes a second protective shell and a second filter screen, wherein the second filter screen is disposed inside the second protective shell;

[0106] The filtration diameter of the second filter screen is smaller than that of the first filter screen;

[0107] like Figure 3 As shown, the microbial separator 22 includes a separation chamber 221, a diluent storage chamber 222, a vacuum pump 223, a magnet 224, and magnetic beads 225;

[0108] The magnetic bead 225 is disposed inside the separation chamber 221, and the magnet 224 is disposed on the lower surface of the separation chamber 221, corresponding to the position of the magnetic bead 225.

[0109] The outlet end of the filtration pump 223 is suspended, the inlet end of the filtration pump 223 extends into the interior of the separation chamber 221, and the control end of the filtration pump 223 is connected to the control end of the central control unit 6.

[0110] The diluent storage chamber 222 is filled with diluent, and the diluent storage chamber 222 is connected to the upper surface of the separation chamber 221 through a pipe.

[0111] The microbial culture is pumped into the first filter 21 to remove large particulate impurities, such as humic substances, to obtain a primary filtered microbial culture. This culture then enters the microbial separator 22 for dilution and separation. After the primary filtered microbial culture enters the separation chamber 221 of the microbial separator 22, the diluent storage chamber 222 is opened, and the diluent enters the separation chamber 221 to dilute the primary filtered microbial culture according to a preset ratio, obtaining a diluted microbial culture. The diluent is mainly used to dilute the culture and protect the cells from damage, and also provides some nutrients. The magnet 224 and magnetic beads 225 are used to rotate the diluted microbial culture in the separation chamber 221. The sediment and cell-bound portions are separated to obtain a diluted microbial solution. This diluted microbial solution then enters a second filter 23 to remove sediment larger than the microbial diameter, resulting in a secondary filtered microbial solution. The first separation filter 24 has a larger filtration diameter than the second separation filter 25. The secondary filtered microbial solution first enters the first separation filter 24 for primary separation. Microbial solutions that fail to pass through the first separation filter 24 enter the first input of the multi-channel single-cell sorter 26 via the first output of the first separation filter 24. Microbial solutions that pass through the first separation filter 24... The microbial solution that fails to pass through the second output end enters the second filter 23 for secondary separation. The microbial solution that passes through the second separation filter 25 enters the second input end of the multi-channel single-cell sorter 26 through the first output end of the second separation filter 25. The microbial solution that passes through the second separation filter 25 enters the third input end of the multi-channel single-cell sorter 26 through the second output end of the second separation filter 25, thus completing the multi-stage separation of cells of different sizes from the microbial solution. The first separation filter 24 and the second separation filter 25 are used to separate cells of different diameters. Since the filtration diameter of the second filter 252 is smaller than that of the first filter 242, the microbial solution filtered in the secondary stage first enters the first separation filter 24. The microbial culture that fails to pass through the first filter screen 242 has the largest cell size and enters the multi-channel single-cell sorter 26 through the first output end of the first separation filter 24. The microbial culture that passes through the first filter screen enters the second separation filter 25 through the second output end of the first separation filter 24 for secondary separation. The microbial culture that fails to pass through the second filter screen 252 has the next smallest cell size and enters the multi-channel single-cell sorter 26 through the first output end of the second separation filter 25. The microbial culture that passes through the second filter screen 252 has the smallest cell size and enters the multi-channel single-cell sorter 26 through the second output end of the second separation filter 25.After entering the multi-channel single-cell sorter 26, the state and type of the single cells entering from each input end are observed through the window on the multi-channel single-cell sorter 26 to determine whether the target microbial cells have been obtained. If the target microbial cells have not been obtained, the microbial liquid filtered in the separation chamber 221 is extracted and discharged by the vacuum pump 223 to refresh the microbial liquid filtered in the microbial separator 22. The above steps are repeated until the target microbial cells are obtained.

[0112] The monitoring and culture unit 3 includes a culture chamber 31, a support frame 32, several culture cups 33, a handle 34, an environmental indicator sensor 35, a first injection pump 36, and a fixed liquid container 37.

[0113] The environmental indicator sensor 35, support frame 32 and several culture cups 33 are arranged in the culture chamber 31, and the data output terminal of the environmental indicator sensor 35 is connected to the data input terminal of the central control unit 6.

[0114] like Figure 4 As shown, the plurality of culture cups 33 are evenly arranged on the upper surface of the support frame 32 along the circumference; the handle 34 is vertically arranged, with one end connected to the lower surface of the support frame 32 and the other end extending from the bottom surface of the culture chamber 31.

[0115] The upper surface of the culture chamber 31 is provided with a first through hole and a second through hole, and the positions of the first through hole and the second through hole correspond to any two culture cups 33.

[0116] The first output end, the second output end, and the third output end of the multi-channel single-cell sorter 26 are connected to the first through hole; the output end of the fixative liquid container 37 is connected to one end of the first injection pump 36, and the other end of the first injection pump 36 is connected to the second through hole.

[0117] The environmental indicator sensor 35 includes an environmental pressure sensor, an environmental temperature sensor, and a gas concentration detector;

[0118] The culture chamber 31 is cylindrical, and the support frame 32 is circular. Several culture cups 33 are evenly arranged along the circumference on the upper surface of the support frame 32. The handle 34 is vertically connected to the lower surface of the support frame 32. The upper surface of the culture chamber 31 is provided with a first through hole and a second through hole, which correspond to any two culture cups 33 respectively. In use, the output end of the multi-channel single-cell sorter 26 corresponding to the target microbial cell is opened, and the cells enter the culture cup 33 through the first through hole for culture. The handle 34 is rotated to make the next culture cup 33 correspond to the first through hole. After a period of culture, the handle 34 is rotated to make the culture cup 33 containing the cultured target microbial cells correspond to the second through hole, and the first injection pump 36 is turned on to make the fixative enter the culture cup 33, thereby completing the fixation of the cultured target microbial cells and obtaining a fixed sample.

[0119] This embodiment enables continuous separation of marine microorganisms of different sizes under in-situ high pressure in the ocean for characteristic detection. It is simple to operate, improves separation efficiency, and provides accurate detection results that are consistent with the actual environment.

[0120] Example 3

[0121] This embodiment provides a method for the continuous separation of marine microorganisms under high pressure, applied to the continuous separation apparatus described in Embodiment 1 or 2, such as... Figure 3 As shown, it includes:

[0122] S1: Sterilize the microbial enrichment unit, multi-stage separation unit and monitoring culture unit; add the substrate to be cultured to the microbial enrichment vessel, add culture gas or inert gas to the gas storage tank, and add nutrient solution to the nutrient bottle;

[0123] 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 gas injection unit, and input it into the microbial enrichment vessel; 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 microbial enrichment vessel, and form microbial liquid.

[0124] S3: Control the magnetic stirrer to stir the microbial liquid. After a first preset time, control the injection pump to pump the microbial liquid into the first filter to filter out large particulate impurities and obtain a first-stage filtered microbial liquid, which then enters the separation chamber of the microbial separator.

[0125] S4: Control the opening of the diluent storage chamber, the diluent enters the separation chamber, and dilutes the microbial solution filtered in the first stage according to the preset ratio to obtain the diluted microbial solution; control the opening of the magnet, the magnetic beads drive the diluted microbial solution in the separation chamber to rotate, and obtain the separated diluted microbial solution.

[0126] S5: The separated diluted microbial solution enters the second filter to remove sediment and obtain a secondary filtered microbial solution;

[0127] S6: The microbial culture from the secondary filtration enters the first separation filter. The microbial culture that does not pass through the first filter enters the first input of the multi-channel single-cell sorter through the first output of the first separation filter. The microbial culture that passes through the first filter enters the second filter through the second output of the first separation filter. The microbial culture that does not pass through the second filter enters the second input of the multi-channel single-cell sorter through the first output of the second separation filter. The microbial culture that passes through the second filter enters the third input of the multi-channel single-cell sorter through the second output of the second separation filter.

[0128] S7: Observe the state and type of the single cells entering from each input end through the window on the multi-channel single-cell sorter to determine whether the target microbial cells have been obtained; if not, proceed to step S8; otherwise, proceed to step S9.

[0129] S8: Control the filtration pump to start, extract and discharge the microbial liquid in the separation chamber, and repeat steps S3-S7.

[0130] S9: Control the output end of the multi-channel single-cell sorter corresponding to the target microbial cell to open, rotate the handle to make the culture cup correspond to the first through hole, and the target microbial cell enters the culture cup for culture; after the second preset time, rotate the handle to make the culture cup correspond to the second through hole, control the first injection pump to open, and make the fixation solution enter the culture cup to complete the fixation of the cultured target microbial cell and obtain a fixed sample;

[0131] S10: The fixed sample is transferred to an external multi-omics unit for cell characterization.

[0132] The external multi-omics unit includes an automated DNA extraction instrument and a single-cell sequencer.

[0133] The automated DNA extraction instrument and single-cell sequencer can obtain the genetic characteristics of individual microorganisms, improving the detection efficiency and genome understanding of isolated individual microorganisms.

[0134] The same or similar labels correspond to the same or similar parts;

[0135] The terms used to describe positional relationships in the accompanying drawings are for illustrative purposes only and should not be construed as limiting this patent.

[0136] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A continuous separation device for marine microorganisms under high pressure, characterized in that, include: The microbial enrichment unit (1), multi-stage separation unit (2), monitoring and culture unit (3), gas injection unit (4), multi-sequence nutrient solution supply unit (5), and central control unit (6) are included. The air inlet of the microbial enrichment unit (1) is connected to the air outlet of the gas injection unit (4), and the liquid inlet of the microbial enrichment unit (1) is connected to the liquid outlet of the multi-sequence nutrient solution supply unit (5). The input end of the multi-stage separation unit (2) is connected to the outlet end of the microbial enrichment unit (1), and the output end of the multi-stage separation unit (2) is connected to the input end of the monitoring and culture unit (3). The data output terminals of the microbial enrichment unit (1) and the monitoring and culture unit (3) are connected to the data input terminal of the central control unit (6), and the control terminals of the microbial enrichment unit (1), the gas injection unit (4), the multi-sequence nutrient solution supply unit (5), the multi-stage separation unit (2), and the monitoring and culture unit (3) are connected to the control terminal of the central control unit (6). The multi-stage separation unit (2) includes a first filter (21), a microbial separator (22), a second filter (23), a first separation filter (24), a second separation filter (25), and a multi-channel single-cell sorter (26); The first filter (21), the microbial separator (22), the second filter (23), and the first separation filter (24) are connected in sequence; the first output end of the first separation filter (24) is connected to the first input end of the multi-channel single-cell sorter (26), the second output end of the first separation filter (24) is connected to the input end of the second separation filter (25), the first output end of the second separation filter (25) is connected to the second input end of the multi-channel single-cell sorter (26), and the second output end of the second separation filter (25) is connected to the third input end of the multi-channel single-cell sorter (26). The input end of the first filter (21) is used as the input end of the multi-stage separation unit (2) and is connected to the liquid outlet of the microbial enrichment unit (1). The first output end, the second output end, and the third output end of the multi-channel single-cell sorter (26) are all used as the output ends of the multi-stage separation unit (2) and are connected to the input end of the monitoring culture unit (3). The microbial separator (22) includes a separation chamber (221), a diluent storage chamber (222), a vacuum pump (223), a magnet (224), and magnetic beads (225); The magnetic bead (225) is disposed inside the separation chamber (221), and the magnet (224) is disposed on the lower surface of the separation chamber (221), corresponding to the position of the magnetic bead (225); The outlet end of the filter pump (223) is suspended, the inlet end of the filter pump (223) extends into the interior of the separation chamber (221), and the control end of the filter pump (223) is connected to the control end of the central control unit (6). The diluent storage chamber (222) is filled with diluent, and the diluent storage chamber (222) is connected to the upper surface of the separation chamber (221) through a pipe.

2. The continuous separation device for marine microorganisms under high pressure environment according to claim 1, characterized in that, The first filter (21) includes a first filter frame and a first filter membrane, wherein the surface of the first filter frame is covered with the first filter membrane; The second filter (23) includes a second filter frame and a second filter membrane, wherein the surface of the second filter frame is covered with the second filter membrane; The filtration diameter of the second filter membrane is smaller than that of the first filter membrane.

3. The continuous separation device for marine microorganisms under high pressure environment according to claim 1, characterized in that, The first separation filter (24) includes a first protective shell and a first filter screen, wherein the first filter screen is disposed inside the first protective shell; The second separation filter (25) includes a second protective shell and a second filter screen, wherein the second filter screen is disposed inside the second protective shell; The filtration diameter of the second filter screen is smaller than that of the first filter screen.

4. The continuous separation device for marine microorganisms under high pressure environment according to any one of claims 1-3, characterized in that, The monitoring and culture unit (3) includes a culture chamber (31), a support frame (32), several culture cups (33), a handle (34), an environmental indicator sensor (35), a first injection pump (36), and a fixed liquid container (37); The environmental indicator sensor (35), support frame (32) and several culture cups (33) are set in the culture chamber (31), and the data output terminal of the environmental indicator sensor (35) is connected to the data input terminal of the central control unit (6); The plurality of culture cups (33) are evenly arranged on the upper surface of the support frame (32) along the circumference; the handle (34) is vertically arranged, with one end connected to the lower surface of the support frame (32) and the other end extending from the bottom surface of the culture chamber (31); The upper surface of the culture chamber (31) is provided with a first through hole and a second through hole, the positions of the first through hole and the second through hole correspond to any two culture cups (33); The first output end, the second output end, and the third output end of the multi-channel single-cell sorter (26) are connected to the first through hole; the output end of the fixative container (37) is connected to one end of the first injection pump (36), and the other end of the first injection pump (36) is connected to the second through hole.

5. The continuous separation device for marine microorganisms under high pressure environment according to claim 4, characterized in that, The microbial enrichment unit (1) includes a microbial enrichment vessel (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), and an air inlet valve (18). The side wall of the microbial enrichment vessel (11) is provided with a liquid outlet hole, which is connected to the input end of the first filter (21) as the liquid outlet end of the microbial enrichment unit (1). The microbial enrichment vessel (11) is set inside the water bath (17), and a magnetic stirrer (12) is provided on the lower surface of the microbial enrichment vessel (11). The control terminals of the water bath (17) and the magnetic stirrer (12) are connected to the control terminal of the central control unit (6). One end of the vent valve (13) is connected to the upper surface of the microbial enrichment vessel (11), and the other end of the vent valve (13) is suspended. 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 microbial enrichment vessel (11), and the other end of the air inlet valve (18) is connected to the air outlet of the gas injection unit (4) as the air inlet of the microbial enrichment unit (1). 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 inlet valve (14) is connected to the side wall of the microbial enrichment vessel (11), and the other end of the inlet valve (14) is connected to the outlet of the multi-sequence nutrient solution supply unit (5) as the inlet end of the microbial enrichment unit (1). The control end of the inlet valve (14) is connected to the control end of the central control unit (6). The temperature sensor (16) and pressure sensor (15) are both located on the upper surface of the microbial enrichment vessel (11), and the data output terminals of the temperature sensor (16) and pressure sensor (15) are connected to the data input terminal of the central control unit (6).

6. The continuous separation device for marine microorganisms under high pressure environment according to claim 4, characterized in that, The gas injection unit (4) includes a booster pump (41), an air compressor (42), an air tank (43), and a pressure regulating valve (44) connected in sequence; The other end of the pressure regulating valve (44) is connected to the other end of the gas injection unit (4) as the outlet end of the gas injection unit (4); the control ends of the booster pump (41), air compressor (42), and pressure regulating valve (44) are connected to the control end of the central control unit (6). The gas storage tank (43) is filled with a culture gas or an inert gas.

7. The continuous separation device for marine microorganisms under high pressure environment according to claim 4, characterized in that, The multi-sequence nutrient solution supply unit (5) includes several nutrient bottles (51), several dispensing valves (52), and a second dispensing pump (53); Each of the nutrient bottles (51) has its outlet connected to one end of an outlet valve (52), and the other end of each outlet valve (52) is connected to one end of a second injection pump (53). The other end of the second injection pump (53) serves as the outlet of the multi-sequence nutrient solution supply unit (5) and is connected to the other end of the inlet valve (14). The control end of each outlet valve (52) and the control end of the second injection pump (53) are connected to the control end of the central control unit (6). Each of the nutrient bottles (51) contains a nutrient solution.

8. A method for continuous separation of marine microorganisms under high pressure, applied to the continuous separation apparatus according to any one of claims 1-7, characterized in that, include: S1: Sterilize the microbial enrichment unit, multi-stage separation unit and monitoring culture unit; add the substrate to be cultured to the microbial enrichment vessel, add culture gas or inert gas to the gas storage tank, and add nutrient solution to 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 gas injection unit, and input it into the microbial enrichment vessel; 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 microbial enrichment vessel, and form microbial liquid. S3: Control the magnetic stirrer to stir the microbial liquid. After a first preset time, control the injection pump to pump the microbial liquid into the first filter to filter out large particulate impurities and obtain a first-stage filtered microbial liquid, which then enters the separation chamber of the microbial separator. S4: Control the opening of the diluent storage chamber, the diluent enters the separation chamber, and dilutes the microbial solution filtered in the first stage according to the preset ratio to obtain the diluted microbial solution; control the opening of the magnet, the magnetic beads drive the diluted microbial solution in the separation chamber to rotate, and obtain the separated diluted microbial solution. S5: The separated diluted microbial solution enters the second filter to remove sediment and obtain a secondary filtered microbial solution; S6: The microbial culture from the secondary filtration enters the first separation filter. The microbial culture that does not pass through the first filter enters the first input of the multi-channel single-cell sorter through the first output of the first separation filter. The microbial culture that passes through the first filter enters the second filter through the second output of the first separation filter. The microbial culture that does not pass through the second filter enters the second input of the multi-channel single-cell sorter through the first output of the second separation filter. The microbial culture that passes through the second filter enters the third input of the multi-channel single-cell sorter through the second output of the second separation filter. S7: Observe the state and type of the single cells entering from each input end through the window on the multi-channel single-cell sorter to determine whether the target microbial cells have been obtained; If not obtained, proceed to step S8; otherwise, proceed to step S9. S8: Control the filtration pump to start, extract and discharge the microbial liquid in the separation chamber, and repeat steps S3-S7. S9: Control the output end of the multi-channel single-cell sorter corresponding to the target microbial cell to open, rotate the handle to make the culture cup correspond to the first through hole, and the target microbial cell enters the culture cup for culture; after the second preset time, rotate the handle to make the culture cup correspond to the second through hole, control the first injection pump to open, and make the fixation solution enter the culture cup to complete the fixation of the cultured target microbial cell and obtain a fixed sample; S10: The fixed sample is transferred to an external multi-omics unit for cell characterization.

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