WO2023173486A1 - 一种海洋原位环境单细胞高通量分选装置及方法 - Google Patents

一种海洋原位环境单细胞高通量分选装置及方法 Download PDF

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WO2023173486A1
WO2023173486A1 PCT/CN2022/083914 CN2022083914W WO2023173486A1 WO 2023173486 A1 WO2023173486 A1 WO 2023173486A1 CN 2022083914 W CN2022083914 W CN 2022083914W WO 2023173486 A1 WO2023173486 A1 WO 2023173486A1
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pressure
chamber
sorting
microorganisms
resistant visual
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PCT/CN2022/083914
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English (en)
French (fr)
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冯景春
张偲
杨志峰
王屹
蔡宴朋
钟松
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南方海洋科学与工程广东省实验室(广州)
广东工业大学
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Priority to US18/019,790 priority Critical patent/US20240052290A1/en
Publication of WO2023173486A1 publication Critical patent/WO2023173486A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure

Definitions

  • the invention relates to the technical field of marine microorganisms, and in particular to a marine in-situ environment single cell high-throughput sorting device and method.
  • Marine microorganisms are important marine biological resources. Metabolites of marine bacteria, marine fungi, marine actinomycetes, marine archaea, etc. in seawater and marine sediments contain a large number of biologically active substances, which have important application prospects in the fields of energy, materials, environment, medicine and other fields. For example, autotrophic microorganisms that can produce bioenergy have been discovered in deep sea cold springs and hydrothermal environments; marine microorganisms that can degrade plastics have been discovered; scientists have isolated effective antibiotics from marine bacteria and actinomycetes; marine methanophiles Archaea have strong methane metabolism capabilities. As primary producers in extreme marine ecological environments, they symbiosis with metazoans through chemical energy synthesis and provide them with important carbon sources and energy. Therefore, marine microorganisms are important biological resources and have important development and utilization value.
  • Isolation and culture are important prerequisites for the development and utilization of marine microorganisms.
  • most of the isolation of marine microorganisms uses plate streaking or single-cell sorting instruments in a normal pressure environment.
  • the number of marine microorganisms that have been isolated is still less than 1%, and the physiology, biogeochemistry and Mechanisms and characteristics such as ecology are not easy to obtain directly from nature.
  • Isolating microorganisms from the natural environment and establishing pure cultures is an important basis for studying their gene sequences, morphological characteristics, physiological characteristics and ecological characteristics.
  • the prior art discloses a method for detecting aerobic and anoxygenic photosynthetic bacteria based on single-cell Raman spectroscopy, which realizes single-cell detection of aerobic and anoxygenic photosynthetic bacteria in environmental water bodies. And its use of Raman spectroscopy is a non-destructive detection, and the detected aerobic and non-oxygenic photosynthetic bacteria can be used for single cell sorting and sequencing.
  • Raman spectroscopy is a non-destructive detection, and the detected aerobic and non-oxygenic photosynthetic bacteria can be used for single cell sorting and sequencing.
  • the above scheme is not suitable for the identification and sorting of deep-sea microorganisms.
  • the present invention provides a marine in-situ environment single cell high-throughput sorting device and method, which realizes high-throughput single cell identification of marine microorganisms through optical and spectral detection in a high-pressure environment. and sorting to improve the culturability of marine microorganisms.
  • a marine in-situ environmental single-cell high-throughput sorting device including an enriched microorganism injection system, a pressure-resistant visual sorting chamber, a surrounding wall temperature control system, a pressurizing system, annular pressure control systems, an optical identification system, and an automatic Sorting system and data acquisition and processing system; wherein: the enriched microorganism injection system is used to cultivate and inject bacterial liquid containing microorganisms into the pressure-resistant visual sorting cabin; the pressure-resistant visual sorting cabin is provided with The carrier chip is made of visible materials and embedded with etched microfluidic channels, which are used to realize the dispersion of enriched microorganisms in the channel; when the enriched microorganisms pass through the carrier chip, they are identified through optical The system observes and identifies microorganisms; the outlet end of the pressure-resistant visual sorting cabin is connected to the automatic sorting system, and the automatic sorting system automatically sorts the microorganisms based on the results of microorganism identification by the optical identification system;
  • a micro-inlet and outlet channel is provided on the carrier chip.
  • the inlet channel is mainly used to pump the microorganism-containing bacterial liquid from the enriched microorganism injection system, and to inject gas and liquid from the pressurization system to pressurize.
  • a carrier chip is proposed to realize the dispersed passage of microorganisms.
  • the automatic sorting system intelligently sorts them, achieving optical and spectral detection in a high-pressure environment.
  • the high-throughput single-cell identification and sorting process of marine microorganisms effectively improves the culturability of marine microorganisms.
  • this program proposes devices and technologies for high-throughput single-cell sorting in high-pressure environments. Compared with the existing normal-pressure separation culture, it can enrich and separate microorganisms in the deep-sea in-situ high-pressure environment, and solve the problems of deep-sea in-situ pressure-loving bacteria that cannot survive or have expression differences when cultured in the normal-pressure environment.
  • this solution provides an idea and method for high-throughput screening of microorganisms based on specific morphological and metabolic characteristics, which not only solves the problem of microorganisms in marine high-pressure environments that are enriched after leaving the high-pressure environment, It solves the difficult problems of collection, separation and culture, and can also solve the problem of high-throughput identification and screening of marine microorganisms at the single-cell scale under high pressure, improving separation efficiency.
  • the enriched microorganism injection system includes a microfluidic pump, a high-pressure microorganism enriched culture chamber and an inlet pressure detection device; wherein: the microfluidic pump control end is electrically connected to the data acquisition and processing system; the microfluidic pump The input end of the flow pump is connected to the liquid outlet end of the high-pressure microorganism enrichment culture chamber, and the output end is connected to the inlet end of the pressure-resistant visual sorting chamber through an inlet pressure detection device; the high-pressure microorganism enrichment culture chamber is used for The bacterial liquid containing microorganisms is cultured and injected into the pressure-resistant visual sorting chamber through the microfluidic pump.
  • the pressure-resistant visual sorting cabin also includes a pressure-resistant visual chamber, a surrounding wall cold/heat chamber and a surrounding wall high-pressure chamber; wherein: the pressure-resistant visual chamber is made of pressure-resistant and anti-corrosion metal materials, and The front and back are inlaid with pressure-resistant visual materials, and the whole can withstand the pressure of 5,000 meters of water depth, and is connected to the pressurization system; the cargo chip is set in the center of the pressure-resistant visual chamber; the microfluidic channel entrance of the cargo chip It is connected to the enriched microorganism injection system, and its outlet is the outlet end of the pressure-resistant visual sorting chamber, which is connected to the automatic sorting system; the ring-wall high-pressure chamber is arranged in the outer ring of the pressure-resistant visual chamber, with To protect the load-carrying chip from damage in the pressure-resistant visual chamber; the ring-wall high-pressure chamber is connected to the pressurization system and the ring pressure control system; the ring-wall cold/hot cavity is wrapped around
  • the pressure-resistant visual chamber is equipped with a vent valve, and its output end is electrically connected to the data acquisition and processing system to facilitate pressure adjustment in the chamber.
  • the pressure-resistant visual chamber is equipped with a ring-wall high-pressure chamber, which is pressurized at the same time in the outer ring of the pressure-resistant visual chamber, and is equipped with a ring pressure control system. According to The pressure of the pressure-resistant visual chamber automatically increases or decreases the pressure of the surrounding wall high-pressure chamber to achieve a pressure balance between the pressure-resistant visual chamber and the surrounding wall high-pressure chamber, ensuring that the carrier chip can withstand the smallest pressure difference without being damaged.
  • the ring wall temperature control system adopts a circulating refrigeration/heating device and a temperature sensor; the control end of the circulating refrigeration/heating device is electrically connected to the data acquisition and processing system for cooling/heating and loading the ring wall.
  • the cold/hot fluid in the cold/hot chamber circulates and flows; the temperature sensor probe is arranged in the pressure-resistant visual chamber, and its signal output end is electrically connected to the data acquisition and processing system.
  • the temperature of the pressure-resistant visual cavity is mainly determined by injecting cold/hot fluid into the ring wall cold/hot cavity, and by circulating the fluid to cool or heat the fluid to ensure the temperature of the fluid in the ring wall cold/hot cavity.
  • the low temperature or high temperature state is then ensured by the heat exchange between the cold/hot fluid and the pressure-resistant visual chamber to ensure the low-temperature or high-temperature state in the pressure-resistant visual chamber.
  • the boosting system includes an air compressor, a boosting pump, a gas storage tank, a pressure regulating valve and a pressure sensor; after the air compressor, boosting pump, gas storage tank and pressure regulating valve are connected in sequence, they are connected to all The pressure-resistant visual chamber and the surrounding wall high-pressure chamber are connected respectively; the pressure sensor probe is arranged in the pressure-resistant visual chamber, and its signal output end is electrically connected to the data acquisition and processing system.
  • the temperature sensor and pressure sensor are set up to measure and monitor the temperature and pressure of the pressure-resistant visual chamber during the entire microorganism sorting process.
  • the ring pressure control system includes a ring pressure detection device, a first back pressure tracking pump, a back pressure detection device, a back pressure valve, a buffer tank and a second back pressure tracking pump; wherein: the ring pressure detection device probe is configured In the ring-wall high-pressure chamber, its output end is electrically connected to the data acquisition and processing system; the back-pressure tracking pump is connected to the ring-wall high-pressure chamber, and its control end is connected to the data acquisition and processing system.
  • the detection end of the back pressure detection device is connected to the pressure-resistant visual chamber, and its signal output end is electrically connected to the data acquisition and processing system; one end of the back pressure valve is connected to the automatic sorting The other end is connected to the second back pressure tracking pump through a buffer tank; the control end of the second back pressure tracking pump is electrically connected to the data acquisition and processing system.
  • the optical identification system uses a spectrum/optical observation module.
  • the microorganisms pass through the carrier chip, the microorganisms are observed and identified through the spectrum/optical observation module, and the identification results are sent to the data collection and processing system.
  • the spectral/optical observation module is used to observe and identify the microorganisms.
  • the morphology of single cells can be identified through high-resolution optical microscope observation above the chip, and intracellular marker biological compounds can be identified through Raman spectroscopy. Combining optical and spectroscopic identification signals, it can be determined whether the microorganisms in the chip are for research. Target microorganisms required by personnel.
  • the automatic sorting system includes an intelligent control tee module, a target microorganism storage module and a non-target microorganism storage module; wherein, the target microorganism storage module and the non-target microorganism storage module are respectively connected to the two connections of the intelligent control tee module.
  • the other connection end of the intelligent control tee module is connected to the outlet end of the pressure-resistant visual sorting cabin; the control end of the intelligent control tee module is electrically connected to the data acquisition and processing system.
  • an automatic sorting system is installed at the outlet of the pressure-resistant visual chamber to perform directional sorting of the identified microorganisms.
  • the automatic sorting system is mainly controlled by the intelligent control tee module.
  • the intelligent control tee module is an automatically opening and closing tee.
  • the valve of the target microorganism storage module channel is opened, and the single cell Enter the target microorganism storage module.
  • the target microorganism storage module pathway is opened, and the cell enters the target microorganism storage module, thereby achieving the purpose of high-throughput single cell sorting.
  • the target microorganism storage module can select an atmospheric pressure container or a high-pressure container according to experimental needs.
  • the containers are equipped with corresponding culture media to meet the needs of continued cultivation of the sorted microorganisms.
  • This solution also provides a marine in-situ environment single cell high-throughput sorting method, which is implemented using a marine in-situ environment single-cell high-throughput sorting device, which specifically includes the following steps:
  • S1 Determine the pressure value in the pressure-resistant visual sorting chamber according to the pressure value of the enriched microorganism injection system; inject gas into the pressure-resistant visual chamber through the pressurization system, so that the pressure value in the pressure-resistant visual chamber is consistent with the enriched Integrated microbial injection system;
  • S3 Determine the temperature value in the pressure-resistant visual chamber based on the temperature value in the enriched microorganism injection system. By turning on the ring wall temperature control system, the temperature value in the ring-wall cold/hot chamber is consistent with the temperature value in the pressure-resistant visual chamber. The temperature values are consistent;
  • S5 Inject the bacterial liquid containing microorganisms from the enriched microorganism injection system into the pressure-resistant visual chamber through the microfluidic pump, so that the bacterial liquid slowly passes through the carrier chip, allowing it to pass through the etching channel in the form of single cells; Turn on the ring wall temperature control system to keep the pressure at the outlet of the pressure-resistant visual chamber constant when the liquid flows out of the pressure-resistant visual chamber;
  • the automatic sorting system intelligently turns on the intelligent control tee module according to the recognition results, sends the target microorganisms to the target microorganism storage module, and sends the non-target microorganisms to the non-target microorganism storage module;
  • the marine in-situ environment single-cell high-throughput sorting device before performing step S1, the marine in-situ environment single-cell high-throughput sorting device also needs to be pre-processed, specifically: open the outlet end of the pressure-resistant visualization chamber, and pump in deionized water to perform pressure-resistant visualization. Clean the cavity repeatedly; after it is rinsed clean, pump in 75% alcohol; after the alcohol in the pressure-resistant visual cavity is completely filled, close the pressure-resistant visual cavity and let it stand for 24 hours, and then put the pressure-resistant visual cavity in The pretreatment is completed when the alcohol is vented.
  • the pressure and temperature values in the pressure-resistant visual chamber are kept consistent with the pressure and temperature environment in the enriched microorganism injection system where the microorganisms were originally located, so that the microorganisms can be sorted under in-situ high pressure.
  • the ring pressure control system is turned on, and the pressure value in the high-pressure chamber of the ring wall is kept consistent with the pressure value change in the pressure-resistant visual chamber, so that the load-carrying chip does not bear the pressure difference and does not undergo deformation or damage.
  • the high-throughput single-cell sorting chip and sorting technology for marine microorganisms in high-pressure environments proposed in this plan can realize the identification and sorting of microorganisms in high-pressure marine environments and meet the needs of subsequent purification and culture.
  • it can effectively solve the problem of low survival rate of marine pressure-resistant bacteria and pressure-loving bacteria in normal pressure environment, and the inability of deep-sea indigenous characteristics to be effectively expressed in normal pressure environment. and other problems, to solve the current problem of low culture of marine microorganisms and difficulty in cultivating pure bacteria.
  • this solution can achieve high-throughput identification and automatic sorting at the single-cell scale in a high-pressure environment.
  • this solution effectively improves the efficiency of microbial culture and purification.
  • the present invention proposes a device and method for high-throughput sorting of single cells in a marine in-situ environment. It also proposes a carrier chip to realize the dispersed passage of microorganisms. After observation and identification through an optical recognition system, an automatic sorting system intelligently Sorting on the ground realizes the high-throughput single-cell identification and sorting process of marine microorganisms through optical and spectral detection in a high-pressure environment, effectively improving the culturability of marine microorganisms.
  • Figure 1 is a schematic structural diagram of the device according to the present invention.
  • FIG. 2 is a schematic connection diagram of the circuit modules of the data acquisition and processing system of the present invention.
  • Figure 3 is a schematic flow chart of the method of the present invention.
  • Microbial enrichment injection system 11. Microfluidic pump; 12. High-pressure microbial enrichment culture chamber; 13. Imported pressure detection device; 2. Pressure-resistant visual sorting cabin; 21. Carrier chip; 22. Ring wall carrying cooling/heat chamber; 23. Vent valve; 3. Ring wall temperature control system; 31. Circulating refrigeration/heating device; 32. Temperature sensor; 4. Pressurization system; 41. Air compressor; 42. Pressurization Pump; 43. Gas storage tank; 44. Pressure regulating valve; 45. Pressure sensor; 5. Ring pressure control system; 51. Ring pressure detection device; 52. First back pressure tracking pump; 53. Back pressure detection device; 54 , Back pressure valve; 55. Buffer tank; 56. Second back pressure tracking pump; 6. Optical identification system; 7. Automatic sorting system; 71. Intelligent control tee module; 72. Target microorganism storage module; 73. Non- Target microorganism storage module; 8. Data acquisition and processing system.
  • This embodiment is a complete usage example with rich content.
  • this embodiment proposes a marine in-situ environmental single-cell high-throughput sorting device, including an enriched microorganism injection system 1, a pressure-resistant visual sorting chamber 2, and a wall temperature control system 3. Pressurization system 4, annular pressure control system 5, optical identification system 6, automatic sorting system 7 and data acquisition and processing system 8; wherein: the enriched microorganism injection system 1 is used for culture and can be viewed against pressure
  • the sorting cabin 2 is injected with bacterial liquid containing microorganisms; the pressure-resistant visual sorting cabin 2 is provided with a carrier chip 21.
  • the carrier chip 21 is made of visible materials and embedded with etched microfluidic channels.
  • the enriched microorganisms used to realize the dispersed passage of enriched microorganisms in the channel; when the enriched microorganisms pass through the carrier chip 21, the microorganisms are observed and identified through the optical identification system 6; the outlet end of the pressure-resistant visual sorting cabin 2 is connected to the automatic The sorting system 7 is connected, and the automatic sorting system 7 automatically sorts microorganisms according to the results of microorganism identification by the optical identification system 6; the surrounding wall temperature control system 3 is used to ensure that the internal temperature of the pressure-resistant visual sorting cabin 2 is consistent ;
  • the pressurizing system 4 is used to make the internal pressure of the pressure-resistant visual sorting chamber 2 consistent with the internal pressure of the enriched microorganism injection system 1; the annular pressure control system 5 is used to adjust the internal pressure of the automatic sorting system 7 according to The value changes to keep the internal pressure of the pressure-resistant visual sorting cabin 2 consistent with it, so as to avoid deformation or damage of the load-carrying chip 21 due to the pressure
  • the carrier chip 21 is provided with a fine inlet and outlet channel.
  • the inlet channel mainly pumps the microorganism-containing bacterial liquid from the enriched microorganism injection system 1, and injects gas and liquid from the pressurization system 4 for pressurization. .
  • this embodiment proposes a carrier chip 21 to realize the dispersed passage of microorganisms.
  • the automatic sorting system 7 intelligently sorts, realizing high-pressure microorganisms.
  • the high-throughput single-cell identification and sorting process of marine microorganisms through optical and spectral detection can effectively improve the culturability of marine microorganisms.
  • This embodiment aims at the current problem of difficult separation of marine microorganisms, and proposes a device and technology for high-throughput single cell sorting in a high-pressure environment. Compared with the existing normal-pressure separation culture, it can enrich and separate microorganisms in the deep-sea in-situ high-pressure environment, and solve the problems of deep-sea in-situ pressure-loving bacteria that cannot survive or have expression differences when cultured in the normal-pressure environment.
  • this embodiment provides an idea and method for high-throughput screening of microorganisms based on specific morphology and metabolic characteristics, which not only solves the problem of microorganisms in marine high-pressure environments when they escape from high-pressure environments. It solves the difficult problems of enrichment, separation, and culture, and can solve the problem of high-throughput identification and screening of marine microorganisms at the single-cell scale under high pressure conditions, and improve the separation efficiency.
  • the enriched microorganism injection system 1 includes a microfluidic pump 11, a high-pressure microorganism enriched culture chamber 12 and an inlet pressure detection device 13; wherein: the microfluidic pump 11 control end and the data acquisition and processing system 8. Electrical connection; the input end of the microfluidic pump 11 is connected to the liquid outlet end of the high-pressure microbial enrichment culture chamber 12, and the output end is connected to the inlet end of the pressure-resistant visual sorting chamber 2 through the inlet pressure detection device 13 ;
  • the high-pressure microorganism enrichment culture chamber 12 is used to cultivate bacterial liquid containing microorganisms, and injects it into the pressure-resistant visual sorting chamber 2 through the microfluidic pump 11.
  • the pressure-resistant visual sorting cabin 2 also includes a pressure-resistant visual chamber, a surrounding wall cold/heat chamber 22 and a surrounding wall high-pressure chamber; wherein: the pressure-resistant visual chamber is made of pressure-resistant and anti-corrosion metal materials It is made, its front and back are inlaid with pressure-resistant visual materials, the whole can withstand the pressure of 5000 meters of water depth, and it is connected to the pressurization system 4; the load-carrying chip 21 is set in the center of the pressure-resistant visual cavity; The microfluidic channel inlet of the chip 21 is connected to the enriched microorganism injection system 1, and its outlet is the outlet end of the pressure-resistant visual sorting chamber 2, connected to the automatic sorting system 7; the ring wall high pressure chamber is provided The outer ring of the pressure-resistant visual chamber is used to protect the carrier chip 21 from damage in the pressure-resistant visual chamber; the ring wall high-pressure chamber is connected to the pressurization system 4 and the ring pressure control system 5; the ring wall The cold/hot cavity
  • the pressure-resistant visual chamber is provided with a vent valve 23, the output end of which is electrically connected to the data acquisition and processing system 8 to facilitate pressure adjustment in the chamber.
  • the pressure-resistant visual chamber is provided with a ring-wall high-pressure chamber, which is pressurized at the same time in the outer ring of the pressure-resistant visual chamber, and is provided with a ring pressure control system 5 , automatically increases or decreases the pressure of the surrounding wall high-pressure chamber according to the pressure change of the pressure-resistant visual chamber, achieving a pressure balance between the pressure-resistant visual chamber and the surrounding wall high-pressure chamber, ensuring that the carrier chip 21 can withstand the minimum pressure difference without being damaged.
  • the surrounding wall temperature control system 3 uses a circulating refrigeration/heating device 31 and a temperature sensor 32; the control end of the circulating refrigeration/heating device 31 is electrically connected to the data acquisition and processing system 8, using It is used for cooling/heating and circulating the cooling/heating fluid in the surrounding wall cooling/heating cavity 22; the temperature sensor 32 probe is arranged in the pressure-resistant visual cavity, and its signal output end is connected to the data collection and processing System 8 is electrically connected.
  • the temperature of the pressure-resistant visual cavity is mainly determined by injecting cold/hot fluid into the ring wall cold/hot cavity 22, and by circulating the fluid to cool or heat the ring wall cold/hot cavity to ensure 22, and then ensure the low temperature or high temperature state in the pressure-resistant visual chamber through heat exchange between the cold/hot fluid and the pressure-resistant visual chamber.
  • the boosting system 4 includes an air compressor 41, a boosting pump 42, a gas storage tank 43, a pressure regulating valve 44 and a pressure sensor 45; the air compressor 41, the boosting pump 42, the gas storage tank 43.
  • the pressure regulating valve 44 is connected in sequence, it is connected to the pressure-resistant visual chamber and the surrounding wall high-pressure chamber respectively; the pressure sensor 45 probe is set in the pressure-resistant visual chamber, and its signal output end is connected to the data collection It is electrically connected to the processing system 8 .
  • the temperature sensor 32 and the pressure sensor 45 are provided to measure and monitor the temperature and pressure of the pressure-resistant visual chamber during the entire microorganism sorting process.
  • the annular pressure control system 5 includes an annular pressure detection device 51, a first back pressure tracking pump 52, a back pressure detection device 53, a back pressure valve 54, a buffer tank 55 and a second back pressure tracking pump 56; where :
  • the probe of the ring pressure detection device 51 is arranged in the ring wall high-pressure chamber, and its output end is electrically connected to the data acquisition and processing system 8;
  • the back pressure tracking pump is connected to the ring wall high-pressure chamber, Its control end is electrically connected to the data acquisition and processing system 8;
  • the detection end of the back pressure detection device 53 is connected to the pressure-resistant visual chamber, and its signal output end is electrically connected to the data acquisition and processing system 8.
  • one end of the back pressure valve 54 is connected to the automatic sorting system 7, and the other end is connected to the second back pressure tracking pump 56 through the buffer tank 55; the control end of the second back pressure tracking pump 56 is connected to the data
  • the collection and processing system 8 is electrically connected.
  • the optical identification system 6 uses a spectrum/optical observation module.
  • the microorganism passes through the carrier chip 21, the microorganism is observed and identified through the spectrum/optical observation module, and the identification result is sent to the Data acquisition and processing system 8.
  • the spectrum/optical observation module is used to observe and identify the microorganisms.
  • the morphology of single cells can be identified through high-resolution optical microscope observation above the chip, and intracellular marker biological compounds can be identified through Raman spectroscopy. Combining optical and spectroscopic identification signals, it can be determined whether the microorganisms in the chip are for research. Target microorganisms required by personnel.
  • the automatic sorting system 7 includes an intelligent control tee module 71, a target microorganism storage module 72 and a non-target microorganism storage module 7372; wherein the target microorganism storage module 72 and the non-target microorganism storage module 7372 are respectively connected to the intelligent control system.
  • the other connection end of the intelligent control tee module 71 is connected to the outlet end of the pressure-resistant visual sorting cabin 2; the control end of the intelligent control tee module 71 is connected to the data collection It is electrically connected to the processing system 8 .
  • an automatic sorting system 7 is provided at the outlet of the pressure-resistant visual chamber to perform directional sorting of the identified microorganisms.
  • the automatic sorting system 7 is mainly controlled by the intelligent control tee module 71.
  • the intelligent control tee module 71 is an automatically opening and closing tee.
  • the valve of the target microorganism storage module 72 passes. Open, single cells enter the target microorganism storage module 72.
  • the path of the target microorganism storage module 72 is opened, and the cell enters the target microorganism storage module 72, thereby achieving the purpose of high-throughput single cell sorting.
  • the target microorganism storage module 72 can select an atmospheric pressure container or a high-pressure container according to experimental needs, and the containers are equipped with corresponding culture media to meet the needs of continued cultivation of the sorted microorganisms.
  • this solution also provides a marine in-situ environment single-cell high-throughput sorting method, which is implemented using a marine in-situ environment single-cell high-throughput sorting device, which specifically includes the following steps :
  • S1 Determine the pressure value in the pressure-resistant visual sorting chamber 2 according to the pressure value of the enriched microorganism injection system 1; inject gas into the pressure-resistant visual sorting chamber through the pressurization system 4, so that the pressure in the pressure-resistant visual sorting chamber The value is consistent with the enriched microorganism injection system 1;
  • S3 Determine the temperature value in the pressure-resistant visual chamber according to the temperature value in the enriched microorganism injection system 1.
  • the temperature value in the ring wall cold/hot chamber 22 can be consistent with the pressure resistance.
  • the temperature value in the visual cavity is consistent;
  • S5 Inject the bacterial liquid containing microorganisms from the enriched microorganism injection system 1 into the pressure-resistant visual chamber through the microfluidic pump 11, so that the bacterial liquid slowly passes through the carrier chip 21, so that it can be in the form of single cells in the etched channel. Pass through; open the ring wall temperature control system 3 to keep the pressure at the outlet of the pressure-resistant visual chamber constant when the liquid flows out of the pressure-resistant visual chamber;
  • the automatic sorting system 7 intelligently opens the intelligent control tee module 71 according to the recognition result, sends the target microorganisms to the target microorganism storage module 72, and sends the non-target microorganisms to the non-target microorganism storage module 7372;
  • the marine in-situ environmental single-cell high-throughput sorting device before performing step S1, the marine in-situ environmental single-cell high-throughput sorting device also needs to be pre-processed, specifically: open the outlet end of the pressure-resistant visual chamber, and pump deionized water into the resistant Repeatedly clean the pressure-resistant visual chamber; after it is rinsed, pump in 75% alcohol; after the pressure-resistant visual chamber is completely filled with alcohol, close the pressure-resistant visual chamber and let it stand for 24 hours, and then put the pressure-resistant visual chamber into place.
  • the pretreatment is completed when the alcohol in the cavity is emptied.
  • the pressure and temperature values in the pressure-resistant visual chamber are kept consistent with the pressure and temperature environment in the enriched microorganism injection system 1 where the microorganisms are initially located, so that the microorganisms can be sorted under in-situ high pressure.
  • the ring pressure control system 5 is turned on, and the pressure value in the high-pressure chamber of the ring wall is kept consistent with the pressure value change in the pressure-resistant visual chamber, so that the carrier chip 21 does not bear the pressure difference and does not produce deformation and destroy.
  • the high-throughput single-cell sorting chip and sorting technology for marine microorganisms in high-pressure environments proposed in this embodiment can realize the identification and sorting of microorganisms in high-pressure marine environments and meet the needs of subsequent purification and culture.
  • it can effectively solve the problem of low survival rate of marine pressure-resistant bacteria and pressure-loving bacteria in normal pressure environment, and the inability of deep-sea indigenous characteristics to be effectively expressed in normal pressure environment. and other problems, to solve the current problem of low culture of marine microorganisms and difficulty in cultivating pure bacteria.
  • this solution can achieve high-throughput identification and automatic sorting at the single-cell scale in a high-pressure environment. Compared with conventional microbial isolation and culture technology, this solution effectively improves the efficiency of microbial culture and purification.
  • the high-throughput single-cell sorting of deep-sea methanophilic bacteria involved in this embodiment can be implemented in a high-pressure environment using a microfluidic chip in an in-situ high-pressure environment.
  • High-throughput single-cell sorting of enriched deep-sea methanophilic bacteria is carried out to meet the needs of subsequent culture and functional determination.
  • the core of this example is the pressure-resistant visual sorting cabin 2 that is high-pressure resistant and visible.
  • Other parts mainly include the boosting system 4, the ring pressure control system 5, the optical identification system 6, the automatic sorting system 7 and the data acquisition and processing system 8.
  • the core component of the pressure-resistant visual sorting cabin 2 mainly includes a pressure-resistant visual chamber, a load-carrying chip 21, a ring-wall cold/heat chamber 22 and a ring-wall high-pressure chamber.
  • the pressure-resistant visual chamber is made of pressure-resistant and anti-corrosion titanium alloy material.
  • the front and back of the chamber are inlaid with pressure-resistant visible sapphire material.
  • the entire chamber can withstand the pressure of 5,000 meters of water depth.
  • the center of the pressure-resistant visual chamber is provided with a carrier chip 21, and a microfluidic channel is provided on the carrier chip 21.
  • the bacterial liquid containing microorganisms is injected from the high-pressure microorganism enrichment culture chamber 12 into the pressure-resistant visual chamber through the microfluidic pump 11.
  • An inlet pressure detection device 13 is provided between the enriched microorganism injection system 1 and the pressure-resistant visual sorting chamber 2 .
  • the outlet end of the pressure-resistant visual chamber is mainly used for the sorted fluid containing the deep-sea methanophile enrichment solution to leave the pressure-resistant visual chamber and enter the automatic sorting system 7 .
  • An annular pressure control system 5 is provided at the outlet end for back pressure control, which mainly includes a back pressure detection device 53, a back pressure valve 54, a buffer tank 55 and a second back pressure tracking pump 56 to ensure that microbial-containing fluids flow out under set pressure conditions.
  • the pressure in the pressure-resistant visual chamber remains constant during the entire sorting process.
  • the pressure-resistant visual chamber is provided with a vent valve 23 to facilitate pressure adjustment in the chamber.
  • the pressure-resistant visual chamber is provided with a temperature sensor 32 and a pressure sensor 45 to measure and monitor the temperature and pressure in the chamber during the sorting process of deep-sea methanophiles.
  • the object-carrying chip 21 is made of visible materials and is embedded with etched microfluidic channels, which facilitates the enriched deep-sea methanophilic bacteria liquid to enter the pressure-resistant visual sorting chamber and pass through the chip at a smaller flow rate to achieve single Cells disperse through the channel.
  • the pressure-resistant visual chamber is provided with a ring-wall high-pressure chamber, which is pressurized at the same time in the outer ring of the pressure-resistant visual chamber, and is provided with a ring pressure control system 5 , automatically increases or decreases the pressure of the surrounding wall high-pressure chamber according to the pressure change of the pressure-resistant visual chamber, achieving a pressure balance between the pressure-resistant visual chamber and the surrounding wall high-pressure chamber, ensuring that the carrier chip 21 can withstand the minimum pressure difference without being damaged.
  • the temperature of the pressure-resistant visual chamber is mainly determined by injecting cold/hot fluid, such as a refrigeration solution containing ethylene glycol, into the ring wall cold/heat chamber 22, and by circulating the fluid through the refrigeration/heat device 31 to ensure that the ring wall
  • cold/hot fluid such as a refrigeration solution containing ethylene glycol
  • the fluid in the cavity is kept at a low temperature of 4°C, and then the low temperature state in the pressure-resistant visual cavity is ensured through heat exchange between the cooling fluid and the pressure-resistant visual cavity.
  • the spectral/optical observation module is used to observe and identify the microorganisms.
  • the morphology of single cells can be identified through high-resolution optical microscope observation above the chip, and intracellular marker biological compounds can be identified through Raman spectroscopy.
  • Combining optical and spectroscopic identification signals can determine whether the microorganisms in the chip are Deep-sea methanophiles.
  • An automatic sorting system 7 is provided at the outlet of the pressure-resistant visual chamber to perform directional sorting of the identified microorganisms.
  • the automatic sorting system is provided with an intelligent control tee module 71.
  • the intelligent control tee module 71 is an automatic opening and closing tee.
  • the valve of the target microorganism storage module 72 passes. Open, single cells enter the target microorganism storage module 72.
  • the non-target microorganism storage module 73 collection path is opened, and the cells enter the non-target microorganism storage module 73, thereby achieving the purpose of high-throughput single cell sorting.
  • the target microorganism storage module 72 can be a normal pressure container or a high-pressure container to meet the need for the sorted deep-sea methanophilic bacteria to continue to be cultured in a high-pressure environment.
  • the high-throughput single-cell sorting technology of marine microorganisms in high-pressure environment involved in this example mainly requires the construction of a high-pressure environment in a pressure-resistant visual chamber similar to that of deep-sea methanophiles living in the deep-sea environment.
  • First clean the pressure-resistant visual chamber open the inlet and outlet, pump in deionized water and rinse repeatedly. After flushing, pump in 75% alcohol. After the pressure-resistant visual chamber is completely filled with alcohol, close the pressure-resistant visual chamber. , let it sit for 24 hours and let it air out.
  • the pressure-resistant visual chamber determines the pressure value in the pressure-resistant visual chamber according to the initial pressure value 12MPa in the enriched microorganism injection system 1, and inject CH 4 gas into the pressure-resistant visual chamber through the pressurization system 4, so that the pressure-resistant visual chamber
  • the pressure is increased to 12MPa; and the ring pressure control system 5 is opened so that the pressure value in the high-pressure chamber of the ring wall is consistent with the pressure in the pressure-resistant visual chamber.
  • gas is injected into the ring-wall high-pressure chamber through the first back-pressure tracking pump 52, or the valve is opened to relieve pressure, so that the pressure of the ring-wall high-pressure chamber is consistent with the pressure-resistant chamber.
  • the pressure in the visual chamber remains consistent. Then, the temperature value in the pressure-resistant visual chamber is determined according to the temperature value 4°C in the enriched microorganism injection system 1. By turning on the ring wall temperature control system 3, the temperature value in the ring wall cold/hot chamber is consistent with the pressure resistance. The temperature inside the visual cavity is consistent. Then, adjust the spectrum/optical observation module so that it can clearly observe the situation inside the object chip 21 .
  • the bacterial liquid containing deep-sea methanophage bacteria is injected from the enriched microorganism injection system 1 through the microfluidic pump 11 into the pressure-resistant visual chamber, and the outlet back pressure is turned on, and the outlet pressure is set to 11.5MPa, so that the bacterial liquid slowly passes through the load
  • the chip 21 allows it to pass through the etching channel in the form of single cells.
  • the spectrum/optical observation module is turned on to fully observe and collect the morphology of the cells and the spectrum of the single-cell microorganisms. Learn the information to determine whether the cell is a methanophile.
  • the sorting process ends. During the entire sorting process, the pressure and temperature values in the pressure-resistant visual chamber are kept consistent with the pressure and temperature environment in the enriched microorganism injection system 1 where the deep-sea methanophiles were originally located, so that the microorganisms can achieve high-pressure in situ. sorting.
  • the ring pressure control system 5 is turned on, and the pressure value in the high-pressure chamber of the ring wall is kept consistent with the pressure value change in the pressure-resistant visual chamber, so that the carrier chip 21 does not bear the pressure difference and does not produce deformation and destroy.

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Abstract

一种海洋原位环境单细胞高通量分选装置，包括耐压可视分选舱（2）、光学识别系统（6）和自动分选系统（7）；通过向耐压可视分选舱（2）注入含微生物的菌液；由设置在耐压可视分选舱（2）内载物芯片（21）实现富集微生物在通道内分散通过；富集微生物微通过载物芯片（21）时，通过光学识别系统（6）对微生物进行观察和识别；自动分选系统（7）根据微生物识别的结果对微生物进行自动分选。还提出一种海洋原位环境单细胞高通量分选方法，通过载物芯片（21）实现微生物的单细胞分散后，通过光学识别系统（6）进行观察识别，并由自动分选系统（7）智能地进行分选，实现了在高压和极端温度环境下，通过光学和光谱检测对海洋微生物的高通量单细胞识别和分选过程，有效提高海洋微生物的可培养性。

Description

一种海洋原位环境单细胞高通量分选装置及方法 技术领域
本发明涉及海洋微生物技术领域，特别是涉及一种海洋原位环境单细胞高通量分选装置及方法。
背景技术
浩瀚的海洋是地球生命的摇篮，蕴含着资源丰富、种类繁多的微生物。海洋微生物是重要的海洋生物资源。海水、海洋沉积物里面的海洋细菌、海洋真菌、海洋放线菌、海洋古菌等的代谢产物存在大量的生物活性物质，在能源、材料、环境、医药等领域有着重要的应用前景。比如，深海冷泉、热液环境发现的能够产生生物能源的自养型微生物；能够降解塑料的海洋微生物已经被发现；科学家从海洋细菌和放线菌中分离出了有效的抗生素；海洋嗜甲烷菌等古菌具有较强的甲烷代谢能力，作为海洋极端生态环境的初级生产者，通过化能合成，与后生动物共生，为它们提供重要的碳源和能源。因此，海洋微生物是重要生物资源，具有重要的开发利用价值。
分离和培养是开发利用海洋微生物的重要前提。目前，对于海洋微生物的分离，多数是在常压环境下采用平板划线或者单细胞分选仪，但已被分离出的海洋微生物仍然很少于1％，而微生物的生理学、生物地球化学和生态学等机理和特性不容易从自然界中直接获得，将微生物从自然环境中分离出来并建立纯培养，是研究其基因序列、形态特征、生理特征和生态特征的重要基础。然而，由于海洋微生物多生活在极端环境，例如嗜压微生物几乎不能在常压环境进分离和培养得到，这限制了我们对海洋微生物的认识和开发利用价值。因此，亟需开发针对海洋高压环境下的微生物有效识别和分选技术。
现有技术公开了一种基于单细胞拉曼光谱的好氧不产氧光合细菌检测方法，实现了环境水体的好氧不产氧光合细菌单细胞检测。且其采用拉曼光谱检测是非破坏性检测，检测出的好氧不产氧光合细菌可用于单细胞分选和测序。但由于深海微生物所生活的环境特殊，上述方案并不适用于深海微生物的识别与分选。
发明内容
本发明为了解决以上至少一种技术缺陷，提供一种海洋原位环境单细胞高通量分选装置及方法，在高压环境下，通过光学和光谱检测，实现海洋微生物的高通量单细胞识别和分选，提高海洋微生物的可培养性。
为解决上述技术问题，本发明的技术方案如下：
一种海洋原位环境单细胞高通量分选装置，包括富集微生物注入系统、耐压可视分选舱、环壁温度控制系统、增压系统、环压控制系统、光学识别系统、自动分选系统和数据采集与处理系统；其中：所述富集微生物注入系统用于培养并向耐压可视分选舱注入含微生物的菌液；所述耐压可视分选舱内设置有载物芯片，所述载物芯片由可视材料制成，内嵌有刻蚀微流体通道，用于实现富集微生物在通道内分散通过；富集微生物微通过载物芯片时，通过光学识别系统对微生物进行观察和识别；耐压可视分选舱出口端与所述自动分选系统连接，自动分选系统根据光学识别系统对微生物识别的结果对微生物进行自动分选；所述环壁温度控制系统用于保证耐压可视分选舱内部温度一致；所述增压系统用于满足耐压可视分选舱内部压力与所述富集微生物注入系统内部压力一致的功能需求；所述环压控制系统用于根据自动分选系统内压力值变化保持耐压可视分选舱内部压力与其一致，避免载物芯片承受压差产生形变或破坏；所述富集微生物注入系统、环壁温度控制系统、增压系统、环压控制系统、光学识别系统、自动分选系统均与所述数据采集与处理系统电性连接。
上述方案中，载物芯片上设置有微细进出通道，进入通道主要是从富集微生物注入系统里面泵入含微生物的菌液，以及从增压系统里面注入气体和液体增压。
上述方案中，提出了一种载物芯片，实现微生物的分散通过，通过光学识别系统进行观察识别后，由自动分选系统智能地进行分选，实现了在高压环境下，通过光学和光谱检测对海洋微生物的高通量单细胞识别和分选过程，有效提高海洋微生物的可培养性。
本方案针对目前海洋微生物难分离的难题，提出了针对高压环境进行高通量单细胞分选的装置与技术。相对于现有的常压分离培养，能够满足微生物在深海原位高压环境进行富集、分离，解决深海原位嗜压菌在常压环境培养不能存活或者表达差异等难题。另一方面，本方案相比常规的富集、平板划线分离技术，提 供了一种根据特定形态和代谢特征高通量筛选微生物的思路与方法，既解决海洋高压环境微生物在脱离高压环境富集、分离、培养困难的问题，又可以实现高压情况下，单细胞尺度的海洋微生物高通量识别和筛选难题，提高分离效率。
其中，所述富集微生物注入系统包括微流泵、高压微生物富集培养室和进口压力检测装置；其中：所述微流泵控制端与所述数据采集与处理系统电性连接；所述微流泵输入端与所述高压微生物富集培养室的出液端连接，输出端通过进口压力检测装置与所述耐压可视分选舱入口端连接；所述高压微生物富集培养室用于培养含微生物的菌液，并通过所述微流泵注入耐压可视分选舱中。
其中，所述耐压可视分选舱还包括耐压可视腔、环壁载冷/热腔和环壁高压腔；其中：耐压可视腔由耐压和防腐金属材料制成，其正面和背面镶嵌耐压可视材料，整个可承受5000米水深的压力，其与增压系统连接；所述载物芯片设置在所述耐压可视腔中央；载物芯片的微流体通道入口与所述富集微生物注入系统连接，其出口为耐压可视分选舱的出口端，与所述自动分选系统连接；所述环壁高压腔设置在耐压可视腔外环，用于保护载物芯片在耐压可视腔中不受损坏；所述环壁高压腔与增压系统、环压控制系统连接；所述环壁载冷/热腔包裹在耐压可视腔外壁，用于装载载冷/热流体，并通过载冷/热流体与环壁温度控制系统连接。
上述方案中，耐压可视腔设置有放空阀，其输出端与数据采集与处理系统电性连接，方便进行腔内的压力调节。为了保护载物芯片在耐压可视腔内不受损坏，耐压可视腔设置有环壁高压腔，在耐压可视腔的外环同时增压，并且设置有环压控制系统，根据耐压可视腔压力变化自动增减环壁高压腔的压力，实现耐压可视腔和环壁高压腔的压力平衡，保证载物芯片承受最小的压力差，而不受破坏。
其中，所述环壁温度控制系统采用循环制冷/热装置和温度传感器；所述循环制冷/热装置控制端与所述数据采集与处理系统电性连接，用于制冷/热并使环壁载冷/热腔内的载冷/热流体循环流动；所述温度传感器探头设置在耐压可视腔内，其信号输出端与所述数据采集与处理系统电性连接。
上述方案中，耐压可视腔的温度主要是通过在环壁载冷/热腔内注入载冷/热流体，并且通过将流体进行循环制冷或者加热保证环壁载冷/热腔内流体的低温或者高温状态，然后通过载冷/热流体与耐压可视腔内的热交换保证耐压可视腔内的低温或者高温状态。
其中，所述增压系统包括空气压缩机、增压泵、储气罐、调压阀和压力传感 器；所述空气压缩机、增压泵、储气罐、调压阀依次连接后，与所述耐压可视腔、环壁高压腔分别连接；所述压力传感器探头设置在耐压可视腔内，其信号输出端与所述数据采集与处理系统电性连接。
上述方案中，温度传感器、压力传感器的设置，用于在整个微生物分选过程中，对耐压可视腔的温度和压力进行测量和监控。
其中，所述环压控制系统包括环压检测装置、第一回压跟踪泵、回压检测装置、回压阀、缓冲罐和第二回压跟踪泵；其中：所述环压检测装置探头设置在所述环壁高压腔内，其输出端与所述数据采集与处理系统电性连接；所述回压跟踪泵与所述环壁高压腔连接，其控制端与所述数据采集与处理系统电性连接；所述回压检测装置检测端与所述耐压可视腔连接，其信号输出端与所述数据采集与处理系统电性连接；所述回压阀一端与所述自动分选系统连接，另一端通过缓冲罐与第二回压跟踪泵连接；所述第二回压跟踪泵控制端与所述数据采集与处理系统电性连接。
其中，所述光学识别系统采用光谱/光学观察模块，在富集微生物微通过载物芯片时，通过光谱/光学观察模块对微生物进行观察和识别，并将识别结果发送至所述数据采集与处理系统。
在富集的微生物通过载物芯片的过程中，利用光谱/光学观察模块对微生物进行观察和识别。可以通过高分辨光学显微镜在芯片上方观测对单细胞的形态进行识别，以及通过拉曼光谱对胞内的标志生物化合物进行识别，结合光学和光谱学识别信号，可判定芯片中的微生物是否为研究人员需要的目标微生物。
其中，所述自动分选系统包括智能控制三通模块、目标微生物存储模块和非目标微生物存储模块；其中，目标微生物存储模块、非目标微生物存储模块分别连接在智能控制三通模块的两个连接端上，智能控制三通模块另一个连接端与所述耐压可视分选舱出口端连接；智能控制三通模块控制端与所述数据采集与处理系统电性连接。
上述方案中，在耐压可视腔的出口端设置有自动分选系统，对识别的微生物进行定向分选。自动分选系统主要是通过智能控制三通模块控制，智能控制三通模块为自动启闭的三通，当识别的单细胞被判定为目标微生物时，目标微生物存储模块通路的阀门打开，单细胞进入目标微生物存储模块。当识别的单细胞被判定为非目标微生物时，则开启目标微生物存储模块通路，该细胞进入目标微生物 存储模块，以此达到高通量单细胞分选的目的。目标微生物存储模块可根据实验需要，选择常压容器或高压容器，容器内均装有相应培养基，满足分选后的微生物继续培养的需求。
本方案还提供一种海洋原位环境单细胞高通量分选方法，采用一种海洋原位环境单细胞高通量分选装置实现，具体包括以下步骤：
S1：根据富集微生物注入系统的压力值确定耐压可视分选舱内的压力值；通过增压系统往耐压可视腔内注入气体，使得耐压可视腔内的压力值与富集微生物注入系统一致；
S2：打开环压控制系统，使得耐压可视腔与环壁高压腔的压力一致；
S3：根据富集微生物注入系统内的温度值确定耐压可视腔内的温度值，通过开启环壁温度控制系统，使得环壁载冷/热腔内的温度值与耐压可视腔内的温度值一致；
S4：对光学识别系统进行调试，使其能清楚的观测到载物芯片内的情况；
S5：将含微生物的菌液从富集微生物注入系统通过微流泵注入耐压可视腔中，使得菌液缓慢通过载物芯片，使其能以单细胞的形式在刻蚀通道内通过；打开环壁温度控制系统，保持液体流出耐压可视腔时，耐压可视腔出口端压力恒定；
S6：在菌液通过载物芯片过程中，通过光学识别系统充分观测采集细胞的形态，以及胞内和胞外代谢化合物的光谱学信息，判断该细胞是否为目标微生物，并将识别结果发送至数据采集与处理系统；
S7：自动分选系统根据识别结果智能开启智能控制三通模块，将目标微生物送至目标微生物存储模块中，将非目标微生物送至非目标微生物存储模块中；
S8：待目标微生物存储模块中的目标微生物数量符合，结束分选过程。
其中，在执行步骤S1之前，还需要对海洋原位环境单细胞高通量分选装置进行预处理，具体为：打开耐压可视腔出口端，通过泵入去离子水对耐压可视腔进行反复清洗；待冲洗干净后，泵入75％酒精；待耐压可视腔内酒精完全注满后，关闭耐压可视腔，静置24小时，再将耐压可视腔中的酒精放空即完成预处理。
在整个分选过程中，保持耐压可视腔内的压力、温度值与微生物最初所在的富集微生物注入系统内的压力、温度环境一致，使得微生物在原位高压情况内实现分选。在分选过程中，打开环压控制系统，根据耐压可视腔内的压力值变化保持环壁高压腔内的压力值与其一致，使得载物芯片不承受压差，不产生形变和破 坏。
本方案提出的海洋微生物高压环境高通量单细胞分选芯片及分选技术，能够实现海洋高压环境下，微生物的识别和分选，满足后续纯化培养的需求。相比目前传统的常压环境富集、分离海洋微生物的技术，可有效的解决海洋耐压菌、嗜压菌在常压环境下存活率低，且深海土著特征在常压环境下不能有效表达等难题，解决目前海洋微生物培养度低，难培养纯菌的问题。同时，本方案可以实现高压环境下，单细胞尺度的高通量识别和自动分选，相比常规的微生物分离培养技术，有效的提高了微生物培养、纯化的效率。
与现有技术相比，本发明技术方案的有益效果是：
本发明提出了一种海洋原位环境单细胞高通量分选装置及方法，提出了一种载物芯片，实现微生物的分散通过，通过光学识别系统进行观察识别后，由自动分选系统智能地进行分选，实现了在高压环境下，通过光学和光谱检测对海洋微生物的高通量单细胞识别和分选过程，有效提高海洋微生物的可培养性。
附图说明
图1为本发明所述装置的结构示意图；
图2为本发明所述数据采集与处理系统的电路模块连接示意图；
图3为本发明所述方法的流程示意图；
其中：1、富集微生物注入系统；11、微流泵；12、高压微生物富集培养室；13、进口压力检测装置；2、耐压可视分选舱；21、载物芯片；22、环壁载冷/热腔；23、放空阀；3、环壁温度控制系统；31、循环制冷/热装置；32、温度传感器；4、增压系统；41、空气压缩机；42、增压泵；43、储气罐；44、调压阀；45、压力传感器；5、环压控制系统；51、环压检测装置；52、第一回压跟踪泵；53、回压检测装置；54、回压阀；55、缓冲罐；56、第二回压跟踪泵；6、光学识别系统；7、自动分选系统；71、智能控制三通模块；72、目标微生物存储模块；73、非目标微生物存储模块；8、数据采集与处理系统。
具体实施方式
附图仅用于示例性说明，不能理解为对本专利的限制；
本实施例为完整的使用示例，内容较丰富
为了更好说明本实施例，附图某些部件会有省略、放大或缩小，并不代表实际产品的尺寸；
对于本领域技术人员来说，附图中某些公知结构及其说明可能省略是可以理解的。
下面结合附图和实施例对本发明的技术方案做进一步的说明。
实施例1
如图1、图2所示，本实施例提出一种海洋原位环境单细胞高通量分选装置，包括富集微生物注入系统1、耐压可视分选舱2、环壁温度控制系统3、增压系统4、环压控制系统5、光学识别系统6、自动分选系统7和数据采集与处理系统8；其中：所述富集微生物注入系统1用于培养并向耐压可视分选舱2注入含微生物的菌液；所述耐压可视分选舱2内设置有载物芯片21，所述载物芯片21由可视材料制成，内嵌由刻蚀微流体通道，用于实现富集微生物在通道内分散通过；富集微生物微通过载物芯片21时，通过光学识别系统6对微生物进行观察和识别；耐压可视分选舱2出口端与所述自动分选系统7连接，自动分选系统7根据光学识别系统6对微生物识别的结果对微生物进行自动分选；所述环壁温度控制系统3用于保证耐压可视分选舱2内部温度一致；所述增压系统4用于令耐压可视分选舱2内部压力与所述富集微生物注入系统1内部压力一致；所述环压控制系统5用于根据自动分选系统7内压力值变化保持耐压可视分选舱2内部压力与其一致，避免载物芯片21承受压差产生形变或破坏；所述富集微生物注入系统1、环壁温度控制系统3、增压系统4、环压控制系统5、光学识别系统6、自动分选系统7均与所述数据采集与处理系统8电性连接。
在具体实施过程中，载物芯片21上设置有微细进出通道，进入通道主要是从富集微生物注入系统1里面泵入含微生物的菌液，以及从增压系统4里面注入气体和液体增压。
在具体实施过程中，本实施例提出了一种载物芯片21，实现微生物的分散通过，通过光学识别系统6进行观察识别后，由自动分选系统7智能地进行分选，实现了在高压环境下，通过光学和光谱检测对海洋微生物的高通量单细胞识别和分选过程，有效提高海洋微生物的可培养性。
本实施例针对目前海洋微生物难分离的难题，提出了针对高压环境进行高通量单细胞分选的装置与技术。相对于现有的常压分离培养，能够满足微生物在深海原位高压环境进行富集、分离，解决深海原位嗜压菌在常压环境培养不能存活或者表达差异等难题。另一方面，本实施例相比常规的富集、平板划线分离技术， 提供了一种根据特定形态和代谢特征高通量筛选微生物的思路与方法，既解决海洋高压环境微生物在脱离高压环境富集、分离、培养困难的问题，又可以实现高压情况下，单细胞尺度的海洋微生物高通量识别和筛选难题，提高分离效率。
更具体的，所述富集微生物注入系统1包括微流泵11、高压微生物富集培养室12和进口压力检测装置13；其中：所述微流泵11控制端与所述数据采集与处理系统8电性连接；所述微流泵11输入端与所述高压微生物富集培养室12出液端连接，输出端通过进口压力检测装置13与所述耐压可视分选舱2入口端连接；所述高压微生物富集培养室12用于培养含微生物的菌液，并通过所述微流泵11注入耐压可视分选舱2中。
更具体的，所述耐压可视分选舱2还包括耐压可视腔、环壁载冷/热腔22和环壁高压腔；其中：耐压可视腔由耐压和防腐金属材料制成，其正面和背面镶嵌耐压可视材料，整个可承受5000米水深的压力，其与增压系统4连接；所述载物芯片21设置在所述耐压可视腔中央；载物芯片21的微流体通道入口与所述富集微生物注入系统1连接，其出口为耐压可视分选舱2的出口端，与所述自动分选系统7连接；所述环壁高压腔设置在耐压可视腔外环，用于保护载物芯片21在耐压可视腔中不受损坏；所述环壁高压腔与增压系统4、环压控制系统5连接；所述环壁载冷/热腔22包裹在耐压可视腔外壁，用于填充载冷/热流体，并通过载冷/热流体与环壁温度控制系统3连接。
在具体实施过程中，耐压可视腔设置有放空阀23，其输出端与数据采集与处理系统8电性连接，方便进行腔内的压力调节。为了保护载物芯片21在耐压可视腔内不受损坏，耐压可视腔设置有环壁高压腔，在耐压可视腔的外环同时增压，并且设置有环压控制系统5，根据耐压可视腔压力变化自动增减环壁高压腔的压力，实现耐压可视腔和环壁高压腔的压力平衡，保证载物芯片21承受最小的压力差，而不受破坏。
在具体实施过程中，所述环壁温度控制系统3采用循环制冷/热装置31和温度传感器32；所述循环制冷/热装置31控制端与所述数据采集与处理系统8电性连接，用于制冷/热并使环壁载冷/热腔22内的载冷/热流体循环流动；所述温度传感器32探头设置在耐压可视腔内，其信号输出端与所述数据采集与处理系统8电性连接。
在具体实施过程中，耐压可视腔的温度主要是通过在环壁载冷/热腔22内注 入载冷/热流体，并且通过将流体进行循环制冷或者加热保证环壁载冷/热腔22内流体的低温或者高温状态，然后通过载冷/热流体与耐压可视腔内的热交换保证耐压可视腔内的低温或者高温状态。
更具体的，所述增压系统4包括空气压缩机41、增压泵42、储气罐43、调压阀44和压力传感器45；所述空气压缩机41、增压泵42、储气罐43、调压阀44依次连接后，与所述耐压可视腔、环壁高压腔分别连接；所述压力传感器45探头设置在耐压可视腔内，其信号输出端与所述数据采集与处理系统8电性连接。
在具体实施过程中，温度传感器32、压力传感器45的设置，用于在整个微生物分选过程中，对耐压可视腔的温度和压力进行测量和监控。
更具体的，所述环压控制系统5包括环压检测装置51、第一回压跟踪泵52、回压检测装置53、回压阀54、缓冲罐55和第二回压跟踪泵56；其中：所述环压检测装置51探头设置在所述环壁高压腔内，其输出端与所述数据采集与处理系统8电性连接；所述回压跟踪泵与所述环壁高压腔连接，其控制端与所述数据采集与处理系统8电性连接；所述回压检测装置53检测端与所述耐压可视腔连接，其信号输出端与所述数据采集与处理系统8电性连接；所述回压阀54一端与所述自动分选系统7连接，另一端通过缓冲罐55与第二回压跟踪泵56连接；所述第二回压跟踪泵56控制端与所述数据采集与处理系统8电性连接。
更具体的，所述光学识别系统6采用光谱/光学观察模块，在富集微生物微通过载物芯片21时，通过光谱/光学观察模块对微生物进行观察和识别，并将识别结果发送至所述数据采集与处理系统8。
在富集的微生物通过载物芯片21的过程中，利用光谱/光学观察模块对微生物进行观察和识别。可以通过高分辨光学显微镜在芯片上方观测对单细胞的形态进行识别，以及通过拉曼光谱对胞内的标志生物化合物进行识别，结合光学和光谱学识别信号，可判定芯片中的微生物是否为研究人员需要的目标微生物。
更具体的，所述自动分选系统7包括智能控制三通模块71、目标微生物存储模块72和非目标微生物存储模块7372；其中，目标微生物存储模块72、非目标微生物存储模块7372分别连接在智能控制三通模块71的两个连接端上，智能控制三通模块71另一个连接端与所述耐压可视分选舱2出口端连接；智能控制三通模块71控制端与所述数据采集与处理系统8电性连接。
在具体实施过程中，在耐压可视腔的出口端设置有自动分选系统7，对识别的微生物进行定向分选。自动分选系统7主要是通过智能控制三通模块71控制，智能控制三通模块71为自动启闭的三通，当识别的单细胞被判定为目标微生物时，目标微生物存储模块72通路的阀门打开，单细胞进入目标微生物存储模块72。当识别的单细胞被判定为非目标微生物时，则开启目标微生物存储模块72通路，该细胞进入目标微生物存储模块72，以此达到高通量单细胞分选的目的。目标微生物存储模块72可根据实验需要，选择常压容器或高压容器，容器内均装有相应培养基，满足分选后的微生物继续培养的需求。
实施例2
更具体的，如图3所示，本方案还提供一种海洋原位环境单细胞高通量分选方法，采用一种海洋原位环境单细胞高通量分选装置实现，具体包括以下步骤：
S1：根据富集微生物注入系统1的压力值确定耐压可视分选舱2内的压力值；通过增压系统4往耐压可视腔内注入气体，使得耐压可视腔内的压力值与富集微生物注入系统1一致；
S2：打开环压控制系统5，使得耐压可视腔与环壁高压腔的压力一致；
S3：根据富集微生物注入系统1内的温度值确定耐压可视腔内的温度值，通过开启环壁温度控制系统3，使得环壁载冷/热腔22内的温度值与耐压可视腔内的温度值一致；
S4：对光学识别系统6进行调试，使其能清楚的观测到载物芯片21内的情况；
S5：将含微生物的菌液从富集微生物注入系统1通过微流泵11注入耐压可视腔中，使得菌液缓慢通过载物芯片21，使其能以单细胞的形式在刻蚀通道内通过；打开环壁温度控制系统3，保持液体流出耐压可视腔时，耐压可视腔出口端压力恒定；
S6：在菌液通过载物芯片21过程中，通过光学识别系统6充分观测采集细胞的形态，以及胞内和胞外代谢化合物的光谱学信息，判断该细胞是否为目标微生物，并将识别结果发送至数据采集与处理系统8；
S7：自动分选系统7根据识别结果智能开启智能控制三通模块71，将目标微生物送至目标微生物存储模块72中，将非目标微生物送至非目标微生物存储模块7372中；
S8：待目标微生物存储模块72中的目标微生物数量符合，结束分选过程。
更具体的，在执行步骤S1之前，还需要对海洋原位环境单细胞高通量分选装置进行预处理，具体为：打开耐压可视腔的出口端，通过泵入去离子水对耐压可视腔进行反复清洗；待冲洗干净后，泵入75％酒精；待耐压可视腔内酒精完全注满后，关闭耐压可视腔，静置24小时，再将耐压可视腔中的酒精放空即完成预处理。
在整个分选过程中，保持耐压可视腔内的压力、温度值与微生物最初所在的富集微生物注入系统1内的压力、温度环境一致，使得微生物在原位高压情况内实现分选。在分选过程中，打开环压控制系统5，根据耐压可视腔内的压力值变化保持环壁高压腔内的压力值与其一致，使得载物芯片21不承受压差，不产生形变和破坏。
本实施例提出的海洋微生物高压环境高通量单细胞分选芯片及分选技术，能够实现海洋高压环境下，微生物的识别和分选，满足后续纯化培养的需求。相比目前传统的常压环境富集、分离海洋微生物的技术，可有效的解决海洋耐压菌、嗜压菌在常压环境下存活率低，且深海土著特征在常压环境下不能有效表达等难题，解决目前海洋微生物培养度低，难培养纯菌的问题。同时，本方案可以实现高压环境下，单细胞尺度的高通量识别和自动分选，相比常规的微生物分离培养技术，有效的提高了微生物培养、纯化的效率。
实施例3
更具体的，为了进一步说明本方案的技术实现过程和技术效果，本实施例涉及的深海嗜甲烷菌的高通量单细胞分选的高压环境的微流控芯片可以实现在原位的高压环境下对富集的深海嗜甲烷菌进行高通量的单细胞分选，满足后续的培养及功能坚定等工作。本实例的核心是耐高压且可视的耐压可视分选舱2。其它部分主要包括增压系统4、环压控制系统5、光学识别系统6、自动分选系统7和数据采集与处理系统8。
核心部件耐压可视分选舱2主要包括耐压可视腔、载物芯片21、环壁载冷/热腔22和环壁高压腔。耐压可视腔由耐压和防腐钛合金材料制成，腔体的正面和背面镶嵌耐压可视蓝宝石材料，整个可承受5000米水深的压力。耐压可视腔的中央设有载物芯片21，载物芯片21上设置有微流体通道，将含微生物的菌液从高压微生物富集培养室12通过微流泵11注入耐压可视腔，使得菌液缓慢通过 载物芯片21，以及从增压系统4注入甲烷。在富集微生物注入系统1和耐压可视分选舱2之间设置有进口压力检测装置13。耐压可视腔出口端主要是用于分选后的含深海嗜甲烷菌富集液的流体离开耐压可视腔进入自动分选系统7。在出口端设置环压控制系统5进行回压控制，主要包括回压检测装置53、回压阀54、缓冲罐55和第二回压跟踪泵56，保证含微生物流体在设定压力条件下流出分选系统，在整个分选过程中，耐压可视腔内的压力保持恒定。耐压可视腔设置有放空阀23，方便进行腔内的压力调节。耐压可视腔设置有温度传感器32和压力传感器45，对深海嗜甲烷菌分选过程中，腔内的温度和压力进行测量和监控。载物芯片21由可视材料做成，内嵌有刻蚀微流体通道，便于富集的深海嗜甲烷菌液进入耐压可视分选腔后在芯片内以较小的流速通过，实现单细胞在通道内进行分散通过。为了保护载物芯片21在耐压可视腔内不受损坏，耐压可视腔设置有环壁高压腔，在耐压可视腔的外环同时增压，并且设置有环压控制系统5，根据耐压可视腔压力变化自动增减环壁高压腔的压力，实现耐压可视腔和环壁高压腔的压力平衡，保证载物芯片21承受最小的压力差，而不受破坏。耐压可视腔的温度主要是通过在环壁载冷/热腔22内注入载冷/热流体，如含乙二醇的制冷溶液，并且通过将流体进行循环制冷/热装置31保证环壁腔内流体保持4℃低温状态，然后通过载冷流体与耐压可视腔内的热交换保证耐压可视腔内的低温状态。
在富集的微生物通过分选芯片的过程中，利用光谱/光学观察模块对微生物进行观察和识别。如可以通过高分辨光学显微镜在芯片上方观测对单细胞的形态进行识别，以及通过拉曼光谱对胞内的标志生物化合物进行识别，结合光学和光谱学识别信号，可判定芯片中的微生物是否为深海嗜甲烷菌。在耐压可视腔的出口设置有自动分选系统7，对识别的微生物进行定向分选。自动分选系统上设置有智能控制三通模块71，智能控制三通模块71是自动启闭的三通，当识别的单细胞被判定为深海嗜甲烷菌时，目标微生物存储模块72通路的阀门打开，单细胞进入目标微生物存储模块72。当识别的单细胞被判定为非深海嗜甲烷菌时，则开启非目标微生物存储模块73收集通路，该细胞进入非目标微生物存储模块73，以此达到高通量单细胞分选的目的。目标微生物存储模块72可以为常压容器，也可以为高压容器，满足分选后的深海嗜甲烷菌继续在高压环境内培养的需求。
本实例涉及的高通量的海洋微生物高压环境单细胞分选技术主要是需要在耐压可视腔内构建与深海嗜甲烷菌在深海环境中生活一样的高压环境。首先清洗 耐压可视腔，打开进出口，泵入去离子水反复冲洗，待冲洗干净后，泵入75％酒精，待耐压可视腔内酒精完全注满后，关闭耐压可视腔，静置24小时，放空。然后根据富集微生物注入系统1内的初始压力值12MPa确定耐压可视腔内的压力值，通过增压系统4，往耐压可视腔内注入CH 4气体，使耐压可视腔内的压力增至12MPa；并打开环压控制系统5，使得环壁高压腔内的压力值与耐压可视腔内的压力一致。在分选过程中，倘若耐压可视腔内有压力变化，通过第一回压跟踪泵52向环壁高压腔内注入气体，或者打开阀门泄压，使得环壁高压腔的压力与耐压可视腔的压力保持一致。然后，根据富集微生物注入系统1内的温度值4℃确定耐压可视腔内的温度值，通过开启环壁温度控制系统3，使得环壁载冷/热腔内的温度值与耐压可视腔内的温度一致。然后，调试好光谱/光学观察模块，使其能清楚的观测到载物芯片21内情况。然后将含深海噬甲烷菌的菌液从富集微生物注入系统1通过微流泵11注入耐压可视腔，并且打开出口回压，设定出口压力为11.5MPa，使得菌液缓慢通过载物芯片21，使其能以单细胞的形式在刻蚀通道内通过，在菌液通过载物芯片21的过程中，开启谱/光学观察模块，充分观测采集细胞的形态，以及单细胞微生物的光谱学信息，判定该细胞是否为嗜甲烷菌，若为嗜甲烷菌，开启自动控制分选系统7的阀门，使其进入嗜甲烷菌收集模块，倘若不是，进入非目标菌收集模块。当富集微生物注入系统1内的流体全部被分选识别后，分选过程结束。在整个分选过程中，保持耐压可视腔内的压力、温度值与深海嗜甲烷菌最初所在的富集微生物注入系统1内的压力、温度环境一致，使得微生物在原位高压情况内实现分选。在分选过程中，打开环压控制系统5，根据耐压可视腔内的压力值变化保持环壁高压腔内的压力值与其一致，使得载物芯片21不承受压差，不产生形变和破坏。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (10)

  1. 一种海洋原位环境单细胞高通量分选装置，其特征在于，包括富集微生物注入系统(1)、耐压可视分选舱(2)、环壁温度控制系统(3)、增压系统(4)、环压控制系统(5)、光学识别系统(6)、自动分选系统(7)和数据采集与处理系统(8)；其中：
    所述富集微生物注入系统(1)用于培养并向耐压可视分选舱(2)注入含微生物的菌液；
    所述耐压可视分选舱(2)内设置有载物芯片(21)，所述载物芯片(21)由可视材料制成，内嵌由刻蚀微流体通道，用于实现富集微生物在通道内分散通过；富集微生物微通过载物芯片(21)时，通过光学识别系统(6)对微生物进行观察和识别；
    耐压可视分选舱(2)出口端与所述自动分选系统(7)连接，自动分选系统(7)根据光学识别系统(6)对微生物识别的结果对微生物进行自动分选；
    所述环壁温度控制系统(3)用于保证耐压可视分选舱(2)内部温度一致；
    所述增压系统(4)用于令耐压可视分选舱(2)内部压力与所述富集微生物注入系统(1)内部压力一致；
    所述环压控制系统(5)用于根据自动分选系统(7)内压力值变化保持耐压可视分选舱(2)内部压力与其一致，避免载物芯片(21)承受压差产生形变或破坏；
    所述富集微生物注入系统(1)、环壁温度控制系统(3)、增压系统(4)、环压控制系统(5)、光学识别系统(6)、自动分选系统(7)均与所述数据采集与处理系统(8)电性连接。
  2. 根据权利要求1所述的一种海洋原位环境单细胞高通量分选装置，其特征在于，所述富集微生物注入系统(1)包括微流泵(11)、高压微生物富集培养室(12)和进口压力检测装置(13)；其中：
    所述微流泵(11)控制端与所述数据采集与处理系统(8)电性连接；
    所述微流泵(11)输入端与所述高压微生物富集培养室(12)出液端连接，输出端通过进口压力检测装置(13)与所述耐压可视分选舱(2)入口端连接；
    所述高压微生物富集培养室(12)用于培养含微生物的菌液，并通过所述微 流泵(11)注入耐压可视分选舱(2)中。
  3. 根据权利要求1所述的一种海洋原位环境单细胞高通量分选装置，其特征在于，所述耐压可视分选舱(2)还包括耐压可视腔、环壁载冷/热腔(22)和环壁高压腔；其中：
    耐压可视腔由耐压和防腐金属材料制成，其正面和背面镶嵌耐压可视材料，其与增压系统(4)连接；
    所述载物芯片(21)设置在所述耐压可视腔中央；载物芯片(21)的微流体通道入口与所述富集微生物注入系统(1)连接，其出口为耐压可视分选舱(2)的出口端，与所述自动分选系统(7)连接；
    所述环壁高压腔设置在耐压可视腔外环，用于保护载物芯片(21)在耐压可视腔中不受损坏；所述环壁高压腔与增压系统(4)、环压控制系统(5)连接；
    所述环壁载冷/热腔(22)包裹在耐压可视腔外壁，用于装载载冷/热流体，并通过载冷/热流体与环壁温度控制系统(3)连接。
  4. 根据权利要求3所述的一种海洋原位环境单细胞高通量分选装置，其特征在于，所述环壁温度控制系统(3)采用循环制冷/热装置(31)和温度传感器(32)；所述循环制冷/热装置(31)控制端与所述数据采集与处理系统(8)电性连接，用于制冷/热并使环壁载冷/热腔(22)内的载冷/热流体循环流动；所述温度传感器(32)探头设置在耐压可视腔内，其信号输出端与所述数据采集与处理系统(8)电性连接。
  5. 根据权利要求3所述的一种海洋原位环境单细胞高通量分选装置，其特征在于，所述增压系统(4)包括空气压缩机(41)、增压泵(42)、储气罐(43)、调压阀(44)和压力传感器(45)；所述空气压缩机(41)、增压泵(42)、储气罐(43)、调压阀(44)依次连接后，与所述耐压可视腔、环壁高压腔分别连接；所述压力传感器(45)探头设置在耐压可视腔内，其信号输出端与所述数据采集与处理系统(8)电性连接。
  6. 根据权利要求3所述的一种海洋原位环境单细胞高通量分选装置，其特征在于，所述环压控制系统(5)包括环压检测装置(51)、第一回压跟踪泵(52)、回压检测装置(53)、回压阀(54)、缓冲罐(55)和第二回压跟踪泵(56)；其中：
    所述环压检测装置(51)探头设置在所述环壁高压腔内，其输出端与所述数 据采集与处理系统(8)电性连接；所述回压跟踪泵(52)与所述环壁高压腔连接，其控制端与所述数据采集与处理系统(8)电性连接；
    所述回压检测装置(53)检测端与所述耐压可视腔连接，其信号输出端与所述数据采集与处理系统(8)电性连接；
    所述回压阀(54)一端与所述自动分选系统(7)连接，另一端通过缓冲罐(55)与第二回压跟踪泵(56)连接；
    所述第二回压跟踪泵(56)控制端与所述数据采集与处理系统(8)电性连接。
  7. 根据权利要求3所述的一种海洋原位环境单细胞高通量分选装置，其特征在于，所述光学识别系统(6)采用光谱/光学观察模块，在富集微生物微通过载物芯片(21)时，通过光谱/光学观察模块对微生物进行观察和识别，并将识别结果发送至所述数据采集与处理系统(8)。
  8. 根据权利要求3所述的一种海洋原位环境单细胞高通量分选装置，其特征在于，所述自动分选系统(7)包括智能控制三通模块(71)、目标微生物存储模块(72)和非目标微生物存储模块(73)；其中，目标微生物存储模块(72)、非目标微生物存储模块(73)分别连接在智能控制三通模块(71)的两个连接端上，智能控制三通模块(71)另一个连接端与所述耐压可视分选舱(2)出口端连接；智能控制三通模块(71)控制端与所述数据采集与处理系统(8)电性连接。
  9. 一种海洋原位环境单细胞高通量分选方法，其特征在于，采用如权利要求1～8所述的一种海洋原位环境单细胞高通量分选装置实现，具体包括以下步骤：
    S1：根据富集微生物注入系统(1)的压力值确定耐压可视分选舱(2)内的压力值；通过增压系统(4)往耐压可视腔内注入气体，使得耐压可视腔内的压力值与富集微生物注入系统(1)一致或者存在微小压差；
    S2：打开环压控制系统(5)，使得耐压可视腔与环壁高压腔的压力一致；
    S3：根据富集微生物注入系统(1)内的温度值确定耐压可视腔内的温度值，通过开启环壁温度控制系统(3)，使得环壁载冷/热腔(22)内的温度值与耐压可视腔内的温度值一致；
    S4：对光学识别系统(6)进行调试，使其能清楚的观测到载物芯片(21)内的情况；
    S5：将含微生物的菌液从富集微生物注入系统(1)通过微流泵(11)注入耐压可视腔中，使得菌液缓慢通过载物芯片(21)，使其能以单细胞的形式在刻蚀通道内通过；打开环壁温度控制系统(3)，保持液体流出耐压可视腔时，耐压可视腔出口端压力恒定；
    S6：在菌液通过载物芯片(21)过程中，通过光学识别系统(6)充分观测采集细胞的形态，以及单细胞微生物的光谱学信息，判断该细胞是否为目标微生物，并将识别结果发送至数据采集与处理系统(8)；
    S7：自动分选系统(7)根据识别结果智能开启智能控制三通模块(71)，将目标微生物送至目标微生物存储模块(72)中，将非目标微生物送至非目标微生物存储模块(73)中；
    S8：待目标微生物存储模块(72)中的目标微生物数量符合，结束分选过程。
  10. 根据权利要求9所述的一种海洋原位环境单细胞高通量分选方法，其特征在于，在执行步骤S1之前，还需要对海洋原位环境单细胞高通量分选装置进行预处理，具体为：
    打开耐压可视腔出口端，通过泵入去离子水对耐压可视腔进行反复清洗；待冲洗干净后，泵入75％酒精；待耐压可视腔内酒精完全注满后，关闭耐压可视腔，静置24小时，再将耐压可视腔中的酒精放空即完成预处理。
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