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WO2023173495A1 - 高压环境海洋微生物富集培养与重力式分离装置 - Google Patents

高压环境海洋微生物富集培养与重力式分离装置 Download PDF

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Publication number
WO2023173495A1
WO2023173495A1 PCT/CN2022/084121 CN2022084121W WO2023173495A1 WO 2023173495 A1 WO2023173495 A1 WO 2023173495A1 CN 2022084121 W CN2022084121 W CN 2022084121W WO 2023173495 A1 WO2023173495 A1 WO 2023173495A1
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culture
enrichment
liquid
pressure
separation
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PCT/CN2022/084121
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English (en)
French (fr)
Inventor
冯景春
张偲
杨志峰
王屹
蔡宴朋
钟松
Original Assignee
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 US18/020,266 priority Critical patent/US20240124827A1/en
Publication of WO2023173495A1 publication Critical patent/WO2023173495A1/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
    • 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
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • 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
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/20—Degassing; Venting; Bubble traps
    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/22—Settling tanks; Sedimentation by gravity
    • 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
    • C12M41/48—Automatic or computerized control
    • 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
    • C12M47/12—Purification

Definitions

  • the invention relates to the technical field of marine microorganisms, and in particular to a high-pressure environment marine microorganism enrichment culture and gravity separation device.
  • Marine microbial resources are important strategic resources in the world and have the largest biodiversity. Technologies such as molecular ecology and metagenomics have significantly increased our understanding of marine microbial diversity. However, currently 99% of marine microorganisms have not been purely cultured, and many genes with unknown functions have not been annotated or may have been incorrectly annotated in the database. Therefore, modern omics technology is not enough to provide enough information to understand all microorganisms, especially are microorganisms that have not yet been classified. Improving the culturability of uncultured microorganisms and innovating pure culture methods are important ways to explain the phenotypes and genotypes of these microorganisms.
  • the present invention provides a high-pressure environment marine microorganism enrichment culture and gravity separation device in view of the extreme environmental characteristics of marine microbial life, and carries out the enrichment culture and gravity separation of marine microorganisms by reshaping its in-situ environment. Separation can effectively improve the culturability of marine microorganisms and provide an important basic means for the development and utilization of deep-sea microbial resources.
  • This solution provides a high-pressure environment marine microorganism enrichment culture and gravity separation device, including an enrichment and multi-level purification unit, a gravity separation culture unit, a temperature control system, a pressure control system and a central control system; enrichment and multi-level
  • the control end and signal detection end of the purification unit and the gravity separation and culture unit are all electrically connected to the central control system; the control ends of the temperature control system and the pressure control system are all electrically connected to the central control system; among them:
  • the enrichment and multi-level purification unit is used to realize the enrichment and multi-level purification process of marine microorganisms, obtain the marine microorganism-enriched bacterial liquid and inject the marine microorganism-enriched bacterial liquid into the gravity separation and culture unit;
  • the gravity-type separation and culture unit is used to automatically draw lines using gravity in a high-pressure environment to achieve solid separation and culture of marine microorganisms, effectively improving the cultivability of marine microorganisms;
  • the temperature control system and the pressure control system are respectively related to
  • the enrichment and multi-level purification unit and gravity separation and culture unit are connected to build a high-pressure and low-temperature environment consistent with the marine environment in the enrichment and multi-level purification unit and gravity separation and culture unit to ensure enriched deep sea Microorganisms are enriched, purified, isolated and cultured under in situ environmental conditions.
  • the enrichment and multi-level purification unit can realize the enrichment and culture of microorganisms under the in-situ temperature and pressure environmental conditions of the ocean, and the gravity separation and culture unit can realize the isolation and cultivation of marine microorganisms.
  • the gravity separation and culture unit can realize the isolation and cultivation of marine microorganisms.
  • the central control system is used to monitor changes in various environmental data information during the enrichment, separation, and purification of microbial enrichment bacteria in a high-pressure environment, as well as real-time collection, processing, storage, and image output.
  • the high-pressure environment theoretically considers the pressure environment with a deep seawater depth greater than 200 meters to be a high-pressure environment
  • the low-temperature environment theoretically considers the deep seawater temperature environment to be lower than 4°C to be a low-temperature environment.
  • a corresponding high-pressure and low-temperature environment is constructed based on the marine microorganism culture environment required to be cultured.
  • the enrichment and multi-level purification unit is composed of a plurality of microbial liquid enrichment culture chambers connected in series; the microbial liquid enrichment culture chamber is provided with a removable sealing cover and a connected sampling valve group, and a sensor is provided inside it Group; each microbial liquid enrichment culture chamber is placed in a temperature control system; wherein: the detachable sealing cover is used to facilitate the sterilization operation and placement of culture substrate inside the microbial liquid enrichment culture chamber; the The connection sampling valve group is used for connecting and sampling each microbial liquid enrichment culture chamber. It is connected to the pressure control system and is used to input liquid or gas into the microbial liquid enrichment culture chamber to increase the pressure in the microbial liquid enrichment culture chamber.
  • the sensor group is used to real-time the temperature and pressure changes in the microbial liquid enrichment culture chamber, and transmits signals to the central control system;
  • the The last microbial liquid enrichment culture chamber of the enrichment and multi-level purification unit is connected to the gravity separation culture unit through a sampling valve group.
  • the microbial liquid enrichment culture chamber is also provided with a stirring rod, and the stirring rod is used to enhance the reaction process of the matrix during the cultivation process of the microbial liquid enrichment culture chamber.
  • the stirring rod is a manual stirring rod, which can enhance mass transfer through intermittent manual stirring.
  • An enhanced continuous or intermittent stirring rod can also be placed as needed to enhance the reaction process of the matrix during the culture process. Increase the energy and nutrient supply of microorganisms and improve culture efficiency.
  • the sensor group includes a temperature sensor and a pressure sensor; the temperature sensor is used for real-time monitoring of temperature changes in the microbial liquid enrichment culture chamber; and the pressure sensor is used for real-time monitoring of pressure changes in the microbial liquid enrichment culture chamber. ;
  • the temperature sensor signal output end and the pressure sensor signal output end are both electrically connected to the central control system.
  • connection sampling valve group includes a liquid inlet valve, an air inlet valve, a sampling valve and a liquid outlet valve; wherein: the microbial liquid enrichment culture chambers are connected in series through the liquid outlet valve and liquid inlet valve, and the previous stage The liquid outlet valve of the microbial liquid enrichment culture chamber is connected to the liquid outlet valve of the subsequent microbial liquid enrichment culture room; the liquid outlet valve of the last microbial liquid enrichment culture room is connected to the gravity separation culture unit; the air inlet valve is connected to the gravity separation culture unit.
  • the pressure control system connection is used to input gas into the microbial liquid enrichment culture chamber to increase the pressure in the microbial liquid enrichment culture chamber, so that the pressure value in the microbial liquid enrichment culture chamber is consistent with the actual situation in the deep sea;
  • the sampling valve is used to measure
  • the microorganisms in the microbial liquid enrichment culture room are sampled and analyzed in real time in order to adjust the corresponding environmental parameters and optimize the enrichment culture process.
  • multiple microbial liquid enrichment culture chambers are connected in series to form an enrichment and multi-level purification unit.
  • the bacterial liquid in the front-stage microbial liquid enrichment culture chamber is transferred to the next-level microbial liquid enrichment culture chamber by maintaining pressure.
  • the microbial liquid obtained in the last microbial liquid enrichment culture chamber will provide enriched and highly purified functional microorganisms under stress in a high-pressure environment and directional nutritional conditions.
  • the pressure-maintaining transfer can be realized by using a microinjection pump to take out the enriched liquid in the front-stage microbial liquid enrichment culture chamber through the sampling valve and then pump it into the subsequent stage microbial liquid enrichment culture chamber.
  • the microbial enrichment liquid will automatically flow from the front-stage microbial liquid enrichment culture chamber into the rear-stage microbial liquid enrichment culture chamber for dilution and culture under the condition of slight pressure difference.
  • the temperature and pressure environmental conditions in the microbial liquid enrichment culture chamber are consistent with the environmental conditions of the microorganisms in the deep sea, ensuring the effectiveness of the enrichment culture.
  • the gravity separation and culture unit includes a separation culture chamber, a liquid injection unit and an environmental parameter detection unit; wherein: the gravity separation and culture unit communicates with the last microbial liquid of the enrichment and multi-level purification unit through the liquid injection unit
  • the enrichment culture chamber is connected;
  • the separation culture chamber is equipped with a microbial separation branch, which is used to separate microorganisms and maximize the area for microbial culture;
  • the top of the separation culture chamber is provided with a central liquid injection pipe for separation and culture
  • the chamber is connected to the liquid injection unit through a central liquid injection pipe;
  • the liquid injection unit injects marine microorganism-enriched bacteria liquid into the microbial separation branch of the separation culture chamber;
  • the separation culture chamber is placed in a temperature control system and is connected to the pressure control system.
  • System connection is used to construct a high-pressure and low-temperature environment consistent with the marine environment in the isolation and cultivation chamber to ensure that the enriched deep-sea microorganisms are isolated and cultured under in-situ environmental conditions;
  • the environmental parameter detection unit is used to detect the separation and cultivation chamber in real time changes in temperature and pressure, and transmits the detected data to the central control system;
  • the microbial separation branch and the liquid injection unit are electrically connected to the central control system.
  • the microorganism separation branch includes a movable liquid storage tank, a small ball, a reciprocating pull rod, a guide groove and a cavity; wherein: the movable liquid storage tank is arranged in the center of the top of the cavity and falls in the center Directly below the liquid injection pipe, it is used to store the marine microorganism-enriched bacterial liquid injected by the liquid injection unit; the small ball is placed in the movable liquid storage tank and is submerged by the injected marine microorganism-enriched bacterial liquid; The bottom of the movable liquid storage tank is provided with a through hole, which is used to fix the position of the small ball and ensure that the small ball can pass through it; the movable end of the reciprocating pull rod is fixedly connected to the movable liquid storage tank; the cavity A gear box is provided on the side of the top of the body; the control end of the reciprocating pull rod is electrically connected to the central control system; the diversion trough is fixedly installed inside the cavity; the diversion trough is filled with
  • a small slot hole is provided in the center of the top of the cavity, where the initial position of the movable liquid reservoir coincides with the through hole, for fixing the position of the small ball.
  • the liquid injection unit includes a micro-injection pump and a liquid injection pipeline; wherein: the liquid inlet end of the micro-injection pump is connected to the last microbial liquid enrichment culture chamber of the enrichment and multi-level purification unit, and its liquid outlet end It is connected to the liquid injection pipe; the liquid injection pipe outlet is connected to the central liquid injection pipe; the micro-injection pump control end is electrically connected to the central control system.
  • the environmental parameter detection unit includes a second temperature sensor and a second pressure sensor; wherein: the second temperature sensor and the second pressure sensor probe are arranged inside the separation culture chamber, and their signal output terminals are connected to the The central control system is electrically connected.
  • the temperature and pressure in the microbial liquid enrichment culture chamber and the separation culture chamber are monitored in real time through the temperature sensor, the pressure sensor, the second temperature sensor and the second pressure sensor respectively; if the temperature/pressure needs to be adjusted, then The temperature control system and pressure control system are controlled by the central control system to work, and the temperature and pressure values in the microbial liquid enrichment culture chamber and separation culture chamber are always consistent with the marine environmental conditions where the microorganisms grow.
  • the top of the separation culture chamber is provided with a quick-opening kettle lid;
  • the central liquid injection pipe is arranged on the quick-opening kettle lid;
  • the quick-opening kettle lid is also provided with a gas injection channel and a sensor placement channel;
  • the pressure control The system is connected to the separation culture chamber through an air injection channel; the second temperature sensor and the second pressure sensor are arranged in the sensor placement channel.
  • the separation culture chamber utilizes the principle of natural release of gravity, and small balls are used to carry the bacterial liquid to move freely under the action of gravity in the separation culture chamber to achieve effective separation of microorganisms on the solid culture medium.
  • the separation culture chamber is designed with a top quick-opening kettle lid to facilitate and quickly load samples into the culture chamber.
  • this solution uses a circular table or cylindrical cavity, which maximizes the area for microbial isolation and culture in a limited space.
  • a rotating serpentine guide groove is provided from top to bottom in the cavity, and the guide groove is flatly filled with a solid culture medium of culture substrate to provide nutrients for the isolation and culture of microorganisms.
  • a movable liquid storage tank is placed on the top of the truncated cone/cylindrical cavity to store the microbial enrichment liquid and pellets to be separated.
  • the diameter of the guide trough is larger than that of the pellets, ensuring that the pellets released from the movable liquid storage tank can enter the guide trough completely and smoothly.
  • a central liquid injection pipe is provided in the center of the quick-opening kettle lid for injecting microbial enrichment liquid into the movable liquid storage tank.
  • the center of the bottom of the movable liquid storage tank is provided with a hole slightly larger in diameter than the pellet.
  • a reciprocating pull rod is provided on the side wall of the separation culture chamber, and the pull rod can realize reciprocating push-pull movement through control methods such as a reciprocating piston.
  • the reciprocating pull rod can be used to drag the movable liquid storage tank from the center to the edge of the cavity. Since the bottom diameter of the movable liquid storage tank is larger than the ball, the small ball will be released from the central hole at the bottom of the movable liquid storage tank and enter the diversion tank. , and then spirally descend from top to bottom in the diversion trough under the action of gravity, and migrate to the bottom of the cavity.
  • the microbial enrichment solution is diluted and dispersed in a concentration gradient in the diversion tank, meeting the requirements for the dispersed growth of single colonies.
  • each isolation and culture chamber is connected to an independent environmental parameter detection unit, using Changes in temperature and pressure in each separation culture chamber are detected in real time, and the detected data are transmitted to the central control system.
  • the microbial separation process involved in the above scheme mainly involves placing several separation and culture chambers in parallel.
  • the inlets of all separation and culture chambers are connected in parallel through pipelines and connected to the microinjection pump and the last-stage microbial liquid enrichment culture chamber.
  • different culture chambers can be placed with different formulas of culture media.
  • the pressure control system includes a purge valve, an air compressor, a booster pump, a gas storage tank, a pressure regulating valve, a regulating valve and a vent pipe; wherein: the purge valve communicates with the enrichment and enrichment system through a vent pipe.
  • the multi-level purification unit and the gravity separation and culture unit are connected, and the control end thereof is electrically connected to the central control system for discharging the gas from the enrichment and multi-level purification unit and the gravity separation and culture unit.
  • the purification unit and the gravity separation and culture unit are depressurized inside; the air compressor, booster pump, gas storage tank, pressure regulating valve, and regulating valve are connected in sequence through the ventilation pipeline, and finally are connected to the enrichment and multi-organizer through the ventilation pipeline.
  • the hierarchical purification unit and the gravity separation and culture unit are connected to inject the gas (or inert gas) required for culture into the enrichment and multi-level purification unit and the gravity separation and culture unit for pressurization;
  • the pressure regulating valve is used for Adjust the internal pressure of the enrichment and multi-level purification unit and the gravity separation and culture unit.
  • the regulating valve is used to adjust the speed of the injected gas; the air compressor control end, the booster pump control end, the pressure regulating valve control end, The control ends of the regulating valve are all electrically connected to the central control system.
  • the temperature control system includes a water bath jacket and a refrigeration/heating device; the water bath jacket is wrapped around the outer wall of the enrichment and multi-level purification unit, the gravity separation and culture unit, and the refrigeration/heating device Connection; the control end of the refrigeration/heating device is electrically connected to the central control system.
  • the constant temperature conditions in the microbial liquid enrichment culture chamber and the separation culture chamber are mainly maintained by placing the microbial liquid enrichment culture chamber and the separation culture chamber in the high/low temperature water bath of the water bath jacket and through the temperature sensor and the second temperature
  • the sensor monitors and displays, and maintains a constant temperature state in the culture room through heat exchange with the refrigeration/heating device.
  • the temperature conditions of the microbial liquid enrichment culture room and separation culture room are mainly controlled through the temperature control system.
  • a cold/hot fluid is injected into the ring wall cavity of a microbial liquid enrichment culture chamber or a separation culture chamber, and the fluid is circulated, refrigerated or heated to ensure the low or high temperature state of the fluid in the ring wall cavity, and then the fluid is circulated through the cold wall cavity.
  • the heat exchange between the hot fluid and the built-in cavity ensures the low or high temperature state in the built-in cavity.
  • microbial liquid enrichment culture room and separation culture room in a low/high temperature water bath/oil bath environment to ensure the special temperature conditions required in the microbial liquid enrichment culture room and separation culture room.
  • the high-pressure environment marine microorganism enrichment culture and gravity separation device also includes a mobile platform, where the enrichment and multi-level purification unit, gravity separation culture unit, temperature control system, pressure control system, and central control system are all placed On the mobile platform, it is used to increase the universality of training scenarios.
  • the high-pressure and extreme temperature environmental conditions in which microorganisms live in the marine environment are constructed in the microbial liquid enrichment culture chamber and the separation culture chamber to improve their survival activity.
  • multi-stage enrichment and liquid dilution culture functional microorganisms with higher purity under the stress of directional environmental conditions are obtained.
  • culture and separation are performed to obtain single microorganisms.
  • multiple sets of selected microbial liquid enrichment culture chambers and separation culture chamber combinations can be used to form an efficient separation process with different culture medium combinations.
  • the high-pressure environment marine microorganism enrichment culture and separation technology involved in the device proposed in this plan mainly includes two steps: enrichment and separation.
  • enrichment and separation First, through enrichment and multi-level purification unit enrichment and culture, after obtaining higher purity bacterial flora, it enters the gravity separation culture unit under pressure maintenance for solid culture separation, and through a combination of different culture media and environmental conditions Screen at the same time to obtain pure culture strains.
  • the specific implementation principle is:
  • Enrichment process First, the microbial liquid enrichment culture chamber and its attached pipes and valves are sterilized, and then the substrates to be cultured, such as deep-sea sediments, macroorganism tissues symbiotic with microorganisms, and extracts, are sequentially loaded. Then fill the nutrient solution needed for culture from the liquid inlet valve, and then inject the gas needed for culture from the air inlet valve (inert gas can be injected if not needed) to increase the pressure value in the microbial liquid enrichment culture chamber to be consistent with the actual environmental conditions of the deep sea. . During the culture process, stirring is performed through the manual stirring rod on the top to increase mass transfer and optimize the culture process.
  • the nutrient solution required for culture is injected into the second-level microbial liquid enrichment culture chamber, and air is injected into the second-level microbial liquid enrichment culture chamber through the pressure control system.
  • the amount of culture liquid injected into the second-level microbial liquid enrichment culture chamber needs to ensure that the dilution ratio of the enrichment liquid from the concentration of the first-level microbial liquid enrichment culture chamber to the second-level microbial liquid enrichment culture chamber meets the purification requirements. , and then transfer the microbial liquid in the first-level microbial liquid enrichment culture chamber to the second-level microbial liquid enrichment culture chamber through pressure-maintaining transfer.
  • the microorganisms in the last-level microbial liquid enrichment culture chamber will reach a highly purified state.
  • concentration of deep-sea microbial liquid in the last-level microbial liquid enrichment culture chamber reaches more than 10 6 cells/mL, it can be considered A better purification state was achieved.
  • the dilution ratio of each level can be adjusted according to the specific cultured microbial groups.
  • Separation process First, sterilize the separation culture chamber and all internal devices and related pipes and valves to maintain sterility. Then fill the diversion trough on the surface of the microorganism separation branch with the culture medium required for culture and install it into the cavity, and then place the movable liquid storage tank on the top of the microorganism separation branch. Then install the reciprocating tie rod. Then install the quick-open kettle lid of the culture room to ensure that the central injection pipe is unobstructed. Then, gas is injected into the separation culture chamber through the pressure control system to pressurize the separation culture chamber to make the pressure conditions in the separation culture chamber consistent with the pressure conditions in the microbial liquid enrichment culture chamber.
  • the diversion trough Released into the diversion trough, under the action of gravity, it will spirally migrate from top to bottom in the diversion trough to the bottom.
  • the enriched bacterial liquid on the beads will be dispersed in the diversion tank to meet the separation, purification and culture process.
  • the streaked trajectory of the bacterial solution will be gradient diluted, and the separated colonies will grow along the diversion trajectory. At this point, the separation process is completed.
  • This solution also involves the automatic separation and purification process of microorganisms, which mainly involves placing several separation and culture chambers in parallel.
  • the inlets of all separation and culture chambers are connected in parallel through pipelines and connected to the microinjection pump and the last-stage microbial liquid enrichment culture chamber.
  • different culture chambers can be placed with different formulas of culture media. Then all separation culture chambers and pipe valves involved in the culture process are sterilized as a whole, and then it is checked that the microbial separation branches, pellets, reciprocating pull rods, diversion troughs, central injection pipes and their ancillary systems in all culture chambers are installed intact.
  • This plan involves the enrichment and cultivation of marine microorganisms in high-pressure environments and gravity separation devices and processes. It proposes multi-level enrichment and cultivation of marine microorganisms and automatic separation and purification of multi-culture media under high-pressure and extreme temperature environmental conditions, solving the current problem of There is a problem that the indoor pure culture technology method is separated from the high pressure and extreme temperature environmental conditions where marine microorganisms survive, resulting in the inability to achieve pure culture of a large number of microorganisms. It does not require professional operators and can be used in multiple culture scenarios such as research laboratories and scientific research ships. It has greater adaptability. Wide; This solution does not require manual enrichment and streaking separation operations by professionals. It can carry out large-scale enrichment and sorting, reduce labor costs, and realize the automated separation and culture of marine microorganisms under in-situ pressure and temperature environmental conditions, providing marine Pure culture of microorganisms in situ provides important technical means.
  • this program proposes a high-pressure pure culture technology to enrich and separate and culture marine microorganisms under the high-pressure and extreme temperature environmental conditions of the ocean in situ, solving the problem of the existing normal pressure separation and culture technology.
  • the temperature and pressure environmental conditions in which marine microorganisms survive in situ have caused the problem that most microorganisms cannot be separated and cultured purely.
  • this solution can effectively reduce the investment of professionals and can carry out large-scale enrichment. and isolation culture to improve the screening efficiency of difficult-to-cultivate microorganisms and improve the screening and cultivation efficiency of engineering bacteria with special functions.
  • the invention proposes a high-pressure environment marine microorganism enrichment culture and gravity separation device.
  • the enrichment and multi-level purification unit Through the enrichment and multi-level purification unit, the enrichment culture of microorganisms under the temperature and pressure environment conditions of the ocean in situ is realized.
  • Through the gravity separation and culture unit Realize the isolation and culture of marine microorganisms.
  • By reshaping the in-situ environment for enrichment, culture and separation of marine microorganisms it solves the problem of isolating and cultivating microorganisms in marine high-pressure environments, effectively improves the culturability of marine microorganisms, and provides an important basic means for the development and utilization of deep-sea microbial resources.
  • Figure 1 is a schematic structural diagram of the device according to the present invention.
  • Figure 2 is a schematic structural diagram of a microbial liquid enrichment culture chamber according to the present invention.
  • Figure 3 is a schematic structural diagram of the separation culture chamber and pressure control system of the present invention.
  • FIG. 4 is a schematic connection diagram of the circuit modules of the central control system according to the present invention.
  • Figure 5 is a schematic connection diagram of the automatic separation and purification process of microorganisms in one embodiment of the present invention.
  • Liquid injection pipeline 23. Environmental parameter detection unit; 231. Second temperature sensor; 232. Second pressure sensor; 3. Temperature control system; 31. Water bath jacket; 32. Refrigeration/heating device; 33. Water bath temperature detection device; 4. Pressure control system; 41. Release valve; 42. Air compressor; 43. Booster pump; 44. Gas storage tank; 45. Pressure regulating valve; 46. Regulating valve; 47. Ventilation duct; 5. Central control system; 6. Mobile platform.
  • This embodiment is a complete usage example with rich content.
  • this solution provides a high-pressure environment marine microorganism enrichment culture and gravity separation device, including enrichment and multi-level purification units: 1. Gravity separation culture unit 2. Temperature control system 3. Pressure control system 4 and the central control system 5; the control end and signal detection end of the enrichment and multi-level purification unit 1 and the gravity separation and culture unit 2 are electrically connected to the central control system 5; the control ends of the temperature control system 3 and the pressure control system 4 Both central control systems 5 are electrically connected; wherein: the enrichment and multi-level purification unit 1 is used to realize the enrichment and multi-level purification process of marine microorganisms, obtain the marine microorganism-enriched bacteria liquid, and enrich the marine microorganisms with bacteria.
  • the liquid is injected into the gravity-type separation and culture unit 2; the gravity-type separation and culture unit 2 is used to automatically draw lines using gravity in a high-pressure environment to achieve solid separation and cultivation of marine microorganisms, effectively improving the sustainability of marine microorganisms. Cultivability; the temperature control system 3 and the pressure control system 4 are respectively connected to the enrichment and multi-level purification unit 1 and the gravity separation and culture unit 2 for use in the enrichment and multi-level purification unit 1 and gravity separation.
  • a high-pressure and low-temperature environment consistent with the marine environment is constructed in the culture unit 2 to ensure that the enriched deep-sea microorganisms are enriched, purified, separated and cultured under in-situ environmental conditions.
  • the enrichment and multi-level purification unit 1 can realize the enrichment and culture of microorganisms under the in-situ temperature and pressure environmental conditions of the ocean, and the gravity separation and culture unit 2 can realize the isolation and cultivation of marine microorganisms.
  • the gravity separation and culture unit 2 can realize the isolation and cultivation of marine microorganisms.
  • the central control system 5 is used to monitor the changes in various environmental data information during the enrichment, separation, and purification of microbial enrichment bacteria in a high-pressure environment, as well as real-time collection, processing, storage, and image output functions. .
  • the enrichment and multi-level purification unit 1 is composed of a plurality of microbial liquid enrichment culture chambers 11 connected in series; A detachable sealing cover 111 and a connected sampling valve group are provided, and a sensor group 112 is provided inside; each microbial liquid enrichment culture chamber 11 is placed in the temperature control system 3; wherein: the detachable sealing cover 111 is used for It is convenient to sterilize the inside of the microbial liquid enrichment culture chamber 11 and put the culture substrate; the connection sampling valve group is used for connecting and sampling each microbial liquid enrichment culture chamber 11, and it is connected with the pressure control system 4 The connection is used to input liquid or gas into the microbial liquid enrichment culture chamber 11 to increase the pressure in the microbial liquid enrichment culture chamber 11 to ensure that the pressure value in the microbial liquid enrichment culture chamber 11 is consistent with the actual situation in the deep sea; The sensor group 112 is used for real-time temperature and pressure changes in the microbial liquid enrichment culture chamber 11, and
  • the microbial liquid enrichment culture chamber 11 is also provided with a stirring rod 113 , and the stirring rod 113 is used to enhance the reaction process of the substrate during the culture process of the microbial liquid enrichment culture chamber 11 .
  • the stirring rod 113 is a manual stirring rod, which can enhance mass transfer through intermittent manual stirring.
  • An enhanced continuous or intermittent stirring rod 113 can also be placed as needed to enhance the matrix during the culture process.
  • the reaction process increases the energy and nutrient supply of microorganisms and improves culture efficiency.
  • the sensor group 112 includes a temperature sensor 1121 and a pressure sensor 1122; the temperature sensor 1121 is used for real-time monitoring of temperature changes in the microbial liquid enrichment culture chamber 11; and the pressure sensor 1122 is used for real-time monitoring of the microbial liquid enrichment.
  • the pressure changes in the culture room 11 are collected; the signal output end of the temperature sensor 1121 and the signal output end of the pressure sensor 1122 are both electrically connected to the central control system 5 .
  • connection sampling valve group includes a liquid inlet valve 1141, an air inlet valve 1142, a sampling valve 1143 and a liquid outlet valve 1144; wherein: the microbial liquid enrichment culture chamber 11 is connected through the liquid outlet valve 1144 and the inlet valve 1144.
  • the liquid valves 1141 are connected in series, and the liquid outlet valve 1144 of the previous stage of microbial liquid enrichment culture chamber 11 is connected with the liquid outlet valve 1144 of the subsequent stage of microbial liquid enrichment culture room 11; the liquid outlet valve of the last stage of microbial liquid enrichment culture room 11 1144 is connected to the gravity separation culture unit 2; the air inlet valve 1142 is connected to the pressure control system 4 and is used to input gas into the microbial liquid enrichment culture chamber 11 to increase the pressure in the microbial liquid enrichment culture chamber 11 , so that the pressure value in the microbial liquid enrichment culture chamber 11 is consistent with the actual situation in the deep sea; the sampling valve 1143 is used for real-time sampling and analysis of the microorganisms in the microbial liquid enrichment culture chamber 11 .
  • multiple microbial liquid enrichment culture chambers 11 are connected in series to form an enrichment and multi-level purification unit 1, and the bacterial liquid in the front-stage microbial liquid enrichment culture chamber 11 is transferred to the next-level microbial liquid by maintaining pressure.
  • the enrichment culture chamber 11 by analogy, according to the concentration gradient dilution, the microbial liquid obtained in the last microbial liquid enrichment culture chamber 11 will provide enriched and highly purified functions under stress in a high-pressure environment and directional nutritional conditions. type microorganisms.
  • the pressure-maintaining transfer can be realized by using a microinjection pump to take out the enriched liquid in the front-stage microbial liquid enrichment culture chamber 11 through the sampling valve 1143 and then pump it into the subsequent stage microbial liquid enrichment culture chamber 11 . It is also possible to pressurize the pressure of the rear-stage microbial liquid enrichment culture chamber 11 to a level slightly smaller than that of the front-stage microbial liquid enrichment culture chamber 11, and then open the outlet valve 1144 of the front-stage microbial liquid enrichment culture chamber 11 and the pressure of the rear-stage microbial liquid enrichment culture chamber 11.
  • the microbial enriched liquid will automatically flow from the front-stage microbial liquid enriched culture chamber 11 into the rear-stage microbial liquid enriched culture chamber 11 for dilution and culture under the condition of slight pressure difference.
  • the temperature and pressure environmental conditions in the microbial liquid enrichment culture chamber 11 are consistent with the environmental conditions of the microorganisms in the deep sea, ensuring the effectiveness of the enrichment culture.
  • the gravity-type separation and culture unit 2 includes a separation culture chamber 21, a liquid injection unit 22, and an environmental parameter detection unit 23; wherein: the gravity-type separation and culture unit 2 passes through injection.
  • the liquid unit 22 is connected to the last microbial liquid enrichment culture chamber 11 of the enrichment and multi-level purification unit 1; a microorganism separation branch 211 is provided in the separation culture chamber 21, which is used to separate microorganisms to the greatest extent possible.
  • a central liquid injection pipe 212 is provided on the top of the separation culture chamber 21, and the separation culture chamber 21 is connected to the liquid injection unit 22 through the central liquid injection pipe 212; from the liquid injection unit 22 to the separation culture chamber
  • the microorganism separation branch 211 of 21 is injected into the marine microorganism-enriched bacterial liquid; the separation culture chamber 21 is placed in the temperature control system 3 and connected to the pressure control system 4, for building a marine environment consistent with the marine environment in the separation culture chamber 21
  • the high-pressure and low-temperature environment ensures that the enriched deep-sea microorganisms are separated and cultured under in-situ environmental conditions; the environmental parameter detection unit 23 is used to detect changes in temperature and pressure in the separation culture chamber 21 in real time, and will The data is transmitted to the central control system 5; the microorganism separation branch 211 and the liquid injection unit 22 are both electrically connected to the central control system 5.
  • the microorganism separation branch 211 includes a movable liquid storage tank 2111, a small ball 2112, a reciprocating pull rod 2113, a guide groove 2114 and a cavity 2115; wherein:
  • the movable liquid storage tank 2111 is provided in the center of the top of the cavity 2115, directly below the central liquid injection pipe 212, and is used to store the marine microorganism-enriched bacterial liquid injected by the liquid injection unit 22; the small ball 2112 Placed in the movable liquid storage tank 2111, the injected marine microorganism-enriched bacteria liquid is immersed; the bottom of the movable liquid storage tank 2111 is provided with a through hole for fixing the position of the small ball 2112 and ensuring that the small ball 2112 can pass through it; the movable end of the reciprocating pull rod 2113 is fixedly connected to the movable liquid storage tank 2111; a gear box 2116 is provided on the top side of the cavity 2115; the control end of the reciprocating pull rod 2113 is connected to the movable liquid storage tank 2111.
  • the central control system 5 is electrically connected; the diversion tank 2114 is fixedly arranged inside the cavity 2115; the diversion tank 2114 is filled with culture medium, and its inlet is connected with the gear box 2116; when the ball 2112 is immersed in the marine microbial enrichment bacteria
  • the movable liquid storage tank 2111 moves toward the edge of the cavity 2115; when the through hole of the movable liquid storage tank 2111 leaves the cavity 2115, due to gravity, the small ball 2112 will move from The through hole passes through the baffle box 2116 that falls in the cavity 2115, and enters the guide groove 2114 inside the cavity 2115; the small balls 2112 carrying the marine microorganism-enriched bacterial liquid move along the guide groove 2114 from top to bottom.
  • the marine microorganism-enriched bacteria liquid is diluted and dispersed in a concentration gradient to maximize the area required for microbial isolation and culture.
  • a small slot hole is provided in the center of the top of the cavity 2115, where the initial position of the movable liquid reservoir 2111 coincides with the through hole, for fixing the position of the small ball 2112.
  • the liquid injection unit 22 includes a micro-injection pump 221 and a liquid injection pipeline 222; wherein: the liquid inlet end of the micro-injection pump 221 is connected to the last microbial liquid enrichment culture of the enrichment and multi-level purification unit 1.
  • the chamber 11 is connected, and its liquid outlet is connected to the liquid injection pipe 222; the liquid outlet of the liquid injection pipe 222 is connected to the central liquid injection pipe 212; the control end of the micro-injection pump 221 is connected to the central control system 5 electrical connection.
  • the environmental parameter detection unit 23 includes a second temperature sensor 231 and a second pressure sensor 232; wherein the probes of the second temperature sensor 231 and the second pressure sensor 232 are both arranged inside the separation culture chamber 21. , and its signal output terminals are all electrically connected to the central control system 5 .
  • real-time monitoring of the temperature and pressure in the microbial liquid enrichment culture chamber 11 and the separation culture chamber 21 is realized through the temperature sensor 1121, the pressure sensor 1122, the second temperature sensor 231, and the second pressure sensor 232 respectively; If the temperature/pressure needs to be adjusted, the temperature control system 3 and the pressure control system 4 are controlled through the central control system 5 to maintain the temperature and pressure values in the microbial liquid enrichment culture chamber and the separation culture chamber and the marine environment for microbial growth at all times. The conditions are the same.
  • the top of the separation culture chamber 21 is provided with a quick-opening kettle cover 213; the central liquid injection pipe 212 is provided on the quick-opening kettle cover 213; the quick-opening kettle cover 213 is also provided with a gas injection channel and a sensor. placement channel; the pressure control system 4 is connected to the separation culture chamber 21 through an air injection channel; the second temperature sensor 231 and the second pressure sensor 232 are arranged in the sensor placement channel.
  • the separation culture chamber 21 utilizes the principle of natural release of gravity, and small balls 2112 are used to carry the bacterial liquid to move freely under the action of gravity in the separation culture chamber 21 to achieve effective separation of microorganisms on the solid culture medium.
  • the separation culture chamber 21 is designed with a top quick-opening cauldron lid 213 to facilitate and quickly load samples into the culture chamber.
  • this embodiment adopts a truncated table or cylindrical cavity 2115, which maximizes the area for microbial separation and culture in a limited space.
  • a rotating serpentine guide groove 2114 is provided from top to bottom in the cavity 2115.
  • the guide groove 2114 is flatly filled with a solid culture medium of a culture substrate to provide nutrients for the isolation and culture of microorganisms.
  • a movable liquid storage tank 2111 is placed on the top of the truncated cone/cylindrical cavity 2115 for storing the microbial enrichment liquid and pellets 2112 to be separated.
  • the diameter of the guide groove 2114 is larger than that of the small ball 2112, ensuring that the small ball 2112 released from the movable liquid storage tank 2111 can enter the guide groove 2114 completely and smoothly.
  • a central liquid injection pipe 212 is provided in the center of the quick-opening kettle lid 213 for injecting the microbial enriched liquid into the movable liquid storage tank 2111.
  • a hole slightly larger in diameter than the ball 2112 is provided in the center of the bottom of the movable liquid storage tank 2111.
  • a reciprocating pull rod 2113 is provided on the side wall of the separation culture chamber 21, and the pull rod can realize reciprocating push-pull movement through a control method such as a reciprocating piston.
  • the reciprocating pull rod 2113 can be used to drag the movable liquid storage tank 2111 from the center to the edge of the cavity 2115. Since the bottom diameter of the movable liquid storage tank 2111 is larger than the diameter of the ball 2112, the small ball 2112 will move from the center of the bottom of the movable liquid storage tank 2111.
  • the hole is released, enters the guide groove 2114, and then spirally descends from top to bottom in the guide groove 2114 under the action of gravity, and moves to the bottom of the cavity 2115.
  • the microbial enriched liquid is diluted and dispersed in a concentration gradient in the diversion tank 2114, meeting the requirements for the dispersed growth of a single colony.
  • the pressure control system 4 includes a bleed valve 41, an air compressor 42, a booster pump 43, a gas storage tank 44, a pressure regulating valve 45, a regulating valve 46 and a ventilation pipe 47; wherein: the bleed The valve 41 is connected to the enrichment and multi-level purification unit 1 and the gravity separation and culture unit 2 through a ventilation pipe 47, and its control end is electrically connected to the central control system 5 for discharging the enrichment and multi-level purification unit. 1.
  • the gas in the gravity separation and culture unit 2 is depressurized inside the enrichment and multi-level purification unit 1 and the gravity separation and culture unit 2; the air compressor 42, booster pump 43, gas storage tank 44, regulator The pressure valve 45 and the regulating valve 46 are connected in sequence through the ventilation pipe 47, and finally connected to the enrichment and multi-level purification unit 1 and the gravity separation and culture unit 2 through the ventilation pipe 47, for supplying the enrichment and multi-level purification unit 1 Inject the gas (or inert gas) required for culture into the gravity separation and culture unit 2 for pressurization; the pressure regulating valve 45 is used to adjust the internal pressure of the enrichment and multi-level purification unit 1 and the gravity separation and culture unit 2 , the regulating valve 46 is used to adjust the speed of the injected gas; the control end of the air compressor 42, the control end of the booster pump 43, the control end of the pressure regulating valve 45, and the control end of the regulating valve 46 are all connected with the central control system 5 electrical connection.
  • the temperature control system 3 includes a water bath jacket 31 and a refrigeration/heating device 32; the water bath jacket 31 is wrapped around the outer walls of the enrichment and multi-level purification unit 1 and the gravity separation and culture unit 2, It is connected to the refrigeration/heating device 32; the control end of the refrigeration/heating device 32 is electrically connected to the central control system 5.
  • the constant temperature conditions in the microbial liquid enrichment culture chamber 11 and the separation culture chamber 21 are mainly maintained by placing the microorganism liquid enrichment culture chamber 11 and the separation culture chamber 21 in the high/low temperature water bath of the water bath jacket 31
  • the temperature sensor 1121 and the second temperature sensor 231 are used for monitoring and display, and the constant temperature state in the culture room is maintained through heat exchange with the refrigeration/heating device.
  • a water bath temperature detection device 33 is also provided, the output end of which is electrically connected to the central processing system 5 for real-time detection of the water bath temperature in the water bath jacket 31 to facilitate real-time temperature adjustment.
  • the temperature conditions of the microbial liquid enrichment culture chamber 11 and the separation culture chamber 21 are mainly controlled through the temperature control system 3 .
  • the cold/hot fluid is injected into the ring wall cavities of the microbial liquid enrichment culture chamber 11 and the separation culture chamber 21, and the low or high temperature state of the fluid in the ring wall cavities is ensured by circulating the fluid to refrigeration or heating, and then through The heat exchange between the cold/hot fluid and the built-in cavity ensures the low or high temperature state in the built-in cavity.
  • the microbial liquid enrichment culture chamber 11 and the separation culture chamber 21 are placed in a low/high temperature water bath/oil bath environment to ensure the special temperature conditions required in the microbial liquid enrichment culture room 11 and the separation culture chamber 21.
  • the microbial liquid enrichment culture chamber 11 and the separation culture chamber 21 are placed in a refrigeration/heating room or box with a constant temperature guaranteed by air heat exchange. To maintain some extreme temperature conditions, several of the above temperature control methods can be used at the same time.
  • the high-pressure environment marine microorganism enrichment culture and gravity separation device also includes a mobile platform 6, the enrichment and multi-level purification unit 1, a gravity separation culture unit 2, a temperature control system 3, and a pressure control system. 4.
  • the central control system 5 is placed on the mobile platform 6 to increase the universality of the training scene.
  • the high-pressure and extreme temperature environmental conditions in which microorganisms live in the marine environment are constructed in the microbial liquid enrichment culture chamber 11 and the separation culture chamber 21 to improve their survival activity.
  • the separation culture chamber 21 Through multi-stage enrichment and liquid dilution culture, functional microorganisms with higher purity under the stress of directional environmental conditions are obtained, and at the same time, combined with the separation culture chamber 21, culture and separation are performed to obtain single microorganisms.
  • multiple sets of selected microbial liquid enrichment culture chambers 11 and separation culture chambers 21 can be combined to form an efficient separation process with different culture medium combinations, as shown in Figure 5.
  • the high-pressure environment marine microorganism enrichment culture and separation technology involved in the device proposed in this solution mainly includes two steps: enrichment and separation.
  • enrichment and separation First, after enrichment and culture in the enrichment and multi-level purification unit 1, a higher purity bacterial population is obtained, and then enters the gravity separation and culture unit 2 under pressure maintenance for solid culture separation, and through different culture media and environmental conditions The combined process is simultaneously screened to obtain pure culture strains.
  • the specific implementation principle is:
  • Enrichment process First, the microbial liquid enrichment culture chamber 11 and its attached pipes and valves are sterilized, and then the substrates to be cultured, such as deep-sea sediments, macroorganism tissues symbiotic with microorganisms, and extracts, etc., are loaded in sequence. , then fill in the nutrient solution required for culture from the liquid inlet valve 1141, and then inject the gas required for culture from the air inlet valve 1142 (inert gas can be injected if not required) so that the pressure value in the microbial liquid enrichment culture chamber 11 increases to The actual environmental conditions in the deep sea are consistent. During the culture process, stirring is performed through the manual stirring rod 113 on the top to increase mass transfer and optimize the culture process.
  • the substrates to be cultured such as deep-sea sediments, macroorganism tissues symbiotic with microorganisms, and extracts, etc.
  • the nutrient solution required for culture is injected into the second-level microbial liquid enrichment culture chamber 11, and is supplied to the second-level microbial liquid enrichment culture chamber 11 through the pressure control system 4. 11. Gas injection and pressurization.
  • the amount of culture liquid injected into the second-stage microbial liquid enrichment culture chamber 11 needs to ensure that the concentration of the enrichment liquid is from the concentration of the first-stage microbial liquid enrichment culture chamber 11 to the second-stage microbial liquid enrichment culture chamber.
  • the dilution ratio in 11 meets the purification requirements, and then the microbial liquid in the first-stage microbial liquid enrichment culture chamber 11 is transferred to the second-stage microbial liquid enrichment culture chamber 11 through pressure-maintaining transfer.
  • the microorganisms in the last-level microbial liquid enrichment culture chamber 11 will reach a highly purified state.
  • concentration of the deep-sea microbial liquid in the last-level microbial liquid enrichment culture chamber 11 reaches more than 10 6 cells/mL, It can be considered that a better purification state has been achieved.
  • the dilution ratio of each level can be adjusted according to the specific cultured microbial groups.
  • Separation process First, sterilize the separation culture chamber 21 and all internal devices and related pipes and valves to maintain sterility. Then, the diversion groove 2114 on the surface of the microorganism separation branch 211 is filled with the culture medium required for culture. Then place the movable liquid storage tank 2111 on the top of the microorganism separation support 211. Then install the reciprocating tie rod 2113. Then install the quick-opening cauldron cover 213 of the culture room to ensure that the central liquid injection pipe 212 is unobstructed. Then, gas is injected into the separation culture chamber 21 to pressurize the gas through the pressure control system 4, so that the pressure conditions in the separation culture chamber 21 are consistent with the pressure conditions in the microbial liquid enrichment culture chamber 11.
  • the small ball 2112 Since the diameter of the small ball 2112 is smaller than the aperture of the bottom of the movable liquid storage tank 2111, the small ball 2112 will It is released from the movable liquid storage tank 2111 and enters the guide groove 2114. Under the action of gravity, it will spirally move from top to bottom in the guide groove 2114 to the bottom. The enriched bacterial liquid on the beads 2112 will be dispersed in the diversion tank 2114 to meet the separation, purification and culture process. Along the movement trajectory of the ball 2112, the streaked trajectory of the bacterial liquid will be gradient diluted, and the separated bacterial colonies will grow along the diversion trajectory. At this point, the separation process is completed.
  • each separation culture chamber 21 is arranged in sequence, and the liquid injection unit 22 is connected to the central liquid injection pipe 212 of all separation culture chambers 21; the temperature control system 3, pressure control System 4 is connected to all separation and culture chambers 21 respectively, and is used to build a high-pressure and low-temperature environment consistent with the marine environment in each separation and culture chamber 21 to ensure that the enriched deep-sea microorganisms are separated and cultured under in-situ environmental conditions; each Each separation culture chamber 21 is connected to an independent environmental parameter detection unit 23 for real-time detection of temperature and pressure changes in each separation culture chamber 21 and transmitting the detected data to the central control system 5 .
  • the microorganism separation process involved in this embodiment mainly places several separation culture chambers 21 in parallel.
  • the inlets of all separation culture chambers 21 are connected in parallel through pipelines and are connected with the microinjection pump 221 and the last stage of microbial liquid enrichment.
  • the cultivation rooms 11 are connected.
  • different culture chambers can be placed with different formulas of culture media. Then all separation culture chambers 21 and pipe valves involved in the culture process are sterilized as a whole, and then the microbial separation branches 211, small balls 2112, reciprocating pull rods 2113, diversion troughs 2114, central injection pipes 212 and Its ancillary systems are well installed. Then, through temperature and pressure monitoring, it is ensured that the temperature and pressure environmental conditions in all separation culture chambers 21 are consistent with the temperature and pressure environmental conditions of the marine environment where the microorganisms are located.
  • This embodiment relates to a high-pressure environment marine microorganism enrichment culture and gravity separation device and process. It proposes to carry out multi-level enrichment culture and multi-medium automatic separation and purification culture of marine microorganisms under high pressure and extreme temperature environmental conditions, solving the problem of
  • the existing indoor pure culture technology method is out of the high-pressure and extreme temperature environment conditions in which marine microorganisms survive, resulting in the difficulty of pure culture of a large number of microorganisms. It does not require professional operators and can be used in multiple culture scenarios such as research laboratories and scientific research ships.
  • This embodiment does not require manual enrichment and streak separation operations by professionals, and can carry out large-scale enrichment and sorting, reduce labor costs, and realize automated separation and culture of marine microorganisms under in-situ pressure and temperature environmental conditions. Provides important technical means for pure culture of marine microorganisms in situ.
  • this embodiment proposes a high-pressure pure culture technology to enrich and separate and culture marine microorganisms under the high-pressure and extreme temperature environmental conditions of the ocean in situ, which solves the problem of the existing normal pressure separation and culture technology. Being separated from the temperature and pressure environmental conditions in which marine microorganisms survive in situ has caused the problem that most microorganisms cannot be separated and cultured purely. Compared with the existing isolation and culture technology, this solution can effectively reduce the investment of professionals and can carry out large-scale enrichment. Integration and separation culture can improve the screening efficiency of difficult-to-cultivate microorganisms and improve the screening and cultivation efficiency of engineering bacteria with special functions.
  • this embodiment proposes a high-pressure environment deep-sea methanophilic bacteria enrichment culture and separation device.
  • the high-pressure environment deep-sea methanophilic bacteria enrichment culture and separation device involved in this example mainly includes two steps: enrichment and separation. First, enrich deep-sea methanophilic bacteria through the microbial liquid enrichment culture chamber 11, and then pass through the next-level microbial liquid enrichment culture chamber 11 while maintaining pressure to obtain deep-sea methanophilic bacteria with higher purity, and then enter The isolation and culture chamber 21 performs isolation and culture, and finally obtains a single strain.
  • the high-pressure environment deep-sea methanophile enrichment culture method involved in this example first sterilizes the microbial liquid enrichment culture chamber 11 and its attached pipes and valves. After the sterilization is completed, each device is connected in sequence, and then the deep-sea methane The sediment in the leakage area is loaded into the microbial liquid enrichment culture chamber 11, and then the nutrient solution required for culture is loaded, and then the air inlet valve 1142 is opened to inject the methane gas required for culture through the pressure control system 4, so that the microbial liquid enrichment culture chamber The pressure value in 11 increases to 14Mpa and is monitored by the pressure sensor 1122.
  • the entire device is placed in the water bath jacket 31 at 4°C, and is displayed and exchanged with the refrigeration/heating device 32 through the water bath temperature detection device 33.
  • the water bath system Fill the refrigerant such as ethylene glycol to maintain the low temperature state in the microbial liquid enrichment culture chamber 11 , and the temperature in the microorganism liquid enrichment culture chamber 11 passes through the temperature sensor 1121 .
  • methane can be continuously vented for 5-10 minutes by opening the vent valve 41.
  • stirring is performed through the stirring rod 113 on the top of the microbial liquid enrichment culture chamber 11 to increase mass transfer and optimize the culture process.
  • the nutrient solution required for culture is injected into the next-level microbial liquid enrichment culture chamber 11 and pressurized using the same method as the microbial liquid enrichment culture chamber 11 .
  • the amount of culture liquid injected into the microbial liquid enrichment culture chamber 11 needs to ensure that the dilution ratio of the enrichment liquid from the concentration of the first-level microbial liquid enrichment culture chamber 11 to the next-level microbial liquid enrichment culture chamber 11 is 1:10.
  • the microbial enrichment liquid will automatically enter the rear-stage microbial liquid enrichment culture from the front-stage microbial liquid enrichment culture chamber 11 under the condition of slight pressure difference.
  • Purification culture is performed in chamber 11. By analogy, it is transferred to the third and fourth-level microbial liquid enrichment culture chambers 11.
  • the number of cells in the fourth-level microbial liquid enrichment culture chamber 11 reaches more than 10 6 /mL and the deep-sea methanophilic bacteria When the abundance is above 50%, it can be considered that a better purification state has been achieved.
  • the dilution ratio of each level can be adjusted according to the specific cultured microbial groups. During the experiment, the bacterial liquid required for sampling and monitoring is carried out by opening the sampling valve 1143.
  • the separation and culture process mainly includes: first, sterilizing the separation and culture chamber 21 and all internal devices and related pipes and valves. Then, the diversion groove 2114 of the microorganism separation branch 211 is filled with the culture medium required for culture. Place the movable liquid storage tank 2111 on the top of the microorganism separation support 211. And place the small ball 2112 in the groove inside the movable liquid storage tank 2111 to prevent it from sliding. Then install the reciprocating tie rod 2113. Then install the upper cover of the separation culture chamber 21 and the central liquid injection pipe 212.
  • gas is injected into the separation culture chamber 21 to pressurize the gas through the pressure control system 4, so that the pressure conditions in the separation culture chamber 21 are consistent with the pressure conditions in the microbial liquid enrichment culture chamber 11.
  • the small ball 2112 Since the diameter of the small ball 2112 is smaller than the hole diameter at the bottom of the movable liquid storage tank 2111, the small ball 2112 will move from The liquid is released from the movable storage tank 2111 and enters the guide groove 2114. Under the action of gravity, it will spirally move from top to bottom in the guide groove 2114 to the bottom.
  • the streaked track of the enriched bacterial liquid on the small ball 2112 is a gradient dilution, and the microorganisms will grow separated colonies along the diversion track.
  • the automatic separation and purification process of deep-sea methanophiles involved in this embodiment mainly involves placing several separation and culture chambers 21 in parallel.
  • the inlets of all separation and culture chambers 21 are connected in parallel through pipelines and connected to the microinjection pump 221 and the enrichment and multi-level purification unit 1 connected.
  • different culture chambers can be placed with different formulas of culture media.
  • all culture chambers and pipe valves involved in the culture process are sterilized as a whole, and then the separation culture branches 211, small balls 2112, reciprocating pull rods 2113, diversion troughs 2114, central injection pipe 212 and their accessories in all culture chambers are inspected.
  • the system is installed well.
  • the temperature and pressure environmental conditions in all culture chambers are guaranteed to be micro 4°C and 14MPa.
  • the small balls 2112 will carry the bacterial liquid in the diversion grooves 2113 of the microorganism separation branches 211 in each separation culture chamber 21.
  • Mobile, through the automated separation and combination of a large number of culture chambers, automatic sorting processes can be realized in different culture media environments, effectively ensuring the separation, culture and purification of microorganisms in high-pressure environments.
  • Various parameter conditions during the entire cultivation process will be data collected, integrated and displayed through the central control system 5.
  • a mobile platform 6 is integrally installed at the bottom of the enrichment culture and separation device to increase the universality of culture scenarios.
  • the enrichment and multi-level purification unit 1 can realize the enrichment and culture of microorganisms under the in-situ temperature and pressure environmental conditions of the ocean, and the gravity separation and culture unit 2 can realize the isolation and cultivation of marine microorganisms.
  • the gravity separation and culture unit 2 can realize the isolation and cultivation of marine microorganisms.

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Abstract

一种高压环境海洋微生物富集培养与重力式分离装置，包括富集与多层级纯化单元（1）和重力式分离培养单元（2）；在构建与海洋环境一致的高压、低温环境下，富集与多层级纯化单元（1）用于实现对海洋微生物的富集与多层级纯化过程，得到海洋微生物富集菌液并将海洋微生物富集菌液注入重力式分离培养单元（2）中；重力式分离培养单元（2）用于在高压环境进行利用重力作用进行自动划线，实现海洋微生物的固体分离和培养，有效地提高海洋微生物的可培养性。所述装置通过重塑原位环境进行海洋微生物的富集培养与分离，解决分离纯培养海洋高压环境微生物的难题，有效的提高海洋微生物的可培养性，为开发利用深海微生物资源提供重要的基础手段。

Description

高压环境海洋微生物富集培养与重力式分离装置 技术领域
本发明涉及海洋微生物技术领域，特别是涉及一种高压环境海洋微生物富集培养与重力式分离装置。
背景技术
海洋微生物资源是全球重要的战略资源，具有最多的生物多样性。分子生态学和宏基因组学等技术显著的增加了我们对海洋微生物多样性的认识。但是，目前99％的海洋微生物未被纯培养，同时很多基因存在具有未知功能未被注释或可能被错误注释在数据库中，因此现代组学技术不足以提供足够的信息去理解所有的微生物，尤其是尚未被分类的微生物。提高尚未培养的微生物的可培养性，创新纯培养方法是解释这些微生物表型和基因型的重要路径。
现有技术公开的方案虽然可实现深海原位状态的微生物富集培养，但并未将海洋微生物进行分离培养，无法有效提高培养的成功率。
发明内容
本发明为了解决以上至少一种技术缺陷，针对海洋微生物生活的极端环境特征提供一种高压环境海洋微生物富集培养与重力式分离装置，通过重塑其原位环境进行海洋微生物的富集培养与分离，有效的提高海洋微生物的可培养性，为开发利用深海微生物资源提供重要的基础手段。
为解决上述技术问题，本发明的技术方案如下：
本方案提供一种高压环境海洋微生物富集培养与重力式分离装置，包括富集与多层级纯化单元、重力式分离培养单元、温度控制系统、压力控制系统和中央控制系统；富集与多层级纯化单元与重力式分离培养单元的控制端与信号检测端均中央控制系统电性连接；温度控制系统、压力控制系统的控制端均中央控制系统电性连接；其中：
所述富集与多层级纯化单元用于实现对海洋微生物的富集与多层级纯化过程，得到海洋微生物富集菌液并将海洋微生物富集菌液注入所述重力式分离培养单元中；所述重力式分离培养单元用于在高压环境进行利用重力作用进行自动划 线，实现海洋微生物的固体分离和培养，有效地提高海洋微生物的可培养性；所述温度控制系统、压力控制系统分别与所述富集与多层级纯化单元、重力式分离培养单元连接，用于在富集与多层级纯化单元、重力式分离培养单元内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行富集、纯化、分离和培养。
上述方案中，富集与多层级纯化单元可以实现海洋原位的温度和压力环境条件下微生物的富集培养，重力式分离培养单元可以实现对海洋微生物的分离培养。通过重塑其原位环境进行海洋微生物的富集培养与分离，解决分离纯培养海洋高压环境微生物的难题，有效的提高海洋微生物的可培养性，为开发利用深海微生物资源提供重要的基础手段。
上述方案中，中央控制系统用于实现微生物富集菌在高压环境进行富集、分离、纯化过程中各项环境数据信息变化的监控、以及实时采集、处理、存储和图像输出等功能。
上述方案中，高压环境理论上认为深海水深大于200米的压力环境就是高压环境，而低温环境理论上认为深海水温环境低于4℃就为低温环境。在本方案中依据所需培养的海洋微生物培养环境构建对应的高压、低温环境。
其中，所述富集与多层级纯化单元由多个串联的微生物液体富集培养室组成；所述微生物液体富集培养室上设置有可拆卸密封盖和连接取样阀组，其内部设置有传感器组；每个微生物液体富集培养室均安置在温度控制系统中；其中：所述可拆卸密封盖用于方便对微生物液体富集培养室内部进行灭菌操作和放入培养底物；所述连接取样阀组用于各个微生物液体富集培养室的连接与取样，其与所述压力控制系统连接，用于向微生物液体富集培养室内输入液体或气体以增加微生物液体富集培养室内的压力，令微生物液体富集培养室内的压力值与深海实际情况一致；所述传感器组用于实时微生物液体富集培养室内的温度和压力变化情况，并将信号输送至所述中央控制系统；所述富集与多层级纯化单元最后一个微生物液体富集培养室通过连接取样阀组与所述重力式分离培养单元连接。
其中，在所述微生物液体富集培养室上还设置有搅拌杆，所述搅拌杆用于微生物液体富集培养室在培养过程增强基质的反应过程。
上述方案中，所述搅拌杆为手动搅拌杆，可通过间歇式手动搅拌增强传质，也可根据需要放置增强型连续或者间歇式搅拌杆，用于在培养过程中，增强基质 的反应过程，增加微生物的能量和营养供给，提高培养效率。
其中，所述传感器组包括温度传感器和压力传感器；所述温度传感器用于实时监测微生物液体富集培养室内的温度变化情况；所述压力传感器用于实时监测微生物液体富集培养室内的压力变化情况；温度传感器信号输出端、压力传感器信号输出端均与所述中央控制系统电性连接。
其中，所述连接取样阀组包括进液阀、进气阀、取样阀和出液阀；其中：微生物液体富集培养室之间通过所述出液阀、进液阀进行串联，前一级微生物液体富集培养室出液阀与后一级微生物液体富集培养室出液阀连接；最后一级微生物液体富集培养室出液阀与所述重力式分离培养单元连接；进气阀与所述压力控制系统连接，用于向微生物液体富集培养室内输入气体以增加微生物液体富集培养室内的压力，令微生物液体富集培养室内的压力值与深海实际情况一致；取样阀用于对微生物液体富集培养室内的微生物进行实时取样分析，以便进行相应的环境参数调整，优化富集培养的流程。
上述方案中，多个微生物液体富集培养室串联构成富集与多层级纯化单元，将前级微生物液体富集培养室内的菌液通过保压转移至下一级微生物液体富集培养室中，以此类推，按照浓度梯度稀释，最后一个微生物液体富集培养室中获得的微生物菌液将为高压环境，定向营养条件供给胁迫下富集的、高度纯化的功能型微生物。保压转移可以通过微注泵将前级微生物液体富集培养室中的富集液通过取样阀取出后泵入后级的微生物液体富集培养室中实现。也可以将后级微生物液体富集培养室的压力增压至略小于前级微生物液体富集培养室，然后开启前级微生物液体富集培养室的出液阀和后级室的进液阀，微生物富集液将在微小压力差条件下，自动地从前级微生物液体富集培养室进入后级微生物液体富集培养室中进行稀释培养。在整个富集培养的过程中，微生物液体富集培养室内的温度、压力环境条件都与微生物在深海的环境条件一致，保证富集培养的有效性。
其中，所述重力式分离培养单元包括分离培养室、注液单元和环境参数检测单元；其中：所述重力式分离培养单元通过注液单元与所述富集与多层级纯化单元最后一个微生物液体富集培养室连接；所述分离培养室内设置有微生物分离支体，用于对微生物进行分离操作，最大程度的为微生物培养提供面积；所述分离培养室顶部设置有中央注液管，分离培养室通过中央注液管与所述注液单元连接；由注液单元向分离培养室的微生物分离支体注入海洋微生物富集菌液；所述 分离培养室安置在温度控制系统中并与压力控制系统连接，用于在分离培养室内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行分离培养；所述环境参数检测单元用于实时检测分离培养室内的温度、压力的变化情况，并将检测的数据传输至所述中央控制系统；所述微生物分离支体、注液单元均与所述中央控制系统电性连接。
其中，所述微生物分离支体包括可移动储液槽、小球、往复式拉杆、导流槽和腔体；其中：所述可移动储液槽设置在所述腔体顶部中央，落在中央注液管正下方，用于存储由注液单元注入的海洋微生物富集菌液；所述小球放置在所述可移动储液槽中，被注入的海洋微生物富集菌液浸没；所述可移动储液槽底部设置有通孔，用于固定小球的位置和保证小球能从其中穿过；所述往复式拉杆可移动端与所述可移动储液槽固定连接；所述腔体顶部侧面设置有档盒；所述往复式拉杆控制端与所述中央控制系统电性连接；导流槽固定设置在腔体内部；导流槽中填充有培养基，其进口与档盒连通；当小球浸没在海洋微生物富集菌液后，在往复式拉杆的作用下，可移动储液槽向腔体边缘移动；当可移动储液槽的通孔离开腔体时，由于重力作用，小球将从通孔中穿过落在腔体的档盒中，进入到腔体内部的导流槽中；携带海洋微生物富集菌液的小球沿着导流槽由上向下滑动的过程中，将海洋微生物富集菌液呈浓度梯度稀释分散，最大程度的为微生物的分离培养提供面积。
其中，在所述腔体顶部中央，即可移动储液槽初始位与通孔重合的位置，设置有小槽孔，用于固定所述小球的位置。
其中，所述注液单元包括微注泵和注液管道；其中：所述微注泵入液端与所述富集与多层级纯化单元最后一个微生物液体富集培养室连接，其出液端与所述注液管道连接；所述注液管道出液口与所述中央注液管连接；所述微注泵控制端与所述中央控制系统电性连接。
其中，所述环境参数检测单元包括第二温度传感器和第二压力传感器；其中：所述第二温度传感器、第二压力传感器探头均设置在所述分离培养室内部，其信号输出端均与所述中央控制系统电性连接。
上述方案中，通过温度传感器、压力传感器、第二温度传感器、第二压力传感器分别实现对微生物液体富集培养室、分离培养室中的温度、压力进行实时监控；若温度/压力需要调整，则通过中央控制系统控制温度控制系统、压力控制 系统进行工作，时刻保持微生物液体富集培养室、分离培养室内的温度值、压力值与微生物生长的海洋环境条件一致。
其中，所述分离培养室顶部设置为快开釜盖；所述中央注液管设置在所述快开釜盖上；快开釜盖还设置有注气通道和传感器放置通道；所述压力控制系统通过注气通道与所述分离培养室连接；所述第二温度传感器、第二压力传感器设置在所述传感器放置通道中。
上述方案中，所述分离培养室是利用重力自然释放原理，在分离培养室内利用小球在重力作用下携带菌液自由移动，实现微生物在固体培养基上的有效分离。分离培养室设计有顶部快开釜盖，方便迅速地往培养室内装样。为了有效的利用空间，本方案采用圆台式或圆柱式腔体，这样在有限的空间内最大程度的增加了能够供微生物分离培养的面积。在腔体内上从上到下设置有旋转蛇形导流槽，导流槽内平整填充有培养基质的固体培养基，为微生物的分离培养提供营养。本方案在圆台/圆柱腔体的顶部放置有可移动储液槽，用于储存待分离的微生物富集液和小球。导流槽直径大于小球，确保从可移动储液槽内释放出来的小球能完全顺利进入导流槽。在快开釜盖的中央设置有中央注液管，用于将微生物富集液注入可移动储液槽。可移动储液槽的底部中央设置有直径略大于小球的孔。在分离培养室的侧壁设置有往复式拉杆，拉杆可通过往复式活塞等控制方法实现往复式推拉移动。可用往复式拉杆实现将可移动储液槽从腔体的中央拖拉至边缘，由于可移动储液槽底部直径大于小球，小球将从可移动储液槽底部中央孔释放，进入导流槽，然后在重力作用下，在导流槽内从上往下螺旋形下降，运移至腔体底部。在小球的运动过程中，微生物富集液在导流槽内呈浓度梯度稀释分散，满足单菌落分散生长要求。
优选的，所述分离培养室依次设置有若干个，所述注液单元与所有分离培养室的中央注液管连接；所述温度控制系统、压力控制系统分别与所有分离培养室连接，用于在每个分离培养室内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行分离培养；每个分离培养室均与独立的环境参数检测单元连接，用于实时检测每个分离培养室内的温度、压力的变化情况，并将检测的数据传输至所述中央控制系统。
上述方案涉及的微生物分离工艺主要是将若干分离培养室并联放置，所有分离培养室的进口通过管线并联连接并且与微注泵和最后一级微生物液体富集培 养室相连。为了便于筛选最优的培养方式，不同的培养室可以放置不同配方的培养基。
其中，所述压力控制系统包括放气阀、空压机、增压泵、储气罐、调压阀、调节阀和通气管道；其中：所述放气阀通过通气管道与所述富集与多层级纯化单元、重力式分离培养单元连接，其控制端与所述中央控制系统电性连接，用于排出富集与多层级纯化单元、重力式分离培养单元的气体，对富集与多层级纯化单元、重力式分离培养单元内部进行降压；所述空压机、增压泵、储气罐、调压阀、调节阀通过通气管道依次连接，最后通过通气管道与所述富集与多层级纯化单元、重力式分离培养单元连接，用于向富集与多层级纯化单元、重力式分离培养单元内注入培养所需要的气体(或惰性气体)进行增压；所述调压阀用于调整富集与多层级纯化单元、重力式分离培养单元的内部压力，所述调节阀用于调整注入气体的速度；所述空压机控制端、增压泵控制端、调压阀控制端、调节阀控制端均与所述中央控制系统电性连接。
其中，所述温度控制系统包括水浴夹套和制冷/制热装置；所述水浴夹套包裹在所述富集与多层级纯化单元、重力式分离培养单元外壁，与所述制冷/制热装置连接；所述制冷/制热装置控制端与所述中央控制系统电性连接。
上述方案中，微生物液体富集培养室、分离培养室内的恒温条件维持主要是将微生物液体富集培养室、分离培养室放置于水浴夹套的高/低温水浴中并通过温度传感器、第二温度传感器进行监控显示，通过于制冷/制热装置的热交换作用，维持培养室内的恒温状态。
上述方案中，微生物液体富集培养室、分离培养室的温度条件控制主要是通过温度控制系统。例如，在微生物液体富集培养室、分离培养室的环壁腔内注入载冷/热流体，并且通过将流体进行循环制冷或者加热保证环壁腔内流体的低温或者高温状态，然后通过载冷/热流体与内置腔内的热交换保证内置腔内的低温或者高温状态。或者将微生物液体富集培养室、分离培养室置于低温/高温的水浴/油浴环境，来保证微生物液体富集培养室、分离培养室内需要的特殊温度条件。或者将微生物液体富集培养室和分离培养室放于通过空气换热保证的温度恒定的制冷/制热房间或者箱内。一些极端的温度条件保持可同时采用上述几种温度控制方式。
其中，所述高压环境海洋微生物富集培养与重力式分离装置还包括移动平 台，所述富集与多层级纯化单元、重力式分离培养单元、温度控制系统、压力控制系统、中央控制系统均放置在所述移动平台上，用于增加培养场景的普适性。
上述方案中，通过在微生物液体富集培养室、分离培养室里面构建微生物在海洋环境生活的高压与极端温度环境条件，提高其生存活性。通过多级富集和液体稀释培养，得到定向环境条件胁迫下纯度较高的功能性微生物，同时结合分离培养室进行培养分离，获得单个微生物。同时，可通过多套选择微生物液体富集培养室和分离培养室组合，形成不同培养基组合的高效分离工艺。
本方案所提出的装置涉及的高压环境海洋微生物富集培养与分离技术主要包括富集和分离两个步骤。首先，通过富集与多层级纯化单元富集培养，得到纯度较高的菌群后，在保压情况下进入重力式分离培养单元进行固体培养分离，并且通过不同培养基和环境条件的组合工艺同时筛选，得到纯培养菌株，其实现原理具体为：
富集过程：首先是将微生物液体富集培养室及其附带管阀件进行灭菌处理，然后依次装入待培养的底物如深海沉积物、与微生物共生的宏生物组织及提取液等，然后从进液阀装入培养需要的营养液，然后从进气阀注入培养需要的气体(若不需要可注入惰性气体)使得微生物液体富集培养室内的压力值增加至与深海实际环境条件一致。在培养的过程中，通过顶部的手动搅拌杆进行搅拌，增加传质作用，优化培养进程。待首级微生物液体富集培养室培养过程完成后，在第二级微生物液体富集培养室中注入培养需要的营养液，并通过压力控制系统向第二级微生物液体富集培养室注气增压，第二级微生物液体富集培养室内注入的培养液体的量需要保证富集液从首级微生物液体富集培养室的浓度到第二级微生物液体富集培养室中的稀释比例满足纯化需求，然后通过保压转移，将首级微生物液体富集培养室内的微生物菌液转移到第二级微生物液体富集培养室中。以此类推，在最后一级微生物液体富集培养室中的微生物将达到高度纯化状态，当最后一级微生物液体富集培养室中的深海微生物菌液浓度达到10 6个/mL以上，可认为达到了较好的纯化状态。针对具体培养的微生物类群，可调整各层级的稀释比例。
当富集过程的菌液浓度经鉴定达到需求后，进入分离培养过程。
分离过程：首先，将分离培养室及其内部所有器件及相关的管阀件进行灭菌处理，保持无菌状态。然后在微生物分离支体的表面的导流槽内填充好培养需要 的培养基并安装至腔体内，然后放好可移动储液槽在微生物分离支体的顶部。然后安装好往复式拉杆。然后安装好培养室的快开釜盖，保证中央注液管畅通。然后通过压力控制系统往分离培养室内注入气体增压，使分离培养室内的压力条件与微生物液体富集培养室内的压力条件一致。确保所有系统部件工作正常后，开启微注泵通过中央注液管从最后一级微生物液体富集培养室向微生物分离支体上的可移动储液槽内注入微量海洋微生物富集菌液，使得海洋微生物富集菌液均匀分散在小球上。然后，开启往复式拉杆拖拉可移动储液槽于圆台侧面处的导流槽的档盒处，由于小球的直径小于可移动储液槽底部的孔径，小球将从可移动储液槽内释放进入导流槽，在重力作用下，将在导流槽内从上往下螺旋形运移至底部。小球上富集的菌液将在导流槽内得到分散，满足分离和纯化培养的过程。沿着小球的运动轨迹，菌液的划线轨迹将呈梯度稀释，将沿着导流的轨迹长出分离后的菌落，至此，完成分离过程。
本方案还涉及微生物自动分离纯化工艺，其主要是将若干分离培养室并联放置，所有分离培养室的进口通过管线并联连接并且与微注泵和最后一级微生物液体富集培养室相连。为了便于筛选最优的培养方式，不同的培养室可以放置不同配方的培养基。然后培养工艺涉及的所有分离培养室和管阀件整体灭菌，然后检查所有培养室内的微生物分离支体、小球、往复式拉杆、导流槽、中央注液管及其附属系统安装完好。然后通过温度和压力监控，保证所有分离培养室内的温度和压力环境条件与微生物所在海洋环境的温度和压力环境条件一致。依次打开微注泵、调节阀、中央注液管和往复式拉杆，小球将在各个分离培养室内的微生物分离支体的的导流槽内携带菌液移动，通过大量培养室的自动化分离组合，可以实现不同培养基环境下的自动分选工艺，有效的保证高压环境微生物的分离培养和纯化，为海洋微生物的高效利用和高压环境分选工艺提供关键技术。整个培养过程中的各项参数条件将通过中央控制系统进行数据采集、集成和显示。
本方案涉及高压环境海洋微生物富集培养与重力式分离装置与工艺，提出了在高压和极端温度环境条件下，对海洋微生物进行多层级富集培养和多培养基自动分离纯化培养，解决了现有室内纯培养技术方法脱离海洋微生物生存的高压与极端温度环境条件而导致大量微生物不能实现纯培养的难题，不需要专业操作人员，可用于研究室、科考船等多培养场景，适应性较广；本方案不需要专业人员手动富集和划线分离操作，可进行规模化富集和分选，减少人力成本，实现海洋 微生物在原位压力和温度环境条件下的自动化分离培养，为海洋微生物原位条件纯培养提供重要技术手段。
本方案相对于现有的纯培养技术，提出了在海洋原位的高压和极端温度环境条件下对海洋微生物进行富集和分离培养的高压纯培养技术，解决了现有常压分离培养技术脱离海洋微生物原位生存的温度和压力环境条件，造成绝大多数微生物不能分离纯培养的问题；本方案相对于现有的分离培养技术，可有效的减少专业人员投入，并且可进行规模化富集和分离培养，提高难培养微生物的筛选效率，提高有特殊功能的工程菌的筛选和培育效率。
与现有技术相比，本发明技术方案的有益效果是：
本发明提出了一种高压环境海洋微生物富集培养与重力式分离装置，通过富集与多层级纯化单元实现海洋原位的温度和压力环境条件下微生物的富集培养，通过重力式分离培养单元实现对海洋微生物的分离培养。其通过重塑原位环境进行海洋微生物的富集培养与分离，解决分离纯培养海洋高压环境微生物的难题，有效的提高海洋微生物的可培养性，为开发利用深海微生物资源提供重要的基础手段。
附图说明
图1为本发明所述装置的结构示意图；
图2为本发明所述微生物液体富集培养室的结构示意图；
图3为本发明所述分离培养室与压力控制系统的结构示意图；
图4为本发明所述中央控制系统电路模块连接示意图；
图5为本发明一实施例中微生物自动分离纯化工艺连接示意图；
其中：1、富集与多层级纯化单元；11、微生物液体富集培养室；111、可拆卸密封盖；112、传感器组；1121、温度传感器；1122、压力传感器；113、搅拌杆；1141、进液阀；1142、进气阀；1143、取样阀；1144、出液阀；2、重力式分离培养单元；21、分离培养室；211、微生物分离支体；2111、可移动储液槽；2112、小球；2113、往复式拉杆；2114、导流槽；2115、腔体；2116、档盒；212、中央注液管；213、快开釜盖；22、注液单元；221、微注泵；222、注液管道；23、环境参数检测单元；231、第二温度传感器；232、第二压力传感器；3、温度控制系统；31、水浴夹套；32、制冷/制热装置；33、水浴温度检测装置；4、压力控制系统；41、放气阀；42、空压机；43、增压泵；44、储气罐；45、调压 阀；46、调节阀；47、通气管道；5、中央控制系统；6、移动平台。
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所示，本方案提供一种高压环境海洋微生物富集培养与重力式分离装置，包括富集与多层级纯化单元：1、重力式分离培养单元2、温度控制系统3、压力控制系统4和中央控制系统5；富集与多层级纯化单元1与重力式分离培养单元2的控制端与信号检测端均中央控制系统5电性连接；温度控制系统3、压力控制系统4的控制端均中央控制系统5电性连接；其中：所述富集与多层级纯化单元1用于实现对海洋微生物的富集与多层级纯化过程，得到海洋微生物富集菌液并将海洋微生物富集菌液注入所述重力式分离培养单元2中；所述重力式分离培养单元2用于在高压环境进行利用重力作用进行自动划线，实现海洋微生物的固体分离和培养，有效地提高海洋微生物的可培养性；所述温度控制系统3、压力控制系统4分别与所述富集与多层级纯化单元1、重力式分离培养单元2连接，用于在富集与多层级纯化单元1、重力式分离培养单元2内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行富集、纯化、分离和培养。
在具体实施过程中，富集与多层级纯化单元1可以实现海洋原位的温度和压力环境条件下微生物的富集培养，重力式分离培养单元2可以实现对海洋微生物的分离培养。通过重塑其原位环境进行海洋微生物的富集培养与分离，解决分离纯培养海洋高压环境微生物的难题，有效的提高海洋微生物的可培养性，为开发利用深海微生物资源提供重要的基础手段。
在具体实施过程中，中央控制系统5用于实现微生物富集菌在高压环境进行富集、分离、纯化过程中各项环境数据信息变化的监控、以及实时采集、处理、 存储和图像输出等功能。
更具体的，如图1、图2、图4所示，所述富集与多层级纯化单元1由多个串联的微生物液体富集培养室11组成；所述微生物液体富集培养室11上设置有可拆卸密封盖111和连接取样阀组，其内部设置有传感器组112；每个微生物液体富集培养室11均安置在温度控制系统3中；其中：所述可拆卸密封盖111用于方便对微生物液体富集培养室11内部进行灭菌操作和放入培养底物；所述连接取样阀组用于各个微生物液体富集培养室11的连接与取样，其与所述压力控制系统4连接，用于向微生物液体富集培养室11内输入液体或气体以增加微生物液体富集培养室11内的压力，保障微生物液体富集培养室11内的压力值与深海实际情况一致；所述传感器组112用于实时微生物液体富集培养室11内的温度和压力变化情况，并将信号输送至所述中央控制系统5；所述富集与多层级纯化单元1最后一个微生物液体富集培养室11通过连接取样阀组与所述重力式分离培养单元2连接。
更具体的，在所述微生物液体富集培养室11上还设置有搅拌杆113，所述搅拌杆113用于微生物液体富集培养室11在培养过程增强基质的反应过程。
在具体实施过程中，所述搅拌杆113为手动搅拌杆，可通过间歇式手动搅拌增强传质，也可根据需要放置增强型连续或者间歇式搅拌杆113，用于在培养过程中，增强基质的反应过程，增加微生物的能量和营养供给，提高培养效率。
其中，所述传感器组112包括温度传感器1121和压力传感器1122；所述温度传感器1121用于实时监测微生物液体富集培养室11内的温度变化情况；所述压力传感器1122用于实时监测微生物液体富集培养室11内的压力变化情况；温度传感器1121信号输出端、压力传感器1122信号输出端均与所述中央控制系统5电性连接。
更具体的，所述连接取样阀组包括进液阀1141、进气阀1142、取样阀1143和出液阀1144；其中：微生物液体富集培养室11之间通过所述出液阀1144、进液阀1141进行串联，前一级微生物液体富集培养室11出液阀1144与后一级微生物液体富集培养室11出液阀1144连接；最后一级微生物液体富集培养室11出液阀1144与所述重力式分离培养单元2连接；进气阀1142与所述压力控制系统4连接，用于向微生物液体富集培养室11内输入气体以增加微生物液体富集培养室11内的压力，令微生物液体富集培养室11内的压力值与深海实际情况一 致；取样阀1143用于对微生物液体富集培养室11内的微生物进行实时取样分析。
在具体实施过程中，多个微生物液体富集培养室11串联构成富集与多层级纯化单元1，将前级微生物液体富集培养室11内的菌液通过保压转移至下一级微生物液体富集培养室11中，以此类推，按照浓度梯度稀释，最后一个微生物液体富集培养室11中获得的微生物菌液将为高压环境，定向营养条件供给胁迫下富集的、高度纯化的功能型微生物。保压转移可以通过微注泵将前级微生物液体富集培养室11中的富集液通过取样阀1143取出后泵入后级的微生物液体富集培养室11中实现。也可以将后级微生物液体富集培养室11的压力增压至略小于前级微生物液体富集培养室11，然后开启前级微生物液体富集培养室11的出液阀1144和后级室的进液阀1141，微生物富集液将在微小压力差条件下，自动地从前级微生物液体富集培养室11进入后级微生物液体富集培养室11中进行稀释培养。在整个富集培养的过程中，微生物液体富集培养室11内的温度、压力环境条件都与微生物在深海的环境条件一致，保证富集培养的有效性。
更具体的，如图1、图3、图4所述重力式分离培养单元2包括分离培养室21、注液单元22和环境参数检测单元23；其中：所述重力式分离培养单元2通过注液单元22与所述富集与多层级纯化单元1最后一个微生物液体富集培养室11连接；所述分离培养室21内设置有微生物分离支体211，用于对微生物进行分离操作，最大程度的为微生物培养提供面积；所述分离培养室21顶部设置有中央注液管212，分离培养室21通过中央注液管212与所述注液单元22连接；由注液单元22向分离培养室21的微生物分离支体211注入海洋微生物富集菌液；所述分离培养室21安置在温度控制系统3中并与压力控制系统4连接，用于在分离培养室21内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行分离培养；所述环境参数检测单元23用于实时检测分离培养室21内的温度、压力的变化情况，并将检测的数据传输至所述中央控制系统5；所述微生物分离支体211、注液单元22均与所述中央控制系统5电性连接。
更具体的，所述微生物分离支体211包括可移动储液槽2111、小球2112、往复式拉杆2113、导流槽2114和腔体2115；其中：
所述可移动储液槽2111设置在所述腔体2115顶部中央，落在中央注液管212正下方，用于存储由注液单元22注入的海洋微生物富集菌液；所述小球2112放 置在所述可移动储液槽2111中，被注入的海洋微生物富集菌液浸没；所述可移动储液槽2111底部设置有通孔，用于固定小球2112的位置和保证小球2112能从其中穿过；所述往复式拉杆2113可移动端与所述可移动储液槽2111固定连接；所述腔体2115顶部侧面设置有档盒2116；所述往复式拉杆2113控制端与所述中央控制系统5电性连接；导流槽2114固定设置在腔体2115内部；导流槽2114中填充有培养基，其进口与档盒2116连通；当小球2112浸没在海洋微生物富集菌液后，在往复式拉杆2113的作用下，可移动储液槽2111向腔体2115边缘移动；当可移动储液槽2111的通孔离开腔体2115时，由于重力作用，小球2112将从通孔中穿过落在腔体2115的档盒2116中，进入到腔体2115内部的导流槽2114中；携带海洋微生物富集菌液的小球2112沿着导流槽2114由上向下滑动的过程中，将海洋微生物富集菌液呈浓度梯度稀释分散，最大程度满足微生物分离培养需要的面积。
更具体的，在所述腔体2115顶部中央，即可移动储液槽2111初始位与通孔重合的位置，设置有小槽孔，用于固定所述小球2112的位置。
更具体的，所述注液单元22包括微注泵221和注液管道222；其中：所述微注泵221入液端与所述富集与多层级纯化单元1最后一个微生物液体富集培养室11连接，其出液端与所述注液管道222连接；所述注液管道222出液口与所述中央注液管212连接；所述微注泵221控制端与所述中央控制系统5电性连接。
更具体的，所述环境参数检测单元23包括第二温度传感器231和第二压力传感器232；其中：所述第二温度传感器231、第二压力传感器232探头均设置在所述分离培养室21内部，其信号输出端均与所述中央控制系统5电性连接。
在具体实施过程中，通过温度传感器1121、压力传感器1122、第二温度传感器231、第二压力传感器232分别实现对微生物液体富集培养室11、分离培养室21中的温度、压力进行实时监控；若温度/压力需要调整，则通过中央控制系统5控制温度控制系统3、压力控制系统4进行工作，时刻保持微生物液体富集培养室、分离培养室内的温度值、压力值与微生物生长的海洋环境条件一致。
更具体的，所述分离培养室21顶部设置为快开釜盖213；所述中央注液管212设置在所述快开釜盖213上；快开釜盖213还设置有注气通道和传感器放置通道；所述压力控制系统4通过注气通道与所述分离培养室21连接；所述第二温度传感器231、第二压力传感器232设置在所述传感器放置通道中。
在具体实施过程中，所述分离培养室21是利用重力自然释放原理，在分离培养室21内利用小球2112在重力作用下携带菌液自由移动，实现微生物在固体培养基上的有效分离。分离培养室21设计有顶部快开釜盖213，方便迅速地往培养室内装样。为了有效的利用空间，本实施例采用圆台式或圆柱式腔体2115，这样在有限的空间内最大程度的增加了能够供微生物分离培养的面积。在腔体2115内上从上到下设置有旋转蛇形导流槽2114，导流槽2114内平整填充有培养基质的固体培养基，为微生物的分离培养提供营养。本实施例在圆台/圆柱腔体2115的顶部放置有可移动储液槽2111，用于储存待分离的微生物富集液和小球2112。导流槽2114直径大于小球2112，确保从可移动储液槽2111内释放出来的小球2112能完全顺利进入导流槽2114。在快开釜盖213的中央设置有中央注液管212，用于将微生物富集液注入可移动储液槽2111。可移动储液槽2111的底部中央设置有直径略大于小球2112的孔。在分离培养室21的侧壁设置有往复式拉杆2113，拉杆可通过往复式活塞等控制方法实现往复式推拉移动。可用往复式拉杆2113实现将可移动储液槽2111从腔体2115的中央拖拉至边缘，由于可移动储液槽2111底部直径大于小球2112，小球2112将从可移动储液槽2111底部中央孔释放，进入导流槽2114，然后在重力作用下，在导流槽2114内从上往下螺旋形下降，运移至腔体2115底部。在小球2112的运动过程中，微生物富集液在导流槽2114内呈浓度梯度稀释分散，满足单菌落分散生长要求。
更具体的，所述压力控制系统4包括放气阀41、空压机42、增压泵43、储气罐44、调压阀45、调节阀46和通气管道47；其中：所述放气阀41通过通气管道47与所述富集与多层级纯化单元1、重力式分离培养单元2连接，其控制端与所述中央控制系统5电性连接，用于排出富集与多层级纯化单元1、重力式分离培养单元2的气体，对富集与多层级纯化单元1、重力式分离培养单元2内部进行降压；所述空压机42、增压泵43、储气罐44、调压阀45、调节阀46通过通气管道47依次连接，最后通过通气管道47与所述富集与多层级纯化单元1、重力式分离培养单元2连接，用于向富集与多层级纯化单元1、重力式分离培养单元2内注入培养所需要的气体(或惰性气体)进行增压；所述调压阀45用于调整富集与多层级纯化单元1、重力式分离培养单元2的内部压力，所述调节阀46用于调整注入气体的速度；所述空压机42控制端、增压泵43控制端、调压阀45控制端、调节阀46控制端，均与所述中央控制系统5电性连接。
更具体的，所述温度控制系统3包括水浴夹套31和制冷/制热装置32；所述水浴夹套31包裹在所述富集与多层级纯化单元1、重力式分离培养单元2外壁，与所述制冷/制热装置32连接；所述制冷/制热装置32控制端与所述中央控制系统5电性连接。
在具体实施过程中，微生物液体富集培养室11、分离培养室21内的恒温条件维持主要是将微生物液体富集培养室11、分离培养室21放置于水浴夹套31的高/低温水浴中并通过温度传感器1121、第二温度传感器231进行监控显示，通过于制冷/制热装置的热交换作用，维持培养室内的恒温状态。同时，还设置有水浴温度检测装置33，其输出端与所述中央处理系统5电性连接，用于实时检测水浴夹套31中的水浴温度，便于实时进行温度调整。
在具体实施过程中，微生物液体富集培养室11、分离培养室21的温度条件控制主要是通过温度控制系统3。例如，在微生物液体富集培养室11、分离培养室21的环壁腔内注入载冷/热流体，并且通过将流体进行循环制冷或者加热保证环壁腔内流体的低温或者高温状态，然后通过载冷/热流体与内置腔内的热交换保证内置腔内的低温或者高温状态。或者将微生物液体富集培养室11、分离培养室21置于低温/高温的水浴/油浴环境，来保证微生物液体富集培养室11、分离培养室21内需要的特殊温度条件。或者将微生物液体富集培养室11和分离培养室21放于通过空气换热保证的温度恒定的制冷/制热房间或者箱内。一些极端的温度条件保持可同时采用上述几种温度控制方式。
更具体的，所述高压环境海洋微生物富集培养与重力式分离装置还包括移动平台6，所述富集与多层级纯化单元1、重力式分离培养单元2、温度控制系统3、压力控制系统4、中央控制系统5均放置在所述移动平台6上，用于增加培养场景的普适性。
在具体实施过程中，通过在微生物液体富集培养室11、分离培养室21里面构建微生物在海洋环境生活的高压与极端温度环境条件，提高其生存活性。通过多级富集和液体稀释培养，得到定向环境条件胁迫下纯度较高的功能性微生物，同时结合分离培养室21进行培养分离，获得单个微生物。同时，可通过多套选择微生物液体富集培养室11和分离培养室21组合，形成不同培养基组合的高效分离工艺，如图5所示。
实施例2
更具体的，在实施例1的基础上，本方案所提出的装置涉及的高压环境海洋微生物富集培养与分离技术主要包括富集和分离两个步骤。首先，通过富集与多层级纯化单元1富集培养，得到纯度较高的菌群后，在保压情况下进入重力式分离培养单元2进行固体培养分离，并且通过不同培养基和环境条件的组合工艺同时筛选，得到纯培养菌株，其实现原理具体为：
富集过程：首先是将微生物液体富集培养室11及其附带管阀件进行灭菌处理，然后依次装入待培养的底物如深海沉积物、与微生物共生的宏生物组织及提取液等，然后从进液阀1141装入培养需要的营养液，然后从进气阀1142注入培养需要的气体(若不需要可注入惰性气体)使得微生物液体富集培养室11内的压力值增加至与深海实际环境条件一致。在培养的过程中，通过顶部的手动搅拌杆113进行搅拌，增加传质作用，优化培养进程。待首级微生物液体富集培养室11培养过程完成后，在第二级微生物液体富集培养室11中注入培养需要的营养液，并通过压力控制系统4向第二级微生物液体富集培养室11注气增压，第二级微生物液体富集培养室11内注入的培养液体的量需要保证富集液从首级微生物液体富集培养室11的浓度到第二级微生物液体富集培养室11中的稀释比例满足纯化需求，然后通过保压转移，将首级微生物液体富集培养室11内的微生物菌液转移到第二级微生物液体富集培养室11中。以此类推，在最后一级微生物液体富集培养室11中的微生物将达到高度纯化状态，当最后一级微生物液体富集培养室11中的深海微生物菌液浓度达到10 6个/mL以上，可认为达到了较好的纯化状态。针对具体培养的微生物类群，可调整各层级的稀释比例。
当富集过程的菌液浓度经鉴定达到需求后，进入分离培养过程。
分离过程：首先，将分离培养室21及其内部所有器件及相关的管阀件进行灭菌处理，保持无菌状态。然后在微生物分离支体211的表面的导流槽2114内填充好培养需要的培养基。然后放好可移动储液槽2111在微生物分离支体211的顶部。然后安装好往复式拉杆2113。然后安装好培养室的快开釜盖213，保证中央注液管212畅通。然后通过压力控制系统4往分离培养室21内注入气体增压，使分离培养室21内的压力条件与微生物液体富集培养室11内的压力条件一致。确保所有系统部件工作正常后，开启微注泵221通过中央注液管212从最后一级微生物液体富集培养室11向微生物分离支体211上的可移动储液槽2111内注入微量海洋微生物富集菌液，使得海洋微生物富集菌液均匀分散在小球2112 上。然后，开启往复式拉杆2113拖拉可移动储液槽2111于圆台侧面处的导流槽2114的档盒2116处，由于小球2112的直径小于可移动储液槽2111底部的孔径，小球2112将从可移动储液槽2111内释放进入导流槽2114，在重力作用下，将在导流槽2114内从上往下螺旋形运移至底部。小球2112上富集的菌液将在导流槽2114内得到分散，满足分离和纯化培养的过程。沿着小球2112的运动轨迹，菌液的划线轨迹将呈梯度稀释，将沿着导流的轨迹长出分离后的菌落，至此，完成分离过程。
实施例3
优选的，如图5所示，所述分离培养室21依次设置有若干个，所述注液单元22与所有分离培养室21的中央注液管212连接；所述温度控制系统3、压力控制系统4分别与所有分离培养室21连接，用于在每个分离培养室21内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行分离培养；每个分离培养室21均与独立的环境参数检测单元23连接，用于实时检测每个分离培养室21内的温度、压力的变化情况，并将检测的数据传输至所述中央控制系统5。
在具体实施过中，本实施例涉及的微生物分离工艺主要是将若干分离培养室21并联放置，所有分离培养室21的进口通过管线并联连接并且与微注泵221和最后一级微生物液体富集培养室11相连。
为了便于筛选最优的培养方式，不同的培养室可以放置不同配方的培养基。然后培养工艺涉及的所有分离培养室21和管阀件整体灭菌，然后检查所有培养室内的微生物分离支体211、小球2112、往复式拉杆2113、导流槽2114、中央注液管212及其附属系统安装完好。然后通过温度和压力监控，保证所有分离培养室21内的温度和压力环境条件与微生物所在海洋环境的温度和压力环境条件一致。依次打开微注泵221、调节阀46、中央注液管212和往复式拉杆2113，小球2112将在各个分离培养21室内的微生物分离支体211的的导流槽2114内携带菌液移动，通过大量培养室的自动化分离组合，可以实现不同培养基环境下的自动分选工艺，有效的保证高压环境微生物的分离培养和纯化，为海洋微生物的高效利用和高压环境分选工艺提供关键技术。整个培养过程中的各项参数条件将通过中央控制系统进行数据采集、集成和显示。
本实施例涉及高压环境海洋微生物富集培养与重力式分离装置与工艺，提出 了在高压和极端温度环境条件下，对海洋微生物进行多层级富集培养和多培养基自动分离纯化培养，解决了现有室内纯培养技术方法脱离海洋微生物生存的高压与极端温度环境条件而导致大量微生物不能实现纯培养的难题，不需要专业操作人员，可用于研究室、科考船等多培养场景，适应性较广；本实施例不需要专业人员手动富集和划线分离操作，可进行规模化富集和分选，减少人力成本，实现海洋微生物在原位压力和温度环境条件下的自动化分离培养，为海洋微生物原位条件纯培养提供重要技术手段。
本施例相对于现有的纯培养技术，提出了在海洋原位的高压和极端温度环境条件下对海洋微生物进行富集和分离培养的高压纯培养技术，解决了现有常压分离培养技术脱离海洋微生物原位生存的温度和压力环境条件，造成绝大多数微生物不能分离纯培养的问题；本方案相对于现有的分离培养技术，可有效的减少专业人员投入，并且可进行规模化富集和分离培养，提高难培养微生物的筛选效率，提高有特殊功能的工程菌的筛选和培育效率。
实施例4
为了进一步说明本方案的技术实现过程和技术效果，本实施例提出一种高压环境深海嗜甲烷菌富集培养与分离装置。本实例涉及的高压环境深海嗜甲烷菌富集培养与分离装置主要包括富集和分离两个步骤。首先，通过微生物液体富集培养室11进行富集深海嗜甲烷菌，在保压情况下再通过下一级微生物液体富集培养室11，得到纯度较高的深海嗜甲烷菌群后，再进入分离培养室21进行分离培养，最终得到单个菌株。
本实例涉及的高压环境深海嗜甲烷菌富集培养方法首先是将微生物液体富集培养室11及其附带管阀件进行灭菌处理，灭菌处理完成后，依次连接各个装置，再将深海甲烷渗漏区的沉积物装入微生物液体富集培养室11，然后装入培养需要的营养液，再打开进气阀1142通过压力控制系统4注入培养需要的甲烷气体，使得微生物液体富集培养室11内的压力值增加至14Mpa，并通过压力传感器1122监控，同时将整套装置置于4℃的水浴夹套31，通过水浴温度检测装置33显示和制冷/制热装置32进行交换，水浴系统内填充制冷剂如乙二醇等，维持微生物液体富集培养室11内的低温状态，微生物液体富集培养室11内的温度均通过温度传感器1121。为保证培养环境为厌氧环境，可通过打开放气阀41持续通甲烷5-10min。在培养的过程中，通过微生物液体富集培养室11顶部的搅 拌杆113进行搅拌，增加传质作用，优化培养进程。待微生物液体富集培养室11内的富集培养过程完成后，在下一级微生物液体富集培养室11注入培养需要的营养液，并采用与微生物液体富集培养室11一样的方法增压。微生物液体富集培养室11内注入的培养液体的量需要保证富集液从首级微生物液体富集培养室11的浓度到下一级微生物液体富集培养室11中的稀释比例为1:10，然后将第二级微生物液体富集培养室11的压力增压至小于首级微生物液体富集培养室11的压力0.2-0.5MPa，然后开启首级微生物液体富集培养室11的出液阀1144和第二级微生物液体富集培养室11的进液阀1141，微生物富集液将在微小压力差的条件下，自动地从前级微生物液体富集培养室11进入后级微生物液体富集培养室11中进行纯化培养。以此类推，再转入第三级和第四级微生物液体富集培养室11中，当第四级微生物液体富集培养室11中的细胞数量达到10 6个/mL以上且深海嗜甲烷菌丰度在50％以上，可认为达到了较好的纯化状态。针对具体培养的微生物类群，可调整各层级的稀释比例。实验过程，取样监控需要的菌液通过打开取样阀1143进行。
当第四级微生物液体富集培养室11的菌液浓度经鉴定达到需求后，进入分离培养过程。分离培养过程主要包括，首先，将分离培养室21及其内部所有器件及相关的管阀件进行灭菌处理。然后在微生物分离支体211的导流槽2114内填充好培养需要的培养基。将可移动储液槽2111置于微生物分离支体211的顶部。并将小球2112放于可移动储液槽2111内部的凹槽处，使其不滑动。然后安装好往复式拉杆2113。再安装好分离培养室21的上盖和中央注液管212。然后通过压力控制系统4往分离培养室21内注入气体增压，使分离培养室21内的压力条件与微生物液体富集培养室11内的压力条件一致。确保所有系统部件工作正常后，开启微注泵221通过中央注液管212从最后一级微生物液体富集培养室向微生物分离支体211上的可移动储液槽2111内注入10微升微生物富集液，使得微生物富集液均匀分散在小球2112上。然后，开启往复式拉杆2113拖拉可移动储液槽2111于微生物分离支体211侧面处的导流槽2114，由于小球2112的直径小于可移动储液槽2111底部的孔径，小球2112将从可移动储液槽2111内释放进入导流槽2114，在重力作用下，将在导流槽2114内从上往下螺旋形运移至底部。小球2112上富集菌液的划线轨迹呈梯度稀释，微生物将沿着导流的轨迹长出分离后的菌落。
本实施例涉及的深海嗜甲烷菌自动分离纯化工艺主要是将若干分离培养室21并联放置，所有分离培养室21的进口通过管线并联连接并且与微注泵221和富集与多层级纯化单元1相连。为了便于筛选最优的培养方式，不同的培养室可以放置不同配方的培养基。然后培养工艺涉及的所有培养室和管阀件整体灭菌，然后检查所有培养室内的分离培养支体211、小球2112、往复式拉杆2113、导流槽2114、中央注液管212及其附属系统安装完好。通过温度和压力监控，保证所有培养室内的温度和压力环境条件微4℃和14MPa。依次打开微注泵221、调节阀46、中央注液管212、和往复式拉杆2113，小球2112将在各个分离培养室21内的微生物分离支体211的的导流槽2113内携带菌液移动，通过大量培养室的自动化分离组合，可以实现不同培养基环境下的自动分选工艺，有效的保证高压环境微生物的分离培养和纯化。为海洋微生物的高效利用和高压环境分选工艺提供关键技术。整个培养过程中的各项参数条件将通过中央控制系统5进行数据采集、集成和显示。富集培养与分离装置底部整体加装移动平台6，增加培养场景的普适性。
在具体实施过程中，富集与多层级纯化单元1可以实现海洋原位的温度和压力环境条件下微生物的富集培养，重力式分离培养单元2可以实现对海洋微生物的分离培养。通过重塑其原位环境进行海洋微生物的富集培养与分离，解决分离纯培养海洋高压环境微生物的难题，有效的提高海洋微生物的可培养性，为开发利用深海微生物资源提供重要的基础手段。
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 (13)

  1. 高压环境海洋微生物富集培养与重力式分离装置，其特征在于，包括富集与多层级纯化单元(1)、重力式分离培养单元(2)、温度控制系统(3)、压力控制系统(4)和中央控制系统(5)；富集与多层级纯化单元(1)与重力式分离培养单元(2)的控制端与信号检测端均中央控制系统(5)电性连接；温度控制系统(3)、压力控制系统(4)的控制端均与中央控制系统(5)电性连接；其中：
    所述富集与多层级纯化单元(1)用于实现对海洋微生物的富集与多层级纯化过程，得到海洋微生物富集菌液并将海洋微生物富集菌液注入所述重力式分离培养单元(2)中；
    所述重力式分离培养单元(2)用于在高压环境进行利用重力作用进行自动划线，实现海洋微生物的固体分离和培养，有效地提高海洋微生物的可培养性；
    所述温度控制系统(3)、压力控制系统(4)分别与所述富集与多层级纯化单元(1)、重力式分离培养单元(2)连接，用于在富集与多层级纯化单元(1)、重力式分离培养单元(2)内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行富集、纯化、分离和培养；
    其中，所述压力控制系统(4)包括放气阀(41)、空压机(42)、增压泵(43)、储气罐(44)、调压阀(45)、调节阀(46)和通气管道(47)；其中：
    所述放气阀(41)通过通气管道(47)与所述富集与多层级纯化单元(1)、重力式分离培养单元(2)连接，其控制端与所述中央控制系统(5)电性连接，用于排出富集与多层级纯化单元(1)、重力式分离培养单元(2)的气体，对富集与多层级纯化单元(1)、重力式分离培养单元(2)内部进行降压；
    所述空压机(42)、增压泵(43)、储气罐(44)、调压阀(45)、调节阀(46)通过通气管道(47)依次连接，最后通过通气管道(47)与所述富集与多层级纯化单元(1)、重力式分离培养单元(2)连接，用于向富集与多层级纯化单元(1)、重力式分离培养单元(2)内注入气体进行增压；所述调压阀(45)用于调整富集与多层级纯化单元(1)、重力式分离培养单元(2)的内部压力，所述调节阀(46)用于调整注入气体的速度；
    所述空压机(42)控制端、增压泵(43)控制端、调压阀(45)控制端、调 节阀(46)控制端均与所述中央控制系统(5)电性连接；
    所述温度控制系统(3)包括水浴夹套(31)和制冷/制热装置(32)；所述水浴夹套(31)包裹在所述富集与多层级纯化单元(1)、重力式分离培养单元(2)外壁，与所述制冷/制热装置(32)连接；所述制冷/制热装置(32)控制端与所述中央控制系统(5)电性连接。
  2. 根据权利要求1所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，所述富集与多层级纯化单元(1)由多个串联的微生物液体富集培养室(11)组成；所述微生物液体富集培养室(11)上设置有可拆卸密封盖(111)和连接取样阀组，其内部设置有传感器组(112)；每个微生物液体富集培养室(11)均安置在温度控制系统(3)中；其中：
    所述可拆卸密封盖(111)用于方便对微生物液体富集培养室(11)内部进行灭菌操作和放入培养底物；
    所述连接取样阀组用于各个微生物液体富集培养室(11)的连接与取样，其与所述压力控制系统(4)连接，用于向微生物液体富集培养室(11)内输入液体或气体以增加微生物液体富集培养室(11)内的压力，令微生物液体富集培养室(11)内的压力值与深海实际情况一致；
    所述传感器组(112)用于实时微生物液体富集培养室(11)内的温度和压力变化情况，并将信号输送至所述中央控制系统(5)；
    所述富集与多层级纯化单元(1)最后一个微生物液体富集培养室(11)通过连接取样阀组与所述重力式分离培养单元(2)连接。
  3. 根据权利要去2所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，在所述微生物液体富集培养室(11)上还设置有搅拌杆(113)，所述搅拌杆(113)用于微生物液体富集培养室(11)在培养过程增强基质的反应过程。
  4. 根据权利要求2所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，所述传感器组(112)包括温度传感器(1121)和压力传感器(1122)；所述温度传感器(1121)用于实时监测微生物液体富集培养室(11)内的温度变化情况；所述压力传感器(1122)用于实时监测微生物液体富集培养室(11)内的压力变化情况；温度传感器(1121)信号输出端、压力传感器(1122)信号输出端均与所述中央控制系统(5)电性连接。
  5. 根据权利要求2所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，所述连接取样阀组包括进液阀(1141)、进气阀(1142)、取样阀(1143)和出液阀(1144)；其中：
    微生物液体富集培养室(11)之间通过所述出液阀(1144)、进液阀(1141)进行串联，前一级微生物液体富集培养室(11)出液阀(1144)与后一级微生物液体富集培养室(11)出液阀(1144)连接；最后一级微生物液体富集培养室(11)出液阀(1144)与所述重力式分离培养单元(2)连接；
    进气阀(1142)与所述压力控制系统(4)连接，用于向微生物液体富集培养室(11)内输入气体以增加微生物液体富集培养室(11)内的压力，令微生物液体富集培养室(11)内的压力值与深海实际情况一致；
    取样阀(1143)用于对微生物液体富集培养室(11)内的微生物进行实时取样分析。
  6. 根据权利要求2所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，所述重力式分离培养单元(2)包括分离培养室(21)、注液单元(22)和环境参数检测单元(23)；其中：
    所述重力式分离培养单元(2)通过注液单元(22)与所述富集与多层级纯化单元(1)最后一个微生物液体富集培养室(11)连接；
    所述分离培养室(21)内设置有微生物分离支体(211)，用于对微生物进行分离操作，最大程度的为微生物培养提供面积；
    所述分离培养室(21)顶部设置有中央注液管(212)，分离培养室(21)通过中央注液管(212)与所述注液单元(22)连接；由注液单元(22)向分离培养室(21)的微生物分离支体(211)注入海洋微生物富集菌液；
    所述分离培养室(21)安置在温度控制系统(3)中并与压力控制系统(4)连接，用于在分离培养室(21)内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行分离培养；
    所述环境参数检测单元(23)用于实时检测分离培养室(21)内的温度、压力的变化情况，并将检测的数据传输至所述中央控制系统(5)；
    所述微生物分离支体(211)、注液单元(22)均与所述中央控制系统(5)电性连接。
  7. 根据权利要求6所述的高压环境海洋微生物富集培养与重力式分离装置， 其特征在于，所述微生物分离支体(211)包括可移动储液槽(2111)、小球(2112)、往复式拉杆(2113)、导流槽(2114)和腔体(2115)；其中：
    所述可移动储液槽(2111)设置在所述腔体(2115)顶部中央，落在中央注液管(212)正下方，用于存储由注液单元(22)注入的海洋微生物富集菌液；所述小球(2112)放置在所述可移动储液槽(2111)中，被注入的海洋微生物富集菌液浸没；所述可移动储液槽(2111)底部设置有通孔，用于固定小球(2112)的位置和保证小球(2112)能从其中穿过；所述往复式拉杆(2113)可移动端与所述可移动储液槽(2111)固定连接；所述腔体(2115)顶部侧面设置有档盒(2116)；所述往复式拉杆(2113)控制端与所述中央控制系统(5)电性连接；
    导流槽(2114)固定设置在腔体(2115)内部；导流槽(2114)中填充有培养基，其进口与档盒(2116)连通；
    当小球(2112)浸没在海洋微生物富集菌液后，在往复式拉杆(2113)的作用下，可移动储液槽(2111)向腔体(2115)边缘移动；当可移动储液槽(2111)的通孔离开腔体(2115)时，由于重力作用，小球(2112)将从通孔中穿过落在腔体(2115)的档盒(2116)中，进入到腔体(2115)内部的导流槽(2114)中；
    携带海洋微生物富集菌液的小球(2112)沿着导流槽(2114)由上向下滑动的过程中，将海洋微生物富集菌液呈浓度梯度稀释分散，最大程度的为微生物的分离培养提供面积。
  8. 根据权利要求7所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，在所述腔体(2115)顶部中央，即可移动储液槽(2111)初始位与通孔重合的位置，设置有小槽孔，用于固定所述小球(2112)的位置。
  9. 根据权利要求7所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，所述注液单元(22)包括微注泵(221)和注液管道(222)；其中：所述微注泵(221)入液端与所述富集与多层级纯化单元(1)最后一个微生物液体富集培养室(11)连接，其出液端与所述注液管道(222)连接；所述注液管道(222)出液口与所述中央注液管(212)连接；所述微注泵(221)控制端与所述中央控制系统(5)电性连接。
  10. 根据权利要求7所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，所述环境参数检测单元(23)包括第二温度传感器(231)和第二压力传感器(232)；其中：所述第二温度传感器(231)、第二压力传感器 (232)探头均设置在所述分离培养室(21)内部，其信号输出端均与所述中央控制系统(5)电性连接。
  11. 根据权利要求10所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，所述分离培养室(21)顶部设置为快开釜盖(213)；所述中央注液管(212)设置在所述快开釜盖(213)上；快开釜盖(213)还设置有注气通道和传感器放置通道；所述压力控制系统(4)通过注气通道与所述分离培养室(21)连接；所述第二温度传感器(231)、第二压力传感器(232)设置在所述传感器放置通道中。
  12. 根据权利要求6～11任一项所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，所述分离培养室(21)依次设置有若干个，所述注液单元(22)与所有分离培养室(21)的中央注液管(212)连接；所述温度控制系统(3)、压力控制系统(4)分别与所有分离培养室(21)连接，用于在每个分离培养室(21)内构建与海洋环境一致的高压、低温环境，保证富集的深海微生物在原位的环境条件下进行分离培养；每个分离培养室(21)均与独立的环境参数检测单元(23)连接，用于实时检测每个分离培养室(21)内的温度、压力的变化情况，并将检测的数据传输至所述中央控制系统(5)。
  13. 根据权利要求12所述的高压环境海洋微生物富集培养与重力式分离装置，其特征在于，还包括移动平台(6)，所述富集与多层级纯化单元(1)、重力式分离培养单元(2)、温度控制系统(3)、压力控制系统(4)、中央控制系统(5)均放置在所述移动平台(6)上，用于增加培养场景的普适性。
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