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WO2023173494A1 - 高压环境生物富集与喷洒式固体分离培养装置 - Google Patents

高压环境生物富集与喷洒式固体分离培养装置 Download PDF

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Publication number
WO2023173494A1
WO2023173494A1 PCT/CN2022/084116 CN2022084116W WO2023173494A1 WO 2023173494 A1 WO2023173494 A1 WO 2023173494A1 CN 2022084116 W CN2022084116 W CN 2022084116W WO 2023173494 A1 WO2023173494 A1 WO 2023173494A1
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Prior art keywords
culture
pressure
spray
enrichment
separation
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PCT/CN2022/084116
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English (en)
French (fr)
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冯景春
张偲
杨志峰
王屹
蔡宴朋
钟松
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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Publication of WO2023173494A1 publication Critical patent/WO2023173494A1/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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/04—Apparatus for enzymology or microbiology with gas introduction 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control

Definitions

  • the invention relates to the technical field of marine microorganisms, and in particular to a high-pressure environment biological enrichment and spray solid separation and cultivation device.
  • High-pressure habitats account for a large proportion of the earth. Habitats with pressures greater than 30MPa are widely distributed in marine environments. More than 77% of marine environments have water depths exceeding 3,000 meters. Deep-sea sediments, deep-sea basins, trenches, and deep continents are all high-pressure environments. Microorganisms in high-pressure habitats change with changes in depth, salinity, pH, oxygen content, and nutrients, showing significant diversity.
  • the existing microbial enrichment and separation culture technologies are mainly performed in normal pressure environments, and the solid separation culture technology in particular lacks precedent for use in high-pressure environments, currently only less than 1% of microorganisms in high-pressure environments have been purely cultured. This brings difficulties to correctly understand the development and utilization value of these microorganisms' phenotypes, genes, and functions.
  • the prior art discloses a deep-sea microbial culture cabin, which includes: a linear bearing, a tension spring, a pressure compensation chamber, a fixed top plate, a deep-sea motor assembly, a fixed bottom plate, a hose, and a culture cabin body; this solution uses a deep-sea motor assembly Rotate and open the end cover of the culture cabin body to perform microbial enrichment culture in a completely open state. During the deployment and recovery process, close the end cover of the culture cabin body to achieve sealing of the microbial culture cabin. Although it can realize the enrichment and culture of microorganisms in the deep sea in situ, it does not isolate and culture marine microorganisms, and cannot effectively improve the success rate of culture.
  • the present invention provides a high-pressure environment biological enrichment and spray solid separation and cultivation device, which can enrich, cultivate and separate marine microorganisms by reshaping its in-situ environment and improve the cultivability of marine microorganisms. properties, forming pure culture technology, providing an important basic means for the development and utilization of microbial resources in high-pressure environments.
  • This plan proposes a high-pressure environmental biological enrichment and spray solid separation culture device, including a spray solid separation culture unit, a biological enrichment unit, a pressurization system, a temperature environment control system and a control collection terminal; wherein: the biological enrichment The collection unit is used to realize the enrichment and multi-level purification process of marine microorganisms, obtain the biological enrichment liquid and inject the biological enrichment liquid into the spray-type solid separation and culture unit; the spray-type solid separation and culture unit is used to The biological enrichment liquid is converted into fine beads, allowing it to be separated and cultured in a dispersed state, effectively improving the culturability of marine microorganisms; the pressurization system and the temperature environment control system are respectively connected with the spray-type solid separation and culture unit, The connection of the bioconcentration unit is used to build a high-pressure and low-temperature environment consistent with the marine environment in the bioconcentration unit and spray solid separation culture unit to ensure that the enriched deep-sea microorganisms are enriched and purified
  • the bioconcentration unit can realize the enrichment and culture of microorganisms under the in-situ temperature and pressure environmental conditions of the ocean, and the spray-type solid separation culture unit can convert the bioconcentration liquid into fine beads to make it in a dispersed state. Achieve isolation and culture.
  • the spray-type solid separation culture unit can convert the bioconcentration liquid into fine beads to make it in a dispersed state. Achieve isolation and culture.
  • control acquisition terminal includes a data collector, a data central processor, an operating computer, etc. to realize the monitoring of 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 spray-type solid separation and culture unit includes a spray-type separation and culture chamber, a liquid injection module, an environmental parameter monitoring unit, a multi-micropore nozzle, an air injection valve and an air outlet valve; wherein: the spray-type separation and culture chamber is provided with a A solid cultivation plate is used to separate and culture microorganisms; the spray-type separation culture chamber is placed in a temperature environment control system, and the temperature environment control system ensures that the temperature in the spray-type separation culture chamber is kept consistent with the seabed temperature in the deep sea cold spring area; The environmental parameter monitoring unit is used to monitor the temperature and pressure changes inside the spray-type separation culture chamber, and transmit the data to the control collection terminal; the multi-micropore nozzle is installed in the spray-type separation culture chamber, and is connected with the spray-type separation culture chamber.
  • the liquid injection module installed outside the spray separation culture chamber is connected; the input end of the liquid injection module is connected with the liquid output end of the biological enrichment unit, and is used to inject biological enrichment liquid into the spray separation culture chamber, and the biological enrichment liquid passes through multiple After the micro-hole nozzle is installed, the particles will be dispersed into fine beads on the solid cultivation plate; the air injection valve and the air outlet valve are both arranged on the spray-type separation culture chamber, and the air injection valve is connected to the pressurization system. It is used to inject gas into the spray-type separation and culture chamber to increase its internal pressure; the control end of the air outlet valve is electrically connected to the control collection terminal and is used to control the gas to be discharged from the spray-type separation and culture chamber to reduce its internal pressure.
  • the liquid injection module includes a microfluidic pump, a liquid injection port and a liquid delivery pipe; wherein: the input end of the microfluidic pump is connected to the liquid output end of the biological enrichment unit, and its output end is connected to the liquid delivery pipe through the liquid delivery pipe.
  • the liquid injection port is connected; the liquid injection port is connected with the multi-microhole nozzle.
  • the spray-type separation culture chamber is provided with a quick-opening clamp.
  • the spray-type separation culture chamber involved in this scheme uses the principle of spray-type separation to inject the microorganism-enriched culture solution of the bioconcentration unit into the spray-type separation culture chamber through a microfluidic pump, and passes through the multi-micropore nozzle to make it Disperse into ultra-fine strains and scatter them on the solid medium. After individual tiny droplets are small enough to adhere to the solid medium, the process of isolation and culture will be realized.
  • the spray-type separation culture chamber involved in this solution is equipped with a liquid injection port for injecting microorganism-enriched bacterial liquid into the spray-type separation culture chamber; it is also equipped with an air injection valve for introducing microbial growth needs into the spray-type separation culture chamber.
  • the gas or inert gas pressurizes the culture chamber; a high-strength solid culture plate is placed in the spray-type separation culture room to fill the solid culture medium to meet the needs of microbial implantation and growth; a spray-type separation culture chamber is placed with a solid culture plate to disperse the bacterial liquid
  • the key component multi-micropore nozzle, which can be spherical, horn-shaped or lotus-shaped, etc., which can allow the microorganism-enriched bacterial liquid to come out of the liquid injection port and disperse into uniform fine droplets in a high-pressure environment.
  • the aperture on the nozzle It is fine enough and numerous enough so that the bacterial liquid can separate and grow after being sprayed out.
  • the distance between the multi-micropore nozzle and the solid culture plate should be such that the bacterial liquid is evenly attached to the solid culture plate after being sprayed, and does not scatter to the inner wall of the spray-type separation culture chamber.
  • the biological enrichment unit is composed of multiple high-pressure microorganism enrichment culture kettles connected in series; the high-pressure microorganism enrichment culture kettle is provided with a detachable sealing cover and a connected sampling valve group, and a temperature and pressure sensor group is provided inside it ; Each high-pressure microorganism enrichment culture kettle is installed in a temperature environment control system; wherein: the detachable sealing cover is used to facilitate the sterilization operation and placement of culture substrate inside the high-pressure microorganism enrichment culture kettle; the The connecting sampling valve group is used to connect and sample each high-pressure microbial enrichment culture kettle.
  • the connecting sampling valve group is connected to the pressurization system and is used to input liquid or gas into the high-pressure microbial enrichment culture kettle to increase the high-pressure microbial enrichment.
  • the pressure in the culture kettle makes the pressure value in the high-pressure microorganism enrichment culture kettle consistent with the actual situation in the deep sea; the temperature and pressure sensor group is used to monitor the temperature and pressure changes in the high-pressure microorganism enrichment culture kettle in real time, and The signal is transmitted to the control acquisition terminal.
  • the high-pressure microorganism enrichment culture kettle is also provided with a stirring rod; the stirring rod is used to enhance the reaction process of the matrix during the cultivation process of the high-pressure microorganism enrichment culture kettle.
  • a stirring rod is designed on the top of the high-pressure microbial enrichment culture kettle, which can enhance mass transfer through manual or mechanical stirring, enhance the reaction process of the matrix during the culture process, and increase the energy and nutrient utilization efficiency of microorganisms.
  • connection sampling valve group includes a liquid inlet valve, an air inlet valve, a sampling valve, a vent valve and a liquid outlet valve; wherein: the high-pressure microorganism enrichment culture kettles are connected in series through the liquid outlet valve and the liquid inlet valve, The outlet valve of the former high-pressure microbial enrichment culture kettle is connected to the liquid inlet valve of the subsequent high-pressure microbial enrichment culture kettle; the air inlet valve is used to input gas into the high-pressure microbial enrichment culture kettle to increase the volume of the high-pressure microbial enrichment culture kettle.
  • the pressure in the high-pressure microbial enrichment culture kettle makes the pressure value in the high-pressure microbial enrichment culture kettle consistent with the actual situation in the deep sea;
  • the vent valve is used to discharge the gas in the high-pressure microbial enrichment culture kettle to reduce the pressure in the high-pressure microbial enrichment culture kettle, and its control end It is electrically connected to the control collection terminal;
  • the sampling valve is used for real-time sampling and analysis of the microorganisms in the high-pressure microorganism enrichment culture kettle.
  • the high-pressure microorganism enrichment culture kettle is equipped with a sampling valve, which is used to analyze and detect samples taken during the enrichment process in order to adjust the corresponding environmental parameters and optimize the enrichment culture process.
  • multiple high-pressure microbial enrichment culture kettles are connected to form a bioconcentration unit, and the bacterial liquid in the previous-level high-pressure microbial enrichment culture kettle is transferred to the next-level high-pressure microbial enrichment culture kettle by maintaining pressure.
  • the microbial liquid obtained in the final high-pressure microbial enrichment culture kettle will provide enriched and highly purified functional microorganisms under stress under directional nutrient conditions in a high-pressure environment.
  • the pressure-maintaining transfer can be realized by using a micro-injection pump to take out the enriched liquid in the front-stage high-pressure microbial enrichment culture kettle through the sampling valve and pump it into the subsequent high-pressure microbial enrichment culture kettle. You can also pressurize the pressure of the rear-stage high-pressure microorganism enrichment culture kettle to be slightly less than that of the front-stage high-pressure microorganism enrichment culture kettle, and then open the outlet valve of the front-stage high-pressure microorganism enrichment culture kettle and the pressure of the rear-stage high-pressure microorganism enrichment culture kettle.
  • the microbial enrichment liquid will automatically enter the front-stage high-pressure microbial enrichment and culture kettle into the rear-stage high-pressure microbial enrichment and culture kettle under the condition of slight pressure difference for purification and culture.
  • the temperature and pressure environmental conditions of the high-pressure microbial enrichment culture kettle are consistent with the environmental conditions of microorganisms in the deep sea, ensuring the effectiveness of enrichment culture.
  • the temperature environment control system includes a low/high temperature environment system, a refrigeration/heat system and a water bath temperature monitoring system; the spray solid separation culture unit and biological enrichment unit are placed in a low/high temperature environment system for water bathing,
  • the low/high temperature environmental system is connected to the refrigeration/heating system to realize heat exchange;
  • the water bath temperature monitoring system is used to monitor temperature changes in the low/high temperature environmental system and transmit the monitoring data to the control collection terminal; so
  • the refrigeration/heating system control terminal is electrically connected to the control collection terminal.
  • the constant temperature conditions of the spray-type separation culture chamber and high-pressure microorganism enrichment culture kettle are mainly maintained by placing the spray-type separation culture chamber and high-pressure microorganism enrichment culture kettle in a water bath in a temperature-monitored low/high temperature environmental system, and through The heat exchange effect of the low/high temperature environmental system maintains a constant temperature state in the spray-type separation culture chamber and the high-pressure microbial enrichment culture kettle. Or place the spray separation culture chamber and high-pressure microbial enrichment culture kettle in an air heat exchange constant temperature room.
  • the temperature conditions in the spray-type separation culture chamber and high-pressure microbial enrichment culture kettle involved in the above plan are mainly controlled through the low/high temperature environment system. For example, inject cold/hot fluid into the ring wall cavity of a spray-type separation culture chamber or a high-pressure microorganism enrichment culture kettle, and circulate the fluid to cool or heat it to ensure the low or high temperature state of the fluid in the ring wall cavity, and then pass it 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 boosting system includes an air compressor, a boosting pump, a gas storage tank, a pressure regulating valve and a ventilation pipe; wherein: the air compressor, boosting pump, gas storage tank and pressure regulating valve pass through the ventilation pipe in sequence. connection, and finally connected to the spray-type solid separation and culture unit and the biological enrichment unit in sequence through the ventilation pipe, for injecting gas into the spray-type solid separation and culture unit and the biological enrichment unit for pressurization; the pressure regulating valve is used To adjust the internal pressure of the spray solid separation culture unit and the biological enrichment unit; the air compressor control end, the booster pump control end, and the pressure regulating valve control end are all electrically connected to the control collection terminal.
  • the pressurization system involved in the above scheme is mainly used to inject gas to pressurize the high-pressure microorganism enrichment culture kettle and spray-type separation culture chamber, so that the pressure environment in the high-pressure microorganism enrichment culture kettle and spray-type separation culture chamber is in line with the microorganisms in the ocean.
  • the pressure value in the original position is consistent.
  • the pressure changes in the culture room are monitored in real time through the environmental parameter monitoring unit and the temperature and pressure sensor group, and the culture room is pressurized and decompressed through active inflation/deflation, so that the pressure value in the culture room is kept consistent with the ocean in which microorganisms grow. Environmental conditions are consistent.
  • the high-pressure environment bioconcentration and spray solid separation culture device also includes a movable platform, where the spray solid separation culture unit, bioconcentration unit, pressurization system, temperature environment control system, and control collection terminal are placed On the movable platform, it is used to increase the universality of culture scenarios.
  • the spray-type solid separation and culture unit there are multiple micropore nozzles and solid cultivation plates in the spray-type solid separation and culture unit; the liquid injection module is connected to each micropore nozzle at the same time; and the solid cultivation plate is arranged horizontally. At the bottom of the spray-type solid separation and culture unit, horizontal separation and culture of microorganisms under the same temperature and pressure conditions is realized.
  • the spray-type separation and culture chamber is provided with a plurality of communicating partitions in the vertical direction, dividing the spray-type separation and culturing chamber into a plurality of connected culture chambers; wherein: each communicating partition is provided with On the solid cultivation plate, each culture cavity is equipped with a multi-micropore nozzle; the liquid injection module is connected to each multi-micropore nozzle at the same time to achieve vertical separation and cultivation of microorganisms under the same temperature and pressure conditions.
  • the spray-type solid separation and culture unit includes multiple spray-type separation and culture chambers, each spray-type separation and culture chamber is equipped with an independent multi-micropore nozzle, a solid culture plate and an air injection valve; all spray-type separation and culture chambers They are all placed in the same temperature environment control system; the liquid injection module is simultaneously connected to the multi-micropore nozzle in each spray-type separation culture chamber to realize the separation and culture of microorganisms under the same temperature and different pressure conditions.
  • the spray-type solid separation culture unit includes a plurality of spray-type separation culture chambers, each spray-type separation culture chamber is equipped with an independent multi-micropore nozzle, a solid culture plate and an air injection valve; each spray-type separation culture chamber The chamber is placed in a correspondingly set independent temperature environment control system; the liquid injection module is simultaneously connected to the multi-micropore nozzle in each spray-type separation culture chamber, and all air injection valves are connected to achieve the same treatment of microorganisms at different temperatures. Isolation culture under stress conditions.
  • the microbial separation process in a high-pressure environment involved in this plan uses a single spray-type separation culture chamber for multi-level combined culture to form a culture process.
  • multiple solid culture plates can be arranged in the horizontal culture chamber.
  • Each solid culture plate can be filled with culture media of different nutrients.
  • Each plate is provided with a Multi-micropore nozzles improve separation and culture efficiency. It can also be set up in a tower structure, with multiple solid cultivation plates and multi-microhole nozzles stacked vertically, or multiple groups of horizontal and vertical combinations can be combined simultaneously for large-scale cultivation.
  • the entire spray separation culture chamber was set to the same temperature and pressure environmental conditions.
  • different spray-type separation culture chambers can also be combined in parallel.
  • the temperature and pressure environment of each spray-type separation culture chamber can be individually controlled according to the purpose of sorting. Gradient setting temperature and pressure parameter conditions. Multiple spray-type separation culture chambers are combined in parallel and connected to a microfluidic pump and a final high-pressure microbial enrichment culture kettle. Form a sorting process under different temperature and pressure environmental conditions.
  • This program improves the cultivability of microorganisms by constructing a high-pressure microorganism enrichment culture kettle and a spray-type separation culture chamber for high-pressure environment microorganisms to live in high-pressure environments, such as the high-pressure and extreme temperature environmental conditions of marine environments.
  • high-pressure environments such as the high-pressure and extreme temperature environmental conditions of marine environments.
  • multi-stage enrichment and liquid dilution culture functional microorganisms with high purity under the stress of directional environmental conditions are obtained.
  • spray solid culture separation monoclonal microbial colonies are obtained.
  • the entire enrichment and separation process is carried out under the microbial in-situ pressure and temperature environmental conditions.
  • multiple sets of separation culture chambers can be combined to form an efficient separation process with different culture medium combinations.
  • the high-pressure environment marine microbial enrichment culture and separation technology involved in this program mainly includes two steps: enrichment and separation. First, after enrichment and culture in the bioconcentration unit, high-purity bacterial colonies are obtained, and then enter the spray separation culture chamber under pressure maintenance for solid culture separation, and simultaneously screen through a combination of different culture media and environmental conditions. Pure culture strains were obtained. Specifically:
  • the high-pressure microbial enrichment culture kettle 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 into the liquid.
  • the valve is filled with the nutrient solution required for culture, and then the gas required for culture is injected from the air inlet valve (inert gas can be injected if not required) to increase the pressure value in the high-pressure microbial enrichment culture kettle to be consistent with the actual environmental conditions of the deep sea.
  • stirring is performed through the 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-stage high-pressure microorganism enrichment culture kettle, and gas is injected into the second-stage high-pressure microorganism enrichment culture kettle through the pressurization system. And the culture liquid is pressurized.
  • the amount of culture liquid injected into the second-stage high-pressure microbial enrichment culture kettle needs to be based on the sorting needs to ensure that the concentration of the enriched liquid is from the concentration of the first-stage high-pressure microbial enrichment culture kettle to the second-stage high-pressure microbial enrichment.
  • the dilution ratio in the culture kettle and then transfer the microbial liquid in the first-stage high-pressure microbial enrichment culture kettle to the second-stage high-pressure microbial enrichment culture kettle through pressure-maintaining transfer.
  • the microorganisms in the final high-pressure microbial enrichment culture kettle will reach a highly purified state.
  • concentration of the deep-sea microbial liquid in the final high-pressure microbial enrichment culture kettle reaches more than 106 cells/mL, it can be considered to have reached a relatively high level. Good purified condition.
  • the dilution ratio of each level can be adjusted according to the specific cultured microbial groups.
  • the solid separation culture process will be entered.
  • the spray-type separation culture chamber sterilizes the spray-type separation culture chamber and all its internal components and related pipes and valves to maintain sterility. Then fill the plate with sterilized solid culture medium.
  • the solid culture plate can be filled with alumina powder, fine glass beads/steel balls, steel wire balls, etc. as supports to meet the needs of solid separation culture. Then, turn on the temperature environment control system to ensure that the temperature in the spray separation culture chamber is consistent with the temperature conditions of the microorganisms in the marine environment.
  • gas is injected into the spray-type separation culture chamber through the gas injection valve to pressurize it, so that the pressure conditions in the spray-type separation culture chamber are consistent with the pressure conditions of microorganisms living in the marine environment.
  • the microbial enrichment liquid will be divided into ultra-high-pressure microorganisms. Fine uniform droplets and ultra-fine droplets will be transported to the solid culture plate at the bottom of the spray separation culture chamber under the action of gravity. The ultrafine droplets implant and grow in the solid culture medium. After a sufficient culture period, isolated single colonies will grow.
  • the automatic microbial culture process involved in this program mainly includes two types: the same temperature and pressure conditions and different temperature and pressure conditions.
  • the purpose of using the same temperature and pressure conditions is mainly to meet the needs of simultaneous screening and separation of different culture media on a large scale.
  • a number of solid culture plates and multi-micropore nozzles are combined horizontally or vertically in a spray-type separation culture chamber.
  • the inlets of all micro-pore nozzles are connected in parallel through pipelines and connected with the micro-injection pump and the final high-pressure microbial enrichment culture.
  • the cauldrons are connected.
  • Different temperature and pressure conditions mainly involve the parallel combination of different spray-type separation and culture chambers.
  • each spray-type separation and culture chamber are individually controlled, and parameter conditions can be set in gradients according to the purpose of sorting.
  • Multiple spray-type separation culture chambers are combined in parallel and connected to a microfluidic pump and a final high-pressure microbial enrichment culture kettle. After the entire process is assembled and debugged, all culture chambers and pipe valves involved in the culture process will be sterilized as a whole. Then, through temperature and pressure monitoring, it is ensured that the temperature and pressure environmental conditions in all spray-type separation culture chambers are consistent with the temperature and pressure environmental conditions of the marine environment where the microorganisms are located. When the microinjection pump is turned on, the finely dispersed droplets will be implanted on the solid culture plate for separation and culture. This can realize the automatic sorting process under different culture media environments and effectively ensure the separation, culture and purification of microorganisms in high-pressure environments. Provide key technologies for the efficient utilization and sorting process of microorganisms in high-pressure environments.
  • the present invention mainly relates to devices and processes for continuous enrichment and spray-type separation and cultivation of microorganisms in high-pressure environments. It proposes multi-level enrichment cultivation and multi-medium spray-type automatic separation and purification of marine microorganisms under high-pressure and extreme temperature environmental conditions. Cultivation equipment and technical methods.
  • This solution solves the problem that existing indoor pure culture technology methods are separated from the high-pressure and extreme temperature environmental conditions where microorganisms survive, resulting in a large number of microorganisms with poor survival activity and unable to achieve pure culture; it does not require professional operators and can be used in research laboratories and scientific expeditions. There are many training scenarios such as boats, and it has wide adaptability.
  • 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 microorganisms in high-pressure environments under in-situ pressure and temperature environmental conditions. It is suitable for high-pressure environments. Under this condition, pure culture of microorganisms under in situ conditions provides an important technical means.
  • this solution proposes a high-pressure pure culture technology that enriches and separates microorganisms in extreme environmental conditions under high-pressure environmental conditions.
  • the temperature and pressure environmental conditions for in-situ survival have caused the majority of microorganisms to have poor activity, or have a large difference in phenotype from the in-situ environmental conditions, and cannot be isolated and cultured in pure form.
  • this solution can effectively reduce the investment of professionals, and can carry out large-scale enrichment and separation culture, improve the screening efficiency of difficult-to-cultivate microorganisms, and improve the screening and cultivation efficiency of functional bacteria in high-pressure environments.
  • the invention proposes a high-pressure environment biological enrichment and spray-type solid separation and culture device.
  • the biological enrichment unit realizes the enrichment and culture of microorganisms under the temperature and pressure environmental conditions of the ocean in situ; the spray-type solid separation and culture unit
  • the enriched solution is transformed into fine beads, enabling separation and culture in a dispersed state.
  • the present invention carries out enrichment culture and separation of marine microorganisms by reshaping their in-situ environment, solves the problem of isolating and purely cultivating marine microorganisms in high-pressure environments, improves the culturability of marine microorganisms, and forms pure culture technology, which provides a basis for the development and utilization of microbial resources in high-pressure environments. Provide important basic means.
  • Figure 1 is a schematic structural diagram of the device according to the present invention.
  • Figure 2 is a schematic structural diagram of the spray separation culture chamber according to the present invention.
  • Figure 3 is a schematic structural diagram of a high-pressure microorganism enrichment culture kettle according to the present invention.
  • FIG. 4 is a schematic connection diagram of the control acquisition terminal circuit module according to the present invention.
  • Figure 5 is a schematic structural diagram of the spray-type solid separation and culture unit in one embodiment of the present invention to achieve lateral separation and culture of microorganisms under the same temperature and pressure conditions;
  • Figure 6 is a schematic structural diagram of the spray-type solid separation and culture unit in one embodiment of the present invention to achieve vertical separation and culture of microorganisms under the same temperature and pressure conditions;
  • Figure 7 is a schematic structural diagram of a spray-type solid separation culture unit according to an embodiment of the present invention for cultivating microorganisms in different media under the same temperature and pressure conditions.
  • Spray-type solid separation and culture unit 11. Spray-type separation and culture chamber; 111. Solid culture plate; 112. Quick-open clamp; 113. Connecting partition; 114. Culture chamber; 12. Liquid injection module; 121. Microfluidic pump; 122. Liquid injection port; 123. Liquid delivery pipeline; 13. Environmental parameter monitoring unit; 14. Multi-microhole nozzle; 15. Gas injection valve; 16. Gas outlet valve; 2. Bioconcentration unit; 21. High-pressure microbial enrichment culture kettle; 211. Removable sealing cover; 212. Temperature and pressure sensor group; 213. Stirring rod; 22. Liquid inlet valve; 23. Air inlet valve; 24. Sampling valve; 25. Vent valve; 26. Liquid outlet valve; 3.
  • Boosting system 31. Air compressor; 32. Boosting pump; 33. Gas storage tank; 34. Pressure regulating valve; 35. Ventilation pipeline; 4. Temperature environment control system; 41. Low/high temperature environment system; 42. Refrigeration/heating system; 43. Water bath temperature monitoring system; 5. Control acquisition terminal; 6. Movable platform.
  • This embodiment is a complete usage example with rich content.
  • this embodiment proposes a high-pressure environment bioconcentration and spray solid separation culture device, including a spray solid separation culture unit 1, a bioconcentration unit 2, a pressurization system 3, and a temperature environment Control system 4 and control collection terminal 5; wherein: the biological enrichment unit 2 is used to realize the enrichment and multi-level purification process of marine microorganisms, obtain biological enrichment liquid and inject the biological enrichment liquid into the spray solid In the separation and culture unit 1; the spray-type solid separation and culture unit 1 is used to convert the biological enrichment liquid into fine beads, so that it can be separated and cultured in a dispersed state, effectively improving the cultivability of marine microorganisms; so
  • the pressurization system 3 and the temperature environment control system 4 are connected to the spray solid separation and culture unit 1 and the bioconcentration unit 2 respectively, and are used to construct structures in the bioconcentration unit 2 and the spray solid separation and culture unit 1 that are consistent with the marine environment.
  • the high-pressure and low-temperature environment ensures that the enriched deep-sea microorganisms are enriched, purified, separated and cultured under in-situ environmental conditions; the control end and signal detection of the spray-type solid separation culture unit 1 and the biological enrichment unit 2
  • the terminals are all electrically connected to the control acquisition terminal 5.
  • control collection terminal 5 includes a data collector, a data central processor, an operating computer, etc. to monitor changes in various environmental data information during the enrichment, separation, and purification of microbial enrichment bacteria in a high-pressure environment, and Real-time collection, processing, storage and image output functions.
  • the spray-type solid separation culture unit 1 includes a spray-type separation culture chamber 11, a liquid injection module 12, an environmental parameter monitoring unit 13, a multi-micropore nozzle 14, and an air injection valve. 15 and an air outlet valve 16; wherein: a solid cultivation plate 111 is provided inside the spray-type separation and culture chamber 11 for separating and cultivating microorganisms; the spray-type separation and culture chamber 11 is placed in the temperature environment control system 4, and is controlled by The temperature environment control system 4 ensures that the temperature in the spray separation culture chamber 11 is maintained consistent with the seafloor temperature in the deep sea cold spring area; the environmental parameter monitoring unit 13 is used to monitor the temperature and pressure changes inside the spray separation culture chamber 11, and The data is transmitted to the control acquisition terminal 5; the multi-micropore nozzle 14 is arranged in the spray-type separation and culture chamber 11, and is connected to the liquid injection module 12 arranged outside the spray-type separation and culture chamber 11; the liquid injection module The input end 12 is connected to the liquid output end of the bioconcentration unit 2, and
  • the air injection valve 15 and the air outlet valve 16 are both arranged on the spray separation culture chamber 11, and the air injection valve 15 is connected to the pressurization system 3 for spray separation. Gas is injected into the culture chamber 11 to increase its internal pressure; the control end of the gas outlet valve 16 is electrically connected to the control collection terminal 5 for controlling gas discharge from the spray separation culture chamber 11 to reduce its internal pressure.
  • the liquid injection module 12 includes a microfluidic pump 121, a liquid injection port 122 and a liquid delivery pipe 123; wherein: the input end of the microfluidic pump 121 is connected to the liquid output end of the biological enrichment unit 2, and The output end is connected to the liquid injection port 122 through a liquid delivery pipe 123; the liquid injection port 122 is connected to the multi-microhole nozzle 14.
  • the spray-type separation culture chamber 11 is provided with a quick-opening clamp 112 .
  • the spray separation culture chamber 11 involved in this embodiment uses the spray separation principle to inject the microorganism enrichment culture solution of the bioconcentration unit 2 into the spray separation culture chamber 11 through the microfluidic pump 121.
  • the multi-micropore nozzle 14 disperses the bacteria into ultra-fine strains and scatters them on the solid medium. After a single tiny droplet is small enough to adhere to the solid medium, the separation and culture process will be realized.
  • the spray-type separation culture chamber 11 involved in this solution is provided with a liquid injection port 122, which is used to inject microorganism-enriched bacterial liquid into the spray-type separation culture chamber 11; it is also provided with an air injection valve 15, which is used to inject the spray-type separation culture chamber 11 with a liquid injection port.
  • the gas or inert gas required for microbial growth is introduced into 11 to pressurize the culture chamber; a high-strength solid culture plate 111 is placed in the spray-type separation culture chamber 11, which is used to fill the solid culture medium to meet the needs of microbial implantation and growth; spray
  • the key component for dispersing the bacterial liquid is placed in the separation culture chamber 11: a multi-micropore nozzle 14, which can be a ball type, a trumpet type, a lotus type, etc., which can allow the microorganisms to enrich the bacterial liquid after it comes out of the liquid injection port 122.
  • the distance between the multi-microporous nozzle 14 and the solid culture plate 111 is such that the bacterial liquid is sprayed and evenly attached to the solid culture plate 111 without being scattered to the inner wall of the spray-type separation culture chamber 11 .
  • the biological enrichment unit 2 is composed of multiple high-pressure microorganism enrichment culture kettles 21 connected in series; the high-pressure microorganism enrichment culture kettle 21 is provided with a removable sealing cover. 211 and connect the sampling valve 24 groups, which are equipped with a temperature and pressure sensor group 212; each high-pressure microorganism enrichment culture kettle 21 is installed in the temperature environment control system 4; wherein: the removable sealing cover 211 is used to facilitate The high-pressure microorganism enrichment culture kettle 21 performs sterilization operations and puts culture substrate inside; the 24 sets of connecting sampling valves are used for connection and sampling of each high-pressure microorganism enrichment culture kettle 21, and the 24 sets of connecting sampling valves are connected to the The pressure system 3 is connected, which is used to input liquid or gas into the high-pressure microorganism enrichment culture kettle 21 to increase the pressure in the high-pressure microorganism enrichment culture kettle 21, so that the pressure value in the high-pressure microorganism enrichment culture kettle 21 is consistent with the
  • the high-pressure microorganism enrichment culture kettle 21 is also provided with a stirring rod 213; the stirring rod 213 is used to enhance the reaction process of the matrix during the culture process of the high-pressure microorganism enrichment culture kettle 21.
  • the top of the high-pressure microorganism enrichment culture kettle 21 is designed with a stirring rod 213, which can enhance mass transfer through manual or mechanical stirring, enhance the reaction process of the substrate during the culture process, and increase the energy and nutrient utilization efficiency of the microorganisms.
  • connection sampling valve group 24 includes a liquid inlet valve 22, an air inlet valve 23, a sampling valve 24, a vent valve 25 and a liquid outlet valve 26; wherein: the high-pressure microorganism enrichment culture kettle 21 passes through the outlet valve.
  • the liquid valve 26 and the liquid inlet valve 22 are connected in series.
  • the liquid outlet valve 26 of the former high-pressure microorganism enrichment culture kettle 21 is connected to the liquid inlet valve 22 of the latter stage high-pressure microorganism enrichment culture kettle 21; the air inlet valve 23 is used to supply the high-pressure Gas is input into the microorganism enrichment culture kettle 21 to increase the pressure in the high-pressure microorganism enrichment culture kettle 21, so that the pressure value in the high-pressure microorganism enrichment culture kettle 21 is consistent with the actual situation in the deep sea; the vent valve 25 is used to discharge the high-pressure microorganism enrichment The gas in the culture kettle 21 is used to reduce the pressure in the high-pressure microorganism enrichment culture kettle 21, and its control end is electrically connected to the control collection terminal 5; the sampling valve 24 is used to measure the microorganisms in the high-pressure microorganism enrichment culture kettle 21. Real-time sampling analysis.
  • the high-pressure microorganism enrichment culture kettle 21 is provided with a sampling valve 24, which is used to analyze and detect the samples taken during the enrichment process, so as to adjust the corresponding environmental parameters and optimize the enrichment culture process.
  • multiple high-pressure microbial enrichment culture kettles 21 are connected to form a bioconcentration unit 2, and the bacterial liquid in the previous high-pressure microbial enrichment culture kettle 21 is transferred to the next-level high-pressure microbial enrichment by maintaining pressure.
  • the microbial liquid obtained in the final high-pressure microorganism enrichment culture kettle 21 will provide enriched and highly purified functional types under stress in a high-pressure environment and directional nutrient conditions. microorganism.
  • the pressure-maintaining transfer can be realized by using a micro-injection pump to take out the enriched liquid in the front-stage high-pressure microorganism enrichment culture kettle 21 through the sampling valve 24 and pump it into the latter-stage high-pressure microorganism enrichment culture kettle 21. It is also possible to pressurize the pressure of the rear-stage high-pressure microorganism enrichment culture kettle 21 to be slightly smaller than that of the front-stage high-pressure microorganism enrichment culture kettle 21, and then open the liquid outlet valve 26 of the front-stage high-pressure microorganism enrichment culture kettle 21 and the rear-stage high-pressure microorganism enrichment culture kettle 21.
  • the microbial enrichment liquid will automatically enter the front-stage high-pressure microbial enrichment culture kettle 21 into the rear-stage high-pressure microbial enrichment culture kettle 21 for purification and culture under the condition of slight pressure difference.
  • the temperature and pressure environmental conditions of the high-pressure microorganism enrichment culture kettle 21 are consistent with the environmental conditions of the microorganisms in the deep sea, ensuring the effectiveness of the enrichment culture.
  • the temperature environment control system 4 includes a low/high temperature environment system 41, a refrigeration/heat system 42 and a water bath temperature monitoring system 43; the spray solid separation culture unit 1 and the biological enrichment unit 2 are both placed at low temperature.
  • a water bath is carried out in the high/high temperature environment system 41, and the low/high temperature environment system 41 is connected to the refrigeration/heating system 42 to realize heat exchange; the water bath temperature monitoring system 43 is used to monitor the temperature changes in the low/high temperature environment system 41, And the monitoring data is transmitted to the control collection terminal 5; the control end of the refrigeration/heating system 42 is electrically connected to the control collection terminal 5.
  • maintaining the constant temperature conditions of the spray-type separation and culture chamber 11 and the high-pressure microorganism enrichment and culture kettle 21 mainly means placing the spray-type separation and culture chamber 11 and the high-pressure microorganism enrichment and culture kettle 21 in a temperature-monitored low/high temperature environment.
  • the water bath in the system 41 maintains a constant temperature state in the spray separation culture chamber 11 and the high-pressure microorganism enrichment culture kettle 21 through heat exchange with the low/high temperature environment system 41.
  • the temperature conditions in the spray-type separation culture chamber 11 and the high-pressure microorganism enrichment culture kettle 21 involved in this embodiment are mainly controlled through the low/high temperature environment system 41 .
  • the low or high temperature state in the built-in cavity is ensured through heat exchange between the cold/hot fluid and the built-in cavity.
  • the boosting system 3 includes an air compressor 31, a boosting pump 32, a gas storage tank 33, a pressure regulating valve 34 and a ventilation pipe 35; wherein: the air pressure The machine 31, the booster pump 32, the gas storage tank 33, and the pressure regulating valve 34 are connected in sequence through the ventilation pipe 35, and finally connected to the spray solid separation culture unit 1 and the biological enrichment unit 2 through the ventilation pipe 35, for Gas is injected into the spray-type solid separation and culture unit 1 and the biological enrichment unit 2 for pressurization; the pressure regulating valve 34 is used to adjust the internal pressure of the spray-type solid separation and culture unit 1 and the biological enrichment unit 2; the air The control end of the press 31 , the control end of the booster pump 32 , and the control end of the pressure regulating valve 34 are all electrically connected to the control collection terminal 5 .
  • the pressurizing system 3 involved in this embodiment is mainly used to inject gas into the high-pressure microorganism enrichment culture kettle 21 and the spray separation culture chamber 11 to pressurize the high-pressure microorganism enrichment culture kettle 21,
  • the pressure environment in the spray separation culture chamber 11 is consistent with the pressure value of the microorganisms in situ in the ocean.
  • the pressure changes in the culture room are monitored in real time through the environmental parameter monitoring unit 13 and the temperature and pressure sensor group 212, and the culture room is pressurized and decompressed through active inflation/deflation to keep the pressure value in the culture room consistent with The marine environmental conditions for microbial growth are consistent.
  • the high-pressure environment biological enrichment and spray solid separation culture device also includes a movable platform 6, the spray solid separation culture unit 1, the biological enrichment unit 2, the pressurization system 3, and the temperature environment control system. 4.
  • the control collection terminals 5 are placed on the movable platform 6 to increase the universality of the culture scenario.
  • the bioconcentration unit 2 can realize the enrichment and culture of microorganisms under the in-situ temperature and pressure environmental conditions of the ocean.
  • the spray-type solid separation culture unit 1 can convert the bioconcentration liquid into a fine bead state to make it Isolation and culture are achieved in a dispersed state.
  • Embodiment 1 More specifically, on the basis of Embodiment 1, as shown in Figure 5, multiple microporous nozzles 14 and solid cultivation plates 111 are provided in the spray solid separation and cultivation unit 1; the liquid injection The module 12 is connected to each multi-micropore nozzle 14 at the same time; the solid culture plate 111 is arranged transversely at the bottom of the spray solid separation culture unit 1 to realize the lateral separation and culture of microorganisms under the same temperature and pressure conditions.
  • the spray-type separation culture chamber 11 is provided with a plurality of communicating partitions 113 in the vertical direction, dividing the spray-type separation culture chamber 11 into multiple There are three connected culture chambers 114; wherein: each connected partition 113 is provided with a solid culture plate 111, and each culture chamber 114 is provided with a multi-microhole nozzle 14; the liquid injection module 12 is simultaneously connected with each Two multi-micropore nozzles 14 are connected to realize vertical separation and culture of microorganisms under the same temperature and pressure conditions.
  • the spray-type solid separation culture unit 1 includes multiple spray-type separation culture chambers 11, each spray-type separation culture chamber 11 is provided with an independent multi-micropore nozzle 14, solid The cultivation plate 111 and the air injection valve 15; all spray-type separation culture chambers 11 are placed in the same temperature environment control system 4; the liquid injection module 12 simultaneously communicates with the multi-microhole nozzles in each spray-type separation culture chamber 11 14 connections to realize the isolation and cultivation of microorganisms under the same temperature and different pressure conditions.
  • the spray-type solid separation culture unit 1 includes multiple spray-type separation culture chambers 11, each spray-type separation culture chamber 11 is provided with an independent multi-micropore nozzle 14, solid The cultivation plate 111 and the air injection valve 15; each spray-type separation culture chamber 11 is placed in a correspondingly set independent temperature environment control system 4; the liquid injection module 12 simultaneously communicates with multiple spray-type separation culture chambers 11
  • the micropore nozzles 14 are connected, and all the air injection valves 15 are connected to realize the separation and culture of microorganisms under different temperatures and the same pressure conditions.
  • the microorganism separation process in a high-pressure environment involved in this embodiment uses a single spray-type separation culture chamber 11 to perform multi-level combined culture to form a culture process.
  • multiple solid culture plates 111 can be arranged in the horizontal culture chamber.
  • Each solid culture plate 111 can be filled with culture media of different nutrients.
  • Each plate A multi-micropore nozzle 14 is provided to improve separation and culture efficiency. It can also be set up in a tower structure, and multiple solid cultivation plates 111 and multi-microhole nozzles 14 can be combined and stacked vertically, or multiple groups can be combined horizontally and vertically at the same time for large-scale cultivation.
  • the entire spray separation culture chamber 11 is set to the same temperature and pressure environmental conditions. If in order to screen bacterial strains under different pressure and temperature environmental conditions, different spray-type separation culture chambers 11 can also be combined in parallel, and the temperature and pressure environment of each spray-type separation culture chamber 11 can be individually controlled, and can be sorted according to The purpose is to set the temperature and pressure parameter conditions in a gradient manner. Multiple spray separation culture chambers 11 are combined in parallel and connected to the microfluidic pump 121 and the final high-pressure microorganism enrichment culture kettle 21 . Form a sorting process under different temperature and pressure environmental conditions.
  • This embodiment improves the cultivability of microorganisms in high-pressure environments, such as the high-pressure and extreme temperature environmental conditions of marine environments, by constructing a high-pressure microorganism enrichment culture kettle 21 and a spray-type separation culture chamber 11 for microorganisms in a high-pressure environment.
  • a high-pressure microorganism enrichment culture kettle 21 and a spray-type separation culture chamber 11 for microorganisms in a high-pressure environment Through multi-stage enrichment and liquid dilution culture, functional microorganisms with high purity under the stress of directional environmental conditions are obtained.
  • spray solid culture separation monoclonal microbial colonies are obtained.
  • the entire enrichment and separation process is carried out under the microbial in-situ pressure and temperature environmental conditions.
  • multiple sets of separation culture chambers can be combined to form an efficient separation process with different culture medium combinations.
  • microorganisms can be cultured in different culture media under the same temperature and pressure conditions.
  • the high-pressure environment marine microorganism enrichment culture and separation technology involved in this embodiment mainly includes two steps: enrichment and separation. First, after enrichment and culture in the bioconcentration unit 2, the bacterial flora with higher purity is obtained, and then enters the spray separation culture chamber 11 under pressure maintenance for solid culture separation, and simultaneously through a combination of different culture media and environmental conditions. Screen and obtain pure culture strains. Specifically:
  • the high-pressure microbial enrichment culture kettle 21 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 into the kettle.
  • the liquid valve 22 is filled with the nutrient solution required for culture, and then the gas required for culture is injected from the air inlet valve 23 (inert gas can be injected if not required), so that the pressure value in the high-pressure microbial enrichment culture kettle 21 is increased to be consistent with the actual environmental conditions of the deep sea. consistent.
  • stirring is performed through the top stirring rod 213 to increase mass transfer and optimize the culture process.
  • the nutrient solution required for culture is injected into the second-stage high-pressure microorganism enrichment culture kettle 21, and is supplied to the second-stage high-pressure microorganism enrichment culture kettle 21 through the pressurizing system 3.
  • 21 injects gas and pressurizes the culture liquid.
  • the amount of culture liquid injected into the second-stage high-pressure microorganism enrichment culture kettle 21 needs to be based on the sorting needs to ensure that the concentration of the enrichment liquid is from the concentration of the first-stage high-pressure microorganism enrichment culture kettle 21 to the concentration of the second-stage high-pressure microorganism enrichment culture kettle 21.
  • the dilution ratio in the second-stage high-pressure microorganism enrichment culture kettle 21 is then transferred to the second-stage high-pressure microorganism enrichment culture kettle 21 by transferring the microbial liquid in the first-stage high-pressure microorganism enrichment culture kettle 21 .
  • the microorganisms in the final high-pressure microbial enrichment culture kettle 21 will reach a highly purified state.
  • concentration of the deep-sea microbial liquid in the final high-pressure microbial enrichment culture kettle 21 reaches more than 106/mL, it can be considered to have reached A better purification state.
  • the dilution ratio of each level can be adjusted according to the specific cultured microbial groups.
  • the solid separation culture process will be entered.
  • the spray-type separation culture chamber 11 and all its internal components and related pipes and valves are sterilized to maintain a sterile state. Then fill the plate with sterilized solid culture medium.
  • the solid culture plate 111 can be filled with alumina powder, fine glass beads/steel balls, steel wire balls, etc. as supports to meet the needs of solid separation culture.
  • the temperature environment control system 4 is turned on to ensure that the temperature in the spray separation culture chamber 11 is consistent with the temperature conditions of the microorganisms in the marine environment.
  • gas is injected into the spray separation culture chamber 11 through the gas injection valve 15 to pressurize the gas, so that the pressure conditions in the spray separation culture chamber 11 are consistent with the pressure conditions of microorganisms living in the marine environment.
  • the microbial enrichment liquid passes through the multi-micropore nozzle 14, will be divided into ultra-fine uniform droplets, and the ultra-fine droplets will be transported to the solid cultivation plate 111 at the bottom of the spray separation culture chamber 11 under the action of gravity.
  • the ultrafine droplets implant and grow in the solid medium, and after a sufficient culture period, isolated single colonies will grow.
  • the automatic cultivation process of microorganisms involved in this embodiment mainly includes two conditions: same temperature and pressure conditions and different temperature and pressure conditions.
  • the purpose of using the same temperature and pressure conditions is mainly to meet the needs of simultaneous screening and separation of different culture media on a large scale.
  • several solid culture plates 111 and multi-microhole nozzles 14 are placed horizontally or vertically in a spray-type separation culture chamber 11.
  • the inlets of all multi-microhole nozzles 14 are connected in parallel through pipelines and connected to the microinjection pump and the final stage.
  • the high-pressure microorganism enrichment culture kettle 21 is connected.
  • Different temperature and pressure conditions mainly involve parallel combination of different spray-type separation culture chambers 11.
  • each spray-type separation culture chamber 11 The temperature and pressure environment of each spray-type separation culture chamber 11 are individually controlled, and parameter conditions can be set in gradients according to the purpose of sorting. Multiple spray separation culture chambers 11 are combined in parallel and connected to the microfluidic pump 121 and the final high-pressure microorganism enrichment culture kettle 21 . After the entire process is assembled and debugged, all culture chambers and pipe valves involved in the culture process will be sterilized as a whole. Then, through temperature and pressure monitoring, it is ensured that the temperature and pressure environmental conditions in all spray-type separation culture chambers 11 are consistent with the temperature and pressure environmental conditions of the marine environment where the microorganisms are located.
  • the finely dispersed droplets When the microinjection pump is turned on, the finely dispersed droplets will be implanted on the solid culture plate 111 for separation and culture, which can realize the automatic sorting process under different culture medium environments and effectively ensure the separation, culture and purification of microorganisms in high-pressure environments.
  • This embodiment mainly relates to devices and processes for continuous enrichment and spray-type separation and cultivation of microorganisms in high-pressure environments. It proposes multi-level enrichment cultivation and multi-culture medium spray-type automatic separation of marine microorganisms under high-pressure and extreme temperature environmental conditions. Purification culture equipment and technical methods.
  • This embodiment solves the problem that the existing indoor pure culture technology method is separated from the high pressure and extreme temperature environmental conditions where microorganisms survive, resulting in a large number of microorganisms with poor survival activity and unable to achieve pure culture; it does not require professional operators and can be used in research laboratories and departments. There are many training scenarios such as ship examination, and it has wide adaptability.
  • This embodiment 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 cultivation of high-pressure environmental microorganisms under in-situ pressure and temperature environmental conditions. It is a high-pressure Environment, pure culture of microorganisms in situ conditions provides important technical means.
  • this embodiment proposes a high-pressure pure culture technology for enriching and separating and cultivating microorganisms under extreme environmental conditions under high-pressure environmental conditions, which solves the problem of the existing normal pressure separation and culture technology being separated from the high-pressure environment.
  • the temperature and pressure environmental conditions for the survival of microorganisms in situ have caused the problem that most microorganisms have poor survival activity, or their phenotypes are greatly different from the in situ environmental conditions, and they cannot be isolated and cultured purely.
  • this solution can effectively reduce the investment of professionals, and can carry out large-scale enrichment and separation culture, improve the screening efficiency of difficult-to-cultivate microorganisms, and improve the screening and cultivation efficiency of functional bacteria in high-pressure environments.
  • this embodiment proposes a multi-level enrichment and separation device for directional enrichment and separation of deep-sea methanophilic bacteria in a high-pressure environment.
  • the high-pressure microorganism enrichment culture kettle 21 and the spray separation culture chamber 11 are the core of this example.
  • Other auxiliary systems include the pressurization system 3, the temperature environment control system 4 and the control collection terminal 5.
  • This example uses multi-stage enrichment and liquid dilution culture to obtain high-purity deep-sea methanophilic bacteria under the stress of high pressure, low temperature, and methane as the only carbon source. At the same time, it is combined with spray solid culture separation to obtain monoclonal microbial colonies. The entire enrichment and separation process was carried out under the conditions of in-situ pressure (14MPa) and temperature environment (4°C) in the Haima Cold Spring Area in the South China Sea. At the same time, multiple sets of separation culture chambers can be combined to form an efficient separation process with different culture medium combinations.
  • the key to this example is the successful enrichment of deep-sea methanophilic bacteria in the bioconcentration unit, and the successful purification, separation and culture of the enriched bacterial liquid on the spray solid separation and culture unit 1.
  • a movable platform 6 is integrally installed at the bottom of the enrichment culture and separation culture chamber to increase the universality of culture scenarios.
  • the biological enrichment unit involved in this embodiment is mainly composed of multiple high-pressure microbial enrichment culture kettles 21 connected in series.
  • the high-pressure microorganism enrichment culture kettle 21 is designed with an openable lid structure, which is convenient for placing culture substrates and sterilizing operations.
  • the top of the high-pressure microorganism enrichment culture kettle 21 is designed with a stirring rod 213, which is used to enhance the reaction process of the substrate during the culture process and increase the carbon source and energy utilization efficiency of the deep-sea methanophilic bacteria.
  • the high-pressure microorganism enrichment culture kettle 21 is equipped with a temperature and pressure sensor group 212 to monitor the temperature and pressure changes in the high-pressure microorganism enrichment culture kettle 21 in real time.
  • the constant temperature condition of the high-pressure microorganism enrichment culture kettle 21 is mainly maintained by placing the high-pressure microorganism enrichment culture kettle 21 in a low-temperature water bath of the low/high temperature environment system 41. Through heat exchange with the water bath system, the low/high temperature environment system 41 Fill with refrigerant such as ethylene glycol to maintain low temperature in the enrichment culture chamber.
  • the top of the high-pressure microbial enrichment culture kettle 21 is provided with an air inlet valve 23 and a liquid inlet valve 22. The air inlet valve 23 and the liquid inlet valve 22 are used to inject the methane gas and nutrient solution required for culture into the closed high-pressure microorganism enrichment culture.
  • the pressure in the kettle 21 is increased to achieve high-pressure microbial enrichment and culture.
  • the pressure value in the kettle 21 is maintained at about 14MPa.
  • the high-pressure microorganism enrichment culture kettle 21 is provided with a sampling valve 24, which is used to analyze and detect samples taken during the enrichment process in order to adjust corresponding environmental parameters and optimize the enrichment culture process.
  • this example consists of four high-pressure microbial enrichment culture kettles 21 connected in series to form a bioconcentration unit 2.
  • the bacterial liquid in the high-pressure microbial enrichment culture kettle 21 is transferred to the next level of high-pressure microbial enrichment culture by maintaining pressure.
  • the microbial liquid obtained in the final high-pressure microbial enrichment culture kettle 21 will highly purify deep-sea methanophilic bacteria in a high-pressure environment.
  • the rear-stage high-pressure microorganism enrichment culture kettle 21 By pressurizing the pressure of the rear-stage high-pressure microorganism enrichment culture kettle 21 to 0.2-0.5MPa less than the pressure of the front-stage high-pressure microorganism enrichment culture kettle 21, and then opening the sampling valve 24 and the rear-stage high-pressure microorganism enrichment culture kettle 21.
  • the liquid inlet valve 22 of the first-stage high-pressure microbial enrichment culture kettle 21 the microbial enrichment liquid will automatically enter the second-stage high-pressure microbial enrichment culture kettle 21 from the front-stage high-pressure microbial enrichment culture kettle 21 for purification under the condition of slight pressure difference. nourish.
  • the temperature and pressure environmental conditions in the high-pressure microorganism enrichment culture kettle 21 are maintained at 4°C and 14MPa to ensure the effectiveness of the enrichment culture.
  • the spray-type solid separation and culture unit 1 involved in this embodiment utilizes the spray-type release principle.
  • the solid culture plate 11 is placed in the spray-type separation and culture chamber 11, and the philobacteria in the final high-pressure microorganism enrichment culture kettle 21 are
  • the methanogen-enriched culture solution is injected into the spray-type separation culture chamber 11 through the microfluidic pump 121, and passes through the multi-micropore nozzle 14 to disperse it into ultra-fine strains, which are scattered on the solid culture medium. Individual tiny droplets are small enough to form After attachment to the solid medium, the process of separation and culture will be realized.
  • the spray-type separation culture chamber 11 involved in this embodiment is provided with a liquid injection port 122 for injecting the methanophile enriched liquid into the solid culture chamber; it is also provided with an air injection valve 15 for injecting the spray-type separation culture chamber 11 into the solid culture chamber.
  • the methane gas required for the growth of methanophilic bacteria is introduced to pressurize the culture chamber to 14MPa; a high-strength solid culture plate 111 is placed in the spray-type separation culture chamber 11 for filling the solid culture medium to meet the needs of methanophilic bacteria for implantation and growth; spraying
  • the key component for dispersing the bacterial liquid is placed in the separation culture chamber 11: a trumpet-shaped multi-micropore nozzle 14, which allows the methanophilic bacteria enriched liquid to be dispersed into a uniform fine liquid in a high-pressure environment after coming out of the liquid injection port 122
  • the holes on the nozzle are fine enough and numerous enough to allow the bacterial liquid to separate and grow after it is sprayed out.
  • the distance between the multi-microporous nozzle 14 and the solid culture plate 111 is 100 mm, so that the bacterial solution after being sprayed can just be evenly attached to the solid culture plate 111 without being scattered to the inner wall of the spray-type separation culture chamber 11 .
  • the pressurizing system 3 involved in this embodiment is mainly used to inject methane gas into the microorganism enrichment and separation culture chamber to pressurize it, so that the pressure environment in the spray separation culture chamber 11 and the high-pressure microorganism enrichment culture kettle 21 is maintained at 14MPa.
  • the enrichment culture and separation technology of deep-sea methanophiles in high-pressure environment involved in this example mainly includes two steps: enrichment and separation.
  • enrichment and separation First, through multi-level high-pressure environment enrichment culture in the bioconcentration unit 2, after obtaining high-purity deep-sea methanophilic bacteria, it enters the spray separation culture chamber 11 under pressure maintenance for solid culture separation, and through different The combined process of culture medium and environmental conditions is simultaneously screened to obtain pure culture strains.
  • the high-pressure environment deep-sea methanophile enrichment culture method involved in this example first sterilizes the high-pressure microorganism enrichment culture kettle 21 and its attached pipes and valves, and then sequentially loads the seahorses to be cultured into the high-pressure microorganism enrichment culture kettle 21
  • the sediments in the cold spring methane leakage area are then filled with the nutrient solution required for culture from the liquid inlet valve 22, and the methane gas required for culture is injected from the air inlet valve 23 to increase the pressure value in the high-pressure microbial enrichment culture kettle 21 to 14MPa.
  • stirring is performed through the top stirring rod 213 to increase mass transfer and optimize the culture process.
  • the nutrient solution required for culture is injected into the second-stage high-pressure microorganism enrichment culture kettle 21, and air is injected and pressurized into the second-stage high-pressure microorganism enrichment culture kettle 21 through the pressurizing system 3.
  • the amount of culture liquid injected into the first-stage high-pressure microorganism enrichment culture kettle 21 needs to ensure that the dilution ratio of the enrichment liquid from the concentration of the first-stage high-pressure microorganism enrichment culture kettle 21 to the second-stage high-pressure microorganism enrichment culture kettle 21 is 1:10.
  • the microorganisms in the final high-pressure microbial enrichment culture kettle 21 will reach a highly purified state.
  • concentration of the deep-sea methanophilic bacteria in the final high-pressure microbial enrichment culture kettle 21 reaches more than 106/mL, it can be considered to have reached A better purification state.
  • the dilution ratio of each level can be adjusted according to the specific cultured microbial groups.
  • the isolation and culture process mainly includes: first, sterilizing the spray-type isolation and culture chamber 11 and all internal devices and related pipes and valves to maintain a sterile state. Then, the solid culture plate 111 is filled with the sterilized solid culture medium. In order to prevent the solid agar culture medium from liquefying under high pressure conditions, fine glass beads can be filled in the solid culture plate 111 as a support to meet the needs of solid separation culture.
  • the temperature environment control system 4 is turned on to ensure that the temperature in the spray separation culture chamber 11 is consistent with the seabed temperature conditions of the methanophilic bacteria in the Haima Cold Spring area. Then, methane gas is injected into the spray-type separation culture chamber 11 through the gas injection valve 15 to increase the pressure in the spray-type separation culture chamber 11 to 14 MPa. After ensuring that all system components are working properly, start the microfluidic pump 121 and inject the purified methanophilic bacteria enrichment solution from the final high-pressure microorganism enrichment culture kettle 21 into the spray separation culture chamber 11.
  • the methanophile bacteria enrichment solution passes through multiple After the microhole nozzle 14 is installed, it will be divided into ultra-fine uniform droplets, and the ultra-fine droplets will be transported to the solid cultivation plate 111 at the bottom of the spray separation culture chamber 11 under the action of gravity. The ultrafine droplets implant and grow in the solid culture medium, growing into isolated single colonies.
  • the automatic cultivation process of methanophilic bacteria involved in this embodiment mainly includes two conditions: same temperature and pressure conditions and different temperature and pressure conditions.
  • the purpose of using the same temperature and pressure conditions is mainly to meet the needs of simultaneous screening and separation of different culture media on a large scale.
  • First, several solid culture plates 111 and multi-microhole nozzles 14 are placed horizontally or vertically in a high-spray separation culture chamber 11.
  • the inlets of all multi-microhole nozzles 14 are connected in parallel through pipelines and connected to the microinjection pump 121 and
  • the final high-pressure microbial enrichment culture kettle 21 is connected, and all the solid culture plates 111 during the entire process screening process are in the environmental conditions of 14MPa and 4°C.
  • Different temperature and pressure conditions mainly involve parallel combination of different culture chambers.
  • each high-spray separation culture chamber 11 The temperature and pressure environment of each high-spray separation culture chamber 11 are individually controlled, and parameter conditions can be set in a gradient according to the purpose of sorting. For example, according to the methanophilic bacteria found in different water depth environments, the pressure conditions in multiple high-spray separation culture chambers 11 are gradient set from 6MPa to 20MPa, and the temperature environment response is set to the seafloor temperature of the depth environment. And multiple high-spray separation culture chambers 11 are combined in parallel and connected to the microfluidic pump 121 and the final high-pressure microorganism enrichment culture kettle 21. After the entire process has been assembled and debugged, all final-stage high-pressure microbial enrichment culture kettles 21 and pipe valves involved in the culture process will be sterilized as a whole.

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Abstract

一种高压环境生物富集与喷洒式固体分离培养装置，包括喷洒式固体分离培养单元（1）、生物富集单元（2）；在构建与海洋环境一致的高压、低温环境的情况下，生物富集单元（2）用于实现对海洋微生物的富集与多层级纯化过程，得到生物富集液并将生物富集液注入喷洒式固体分离培养单元（1）中；喷洒式固体分离培养单元（1）用于将生物富集液进行微细珠状态化，使其在分散状态下实现分离和培养。所述的一种高压环境生物富集与喷洒式固体分离培养装置，通过重塑其原位环境进行海洋微生物的富集培养与分离，解决分离纯培养海洋高压环境微生物的难题，提高海洋微生物的可培养性，形成纯培养技术，为开发利用高压环境微生物资源提供重要的基础手段。

Description

高压环境生物富集与喷洒式固体分离培养装置 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生物富集与喷洒式固体分离培养装置。
背景技术
高压生境占地球上很大比例，压力大于30MPa的生境在海洋环境内广泛分布，77％以上的海洋环境的水深度超过3000米。深海沉积层、深海盆地、海沟和陆地深层都是高压环境。高压生境的微生物随着深度、盐度、pH值、含氧量和营养物质的改变而改变，呈现显著的多样性。然而，由于现有的微生物富集和分离培养技术主要在常压环境下进行，尤其是固体分离培养技术缺乏高压环境使用的先例，导致目前仅小于1％的高压环境的微生物得到了纯培养，为正确认识这些微生物的表型、基因、功能开发利用价值带来困难。
现有技术公开了技术公开了一种深海微生物培养舱，包括：直线轴承、拉簧、压力补偿腔、固定顶板、深海电机组件、固定底板、软管和培养舱本体；该方案通过深海电机组件转动顶开培养舱本体的端盖，在完全开放的状态下进行微生物富集培养，在布放和回收过程中，关闭培养舱本体的端盖实现微生物培养舱体密封。其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，提供一种高压环境生物富集与喷洒式固体分离培养装置，通过重塑其原位环境进行海洋微生物的富集培养与分离，提高海洋微生物的可培养性，形成纯培养技术，为开发利用高压环境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：
本方案提出一种高压环境生物富集与喷洒式固体分离培养装置，包括喷洒式固体分离培养单元、生物富集单元、增压系统、温度环境控制系统和控制采集终端；其中：所述生物富集单元用于实现对海洋微生物的富集与多层级纯化过程， 得到生物富集液并将生物富集液注入所述喷洒式固体分离培养单元中；所述喷洒式固体分离培养单元用于将生物富集液进行微细珠状态化，使其在分散状态下实现分离和培养，有效地提高海洋微生物的可培养性；所述增压系统、温度环境控制系统分别与喷洒式固体分离培养单元、生物富集单元连接，用于在生物富集单元、喷洒式固体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；所述喷洒式固体分离培养单元、生物富集单元的控制端与信号检测端均与控制采集终端电性连接。
上述方案中，生物富集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，喷洒式固体分离培养单元可以将生物富集液进行微细珠状态化，使其在分散状态下实现分离和培养。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，提高海洋微生物的可培养性，形成纯培养技术，为开发利用高压环境微生物资源提供重要的基础手段。
上述方案中，控制采集终端包括数据采集器、数据中央处理器、操作电脑等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。
其中，所述喷洒式固体分离培养单元包括喷洒式分离培养室、液体注入模块、环境参数监测单元、多微孔喷头、注气阀和出气阀；其中：所述喷洒式分离培养室内部设置有固体培育平板，用于对微生物进行分离培养；所述喷洒式分离培养室置于温度环境控制系统中，通过温度环境控制系统保证喷洒式分离培养室内的温度恒温保持与深海冷泉区海底温度一致；所述环境参数监测单元用于监测喷洒式分离培养室内部的温度和压力变化情况，并将数据传输至所述控制采集终端；所述多微孔喷头设置在所述喷洒式分离培养室内，与设置在喷洒式分离培养室外的液体注入模块连接；液体注入模块输入端与所述生物富集单元液体输出端连接，用于向喷洒式分离培养室注入生物富集液，生物富集液经过多微孔喷头后，将分散为微细珠状态散落在所述固体培育平板；所述注气阀、出气阀均设置在所述喷洒式分离培养室上，注气阀与所述增压系统连接，用于对喷洒式分离培养室注入气体，增加其内部压力；出气阀控制端与所述控制采集终端电性连接，用于控制气体排出喷洒式分离培养室，降低其内部压力。
其中，所述液体注入模块包括微流泵、注液口和液体输送管道；其中：所述 微流泵输入端与所述生物富集单元液体输出端连接，其输出端通过液体输送管道与所述注液口连接；所述注液口与所述多微孔喷头连接。
其中，所述喷洒式分离培养室设置有快开卡箍。
上述方案中，本方案涉及的喷洒式分离培养室是利用喷洒式分离原理，将生物富集单元的微生物富集培养液通过微流泵注入喷洒式分离培养室，经过多微孔喷头，使其分散成超微细菌株，散落在固体培养基上，足够小的单个微小液珠在固体培养基上附着后，将实现分离培养的过程。本方案涉及的喷洒式分离培养室设置有注液口，用于往喷洒式分离培养室内注入微生物富集菌液；还设置有注气阀，用于往喷洒式分离培养室内通入微生物生长需要的气体或者惰性气体使培养室增压；喷洒式分离培养室内安放有高强度的固体培育平板，用于填充固体培养基，满足微生物着床生长需要；喷洒式分离培养室内安放有使菌液分散的关键元件：多微孔喷头，其可以为球型、喇叭型或者莲蓬型等能让微生物富集菌液从注液口内出来后在高压环境中分散成均匀的微细液滴，喷头上的孔径足够细且足够多，使得菌液喷出后能分离生长。多微孔喷头与固体培育平板的安放距离要使得菌液喷洒出来后刚好均匀的附着在固体培育平板上，而不会散落到喷洒式分离培养室的内壁。
其中，所述生物富集单元由多个串联的高压微生物富集培养釜组成；所述高压微生物富集培养釜上设置有可拆卸密封盖和连接取样阀组，其内部设置有温压传感器组；每个高压微生物富集培养釜均设置在温度环境控制系统中；其中：所述可拆卸密封盖用于方便对高压微生物富集培养釜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高压微生物富集培养釜的连接与取样，连接取样阀组与所述增压系统连接，用于向高压微生物富集培养釜内输入液体或气体以增加高压微生物富集培养釜内的压力，令高压微生物富集培养釜内的压力值与深海实际情况一致；所述温、压传感器组用于实时监测高压微生物富集培养釜内的温度、压力变化情况，并将信号输送至所述控制采集终端。
其中，在所述高压微生物富集培养釜上还设置有搅拌杆；所述搅拌杆用于高压微生物富集培养釜在培养过程增强基质的反应过程。
上述方案中，高压微生物富集培养釜顶部设计有搅拌杆，可通过手动或者机械搅拌增强传质，增强培养过程中基质的反应过程，增加微生物的能量和营养利用效率。
其中，所述连接取样阀组包括进液阀、进气阀、取样阀、放空阀和出液阀；其中：高压微生物富集培养釜之间通过所述出液阀、进液阀进行串联，前一级高压微生物富集培养釜出液阀与后一级高压微生物富集培养釜进液阀连接；进气阀用于向高压微生物富集培养釜内输入气体以增加高压微生物富集培养釜内的压力，令高压微生物富集培养釜内的压力值与深海实际情况一致；放空阀用于排出高压微生物富集培养釜内的气体以降低高压微生物富集培养釜内的压力，其控制端与所述控制采集终端电性连接；取样阀用于对高压微生物富集培养釜内的微生物进行实时取样分析。
上述方案中，高压微生物富集培养釜设置有取样阀，用于对富集过程中取样进行分析检测，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，多个高压微生物富集培养釜联构成生物富集单元，将前一级高压微生物富集培养釜内的菌液通过保压转移至下一级高压微生物富集培养釜中，以此类推，按照浓度梯度稀释，末级高压微生物富集培养釜中获得的微生物菌液将为高压环境下，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高压微生物富集培养釜中的富集液通过取样阀取出泵入后级的高压微生物富集培养釜中实现。也可以将后级高压微生物富集培养釜的压力增压至略小于前级高压微生物富集培养釜，然后开启前级高压微生物富集培养釜出液阀和后级高压微生物富集培养釜的进液阀，微生物富集液将在微小压力差条件下，自动地前级高压微生物富集培养釜进入后级高压微生物富集培养釜中进行纯化培养。在整个多级富集与液体分离纯化培养的过程中，高压微生物富集培养釜的温度、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，所述温度环境控制系统包括低/高温环境系统、制冷/热系统和水浴温度监测系统；所述喷洒式固体分离培养单元、生物富集单元均置于低/高温环境系统中进行水浴，低/高温环境系统与所述制冷/热系统连接，实现热量交换；所述水浴温度监测系统用于监测低/高温环境系统中的温度变化，并将监测数据传输至所述控制采集终端；所述制冷/热系统控制端与所述控制采集终端电性连接。
上述方案中，喷洒式分离培养室、高压微生物富集培养釜的恒温条件维持主要是将喷洒式分离培养室、高压微生物富集培养釜安放于温度监控的低/高温环境系统中水浴，通过与低/高温环境系统的热交换作用，维持喷洒式分离培养室、 高压微生物富集培养釜内的恒温状态。或者将喷洒式分离培养室、高压微生物富集培养釜放置于空气换热式恒温房中。
上述方案中涉及的喷洒式分离培养室、高压微生物富集培养釜内的温度条件控制主要是通过低/高温环境系统。例如，在喷洒式分离培养室、高压微生物富集培养釜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。或者将喷洒式分离培养室、高压微生物富集培养釜置于低温/高温的水浴/油浴环境，来保证喷洒式分离培养室、高压微生物富集培养釜内需要的特殊温度条件。或者将喷洒式分离培养室、高压微生物富集培养釜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。其通过环境参数监测单元、温压传感器组对温度进行实时监测。
其中，所述增压系统包括空压机、增压泵、储气罐、调压阀和通气管道；其中：所述空压机、增压泵、储气罐、调压阀通过通气管道依次连接，最后通过通气管道依次与所述喷洒式固体分离培养单元、生物富集单元连接，用于向喷洒式固体分离培养单元、生物富集单元内注入气体进行增压；所述调压阀用于调整喷洒式固体分离培养单元、生物富集单元的内部压力；所述空压机控制端、增压泵控制端、调压阀控制端均与所述控制采集终端电性连接。
上述方案所涉及的增压系统主要是用于向高压微生物富集培养釜、喷洒式分离培养室内注入气体增压，使得高压微生物富集培养釜、喷洒式分离培养室内的压力环境与微生物在海洋原位所处的压力值一致。
上述方案中，通过环境参数监测单元、温压传感器组实时监测培养室内的压力变化，通过主动充气/放气进行培养室的增压、减压，使培养室内的压力值保持与微生物生长的海洋环境条件一致。
其中，所述高压环境生物富集与喷洒式固体分离培养装置还包括可移动平台，所述喷洒式固体分离培养单元、生物富集单元、增压系统、温度环境控制系统、控制采集终端均放置在所述可移动平台上，用于增加培养场景的普适性。
优选的，所述多微孔喷头、固体培育平板在所述喷洒式固体分离培养单元均设置有多个；所述液体注入模块同时与每个多微孔喷头连接；所述固体培育平板横向设置在所述喷洒式固体分离培养单元底部，实现对微生物在相同温、压条件 的横向分离培养。
优选的，所述喷洒式分离培养室在竖直方向上设置有多个连通隔板，将喷洒式分离培养室分隔为多个连通的培养腔体；其中：每个连通隔板上均设置有固体培育平板，每个培养腔体内均设置有多微孔喷头；所述液体注入模块同时与每个多微孔喷头连接，实现对微生物在相同温、压条件下的竖向分离培养。
优选的，所述喷洒式固体分离培养单元包括多个喷洒式分离培养室，每个喷洒式分离培养室设置有独立的多微孔喷头、固体培育平板和注气阀；所有喷洒式分离培养室均置于同一个温度环境控制系统中；所述液体注入模块同时与每个喷洒式分离培养室内的多微孔喷头连接，实现对微生物在相同温度不同压力条件下的分离培养。
优选的，所述喷洒式固体分离培养单元包括多个喷洒式分离培养室，每个喷洒式分离培养室设置有独立的多微孔喷头、固体培育平板和注气阀；每个喷洒式分离培养室置于对应设置的独立的温度环境控制系统中；所述液体注入模块同时与每个喷洒式分离培养室内的多微孔喷头连接，所有注气阀之间连通，实现对微生物在不同温度相同压力条件下的分离培养。
在上述方案基础上，将所有注气阀之间的连通切断，分别进行控制，便可实现对微生物在不同温度不同压力条件下的分离培养。
为了提高微生物的筛选效率，本方案涉及的高压环境下微生物分离工艺将单个喷洒式分离培养室进行多层级组合培养形成培养工艺。在相同的温度和压力环境条件下，为了增加培养面积和分选量，可以通过在横向培养室内布置多个固体培育平板，每个固体培育平板可以填充不同营养的培养基，每个平板设置一个多微孔喷头，提高分离培养效率。也可以设置成塔式结构，将多个固体培育平板和多微孔喷头组合进行竖向堆叠安放，也可以进行多组横向和竖向的同时组合，进行规模化培养。整个喷洒式分离培养室设置成相同的温度和压力环境条件。如果为了筛选不同压力和温度环境条件下的菌种，也可以将不同的喷洒式分离培养室进行并联组合，每个喷洒式分离培养室的温度和压力环境进行单独控制，可以按照分选目的进行梯度设置温度、压力参数条件。多个喷洒式分离培养室通过并联组合并且与微流泵和末级高压微生物富集培养釜相连。形成不同温度、压力环境条件下的分选工艺。
本方案通过在高压环境微生物的高压微生物富集培养釜与喷洒式分离培养 室里面构建微生物在高压环境，如海洋环境生活的高压与极端温度环境条件，提高其可培养性。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，同时结合喷洒式固体培养分离，获得单克隆的微生物菌落。整个富集和分离的全过程都在微生物原位压力、和温度环境条件下进行。同时，可通过多套分离培养室组合，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，通过生物富集单元富集培养，得到纯度较高的菌群后，在保压情况下进入喷洒式分离培养室进行固体培养分离，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。具体为：
富集培养过程：
首先是将高压微生物富集培养釜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)使得高压微生物富集培养釜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，通过顶部的搅拌杆进行搅拌，增加传质作用，优化培养进程。待首级高压微生物富集培养釜培养过程完成后，在第二级高压微生物富集培养釜中注入培养需要的营养液，并通过增压系统向第二级高压微生物富集培养釜内注气体和培养液体增压，第二级高压微生物富集培养釜内注入的培养液体的量需要根据分选需要保证富集液从首级高压微生物富集培养釜的浓度到第二级高压微生物富集培养釜中的稀释比例，然后通过保压转移，将首级高压微生物富集培养釜内的微生物菌液转移到第二级高压微生物富集培养釜中。以此类推，在末级高压微生物富集培养釜中的微生物将达到高度纯化状态，当末级高压微生物富集培养釜中的深海微生物菌液浓度达到106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。
当富集培养过程的菌液浓度经鉴定达到需求后，进入固体分离培养过程。
固体分离培养过程：
首先，将喷洒式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。然后在平板上填充好灭菌后的固体培养基。为了避免固体琼脂培养基在高压条件下液化或形成多孔结构，可在固体培育平板内填充氧化铝粉末、微细玻璃珠/钢球、钢丝球等作为支撑，满足固体分离培养的需求。然后， 开启温度环境控制系统，保证喷洒式分离培养室内的温度与微生物在海洋环境的温度条件一致。然后通过注气阀往喷洒式分离培养室内注入气体增压，使喷洒式分离培养室内的压力条件与微生物在海洋环境生活的压力条件一致。确保所有系统部件工作正常后，开启微流泵通过从末级高压微生物富集培养釜向喷洒式分离培养室内注入微生物富集液，微生物富集液通过多微孔喷头后，将被分割成超微细均匀液滴，超微细液滴将在重力作用下，运移至喷洒式分离培养室底部的固体培育平板上。超微细液滴在固体培养基内着床生长，待足够培养周期后，将长出分离后的单菌落。
本方案涉及的微生物自动培养工艺主要包括相同温压条件和不同温压条件两种。在相同温压条件主要是为了满足大规模不同培养基同时筛选分离的需求。首先，在喷洒式分离培养室内将若干固体培育平板和多微孔喷头在横向或者竖向组合放置，所有多微孔喷头的进口通过管线并联连接并且与微注泵和末级高压微生物富集培养釜相连。不同的温压条件主要是将不同的喷洒式分离培养室进行并联组合，每个喷洒式分离培养室的温度和压力环境进行单独控制，可以按照分选目的进行梯度设置参数条件。多个喷洒式分离培养室通过并联组合并且与微流泵和末级高压微生物富集培养釜相连。当整体工艺环节组装调试好以后，将培养工艺涉及的所有培养室和管阀件整体灭菌。然后通过温度和压力监控，保证所有喷洒式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。打开微注泵，微细分散液滴将在固体培育平板上着床，进行分离培养，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。为高压环境微生物的高效利用和分选工艺提供关键技术。
本发明主要涉及高压环境下微生物的连续富集与喷洒式分离培养装置与工艺，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喷洒式自动分离纯化培养装置与技术方法。
本方案解决了现有室内纯培养技术方法脱离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生存活性差，不能实现纯培养的难题；其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，实现高压环境微生物在原位压力和温度环境条件下的自动化分离培养，为高压环境下，微生物在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，提出了在高压环境条件下，对极端环境条件的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高压环境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活性差，或者表型与原位环境条件差异大，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，提高高压环境功能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生物富集与喷洒式固体分离培养装置，通过生物富集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；通过喷洒式固体分离培养单元将生物富集液进行微细珠状态化，使其在分散状态下实现分离和培养。本发明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，提高海洋微生物的可培养性，形成纯培养技术，为开发利用高压环境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为本发明所述喷洒式分离培养室结构示意图；
图3为本发明所述高压微生物富集培养釜结构示意图；
图4为本发明所述控制采集终端电路模块连接示意图；
图5为本发明一实施例中所述喷洒式固体分离培养单元实现对微生物在相同温、压条件的横向分离培养的结构示意图；
图6为本发明一实施例中所述喷洒式固体分离培养单元实现对微生物在相同温、压条件的竖向分离培养的结构示意图；
图7为本发明一实施例中所述喷洒式固体分离培养单元实现对微生物在相同温、压条件的进行不同培养基培养的结构示意图。
其中：1、喷洒式固体分离培养单元；11、喷洒式分离培养室；111、固体培育平板；112、快开卡箍；113、连通隔板；114、培养腔体；12、液体注入模块；121、微流泵；122、注液口；123、液体输送管道；13、环境参数监测单元；14、多微孔喷头；15、注气阀；16、出气阀；2、生物富集单元；21、高压微生物富集培养釜；211、可拆卸密封盖；212、温压传感器组；213、搅拌杆；22、进液阀；23、进气阀；24、取样阀；25、放空阀；26、出液阀；3、增压系统；31、 空压机；32、增压泵；33、储气罐；34、调压阀；35、通气管道；4、温度环境控制系统；41、低/高温环境系统；42、制冷/热系统；43、水浴温度监测系统；5、控制采集终端；6、可移动平台。
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、图4所示，本实施例提出一种高压环境生物富集与喷洒式固体分离培养装置，包括喷洒式固体分离培养单元1、生物富集单元2、增压系统3、温度环境控制系统4和控制采集终端5；其中：所述生物富集单元2用于实现对海洋微生物的富集与多层级纯化过程，得到生物富集液并将生物富集液注入所述喷洒式固体分离培养单元1中；所述喷洒式固体分离培养单元1用于将生物富集液进行微细珠状态化，使其在分散状态下实现分离和培养，有效地提高海洋微生物的可培养性；所述增压系统3、温度环境控制系统4分别与喷洒式固体分离培养单元1、生物富集单元2连接，用于在生物富集单元2、喷洒式固体分离培养单元1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；所述喷洒式固体分离培养单元1、生物富集单元2的控制端与信号检测端均与控制采集终端5电性连接。
在具体实施过程中，控制采集终端5包括数据采集器、数据中央处理器、操作电脑等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2、图4所示，所述喷洒式固体分离培养单元1包括喷洒式分离培养室11、液体注入模块12、环境参数监测单元13、多微孔喷头14、注气阀15和出气阀16；其中：所述喷洒式分离培养室11内部设置有固体培育平板111，用于对微生物进行分离培养；所述喷洒式分离培养室11置于温度环境控制 系统4中，通过温度环境控制系统4保证喷洒式分离培养室11内的温度恒温保持与深海冷泉区海底温度一致；所述环境参数监测单元13用于监测喷洒式分离培养室11内部的温度和压力变化情况，并将数据传输至所述控制采集终端5；所述多微孔喷头14设置在所述喷洒式分离培养室11内，与设置在喷洒式分离培养室11外的液体注入模块12连接；液体注入模块12输入端与所述生物富集单元2液体输出端连接，用于向喷洒式分离培养室11注入生物富集液，生物富集液经过多微孔喷头14后，将分散为微细珠状态散落在所述固体培育平板111；所述注气阀15、出气阀16均设置在所述喷洒式分离培养室11上，注气阀15与所述增压系统3连接，用于对喷洒式分离培养室11注入气体，增加其内部压力；出气阀16控制端与所述控制采集终端5电性连接，用于控制气体排出喷洒式分离培养室11，降低其内部压力。
更具体的，所述液体注入模块12包括微流泵121、注液口122和液体输送管道123；其中：所述微流泵121输入端与所述生物富集单元2液体输出端连接，其输出端通过液体输送管道123与所述注液口122连接；所述注液口122与所述多微孔喷头14连接。
更具体的，所述喷洒式分离培养室11设置有快开卡箍112。
在具体实施过程中，本实施例涉及的喷洒式分离培养室11是利用喷洒式分离原理，将生物富集单元2的微生物富集培养液通过微流泵121注入喷洒式分离培养室11，经过多微孔喷头14，使其分散成超微细菌株，散落在固体培养基上，足够小的单个微小液珠在固体培养基上附着后，将实现分离培养的过程。本方案涉及的喷洒式分离培养室11设置有注液口122，用于往喷洒式分离培养室11内注入微生物富集菌液；还设置有注气阀15，用于往喷洒式分离培养室11内通入微生物生长需要的气体或者惰性气体使培养室增压；喷洒式分离培养室11内安放有高强度的固体培育平板111，用于填充固体培养基，满足微生物着床生长需要；喷洒式分离培养室11内安放有使菌液分散的关键元件：多微孔喷头14，其可以为球型、喇叭型或者莲蓬型等能让微生物富集菌液从注液口122内出来后在高压环境中分散成均匀的微细液滴，喷头上的孔径足够细且足够多，使得菌液喷出后能分离生长。多微孔喷头14与固体培育平板111的安放距离要使得菌液喷洒出来后刚好均匀的附着在固体培育平板111上，而不会散落到喷洒式分离培养室11的内壁。
更具体的，如图3、图4所示，所述生物富集单元2由多个串联的高压微生物富集培养釜21组成；所述高压微生物富集培养釜21上设置有可拆卸密封盖211和连接取样阀24组，其内部设置有温压传感器组212；每个高压微生物富集培养釜21均设置在温度环境控制系统4中；其中：所述可拆卸密封盖211用于方便对高压微生物富集培养釜21内部进行灭菌操作和放入培养底物；所述连接取样阀24组用于各个高压微生物富集培养釜21的连接与取样，连接取样阀24组与所述增压系统3连接，用于向高压微生物富集培养釜21内输入液体或气体以增加高压微生物富集培养釜21内的压力，令高压微生物富集培养釜21内的压力值与深海实际情况一致；所述温压传感器组212用于实时监测高压微生物富集培养釜21内的温度、压力变化情况，并将信号输送至所述控制采集终端5。
更具体的，在所述高压微生物富集培养釜21上还设置有搅拌杆213；所述搅拌杆213用于高压微生物富集培养釜21在培养过程增强基质的反应过程。
在具体实施过程中，高压微生物富集培养釜21顶部设计有搅拌杆213，可通过手动或者机械搅拌增强传质，增强培养过程中基质的反应过程，增加微生物的能量和营养利用效率。
更具体的，所述连接取样阀24组包括进液阀22、进气阀23、取样阀24、放空阀25和出液阀26；其中：高压微生物富集培养釜21之间通过所述出液阀26、进液阀22进行串联，前一级高压微生物富集培养釜21出液阀26与后一级高压微生物富集培养釜21进液阀22连接；进气阀23用于向高压微生物富集培养釜21内输入气体以增加高压微生物富集培养釜21内的压力，令高压微生物富集培养釜21内的压力值与深海实际情况一致；放空阀25用于排出高压微生物富集培养釜21内的气体以降低高压微生物富集培养釜21内的压力，其控制端与所述控制采集终端5电性连接；取样阀24用于对高压微生物富集培养釜21内的微生物进行实时取样分析。
在具体实施过程中，高压微生物富集培养釜21设置有取样阀24，用于对富集过程中取样进行分析检测，以便进行相应的环境参数调整，优化富集培养流程。
在具体实施过程中，多个高压微生物富集培养釜21联构成生物富集单元2，将前一级高压微生物富集培养釜21内的菌液通过保压转移至下一级高压微生物富集培养釜21中，以此类推，按照浓度梯度稀释，末级高压微生物富集培养釜21中获得的微生物菌液将为高压环境下，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高压微生物富集培养釜21中的富集液通过取样阀24取出泵入后级的高压微生物富集培养釜21中实现。也可以将后级高压微生物富集培养釜21的压力增压至略小于前级高压微生物富集培养釜21，然后开启前级高压微生物富集培养釜21出液阀26和后级高压微生物富集培养釜21的进液阀22，微生物富集液将在微小压力差条件下，自动地前级高压微生物富集培养釜21进入后级高压微生物富集培养釜21中进行纯化培养。在整个多级富集与液体分离纯化培养的过程中，高压微生物富集培养釜21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，所述温度环境控制系统4包括低/高温环境系统41、制冷/热系统42和水浴温度监测系统43；所述喷洒式固体分离培养单元1、生物富集单元2均置于低/高温环境系统41中进行水浴，低/高温环境系统41与所述制冷/热系统42连接，实现热量交换；所述水浴温度监测系统43用于监测低/高温环境系统41中的温度变化，并将监测数据传输至所述控制采集终端5；所述制冷/热系统42控制端与所述控制采集终端5电性连接。
在具体实施过程中，喷洒式分离培养室11、高压微生物富集培养釜21的恒温条件维持主要是将喷洒式分离培养室11、高压微生物富集培养釜21安放于温度监控的低/高温环境系统41中水浴，通过与低/高温环境系统41的热交换作用，维持喷洒式分离培养室11、高压微生物富集培养釜21内的恒温状态。或者将喷洒式分离培养室11、高压微生物富集培养釜21放置于空气换热式恒温房中。
在具体实施过程中，本实施例涉及的喷洒式分离培养室11、高压微生物富集培养釜21内的温度条件控制主要是通过低/高温环境系统41。例如，在喷洒式分离培养室11、高压微生物富集培养釜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。或者将喷洒式分离培养室11、高压微生物富集培养釜21置于低温/高温的水浴/油浴环境，来保证喷洒式分离培养室11、高压微生物富集培养釜21内需要的特殊温度条件。或者将喷洒式分离培养室11、高压微生物富集培养釜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。其通过环境参数监测单元13、温压传感器组212对温度 进行实时监测。
更具体的，如图1、图4所示，所述增压系统3包括空压机31、增压泵32、储气罐33、调压阀34和通气管道35；其中：所述空压机31、增压泵32、储气罐33、调压阀34通过通气管道35依次连接，最后通过通气管道35依次与所述喷洒式固体分离培养单元1、生物富集单元2连接，用于向喷洒式固体分离培养单元1、生物富集单元2内注入气体进行增压；所述调压阀34用于调整喷洒式固体分离培养单元1、生物富集单元2的内部压力；所述空压机31控制端、增压泵32控制端、调压阀34控制端均与所述控制采集终端5电性连接。
在具体实施过程中，本实施例所涉及的增压系统3主要是用于向高压微生物富集培养釜21、喷洒式分离培养室11内注入气体增压，使得高压微生物富集培养釜21、喷洒式分离培养室11内的压力环境与微生物在海洋原位所处的压力值一致。
在具体实施过程中，通过环境参数监测单元13、温压传感器组212实时监测培养室内的压力变化，通过主动充气/放气进行培养室的增压、减压，使培养室内的压力值保持与微生物生长的海洋环境条件一致。
更具体的，所述高压环境生物富集与喷洒式固体分离培养装置还包括可移动平台6，所述喷洒式固体分离培养单元1、生物富集单元2、增压系统3、温度环境控制系统4、控制采集终端5均放置在所述可移动平台6上，用于增加培养场景的普适性。
在具体实施过程中，生物富集单元2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，喷洒式固体分离培养单元1可以将生物富集液进行微细珠状态化，使其在分散状态下实现分离和培养。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，提高海洋微生物的可培养性，形成纯培养技术，为开发利用高压环境微生物资源提供重要的基础手段。
实施例2
更具体的，在实施例1的基础上，如图5所示，所述多微孔喷头14、固体培育平板111在所述喷洒式固体分离培养单元1均设置有多个；所述液体注入模块12同时与每个多微孔喷头14连接；所述固体培育平板111横向设置在所述喷洒式固体分离培养单元1底部，实现对微生物在相同温、压条件的横向分离培养。
更具体的，在实施例1的基础上，如图6所示，所述喷洒式分离培养室11在竖直方向上设置有多个连通隔板113，将喷洒式分离培养室11分隔为多个连通的培养腔体114；其中：每个连通隔板113上均设置有固体培育平板111，每个培养腔体114内均设置有多微孔喷头14；所述液体注入模块12同时与每个多微孔喷头14连接，实现对微生物在相同温、压条件下的竖向分离培养。
更具体的，在实施例1的基础上，所述喷洒式固体分离培养单元1包括多个喷洒式分离培养室11，每个喷洒式分离培养室11设置有独立的多微孔喷头14、固体培育平板111和注气阀15；所有喷洒式分离培养室11均置于同一个温度环境控制系统4中；所述液体注入模块12同时与每个喷洒式分离培养室11内的多微孔喷头14连接，实现对微生物在相同温度不同压力条件下的分离培养。
更具体的，在实施例1的基础上，所述喷洒式固体分离培养单元1包括多个喷洒式分离培养室11，每个喷洒式分离培养室11设置有独立的多微孔喷头14、固体培育平板111和注气阀15；每个喷洒式分离培养室11置于对应设置的独立的温度环境控制系统4中；所述液体注入模块12同时与每个喷洒式分离培养室11内的多微孔喷头14连接，所有注气阀15之间连通，实现对微生物在不同温度相同压力条件下的分离培养。
在具体实施过程中，将所有注气阀15之间的连通切断，分别进行控制，便可实现对微生物在不同温度不同压力条件下的分离培养。
为了提高微生物的筛选效率，本实施例涉及的高压环境下微生物分离工艺将单个喷洒式分离培养室11进行多层级组合培养形成培养工艺。在相同的温度和压力环境条件下，为了增加培养面积和分选量，可以通过在横向培养室内布置多个固体培育平板111，每个固体培育平板111可以填充不同营养的培养基，每个平板设置一个多微孔喷头14，提高分离培养效率。也可以设置成塔式结构，将多个固体培育平板111和多微孔喷头14组合进行竖向堆叠安放，也可以进行多组横向和竖向的同时组合，进行规模化培养。整个喷洒式分离培养室11设置成相同的温度和压力环境条件。如果为了筛选不同压力和温度环境条件下的菌种，也可以将不同的喷洒式分离培养室11进行并联组合，每个喷洒式分离培养室11的温度和压力环境进行单独控制，可以按照分选目的进行梯度设置温度、压力参数条件。多个喷洒式分离培养室11通过并联组合并且与微流泵121和末级高压微生物富集培养釜21相连。形成不同温度、压力环境条件下的分选工艺。
本实施例通过在高压环境微生物的高压微生物富集培养釜21与喷洒式分离培养室11里面构建微生物在高压环境，如海洋环境生活的高压与极端温度环境条件，提高其可培养性。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，同时结合喷洒式固体培养分离，获得单克隆的微生物菌落。整个富集和分离的全过程都在微生物原位压力、和温度环境条件下进行。同时，可通过多套分离培养室组合，形成不同培养基组合的高效分离工艺，如图7所示的实现对微生物在相同温、压条件的进行不同培养基培养。
实施例3
本实施例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，通过生物富集单元2富集培养，得到纯度较高的菌群后，在保压情况下进入喷洒式分离培养室11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。具体为：
富集培养过程：
首先是将高压微生物富集培养釜21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22装入培养需要的营养液，然后从进气阀23注入培养需要的气体(若不需要可注入惰性气体)使得高压微生物富集培养釜21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，通过顶部的搅拌杆213进行搅拌，增加传质作用，优化培养进程。待首级高压微生物富集培养釜21培养过程完成后，在第二级高压微生物富集培养釜21中注入培养需要的营养液，并通过增压系统3向第二级高压微生物富集培养釜21内注气体和培养液体增压，第二级高压微生物富集培养釜21内注入的培养液体的量需要根据分选需要保证富集液从首级高压微生物富集培养釜21的浓度到第二级高压微生物富集培养釜21中的稀释比例，然后通过保压转移，将首级高压微生物富集培养釜21内的微生物菌液转移到第二级高压微生物富集培养釜21中。以此类推，在末级高压微生物富集培养釜21中的微生物将达到高度纯化状态，当末级高压微生物富集培养釜21中的深海微生物菌液浓度达到106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。
当富集培养过程的菌液浓度经鉴定达到需求后，进入固体分离培养过程。
固体分离培养过程：
首先，将喷洒式分离培养室11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。然后在平板上填充好灭菌后的固体培养基。为了避免固体琼脂培养基在高压条件下液化，可在固体培育平板111内填充氧化铝粉末、微细玻璃珠/钢球、钢丝球等作为支撑，满足固体分离培养的需求。然后，开启温度环境控制系统4，保证喷洒式分离培养室11内的温度与微生物在海洋环境的温度条件一致。然后通过注气阀15往喷洒式分离培养室11内注入气体增压，使喷洒式分离培养室11内的压力条件与微生物在海洋环境生活的压力条件一致。确保所有系统部件工作正常后，开启微流泵121通过从末级高压微生物富集培养釜21向喷洒式分离培养室11内注入微生物富集液，微生物富集液通过多微孔喷头14后，将被分割成超微细均匀液滴，超微细液滴将在重力作用下，运移至喷洒式分离培养室11底部的固体培育平板111上。超微细液滴在固体培养基内着床生长，待足够的培养周期后，将长出分离后的单菌落。
本实施例涉及的微生物自动培养工艺主要包括相同温压条件和不同温压条件两种。在相同温压条件主要是为了满足大规模不同培养基同时筛选分离的需求。首先，在喷洒式分离培养室11内将若干固体培育平板111和多微孔喷头14在横向或者竖向组合放置，所有多微孔喷头14的进口通过管线并联连接并且与微注泵和末级高压微生物富集培养釜21相连。不同的温压条件主要是将不同的喷洒式分离培养室11进行并联组合，每个喷洒式分离培养室11的温度和压力环境进行单独控制，可以按照分选目的进行梯度设置参数条件。多个喷洒式分离培养室11通过并联组合并且与微流泵121和末级高压微生物富集培养釜21相连。当整体工艺环节组装调试好以后，将培养工艺涉及的所有培养室和管阀件整体灭菌。然后通过温度和压力监控，保证所有喷洒式分离培养室11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。打开微注泵，微细分散液滴将在固体培育平板111上着床，进行分离培养，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。为高压环境微生物的高效利用和分选工艺提供关键技术。
本实施例主要涉及高压环境下微生物的连续富集与喷洒式分离培养装置与工艺，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喷洒式自动分离纯化培养装置与技术方法。
本实施例解决了现有室内纯培养技术方法脱离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生存活性差，不能实现纯培养的难题；其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，实现高压环境微生物在原位压力和温度环境条件下的自动化分离培养，为高压环境下，微生物在原位条件纯培养提供重要技术手段。
本实施例相对于现有的纯培养技术，提出了在高压环境条件下，对极端环境条件的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高压环境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生存活性差，或者表型与原位环境条件差异大，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，提高高压环境功能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，本实施例提出一种在高压环境下定向富集和分离深海嗜甲烷菌的多层级富集与分离装置。高压微生物富集培养釜21与喷洒式分离培养室11是本实例的核心，其它辅助系统包括增压系统3、温度环境控制系统4和控制采集终端5。通过在高压环境微生物的高压微生物富集培养釜21与喷洒式分离培养室11里面构建深海冷泉区嗜甲烷菌在深海环境生活的高压与低温环境条件，提高其生存活性。本实例通过多级富集和液体稀释培养，得到高压、低温、甲烷为唯一碳源胁迫下纯度较高的深海嗜甲烷菌，同时结合喷洒式固体培养分离，获得单克隆的微生物菌落。整个富集和分离的全过程都在我国南海海马冷泉区原位压力(14MPa)、和温度环境(4℃)条件下进行。同时，可通过多套分离培养室组合，形成不同培养基组合的高效分离工艺。
在生物富集单元内成功实现深海嗜甲烷菌的富集，和富集后的菌液在喷洒式固体分离培养单元1上成功纯化和分离培养，是本实例的关键。富集培养与分离培养室底部整体加装可移动平台6，增加培养场景的普适性。
本实施例涉及的生物富集单元主要是通过多个高压微生物富集培养釜21串联而成。高压微生物富集培养釜21为可开盖式结构设计，方便放入培养底物和灭菌操作处理。高压微生物富集培养釜21顶部设计有搅拌杆213，用于在培养过程中，增强基质的反应过程，增加深海嗜甲烷菌的碳源和能量利用效率。高压 微生物富集培养釜21安装有温压传感器组212，实时监测高压微生物富集培养釜21内的温度和压力变化。高压微生物富集培养釜21的恒温条件维持主要是将高压微生物富集培养釜21安放于低/高温环境系统41的低温水浴中，通过与水浴系统的热交换作用，低/高温环境系统41内填充制冷剂如乙二醇等，维持富集培养室内的低温状态。高压微生物富集培养釜21的顶部设置有进气阀23和进液阀22，通过进气阀23和进液阀22内注入培养需要的甲烷气体和营养液来向密闭式高压微生物富集培养釜21内增压，实现高压微生物富集培养釜21内的压力值维持在14MPa左右。高压微生物富集培养釜21设置有取样阀24，用于对富集过程中取样进行分析检测，以便进行相应的环境参数调整，优化富集培养的流程。
如图1所示，本实例由4个高压微生物富集培养釜21串联构成生物富集单元2，将高压微生物富集培养釜21的菌液通过保压转移至下一级高压微生物富集培养釜2121中，以此类推，按照浓度梯度稀释，末级高压微生物富集培养釜21中获得的微生物菌液将为高压环境高度纯化深海嗜甲烷菌。通过将后级高压微生物富集培养釜21的压力增压至小于前级高压微生物富集培养釜21的压力0.2-0.5MPa，然后开启前级高压微生物富集培养釜21的取样阀24和后级高压微生物富集培养釜21的进液阀22，微生物富集液将在微小压力差条件下，自动地从前级高压微生物富集培养釜21进入后级高压微生物富集培养釜21中进行纯化培养。在4个多级富集与纯化培养的过程中，高压微生物富集培养釜21内的温度、压力环境条件都保持4℃和14MPa，保证富集培养的有效性。
本实施例涉及的喷洒式固体分离培养单元1是利用喷洒式释放原理，本实施例将固体培育平板11放置于喷洒式分离培养室11内，将末级高压微生物富集培养釜21内的嗜甲烷菌富集培养液通过微流泵121注入喷洒式分离培养室11，经过多微孔喷头14，使其分散成超微细菌株，散落在固体培养基上，足够小的单个微小液珠在固体培养基上附着后，将实现分离培养的过程。本实施例涉及的喷洒式分离培养室11设置有注液口122，用于往固体培养室内注入嗜甲烷菌富集液；还设置有注气阀15，用于往喷洒式分离培养室11内通入嗜甲烷菌生长需要的甲烷气体使培养室增压至14MPa；喷洒式分离培养室11内安放有高强度固体培育平板111用于填充固体培养基，满足嗜甲烷菌着床生长需要；喷洒式分离培养室11内安放有使菌液分散的关键元件：喇叭状多微孔喷头14，能让嗜甲烷菌 富集液从注液口122内出来后在高压环境中分散成均匀的微细液滴，喷头上的孔径足够细且足够多，使得菌液喷出后能分离生长。多微孔喷头14与固体培育平板111的安放距离为100㎜，使得菌液喷洒出来后刚好均匀的附着在固体培育平板111上，而不会散落到喷洒式分离培养室11的内壁。
本实施例涉及的增压系统3主要是用于向微生物富集和分离培养室内注入甲烷气体增压，使得喷洒式分离培养室11和高压微生物富集培养釜21内的压力环境维持14MPa。
本实例涉及的高压环境深海嗜甲烷菌富集培养与分离技术主要包括富集和分离两个步骤。首先，通过在生物富集单元2进行多层级高压环境富集培养，得到纯度较高的深海嗜甲烷菌群后，在保压情况下进入喷洒式分离培养室11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。
本实例涉及的高压环境深海嗜甲烷富集培养方法首先是将高压微生物富集培养釜21及其附带管阀件进行灭菌处理，然后在高压微生物富集培养釜21依次装入待培养的海马冷泉甲烷渗漏区的沉积物，然后从进液阀22装入培养需要的营养液，从进气阀23注入培养需要的甲烷气体使得高压微生物富集培养釜21内的压力值增加至14MPa。在培养的过程中，通过顶部的搅拌杆213进行搅拌，增加传质作用，优化培养进程。待富集培养过程完成后，在第二级高压微生物富集培养釜21注入培养需要的营养液，并通过增压系统3向第二级高压微生物富集培养釜21注气增压，第二级高压微生物富集培养釜21内注入的培养液体的量需要保证富集液从首级高压微生物富集培养釜21的浓度到第二级高压微生物富集培养釜21的稀释比例为1:10，然后通过保压转移，将首级高压微生物富集培养釜21内的微生物菌液转移到第二级高压微生物富集培养釜21中。以此类推，在末级高压微生物富集培养釜21的微生物将达到高度纯化状态，当末级高压微生物富集培养釜21中的深海嗜甲烷菌液浓度达到106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，进入喷洒式固体分离培养单元1进行固体分离培养过程。分离培养过程主要包括，首先，将喷洒式分离培养室11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。然后在固体培育平板111填充好灭菌后的固体培养基。为了避免固体琼脂培养基在高压条 件下液化，可在固体培育平板111内填充微细玻璃珠作为支撑，满足固体分离培养的需求。然后，开启温度环境控制系统4，保证喷洒式分离培养室11内的温度与嗜甲烷菌在海马冷泉区海底温度条件一致。然后通过注气阀15往喷洒式分离培养室11内注入甲烷气体增压，使喷洒式分离培养室11内的压力增加至14MPa。确保所有系统部件工作正常后，开启微流泵121通过从末级高压微生物富集培养釜21向喷洒式分离培养室11内注入纯化的嗜甲烷菌富集液，嗜甲烷菌富集液通过多微孔喷头14后，将被分割成超微细均匀液滴，超微细液滴将在重力作用下，运移至喷洒式分离培养室11底部的固体培育平板111上。超微细液滴在固体培养基内着床生长，长出分离后的单菌落。
本实施例涉及的嗜甲烷菌自动培养工艺主要包括相同温压条件和不同温压条件两种。在相同温压条件主要是为了满足大规模不同培养基同时筛选分离的需求。首先，在高喷洒式分离培养室11内将若干固体培育平板111和多微孔喷头14在横向或者竖向组合放置，所有多微孔喷头14的进口通过管线并联连接并且与微注泵121和末级高压微生物富集培养釜21相连，整个工艺筛选过程所有的固体培育平板111都处于14MPa和4℃温度环境条件中。不同的温压条件主要是将不同的培养室进行并联组合，每个高喷洒式分离培养室11的温度和压力环境进行单独控制，可以按照分选目的进行梯度设置参数条件。如根据不同水深环境发现的嗜甲烷菌，将多个高喷洒式分离培养室11内的压力条件从6MPa至20MPa进行梯度设施，温度环境响应的设置为该深度环境的海底温度。且多个高喷洒式分离培养室11通过并联组合并且与微流泵121和末级高压微生物富集培养釜21相连。当整体工艺环节组装调试好以后，将培养工艺涉及的所有末级高压微生物富集培养釜21和管阀件整体灭菌。然后通过温度和压力监控，保证所有末级高压微生物富集培养釜21内的温度和压力环境条件与嗜甲烷菌所在海洋环境的温度和压力环境条件一致。打开微流泵121，微细分散液滴将在固体培育平板111上着床，进行分离培养，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嗜甲烷菌的分离培养和纯化。为深海嗜甲烷菌的高效利用和高压环境分选工艺提供关键技术。
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 (15)

  1. 高压环境生物富集与喷洒式固体分离培养装置，其特征在于，包括喷洒式固体分离培养单元(1)、生物富集单元(2)、增压系统(3)、温度环境控制系统(4)和控制采集终端(5)；其中：
    所述生物富集单元(2)用于实现对海洋微生物的富集与多层级纯化过程，得到生物富集液并将生物富集液注入所述喷洒式固体分离培养单元(1)中；
    所述喷洒式固体分离培养单元(1)用于将生物富集液进行微细珠状态化，使其在分散状态下实现分离和培养，有效地提高海洋微生物的可培养性；
    所述增压系统(3)、温度环境控制系统(4)分别与喷洒式固体分离培养单元(1)、生物富集单元(2)连接，用于在生物富集单元(2)、喷洒式固体分离培养单元(1)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；
    所述喷洒式固体分离培养单元(1)、生物富集单元(2)的控制端与信号检测端均与控制采集终端(5)电性连接。
  2. 根据权利要求1所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述喷洒式固体分离培养单元(1)包括喷洒式分离培养室(11)、液体注入模块(12)、环境参数监测单元(13)、多微孔喷头(14)、注气阀(15)和出气阀(16)；其中：
    所述喷洒式分离培养室(11)内部设置有固体培育平板(111)，用于对微生物进行分离培养；
    所述喷洒式分离培养室(11)置于温度环境控制系统(4)中，通过温度环境控制系统(4)保证喷洒式分离培养室(11)内的温度恒温保持与深海冷泉区海底温度一致；
    所述环境参数监测单元(13)用于监测喷洒式分离培养室(11)内部的温度和压力变化情况，并将数据传输至所述控制采集终端(5)；
    所述多微孔喷头(14)设置在所述喷洒式分离培养室(11)内，与设置在喷洒式分离培养室(11)外的液体注入模块(12)连接；液体注入模块(12)输入端与所述生物富集单元(2)液体输出端连接，用于向喷洒式分离培养室(11)注入生物富集液，生物富集液经过多微孔喷头(14)后，将分散为微细珠状态散 落在所述固体培育平板(111)；
    所述注气阀(15)、出气阀(16)均设置在所述喷洒式分离培养室(11)上，注气阀(15)与所述增压系统(3)连接，用于对喷洒式分离培养室(11)注入气体，增加其内部压力；出气阀(16)控制端与所述控制采集终端(5)电性连接，用于控制气体排出喷洒式分离培养室(11)，降低其内部压力。
  3. 根据权利要求2所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述液体注入模块(12)包括微流泵(121)、注液口(122)和液体输送管道(123)；其中：所述微流泵(121)输入端与所述生物富集单元(2)液体输出端连接，其输出端通过液体输送管道(123)与所述注液口(122)连接；所述注液口(122)与所述多微孔喷头(14)连接。
  4. 根据权利要求2所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述喷洒式分离培养室(11)设置有快开卡箍(112)。
  5. 根据权利要求2所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述生物富集单元(2)由多个串联的高压微生物富集培养釜(21)组成；所述高压微生物富集培养釜(21)上设置有可拆卸密封盖(211)和连接取样阀组，其内部设置有温压传感器组(212)；每个高压微生物富集培养釜(21)均设置在温度环境控制系统(4)中；其中：
    所述可拆卸密封盖(211)用于方便对高压微生物富集培养釜(21)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高压微生物富集培养釜(21)的连接与取样，连接取样阀组与所述增压系统(3)连接，用于向高压微生物富集培养釜(21)内输入液体或气体以增加高压微生物富集培养釜(21)内的压力，令高压微生物富集培养釜(21)内的压力值与深海实际情况一致；
    所述温压传感器组(212)用于实时监测高压微生物富集培养釜(21)内的温度、压力变化情况，并将信号输送至所述控制采集终端(5)。
  6. 根据权利要求5所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，在所述高压微生物富集培养釜(21)上还设置有搅拌杆(213)；所述搅拌杆(213)用于高压微生物富集培养釜(21)在培养过程增强基质的反应过程。
  7. 根据权利要求6所述的高压环境生物富集与喷洒式固体分离培养装置，其 特征在于，所述连接取样阀组包括进液阀(22)、进气阀(23)、取样阀(24)、放空阀(25)和出液阀(26)；其中：
    高压微生物富集培养釜(21)之间通过所述出液阀(26)、进液阀(22)进行串联，前一级高压微生物富集培养釜(21)出液阀(26)与后一级高压微生物富集培养釜(21)进液阀(22)连接；
    进气阀(23)用于向高压微生物富集培养釜(21)内输入气体以增加高压微生物富集培养釜(21)内的压力，令高压微生物富集培养釜(21)内的压力值与深海实际情况一致；
    放空阀(25)用于排出高压微生物富集培养釜(21)内的气体以降低高压微生物富集培养釜(21)内的压力，其控制端与所述控制采集终端(5)电性连接；
    取样阀(24)用于对高压微生物富集培养釜(21)内的微生物进行实时取样分析。
  8. 根据权利要求2所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述温度环境控制系统(4)包括低/高温环境系统(41)、制冷/热系统(42)和水浴温度监测系统(43)；所述喷洒式固体分离培养单元(1)、生物富集单元(2)均置于低/高温环境系统(41)中进行水浴，低/高温环境系统(41)与所述制冷/热系统(42)连接，实现热量交换；所述水浴温度监测系统(43)用于监测低/高温环境系统(41)中的温度变化，并将监测数据传输至所述控制采集终端(5)；所述制冷/热系统(42)控制端与所述控制采集终端(5)电性连接。
  9. 根据权利要求2所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述增压系统(3)包括空压机(31)、增压泵(32)、储气罐(33)、调压阀(34)和通气管道(35)；其中：
    所述空压机(31)、增压泵(32)、储气罐(33)、调压阀(34)通过通气管道(35)依次连接，最后通过通气管道(35)依次与所述喷洒式固体分离培养单元(1)、生物富集单元(2)连接，用于向喷洒式固体分离培养单元(1)、生物富集单元(2)内注入气体进行增压；所述调压阀(34)用于调整喷洒式固体分离培养单元(1)、生物富集单元(2)的内部压力；
    所述空压机(31)控制端、增压泵(32)控制端、调压阀(34)控制端均与所述控制采集终端(5)电性连接。
  10. 根据权利要求2所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，还包括可移动平台(6)，所述喷洒式固体分离培养单元(1)、生物富集单元(2)、增压系统(3)、温度环境控制系统(4)、控制采集终端(5)均放置在所述可移动平台(6)上，用于增加培养场景的普适性。
  11. 根据权利要求2～10任一项所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述多微孔喷头(14)、固体培育平板(111)在所述喷洒式固体分离培养单元(1)均设置有多个；所述液体注入模块(12)同时与每个多微孔喷头(14)连接；所述固体培育平板(111)横向设置在所述喷洒式固体分离培养单元(1)底部，实现对微生物在相同温、压条件的横向分离培养。
  12. 根据权利要求2～10任一项所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述喷洒式分离培养室(11)在竖直方向上设置有多个连通隔板(113)，将喷洒式分离培养室(11)分隔为多个连通的培养腔体(114)；其中：每个连通隔板(113)上均设置有固体培育平板(111)，每个培养腔体(114)内均设置有多微孔喷头(14)；所述液体注入模块(12)同时与每个多微孔喷头(14)连接，实现对微生物在相同温、压条件下的竖向分离培养。
  13. 根据权利要求2～10任一项所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述喷洒式固体分离培养单元(1)包括多个喷洒式分离培养室(11)，每个喷洒式分离培养室(11)设置有独立的多微孔喷头(14)、固体培育平板(111)和注气阀(15)；所有喷洒式分离培养室(11)均置于同一个温度环境控制系统(4)中；所述液体注入模块(12)同时与每个喷洒式分离培养室(11)内的多微孔喷头(14)连接，实现对微生物在相同温度不同压力条件下的分离培养。
  14. 根据权利要求2～10任一项所述的高压环境生物富集与喷洒式固体分离培养装置，其特征在于，所述喷洒式固体分离培养单元(1)包括多个喷洒式分离培养室(11)，每个喷洒式分离培养室(11)设置有独立的多微孔喷头(14)、固体培育平板(111)和注气阀(15)；每个喷洒式分离培养室(11)置于对应设置的独立的温度环境控制系统(4)中；所述液体注入模块(12)同时与每个喷洒式分离培养室(11)内的多微孔喷头(14)连接，所有注气阀(15)之间连通，实现对微生物在不同温度相同压力条件下的分离培养。
  15. 根据权利要求14所述的高压环境生物富集与喷洒式固体分离培养装置， 其特征在于，将所有注气阀(15)之间的连通切断，分别进行控制，便可实现对微生物在不同温度不同压力条件下的分离培养。
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