WO2023173493A1 - 一种深海原位环境的单菌落分离装置及分离方法 - Google Patents

一种深海原位环境的单菌落分离装置及分离方法 Download PDF

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WO2023173493A1
WO2023173493A1 PCT/CN2022/084115 CN2022084115W WO2023173493A1 WO 2023173493 A1 WO2023173493 A1 WO 2023173493A1 CN 2022084115 W CN2022084115 W CN 2022084115W WO 2023173493 A1 WO2023173493 A1 WO 2023173493A1
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pressure
separation operation
sampling
incubator
separation
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PCT/CN2022/084115
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English (en)
French (fr)
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冯景春
钟松
张偲
杨志峰
王屹
蔡宴朋
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广东工业大学
南方海洋科学与工程广东省实验室(广州)
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Publication of WO2023173493A1 publication Critical patent/WO2023173493A1/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
    • C12M1/04—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 present invention relates to the technical field of marine microorganisms, and in particular to a single colony separation device and separation method in a deep sea in-situ environment.
  • the marine environment is different from the land. It is a unique ecological environment with characteristics such as high salinity, high osmosis, high pressure, and oligonutrients. In some sea areas, there are also special conditions such as high acidity, high alkali, hypoxia, low temperature, high temperature, and high radiation. . Compared with some ecological environments on land, the activity of microorganisms is not intense, but there are very rich microbial resources from the sea surface to the seabed sediments. Marine microorganisms are extremely innovative and diverse in terms of genes, physiological metabolism, etc., and play an irreplaceable role in promoting ocean energy transfer and material circulation, and maintaining the stability of ocean and even global ecosystems.
  • the isolation of microorganisms in the ocean mainly involves the isolation of single colonies under normal pressure after the pressure is released.
  • special bacterial species originally existing in the marine environment have not yet been isolated, such as pressure-loving bacteria, etc., and those that have been isolated in the ocean have not yet been isolated.
  • Microorganisms are less abundant.
  • special target bacteria with high abundance can be obtained, and single colonies can be streaked and separated under pressure maintaining conditions.
  • how to select and successfully cultivate single colonies has not yet been solved. This further limits our understanding of marine microorganisms and the development and utilization of resources.
  • the prior art discloses a deep-sea microbial culture cabin, including: 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 cultivation of microorganisms in the deep sea in situ, it still isolates and cultivates marine microorganisms, which cannot effectively improve the success rate of culture.
  • the present invention provides a single bacterial colony separation device and separation method in a deep-sea in-situ environment, which realizes the isolation and cultivation of a single bacterial colony in a high-pressure environment and overcomes the difficulty that deep-sea microorganisms are difficult to separate and cultivate. .
  • a single colony separation device for deep sea in situ environment including a central control system, a separation operation incubator, an environmental parameter detection unit, a pressure control unit, a temperature control unit, a liquid injection unit and a sampling unit; wherein: the separation operation incubator It includes a base and a cover fixedly connected to the base; the cover serves as an observation area and a separation operation area, and the base serves as a culture area; the observation area is used to observe the separation and culture process; the separation operation area is based on The separation operation requires an inner slide rail, and a sampling probe device is installed on the inner slide rail.
  • the sampling probe device can slide on the inner slide rail to realize the scribing operation and sampling operation of microorganisms; the culture area is provided with The culture medium is used to cultivate the streaked microbial colonies; the environmental parameter detection unit is set inside the separation operation incubator, used to detect the temperature and pressure changes in the separation operation incubator in real time, and transmit the data to Central control system; the pressure control unit and the temperature control unit are respectively connected to the separation operation incubator to ensure that the pressure and temperature in the separation operation incubator are consistent with the growth and culture environment of microorganisms; the liquid injection unit is used to Inject enriched microbial liquid into the separation operation incubator for dipping and marking by the sampling probe device; the sampling unit is used for pressure-maintaining sampling of microorganisms; the control end of the sampling probe device, the control end of the pressure control unit, The control end of the temperature control unit, the control end of the liquid injection unit and the control end of the sampling unit are all electrically connected to the central control system; the data output end of the observation area is electrically connected
  • the base and the cover are sealed and connected through buckles.
  • the cover is similar to an "L" shape and the base is similar to a cylinder.
  • the inner slide rail is a three-dimensional slide rail.
  • the sampling probe device can slide up, down, left, and right on the inner slide rail in all directions. This is mainly to ensure that the sampling probe device can contact the surface of the culture medium in the base. At the same time, It can also be located above the culture medium in the base to facilitate the sampling probe device to perform various operations on the culture medium.
  • the control of the sampling probe device is mainly controlled and displayed on the central control system through the pressure holding chip of the sampling probe device.
  • the central control system includes servers, computers, etc., which are used to realize changes in various environmental data information during the separation and cultivation of enriched marine microorganisms in high-pressure environments, as well as real-time collection, processing, storage and image output functions. .
  • the single colony is separated, selected and cultured, and finally the target bacteria can be obtained, effectively improving the culturability of marine microorganisms and providing a good foundation for the ocean.
  • the above solution can realize the pressure-maintaining separation of enriched marine microorganisms in an in-situ high-pressure environment to meet the needs of subsequent culture and functional identification.
  • the observation area includes an outer slide rail provided on the outer surface of the cover body, a connecting bracket slidably connected to the outer slide rail, an observation device fixed on the end of the connecting bracket and a viewing window provided on the surface of the cover body;
  • the data output end of the observation device is electrically connected to the central control system.
  • the viewing window is a circular viewing window, which is arranged in the center of the cover body to facilitate the observation device to observe the inside of the separation operation incubator.
  • the outer slide rail is a horizontal slide rail, and a telescopic "L"-shaped connecting bracket is installed on the slide rail.
  • An observation device is provided at the end of the connecting bracket to facilitate the observation and identification of microorganisms. If the optical observation module of a high-resolution optical microscope is used as an observation device to observe and identify microorganisms, it can be preliminarily determined whether the single colony is the target microorganism required by the researcher or whether the colony is a single colony. It can also be used to photograph high-pressure orders. The colony formation process (such as color, shape, size, etc.) facilitates the subsequent selection of individual colonies.
  • the environmental parameter detection unit includes a temperature sensor and a pressure sensor; the temperature sensor and the pressure sensor are both arranged inside the separation operation incubator, and are used to detect the temperature and pressure changes in the separation operation incubator in real time, and store the data transmitted to the central control system.
  • the temperature sensor and the pressure sensor are both arranged on the inner edge of the cover, and are mainly used to monitor the temperature and pressure changes in the separation operation incubator, so as to facilitate macro-control of the entire culture environment during actual application.
  • the pressure control unit includes an air compressor, a booster pump, a gas storage tank, a pressure regulating valve, an air intake valve and an air supply pipeline; wherein: the air compressor, booster pump, gas storage tank, pressure regulating valve The air supply pipeline is connected in sequence, and finally the air inlet valve is connected to the separation operation incubator; the control end of the air compressor, the control end of the booster pump, the control end of the pressure regulating valve, and the air inlet valve All terminals are electrically connected to the central control system.
  • the pressure control unit is mainly used to inject gas to pressurize the separation operation incubator.
  • the temperature control unit adopts a water bath jacket device, which is wrapped on the outer wall of the separation operation incubator, and the control end is electrically connected to the central control system.
  • the liquid injection unit includes a microorganism enrichment kettle, a liquid delivery pipeline and a micro-injection pump; wherein: the microorganism enrichment kettle is used for enrichment of microbial liquid, and its output end is connected to a separation operation incubator through a liquid delivery pipeline Connection; the micro-injection pump is arranged on the liquid delivery pipe, and the control end is electrically connected to the central control system.
  • the liquid injection unit further includes a groove provided on the inner bottom surface of the cover body of the separation operation incubator; the liquid outlet of the liquid supply pipe inside the separation operation incubator is provided in the groove.
  • the groove is mainly used to hold the microbial liquid injected through the micro-injection pump, so that the microbial liquid cannot flow freely after being injected into the separation operation incubator, ensuring that subsequent separation and marking operations can be realized.
  • the liquid injection unit further includes a vent valve provided on the liquid supply pipeline, and the control end of the vent valve is electrically connected to the central control system.
  • the excess bacterial liquid can be slowly drained through the vent valve so that the excess bacterial liquid does not enter the culture medium of the base.
  • the sampling unit includes a reaction kettle, a sampling pipeline and a sampling valve; wherein: the reaction kettle is connected to the separation operation incubator through a sampling pipeline; the sampling valve is provided on the sampling pipeline, and its control end is connected to The central control system is electrically connected.
  • the inner diameters of the sampling pipeline and the sampling valve are both larger than the diameter of the sampling probe device, mainly so that the sampling probe device can extend into the sampling pipe and transfer the dipped single colony to the reaction chamber containing the liquid culture medium while maintaining pressure.
  • the sampling valve can be closed and replaced with the next reaction kettle.
  • the reaction kettle is filled with 75% alcohol.
  • the sampling probe device is sterilized, it can be replaced.
  • the upper and lower reactors are filled with liquid culture medium, and so on to complete the isolation and culture of single colonies under pressure. The pressure difference that occurs every time the reactor is replaced can be controlled and supplemented with the corresponding pressure through the pressure control unit.
  • This solution also provides a single colony isolation method in a deep sea in situ environment, which is implemented by using a single colony isolation device in a deep sea in situ environment as described above, which specifically includes the following steps:
  • S1 Clean and sterilize the single colony isolation device in the deep sea in-situ environment and load it with culture medium;
  • the liquid injection unit injects the microbial liquid into the separation operation incubator, controls the sampling probe device to slide on the inner slide rail, first dips the microbial liquid, and then positions the sampling probe device on the surface of the culture medium to allow it to slide on the culture medium.
  • the surface is separated according to a certain trajectory;
  • S5 The isolated microorganisms will grow according to the marked trajectory and be observed in real time through the observation area;
  • S7 Control the sampling probe device to pick a single colony on the culture medium, and send the colony into the sampling unit containing the liquid culture medium to realize pressure-maintaining transfer culture of a single colony.
  • the above-mentioned scheme mainly builds a high-pressure environment in the separation operation incubator that is the same as the microorganisms living in the marine environment.
  • the separation operation incubator determines the temperature value in the separation operation incubator according to the temperature value in the microorganism enrichment kettle, and obtain a temperature consistent with that in the microorganism enrichment kettle by placing the separation operation incubator in the water bath environment of the water bath jacket device. Then, adjust the observation device so that it can clearly observe the situation on the culture medium inside the base. Then, the bacterial liquid containing microorganisms is injected from the microorganism enrichment kettle into the groove through a microinjection pump, so that the bacterial liquid forms droplets and is located in the groove.
  • the sampling probe device By controlling the sampling probe device to dip into the bacterial liquid in the groove, and then positioning the bottom of the sampling probe device on the surface of the culture medium, the sampling probe device draws lines on the surface of the culture medium according to a certain trajectory, and the finally separated microorganisms will follow the marking trajectory. growth to achieve isolation of individual colonies. Observe the whole process through the visual window on the cover. When a single colony is formed on the culture medium, the streaking and isolation work can be started.
  • sampling pipe Before isolating a single colony, first connect the end of the sampling pipe to the reaction kettle and open the sampling valve.
  • the reaction kettle is filled with 75% alcohol.
  • the pressure of the entire device is maintained constant through the pressure control unit.
  • the sampling probe device is controlled through the central control system.
  • the sampling probe device is allowed to pass through the sampling pipe and enter the reaction kettle to realize the sterilization process of the sampling probe device.
  • close the sampling valve replace the reaction kettle containing 75% alcohol with a reaction kettle containing liquid culture medium, and maintain the pressure of the entire device constant through the pressure control unit.
  • the sampling probe device is controlled through the central control system, and successfully A single colony on the culture medium is picked, and then the sampling probe device is passed through the sampling pipe into the reaction kettle containing the liquid culture medium, and finally the pressure-maintaining transfer culture of the single colony is realized.
  • the subsequent isolation of single colonies can be deduced in the same way.
  • the pressure and temperature values in the separation operation incubator are kept consistent with the pressure and temperature environment in the high-pressure enrichment system where the microorganisms were originally located, so that the microorganisms can be separated under in-situ high pressure.
  • the above-mentioned plan aims at the current problem of difficult separation of marine microorganisms, and proposes a single colony separation device and separation method for high-pressure environments.
  • the existing normal-pressure separation culture it can enrich, separate and culture microorganisms in a deep-sea in-situ high-pressure environment, and solve the problems of deep-sea in-situ pressure-loving bacteria being unable to survive or expressing differences when cultured in a normal-pressure environment.
  • the existing high-pressure enrichment isolation culture can select a single colony while maintaining pressure and successfully culture it.
  • the present invention proposes a single colony separation device and a separation method in a deep sea in-situ environment.
  • a single colony separation device By establishing a single colony separation device and maintaining pressure, the isolation, selection and cultivation of single colonies can be achieved. Finally, it can Obtain target bacteria, effectively improve the culturability of marine microorganisms, and provide basic devices and methods for the isolation and cultivation of marine microorganisms.
  • Figure 1 is a schematic structural diagram of the device according to the present invention.
  • FIG. 2 is a schematic connection diagram of the circuit modules of the central control system according to the present invention.
  • Figure 3 is a schematic flow chart of the method of the present invention.
  • This embodiment is a complete usage example with rich content.
  • this embodiment provides a single colony separation device for a deep sea in-situ environment, including a central control system 1, a separation operation incubator 2, an environmental parameter detection unit 3, a pressure control unit 4, and a temperature control unit.
  • Unit 5 liquid injection unit 6 and sampling unit 7;
  • the separation operation incubator 2 includes a base 21 and a cover 22 fixedly connected to the base 21; the cover 22 serves as an observation area 221 and a separation operation area 222,
  • the base 21 serves as the culture area 211; the observation area 21 is used for observing the separation and culture process;
  • the separation operation area 222 is provided with an inner slide rail 2221 according to the needs of the separation operation, and a sampling probe is installed on the inner slide rail 2221.
  • the sampling probe device 2222 can slide on the inner slide rail 2221, and is used to realize the marking operation and sampling operation of microorganisms;
  • the culture area 211 is provided with a culture medium for carrying out the marking of the microbial colonies after marking.
  • Culture the environmental parameter detection unit 3 is arranged inside the separation operation incubator 2, used to detect the temperature and pressure changes in the separation operation incubator 2 in real time, and transmit the data to the central control system 1;
  • the temperature control unit 5 is connected to the separation operation incubator 2 respectively, and is used to ensure that the pressure and temperature in the separation operation incubator 2 are consistent with the growth and culture environment of the microorganisms;
  • the liquid injection unit 6 is used to inject the liquid into the separation operation incubator 2.
  • the enriched microbial liquid is injected into the device 2 for dipping and marking by the sampling probe device 2222; the sampling unit 7 is used for pressure-maintaining sampling of microorganisms; the control end of the sampling probe device 2222 and the control end of the pressure control unit 4 , the control end of the temperature control unit 5, the control end of the liquid injection unit 6 and the control end of the sampling unit 7 are all electrically connected to the central control system 1; the data output end of the observation area 221 is electrically connected to the central control system 1 connect.
  • the base 21 and the cover 22 are sealed and connected through buckles.
  • the cover is similar to an "L" shape and the base is similar to a cylinder.
  • the inner slide rail 2221 is a three-dimensional slide rail.
  • the sampling probe device 2222 can slide up, down, left, and right on the inner slide rail 2221, mainly to ensure that the sampling probe device 2222 can contact the inside of the base 21.
  • the surface of the culture medium can also be located above the culture medium in the base 21 to facilitate the sampling probe device 2222 to perform various operations on the culture medium.
  • the control of the sampling probe device 2222 is mainly controlled and displayed on the central control system 1 through the pressure holding chip of the sampling probe device 2222.
  • the central control system 1 includes a server, a computer, etc., and is used to realize changes in various environmental data information during the separation and cultivation of enriched marine microorganisms in a high-pressure environment, as well as real-time collection, processing, storage and imaging. Output and other functions.
  • the observation area 221 includes an outer slide rail 2211 provided on the outer surface of the cover 22, a connection bracket 2212 slidably connected to the outer slide rail 2211, an observation device 2213 fixed on the end of the connection bracket 2212, and an observation device 2213 provided on the end of the connection bracket 2212.
  • the viewing window 2214 on the surface of the cover 22; the data output end of the observation device 2213 is electrically connected to the central control system 1.
  • the viewing window 2214 is a circular viewing window, which is arranged in the center of the cover 22 to facilitate the observation device 2213 to observe the inside of the separation operation culture vessel 2.
  • the outer slide rail 2211 is a horizontal slide rail, and a telescopic "L"-shaped connecting bracket 2212 is installed on the slide rail.
  • the end of the connecting bracket 2212 is provided with an observation device 2213, which can facilitate the observation and identification of microorganisms. If the optical observation module of a high-resolution optical microscope is used as the observation device 2213 to observe and identify microorganisms, it can be preliminarily determined whether the single colony is the target microorganism required by the researcher or whether the colony is a single colony. It can also be used to photograph under high pressure. The formation process of single colonies (such as color, shape, size, etc.) facilitates the subsequent selection of individual colonies.
  • the environmental parameter detection unit 3 includes a temperature sensor 31 and a pressure sensor 32; the temperature sensor 31 and the pressure sensor 32 are both arranged inside the separation operation incubator 2 for real-time detection of the temperature in the separation operation incubator 2. temperature and pressure changes, and transmit the data to the central control system 1.
  • the temperature sensor 31 and the pressure sensor 32 are both arranged on the inner edge of the cover 22, and are mainly used to monitor the temperature and pressure changes in the separation operation incubator 2, so as to facilitate the entire culture environment in the actual application process. Macro-control.
  • the pressure control unit 4 includes an air compressor 41, a booster pump 42, a gas storage tank 43, a pressure regulating valve 44, an air intake valve 45 and an air supply pipeline 46; wherein: the air compressor 41, booster pump The pressure pump 42, the gas storage tank 43, and the pressure regulating valve 44 are connected in sequence through the air supply pipe 46, and finally connected to the separation operation incubator 2 through the air inlet valve 45; the control end of the air compressor 41, the booster The control end of the pump 42 , the control end of the pressure regulating valve 44 , and the control end of the air intake valve 45 are all electrically connected to the central control system 1 .
  • the pressure control unit 4 is mainly used to inject gas into the separation operation incubator 1 for pressurization.
  • the temperature control unit 5 adopts a water bath jacket device, which is wrapped around the outer wall of the separation operation incubator 2 , and the control end is electrically connected to the central control system 1 .
  • the liquid injection unit 6 includes a microorganism enrichment kettle 61, a liquid delivery pipe 62 and a microinjection pump 63; wherein: the microorganism enrichment kettle 61 is used for enrichment of microbial liquid, and its output end passes through
  • the liquid pipeline 62 is connected to the separation operation incubator 2; the micro-injection pump 63 is provided on the liquid supply pipeline 62, and the control end is electrically connected to the central control system 1.
  • the liquid injection unit 6 also includes a groove 64 provided on the inner bottom surface of the cover 22 of the separation operation incubator 2; the liquid outlet of the liquid supply pipe 62 inside the separation operation incubator 2 is provided in the groove. Within 64.
  • the groove 64 is mainly used to hold the microbial liquid injected through the micro-injection pump 63 so that the microbial liquid cannot flow freely after being injected into the separation operation incubator 2 to ensure the subsequent separation and marking operation. Can achieve.
  • the liquid injection unit 6 further includes a vent valve 65 provided on the liquid supply pipe 62 , and the control end of the vent valve 65 is electrically connected to the central control system 1 .
  • the excess bacterial liquid can be slowly drained through the vent valve 65 so that the excess bacterial liquid does not enter the base 21 within the culture medium.
  • the sampling unit 7 includes a reaction kettle 71, a sampling pipe 72 and a sampling valve 73; wherein: the reaction kettle 71 is connected to the separation operation incubator 2 through a sampling pipe 72; the sampling valve 73 is provided at The control end of the sampling pipe 72 is electrically connected to the central control system 1 .
  • the inner diameters of the sampling pipe 72 and the sampling valve 73 are both larger than the diameter of the sampling probe device 2222, mainly so that the sampling probe device 2222 can extend into the sampling pipe 72 and transfer the dipped single bacterial colony to the In the reaction kettle 71 filled with liquid culture medium.
  • the sampling valve 73 can be closed and replaced with the next reaction kettle 71. At this time, the reaction kettle 71 is filled with 75% alcohol until the disinfection of the sampling probe device 2222 is completed.
  • the reactor 71 is replaced with the next reactor 71 containing liquid culture medium, and by analogy, the isolation and cultivation of a single colony in a pressure-maintaining state can be completed.
  • the pressure difference that occurs every time the reactor 71 is replaced can be controlled 4 by the pressure control unit and the corresponding pressure can be supplemented.
  • this embodiment establishes a single colony separation device and realizes the isolation, selection and cultivation of single colonies while maintaining pressure. Finally, the target bacteria can be obtained, effectively improving the sustainability of marine microorganisms. Cultivation, providing basic solutions for the isolation and cultivation of marine microorganisms. The above solution can realize the pressure-maintaining separation of enriched marine microorganisms in an in-situ high-pressure environment to meet the needs of subsequent culture and functional identification.
  • this embodiment provides a single colony separation method in a deep sea in situ environment, which is implemented by a single colony separation device in a deep sea in situ environment, specifically including: Following steps:
  • S1 Clean and sterilize the single colony isolation device in the deep sea in-situ environment and load it with culture medium;
  • S2 Inject gas into the separation operation incubator 2 through the pressure control unit 4, so that the pressure value in the separation operation incubator 2 is consistent with the pressure value of the microorganism enrichment environment;
  • S5 The isolated microorganisms will grow according to the marked trajectory and be observed in real time through the observation area 221;
  • S7 Control the sampling probe device 2222 to pick out a single colony on the culture medium, and send the colony into the sampling unit 7 containing the liquid culture medium to realize pressure-maintaining transfer culture of a single colony.
  • This embodiment mainly constructs the same high-pressure environment as the microorganisms living in the marine environment in the separation operation incubator 2 .
  • the separation operation incubator 2 determines the temperature value in the separation operation incubator 2 according to the temperature value in the microorganism enrichment kettle 61, and obtain the same temperature as in the microorganism enrichment kettle 61 by placing the separation operation incubator 2 in the water bath environment of the water bath jacket device. temperature. Then, adjust the observation device 2213 so that it can clearly observe the conditions on the culture medium inside the base 21. Then, the bacterial liquid containing microorganisms is injected from the microorganism enrichment kettle 61 into the groove 64 through the microinjection pump 63, so that the bacterial liquid forms droplets and is located in the groove 64.
  • the sampling probe device 2222 By controlling the sampling probe device 2222 to dip into the bacterial liquid in the groove 64, and then positioning the bottom of the sampling probe device 2222 on the surface of the culture medium, the sampling probe device 2222 draws lines on the surface of the culture medium according to a certain trajectory, and the finally separated microorganisms will Grow according to the streaked trajectory to achieve the isolation of individual colonies. Observe through the visual window 2214 on the cover 22 during the entire process. When a single colony is formed on the culture medium, the streaking and separation work can be started.
  • the probe device 2222 is used to successfully pick a single colony on the culture medium, and then the sampling probe device 2222 is passed through the sampling pipe 72 into the reaction kettle 71 containing the liquid culture medium, and finally the pressure-maintaining transfer culture of a single colony is realized. The subsequent isolation of single colonies can be deduced in the same way.
  • the pressure and temperature values in the separation operation incubator 2 are kept consistent with the pressure and temperature environment in the high-pressure enrichment system where the microorganisms are initially located, so that the microorganisms can be separated under in-situ high pressure conditions.
  • This embodiment aims at the current problem of difficult separation of marine microorganisms, and proposes a single colony separation device and separation method for high-pressure environments.
  • the existing normal-pressure separation culture it can enrich, separate and culture microorganisms in the deep-sea in-situ high-pressure environment, and solve the problems of deep-sea in-situ pressure-loving bacteria being unable to survive or expressing differences when cultured in the normal-pressure environment.
  • the existing high-pressure enrichment isolation culture can select a single colony while maintaining pressure and successfully culture it.
  • this embodiment proposes a method of isolating and cultivating enriched deep-sea methanophilic bacteria in an in-situ high-pressure environment to meet the needs of subsequent library construction and functional identification.
  • the core of this embodiment is the isolation operation technology of a single colony, and other parts mainly include the pressure control unit 4, the isolation operation incubator 2 and the central control system 1.
  • the separation operation incubator 2 mainly includes a cover 22 and a base 21 .
  • the cover 22 and the base 21 are connected through buckles.
  • the cover 22 is similar to an "L" shape, and the base 21 is similar to a cylinder.
  • the upper edge of the cover 22 is provided with a temperature sensor 31 and a pressure sensor 32, which are mainly used to monitor changes in temperature and pressure in the separation operation incubator 2.
  • a circular viewing window 2215 is provided in the middle of the upper part of the cover 22, which is mainly used for the observation device 2213.
  • the cover 22 is provided with a three-dimensional inner slide rail 2221, mainly to ensure that the sampling probe device 2222 can contact the surface of the culture medium in the base 21, and at the same time, it can be located above the culture medium in the base 21, making it convenient for the sampling probe device 2222 to be placed on the culture medium.
  • Various operations are performed on the surface, which operations are mainly controlled on the central control system 1 through the pressure maintaining chip on the sampling probe device 2222 and displayed on the computer.
  • the outer wall on the left side of the cover 22 is provided with a horizontal outer slide rail 2211.
  • a telescopic "L"-shaped connecting bracket 2212 is installed on the outer sliding rail 2211.
  • the other end of the "L"-shaped connecting bracket 2212 is provided with an observation device 2213, such as Using the optical observation module of a high-resolution optical microscope to observe and identify microorganisms can initially determine whether the single colony is the target microorganism required by the researcher or whether the colony is a single colony. It is also used to photograph the formation process of a single colony under high pressure (such as color, shape, size, etc.) to facilitate the subsequent selection of individual colonies.
  • a groove 64 is provided in the lower left portion of the cover 22 , and the groove 64 is mainly used to hold the microbial liquid injected through the microinjection pump 63 . If there is excess bacterial liquid left after the sampling probe device 2222 has drawn lines on the culture medium, the excess bacterial liquid can be slowly drained through the vent valve 65 so that the excess bacterial liquid does not enter the culture medium of the base 21 .
  • the bottom of the right rear of the cover 22 is provided with a sampling pipe 72.
  • the internal diameters of the sampling pipe 72 and the sampling valve 73 are both larger than the diameter of the sampling probe device 2222, mainly so that the sampling probe device 2222 can extend into the sampling pipe 72, and the sampling probe
  • the single colony dipped in the device 2222 is transferred to the reaction kettle 71 filled with liquid culture medium while maintaining pressure.
  • the sampling pipe 72 is provided with a sampling valve 73. After each single colony is transferred, the sampling valve 73 can be closed and replaced with the next reaction kettle 71.
  • reaction kettle 71 is filled with 75% of Alcohol, after the sterilization of the sampling probe device 2222 is completed, the next reaction kettle 71 containing the liquid culture medium can be replaced, and by analogy, the single colony isolation and culture in the re-pressure state can be completed.
  • the pressure difference that occurs every time the reactor 71 is replaced is controlled by the pressure control unit 4 and the corresponding pressure is supplemented.
  • the pressure control unit 4 involved in this embodiment is mainly used to inject gas into the culture kettle to pressurize it, and includes a gas storage tank 43, a pressure regulating valve 44, an air compressor 41 and a boosting pump 42.
  • the central control system 1 involved in this embodiment includes a server, a computer, etc., and realizes the functions of various environmental data information changes during the separation and cultivation of enriched marine microorganisms in a high-pressure environment, as well as real-time collection, processing, storage, and image output.
  • the device and technology for high-pressure single-colony separation and cultivation of marine microorganisms involved in this embodiment mainly construct a high-pressure environment in the separation operation incubator 2 that is the same as the life of methanophilic bacteria in the marine environment.
  • the temperature value in the separation operation incubator 2 is determined based on the temperature value in the microorganism enrichment kettle 61, and the temperature consistent with the temperature in the microorganism enrichment kettle 61 is obtained by placing the separation operation incubator 2 in a water bath environment. Then, adjust the observation device 2213 so that it can clearly observe the conditions on the culture medium inside the base 21. Then, the bacterial liquid containing microorganisms is injected from the microorganism enrichment kettle 61 into the groove 64 through the microinjection pump 63, so that the bacterial liquid forms droplets and is located in the groove 64.
  • the sampling probe device 2222 By controlling the sampling probe device 2222 to dip into the bacterial liquid in the groove 64, and then positioning the bottom of the sampling probe device 2222 on the surface of the culture medium, the sampling probe device 2222 draws lines on the surface of the culture medium according to a certain trajectory, and the finally separated microorganisms will Grow according to the streaked trajectory to achieve the isolation of individual colonies. Observe through the visual window 2214 of the cover 22, and when a single bacterial colony is formed on the culture medium, the streaking and separation work can be started. Before isolating a single colony, first connect the end of the sampling pipe 72 to the reaction kettle 71 and open the sampling valve 73. The reaction kettle 71 is filled with 75% alcohol.
  • the pressure of the entire device is maintained constant through the pressure control unit 4, and finally through the central control system 1
  • the sampling probe device 2222 is controlled so that the sampling probe device 2222 passes through the sampling pipe 72 and enters the reaction kettle 71 to realize the sterilization process of the sampling probe device 2222.
  • close the sampling valve 73 replace the reactor 71 containing 75% alcohol with the reactor 71 containing liquid culture medium, maintain the pressure of the entire device constant through the pressure control unit 4, and finally control the sampling through the central control system 1
  • the probe device 2222 is used to successfully pick a single colony on the culture medium, and then the sampling probe device 2222 is passed through the sampling pipe 72 into the reaction kettle 71 containing the liquid culture medium, and finally the pressure-maintaining transfer culture of a single colony is realized.
  • the subsequent isolation of single colonies can be deduced in the same way.
  • the pressure and temperature values in the culture kettle are kept consistent with the pressure and temperature environment in the microorganism enrichment kettle 61 where the microorganisms are initially located, so that the microorganisms can be separated under in-situ high pressure.
  • the device and technology for separation and cultivation of marine microorganisms in a high-pressure environment proposed in this embodiment can realize the formation of a single microbial colony and the cultivation of a single colony in a marine high-pressure environment, meeting the needs for the growth and cultivation of special bacterial species in the deep sea.
  • it can effectively solve the problem of low survival rate of marine pressure-resistant bacteria and pressure-loving bacteria in normal pressure environment, and the inability of deep-sea indigenous characteristics to be effectively expressed in normal pressure environment. and other problems, to solve the current problem of low culture of marine microorganisms and difficulty in cultivating pure bacteria.
  • This embodiment can realize the artificial separation, selection and culture process of a single bacterial colony in a high-pressure environment. Compared with conventional microbial isolation and culture technology, it effectively improves the efficiency of microbial culture and purification.

Abstract

一种深海原位环境的单菌落分离装置，包括分离操作培养器（2）、注液单元（6）、取样单元（7）和取样探头装置（2222）；还提供一种深海原位环境的单菌落分离方法，在保持分离操作培养器（2）内部压力、温度与深海微生物培养的环境一致的情况下，通过注液单元（6）向分离操作培养器（2）注入微生物菌液，由取样探头装置（2222）进行蘸取和划线操作再进行分离培养，最后通过取样单元（7）将单个菌落挑选出来，实现单菌落的分离。通过建立一种单菌落的分离装置，并在保压的情况下，实现单菌落的分离与挑选并进行培养，最终可以获得目标菌，有效提高海洋微生物的可培养性，为海洋微生物的分离和培养提供基础方案。

Description

一种深海原位环境的单菌落分离装置及分离方法 技术领域
本发明涉及海洋微生物技术领域，特别是涉及一种深海原位环境的单菌落分离装置及分离方法。
背景技术
海洋环境不同于陆地，是一个独特的生态环境，有高盐、高渗、高压、寡营养等特点，某些海域还存在高酸、高碱、缺氧、低温、高温、高辐射等特殊条件。与陆地的一些生态环境相比微生物的活动并不剧烈，但从海水表面到海底沉积物都存在非常丰富的微生物资源。海洋微生物在基因、生理代谢等方面具有极高的创新性和多样性，在促进海洋能量传递和物质循环及维护海洋乃至全球生态系统稳定等方面具有不可取代的作用。
目前，海洋中微生物的分离主要是释压后，在常压下进行分离单个菌落，导致海洋环境中原本存在的特殊菌种尚未被分离，如嗜压菌等，且海洋中已被分离出的微生物数量较少。此外，通过长周期的富集培养已经可以得到丰度较高的特殊目标菌，实现了在保压条件下的单菌落划线分离，但如何实现单菌落的挑选并成功培养仍未解决，这进一步限制了我们对海洋微生物的了解以及资源的开发与利用。
对于此，现有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，但还是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，提供一种深海原位环境的单菌落分离装置及分离方法，在高压环境下，实现单个菌落的分离与培养，克服深海微生物不易分离且不易培养的难点。
为解决上述技术问题，本发明的技术方案如下：
一种深海原位环境的单菌落分离装置，包括中央控制系统、分离操作培养器、环境参数检测单元、压力控制单元、温度控制单元、注液单元和取样单元；其中：所述分离操作培养器包括底座和与底座固定连接的盖体；所述盖体作为观察区和分离操作区，所述底座作为培养区；所述观察区用于对分离培养过程的观察；所述分离操作区中根据分离操作需要设置有内滑轨，内滑轨上安装有取样探头装置，取样探头装置可在内滑轨上进行滑动，用于实现微生物的划线操作及取样操作；所述培养区中设置有培养基，用于对划线后的微生物菌落进行培养；所述环境参数检测单元设置在分离操作培养器内部，用于实时检测分离操作培养器内的温度、压力变化情况，并将数据传输至中央控制系统；所述压力控制单元、温度控制单元分别与所述分离操作培养器连接，用于保证分离操作培养器内的压力、温度与微生物的生长培养环境一致；所述注液单元用于向分离操作培养器内注入富集微生物菌液，供取样探头装置蘸取和划线；所述取样单元用于对微生物进行保压取样；所述取样探头装置控制端、压力控制单元控制端、温度控制单元控制端、注液单元控制端和取样单元控制端均与所述中央控制系统电性连接；所述观察区的数据输出端与所述中央控制系统电性连接。
上述方案中，底座和盖体通过卡扣进行密封连接，盖体类似“L”型，底座类似圆柱体。所述内滑轨为三维式的滑轨，取样探头装置可以在内滑轨上做上、下、左、右各个方向的滑动，主要为了确保取样探头装置可以接触到底座内培养基表面，同时又可以位于底座内培养基的上方，方便取样探头装置在培养基上进行各种操作。对于取样探头装置的控制主要是通过取样探头装置的保压芯片在中央控制系统上控制并进行显示。
上述方案中，所述中央控制系统包括服务器、计算机等，用于实现富集的海洋微生物在高压环境分离和培养过程中各项环境数据信息变化、以及实时采集、处理、存储和图像输出等功能。
上述方案中，通过建立一种单菌落的分离装置，并在保压的情况下，实现单菌落的分离与挑选并进行培养，最终可以获得目标菌，有效提高海洋微生物的可培养性，为海洋微生物的分离和培养提供基础方案。上述方案可以实现在原位的高压环境下对富集海洋微生物进行保压分离，满足后续的培养及功能鉴定等工作。
其中，所述观察区包括设置在盖体外侧表面的外滑轨、与外滑轨可滑动连接的连接支架、固定在连接支架端部的观察装置和设置在盖体表面的可视窗；所述观察装置的数据输出端与所述中央控制系统电性连接。
上述方案中，所述可视窗为圆形视窗，设置在盖体中央，方便观察装置对于分离操作培养器内部进行观察。所述外滑轨为水平滑轨，滑轨上安装有可伸缩的“L”型连接支架，连接支架的端部设置有观察装置，可方便对微生物进行观察和识别。如利用高分辨率光学显微镜的光学观察模块作为观察装置对微生物进行观察和识别，可初步判定该单菌落是否为研究人员需要的目标微生物或该菌落是否为单个菌落，同时用于拍摄高压下单菌落形成过程(如颜色、形态、大小等)，方便后续对单个菌落进行挑选。
其中，所述环境参数检测单元包括温度传感器和压力传感器；所述温度传感器、压力传感器均设置在分离操作培养器内部，用于实时检测分离操作培养器内的温度、压力变化情况，并将数据传输至中央控制系统。
上述方案中，温度传感器和压力传感器均设置在盖体内部边缘，主要用于监测分离操作培养器内的温度和压力变化情况，便于在实际应用过程中对于整个培养环境进行宏观调控。
其中，所述压力控制单元包括空气压缩机、增压泵、储气罐、调压阀、进气阀和送气管道；其中：所述空气压缩机、增压泵、储气罐、调压阀通过送气管道依次连接，最后通过所述进气阀与所述分离操作培养器连接；所述空气压缩机的控制端、增压泵的控制端、调压阀的控制端、进气阀的控制端均与所述中央控制系统电性连接。
上述方案中，所述压力控制单元主要用于向分离操作培养器中注入气体增压。
其中，所述温度控制单元采用水浴夹套装置，其包裹在所述分离操作培养器外壁上，控制端与所述中央控制系统电性连接。
其中，所述注液单元包括微生物富集釜、送液管道和微注泵；其中：所述微生物富集釜用于微生物菌液的富集，其输出端通过送液管道与分离操作培养器连接；所述微注泵设置在所述送液管道上，控制端与所述中央控制系统电性连接。
其中，所述注液单元还包括设置在分离操作培养器盖体内部底面的凹槽；所述送液管道在分离操作培养器内部的出液口设置在凹槽内。
上述方案中，所述凹槽主要用于盛放通过微注泵注入的微生物菌液，使微生物菌液在注入分离操作培养器后不能随意流动，保证后续分离划线操作的可实现。
其中，所述注液单元还包括设置在送液管道上的放空阀，所述放空阀的控制端与所述中央控制系统电性连接。
上述方案中，若取样探头装置在培养基上划线完成后，还剩有多余菌液，则可通过放空阀将多余菌液缓慢放掉，使其多余菌液不进入底座的培养基内。
其中，所述取样单元包括反应釜、取样管道和取样阀门；其中：所述反应釜通过取样管道与所述分离操作培养器连接；所述取样阀门设置在所述取样管道上，其控制端与所述中央控制系统电性连接。
上述方案中，所述取样管道、取样阀门的内径均大于取样探头装置的直径，主要为了取样探头装置可以伸入取样管道内，将蘸取的单个菌落保压转移到装有液体培养基的反应釜中。为了方便转移且不相互交叉污染，每转移完一个单菌落，即可关闭取样阀门，换上下一个反应釜，此时的反应釜内装有75％的酒精，待取样探头装置消毒完成后，再换上下一个装有液体培养基的反应釜，以此类推就可完成在保压状态下的单菌落分离培养。每次更换反应釜所出现的压力差，均可通过压力控制单元控制并补充相应的压力。
本方案还提供一种深海原位环境的单菌落分离方法，应用如上文所述的一种深海原位环境的单菌落分离装置实现，具体包括以下步骤：
S1：对深海原位环境的单菌落分离装置进行清洗灭菌，并装入培养基；
S2：通过压力控制单元往分离操作培养器内注入气体，使得分离操作培养器中的压力值与微生物富集环境的压力值一致；
S3：开启温度控制单元，使得分离操作培养器获得与微生物富集环境一致的温度；
S4：由注液单元向分离操作培养器内注入微生物菌液，控制取样探头装置在内滑轨上滑动，先蘸取微生物菌液，然后使取样探头装置位于培养基表面，令其在培养基的表面按照一定轨迹划线分离；
S5：分离的微生物将按照划线轨迹生长，通过观察区进行实时观察；
S6：当培养基上有单个菌落形成时，开启取样单元，令其与分离操作培养器的温度、压力保持一致；
S7：控制取样探头装置挑取培养基上的单个菌落，并将菌落送入装有液体培养基的取样单元内，实现单个菌落的保压转移培养。
上述方案主要是在分离操作培养器内构建与微生物在海洋环境中生活一样的高压环境。首先清洗分离操作培养器，打开盖体和底座，分别用75％酒精擦拭，待酒精完全挥发后，再置于紫外下灭菌15min。待灭菌结束，在底座内加入事先灭菌完成的固体培养基，最后合上盖体。然后根据微生物富集釜内的压力值确定分离操作培养器内的压力值，通过压力控制单元往分离操作培养器内注入气体，使分离操作培养器内的压力值与微生物富集釜一致。
然后，根据微生物富集釜内的温度值确定分离操作培养器内的温度值，通过将分离操作培养器置于水浴夹套装置的水浴环境内获得与微生物富集釜内一致的温度。然后，调试好观察装置，使其能清楚的观测到底座内部培养基上的情况。然后将含微生物的菌液从微生物富集釜通过微注泵注入凹槽内，使得菌液形成液滴位于凹槽内。通过控制取样探头装置蘸取凹槽内的菌液，然后使取样探头装置的底部位于培养基表面，使得取样探头装置在培养基的表面按照一定轨迹划线，最后分离的微生物将按照划线轨迹生长，实现单个菌落的分离。在整个过程中通过盖体上的可视窗进行观察，当培养基上有单个菌落形成时，即可开始进行划线分离工作。
在分离单个菌落之前，首先将取样管道的末端连接反应釜并打开取样阀门，反应釜内装有75％酒精，通过压力控制单元维持整个装置的压力保持恒定，最后通过中央控制系统控制取样探头装置，使得取样探头装置穿过取样管道进入反应釜，实现取样探头装置的灭菌过程。再关闭取样阀门，将装有75％酒精的反应釜更换为装有液体培养基的反应釜，并通过压力控制单元维持整个装置的压力保持恒定，最后通过中央控制系统控制取样探头装置，并成功挑取培养基上的单个菌落，然后使取样探头装置穿过取样管道进入装有液体培养基的反应釜，最终实现单个菌落的保压转移培养。后续单菌落的分离以此类推即可。在整个挑选和培养过程中，保持分离操作培养器内的压力、温度值与微生物最初所在的高压富集系统内的压力、温度环境一致，使得微生物在原位高压情况内实现分离。
上述方案针对目前海洋微生物难分离的难题，提出了针对高压环境进行单菌落分离装置及分离方法。相对于现有的常压分离培养，能够满足微生物在深海原位高压环境进行富集、分离和培养，解决深海原位嗜压菌在常压环境培养不能存 活或者表达差异等难题。对于现有的高压富集分离培养，能够满足在保压的情况下实现单个菌落的挑选，并成功培养。
与现有技术相比，本发明技术方案的有益效果是：
本发明提出了一种深海原位环境的单菌落分离装置及分离方法，通过建立一种单菌落的分离装置，并在保压的情况下，实现单菌落的分离与挑选并进行培养，最终可以获得目标菌，有效提高海洋微生物的可培养性，为海洋微生物的分离和培养提供基础装置和方法。
附图说明
图1为本发明所述装置的结构示意图；
图2为本发明所述中央控制系统电路模块连接示意图；
图3为本发明所述方法流程示意图；
其中：1、中央控制系统；2、分离操作培养器；21、底座；211、培养区；22、盖体；221、观察区；2211、外滑轨；2212、连接支架；2213、观察装置；2214、可视窗；222、分离操作区；2221、内滑轨；2222、取样探头装置；3、环境参数检测单元；31、温度传感器；32、压力传感器；4、压力控制单元；41、空气压缩机；42、增压泵；43、储气罐；44、调压阀；45、进气阀；46、送气管道；5、温度控制单元；6、注液单元；61、微生物富集釜；62、送液管道；63、微注泵；64、凹槽；65、放空阀；7、取样单元；71、反应釜；72、取样管道；73、取样阀门。
具体实施方式
附图仅用于示例性说明，不能理解为对本专利的限制；
本实施例为完整的使用示例，内容较丰富
为了更好说明本实施例，附图某些部件会有省略、放大或缩小，并不代表实际产品的尺寸；
对于本领域技术人员来说，附图中某些公知结构及其说明可能省略是可以理解的。
下面结合附图和实施例对本发明的技术方案做进一步的说明。
实施例1
如图1、图2所示，本实施例提供一种深海原位环境的单菌落分离装置，包括中央控制系统1、分离操作培养器2、环境参数检测单元3、压力控制单元4、 温度控制单元5、注液单元6和取样单元7；其中：所述分离操作培养器2包括底座21和与底座21固定连接的盖体22；所述盖体22作为观察区221和分离操作区222，所述底座21作为培养区211；所述观察区21用于对分离培养过程的观察；所述分离操作区222中根据分离操作需要设置有内滑轨2221，内滑轨2221上安装有取样探头装置2222，取样探头装置2222可在内滑轨2221上进行滑动，用于实现微生物的划线操作及取样操作；所述培养区211中设置有培养基，用于对划线后的微生物菌落进行培养；所述环境参数检测单元3设置在分离操作培养器2内部，用于实时检测分离操作培养器2内的温度、压力变化情况，并将数据传输至中央控制系统1；所述压力控制单元4、温度控制单元5分别与所述分离操作培养器2连接，用于保证分离操作培养器2内的压力、温度与微生物的生长培养环境一致；所述注液单元6用于向分离操作培养器2内注入富集微生物菌液，供取样探头装置2222蘸取和划线；所述取样单元7用于对微生物进行保压取样；所述取样探头装置2222控制端、压力控制单元4控制端、温度控制单元5控制端、注液单元6控制端和取样单元7控制端均与所述中央控制系统1电性连接；所述观察区221的数据输出端与所述中央控制系统1电性连接。
在具体实施过程中，底座21和盖体22通过卡扣进行密封连接，盖体类似“L”型，底座类似圆柱体。所述内滑轨2221为三维式的滑轨，取样探头装置2222可以在内滑轨2221上做上、下、左、右各个方向的滑动，主要为了确保取样探头装置2222可以接触到底座21内培养基表面，同时又可以位于底座21内培养基的上方，方便取样探头装置2222在培养基上进行各种操作。对于取样探头装置2222的控制主要是通过取样探头装置2222的保压芯片在中央控制系统1上控制并进行显示。
在具体实施过程中，所述中央控制系统1包括服务器、计算机等，用于实现富集的海洋微生物在高压环境分离和培养过程中各项环境数据信息变化、以及实时采集、处理、存储和图像输出等功能。
更具体的，所述观察区221包括设置在盖体22外侧表面的外滑轨2211、与外滑轨2211可滑动连接的连接支架2212、固定在连接支架2212端部的观察装置2213和设置在盖体22表面的可视窗2214；所述观察装置2213的数据输出端与所述中央控制系统1电性连接。
在具体实施过程中，所述可视窗2214为圆形视窗，设置在盖体22中央，方 便观察装置2213对于分离操作培养器2内部进行观察。所述外滑轨2211为水平滑轨，滑轨上安装有可伸缩的“L”型连接支架2212，连接支架2212的端部设置有观察装置2213，可方便对微生物进行观察和识别。如利用高分辨率光学显微镜的光学观察模块作为观察装置2213对微生物进行观察和识别，可初步判定该单菌落是否为研究人员需要的目标微生物或该菌落是否为单个菌落，同时用于拍摄高压下单菌落形成过程(如颜色、形态、大小等)，方便后续对单个菌落进行挑选。
更具体的，所述环境参数检测单元3包括温度传感器31和压力传感器32；所述温度传感器31、压力传感器32均设置在分离操作培养器2内部，用于实时检测分离操作培养器2内的温度、压力变化情况，并将数据传输至中央控制系统1。
在具体实施过程中，温度传感器31和压力传感器32均设置在盖体22内部边缘，主要用于监测分离操作培养器2内的温度和压力变化情况，便于在实际应用过程中对于整个培养环境进行宏观调控。
更具体的，所述压力控制单元4包括空气压缩机41、增压泵42、储气罐43、调压阀44、进气阀45和送气管道46；其中：所述空气压缩机41、增压泵42、储气罐43、调压阀44通过送气管道46依次连接，最后通过所述进气阀45与所述分离操作培养器2连接；所述空气压缩机41的控制端、增压泵42的控制端、调压阀44的控制端、进气阀45的控制端均与所述中央控制系统1电性连接。
在具体实施过程中，所述压力控制单元4主要用于向分离操作培养器1中注入气体增压。
更具体的，所述温度控制单元5采用水浴夹套装置，其包裹在所述分离操作培养器2外壁上，控制端与所述中央控制系统1电性连接。
更具体的，所述注液单元6包括微生物富集釜61、送液管道62和微注泵63；其中：所述微生物富集釜61用于微生物菌液的富集，其输出端通过送液管道62与分离操作培养器2连接；所述微注泵63设置在所述送液管道62上，控制端与所述中央控制系统1电性连接。
更具体的，所述注液单元6还包括设置在分离操作培养器2盖体22内部底面的凹槽64；所述送液管道62在分离操作培养器2内部的出液口设置在凹槽64内。
在具体实施过程中，所述凹槽64主要用于盛放通过微注泵63注入的微生物菌液，使微生物菌液在注入分离操作培养器2后不能随意流动，保证后续分离划线操作的可实现。
更具体的，所述注液单元6还包括设置在送液管道62上的放空阀65，所述放空阀65的控制端与所述中央控制系统1电性连接。
在具体实施过程中，若取样探头装置2222在培养基上划线完成后，还剩有多余菌液，则可通过放空阀65将多余菌液缓慢放掉，使其多余菌液不进入底座21的培养基内。
更具体的，所述取样单元7包括反应釜71、取样管道72和取样阀门73；其中：所述反应釜71通过取样管道72与所述分离操作培养器2连接；所述取样阀门73设置在所述取样管道72上，其控制端与所述中央控制系统1电性连接。
在具体实施过程中，所述取样管道72、取样阀门73的内径均大于取样探头装置2222的直径，主要为了取样探头装置2222可以伸入取样管道72内，将蘸取的单个菌落保压转移到装有液体培养基的反应釜71中。为了方便转移且不相互交叉污染，每转移完一个单菌落，即可关闭取样阀门73，换上下一个反应釜71，此时的反应釜71内装有75％的酒精，待取样探头装置2222消毒完成后，再换上下一个装有液体培养基的反应釜71，以此类推就可完成在保压状态下的单菌落分离培养。每次更换反应釜71所出现的压力差，均可通过压力控制单元控制4并补充相应的压力。
在具体实施过程中，本实施例通过建立一种单菌落的分离装置，并在保压的情况下，实现单菌落的分离与挑选并进行培养，最终可以获得目标菌，有效提高海洋微生物的可培养性，为海洋微生物的分离和培养提供基础方案。上述方案可以实现在原位的高压环境下对富集海洋微生物进行保压分离，满足后续的培养及功能鉴定等工作。
实施例2
更具体的，在实施例1的基础上，如图3所示，本实施例提供一种深海原位环境的单菌落分离方法，应用一种深海原位环境的单菌落分离装置实现，具体包括以下步骤：
S1：对深海原位环境的单菌落分离装置进行清洗灭菌，并装入培养基；
S2：通过压力控制单元4往分离操作培养器2内注入气体，使得分离操作培 养器2中的压力值与微生物富集环境的压力值一致；
S3：开启温度控制单元5，使得分离操作培养器2获得与微生物富集环境一致的温度；
S4：由注液单元6向分离操作培养器2内注入微生物菌液，控制取样探头装置2222在内滑轨2221上滑动，先蘸取微生物菌液，然后使取样探头装置2222位于培养基表面，令其在培养基的表面按照一定轨迹划线分离；
S5：分离的微生物将按照划线轨迹生长，通过观察区221进行实时观察；
S6：当培养基上有单个菌落形成时，开启取样单元7，令其与分离操作培养器2的温度、压力保持一致；
S7：控制取样探头装置2222挑取培养基上的单个菌落，并将菌落送入装有液体培养基的取样单元7内，实现单个菌落的保压转移培养。
本实施例主要是在分离操作培养器2内构建与微生物在海洋环境中生活一样的高压环境。首先清洗分离操作培养器2，打开盖体22和底座21，分别用75％酒精擦拭，待酒精完全挥发后，再置于紫外下灭菌15min。待灭菌结束，在底座21内加入事先灭菌完成的固体培养基，最后合上盖体22。然后根据微生物富集釜61内的压力值确定分离操作培养器1内的压力值，通过压力控制单元4往分离操作培养器2内注入气体，使分离操作培养器2内的压力值与微生物富集釜61一致。
然后，根据微生物富集釜61内的温度值确定分离操作培养器2内的温度值，通过将分离操作培养器2置于水浴夹套装置的水浴环境内获得与微生物富集釜61内一致的温度。然后，调试好观察装置2213，使其能清楚的观测到底座21内部培养基上的情况。然后将含微生物的菌液从微生物富集釜61通过微注泵63注入凹槽64内，使得菌液形成液滴位于凹槽64内。通过控制取样探头装置2222蘸取凹槽64内的菌液，然后使取样探头装置2222的底部位于培养基表面，使得取样探头装置2222在培养基的表面按照一定轨迹划线，最后分离的微生物将按照划线轨迹生长，实现单个菌落的分离。在整个过程中通过盖体22上的可视窗2214进行观察，当培养基上有单个菌落形成时，即可开始进行划线分离工作。
在分离单个菌落之前，首先将取样管道72的末端连接反应釜71并打开取样阀门73，反应釜71内装有75％酒精，通过压力控制单元4维持整个装置的压力保持恒定，最后通过中央控制系统1控制取样探头装置2222，使得取样探头装 置2222穿过取样管道72进入反应釜71，实现取样探头装置2222的灭菌过程。再关闭取样阀门73，将装有75％酒精的反应釜71更换为装有液体培养基的反应釜71，并通过压力控制单元4维持整个装置的压力保持恒定，最后通过中央控制系统1控制取样探头装置2222，并成功挑取培养基上的单个菌落，然后使取样探头装置2222穿过取样管道72进入装有液体培养基的反应釜71，最终实现单个菌落的保压转移培养。后续单菌落的分离以此类推即可。在整个挑选和培养过程中，保持分离操作培养器2内的压力、温度值与微生物最初所在的高压富集系统内的压力、温度环境一致，使得微生物在原位高压情况内实现分离。
本实施例针对目前海洋微生物难分离的难题，提出了针对高压环境进行单菌落分离装置及分离方法。相对于现有的常压分离培养，能够满足微生物在深海原位高压环境进行富集、分离和培养，解决深海原位嗜压菌在常压环境培养不能存活或者表达差异等难题。对于现有的高压富集分离培养，能够满足在保压的情况下实现单个菌落的挑选，并成功培养。
实施例3
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原位的高压环境下对富集的深海嗜甲烷菌进行分离与培养，满足后续的建库及功能鉴定等工作。本实施例的核心是单菌落的分离操作技术，其它部分主要包括压力控制单元4、分离操作培养器2和中央控制系统1。
分离操作培养器2主要包括盖体22和底座21。盖体22和底座21通过卡扣连接，盖体22类似“L”型，底座21类似圆柱形。盖体22的上部边缘设置有温度传感器31和压力传感器32，主要用于监测分离操作培养器2内的温度和压力的变化。盖体22上部的正中间设置有圆形可视窗2215，其主要用于观察装置2213的使用。盖体22内部设置有立体式的内滑轨2221，主要为了确保取样探头装置2222可接触底座21内培养基表面，同时又可以位于底座21内培养基的上方，方便取样探头装置2222在培养基表面进行各种操作，该操作主要通过取样探头装置2222上的保压芯片在中央控制系统1上控制并在在计算机上显示。盖体22左侧的外壁设置有水平外滑轨2211，外滑轨2211上安装有可伸缩的“L”型连接支架2212，“L”型连接支架2212的另一端设有观察装置2213，如利用高分辨光学显微镜的光学观察模块对微生物进行观察和识别，可初步判定该单菌落是否为研究人员需要的目标微生物或该菌落是否为单个菌落，同时用于拍摄高压下 单菌落的形成过程(如颜色、形态、大小等)，方便后续对单个菌落的挑选。
盖体22左下方的内部设有凹槽64，该凹槽64主要用于盛放通过微注泵63注入的微生物菌液。若取样探头装置2222在培养基上划线完成后，还剩有多余菌液，则可通过放空阀65将多余菌液缓慢放掉，使其多余菌液不进入底座21的培养基内。盖体22右后方的底部设有取样管道72，该取样管道72和取样阀门73内部的直径均大于取样探头装置2222的直径，主要为了取样探头装置2222可以伸入该取样管道72内，取样探头装置2222中蘸取的单个菌落保压转移到装有液体培养基的反应釜71中。为方便转移且不相互交叉污染，取样管道72上设置有取样阀门73，每转移完一个单菌落，即可关闭取样阀门73，换上下一个反应釜71，此时的反应釜71内装75％的酒精，待取样探头装置2222消毒完成后，再换上下一个装有液体培养基的反应釜71，以此类推就可完成再保压状态下的单菌落分离培养。每次更换反应釜71所出现的压力差，均通过压力控制单元4控制并补充相应的压力。
本实施例涉及的压力控制单元4主要是用于培养釜内注入气体增压，包括储气罐43、调压阀44、空气压缩机41和增压泵42组成。本实施例涉及的中央控制系统1包括服务器、计算机等，实现富集的海洋微生物在高压环境分离和培养过程中各项环境数据信息变化、以及实时采集、处理、存储和图像输出等功能。
本实施例涉及的海洋微生物高压单菌落分离培养装置与技术主要是在分离操作培养器2内构建与嗜甲烷菌在海洋环境中生活一样的高压环境。首先清洗分离操作培养器2，打开盖体22和底座21，分别用75％酒精擦拭，待酒精完全挥发后，再置于紫外下灭菌15min。待灭菌结束，在底座21内加入事先灭菌完成的固体培养基，最后合上盖体22。然后根据微生物富集釜61内的压力值确定培养皿内的压力值，通过压力控制单元4，往分离操作培养器2内注入甲烷气体，使分离操作培养器2内的压力值与微生物富集釜61一致。
然后，根据微生物富集釜61内的温度值确定分离操作培养器2内的温度值，通过将分离操作培养器2置于水浴环境内获得与微生物富集釜61内一致的温度。然后，调试好观察装置2213，使其能清楚的观测到底座21内部培养基上的情况。然后将含微生物的菌液从微生物富集釜61通过微注泵63注入凹槽64内，使得菌液形成液滴位于凹槽64内。通过控制取样探头装置2222蘸取凹槽64内的菌液，然后使取样探头装置2222的底部位于培养基表面，使得取样探头装置2222 在培养基的表面按照一定轨迹划线，最后分离的微生物将按照划线轨迹生长，实现单个菌落的分离。通过盖体22的可视窗2214观察，当培养基上有单个菌落形成时，即可开始进行划线分离工作。分离单个菌落之前，首先将取样管道72的末端连接反应釜71并打开取样阀门73，反应釜71内装有75％酒精，通过压力控制单元4维持整个装置的压力保持恒定，最后通过中央控制系统1控制取样探头装置2222，使得取样探头装置2222穿过取样管道72进入反应釜71，实现取样探头装置2222的灭菌过程。再关闭取样阀门73，将装有75％酒精的反应釜71更换为装有液体培养基的反应釜71，并通过压力控制单元4维持整个装置的压力保持恒定，最后通过中央控制系统1控制取样探头装置2222，并成功挑取培养基上的单个菌落，然后使取样探头装置2222穿过取样管道72进入装有液体培养基的反应釜71，最终实现单个菌落的保压转移培养。后续单菌落的分离以此类推即可。在整个挑选和培养过程中，保持培养釜内的压力、温度值与微生物最初所在的微生物富集釜61内的压力、温度环境一致，使得微生物在原位高压情况内实现分离。
在具体实施过程中，本实施例提出的海洋微生物高压环境分离培养装置与技术，能够实现海洋高压环境下，微生物单菌落形成以及单个菌落的培养，满足深海特殊菌种生长培养的需求。相比目前传统的常压环境富集、分离海洋微生物的技术，可有效的解决海洋耐压菌、嗜压菌在常压环境下存活率低，且深海土著特征在常压环境下不能有效表达等难题，解决目前海洋微生物培养度低，难培养纯菌的问题。本实施例可以实现高压环境下，人为实现单个菌落的分离、挑选和培养的过程，相比常规的微生物分离培养技术，有效的提高了微生物培养、纯化的效率。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (10)

  1. 一种深海原位环境的单菌落分离装置，其特征在于，包括中央控制系统(1)、分离操作培养器(2)、环境参数检测单元(3)、压力控制单元(4)、温度控制单元(5)、注液单元(6)和取样单元(7)；其中：
    所述分离操作培养器(2)包括底座(21)和与底座(21)固定连接的盖体(22)；所述盖体(22)作为观察区(221)和分离操作区(222)，所述底座(21)作为培养区(211)；所述观察区(21)用于对分离培养过程的观察；所述分离操作区(222)中根据分离操作需要设置有内滑轨(2221)，内滑轨(2221)上安装有取样探头装置(2222)，取样探头装置(2222)可在内滑轨(2221)上进行滑动，用于实现微生物的划线操作及取样操作；所述培养区(211)中设置有培养基，用于对划线后的微生物菌落进行培养；
    所述环境参数检测单元(3)设置在分离操作培养器(2)内部，用于实时检测分离操作培养器(2)内的温度、压力变化情况，并将数据传输至中央控制系统(1)；
    所述压力控制单元(4)、温度控制单元(5)分别与所述分离操作培养器(2)连接，用于保证分离操作培养器(2)内的压力、温度与微生物的生长培养环境一致；
    所述注液单元(6)用于向分离操作培养器(2)内注入富集微生物菌液，供取样探头装置(2222)蘸取和划线；
    所述取样单元(7)用于对微生物进行保压取样；
    所述取样探头装置(2222)控制端、压力控制单元(4)控制端、温度控制单元(5)控制端、注液单元(6)控制端和取样单元(7)控制端均与所述中央控制系统(1)电性连接；所述观察区(221)的数据输出端与所述中央控制系统(1)电性连接。
  2. 根据权利要求1所述的一种深海原位环境的单菌落分离装置，其特征在于，所述观察区(221)包括设置在盖体(22)外侧表面的外滑轨(2211)、与外滑轨(2211)可滑动连接的连接支架(2212)、固定在连接支架(2212)端部的观察装置(2213)和设置在盖体(22)表面的可视窗(2214)；所述观察装置(2213)的数据输出端与所述中央控制系统(1)电性连接。
  3. 根据权利要求1所述的一种深海原位环境的单菌落分离装置，其特征在于，所述环境参数检测单元(3)包括温度传感器(31)和压力传感器(32)；所述温度传感器(31)、压力传感器(32)均设置在分离操作培养器(2)内部，用于实时检测分离操作培养器(2)内的温度、压力变化情况，并将数据传输至中央控制系统(1)。
  4. 根据权利要求1所述的一种深海原位环境的单菌落分离装置，其特征在于，所述压力控制单元(4)包括空气压缩机(41)、增压泵(42)、储气罐(43)、调压阀(44)、进气阀(45)和送气管道(46)；其中：
    所述空气压缩机(41)、增压泵(42)、储气罐(43)、调压阀(44)通过送气管道(46)依次连接，最后通过所述进气阀(45)与所述分离操作培养器(2)连接；
    所述空气压缩机(41)的控制端、增压泵(42)的控制端、调压阀(44)的控制端、进气阀(45)的控制端均与所述中央控制系统(1)电性连接。
  5. 根据权利要求1所述的一种深海原位环境的单菌落分离装置，其特征在于，所述温度控制单元(5)采用水浴夹套装置，其包裹在所述分离操作培养器(2)外壁上，控制端与所述中央控制系统(1)电性连接。
  6. 根据权利要求1所述的一种深海原位环境的单菌落分离装置，其特征在于，所述注液单元(6)包括微生物富集釜(61)、送液管道(62)和微注泵(63)；其中：
    所述微生物富集釜(61)用于微生物菌液的富集，其输出端通过送液管道(62)与分离操作培养器(2)连接；所述微注泵(63)设置在所述送液管道(62)上，控制端与所述中央控制系统(1)电性连接。
  7. 根据权利要求6所述的一种深海原位环境的单菌落分离装置，其特征在于，所述注液单元(6)还包括设置在分离操作培养器(2)盖体(22)内部底面的凹槽(64)；所述送液管道(62)在分离操作培养器(2)内部的出液口设置在凹槽(64)内。
  8. 根据权利要求7所述的一种深海原位环境的单菌落分离装置，其特征在于，所述注液单元(6)还包括设置在送液管道(62)上的放空阀(65)，所述放空阀(65)的控制端与所述中央控制系统(1)电性连接。
  9. 根据权利要求1所述的一种深海原位环境的单菌落分离装置，其特征在于， 所述取样单元(7)包括反应釜(71)、取样管道(72)和取样阀门(73)；其中：
    所述反应釜(71)通过取样管道(72)与所述分离操作培养器(2)连接；所述取样阀门(73)设置在所述取样管道(72)上，其控制端与所述中央控制系统(1)电性连接。
  10. 一种深海原位环境的单菌落分离方法，其特征在于，应用如权利要求1～9任一项所述的一种深海原位环境的单菌落分离装置实现，具体包括以下步骤：
    S1：对深海原位环境的单菌落分离装置进行清洗灭菌，并装入培养基；
    S2：通过压力控制单元(4)往分离操作培养器(2)内注入气体，使得分离操作培养器(2)中的压力值与微生物富集环境的压力值一致；
    S3：开启温度控制单元(5)，使得分离操作培养器(2)获得与微生物富集环境一致的温度；
    S4：由注液单元(6)向分离操作培养器(2)内注入微生物菌液，控制取样探头装置(2222)在内滑轨(2221)上滑动，先蘸取微生物菌液，然后使取样探头装置(2222)位于培养基表面，令其在培养基的表面按照一定轨迹划线分离；
    S5：分离的微生物将按照划线轨迹生长，通过观察区(221)进行实时观察；
    S6：当培养基上有单个菌落形成时，开启取样单元(7)，令其与分离操作培养器(2)的温度、压力保持一致；
    S7：控制取样探头装置(2222)挑取培养基上的单个菌落，并将菌落送入装有液体培养基的取样单元(7)内，实现单个菌落的保压转移培养。
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