Device and method for high-throughput identification and separation of marine anaerobic microorganisms difficult to culture

Abstract

The invention discloses a device and a method for high-throughput identification and separation of marine anaerobic microorganisms difficult to culture, which are applied to the field of identification and separation culture of marine anaerobic microorganisms difficult to culture. The device comprises a high-pressure incubation bin for providing a high-pressure environment for microbial culture; the central control system is electrically connected with the temperature control unit and the pressure control unit and controls the temperature control unit and the pressure control unit to operate, and the temperature control unit and the pressure control unit are communicated with the high-pressure incubation bin through pipelines and adjust the temperature and the pressure in the bin; the microorganism enrichment system is connected with the high-pressure incubation bin through a liquid outlet pipeline and is used for enriching microorganisms to be separated; the high-throughput module comprises a high-throughput recognition module and a high-throughput separation module which are arranged on the high-pressure incubation bin and connected with the central control system, and the high-throughput recognition module and the high-throughput separation module are used for recognizing and separating microorganisms in the high-pressure incubation bin and picking out target microorganisms.

Description

Technical Field

[0001] This invention relates to the field of identification and isolation of marine anaerobic microorganisms that are difficult to culture, and specifically to a device and method for high-throughput identification and isolation of anaerobic marine microorganisms that are difficult to culture. Background Technology

[0002] The ocean is the largest ecosystem on Earth, harboring abundant microbial resources. Research indicates that a large number of uncultured microorganisms with unknown phylogenetic pathways still exist in the marine biosphere. The Illumina high-throughput DNA sequencing technology, introduced in the 20th century, has enabled the construction of microbial genome sequence databases. Although high-throughput sequencing technology has facilitated the analysis of microbial community information, the isolation and culture of microorganisms remains a crucial step in microbiological research, holding significant importance for a deeper understanding of the ecology, physiology, and genetics of specific microbial communities.

[0003] Marine microorganisms are characterized by their comprehensive adaptability to extreme environmental factors such as high pressure, low temperature, darkness, and oligotrophic conditions. This adaptability is reflected in multiple aspects, including cell structure, metabolic pathways, energy acquisition methods, and growth strategies. Currently, less than 1% of marine microorganisms can be cultured under laboratory conditions, with difficult-to-culture anaerobic microorganisms being particularly scarce. Existing marine microbial isolation technologies are mainly divided into atmospheric pressure and high pressure techniques, but both have certain limitations in isolating marine microorganisms. For example, atmospheric pressure isolation techniques cannot isolate barophilic bacteria, while high pressure isolation techniques struggle to accurately identify and isolate individual microorganisms at high throughput. Summary of the Invention

[0004] The purpose of this invention is to provide a device and method for high-throughput identification and separation of difficult-to-culture marine anaerobic microorganisms. This technology achieves high-throughput online identification and separation of difficult-to-culture anaerobic single colonies by reshaping their in-situ environment, while simultaneously detecting the growth of individual colonies online, thereby improving the reliability of microbial enrichment culture.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a device for high-throughput identification and separation of recalcitrant marine anaerobic microorganisms, comprising:

[0007] High-pressure incubation chambers are used to provide a high-pressure environment for microbial culture;

[0008] The system includes a central control system, a temperature control unit, and a pressure control unit. The central control system is electrically connected to the temperature control unit and the pressure control unit, respectively, and is used to control the operation of the temperature control unit and the pressure control unit. The temperature control unit is connected to the high-pressure incubation chamber through a pipeline and is used to regulate the temperature inside the high-pressure incubation chamber. The pressure control unit is connected to the high-pressure incubation chamber through a pipeline and is used to regulate the pressure inside the high-pressure incubation chamber.

[0009] A microbial enrichment system, wherein the microbial enrichment system is connected to a high-pressure incubation chamber via an outlet pipe, and is used to enrich microorganisms to be separated;

[0010] The high-throughput module includes a high-throughput identification module and a high-throughput separation module. The high-throughput identification module is installed on the high-pressure incubation chamber and connected to the central control system for identifying microorganisms in the high-pressure incubation chamber. The high-throughput separation module is installed on the high-pressure incubation chamber and connected to the central control system for separating microorganisms in the high-pressure incubation chamber and selecting the identified target microorganisms.

[0011] In one possible implementation, the microbial enrichment system includes a microbial enrichment vessel, which includes a lid, a vessel body, a magnetic stirrer, and a dissolved oxygen sensor.

[0012] The lid and the body of the vessel are detachably connected;

[0013] The magnetic stirrer is located at the bottom of the vessel and is used to stir the microbial culture medium inside the vessel.

[0014] The dissolved oxygen sensor is installed in the vessel body, with its detection end extending into the vessel body and its signal end electrically connected to the central control system to detect the dissolved oxygen content of the culture medium inside the vessel body.

[0015] The vessel body is also equipped with a pressure sensor and a temperature sensor. The detection ends of the pressure sensor and the temperature sensor extend into the interior of the vessel body, and the signal ends of the pressure sensor and the temperature sensor are electrically connected to the central control system.

[0016] The lower part of the vessel is connected to a liquid outlet pipe, which is equipped with a micro-injection pump. The end of the liquid outlet pipe away from the vessel is connected to a high-pressure incubation chamber. The liquid outlet pipe is used to transport the enriched bacterial solution in the vessel, and the micro-injection pump is used to control the bacterial solution delivery rate.

[0017] In one possible implementation, the high-pressure incubation chamber includes a top cover, a chamber body, and a tray, wherein the top cover is connected to the chamber body; and the tray is disposed at the lower part of the chamber body.

[0018] The upper cover is equipped with a temperature sensor, a pressure sensor, and a dissolved oxygen sensor. The detection ends of the temperature sensor, pressure sensor, and dissolved oxygen sensor extend into the interior of the chamber, and the signal ends of the temperature sensor, pressure sensor, and dissolved oxygen sensor are electrically connected to the central control system.

[0019] The tray is equipped with a stacking box and a bacterial solution holding device. The stacking box contains shelves for placing culture dishes in layers, which are used to culture microorganisms. The bacterial solution holding device includes a bacterial solution cup and a dilution cup. The bacterial solution cup is connected to an outlet pipe and is used to hold the bacterial solution to be separated. The dilution cup is connected to a diluent outside the high-pressure incubation chamber via a micro-injection pump, which is used to control the rate of addition of ultrapure water.

[0020] The top cover has a viewing window in the center for observing the growth of microorganisms inside the chamber; a high-throughput identification module is located above the viewing window.

[0021] In one possible implementation, the high-throughput identification module includes a high-definition camera and a colony identification device, wherein the colony identification device includes a high-throughput data preprocessing module, a high-throughput feature extraction module, a high-throughput identification module, and a high-throughput data calculation module.

[0022] The high-definition camera is connected to the high-pressure incubation chamber via a bracket, and the lens of the high-definition camera is set facing the viewing window to capture microbial colonies inside the chamber.

[0023] The high-definition camera, high-throughput data preprocessing module, high-throughput data preprocessing module, high-throughput recognition module, high-throughput data calculation module, and central control system are electrically connected in sequence.

[0024] The high-throughput data preprocessing module is used to preprocess the captured images; the high-throughput feature extraction module is used to extract the features of microbial colonies from the preprocessed images; the high-throughput identification module is used to identify the type and attributes of the colonies based on the extracted features; and the high-throughput data calculation module is used to calculate and analyze the identification results and transmit the results to the central control system.

[0025] In one possible implementation, the high-throughput separation module includes a moving axis, a needle-dipping box, a culture cup, a measuring cup, and a dipping handle;

[0026] The movable axis is set on the tray and includes an X-axis, a Y-axis and a Z-axis. A sleeve is provided on the Z-axis. The movable axis is used to drive the sleeve to move in the X, Y and Z directions. The sleeve is used to hold the culture cup, the measuring cup and the dipping handle.

[0027] The needle box is set on the tray, and the needle box contains a needle. One end of the dipping handle is detachably connected to the needle, and the other end can be held by a clip.

[0028] Both the culture cup and the measuring cup are placed on the tray and are compatible with the jacket;

[0029] Mixing balls for use with dilution cups, culture cups, and measuring cups;

[0030] The cotton balls are used with the culture cups and measuring cups and are located inside the cotton ball box.

[0031] A height measuring device is provided on the Z-axis. The height measuring device is connected to the central control system and is used to measure the distance from the surface of a single colony to the bottom of the dip needle.

[0032] In one possible implementation, a measurement module is also included, which comprises a measurement pool, a light source, and a data processing center. A measurement cup is placed inside the measurement pool, and the measurement cup is connected to a diluent outside the high-pressure incubation chamber via a micro-injection pump. The micro-injection pump is used to control the addition rate of ultrapure water. A circular hole is provided on one side of the measurement pool for the light from the light source to pass through. The data processing center is electrically connected to the central control system, and the central control system is electrically connected to the alarm device.

[0033] In one possible implementation, an automatic sterilization controller is also included for performing microbial sterilization operations inside the high-pressure incubation chamber. The automatic sterilization controller includes a UV lamp and a lifting platform. The UV lamp is located on the lifting platform. The high-pressure incubation chamber is provided with an automatic opening and closing perforation. The UV lamp can enter the interior of the high-pressure incubation chamber and perform microbial sterilization operations by the rise of the lifting platform. After sterilization is completed, the UV lamp is removed from the interior of the high-pressure incubation chamber by the lifting platform, and the automatic opening and closing perforation of the high-pressure incubation chamber is closed at the same time.

[0034] In one possible implementation, the pressure control unit includes an air compressor, a booster pump, an air tank, and a pressure regulating valve;

[0035] The output end of the air compressor is connected to the air inlet pipe of the high-pressure incubation chamber and the microbial enrichment system through an air supply pipe, which is used to control the air supply to the high-pressure incubation chamber and the microbial enrichment system.

[0036] A booster pump is used to pressurize gas, a gas storage tank is used to store high-pressure gas, and a pressure regulating valve is used to regulate gas pressure.

[0037] In one possible implementation, the height measuring device includes an ultrasonic rangefinder and an algorithm control center. The ultrasonic rangefinder is used to detect the distance from the surface of a single colony to the bottom of the dip needle in the swivel. The algorithm control center is electrically connected to both the ultrasonic rangefinder and the central control system to process and feed back the detection data.

[0038] Secondly, the present invention provides a method for high-throughput identification and separation of refractory marine anaerobic microorganisms, comprising the steps of:

[0039] Sterilize the microbial enrichment vessel, load it with the substrate to be cultured and nutrient solution, inject the required gas through the gas injection port to adjust the pressure, and culture the microorganisms under the set temperature and pressure conditions.

[0040] After the preset time has elapsed, the bacterial solution in the microbial enrichment vessel is transported to the bacterial solution cup in the high-pressure incubation chamber through the outlet pipe.

[0041] The central control system controls the moving shaft to drive the dip needle to dip into the bacterial solution in the bacterial solution cup. The dip needle with bacterial solution is moved to the dilution cup and the bacterial solution is released. Then, ultrapure water is injected into the dilution cup through the injection pump to complete the dilution of the bacterial solution.

[0042] After each dilution, the mixing ball is inserted into the dilution cup via the moving shaft to mix the diluted solution.

[0043] The culture dish is removed from the shelf and fixed by moving the axis. The dipping needle is controlled to draw lines on the surface of the culture dish to separate the colonies into individual colonies. Then the culture dish is put back on the shelf.

[0044] After the streaking is completed, a camera is used to periodically photograph the surface of the petri dish. The captured images are processed by the central control system to identify colony characteristics and obtain colony growth data.

[0045] Select target single colonies based on colony characteristics, use a moving shaft to drive the dip needle to select target single colonies and transfer them to culture cups, and seal the culture cups with cotton balls;

[0046] The moving shaft drives the dip needle to pick up the bacterial solution in the culture cup and transfer it to the measuring cup. After dilution with ultrapure water, the measuring cup is placed in the measuring pool. The OD600 value of the bacterial solution is measured by irradiating it with a light source, and the measurement result is recorded by the central control system.

[0047] After the measurement is completed, reset the measuring cup and repeat the above steps to complete the measurement of other bacterial solutions.

[0048] Compared with the prior art, the advantages of this invention are as follows:

[0049] 1. Compared to existing in situ microbial culture techniques, streak isolation of extremophiles faces the challenge of high-throughput and precise identification. This invention proposes a method that, under high-pressure conditions, solves the problem of requiring manual identification of individual colonies in existing technologies, and also addresses the difficulty of understanding the growth rate of each individual colony.

[0050] 2. Compared with existing in situ microbial culture technology, it can realize a series of processes such as single-cell isolation, culture and monitoring of microorganisms in an in situ environment, solving the problem that the above operations can only be completed by releasing pressure.

[0051] 3. Compared with existing isolation and culture technologies, this invention can identify, isolate and detect the growth of microorganisms at various stages with high throughput, providing basic data support for the subsequent growth of microorganisms. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 This is a schematic diagram of the device for high-throughput identification and separation of refractory marine anaerobic microorganisms in an embodiment of the present invention;

[0054] Figure 2 This is a schematic diagram of the moving axis and measuring module in an embodiment of the present invention;

[0055] Figure 3 This is a schematic diagram of the planar structure of the dipping handle and dipping needle in an embodiment of the present invention;

[0056] Figure 4 This is a schematic diagram of the planar structure of the mixing ball and the culture dish in an embodiment of the present invention;

[0057] Figure 5 This is a three-dimensional structural diagram of the stacking box in an embodiment of the present invention;

[0058] Figure 6 This is a schematic diagram of the connection of the central control system circuit module in an embodiment of the present invention.

[0059] In the attached diagram: 1. Central control system; 2. Microbial enrichment vessel; 21. Handle; 22. Vessel lid; 23. Vessel body; 24. Stirring shaft; 25. Dissolved oxygen sensor; 3. Temperature sensor; 4. Pressure sensor; 5. High-pressure incubation chamber; 51. Viewing window; 52. Colony identification device; 521. High-definition camera; 522. High-throughput data preprocessing module; 523. High-throughput feature extraction module; 524. High-throughput identification module; 525. High-throughput data calculation module; 53. Top cover; 54. Chamber body; 55. Tray; 551. Hole; 56. Moving axis; 561. "X" axis; 562. "Y" axis; 563. "Z" axis; 5631. Jacket; 5632. Height measuring device; 56321. Ultrasonic rangefinder; 5632 2. Algorithm Control Center; 57. High-Throughput Separation Module; 571. Bacterial Liquid Storage Device; 5711. Bacterial Liquid Cup; 5712. Dipping Handle; 5713. Dilution Cup; 572. Mixing Ball; 573. Culture Cup; 574. Fixing Frame; 575. Needle Dipping Box; 5751. Needle Dipping; 576. Stacking Box; 5761. Petri Dish; 5762. Shelf; 577. Cotton Ball Box; 58. Measurement Module; 581. Measurement Pool; 582. Light Source; 583. Data Processing Center; 59. Automatic Sterilization Controller; 591. Ultraviolet Lamp; 592. Lifting Platform; 593. Automatic Opening and Closing Perforator; 6. Diluter; 7. Pressure Control Unit; 71. Air Compressor; 72. Booster Pump; 73. Air Tank; 74. Pressure Regulating Valve; 8. Alarm Device; 9. Micro-Injection Pump. Detailed Implementation

[0060] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0061] Example:

[0062] It should be noted that the terms "comprising" and "having" and any variations thereof in the embodiments of the present invention are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such processes, methods, products, or devices.

[0063] See also Figures 1 to 6This invention relates to a high-throughput device for identifying and separating recalcitrant marine anaerobic microorganisms. The core component of the device is a high-pressure incubation chamber 5, and other auxiliary systems include a central control system 1, a temperature control unit, and a pressure control unit 7. By constructing high-pressure and extreme temperature conditions simulating the marine environment within a high-pressure microbial enrichment system, the culturability of microorganisms is effectively improved. The high-throughput module consists of a high-throughput identification module 524 and a high-throughput separation module 57. A micro-injection pump 9 injects the recalcitrant anaerobic microbial culture into the high-pressure incubation chamber 5, and the moving shaft 56 of the high-throughput separation module 57 separates the recalcitrant anaerobic microorganisms into individual colonies. Using the microbial colony identification device 52 and the height measurement device 5632 of the high-throughput identification module 524, recalcitrant anaerobic single bacteria are accurately selected, and their growth characteristics are automatically monitored online, completing the screening of recalcitrant anaerobic single bacteria. The entire process of microbial enrichment, separation, cultivation, and detection is carried out in situ under marine pressure and temperature conditions, or suitable pressure and temperature conditions can be set according to experimental needs.

[0064] In one embodiment, the microbial enrichment system mainly includes a microbial enrichment vessel 2. The microbial enrichment vessel 2 includes the following components: a handle 21, a lid 22, a vessel body 23, a stirring shaft 24, and a dissolved oxygen sensor 25. The vessel adopts a removable lid structure for easy placement of culture substrates and sterilization operations. A magnetic stirrer is equipped at the bottom of the vessel body to enhance the mass transfer process within the vessel and improve the energy and nutrient utilization efficiency of the microorganisms.

[0065] For anaerobic microorganisms that are difficult to cultivate in the ocean, the microbial enrichment vessel 2 is equipped with pressure sensor 4, temperature sensor 3, vent valve, and dissolved oxygen sensor 25 to monitor changes in temperature and pressure inside the vessel in real time. Maintaining constant temperature conditions is achieved by placing the vessel in a temperature-monitored high / low temperature water bath, utilizing the heat exchange effect of the water bath system, or by placing it in an air-heat exchange-type constant temperature chamber, ensuring that the difficult-to-cultivate anaerobic microorganisms are in a suitable temperature environment.

[0066] The top of the vessel body 23 is equipped with an air inlet pipe and a regulating valve. This pipe allows the injection of the gas (or inert gas) required for the cultivation of anaerobic microorganisms, enabling them to survive in a suitable high-pressure environment. The bottom of the vessel body 23 has an outlet pipe connected to the high-pressure incubation chamber 5 via a micro-injection pump 9. The dissolved oxygen sensor 25 is primarily used to monitor the oxygen concentration inside the vessel; once oxygen is detected, gas replacement is required.

[0067] The gas replacement process involves injecting the original gas through a pressurization system and releasing the gas inside the vessel through a vent valve, ensuring that the pressure and temperature inside the vessel remain stable throughout the process, ultimately providing a continuous and suitable environmental condition for difficult-to-cultivate anaerobic microorganisms.

[0068] In one embodiment, the high-pressure incubation chamber 5 mainly includes a viewing window 51, a colony identification device 52, a top cover 53, a chamber body 54, a tray 55, holes 551, a high-throughput separation module 57, and a height measuring device 5632. The top cover 53 of the high-pressure incubation chamber 5 is equipped with a temperature sensor 3, a pressure sensor 4, and a dissolved oxygen sensor 25. The temperature sensor 3, pressure sensor 4, and dissolved oxygen sensor 25 monitor the temperature, pressure, and dissolved oxygen concentration inside the high-pressure incubation chamber 5, respectively. When the oxygen concentration can be detected, the gas inside the high-pressure incubation chamber 5 needs to be replaced. The gas replacement method inside the high-pressure incubation chamber 5 involves injecting the original gas through a pressurization system while releasing the gas inside the high-pressure incubation chamber 5 through a vent valve. Throughout the process, the pressure and temperature inside the high-pressure incubation chamber 5 remain constant, ultimately ensuring that the difficult-to-culture anaerobic microorganisms survive in suitable environmental conditions.

[0069] A viewing window 51 is provided in the center of the top cover 53, and a high-throughput microbial colony identification module 524 is provided above the viewing window 51. The high-throughput microbial colony identification module 524 includes a high-definition camera 53, a high-throughput data preprocessing module 522, a high-throughput feature extraction module, a high-throughput colony identification module 524, and a high-throughput data calculation module 525. The high-definition camera 53 is connected to the side wall of the high-pressure incubation chamber 5 through a bracket.

[0070] The high-definition camera 53 automatically captures the microbial state on the plate at different times using high throughput periodicly. Through the high-throughput data preprocessing module 522, feature module high-throughput extraction module 523, colony high-throughput identification module 524, and data high-throughput calculation module 525 in the central control system 1, it finally obtains the number of difficult-to-culture anaerobic single bacteria on each plate at each shooting time, the colony characteristics (shape, color, and size, etc.), and the growth rate of each difficult-to-culture anaerobic single bacteria in high throughput. The high-throughput data preprocessing module 522 removes the original base color of the culture medium on the plate, thereby better identifying the color of difficult-to-culture anaerobic single colonies. It can process at least one photograph. The high-throughput feature extraction module 523 uses image recognition technology to extract the features (shape, color, and size, etc.) of difficult-to-culture anaerobic single colonies on the surface of the culture medium. It can identify single colony feature values ​​greater than or equal to 1. The high-throughput colony identification module 524 identifies the growth range of difficult-to-culture anaerobic single bacteria. It can identify single colony diameter values ​​greater than or equal to 0.1 mm. The high-throughput data calculation module 525 calculates the lateral growth rate of difficult-to-culture anaerobic single bacteria and the time of their first appearance. It can simultaneously calculate the lateral growth rate of different colonies, the total number of single colonies on the plate at each time point, and the total number of characteristic colonies, among other basic characteristics. The results of photographs taken by the high-definition camera 53 of the high-throughput microbial colony identification module 524 must be used in conjunction with the high-throughput identification module 524.

[0071] See Figures 2 to 5 The lower part of the chamber body 54 of the high-pressure incubation chamber 5 is equipped with a tray 55, a high-throughput separation module 57, a measurement module 58 and an automatic sterilization controller 69. The tray 55 has holes 551, which are square and have the same diameter as the bottom diameter of the measuring cup. The number of holes 551 can be set according to the requirements.

[0072] A high-throughput separation module 57 is mounted on tray 55. The high-throughput separation module 57 includes a moving axis 56, a needle holder 575, a culture cup 573, a measuring cup, a dipping handle 5712, and a height measuring device 5632. The moving axis 56 includes an X-axis 561, a Y-axis 562, and a Z-axis 563. The X-axis 561 is U-shaped, and the lower part of the Z-axis 563 has a clip 5631 that can move freely in the vertical direction. This moving axis 56 has six degrees of freedom. The automatic movement of the moving axis 56 allows the clip 5631 to move rapidly to any position in three-dimensional space, ultimately achieving high-throughput automatic separation and extraction of microorganisms. The needles 5751 used for the automatic separation and extraction of microorganisms are located inside the needle holder 575, and the number of needles 5751 is greater than or equal to four. The automatic separation and extraction of microorganisms by the high-throughput separation module 57 requires the operation of the high-throughput identification module 524. Specifically, when the high-throughput identification module 524 needs to extract difficult-to-culture anaerobic single bacteria, it needs to automatically extract suitable difficult-to-culture anaerobic single bacteria based on its calculation results. The clip 5631 in the six-degree-of-freedom moving axis 56 is mainly used to grip the culture cup 573, measuring cup, and dipping handle 5712. A fixed height measuring device 5632 is provided on the upper part of the side wall of the "Z" axis 563.

[0073] The height measuring device 5632 includes an ultrasonic rangefinder 56321 and an algorithm control center 56322. This instrument is mainly used to detect the distance from the surface of a single colony to the bottom of the dipping needle 5751 in the dipping handle 5712. A bacterial solution holding device 571 is located on the upper left of the tray 55. The bacterial solution holding device 571 is connected to the outlet pipe via a micro-injection pump 9. The bacterial solution holding device 571 includes a bacterial solution cup 5711, a dilution cup 5713, and a dipping handle 5712. There is one bacterial solution cup 5711. The number of dilution cups 5713 can be set according to experimental needs, and the number can be greater than or equal to one. The dilution cup 5713 is connected to the diluter 6 outside the autoclave 5 via a micro-injection pump 9. Its main function is to use the dipping needle 5751 on the jacket 5631 to draw bacterial solution from the bacterial solution container and place it in the dilution cup. The ultrapure water in the diluter 6 is then used to dilute the bacterial solution, maintaining it within a specific concentration range for subsequent bacterial separation. The measuring cup is also connected to the diluter 6 outside the autoclave 5 via a micro-injection pump 9, ensuring that the concentration of difficult-to-culture anaerobic single bacteria can be measured subsequently. The number of measuring cups is greater than or equal to one.

[0074] The dipping handle 5712 is cross-shaped, and its bottom is paired with the dipping needle 5751 to prevent it from easily falling off. The number of dipping handles 5712 is consistent with the number of dilution cups 5713. To facilitate thorough mixing of the diluent and bacterial culture, a mixing ball 572 is used in conjunction with the dilution cup 5713, culture cup 573, and measuring cup. There is one mixing ball 572. The main purpose of the mixing ball 572 is to thoroughly mix any unmixed bacterial culture by blowing air into it. A fixing frame 574 is provided at the rear of the tray 55. The fixing frame 574 is mainly used to fix the culture dish 5761 and prevent it from moving during the selection of difficult-to-culture anaerobic single colonies. The dipping needle 5751 is paired with the dipping handle 5712 and is located inside the dipping needle box 575, which is located between the stacking box 576 and the fixing frame 574. The needle-dipping box 575 contains rows of sterilized needles 5751, designed so that the next needle 5751 can be replaced after each single colony extraction. The stacking box 576 contains petri dishes 5761 and shelves 5762. Each petri dish 5761 is located on a shelf 5762, and the number of petri dishes 5761 is consistent with the number of shelves 5762, and the number of each is greater than or equal to 1.

[0075] Petri dish 5761 is paired with fixing frame 574, and the edge of petri dish 5761 has a handle that can be gripped using clip 5631 at the lower part of "Z" axis 563. The upper part of culture cup 573 and measuring cup are circular, and the lower part is square, made of optical glass. Two opposite sides of the square sidewalls are smooth, while the others are matte, which facilitates subsequent optical measurement of the growth rate of recalcitrant anaerobic bacteria. The circular part of the upper part of culture cup 573 and measuring cup is paired with a cotton ball, which is located inside cotton ball box 577. The number of cotton balls is consistent with the number of culture cups 573 and measuring cups. The cotton balls can seal the upper opening of culture cup 573, preventing contamination of the recalcitrant anaerobic bacteria in culture cup 573 by atmospheric bacteria after the experiment.

[0076] In addition, a measurement module 58 is located in the middle section of the front of the "X" axis 561. This measurement module 58 is fixedly connected to the tray 55. The measurement module 58 contains a measurement pool 581, a light source 582, and a data processing center 583. The measurement pool 581 is mainly used to detect the growth rate of recalcitrant anaerobic bacteria in the measurement cup. The interior of the measurement pool 581 is just large enough to hold the measurement cup. A circular hole is provided on the left side of the measurement pool 581 to allow light from the left side to pass through. The light is 600nm monochromatic light. The measurement results are returned to the central control system 1 for display and processing. When the growth rate of recalcitrant anaerobic bacteria is found to be decreasing during the measurement process, the alarm device 8 is activated to stop the culture of recalcitrant anaerobic bacteria.

[0077] In one embodiment, the sterilization of the entire device is achieved using an automatic sterilization controller 69, located in the lower left part of the high-pressure incubation chamber 5. The automatic sterilization controller 69 includes an ultraviolet lamp 591 and a lifting platform 592, with the ultraviolet lamp 591 located on the lifting platform 592. The lower left part of the high-pressure incubation chamber 5 has an automatically opening and closing perforation 593. The ultraviolet lamp 591 can enter the interior of the high-pressure incubation chamber 5 and perform microbial sterilization by the rise of the lifting platform 592. After sterilization, the ultraviolet lamp 591 is withdrawn from the interior of the high-pressure incubation chamber 5 by the lifting platform 592, and the automatically opening and closing perforation 593 of the high-pressure incubation chamber 5 closes simultaneously.

[0078] In one embodiment, the pressure control system is mainly used to inject gas into the microbial enrichment system and the high-pressure incubation chamber 5 to pressurize them, ensuring that the pressure environment within the microbial enrichment system and the high-pressure incubation chamber 5 is consistent with the pressure value of the microorganisms in situ at sea (or pressurized according to experimental requirements). Simultaneously, it monitors pressure changes within the microbial enrichment system and the online detection system. Pressure changes within the culture chamber are monitored in real time by a pressure sensor 4. The pressure is increased or decreased by actively inflating / deflating the culture chamber, maintaining the pressure values ​​in the microbial enrichment system and the high-pressure incubation chamber 5 consistent with the marine environmental conditions for microbial growth. The pressurization system mainly consists of an air compressor 71, a booster pump 72, an air storage tank 73, a pressure regulating valve 74, an air inlet valve, air delivery pipes, and other fittings.

[0079] The central control system 1 of the present invention includes a data acquisition unit, a data central processing unit, an operating computer, etc., to monitor the changes of various environmental data information during the enrichment and detection of microbial enrichment bacteria in a high-pressure environment, as well as to collect, process, store and output images.

[0080] The present invention relates to a method for high-throughput identification and isolation culture of marine microorganisms, which mainly includes two steps: enrichment and high-throughput identification and isolation culture. First, microorganisms are enriched using a microbial enrichment system, and then high-throughput precise isolation, culture, and monitoring of microorganisms are performed in a high-pressure incubation chamber 5. This process is mainly performed by a central control system 1 for calculation and image processing, and the above operations are completed using a moving axis 56, which improves the efficiency of microbial isolation and culture under high pressure.

[0081] This invention relates to a high-throughput identification and isolation culture technology for marine microorganisms. First, the microbial enrichment vessel 2 and its associated pipes and valves are sterilized. Then, the substrate to be cultured, such as deep-sea sediment, macrobial tissues symbiotic with the microorganisms, and extracts, are sequentially loaded. Next, the nutrient solution required for culture is added through the injection port, and the gas required for culture (or an inert gas if not needed) is injected through the gas injection port to increase the pressure within the culture chamber to match the actual deep-sea environmental conditions. During the culture process, a magnetic stirrer at the bottom is used to agitate the mixture, increasing mass transfer and optimizing the culture process.

[0082] A certain amount of liquid culture medium is added to culture cup 573, and the corresponding fixed culture medium is added to culture dish 5761. The automatic sterilization device is then activated through the central control system 1 to ensure the interior of the high-pressure incubation chamber 5 is sterile. According to experimental needs, after a certain period of cultivation, the valve is first opened to allow the bacterial solution in the microbial enrichment culture system to automatically enter the outlet pipe. The bacterial solution in the outlet pipe enters the injection pump 9, and finally, the bacterial solution enters the bacterial solution cup 5711 within the high-pressure incubation chamber 5.

[0083] The central control system 1 controls the automatic movement of the moving shaft 56 in three-dimensional space, causing the jacket 5631 to clamp the dipping handle 5712. Through the automatic movement of the moving shaft 56, the dipping handle 5712 can be inserted into the dipping needle 5751, at which point the bacterial solution dilution can begin. Through the automatic movement of the moving shaft 56, the bottom of the dipping needle 5751 is used to pick up the bacterial solution from the bacterial solution cup 5711. The dipped needle 5751 is then released into the dilution cup 5713 (this release process involves placing the dipped needle 5751 at the bottle opening and then automatically detaching it under force through the left and right movement of the moving shaft 56). Then, the ultrapure water in the diluter 6 is pumped into the dilution cup 5713 by the external injection pump 9 of the high-pressure incubation chamber 5, thus completing the dilution of the bacterial solution. Finally, the moving shaft 56 automatically returns the dipping handle 5712 to its original position.

[0084] After each dilution, the mixing ball 572 is grasped by the jacket 5631 and inserted into the dilution cup 5713. The back-and-forth clamping and pulling of the mixing ball 572 by the jacket 5631 causes gas flow inside the mixing ball 572, thereby mixing the bacterial solution in the diluent. Repeating the above operation multiple times completes the multi-stage dilution of the bacterial solution.

[0085] The culture dish 5761 is automatically removed from the shelf 5762 by the clamp 5631 via the moving shaft 56, and the clamp 5631 is released so that the culture dish 5761 is placed in the fixed frame 574. The clamp 5631 of the moving shaft 56 "Z" axis 563 is then automatically moved again to clamp the dipping handle 5712, and the dipping handle 5712 is connected to the dipping needle 5751 via the moving shaft 56. The automatic movement of the moving shaft 56 causes the dipping needle 5751 to streak on the surface of the culture dish 5761 to separate the colonies on the dipping needle 5751, forming individual colonies on the surface of the culture dish 5761. After the streak separation is completed, the culture dish 5761 is returned to its original position, and the above steps are repeated to perform streak separation again.

[0086] After streaking, a high-definition camera 53 automatically captures images of the surface of each petri dish 5761, providing raw data for subsequent determination of the growth rate of colonies on the surface of the petri dishes 5761. At regular intervals, the high-definition camera 53 automatically captures images of the colony growth on the surface of each petri dish 5761. Through the high-throughput data preprocessing module 522, the high-throughput feature extraction module 523, the high-throughput recognition module 524, and the high-throughput data calculation module 525, the lateral growth rate and lateral area of ​​colonies under each plate can be clearly determined. Based on the color differences of the colonies formed after the images are captured, the desired single colony types are manually selected for single colony selection.

[0087] The automatic movement of the movable shaft 56 clamps the dipping handle 5712 with the clip 5631, and connects the dipping handle 5712 to the dipping needle 5751 for single colony selection. To prevent the dipping needle 5751 from puncturing the culture medium or failing to contact the colony, the downward movement of the dipping needle 5751 is within the range of (BD) to (CD) mm, where B (mm) is the distance from the ultrasonic wave emitted to the culture medium next to the colony; C (mm) is the distance from the ultrasonic wave emitted to the center of the colony; and D (mm) is the distance from the dipping needle 5751 to the bottom of the ultrasonic transmitter. The single colony after dipping is placed in the culture cup 573 for incubation. The automatic movement of the movable shaft 56 and the clip 5631 insert the cotton balls in the cotton ball box 577 into the culture cup 573.

[0088] During the cultivation process, to determine the growth rate of a single colony, an optical method is used to measure the OD600 value of the bacterial solution. The automatic movement of the movable shaft 56 grips the dipping handle 5712 and connects it to the dipping needle 5751, then dips into the bacterial solution in the culture cup 573. The dipped bacterial solution is placed in a measuring cup, and a certain amount of ultrapure water is injected into the measuring cup through the injection pump 9 of the high-pressure incubation chamber 5 to dilute the bacterial solution. The measuring cup is then placed into the measuring pool 581 using the jacket 5631, and the light source 582 is turned on to measure the OD600 value (i.e., bacterial concentration) of the bacterial solution. The measured value is processed and recorded by the central control system 1. After the measurement is completed, the movable shaft 56 automatically moves the measuring cup back to its original position, and the above operation is repeated to continue testing the next bacterial solution.

[0089] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0090] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A device for high-throughput identification and separation of refractory marine anaerobic microorganisms, characterized in that, include: High-pressure incubation chambers are used to provide a high-pressure environment for microbial culture; The system includes a central control system, a temperature control unit, and a pressure control unit. The central control system is electrically connected to the temperature control unit and the pressure control unit, respectively, and is used to control the operation of the temperature control unit and the pressure control unit. The temperature control unit is connected to the high-pressure incubation chamber through a pipeline and is used to regulate the temperature inside the high-pressure incubation chamber. The pressure control unit is connected to the high-pressure incubation chamber through a pipeline and is used to regulate the pressure inside the high-pressure incubation chamber. A microbial enrichment system, wherein the microbial enrichment system is connected to a high-pressure incubation chamber via an outlet pipe, and is used to enrich microorganisms to be separated; The high-throughput module includes a high-throughput identification module and a high-throughput separation module. The high-throughput identification module is installed on the high-pressure incubation chamber and connected to the central control system for identifying microorganisms in the high-pressure incubation chamber. The high-throughput separation module is installed on the high-pressure incubation chamber and connected to the central control system for separating microorganisms in the high-pressure incubation chamber and selecting the identified target microorganisms.

2. The device for high-throughput identification and separation of difficult-to-culture marine anaerobic microorganisms according to claim 1, characterized in that, The microbial enrichment system includes a microbial enrichment vessel, which includes a lid, a vessel body, a magnetic stirrer, and a dissolved oxygen sensor. The lid and the body of the vessel are detachably connected; The magnetic stirrer is located at the bottom of the vessel and is used to stir the microbial culture medium inside the vessel. The dissolved oxygen sensor is installed in the vessel body, with its detection end extending into the vessel body and its signal end electrically connected to the central control system to detect the dissolved oxygen content of the culture medium inside the vessel body. The vessel body is also equipped with a pressure sensor and a temperature sensor. The detection ends of the pressure sensor and the temperature sensor extend into the interior of the vessel body, and the signal ends of the pressure sensor and the temperature sensor are electrically connected to the central control system. The lower part of the vessel is connected to a liquid outlet pipe, which is equipped with a micro-injection pump. The end of the liquid outlet pipe away from the vessel is connected to a high-pressure incubation chamber. The liquid outlet pipe is used to transport the enriched bacterial solution in the vessel, and the micro-injection pump is used to control the bacterial solution delivery rate.

3. The device for high-throughput identification and separation of difficult-to-culture marine anaerobic microorganisms according to claim 1, characterized in that, The high-pressure incubation chamber includes a top cover, a chamber body, and a tray; the top cover is connected to the chamber body; the tray is located at the bottom of the chamber body. The upper cover is equipped with a temperature sensor, a pressure sensor, and a dissolved oxygen sensor. The detection ends of the temperature sensor, pressure sensor, and dissolved oxygen sensor extend into the interior of the chamber, and the signal ends of the temperature sensor, pressure sensor, and dissolved oxygen sensor are electrically connected to the central control system. The tray is equipped with a stacking box and a bacterial solution holding device. The stacking box contains shelves for placing culture dishes in layers, which are used to culture microorganisms. The bacterial solution holding device includes a bacterial solution cup and a dilution cup. The bacterial solution cup is connected to an outlet pipe and is used to hold the bacterial solution to be separated. The dilution cup is connected to a diluent outside the high-pressure incubation chamber via a micro-injection pump, which is used to control the rate of addition of ultrapure water. The top cover has a viewing window in the center for observing the growth of microorganisms inside the chamber; a high-throughput identification module is located above the viewing window.

4. The device for high-throughput identification and separation of refractory marine anaerobic microorganisms according to claim 3, characterized in that, The high-throughput identification module includes a high-definition camera and a colony identification device. The colony identification device includes a high-throughput data preprocessing module, a high-throughput feature extraction module, a high-throughput identification module, and a high-throughput data calculation module. The high-definition camera is connected to the high-pressure incubation chamber via a bracket, and the lens of the high-definition camera is set facing the viewing window to capture microbial colonies inside the chamber. The high-definition camera, high-throughput data preprocessing module, high-throughput data preprocessing module, high-throughput recognition module, high-throughput data calculation module, and central control system are electrically connected in sequence. The high-throughput data preprocessing module is used to preprocess the captured images; the high-throughput feature extraction module is used to extract the features of microbial colonies from the preprocessed images; the high-throughput identification module is used to identify the type and attributes of the colonies based on the extracted features; and the high-throughput data calculation module is used to calculate and analyze the identification results and transmit the results to the central control system.

5. The device for high-throughput identification and separation of refractory marine anaerobic microorganisms according to claim 4, characterized in that, The high-throughput separation module includes a moving shaft, a needle-dipping box, a culture cup, a measuring cup, and a dipping handle; The movable axis is set on the tray and includes an X-axis, a Y-axis and a Z-axis. A sleeve is provided on the Z-axis. The movable axis is used to drive the sleeve to move in the X, Y and Z directions. The sleeve is used to hold the culture cup, the measuring cup and the dipping handle. The needle box is set on the tray, and the needle box contains a needle. One end of the dipping handle is detachably connected to the needle, and the other end can be held by a clip. Both the culture cup and the measuring cup are placed on the tray and are compatible with the jacket; Mixing balls for use with dilution cups, culture cups, and measuring cups; The cotton balls are used with the culture cups and measuring cups and are located inside the cotton ball box. A height measuring device is provided on the Z-axis. The height measuring device is connected to the central control system and is used to measure the distance from the surface of a single colony to the bottom of the dip needle.

6. The device for high-throughput identification and separation of recalcitrant marine anaerobic microorganisms according to claim 5, characterized in that, It also includes a measurement module mounted on a tray. The measurement module includes a measurement pool, a light source, and a data processing center. A measurement cup is placed inside the measurement pool. The measurement cup is connected to a diluent outside the high-pressure incubation chamber via a micro-injection pump. The micro-injection pump is used to control the addition rate of ultrapure water. A circular hole is provided on one side of the measurement pool for the light from the light source to pass through. The data processing center is electrically connected to the central control system, and the central control system is electrically connected to the alarm device.

7. The device for high-throughput identification and separation of difficult-to-culture marine anaerobic microorganisms according to claim 5, characterized in that, It also includes an automatic sterilization controller for performing microbial sterilization operations inside the high-pressure incubation chamber. The automatic sterilization controller includes a UV lamp and a lifting platform. The UV lamp is located on the lifting platform. The high-pressure incubation chamber is equipped with an automatic opening and closing perforation. The UV lamp can enter the interior of the high-pressure incubation chamber and perform microbial sterilization operations by the rise of the lifting platform. After sterilization, the UV lamp is removed from the interior of the high-pressure incubation chamber by the lifting platform, and the automatic opening and closing perforation of the high-pressure incubation chamber closes at the same time.

8. The device for high-throughput identification and separation of recalcitrant marine anaerobic microorganisms according to claim 5, characterized in that, The pressure control unit includes an air compressor, a booster pump, an air tank, and a pressure regulating valve; The output end of the air compressor is connected to the air inlet pipe of the high-pressure incubation chamber and the microbial enrichment system through an air supply pipe, which is used to control the air supply to the high-pressure incubation chamber and the microbial enrichment system. A booster pump is used to pressurize gas, a gas storage tank is used to store high-pressure gas, and a pressure regulating valve is used to regulate gas pressure.

9. The device for high-throughput identification and separation of refractory marine anaerobic microorganisms according to claim 5, characterized in that, The height measuring device includes an ultrasonic rangefinder and an algorithm control center. The ultrasonic rangefinder is used to detect the distance from the surface of a single colony to the bottom of the dip needle in the dip handle. The algorithm control center is electrically connected to both the ultrasonic rangefinder and the central control system to process and feed back the detection data.

10. A method for high-throughput identification and separation of refractory marine anaerobic microorganisms, characterized in that, Including the following steps: Sterilize the microbial enrichment vessel, load it with the substrate to be cultured and nutrient solution, inject the required gas through the gas injection port to adjust the pressure, and culture the microorganisms under the set temperature and pressure conditions. After the preset time has elapsed, the bacterial solution in the microbial enrichment vessel is transported to the bacterial solution cup in the high-pressure incubation chamber through the outlet pipe. The central control system controls the moving shaft to drive the dip needle to dip into the bacterial solution in the bacterial solution cup. The dip needle with bacterial solution is moved to the dilution cup and the bacterial solution is released. Then, ultrapure water is injected into the dilution cup through the injection pump to complete the dilution of the bacterial solution. After each dilution, the mixing ball is inserted into the dilution cup via the moving shaft to mix the diluted solution. The culture dish is removed from the shelf and fixed by moving the axis. The dipping needle is controlled to draw lines on the surface of the culture dish to separate the colonies into individual colonies. Then the culture dish is put back on the shelf. After the streaking is completed, a camera is used to periodically photograph the surface of the petri dish. The captured images are processed by the central control system to identify colony characteristics and obtain colony growth data. Select target single colonies based on colony characteristics, use a moving shaft to drive the dip needle to select target single colonies and transfer them to culture cups, and seal the culture cups with cotton balls; The moving shaft drives the dip needle to pick up the bacterial solution in the culture cup and transfer it to the measuring cup. After dilution with ultrapure water, the measuring cup is placed in the measuring pool. The OD600 value of the bacterial solution is measured by irradiating it with a light source, and the measurement result is recorded by the central control system. After the measurement is completed, reset the measuring cup and repeat the above steps to complete the measurement of other bacterial solutions.

Images