Device and method for on-line enrichment and automatic sorting of deep-sea microorganisms in high-pressure environment

Abstract

According to the online enrichment and automatic sorting device and method for deep-sea microorganisms in the high-pressure environment, multi-stage enrichment is carried out by simulating the high-pressure and special-temperature environment of deep sea, and meanwhile, based on environment index changes observed online in the enrichment device, the deep-sea microorganisms in the high-pressure environment can be automatically sorted; the optimal time point of microbial passage is automatically judged, and a culture is transferred to a next-stage enrichment device, so that the problems that in a high-pressure environment, microbial enrichment is difficult to transfer at the optimal time point, manual operation is complicated and the like are effectively solved, and the early-stage enrichment efficiency of obtaining a single species of deep-sea microorganisms is improved; besides, automatic streak separation and automatic secondary sorting of the enriched substances are carried out in a culture medium in a high-pressure environment, and specific single colonies are automatically transferred to a pure culture kettle, so that the problems of overlapping of single colonies, low separation degree, complicated manual operation, difficulty in automatic secondary sorting in the high-pressure environment and the like in an existing high-pressure sorting technology are solved; and the high-pressure environment deep-sea microorganism sorting efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of marine microorganism enrichment culture and microorganism individual sorting, and more specifically, to a device and method for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment. Background Art

[0002] Due to its special environmental conditions, the deep-sea ecosystem has bred a variety of special habitats such as cold springs and hydrothermal fluids. In extreme environments such as high pressure and low / high temperatures, deep-sea microorganisms have extremely high uniqueness and diversity in terms of genetic structure, physiological metabolism, etc. These deep-sea microorganisms play an irreplaceable role in promoting ocean energy transfer and material circulation and maintaining the stability of the ocean and even the global ecosystem.

[0003] In order to explore the unique role of a certain deep-sea microorganism in the marine ecosystem, it is necessary to sort and pure culture individual organisms to determine their microbial characteristics, such as physiological characteristics such as growth and metabolism. However, the current technology for sorting and pure culture of deep-sea microorganisms is still immature. After deep-sea microorganisms are collected and transferred to the laboratory, they are separated from their original high-pressure environment, and are prone to cell rupture and difficult to survive. The efficient separation and culture of a single species of microorganism will become extremely difficult. Therefore, microbial enrichment and pure culture using high-pressure culture devices are indispensable means in the field of deep-sea microbial research.

[0004] For the enrichment of deep-sea microorganisms under simulated environmental conditions, the prior art discloses a high-pressure deep-sea microorganism enrichment and multi-level purification device and method. The existing high-pressure enrichment technology mainly collects the enriched material through a sampler for instrument detection and analysis. It is impossible to observe the reaction substances of the enriched bacterial solution inside the device online, and thus it is impossible to transfer the enriched material according to the reaction substances. It is difficult to perform microbial passage at the optimal time point of the inflection point of the metabolic substance change, and there are limitations such as complex manual operation and low microbial sorting activity.

[0005] For the sorting of deep-sea microorganisms under simulated environmental conditions, the prior art also discloses a system and method for high-throughput automatic screening of single colonies in deep-sea heat preservation and pressure maintenance. The existing automatic sorting technology separates the enriched bacterial liquid by spraying, and has problems such as low biological separation and easy overlapping of colonies, which is not conducive to the rapid growth and effective separation of single microbial colonies. In addition, the prior art also discloses a biological coating device. This automatic sorting technology uses a robotic arm to coat the bacterial liquid in the culture medium for separation. However, since the culture medium is uneven or tilted in the culture dish when used in a high-pressure environment, the coating rod head may fall off, resulting in streaking failure and difficulty in recovering the gun head and re-coating. There are problems such as complicated manual sorting operations and difficulty in automatic secondary sorting after automatic coating failure.

[0006] In general, problems such as complex manual operations for microbial enrichment, low separation of enriched materials, low activity of sorted microorganisms, and difficulty in automatic secondary sorting make it difficult to efficiently obtain a single species of deep-sea microorganisms. Summary of the invention

[0007] In order to overcome the defects of the above-mentioned prior art deep-sea microbial enrichment, such as complex manual operation, low separation degree of enriched materials, low activity of sorted microorganisms and difficulty in automatic secondary sorting, the present invention provides a device and method for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment. By online monitoring of the enrichment environment, automatic judgment of the optimal time for microbial propagation, automatic streaking and separation of enriched materials, automatic secondary sorting and automatic transfer of specific single colonies, the cultivability and acquisition efficiency of a single species of deep-sea microorganisms are improved, providing an important basic means for improving the understanding of deep-sea life science laws and the efficiency of biological resource development and application.

[0008] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0009] A high-pressure environment deep-sea microorganism online enrichment device, comprising: a multi-stage enrichment unit, a first pressure control unit, a first temperature control unit, a first data acquisition unit and a first central control unit;

[0010] The multi-stage enrichment unit comprises a liquid injection mechanism, a liquid transfer mechanism and at least two microorganism culture kettles connected in series, wherein the liquid injection mechanism is used to inject microorganism culture liquid into each microorganism culture kettle; the first-stage microorganism culture kettle is filled with an in-situ sediment sample for microorganism enrichment; the liquid transfer mechanism is used to transfer the bacterial liquid in the upper-stage microorganism culture kettle to the lower-stage microorganism culture kettle;

[0011] The first pressure control unit and the first temperature control unit are connected to the first central control unit and each microorganism culture kettle, respectively, and are used to control the pressure and temperature in each kettle, respectively;

[0012] The first data acquisition unit is connected to the first central control unit, and the first data acquisition unit is used to collect environmental data in each microorganism culture kettle in real time and transmit it to the first central control unit in real time;

[0013] The first central control unit is also respectively connected to each microbial culture kettle, liquid injection mechanism, liquid transfer mechanism, first pressure control unit and first temperature control unit. The first central control unit is used to control the high-pressure environment deep-sea microbial online enrichment device according to the data collected by the first data acquisition unit.

[0014] Preferably, in the multi-stage enrichment unit, the injection mechanism includes a continuous injection pump and a plurality of injection valves, each of the injection valves is arranged one by one on the microbial culture solution inlet pipeline of each microbial culture kettle; the continuous injection pump is connected to each injection valve respectively;

[0015] The liquid transfer mechanism includes a sample transfer injection pump, a plurality of groups of filters and sample transfer valves, and a plunger pump; the sample transfer injection pump is respectively connected to all microbial culture kettles except the last stage, and a group of filters and sample transfer valves are arranged on the connecting pipeline between each two stages of microbial culture kettles connected in series; the plunger pump is connected to the last stage of microbial culture kettle;

[0016] Each of the microorganism culture kettles has the same structure, and is provided with a magnetic stirrer and a sampling valve at the bottom;

[0017] In all microbial culture kettles except the last one, a sample transfer piston is arranged on the top and a protruding ring is arranged in the middle; the sample transfer piston is used to reduce the pressure difference required for transferring the bacterial liquid in the upper microbial culture kettle to the lower microbial culture kettle; the protruding ring is used to prevent the sample transfer piston from being over-pressurized.

[0018] Preferably, the first pressure control unit comprises: a gas pressurization system, a plurality of gas injection valves and a plurality of PID control valves;

[0019] Each of the microorganism culture kettles is provided with a gas injection valve; the gas pressurization system is connected to each gas injection valve through a pipeline, and the gas pressurization system is connected to the first central control unit for pressurizing the gas for each kettle;

[0020] Each of the microorganism culture kettles is also provided with a PID control valve, and each of the PID control valves is connected to a central control unit; the PID control valve is used to control the formation of a continuous pressure environment in the microorganism culture kettles connected in series;

[0021] The first temperature control unit includes: an air conditioning system;

[0022] The air conditioning system is respectively connected to the first central control unit and each of the microorganism culture kettles to control the temperature in each kettle;

[0023] The first data acquisition unit includes: a pressure sensing system, a temperature sensing system, a Raman measurement system, a dissolved oxygen measurement system and a pH value measurement system;

[0024] The pressure sensing system and the temperature sensing system are respectively connected to the first central control unit and each of the microorganism culture kettles, and are respectively used to collect the pressure and temperature in each kettle in real time and transmit them to the first central control unit in real time;

[0025] The Raman measurement system, dissolved oxygen measurement system and pH value measurement system are respectively connected to the first central control unit and each of the microbial culture kettles, and are respectively used to observe the changes in the chemical substance content, dissolved oxygen content and pH value in each microbial culture kettle in real time online and transmit them to the first central control unit in real time.

[0026] The present invention also provides an online automatic sorting device for deep-sea microorganisms in a high-pressure environment, comprising: an automatic sorting unit, a second pressure control unit, a second temperature control unit, a second data acquisition unit, and a second central control unit;

[0027] The automatic sorting unit includes a sorting operation kettle and a plurality of pure culture kettles; the sorting operation kettle is connected to each pure culture kettle respectively;

[0028] The sorting operation kettle comprises an internal three-axis moving mechanism and at least one marking pen tip box, wherein a plurality of marking pen tips are placed in the marking pen tip box, and a moving Z-axis of the three-axis moving mechanism is provided with a mechanical arm capable of extracting marking pen tips from the marking pen tip box, and the mechanical arm is used to drive the marking pen tips to perform sorting operations; the X-axis and Y-axis planes of the three-axis moving mechanism are provided with a culture area consisting of a plurality of culture dishes;

[0029] The second pressure control unit and the second temperature control unit are respectively connected to the second central control unit, the sorting operation kettle and each pure culture kettle, and are used to control the pressure and temperature in each kettle respectively;

[0030] The second data acquisition unit is connected to the second central control unit, and the second data acquisition unit is used to collect image data of the culture area in the sorting operation kettle in real time, and transmit it to the second central control unit in real time;

[0031] The second central control unit is also connected to the robotic arm, the second pressure control unit and the second temperature control unit respectively. The second central control unit is used to control the high-pressure environment deep-sea microorganism online automatic sorting device according to the data collected by the second data acquisition unit.

[0032] Preferably, in the automatic sorting unit, the sorting operation kettle comprises an upper kettle body and a lower kettle body which are sealed and connected;

[0033] The upper surface of the upper kettle body is provided with a pressure-resistant window for observing the interior of the sorting operation kettle;

[0034] The lower kettle body is provided with a bacterial liquid pool, a three-axis moving mechanism, at least one scribing pen tip box, a plurality of culture dishes, a pen tip recovery pool and a sorting transfer port;

[0035] The bacterial liquid pool is used to hold the bacterial liquid to be sorted;

[0036] The three-axis moving mechanism is provided with three vertically intersecting slide rails of X-axis, Y-axis and Z-axis, and the mechanical arm is arranged on the Z-axis slide rail;

[0037] The mechanical arm comprises: a vertical rod and a magnetic attraction sensor and a fixed block arranged at both ends of the vertical rod; the magnetic attraction sensor is connected to the second central control unit and the fixed block respectively, the magnetic attraction sensor provides magnetic force for the fixed block, and the fixed block extracts the marking pen tip through the magnetic force;

[0038] The robot arm is used to perform the following operations: extract and install the marking pen tip in the marking pen tip box; drive the marking pen tip to dip into the bacterial solution in the bacterial solution pool and the culture dish; drive the marking pen tip to mark and sort in the culture dish; release the marking pen tip in the pen tip recovery pool and the sorting transfer port;

[0039] The culture dish is pre-filled with a special culture medium for culturing a specific single bacterial colony to separate the desired special microbial species;

[0040] The pen tip recovery pool is used to recover used marking pen tips;

[0041] The sorting transfer port is connected to each of the pure culture kettles through a pipeline and a quick-connect interface, and the sorting transfer port is used to transfer the streaking pen tip dipped in a single bacterial colony after sorting to the pure culture kettle.

[0042] Preferably, the second pressure control unit comprises: a gas pressurizing system, an exhaust valve and a plurality of gas injection valves;

[0043] The sorting operation kettle and each pure culture kettle are respectively provided with a gas injection valve; the gas pressurizing system is respectively connected to each gas injection valve through a pipeline, and the gas pressurizing system is connected to the second central control unit for performing gas pressurization for each kettle;

[0044] The exhaust valve is arranged on the sorting operation kettle and is used for exhausting the sorting operation kettle;

[0045] The second temperature control unit includes: an air conditioning system;

[0046] The air conditioning system is respectively connected to the second central control unit, the sorting operation kettle and each pure culture kettle, and is used to control the temperature in each kettle.

[0047] The second data acquisition unit includes a pressure sensing system, a temperature sensing system and a camera system;

[0048] The pressure sensing system and the temperature sensing system are respectively connected to the second central control unit, the sorting operation kettle and each pure culture kettle, and are respectively used to collect the pressure and temperature in each kettle in real time and transmit them to the second central control unit in real time;

[0049] The camera system is arranged on the top of the sorting operation kettle and is connected to the second central control unit. The camera system is used for taking real-time image data of the culture area in the sorting operation kettle and transmitting the image data to the second central control unit in real time.

[0050] The present invention also provides an online enrichment and automatic sorting device for deep-sea microorganisms in a high-pressure environment, comprising an enrichment device and a sorting device connected in sequence, wherein the enrichment device is specifically the above-mentioned online enrichment device for deep-sea microorganisms in a high-pressure environment; and the sorting device is specifically the above-mentioned online automatic sorting device for deep-sea microorganisms in a high-pressure environment.

[0051] Preferably, the last-stage microorganism culture kettle in the multi-stage enrichment unit is connected to the bacterial liquid pool in the sorting operation kettle;

[0052] The bacterial liquid pool is used to hold the bacterial liquid to be sorted after being enriched in the last stage of microbial culture kettle.

[0053] The present invention also provides a method for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment. Based on the above-mentioned device for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment, the method comprises the following steps:

[0054] S1: Clean and sterilize each of the microorganism culture kettles, the sorting operation kettle, and each of the pure culture kettles, and perform pressure leak detection; install each unit in the device, and perform initial configuration;

[0055] S2: Transfer the collected in-situ sediment samples to the first-stage microbial culture reactor to start microbial enrichment;

[0056] S3: using the first data acquisition unit to collect the environmental data in each stage of the microorganism culture kettle in real time and transmit it to the first central control unit in real time;

[0057] Starting from the first-level microorganism culture kettle, when the environmental data in the previous-level microorganism culture kettle meets the first preset condition, the first central control unit controls the liquid transfer mechanism to transfer the bacterial liquid in the previous-level microorganism culture kettle to the next-level microorganism culture kettle for continued enrichment culture;

[0058] S4: When the environmental data in the last-stage microorganism culture kettle meets the second preset condition, the enrichment operation is completed, and the bacterial liquid in the last-stage microorganism culture kettle is transferred to the sorting operation kettle;

[0059] S5: using the second data acquisition unit to collect image data of the culture area in the sorting operation kettle in real time and transmit it to the second central control unit in real time; the second central control unit controls the robot arm to perform several marking operations in the culture area to perform sorting and culture for a preset time;

[0060] S6: When the second central control unit recognizes from the image data of the culture area that multiple single bacterial colonies are grown in the culture dish, the second central control unit controls the robot arm to transfer each single bacterial colony to each of the pure culture kettles respectively, to complete the sorting operation.

[0061] Preferably, in step S1, the initialization configuration includes: using the injection mechanism to inject pre-prepared microbial culture solution into each microbial culture kettle at a certain flow rate; preparing a special culture medium for each culture dish of the sorting operation kettle in advance, and injecting the pre-prepared special culture solution into each pure culture kettle in advance; using the first and second pressure control units to perform gas pressurization on each kettle to simulate the high-pressure environment of the deep sea; using the first and second temperature control units to control the temperature of each kettle to maintain the special temperature environment of the deep sea;

[0062] In step S5, each scribing operation includes:

[0063] The second central control unit controls the robot arm to move to the marking pen tip box, controls the robot arm to move to the culture dish to mark after extracting the marking pen tip, and controls the robot arm to leave the culture area after marking, moves to the pen tip recovery pool and releases the marking pen tip;

[0064] In step S6, the second central control unit controls the robot arm to transfer each single bacterial colony to each pure culture tank respectively, including:

[0065] The second central control unit controls the robotic arm to extract the streaking pen tip, move it to a single bacterial colony for dipping, transfer it to the sorting transfer port to release the streaking pen tip, and transfer the streaking pen tip dipped in the single bacterial colony to the pure culture kettle.

[0066] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0067] The present invention provides an online enrichment device for deep-sea microorganisms in a high-pressure environment. The device performs multi-stage enrichment by simulating the high pressure and special temperature environment of the deep sea. At the same time, based on the changes in environmental indicators observed online in the enrichment device, the device automatically determines the optimal time point for microbial propagation and transfers the culture to the next-stage enrichment device, thereby effectively solving the problems of difficulty in transferring microbial enrichment at the optimal time point in a high-pressure environment and complex manual operations, and improving the early enrichment efficiency of a single species of deep-sea microorganisms.

[0068] In addition, the present invention also provides an online automatic sorting device for deep-sea microorganisms in a high-pressure environment, which solves the problems of overlapping single colonies and low separation, complex manual operation, and difficulty in automatic secondary sorting in a high-pressure environment in the existing high-pressure sorting technology by automatically streaking and separating the enriched material in the culture medium in the high-pressure environment, automatically sorting, and automatically transferring specific single colonies to a pure culture kettle, thereby greatly improving the efficiency of deep-sea microorganism sorting in a high-pressure environment.

[0069] The present invention also combines the two to form an online enrichment and automatic sorting device, which can carry out continuous, multi-stage enrichment of the culture by dilution and subculture under high-pressure environmental conditions, and monitor the environmental indicators in the culture system online at the same time, realizing the integration of enrichment and sorting devices, transferring the culture under pressure-maintaining and sterile conditions, automatically streaking and sorting the culture medium in the device, and online monitoring of the growth status of single colonies, thereby improving the cultivability and acquisition efficiency of a single species of deep-sea microorganisms, and providing an important basic means for improving the understanding of deep-sea life science laws and the efficiency of biological resource development and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a framework diagram of a high-pressure environment deep-sea microorganism online enrichment device provided in Example 1.

[0071] Figure 2 This is a framework diagram of an online automatic sorting device for deep-sea microorganisms in a high-pressure environment provided in Example 2.

[0072] Figure 3 This is a framework diagram of a high-pressure environment deep-sea microorganism online enrichment and automatic sorting device provided in Example 3.

[0073] Figure 4 This is a mechanical structure diagram of a high-pressure environment deep-sea microorganism online enrichment and automatic sorting device provided in Example 4.

[0074] Figure 5 This is a top view of the interior of the sorting operation kettle provided in Example 4.

[0075] Figure 6 This is a schematic diagram of the connection between the central control unit provided in Example 4 and other components.

[0076] Figure 7 This is a flow chart of a method for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment provided in Example 5.

[0077] Figure 8 This is a specific operation flow chart of the online enrichment and automatic sorting provided in Example 5. DETAILED DESCRIPTION

[0078] The drawings are for illustrative purposes only and should not be construed as limiting the present patent;

[0079] In order to better illustrate the present embodiment, some parts in the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product;

[0080] It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0081] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0082] Example 1

[0083] like Figure 1 As shown, this embodiment provides an online enrichment device for deep-sea microorganisms in a high-pressure environment, including: a multi-stage enrichment unit, a first pressure control unit, a first temperature control unit, a first data acquisition unit and a first central control unit;

[0084] The multi-stage enrichment unit comprises a liquid injection mechanism, a liquid transfer mechanism and at least two microorganism culture kettles connected in series, wherein the liquid injection mechanism is used to inject microorganism culture liquid into each microorganism culture kettle; the first-stage microorganism culture kettle is filled with an in-situ sediment sample for microorganism enrichment; the liquid transfer mechanism is used to transfer the bacterial liquid in the upper-stage microorganism culture kettle to the lower-stage microorganism culture kettle;

[0085] The first pressure control unit and the first temperature control unit are connected to the first central control unit and each microorganism culture kettle, respectively, and are used to control the pressure and temperature in each kettle, respectively;

[0086] The first data acquisition unit is connected to the first central control unit, and the first data acquisition unit is used to collect environmental data in each microorganism culture kettle in real time and transmit it to the first central control unit in real time;

[0087] The first central control unit is also respectively connected to each microbial culture kettle, liquid injection mechanism, liquid transfer mechanism, first pressure control unit and first temperature control unit. The first central control unit is used to control the high-pressure environment deep-sea microbial online enrichment device according to the data collected by the first data acquisition unit.

[0088] In the specific implementation process, before deep-sea microorganism enrichment, each microorganism culture kettle is first cleaned and sterilized, and pressure leak detection is performed; then each unit in the entire device is installed, and all sensors and other electronic instruments in the device are connected to the first central control unit, and the instruments are activated and connected to the software of the first central control unit;

[0089] The collected in situ sediment samples are then transferred to the first-stage microbial culture kettle to start microbial enrichment;

[0090] Using the first data acquisition unit to collect the environmental data in each stage of the microorganism culture kettle in real time and transmit it to the first central control unit in real time;

[0091] Starting from the first-level microorganism culture kettle, when the environmental data in the previous-level microorganism culture kettle meets the first preset condition, the first central control unit controls the liquid transfer mechanism to transfer the bacterial liquid in the previous-level microorganism culture kettle to the next-level microorganism culture kettle for continued enrichment culture;

[0092] When the environmental data in the last-stage microorganism culture kettle meets the second preset condition, the multi-stage enrichment operation is completed;

[0093] This device simulates the high pressure and special temperature environment of the deep sea to perform multi-stage enrichment. At the same time, based on the changes in environmental indicators observed online in the enrichment device, it automatically determines the optimal time point for microbial propagation and transfers the culture to the next-level enrichment device, thereby effectively solving the problems of microbial enrichment in a high-pressure environment being difficult to transfer at the optimal time point and complex manual operations, and improving the initial enrichment efficiency of a single species of deep-sea microorganisms.

[0094] Example 2

[0095] like Figure 2 As shown, this embodiment provides an online automatic sorting device for deep-sea microorganisms in a high-pressure environment, comprising: an automatic sorting unit, a second pressure control unit, a second temperature control unit, a second data acquisition unit, and a second central control unit;

[0096] The automatic sorting unit includes a sorting operation kettle and a plurality of pure culture kettles; the sorting operation kettle is connected to each pure culture kettle respectively;

[0097] The sorting operation kettle comprises an internal three-axis moving mechanism and at least one marking pen tip box, wherein a plurality of marking pen tips are placed in the marking pen tip box, and a moving Z-axis of the three-axis moving mechanism is provided with a mechanical arm capable of extracting marking pen tips from the marking pen tip box, and the mechanical arm is used to drive the marking pen tips to perform sorting operations; the X-axis and Y-axis planes of the three-axis moving mechanism are provided with a culture area consisting of a plurality of culture dishes;

[0098] The second pressure control unit and the second temperature control unit are respectively connected to the second central control unit, the sorting operation kettle and each pure culture kettle, and are used to control the pressure and temperature in each kettle respectively;

[0099] The second data acquisition unit is connected to the second central control unit, and the second data acquisition unit is used to collect image data of the culture area in the sorting operation kettle in real time, and transmit it to the second central control unit in real time;

[0100] The second central control unit is also connected to the robotic arm, the second pressure control unit and the second temperature control unit respectively. The second central control unit is used to control the high-pressure environment deep-sea microorganism online automatic sorting device according to the data collected by the second data acquisition unit.

[0101] In the specific implementation process, before deep-sea microorganism sorting, the sorting operation kettle and each pure culture kettle are first cleaned and sterilized, and pressure leak detection is performed; then the various units in the entire device are installed, and all sensors and other electronic instruments in the device are connected to the central control unit, and the instruments are activated and connected to the software of the second central control unit;

[0102] Transfer the bacterial liquid to be sorted to the sorting operation kettle;

[0103] The second data acquisition unit is used to collect image data of the culture area in the sorting operation kettle in real time and transmit it to the second central control unit in real time; the second central control unit controls the robot arm to perform several marking operations in the culture area to perform sorting and culture for a preset time;

[0104] When the second central control unit recognizes from the image data of the culture area that multiple single bacterial colonies are grown in the culture dish, the second central control unit controls the robot arm to transfer each single bacterial colony to each pure culture kettle to complete the sorting operation;

[0105] The device solves the problems of existing high-pressure sorting technology, such as overlapping single colonies and low separation, complex manual operation and difficulty in automatic secondary sorting in a high-pressure environment, by automatically streaking and separating the enriched materials in the culture medium in a high-pressure environment, automatically performing secondary sorting and automatically transferring specific single colonies to a pure culture kettle. It greatly improves the efficiency of deep-sea microbial sorting in a high-pressure environment.

[0106] Example 3

[0107] like Figure 3 As shown, this embodiment provides an online enrichment and automatic sorting device for deep-sea microorganisms in a high-pressure environment, based on the online enrichment device for deep-sea microorganisms in a high-pressure environment in Example 1 and the online automatic sorting device for deep-sea microorganisms in a high-pressure environment in Example 2, comprising: a multi-stage enrichment unit, an automatic sorting unit, a pressure control unit, a temperature control unit, a data acquisition unit and a central control unit;

[0108] In this embodiment, the pressure control unit, the temperature control unit, the data acquisition unit and the central control unit respectively integrate the functions of the first and second pressure control units, the first and second temperature control units, the first and second data acquisition units, and the first and second central control units, and are respectively used to control the pressure, temperature, data acquisition and specific operations of the entire device (including the enrichment and separation devices);

[0109] The multi-stage enrichment unit comprises a liquid injection mechanism, a liquid transfer mechanism and at least two microorganism culture kettles connected in series, wherein the liquid injection mechanism is used to inject microorganism culture liquid into each microorganism culture kettle; the first-stage microorganism culture kettle is filled with an in-situ sediment sample for microorganism enrichment; the liquid transfer mechanism is used to transfer the bacterial liquid in the upper-stage microorganism culture kettle to the lower-stage microorganism culture kettle;

[0110] The automatic sorting unit includes a sorting operation kettle and a plurality of pure culture kettles; the sorting operation kettle is connected to each pure culture kettle respectively;

[0111] The sorting operation kettle comprises an internal three-axis moving mechanism and at least one marking pen tip box, wherein a plurality of marking pen tips are placed in the marking pen tip box, and a moving Z-axis of the three-axis moving mechanism is provided with a mechanical arm capable of extracting marking pen tips from the marking pen tip box, and the mechanical arm is used to drive the marking pen tips to perform sorting operations; the X-axis and Y-axis planes of the three-axis moving mechanism are provided with a culture area consisting of a plurality of culture dishes;

[0112] The last stage microorganism culture kettle in the multi-stage enrichment unit is connected to the sorting operation kettle;

[0113] The pressure control unit and the temperature control unit are respectively connected to the central control unit, the sorting operation kettle, each microorganism culture kettle and each pure culture kettle, and are used to control the pressure and temperature in each kettle respectively;

[0114] The data acquisition unit is connected to the central control unit, and is used to collect environmental data in each microorganism culture kettle and image data of the culture area in the sorting operation kettle in real time, and transmit them to the central control unit in real time;

[0115] The central control unit is also connected to each microorganism culture kettle, liquid injection mechanism, liquid transfer mechanism, robotic arm, pressure control unit and temperature control unit respectively. The central control unit is used to control the deep-sea microorganism online enrichment and automatic sorting device according to the data collected by the data acquisition unit.

[0116] In the specific implementation process, before deep-sea microorganism enrichment, each microorganism culture kettle, sorting operation kettle and each pure culture kettle are first cleaned and sterilized, and pressure leak detection is performed; then the various units in the entire device are installed, and all sensors and other electronic instruments in the device are connected to the central control unit, and the instruments are activated and connected to the software of the central control unit;

[0117] The collected in situ sediment samples are then transferred to the first-stage microbial culture kettle to start microbial enrichment;

[0118] The data acquisition unit is used to collect the environmental data in each stage of the microorganism culture kettle in real time and transmit it to the central control unit in real time;

[0119] Starting from the first-level microorganism culture kettle, when the environmental data in the previous-level microorganism culture kettle meets the first preset condition, the central control unit controls the liquid transfer mechanism to transfer the bacterial liquid in the previous-level microorganism culture kettle to the next-level microorganism culture kettle for continued enrichment culture;

[0120] When the environmental data in the last stage microorganism culture kettle meets the second preset condition, the enrichment operation is completed, and the bacterial liquid in the last stage microorganism culture kettle is transferred to the sorting operation kettle;

[0121] Then, the data acquisition unit is used to collect image data of the culture area in the sorting operation kettle in real time and transmit the image data to the central control unit in real time;

[0122] The central control unit controls the robotic arm to continuously extract and release the marking pen tip, and drives the marking pen tip to perform marking operations in each culture dish in the culture area, and performs sorting and culture for a preset time;

[0123] When the central control unit identifies from the image data of the culture area that multiple single bacterial colonies have grown in the culture dish, the central control unit controls the robotic arm to transfer each single bacterial colony to each of the pure culture kettles to complete the sorting operation;

[0124] This device can carry out continuous, multi-stage enrichment of cultures through dilution and subculture under high-pressure environmental conditions, while online monitoring of environmental indicators within the culture system, realizing the integration of enrichment and sorting devices, transferring cultures under pressure-maintaining, sterile conditions, automatically streaking and sorting the culture medium within the device, and online monitoring of the growth status of single colonies, thereby improving the cultivability and acquisition efficiency of single species of deep-sea microorganisms.

[0125] Example 4

[0126] like Figure 4As shown, based on the high-pressure environment deep-sea microorganism online enrichment and automatic sorting device described in Example 3, this embodiment provides a high-pressure environment deep-sea microorganism online enrichment and automatic sorting device, including: a multi-stage enrichment unit 1, an automatic sorting unit 2, a pressure control unit 3, a temperature control unit 4, a data acquisition unit 5 and a central control unit 6;

[0127] The multi-stage enrichment unit 1 comprises a liquid injection mechanism 11, a liquid transfer mechanism 12 and two microorganism culture kettles connected in series, which are a primary culture kettle 13 and a secondary culture kettle 14 in this embodiment. The liquid injection mechanism 11 is used to inject microorganism culture liquid into each of the microorganism culture kettles; the primary culture kettle 13 is filled with an in-situ sediment sample for microorganism enrichment; the liquid transfer mechanism 12 is used to transfer the bacterial liquid in the primary culture kettle 13 to the secondary culture kettle 14;

[0128] The automatic sorting unit 2 includes a sorting operation kettle 21 and a plurality of pure culture kettles 22; the sorting operation kettle 21 is connected to each pure culture kettle 22 respectively;

[0129] The sorting operation kettle 21 includes an internal three-axis moving mechanism 211 and at least one marking pen tip box 212, wherein a plurality of marking pen tips 213 are placed in the marking pen tip box 212, and a mechanical arm 214 capable of extracting the marking pen tips 213 from the marking pen tip box 212 is provided on the moving Z axis of the three-axis moving mechanism 211, and the mechanical arm 214 is used to drive the marking pen tips 213 to perform sorting operations; the X-axis and Y-axis planes of the three-axis moving mechanism 211 are provided with a culture area consisting of a plurality of culture dishes 215;

[0130] The secondary culture kettle 14 in the multi-stage enrichment unit 1 is connected to the sorting operation kettle 21;

[0131] The pressure control unit 3 and the temperature control unit 4 are respectively connected to the central control unit 6, the sorting operation kettle 21, the first culture kettle 13, the second culture kettle 14 and each pure culture kettle 22, and are used to control the pressure and temperature in each kettle respectively;

[0132] The data acquisition unit 5 is connected to the central control unit 6, and is used to collect the environmental data in the first-level culture kettle 13 and the second-level culture kettle 14 in real time, as well as the image data of the culture area in the sorting operation kettle 21, and transmit it to the central control unit 6 in real time;

[0133] The central control unit 6 is also connected to the first-level culture kettle 13, the second-level culture kettle 14, the injection mechanism 11, the liquid transfer mechanism 12, the mechanical arm 214, the pressure control unit 3 and the temperature control unit 4, respectively, and the central control unit 6 is used to control the deep-sea microorganism online enrichment and automatic sorting device according to the data collected by the data acquisition unit 5;

[0134] In the multi-stage enrichment unit 1, the injection mechanism 11 includes a continuous injection pump 111 and two injection valves 112, each of the injection valves 112 is arranged on the microbial culture liquid inlet pipeline of each microbial culture kettle in a one-to-one correspondence; the continuous injection pump 111 is connected to each injection valve 112 respectively;

[0135] The liquid transfer mechanism 12 includes a sample transfer injection pump 121, a group of filters 122 and a sample transfer valve 123, and a plunger pump 124; the sample transfer injection pump 121 is connected to the primary culture kettle 13 through a pressure-maintaining valve 125, and the pressure-maintaining valve 125 is used to maintain a constant pressure during the liquid transfer process; a group of filters 122 and a sample transfer valve 123 are provided on the connecting pipeline between the primary culture kettle 13 and the secondary culture kettle 14 to realize the transfer and subculture process of the primary enrichment and the secondary enrichment of the culture; the plunger pump 124 is connected to the secondary culture kettle 14; the plunger pump 124 is used to transfer the secondary culture kettle 14 to the sorting operation kettle 21;

[0136] The first-stage culture kettle 13 and the second-stage culture kettle 14 have the same structure, and are both provided with a magnetic stirrer 15 and a sampling valve 16 at the bottom; the magnetic stirrer 15 is used to stir the culture solution in the kettle; the sampling valve 16 is used to regularly collect the culture and perform gene sequencing analysis;

[0137] In the first-stage culture kettle 13, a sample transfer piston 131 is arranged at the top, and a protruding ring 132 with a narrower inner diameter is arranged in the middle, and the protruding ring 132 and the first-stage culture kettle 13 are an integral structure;

[0138] The sample transfer piston 131 is used to reduce the pressure difference required for transferring the bacterial solution to the secondary culture kettle 14; in this embodiment, the upper part of the sample transfer piston 131 is connected to the pressure-maintaining valve 125 and the sample transfer injection pump 121, so as to form a pressure-maintaining layer on the upper part of the primary culture kettle 13, which is used to maintain the pressure of the primary enrichment of the culture and to promote the transfer and subculture process of the culture by using a smaller pressure difference; in addition, a pipeline is also provided in the sample transfer piston 131 for installing a temperature sensor 521 and connecting the gas injection valve 32, so as to facilitate the spatial arrangement of the interface;

[0139] The protruding ring 132 is used to prevent the sample transfer piston 131 from being over-pressurized, thereby damaging the magnetic stirrer 15 at the bottom and the sensor device interface in the data acquisition unit 5;

[0140] In the secondary culture kettle 14, a sorting valve 141 is also provided at the bottom thereof, and the sorting valve 141 is connected to the plunger pump 124, and the plunger pump 124 is further connected to the bacterial liquid pool 217 in the sorting operation kettle 21. The pipeline is used to transfer the enriched culture to the next automatic sorting process under pressure;

[0141] In the automatic sorting unit 2, the sorting operation kettle 21 includes an upper kettle body and a lower kettle body that are sealed; the upper kettle body and the lower kettle body are connected and sealed by a sealing rubber ring and a plurality of screws 23 on the outer ring;

[0142] The upper surface of the upper kettle body is provided with a pressure-resistant window 216, which is relatively large and made of a pressure-resistant material, and is used to observe the interior of the sorting operation kettle 21;

[0143] The lower kettle body is provided with a bacterial liquid pool 217, a three-axis moving mechanism 211, at least one marking pen tip box 212, a plurality of culture dishes 215, a pen tip recovery pool 218 and a sorting transfer port 219. The top view of the lower kettle body is as shown in FIG. Figure 5 As shown;

[0144] The bacterial liquid pool 217 is used to hold the bacterial liquid to be sorted after being enriched in the secondary culture kettle 14;

[0145] The three-axis moving mechanism 211 is provided with three vertically intersecting slide rails of X-axis, Y-axis and Z-axis. The Y-axis slide rail 2112 is installed on the track of the X-axis slide rail 2111 through a connecting block to realize the horizontal movement of the robot arm 214 in the plane; the Z-axis slide rail 2113 is installed on the track of the Y-axis slide rail 2112 through a connecting block to realize the vertical movement of the robot arm 214 in the plane; the robot arm 214 is installed on the track of the Z-axis slide rail 2113 through a connecting block to realize the movement of the robot arm 214 in the direction perpendicular to the plane;

[0146] The culture area is a circular structure, which includes four culture dishes 215, wherein the culture dishes 215 are pre-filled with special culture media for cultivating specific single colonies to separate the required special microbial species;

[0147] The pen tip recovery pool 218 is used to recover the used marking pen tip 213;

[0148] The sorting transfer port 219 is connected to each of the pure culture kettles 22 through a pipeline, a ball valve 24 and a quick-connect interface 25. The sorting transfer port 219 is used to transfer the streaking pen tip 213 dipped with a single bacterial colony after sorting to the pure culture kettle 22;

[0149] The mechanical arm 214 includes: a vertical rod 2141 and a magnetic attraction sensor 2142 and a fixed block 2143 arranged at both ends of the vertical rod 2141; the magnetic attraction sensor 2142 is connected to the central control unit 6 and the fixed block 2143 respectively, and the magnetic attraction sensor 2142 provides magnetic force for the fixed block 2143, and the fixed block 2143 extracts the marking pen head 213 through the magnetic force;

[0150] The robot arm 214 is used to perform the following operations: extract and install the marking pen tip 213 in the marking pen tip box 212; drive the marking pen tip 213 to dip into the bacterial solution in the bacterial solution pool 217 and the culture dish 215; drive the marking pen tip 213 to mark and sort in the culture dish 215; release the marking pen tip 213 in the pen tip recovery pool 218 and the sorting transfer port 219;

[0151] The pure culture kettle 22 in this embodiment is used to provide a variety of special microbial species for pure culture; before the experiment begins, it is necessary to inject a special culture solution and connect to the pressure control unit 3 to pressurize the corresponding gas. The pure culture kettle 22 can be connected to the quick-connect interface 25 corresponding to the sorting transfer port 219 of the sorting operation kettle 21 to achieve rapid transfer of the sorted marking pen tip 213 and the microorganisms contained therein, so as to start pure culture using the pure culture kettle 22;

[0152] The pressure control unit 3 includes: a gas pressurizing system 31, a plurality of gas injection valves 32, a plurality of PID control valves 33 and an exhaust valve 34;

[0153] The first-stage culture kettle 13, the second-stage culture kettle 14, the sorting operation kettle 21 and each pure culture kettle 22 are respectively provided with a gas injection valve 32; the gas pressurizing system 31 is respectively connected to each gas injection valve 32 through a pipeline, and the gas pressurizing system 31 is connected to the central control unit 6 for performing gas pressurization for each kettle;

[0154] A PID control valve 33 is also provided on the first-level culture kettle 13 and the second-level culture kettle 14, respectively. Each of the PID control valves 33 is connected to the central control unit 6; the PID control valve 33 is used to set a certain pressure threshold to realize the construction of a continuous reaction system in the first-level culture kettle 13 and the second-level culture kettle 14, so as to realize uniform mass transfer and continuous renewal of the culture solution and improve the enrichment efficiency of the culture in the kettle;

[0155] The exhaust valve 34 is disposed on the sorting operation kettle 21 and is used for exhausting the sorting operation kettle 21;

[0156] The temperature control unit 4 includes: an air conditioning system 41;

[0157] The air conditioning system 41 is respectively connected to the central control unit 6, the first culture kettle 13, the second culture kettle 14, the sorting operation kettle 21 and each pure culture kettle 22, and is used to control the temperature in each kettle;

[0158] The data acquisition unit 5 includes: a pressure sensing system 51, a temperature sensing system 52, a Raman measurement system 53, a dissolved oxygen measurement system 54, a pH value measurement system 55 and a camera system 56;

[0159] The pressure sensing system 51 and the temperature sensing system 52 are respectively connected to the central control unit 6, the first-level culture kettle 13, the second-level culture kettle 14, the sorting operation kettle 21 and each pure culture kettle 22, and are respectively used to collect the pressure and temperature in each kettle in real time and transmit them to the central control unit 6 in real time; in this embodiment, the pressure sensing system 51 includes three pressure sensors 511, and the temperature sensing system 52 includes three temperature sensors 521, and a pressure sensor 511 and a temperature sensor 521 are respectively arranged inside the first-level culture kettle 13, the second-level culture kettle 14 and the sorting operation kettle 21;

[0160] The Raman measurement system 53, the dissolved oxygen measurement system 54 and the pH value measurement system 55 are connected to the central control unit 6, the first culture tank 13 and the second culture tank 14 respectively, and are used to respectively observe the changes in the chemical substance content, the dissolved oxygen content and the pH value in each microbial culture tank in real time online and transmit them to the central control unit 6 in real time;

[0161] In this embodiment, the Raman measurement system 53 includes: two groups of pressure-resistant observation cabins 531, two groups of observation valves 532 and two groups of drain valves 533. The two groups of equipment are respectively arranged in the first-level culture kettle 13 and the second-level culture kettle 14. A Raman measurement probe is arranged in the pressure-resistant observation cabin 531. Every other period of time, samples are taken through the observation valve 532 and transferred to the pressure-resistant observation cabin 531. The Raman measurement probe is used to measure the change of the chemical substance content and transmit it to the central control unit 6 in real time. After the observation is completed, the drain valve 533 is used to drain the sample in the pressure-resistant observation cabin 531, waiting for the next sampling.

[0162] The dissolved oxygen measuring system 54 and the pH value measuring system 55 are used to measure the dissolved oxygen content and the pH value of the primary culture tank 13 and the secondary culture tank 14, respectively, so as to identify the progress of the anaerobic oxidation reaction of the microorganisms;

[0163] The camera system 56 includes a camera 561 and a ring-shaped lighting lamp 562. The camera 561 is arranged on the upper part of the pressure-resistant window 216 on the top of the sorting operation kettle 21 and is connected to the central control unit 6. The camera 561 is used to take real-time image data of the culture area in the sorting operation kettle 21 and transmit it to the central control unit 6 in real time. In addition, a ring-shaped lighting lamp 562 is also arranged inside the upper kettle body to facilitate observation of single bacterial colonies and improve imaging quality.

[0164] The central control unit 6 includes: a computer host 61;

[0165] like Figure 6As shown, the computer host 61 establishes a communication connection with the pressure sensing system 51, the temperature sensing system 52, the Raman measurement system 53, the dissolved oxygen measurement system 54, the pH value measurement system 55 and the camera system 56 in the data acquisition unit 5, so as to receive the data collected by the data acquisition unit 5;

[0166] The computer host 61 is also connected to the liquid injection mechanism 11, the liquid transfer mechanism 12, the magnetic stirrer 15 in the first-level culture kettle 13 and the second-level culture kettle 14, the robotic arm 214, the pressure control unit 3 and the temperature control unit 4, and is used to control the deep-sea microorganism online enrichment and automatic sorting device according to the data collected by the data acquisition unit 5.

[0167] In the specific implementation process, before deep-sea microorganism enrichment, the first-level culture kettle 13, the second-level culture kettle 14, the sorting operation kettle 21 and each of the pure culture kettles 22 are first cleaned and sterilized, and pressure leak detection is performed; then the various units in the entire device are installed, and all sensors and other electronic instruments in the device are connected to the central control unit 6, and the instruments are activated and connected to the software of the computer host 61;

[0168] Then, the injection mechanism 11 is used to inject the pre-prepared microbial culture solution into each microbial culture kettle at a certain flow rate; a special culture medium is prepared in advance for each culture dish 215 of the sorting operation kettle 21, and the pre-prepared special culture solution is injected into each pure culture kettle 22; the gas pressurization system 31 in the pressure control unit 3 is used in combination with the pressure sensing system 51 to perform gas pressurization on each kettle to simulate the high-pressure environment of the deep sea, and the PID control valve 33 is opened to set the pressure to form a continuous environmental system; the temperature control unit 4 is used in combination with the temperature sensing system 52 to control the temperature of each kettle to maintain the special temperature environment of the deep sea;

[0169] Then, the collected in-situ sediment sample is transferred to the primary culture kettle 13, and the magnetic stirrer 15 is turned on to start microbial enrichment;

[0170] The Raman measurement system 53, the dissolved oxygen measurement system 54 and the pH value measurement system 55 are used to measure the chemical substance content change, the dissolved oxygen content and the pH value in the first-level culture tank 14 respectively and transmit them to the computer host 61 in the central control unit 6 in real time;

[0171] When the environmental data in the primary culture kettle 13 meets the first preset condition, the central control unit 6 controls the liquid transfer mechanism 12 to transfer the bacterial liquid in the primary culture kettle 13 to the secondary culture kettle 14 for continued enrichment culture; specifically, the sample transfer injection pump 121, the pressure-maintaining valve 125 and the sample transfer valve 123 are opened, and pure water is injected to push the sample transfer piston 131, so that the enriched bacterial liquid enters the secondary culture kettle 14 for continued culture;

[0172] When the environmental data in the secondary culture kettle 14 meets the second preset condition, the enrichment operation is completed, the plunger pump 124 and the sorting valve 141 are opened, and the bacterial liquid in the secondary culture kettle 14 is transferred to the bacterial liquid pool 217 in the sorting operation kettle 21;

[0173] Then, the camera system 56 is used to collect image data of the culture area in the sorting operation kettle 21 in real time and transmit it to the central control unit 6 in real time;

[0174] The central control unit 6 controls the mechanical arm 214 to continuously extract and release the marking pen head 213, and drives the marking pen head 213 to perform marking operations in each culture dish 215 in the culture area, and performs sorting and culture for a preset time;

[0175] The central control unit 6 drives the robot arm 214 to move by controlling the X-axis slide rail 2111, the Y-axis slide rail 2112 and the Z-axis slide rail 2113. The specific process of each marking operation is as follows:

[0176] The central control unit 6 controls the robot arm 214 to move to the marking pen tip box 212, and controls the robot arm 214 to move to the culture dish 215 to mark after extracting the marking pen tip 213. After marking, the robot arm 214 is controlled to leave the culture area, move to the pen tip recovery pool 218, and release the used marking pen tip 213.

[0177] After the bacterial liquid is cultured in the culture area for a period of time, when the central control unit 6 recognizes from the image data of the culture area that multiple single bacterial colonies have grown in the culture dish 215, the central control unit 6 controls the robot arm to transfer each single bacterial colony to each of the pure culture kettles 22 to complete the sorting operation;

[0178] Specifically, the transfer process is as follows:

[0179] The central control unit 6 controls the mechanical arm 214 to extract the streaking pen tip 213, move it to a single bacterial colony for dipping, and transfer it to the sorting transfer port 219 to release the streaking pen tip 213, and transfer the streaking pen tip 213 dipped with the single bacterial colony to the pure culture kettle 22; repeat this step until all single bacterial colonies are transferred to the pure culture kettle 22 for continued pure culture and preservation;

[0180] This device can carry out continuous, multi-stage enrichment of cultures through dilution and subculture under high-pressure environmental conditions, while online monitoring of environmental indicators within the culture system, realizing the integration of enrichment and sorting devices, transferring cultures under pressure-maintaining, sterile conditions, automatically streaking and sorting the culture medium within the device, and online monitoring of the growth status of single colonies, thereby improving the cultivability and acquisition efficiency of single species of deep-sea microorganisms.

[0181] Example 5

[0182] like Figure 7 As shown, this embodiment provides a method for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment, based on a device for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment described in Example 4, comprising the following steps:

[0183] S1: Clean and sterilize each of the microorganism culture kettles, the sorting operation kettle, and each of the pure culture kettles, and perform pressure leak detection; install each unit in the device, and perform initial configuration;

[0184] S2: Transfer the collected in-situ sediment samples to the first-stage microbial culture reactor to start microbial enrichment;

[0185] S3: using the data acquisition unit to collect the environmental data in each stage of the microorganism culture kettle in real time and transmit it to the central control unit in real time;

[0186] Starting from the first-level microorganism culture kettle, when the environmental data in the previous-level microorganism culture kettle meets the first preset condition, the central control unit controls the liquid transfer mechanism to transfer the bacterial liquid in the previous-level microorganism culture kettle to the next-level microorganism culture kettle for continued enrichment culture;

[0187] S4: When the environmental data in the last-stage microorganism culture kettle meets the second preset condition, the enrichment operation is completed, and the bacterial liquid in the last-stage microorganism culture kettle is transferred to the sorting operation kettle;

[0188] S5: using the data acquisition unit to collect image data of the culture area in the sorting operation kettle in real time and transmit it to the central control unit in real time; the central control unit controls the robot arm to perform several marking operations in the culture area to perform sorting and culture for a preset time;

[0189] S6: When the central control unit recognizes from the image data of the culture area that multiple single bacterial colonies grow in the culture dish, the central control unit controls the robot arm to transfer each single bacterial colony to each of the pure culture kettles to complete the sorting operation;

[0190] In step S1, the initialization configuration includes: using the injection mechanism to inject pre-prepared microbial culture solution into each microbial culture kettle at a certain flow rate; preparing a special culture medium for each culture dish of the sorting operation kettle in advance, and injecting the pre-prepared special culture solution into each pure culture kettle in advance; using the pressure control unit to pressurize each kettle with gas to simulate the high-pressure environment of the deep sea; using the temperature control unit to control the temperature of each kettle to maintain the special temperature environment of the deep sea;

[0191] In step S5, each scribing operation includes:

[0192] The central control unit controls the robot arm to move to the marking pen tip box, controls the robot arm to move to the culture dish to mark after extracting the marking pen tip, and controls the robot arm to leave the culture area after marking, moves to the pen tip recovery pool and releases the marking pen tip;

[0193] In step S6, the central control unit controls the robotic arm to transfer each single bacterial colony to each pure culture tank respectively, including:

[0194] The central control unit controls the robotic arm to extract the streaking pen tip, move it to a single bacterial colony for dipping, transfer it to the sorting transfer port to release the streaking pen tip, and transfer the streaking pen tip dipped in the single bacterial colony to the pure culture kettle.

[0195] In the specific implementation process, before deep-sea microorganism enrichment, the first-level culture kettle 13, the second-level culture kettle 14, the sorting operation kettle 21 and each of the pure culture kettles 22 are first cleaned and sterilized, and pressure leak detection is performed; then the various units in the entire device are installed, and all sensors and other electronic instruments in the device are connected to the central control unit 6, and the instruments are activated and connected to the software of the computer host 61;

[0196] Then, the injection mechanism 11 is used to inject the pre-prepared microbial culture solution into each microbial culture kettle at a certain flow rate; a special culture medium is prepared in advance for each culture dish 215 of the sorting operation kettle 21, and the pre-prepared special culture solution is injected into each pure culture kettle 22; the gas pressurization system 31 in the pressure control unit 3 is used in combination with the pressure sensing system 51 to perform gas pressurization on each kettle to simulate the high-pressure environment of the deep sea, and the PID control valve 33 is opened to set the pressure to form a continuous environmental system; the temperature control unit 4 is used in combination with the temperature sensing system 52 to control the temperature of each kettle to maintain the special temperature environment of the deep sea;

[0197] Then, the collected in-situ sediment sample is transferred to the primary culture kettle 13, and the magnetic stirrer 15 is turned on to start microbial enrichment;

[0198] like Figure 8 As shown, after the primary culture kettle 13 is loaded with the enriched material, the primary enrichment starts, and the environmental indicators are identified by the Raman measurement system 53, the dissolved oxygen measurement system 54, and the pH value measurement system 55. If the environmental indicators meet the following conditions at the same time: dissolved oxygen content <1 μmol / kg, sulfide content >5 mmol / L (sulfide content comes from the Raman measurement system 53), and pH value >8.5, the sample can be transferred, and the sample transfer valve 123 and the sample transfer injection pump 121 are opened to transfer the enriched material into the secondary culture kettle 14 to start the secondary enrichment; if any of these environmental indicators is not met, the primary enrichment continues;

[0199] When the secondary enrichment begins, the environmental indicators are also identified by the Raman measurement system 53, the dissolved oxygen measurement system 54, and the pH value measurement system 55. If the environmental indicators simultaneously meet the conditions of dissolved oxygen content <1 μmol / kg, sulfide content >5 mmol / L, and pH value >8.5, sorting can be performed, the sorting valve 141 is opened, the plunger pump 124 is turned on, and the piston container thereof absorbs 20 mL of liquid and then injects liquid, and the bacterial liquid enters the bacterial liquid pool 217 through the pipeline;

[0200] When the secondary enriched product enters the bacterial liquid pool 217 and starts to be sorted, the central control unit 6 controls the X-axis slide rail 2111, the Y-axis slide rail 2112, and the Z-axis slide rail 2113 of the three-axis moving mechanism 211 to drive the robot 214 to move. The activity range of the three-axis slide rail is from (0, 0, 0) to (100, 100, 100). The position of the robot 214 is represented by the coordinates of the activity range in the three-axis moving mechanism 211.

[0201] Then, the mechanical arm 214 is moved to the position (50, 0) to (80, 5) of the marking pen box 212, and the coil of the magnetic attraction sensor 2142 in the mechanical arm 214 is controlled to be electromagnetically conductive, so as to absorb and install the marking pen 213, which is equipped with 12 pen heads in total, and each pen head is distributed at a distance of 5 coordinate points;

[0202] Then, the robot arm 214 is moved to the position (0, 0) of the bacterial liquid pool 217, and moves back and forth in a small range from (0, 0) to (5, 5) to dip the bacterial liquid; the slide rail is controlled to move the robot arm 214 to the four culture dishes 215 in the culture area to draw lines according to the preset coordinate route, that is, the coordinate system length of each line is 20, the interval length is 3, and each culture dish 215 can draw 8 lines. Therefore, the drawing range of the first culture dish 215 is (20, 40-60), (23, 40-60) ... (38, 40-60), (41, 40 ~60); the marking range of the second culture dish 215 is (45, 60~80), (48, 60~80)…(63, 60~80), (66, 60~80); the marking range of the third culture dish 215 is (45, 20~40), (48, 20~40)…(63, 20~40), (66, 20~40); the marking range of the fourth culture dish 215 is (70, 40~60), (73, 40~60)…(88, 40~60), (91, 40~60);

[0203] During the sorting and marking process, the magnetic attraction sensor 2142 on the robot 214 can sense the magnetic attraction. If the magnetic attraction is 0, it is determined that the marking tip 213 has fallen off. The central control unit 6 records the coordinate point, controls the robot 214 to re-walk the route of the coordinate point to recover the fallen tip, moves the robot 214 to the position (0, 50) of the tip recovery pool 218, and adjusts the electromagnetic magnitude of the robot 214 to zero to release and discard the marking tip 214; if the magnetic attraction is not 0, it is determined that the marking tip 213 is in normal use, and the marking procedure is continued until it is recognized that the robot 214 enters the coordinate interval in each culture dish 215, that is, (41 , 60), (66, 40), (66, 80) and (91, 60), the streaking operation of the culture dish 215 is completed, the robot arm 214 is moved to the position (0, 50) of the pen tip recovery pool 218, and the electromagnetic size of the robot arm 214 is adjusted to zero to release and discard the streaking pen tip 213; finally, the central control unit 6 determines whether the robot arm 214 enters the last streaking coordinate position (91, 60) of the fourth culture dish 215. If the robot arm 214 has not reached the last position, the above streaking operation is continued; if the robot arm 214 has reached the last position, the streaking operation is ended, the sorting operation is completed, and the single colony culture is started;

[0204] During the single colony culture process, based on the image acquisition of the high-definition camera system 56 and the image recognition function of the central control unit 6, when the diameter of the single colony is identified to be greater than 1 mm, the central control unit 6 records the coordinate point of the colony, moves the mechanical arm 214 to the marking pen box 212 to assemble the marking pen 213 and then moves to the coordinate point, the mechanical arm 214 makes the marking pen 213 contact the colony and then moves back and forth twice within a range of 2 mm in the X and Y directions of the position, moves the mechanical arm 214 to the position (50, 100) of the sorting transfer port 219, adjusts the electromagnetic size of the mechanical arm 214 to zero, opens the ball valve 24, and releases the marking pen 213 to make it fall into the connected pure culture kettle 22 for subsequent separate cultivation and preservation, thereby completing the microbial sorting;

[0205] This method can carry out continuous, multi-stage enrichment of cultures through dilution and subculture under high-pressure environmental conditions, while online monitoring of environmental indicators within the culture system, realizing the integration of enrichment and sorting devices, transferring cultures under pressure-maintaining, aseptic conditions, automatically streaking and sorting the culture medium within the device, and online monitoring of the growth status of single colonies, thereby improving the cultivability and acquisition efficiency of single species of deep-sea microorganisms.

[0206] The same or similar reference numerals correspond to the same or similar components;

[0207] The terms used in the drawings to describe positional relationships are only used for illustrative purposes and should not be construed as limiting this patent;

[0208] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A high-pressure environment deep-sea microorganism online enrichment device, characterized in that: include: A multi-stage enrichment unit, a first pressure control unit, a first temperature control unit, a first data acquisition unit and a first central control unit; The multi-stage enrichment unit comprises a liquid injection mechanism, a liquid transfer mechanism and at least two microorganism culture kettles connected in series, wherein the liquid injection mechanism is used to inject microorganism culture liquid into each microorganism culture kettle; the first-stage microorganism culture kettle is filled with an in-situ sediment sample for microorganism enrichment; the liquid transfer mechanism is used to transfer the bacterial liquid in the upper-stage microorganism culture kettle to the lower-stage microorganism culture kettle; The first pressure control unit and the first temperature control unit are connected to the first central control unit and each microorganism culture kettle, respectively, and are used to control the pressure and temperature in each kettle, respectively; The first data acquisition unit is connected to the first central control unit, and the first data acquisition unit is used to collect environmental data in each microorganism culture kettle in real time and transmit it to the first central control unit in real time; The first central control unit is also respectively connected to each microbial culture kettle, liquid injection mechanism, liquid transfer mechanism, first pressure control unit and first temperature control unit. The first central control unit is used to control the high-pressure environment deep-sea microbial online enrichment device according to the data collected by the first data acquisition unit.

2. The device for online enrichment of deep-sea microorganisms in a high-pressure environment according to claim 1, characterized in that: In the multi-stage enrichment unit, the injection mechanism includes a continuous injection pump and a plurality of injection valves, each of which is arranged on the microbial culture liquid inlet pipeline of each microbial culture kettle in a one-to-one correspondence; the continuous injection pump is connected to each injection valve respectively; The liquid transfer mechanism includes a sample transfer injection pump, a plurality of groups of filters and sample transfer valves, and a plunger pump; the sample transfer injection pump is respectively connected to all microbial culture kettles except the last stage, and a group of filters and sample transfer valves are arranged on the connecting pipeline between each two stages of microbial culture kettles connected in series; the plunger pump is connected to the last stage of microbial culture kettle; Each of the microorganism culture kettles has the same structure, and is provided with a magnetic stirrer and a sampling valve at the bottom; In all microbial culture kettles except the last one, a sample transfer piston is arranged on the top and a protruding ring is arranged in the middle; the sample transfer piston is used to reduce the pressure difference required for transferring the bacterial liquid in the upper microbial culture kettle to the lower microbial culture kettle; the protruding ring is used to prevent the sample transfer piston from being over-pressurized.

3. The device for online enrichment of deep-sea microorganisms in a high-pressure environment according to claim 1, characterized in that: The first pressure control unit includes: a gas pressurization system, a plurality of gas injection valves and a plurality of PID control valves; Each of the microorganism culture kettles is provided with a gas injection valve; the gas pressurization system is connected to each gas injection valve through a pipeline, and the gas pressurization system is connected to the first central control unit for pressurizing the gas for each kettle; Each of the microorganism culture kettles is also provided with a PID control valve, and each of the PID control valves is connected to a central control unit; the PID control valve is used to control the formation of a continuous pressure environment in the microorganism culture kettles connected in series; The first temperature control unit includes: an air conditioning system; The air conditioning system is respectively connected to the first central control unit and each of the microorganism culture kettles to control the temperature in each kettle; The first data acquisition unit includes: a pressure sensing system, a temperature sensing system, a Raman measurement system, a dissolved oxygen measurement system and a pH value measurement system; The pressure sensing system and the temperature sensing system are respectively connected to the first central control unit and each of the microorganism culture kettles, and are respectively used to collect the pressure and temperature in each kettle in real time and transmit them to the first central control unit in real time; The Raman measurement system, dissolved oxygen measurement system and pH value measurement system are respectively connected to the first central control unit and each of the microbial culture kettles, and are respectively used to observe the changes in the chemical substance content, dissolved oxygen content and pH value in each microbial culture kettle in real time online and transmit them to the first central control unit in real time.

4. An online automatic sorting device for deep-sea microorganisms in a high-pressure environment, characterized in that: include: Automatic sorting unit, second pressure control unit, second temperature control unit, second data acquisition unit and second central control unit; The automatic sorting unit includes a sorting operation kettle and a plurality of pure culture kettles; the sorting operation kettle is connected to each pure culture kettle respectively; The sorting operation kettle comprises an internal three-axis moving mechanism and at least one marking pen tip box, wherein a plurality of marking pen tips are placed in the marking pen tip box, and a moving Z-axis of the three-axis moving mechanism is provided with a mechanical arm capable of extracting marking pen tips from the marking pen tip box, and the mechanical arm is used to drive the marking pen tips to perform sorting operations; the X-axis and Y-axis planes of the three-axis moving mechanism are provided with a culture area consisting of a plurality of culture dishes; The second pressure control unit and the second temperature control unit are respectively connected to the second central control unit, the sorting operation kettle and each pure culture kettle, and are used to control the pressure and temperature in each kettle respectively; The second data acquisition unit is connected to the second central control unit, and the second data acquisition unit is used to collect image data of the culture area in the sorting operation kettle in real time, and transmit it to the second central control unit in real time; The second central control unit is also connected to the robotic arm, the second pressure control unit and the second temperature control unit respectively. The second central control unit is used to control the high-pressure environment deep-sea microorganism online automatic sorting device according to the data collected by the second data acquisition unit.

5. The device for automatically sorting deep-sea microorganisms in a high-pressure environment according to claim 4, characterized in that: In the automatic sorting unit, the sorting operation kettle comprises an upper kettle body and a lower kettle body which are sealed and connected; The upper surface of the upper kettle body is provided with a pressure-resistant window for observing the interior of the sorting operation kettle; The lower kettle body is provided with a bacterial liquid pool, a three-axis moving mechanism, at least one scribing pen tip box, a plurality of culture dishes, a pen tip recovery pool and a sorting transfer port; The bacterial liquid pool is used to hold the bacterial liquid to be sorted; The three-axis moving mechanism is provided with three vertically intersecting slide rails of X-axis, Y-axis and Z-axis, and the mechanical arm is arranged on the Z-axis slide rail; The mechanical arm comprises: a vertical rod and a magnetic attraction sensor and a fixed block arranged at both ends of the vertical rod; the magnetic attraction sensor is connected to the second central control unit and the fixed block respectively, the magnetic attraction sensor provides magnetic force for the fixed block, and the fixed block extracts the marking pen tip through the magnetic force; The robot arm is used to perform the following operations: extract and install the marking pen tip in the marking pen tip box; drive the marking pen tip to dip into the bacterial solution in the bacterial solution pool and the culture dish; drive the marking pen tip to mark and sort in the culture dish; release the marking pen tip in the pen tip recovery pool and the sorting transfer port; The culture dish is pre-filled with a special culture medium for culturing a specific single bacterial colony to separate the desired special microbial species; The pen tip recovery pool is used to recover used marking pen tips; The sorting transfer port is connected to each of the pure culture kettles through a pipeline and a quick-connect interface, and the sorting transfer port is used to transfer the streaking pen tip dipped in a single bacterial colony after sorting to the pure culture kettle.

6. The device for automatically sorting deep-sea microorganisms in a high-pressure environment according to claim 4, characterized in that: The second pressure control unit includes: a gas pressurizing system, an exhaust valve and a plurality of gas injection valves; The sorting operation kettle and each pure culture kettle are respectively provided with a gas injection valve; the gas pressurizing system is respectively connected to each gas injection valve through a pipeline, and the gas pressurizing system is connected to the second central control unit for performing gas pressurization for each kettle; The exhaust valve is arranged on the sorting operation kettle and is used for exhausting the sorting operation kettle; The second temperature control unit includes: an air conditioning system; The air conditioning system is respectively connected to the second central control unit, the sorting operation kettle and each pure culture kettle, and is used to control the temperature in each kettle. The second data acquisition unit includes a pressure sensing system, a temperature sensing system and a camera system; The pressure sensing system and the temperature sensing system are respectively connected to the second central control unit, the sorting operation kettle and each pure culture kettle, and are respectively used to collect the pressure and temperature in each kettle in real time and transmit them to the second central control unit in real time; The camera system is arranged on the top of the sorting operation kettle and is connected to the second central control unit. The camera system is used for taking real-time image data of the culture area in the sorting operation kettle and transmitting the image data to the second central control unit in real time.

7. A device for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment, comprising an enrichment device and a sorting device connected in sequence, characterized in that: The enrichment device is specifically the high-pressure environment deep-sea microorganism online enrichment device described in any one of claims 1 to 3; the sorting device is specifically the high-pressure environment deep-sea microorganism online automatic sorting device described in any one of claims 4 to 6.

8. The device for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment according to claim 7, characterized in that: The last stage microorganism culture kettle in the multi-stage enrichment unit is connected to the bacterial liquid pool in the sorting operation kettle; The bacterial liquid pool is used to hold the bacterial liquid to be sorted after being enriched in the last stage of microbial culture kettle.

9. A method for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment, based on a device for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment as claimed in any one of claims 7 to 8, characterized in that: The following steps are involved: S1: Clean and sterilize each of the microorganism culture kettles, the sorting operation kettle, and each of the pure culture kettles, and perform pressure leak detection; install each unit in the device, and perform initial configuration; S2: Transfer the collected in-situ sediment samples to the first-stage microbial culture reactor to start microbial enrichment; S3: using the first data acquisition unit to collect the environmental data in each stage of the microorganism culture kettle in real time and transmit it to the first central control unit in real time; Starting from the first-level microorganism culture kettle, when the environmental data in the previous-level microorganism culture kettle meets the first preset condition, the first central control unit controls the liquid transfer mechanism to transfer the bacterial liquid in the previous-level microorganism culture kettle to the next-level microorganism culture kettle for continued enrichment culture; S4: When the environmental data in the last-stage microorganism culture kettle meets the second preset condition, the enrichment operation is completed, and the bacterial liquid in the last-stage microorganism culture kettle is transferred to the sorting operation kettle; S5: using the second data acquisition unit to collect image data of the culture area in the sorting operation kettle in real time and transmit it to the second central control unit in real time; the second central control unit controls the robot arm to perform several marking operations in the culture area to perform sorting and culture for a preset time; S6: When the second central control unit recognizes from the image data of the culture area that multiple single bacterial colonies are grown in the culture dish, the second central control unit controls the robot arm to transfer each single bacterial colony to each of the pure culture kettles respectively, to complete the sorting operation.

10. A method for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment according to claim 9, characterized in that: In step S1, the initialization configuration includes: using the injection mechanism to inject pre-prepared microbial culture solution into each microbial culture kettle at a certain flow rate; preparing a special culture medium for each culture dish of the sorting operation kettle in advance, and injecting the pre-prepared special culture solution into each pure culture kettle in advance; using the first and second pressure control units to perform gas pressurization on each kettle to simulate the high-pressure environment of the deep sea; using the first and second temperature control units to control the temperature of each kettle to maintain the special temperature environment of the deep sea; In step S5, each scribing operation includes: The second central control unit controls the robot arm to move to the marking pen tip box, controls the robot arm to move to the culture dish to mark after extracting the marking pen tip, and controls the robot arm to leave the culture area after marking, moves to the pen tip recovery pool and releases the marking pen tip; In step S6, the second central control unit controls the robot arm to transfer each single bacterial colony to each pure culture tank respectively, including: The second central control unit controls the robotic arm to extract the streaking pen tip, move it to a single bacterial colony for dipping, transfer it to the sorting transfer port to release the streaking pen tip, and transfer the streaking pen tip dipped in the single bacterial colony to the pure culture kettle.

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