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

Abstract

This invention provides an online enrichment and automatic sorting device and method for deep-sea microorganisms under high pressure. It simulates the high pressure and unique temperature environment of the deep sea for multi-stage enrichment. Simultaneously, based on changes in environmental indicators observed online within the enrichment device, it automatically determines the optimal time point for microbial passage and transfers the culture to the next stage of the enrichment device. This effectively solves the problems of difficulty in transferring microorganisms at the optimal time point and complex manual operation in high-pressure environments, thus improving the initial enrichment efficiency for obtaining single species of deep-sea microorganisms. Furthermore, this invention solves the problems of overlapping single colonies and low separation, complex manual operation, and difficulty in automatic secondary sorting under high pressure by automatically streaking the enriched material in the culture medium under high pressure, as well as automatically transferring specific single colonies to a pure culture vessel. This significantly improves the efficiency of deep-sea microorganism sorting under high pressure.

Description

Technical Field

[0001] This invention relates to the field of marine microbial enrichment culture and individual microbial sorting technology, and more specifically, to a device and method for online enrichment and automatic sorting of deep-sea microorganisms under high pressure environment. Background Technology

[0002] Deep-sea ecosystems, due to their unique environmental conditions, have fostered a variety of special habitats such as cold seeps and hydrothermal vents. In extreme environments such as high pressure and low / high temperatures, deep-sea microorganisms exhibit extremely high uniqueness and diversity in their genetic structure and physiological metabolism. These deep-sea microorganisms play an irreplaceable role in promoting marine energy transfer and material cycling, as well as maintaining the stability of marine and even global ecosystems.

[0003] To investigate the unique role of a particular 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 features like growth and metabolism. However, current technologies for sorting and pure culturing deep-sea microorganisms are still immature. Once collected and transferred to the laboratory, deep-sea microorganisms are removed from their original high-pressure environment, making them prone to cell rupture and survival. This significantly increases the difficulty of efficiently isolating and culturing single microbial species. Therefore, high-pressure culture is an indispensable tool in the field of deep-sea microbiology research.

[0004] For the enrichment of deep-sea microorganisms under simulated environmental conditions, the existing technology discloses a device and method for enrichment and multi-level purification of deep-sea microorganisms under high pressure environment. 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 in the enriched bacterial solution inside the device online, and therefore it is impossible to transfer the enriched material according to the reaction substances. It is difficult to pass the microorganisms at the optimal time point of the inflection point of the metabolic substance change. It has the limitations of complicated manual operation and low microbial sorting activity.

[0005] For the sorting of deep-sea microorganisms under simulated environmental conditions, existing technologies also disclose a system and method for high-throughput automatic screening of single colonies under heat preservation and pressure in deep-sea environments. Existing automatic sorting technologies separate enriched bacterial solutions by spraying, which has problems such as low bioseparation and easy colony overlap, which is not conducive to the rapid growth and effective separation of single microbial colonies. In addition, existing technologies also disclose a biocoating device. This automatic sorting technology uses a robotic arm to coat the bacterial solution onto the culture medium for separation. However, when applied in a high-pressure environment, the culture medium is uneven or tilted in the petri dish, which may cause the coating rod tip to fall off, resulting in streaking failure and difficulty in recovering the tip for re-coating. This presents problems such as complex manual sorting operations and difficulty in automatic re-sorting after automatic coating failure.

[0006] In general, the artificial operation of microbial enrichment is complicated, the separation degree of the enriched material is low, the activity of sorted microorganisms is low, and the automatic secondary sorting is difficult, making it difficult to efficiently obtain single species of deep-sea microorganisms. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies in deep-sea microbial enrichment, such as complex manual operations, low separation of enriched materials, low activity of sorted microorganisms, and difficulty in automatic secondary sorting, this invention provides a high-pressure environment for online enrichment and automatic sorting of deep-sea microorganisms. Through online monitoring of the enrichment environment, automatic determination of the optimal time for microbial passage, automatic streaking separation of enriched materials, automatic secondary sorting, and automatic transfer of specific single colonies, this invention improves the culturability and acquisition efficiency of single species of deep-sea microorganisms, providing an important foundational means for enhancing the understanding of deep-sea life science laws and the efficiency of biological resource development and application.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] A high-pressure environment deep-sea microbial online enrichment device includes: 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 includes a liquid injection mechanism, a liquid transfer mechanism, and at least two microbial culture vessels connected in series. The liquid injection mechanism is used to inject microbial culture medium into each of the microbial culture vessels. The first-stage microbial culture vessel contains in-situ sediment samples for microbial enrichment. The liquid transfer mechanism is used to transfer the bacterial culture medium in the previous-stage microbial culture vessel to the next-stage microbial culture vessel.

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

[0012] The first data acquisition unit is connected to the first central control unit. The first data acquisition unit is used to collect environmental data in each microbial culture vessel in real time and transmit it to the first central control unit in real time.

[0013] The first central control unit is also connected to each microbial culture vessel, liquid injection mechanism, liquid transfer mechanism, first pressure control unit and first temperature control unit respectively. 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 liquid injection mechanism includes a continuous liquid injection pump and a plurality of liquid injection valves, each of the liquid injection valves being installed one-to-one on the microbial culture medium inlet pipe of each microbial culture vessel; the continuous liquid injection pump is connected to each liquid injection valve respectively;

[0015] The liquid transfer mechanism includes a sample transfer pump, several sets of filters and transfer valves, and a plunger pump; the sample transfer pump is connected to all microbial culture vessels except the last stage, and a set of filters and transfer valves are provided on the connecting pipeline between every two stages of microbial culture vessels connected in series; the plunger pump is connected to the last stage microbial culture vessel.

[0016] Each of the aforementioned microbial culture vessels has the same structure, and each is equipped with a magnetic stirrer and a sampling valve at the bottom;

[0017] In all microbial culture vessels except the last stage, a transfer piston is provided at the top and a protruding ring is provided in the middle. The transfer piston is used to reduce the pressure difference required for the bacterial solution in the previous stage microbial culture vessel to be transferred to the next stage microbial culture vessel. The protruding ring is used to prevent the transfer piston from being pressurized too much.

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

[0019] Each of the aforementioned microbial culture vessels is equipped with a gas injection valve; the gas pressurization system is connected to each gas injection valve via pipelines, and the gas pressurization system is connected to the first central control unit for pressurizing the gas in each vessel.

[0020] Each of the microbial culture vessels is also equipped with a PID control valve, and each of the PID control valves is connected to the central control unit; the PID control valve is used to control the formation of a continuous pressure environment in the series-connected microbial culture vessels.

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

[0022] The air conditioning system is connected to the first central control unit and each of the microbial culture vessels, respectively, and is used to control the temperature inside each vessel;

[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 measurement system;

[0024] The pressure sensing system and temperature sensing system are respectively connected to the first central control unit and each of the microbial culture vessels, and are used to collect the pressure and temperature in each vessel 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 measurement system are all connected to the first central control unit and each of the microbial culture vessels, respectively, and are used to observe the changes in chemical content, dissolved oxygen content, and pH value in each microbial culture vessel in real time 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 vessel and several pure culture vessels; the sorting operation vessel is connected to each pure culture vessel respectively;

[0028] The sorting operation vessel includes an internal three-axis moving mechanism and at least one marking pen tip box. The marking pen tip box contains multiple marking pen tips. The Z-axis of the three-axis moving mechanism is equipped with a robotic arm that can extract the marking pen tips from the marking pen tip box. The robotic 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 zone composed of multiple petri 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 vessel and each pure culture vessel, and are used to control the pressure and temperature in each vessel;

[0030] The second data acquisition unit is connected to the second central control unit. The second data acquisition unit is used to acquire image data of the culture area inside the sorting operation vessel 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. The second central control unit is used to control the high-pressure environment deep-sea microorganism online automatic sorting device based on the data collected by the second data acquisition unit.

[0032] Preferably, in the automatic sorting unit, the sorting operation vessel includes an upper vessel body and a lower vessel body that are sealed together;

[0033] The upper surface of the upper vessel is provided with a pressure-resistant viewing window for observing the inside of the sorting operation vessel;

[0034] The lower vessel is equipped with a bacterial liquid pool, a three-axis moving mechanism, at least one streak pen tip box, multiple petri dishes, a pen tip recycling pool, and a sorting and transfer port.

[0035] The bacterial solution tank is used to hold the bacterial solution to be sorted.

[0036] The three-axis moving mechanism is provided with three vertically intersecting slide rails: the X-axis, the Y-axis, and the Z-axis. The robotic arm is mounted on the Z-axis slide rail.

[0037] The robotic arm includes a vertical rod and magnetic attraction sensors and fixed blocks disposed at both ends of the vertical rod; the magnetic attraction sensors are respectively connected to the second central control unit and the fixed blocks, the magnetic attraction sensors provide magnetic force to the fixed blocks, and the fixed blocks extract the marking pen tip through magnetic force;

[0038] The robotic arm is used to perform the following operations: extracting and installing a streak pen tip from a streak pen tip box; dipping the streak pen tip into the bacterial solution in the bacterial solution pool and petri dish; streak the streak pen tip in the petri dish for sorting; and releasing the streak pen tip in the pen tip recycling pool and sorting transfer port.

[0039] The petri dish contains a special culture medium for culturing specific single colonies in order to sort out the desired special microbial species;

[0040] The pen tip recycling pool is used to recycle used drawing pen tips;

[0041] The sorting and transfer port is connected to each of the pure culture vessels via a pipeline and a quick-connect interface. The sorting and transfer port is used to transfer the streak pen tip dipped with a single colony after sorting to the pure culture vessel.

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

[0043] Each sorting operation vessel and each pure culture vessel are equipped with a gas injection valve; the gas pressurization system is connected to each gas injection valve through pipelines, and the gas pressurization system is connected to the second central control unit for pressurizing gas in each vessel.

[0044] The exhaust valve is installed on the sorting operation vessel and is used for exhausting the wastewater from the sorting operation vessel;

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

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

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

[0049] The camera system is installed on the top of the sorting operation vessel and connected to the second central control unit. The camera system is used to capture image data of the culture area inside the sorting operation vessel in real time and transmit it 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 microbial culture vessel in the multi-stage enrichment unit is connected to the bacterial solution pool in the sorting operation vessel;

[0052] The bacterial solution tank is used to hold the bacterial solution to be sorted after enrichment in the last stage of microbial culture vessel.

[0053] This invention also provides a method for online enrichment and automatic sorting of deep-sea microorganisms under high pressure environment, based on the above-mentioned device for online enrichment and automatic sorting of deep-sea microorganisms under high pressure environment, comprising the following steps:

[0054] S1: Clean and sterilize each of the aforementioned microbial culture vessels, sorting operation vessels, and pure culture vessels, and perform pressure leak testing; 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 vessel to begin microbial enrichment;

[0056] S3: The first data acquisition unit collects environmental data in each stage of the microbial culture vessel in real time and transmits it to the first central control unit in real time.

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

[0058] S4: When the environmental data in the last-stage microbial culture vessel meets the second preset condition, the enrichment operation is completed, and the bacterial solution in the last-stage microbial culture vessel is transferred to the sorting operation vessel.

[0059] S5: The second data acquisition unit collects image data of the culture area inside the sorting operation vessel in real time and transmits it to the second central control unit in real time; the second central control unit controls the robotic arm to perform several marking operations in the culture area to carry out sorting and culture for a preset time.

[0060] S6: When the second central control unit identifies multiple single colonies growing in the culture dish from the image data of the culture area, the second central control unit controls the robotic arm to transfer each single colony to each of the pure culture vessels to complete the sorting operation.

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

[0062] In step S5, each line-drawing operation includes:

[0063] The second central control unit controls the robotic arm to move to the marking pen tip box, extracts the marking pen tip, and then controls the robotic arm to move to the culture dish to mark the lines. After marking the lines, the robotic arm is controlled to leave the culture area, move to the pen tip recycling pool, and release the marking pen tip.

[0064] In step S6, the second central control unit controls the robotic arm to transfer each single colony to each of the pure culture vessels, including:

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

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

[0067] This invention provides an online enrichment device for deep-sea microorganisms under high pressure. It 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 within the enrichment device, it automatically determines the optimal time point for microbial passage and transfers the culture to the next level of enrichment device. This effectively solves the problems of difficulty in transferring microorganisms at the optimal time point and complexity of manual operation in high-pressure environments, and improves the early enrichment efficiency of obtaining 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. By automatically streaking and separating the enriched material in a culture medium under high pressure, automatically sorting it again, and automatically transferring specific single colonies to a pure culture vessel, the present invention solves the problems of single colony overlap and low separation degree, complex manual operation, and difficulty in automatic secondary sorting under high pressure environment in existing high-pressure sorting technologies, and greatly improves the efficiency of sorting deep-sea microorganisms under high pressure environment.

[0069] This invention combines the two to form an online enrichment and automatic sorting device, which can continuously and multi-stage enrich cultures through dilution and subculturing under high-pressure conditions, while simultaneously monitoring environmental indicators within the culture system online. This achieves the integration of enrichment and sorting devices, allowing for the transfer of cultures under pressure and aseptic conditions, automatic streaking and sorting of culture media within the device, and online monitoring of single colony growth status. This improves the culturability and acquisition efficiency of single species of deep-sea microorganisms, providing an important foundational means for enhancing the understanding of deep-sea life science laws and the efficiency of biological resource development and application. Attached Figure Description

[0070] Figure 1 This is a framework diagram of an online enrichment device for deep-sea microorganisms in a high-pressure environment, as 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, as 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 vessel provided in Example 4.

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

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

[0077] Figure 8 This is a flowchart illustrating the specific operation of online enrichment and automatic sorting provided in Example 5. Detailed Implementation

[0078] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the scope of this patent.

[0079] To better illustrate this embodiment, some parts in the accompanying drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions;

[0080] It will be understood by those skilled in the art that certain well-known structures and their descriptions may be omitted in the accompanying drawings.

[0081] The technical solution of the present invention will be further described below with reference to 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 includes a liquid injection mechanism, a liquid transfer mechanism, and at least two microbial culture vessels connected in series. The liquid injection mechanism is used to inject microbial culture medium into each of the microbial culture vessels. The first-stage microbial culture vessel contains in-situ sediment samples for microbial enrichment. The liquid transfer mechanism is used to transfer the bacterial culture medium in the previous-stage microbial culture vessel to the next-stage microbial culture vessel.

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

[0086] The first data acquisition unit is connected to the first central control unit. The first data acquisition unit is used to collect environmental data in each microbial culture vessel in real time and transmit it to the first central control unit in real time.

[0087] The first central control unit is also connected to each microbial culture vessel, liquid injection mechanism, liquid transfer mechanism, first pressure control unit and first temperature control unit respectively. 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 the enrichment of deep-sea microorganisms, each microbial culture vessel is first cleaned and sterilized, and pressure leak detection is performed; then, each unit in the entire device is installed, and all electronic instruments such as sensors in the device are connected to the first central control unit, the instruments are activated online and connected to the software of the first central control unit.

[0089] The collected in-situ sediment samples were then transferred to the first-stage microbial culture vessel to begin microbial enrichment.

[0090] The first data acquisition unit collects environmental data from each stage of the microbial culture vessel in real time and transmits it to the first central control unit in real time.

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

[0092] When the environmental data in the last-stage microbial culture vessel meets the second preset condition, the multi-stage enrichment operation is completed.

[0093] This 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 within the enrichment device, it automatically determines the optimal time point for microbial passage and transfers the culture to the next stage of enrichment device. This effectively solves the problems of difficulty in transferring microbial enrichment at the optimal time point under high pressure environment and the complexity of human operation, and improves the early enrichment efficiency of obtaining 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, including: 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 vessel and several pure culture vessels; the sorting operation vessel is connected to each pure culture vessel respectively;

[0097] The sorting operation vessel includes an internal three-axis moving mechanism and at least one marking pen tip box. The marking pen tip box contains multiple marking pen tips. The Z-axis of the three-axis moving mechanism is equipped with a robotic arm that can extract the marking pen tips from the marking pen tip box. The robotic 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 zone composed of multiple petri 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 vessel and each pure culture vessel, and are used to control the pressure and temperature in each vessel;

[0099] The second data acquisition unit is connected to the second central control unit. The second data acquisition unit is used to acquire image data of the culture area inside the sorting operation vessel 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. The second central control unit is used to control the high-pressure environment deep-sea microorganism online automatic sorting device based on the data collected by the second data acquisition unit.

[0101] In the specific implementation process, before sorting deep-sea microorganisms, the sorting operation vessel and each pure culture vessel are 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 central control unit, the instruments are activated online and connected to the software of the second central control unit.

[0102] Transfer the bacterial solution to be sorted to the sorting operation tank;

[0103] The second data acquisition unit collects image data of the culture area inside the sorting operation vessel in real time and transmits it to the second central control unit in real time; the second central control unit controls the robotic arm to perform several marking operations in the culture area to carry out sorting and culture for a preset time.

[0104] When the second central control unit identifies multiple single colonies growing in the culture dish from the image data of the culture area, the second central control unit controls the robotic arm to transfer each single colony to each pure culture vessel to complete the sorting operation;

[0105] This device solves the problems of existing high-pressure sorting technologies, such as overlapping single colonies and low separation, complex manual operation, and difficulty in automatic secondary sorting in high-pressure environments, by automatically streaking and separating enriched substances in culture media under high pressure, automatically sorting secondary colonies, and automatically transferring specific single colonies to pure culture tanks. It greatly improves the efficiency of high-pressure environment deep-sea microbial sorting.

[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 Embodiment 1 and the online automatic sorting device for deep-sea microorganisms in a high-pressure environment in Embodiment 2, including: 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, temperature control unit, data acquisition unit, and 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 operation of the entire device (including enrichment and sorting devices).

[0109] The multi-stage enrichment unit includes a liquid injection mechanism, a liquid transfer mechanism, and at least two microbial culture vessels connected in series. The liquid injection mechanism is used to inject microbial culture medium into each of the microbial culture vessels. The first-stage microbial culture vessel contains in-situ sediment samples for microbial enrichment. The liquid transfer mechanism is used to transfer the bacterial culture medium in the previous-stage microbial culture vessel to the next-stage microbial culture vessel.

[0110] The automatic sorting unit includes a sorting operation vessel and several pure culture vessels; the sorting operation vessel is connected to each pure culture vessel respectively;

[0111] The sorting operation vessel includes an internal three-axis moving mechanism and at least one marking pen tip box. The marking pen tip box contains multiple marking pen tips. The Z-axis of the three-axis moving mechanism is equipped with a robotic arm that can extract the marking pen tips from the marking pen tip box. The robotic 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 zone composed of multiple petri dishes.

[0112] The last stage of the multi-stage enrichment unit is connected to the sorting operation vessel;

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

[0114] The data acquisition unit is connected to the central control unit. The data acquisition unit is used to collect environmental data and image data of the culture area in each microbial culture vessel in real time and transmit them to the central control unit in real time.

[0115] The central control unit is also connected to each microbial culture vessel, 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 microbial online enrichment and automatic sorting device according to the data collected by the data acquisition unit.

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

[0117] The collected in-situ sediment samples were then transferred to the first-stage microbial culture vessel to begin microbial enrichment.

[0118] The data acquisition unit collects environmental data from each stage of the microbial culture vessel in real time and transmits it to the central control unit in real time.

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

[0120] When the environmental data in the last-stage microbial culture vessel meets the second preset condition, the enrichment operation is completed, and the bacterial solution in the last-stage microbial culture vessel is transferred to the sorting operation vessel.

[0121] Subsequently, the data acquisition unit is used to acquire image data of the culture area inside the sorting operation vessel in real time and transmit it to the central control unit in real time.

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

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

[0124] This device enables continuous, multi-stage enrichment of cultures through dilution and subculturing under high-pressure conditions, while simultaneously monitoring environmental indicators within the culture system online. It integrates enrichment and sorting devices, transfers cultures under pressure and aseptic conditions, automatically performs streaking and sorting on the culture medium within the device, and monitors the growth status of single colonies online, thereby improving the culturability 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 includes a liquid injection mechanism 11, a liquid transfer mechanism 12, and two microbial culture vessels connected in series. In this embodiment, these are a primary culture vessel 13 and a secondary culture vessel 14. The liquid injection mechanism 11 is used to inject microbial culture medium into each of the microbial culture vessels. The primary culture vessel 13 contains in-situ sediment samples for microbial enrichment. The liquid transfer mechanism 12 is used to transfer the bacterial solution in the primary culture vessel 13 to the secondary culture vessel 14.

[0128] The automatic sorting unit 2 includes a sorting operation vessel 21 and several pure culture vessels 22; the sorting operation vessel 21 is connected to each pure culture vessel 22 respectively.

[0129] The sorting operation vessel 21 includes an internal three-axis moving mechanism 211 and at least one marking pen tip box 212. The marking pen tip box 212 contains a plurality of marking pen tips 213. The Z-axis of the three-axis moving mechanism 211 is equipped with a robotic arm 214 that can extract the marking pen tips 213 from the marking pen tip box 212. The robotic 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 zone composed of a plurality of culture dishes 215.

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

[0131] The pressure control unit 3 and temperature control unit 4 are respectively connected to the central control unit 6, the sorting operation vessel 21, the primary culture vessel 13, the secondary culture vessel 14 and each pure culture vessel 22, and are used to control the pressure and temperature in each vessel respectively.

[0132] The data acquisition unit 5 is connected to the central control unit 6. The data acquisition unit 5 is used to collect environmental data in the primary culture vessel 13 and the secondary culture vessel 14 in real time, as well as image data of the culture area in the sorting operation vessel 21, and transmit them to the central control unit 6 in real time.

[0133] The central control unit 6 is also connected to the primary culture vessel 13, the secondary culture vessel 14, the liquid injection mechanism 11, the liquid transfer mechanism 12, the robotic arm 214, the pressure control unit 3, and the temperature control unit 4 respectively. 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 liquid injection mechanism 11 includes a continuous liquid injection pump 111 and two liquid injection valves 112. Each of the liquid injection valves 112 is correspondingly installed on the microbial culture medium inlet pipe of each microbial culture vessel. The continuous liquid injection pump 111 is connected to each liquid injection valve 112.

[0135] The liquid transfer mechanism 12 includes a sample transfer pump 121, a set of filters 122 and a sample transfer valve 123, and a plunger pump 124. The sample transfer pump 121 is connected to the primary culture vessel 13 via a pressure holding valve 125, which is used to maintain a constant pressure during the liquid transfer process. A set of filters 122 and a sample transfer valve 123 are provided on the connecting pipeline between the primary culture vessel 13 and the secondary culture vessel 14 to realize the transfer and subculturing process of primary and secondary enrichment of the culture. The plunger pump 124 is connected to the secondary culture vessel 14 and is used to transfer the secondary culture vessel 14 to the sorting operation vessel 21.

[0136] The primary culture vessel 13 and the secondary culture vessel 14 have the same structure, and both are equipped with a magnetic stirrer 15 and a sampling valve 16 at the bottom; the magnetic stirrer 15 is used to stir the culture medium in the vessel; the sampling valve 16 is used to periodically collect the culture and perform gene sequencing analysis.

[0137] In the primary culture vessel 13, a sample transfer piston 131 is provided at the top, and a protruding ring 132 with a narrower inner diameter is provided in the middle. The protruding ring 132 and the primary culture vessel 13 are an integral structure.

[0138] The sample transfer piston 131 is used to reduce the pressure difference required for transferring the bacterial culture to the secondary culture vessel 14. In this embodiment, the upper part of the sample transfer piston 131 is connected to the pressure holding valve 125 and the sample transfer injection pump 121, thereby forming a pressure holding layer in the upper part of the primary culture vessel 13. This layer is used to maintain the pressure for primary enrichment of the culture and to promote the transfer and subculturing process of the culture using a smaller pressure difference. In addition, the sample transfer piston 131 is also provided with a pipeline for installing a temperature sensor 521 and connecting an air injection valve 32, so as to facilitate a suitable spatial arrangement of the interfaces.

[0139] The protruding ring 132 is used to prevent the sample transfer piston 131 from being pressurized too much, which could damage the magnetic stirrer 15 at the bottom and the sensor interface in the data acquisition unit 5.

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

[0141] In the automatic sorting unit 2, the sorting operation vessel 21 includes an upper vessel body and a lower vessel body that are sealed together; the upper vessel body and the lower vessel body are connected and sealed by a sealing ring and multiple screws 23 on the outer ring.

[0142] The upper surface of the upper vessel body is provided with a pressure-resistant viewing window 216. The pressure-resistant viewing window 216 is large and made of pressure-resistant material, and is used to observe the inside of the sorting operation vessel 21.

[0143] The lower vessel body is equipped with a bacterial culture tank 217, a three-axis moving mechanism 211, at least one streaking pen tip box 212, multiple petri dishes 215, a pen tip recovery tank 218, and a sorting and transfer port 219. A top view of the lower vessel body is shown below. Figure 5 As shown;

[0144] The bacterial solution tank 217 is used to hold the bacterial solution to be sorted after enrichment in the secondary culture vessel 14.

[0145] The three-axis moving mechanism 211 is equipped with three vertically intersecting slide rails: an X-axis, a Y-axis, and a Z-axis. The Y-axis slide rail 2112 is mounted on the X-axis slide rail 2111 via a connecting block to enable the robotic arm 214 to move laterally in the plane. The Z-axis slide rail 2113 is mounted on the Y-axis slide rail 2112 via a connecting block to enable the robotic arm 214 to move vertically in the plane. The robotic arm 214 is mounted on the Z-axis slide rail 2113 via a connecting block to enable the robotic arm 214 to move in a direction perpendicular to the plane.

[0146] The culture area has a circular structure and contains four culture dishes 215. Each culture dish 215 is pre-filled with a special culture medium for culturing specific single colonies in order to sort out the desired special microbial species.

[0147] The pen tip recycling pool 218 is used to recycle used drawing pen tips 213;

[0148] The sorting and transfer port 219 is connected to each of the pure culture vessels 22 via pipelines, ball valves 24 and quick-connect interfaces 25. The sorting and transfer port 219 is used to transfer the streak pen tip 213 with a single colony after sorting to the pure culture vessel 22.

[0149] The robotic arm 214 includes a vertical rod 2141 and magnetic attraction sensors 2142 and fixing blocks 2143 disposed at both ends of the vertical rod 2141; the magnetic attraction sensors 2142 are connected to the central control unit 6 and the fixing blocks 2143 respectively, and the magnetic attraction sensors 2142 provide magnetic force to the fixing blocks 2143, and the fixing blocks 2143 extract the marking pen tip 213 by magnetic force;

[0150] The robotic arm 214 is used to perform the following operations: extracting and installing a streak pen tip 213 in the streak pen tip box 212; driving the streak pen tip 213 to dip into the bacterial solution in the bacterial solution pool 217 and the petri dish 215; driving the streak pen tip 213 to streak and sort in the petri dish 215; and releasing the streak pen tip 213 in the pen tip recycling pool 218 and the sorting transfer port 219.

[0151] In this embodiment, the pure culture vessel 22 is used to provide a variety of special microbial species for pure culture. Before the experiment begins, a special culture medium needs to be injected and connected to the pressure control unit 3 to pressurize the corresponding gas. The pure culture vessel 22 can be connected to the quick-connect interface 25 corresponding to the sorting transfer port 219 of the sorting operation vessel 21 to realize the rapid transfer of the sorted streaking pen tip 213 and the microorganisms contained therein, so as to start pure culture using the pure culture vessel 22.

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

[0153] Each of the primary culture vessel 13, the secondary culture vessel 14, the sorting operation vessel 21, and each pure culture vessel 22 is equipped with a gas injection valve 32; the gas pressurization system 31 is connected to each gas injection valve 32 through pipelines, and the gas pressurization system 31 is connected to the central control unit 6 to pressurize the gas in each vessel.

[0154] Each of the primary culture vessel 13 and the secondary culture vessel 14 is also equipped with a PID control valve 33, and each of the PID control valves 33 is connected to the central control unit 6. The PID control valves 33 are used to set a certain pressure threshold to realize the construction of a continuous reaction system in the primary culture vessel 13 and the secondary culture vessel 14, thereby achieving uniform mass transfer and continuous renewal of the culture medium and improving the enrichment efficiency of the culture in the vessel.

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

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

[0157] The air conditioning system 41 is connected to the central control unit 6, the primary culture vessel 13, the secondary culture vessel 14, the sorting operation vessel 21, and each pure culture vessel 22, respectively, and is used to control the temperature in each vessel.

[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 measurement system 55, and a camera system 56;

[0159] The pressure sensing system 51 and temperature sensing system 52 are respectively connected to the central control unit 6, the primary culture vessel 13, the secondary culture vessel 14, the sorting operation vessel 21, and each pure culture vessel 22, and are used to collect the pressure and temperature in each vessel 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. One pressure sensor 511 and one temperature sensor 521 are respectively installed inside the primary culture vessel 13, the secondary culture vessel 14, and the sorting operation vessel 21.

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

[0161] In this embodiment, the Raman measurement system 53 includes: two sets of pressure-resistant observation chambers 531, two sets of observation valves 532, and two sets of vent valves 533. The two sets of equipment are respectively installed in the primary culture vessel 13 and the secondary culture vessel 14. A Raman measurement probe is installed in the pressure-resistant observation chamber 531. At regular intervals, samples are taken through the observation valves 532 and transferred to the pressure-resistant observation chamber 531. The Raman measurement probe is used to measure the change in the chemical substance content and transmit it to the central control unit 6 in real time. After the observation is completed, the sample in the pressure-resistant observation chamber 531 is emptied using the vent valves 533, waiting for the next sampling.

[0162] The dissolved oxygen measurement system 54 and the pH measurement system 55 are used to measure the dissolved oxygen content and pH value of the primary culture vessel 13 and the secondary culture vessel 14, respectively, to identify the progress of the microbial anaerobic oxidation reaction.

[0163] The camera system 56 includes a camera 561 and a ring-shaped light 562. The camera 561 is located on the upper part of the pressure-resistant viewing window 216 on the top of the sorting operation vessel 21 and is connected to the central control unit 6. The camera 561 is used to capture image data of the culture area inside the sorting operation vessel 21 in real time and transmit it to the central control unit 6 in real time. In addition, a ring-shaped light 562 is also provided inside the upper vessel to facilitate the observation of single colonies and improve the 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, temperature sensing system 52, Raman measurement system 53, dissolved oxygen measurement system 54, pH value measurement system 55 and camera system 56 in the data acquisition unit 5, and is used to receive the data collected by the data acquisition unit 5;

[0166] The computer host 61 is also connected to the magnetic stirrer 15, robotic arm 214, pressure control unit 3, and temperature control unit 4 in the liquid injection mechanism 11, liquid transfer mechanism 12, primary culture vessel 13, and secondary culture vessel 14, respectively, for controlling 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 the enrichment of deep-sea microorganisms, the primary culture vessel 13, the secondary culture vessel 14, the sorting operation vessel 21 and each of the pure culture vessels 22 are first cleaned and sterilized, and pressure leak detection is performed; then, each unit in the entire device is installed, and all electronic instruments such as sensors in the device are connected to the central control unit 6, the instruments are activated online and connected to the software of the computer host 61.

[0168] Subsequently, the injection mechanism 11 injects pre-prepared microbial culture medium into each microbial culture vessel at a certain flow rate; a special culture medium is pre-prepared for each culture dish 215 of the sorting operation vessel 21, and a pre-prepared special culture medium is pre-injected into each pure culture vessel 22; the gas pressurization system 31 in the pressure control unit 3, combined with the pressure sensing system 51, pressurizes each vessel to simulate the high-pressure environment of the deep sea, and the PID control valve 33 is opened to set the pressure, forming a continuous environmental system; the temperature control unit 4, combined with the temperature sensing system 52, controls the temperature of each vessel to maintain the special temperature environment of the deep sea.

[0169] The collected in-situ sediment samples were then transferred to the primary culture vessel 13, and the magnetic stirrer 15 was turned on to begin microbial enrichment.

[0170] The Raman measurement system 53, dissolved oxygen measurement system 54 and pH measurement system 55 are used to measure the changes in chemical content, dissolved oxygen content and pH value in the primary culture vessel 14 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 vessel 13 meets the first preset condition, the central control unit 6 controls the transfer mechanism 12 to transfer the bacterial solution in the primary culture vessel 13 to the secondary culture vessel 14 for continued enrichment and culture. Specifically, the transfer pump 121, the pressure holding valve 125 and the transfer valve 123 are turned on and pure water is injected to push the transfer piston 131, so that the enriched bacterial solution enters the secondary culture vessel 14 for continued culture.

[0172] When the environmental data in the secondary culture vessel 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 solution in the secondary culture vessel 14 is transferred to the bacterial solution pool 217 in the sorting operation vessel 21.

[0173] Subsequently, the camera system 56 is used to collect image data of the culture area inside the sorting operation vessel 21 in real time and transmits it to the central control unit 6 in real time.

[0174] The central control unit 6 controls the robotic arm 214 to continuously extract and release the scribing pen tip 213, and drives the scribing pen tip 213 to perform scribing operations in each culture dish 215 in the culture area for sorting and culture for a preset time.

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

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

[0177] After the bacterial culture has been cultured in the culture zone for a period of time, when the central control unit 6 identifies from the image data of the culture zone that multiple single colonies have grown in the culture dish 215, the central control unit 6 controls the robotic arm to transfer each single colony to each of the pure culture vessels 22 to complete the sorting operation.

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

[0179] The central control unit 6 controls the robotic arm 214 to pick up the streak pen tip 213, move it to a single colony for dipping, and transfer it to the sorting and transfer port 219 to release the streak pen tip 213. The streak pen tip 213 with the single colony is then transferred to the pure culture vessel 22. This step is repeated until all single colonies are transferred to the pure culture vessel 22 for continued pure culture and preservation.

[0180] This device enables continuous, multi-stage enrichment of cultures through dilution and subculturing under high-pressure conditions, while simultaneously monitoring environmental indicators within the culture system online. It integrates enrichment and sorting devices, transfers cultures under pressure and aseptic conditions, automatically performs streaking and sorting on the culture medium within the device, and monitors the growth status of single colonies online, thereby improving the culturability 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 the online enrichment and automatic sorting device for deep-sea microorganisms in a high-pressure environment described in Embodiment 4, including the following steps:

[0183] S1: Clean and sterilize each of the aforementioned microbial culture vessels, sorting operation vessels, and pure culture vessels, and perform pressure leak testing; 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 vessel to begin microbial enrichment;

[0185] S3: The data acquisition unit is used to collect environmental data in each stage of the microbial culture vessel in real time and transmit it to the central control unit in real time.

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

[0187] S4: When the environmental data in the last-stage microbial culture vessel meets the second preset condition, the enrichment operation is completed, and the bacterial solution in the last-stage microbial culture vessel is transferred to the sorting operation vessel.

[0188] S5: The data acquisition unit collects image data of the culture area inside the sorting operation vessel in real time and transmits it to the central control unit in real time; the central control unit controls the robotic arm to perform several marking operations in the culture area to carry out sorting and cultivation for a preset time.

[0189] S6: When the central control unit identifies multiple single colonies growing in the culture dish from the image data of the culture area, the central control unit controls the robotic arm to transfer each single colony to each pure culture vessel to complete the sorting operation;

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

[0191] In step S5, each line-drawing operation includes:

[0192] The central control unit controls the robotic arm to move to the marking pen tip box, extracts the marking pen tip, and then controls the robotic arm to move to the culture dish to mark the lines. After marking the lines, the robotic arm is controlled to leave the culture area, move to the pen tip recycling pool, and release the marking pen tip.

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

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

[0195] In the specific implementation process, before the enrichment of deep-sea microorganisms, the primary culture vessel 13, the secondary culture vessel 14, the sorting operation vessel 21 and each of the pure culture vessels 22 are first cleaned and sterilized, and pressure leak detection is performed; then, each unit in the entire device is installed, and all electronic instruments such as sensors in the device are connected to the central control unit 6, the instruments are activated online and connected to the software of the computer host 61.

[0196] Subsequently, the injection mechanism 11 injects pre-prepared microbial culture medium into each microbial culture vessel at a certain flow rate; a special culture medium is pre-prepared for each culture dish 215 of the sorting operation vessel 21, and a pre-prepared special culture medium is pre-injected into each pure culture vessel 22; the gas pressurization system 31 in the pressure control unit 3, combined with the pressure sensing system 51, pressurizes each vessel to simulate the high-pressure environment of the deep sea, and the PID control valve 33 is opened to set the pressure, forming a continuous environmental system; the temperature control unit 4, combined with the temperature sensing system 52, controls the temperature of each vessel to maintain the special temperature environment of the deep sea.

[0197] The collected in-situ sediment samples were then transferred to the primary culture vessel 13, and the magnetic stirrer 15 was turned on to begin microbial enrichment.

[0198] like Figure 8 As shown, after the enriched material is loaded into the primary culture vessel 13, primary enrichment begins. Environmental indicators are identified using the Raman measurement system 53, dissolved oxygen measurement system 54, and pH measurement system 55. If the environmental indicators simultaneously meet the following criteria: dissolved oxygen content <1 μmol / kg, sulfide content >5 mmol / L (sulfide content is obtained from the Raman measurement system 53), and pH value >8.5, sample transfer can be performed. The sample transfer valve 123 and sample transfer injection pump 121 are opened to transfer the enriched material into the secondary culture vessel 14, thus initiating secondary enrichment. If any of these environmental indicators are not met, primary enrichment continues.

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

[0200] When the secondary enrichment enters the bacterial solution tank 217, sorting begins. The central control unit 6 controls the X-axis slide rail 2111, Y-axis slide rail 2112, and Z-axis slide rail 2113 of the three-axis moving mechanism 211 to move the robotic arm 214. The range of motion of the three-axis slide rail is from (0, 0, 0) to (100, 100, 100). The position of the robotic arm 214 in the three-axis moving mechanism 211 is represented by the coordinates of this range of motion.

[0201] Then, the robotic arm 214 is moved to the position (50, 0) to (80, 5) of the marking pen tip box 212, and the coil of the magnetic attraction sensor 2142 in the robotic arm 214 is electromagnetically turned on to pick up and install the marking pen tip 213. A total of 12 pen tips are assembled, and each pen tip is distributed at a distance of 5 coordinate points apart.

[0202] The robotic arm 214 is then moved to position (0, 0) in the bacterial culture tank 217, and moves back and forth within a small range from (0, 0) to (5, 5) to pick up the bacterial culture. The control rail moves the robotic arm 214 to the four culture dishes 215 in the culture area to draw lines according to a preset coordinate route. Each line has a coordinate system length of 20 and an interval length of 3. Eight lines can be drawn on each culture dish 215. Therefore, the drawing range for the first culture dish 215 is (20, 40–60), (23, 40–60)…(38, 40–60), (41, 40…60)…( ... The streaks on the second petri dish 215 are (45, 60-80), (48, 60-80)...(63, 60-80), (66, 60-80); the streaks on the third petri dish 215 are (45, 20-40), (48, 20-40)...(63, 20-40), (66, 20-40); the streaks on the fourth petri dish 215 are (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 robotic arm 214 can sense the magnetic attraction. If the magnetic attraction is 0, it is determined that the marking pen tip 213 has fallen off. The central control unit 6 records the coordinate point and controls the robotic arm 214 to retrace the route of that coordinate point to retrieve the fallen pen tip. The robotic arm 214 is moved to the position (0, 50) of the pen tip recycling pool 218, and the electromagnetic strength of the robotic arm 214 is adjusted to zero to release and discard the marking pen tip 214. If the magnetic attraction is not 0, it is determined that the marking pen tip 213 is working normally, and the marking program continues until the robotic arm 214 is identified as entering the coordinate interval in each culture dish 215, that is, (41). When the positions (60), (66, 40), (66, 80), and (91, 60) are reached, the streaking operation of the culture dish 215 is completed. The robotic arm 214 is moved to the position (0, 50) of the pen tip recovery pool 218, and the electromagnetic strength of the robotic arm 214 is adjusted to zero to release and discard the streaking pen tip 213. Finally, the central control unit 6 determines whether the robotic arm 214 has entered the last streaking coordinate position (91, 60) of the fourth culture dish 215. If the robotic arm 214 has not reached the last position, the streaking operation continues. If the robotic arm 214 has reached the last position, the streaking operation ends, the sorting operation is completed, and single colony culture begins.

[0204] During single colony culture, based on the image acquisition of the high-definition camera system 56 and the image recognition function of the central control unit 6, when a single colony with a diameter >1mm is detected, the central control unit 6 records the coordinate point of the colony. The robotic arm 214 is moved to the streak pen holder 212 to assemble the streak pen 213 and then moved to the coordinate point. The robotic arm 214 makes the streak pen 213 contact the colony and moves back and forth twice within a 2mm range in the X and Y directions at that position. The robotic arm 214 is moved to the position (50, 100) of the sorting transfer port 219, the electromagnetic magnitude of the robotic arm 214 is adjusted to zero, the ball valve 24 is opened, and the streak pen 213 is released so that it falls into the connected pure culture vessel 22 for subsequent individual cultivation and preservation, thus ending the microbial sorting.

[0205] This method enables continuous, multi-stage enrichment of cultures through dilution and subculturing under high-pressure conditions, while simultaneously monitoring environmental indicators within the culture system online. It integrates enrichment and sorting devices, transfers cultures under pressure and aseptic conditions, automatically performs streaking and sorting on the culture medium within the device, and monitors the growth status of single colonies online, thereby improving the culturability and acquisition efficiency of single species of deep-sea microorganisms.

[0206] The same or similar labels correspond to the same or similar parts;

[0207] The terms used to describe positional relationships in the accompanying drawings are for illustrative purposes only 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 implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A high-pressure environment deep-sea microbial online enrichment device, characterized in that, include: Multi-level enrichment unit, first pressure control unit, first temperature control unit, first data acquisition unit and first central control unit; The multi-stage enrichment unit includes a liquid injection mechanism, a liquid transfer mechanism, and at least two microbial culture vessels connected in series. The liquid injection mechanism is used to inject microbial culture medium into each of the microbial culture vessels. The first-stage microbial culture vessel contains in-situ sediment samples for microbial enrichment. The liquid transfer mechanism is used to transfer the bacterial culture medium in the previous-stage microbial culture vessel to the next-stage microbial culture vessel. The first pressure control unit and the first temperature control unit are respectively connected to the first central control unit and each microbial culture vessel, and are used to control the pressure and temperature in each vessel respectively; The first data acquisition unit is connected to the first central control unit. The first data acquisition unit is used to collect environmental data in each microbial culture vessel in real time and transmit it to the first central control unit in real time. 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 measurement system; The pressure sensing system and temperature sensing system are respectively connected to the first central control unit and each of the microbial culture vessels, and are used to collect the pressure and temperature in each vessel 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 measurement system are all connected to the first central control unit and each of the microbial culture vessels, respectively, and are used to observe the changes in chemical content, dissolved oxygen content, and pH value in each microbial culture vessel in real time and transmit them to the first central control unit in real time. The first central control unit is also connected to each microbial culture vessel, liquid injection mechanism, liquid transfer mechanism, first pressure control unit and first temperature control unit respectively. 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 high-pressure environment deep-sea microbial online enrichment device according to claim 1, characterized in that, In the multi-stage enrichment unit, the liquid injection mechanism includes a continuous liquid injection pump and several liquid injection valves, each of which is correspondingly installed on the inlet pipe of the microbial culture medium in each microbial culture vessel; the continuous liquid injection pump is connected to each liquid injection valve respectively. The liquid transfer mechanism includes a sample transfer pump, several sets of filters and transfer valves, and a plunger pump; the sample transfer pump is connected to all microbial culture vessels except the last stage, and a set of filters and transfer valves are provided on the connecting pipeline between every two stages of microbial culture vessels connected in series; the plunger pump is connected to the last stage microbial culture vessel. Each of the aforementioned microbial culture vessels has the same structure, and each is equipped with a magnetic stirrer and a sampling valve at the bottom; In all microbial culture vessels except the last stage, a transfer piston is provided at the top and a protruding ring is provided in the middle. The transfer piston is used to reduce the pressure difference required for the bacterial solution in the previous stage microbial culture vessel to be transferred to the next stage microbial culture vessel. The protruding ring is used to prevent the transfer piston from being pressurized too much.

3. The high-pressure environment deep-sea microbial online enrichment device according to claim 1, characterized in that, The first pressure control unit includes: a gas boosting system, several gas injection valves, and several PID control valves; Each of the aforementioned microbial culture vessels is equipped with a gas injection valve; the gas pressurization system is connected to each gas injection valve via pipelines, and the gas pressurization system is connected to the first central control unit for pressurizing the gas in each vessel. Each of the microbial culture vessels is also equipped with a PID control valve, and each of the PID control valves is connected to the central control unit; the PID control valve is used to control the formation of a continuous pressure environment in the series-connected microbial culture vessels. The first temperature control unit includes: an air conditioning system; The air conditioning system is connected to the first central control unit and each of the microbial culture vessels, respectively, and is used to control the temperature inside each vessel.

4. A high-pressure environment deep-sea microbial online enrichment and automatic sorting device, 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 microbial online enrichment device as described in any one of claims 1 to 3; the sorting device includes: 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; The automatic sorting unit includes a sorting operation vessel and several pure culture vessels; the sorting operation vessel is connected to each pure culture vessel respectively; The sorting operation vessel includes an internal three-axis moving mechanism and at least one marking pen tip box. The marking pen tip box contains multiple marking pen tips. The Z-axis of the three-axis moving mechanism is equipped with a robotic arm that can extract the marking pen tips from the marking pen tip box. The robotic 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 zone composed of multiple petri dishes. The sorting operation vessel includes an upper vessel body and a lower vessel body that are sealed together. The upper surface of the upper vessel is provided with a pressure-resistant viewing window for observing the inside of the sorting operation vessel; The lower vessel is equipped with a bacterial liquid pool, a three-axis moving mechanism, at least one streak pen tip box, multiple petri dishes, a pen tip recycling pool, and a sorting and transfer port. The bacterial solution tank is used to hold the bacterial solution to be sorted. The three-axis moving mechanism is provided with three vertically intersecting slide rails: the X-axis, the Y-axis, and the Z-axis. The robotic arm is mounted on the Z-axis slide rail. The robotic arm includes a vertical rod and magnetic attraction sensors and fixed blocks disposed at both ends of the vertical rod; the magnetic attraction sensors are respectively connected to the second central control unit and the fixed blocks, the magnetic attraction sensors provide magnetic force to the fixed blocks, and the fixed blocks extract the marking pen tip through magnetic force; The robotic arm is used to perform the following operations: extracting and installing a streak pen tip from a streak pen tip box; dipping the streak pen tip into the bacterial solution in the bacterial solution pool and petri dish; streak the streak pen tip in the petri dish for sorting; and releasing the streak pen tip in the pen tip recycling pool and sorting transfer port. The petri dish is pre-filled with culture medium for culturing single colonies in order to sort the desired microbial species; The pen tip recycling pool is used to recycle used drawing pen tips; The sorting and transfer port is connected to each of the pure culture vessels via a pipeline and a quick-connect interface. The sorting and transfer port is used to transfer the streak pen tip dipped with a single colony to the pure culture vessel after sorting. The second pressure control unit and the second temperature control unit are respectively connected to the second central control unit, the sorting operation vessel and each pure culture vessel, and are used to control the pressure and temperature in each vessel; The second data acquisition unit is connected to the second central control unit. The second data acquisition unit is used to acquire image data of the culture area inside the sorting operation vessel 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. The second central control unit is used to control the high-pressure environment deep-sea microorganism online automatic sorting device based on the data collected by the second data acquisition unit.

5. The online enrichment and automatic sorting device for deep-sea microorganisms under high pressure environment according to claim 4, characterized in that, The second pressure control unit includes: a gas pressurization system, an exhaust valve, and several injection valves; Each sorting operation vessel and each pure culture vessel are equipped with a gas injection valve; the gas pressurization system is connected to each gas injection valve through pipelines, and the gas pressurization system is connected to the second central control unit for pressurizing gas in each vessel. The exhaust valve is installed on the sorting operation vessel and is used for exhausting the wastewater from the sorting operation vessel; The second temperature control unit includes: an air conditioning system; The air conditioning system is connected to the second central control unit, the sorting operation vessel and each pure culture vessel, respectively, and is used to control the temperature inside each vessel; The second data acquisition unit includes a pressure sensing system, a temperature sensing system, and a camera system; The pressure sensing system and temperature sensing system are respectively connected to the second central control unit, the sorting operation vessel and each pure culture vessel, and are used to collect the pressure and temperature in each vessel in real time and transmit them to the second central control unit in real time. The camera system is installed on the top of the sorting operation vessel and connected to the second central control unit. The camera system is used to capture image data of the culture area inside the sorting operation vessel in real time and transmit it to the second central control unit in real time.

6. The high-pressure environment deep-sea microbial online enrichment and automatic sorting device according to claim 4, characterized in that, The last stage of the multi-stage enrichment unit is connected to the bacterial culture tank in the sorting operation tank; The bacterial solution tank is used to hold the bacterial solution to be sorted after enrichment in the last stage of microbial culture vessel.

7. A method for online enrichment and automatic sorting of deep-sea microorganisms in a high-pressure environment, based on the online enrichment and automatic sorting device for deep-sea microorganisms in a high-pressure environment as described in any one of claims 4 to 6, characterized in that, Includes the following steps: S1: Clean and sterilize each of the aforementioned microbial culture vessels, sorting operation vessels, and pure culture vessels, and perform pressure leak testing; install each unit in the device and perform initial configuration; S2: Transfer the collected in-situ sediment samples to the first-stage microbial culture vessel to begin microbial enrichment; S3: The first data acquisition unit collects environmental data in each stage of the microbial culture vessel in real time and transmits it to the first central control unit in real time. Starting from the first-level microbial culture vessel, when the environmental data in the previous-level microbial culture vessel meets the first preset condition, the first central control unit controls the liquid transfer mechanism to transfer the bacterial solution in the previous-level microbial culture vessel to the next-level microbial culture vessel for continued enrichment and culture. S4: When the environmental data in the last-stage microbial culture vessel meets the second preset condition, the enrichment operation is completed, and the bacterial solution in the last-stage microbial culture vessel is transferred to the sorting operation vessel. S5: The second data acquisition unit collects image data of the culture area inside the sorting operation vessel in real time and transmits it to the second central control unit in real time; the second central control unit controls the robotic arm to perform several marking operations in the culture area to carry out sorting and culture for a preset time. S6: When the second central control unit identifies multiple single colonies growing in the culture dish from the image data of the culture area, the second central control unit controls the robotic arm to transfer each single colony to each of the pure culture vessels to complete the sorting operation.

8. The method for online enrichment and automatic sorting of deep-sea microorganisms under high pressure environment according to claim 7, characterized in that, In step S1, the initialization configuration includes: injecting pre-prepared microbial culture medium into each microbial culture vessel at a certain flow rate using the injection mechanism; pre-preparing culture medium for each culture dish in the sorting operation vessel; and pre-injecting pre-prepared culture medium into each pure culture vessel; pressurizing each vessel with gas using the first and second pressure control units to simulate the high-pressure environment of the deep sea; and controlling the temperature of each vessel using the first and second temperature control units to maintain the temperature environment of the deep sea. In step S5, each line-drawing operation includes: The second central control unit controls the robotic arm to move to the marking pen tip box, extracts the marking pen tip, and then controls the robotic arm to move to the culture dish to mark the lines. After marking the lines, the robotic arm is controlled to leave the culture area, move to the pen tip recycling pool, and release the marking pen tip. In step S6, the second central control unit controls the robotic arm to transfer each single colony to each of the pure culture vessels, including: The second central control unit controls the robotic arm to extract the streak pen tip, move it to a single colony for dipping, and transfer it to the sorting and transfer port to release the streak pen tip, transferring the streak pen tip dipped in the single colony to the pure culture vessel.

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