A device and method for in-situ directional culture of deep-sea microorganisms

Abstract

This invention discloses a deep-sea microbial in-situ directional culture device, relating to the field of deep-sea equipment technology. It includes a microbial culture chamber, a nutrient solution storage module, a bidirectional flow path assembly, an intelligent control module, and supporting components. The microbial culture chamber consists of a flow channel, a flow-blocking block, a culture chamber, a movable plate, a cover, and sealing components. The flow channel and flow-blocking block form a structure based on the Tesla valve principle. Through a bidirectional flow path assembly composed of a deep-sea high-precision metering pump and a deep-sea diversion pump, nutrient substrates are injected in reverse with high resistance to achieve efficient mixing, while seawater flows in the forward direction with low resistance to achieve environmental material exchange. Multiple nutrient solution storage modules and microbial culture chambers are independently connected via a deep-sea multi-channel switching valve, enabling culture under various environmental conditions. Based on sensor data calculation and analysis, the pump start-up and shutdown times are intelligently controlled to effectively ensure the stability of nutrient substrate concentration and environmental parameters within the microbial culture chamber, thereby achieving controllable in-situ culture of deep-sea microbial communities.

Description

Technical Field

[0001] This invention relates to the field of deep-sea equipment technology, specifically to a deep-sea microbial in-situ directional culture device and culture method. Background Technology

[0002] It is a consensus among scientists that most deep-sea microorganisms cannot be isolated and cultured in a laboratory; these microorganisms are referred to as "deep-sea microbial dark matter." In-situ culture techniques, which have developed in recent years, place the culture system directly in the natural habitat of the microorganisms, allowing them to grow in their original environment. This maintains the natural interaction between the microorganisms and their environment, thus improving the success rate of culture. Conventional in-situ culture methods for deep-sea microorganisms involve placing a certain amount of solid or gelatinous nutrient substrate in different types of containers, deploying them on the seabed, and then sealing and retrieving the samples or preserving them in situ after a period of cultivation.

[0003] A problem with existing methods is the difficulty in establishing a stable micro-ecological environment during cultivation. It is well known that deep-sea microorganisms interact with their environment. The metabolism, growth, and reproduction of microorganisms consume nutrient substrates and surrounding environmental substances (such as dissolved oxygen and trace elements), altering the original environment or microenvironment. This environmental change can cause the microbial community to evolve in an uncontrollable direction, leading to uncertainty in the final collected microbial samples and potentially biasing scientific research conclusions. For example, to prevent the nutrient substrates and cultured microorganisms from being washed away by seawater, current cultivation containers are typically designed with weak seawater exchange. Dissolved oxygen in the microscale areas where microorganisms grow is continuously consumed without replenishment, causing the microbial community to transform from an aerobic to an anaerobic community.

[0004] Therefore, in order to achieve in situ directed culture of deep-sea microorganisms, more accurately elucidate the mechanisms of deep-sea microorganisms, and efficiently develop and utilize deep-sea genetic resources, it is urgent to develop intelligent environmental control technologies and devices for in situ culture of microorganisms. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and propose a deep-sea microbial in-situ directional culture device and method. By controlling the peristaltic pump through the feedback of monitoring data, the nutrient solution and reagents are injected into the culture chamber and thoroughly mixed. It is also necessary to realize the exchange of substances with the flowing in-situ seawater during the culture process, so as to achieve intelligent regulation and maintenance of stable culture conditions during the metabolism, growth and reproduction of microorganisms, and obtain deep-sea microbial community samples expected in scientific experiments.

[0006] To achieve the above objectives, the technical solution specifically adopted by the present invention is as follows: A deep-sea microbial in-situ directional culture device includes a microbial culture chamber, a nutrient solution storage module, a bidirectional flow path assembly, and a support assembly. The support assembly includes a frame, on which the microbial culture chamber, nutrient solution storage module, and bidirectional flow path assembly are mounted. The microbial culture chamber and nutrient solution storage module are connected through the bidirectional flow path assembly, which has a deep-sea water inlet. The microbial culture chamber includes a culture chamber body and a cover attached to the culture chamber body. The culture chamber body has a flow channel, and the flow channel has several flow-blocking blocks. The flow channel and flow-blocking blocks form a Tesla valve structure. Protrusions extend outward from both sides of the flow channel, and the space within the protrusions is a culture chamber. The culture chamber contains a mesh for microbial attachment. One end of the flow channel is a nutrient solution injection end, and the other end is a seawater injection end.

[0007] Preferably, the flow-blocking block has a teardrop-shaped structure, with the tip of the flow-blocking block facing the nutrient solution injection end.

[0008] Preferably, the tip of the flow-blocking block is located in the center of the flow channel, dividing the flow channel into two paths, with arc-shaped flow paths formed on both sides in sequence.

[0009] Preferably, the culture chamber and the arc-shaped flow path are separated by a movable plate, one end of which is rotatable and leaves a gap when closed.

[0010] Understandably, when the nutrient solution flows into the flow channel through the nutrient solution injection end, the nutrient solution impacts the movable plate, causing the movable plate to open, thus allowing the nutrient solution to flow into the culture chamber as much as possible.

[0011] Preferably, the hatch cover has an annular groove in the radial direction, and a sealing element is provided in the annular groove.

[0012] Preferably, the nutrient solution storage module includes a storage chamber, which is divided into a non-communicating liquid storage chamber and a seawater chamber by a soft membrane. The liquid storage chamber is used to store nutrient solution, and the seawater chamber is used to fill with seawater.

[0013] Preferably, the storage compartment is a hollow cylindrical structure with an annular inner flange in the center of its inner cavity; the soft membrane is pocket-shaped with its pocket edge fixed to the inner flange.

[0014] Preferably, the bidirectional flow path assembly includes a deep-sea high-precision metering pump, a deep-sea diversion pump, a multi-channel switching valve, a reversing valve, a first three-way valve, a second three-way valve, a first one-way valve, a second one-way valve, and connecting pipelines. The deep-sea high-precision quantitative pump and the deep-sea diversion pump are connected to the common port of the multi-channel switching valve through the reversing valve; a first three-way valve and a second three-way valve are provided between the multi-channel switching valve and the microbial culture chamber. The three connection ports of the first three-way valve are respectively connected to the outlet of the multi-channel switching valve, the connection port of the seawater chamber, and the second three-way valve. The first one-way valve is provided between the first three-way valve and the second three-way valve, and the second three-way valve guides the flow unidirectionally to the first three-way valve. The other two connection ports of the second three-way valve are respectively connected to the nutrient solution injection end of the liquid storage chamber and the microbial culture chamber. The second one-way valve is set on the connecting pipeline between the second three-way valve and the liquid storage chamber, and flows unidirectionally from the liquid storage chamber to the second three-way valve.

[0015] Preferably, it also includes an intelligent control module, which includes sensors, a control cabin, a microprocessor, and a deep-sea cable; The sensor is located at the front or rear end of the deep-sea diversion pump. The control cabin is connected to sensors, deep-sea high-precision metering pumps, deep-sea diversion pumps, multi-channel switching valves and reversing valves via deep-sea cables. The control cabin is equipped with a microprocessor for receiving and analyzing data from the sensors, thereby controlling the start-up, shutdown, and opening / closing states of the pumps and valves in the bidirectional flow path assembly; the control cabin is also equipped with a pressure gauge for collecting environmental pressure data.

[0016] Preferably, the sensor is any one or more of the following: microbial density sensor, dissolved oxygen sensor, pH sensor, nutrient salt sensor, and dissolved methane sensor.

[0017] Preferably, the frame is a rectangular frame welded from titanium alloy tubing, with at least two small seawater inlet holes on all closed sections of the titanium tubing for introducing seawater and balancing internal and external pressure.

[0018] Preferably, the support assembly also includes a deep-sea battery. The deep-sea power supply includes a deep-sea pressure-resistant battery compartment and a rechargeable lithium battery pack disposed therein. The deep-sea pressure-resistant battery compartment is equipped with a watertight connector and is connected to the control compartment via a deep-sea cable. The rechargeable lithium battery pack is provided with a power output interface, a charging interface, and overcurrent, overvoltage, overtemperature, reverse connection protection, and surge protection circuits.

[0019] This invention also provides an intelligently controlled in-situ directional culture method for deep-sea microorganisms, applicable to the aforementioned in-situ directional culture device for deep-sea microorganisms. The method includes the following steps: S1. Clean and sterilize the components of the device that come into contact with the microbial sample; S2. Fill the multiple liquid storage chambers with different types of nutrient solution reagents, and fill the multiple microbial culture chambers and the pipes and valves of the bidirectional flow path assembly with distilled water. S3. Formulate a power consumption plan for components, calculate the power consumption based on the expected cultivation process time, and convert it into the working frequency and start-stop time of sensors and pumps / valve. The power consumption plan shall not exceed 1 / 2 of the total power of the deep-sea power supply. S4. Before deployment, obtain water depth data at the work site and set the water depth threshold for starting and stopping the device based on this data; S5. Set parameters for the microprocessor according to the power consumption plan, including time setting, single injection volume setting, sensor operating frequency setting, and threshold or conditions for pump and valve start / stop. S6. The device is mounted on the deep-sea transport platform of the research vessel and lowered to the target area on the seabed. When the pressure gauge detects that the water depth exceeds the set threshold, the device automatically starts the operation program. S7. After the cultivation experiment is completed, the recovery device will automatically stop working and close all valves when the pressure gauge detects that the water depth is less than the set threshold during the upward process. S8. After the device is recovered and loaded onto the ship, it is rinsed with fresh water and dried. The microbial culture chamber is removed, and the sample is exported in a sterile operating table.

[0020] First, this invention proposes an innovative method that, through a unique microbial culture chamber structure and intelligent control design, resolves the contradiction between nutrient solution input and seawater exchange, effectively maintaining the material concentration within the microbial culture chamber, thereby ensuring the stability and controllability of the in-situ cultured deep-sea microbial community. Simultaneously, it enables the use of liquid nutrient substrate reagents for in-situ culture of deep-sea microorganisms, overcoming the limitations of traditional technologies where nutrient substrates must be pre-prepared in solid or colloidal form. This solves the problems of continuous nutrient substrate consumption and uncontrollable environment, thus providing greater flexibility and convenience for in-situ culture of deep-sea microorganisms. Second, this invention designs a unique passive hybrid microbial culture chamber structure, which not only improves the utilization rate of the nutrient solution but also significantly reduces energy consumption during long-term deep-sea culture experiments. Furthermore, this invention ensures that the microbial culture chamber within the device remains closed before and after in-situ directional culture experiments of deep-sea seabed microorganisms, effectively preventing sample contamination from microorganisms in the upper and middle layers of seawater. Through the bidirectional flow path design and intelligently controlled pump and valve operation, the stability of the in-situ environment within the microbial culture chamber is effectively maintained.

[0021] Another significant advantage of this invention is that it enables in-situ cultivation of deep-sea microorganisms under various conditions during a single deep-sea operation. This greatly improves the efficiency of deep-sea biological equipment and provides a wider variety of samples for scientific research. Finally, the design of this invention fully considers the reliability of deep-sea operation equipment and the safety of onboard operations, ensuring the safety and efficiency of the entire microbial cultivation experiment process. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a deep-sea microbial in-situ directional culture device according to an embodiment of the present invention.

[0023] Figure 2 This is a schematic diagram illustrating the working principle of an in-situ directional culture device for deep-sea microorganisms according to an embodiment of the present invention.

[0024] Figure 3 for Figure 1 A schematic diagram of the microbial culture chamber and nutrient solution injection process.

[0025] Figure 4 for Figure 1 A schematic diagram of the seawater exchange process in the microbial culture chamber.

[0026] Figure 5 for Figure 1 Schematic diagram of the nutrient solution storage module.

[0027] Figure label: 1-Microbial culture chamber, 11-Flow channel, 12-Baffle block, 13-Cultivation chamber, 14-Movable plate, 15-Cover, 16-Sealing component; 2-Nutrient solution storage module, 21-Storage chamber, 22-Soft membrane, 23-Reservoir, 24-Seawater chamber, 211-Inner flange; 3-Bidirectional flow path assembly; 31-Deep-sea high-precision metering pump; 32-Deep-sea diverting pump; 33-Multi-channel switching valve; 34-Reversing valve. 35-First three-way valve, 36-Second three-way valve, 37-First check valve, 38-Second check valve 4-Intelligent control module, 41-Sensor, 42-Control cabin; 5-Supporting components, 51-Rack, 52-Deep-sea power supply, 53-Small hole. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. Example

[0029] This embodiment provides a device for in-situ directional culture of deep-sea microorganisms, such as... Figure 1 As shown, it includes a microbial culture chamber 1, a nutrient solution storage module 2, a bidirectional flow path assembly 3, and a support assembly 5.

[0030] Among them, such as Figure 3 and Figure 4 As shown, the microbial culture chamber 1 includes a culture chamber body and a cover 15 attached to the culture chamber body. The culture chamber body is provided with a flow channel 11, and the flow channel 11 is provided with a number of flow-blocking blocks 12. The flow channel 11 and the flow-blocking blocks 12 constitute a Tesla valve structure. This structure has special advantages in fluid control such as nutrient solution injection and in-situ seawater exchange.

[0031] The flow channel 11 has outwardly extending protrusions on both sides, and the space within these protrusions houses a culture chamber 13. This design facilitates the growth of microorganisms in a relatively stable and suitable environment. The culture chamber 13 contains a mesh-like material for microbial attachment, providing a large amount of substrate for microbial growth. One end of the flow channel 11 is a nutrient solution injection end, and the other end is a seawater injection end.

[0032] Understandably, the opening to the outside pointed to by the tip of the flow-blocking block 12 is clearly designated as the nutrient solution injection end, through which one or more nutrient solutions required for microbial growth can be injected into the device; while the opening to the outside in the opposite direction is the seawater injection end, through which seawater can enter the device, providing a deep-sea in-situ living environment for microorganisms.

[0033] Furthermore, in this embodiment, the flow-blocking block 12 has a teardrop-shaped structure, with the tip of the flow-blocking block 12 facing the nutrient solution injection end. It can be understood that the tip of the flow-blocking block 12, located in the center of the flow channel 11, divides the flow channel into two paths, with arc-shaped flow paths forming on both sides in sequence.

[0034] In a further embodiment, the culture chamber 13 is separated from the arc-shaped flow path by a movable plate 14, one end of which is rotatable. When the nutrient solution flows into the flow channel through the nutrient solution injection end, the nutrient solution impacts the movable plate 14, causing the movable plate to open and allowing the nutrient solution to flow into the culture chamber as much as possible.

[0035] It should be noted that when the flow channel 11 is in a static state and the movable plate 14 is in a closed partition state, there is still a gap between the movable plate 14 and the inner wall of the culture chamber.

[0036] Understandably, the culture chamber 13 and the arc-shaped flow path 11 are separated by a movable plate 18. When the movable plate 14 is opened, the nutrient solution reagent can be introduced into the culture chamber 13. When the movable plate 14 is closed, a gap will be left. This gap can ensure a certain degree of material exchange and control the environment inside the culture chamber 13.

[0037] In a further embodiment, the upper surface of the microbial culture chamber 1 is designed as an inwardly recessed structure; additionally, an O-ring groove is specially designed in the radial position of the chamber cover 15. During installation, the chamber cover 15 is embedded into this inwardly recessed area, ensuring a radial seal between the chamber cover and the chamber body. To further enhance the reliability of fixation and sealing, the chamber cover 15 is fixed to the chamber body with screws.

[0038] Through this design, the microbial culture chamber 1 cleverly utilizes the Tesla valve principle, achieving efficient mixing through reverse high-resistance injection of nutrient solution and achieving environmental substance exchange through forward low-resistance seawater flow. This effectively ensures the stability of substance concentration and environmental parameters near the culture chamber, thereby directionally cultivating stable deep-sea microbial communities required for scientific research. Figure 4 As shown, the nutrient solution enters the flow channel 11 through the injection port. The flow path is divided into two paths by the flow-blocking block. One path changes direction after passing through the arc-shaped flow path 17, creating turbulence by opposing the original flow path, thus ensuring thorough mixing of the nutrient solution within the microbial culture chamber 1. When the injected liquid flows through the arc-shaped flow path 17, it can push aside the movable plate 14 at a certain angle, guiding the nutrient solution into the culture chamber 13. After the nutrient solution input is completed, the flow direction is changed to allow external seawater to enter the microbial culture chamber 1, as shown in the attached diagram. Figure 3 As shown, due to the low resistance of seawater flow, a small amount of ambient seawater can be exchanged with relatively little power. At this time, the movable plate 14 is closed, effectively preventing the nutrient solution and cultured microorganisms in the culture chamber 13 from being washed away by the water flow. When the movable plate 14 is closed, a gap is left, allowing substances in the fresh seawater in the flow channel 11 to diffuse into the culture chamber 13 due to the concentration difference. The microbial culture chamber 1 and the culture chamber 13 maintain their in-situ environment through the internal seawater exchange.

[0039] Further settings in this embodiment, such as Figure 5 As shown, the nutrient solution storage module 2 includes a storage chamber 21, which is divided into a non-communicating liquid storage chamber 23 and a seawater chamber 24 by a soft membrane 22. The liquid storage chamber 23 is used to store liquid nutrient solution, and the seawater chamber 24 is used to fill seawater.

[0040] Specifically, the storage compartment 21 is a hollow cylindrical structure with an annular inner flange 211 in the center of its inner cavity; the soft membrane 22 is pocket-shaped with its pocket edge fixed to the inner flange 211.

[0041] Understandably, through the above technical solution, the nutrient solution storage module 2 can achieve high-precision control of nutrient solution output. When a fixed amount of seawater is injected into the seawater chamber 24, the pressure inside the seawater chamber is transmitted to the storage chamber 23 through the soft membrane 22. Due to the elastic properties of the soft membrane 22, it transmits the pressure to the nutrient solution in the storage chamber, thereby squeezing out a corresponding volume of nutrient solution from the storage chamber. By precisely controlling the seawater flow rate injected into the seawater chamber 24, the volume of nutrient solution squeezed out of the storage chamber 23 can be indirectly controlled, thereby controlling the amount of nutrient solution input into the microbial culture chamber. This control mechanism provides a more precise and stable environment for the cultivation of deep-sea microorganisms, significantly improving the controllability of the in-situ cultivation process of deep-sea microorganisms.

[0042] In a further embodiment, the bidirectional flow path assembly 3 includes a deep-sea high-precision metering pump 31, a deep-sea diversion pump 32, a multi-channel switching valve 33, a reversing valve 34, a first three-way valve 35, a second three-way valve 36, a first one-way valve 37, a second one-way valve 38, and connecting pipelines. The deep-sea high-precision quantitative pump 31 and the deep-sea diversion pump 32 are connected to the common port of the multi-channel switching valve 33 through the reversing valve 34; a first three-way valve 35 and a second three-way valve 36 are provided between the multi-channel switching valve 33 and the microbial culture chamber 1. The three connection ports of the first three-way valve 35 are respectively connected to the outlet of the multi-channel switching valve 33, the connection port of the seawater chamber 24, and the second three-way valve 36. The first one-way valve 37 is provided between the first three-way valve 35 and the second three-way valve, and it unidirectionally flows from the first three-way valve 35 to the second three-way valve 36. The other two connection ports of the second three-way valve 36 are respectively connected to the liquid storage chamber 23 and the nutrient solution injection end of the microbial culture chamber 1. The second one-way valve 38 is set on the connection pipeline between the second three-way valve 36 and the liquid storage chamber 23, and flows unidirectionally from the liquid storage chamber 23 to the second three-way valve 36.

[0043] Based on the above technical solution, the deep-sea microbial in-situ directional culture device provided in this embodiment is used as an example to cultivate a new species of degradable heavy metal microorganisms that have not been discovered in the deep sea: First, this embodiment provides a deep-sea in-situ directional culture device. Therefore, the seawater injection end of the microbial culture chamber 1 is directly connected to the deep sea, and the channel is filled with seawater. It is understood that deep-sea water contains various different microorganisms. In this embodiment, the microorganisms to be cultured are those capable of degrading heavy metals; therefore, a nutrient solution containing heavy metals needs to be injected.

[0044] Specifically, in this embodiment, a sufficient amount of nutrient solution containing heavy metals is first stored in the storage chamber 23.

[0045] When nutrient solution needs to be replenished, the switching valve 34 is activated, connecting the deep-sea high-precision metering pump 31 to the multi-channel switching valve 33. The deep-sea high-precision metering pump 31 then starts, drawing a measured amount of seawater. After the seawater enters the connecting pipe, the first one-way valve 37 closes under the pressure of the seawater, blocking the passage between the first three-way valve 35 and the second three-way valve 36. At this time, seawater flows into the seawater chamber 24 through the first three-way valve 35. As the amount of seawater flowing in increases, the water pressure in the seawater chamber 24 continuously increases, thus squeezing the soft membrane 22. This causes the nutrient solution in the storage chamber 23 on the other side of the soft membrane to be squeezed out. Simultaneously, the second one-way valve 38 opens under the pressure of the nutrient solution, allowing the nutrient solution to enter the flow channel 11 of the Tesla valve structure through the nutrient solution injection end. After the nutrient solution enters the flow channel 11, it is divided into two paths by the tip of the flow-blocking block 12. One path enters the arc-shaped flow path. The nutrient solution passing through the arc-shaped flow path changes the direction of the water flow and counteracts the nutrient solution in the original flow path, forming turbulence. At this time, the nutrient solution is fully mixed with the seawater in the flow head 11 and will not be quickly flushed out of the seawater injection end, reducing losses. At the same time, the nutrient solution in the arc-shaped flow path impacts the movable plate 14. The movable plate 14 rotates and opens under the impact of the nutrient solution, allowing the nutrient solution to fully enter the culture chamber.

[0046] After the injection is completed, the cultured microorganisms require other substances from the in-situ seawater to provide the necessary nutrients for their growth. Switching valve 34 connects the deep-sea diversion pump 32 to the multi-channel switching valve 33. The deep-sea diversion pump 32 then draws seawater from the connecting pipe. At this time, the side connected to the first one-way valve 37 and the first three-way valve 35 is under negative pressure, thus opening the first one-way valve. Simultaneously, negative pressure is also formed in the storage chamber 25 on the other side of the soft membrane 22, causing the second one-way valve 38 to close, preventing seawater from flowing into the storage chamber 23 and contaminating the nutrient solution. At this point, the microbial culture chamber 1 is connected to the multi-channel switching valve 33, drawing seawater from the microbial culture chamber 1. Simultaneously, seawater flows into the other end of the microbial culture chamber 1 to replenish the nutrients, and the in-situ seawater from the outside enters the flow channel 11. At this time, dissolved oxygen and other elements required for microbial growth in the flow channel 11 diffuse to the nutrient chamber due to the concentration difference. Understandably, due to the special structure of the Tesla valve, the heavy metal-containing nutrient solution and cultured microorganisms in the culture chamber 13 will be retained within the chamber as much as possible, unaffected by the impact of seawater flow. It should be noted that a microbial culture chamber 1 and a nutrient solution storage module 2 constitute a culture unit, and one outlet of the multi-channel switching valve 33 connects to one culture unit. Depending on the actual situation, the multi-channel switching valve 33 may have multiple outlets, which can be connected to multiple culture units respectively, thereby enabling multiple culture units to perform culture operations simultaneously.

[0047] In a further embodiment, the support component 5 includes a frame 51. The microbial culture chamber 1, the nutrient solution storage module 2, and the bidirectional flow path component 3 are mounted on the frame 3. The frame 51 is a rectangular frame welded from titanium alloy tubes. At least two small holes 53 are provided on all closed sections of the titanium tubes for introducing seawater and balancing internal and external pressure.

[0048] Furthermore, the support component 5 also includes a deep-sea battery 52, which includes a deep-sea pressure-resistant battery compartment and a rechargeable lithium battery pack disposed therein. The deep-sea pressure-resistant battery compartment is equipped with a watertight connector and is connected to the control compartment 42 via a deep-sea cable. The rechargeable lithium battery pack is provided with a power output interface, a charging interface, and overcurrent, overvoltage, overtemperature, reverse connection protection, and surge protection circuits.

[0049] Through the above technical solutions, the device can not only meet the reliability requirements under the high pressure conditions of the deep sea, but also fully consider the harsh environment that may be encountered when it is deployed and debugged on an oceanographic research vessel. This ensures that the device can still guarantee its safety even in the event of misoperation, thus providing reliable technical support for deep-sea scientific research missions.

[0050] In a further embodiment, the deep-sea microbial directional culture device also includes an intelligent control module 4, which includes a sensor 41, a control cabin 42, a microprocessor, and a deep-sea cable. The sensor 41 is disposed at the front or rear end of the deep-sea diversion pump 32. In this embodiment, the sensor 41 is any one or more of the following: microbial density sensor, dissolved oxygen sensor, pH sensor, nutrient salt sensor, and dissolved methane sensor.

[0051] Understandably, sensors are used to detect the content of microorganisms in the microbial culture chamber, thereby determining whether it is necessary to replenish nutrient solution or re-inject seawater.

[0052] The control cabin 42 is connected to the sensor 41, the deep-sea high-precision quantitative pump 31, the deep-sea diversion pump 32, the multi-channel switching valve 33, and the reversing valve 34 via deep-sea cables. The control compartment 42 is equipped with a microprocessor for receiving and analyzing data from the sensor 41, thereby controlling the start-up, shutdown, and opening / closing states of the pumps and valves in the bidirectional flow path assembly 3; the control compartment 42 is also equipped with a pressure gauge for collecting environmental pressure data.

[0053] Understandably, the pressure gauge transmits data to the microprocessor in real time, which then collects the data. The microprocessor compares the data with a set water depth threshold to control the start or stop of the device. When the device is deployed, the microbial culture program automatically starts when the pressure count exceeds the set water depth threshold. When the device is retrieved, the microbial culture program automatically shuts down and closes all valves when the pressure count falls below the set water depth threshold to prevent seawater from the upper and middle layers from contaminating the cultured microbial samples.

[0054] Through the aforementioned technical solution, the device can effectively collect and monitor the number of microorganisms and related environmental data at the seawater outlet. After complex processing and computational analysis, this data can automatically assess the biological status and environmental conditions within the microbial culture chamber 1. Based on pre-set thresholds or specific conditions, the system can accurately calculate the required nutrient solution injection volume and seawater exchange volume, thereby precisely controlling the start-up and stop times of the high-precision metering pump 31 and the deep-sea diversion pump 32. This enables intelligent adjustment and maintenance of the culture environment within the microbial culture chamber 1, ensuring that microbial growth and reproduction occur under optimal conditions. Furthermore, the intelligent control circuitry allows the device to start operating near the seabed and shut down the closed pipeline after ascent, preventing the introduction of non-bottom-layer microorganisms during the early stages of culture and preventing contamination of the cultured microbial samples.

[0055] In this embodiment, the microprocessor used is ARMv7. It should be noted that the microprocessor is a conventional control component, so it will not be described in detail in this embodiment. Example

[0056] This embodiment provides an intelligently controlled in-situ directional culture method for deep-sea microorganisms, applicable to the in-situ directional culture device for deep-sea microorganisms described in Embodiment 1. The method includes the following steps: S1. Clean and sterilize the components of the device that come into contact with the microbial sample; S2. Fill the multiple liquid storage chambers 23 with different kinds of nutrient solution reagents, and fill the multiple microbial culture chambers 1 and the pipes and valves of the bidirectional flow path assembly 3 with distilled water. S3. Formulate a power consumption plan for components, calculate the power consumption based on the expected cultivation process time, and convert it into the working frequency and start-stop time of sensors and pumps / valve. The power consumption plan shall not exceed 1 / 2 of the total power of the deep-sea power supply 52. S4. Before deployment, obtain water depth data at the work site and set the water depth threshold for starting and stopping the device based on this data; S5. Set parameters for the microprocessor according to the power consumption plan, including time setting, single injection volume setting, sensor operating frequency setting, and threshold or conditions for pump and valve start / stop. S6. The device is mounted on the deep-sea transport platform of the research vessel and lowered to the target area on the seabed. When the pressure gauge detects that the water depth exceeds the set threshold, the device automatically starts the fully automatic operation program. Specifically, in this embodiment, the fully automated cultivation procedure is as follows: The reversing valve 34 is placed in the open position between the deep-sea diversion pump 32 and the multi-channel switching valve 33; the deep-sea diversion pump 32 is started and runs for a period of time to displace the distilled water in the microbial culture chamber 1 with external seawater; the deep-sea diversion pump 32 is stopped, and the reversing valve 34 is placed in the open position between the deep-sea high-precision quantitative pump 31 and the multi-channel switching valve 33. Through time control, a certain amount of seawater is injected into the seawater chamber 24 of the nutrient solution storage module 2, and the same amount of nutrient solution in the storage chamber 23 is squeezed out and injected into the microbial culture chamber 1; The nutrient solution entering the microbial culture chamber 1 is fully mixed under the action of the Tesla valve structure and enters the culture chamber 13 under the guidance of the movable plate 14. The reversing valve 34 is then placed in the conducting position between the deep-sea diversion pump 32 and the multi-channel switching valve 33, and the deep-sea diversion pump 32 is started to guide the external seawater to flow through the microbial culture chamber 1 for environmental information exchange. At the same time, the sensor 41 monitors the relevant data of the outflowing seawater. The microprocessor analyzes and judges the data from the sensor 41 and intelligently controls the relevant actions of liquid injection or seawater exchange to maintain the environmental stability of the microbial culture chamber 1 until the culture is completed.

[0057] S7. After the cultivation experiment is completed, the recovery device will automatically stop working and close all valves when the pressure gauge detects that the water depth is less than the set threshold during the upward process. S8. After the device is recovered and loaded onto the ship, it is rinsed with fresh water and dried. The microbial culture chamber 1 is then removed, and the samples are exported in a sterile operating table.

[0058] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A device for in-situ directional culture of deep-sea microorganisms, characterized in that, The system includes a microbial culture chamber (1), a nutrient solution storage module (2), a bidirectional flow path assembly (3), and a support assembly (5). The support assembly (5) includes a frame (51). The microbial culture chamber (1), the nutrient solution storage module (2), and the bidirectional flow path assembly (3) are mounted on the frame (51). The microbial culture chamber (1) and the nutrient solution storage module (2) are connected through the bidirectional flow path assembly (3). The bidirectional flow path assembly (3) is provided with a deep-sea inlet. The microbial culture chamber (1) includes a culture chamber body and a cover (15) attached to the culture chamber body. The culture chamber body is provided with a flow channel (11). The flow channel (11) is provided with several flow-blocking blocks (12). The flow channel (11) and the flow-blocking blocks (12) constitute a Tesla valve structure. There are protrusions extending outward on both sides of the flow channel (11). The space inside the protrusions is provided with a culture chamber (13). The culture chamber (13) contains a mesh for microbial attachment. One end of the flow channel (11) is the nutrient solution injection end, and the other end is the seawater injection end. The tip of the flow-blocking block (12) is located in the center of the flow channel (11), dividing the flow channel into two paths, forming arc-shaped flow paths on both sides. The culture chamber (13) and the arc-shaped flow paths are separated by a movable plate (14). The movable plate (14) is pivotally rotatable at one end, so that when the nutrient solution flows in from the nutrient solution injection end, the nutrient solution impacts the movable plate (14) to open it, guiding the nutrient solution into the culture chamber (13). When the seawater flows in from the seawater injection end, the movable plate (14) closes to prevent the substances in the culture chamber (13) from being washed away by the water flow. At the same time, a gap is left between the movable plate (14) and the inner wall of the culture chamber (13) to allow for the exchange of substances. The nutrient solution storage module (2) includes a storage chamber (21), which is divided into a non-communicating liquid storage chamber (23) and a seawater chamber (24) by a soft membrane (22). The liquid storage chamber (23) is used to store nutrient solution, and the seawater chamber (24) is used to fill seawater. The bidirectional flow path assembly (3) includes a deep-sea high-precision metering pump (31), a deep-sea diversion pump (32), a multi-channel switching valve (33), a reversing valve (34), a first three-way valve (35), a second three-way valve (36), a first one-way valve (37), a second one-way valve (38), and connecting pipelines. The deep-sea high-precision quantitative pump (31) and the deep-sea diversion pump (32) are connected to the common port of the multi-channel switching valve (33) through the reversing valve (34); a first three-way valve (35) and a second three-way valve (36) are provided between the multi-channel switching valve (33) and the microbial culture chamber (1). The three connection ports of the first three-way valve (35) are respectively connected to the outlet of the multi-channel switching valve (33), the connection port of the seawater chamber (24) and the second three-way valve (36). The first one-way valve (37) is provided between the first three-way valve (35) and the second three-way valve (36), and is unidirectionally guided from the second three-way valve (36) to the first three-way valve (35). The other two connection ports of the second three-way valve (36) are respectively connected to the nutrient solution injection end of the liquid storage chamber (23) and the microbial culture chamber (1). The second one-way valve (38) is set on the connecting pipeline between the second three-way valve (36) and the liquid storage chamber (23), and is unidirectionally connected from the liquid storage chamber (23) to the second three-way valve (36).

2. The in-situ directional culture device for deep-sea microorganisms according to claim 1, characterized in that, The flow-blocking block (12) has a teardrop-shaped structure, with the tip of the flow-blocking block (12) facing the nutrient solution injection end.

3. The in-situ directional culture device for deep-sea microorganisms according to claim 1, characterized in that, The storage compartment (21) is a hollow cylindrical structure with an annular inner flange (211) in the middle of its inner cavity; the soft membrane (22) is pocket-shaped with its pocket edge fixed to the inner flange (211).

4. The in-situ directional culture device for deep-sea microorganisms according to claim 3, characterized in that, It also includes an intelligent control module (4), which includes a sensor (41), a control cabin (42), a microprocessor, and a deep-sea cable; The sensor (41) is located at the front or rear end of the deep-sea diversion pump (32). The control cabin (42) is connected to the sensor (41), the deep-sea high-precision metering pump (31), the deep-sea diversion pump (32), the multi-channel switching valve (33), and the reversing valve (34) via deep-sea cables; The control cabin (42) is equipped with a microprocessor for receiving and analyzing data from the sensor (41) and then controlling the start-up, shutdown and opening / closing status of the pump and valve in the bidirectional flow path assembly (3); the control cabin (42) is also equipped with a pressure gauge for collecting seawater depth data.

5. The deep-sea microbial in-situ directional culture device according to claim 4, characterized in that, The sensor (41) is any one or more of the following: microbial density sensor, dissolved oxygen sensor, pH sensor, nutrient salt sensor, and dissolved methane sensor.

6. The in-situ directional culture device for deep-sea microorganisms according to claim 1, characterized in that, The frame (51) is a rectangular frame welded from titanium alloy tubes, and at least two small holes (53) are provided on all closed sections of the titanium tubes for introducing seawater and balancing internal and external pressure.

7. The deep-sea microbial in-situ directional culture device according to claim 5, characterized in that, The support component (5) also includes a deep-sea power supply (52), which includes a deep-sea pressure-resistant battery compartment and a rechargeable lithium battery pack disposed therein. The deep-sea pressure-resistant battery compartment is equipped with a watertight connector and is connected to the control compartment (42) via a deep-sea cable. The rechargeable lithium battery pack is provided with a power output interface, a charging interface, and overcurrent, overvoltage, overtemperature, reverse connection protection, and surge protection circuits.

8. A method for intelligently controlled in-situ directional culture of deep-sea microorganisms, applicable to the in-situ directional culture device for deep-sea microorganisms as described in any one of claims 1 to 7, characterized in that, The method includes the following steps: S1. Clean and sterilize the components of the device that come into contact with the microbial sample; S2. Fill the multiple liquid storage chambers (23) with different kinds of nutrient solution reagents, and fill the multiple microbial culture chambers (1) and the pipes and valves of the bidirectional flow path assembly (3) with distilled water; S3. Formulate a power consumption plan for components, calculate the power consumption based on the expected cultivation process time, and convert it into the working frequency and start-stop time of sensors and pumps / valve. The power consumption plan shall not exceed 1 / 2 of the total power of the deep-sea power supply (52). S4. Before deployment, obtain water depth data at the work site and set the water depth threshold for starting and stopping the device based on this data; S5. Set initial parameters for the microprocessor according to the power consumption plan, including time setting, single injection volume setting, sensor operating frequency setting, and threshold or conditions for pump and valve start / stop. S6. The device is mounted on the deep-sea transport platform of the research vessel and lowered to the target area on the seabed. When the pressure gauge detects that the water depth exceeds the set threshold, the device automatically starts the fully automatic cultivation program. S7. After the cultivation experiment is completed, the recovery device will automatically stop working and close all valves when the pressure gauge detects that the water depth is less than the set threshold during the upward process. S8. After the device is recovered and loaded onto the ship, it is rinsed with fresh water and dried. The microbial culture chamber (1) is removed and the sample is exported in a sterile operating table.

9. A method for intelligently controlled in-situ directional culture of deep-sea microorganisms according to claim 8, characterized in that, In step S7, the fully automated culture program is as follows: Place the reversing valve (34) in the open position between the deep-sea diversion pump (32) and the multi-channel switching valve (33), start the deep-sea diversion pump (32) to run for a period of time, so that the outside seawater replaces the distilled water in the microbial culture chamber (1); stop the deep-sea diversion pump (32), place the reversing valve (34) in the open position between the deep-sea high-precision quantitative pump (31) and the multi-channel switching valve (33), inject a certain amount of seawater into the seawater chamber (24) in the nutrient solution storage module (2), and the same amount of nutrient solution in the storage chamber (23) is squeezed out and injected into the microbial culture chamber (1); inject into the microbial culture chamber (1) The nutrient solution is fully mixed under the action of the Tesla valve structure and enters the culture chamber (13) under the guidance of the movable plate (14); the reversing valve (34) is placed in the conduction position of the deep-sea diversion pump (32) and the multi-channel switching valve (33) again, the deep-sea diversion pump (32) is started, and the external seawater is guided to flow through the microbial culture chamber (1) to exchange environmental information. At the same time, the sensor (41) monitors the relevant data of the outflowing seawater; the microprocessor analyzes and judges the data of the sensor (41), and intelligently controls the relevant actions of liquid injection or seawater exchange to maintain the environmental stability of the microbial culture chamber (1) until the end of the culture.

Images