Method and system for whole-course pressure and heat preservation sample transfer-incubation marking of barophilic bacteria in deep sea based on in-situ reconstruction

Abstract

The invention discloses a method and a system for marking barophile in deep sea through whole-course pressure and heat preservation sample transfer-incubation based on in-situ reconstruction, and relates to the technical field of marking of barophile in deep sea. In the transferring step, the sample is transferred from the sampler to a transferring device in a flowing manner under the action of gravity in a pressure-maintaining and heat-preserving environment; between the step of transferring and the step of incubating, small tubes, containing samples, of a transferring device automatically fall into an incubating device in a pressure-maintaining and heat-preserving environment; in the incubation step, a heavy water culture medium is added into an incubator under the conditions of pressure and heat preservation, so that a sample in a small tube and the heavy water culture medium are co-incubated; in the marking step, heavy water marking is carried out on living cells of barophilic bacteria in a sample through a heavy water culture medium in the incubation process, in-situ environment conditions for survival of deep sea barophilic bacteria are kept to the maximum extent, the living cells of barophilic bacteria are marked and obtained, and deep research on barophilic bacteria is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of labeling deep-sea piezophiles, and particularly relates to a method and system for labeling deep-sea piezophiles by whole-process pressure and temperature preservation sample transfer-incubation based on in-situ reconstruction. Background Art

[0002] The deep sea is usually defined as the sea area with a depth below 1000 m below sea level. Due to its characteristics of low temperature, high pressure, darkness, and oligotrophy, it is called an extreme environment. In fact, it is one of the aquatic environments with the most abundant microorganisms on the earth. During the long-term evolution process, deep-sea microorganisms have gradually formed the characteristics of pressure resistance or piezophily to adapt to the extreme high-pressure environment. Among them, deep-sea piezophiles have become important model organisms for studying the adaptation of microorganisms to extreme environments due to their unique pressure adaptation mechanisms. The high-pressure environment is a key factor for the survival of piezophiles. Under normal pressure conditions, their activity may decrease or even they cannot survive. Research shows that high pressure can stabilize the hydrogen bonds and base stacking of piezophile DNA, while normal pressure may cause conformational changes in proteins and nucleic acids, thereby affecting their functions. For example, 99% of the single-stranded DNA-binding protein (SSB protein) of piezophiles is more stable under high pressure than under normal pressure. Therefore, the high-pressure environment can more truly simulate the natural survival state of piezophiles and help to reveal their adaptation mechanisms. In addition, to adapt to high pressure, piezophiles have evolved unique molecular and cellular regulatory strategies, such as increasing the content of unsaturated fatty acids in the cell membrane to maintain membrane fluidity and stability. Although some piezophiles can survive under normal pressure, the expression of their high-pressure adaptation-related genes and proteins usually depends on the high-pressure environment. Therefore, high-pressure culture is crucial for analyzing their pressure adaptation mechanisms. Shewanella PT In the cold seep fluid, the presence of a large amount of solid combustible ice provides carbon and nitrogen sources for chemolithoautotrophic organisms, thus forming a unique cold seep ecosystem. However, due to the fact that changes in both temperature and pressure can cause the decomposition of combustible ice, it will also damage the growth of extremely psychrophilic and piezophilic microorganisms that are highly dependent on deep-sea high pressure (up to dozens of megapascals) and low temperature (about 2-4 °C) for metabolic activity. And traditional sampling methods are prone to cause the inactivation of the microbial community or the change of metabolic pathways due to sudden changes in temperature and pressure. Therefore, when studying these extreme microorganisms, it is necessary to adopt a technology that combines a whole-process non-pressure-relief transfer device system with an in-situ reconstructed experimental environment in the deep sea.

[0003] Based on this situation, the research on deep-sea piezophiles is very limited. To further study the characteristics of piezophiles such as high-pressure resistance and in-situ special metabolic pathways, and to explore the key roles they play in the material cycle and energy conversion processes of the deep-sea ecosystem, therefore, it is particularly crucial to identify and isolate single living cells of piezophiles, identify and analyze piezophiles, and obtain the whole-genome gene information of piezophiles for further research.

[0004]

[0005] ​In the prior art, some acquisition methods, separation and purification methods, and related devices related to piezophiles have been disclosed. For example, in the patent application with the publication number "CN114317249A" and the title "An in-situ marine environmental single-cell high-throughput sorting device and method", in this solution, there is a pressure relief process when transferring the deep-sea fidelity sample containing piezophiles to the enrichment microorganism injection system, and the change in pressure during this process has an adverse effect on the survival and metabolism of piezophiles; another example is the patent application with the publication number "CN114456918A" and the title "A deep-sea microorganism enrichment and multi-level purification device and method in a high-pressure environment", in the multi-level purification process of this solution, the purpose of transferring the culture medium is achieved by using the pressure difference. It can be seen that this treatment step also involves the change in pressure. Similarly, the change in pressure will have an adverse effect on the survival and metabolism of piezophiles; another example is the invention patent application with the publication number "CN117158370A" and the title "A deep-sea biological culture test device and test method", this method is for the enrichment analysis of all culturable microorganisms in the sample, and for more than 99% of unculturable microorganisms such as piezophiles, this method cannot achieve the separation and purification of its single functional active microorganisms and their subsequent research, and only metagenomic information can be obtained in downstream tests, and the whole-genome information of a single piezophile cannot be obtained.

[0006] In summary, due to the imperfect pressure maintenance technology and sample fidelity transfer technology, it is difficult to accurately maintain a stable pressure during the process of transferring deep-sea samples to the laboratory, and it is impossible to effectively ensure the original state of the samples, which is likely to cause damage and variation of piezophiles. In the prior art, most are methods for quickly labeling and detecting functional microorganisms in the laboratory normal pressure environment, and there are few relevant research reports on the labeling and sorting technology of deep-sea piezophiles in the in-situ high-pressure environment.

[0007] Therefore, it is necessary to reconstruct the in-situ environment where deep-sea piezophiles live, and develop a new culture-free method under the condition of maintaining the whole process pressure in the laboratory environment, so as to realize the functional analysis of deep-sea piezophiles and the preliminary exploration of the high-pressure resistance mechanism. Summary of the Invention

[0008] The purpose of the present invention is to provide a method and system for transferring-incubating-labeling deep-sea piezophiles with full-pressure and full-temperature preservation based on in-situ reconstruction, which can reconstruct the in-situ environment for the growth and metabolism of piezophiles in the laboratory environment, and realize the identification and analysis of piezophiles in complex samples of deep-sea extreme environments under culture-free conditions, and maximize the preservation of the in-situ environmental conditions for the survival of deep-sea piezophiles.

[0009] The present invention is realized through the following technical solutions: A method for transferring-incubating-labeling deep-sea piezophiles with full-pressure and full-temperature preservation based on in-situ reconstruction includes transfer, incubation, and labeling steps. a. The transfer step is to flow and transfer the sample from the sampler to the transfer device under the action of gravity in an environment of pressure holding and heat preservation. b. Between the transfer and incubation steps, the small tube containing the sample in the transfer device is automatically detached into the incubator in an environment of pressure holding and heat preservation. c. The incubation step is to co-incubate the sample in the small tube with the heavy water culture medium by adding the heavy water culture medium into the incubator under the conditions of pressure holding and heat preservation. d. The labeling step refers to labeling the live cells of piezophilic bacteria in the sample with heavy water through the heavy water culture medium during the incubation process. e. The pressure holding and heat preservation means that the whole process of the transfer and incubation steps maintains the same pressure and temperature as the sampling environment to ensure that the growth and metabolism of piezophilic bacteria are not affected.

[0010] Further, the transfer device is docked with the interface Ⅰ of the sampler through its interface and is vertically installed below the sampler. The transfer device is provided with an interface Ⅱ connected to the pressurizing device. After the internal pressure of the transfer device is increased to the in-situ pressure of the sample through the pressurizing device, the connection valves on the sampler and the transfer device are opened. The interface Ⅲ of the transfer device connected to the driving mechanism is connected to the servo motor. The small tube connected to the telescopic rod is driven to extend into the inner cavity of the sampler through the operation of the control panel equipped with Ⅲ, so that the sample in the sampler naturally falls into the small tube. After the sample transfer is completed, the control panel is operated to drive the small tube to retract into the transfer device, and then the ball valve is closed. During the sample transfer process, the transfer device maintains the in-situ pressure of the sample through the pressurizing device.

[0011] Further, the incubator is docked with the interface of the transfer device through its interface. The incubator is provided with a pressure regulating valve. The pressure regulating valve is a piston structure connected by a long threaded rod. The position of the piston is adjusted by turning the threaded rod to adjust the pressure in the inner cavity of the incubator. The internal pressure of the incubator is set to the same in-situ pressure as the transfer device. Then the connection valves on the incubator and the transfer device are opened. The driving mechanism on the transfer device drives the small tube to extend into the incubator, and when retracting, the small tube automatically falls into the incubation cavity under the action of the one-way snap ring.

[0012] Further, the pressure holding is to maintain the in-situ pressure of piezophilic bacteria, and the pressure range is 0.1 - 200 MPa. The heat preservation is to maintain the in-situ temperature of piezophilic bacteria, and the temperature range is 2 - 4 °C.

[0013] Further, in the incubation step, 2216E heavy water culture medium is used as the incubation medium, and the incubation is carried out with 2216E heavy water culture medium at a concentration of 50 - 75% for 12 - 72 h under the conditions of pressure holding and heat preservation.

[0014] A system applicable to the method for whole-process pressure and temperature maintenance sample transfer-incubation labeling of deep-sea piezophiles based on in-situ reconstruction as described above, characterized in that: it includes a transfer device and an incubation device, The transfer device includes a transfer device body, a small tube, and a pressure gauge for monitoring the internal pressure of the transfer device. One end of the transfer device body is provided with an interface III connected to a driving mechanism; the other end is provided with an interface I, and a connection valve is provided at the interface I; the output end of the driving mechanism is connected to a telescopic rod, and the other end of the telescopic rod is detachably connected to the small tube. The driving mechanism drives the small tube connected to the telescopic rod to move in the inner cavity of the transfer device body, and the small tube can extend out of the transfer device body along the interface I of the transfer device body. An interface II for connecting a pressurizing device is provided on the transfer device body; The incubation device includes an incubation device body and a pressure gauge for detecting the internal pressure of the incubation device. One end of the incubation device body is provided with an interface communicating with the transfer device, and a connection valve is provided at the interface. An incubation cavity and a one-way snap ring are provided inside the incubation device body. The one-way snap ring is used to drop the small tube extending into the incubation cavity into the chamber. The incubation device is connected with a pressure regulating valve. The pressure regulating valve is a piston structure connected by a long threaded rod, and the position of the piston is adjusted by screwing the threaded rod to adjust the pressure in the inner cavity of the incubation device.

[0015] Further, the incubation cavity is made of peek material.

[0016] Further, the driving mechanism is a servo motor.

[0017] Further, the connection valve is a ball valve.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: First, in the present invention, by using the method for whole-process pressure and temperature maintenance sample transfer-incubation labeling of deep-sea piezophiles based on in-situ reconstruction, the fidelity transfer of piezophiles from a deep-sea sampling device to a culture incubation chamber and the precise labeling of heavy water can be realized. By using Raman spectroscopy detection and sorting technology, the identification and analysis of piezophiles in complex samples in the deep-sea extreme environment can be realized under the condition of non-culture, maximizing the preservation of the in-situ environmental conditions for the survival of deep-sea piezophiles, further deeply studying the characteristics of piezophiles such as high-pressure resistance and in-situ special metabolic pathways, and further exploring the key roles they play in the material cycle and energy conversion processes of the deep-sea ecosystem.

[0019] Second, in the present invention, a system matching the method for transferring, incubating, and labeling deep-sea piezophiles with full-pressure and full-temperature preservation based on in-situ reconstruction is proposed. Through the transfer device and incubator in the system, the full-pressure and full-temperature conditions for analyzing deep-sea samples after collection and transfer to the laboratory environment are realized. This system can avoid the problem of piezophile inactivation caused by pressure changes in traditional methods, maintain the in-situ state of deep-sea microorganisms to the greatest extent, avoid damage to cells caused by pressure fluctuations, and thus maintain the cell and genomic integrity of piezophiles for subsequent sorting and sequencing research.

[0020] Third, in the present invention, by using the method for transferring, incubating, and labeling deep-sea piezophiles with full-pressure and full-temperature preservation based on in-situ reconstruction, the survival of piezophiles can be ensured as much as possible, and the live cells of piezophiles can be successfully labeled with heavy water. This method uses heavy water labeling under high-pressure conditions. Compared with normal-pressure labeling, the high-pressure environment can more realistically reflect the metabolic state of deep-sea piezophiles. To avoid labeling deviation caused by metabolic differences due to pressure changes and significantly improve the labeling efficiency and accuracy, this technology specifically designs a pressure compensation module for the heavy water labeling stage to ensure that the pressure remains constant during the incubation of the culture medium containing heavy water (D2O) and is consistent with the in-situ high-pressure environment. Through synchronous high-pressure labeling and sorting, piezophiles complete metabolic labeling and activity verification under pressures close to their natural habitats for subsequent research.

[0021] Fourth, in the present invention, by using the full-pressure and full-temperature sampling-transfer method and combining it with pressure-preserved heavy water incubation, the in-situ active state labeling of deep-sea microorganisms is realized. Then, using Raman spectroscopy detection method, the C-D characteristic peak (2000~2400 cm -1 -1) labeled with heavy water in active microorganisms and the C-H stretching vibration peak (~3000 cm -1 -1) of long-chain unsaturated fatty acids in piezophiles are detected to specifically analyze the metabolic characteristics of low-abundance active piezophiles in deep-sea samples. It can also be combined with optical tweezers sorting technology to further realize the separation of labeled piezophiles, and through single-cell whole-genome sequencing technology, genomic and functional analysis of single cells can be carried out, so as to combine metabolic activity with genomic information, preliminarily explore its environmental adaptation mechanism, and more comprehensively analyze the functions of piezophiles and their roles in the deep-sea ecosystem.

[0022] Fifth, in the present invention, stable isotope labeling combined with Raman spectroscopy is adopted. Without pure culture, through stable isotope tracing and Raman spectroscopy feature recognition, based on Raman-activated cell sorting method, optical tweezers are used to directly capture target single cells in complex samples, realizing non-destructive sorting and functional analysis of living single cells. This method breaks through the traditional culture limitation, can directly analyze functional individuals in complex samples, and forms a closed loop of "metabolic phenotype recognition - precise sorting - functional verification". BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the docking process between the transfer device and the sampler and the incubator respectively.

[0024] Figure 2 It is a schematic structural diagram of the transfer device assembled with the incubator.

[0025] Figure 3 It is Figure 2 a sectional view of.

[0026] Figure 4 It is a physical diagram of the transfer device.

[0027] Figure 5 It is a physical diagram of the incubator.

[0028] Figure 6 It is a schematic diagram of the internal structure of the incubator.

[0029] Figure 7 It is the average cell atlas under different incubation media.

[0030] Figure 8 It is the difference in the proportion of living cells under different heavy water concentrations.

[0031] Figure 9 It is the proportion of living cells at different incubation times.

[0032] Figure 10 It is a comparison chart of the difference in the proportion of living cells detected by Raman in the TSC and PSC groups.

[0033] Figure 11 It is the average Raman atlas of living cells in the TSC and PSC groups.

[0034] Among them, 1. Sampler; 2. Transfer device; 3. Incubator; 4. Pressure gauge; 5. Connection valve; 6. Driving mechanism; 7. Pressure regulating valve; 8. Control panel; 1.1. Sampler interface; 2.1. Transfer device body; 2.2. Small tube; 2.3. Interface III; 2.4. Interface I; 2.5. Telescopic rod; 2.6. Interface II; 3.1. Incubator body; 3.2. Incubator interface; 3.3. Incubation cavity; 3.4. One-way snap ring; 7.1. Threaded rod. Specific implementation mode

[0035] The present invention will be further described in detail below in conjunction with the embodiments, but the implementation modes of the present invention are not limited thereto.

[0036] Embodiment 1 A method for transferring-incubating and labeling deep-sea piezophiles with full-pressure and full-temperature preservation based on in-situ reconstruction, which relates to the technical field of deep-sea piezophile labeling, and includes the steps of transfer, incubation and labeling. a. The transfer step is to flow and transfer the sample from the sampler to the transfer device 2 by gravity in an environment of constant pressure and temperature. b. Between the transfer and incubation steps, in an environment of constant pressure and temperature, the small tube 2.2 containing the sample in the transfer device 2 automatically falls off into the incubator 3. c. The incubation step is to co-incubate the sample in the small tube 2.2 with the heavy water culture medium by adding the heavy water culture medium into the incubator 3 under the conditions of constant pressure and temperature. d. The labeling step refers to labeling the live cells of piezophilic bacteria in the sample with heavy water through the heavy water culture medium during the incubation process. e. The constant pressure and temperature means that the transfer and incubation steps are kept under the same pressure and temperature as the sampling environment throughout to ensure that the growth and metabolism of piezophilic bacteria are not affected.

[0037] Preferably, in the incubation step, the 2216E heavy water culture medium is used as the incubation medium, and the incubation is carried out with the 2216E heavy water culture medium at a concentration of 50 - 75% for 12 - 72 h under the conditions of constant pressure and temperature.

[0038] For the convenience of the public to understand this solution, in this embodiment, a relatively optimal system applicable to the method for transferring-incubating and labeling deep-sea piezophilic bacteria with constant pressure and temperature throughout based on in-situ reconstruction is taken as an example to further illustrate this solution.

[0039] Reference Figures 1 - 3 , this system includes a transfer device 2 and an incubator 3.

[0040] The transfer device 2 includes a transfer device body 2.1, a small tube 2.2, and a pressure gauge 4 for monitoring the internal pressure of the transfer device 2. Refer to Figure 4 , one end of the transfer device body 2.1 is provided with an interface III 2.3 connected to the driving mechanism 6. In this embodiment, the driving mechanism 6 uses a servo motor; the other end of the transfer device body 2.1 is provided with an interface I 2.4, and a connection valve 5 is provided at the interface I 2.4. In this embodiment, the connection valves 5 all use ball valves; the output end of the servo motor is connected to a telescopic rod 2.5, and the other end of the telescopic rod 2.5 is detachably connected to the small tube 2.2. The servo motor drives the small tube 2.2 connected to the telescopic rod 2.5 to move in the inner cavity of the transfer device body 2.1, and the small tube 2.2 can extend out of the transfer device body 2.1 along the interface I 2.4 of the transfer device body 2.1. The transfer device body 2.1 is provided with an interface II 2.6 for connecting the pressurizing device. The incubator 3 includes an incubator body 3.1 and a pressure gauge 4 for detecting the internal pressure of the incubator 3. Refer to Figure 5 、 6, one end of the incubator body 3.1 is provided with an incubator interface 3.2 communicating with the transfer device 2, and a connection valve 5 (ball valve) is provided at the interface. Inside the incubator body 3.1, there are an incubation chamber 3.3 and a one-way snap ring 3.4. The one-way snap ring 3.4 is used to drop the small tube 2.2 extending into the incubation chamber 3.3 into this chamber. The pressure regulating valve 7 is a piston structure connected by a long threaded rod 7.1. The position of the piston is adjusted by turning the threaded rod 7.1 to adjust the pressure inside the inner cavity of the incubator 3.

[0041] During use, the transfer device 2 is docked with the sampler interface 1.1 of the sampler 1 through its interface Ⅰ 2.4 and is vertically installed below the sampler 1. The transfer device 2 is connected with a pressurizing device. After the internal pressure of the transfer device 2 is increased to the in-situ pressure of the sample by the pressurizing device, then the ball valves on the sampler and the transfer device 2 are opened. The transfer device 2 is connected to a servo motor through its interface Ⅲ 2.3 connected to the servo motor. The servo motor is connected to the control panel 8. By operating the control panel 8, the servo motor is controlled to drive the small tube 2.2 connected to it to extend into the inner cavity of the sampler, so that the sample in the sampler 1 naturally falls into the small tube 2.2. After the sample transfer is completed, operate the control panel 8 to make the servo motor drive the small tube 2.2 connected to the telescopic rod 2.5 to retract into the transfer device 2, and then close the ball valve. During the sample transfer process, the transfer device 2 maintains the in-situ pressure of the sample through the pressurizing device.

[0042] During use, the incubator 3 is docked with the interface Ⅰ 2.4 of the transfer device 2 through its interface. The incubator 3 also maintains the in-situ pressure of the piezophilic bacteria (the same pressure as in the sampler and the transfer device 2) through the pressurizing device connected to the transfer device 2. At the same time, a pressure regulating valve 7 is connected to the incubator 3. The pressure regulating valve 7 is a piston structure connected by a long threaded rod 7.1. The position of the piston is adjusted by turning the threaded rod 7.1 to finely adjust the pressure inside the inner cavity of the incubator 3, so that the sample remains at a constant pressure during the incubation process. After the internal pressure of the incubator 3 is increased to the in-situ pressure of the sample by the pressurizing device connected to the transfer device 2, then the connection valve 5 on the incubator 3 and the transfer device 2 is opened. The servo motor installed on the transfer device 2 drives the small tube 2.2 to extend and automatically fall off into the inner cavity of the incubator 3. During the sample transfer process, the incubator 3 maintains the in-situ pressure of the sample through the pressurizing device.

[0043] More preferably, in this system, the incubation chamber 3.3 of the incubator 3 is made of peek material.

[0044] Specifically, in this embodiment, the operation steps of the method for whole-process in-situ pressure and temperature preservation transfer-incubation labeling of deep-sea piezophilic bacteria based on in-situ reconstruction are as follows: S1. Take the transfer device 2 and the incubator 3 in the above system. First, disinfect the interface Ⅰ 2.4 of the transfer device 2 and the small tube 2.2 in the transfer device 2, and at the same time disinfect the interface of the sampler 1. The sampler 1 is a sampling device that can maintain the in-situ pressure and temperature of deep-sea samples. And the entire system (all experimental devices) should be carried out in a low-temperature environment (2 - 4 °C). S2. Connect the pressurizing device through the interface Ⅱ 2.6 of the transfer device 2. In this embodiment, the interface Ⅱ 2.6 is a high-pressure quick-release male interface, and the pressurizing device also has a high-pressure quick-release female interface matching the interface Ⅱ 2.6. The pressurizing device pressurizes the inner cavity of the transfer device 2 until the internal pressure of the transfer device 2 is the same as the in-situ pressure of the sediment inside the sampler. S3. Then dock the interface Ⅰ 2.4 of the transfer device 2 with the sampler 1 through the corresponding interfaces, and then open the ball valves on both of them respectively. Operate the control panel 8 connected to the servo motor. The servo motor drives the telescopic rod 2.5 to drive the small tube 2.2 into the sampler 1. Invert the sampler 1, and use gravity to transfer the sample in the sampler 1 to the small tube 2.2 in the transfer device 2 to complete the sampling. The pressure gauge 4 on the transfer device 2 monitors its internal pressure, and supplements the pressure through the pressurizing device to ensure that the internal pressures of the transfer device 2 and the sampler 1 are the same during the entire transfer process, that is, the in-situ pressure of the deep-sea sediment sample is still maintained during this process. S4. Then operate the servo motor connected to the transfer device 2 to drive the telescopic rod 2.5 to retract, so that its small tube 2.2 carries 10 mL of sample (the amount of the sample is related to the volume of the small tube 2.2) and returns to the inside of the transfer device 2. S5. Close the ball valves on the transfer device 2 and the sampler 1, and disassemble the transfer device 2 and the sampler 1. S6. Disinfect the incubation chamber 3.3 of the incubator 3 and its interface, and then add 10 mL of the 2216E marine bacteria special medium prepared with sterilized heavy water to the incubation chamber 3.3. S7. Operate the pressure regulating valve 7 connected to the incubator 3, and adjust the position of the piston by manually turning the thread, so as to change the internal volume of the incubation chamber 3.3 to adjust the internal pressure of the incubator 3, and set its internal pressure to the same in-situ pressure as the transfer device 2. S8. Then dock the transfer device 2 with the incubator 3, and then open the ball valves on both the transfer device 2 and the incubator 3 respectively. Drive the servo motor of the transfer device 2 to drive the telescopic rod 2.5 to drive the small tube 2.2 into the incubation chamber 3.3, and send the sampling tube containing the deep-sea sediment sample in the transfer device 2 into the inside of the incubator 3. S9. Drive the servo motor on the transfer device 2 to retract the telescopic rod 2.5, and the one-way snap ring 3.4 inside the incubator 3 disengages the small tube 2.2 of the transfer device 2 from the telescopic rod 2.5, so that the small tube 2.2 containing the sediment sample remains in the incubation chamber 3.3. S10. The entire system of the transfer device 2 and the incubator 3 is connected to the pressure-maintaining coring laboratory simulation test and analysis system with a pressure sensor through the interface connecting the pressure regulating valve 7. After setting the target pressure, the PID control of the pressure is automatically carried out, the pressure change is monitored throughout the process, and the pressure is replenished in a timely manner. Incubation is carried out for 48 h under the in-situ pressure and temperature conditions of deep-sea piezophiles, and live cell heavy water labeling is carried out; the pressure is maintained stable throughout the process, and the co-incubation of deep-sea sediment samples and heavy water culture medium is completed.

[0045] In this embodiment, the influence of process parameters in each step on piezophiles is further investigated.

[0046] 1. Investigate the influence of the incubation medium on piezophiles.

[0047] In this experiment, two incubation media, heavy water and 2216E heavy water culture medium, were used respectively, and the average cell maps of the two groups are as attached Figure 7 shown.

[0048] As Figure 7 can be seen, in the incubation group with only heavy water added, no peak appears in the C-D peak shift section (2000 - 2400 cm -1 ), and no live cells are detected; while in the incubation group with 2216E heavy water culture medium added, a peak appears in the C-D peak shift section, that is, live cells are detected. Therefore, in this scheme, 2216E heavy water culture medium is selected as the incubation medium for live cell labeling.

[0049] 2. Investigate the influence of the incubation heavy water concentration on piezophiles.

[0050] According to known literature, too high a heavy water concentration will have a certain adverse effect on cell growth. Therefore, heavy water culture media with concentrations of 50% and 75% were used, and incubation was carried out under the condition of using 2216E heavy water culture medium as the incubation medium. The proportion of live cells is as attached Figure 8 .

[0051] As Figure 8 can be seen, there is no significant difference in the proportion of live cells in samples with different heavy water concentrations, and all can effectively label live cells. In order to reduce costs, in this scheme, 50% heavy water final concentration is selected as the incubation concentration for live cell labeling.

[0052] 3. Investigate the influence of the incubation time on piezophiles.

[0053] Under the condition of using 2216E heavy water culture medium as the incubation medium and 50% heavy water concentration as the incubation concentration, the samples were incubated for 6 h, 12 h, and 48 h, and then the proportion of live cells was detected. The results are as attached Figure 10 shown.

[0054] As Figure 9It can be seen that no live cells were detected after 6 h of incubation. As the incubation time increased, the proportion of detected live cells increased, and the results of significance analysis showed that the proportion of live cells after 48 h of incubation was significantly higher than that after 6 h. Therefore, an incubation time of 48 h was finally adopted.

[0055] Based on the above investigation results, the following conditions were determined as the preferred incubation conditions for labeling live cells of piezophilic bacteria in this protocol: using 2216E heavy water medium as the incubation medium, incubating with 2216E heavy water medium at a final concentration of 50% (i.e., mixing 10 mL of 2216E medium containing 100% heavy water with 10 mL of the sample equally and incubating), and incubating for 48 h under the conditions of maintaining pressure and temperature. Method validation The deep-sea sediment samples were co-incubated with heavy water medium under in-situ pressure and temperature conditions. During the active metabolism of piezophilic bacteria, deuterium (²H) can be incorporated into biological macromolecules such as fatty acids, proteins, and nucleic acids to form stable C-D bonds. After 48 h of incubation, a single-cell Raman optical tweezers cell sorter (RACS-Seq RC24) was used to identify the Raman characteristic peaks (2000 - 2400 cm -1 ) of C-D bonds through Raman spectral characteristics to detect whether the live cells of piezophilic bacteria in the sample were labeled; based on Raman-activated Cell Sorting (RACS), optical tweezers were used to directly capture single target piezophilic bacteria cells in complex samples, and then subsequent single-cell whole-genome analysis was carried out to achieve non-destructive sorting and functional analysis of living single cells.

[0056] The samples incubated under pressure and temperature using the whole-process pressure and temperature maintenance sampling - transfer - incubation system in this protocol were the experimental group (PSC), and the samples incubated under normal pressure and low temperature conditions in a traditional laboratory were used as the control group (TSC). The proportion of live cells in both groups was detected using Raman spectroscopy, and the results are shown in Table 1 and Figure 10 、 11 as shown. Figure 10 is a comparison chart of the difference in the proportion of live cells detected by Raman in the TSC and PSC groups, Figure 11 is the average Raman spectrum of live cells in the TSC and PSC groups.

[0057] The specific operation steps are as follows: 1. Sediment sample pretreatment steps: (1) Take 1 mL of the incubated deep-sea sediment sample into a sterile centrifuge tube; (2) Add 1 mL of SM solution and 0.5 g of glass beads, and shake for 15 min. (SM solution: 5.8 g of NaCl, 50 mL of 1 mol / L Tris-HCl (pH 7.5), 2 g of MgSO4•7H2O, made up to 1 L with ddH2O); (3) Centrifuge at 100 g for 5 min at 4°C, and collect the supernatant; (4) Add 1 mL of SM solution to the precipitate to resuspend it, and repeat 3 times; (5) Combine the supernatants collected 3 times, and centrifuge at 300 g for 5 min at 4°C; (6) Collect the supernatant, and centrifuge at 5000 g for 30 min at 4°C; (7) Discard the supernatant, wash the precipitate with 1 mL of sterile saline 2 times (5000 r / min, 10 min), and directly freeze the precipitate.

[0058] 2. Raman test sorting method: (1) Thaw the cell precipitate, resuspend it with buffer and dilute appropriately so that the bacterial injection concentration is about 5.0×10 6 ~5.0×10 7 cells / mL; (2) Take 20 μL of the sample, suck it into the chip by applying negative pressure, then install the chip on the single-cell Raman optical tweezers sorter (RACS-Seq RC24), connect the syringe and capillary, open the injection pump flow rate at 9 mL / min, and rinse the sorting channel for 10 min; (3) Use a 532 nm laser to collect Raman spectra, select the cells with C-D peaks, and then use a 1032 nm laser to capture and sort single target cells.

[0059] Table 1 Detection of the number and proportion of live cell spectra.

[0060] From Table 1 and Figure 10 、 11 it can be seen that the proportion of live cells of piezophilic bacteria in the experimental group is significantly higher than that in the control group. It shows that this method can complete the labeling of live piezophilic bacteria, and compared with the traditional atmospheric pressure labeling method, this method can significantly increase the proportion of live cells.

[0061] 3. Single-cell whole-genome amplification and sequencing: (1) Add 2 μL of mixed lytic enzymes to the sorted single-cell oil droplets, and incubate at 37°C for 30 min.

[0062] (2) Add 4 μL of the starting cell sample solution (stored in PBS) to a microcentrifuge tube. If the volume of the starting cell solution is less than 4 μL, adjust the final volume to 4 μL by adding PBS solution.

[0063] (3) Add 3 μL of Buffer D2, mix well, and incubate at 65 °C for 10 min.

[0064] (4) Add 3 μL of Stop Solution, mix well, and place on ice.

[0065] (5) Add 40 μL of the premix containing REPLI-g sc DNA Polymerase, and incubate at 30 °C for 8 h.

[0066] (6) Inactivate REPLI-g sc DNA Polymerase at 65 °C for 3 min.

[0067] (7) Dilute the amplified DNA at a ratio of 1:100, and take 2 μL of the diluted DNA for downstream sequencing analysis.

[0068] (8) Send the amplified DNA product to the Illumina NovaSeq 6000 platform of Novogene Bioinformatics Technology Co., Ltd. for next-generation sequencing.

[0069] After detection, this method can obtain the whole-genome map of piezophilic bacteria single cells, thus enabling further research on the special metabolic functions of piezophilic bacteria.

[0070] In this protocol, stable isotope labeling combined with Raman spectroscopy is used. Without pure culture, through stable isotope tracing and Raman spectroscopy feature recognition, based on Raman-activated cell sorting method, optical tweezers are used to directly capture target single cells in complex samples, realizing non-destructive sorting and functional analysis of living single cells. This method breaks through the traditional cultivation limitations, can directly analyze functional individuals in complex samples, and forms a closed loop of "metabolic phenotype recognition - precise sorting - function verification". Combined with single-cell amplification and sequencing methods, it can also achieve precise association between phenotype and genotype, transitioning from "metabolic function screening" to "gene mechanism analysis".

[0071] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any simple modification or equivalent change made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for transferring, incubating, and labeling deep-sea piezophilic bacteria with whole-process pressure and temperature preservation based on in-situ reconstruction, characterized in that, It includes transfer, incubation, and labeling steps. a. In the transfer step, under the action of gravity in an environment with pressure maintained and temperature kept constant, the sample is flowed from the sampler (1) to the transfer device (2). b. Between the transfer and incubation steps, in an environment with pressure maintained and temperature kept constant, the small tube (2.2) containing the sample in the transfer device (2) automatically falls off into the incubator (3). c. In the incubation step, under the conditions of pressure maintained and temperature kept constant, by adding heavy water culture medium into the incubator (3), the sample in the small tube (2.2) is co-incubated with the heavy water culture medium. d. The labeling step refers to labeling the live cells of piezophilic bacteria in the sample with heavy water through the heavy water culture medium during incubation. e. The pressure-maintaining and temperature-keeping means that the transfer and incubation steps are kept at the same pressure and temperature as the sampling environment throughout to ensure that the growth and metabolism of piezophilic bacteria are not affected.

2. The method according to claim 1, characterized in that: The transfer device (2) is docked with the interface of the sampler (1) through its interface I (2.4) and is vertically installed below the sampler (1). The transfer device (2) is provided with an interface II (2.6) connected to the pressurizing device. After the internal pressure of the transfer device (2) is increased to the in-situ pressure of the sample through the pressurizing device, the connection valve (5) on the sampler (1) and the transfer device (2) is opened. The small tube (2.2) connected to the telescopic rod (2.5) is driven to extend into the inner cavity of the sampler (1) through the interface III (2.3) of the transfer device (2) connected to the driving mechanism (6). By operating the control panel (8) equipped with the driving mechanism (6), the sample in the sampler (1) naturally falls into the small tube (2.2). After the sample transfer is completed, the control panel (8) is operated to drive the small tube (2.2) to retract into the transfer device (2). Subsequently, the ball valve is closed. During the sample transfer process, the transfer device (2) maintains the in-situ pressure of the sample through the pressurizing device.

3. The method according to claim 1, characterized in that: The incubator (3) is docked with the interface I (2.4) of the transfer device (2) through its interface. The incubator (3) is provided with a pressure regulating valve (7). The pressure regulating valve (7) is a piston structure connected by a long threaded rod (7.1). By turning the threaded rod (7.1), the position of the piston is adjusted to regulate the pressure in the inner cavity of the incubator (3). The internal pressure of the incubator (3) is set to the same in-situ pressure as that of the transfer device (2). Then the connection valve (5) on the incubator (3) and the transfer device (2) is opened. The driving mechanism (6) on the transfer device (2) drives the small tube (2.2) to extend into the incubation cavity (3.3). When retracting, the small tube (2.2) automatically falls off into the incubation cavity (3.3) under the action of the one-way snap ring (3.4).

4. The method according to claim 1, characterized in that: The pressure-maintaining is to maintain the in-situ pressure of piezophilic bacteria, and the pressure range is 0.1~200 MPa. The temperature-keeping is to maintain the in-situ temperature of piezophilic bacteria, and the temperature range is 2~4 °C.

5. The method according to claim 1, characterized in that: In the incubation step, 2216E heavy water culture medium is used as the incubation medium, and incubation is carried out with 2216E heavy water culture medium at a concentration of 50~75% for 12~72 h under the conditions of pressure maintained and temperature kept constant.

6. A system applicable to the method for transferring, incubating, and labeling deep-sea piezophiles with in-situ reconstruction-based full-process pressure and temperature maintenance as described in claim 1, characterized in that: It includes a transfer device (2) and an incubator (3). The transfer device (2) includes a transfer device body (2.1), a small tube (2.2), and a pressure gauge (4) for monitoring the internal pressure of the transfer device (2). One end of the transfer device body (2.1) is provided with an interface III (2.3) connected to the drive mechanism (6); the other end is provided with an interface I (2.4), and a connection valve (5) is provided at the interface I (2.4); the output end of the drive mechanism (6) is connected to the telescopic rod (2.5), and the other end of the telescopic rod (2.5) is detachably connected to the small tube (2.2). The drive mechanism (6) drives the small tube (2.2) connected to the telescopic rod (2.5) to move in the inner cavity of the transfer device body (2.1), and the small tube (2.2) can extend out of the transfer device body (2.1) along the interface I (2.4) of the transfer device body (2.1). An interface II (2.6) for connecting a pressurizing device is provided on the transfer device body (2.1); The incubator (3) includes an incubator body (3.1) and a pressure gauge (4) for detecting the internal pressure of the incubator (3). One end of the incubator body (3.1) is provided with an interface communicating with the transfer device (2), and a connection valve (5) is provided at the interface. An incubation chamber (3.3) and a one-way snap ring (3.4) are provided inside the incubator body (3.1). The one-way snap ring (3.4) is used to drop the small tube (2.2) extending into the incubation chamber (3.3) into this chamber. The incubator (3) is connected with a pressure regulating valve (7). The pressure regulating valve (7) is a piston structure connected by a long threaded rod (7.1). The position of the piston is adjusted by turning the threaded rod (7.1) to adjust the pressure in the inner cavity of the incubator (3).

7. The system according to claim 6, wherein: The incubation chamber (3.3) is made of peek material.

8. The system according to claim 6, wherein: The drive mechanism (6) is a servo motor.

9. The system according to claim 6, characterized in that: The connection valve (5) is a ball valve.

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