A method and system for sample transfer and incubation labeling of deep-sea barophiles based on in-situ reconstruction and full-process pressure and temperature preservation

Abstract

The present invention discloses a method and system for labeling deep-sea barophiles by transferring and incubating samples under full pressure and heat preservation based on in-situ reconstruction, which relates to the technical field of labeling deep-sea barophiles, including transfer, incubation and labeling steps. The transfer step is to utilize gravity to transfer the sample from a sampler to a transferor in a pressure-maintaining and heat-maintaining environment; between the transfer and incubation steps, the small tube containing the sample in the transferor is automatically dropped into the incubator in the pressure-maintaining and heat-maintaining environment; the incubation step is to add heavy water culture medium to the incubator under the pressure-maintaining and heat-maintaining conditions so that the sample in the small tube is co-incubated with the heavy water culture medium; the labeling step refers to labeling the living cells of the barophiles in the sample with heavy water by using the heavy water culture medium during the incubation process, thereby maximally maintaining the in-situ environmental conditions for the survival of the deep-sea barophiles, labeling and obtaining the living cells of the barophiles, and facilitating in-depth research on the barophiles.

Description

Technical Field

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

[0002] The deep sea is usually defined as the sea area with a depth of 1000m below sea level. It is called an extreme environment due to its low temperature, high pressure, darkness and oligotrophic characteristics, but in fact it is one of the aquatic water environments with the richest number of microorganisms on Earth. In the long-term evolution process, deep-sea microorganisms have gradually developed pressure-resistant or barophilic characteristics in order to adapt to the extreme high-pressure environment. Among them, deep-sea barophiles have become important model organisms for studying the adaptation of microorganisms to extreme environments due to their unique pressure adaptation mechanism. High-pressure environment is a key factor for the survival of barophiles. Under normal pressure conditions, their activity may be reduced or even unable to survive. Studies have shown that high pressure can stabilize the hydrogen bonds and base stacking of barophilic DNA, while normal pressure may cause conformational changes in proteins and nucleic acids, thereby affecting their functions. For example, barophiles Shewanella PT 99's single-stranded DNA binding protein (SSB protein) is more stable under high pressure than under normal pressure. Therefore, high-pressure environments can more realistically simulate the natural living conditions of barophiles, helping to reveal their adaptation mechanisms. Furthermore, to adapt to high pressure, barophiles 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 barophiles can survive under normal pressure, the expression of genes and proteins related to high-pressure adaptation is often dependent on high-pressure environments. Therefore, high-pressure cultivation is crucial for understanding their pressure adaptation mechanisms.

[0003] The presence of large amounts of solid methane hydrate in cold seep fluids provides carbon and nitrogen sources for chemoautotrophic organisms, thus forming a unique cold seep ecosystem. However, fluctuations in temperature and pressure can cause methane hydrate to decompose, impairing the growth of extreme psychrophilic and barophilic microorganisms whose metabolic activity is highly dependent on the high pressures (up to tens of MPa) and low temperatures (approximately 2-4°C) of the deep sea. Traditional sampling methods are susceptible to inactivation of bacterial communities or alteration of metabolic pathways due to sudden changes in temperature and pressure. Therefore, the study of these extreme microorganisms requires a combination of a fully non-depressurized transfer system and a deep-sea in situ reconstructed experimental environment.

[0004] Given this current situation, research on deep-sea piezophiles is very limited. To further investigate their properties, such as their high-pressure tolerance and specialized metabolic pathways, and to explore their key roles in the material circulation and energy conversion processes of deep-sea ecosystems, further research is crucial, including identifying and isolating living individual piezophile cells, identifying and analyzing them, and obtaining their complete genome information.

[0005] In the prior art, some methods for obtaining, separating and purifying barophiles and related devices are disclosed. For example, the patent application with the publication number "CN114317249A" and the name "A high-throughput sorting device and method for single cells in an in-situ marine environment" has a pressure relief process when transferring the deep-sea fidelity sample containing barophiles to the enrichment microorganism injection system. The pressure change in this process has an adverse effect on the survival and metabolism of barophiles; another example is the patent application with the publication number "CN114456918A" and the name "A high-pressure environment deep-sea microorganism enrichment and multi-stage purification device and method". The multi-stage purification process in this scheme uses the pressure difference to achieve the purpose of transferring the culture medium, which can be used for the enrichment of barophiles. It can be seen that this processing step also involves changes in pressure. Similarly, changes in pressure will have an adverse effect on the survival and metabolism of barophiles. For example, the invention patent application with publication number "CN117158370A" and titled "A Deep-sea Biological Cultivation Test Device and Test Method" is an invention patent application that performs enrichment analysis on all culturable microorganisms in the sample. However, for more than 99% of unculturable microorganisms such as barophiles, this method cannot achieve the separation and purification of individual functionally active microorganisms and their subsequent research. Downstream testing can only obtain macro-omics information, and cannot obtain the whole genome information of a single barophile.

[0006] In summary, due to the imperfections of pressure maintenance and sample fidelity transfer technologies, it is difficult to accurately maintain stable pressure during the transfer of deep-sea samples to the laboratory, making it impossible to effectively preserve the original state of the samples, which can easily cause damage and mutation of barophiles. Existing technologies mostly focus on rapid labeling and detection of functional microorganisms under normal laboratory pressure, while few studies have reported on labeling and sorting deep-sea barophiles under in situ high-pressure conditions.

[0007] Therefore, it is necessary to reconstruct the in situ environment in which deep-sea barophiles live and develop a new culture-free method under full-time pressure maintenance in a laboratory environment, so as to achieve functional analysis of deep-sea barophiles and 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 the full-process pressure-maintained and temperature-maintained sample transfer-incubation labeling of deep-sea barophiles based on in situ reconstruction, which can reconstruct the in situ environment for the growth and metabolism of barophiles in a laboratory environment, realize the identification and analysis of barophiles in complex samples in deep-sea extreme environments under culture-free conditions, and maximize the maintenance of the in situ environmental conditions for the survival of deep-sea barophiles.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for transferring and incubating deep-sea barophiles based on in-situ reconstruction and full-process pressure-maintaining and temperature-maintaining sample transfer and labeling, comprising the steps of transfer, incubation and labeling.

[0011] a. The transfer step is to transfer the sample from the sampler to the transfer device by gravity in a pressure-maintaining and temperature-maintaining environment;

[0012] b. Between the transfer and incubation steps, the small tube containing the sample in the transfer device is automatically dropped into the incubator in a pressure-maintaining and temperature-maintaining environment;

[0013] c. The incubation step is to add heavy water culture medium to the incubator under pressure and temperature maintenance conditions, so that the sample in the small tube is co-incubated with the heavy water culture medium;

[0014] d. The labeling step refers to labeling the living cells of the barophilic bacteria in the sample with heavy water using a heavy water culture medium during the incubation process.

[0015] e. The pressure and temperature maintenance refers to maintaining the same pressure and temperature as the sampling environment throughout the transfer and incubation steps to ensure that the growth and metabolism of barophilic bacteria are not affected.

[0016] Furthermore, the transferor is docked with the interface I of the sampler through its interface and is vertically installed below the sampler. The transferor is provided with an interface II connected to a pressurizing device. After the transferor increases the internal pressure of the transferor to the in-situ pressure of the sample through the pressurizing device, the connecting valve on the sampler and the transferor is opened, and the servo motor is connected through the interface III connected to the driving mechanism on the transferor. The control panel equipped with Ⅲ is operated to drive the small tube connected to the telescopic rod into the inner cavity of the sampler, 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 transferor, and then the ball valve is closed. During the sample transfer process, the transferor maintains the in-situ pressure of the sample through the pressurizing device.

[0017] Furthermore, the incubator is docked with the interface of the transferor through its interface. The incubator is provided with a pressure regulating valve, which is a piston structure connected by a long threaded rod. The pressure in the inner cavity of the incubator is adjusted by twisting the threaded rod to adjust the position of the piston, and the internal pressure of the incubator is set to the same in-situ pressure as that of the transferor. Then, the connecting valves on the incubator and the transferor are opened, and the driving mechanism on the transferor drives the small tube into the incubator, and when retracted, the small tube automatically falls off into the incubation cavity under the action of the one-way clamp.

[0018] Furthermore, the pressure maintenance is to maintain the in-situ pressure of the barophilic bacteria, and the pressure range is 0.1~200MPa. The heat preservation is to maintain the in-situ temperature of the barophilic bacteria, and the temperature range is 2~4°C.

[0019] Furthermore, in the incubation step, 2216E heavy water culture medium is used as the incubation medium, and the incubation is performed using 2216E heavy water culture medium with a concentration of 50-75%, and the incubation is performed under pressure and temperature maintenance conditions for 12-72 hours.

[0020] A system suitable for the above-mentioned method of transferring and incubating deep-sea barophiles by pressure-maintaining and temperature-maintaining samples based on in situ reconstruction, characterized by comprising a transfer device and an incubator,

[0021] 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 drive mechanism; the other end is provided with an interface I, and an interface I is provided with a connecting valve; the output end of the drive mechanism is connected to a telescopic rod, and the other end of the telescopic rod is detachably connected to the small tube. The drive mechanism drives the small tube connected to the telescopic rod to move within 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. The transfer device body is provided with an interface II for connecting to a pressurizing device;

[0022] The incubator includes an incubator body and a pressure gauge for detecting the internal pressure of the incubator. One end of the incubator body is provided with an interface connected to the transferor, and a connecting valve is provided at the interface. An incubation cavity and a one-way clamp are provided inside the incubator body. The one-way clamp is used to drop the small tube inserted into the incubation cavity into the cavity. The incubator is connected to a pressure regulating valve, which is a piston structure connected by a long threaded rod. The pressure in the inner cavity of the incubator is adjusted by twisting the threaded rod to adjust the position of the piston.

[0023] Furthermore, the incubation chamber is made of peek material.

[0024] Furthermore, the driving mechanism is a servo motor.

[0025] Furthermore, the connecting valve is a ball valve.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] 1. In the present invention, the method of in-situ reconstruction-based full-process pressure-maintained and temperature-maintained sample transfer-incubation labeling of deep-sea barophiles is adopted, which can realize the fidelity transfer of barophiles from the deep-sea sampling device to the culture and incubation chamber and the precise labeling of heavy water. In addition, the use of Raman spectroscopy detection and sorting technology can realize the identification and analysis of barophiles in complex samples of deep-sea extreme environments under culture-free conditions, thereby maximally maintaining the in-situ environmental conditions for the survival of deep-sea barophiles. Further in-depth research is conducted on the characteristics of barophiles such as high pressure resistance and in-situ special metabolic pathways, and further exploration of their key role in the material circulation and energy conversion process of deep-sea ecosystems.

[0028] Second, this invention also proposes a system compatible with this in situ reconstruction-based method for sample transfer and incubation labeling of deep-sea barophiles. Through the system's transfer and incubator, pressure and temperature are maintained throughout the entire process of transferring deep-sea samples from collection to a laboratory environment for analysis. This system avoids the inactivation of barophiles due to pressure fluctuations encountered in traditional methods, maintaining the deep-sea microorganisms in situ to the greatest extent possible and preventing cell damage from pressure fluctuations. This system thus maintains the cellular and genomic integrity of the barophiles, facilitating subsequent sorting and sequencing studies.

[0029] Third, the present invention utilizes a method for labeling deep-sea barophiles using in situ reconstruction-based, fully pressurized and heat-maintained sample transfer and incubation, ensuring their survival and enabling successful heavy water labeling of viable cells. This method utilizes heavy water labeling under high pressure. Compared to labeling at normal pressure, this high-pressure environment more accurately reflects the metabolic state of deep-sea barophiles. To avoid labeling deviations caused by metabolic differences due to pressure fluctuations and significantly improve labeling efficiency and accuracy, this technology incorporates a pressure replenishment module specifically designed for the heavy water labeling stage. This ensures that the pressure of the heavy water (D2O)-containing culture medium remains constant during incubation, consistent with the in situ high-pressure environment. By simulating high-pressure labeling and sorting, barophiles can be metabolically labeled and their activity verified under pressures close to their natural habitat, facilitating subsequent research.

[0030] Fourth, the present invention utilizes a full-process pressure-maintained temperature-maintained sampling-transfer method, combined with pressure-maintained heavy water incubation, to achieve in-situ labeling of the active state of deep-sea microorganisms. Raman spectroscopy is then used to detect the CD characteristic peak (2000-2400 cm) labeled by heavy water in the active microorganisms. -1 ) and the CH stretching vibration peaks of long-chain unsaturated fatty acids in barophilic bacteria (~3000 cm -1 ), specifically analyzing the metabolic characteristics of low-abundance active piezophiles in deep-sea samples. Optical tweezers sorting can also be combined to further isolate labeled piezophiles, and single-cell whole-genome sequencing can be used to perform genomic and functional analyses on individual cells. This, combined with metabolic activity and genomic information, allows for preliminary exploration of their environmental adaptation mechanisms and a more comprehensive understanding of the functions of piezophiles and their role in deep-sea ecosystems.

[0031] Fifth, this invention utilizes stable isotope labeling combined with Raman spectroscopy, eliminating the need for pure culture. By combining stable isotope tracing with Raman spectroscopy signature recognition, and based on Raman-activated cell sorting (RAC), optical tweezers are used to directly capture target single cells from complex samples, enabling non-destructive sorting and functional analysis of living single cells. This method transcends the limitations of traditional culture and enables direct analysis of functional individuals within complex samples, forming a closed loop of "metabolic phenotype identification - precise sorting - functional verification." BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the docking process of the transferor with the sampler and incubator respectively.

[0033] Figure 2 It is a schematic diagram of the structure in which the transfer device and the incubator are assembled together.

[0034] Figure 3 yes Figure 2 sectional view of .

[0035] Figure 4 This is a physical picture of the transfer device.

[0036] Figure 5 This is a physical picture of the incubator.

[0037] Figure 6 It is a schematic diagram of the internal structure of the incubator.

[0038] Figure 7 It is the average spectrum of cells in different incubation media.

[0039] Figure 8 It is the difference in the proportion of living cells under different heavy water concentrations.

[0040] Figure 9 is the proportion of living cells at different incubation times.

[0041] Figure 10 This is a comparison chart of the difference in the proportion of living cells detected by Raman in the TSC and PSC groups.

[0042] Figure 11 is the average Raman spectrum of living cells in the TSC and PSC groups.

[0043] Among them, 1. Sampler; 2. Transferr; 3. Incubator; 4. Pressure gauge; 5. Connecting valve; 6. Driving mechanism; 7. Pressure regulating valve; 8. Control panel; 1.1. Sampler interface; 2.1. Transferr 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 chamber; 3.4. One-way clamp; 7.1. Threaded rod. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.

[0045] Example 1

[0046] A method for transferring and incubating deep-sea barophiles based on in-situ reconstruction and full-process pressure-maintaining and heat-maintaining sample labeling, which relates to the technical field of deep-sea barophiles labeling, includes the steps of transfer, incubation and labeling.

[0047] a. The transfer step is to transfer the sample from the sampler to the transfer device 2 by gravity in a pressure-maintaining and temperature-maintaining environment;

[0048] b. Between the transfer and incubation steps, the small tube 2.2 containing the sample in the transfer device 2 is automatically dropped into the incubator 3 in a pressure-maintaining and temperature-maintaining environment;

[0049] c. The incubation step is to add heavy water culture medium to the incubator 3 under pressure and temperature maintenance conditions, so that the sample in the small tube 2.2 is co-incubated with the heavy water culture medium;

[0050] d. The labeling step refers to labeling the living cells of the barophilic bacteria in the sample with heavy water using a heavy water culture medium during the incubation process;

[0051] e. The pressure and temperature maintenance refers to maintaining the same pressure and temperature as the sampling environment throughout the transfer and incubation steps to ensure that the growth and metabolism of barophilic bacteria are not affected.

[0052] Preferably, in the incubation step, 2216E heavy water culture medium is used as the incubation medium, and the incubation is performed using 2216E heavy water culture medium with a concentration of 50-75%, and the incubation is performed under pressure and temperature maintenance conditions for 12-72 hours.

[0053] To facilitate public understanding of the present solution, this embodiment takes a system that is preferably suitable for the method of in-situ reconstruction-based full-process pressure-maintained temperature-maintained sample transfer-incubation labeling of deep-sea barophiles as an example to further illustrate the present solution.

[0054] refer to Figures 1-3 The system includes a transferor 2 and an incubator 3.

[0055] 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. Figure 4 One end of the transfer body 2.1 is provided with an interface III 2.3 connected to the drive mechanism 6. In this embodiment, the drive mechanism 6 adopts a servo motor; the other end of the transfer body 2.1 is provided with an interface I 2.4, and a connecting valve 5 is provided at the interface I 2.4. In this embodiment, the connecting valve 5 adopts a ball valve; the output end of the servo motor 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 servo motor drives the small tube 2.2 connected to the telescopic rod 2.5 to move in the inner cavity of the transfer body 2.1, and the small tube 2.2 can extend out of the transfer body 2.1 along the interface I 2.4 of the transfer body 2.1. The transfer body 2.1 is provided with an interface II 2.6 for connecting to a pressurizing device;

[0056] The incubator 3 includes an incubator body 3.1 and a pressure gauge 4 for detecting the internal pressure of the incubator 3. Figure 5 、 6 One end of the incubator body 3.1 is provided with an incubator interface 3.2 connected to the transferor 2, and a connecting valve 5 (ball valve) is provided at the interface. An incubation chamber 3.3 and a one-way clamp 3.4 are provided inside the incubator body 3.1. The one-way clamp 3.4 is used to drop the small tube 2.2 inserted into the incubation chamber 3.3 into the chamber. The pressure regulating valve 7 is a piston structure connected by a long threaded rod 7.1. The pressure in the inner cavity of the incubator 3 is adjusted by twisting the threaded rod 7.1 to adjust the position of the piston.

[0057] During use, the transferor 2 is docked with the sampler interface 1.1 of the sampler 1 via its interface I 2.4 and is vertically mounted below the sampler 1. A pressurizing device is connected to the transferor 2, which raises the internal pressure of the transferor 2 to the sample's in-situ pressure. The ball valves on the sampler and transferor 2 are then opened. A servo motor is connected via interface III 2.3 on the transferor 2, which is connected to the servo motor. The servo motor is connected to a control panel 8. The servo motor is controlled by operating the control panel 8 to drive the small tube 2.2 connected to the servo motor into the inner cavity of the sampler, allowing the sample in the sampler 1 to naturally fall into the small tube 2.2. After sample transfer is complete, the control panel 8 is operated to cause the servo motor to retract the small tube 2.2 connected to the telescopic rod 2.5 into the transferor 2, and the ball valve is then closed. During sample transfer, the pressurizing device maintains the sample's in-situ pressure.

[0058] When in use, the incubator 3 is docked with the interface Ⅰ2.4 of the transferor 2 through its interface. The incubator 3 also maintains the in-situ pressure of the barophilic bacteria (the same as the pressure in the sampler and transferor 2) through the pressurizing device connected to the transferor 2. At the same time, 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 twisting the threaded rod 7.1 to fine-tune the pressure in the inner cavity of the incubator 3 so that the pressure of the sample remains unchanged during the incubation process. After the incubator 3 increases the internal pressure of the incubator 3 to the in-situ pressure of the sample through the pressurizing device connected to the transferor 2, the connecting valve 5 on the incubator 3 and the transferor 2 is opened. The servo motor installed on the transferor 2 drives the small tube 2.2 to extend into 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.

[0059] More preferably, in the system, the incubation chamber 3.3 of the incubator 3 is made of peek material.

[0060] Specifically, the steps of the method for in-situ pressure-maintaining and temperature-maintaining transfer-incubation labeling of deep-sea barophiles based on in-situ reconstruction in this embodiment are as follows:

[0061] S1. Take the transferor 2 and incubator 3 from the above system and first disinfect the interface I 2.4 of the transferor 2 and the small tube 2.2 in the transferor 2. Simultaneously disinfect the interface of the sampler 1. The sampler 1 is a sampling device that can maintain pressure and temperature of deep-sea samples in situ. The entire system (all experimental devices) should be operated in a low temperature environment (2-4°C).

[0062] S2. Connect the pressurizing device via port II 2.6 of the transferor 2. In this embodiment, port II 2.6 is a high-pressure quick-pull male port, and the pressurizing device also has a high-pressure quick-pull female port that matches port II 2.6. The pressurizing device pressurizes the inner cavity of the transferor 2 until the pressure inside the transferor 2 matches the in-situ pressure of the sediment inside the sampler.

[0063] S3. Then, connect the interface I 2.4 of the transferor 2 to the sampler 1 through the corresponding interface, open the ball valves on both, operate the control panel 8 connected to the servo motor, and the servo motor drives the telescopic rod 2.5 to drive the small tube 2.2 deep 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 of the transferor 2 to complete the sampling. The pressure gauge 4 on the transferor 2 monitors its internal pressure, and the pressure is replenished by the pressure device to ensure that the internal pressure of the transferor 2 and the sampler 1 are consistent throughout the transfer process. That is, the in-situ pressure of the deep-sea sediment sample is still maintained during this process;

[0064] S4. The servo motor connected to the transfer device 2 is operated again to drive the telescopic rod 2.5 to retract, so that the small tube 2.2 carrying 10 mL of sample (the amount of sample is related to the volume of the small tube 2.2) returns to the interior of the transfer device 2;

[0065] S5. Close the ball valves on the transferor 2 and the sampler 1, and separate the transferor 2 and the sampler 1;

[0066] S6. Disinfect the incubation chamber 3.3 of the incubator 3 and its interface, and then add 10 mL of sterilized 2216E marine bacteria culture medium prepared with heavy water into the incubation chamber 3.3;

[0067] S7, operate the pressure regulating valve 7 connected to the incubator 3, manually adjust the position of the piston by twisting the thread, thereby changing 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 transferor 2;

[0068] S8, then docking the transferor 2 with the incubator 3, then opening the ball valves on both the transferor 2 and the incubator 3, respectively, driving the servo motor of the transferor 2 to drive the telescopic rod 2.5 to drive the small tube 2.2 deep into the incubation chamber 3.3, and delivering the sampling tube of the deep-sea sediment sample in the transferor 2 into the interior of the incubator 3;

[0069] S9, driving the servo motor on the transfer device 2 to retract the telescopic rod 2.5, and the one-way clamp 3.4 inside the incubator 3 detaches 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;

[0070] The entire system of S10, transferor 2 and incubator 3 is connected to a pressure-maintaining coring laboratory simulation test and analysis system with a pressure sensor through an interface connected to a pressure regulating valve 7. After setting the target pressure, PID control of the pressure is automatically performed, and the pressure changes are monitored throughout the process and the pressure is replenished in time. The deep-sea barophile bacteria are incubated for 48 hours under the in situ pressure and temperature conditions to carry out heavy water labeling of living cells; the pressure is maintained stable throughout the process to complete the co-incubation of deep-sea sediment samples and heavy water culture medium.

[0071] In this example, the effects of the process parameters in each step on the piezophilic bacteria were further investigated.

[0072] 1. Investigate the effect of incubation medium on barophilic bacteria.

[0073] In this experiment, two incubation media, heavy water and 2216E heavy water medium, were used. The average atlas of the two groups of cells is shown in the attached figure. Figure 7 shown.

[0074] Depend on Figure 7 It can be seen that in the incubation group with only heavy water added, the CD peak shift section (2000-2400 cm -1 ) showed no peak, indicating no live cells were detected. However, in the group incubated with 2216E heavy water medium, a peak appeared in the CD peak shift region, indicating live cells were detected. Therefore, in this protocol, 2216E heavy water medium was selected as the incubation medium for live cell labeling.

[0075] 2. Investigate the effect of incubation heavy water concentration on barophilic bacteria.

[0076] According to known literature, excessively high concentrations of heavy water will have a certain adverse effect on cell growth. Therefore, 50% and 75% concentrations of heavy water medium were used. The cells were incubated under the conditions of 2216E heavy water medium as the incubation medium. The ratio of living cells to attached cells was 2. Figure 8 .

[0077] Depend on Figure 8 It can be seen that there is no significant difference in the proportion of live cells in samples with different heavy water concentrations, and all of them can effectively label live cells. In order to reduce costs, in this scheme, a final heavy water concentration of 50% is selected as the incubation concentration for live cell labeling.

[0078] 3. Investigate the effect of incubation time on barophilic bacteria.

[0079] The samples were incubated for 6 h, 12 h, and 48 h using 2216E heavy water medium as the incubation medium and 50% heavy water as the incubation concentration. The proportion of live cells was then detected. The results are shown in the attached table. Figure 10 shown.

[0080] Depend on Figure 9 It can be seen that no living cells were detected after 6 hours of incubation. As time went on, the proportion of living cells detected increased, and the significance analysis results showed that the proportion of living cells under 48 hours of incubation was significantly greater than that under 6 hours. Therefore, the incubation time of 48 hours was finally adopted.

[0081] Based on the above investigation results, the following conditions were determined in this protocol as the preferred incubation conditions for labeling live cells of barophilic bacteria: 2216E heavy water medium was used as the incubation medium, and a final concentration of 50% 2216E heavy water medium was used for incubation (i.e., 10 mL of 2216E medium containing 100% heavy water was mixed with 10 mL of sample in equal amounts for incubation), and the cells were incubated under pressure and temperature maintenance conditions for 48 h.

[0082] Method validation

[0083] Deep-sea sediment samples were incubated with heavy water culture medium under in-situ pressure and temperature conditions. During the active metabolism of barophiles, deuterium (²H) was incorporated into biomacromolecules such as fatty acids, proteins, and nucleic acids, forming stable CD bonds. After 48 hours of incubation, the Raman spectral characteristics of CD bonds (2000-2400 cm) were identified using a single-cell Raman optical tweezers sorter (RACS-Seq RC24). -1 ), detect whether the live piezophilic cells in the sample are labeled; based on Raman-activated cell sorting (RACS) technology, optical tweezers are used to directly capture the target piezophilic single cells in complex samples, followed by subsequent single-cell whole genome analysis to achieve non-destructive sorting and functional analysis of living single cells.

[0084] The samples incubated under pressure and temperature using the full pressure and temperature preservation sampling-transfer-incubation system in this protocol were the experimental group (PSC), and the samples incubated under normal pressure and low temperature conditions in the traditional laboratory were the control group (TSC). Raman spectroscopy was used to detect the proportion of living cells in the two groups, and the results are shown in Tables 1 and 2. Figure 10 、 11 shown. Figure 10 This is a comparison chart of the difference in the proportion of living cells detected by Raman in the TSC and PSC groups. Figure 11 is the average Raman spectrum of living cells in the TSC and PSC groups.

[0085] The specific steps are as follows:

[0086] 1. Sediment sample pretreatment steps:

[0087] (1) Take 1 mL of incubated deep-sea sediment sample into a sterile centrifuge tube;

[0088] (2) Add 1 mL of SM solution and 0.5 g of glass beads and shake for 15 min. (SM solution: 5.8 g NaCl, 50 mL of 1 mol / L Tris-HCl (pH 7.5), 2 g of MgSO4·7H2O, add ddH2O to 1 L)

[0089] (3) Centrifuge at 100 g for 5 min at 4°C and collect the supernatant;

[0090] (4) Add 1 mL of SM solution to the precipitate and resuspend it. Repeat this process three times.

[0091] (5) Combine the supernatants collected three times and centrifuge at 300 g for 5 min at 4°C;

[0092] (6) Collect the supernatant and centrifuge at 5000 g for 30 min at 4°C;

[0093] (7) Discard the supernatant and wash the pellet twice with 1 mL of sterile sea salt water (5000 rpm, 10 min) and freeze the pellet directly.

[0094] 2. Raman test sorting method:

[0095] (1) Thaw the cell pellet, resuspend it in buffer and dilute it appropriately so that the bacterial injection concentration is 5.0×10 6 ~5.0×10 7 cells / mL;

[0096] (2) Take 20 μL of sample and aspirate 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, turn on the syringe pump flow rate of 9 mL / min, and flush the sorting channel for 10 minutes;

[0097] (3) Use a 532 nm laser to collect Raman spectra, select cells with CD peaks, and then use a 1032 nm laser to capture and sort single target cells.

[0098] Table 1. Number and ratio of detected live cell patterns.

[0099]

[0100] From Table 1 and Figure 10 、 11It can be seen that the proportion of live cells of barophiles in the experimental group was significantly higher than that in the control group, indicating that this method can complete the labeling of live cells of barophiles and can significantly increase the proportion of live cells compared to the traditional normal pressure labeling method.

[0101] 3. Single-cell whole genome amplification and sequencing:

[0102] (1) Add 2 μL of mixed lysing enzyme to the sorted single-cell oil droplets and incubate at 37°C for 30 min.

[0103] (2) Add 4 μL of the starting cell sample (preserved in PBS) to a microcentrifuge tube. If the starting cell sample volume is less than 4 μL, add PBS solution to bring the final volume to 4 μL.

[0104] (3) Add 3 μL of Buffer D2, mix well, and incubate at 65°C for 10 min.

[0105] (4) Add 3 μL of Stop Solution, mix well and place on ice.

[0106] (5) Add 40 μL of premix containing REPLI-g sc DNA Polymerase and incubate at 30°C for 8 h.

[0107] (6) Inactivate REPLI-g sc DNA Polymerase at 65°C for 3 min.

[0108] (7) The amplified DNA was diluted 1:100 and 2 μL of the diluted DNA was used for downstream sequencing analysis.

[0109] (8) The amplified DNA products were sent to Novogene Biotechnology's IlluminaNovaSeq 6000 platform for next-generation sequencing.

[0110] After testing, it was found that this method can obtain the whole genome map of single cells of barophiles, thereby enabling further research on the special metabolic functions of barophiles.

[0111] In this protocol, stable isotope labeling combined with Raman spectroscopy is used, eliminating the need for pure culture. Through stable isotope tracing and Raman spectroscopy feature recognition, optical tweezers are used to directly capture target single cells in complex samples based on Raman-activated cell sorting, enabling non-destructive sorting and functional analysis of living single cells. This method breaks through the limitations of traditional culture and can directly analyze functional individuals in complex samples, forming a closed loop of "metabolic phenotype identification-precise sorting-functional verification." Combined with single-cell amplification and sequencing methods, it can also achieve precise correlation between phenotype and genotype, transitioning from "metabolic function screening" to "gene mechanism analysis."

[0112] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for labeling deep-sea barophiles by sample transfer and incubation based on in situ reconstruction and full-process pressure and temperature preservation, characterized in that: Includes transfer, incubation, and labeling steps: a. The transfer step is to transfer the sample from the sampler (1) to the transfer device (2) by gravity in a pressure-maintaining and temperature-maintaining environment in the following manner: the transfer device (2) is docked with the interface of the sampler (1) through its interface I (2.4) and vertically installed below the sampler (1); after the internal pressure of the transfer device (2) is increased to the sample in-situ pressure by a pressurizing device, the connecting valve (5) on the sampler (1) and the transfer device (2) is opened, and the small tube (2.2) connected to the telescopic rod (2.5) is driven by the driving mechanism (6) to extend into the inner cavity of the sampler (1), so that the sample in the sampler (1) naturally falls into the small tube (2.2) under the action of gravity; after the sample transfer is completed, the small tube (2.2) is driven to retract into the transfer device (2) and the connecting valve (5) is closed; during the transfer process, the transfer device (2) maintains the in-situ pressure by the pressurizing device; b. Between the transfer and incubation steps, the small tube (2.2) containing the sample of the transferor (2) is automatically dropped into the incubator (3) in a pressure-maintaining and temperature-maintaining environment by the following method: the incubator (3) is docked with the interface I (2.4) of the transferor (2) through its interface, the internal pressure of the incubator (3) is adjusted to the same in-situ pressure as that of the transferor (2) through the pressure regulating valve (7) of the incubator (3), the connecting valve (5) of the two is opened, and the small tube (2.2) is driven by the driving mechanism (6) to extend into the incubation chamber (3.3) of the incubator (3), and when retracted, the small tube (2.2) automatically drops off under the action of the one-way clamp (3.4) in the incubation chamber (3.3), and the sample in the small tube (2.2) remains in the incubation chamber (3.3) with the small tube (2.2), and the in-situ pressure and temperature are maintained during the dropping process; c. The incubation step is to add heavy water culture medium to the incubator (3) under pressure and temperature conditions, so that the sample in the small tube (2.2) is co-incubated with the heavy water culture medium; d. The labeling step refers to the heavy water labeling of live cells of barophilic bacteria in the sample during incubation by heavy water medium; e. The pressure and temperature maintenance refers to maintaining the same pressure and temperature as the sampling environment throughout the transfer and incubation steps to ensure that the growth and metabolism of barophilic bacteria are not affected.

2. The method according to claim 1, wherein: The transfer device (2) is provided with an interface II (2.6) connected to the pressurizing device. The transfer device (2) is provided with an interface III (2.3). The transfer device (2) is connected to the driving mechanism (6) via the interface III (2.3). The control panel (8) equipped with the driving mechanism (6) is operated to drive the telescopic rod (2.5) to telescopic movement.

3. The method according to claim 1, wherein: The pressure maintenance is to maintain the in-situ pressure of the barophilic bacteria, and the pressure range is 0.1~200MPa. The heat preservation is to maintain the in-situ temperature of the barophilic bacteria, and the temperature range is 2~4°C.

4. The method according to claim 1, wherein: In the incubation step, 2216E heavy water medium is used as the incubation medium, and the incubation is performed using 2216E heavy water medium with a concentration of 50-75%, and the incubation is performed under pressure and temperature maintenance conditions for 12-72 hours.

5. A system suitable for the method of sample transfer and incubation labeling of deep-sea barophiles based on in situ reconstruction and full pressure and temperature preservation as claimed in claim 1, characterized in that: comprising a transfer device (2) and an incubator (3), The transfer device (2) comprises 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 a driving mechanism (6); the other end is provided with an interface I (2.4), and a connecting valve (5) is provided at the interface I (2.4); the output end of the driving mechanism (6) 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 driving 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); the transfer device body (2.1) is provided with an interface II (2.6) for connecting to a pressurizing device; The incubator (3) comprises 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 connected to the transfer device (2); a connecting valve (5) is provided at the interface; an incubation cavity (3.3) and a one-way clamp (3.4) are provided inside the incubator body (3.1); the one-way clamp (3.4) is used to drop a small tube (2.2) extending into the incubation cavity (3.3) into the cavity; the incubator (3) is connected to a pressure regulating valve (7); the pressure regulating valve (7) is a piston structure connected to a long threaded rod (7.1); the pressure in the inner cavity of the incubator (3) is regulated by adjusting the position of the piston by twisting the threaded rod (7.1).

6. The system according to claim 5, characterized in that: The incubation chamber (3.3) is made of peek material.

7. The system according to claim 5, characterized in that: The driving mechanism (6) is a servo motor.

8. The system according to claim 5, characterized in that: The connecting valve (5) is a ball valve.

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