Long-period high-pressure enrichment culture device and method for deep-sea microorganisms and online monitoring device and method

Abstract

The application provides a deep-sea microorganism long-period high-pressure enrichment culture and online monitoring device and method, the device can simulate high pressure, low temperature and oligotrophic and other deep-sea extreme environment, through pre-culture and grading series culture, the stable growth and reproduction of deep-sea microorganism long-period are ensured, and the abundance, activity and culture condition of microorganism are monitored in real time by using an online system to determine the appropriate strain subculture time, and interactive subculture and culture are carried out in time, and the stable supply of high-abundance, high-activity microorganism enrichment culture solution is realized; the application can carry out accurate microorganism pressure-keeping abundance counting under the condition of keeping in-situ pressure environment, the accurate control of microorganism growth period in the enrichment culture process is realized, the timeliness and cultivation efficiency of microorganism enrichment culture solution are effectively improved, and high-abundance, high-activity microorganism strain solution can be obtained for a long time, which provides a solid basic strain source for the development of subsequent deep-sea microorganism resources.

Description

Technical Field

[0001] The present invention relates to the technical field of marine microbial enrichment culture, and more specifically, to a device and method for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms. Background Art

[0002] The deep sea is home to a diverse array of complex geological features, including seamounts, ridges, abyssal plains, hadals, and trenches, as well as unique chemosynthetic ecosystems such as hydrothermal vents and cold seeps. Due to its unique environment of high pressure, low temperature, darkness, and oligotrophic conditions, the deep sea fosters a rich diversity of extreme microorganisms. Intense environmental pressures have necessitated the evolution of specialized physiological structures and functions, enabling deep-sea microorganisms to produce a variety of active substances with specialized physiological functions during growth and metabolism to sustain their life. Consequently, deep-sea microorganisms possess exceptional abundance, activity, and compositional and functional diversity, resulting in unique species, gene types, and metabolites through natural evolution, possessing significant scientific and economic value. Exploiting new microbial resources from the deep sea, conducting in-depth research on their biological characteristics, metabolic mechanisms, and ecological functions, and obtaining specialized bioactive substances or genetic resources, is a cutting-edge direction in international research and development of new resources. However, due to the limitations of current traditional deep-sea microbial sampling devices, the microorganisms used for research often cannot maintain their original growth and metabolic activity. Microbial samples need to be activated and enriched under laboratory conditions to simulate the in situ extreme environment, thereby providing high-abundance, high-activity and sufficient bacterial species support for downstream microbial resource mining.

[0003] Due to the limitations of the extreme environment, the growth and metabolic rates of most deep-sea microorganisms are very slow. The consumption of nutrients and the generation of intermediate metabolic waste during the culture process will also inhibit the growth and reproduction of microorganisms. The most effective method is to transfer the microorganisms to a new culture environment. Microbial growth is divided into hysteresis phase, logarithmic phase, plateau phase and decay phase. Determining the growth status of microorganisms and selecting enrichment culture fluid from the late logarithmic growth phase to the early plateau phase for subculture is the key to obtaining highly active and high-abundance bacterial culture fluid. If the optimal subculture time is missed, not only will the enrichment culture fluid enter the decay phase and a large number of microorganisms die, making the enrichment culture fluid unsuitable for subsequent microbial resource development, but the enrichment fluid after subculture will also take longer to enter logarithmic growth, extending the enrichment culture cycle.

[0004] Some existing deep-sea microbial enrichment devices are already capable of simulating deep-sea in-situ environments under laboratory conditions to enrich and culture microorganisms under high pressure. For example, existing patent documents disclose "a device and method for achieving rapid enrichment and culture of marine anaerobic ammonium-oxidizing bacteria." This method can detect the real-time ammonia nitrogen concentration of the culture medium in the reactor to determine the growth and metabolic activity of the anaerobic ammonium-oxidizing bacteria, and by adjusting the injection volume of the low-salinity seawater tank, the anaerobic ammonium-oxidizing bacteria can always maintain a high ammonia nitrogen metabolism capacity. However, traditional monitoring methods, including this scheme, can only rely on monitoring environmental parameters such as carbon source, pH, electron acceptor, and metabolite concentration to determine the timing of subculture. The monitoring of these environmental parameters cannot directly reflect the growth of microorganisms and has low monitoring accuracy. Currently, the most effective way to determine microbial growth is to count the cells of the microorganisms in the enrichment culture medium. The traditional counting method is based on optical microscopy to count cell abundance, but the cell abundance counting method is not suitable for counting cells in deep-sea high-pressure enrichment culture medium because the decompression process will cause pressure shock to many deep-sea barophiles and cause them to die, making it difficult to obtain a true and effective microbial growth condition under normal pressure. Therefore, developing monitoring methods for high-pressure microbial abundance is the key to deep-sea microbial enrichment and cultivation. Summary of the Invention

[0005] In order to overcome the defects of low abundance, activity and timeliness of the deep-sea microbial strain enrichment liquid cultivated by the above-mentioned prior art, the present invention provides a deep-sea microbial long-term high-pressure enrichment culture and online monitoring device and method. The device can simulate deep-sea extreme environments such as high pressure, low temperature and oligotrophy, ensure the long-term stable growth and reproduction of deep-sea microorganisms through pre-culture and graded serial culture, and use the online system to monitor the abundance, activity and culture conditions of microorganisms in real time to determine the appropriate time for strain propagation. At the same time, interactive propagation and cultivation are carried out, so that the culture device always maintains a reserve microbial inoculum that meets the requirements of subsequent experiments, providing important technical support for the development and utilization of deep-sea microbial resources.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] A deep-sea microbial long-term high-pressure enrichment culture and online monitoring device, comprising: a microbial enrichment system, an online monitoring system, a temperature control system, a pressure control system and a central control system;

[0008] The microbial enrichment system is provided with a plurality of reactors, and is used for long-term enrichment culture of deep-sea microorganisms and for alternating passage;

[0009] The online monitoring system includes: an environmental parameter monitoring unit and a microbial abundance counting unit respectively connected to the microbial enrichment system, the environmental parameter monitoring unit and the microbial abundance counting unit respectively used to monitor the environmental parameters and biological abundance indicators of the culture solution in the microbial enrichment system in real time;

[0010] The temperature control system and the pressure control system are respectively connected to the microorganism enrichment system, and are used to adjust the temperature and pressure in the microorganism enrichment system respectively; the pressure control system is also connected to the microorganism abundance counting unit, and is used to realize the pressure-maintaining counting of microorganisms;

[0011] The signal output end of the environmental parameter monitoring unit, the control end of the temperature control system and the control end of the pressure control system are respectively connected to the central control system.

[0012] Preferably, the microorganism enrichment system comprises: a plurality of reactors connected in series and having the same structure, each reactor having a detachable upper cover structure, and a sample transfer unit being provided between two reactors connected in series;

[0013] Each of the reactors is provided with a microbial culture solution and a magnetic stirrer, and a magnetic stirrer is provided at the bottom of each reactor, and the magnetic stirrer is used to drive the magnetic stirrer to rotate mechanically;

[0014] The probe of the environmental parameter monitoring unit is in contact with the culture solution in each reactor respectively;

[0015] A sampling valve is provided on the outside of each reactor, and the sampling valve is connected to the microfluidic chip in the microbial abundance counting unit;

[0016] An air injection valve and an air release valve are provided on the top of each reactor, and the air injection valve is connected to the pressure control system; each reactor is connected to a temperature control system.

[0017] Preferably, the sample transfer unit comprises: a regulating valve, an injection pump and a filter connected in sequence;

[0018] The filter is specifically a stainless steel one-way valve, and the stainless steel one-way valve has a built-in stainless steel metal filter membrane.

[0019] Preferably, the environmental parameter monitoring unit includes: a dissolved oxygen sensor, a pH sensor and a Raman sensor;

[0020] The probes of the dissolved oxygen sensor, pH sensor and Raman sensor are respectively in contact with the culture solution in each reactor;

[0021] The signal output ends of the dissolved oxygen sensor, pH sensor and Raman sensor are electrically connected to the central control system respectively.

[0022] Preferably, the microbial abundance counting unit comprises: an observer, a microfluidic chip and a fluorescence microscope;

[0023] The top and bottom of the observer are both sapphire windows to allow the excitation fluorescence of the fluorescence microscope to pass through the microfluidic chip; the top of the observer is also provided with an air inlet valve and an exhaust valve, and the air inlet valve is connected to the pressure control system;

[0024] The microfluidic chip is fixed to the center of the observer via a slot. The microfluidic chip is provided with a counting chamber, and a lower injection valve and an upper exhaust valve connected to the counting chamber; the lower injection valve is connected to the sampling valve on the outside of each reactor and is used to inject the bacterial solution in the reactor into the counting chamber; the upper exhaust valve is used to exhaust the gas in the counting chamber when the bacterial solution is injected into the counting chamber;

[0025] A fluorescent dye for microbial staining is pre-added in the counting chamber, and the bacterial solution in the reactor is mixed with the fluorescent dye for a certain period of time to complete the fluorescent staining; the observer is placed on a fluorescent microscope, and the microbial abundance is counted using the fluorescent microscope.

[0026] Preferably, a grid for cell counting is engraved on the bottom surface of the counting chamber, and the grid includes a plurality of grids of different sizes.

[0027] Preferably, the grid includes a large squares of the same size, any large square in the grid is further divided into b medium squares, and each medium square is divided into c small squares; wherein a, b and c are the first, second and third positive integers respectively;

[0028] The equally divided large squares are used for cell counting, and the concentration of microorganisms in the bacterial solution is calculated based on the number of cells in the large squares.

[0029] Preferably, the temperature control system comprises: a refrigeration unit, a refrigeration jacket and a temperature sensor;

[0030] The control end of the refrigeration unit is connected to the central control system, the output end of the refrigeration unit is connected to the refrigeration jacket, and each reactor is wrapped in the refrigeration jacket; a temperature sensor is provided inside each reactor; and the signal output end of the temperature sensor is connected to the central control system.

[0031] Preferably, the pressure control system comprises: an air compressor, a booster pump, an air storage tank and a pressure regulating valve, and a pressure sensor connected in sequence;

[0032] The control ends of the air compressor, booster pump and pressure regulating valve are connected to the central control system, and the output end of the pressure regulating valve is connected to the air injection valve of each reactor and the air inlet valve of the observer respectively;

[0033] A pressure sensor is provided inside each reactor and inside the observer, and a signal output end of the pressure sensor is connected to the central control system.

[0034] The present invention also provides a method for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms, based on the above-mentioned long-term high-pressure enrichment culture and online monitoring device for deep-sea microorganisms, comprising the following steps:

[0035] S1: All reactors of the microbial enrichment system and various components of the online monitoring system are sterile operated, and the deep-sea microbial long-term high-pressure enrichment culture and online monitoring device is installed;

[0036] S2: adding a deep-sea microbial sample and an activation culture medium into each reactor of the microbial enrichment system in sequence, and adjusting the pressure and temperature in the microbial enrichment system using a pressure control system and a temperature control system to enrich and culture the microorganisms under a preset pressure and temperature environment;

[0037] S3: Using the environmental parameter monitoring unit to monitor the environmental parameter changes of the culture solution in each reactor in real time, and when the environmental parameters meet the preset conditions, injecting the microbial liquid in the corresponding reactor into the microbial abundance counting unit;

[0038] S4: pressurizing the microbial abundance counting unit using a pressure control system, counting the microorganisms in the microbial abundance counting unit while maintaining the pressure, and drawing a growth curve of the microbial community;

[0039] S5: determining the optimal passage time of the microorganism according to the growth curve, and transferring the bacterial solution of the upper reactor to the lower reactor while maintaining pressure at the optimal passage time;

[0040] S6: Repeat steps S3 to S5 to perform long-term enrichment culture and cross-passaging on the deep-sea microbial samples in the microbial enrichment system.

[0041] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0042] The present invention provides a device and method for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms. The device can simulate extreme deep-sea environments such as high pressure, low temperature, and oligotrophic conditions. Pre-culture and graded serial culture ensure the long-term stable growth and reproduction of deep-sea microorganisms. An online system is used to monitor the abundance, activity, and culture conditions of microorganisms in real time to determine the appropriate time for bacterial subculture. Simultaneously, alternating subculture and culture are performed, achieving a stable supply of high-abundance, high-activity microbial enrichment culture fluid.

[0043] The present invention determines the optimal timing for enrichment culture and subculture by directly counting microbial abundance and combining it with environmental auxiliary indicators. This solves the difficulty of controlling the subculture timing during deep-sea microbial enrichment culture. A reserve microbial inoculum that meets the requirements of subsequent experiments can be obtained from the enrichment kettle over a long period of time, providing important strain support for the development and utilization of downstream deep-sea microbial resources.

[0044] The present invention has a wide range of applications and can be used for the enrichment and cultivation of various deep-sea microorganisms; the present invention does not require professional operation training, and the pressure-maintained counting of microbial abundance is similar to the conventional blood cell counting method, which reduces manpower and training costs; compared with existing enrichment and cultivation technologies, the invention achieves precise control of the microbial growth cycle during the enrichment process, so that the microbial culture liquid in the enrichment culture kettle always maintains a high growth rate and metabolic activity, ensuring the timeliness and cultivation efficiency of the microbial enrichment culture liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic diagram of the architecture of a long-term high-pressure enrichment culture and online monitoring device for deep-sea microorganisms provided in Example 1.

[0046] Figure 2 This is a mechanical structure diagram of a long-term high-pressure enrichment culture and online monitoring device for deep-sea microorganisms provided in Example 2.

[0047] Figure 3 Schematic diagram of the structure of the observer provided in Example 2.

[0048] Figure 4 This is a front view of the microfluidic chip provided in Example 2.

[0049] Figure 5 1 is a top view of the microfluidic chip provided in Example 2 and a schematic diagram of the grid structure.

[0050] Figure 6 This is a schematic diagram of the connection relationship between the central control system provided in Example 2 and other components.

[0051] Figure 7This is a flow chart for the long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms provided in Example 3. DETAILED DESCRIPTION

[0052] The accompanying drawings are for illustrative purposes only and are not to be construed as limiting this patent;

[0053] In order to better illustrate this embodiment, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product size;

[0054] It is understandable to those skilled in the art that some well-known structures and descriptions thereof may be omitted in the drawings.

[0055] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0056] Example 1

[0057] like Figure 1 As shown, this embodiment provides a deep-sea microbial long-term high-pressure enrichment culture and online monitoring device, including: a microbial enrichment system, an online monitoring system, a temperature control system, a pressure control system and a central control system;

[0058] The microbial enrichment system is provided with a plurality of reactors, and is used for long-term enrichment culture of deep-sea microorganisms and for alternating passage;

[0059] The online monitoring system includes: an environmental parameter monitoring unit and a microbial abundance counting unit respectively connected to the microbial enrichment system, the environmental parameter monitoring unit and the microbial abundance counting unit respectively used to monitor the environmental parameters and biological abundance indicators of the culture solution in the microbial enrichment system in real time;

[0060] The temperature control system and the pressure control system are respectively connected to the microorganism enrichment system, and are used to adjust the temperature and pressure in the microorganism enrichment system respectively; the pressure control system is also connected to the microorganism abundance counting unit, and is used to realize the pressure-maintaining counting of microorganisms;

[0061] The signal output end of the environmental parameter monitoring unit, the control end of the temperature control system and the control end of the pressure control system are respectively connected to the central control system.

[0062] During the specific implementation process, all reactors in the microbial enrichment system and various components in the online monitoring system are first sterilized, and the deep-sea microbial long-cycle high-pressure enrichment culture and online monitoring equipment are installed;

[0063] Deep-sea microbial samples and activation culture medium are sequentially added to each reactor of the microbial enrichment system, and the pressure and temperature in the microbial enrichment system are adjusted using a pressure control system and a temperature control system to enrich and culture the microorganisms under a preset pressure and temperature environment;

[0064] The environmental parameter monitoring unit is used to monitor the environmental parameter changes of the culture solution in each reactor in real time. When the environmental parameters meet the preset conditions, the microbial liquid in the corresponding reactor is injected into the microfluidic chip of the microbial abundance counting unit;

[0065] The microbial abundance counting unit is pressurized using a pressure control system, microorganisms are counted under a fluorescence microscope while maintaining pressure, and a growth curve of the microbial community is drawn;

[0066] Determine the optimal passage time for microorganisms based on the growth curve, and transfer the bacterial solution from the upper reactor to the lower reactor at the optimal passage time while maintaining pressure;

[0067] Repeat the above steps to carry out long-term enrichment culture and cross-passaging of the deep-sea microbial samples in the microbial enrichment system;

[0068] This device can perform precise pressure-maintained abundance counting of microorganisms while maintaining an in-situ pressure environment, thereby achieving accurate control of the microbial growth cycle during the enrichment culture process, solving the difficulty of controlling the timing of subculture during the enrichment culture process, and effectively improving the timeliness and cultivation efficiency of the microbial enrichment culture solution. It can obtain high-abundance and high-activity microbial strains for a long time, providing a solid basic strain source for the subsequent development of deep-sea microbial resources.

[0069] Example 2

[0070] like Figure 2 As shown, this embodiment provides a deep-sea microbial long-term high-pressure enrichment culture and online monitoring device, including: a microbial enrichment system 1, an online monitoring system 2, a temperature control system 3, a pressure control system 4 and a central control system 5;

[0071] The microorganism enrichment system 1 is provided with a plurality of reactors 11, and the microorganism enrichment system 1 is used for long-term enrichment culture of deep-sea microorganisms and for alternating passage;

[0072] The online monitoring system 2 includes: an environmental parameter monitoring unit 21 and a microbial abundance counting unit 22 respectively connected to the microbial enrichment system 1, and the environmental parameter monitoring unit 21 and the microbial abundance counting unit 22 are respectively used to monitor the environmental parameters and biological abundance indicators of the culture solution in the microbial enrichment system 1 in real time;

[0073] The microbial abundance counting unit 22 is provided with a microfluidic chip 222 and a fluorescence microscope 223. A fluorescent dye for microbial staining is pre-added in the microfluidic chip 222. After the bacterial solution in the microbial enrichment system 1 is fluorescently stained, the microbial abundance is counted using the fluorescence microscope 223.

[0074] The temperature control system 3 and the pressure control system 4 are respectively connected to the microorganism enrichment system 1, and are used to adjust the temperature and pressure in the microorganism enrichment system 1 respectively; the pressure control system 4 is also connected to the microorganism abundance counting unit 22, for realizing the microorganism pressure-maintaining counting;

[0075] The signal output end of the environmental parameter monitoring unit 21, the control end of the temperature control system 3 and the control end of the pressure control system 4 are respectively connected to the central control system 5;

[0076] The microorganism enrichment system 1 comprises: a plurality of reactors 11 of the same structure connected in series, each reactor 11 being a high-pressure resistant detachable upper cover structure 12, which is conducive to the placement of nutrient substrates and simplifies operation;

[0077] A sample transfer unit 13 is provided between the two reactors 11 connected in series, for alternate subculturing of the microbial culture liquid between the reactors 11 in the later stage of cultivation. In this embodiment, the sample transfer unit 13 includes: a regulating valve 131, an injection pump 132, and a filter 133 connected in sequence;

[0078] The filter 133 is specifically a stainless steel one-way valve, which has a built-in 10 μm stainless steel metal filter membrane, which can effectively intercept particulate sediments and certain particulate metabolites in the original sample without affecting the passage of microorganisms, thereby improving the inoculation efficiency of microorganisms during the passage process;

[0079] In order to increase the nutrient utilization efficiency of microorganisms during the cultivation process, each of the reactors 11 is provided with a microbial culture solution and a magnetic stirrer 14. A magnetic stirrer 15 is provided at the bottom of each reactor 11. The magnetic stirrer 15 is used to drive the magnetic stirrer 14 to mechanically rotate, thereby enhancing the mass transfer between the microorganisms and the nutrient solution. This solves the problem that a manual stirring rod can only stir indirectly and is not conducive to the sealing of the reactor, thereby achieving real-time homogenization of the enriched culture solution during the cultivation process.

[0080] The probe of the environmental parameter monitoring unit 21 is in contact with the culture solution in each reactor 11;

[0081] A sampling valve 16 is provided on the outside of each reactor 11. The sampling valve 16 is connected to the microfluidic chip 222 in the microbial abundance counting unit 22. Its function is to collect the microbial liquid in the reactor 11 and perform subsequent pressure-maintained biological abundance counting.

[0082] Each reactor 11 is provided with an air injection valve 17 and an air release valve 18 on the top. The air injection valve 17 is connected to the pressure control system 4 and is used to control the pressurization and depressurization of the culture system, so that the pressure in the reactor 11 reaches the in-situ environmental pressure of the culture sample.

[0083] Each of the reactors 11 is connected to a temperature control system 3, and the temperature inside the reactor 11 is adjusted by the temperature control system 3 to be consistent with the temperature of the deep-sea in-situ environment;

[0084] The environmental parameter monitoring unit 21 includes: a dissolved oxygen sensor 211, a pH sensor 212 and a Raman sensor 213;

[0085] The probes of the dissolved oxygen sensor 211, the pH sensor 212 and the Raman sensor 213 are respectively in contact with the culture solution in each reactor 11, and can be used to monitor the concentration changes of dissolved oxygen, pH value and organic nutrients in the enriched culture solution;

[0086] The signal output ends of the dissolved oxygen sensor 211, pH sensor 212 and Raman sensor 213 are electrically connected to the central control system 5, and the specific values ​​are directly displayed on the central control system 5;

[0087] By monitoring the above indicators, we can ensure that the culture process is in an anaerobic environment, and reflect the consumption of organic matter in the culture medium and the growth of acidic metabolic waste, which can be used as an indirect signal for subculture inoculation;

[0088] The microbial abundance counting unit 22 includes: a high-pressure stainless steel observer 221, a microfluidic chip 222 and a fluorescence microscope 223;

[0089] like Figure 3 As shown, the top and bottom of the observer 221 are both sapphire windows to allow the excitation fluorescence of the fluorescence microscope 223 to pass through the microfluidic chip 222; the observer 221 should not be too thick, preferably less than 5 cm, to prevent the fluorescence microscope 223 from being unable to focus on the microfluidic chip 222;

[0090] To allow the microbial counting process to be performed in a high-pressure environment, an air inlet valve 224 and an air exhaust valve 225 are further provided on the top of the observer 221. The air inlet valve 224 is connected to the pressure control system 4. During counting, the air inlet valve 224 is used to increase the pressure until the pressure value in the observer 221 is consistent with the pressure value of the reactor 11. After the counting is completed, the air exhaust valve 225 is used to relieve the pressure in the observer 221. The pressure is maintained throughout the counting process to prevent certain barophilic bacteria from dying after pressure loss, thereby ensuring the reliability of the microbial counting.

[0091] The microfluidic chip 222 is fixed to the center of the observer 221 through a slot 226 to prevent disturbances caused by gas and liquid injection.

[0092] like Figure 4 As shown, the microfluidic chip 222 is provided with a counting chamber 227, and a lower injection valve 228 and an upper exhaust valve 229 connected to the counting chamber 227; the lower injection valve 228 is connected to the sampling valve 16 outside each reactor 11, and is used to inject the bacterial liquid in the reactor 11 into the counting chamber 227; the upper exhaust valve 229 is used to empty the gas in the counting chamber 227 when the bacterial liquid is injected into the counting chamber 227, so that the enriched liquid can be successfully and stably injected;

[0093] A fluorescent dye for microbial staining is pre-added in the counting chamber 227. After the bacterial solution in the reactor 11 is mixed with the fluorescent dye for a certain period of time, fluorescent staining is completed. The observer 221 is placed on the fluorescent microscope 223, and the microbial abundance is counted using the fluorescent microscope 223.

[0094] like Figure 5 As shown, the bottom surface of the counting chamber 227 is engraved with a grid for cell counting, and the grid includes a plurality of grids of different sizes;

[0095] In this embodiment, the counting chamber 227 has a thickness of 0.1 mm, a bottom dimension of 2 cm×2 cm, and a volume of 40 μL, which can accommodate 40 μL of staining solution and microbial culture solution;

[0096] The grid consists of 9 large squares of the same size, each of which has a side length of 1 mm and a volume of 0.1 mm. 3 ;

[0097] The large square in the middle of the grid is further divided into 16 medium squares, and each medium square is divided into 25 small squares;

[0098] The large square in the middle of the grid is used for cell counting, and the concentration of microorganisms in the bacterial solution is calculated based on the number of all cells in the large square in the middle of the grid and the volume of the large square;

[0099] The temperature control system 3 includes: a refrigeration unit 31, a refrigeration jacket 32 ​​and a temperature sensor 33;

[0100] The control end of the refrigeration unit 31 is connected to the central control system 5, and the output end of the refrigeration unit 31 is connected to the refrigeration jacket 32. Each of the reactors 11 is wrapped in the refrigeration jacket 32. The refrigeration jacket 32 ​​in this embodiment is specifically a water bath jacket. The use of water bath refrigeration allows for more precise and uniform temperature control. A temperature sensor 33 is provided inside each reactor 11. The signal output end of the temperature sensor 33 is connected to the central control system 5.

[0101] The pressure control system 4 includes: an air compressor 41, a booster pump 42, an air storage tank 43, a pressure regulating valve 44, and a pressure sensor 45 connected in sequence;

[0102] The control ends of the air compressor 41, the booster pump 42 and the pressure regulating valve 44 are connected to the central control system 5, and the output end of the pressure regulating valve 44 is respectively connected to the gas injection valve 17 of each reactor 11 and the gas inlet valve 224 of the observer 221;

[0103] A pressure sensor 45 is provided inside each of the reactors 11 and the observer 221, and a signal output end of the pressure sensor 45 is connected to the central control system 5;

[0104] In this embodiment, if Figure 6 As shown, the central control system 5 is electrically connected to the air compressor 41 control end, the booster pump 42 control end, the pressure regulating valve 44 control end and the pressure sensor 45 signal output end of the pressure control system 4, is electrically connected to the refrigeration system 31 control end and the temperature sensor 33 signal output end of the temperature control system 3, and is electrically connected to the signal output ends of each sensor of the environmental parameter monitoring unit 21, and is used to control the temperature and pressure of the reactor 11 and to collect, process and store real-time data on changes in environmental parameters of the enriched culture medium.

[0105] During the specific implementation process, firstly, all the reactors 11 of the microbial enrichment system 1 and the various components in the online monitoring system 2 are sterile operated, and the deep-sea microbial long-cycle high-pressure enrichment culture and online monitoring device are installed;

[0106] Deep-sea microbial samples and activation culture medium are sequentially added to each reactor 11 of the microbial enrichment system 1, and the pressure and temperature in the microbial enrichment system 1 are adjusted using the pressure control system 4 and the temperature control system 3 to enrich and culture the microorganisms under high pressure and low temperature conditions;

[0107] The environmental parameter monitoring unit 21 is used to monitor the environmental parameter changes of the culture solution in each reactor 11 in real time. When the environmental parameters meet the preset conditions, the microbial liquid in the corresponding reactor 11 is injected into the microfluidic chip 222 of the microbial abundance counting unit 22;

[0108] The pressure control system 4 is used to pressurize the observer 221 of the microbial abundance counting unit 22, and the microorganisms are counted under the fluorescence microscope 223 while maintaining the pressure, and a growth curve of the microbial community is drawn;

[0109] Determine the optimal passage time of the microorganisms according to the growth curve, and transfer the bacterial solution in the upper reactor 11 to the lower reactor 11 while maintaining pressure at the optimal passage time;

[0110] Repeat the above steps to perform long-term enrichment culture and cross-passaging on the deep-sea microbial sample in the microbial enrichment system 1;

[0111] This device can perform precise pressure-maintained abundance counting of microorganisms while maintaining an in-situ pressure environment, thereby achieving accurate control of the microbial growth cycle during the enrichment culture process, solving the difficulty of controlling the timing of subculture during the enrichment culture process, and effectively improving the timeliness and cultivation efficiency of the microbial enrichment culture solution. It can obtain high-abundance and high-activity microbial strains for a long time, providing a solid basic strain source for the subsequent development of deep-sea microbial resources.

[0112] Example 3

[0113] like Figure 7 As shown, this embodiment provides a method for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms, based on a long-term high-pressure enrichment culture and online monitoring device for deep-sea microorganisms described in Example 2, comprising the following steps:

[0114] S1: All reactors of the microbial enrichment system and various components of the online monitoring system are sterile operated, and the deep-sea microbial long-term high-pressure enrichment culture and online monitoring device is installed;

[0115] S2: adding a deep-sea microbial sample and an activation culture medium into each reactor of the microbial enrichment system in sequence, and adjusting the pressure and temperature in the microbial enrichment system using a pressure control system and a temperature control system to enrich and culture the microorganisms under a preset pressure and temperature environment;

[0116] S3: Using the environmental parameter monitoring unit to monitor the environmental parameter changes of the culture solution in each reactor in real time, and when the environmental parameters meet the preset conditions, injecting the microbial liquid in the corresponding reactor into the microbial abundance counting unit;

[0117] S4: pressurizing the microbial abundance counting unit using a pressure control system, counting the microorganisms in the microbial abundance counting unit while maintaining the pressure, and drawing a growth curve of the microbial community;

[0118] S5: determining the optimal passage time of the microorganism according to the growth curve, and transferring the bacterial solution of the upper reactor to the lower reactor while maintaining pressure at the optimal passage time;

[0119] S6: Repeat steps S3 to S5 to perform long-term enrichment culture and cross-passaging on the deep-sea microbial samples in the microbial enrichment system.

[0120] In the specific implementation process, step 1: before cultivation, the reactor 11 and its associated pipe and valve components are sterilized by using 75% ethanol or ultraviolet light irradiation, and then the reactor 11 and its attached components are transferred to a clean room for assembly in a sterile environment. Generally, before selecting to enrich a specific microbial group, the microbial community in the deep-sea sample must be activated and cultured to allow most of the microbial groups in the sample to re-metabolize functionally. The activated bacterial liquid is then pressure-maintained and inoculated into a new enrichment culture reactor to enrich and culture the specific microbial group. Therefore, in a sterile environment, the deep-sea microbial sample (deep-sea sediment or seawater, etc.), microbial activation culture medium (depending on the sample and the desired microbial group), and magnetic stirrer 14 are sequentially added to the reactor 11, and then various valves and sensor components are installed.

[0121] Step 2: Connect the air injection valve 17 of the reactor 11 to the pressure regulating valve 44 of the pressure control system 4, adjust the air compressor 41, the booster pump 42 and the pressure regulating valve 44 in sequence, and open the air injection valve 17 so that the pressure value of the pressure sensor 45 in the reactor 11 reaches a preset value; then wrap the refrigeration jacket 32 ​​of the temperature control system 3 outside the reactor 11, start the refrigeration system 31 so that the temperature of the temperature sensor 33 in the reactor 11 reaches a preset value; finally, start the magnetic stirrer 15 to drive the internal magnetic stirrer 14 to stir; the entire activation culture process is carried out in a high-pressure and low-temperature environment;

[0122] Step 3: During the culture process, the dissolved oxygen sensor 211, pH sensor 212, and Raman sensor 213 are used to monitor the changes in dissolved oxygen, pH, and organic nutrient concentrations in the enriched culture medium in real time. The values ​​are displayed in real time on the central control system 5, indirectly reflecting the growth of the microorganisms. When the pH value of the culture medium drops by 2 to 3 orders of magnitude and the concentrations of organic nutrients such as glucose and acetic acid decrease significantly, it means that the nutrients in the culture medium are being consumed in large quantities and acidic metabolites are being generated. Inoculation and subculture can then be considered.

[0123] Step 4: Count the cells of the microbial culture medium every 1 to 2 days; specifically, first inject 20 μL of DAPI fluorescent dye into the microfluidic chip 222, then place the microfluidic chip 222 in the observer 221 and fix it with the card slot 226; connect the air inlet valve 224 of the observer 221 to the pressure regulating valve 44 of the pressure control system 4, and control the air compressor 41, the booster pump 42, the pressure regulating valve 44 and the air inlet valve 224 in sequence to increase the internal pressure of the observer 221 to the same pressure condition as the reactor 11; then connect the lower injection valve 228 of the observer 221 to the injection pump 132 and the sampling valve 16 of the reactor 11 in sequence. Connect, start the injection pump 132, and slowly and quantitatively inject 20 μL of culture solution into the microfluidic chip 222; incubate in a low-temperature environment for 15 to 20 minutes to allow the fluorescent label to be attached to the microorganisms; after staining, place the microfluidic chip 222 under a fluorescence microscope 223; adjust the focus of the fluorescence microscope 223 to first find the grid in the counting chamber 227 under a low-power microscope, then switch the low-power microscope to a high-power microscope, count the total number of cells in the large square in the middle of the grid within the field of view, and then convert it to the bacterial concentration of the inoculum; draw a growth curve of the microbial community based on the cell counting results, and determine the growth hysteresis phase, logarithmic phase, plateau phase, and decay phase of the microbial community;

[0124] Step 5: Determine the optimal time for subculturing based on the growth curve and changes in environmental parameters, generally from the end of the logarithmic growth phase to the early stage of the plateau phase; before subculturing, repeat steps 1 and 2, but replace the activated culture medium with an enrichment culture medium for the desired microbial group; during subculturing, connect the regulating valve 131, the injection pump 132, and the filter 133 to the bottom of the previous reactor 11 in sequence through a pipeline, and then connect the filter 133 to the regulating valve 131 of the next reactor 11. Open the regulating valves 131 at both ends to connect the two reactors 11, and then the injection pump 132 quantitatively inoculates the culture medium in the previous enrichment reactor 11 into the next reactor 11 while maintaining pressure;

[0125] Step 6: Repeat steps 3 to 6 to perform long-term enrichment culture and cross-culture on the microbial liquid in the reactor 11;

[0126] Through the above steps, the microbial culture liquid in the next-stage reactor 11 can maintain a high growth rate, and a high-abundance and high-activity microbial enrichment liquid can be obtained from the previous-stage reactor 11, providing a sufficient source of bacterial strains for the development and utilization of downstream deep-sea microbial resources in the long term.

[0127] The same or similar reference numerals correspond to the same or similar components;

[0128] The terms used in the drawings to describe positional relationships are for illustrative purposes only and should not be construed as limiting this patent;

[0129] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A device for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms, characterized in that: include: Microbial enrichment system, online monitoring system, temperature control system, pressure control system and central control system; The microbial enrichment system is provided with a plurality of reactors, and is used for long-term enrichment culture of deep-sea microorganisms and for alternating passage; The online monitoring system includes: an environmental parameter monitoring unit and a microbial abundance counting unit respectively connected to the microbial enrichment system, the environmental parameter monitoring unit and the microbial abundance counting unit respectively used to monitor the environmental parameters and biological abundance indicators of the culture solution in the microbial enrichment system in real time; The microbial abundance counting unit includes: an observer, a microfluidic chip and a fluorescence microscope; The top and bottom of the observer are both sapphire windows to allow the excitation fluorescence of the fluorescence microscope to pass through the microfluidic chip; the top of the observer is also provided with an air inlet valve and an exhaust valve, and the air inlet valve is connected to the pressure control system; The microfluidic chip is fixed to the center of the observer via a slot. The microfluidic chip is provided with a counting chamber, and a lower injection valve and an upper exhaust valve connected to the counting chamber; the lower injection valve is connected to the sampling valve on the outside of each reactor and is used to inject the bacterial solution in the reactor into the counting chamber; the upper exhaust valve is used to exhaust the gas in the counting chamber when the bacterial solution is injected into the counting chamber; A fluorescent dye for microbial staining is pre-added in the counting chamber, and the bacterial solution in the reactor is mixed with the fluorescent dye for a certain period of time to complete the fluorescent staining; the observer is placed on a fluorescence microscope, and the microbial abundance is counted using the fluorescence microscope; The temperature control system and the pressure control system are respectively connected to the microorganism enrichment system, and are used to adjust the temperature and pressure in the microorganism enrichment system respectively; the pressure control system is also connected to the microorganism abundance counting unit, and is used to realize the pressure-maintaining counting of microorganisms; The signal output end of the environmental parameter monitoring unit, the control end of the temperature control system and the control end of the pressure control system are respectively connected to the central control system.

2. The device for long-term high-pressure enrichment, cultivation and online monitoring of deep-sea microorganisms according to claim 1, characterized in that: The microorganism enrichment system comprises: a plurality of reactors of the same structure connected in series, each reactor having a detachable upper cover, and a sample transfer unit being provided between two reactors connected in series; Each of the reactors is provided with a microbial culture solution and a magnetic stirrer, and a magnetic stirrer is provided at the bottom of each reactor, and the magnetic stirrer is used to drive the magnetic stirrer to rotate mechanically; The probe of the environmental parameter monitoring unit is in contact with the culture solution in each reactor respectively; A sampling valve is provided on the outside of each reactor, and the sampling valve is connected to the microfluidic chip in the microbial abundance counting unit; An air injection valve and an air release valve are provided on the top of each reactor, and the air injection valve is connected to the pressure control system; each reactor is connected to a temperature control system.

3. The device for long-term high-pressure enrichment, cultivation and online monitoring of deep-sea microorganisms according to claim 2, characterized in that: The sample transfer unit comprises: a regulating valve, an injection pump and a filter connected in sequence; The filter is specifically a stainless steel one-way valve, and the stainless steel one-way valve has a built-in stainless steel metal filter membrane.

4. The device for long-term high-pressure enrichment, cultivation and online monitoring of deep-sea microorganisms according to claim 2, characterized in that: The environmental parameter monitoring unit includes: a dissolved oxygen sensor, a pH sensor and a Raman sensor; The probes of the dissolved oxygen sensor, pH sensor and Raman sensor are respectively in contact with the culture solution in each reactor; The signal output ends of the dissolved oxygen sensor, pH sensor and Raman sensor are electrically connected to the central control system respectively.

5. The device for long-term high-pressure enrichment, cultivation and online monitoring of deep-sea microorganisms according to claim 1, characterized in that: A square grid for cell counting is engraved on the bottom surface of the counting chamber, and the square grid includes a plurality of squares of different sizes.

6. The device for long-term high-pressure enrichment, cultivation and online monitoring of deep-sea microorganisms according to claim 5, characterized in that: The grid includes a large squares of the same size, any large square in the grid is further divided into b medium squares, and each medium square is divided into c small squares; wherein a, b and c are first, second and third positive integers respectively; The equally divided large squares are used for cell counting, and the concentration of microorganisms in the bacterial solution is calculated based on the number of cells in the large squares.

7. The device for long-term high-pressure enrichment, cultivation and online monitoring of deep-sea microorganisms according to claim 2, characterized in that: The temperature control system includes: a refrigeration unit, a refrigeration jacket and a temperature sensor; The control end of the refrigeration unit is connected to the central control system, the output end of the refrigeration unit is connected to the refrigeration jacket, and each reactor is wrapped in the refrigeration jacket; a temperature sensor is provided inside each reactor; and the signal output end of the temperature sensor is connected to the central control system.

8. The device for long-term high-pressure enrichment, cultivation and online monitoring of deep-sea microorganisms according to claim 1, characterized in that: The pressure control system includes: an air compressor, a booster pump, an air storage tank and a pressure regulating valve, and a pressure sensor connected in sequence; The control ends of the air compressor, booster pump and pressure regulating valve are connected to the central control system, and the output end of the pressure regulating valve is respectively connected to the air injection valve of each reactor and the air inlet valve of the observer; A pressure sensor is provided inside each of the reactors and inside the observer, and a signal output end of the pressure sensor is connected to the central control system.

9. A method for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms, based on the device for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1: All reactors of the microbial enrichment system and various components of the online monitoring system are sterile operated, and the deep-sea microbial long-term high-pressure enrichment culture and online monitoring device is installed; S2: adding a deep-sea microbial sample and an activation culture medium into each reactor of the microbial enrichment system in sequence, and adjusting the pressure and temperature in the microbial enrichment system using a pressure control system and a temperature control system to enrich and culture the microorganisms under a preset pressure and temperature environment; S3: Using the environmental parameter monitoring unit to monitor the environmental parameter changes of the culture solution in each reactor in real time, and when the environmental parameters meet the preset conditions, injecting the microbial liquid in the corresponding reactor into the microbial abundance counting unit; S4: pressurizing the microbial abundance counting unit using a pressure control system, counting the microorganisms while maintaining the pressure, and drawing a growth curve of the microbial community; S5: determining the optimal passage time of the microorganism according to the growth curve, and transferring the bacterial solution of the upper reactor to the lower reactor while maintaining pressure at the optimal passage time; S6: Repeat steps S3 to S5 to perform long-term enrichment culture and cross-passaging on the deep-sea microbial samples in the microbial enrichment system.

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