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

Abstract

According to the long-period high-pressure enrichment culture and online monitoring device and method for the deep-sea microorganisms, the device can simulate deep-sea extreme environments such as high pressure, low temperature and oligotrophic environments, and long-period stable growth and reproduction of the deep-sea microorganisms are ensured through pre-culture and graded series culture; an on-line system is used for monitoring the abundance, activity and culture conditions of microorganisms in real time so as to determine a proper strain passage opportunity, and interactive passage and culture are carried out in time, so that stable supply of a high-abundance and high-activity microorganism enrichment culture solution is realized; according to the invention, accurate microbe pressure-maintaining abundance counting can be carried out under an in-situ pressure environment, accurate control of a microbe growth period in an enrichment culture process is realized, the timeliness and the culture efficiency of a microbe enrichment culture solution are effectively improved, a microbe strain solution with high abundance and high activity can be obtained for a long time, and the production cost is reduced. And a solid basic strain source is provided for subsequent development of deep-sea microorganism resources.

Description

Technical Field

[0001] The present invention relates to the technical field of marine microorganism 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 variety of complex geological landforms such as seamounts, sea ridges, abyssal plains, abysses, trenches, and unique chemosynthetic ecosystems such as hydrothermal fluids and cold springs. Due to the unique high pressure, low temperature, darkness, and oligotrophic environment, the deep sea breeds an abundance of extreme microbial resources. The huge environmental pressure has caused deep-sea microorganisms to evolve special physiological structures and functions, and they can produce various active substances with special physiological functions during growth and metabolism to maintain their own life activities. Therefore, deep-sea microorganisms have extremely high abundance, activity, composition and functional diversity, and have formed special species, gene types and metabolites in natural evolution, which have important scientific research and economic value. Developing new microbial resources from the deep sea, conducting in-depth research on their biological characteristics, metabolic mechanisms and ecological functions, and obtaining special biologically active substances or genetic resources are the frontiers of new resource research and development in the world. 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. It is necessary to activate and enrich the microbial samples under laboratory conditions to simulate the in situ extreme environment, thereby providing high-abundance, high-activity and sufficient strain 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. The growth of microorganisms is divided into hysteresis phase, logarithmic phase, platform phase and decay phase. Determining the growth status of microorganisms and selecting enriched culture fluid from the end of logarithmic growth to the early stage of platform for subculture is the key to obtaining highly active and high-abundance bacterial fluid. If the best time for subculture is missed, not only will the enriched culture fluid enter the decay phase and a large number of microorganisms die, making the enriched culture fluid unsuitable for the subsequent development of microbial resources, but it will also take longer for the enriched fluid after subculture to enter logarithmic growth, extending the enrichment culture cycle.

[0004] Some deep-sea microbial enrichment devices in the prior art have been able to simulate the deep-sea in-situ environment under laboratory conditions to carry out high-pressure enrichment and cultivation of microorganisms. For example, the existing patent documents disclose "a device and method for realizing rapid enrichment and cultivation of marine anaerobic ammonia-oxidizing bacteria", which can detect the real-time ammonia nitrogen concentration of the culture solution in the reactor to judge the growth and metabolic activity of anaerobic ammonia-oxidizing bacteria, and adjust the injection volume of the low-salinity seawater tank to make the anaerobic ammonia-oxidizing bacteria 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 the monitoring accuracy is low. At present, the most effective way to determine the growth of microorganisms is to count the cells of the microorganisms in the enriched culture solution. The traditional counting method is the cell abundance counting based on optical microscopy, but the cell abundance counting method is not suitable for the cell counting of deep-sea high-pressure enriched culture solution, because the decompression process will cause pressure shock to many deep-sea barophiles and cause their death, making it difficult to obtain real and effective microbial growth conditions 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 solution cultivated in 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, which 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 series culture, and use an online system to monitor the abundance, activity and culture conditions of microorganisms in real time to determine the appropriate time for strain subculture, while performing interactive subculture and cultivation, 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 solution of the present invention is as follows:

[0007] A deep-sea microorganism long-term high-pressure enrichment culture and online monitoring device, comprising: a microorganism 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 reaction kettles, and the microbial enrichment system is used for long-term enrichment culture of deep-sea microorganisms and for alternating passage;

[0009] The online monitoring system comprises: 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 perform real-time monitoring of environmental parameters and biological abundance indicators of the culture solution in the microbial enrichment system;

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

[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 of the reactors being a detachable upper cover structure, and a sample transfer unit being arranged 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 arranged on the outside of each reactor, and the sampling valve is connected to the microfluidic chip in the microbial abundance counting unit;

[0016] A gas injection valve and a vent valve are provided on the top of each reactor, and the gas 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 stainless steel metal filter membrane built in it.

[0019] Preferably, the environmental parameter monitoring unit comprises: 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, so that the excitation fluorescence of the fluorescence microscope can pass through the microfluidic chip; an air inlet valve and an exhaust valve are also arranged on the top of the observer, and the air inlet valve is connected to the pressure control system;

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

[0025] A fluorescent dye for staining microorganisms 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 abundance of microorganisms 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 square grid includes a large squares of the same size, any large square in the square 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 of the reactors is wrapped in the refrigeration jacket; a temperature sensor is arranged inside each of the reactors; 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 respectively connected to the gas injection valve of each reactor and the gas inlet valve of the observer;

[0033] A pressure sensor is arranged 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.

[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, the method comprises the following steps:

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

[0036] S2: adding deep-sea microbial samples and activation culture medium into each reactor of the microbial enrichment system in turn, and adjusting the pressure and temperature in the microbial enrichment system by using a pressure control system and a temperature control system, and enriching and culturing the microorganisms under a preset pressure and preset 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 parameter meets the preset conditions, injecting the microbial solution in the corresponding reactor into the microbial abundance counting unit;

[0038] S4: pressurizing the microbial abundance counting unit by 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 subculture time of the microorganism according to the growth curve, and transferring the bacterial liquid in the upper reactor to the lower reactor under pressure at the optimal subculture time;

[0040] S6: Repeat steps S3 to S5 to carry out long-term enrichment culture and cross-culture 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 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 an online system to monitor the abundance, activity and culture conditions of microorganisms in real time to determine the appropriate time for bacterial strain propagation, while performing interactive propagation and cultivation, thereby achieving a stable supply of high-abundance and high-activity microbial enrichment culture fluid;

[0043] The present invention can determine the best enrichment culture and subculture timing by directly counting the abundance of microorganisms and combining them with environmental auxiliary indicators, thus solving the difficulty of controlling the subculture timing during deep-sea microbial enrichment culture. The present invention can obtain a reserve microbial inoculum that meets the requirements of subsequent experiments from the enrichment reactor for a long period of time, thus 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 present invention achieves precise control of the microbial growth cycle during the enrichment process, so that the microbial culture in the enrichment and culture kettle always maintains a high growth rate and metabolic activity, ensuring the timeliness and cultivation efficiency of the microbial enrichment culture solution. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0046] Figure 2 This is a mechanical structure diagram of a deep-sea microorganism long-period high-pressure enrichment culture and online monitoring device 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 structure of the grid.

[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 drawings are for illustrative purposes only and should not be construed as limiting the present patent;

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

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

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

[0056] Example 1

[0057] like Figure 1 As shown, this embodiment provides a deep-sea microorganism long-term high-pressure enrichment culture and online monitoring device, including: a microorganism 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 reaction kettles, and the microbial enrichment system is used for long-term enrichment culture of deep-sea microorganisms and for alternating passage;

[0059] The online monitoring system comprises: 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 perform real-time monitoring of environmental parameters and biological abundance indicators of the culture solution in the microbial enrichment system;

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

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

[0063] Adding deep-sea microbial samples and activation culture medium into each reactor of the microbial enrichment system in turn, and adjusting the pressure and temperature in the microbial enrichment system by using a pressure control system and a temperature control system, and enriching and culturing the microorganisms under a preset pressure and preset 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 culture solution in the corresponding reactor is injected into the microfluidic chip of the microbial abundance counting unit;

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

[0066] Determine the best time for microbial subculture according to the growth curve, and transfer the bacterial solution from the previous reactor to the next reactor under pressure at the best time for subculture;

[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-maintaining 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 strain solution 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 microorganism long-term high-pressure enrichment culture and online monitoring device, including: a microorganism 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 reaction kettles 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 comprises: an environmental parameter monitoring unit 21 and a microbial abundance counting unit 22 respectively connected to the microbial enrichment system 1, the environmental parameter monitoring unit 21 and the microbial abundance counting unit 22 are respectively used to perform real-time monitoring of the environmental parameters and biological abundance indicators of the culture solution in the microbial enrichment system 1;

[0073] The microbial abundance counting unit 22 is provided with a microfluidic chip 222 and a fluorescent microscope 223. A fluorescent dye for microbial staining is pre-added in the microfluidic chip 222. After the bacterial liquid in the microbial enrichment system 1 is fluorescently stained, the microbial abundance is counted using the fluorescent 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, and is used to realize 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 connected in series and having the same structure, each of the reactors 11 is 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, and is used for alternate subculture of the microbial culture liquid between the reactors 11 in the later stage of cultivation; in this embodiment, the sample transfer unit 13 comprises: 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 10 μm stainless steel metal filter membrane built in it, which can effectively intercept the particle sediment and certain particle metabolites in the original sample but does not affect the passage of microorganisms, thereby improving the inoculation efficiency of microorganisms in the passage process;

[0079] In order to increase the nutrient utilization efficiency of the microorganisms during the cultivation process, each of the reactors 11 is provided with a microorganism culture solution and a magnetic stirrer 14, and 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, solving the difficulty that the manual stirring rod can only stir indirectly and is not conducive to the sealing of the reactor, and realizing the 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 respectively;

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

[0082] Each of the reactors 11 is provided with an injection valve 17 and an exhaust valve 18 at the top. The 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, the pH sensor 212 and the 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, it can be ensured 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, so that the excitation fluorescence of the fluorescence microscope 223 can 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] In order to carry out the microbial counting process 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. When 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 of the observer 221. The pressure is maintained throughout the counting process, which prevents some barophilic bacteria from dying after the pressure is lost, and ensures the reliability of microbial counting.

[0091] The microfluidic chip 222 is fixed in the center of the observer 221 through a slot 226 to prevent disturbances caused by gas injection 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 communicated with the counting chamber 227; the lower injection valve 228 is connected to the sampling valve 16 outside each of the reactors 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 exhaust 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 microorganism staining is pre-added in the counting chamber 227, and the bacterial solution in the reactor 11 is mixed with the fluorescent dye for a certain period of time to complete the fluorescent staining; the observer 221 is placed on the fluorescent microscope 223, and the abundance of microorganisms 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 square grid for cell counting, and the square grid includes a plurality of squares 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 plus microbial solution;

[0096] The grid includes 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. By using water bath refrigeration, the temperature control is more precise and uniform; each of the reactors 11 is provided with a temperature sensor 33; 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 the changes in the environmental parameters of the enriched culture solution.

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

[0106] A deep-sea microorganism sample and an activation culture medium are sequentially added into each reactor 11 of the microorganism enrichment system 1, and the pressure and temperature in the microorganism enrichment system 1 are adjusted by using the pressure control system 4 and the temperature control system 3, so as to enrich and culture the microorganisms under a high pressure and low temperature environment;

[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 solution 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, 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 best subculture time of the microorganism according to the growth curve, and transfer the bacterial solution of the upper reactor 11 to the lower reactor 11 under pressure at the best subculture time;

[0110] Repeat the above steps to carry out long-term enrichment culture and cross-culture of the deep-sea microbial sample in the microbial enrichment system 1;

[0111] This device can perform precise pressure-maintaining 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 strain solution 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 the reactors of the microbial enrichment system and the various components in the online monitoring system are sterile operated, and the deep-sea microbial long-term high-pressure enrichment culture and online monitoring device are installed;

[0115] S2: adding deep-sea microbial samples and activation culture medium into each reactor of the microbial enrichment system in turn, and adjusting the pressure and temperature in the microbial enrichment system by using a pressure control system and a temperature control system, and enriching and culturing the microorganisms under a preset pressure and preset 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 parameter meets the preset conditions, injecting the microbial solution in the corresponding reactor into the microbial abundance counting unit;

[0117] S4: pressurizing the microbial abundance counting unit by 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 subculture time of the microorganism according to the growth curve, and transferring the bacterial liquid in the upper reactor to the lower reactor under pressure at the optimal subculture time;

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

[0120] In the specific implementation process, step 1: before culturing, the reactor 11 and its attached pipe and valve components are first sterilized by 75% ethanol or ultraviolet light irradiation, and then the reactor 11 and its attached components are transferred to the ultra-clean studio for assembly in a sterile environment; generally, before selecting to enrich a certain specific microbial group, the microbial community in the deep-sea sample needs to be activated and cultured first, so that most of the microbial groups in the sample can re-metabolize functionally, and then the activated bacterial liquid is inoculated into a new enrichment culture kettle under pressure to enrich and culture the specific microbial flora; therefore, deep-sea microbial samples (deep-sea sediments or seawater, etc.), microbial activation culture medium (depending on the sample and the required microbial group) and magnetic stirrer 14 are added to the reactor 11 in a sterile environment, and then various valves and sensor components are installed;

[0121] Step 2: Connect the gas 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 gas 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, the pH sensor 212 and the Raman sensor 213 are used to monitor the concentration changes of dissolved oxygen, pH value and organic nutrients in the enriched culture solution in real time, and the values ​​are displayed in real time on the central control system 5, which indirectly reflects the growth of microorganisms; when the pH value in the culture solution drops by 2 to 3 orders of magnitude, and the concentration of organic nutrients such as glucose and acetic acid is significantly reduced, it means that the nutrients in the culture solution are consumed in large quantities and acidic metabolites are generated, then inoculation and subculture can be considered;

[0123] Step 4: Count the cells of the microbial culture in the 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, so that the internal pressure of the observer 221 increases 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 the staining is completed, place the microfluidic chip 222 under the fluorescence microscope 223; adjust the focal length of the fluorescence microscope 223 to first find the grid in the counting chamber 227 under a low-power microscope, and then convert 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 in the field of view, and then convert it into 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 period, logarithmic period, platform period and decay period of the microbial community;

[0124] Step 5: Determine the best time for subculturing in combination with the growth curve and the changes in environmental parameters, which is generally from the end of the logarithmic growth stage to the early stage of the platform; before subculturing, repeat steps 1 to 2, but replace the activated culture medium with an enriched culture medium for the desired microbial group; during subculturing, connect the regulating valve 131, the injection pump 132 and the filter 133 in sequence at the bottom of the previous reactor 11 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 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 only used for illustrative purposes and should not be construed as limiting this patent;

[0129] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope 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 reaction kettles, and the microbial enrichment system is used for long-term enrichment culture of deep-sea microorganisms and for alternating passage; The online monitoring system comprises: 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 perform real-time monitoring of environmental parameters and biological abundance indicators of the culture solution in the microbial enrichment system; The temperature control system and the pressure control system are respectively connected to the microbial enrichment system, and are used to adjust the temperature and pressure in the microbial enrichment system respectively; the pressure control system is also connected to the microbial abundance counting unit, and is used to realize the microbial pressure-maintaining counting; 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. A deep-sea microorganism long-term high-pressure enrichment culture and online monitoring device according to claim 1, characterized in that: The microorganism enrichment system comprises: a plurality of reactors connected in series and having the same structure, each of the reactors being a detachable upper cover structure, and a sample transfer unit being arranged 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 arranged on the outside of each reactor, and the sampling valve is connected to the microfluidic chip in the microbial abundance counting unit; A gas injection valve and a vent valve are provided on the top of each reactor, and the gas injection valve is connected to the pressure control system; each reactor is connected to a temperature control system.

3. A deep-sea microorganism long-term high-pressure enrichment culture and online monitoring device 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 stainless steel metal filter membrane built in it.

4. A deep-sea microorganism long-term high-pressure enrichment culture and online monitoring device 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 culture and online monitoring of deep-sea microorganisms according to claim 2, characterized in that: The microbial abundance counting unit comprises: an observer, a microfluidic chip and a fluorescence microscope; The top and bottom of the observer are both sapphire windows, so that the excitation fluorescence of the fluorescence microscope can pass through the microfluidic chip; an air inlet valve and an exhaust valve are also arranged on the top of the observer, and the air inlet valve is connected to the pressure control system; The microfluidic chip is fixed in the center of the observer through a card slot, and is provided with a counting chamber, and a lower injection valve and an upper exhaust valve communicated with the counting chamber; the lower injection valve is connected to the sampling valve outside each reactor, and is used to inject the bacterial liquid in the reactor into the counting chamber; the upper exhaust valve is used to exhaust the gas in the counting chamber when the bacterial liquid is injected into the counting chamber; A fluorescent dye for staining microorganisms 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 fluorescent microscope is used to count the abundance of microorganisms.

6. A deep-sea microorganism long-term high-pressure enrichment culture and online monitoring device according to claim 5, 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.

7. A deep-sea microorganism long-term high-pressure enrichment culture and online monitoring device according to claim 6, characterized in that: The square grid includes a large squares of the same size, any large square in the square grid is further divided into b medium squares, and each medium square is divided into c small squares; wherein a, b and c are respectively the first, second and third positive integers; 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.

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

9. The device for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms according to claim 5, characterized in that: 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; 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 gas injection valve of each reactor and the gas inlet valve of the observer; A pressure sensor is arranged 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.

10. A method for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms, based on a device for long-term high-pressure enrichment culture and online monitoring of deep-sea microorganisms as claimed in any one of claims 1 to 9, characterized in that: The following steps are involved: S1: All the reactors of the microbial enrichment system and the components of the online monitoring system are sterile operated, and the deep-sea microbial long-term high-pressure enrichment culture and online monitoring device are installed; S2: adding deep-sea microbial samples and activation culture medium into each reactor of the microbial enrichment system in turn, and adjusting the pressure and temperature in the microbial enrichment system by using a pressure control system and a temperature control system, and enriching and culturing the microorganisms under a preset pressure and preset 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 parameter meets the preset conditions, injecting the microbial solution 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 under pressure, and drawing a growth curve of the microbial community; S5: determining the optimal subculture time of the microorganism according to the growth curve, and transferring the bacterial liquid in the upper reactor to the lower reactor under pressure at the optimal subculture time; S6: Repeat steps S3 to S5 to carry out long-term enrichment culture and cross-culture on the deep-sea microbial samples in the microbial enrichment system.

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