Deep-sea microorganism online purification sampling and visual automatic enrichment device and method

Abstract

The invention provides an online purification sampling and visual automatic enrichment device and method for deep-sea microorganisms, and the method comprises the steps: carrying out online sample pretreatment in a microorganism sampling process, automatically concentrating and purifying organisms, carrying out online monitoring on environmental indexes in a biological enrichment process, carrying out online observation on biomass, and automatically enriching and transferring high-purity enriched substances. The defects that in the prior art, a deep-sea microorganism sample in a high-pressure environment is difficult to purify, manual operation of enrichment and observation is complex, timeliness of biomass observation is low, and real-time cell counting of enriched substances is difficult are overcome, and the enrichment rate and the acquisition efficiency of deep-sea microorganisms difficult to culture are effectively improved; a basic means is provided for improving the deep-sea biology research level and the biological resource development efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of deep-sea microorganism sample purification, enrichment and observation, and more specifically, to a deep-sea microorganism online purification sampling and visual automatic enrichment device and method. Background Art

[0002] The deep sea is considered to be the last unknown frontier on Earth. Extreme conditions such as high pressure, low temperature / high temperature, and oligotrophy in the deep sea environment have created special deep-sea microorganisms. Their evolutionary branches, biochemical reactions, and metabolic functions are still unsolved mysteries, and they have extremely high research and application value. In order to explore the role of deep-sea microorganisms in the marine material cycle and the earth's ecosystem, it is first necessary to pre-treat, enrich and observe deep-sea environmental samples to identify their biological community characteristics and physiological metabolic characteristics. However, the biomass of the deep-sea environment is low, and the current technology for high-pressure environment sample processing and biological enrichment is still immature, making it very difficult to quickly enrich difficult-to-cultivate organisms. After deep-sea microorganisms are collected, it is difficult to purify biological samples in a high-pressure environment, and it is difficult to monitor biomass changes during microbial enrichment. Efficient visualization of microorganisms will be very difficult. Therefore, using a heat-insulating and pressure-maintaining device that simulates a deep-sea environment to purify and visualize microorganisms is an important means to improve the research level and development efficiency of deep-sea microorganisms.

[0003] For the purification and sampling of microorganisms under deep-sea simulation conditions, the purification and sampling technologies disclosed in existing patents, such as "a method for extracting and semi-quantitatively analyzing biofilms on the surface of sediments" and "a method for simultaneously purifying nucleic acids and proteins from environmental samples", mainly involve purification processes such as washing, oscillating and centrifuging sediment samples. However, these purification processes are difficult to implement in a high-pressure environment. Therefore, it is difficult to separate environmental impurities such as gravel and organic matter from microbial cells in deep-sea environmental samples under high-pressure conditions. There are also limitations such as low activity of the obtained enrichment, low efficiency of subsequent biological enrichment, and low purity of the obtained biological samples.

[0004] Regarding the enrichment of microorganisms under deep-sea simulation conditions, the existing patent discloses "a microbial culture device and system", which mainly observes the general situation of the internal enrichment through a visualization window and a light-transmitting culture cavity, but it is difficult to observe the biological abundance of the enrichment in real time in a high-pressure environment, and it is still necessary to obtain the relative microbial abundance through regular multiple sampling and gene sequencing analysis. The process has the problems of cumbersome manual operation, low timeliness, and loss of enriched biomass; in addition, the existing patent also discloses "a high-pressure temperature-controlled online culture observation sample station", which connects the outlet of the culture kettle to the observation window set on the pipeline, and is connected to an external microscope for real-time observation of microbial morphology, but the samples observed in the pipeline are not diluted, separated, and evenly pressed, so it is difficult to obtain spaced, separated, flat and uniform cell distribution images in the microscope for cell counting. This technology has the problems of excessive accumulation of cells in the observed sample, large amount of cell overlap in the observation field of view, and no unit grid division in the observation area for cell counting, making it difficult to obtain the actual enriched biomass.

[0005] In general, existing technologies have problems such as difficulty in purifying deep-sea microbial samples in high-pressure environments, complex enrichment and observation operations, low timeliness of biomass observation, and difficulty in real-time cell counting of enrichments, which make it difficult to quickly obtain high-purity enrichments of deep-sea microorganisms. Summary of the invention

[0006] In order to overcome the defects of the above-mentioned prior art, namely, difficulty in purifying deep-sea microbial samples in a high-pressure environment, complex enrichment and observation manual operations, low timeliness of biomass observation, and difficulty in real-time cell counting of enriched products, the present invention provides a deep-sea microbial online purification sampling and visual automatic enrichment device and method. Through online sample pretreatment during microbial sampling, automatic biological concentration and purification, online monitoring of environmental indicators during biological enrichment, online observation of biomass, and automatic enrichment and transfer of high-purity enriched products, the enrichment rate and acquisition efficiency of deep-sea difficult-to-cultivate microorganisms are improved, providing a basic means for improving the level of deep-sea biological research and the efficiency of biological resource development.

[0007] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0008] A deep-sea microorganism online purification sampling device, comprising: a sampling and purification unit, a first temperature regulating unit, a first pressure regulating unit and a first central control unit;

[0009] The sampling and purification unit comprises: a sample mixing kettle, a sample settling kettle, a sample purification kettle, two sets of pressure-maintaining stirrers, a turbidity meter, a total organic carbon analyzer, a purification piston and a filter membrane;

[0010] The sample mixing kettle is connected to the pipeline of the container to be sampled, and is used to obtain deep-sea microbial samples from the container to be sampled; the lower part of the sample mixing kettle is connected to the upper pipeline of the sample settling kettle; the lower part of the sample settling kettle is connected to the lower pipeline of the sample purification kettle;

[0011] The bottom of the sample settling kettle is a funnel-shaped structure to achieve the impurity sedimentation effect of the enriched sample; two sets of pressure-maintaining agitators are respectively arranged at the top of the sample mixing kettle and the sample purification kettle; the turbidity meter is arranged inside the sample settling kettle; the total organic carbon analyzer is connected to the sample purification kettle; the purification piston is arranged at the lower part of the sample purification kettle and can be pushed upward to achieve pressurized concentrated samples; the filter membrane is arranged on the upper surface of the purification piston;

[0012] The first temperature regulating unit and the first pressure regulating unit are respectively connected to the first central control unit, the sample mixing kettle, the sample settling kettle and the sample purification kettle, and the first temperature regulating unit and the first pressure regulating unit are respectively controlled by the first central control unit to adjust the temperature and pressure in each kettle;

[0013] The first central control unit is also connected to two groups of pressure-maintaining stirrers, a turbidity meter and a total organic carbon analyzer respectively.

[0014] Preferably, the first temperature regulating unit comprises: a temperature regulator and three groups of temperature sensors respectively connected to the first central control unit; the three groups of temperature sensors are respectively arranged inside the sample mixing kettle, the sample settling kettle and the sample purification kettle;

[0015] The first pressure regulating unit comprises: a gas booster pump, three gas injection valves, a purification liquid injection pump, a purification valve, and four groups of pressure sensors; the gas booster pump, the purification liquid injection pump and the four groups of pressure sensors are respectively connected to the first central control unit;

[0016] The gas booster pump is connected to the sample mixing kettle, sample sedimentation kettle and sample purification kettle through three gas injection valves and pipelines respectively; the purification injection pump is connected to the bottom of the sample purification kettle through purification valves and pipelines; a group of pressure sensors are respectively provided at the top of the sample mixing kettle, the top of the sample sedimentation kettle, and the top and bottom of the sample purification kettle. The pressure sensor at the bottom of the sample purification kettle is used to monitor the purification progress and stop it in time.

[0017] The present invention also provides an online visual automatic enrichment device for deep-sea microorganisms, comprising: an enrichment unit, a visual monitoring unit, a second temperature regulating unit, a second pressure regulating unit, and a second central control unit;

[0018] The enrichment unit comprises: an enrichment kettle, a magnetic stirrer and an environmental parameter sensing system; the magnetic stirrer is arranged at the bottom of the enrichment kettle and a magnet is placed inside the enrichment kettle; the environmental parameter sensing system is respectively connected to the enrichment kettle and the second central control unit, and is used to detect the environmental parameters in the enrichment kettle and transmit them to the second central control unit;

[0019] The visual monitoring unit includes: a biological observation kettle, a biological abundance measurement system, two sets of pressure-resistant observation windows, a microscopic camera system, a measurement solenoid valve and a recovery solenoid valve;

[0020] The biological observation kettle is connected to the enrichment kettle pipeline; two sets of pressure-resistant observation windows are respectively arranged at the top and bottom of the biological observation kettle, and are used to observe the morphology of microorganisms in the biological observation kettle; the biological abundance measurement system is respectively connected to the biological observation kettle and the second central control unit, and is used to place the microorganisms to be observed; the microscopic camera system is arranged at the bottom of the biological observation kettle and is connected to the second central control unit, and is used to observe the changes in the abundance of microorganisms in the biological observation kettle from the pressure-resistant observation window at the bottom; the measuring solenoid valve and the recovery solenoid valve are respectively connected to the biological abundance measurement system and the enrichment kettle through pipelines, and are respectively connected to the second central control unit;

[0021] The second temperature regulating unit and the second pressure regulating unit are respectively connected to the second central control unit, the enrichment kettle and the biological observation kettle, and the second temperature regulating unit and the second pressure regulating unit are respectively controlled by the second central control unit to adjust the temperature and pressure in each kettle.

[0022] Preferably, the enrichment unit further comprises: an enrichment ball valve, a plunger pump, a continuous injection pump and an injection valve;

[0023] One end of the enrichment ball valve and the plunger pump are connected to the enrichment kettle through pipelines respectively; the other end of the plunger pump is connected to the measurement solenoid valve and the recovery solenoid valve through pipelines respectively;

[0024] The continuous injection pump is connected to the injection valve and the enrichment kettle in sequence through a pipeline, and is used to inject microbial culture solution into the enrichment kettle;

[0025] The visual monitoring unit also includes a dilution kettle, a dilution valve and an illuminator;

[0026] One end of the plunger pump is also connected to the dilution valve and the dilution kettle in sequence through a pipeline; the dilution kettle is provided with a dilution culture solution;

[0027] The illuminator is arranged on the top of the biological observation kettle and is connected to the second central control unit, and is used to provide illumination for the biological observation kettle from the pressure-resistant observation window on the top.

[0028] Preferably, the environmental parameter sensing system comprises: a dissolved oxygen detector, a pH value detector and a Raman measuring instrument, which are respectively used to detect the dissolved oxygen content, pH value and chemical substance content changes in the enrichment kettle and transmit them to the second central control unit.

[0029] Preferably, the biological abundance measurement system comprises: an observation pool, a cover glass flipper and a cover glass;

[0030] The two ends of the observation pool are respectively connected to a measuring solenoid valve and a recovery solenoid valve through pipelines; the cover glass flipper is connected to the second central control unit, and is used to drive the cover glass to flip and attach it to the observation pool;

[0031] The observation pool is provided with a square groove, which is completely fitted with a preset protruding square on the cover glass; a bidirectional funnel-shaped passage is also provided in the square groove, and a grid for observing and counting cells is engraved in the passage; a plurality of squares of different sizes are provided in the grid for counting cells of different quantities;

[0032] The microscopic camera system comprises: a lens switcher and a plurality of microscope lenses with different magnifications;

[0033] The lens switcher is connected to the second central control unit and is used for switching the microscope lens.

[0034] Preferably, the second temperature regulating unit comprises: a temperature regulator and two groups of temperature sensors respectively connected to the second central control unit; the two groups of temperature sensors are respectively arranged inside the enrichment kettle and the biological observation kettle;

[0035] The second pressure regulating unit comprises: a gas booster pump, a gas injection valve, a PID control valve, an exhaust valve and two groups of pressure sensors respectively connected to the second central control unit;

[0036] The gas booster pump is connected to the gas injection valve and the enrichment kettle in sequence through pipelines; the PID control valve is arranged on the enrichment kettle; the exhaust valve is arranged on the biological observation kettle; and two groups of pressure sensors are respectively arranged inside the enrichment kettle and the biological observation kettle.

[0037] The present invention also provides an online purification sampling and visual automatic enrichment device for deep-sea microorganisms, comprising a purification sampling device and an enrichment device connected in sequence through pipelines, wherein the purification sampling device is specifically the above-mentioned online purification sampling device for deep-sea microorganisms, and the enrichment device is specifically the above-mentioned online visual automatic enrichment device for deep-sea microorganisms.

[0038] The present invention also provides a method for online purification sampling and visualization automatic enrichment of deep-sea microorganisms, based on the above-mentioned online purification sampling and visualization automatic enrichment device of deep-sea microorganisms, comprising the following steps:

[0039] S1: Clean and sterilize the sample mixing kettle, sample settling kettle, sample purification kettle, enrichment kettle and biological observation kettle, and respectively perform culture solution injection, kettle body installation and gas pressurization; install each unit in the deep-sea microorganism online purification sampling and visual automatic enrichment device;

[0040] S2: Obtain a deep-sea microorganism sample from the sampling container and transfer it to a sample mixing kettle, start a pressure-maintaining agitator in the sample mixing kettle, wait for a first preset time for the sample to be completely mixed, and obtain the mixed sample;

[0041] S3: transferring the mixed sample into a sample settling kettle, using a turbidity meter to monitor the settling and separation of sample impurities in real time, and obtaining the settled sample after the turbidity drops to a first preset threshold;

[0042] S4: transferring the settled sample into a sample purification kettle, turning on the pressure-maintaining agitator in the sample purification kettle, pushing the purification piston for purification, and using a total organic carbon analyzer to monitor the sample purification in real time. After the total organic matter content obtained by the total organic carbon analyzer is greater than a second preset threshold, obtaining the purified sample, and transferring the purified sample to an enrichment kettle;

[0043] S5: Turn on the magnetic stirrer to start enrichment, and use the environmental parameter sensing system to monitor the environmental indicators in the enrichment kettle in real time. When the monitored environmental indicators meet the preset conditions, obtain the enriched bacterial solution to complete the enrichment;

[0044] S6: Open the measuring solenoid valve to transfer the enriched bacterial solution to the biological abundance measurement system in the biological observation kettle, and use the microscopic camera system to observe the biological abundance. After the observation is completed, open the recovery solenoid valve to transfer the enriched bacterial solution after observation back to the enrichment kettle for recovery;

[0045] S7: Repeat steps S5 to S6 to perform multiple enrichment and observation until the observed biological abundance reaches a third preset threshold, at which time all the enriched bacterial liquid in the enrichment kettle is collected for further preservation or experiment.

[0046] Preferably, in step S4, when the total organic matter content obtained by the total organic carbon analyzer is greater than the second preset threshold, or when the pressure difference collected by the top and bottom pressure sensors in the sample purification kettle is greater than the fourth preset threshold, the purified sample is obtained;

[0047] In the biological abundance measurement system of step S6, the height of the square groove of the observation pool is 0.1 cm, and the grid size is specifically 1 cm×1 cm;

[0048] After the microscopic camera system acquires the observed image, the biological abundance is calculated according to the following formula:

[0049] N C =N F ×D×F×10000

[0050] Among them, N C Indicates the number of cells per milliliter of sample; N F represents the average number of cells in a square; D represents the dilution factor of the cell sample; F represents the total number of squares of different sizes.

[0051] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0052] The present invention provides a device for online purification and sampling of deep-sea heat-insulated and pressure-maintained microorganisms. Compared with the existing high-pressure sampling technology, the device can realize an automated pretreatment process for effectively separating biological cells from environmental impurities in a microbial sampling process under a simulated deep-sea high-pressure environment under heat-insulated and pressure-maintained conditions, and obtain high-purity biological samples, thereby solving the problems of difficulty in purifying microbial samples in a high-pressure environment during deep-sea microbial sampling, and low activity and purity of the obtained biological samples, and improving the biological purity and processing efficiency of microorganisms that are difficult to culture in a deep-sea environment;

[0053] The present invention also provides an online visualization automatic enrichment device for deep-sea microorganisms, which solves the problems of complex manual operation of biomass observation during enrichment, low observation timeliness, and difficulty in real-time cell counting of enriched products. The device can perform visualization monitoring of biomass abundance changes after automatic enrichment and purification of microorganisms under deep-sea heat preservation and pressure conditions, thereby improving the efficiency of obtaining high-purity enriched products of difficult-to-cultivate microorganisms in deep-sea environments.

[0054] In addition, the present invention combines the two to provide a deep-sea microorganism online purification sampling and visual automatic enrichment device and method. Through online sample pretreatment during microbial sampling, automatic biological concentration and purification, online monitoring of environmental indicators during biological enrichment, online observation of biomass, and automatic enrichment and transfer of high-purity enriched materials, the enrichment rate and acquisition efficiency of deep-sea difficult-to-cultivate microorganisms are improved, providing a basic means for improving the level of deep-sea biological research and the efficiency of biological resource development. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a schematic diagram of the architecture of an online deep-sea microorganism purification and sampling device provided in Example 1.

[0056] Figure 2 This is a schematic diagram of the architecture of an online visual automatic enrichment device for deep-sea microorganisms provided in Example 2.

[0057] Figure 3 This is a schematic diagram of the architecture of a deep-sea microorganism online purification sampling and visual automatic enrichment device provided in Example 3.

[0058] Figure 4 This is a mechanical structure diagram of a deep-sea microorganism online purification sampling and visual automatic enrichment device provided in Example 4.

[0059] Figure 5 This is a specific structural diagram of the biological observation kettle provided in Example 4.

[0060] Figure 6 This is a top view of the internal structure of the biological observation kettle provided in Example 4.

[0061] Figure 7 This is the connection diagram of the central control unit provided in Example 4.

[0062] Figure 8 This is a flow chart of the online purification sampling and visual automatic enrichment method for deep-sea microorganisms provided in Example 5.

[0063] Fig. 9 This is a specific implementation flow chart of the method for online purification, sampling and visual automatic enrichment of deep-sea microorganisms provided in Example 5. DETAILED DESCRIPTION

[0064] The drawings are for illustrative purposes only and should not be construed as limiting the present patent;

[0065] 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;

[0066] It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0067] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0068] Example 1

[0069] like Figure 1 As shown, this embodiment provides a deep-sea microorganism online purification sampling device, including: a sampling and purification unit, a first temperature adjustment unit, a first pressure adjustment unit and a first central control unit;

[0070] The sampling and purification unit comprises: a sample mixing kettle, a sample settling kettle, a sample purification kettle, two sets of pressure-maintaining stirrers, a turbidity meter, a total organic carbon analyzer, a purification piston and a filter membrane;

[0071] The sample mixing kettle is connected to the pipeline of the container to be sampled, and is used to obtain deep-sea microbial samples from the container to be sampled; the lower part of the sample mixing kettle is connected to the upper pipeline of the sample settling kettle; the lower part of the sample settling kettle is connected to the lower pipeline of the sample purification kettle;

[0072] The bottom of the sample settling kettle is a funnel-shaped structure to achieve the impurity sedimentation effect of the enriched sample; two sets of pressure-maintaining agitators are respectively arranged at the top of the sample mixing kettle and the sample purification kettle; the turbidity meter is arranged inside the sample settling kettle; the total organic carbon analyzer is connected to the sample purification kettle; the purification piston is arranged at the lower part of the sample purification kettle and can be pushed upward to achieve pressurized concentrated samples; the filter membrane is arranged on the upper surface of the purification piston;

[0073] The first temperature regulating unit and the first pressure regulating unit are respectively connected to the first central control unit, the sample mixing kettle, the sample settling kettle and the sample purification kettle, and the first temperature regulating unit and the first pressure regulating unit are respectively controlled by the first central control unit to adjust the temperature and pressure in each kettle;

[0074] The first central control unit is also connected to two groups of pressure-maintaining stirrers, a turbidity meter and a total organic carbon analyzer respectively.

[0075] In the specific implementation process, the sample mixing kettle, sample settling kettle and sample purification kettle are first cleaned and sterilized, and the culture solution is injected, the kettle body is installed and the gas is pressurized respectively; the various units in the deep-sea microorganism online purification sampling device are installed;

[0076] Obtain a deep-sea microbial sample from a sampling container and transfer it to a sample mixing kettle, start a pressure-maintaining agitator in the sample mixing kettle, wait for a first preset time for the sample to be completely mixed, and obtain the mixed sample;

[0077] The mixed sample is transferred into a sample settling kettle, and the settling and separation of the sample impurities is monitored in real time by a turbidity meter. After the turbidity is reduced to a first preset threshold, the settled sample is obtained;

[0078] The settled sample is transferred into a sample purification kettle, the pressure-maintaining agitator in the sample purification kettle is turned on, the purification piston is pushed for purification, and the sample purification status is monitored in real time using a total organic carbon analyzer. After the total organic matter content obtained by the total organic carbon analyzer is greater than a second preset threshold, the purified sample is obtained to complete the purification sampling;

[0079] This device can, under the conditions of heat preservation and pressure preservation, realize an automated pretreatment process for effectively separating biological cells from environmental impurities during microbial sampling in a simulated deep-sea high-pressure environment, and obtain high-purity biological samples. It solves the difficulties in purifying microbial samples in a high-pressure environment during deep-sea microbial sampling, as well as the problems of low activity and purity of biological samples, and improves the biological purity and processing efficiency of difficult-to-cultivate microorganisms in deep-sea environments.

[0080] Example 2

[0081] like Figure 2As shown, this embodiment provides an online visual automatic enrichment device for deep-sea microorganisms, including: an enrichment unit, a visual monitoring unit, a second temperature adjustment unit, a second pressure adjustment unit, and a second central control unit;

[0082] The enrichment unit comprises: an enrichment kettle, a magnetic stirrer and an environmental parameter sensing system; the magnetic stirrer is arranged at the bottom of the enrichment kettle and a magnet is placed inside the enrichment kettle; the environmental parameter sensing system is respectively connected to the enrichment kettle and the second central control unit, and is used to detect the environmental parameters in the enrichment kettle and transmit them to the second central control unit;

[0083] The visual monitoring unit includes: a biological observation kettle, a biological abundance measurement system, two sets of pressure-resistant observation windows, a microscopic camera system, a measurement solenoid valve and a recovery solenoid valve;

[0084] The biological observation kettle is connected to the enrichment kettle pipeline; two sets of pressure-resistant observation windows are respectively arranged at the top and bottom of the biological observation kettle, and are used to observe the morphology of microorganisms in the biological observation kettle; the biological abundance measurement system is respectively connected to the biological observation kettle and the second central control unit, and is used to place the microorganisms to be observed; the microscopic camera system is arranged at the bottom of the biological observation kettle and is connected to the second central control unit, and is used to observe the changes in the abundance of microorganisms in the biological observation kettle from the pressure-resistant observation window at the bottom; the measuring solenoid valve and the recovery solenoid valve are respectively connected to the biological abundance measurement system and the enrichment kettle through pipelines, and are respectively connected to the second central control unit;

[0085] The second temperature regulating unit and the second pressure regulating unit are respectively connected to the second central control unit, the enrichment kettle and the biological observation kettle, and the second temperature regulating unit and the second pressure regulating unit are respectively controlled by the second central control unit to adjust the temperature and pressure in each kettle.

[0086] In the specific implementation process, the enrichment kettle and biological observation kettle are first cleaned and sterilized, and the culture solution is injected, the kettle body is installed and the gas is pressurized respectively; the various units in the deep-sea microorganism visualization automatic enrichment device are installed;

[0087] The bacterial solution to be enriched is transferred to the enrichment kettle; the magnetic stirrer is turned on to start enrichment, and the environmental parameter sensing system is used to monitor the environmental indicators in the enrichment kettle in real time. When the environmental indicators to be monitored meet the preset conditions, the enriched bacterial solution is obtained to complete the enrichment;

[0088] Open the measuring solenoid valve to transfer the enriched bacterial solution to the biological abundance measurement system in the biological observation kettle, and use the microscopic camera system to observe the biological abundance. After the observation is completed, open the recovery solenoid valve to transfer the enriched bacterial solution after observation back to the enrichment kettle for recovery;

[0089] Finally, the above steps are repeated for multiple enrichment and observation until the observed biological abundance reaches a third preset threshold, at which time all the enriched bacterial liquid in the enrichment kettle is collected for further preservation or experiment;

[0090] The device solves the problems of complex manual operation of biomass observation during the enrichment process, low observation timeliness, and difficulty in real-time cell counting of enriched products. The device can perform visual monitoring of changes in biomass abundance after automatic enrichment and purification of microorganisms under deep-sea insulation and pressure conditions, thereby improving the efficiency of obtaining high-purity enrichments of difficult-to-cultivate microorganisms in deep-sea environments.

[0091] Example 3

[0092] like Figure 3 As shown, this embodiment provides a deep-sea microorganism online purification sampling and visual automatic enrichment device, including: a sampling and purification unit, an enrichment unit, a visual monitoring unit, a temperature adjustment unit, a pressure adjustment unit and a central control unit;

[0093] In this embodiment, the pressure regulating unit, the temperature regulating unit and the central control unit respectively integrate the functions of the first and second pressure regulating units, the first and second temperature regulating units, and the first and second central control units, and are respectively used to control the pressure, temperature and specific operations of the entire device (including the online purification sampling and enrichment device);

[0094] The sampling and purification unit comprises: a sample mixing kettle, a sample settling kettle, a sample purification kettle, two sets of pressure-maintaining stirrers, a turbidity meter, a total organic carbon analyzer, a purification piston and a filter membrane;

[0095] The sample mixing kettle is connected to the pipeline of the container to be sampled, and is used to obtain deep-sea microbial samples from the container to be sampled; the lower part of the sample mixing kettle is connected to the upper pipeline of the sample settling kettle; the lower part of the sample settling kettle is connected to the lower pipeline of the sample purification kettle;

[0096] The bottom of the sample settling kettle is a funnel-shaped structure to achieve the impurity sedimentation effect of the enriched sample; two sets of pressure-maintaining agitators are respectively arranged at the top of the sample mixing kettle and the sample purification kettle; the turbidity meter is arranged inside the sample settling kettle; the total organic carbon analyzer is connected to the sample purification kettle; the purification piston is arranged at the lower part of the sample purification kettle and can be pushed upward to achieve pressurized concentrated samples; the filter membrane is arranged on the upper surface of the purification piston;

[0097] The central control unit is also connected to two groups of pressure-maintaining stirrers, a turbidity meter and a total organic carbon analyzer respectively;

[0098] The enrichment unit comprises: an enrichment kettle, a magnetic stirrer and an environmental parameter sensing system; the magnetic stirrer is arranged at the bottom of the enrichment kettle and a magnet is placed inside the enrichment kettle; the environmental parameter sensing system is connected to the enrichment kettle and the central control unit respectively, and is used to detect the environmental parameters in the enrichment kettle and transmit them to the central control unit;

[0099] The visual monitoring unit includes: a biological observation kettle, a biological abundance measurement system, two sets of pressure-resistant observation windows, a microscopic camera system, a measurement solenoid valve and a recovery solenoid valve;

[0100] The biological observation kettle is connected to the enrichment kettle pipeline; two sets of pressure-resistant observation windows are respectively arranged at the top and bottom of the biological observation kettle, and are used to observe the morphology of microorganisms in the biological observation kettle; the biological abundance measurement system is respectively connected to the biological observation kettle and the central control unit, and is used to place the microorganisms to be observed; the microscopic camera system is arranged at the bottom of the biological observation kettle and is connected to the central control unit, and is used to observe the changes in the abundance of microorganisms in the biological observation kettle from the pressure-resistant observation window at the bottom; the measuring solenoid valve and the recovery solenoid valve are respectively connected to the biological abundance measurement system and the enrichment kettle through pipelines, and are respectively connected to the central control unit;

[0101] The temperature regulating unit and the pressure regulating unit are respectively connected to the central control unit, the sample mixing kettle, the sample sedimentation kettle, the sample purification kettle, the enrichment kettle and the biological observation kettle. The temperature regulating unit and the pressure regulating unit are respectively controlled by the central control unit to adjust the temperature and pressure in each kettle.

[0102] In the specific implementation process, the sample mixing kettle, sample settling kettle, sample purification kettle, enrichment kettle and biological observation kettle are first cleaned and sterilized, and the culture solution is injected, the kettle body is installed and the gas is pressurized respectively; and each unit in the device is installed;

[0103] Obtain a deep-sea microbial sample from a sampling container and transfer it to a sample mixing kettle, start a pressure-maintaining agitator in the sample mixing kettle, wait for a first preset time for the sample to be completely mixed, and obtain the mixed sample;

[0104] The mixed sample is transferred into a sample settling kettle, and the settling and separation of the sample impurities is monitored in real time by a turbidity meter. After the turbidity is reduced to a first preset threshold, the settled sample is obtained;

[0105] The settled sample is transferred into a sample purification kettle, the pressure-maintaining agitator in the sample purification kettle is turned on, the purification piston is pushed for purification, and the sample purification status is monitored in real time using a total organic carbon analyzer. After the total organic matter content obtained by the total organic carbon analyzer is greater than a second preset threshold, the purified sample is obtained to complete the purification sampling;

[0106] Then, the purified bacterial liquid sample is transferred to the enrichment kettle, the magnetic stirrer is turned on, enrichment begins, and the environmental parameter sensing system is used to monitor the environmental indicators in the enrichment kettle in real time. When the monitored environmental indicators meet the preset conditions, the enriched bacterial liquid is obtained to complete the enrichment;

[0107] Open the measuring solenoid valve to transfer the enriched bacterial solution to the biological abundance measurement system in the biological observation kettle, and use the microscopic camera system to observe the biological abundance. After the observation is completed, open the recovery solenoid valve to transfer the enriched bacterial solution after observation back to the enrichment kettle for recovery;

[0108] Finally, the above steps are repeated for multiple enrichment and observation until the observed biological abundance reaches a third preset threshold, at which time all the enriched bacterial liquid in the enrichment kettle is collected for further preservation or experiment;

[0109] This device can realize the automated pretreatment process of effectively separating biological cells from environmental impurities during microbial sampling under deep-sea heat preservation and pressure conditions, obtain high-purity biological enrichments, real-time detection of biochemical indicators during microbial enrichment, visual monitoring of biomass, and recycling of detected enrichments, effectively improving the sampling purity and enrichment efficiency of deep-sea environmental microorganisms.

[0110] Example 4

[0111] like Figure 4 As shown, this embodiment provides a deep-sea microorganism online purification sampling and visual automatic enrichment device, including: a sampling and purification unit 1, an enrichment unit 2, a visual monitoring unit 3, a temperature adjustment unit 4, a pressure adjustment unit 5 and a central control unit 6;

[0112] The sampling and purification unit 1 comprises: a sample mixing kettle 11, a sample settling kettle 12, a sample purification kettle 13, two sets of pressure-maintaining stirrers 14, a turbidity meter 15, a total organic carbon analyzer 16, a purification piston 17 and a filter membrane 18;

[0113] The sample mixing kettle 11 is connected to the pipeline of the container to be sampled, and a sampling ball valve 19 is provided on the pipeline for obtaining deep-sea microbial samples from the container to be sampled; the lower part of the sample mixing kettle 11 is connected to the upper pipeline of the sample settling kettle 12, and a sample transfer valve 110 is provided in the pipeline; the lower part of the sample settling kettle 12 is connected to the lower pipeline of the sample purification kettle 13, and a sample transfer valve 110 is also provided in the pipeline; the sample transfer valve 110 is used to realize the transfer during the sample purification process;

[0114] The bottom of the sample settling kettle 12 is a funnel-shaped structure to achieve the impurity sedimentation effect of the enriched sample; two sets of pressure-maintaining agitators 14 are respectively arranged at the top of the sample mixing kettle 11 and the sample purification kettle 13 to achieve sample mixing and mass transfer in the kettle; the turbidity meter 15 is arranged inside the sample settling kettle 12 to monitor the turbidity changes in the kettle; the total organic carbon analyzer 16 is connected to the sample purification kettle 13 to monitor the total organic carbon changes in the kettle; the purification piston 17 is arranged at the lower part of the sample purification kettle 13, and can be pushed upward to achieve pressurized concentrated samples; the filter membrane 18 is arranged on the upper surface of the purification piston 17 to filter out the culture medium solution and increase the microbial abundance per unit volume;

[0115] In addition, the upper part of the sample purification kettle 13 of this embodiment is provided with a protruding ring 111 with a smaller inner diameter, and the protruding ring 111 is integrated with the sample purification kettle 13 to prevent the purification piston 17 from increasing the pressure through its lower part and pushing and damaging the pressure-maintaining stirrer 14 and the interface of the total organic carbon analyzer 17 at the top;

[0116] The central control unit 6 is also connected to two groups of pressure-maintaining stirrers 14, a turbidity meter 15 and a total organic carbon analyzer 16 respectively;

[0117] The enrichment unit 2 includes: an enrichment kettle 21, a magnetic stirrer 22 and an environmental parameter sensing system 23; the magnetic stirrer 22 is arranged at the bottom of the enrichment kettle 21 and a magnet is placed inside the enrichment kettle to achieve the stirring of the enriched material in the kettle and enhance the mass transfer effect; the environmental parameter sensing system 23 is connected to the enrichment kettle 21 and the central control unit 6 respectively, and is used to detect the environmental parameters in the enrichment kettle 21 and transmit them to the central control unit 6, so as to regularly measure the biochemical process in the kettle and evaluate the enrichment effect;

[0118] like Figure 5 As shown, the visual monitoring unit 3 includes: a biological observation kettle 31, a biological abundance measurement system 32, two sets of pressure-resistant observation windows 33, a microscopic camera system 34, a measurement solenoid valve 35 and a recovery solenoid valve 36;

[0119] The biological observation kettle 31 is connected to the enrichment kettle 21 by pipeline; two sets of pressure-resistant observation windows 33 are respectively arranged on the top and bottom surfaces of the biological observation kettle 31, which are made of pressure-resistant and highly light-transmitting materials and are used to observe the morphology of microorganisms in the biological observation kettle 31; the biological abundance measurement system 32 is respectively connected to the biological observation kettle 31 and the central control unit 6, and is used to place the microorganisms to be observed; the microscopic camera system 34 is arranged at the bottom of the biological observation kettle 31 and is connected to the central control unit 6, and is used to observe the changes in the abundance of microorganisms in the biological observation kettle 31 from the pressure-resistant observation window 33 at the bottom; the measuring solenoid valve 35 and the recovery solenoid valve 36 are respectively connected to the biological abundance measurement system 32 and the enrichment kettle 21 through pipelines, and are respectively connected to the central control unit 6, and the measuring solenoid valve 35 and the recovery solenoid valve 36 are used to realize sample injection, dilution and recovery during observation;

[0120] The temperature regulating unit 4 and the pressure regulating unit 5 are respectively connected to the central control unit 6, the sample mixing kettle 11, the sample settling kettle 12, the sample purification kettle 13, the enrichment kettle 21 and the biological observation kettle 31, and the temperature regulating unit 4 and the pressure regulating unit 5 are respectively controlled by the central control unit 6 to adjust the temperature and pressure in each kettle;

[0121] In this embodiment, the enrichment unit 2 further includes: an enrichment ball valve 24, a plunger pump 25, a continuous injection pump 26 and an injection valve 27;

[0122] One end of the enrichment ball valve 24 and the plunger pump 25 are respectively connected to the enrichment kettle 21 through pipelines for collecting the enriched material after the cultivation is completed; the other end of the plunger pump 25 is respectively connected to the measurement solenoid valve 35 and the recovery solenoid valve 36 through pipelines;

[0123] The continuous injection pump 26 is connected to the injection valve 27 and the enrichment kettle 21 in sequence through a pipeline, and is used to inject microbial culture solution into the enrichment kettle 21;

[0124] The visual monitoring unit 3 further includes a dilution kettle 37, a dilution valve 38 and an illuminator 39;

[0125] One end of the plunger pump 25 is also connected to the dilution valve 38 and the dilution tank 37 in sequence through a pipeline; the dilution tank 37 is provided with a dilution culture solution for injecting into the observation pool 321 and diluting the enrichment during biological observation, so as to better observe and count biological cells;

[0126] The illuminator 39 is disposed on the top of the biological observation kettle 31 and is connected to the central control unit 6, and is used to provide illumination for the biological observation kettle 31 from the pressure-resistant observation window 33 on the top;

[0127] The environmental parameter sensing system 23 includes: a dissolved oxygen detector 231, a pH value detector 232 and a Raman measuring instrument 233, which are respectively used to detect the dissolved oxygen content, pH value and chemical substance content changes in the enrichment kettle 21 and transmit them to the central control unit 6;

[0128] like Figure 6 As shown, the biological abundance measurement system 32 includes: an observation pool 321, a cover glass flipper 322 and a cover glass 323; the observation pool 321 and the cover glass 323 are both made of highly light-transmitting materials and allow the enriched bacterial liquid sample to be retained between the two for microscopic observation when they are attached;

[0129] The two ends of the observation pool 321 are respectively connected to the measuring solenoid valve 35 and the recovery solenoid valve 36 through pipelines for injecting and recovering samples; the cover glass flipper 322 is connected to the central control unit 6 for driving the cover glass 323 to flip and attach to the observation pool 321;

[0130] The observation pool 321 is provided with a square groove, which is completely fitted with the preset protruding square on the cover glass 323; a bidirectional funnel-shaped passage is also provided in the square groove, and a grid for observing and counting cells is engraved in the passage; in this embodiment, the grid area actually accommodates a volume of about 10 μL of bacterial liquid, that is, the size is 1×1×0.01 cm 3 The grid contains 4 large squares around and 1 small square in the middle to count cells of different numbers. When the number of cells is small, the large squares are used for counting, and when the number of cells is large, the small squares are used for counting.

[0131] The microscopic camera system 34 includes: a lens switcher 341 and three microscope lenses 342 with different magnifications. In this embodiment, the magnifications of the microscope lenses include 50, 100 and 200 times, so as to realize multiple field of view switching during biomass observation;

[0132] The lens switcher 341 is connected to the central control unit 6 and is used to switch the microscope lens 342;

[0133] In addition, in this embodiment, the microscopic camera system 34 is also equipped with image analysis software connected to the central control unit 6, which can automatically identify and count cells. The counting rule is expressed by the following formula:

[0134] N C =N F ×D×F×10000

[0135] Where N C Indicates the number of cells per milliliter of sample; N Frepresents the average number of cells in the grid; D represents the dilution multiple of the cell sample; F represents the number of grids, including the number of two types of grids, 16 for large grids and 25 for small grids;

[0136] The temperature regulating unit 4 comprises: a temperature regulator 41 and five groups of temperature sensors 42 respectively connected to the central control unit 6; the five groups of temperature sensors 42 are respectively arranged inside the sample mixing kettle 11, the sample settling kettle 12, the sample purification kettle 13, the enrichment kettle 21 and the biological observation kettle 31;

[0137] The pressure regulating unit 5 comprises: a gas booster pump 51, four gas injection valves 52, a purification injection pump 53, a purification valve 54, six groups of pressure sensors 55, a PID control valve 56 and an exhaust valve 57; the gas booster pump 51, the purification injection pump 53 and all the pressure sensors 55 are respectively connected to the central control unit 6;

[0138] The gas booster pump 51 is connected to the sample mixing kettle 11, the sample settling kettle 12, the sample purification kettle 13 and the enrichment kettle 21 through four gas injection valves 52 and pipelines to provide gas pressure; the purification injection pump 53 is connected to the bottom of the sample purification kettle 13 through a purification valve 54 and a pipeline to provide liquid pressure in the purification process; a group of pressure sensors 55 are respectively provided at the top of the sample mixing kettle 11, the top of the sample settling kettle 12, and the top and bottom of the sample purification kettle 13. The pressure sensor 55 at the bottom of the sample purification kettle 13 is used to monitor the purification progress and stop in time; the enrichment kettle 21 and the biological observation kettle 31 are also provided with pressure sensors 55; the PID control valve 56 is provided on the enrichment kettle 21 to achieve continuous environment and pressure maintenance in the kettle; the exhaust valve 57 is provided on the biological observation kettle 31 to control the discharge of high-pressure gas;

[0139] like Figure 7 As shown, in this embodiment, the central control unit 6 includes a communication module, a control module and a computer host, wherein the communication module is used to connect various sensing devices to obtain changes in environmental indicators; the communication module is connected and communicated with the computer host through a circuit; the control module is connected to each operating device, and the control module is connected to the computer host through a circuit, and the computer host sends an electrical signal to control the pressure regulation, temperature regulation, filtration and purification, continuous conditions, mixing and mass transfer, abundance observation and other device functions of the control module; the computer host monitors and controls all electronic instruments in the device through the communication module and the control module.

[0140] In the specific implementation process, firstly, the sample mixing kettle 11, the sample settling kettle 12, the sample purification kettle 13, the enrichment kettle 21 and the biological observation kettle 31 are cleaned and sterilized, and the culture solution is injected, the kettle body is installed and the gas is pressurized respectively; and each unit in the device is installed;

[0141] Obtain a deep-sea microorganism sample from the sampling container and transfer it to the sample mixing kettle 11, start the pressure-maintaining agitator 14 in the sample mixing kettle 11, wait for a first preset time for the sample to be completely mixed, and obtain the mixed sample;

[0142] The mixed sample is transferred to the sample settling kettle 12, and the settling and separation of the sample impurities is monitored in real time by using the turbidity meter 15. After the turbidity is reduced to a first preset threshold value, the settled sample is obtained;

[0143] The settled sample is transferred to the sample purification kettle 13, the pressure-maintaining agitator 14 in the sample purification kettle 13 is turned on, the purification piston 17 is pushed for purification, and the sample purification status is monitored in real time using the total organic carbon analyzer 16. When the total organic matter content obtained by the total organic carbon analyzer 16 is greater than the second preset threshold value, or when the pressure difference collected by the top and bottom pressure sensors 55 in the sample purification kettle 13 is greater than the fourth preset threshold value, the purified sample is obtained to complete the purification sampling;

[0144] Then, the purified bacterial liquid sample is transferred to the enrichment kettle 21, the magnetic stirrer 22 is turned on, enrichment begins, and the environmental parameter sensor system 23 is used to monitor the environmental indicators in the enrichment kettle 21 in real time. When the monitored environmental indicators meet the preset conditions, the enriched bacterial liquid is obtained to complete the enrichment;

[0145] When the enrichment environment meets the standard, the cover glass flipper 322 is turned on, the cover glass 323 is turned over, the measurement solenoid valve 35 is opened, the plunger pump 25 is connected to the enrichment kettle 21 and an appropriate amount of enriched bacterial liquid is injected into the biological observation kettle 31;

[0146] Open the dilution valve 38, connect the plunger pump 25 to the dilution tank 37 and inject the diluted culture solution into the biological observation tank 31;

[0147] Control the cover glass flipper 322 to cover the cover glass 323, and close the measuring electromagnetic valve 35; the microscopic camera system 34 automatically identifies and calculates the number of cells per unit area in the grid in the acquired image;

[0148] After each observation, the recovery solenoid valve 36 is opened, the plunger pump 25 is connected to the biological observation kettle 31 and the enriched bacterial solution is injected into the enrichment kettle 21 at a certain flow rate, the observed bacterial solution is recovered to the enrichment kettle 21, and then the recovery solenoid valve 36 is closed, and the total abundance of organisms is calculated by the image recognition software of the microscopic camera system 34;

[0149] Repeat the above steps and observe several times until a certain biological abundance is reached, open the enrichment ball valve 24, and collect all the enriched materials in the enrichment kettle 21 for further preservation and experiments;

[0150] This device can realize the automated pretreatment process of effectively separating biological cells from environmental impurities during microbial sampling under deep-sea heat preservation and pressure conditions, obtain high-purity biological enrichments, real-time detection of biochemical indicators during microbial enrichment, visual monitoring of biomass, and recycling of detected enrichments, effectively improving the sampling purity and enrichment efficiency of deep-sea environmental microorganisms.

[0151] Example 5

[0152] like Figure 8 As shown, this embodiment provides a method for online purification sampling and visualization automatic enrichment of deep-sea microorganisms, based on a deep-sea microorganism online purification sampling and visualization automatic enrichment device described in Example 4, comprising the following steps:

[0153] S1: Clean and sterilize the sample mixing kettle, sample settling kettle, sample purification kettle, enrichment kettle and biological observation kettle, and respectively perform culture solution injection, kettle body installation and gas pressurization; install each unit in the deep-sea microorganism online purification sampling and visual automatic enrichment device;

[0154] S2: Obtain a deep-sea microorganism sample from the sampling container and transfer it to a sample mixing kettle, start a pressure-maintaining agitator in the sample mixing kettle, wait for a first preset time for the sample to be completely mixed, and obtain the mixed sample;

[0155] S3: transferring the mixed sample into a sample settling kettle, using a turbidity meter to monitor the settling and separation of sample impurities in real time, and obtaining the settled sample after the turbidity drops to a first preset threshold;

[0156] S4: transferring the settled sample into a sample purification kettle, turning on the pressure-maintaining agitator in the sample purification kettle, pushing the purification piston for purification, and using a total organic carbon analyzer to monitor the sample purification in real time. When the total organic matter content obtained by the total organic carbon analyzer is greater than a second preset threshold, or when the pressure difference collected by the top and bottom pressure sensors in the sample purification kettle is greater than a fourth preset threshold, obtaining the purified sample, and transferring the purified sample to an enrichment kettle;

[0157] S5: Turn on the magnetic stirrer to start enrichment, and use the environmental parameter sensing system to monitor the environmental indicators in the enrichment kettle in real time. When the monitored environmental indicators meet the preset conditions, obtain the enriched bacterial solution to complete the enrichment;

[0158] S6: Open the measuring solenoid valve to transfer the enriched bacterial solution to the biological abundance measurement system in the biological observation kettle, and use the microscopic camera system to observe the biological abundance. After the observation is completed, open the recovery solenoid valve to transfer the enriched bacterial solution after observation back to the enrichment kettle for recovery;

[0159] S7: repeating steps S5 to S6, performing multiple enrichment and observations, until the observed biological abundance reaches a third preset threshold, at which time all the enriched bacterial liquid in the enrichment kettle is collected for further preservation or experiment;

[0160] In the biological abundance measurement system of step S6, the height of the square groove of the observation pool is 0.1 cm, and the grid size is specifically 1 cm×1 cm;

[0161] After the microscopic camera system acquires the observed image, the biological abundance is calculated according to the following formula:

[0162] N C =N F ×D×F×10000

[0163] Among them, N C Indicates the number of cells per milliliter of sample; N F represents the average number of cells in a square; D represents the dilution factor of the cell sample; F represents the total number of squares of different sizes.

[0164] In the specific implementation process, Fig. 9 As shown, the control method comprises the following steps:

[0165] After all kettles are filled with culture medium solution and high-pressure gas, the sample mixing kettle 11 is connected to the front-end simulation device, sampling begins, the sampling ball valve 19 is opened, the pressure-maintaining agitator 14 of the sample mixing kettle 11 is turned on, and the sample and culture medium solution are completely mixed after waiting for 10 minutes;

[0166] When the sample is completely mixed, the sample transfer valve 110 behind the sample mixing kettle 11 is opened, and the sample is transferred to the sample settling kettle 12, waiting for the impurities and enriched products of the sample to be separated by sedimentation, and the separation status is identified by the turbidity meter 15. If the turbidity measured by the turbidity meter 15 is less than 1500NTU, the sample transfer valve 110 behind the sample settling kettle 12 is opened, and the pressure-maintaining agitator 14 and the PID control valve 56 of the sample purification kettle 13 are opened, otherwise, the sample continues to wait for sedimentation separation;

[0167] After the sample after sedimentation is transferred, the purification valve 54 is opened, the sample transfer valve 110 is closed, the purification injection pump 53 is opened, the purification piston 17 is pushed, and purification is started. The purification situation is identified by the total organic carbon analyzer 16. If the total organic matter content of the total organic carbon analyzer 16 is greater than 600 mM or the lower pressure displayed by the upper and lower pressure sensors 55 of the sample purification kettle 13 is greater than the upper pressure + 5 MPa, the sample transfer valve 110 after the sample purification kettle 13 is opened, and the magnetic stirrer 22 and the PID control valve 56 of the enrichment kettle 21 are opened, otherwise the above purification operation is continued;

[0168] When the sample purification is completed, open the injection valve 27, close the sample transfer valve 110 behind the sample purification kettle 13, turn on the continuous injection pump 26, start enrichment, and identify the environmental indicators in the enrichment kettle 21 through the dissolved oxygen detector 231, the pH value detector 232 and the Raman measuring instrument 233. If the dissolved oxygen content is <1μmol / kg, the sulfide content is >5mmol / L and the pH value is >8.5, it is determined that biological observation can be started, turn on the illuminator 39 of the biological observation kettle 31, turn on the biological abundance measurement system 32, turn on the microscopic camera system 34, otherwise continue to wait for the enrichment environment to meet the standards;

[0169] When the enrichment environment meets the standard, biological observation begins, the coverslip flipper 322 is turned on, the coverslip 323 is turned over, and the measurement electromagnetic valve 35 is opened; the plunger pump 25 is turned on, connected to the enrichment kettle 21, and 0.1 mL of enriched bacterial solution is injected into the biological observation kettle 31; the dilution valve 38 is opened, the plunger pump 25 is connected to the dilution kettle 37, and 0.9 mL of diluted culture solution is injected into the biological observation kettle 31; the coverslip flipper 322 is controlled to cover the coverslip 323, and the measurement electromagnetic valve 35 is closed; the microscopic camera system 34 automatically identifies objects <0.22 μm in the acquired image and records the number of cells per unit area in the grid;

[0170] After each observation, the recovery solenoid valve 36 is opened, the plunger pump 25 is connected to the biological observation kettle 31, and the enriched bacterial solution after the observation is injected into the enrichment kettle 21 at a certain flow rate to realize the recovery of the bacterial solution; then the recovery solenoid valve 36 is closed; the total biological abundance is calculated by the image recognition software of the microscopic camera system 34. If the biological abundance is greater than 107 cell / mL, the enrichment ball valve 24 is opened, the enriched material is collected, and the enrichment is terminated. Otherwise, the above-mentioned enrichment process is continued until the enriched material with high biological abundance can be obtained.

[0171] The same or similar reference numerals correspond to the same or similar components;

[0172] 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;

[0173] 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 deep-sea microorganism online purification sampling device, characterized in that: include: A sampling and purification unit, a first temperature regulating unit, a first pressure regulating unit and a first central control unit; The sampling and purification unit comprises: a sample mixing kettle, a sample settling kettle, a sample purification kettle, two sets of pressure-maintaining stirrers, a turbidity meter, a total organic carbon analyzer, a purification piston and a filter membrane; The sample mixing kettle is connected to the pipeline of the container to be sampled, and is used to obtain deep-sea microbial samples from the container to be sampled; the lower part of the sample mixing kettle is connected to the upper pipeline of the sample settling kettle; the lower part of the sample settling kettle is connected to the lower pipeline of the sample purification kettle; The bottom of the sample settling kettle is a funnel-shaped structure to achieve the impurity sedimentation effect of the enriched sample; two sets of pressure-maintaining agitators are respectively arranged at the top of the sample mixing kettle and the sample purification kettle; the turbidity meter is arranged inside the sample settling kettle; the total organic carbon analyzer is connected to the sample purification kettle; the purification piston is arranged at the lower part of the sample purification kettle and can be pushed upward to achieve pressurized concentrated samples; the filter membrane is arranged on the upper surface of the purification piston; The first temperature regulating unit and the first pressure regulating unit are respectively connected to the first central control unit, the sample mixing kettle, the sample settling kettle and the sample purification kettle, and the first temperature regulating unit and the first pressure regulating unit are respectively controlled by the first central control unit to adjust the temperature and pressure in each kettle; The first central control unit is also connected to two groups of pressure-maintaining stirrers, a turbidity meter and a total organic carbon analyzer respectively.

2. The deep-sea microorganism online purification sampling device according to claim 1, characterized in that: The first temperature regulating unit comprises: a temperature regulator and three groups of temperature sensors respectively connected to the first central control unit; the three groups of temperature sensors are respectively arranged inside the sample mixing kettle, the sample settling kettle and the sample purification kettle; The first pressure regulating unit comprises: a gas booster pump, three gas injection valves, a purification liquid injection pump, a purification valve, and four groups of pressure sensors; the gas booster pump, the purification liquid injection pump and the four groups of pressure sensors are respectively connected to the first central control unit; The gas booster pump is connected to the sample mixing kettle, sample sedimentation kettle and sample purification kettle through three gas injection valves and pipelines respectively; the purification injection pump is connected to the bottom of the sample purification kettle through purification valves and pipelines; a group of pressure sensors are respectively provided at the top of the sample mixing kettle, the top of the sample sedimentation kettle, and the top and bottom of the sample purification kettle. The pressure sensor at the bottom of the sample purification kettle is used to monitor the purification progress and stop it in time.

3. A deep-sea microorganism online visualization automatic enrichment device, characterized in that: include: An enrichment unit, a visual monitoring unit, a second temperature regulating unit, a second pressure regulating unit, and a second central control unit; The enrichment unit comprises: an enrichment kettle, a magnetic stirrer and an environmental parameter sensing system; the magnetic stirrer is arranged at the bottom of the enrichment kettle and a magnet is placed inside the enrichment kettle; the environmental parameter sensing system is respectively connected to the enrichment kettle and the second central control unit, and is used to detect the environmental parameters in the enrichment kettle and transmit them to the second central control unit; The visual monitoring unit includes: a biological observation kettle, a biological abundance measurement system, two sets of pressure-resistant observation windows, a microscopic camera system, a measurement solenoid valve and a recovery solenoid valve; The biological observation kettle is connected to the enrichment kettle pipeline; two sets of pressure-resistant observation windows are respectively arranged at the top and bottom of the biological observation kettle, and are used to observe the morphology of microorganisms in the biological observation kettle; the biological abundance measurement system is respectively connected to the biological observation kettle and the second central control unit, and is used to place the microorganisms to be observed; the microscopic camera system is arranged at the bottom of the biological observation kettle and is connected to the second central control unit, and is used to observe the changes in the abundance of microorganisms in the biological observation kettle from the pressure-resistant observation window at the bottom; the measuring solenoid valve and the recovery solenoid valve are respectively connected to the biological abundance measurement system and the enrichment kettle through pipelines, and are respectively connected to the second central control unit; The second temperature regulating unit and the second pressure regulating unit are respectively connected to the second central control unit, the enrichment kettle and the biological observation kettle, and the second temperature regulating unit and the second pressure regulating unit are respectively controlled by the second central control unit to adjust the temperature and pressure in each kettle.

4. The deep-sea microorganism online visualization automatic enrichment device according to claim 3, characterized in that: The enrichment unit also includes: an enrichment ball valve, a plunger pump, a continuous injection pump and an injection valve; One end of the enrichment ball valve and the plunger pump are connected to the enrichment kettle through pipelines respectively; the other end of the plunger pump is connected to the measurement solenoid valve and the recovery solenoid valve through pipelines respectively; The continuous injection pump is connected to the injection valve and the enrichment kettle in sequence through a pipeline, and is used to inject microbial culture solution into the enrichment kettle; The visual monitoring unit also includes a dilution kettle, a dilution valve and an illuminator; One end of the plunger pump is also connected to the dilution valve and the dilution kettle in sequence through a pipeline; the dilution kettle is provided with a dilution culture solution; The illuminator is arranged on the top of the biological observation kettle and is connected to the second central control unit, and is used to provide illumination for the biological observation kettle from the pressure-resistant observation window on the top.

5. The deep-sea microorganism online visualization automatic enrichment device according to claim 3, characterized in that: The environmental parameter sensing system includes: a dissolved oxygen detector, a pH value detector and a Raman measuring instrument, which are respectively used to detect the dissolved oxygen content, pH value and chemical substance content changes in the enrichment kettle and transmit them to the second central control unit.

6. The deep-sea microorganism online visualization automatic enrichment device according to claim 3, characterized in that: The biological abundance measurement system comprises: an observation pool, a cover glass flipper and a cover glass; The two ends of the observation pool are respectively connected to a measuring solenoid valve and a recovery solenoid valve through pipelines; the cover glass flipper is connected to the second central control unit, and is used to drive the cover glass to flip and attach it to the observation pool; The observation pool is provided with a square groove, which is completely fitted with a preset protruding square on the cover glass; a bidirectional funnel-shaped passage is also provided in the square groove, and a grid for observing and counting cells is engraved in the passage; a plurality of squares of different sizes are provided in the grid for counting cells of different quantities; The microscopic camera system comprises: a lens switcher and a plurality of microscope lenses with different magnifications; The lens switcher is connected to the second central control unit and is used for switching the microscope lens.

7. The deep-sea microorganism online visualization automatic enrichment device according to claim 3, characterized in that: The second temperature regulating unit comprises: a temperature regulator and two groups of temperature sensors respectively connected to the second central control unit; the two groups of temperature sensors are respectively arranged inside the enrichment kettle and the biological observation kettle; The second pressure regulating unit comprises: a gas booster pump, a gas injection valve, a PID control valve, an exhaust valve and two groups of pressure sensors respectively connected to the second central control unit; The gas booster pump is connected to the gas injection valve and the enrichment kettle in sequence through pipelines; the PID control valve is arranged on the enrichment kettle; the exhaust valve is arranged on the biological observation kettle; and two groups of pressure sensors are respectively arranged inside the enrichment kettle and the biological observation kettle.

8. A deep-sea microorganism online purification sampling and visual automatic enrichment device, comprising a purification sampling device and an enrichment device connected in sequence through a pipeline, characterized in that: The purification sampling device is specifically a deep-sea microorganism online purification sampling device as described in any one of claims 1 to 2, and the enrichment device is specifically a deep-sea microorganism online visualization automatic enrichment device as described in any one of claims 3 to 7.

9. A method for online purification sampling and visualization automatic enrichment of deep-sea microorganisms, based on the device for online purification sampling and visualization automatic enrichment of deep-sea microorganisms as claimed in claim 8, characterized in that: The following steps are involved: S1: Clean and sterilize the sample mixing kettle, sample settling kettle, sample purification kettle, enrichment kettle and biological observation kettle, and respectively perform culture solution injection, kettle body installation and gas pressurization; install each unit in the deep-sea microorganism online purification sampling and visual automatic enrichment device; S2: Obtain a deep-sea microorganism sample from the sampling container and transfer it to a sample mixing kettle, start a pressure-maintaining agitator in the sample mixing kettle, wait for a first preset time for the sample to be completely mixed, and obtain the mixed sample; S3: transferring the mixed sample into a sample settling kettle, using a turbidity meter to monitor the settling and separation of sample impurities in real time, and obtaining the settled sample after the turbidity drops to a first preset threshold; S4: transferring the settled sample into a sample purification kettle, turning on the pressure-maintaining agitator in the sample purification kettle, pushing the purification piston for purification, and using a total organic carbon analyzer to monitor the sample purification in real time. After the total organic matter content obtained by the total organic carbon analyzer is greater than a second preset threshold, obtaining the purified sample, and transferring the purified sample to an enrichment kettle; S5: Turn on the magnetic stirrer to start enrichment, and use the environmental parameter sensing system to monitor the environmental indicators in the enrichment kettle in real time. When the monitored environmental indicators meet the preset conditions, obtain the enriched bacterial solution to complete the enrichment; S6: Open the measuring solenoid valve to transfer the enriched bacterial solution to the biological abundance measurement system in the biological observation kettle, and use the microscopic camera system to observe the biological abundance. After the observation is completed, open the recovery solenoid valve to transfer the enriched bacterial solution after observation back to the enrichment kettle for recovery; S7: Repeat steps S5 to S6 to perform multiple enrichment and observation until the observed biological abundance reaches a third preset threshold, at which time all the enriched bacterial liquid in the enrichment kettle is collected for further preservation or experiment.

10. A method for online purification, sampling and visual automatic enrichment of deep-sea microorganisms according to claim 9, characterized in that: In step S4, when the total organic matter content obtained by the total organic carbon analyzer is greater than the second preset threshold, or when the pressure difference collected by the top and bottom pressure sensors in the sample purification kettle is greater than the fourth preset threshold, the purified sample is obtained; In the biological abundance measurement system of step S6, the height of the square groove of the observation pool is 0.1 cm, and the grid size is specifically 1 cm×1 cm; After the microscopic camera system acquires the observed image, the biological abundance is calculated according to the following formula: N C =N F ×D×F×10000 Among them, N C Indicates the number of cells per milliliter of sample; N F represents the average number of cells in a square; D represents the dilution factor of the cell sample; F represents the total number of squares of different sizes.

Images