Deep-sea microbial online purification sampling and visualized automatic enrichment device and method

Abstract

The application provides an online purification sampling and visual automatic enrichment device and method for deep-sea microorganisms, which realizes online sample pretreatment, automatic biological concentration and purification, online monitoring of environmental indexes in the biological enrichment process, online observation of biological mass, automatic enrichment and transfer of high-purity enrichment matters during the sampling process of microorganisms, solves the defects of the prior art, such as the difficulty in purifying deep-sea microorganism samples in a high-pressure environment, the complexity of manual operation for enrichment and observation, the low timeliness of biological mass observation, and the difficulty in real-time cell counting of enrichment matters, and effectively improves the enrichment rate and acquisition efficiency of deep-sea uncultured microorganisms, thereby providing a basic means for improving the level of deep-sea biology research and the efficiency of biological resource development.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of deep-sea microbial sample purification, enrichment and observation, and more particularly to an online deep-sea microbial purification sampling and visualized automatic enrichment device and method. BACKGROUND

[0002] Deep sea is considered as the last frontier of the earth that has not been recognized. The extreme conditions such as high pressure, low / high temperature and oligotrophic in deep sea environment have resulted in special deep-sea microorganisms. The evolutionary branches, biochemical reactions and metabolic functions of the deep-sea microorganisms are still a mystery, which has a very high research and application value. In order to explore the role of deep-sea microorganisms in the process of marine material cycle and the earth's ecological system, it is necessary to pretreat the deep-sea environmental samples, enrich and observe the microorganisms, so as to identify the biological community characteristics and physiological metabolic characteristics. However, the biomass of deep-sea environment is low, and the current technology for high-pressure environmental sample processing and biological enrichment is still not mature. It is very difficult to carry out rapid enrichment of difficult-to-culture organisms. After the deep-sea microorganisms are collected, it is difficult to purify the biological samples in the high-pressure environment, it is difficult to monitor the biomass change in the process of microbial enrichment, and the efficient visualized enrichment of microorganisms will be very difficult. Therefore, using the heat and pressure preservation device simulating deep-sea environment to purify and visually enrich microorganisms is an important means to improve the research level and development efficiency of deep-sea microorganisms.

[0003] For the purification sampling of microorganisms under deep-sea simulation conditions, the existing patents such as "a method for extracting and semi-quantitatively analyzing biological membrane on sediment surface", "a method for simultaneously purifying nucleic acid and protein from environmental samples" and the like mainly purify and sample the sediment samples by water washing, oscillation and centrifugation and the like purification process. However, these purification processes are difficult to realize in high-pressure environment, so that the deep-sea environmental samples are difficult to separate environmental impurities such as gravel and organic matter from microbial cells under the condition of maintaining high pressure. There are also limitations such as low activity of the obtained enrichment, low efficiency of subsequent biological enrichment, low purity of the obtained biological samples, and the like.

[0004] For 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 the visual window and the light-transmitting culture cavity, but it is difficult to observe the biological abundance of the enrichment in a high-pressure environment in real time, and the relative microbial abundance still needs to be obtained through periodic multiple sampling and gene sequencing analysis, which has the problems of complicated 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 table" which is connected with the observation window arranged on the outlet pipeline of the culture kettle and is externally connected with a microscope for real-time observation of the morphology of microorganisms, but the sample observed in the pipeline is not diluted, separated and uniformly pressed, and it is difficult to obtain an image of the cell distribution with interval separation and flatness in the microscope for cell counting, and the technology has the problems of excessive accumulation of cell amount in the observed sample, a large amount of cell overlap in the observed field of view, and no unit grid division in the observed area, which makes it difficult to obtain the actual enriched biomass.

[0005] In general, the existing technology has the problems of difficulty in purifying deep-sea microbial samples in a high-pressure environment, complicated manual operation for enrichment and observation, low timeliness of biomass observation, and difficulty in real-time cell counting of enrichment, which brings difficulties for quickly obtaining high-purity enrichment of deep-sea microorganisms. SUMMARY

[0006] The present application is to overcome the defects of the prior art, such as the difficulty in purifying deep-sea microbial samples in a high-pressure environment, the complicated manual operation for enrichment and observation, the low timeliness of biomass observation, and the difficulty in real-time cell counting of enrichment, and provides an online purification sampling and visual automatic enrichment device and method for deep-sea microorganisms, which improves the enrichment rate and acquisition efficiency of deep-sea uncultured microorganisms by online sample pretreatment, automatic biological concentration and purification, online monitoring of environmental indicators during the biological enrichment process, online observation of biomass, and automatic enrichment and transfer of high-purity enrichment, and provides a basic means for improving the level of deep-sea biology research and the efficiency of biological resource development.

[0007] To solve the above technical problems, the technical scheme of the present application is as follows:

[0008] An online purification sampling device for deep-sea microorganisms, comprising: a sampling and purification unit, a first temperature adjusting unit, a first pressure adjusting 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 groups of pressure maintaining stirrers, a turbidimeter, a total organic carbon analyzer, a purification piston and a filter membrane;

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

[0011] The bottom part of the sample settling kettle is in a funnel shape to realize the function of precipitating impurities in the enriched sample; two groups of pressure maintaining stirrers are respectively arranged at the top of the sample mixing kettle and the sample purifying kettle; the turbidimeter is arranged inside the sample settling kettle; the total organic carbon analyzer is connected with the sample purifying kettle; the purifying piston is arranged below the inside of the sample purifying kettle, and can be pushed from bottom to top to realize the function of pressurized concentration of the sample; the filter membrane is arranged on the upper surface of the purifying piston;

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

[0013] The first central control unit is further connected with the two groups of pressure maintaining stirrers, the turbidimeter and the total organic carbon analyzer.

[0014] Preferably, the first temperature adjusting unit comprises a temperature regulator and three groups of temperature sensors which are respectively connected with 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 purifying kettle.

[0015] The first pressure adjusting unit comprises a gas booster pump, three gas injection valves, a purifying liquid injection pump, a purifying valve and four groups of pressure sensors; the gas booster pump, the purifying liquid injection pump and the four groups of pressure sensors are respectively connected with the first central control unit.

[0016] The gas booster pump is connected with the sample mixing kettle, the sample settling kettle and the sample purifying kettle through the three gas injection valves and the pipelines; the purifying liquid injection pump is connected with the bottom part of the sample purifying kettle through the purifying valve and the pipeline; the top part of the sample mixing kettle, the top part of the sample settling kettle, the top part and the bottom part of the sample purifying kettle are respectively provided with a group of pressure sensors, and the pressure sensor of the bottom part of the sample purifying kettle is used to monitor the purifying progress and stop in time.

[0017] The application further provides a deep-sea microbial online visualized automatic enrichment device, which comprises an enrichment unit, a visualized monitoring unit, a second temperature adjusting unit, a second pressure adjusting 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 magnetic sub is arranged inside the enrichment kettle; the environmental parameter sensing system is connected with the enrichment kettle and the second central control unit respectively, and is used for detecting the environmental parameters in the enrichment kettle and transmitting the environmental parameters to the second central control unit;

[0019] The visual monitoring unit comprises a biological observation kettle, a biological abundance measuring system, two groups of pressure-resistant observation windows, a microscopic camera system, a measuring electromagnetic valve and a recovery electromagnetic valve.

[0020] The biological observation kettle is connected with the enrichment kettle through a pipeline; the two groups of pressure-resistant observation windows are arranged at the top and the bottom of the biological observation kettle respectively, and are used for observing the microbial morphology in the biological observation kettle; the biological abundance measuring system is connected with the biological observation kettle and the second central control unit respectively, and is used for placing the microorganisms to be observed; the microscopic camera system is arranged at the bottom of the biological observation kettle and is connected with the second central control unit, and is used for observing the change of the microbial abundance in the biological observation kettle from the pressure-resistant observation window at the bottom; the measuring electromagnetic valve and the recovery electromagnetic valve are connected with the biological abundance measuring system and the enrichment kettle through pipelines respectively, and are connected with the second central control unit respectively.

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

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

[0023] One end of the enrichment ball valve and the plunger pump is connected with the enrichment kettle through a pipeline respectively; the other end of the plunger pump is connected with the measuring electromagnetic valve and the recovery electromagnetic valve through a pipeline respectively.

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

[0025] The visual monitoring unit further comprises a dilution kettle, a dilution valve and an illuminator.

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

[0027] The illuminator is arranged at the top of the biological observation kettle and is connected with the second central control unit, and is used for providing light for the biological observation kettle from the pressure-resistant observation window at the top.

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

[0029] Preferably, the biological abundance measuring system comprises an observation tank, a cover glass flipper and a cover glass.

[0030] The two ends of the observation tank are connected with a measuring electromagnetic valve and a recovery electromagnetic valve through pipelines respectively; the cover glass flipper is connected with the second central control unit and used to drive the cover glass to flip and adhere to the observation tank.

[0031] A square groove is arranged on the observation tank, which completely adheres to a protruding square on the cover glass; a two-way funnel-shaped passage is further arranged in the square groove, and a grid for observing and counting cells is engraved in the passage; a plurality of different size squares are arranged in the grid and used to count cells with 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 with the second central control unit and used to switch the microscope lenses.

[0034] Preferably, the second temperature adjusting unit comprises a temperature regulator and two groups of temperature sensors which are connected with the second central control unit respectively; the two groups of temperature sensors are arranged inside the enrichment tank and the biological observation tank respectively.

[0035] The second pressure adjusting unit comprises a gas booster pump, an air injection valve, a PID control valve, an exhaust valve and two groups of pressure sensors which are connected with the second central control unit respectively.

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

[0037] The application further provides a deep-sea microorganism online purification sampling and visual automatic enrichment device, which comprises a purification sampling device and an enrichment device connected through pipelines in sequence, wherein the purification sampling device is the above-mentioned deep-sea microorganism online purification sampling device, and the enrichment device is the above-mentioned deep-sea microorganism online visual automatic enrichment device.

[0038] The application further provides a deep-sea microorganism online purification sampling and visual automatic enrichment method based on the above-mentioned deep-sea microorganism online purification sampling and visual automatic enrichment device, which comprises the following steps:

[0039] S1: washing and sterilizing the sample mixing kettle, sample settling kettle, sample purification kettle, enrichment kettle and biological observation kettle, and respectively performing culture solution injection, kettle installation and gas pressurization; installing each unit in the deep-sea microorganism online purification sampling and visualization automatic enrichment device;

[0040] S2: obtaining a deep-sea microorganism sample from a to-be-sampled container and transferring the sample to the sample mixing kettle, starting the pressure-maintaining stirrer in the sample mixing kettle, waiting for the sample to be completely mixed after a first preset time, and obtaining the mixed sample;

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

[0042] S4: transferring the settled sample into the sample purification kettle, starting the pressure-maintaining stirrer in the sample purification kettle, pushing the purification piston to perform purification, and using a total organic carbon analyzer to monitor the purification of the sample in real time, obtaining the purified sample after the total organic carbon analyzer obtains a total organic matter content greater than a second preset threshold, and transferring the purified sample to the enrichment kettle;

[0043] S5: starting the magnetic stirrer to begin enrichment, and using an environmental parameter sensing system to monitor environmental indicators in the enrichment kettle in real time, obtaining the enriched bacterial solution when the monitored environmental indicators meet preset conditions, and completing enrichment;

[0044] S6: starting the measurement electromagnetic valve, transferring the enriched bacterial solution to the biological abundance measurement system in the biological observation kettle, using a microscopic camera system to observe biological abundance, and starting the recovery electromagnetic valve to transfer the observed enriched bacterial solution back to the enrichment kettle for recovery after the observation is completed;

[0045] S7: repeating steps S5-S6 to perform multiple enrichments and observations until the observed biological abundance reaches a third preset threshold, at which time all enriched bacterial solutions in the enrichment kettle are collected for further preservation or experiments.

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

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

[0048] After the microscopic camera system obtains the observation 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 beneficial effects of the technical solution of the present invention are:

[0052] The present invention provides a device for online purification and sampling of deep-sea microorganisms under heat and pressure. Compared with existing high-pressure sampling technologies, this device can achieve an automated pretreatment process for effectively separating biological cells from environmental impurities during microbial sampling in a simulated deep-sea high-pressure environment under heat and pressure conditions, and obtain high-purity biological samples. This solves the problems of difficulty in purifying microbial samples in high-pressure environments during deep-sea microbial sampling, as well as low activity and purity of the obtained biological samples, thereby improving the biological purity and processing efficiency of difficult-to-cultivate microorganisms in deep-sea environments.

[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 the enrichment process, low observation timeliness, and difficulty in real-time cell counting of enriched products. This device can visually monitor changes in biomass abundance after automatic enrichment and purification of microorganisms under deep-sea heat and pressure conditions, thereby improving the efficiency of obtaining high-purity enrichments of difficult-to-cultivate microorganisms in deep-sea environments.

[0054] In addition, the present invention also combines the two to provide an online purification sampling and visual automatic enrichment device and method for deep-sea microorganisms. 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 enrichments, 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 the deep-sea microorganism online purification sampling and visual automatic enrichment device provided in Example 3.

[0058] Figure 4 A mechanical structural diagram of an online purification sampling and visual automatic enrichment device for deep-sea microorganisms provided in Embodiment 4.

[0059] Figure 5 A specific structural diagram of a biological observation kettle provided in Embodiment 4.

[0060] Figure 6 A top view of an internal structure of a biological observation kettle provided in Embodiment 4.

[0061] Figure 7 A connection diagram of a central control unit provided in Embodiment 4.

[0062] Figure 8 A flowchart of an online purification sampling and visual automatic enrichment method for deep-sea microorganisms provided in Embodiment 5.

[0063] Figure 9 A specific implementation flowchart of an online purification sampling and visual automatic enrichment method for deep-sea microorganisms provided in Embodiment 5. DETAILED DESCRIPTION

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

[0065] In order to better illustrate the embodiments, some components in the drawings may be omitted, enlarged or reduced, and do not represent the actual size of the product;

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

[0067] The technical solutions of the present application will be further described below in combination with the drawings and embodiments.

[0068] Embodiment 1

[0069] As shown in the drawings, Figure 1 the present embodiment provides an online purification sampling device for deep-sea microorganisms, comprising: a sampling and purification unit, a first temperature adjusting unit, a first pressure adjusting 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 groups of pressure maintaining stirrers, a turbidimeter, a total organic carbon analyzer, a purification piston and a filter membrane;

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

[0072] The bottom of the sample settling kettle is funnel-shaped to realize the settling of impurities in the enriched sample; two sets of pressure maintaining stirrers are respectively arranged at the top of the sample mixing kettle and the sample purification kettle; the turbidimeter is arranged inside the sample settling kettle; the total organic carbon analyzer is connected with the sample purification kettle; the purification piston is arranged below the inside of the sample purification kettle and can be pushed upwards to realize pressure concentration of the sample; and the filter membrane is arranged on the upper surface of the purification piston;

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

[0074] The first central control unit is also connected with the two sets of pressure maintaining stirrers, the turbidimeter and the total organic carbon analyzer.

[0075] In the specific implementation process, first, the sample mixing kettle, the sample settling kettle and the sample purification kettle are cleaned and sterilized, and the culture solution injection, the kettle body installation and the gas pressurization are respectively performed; and each unit in the deep-sea microbial online purification sampling device is installed.

[0076] The deep-sea microbial sample is obtained from the sampling container and transferred to the sample mixing kettle, the pressure maintaining stirrer in the sample mixing kettle is started, the sample is completely mixed after waiting for a first preset time, and the mixed sample is obtained.

[0077] The mixed sample is transferred to the sample settling kettle, the turbidimeter is used to monitor the settling and separation of the sample impurities in real time, the settled sample is obtained after the turbidity is reduced to a first preset threshold value.

[0078] The settled sample is transferred to the sample purification kettle, the pressure maintaining stirrer in the sample purification kettle is started, the purification piston is pushed to perform purification, and the total organic carbon analyzer is used to monitor the sample purification in real time, the purified sample is obtained after the total organic carbon content obtained by the total organic carbon analyzer is greater than a second preset threshold value, and the purification sampling is completed.

[0079] The device can realize the automatic pretreatment process of effectively separating biological cells and environmental impurities in the microbial sampling process under the simulated deep-sea high-pressure environment under the conditions of heat preservation and pressure maintenance, and obtain high-purity biological samples, solve the problems of difficult purification of microbial samples in the high-pressure environment in the deep-sea microbial sampling process and low activity and purity of obtained biological samples, and improve the biological purity and processing efficiency of the deep-sea environmental difficult-to-culture microorganism.

[0080] Example 2

[0081] As Figure 2The embodiment shown provides a deep-sea microorganism online visual automatic enrichment device, which comprises an enrichment unit, a visual monitoring unit, a second temperature adjusting unit, a second pressure adjusting 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 magnetic sub is arranged inside the enrichment kettle; the environmental parameter sensing system is connected with the enrichment kettle and the second central control unit respectively, and is used for detecting the environmental parameters in the enrichment kettle and transmitting the environmental parameters to the second central control unit.

[0083] The visual monitoring unit comprises a biological observation kettle, a biological abundance measuring system, two groups of pressure-resistant observation windows, a microscopic camera system, a measuring electromagnetic valve and a recovery electromagnetic valve.

[0084] The biological observation kettle is connected with the enrichment kettle through a pipeline; the two groups of pressure-resistant observation windows are arranged at the top and the bottom of the biological observation kettle respectively, and are used for observing the morphology of microorganisms in the biological observation kettle; the biological abundance measuring system is connected with the biological observation kettle and the second central control unit respectively, and is used for placing microorganisms to be observed; the microscopic camera system is arranged at the bottom of the biological observation kettle and is connected with the second central control unit, and is used for observing the change of the abundance of microorganisms in the biological observation kettle from the pressure-resistant observation window at the bottom; the measuring electromagnetic valve and the recovery electromagnetic valve are connected with the biological abundance measuring system and the enrichment kettle through pipelines respectively, and are connected with the second central control unit respectively.

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

[0086] In the specific implementation process, first, the enrichment kettle and the biological observation kettle are cleaned and sterilized, and liquid culture injection, kettle body installation and gas pressurization are performed respectively; each unit in the deep-sea microorganism visual automatic enrichment device is installed;

[0087] The bacteria liquid to be enriched is transferred to the enrichment kettle; the magnetic stirrer is started, the enrichment is started, 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 bacteria liquid is obtained, and the enrichment is completed;

[0088] The measuring electromagnetic valve is started, the enriched bacteria liquid is transferred to the biological abundance measuring system in the biological observation kettle, the biological abundance observation is performed by using the microscopic camera system, and after the observation is completed, the recovery electromagnetic valve is started to transfer the observed enriched bacteria liquid back to the enrichment kettle for recovery;

[0089] Finally, repeat the above, enrichment and observation, until the observed biological abundance reaches the third preset threshold, at this time, collect all the enrichment bacteria liquid in the enrichment tank for further preservation or experiment;

[0090] The device solves the problems of complex manual operation, low timeliness of observation, and difficulty in real-time cell counting of enriched substances during the enrichment process. The device can visually monitor the change in biomass abundance after automatic enrichment and purification of microorganisms under deep-sea temperature and pressure preservation conditions, thereby improving the efficiency of obtaining high-purity enriched substances of deep-sea difficult-to-culture microorganisms.

[0091] Embodiment 3

[0092] As shown in Figure 3 The embodiment provides a deep-sea microbial online purification sampling and visual automatic enrichment device, which comprises a sampling and purification unit, an enrichment unit, a visual monitoring unit, a temperature adjusting unit, a pressure adjusting unit, and a central control unit.

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

[0094] The sampling and purification unit comprises a sample mixing tank, a sample settling tank, a sample purification tank, two groups of pressure maintaining stirrers, a turbidimeter, a total organic carbon analyzer, a purification piston, and a filter membrane.

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

[0096] The bottom of the sample settling tank is in a funnel shape to realize the impurity settling of the enriched sample; the two groups of pressure maintaining stirrers are respectively arranged at the top of the sample mixing tank and the sample purification tank; the turbidimeter is arranged inside the sample settling tank; the total organic carbon analyzer is connected with the sample purification tank; the purification piston is arranged below the inside of the sample purification tank and can be pushed upwards to realize pressure concentration of the sample; and the filter membrane is arranged on the upper surface of the purification piston.

[0097] The central control unit is also connected with the two groups of pressure maintaining stirrers, the turbidimeter, and the total organic carbon analyzer.

[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 magnetic sub is arranged inside the enrichment kettle; the environmental parameter sensing system is connected with the enrichment kettle and the central control unit respectively, and is used for detecting the environmental parameters in the enrichment kettle and transmitting the environmental parameters to the central control unit;

[0099] The visual monitoring unit comprises a biological observation kettle, a biological abundance measuring system, two groups of pressure-resistant observation windows, a microscopic camera system, a measuring electromagnetic valve and a recovery electromagnetic valve.

[0100] The biological observation kettle is connected with the enrichment kettle through pipelines; the two groups of pressure-resistant observation windows are arranged at the top and the bottom of the biological observation kettle respectively, and are used for observing the microbial morphology in the biological observation kettle; the biological abundance measuring system is connected with the biological observation kettle and the central control unit respectively, and is used for placing the microorganisms to be observed; the microscopic camera system is arranged at the bottom of the biological observation kettle and is connected with the central control unit, and is used for observing the change of the microbial abundance in the biological observation kettle from the pressure-resistant observation window at the bottom; the measuring electromagnetic valve and the recovery electromagnetic valve are connected with the biological abundance measuring system and the enrichment kettle through pipelines respectively, and are connected with the central control unit respectively.

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

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

[0103] The deep-sea microbial sample is obtained from the sampling container and is transferred to the sample mixing kettle, the pressure-stabilizing stirrer in the sample mixing kettle is started, the sample is completely mixed after waiting for a first preset time, and the mixed sample is obtained;

[0104] The mixed sample is transferred to the sample settling kettle, the turbidimeter is used to monitor the settling and separation of the impurities in the sample in real time, the settled sample is obtained after the turbidity is reduced to a first preset threshold;

[0105] The settled sample is transferred to the sample purification kettle, the pressure-stabilizing stirrer in the sample purification kettle is started, the purification piston is pushed to perform purification, and the total organic carbon analyzer is used to monitor the purification of the sample in real time, the purified sample is obtained after the total organic carbon analyzer obtains a total organic matter content greater than a second preset threshold, and the purification sampling is completed;

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

[0107] The measurement electromagnetic valve is opened, the enriched bacterial liquid is transferred to the biological abundance measurement system in the biological observation tank, the biological abundance is observed using the microscopic camera system, and after the observation is completed, the recovery electromagnetic valve is opened to retransfer the observed enriched bacterial liquid to the enrichment tank for recovery.

[0108] Finally, the above is repeated for multiple enrichments and observations until the observed biological abundance reaches a third preset threshold. At this time, all enriched bacterial liquids in the enrichment tank are collected for further preservation or experiments.

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

[0110] Example 4

[0111] As shown in Figure 4 , the present embodiment provides a deep-sea microbial online purification sampling and visual automatic enrichment device, which comprises 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 tank 11, a sample settling tank 12, a sample purification tank 13, two sets of pressure maintaining stirrers 14, a turbidimeter 15, a total organic carbon analyzer 16, a purification piston 17, and a filter membrane 18.

[0113] The sample mixing tank 11 is connected with a pipeline of a sample container, and a sampling ball valve 19 is arranged on the pipeline for obtaining a deep-sea microbial sample from the sample container. The lower part of the sample mixing tank 11 is connected with the upper part of the sample settling tank 12 through a pipeline, and a sample transfer valve 110 is arranged in the pipeline. The lower part of the sample settling tank 12 is connected with the lower part of the sample purification tank 13 through a pipeline, and a sample transfer valve 110 is also arranged in the pipeline. The sample transfer valve 110 is used for transferring during the sample purification process.

[0114] The bottom of the sample settling kettle 12 is funnel-shaped to realize the enrichment of the impurities in the sample; two sets of pressure maintaining stirrers 14 are arranged at the top of the sample mixing kettle 11 and the sample purification kettle 13 respectively to realize the mixing and mass transfer of the sample in the kettle; the turbidimeter 15 is arranged inside the sample settling kettle 12 to monitor the turbidity change in the kettle; the total organic carbon analyzer 16 is connected with the sample purification kettle 13 to monitor the total organic carbon change in the kettle; the purification piston 17 is arranged below the inside of the sample purification kettle 13 and can be pushed up from bottom to top to realize the pressurized concentration of the sample; the filter membrane 18 is arranged on the upper surface of the purification piston 17 to filter out the culture medium solution and improve the microbial abundance per unit volume;

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

[0116] The central control unit 6 is also connected with the two sets of pressure maintaining stirrers 14, the turbidimeter 15 and the 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 magnetic sub is placed inside the enrichment kettle to realize the stirring and mass transfer enhancement of the enrichment in the kettle; the environmental parameter sensing system 23 is connected with the enrichment kettle 21 and the central control unit 6 respectively, which is used to detect the environmental parameters in the enrichment kettle 21 and transmit them to the central control unit 6 to regularly measure the biochemical process in the kettle and evaluate the enrichment effect;

[0118] As shown in Figure 5 The visual monitoring unit 3 includes a biological observation kettle 31, a biological abundance measuring system 32, two sets of pressure-resistant observation windows 33, a microscopic camera system 34, a measuring electromagnetic valve 35 and a recovery electromagnetic 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 sedimentation kettle 12, the sample purification kettle 13, the enrichment kettle 21 and the biological observation kettle 31. 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 connected to the enrichment kettle 21 through pipelines, respectively, for collecting the enriched material after the culture is completed; the other end of the plunger pump 25 is 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 liquid 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 a dilution valve 38 and a 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, thereby better observing and counting biological cells;

[0126] The illuminator 39 is provided on the top of the biological observation kettle 31 and is connected to the central control unit 6, and is used to provide light for the biological observation kettle 31 through 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 detector 233, which are used to detect the dissolved oxygen content, the pH value, and the chemical content in the enrichment tank 21, respectively, and transmit the detection results to the central control unit 6.

[0128] As shown in FIG. 3, the biological abundance measuring system 32 includes an observation tank 321, a cover glass flipper 322, and a cover glass 323. The observation tank 321 and the cover glass 323 are both high-transmittance materials and allow the enrichment bacterial liquid sample to be stored therebetween for microscopic observation when they are attached. Figure 6

[0129] The two ends of the observation tank 321 are connected to a measuring electromagnetic valve 35 and a recovery electromagnetic valve 36 through pipelines, respectively, for injecting and recovering the sample. The cover glass flipper 322 is connected to the central control unit 6, and is used to drive the cover glass 323 to flip and attach to the observation tank 321.

[0130] A square recess is arranged on the observation tank 321, and the square recess is completely attached to a protruding square on the cover glass 323. A two-way funnel-shaped passage is further arranged in the square recess, and a grid for observing and counting cells is engraved in the passage. In this embodiment, the grid area actually contains a bacterial liquid volume of about 10 μL, i.e., the size is 1×1×0.01 cm 3 The grid contains four large squares around and one small square in the middle, so as to count different amounts of cells. When the number of cells is small, the large squares are used for counting, and when the number of cells is large, the small square is used for counting.

[0131] The microscopic camera system 34 includes a lens switcher 341 and three microscope lenses 342 with different magnification ratios. In this embodiment, the magnification ratios of the microscope lenses include 50, 100, and 200, so as to realize the multi-view switching during the biological quantity observation.

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

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

[0134] N C = N F × D × F × 10000

[0135] In the formula, N C represents the number of cells per milliliter of sample, and N F ​D represents the dilution factor of the cell sample; F represents the number of squares, including the number of two types of squares, 16 large squares and 25 small squares;

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

[0137] The pressure regulating unit 5 comprises a gas booster pump 51, four gas injection valves 52, a purification liquid 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 liquid injection pump 53 and all the pressure sensors 55 are respectively connected with the central control unit 6;

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

[0139] As shown in Figure 7 In the embodiment, the central control unit 6 comprises a communication module, a control module and a computer host, wherein the communication module is used to connect various sensing devices to obtain changes of environmental indicators; the communication module is connected with the computer host through a circuit for communication; the control module is connected with various operating devices, and the control module is connected with the computer host through a circuit; the computer host sends electrical signals to control the functions of the pressure regulating unit, the temperature regulating unit, the filtration and purification unit, the continuous condition unit, the mixing and mass transfer unit and the abundance observation unit; 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, the sample mixing tank 11, the sample settling tank 12, the sample purification tank 13, the enrichment tank 21 and the biological observation tank 31 are first cleaned and sterilized, and then the culture liquid is injected, the tank body is installed and the gas is boosted; each unit in the device is installed;

[0141] The deep-sea microbial sample is obtained from the container to be sampled and transferred to the sample mixing kettle 11. The pressure maintaining stirrer 14 in the sample mixing kettle 11 is turned on. After waiting for a first preset time, the sample is completely mixed. The mixed sample is obtained.

[0142] The mixed sample is transferred to the sample settling kettle 12. The turbidimeter 15 is used to monitor the settling and separation of impurities in the sample in real time. After the turbidity is reduced to a first preset threshold, the settled sample is obtained.

[0143] The settled sample is transferred to the sample purification kettle 13. The pressure maintaining stirrer 14 in the sample purification kettle 13 is turned on. The purification piston 17 is pushed to perform purification. The total organic carbon analyzer 16 is used to monitor the purification of the sample in real time. When the total organic carbon analyzer 16 obtains a total organic matter content greater than a second preset threshold, or the difference between the pressures collected by the top and bottom pressure sensors 55 in the sample purification kettle 13 is greater than a fourth preset threshold, the purified sample is obtained, and the purification sampling is completed.

[0144] Subsequently, the purified bacterial liquid sample is transferred to the enrichment kettle 21. The magnetic stirrer 22 is turned on to start enrichment. The environmental parameter sensing 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, and the enrichment is completed.

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

[0146] The dilution valve 38 is opened. The plunger pump 25 is connected to the dilution kettle 37 and injects the dilution culture solution into the biological observation kettle 31.

[0147] The cover glass flipper 322 is controlled to cover the cover glass 323. The measurement electromagnetic valve 35 is closed. The microscopic camera system 34 automatically identifies and calculates the number of cells per unit area in the grid in the obtained image.

[0148] After each observation, the recovery electromagnetic valve 36 is opened. The plunger pump 25 is connected to the biological observation kettle 31 and injects the enriched bacterial liquid into the enrichment kettle 21 at a certain flow rate. The observed bacterial liquid is recovered into the enrichment kettle 21. Subsequently, the recovery electromagnetic valve 36 is closed. The image recognition software of the microscopic camera system 34 calculates the total abundance of the organism.

[0149] The above steps are repeated for multiple observations until a certain biological abundance is reached. The enrichment ball valve 24 is opened to collect all the enrichment materials in the enrichment kettle 21 for further preservation and experiments.

[0150] The device can realize the automatic pretreatment process of effectively separating biological cells from environmental impurities in the microbial sampling process under the deep-sea temperature and pressure preservation condition, obtain high-purity biological enrichment, real-time detection of biochemical indicators in the microbial enrichment process, visual monitoring of biomass, and detection of the recycling of the enrichment, thereby effectively improving the sampling purity and enrichment efficiency of deep-sea environmental microorganisms.

[0151] Embodiment 5

[0152] As shown in Figure 8 the embodiment, the deep-sea microbial online purification sampling and visual automatic enrichment method is provided, which is based on the deep-sea microbial online purification sampling and visual automatic enrichment device described in Embodiment 4 and includes the following steps:

[0153] S1: The sample mixing kettle, sample settling kettle, sample purification kettle, enrichment kettle, and biological observation kettle 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 deep-sea microbial online purification sampling and visual automatic enrichment device is installed;

[0154] S2: The deep-sea microbial sample is obtained from the to-be-sampled container and transferred to the sample mixing kettle, the pressure-stabilizing stirrer in the sample mixing kettle is turned on, the sample is completely mixed after waiting for a first preset time, and the mixed sample is obtained;

[0155] S3: The mixed sample is transferred to the sample settling kettle, the turbidimeter is used to monitor the settling separation of the sample impurities in real time, the settled sample is obtained after the turbidity is reduced to a first preset threshold;

[0156] S4: The settled sample is transferred to the sample purification kettle, the pressure-stabilizing stirrer in the sample purification kettle is turned on, the purification piston is pushed to perform purification, and the total organic carbon analyzer is used to monitor the sample purification in real time, the purified sample is obtained after the total organic carbon analyzer obtains a total organic matter content greater than a second preset threshold, or the pressure difference collected by the top and bottom pressure sensors in the sample purification kettle is greater than a fourth preset threshold, and the purified sample is transferred to the enrichment kettle;

[0157] S5: 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, the enrichment bacteria solution is obtained after the monitored environmental indicators meet the preset conditions, and the enrichment is completed;

[0158] S6: The measurement electromagnetic valve is turned on, the enrichment bacteria solution is transferred to the biological abundance measurement system in the biological observation kettle, the biological abundance is observed by using the microscopic camera system, the enrichment bacteria solution after observation is transferred back to the enrichment kettle for recycling after the recycling electromagnetic valve is turned on;

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

[0160] In the biological abundance measurement system of step S6, the square groove height 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 observation image, the biological abundance is calculated according to the following formula:

[0162] N C = N F × D × F × 10000

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

[0164] In the specific implementation process, as shown in Figure 9 The control method comprises the following steps:

[0165] After all the tanks are filled with medium solution and high-pressure gas, the sample mixing tank 11 is connected to the front-end simulation device, sampling begins, the sample mixing tank 11 is opened, and the pressure agitator 14 is opened, and after waiting for 10 min, the sample and the medium solution are completely mixed;

[0166] After the sample is completely mixed, the sample transfer valve 110 behind the sample mixing tank 11 is opened, the sample is transferred into the sample settling tank 12, and the impurities and the enrichment are separated by waiting for the sample to settle, and the separation condition is identified by the turbidimeter 15, if the turbidity measured by the turbidimeter 15 is <1500 NTU, the sample transfer valve 110 behind the sample settling tank 12 is opened, and the pressure agitator 14 and the PID control valve 56 of the sample purification tank 13 are opened, otherwise the separation is continued;

[0167] After the sample is transferred after the settling, the purification valve 54 is opened, the sample transfer valve 110 is closed, the purification liquid pump 53 is opened, the purification piston 17 is pushed, the purification begins, the purification condition is identified by the total organic carbon analyzer 16, if the total organic matter content of the total organic carbon analyzer 16 is >600 mM or the lower pressure of the upper and lower pressure sensors 55 of the sample purification tank 13 is > the upper pressure+5 MPa, the sample transfer valve 110 behind the sample purification tank 13 is opened, and the magnetic agitator 22 and the PID control valve 56 of the enrichment tank 21 are opened, otherwise the above purification operation is continued;

[0168] When the sample purification is completed, the liquid injection valve 27 is opened, the sample transfer valve 110 behind the sample purification kettle 13 is closed, the continuous liquid injection pump 26 is opened, and the enrichment is started, and the environmental indicators in the enrichment kettle 21 are identified through the dissolved oxygen detector 231, the pH value detector 232 and the Raman measuring instrument 233, if the dissolved oxygen content is <1 μmol / kg, the sulfide content is >5 mmol / L and the pH value is >8.5, it is judged that the biological observation can be started, the illuminator 39 of the biological observation kettle 31 is opened, the biological abundance measuring system 32 is opened, and the microscopic camera system 34 is opened, otherwise the enrichment environment is continued to be waited for reaching the standard;

[0169] When the enrichment environment reaches the standard, the biological observation is started, the cover glass flipper 322 is opened, the cover glass 323 is turned over, and the measuring electromagnetic valve 35 is opened; the plunger pump 25 is opened, and is connected to the enrichment kettle 21 and injects 0.1 mL of the enriched bacteria liquid into the biological observation kettle 31; the dilution valve 38 is opened, the plunger pump 25 is connected to the dilution kettle 37 and injects 0.9 mL of the dilution culture solution into the biological observation kettle 31; the cover glass flipper 322 is controlled to cover the cover glass 323, and the measuring electromagnetic valve 35 is closed; the microscopic camera system 34 automatically identifies <0.22 μm objects in the image acquisition, and records the cell quantity per unit area in the grid;

[0170] After each observation is completed, the recovery electromagnetic valve 36 is opened, the plunger pump 25 is connected to the biological observation kettle 31 and injects the completed observation enriched bacteria liquid into the enrichment kettle 21 at a certain flow rate, so that the bacteria liquid recovery is realized; then the recovery electromagnetic valve 36 is closed; the biological total abundance is calculated through the image recognition software of the microscopic camera system 34, if the biological abundance is >107 cell / mL, the enrichment ball valve 24 is opened, the enrichment substance is collected, and the enrichment is ended, otherwise the above enrichment process is continuously executed until the enrichment substance with high biological abundance is obtained, and then the enrichment is ended.

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

[0172] The terms describing the positional relationship in the drawings are only used for example illustration, and cannot be understood as the limitation of the patent;

[0173] Obviously, the above embodiments of the application are only examples for clearly illustrating the application, and are not the limitation of the embodiments of the application. Any modification, equivalent replacement and improvement made on the basis of the above description for those skilled in the art should be included in the protection scope of the claims of the application.

Claims

1. A deep-sea microorganism online purification and sampling device, characterized in that: include: Sampling and purification unit, first temperature regulating unit, first pressure regulating unit and first central control unit; The sampling and purification unit includes: 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 sedimentation of impurities in 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 bottom of the sample purification kettle and can be pushed upward from the bottom to achieve pressurized concentration of the sample; 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 and sampling device according to claim 1, characterized in that: The first temperature regulating unit comprises: a temperature regulator and three sets of temperature sensors respectively connected to the first central control unit; the three sets 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 includes: a gas booster pump, three gas injection valves, a purification liquid injection pump, a purification valve, and four sets of pressure sensors; the gas booster pump, the purification liquid injection pump and the four sets 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; the top of the sample mixing kettle, the top of the sample sedimentation kettle, and the top and bottom of the sample purification kettle are respectively provided with a group of pressure sensors, and the pressure sensor at the bottom of the sample purification kettle is used to monitor the purification progress and stop in time.

3. A deep-sea microorganism online visualization automatic enrichment device, characterized in that: include: Enrichment unit, visual monitoring unit, second temperature regulating unit, second pressure regulating unit and second central control unit; The enrichment unit includes: 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 second central control unit respectively, for detecting the environmental parameters in the enrichment kettle and transmitting 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, for observing 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, for placing the microorganisms to be observed; the microscopic camera system is arranged at the bottom of the biological observation kettle and connected to the second central control unit, for observing the changes in the abundance of microorganisms in the biological observation kettle through the pressure-resistant observation window at the bottom; the measurement 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 further comprises: 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 respectively through pipelines; 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 light for the biological observation kettle through 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 connected to a measuring solenoid valve and a recovery solenoid valve respectively 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 aligned with the pre-set 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 numbers; The microscopic camera system includes: 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 sets of temperature sensors respectively connected to the second central control unit; the two sets 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 sets 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 a pipeline; the PID control valve is arranged on the enrichment kettle; the exhaust valve is arranged on the biological observation kettle; and two sets 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 by pipelines, 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 inject culture solution, install the kettle body, and perform gas pressurization; install each unit of the deep-sea microorganism online purification sampling and visual automatic enrichment device; S2: Obtain a deep-sea microbial sample from the sampling container and transfer it to a sample mixing kettle, start a pressure-maintaining stirrer 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 sedimentation 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 to purify, and using a total organic carbon analyzer to monitor the sample purification status 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 and complete the enrichment; S6: Open the measurement 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 for multiple enrichment and observation until the observed biological abundance reaches a third preset threshold. At this time, all the enriched bacterial liquid in the enrichment kettle is collected for further preservation or experiment.

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