Device for enrichment culture and gravity-type isolation of marine microorganisms in high-pressure environment

1 . A device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment, wherein the high-pressure environment is a pressure environment at a water depth greater than 200 meters in a deep sea, the device comprises an enrichment and multistage purification unit, a gravity isolation and culture unit, a temperature controlling system, a pressure control system, and a central control system, wherein the central control system includes a processor to control the temperature controlling system and the pressure control system, wherein control ends and signal detection ends of the enrichment and multistage purification unit and the gravity isolation and culture unit are electrically connected to the central control system; control ends of the temperature controlling system and the pressure control system are electrically connected to the central controller control system; wherein,

the enrichment and multistage purification unit is used for realizing a process of enrichment and multistage purification of the marine microorganisms, obtaining a marine microorganism enrichment bacteria liquid, and injecting the marine microorganism enrichment bacteria liquid into the gravity isolation and culture unit;

the gravity isolation and culture unit is used for carrying out automatic streaking by means of gravity in the high-pressure environment to achieve solid isolation and culture of the marine microorganisms, such that culturability of the marine microorganisms is effectively improved;

the temperature controlling system and the pressure control system are connected to the enrichment and multistage purification unit and the gravity isolation and culture unit respectively for constructing a high-pressure and low-temperature environment consistent with a marine environment in the enrichment and multistage purification unit and the gravity isolation and culture unit, so as to ensure that enriched deep-sea microorganisms are enriched, purified, separated and cultured under in-situ environmental conditions, wherein the high-pressure and low-temperature environment is environment at a water depth greater than 200 meters and below 4° C. in a deep sea;

wherein, the pressure control system comprises a bleed valve, an air compressor, a booster pump, a gas storage tank, a pressure regulating valve, a regulating valve, and a vent pipeline; wherein,

the bleed valve is connected to the enrichment and multistage purification unit and the gravity isolation and culture unit through the vent pipeline, and a control end of the bleed valve is electrically connected to the central control system for discharging gas from the enrichment and multistage purification unit and the gravity isolation and culture unit, and for depressurizing interior of the enrichment and multistage purification unit and the gravity isolation and culture unit;

the air compressor, the booster pump, the gas storage tank, the pressure regulating valve and the regulating valve are connected in sequence through the vent pipeline, and finally connected to the enrichment and multistage purification unit and the gravity isolation and culture unit through the vent pipeline for filling the enrichment and multistage purification unit and the gravity isolation and culture unit with gas for pressurization; the pressure regulating valve is used for regulating internal pressure of the enrichment and multistage purification unit and the gravity isolation and culture unit; the regulating valve is used for regulating a speed of gas injection;

a control end of the air compressor, a control end of the booster pump, a control end of the pressure regulating valve, and a control end of the regulating valve are electrically connected to the central control system;

the temperature controlling system comprises a water bath jacket and a heat exchanger; the water bath jacket is wrapped on outer walls of the enrichment and multistage purification unit and the gravity isolation and culture unit, and is connected to the water bath heat exchanger; and a control end of the heat exchanger is electrically connected to the central control system; the temperature controlling system further comprises a thermocouple, and an output end of the thermocouple is electrically connected to the central control system for detecting the water bath temperature in the water bath jacket in real time, so as to facilitate real-time temperature regulation;

the enrichment and multistage purification unit comprises a plurality of microbial liquid enrichment culture chambers connected in series; the microbial liquid enrichment culture chambers are provided with removable sealing lids and connection and sampling valve sets, and sensor sets are disposed inside the microbial liquid enrichment culture chambers; each of the microbial liquid enrichment culture chambers is disposed in the temperature controlling system; wherein,

the removable sealing lids are used for facilitating sterilization operations and placement of culture substrates inside the microbial liquid enrichment culture chambers;

the connection and sampling valve sets are used for connection and sampling of the microbial liquid enrichment culture chambers, and are connected to the pressure control system for feeding liquid or gas into the microbial liquid enrichment culture chambers to increase pressure inside the microbial liquid enrichment culture chambers, such that pressure values in the microbial liquid enrichment culture chambers are consistent with pressure values in the pressure environment at the water depth greater than 200 meters in the deep sea;

the sensor sets are used for real-time monitoring of temperature and pressure changes in the microbial liquid enrichment culture chambers, and transmitting signals to the central control system; and

a last microbial liquid enrichment culture chamber of the enrichment and multistage purification unit is connected to the gravity isolation and culture unit through the connection and sampling valve sets;

the gravity isolation and culture unit comprises at least one isolation and culture chamber, a liquid injection unit, and an environmental parameter detection unit; wherein, the liquid injection unit comprises a microinjection pump and a liquid injection pipeline, and the environmental parameter detection unit comprises a second temperature sensor and a second pressure sensor,

the gravity isolation and culture unit is connected to the last microbial liquid enrichment culture chamber of the enrichment and multistage purification unit through the liquid injection unit;

a microorganism isolation branch is disposed in the at least one isolation and culture chamber for carrying out an isolation operation on microorganisms to provide a maximum area for microorganism culture;

a central liquid injection pipe is disposed on a top of the at least one isolation and culture chamber, and the at least one isolation and culture chamber is connected to the liquid injection unit through the central liquid injection pipe; a marine microorganism enrichment bacteria liquid is injected by the liquid injection unit into the microorganism isolation branch of the at least one isolation and culture chamber;

the at least one isolation and culture chamber is disposed in the temperature controlling system and connected to the pressure control system for constructing the high-pressure and low-temperature environment consistent with the marine environment in the at least one isolation and culture chamber, so as to ensure that the enriched deep-sea microorganisms are isolated and cultured in the in-situ environmental condition;

the environmental parameter detection unit is used for real-time detection of temperature and pressure changes in the at least one isolation and culture chamber and transmission of detected data to the central control system; and

the microorganism isolation branch and the liquid injection unit are electrically connected to the central control system;

the microorganism isolation branch comprises a removable reservoir, a pellet, a reciprocating puller, a guide chute, and a cavity; wherein,

the removable reservoir is disposed in a center of a top of the cavity, and located under the central liquid injection pipe for storing the marine microorganism enrichment bacteria liquid injected by the liquid injection unit; the pellet is placed in the removable reservoir and submerged by the injected marine microorganism enrichment bacteria liquid; a through hole is provided in a bottom of the removable reservoir for fixing a position of the pellet and ensuring that the pellet is able to pass through the through hole; a movable end of the reciprocating puller is fixedly connected to the removable reservoir; a stop box is disposed on an outer surface of the top of the cavity; a control end of the reciprocating puller is electrically connected to the central control system

the guide chute is fixedly disposed inside the cavity; the guide chute is filled with a culture medium, and an inlet of the guide chute communicates with the stop box;

after the pellet is submerged in the marine microorganism enrichment bacteria liquid, the removable reservoir moves toward an edge of the cavity under action of the reciprocating puller; when the through hole of the removable reservoir leaves the cavity, the pellet passes through the through hole and falls into the stop box of the cavity due to gravity, and enters the guide chute inside the cavity; and

while the pellet carries the marine microorganism enrichment bacteria liquid to slide along the guide chute from top to bottom, the marine microorganism enrichment bacteria liquid is dispersed through concentration gradient dilution to provide the maximum area for microorganism isolation and culture.

2 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 1 , wherein the microbial liquid enrichment culture chambers are further provided with stirring rods, and the stirring rods are used for enhancing a reaction process of a substrate in a culture process of the microbial liquid enrichment culture chambers.

3 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 1 , wherein the sensor sets comprise second temperature sensors and second pressure sensors; the second temperature sensors are used for real-time monitoring of temperature changes in the microbial liquid enrichment culture chambers; the second pressure sensors are used for real-time monitoring of pressure changes in the microbial liquid enrichment culture chambers; and signal output ends of the second temperature sensors and signal output ends of the second pressure sensors are electrically connected to the central control system.

4 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 1 , wherein the connection and sampling valve sets comprise liquid inlet valves, gas inlet valves, sampling valves, and liquid outlet valves; wherein,

the microbial liquid enrichment culture chambers are connected in series through the liquid outlet valves and the liquid inlet valves, and a liquid outlet valve of a former microbial liquid enrichment culture chamber is connected to a liquid inlet valve of a latter microbial liquid enrichment culture chamber; a liquid outlet valve of the last microbial liquid enrichment culture chamber is connected to the gravity isolation and culture unit;

the gas inlet valves are connected to the pressure control system for feeding gas into the microbial liquid enrichment culture chambers to increase the pressure in the microbial liquid enrichment culture chambers, such that the pressure values in the microbial liquid enrichment culture chambers are consistent with the pressure values in the pressure environment at the water depth greater than 200 meters in the deep sea; and

the sampling valves are used for real-time sampling and analysis of the microorganisms in the microbial liquid enrichment culture chambers.

5 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 1 , wherein a slot is provided in the center of the top of the cavity, that is, a position where an initial position of the removable reservoir coincides with the through hole, for fixing the position of the pellet.

6 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 1 , wherein, a liquid inlet end of the microinjection pump is connected to the last microbial liquid enrichment culture chamber of the enrichment and multistage purification unit, and a liquid outlet end of the microinjection pump is connected to the liquid injection pipeline; a liquid outlet of the liquid injection pipeline is connected to the central liquid injection pipe; and a control end of the microinjection pump is electrically connected to the central control system.

7 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 1 , wherein, probes of the second temperature sensor and the second pressure sensor are disposed inside the at least one isolation and culture chamber, and signal output ends of the second temperature sensor and the second pressure sensor are electrically connected to the central control system.

8 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 7 , wherein a quick-open kettle lid is disposed on the top of the at least one isolation and culture chamber; the central liquid injection pipe is disposed on the quick-open kettle lid; the quick-open kettle lid is further provided with a gas injection channel and a sensor containing channel; the pressure control system is connected to the at least one isolation and culture chamber through the gas injection channel; and the second temperature sensor and the second pressure sensor are disposed in the sensor containing channel.

9 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 1 , wherein the at least one isolation and culture chamber includes a plurality of isolation and culture chambers disposed in sequence, the central liquid injection pipe comprises a plurality of sub-central liquid injection pipes and the liquid injection unit is connected to the plurality of sub-central liquid injection pipes of all the plurality of isolation and culture chambers; the temperature controlling system and the pressure control system are connected to all the plurality of isolation and culture chambers respectively for constructing the high-pressure and low-temperature environment consistent with the marine environment in each of the plurality of isolation and culture chambers, so as to ensure that the enriched deep-sea microorganisms are isolated and cultured under the in-situ environmental condition; and each of the plurality of isolation and culture chambers is connected to an independent environmental parameter detector for real-time detection of temperature and pressure changes in each of the plurality of isolation and culture chambers and transmission of detected data to the central control system.

10 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 9 , wherein the device further comprises a mobile platform, wherein the enrichment and multistage purification unit, the gravity isolation and culture unit, the temperature controlling system, the pressure control system, and the central control system are placed on the mobile platform for improving general applicability to a culture scenario.

11 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 5 , wherein the at least one isolation and culture chamber includes a plurality of isolation and culture chambers disposed in sequence, the central liquid injection pipe comprises a plurality of sub-central liquid injection pipes and the liquid injection unit is connected to the plurality of sub-central liquid injection pipes of all the plurality of isolation and culture chambers; the temperature controlling system and the pressure control system are connected to all the plurality of isolation and culture chambers respectively for constructing the high-pressure and low-temperature environment consistent with the marine environment in each of the plurality of isolation and culture chambers, so as to ensure that the enriched deep-sea microorganisms are isolated and cultured under the in-situ environmental condition; and each of the plurality of isolation and culture chambers is connected to an independent environmental parameter detector for real-time detection of temperature and pressure changes in each of the plurality of isolation and culture chambers and transmission of detected data to the central control system.

12 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 9 , wherein the at least one isolation and culture chamber includes a plurality of isolation and culture chambers disposed in sequence, the central liquid injection pipe comprises a plurality of sub-central liquid injection pipes and the liquid injection unit is connected to the plurality of sub-central liquid injection pipes of all the plurality of isolation and culture chambers; the temperature controlling system and the pressure control system are connected to all the plurality of isolation and culture chambers respectively for constructing the high-pressure and low-temperature environment consistent with the marine environment in each of the plurality of isolation and culture chambers, so as to ensure that the enriched deep-sea microorganisms are isolated and cultured under the in-situ environmental condition; and each of the plurality of isolation and culture chambers is connected to an independent environmental parameter detector for real-time detection of temperature and pressure changes in each of the plurality of isolation and culture chambers and transmission of detected data to the central control system.

13 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 10 , wherein the at least one isolation and culture chamber includes a plurality of isolation and culture chambers disposed in sequence, the central liquid injection pipe comprises a plurality of sub-central liquid injection pipes and the liquid injection unit is connected to the plurality of sub-central liquid injection pipes of all the plurality of isolation and culture chambers; the temperature controlling system and the pressure control system are connected to all the plurality of isolation and culture chambers respectively for constructing the high-pressure and low-temperature environment consistent with the marine environment in each of the plurality of isolation and culture chambers, so as to ensure that the enriched deep-sea microorganisms are isolated and cultured under the in-situ environmental condition; and each of the plurality of isolation and culture chambers is connected to an independent environmental parameter detector for real-time detection of temperature and pressure changes in each of the plurality of isolation and culture chambers and transmission of detected data to the central control system.

14 . The device for enrichment culture and gravity isolation of marine microorganisms in a high-pressure environment according to claim 11 , wherein the at least one isolation and culture chamber includes a plurality of isolation and culture chambers disposed in sequence, the central liquid injection pipe comprises a plurality of sub-central liquid injection pipes and the liquid injection unit is connected to the plurality of sub-central liquid injection pipes of all the plurality of isolation and culture chambers; the temperature controlling system and the pressure control system are connected to all the plurality of isolation and culture chambers respectively for constructing the high-pressure and low-temperature environment consistent with the marine environment in each of the plurality of isolation and culture chambers, so as to ensure that the enriched deep-sea microorganisms are isolated and cultured under the in-situ environmental condition; and each of the plurality of isolation and culture chambers is connected to an independent environmental parameter detector for real-time detection of temperature and pressure changes in each of the plurality of isolation and culture chambers and transmission of detected data to the central control system.