A system and method for in situ microbial cultivation and collection in deep sea

Abstract

The application discloses a deep-sea in-situ microorganism culture and collection system, which comprises a microorganism automatic collecting device, a deep-sea in-situ microorganism culture chamber, a pipeline switching device and a microorganism enrichment and preservation device. The microorganism automatic collecting device is used for collecting seawater and culture mother liquor and conveying the seawater and the culture mother liquor to the deep-sea in-situ microorganism culture chamber respectively, and in-situ microorganism culture is carried out in the deep-sea in-situ microorganism culture chamber. The microorganism enrichment and preservation device comprises a plurality of microorganism enrichment filter discs, and the deep-sea in-situ microorganism culture chamber is connected with each microorganism enrichment filter disc through the pipeline switching device. Through control of the pipeline switching device, the deep-sea in-situ microorganism culture chamber is switched and connected to different microorganism enrichment filter discs at different culture times, and microorganism samples at different culture times are collected by each microorganism enrichment filter disc, so that the deep-sea in-situ microorganism culture, regular sampling, preservation and detection are realized.

Description

A system and method for in-situ cultivation and collection of deep-sea microorganisms Technical Field

[0001] This invention relates to the field of microbial culture and collection technology, and in particular to a deep-sea in-situ microbial culture and collection system. Background Technology

[0002] The deep sea harbors abundant microbial resources, which are of great value in environmental remediation and organic synthesis. Traditional sampling methods significantly impact deep-sea biological samples, limiting the utilization of microorganisms in the extreme environment of the deep sea. Furthermore, the unique nature of the environment makes highly realistic simulation cultivation in the laboratory virtually impossible. Although using deep-sea environment simulation for biological cultivation is challenging, it remains an important method in the field of microbial geochemistry research internationally. Leading institutions in the development of advanced deep-sea microbial cultivation systems include the Department of Geophysics and Geophysics at the University of Minnesota, the University of California, Berkeley, the Woods Hole Oceanographic Institution (WHOI), and the Japan Agency for Marine-Earth Science and Technology (JAMSTEC). Research on laboratory simulation cultivation in China started relatively late. Under laboratory conditions, these culturable microorganisms undergo a "domestication effect" due to altered living conditions and continuous subculturing. The addition of organic carbon sources to the culture medium eventually leads to gradual changes in the genome of chemoautotrophic bacteria, resulting in the lateral transfer of heterotrophic genes and transformation into facultative bacteria. Therefore, laboratory-cultured strains lose many characteristics of the original deep-sea strains. Simulated culture can address issues related to basic physiological characteristics of microorganisms, but it cannot monitor and assess the in-situ ecological functions and dynamic changes of microorganisms. Therefore, in-situ culture in the deep sea has greater significance for the utilization of deep-sea microbial resources.

[0003] Zhejiang University and other institutions have collaborated to successfully develop my country's first deep-sea in-situ microbial culture system, consisting of a control room, culture chambers, and buoyancy materials. However, this device lacks the ability to periodically sample and test, and cannot prevent cell damage caused by changes in environmental pressure. The State Oceanic Administration and Hangzhou Dianzi University have jointly developed a deep-sea in-situ biological experimental platform capable of independent operation in a deep-sea environment. The main body consists of 16 culture chambers, in-situ environmental monitoring sensors, acoustic release devices, buoyancy materials, and gravity anchors. The system is designed to operate at a maximum water depth of 6000 meters and can be deployed at the seawater-sediment interface for long-term in-situ culture of deep-sea microorganisms. However, this device also cannot sample and preserve data from each culture chamber at different time intervals. Summary of the Invention

[0004] The purpose of this invention is to provide a system and method for in-situ culture and collection of deep-sea microorganisms, solving the problem that in-situ culture of deep-sea microorganisms cannot be sampled and tested regularly.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A deep-sea in-situ microbial culture and collection system is provided, comprising an automatic microbial collection device, a deep-sea in-situ microbial culture chamber, a pipeline switching device, and a microbial enrichment and preservation device. The automatic microbial collection device is used to collect seawater and culture stock solution, and respectively deliver them to the deep-sea in-situ microbial culture chamber for in-situ microbial culture. The microbial enrichment and preservation device includes multiple microbial enrichment filter discs. The deep-sea in-situ microbial culture chamber is connected to each microbial enrichment filter disc through the pipeline switching device. By controlling the pipeline switching device, the deep-sea in-situ microbial culture chamber is switched to different microbial enrichment filter discs at different culture times, and each microbial enrichment filter disc collects microbial samples at different culture times.

[0007] In some embodiments of the present invention, the microbial enrichment filter disc includes a cavity and a filter membrane with corresponding pore sizes, on which microorganisms of corresponding sizes are enriched.

[0008] In some embodiments of the present invention, a glycerol addition device is also included, which is connected to each microbial enrichment filter disc via the pipeline switching device to add glycerol protectant to the microbial enrichment filter disc to preserve microorganisms.

[0009] In some embodiments of the present invention, at least one bacterial culture filter plate and a water bag are also included. The deep-sea in-situ microbial culture chamber is connected to the bacterial culture filter plate through the pipeline switching device. The bacterial culture filter plate is connected to the water bag. The cultured bacterial culture is filtered by the bacterial culture filter plate to remove microorganisms of a limited size and then stored in the water bag.

[0010] In some embodiments of the present invention, the pipeline switching device includes a plurality of two-position two-way valves and their valve seats, each two-position two-way valve being connected to a corresponding filter disc.

[0011] In some embodiments of the present invention, a pump is connected between the automatic microbial collection device and the deep-sea in-situ microbial culture chamber, and between the deep-sea in-situ microbial culture chamber and the microbial enrichment and preservation device.

[0012] In some embodiments of the invention, the pump includes a peristaltic pump.

[0013] In some embodiments of the present invention, the culture medium is sodium bicarbonate labeled with C14 and with a final concentration of 0.1M.

[0014] In some embodiments of the present invention, the automatic microbial collection device injects seawater into the deep-sea in-situ microbial culture chamber, and then injects the culture mother liquor.

[0015] The present invention also provides a method for in-situ cultivation and collection of deep-sea microorganisms, wherein the in-situ cultivation and collection system for deep-sea microorganisms is used.

[0016] The present invention has the following beneficial effects:

[0017] This invention collects seawater and culture stock solution using an automated microbial collection device and delivers them separately to a deep-sea in-situ microbial culture chamber. In-situ microbial culture is then conducted in the chamber. A pipeline switching device connects the chamber to different microbial enrichment filter trays in a microbial enrichment and preservation device at different culture times. Each enrichment filter tray collects microbial samples from different culture times, enabling the in-situ culture, periodic sampling, preservation, and detection of deep-sea microorganisms. Because it can preserve cultured microorganisms at different times within a deep-sea culture system, this invention can be used for detecting microbial community changes and preserving microbial strains, allowing for the retention and extraction of microbial changes during the culture process.

[0018] In some embodiments of the present invention, a glycerol addition device can be used to add glycerol protectant to the microbial enrichment filter plate via a pipeline switching device to preserve microorganisms, avoid damage to cells caused by changes in environmental pressure, and thus ensure the reliability of in-situ periodic sampling and testing in the deep sea.

[0019] Other beneficial effects of the embodiments of the present invention will be further described below. Attached Figure Description

[0020] Figure 1 is a structural diagram of the deep-sea in-situ microbial culture and collection system in an embodiment of the present invention;

[0021] Figure 2 is a flowchart of the deep-sea in-situ microbial culture and collection method in an embodiment of the present invention;

[0022] Figure 3 is a physical layout diagram of the deep-sea in-situ microbial culture and collection system in an embodiment of the present invention;

[0023] Figure 4 is a diagram showing the proportion of microbial community structure of deep-sea in situ cultured microorganisms obtained from different samples in the embodiments of the present invention.

[0024] The attached figures are labeled as follows:

[0025] 1 is an automated microbial collection device; 11 is a peristaltic pump A; 12 is a seawater pump; 13 is the culture stock solution; 14 is seawater; 2 is a deep-sea in-situ microbial culture chamber; 3 is a pipeline switching device; 31 is a two-position two-way valve A; 32 is a two-position two-way valve B; 33 is a two-position two-way valve C; 34 is a two-position two-way valve D; 35 is a two-position two-way valve E; 36 is a two-position two-way valve F; 37 is a two-position two-way valve G; 38 is a two-position two-way valve H; 39 is a two-position two-way valve I. 41 is microbial enrichment filter plate A, 42 is microbial enrichment filter plate B, 43 is microbial enrichment filter plate C, 44 is microbial enrichment filter plate D, 45 is microbial enrichment filter plate E, 5 is glycerol addition device, 61 is bacterial solution filter plate A, 62 is bacterial solution filter plate B, 63 is bacterial solution filter plate C, 64 is bacterial solution filter plate D, 71 is water bag A, 72 is water bag B, 73 is water bag C, 74 is water bag D, peristaltic pump B8, peristaltic pump C9, and bacterial culture solution 10. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0027] It should be noted that the directional terms such as left, right, up, down, top, and bottom used in this embodiment are only relative concepts or are based on the normal use of the product, and should not be considered as restrictive.

[0028] Existing deep-sea culture devices are all placed on the seabed for extended periods, making it impossible to monitor changes in the bacterial community during the culture process. Furthermore, it is impossible to inject glycerol into the samples before returning them to the surface to prevent cell damage due to pressure changes. This invention allows for the periodic sampling and preservation of the bacterial community at different time points during the culture process, enabling the detection of changes in the bacterial community during cultivation.

[0029] Existing deep-sea microbial culture methods involve bringing samples to the laboratory in water sample bottles for culture and separation. This invention avoids the damage to samples caused by environmental changes such as pressure and temperature, allowing for direct in-situ culture and screening. Furthermore, the addition of a glycerol protectant further prevents cell damage from environmental pressure changes.

[0030] This invention provides a system and method for in-situ deep-sea microbial culture and collection, as shown in Figure 2. The process includes in-situ culture, in-situ collection, and glycerol protection, all completed within the system of this invention. A control group is also set up to complete in-situ collection and simulated culture. After obtaining the microbial samples, data processing and analysis are performed, including agarose gel electrophoresis to detect the extracted DNA and RNA; precise measurement of the concentrations of extracted DNA and RNA using a Qubit 2.0 fluorometer (Invitrogen, USA); library construction of DNA and RNA; genome sequencing; and analysis of the community structure.

[0031] Deep-sea in-situ microbial culture and collection mainly involves three steps: in-situ culture, in-situ collection, and glycerol preservation. This is accomplished by a deep-sea in-situ microbial culture and collection system provided in this embodiment. The main components of the system are shown in Figure 1, including an automatic microbial collection device 1, a deep-sea in-situ microbial culture chamber 2, a pipeline switching device 3, a microbial enrichment and preservation device, a glycerol addition device 5, at least one bacterial solution filter plate and water bag, several pumps and valves, including peristaltic pumps and two-position two-way valves. The pipeline switching device 3 and the pumps enable continuous collection of microorganisms.

[0032] The automatic microbial collection device 1 is used to collect seawater 14 and culture mother liquor 13, and respectively transport them to the deep-sea in-situ microbial culture chamber 2 for in-situ microbial culture.

[0033] The deep-sea in-situ microbial culture chamber 2 is a stainless steel cavity used to complete the in-situ culture process of microorganisms. After the culture stock solution 13 and seawater 14 are pumped into the chamber via the peristaltic pump A11 and seawater pump 12 respectively, the deep-sea microorganisms are cultured in situ within the cavity of the chamber 2. The deep-sea in-situ microbial culture chamber 2 is connected to various microbial enrichment filter plates in the microbial enrichment and preservation device via a pipeline switching device 3. By controlling the pipeline switching device 3, the deep-sea in-situ microbial culture chamber 2 is switched to different microbial enrichment filter plates at different culture times, and each microbial enrichment filter plate collects microbial samples at different culture times.

[0034] The pipeline switching device 3 mainly consists of several two-position two-way valves and their valve seats. Each two-position two-way valve is connected to a corresponding filter plate. When the control unit (not shown) of the deep-sea in-situ microbial culture chamber 2 sends a command to control a certain two-position two-way valve to open, the culture liquid 10 of the deep-sea in-situ microbial culture chamber 2 enters the corresponding filter plate through the peristaltic pump B8 between the deep-sea in-situ microbial culture chamber 2 and the microbial enrichment and preservation device to complete the microbial enrichment or filtration work. Among them, microbial enrichment is completed by the microbial enrichment filter plate, and microbial filtration is completed by the bacterial liquid filter plate.

[0035] Specifically, in Figure 1 of this embodiment, two-position two-way valves A31, C33, E35, G37, and I39 control microbial enrichment filter plates A41, B42, C43, D44, and E45, respectively; and two-position two-way valves B32, D34, F36, and H38 control bacterial solution filter plates A61, B62, C63, and D64, respectively.

[0036] The microbial enrichment and preservation device includes multiple microbial enrichment filter discs, which in this embodiment are microbial enrichment filter discs A41, B42, C43, D44, and E45. These discs are used for in-situ nucleic acid enrichment by microorganisms. Each microbial enrichment filter disc includes a cavity and a filter membrane with a corresponding pore size. Microorganisms of the corresponding size are enriched on the filter membrane. In subsequent work, glycerol is injected to achieve in-situ fixation of nucleic acids.

[0037] The pipeline switching device 3 is also connected to at least one bacterial solution filter plate and a water bag connected to the bacterial solution filter plate. In Figure 1 of the embodiment of the present invention, the pipeline switching device 3 is connected to bacterial solution filter plates A61, B62, C63, and D64. Bacterial solution filter plates A61, B62, C63, and D64 are respectively connected to water bags A71, B72, C73, and D74, thereby realizing the preservation of the culture bacterial solution 10. After the culture bacterial solution 10 is filtered out by the bacterial solution filter plate to remove large microorganisms, the culture bacterial solution 10 is preserved in the water bag of the corresponding bacterial solution filter plate.

[0038] The glycerol addition device 5 is connected to each microbial enrichment filter plate through the pipeline switching device 3, and the peristaltic pump C9 adds glycerol protectant to the microbial enrichment filter plate to preserve the microorganisms.

[0039] The steps for deep-sea in-situ microbial culture and collection are as follows:

[0040] (1) Turn on the peristaltic pump A11 and the seawater pump 12, and inject 45L of seawater 14 and 5L of culture medium 13 (NaHC) of a certain concentration into the deep-sea in-situ microbial culture chamber 2. 14 O3, where the C element uses C 14 Label it to a final concentration of 0.1M sodium bicarbonate.

[0041] After the automatic microbial collection device 1 injects seawater 14 into the deep-sea in-situ microbial culture chamber 2, it closes the seawater pump 12 inlet pipe of the deep-sea in-situ microbial culture chamber 2, and then injects culture medium 13 and closes the culture medium pipe of peristaltic pump A11, making the deep-sea in-situ microbial culture chamber 2 a closed environment and starting the in-situ culture work. That is, after injecting two kinds of seawater 14 and culture mother liquid 13, the microorganisms in the seawater 14 proliferate and grow through the nutrient solution.

[0042] (2) Open the pipeline switching device 3, and filter the seawater 14 in the deep-sea in-situ microbial culture chamber 2 through the peristaltic pump B8 into several bacterial filter plates to complete the in-situ microbial filtration and collection work at 5 different time points. In this embodiment, the bacterial filter plates are A61, B62, C63, and D64. At this time, the deep-sea microbial culture chamber is always sealed and filled to ensure the growth of carbon-fixing microorganisms and to carry out the in-situ cultivation of seawater microorganisms.

[0043] (3) After each microbial enrichment filter plate has finished filtering, turn on the peristaltic pump C9 to deliver glycerol to the microbial enrichment filter plate to complete the microbial preservation work of the in-situ cultured microorganism water.

[0044] (4) A certain volume of water sample was collected using conventional water sampling techniques (Niskin), and nucleic acid was extracted under laboratory conditions. This sample served as a control group for subsequent experiments.

[0045] 2. Data processing and analysis

[0046] (1) The integrity of the extracted DNA and RNA was detected by agarose gel electrophoresis, and the concentration of the extracted DNA and RNA was accurately measured using a Qubit 2.0 fluorometer (Invitrogen, USA). Library construction was performed using 200 ng of purified sample DNA, followed by metagenomic sequencing. Additionally, the extracted RNA of acceptable quality was used to construct a library and undergo metagenomic sequencing.

[0047] (2) Community structure analysis: Based on metagenomic sequencing results, the community structure of in situ cultured samples and control group samples was analyzed using miTAG combined with QIIME software to analyze the changes in community structure under in situ culture conditions.

[0048] The present invention can preserve cultured microorganisms in a deep-sea culture system at different times for detecting changes in the microbial community and for strain preservation. It can preserve and extract changes in microorganisms during the culture process, and can also add glycerol protectant to the culture medium to prevent these deep-sea microorganisms from dying due to environmental changes.

[0049] The deep-sea in-situ microbial culture and collection system involved in this invention is irreplaceable. This is determined by the inevitable natural death and rapid changes in community structure of deep-sea microorganisms under environmental changes, necessitating the community detection and strain preservation of in-situ cultured microorganisms. The culture of deep-sea bacteria that have not died has long been proven to contain only some heterotrophic bacteria, and the number and types of cultured microorganisms obtained using existing methods and procedures have reached a bottleneck.

[0050] This invention enables the preservation of deep-sea in-situ cultured microorganisms at different time points, and also allows for the cultivation of multiple sequences and substrates during a single dive. The key process lies in the conversion between different stages within the culture system. Existing technologies often require bringing deep-sea water samples to a laboratory for cultivation. However, some deep-sea in-situ culture systems cannot selectively sample and test at specific time points during cultivation, nor can they inject microbial glycerol preservation solutions, causing some cultured microorganisms to die after leaving the water.

[0051] Example

[0052] The deep-sea in-situ microbial culture and collection system of this invention was carried out by the Phoenix lander to carry out deep-sea in-situ microbial culture. It has completed 8 culture tests so far, all of which have achieved the predetermined goals. The on-site layout is shown in Figure 3, and the proportion of the microbial community structure of the deep-sea in-situ cultured microorganisms obtained from different samples is shown in Figure 4.

[0053] In Figure 4, the horizontal axis represents different samples, and the vertical axis represents the proportion of different microorganisms in different samples. In Figure 4, FH21-C is the microbial community structure of deep-sea in-situ cultured in a 40L water bag using 0.1mM sodium bicarbonate; FH18-N and PH22-N are the community structures of water sample bottles; FH21-5, FH21-7, FH21-8, FH21-9, and FH21-10 are the community structures of in-situ filtering microorganisms.

[0054] The amplicon read classification results of this invention showed that Proteobacteria had the highest relative abundance in all samples. For the in situ filtered samples, high relative abundances of Firmicutes (Euryarchaeota), Chloroflexi, and Bacteroidetes were also observed, whereas these were almost absent in the water bottle (FH18-N and FH22-N) and culture (FH21-C) samples. Changes in community structure were also observed among the in situ filtered samples. Between pH 21-6 and pH 21-7, the number of Proteobacteria decreased, while the number of Actinobacteria and Planctomycetes increased, indicating a change in microbial composition during the early morning period. Although the sequencing reads for pH 21-5 and pH 21-8 were low (<1,000), the microbial community structure of these two samples was similar to other MISNAC samples. Therefore, it can be seen that the community structure of the cultured microorganisms is completely different from that of the water sample bottle and in-situ filtered microorganisms, proving the feasibility of the system in the embodiments of the present invention. From March to April 2022, the system, carried by the Phoenix submersible, completed six deep-sea in-situ microbial cultures at depths of 600-3000 meters in the South China Sea, adding sodium bicarbonate and ammonium chloride to screen and cultivate carbon-fixing microorganisms such as ammonia-oxidizing archaea.

[0055] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several equivalent substitutions or obvious modifications can be made without departing from the concept of the present invention, and all such modifications, achieving the same performance or purpose, should be considered within the scope of protection of the present invention.

Claims

1. A deep-sea in-situ microbial culture and collection system, characterized in that, The system includes an automated microbial collection device, a deep-sea in-situ microbial culture chamber, a pipeline switching device, and a microbial enrichment and preservation device. The automated microbial collection device collects seawater and culture stock solution, which are then delivered to the deep-sea in-situ microbial culture chamber. After injecting seawater into the chamber, the automated microbial collection device injects the culture stock solution, which is a sodium bicarbonate culture stock solution labeled with C14 and with a final concentration of 0.1M, for in-situ microbial culture. The microbial enrichment and preservation device includes multiple microbial enrichment filter discs. The deep-sea in-situ microbial culture chamber is connected to each microbial enrichment filter disc via the pipeline switching device. By controlling the pipeline switching device, the deep-sea in-situ microbial culture chamber is switched to different microbial enrichment filter discs at different culture times, and each microbial enrichment filter disc collects microbial samples at different culture times. The system also includes a glycerol addition device, which is connected to each microbial enrichment filter disc via the pipeline switching device, to add glycerol as a preservative to the microbial enrichment filter discs to preserve the microorganisms.

2. The deep-sea in-situ microbial culture and collection system as described in claim 1, characterized in that, The microbial enrichment filter disc includes a cavity and a filter membrane with corresponding pore sizes, on which microorganisms of corresponding sizes are enriched.

3. The deep-sea in-situ microbial culture and collection system as described in any one of claims 1 to 2, characterized in that, It also includes at least one bacterial culture filter plate and a water bag. The deep-sea in-situ microbial culture chamber is connected to the bacterial culture filter plate through the pipeline switching device. The bacterial culture filter plate is connected to the water bag. The cultured bacterial culture is filtered by the bacterial culture filter plate to remove microorganisms of a limited size and then stored in the water bag.

4. The deep-sea in-situ microbial culture and collection system as described in any one of claims 1 to 2, characterized in that, The pipeline switching device includes several two-position two-way valves and their seats, with each two-position two-way valve connected to a corresponding filter disc.

5. The deep-sea in-situ microbial culture and collection system as described in any one of claims 1 to 2, characterized in that, Pumps are connected between the automatic microbial collection device and the deep-sea in-situ microbial culture chamber, and between the deep-sea in-situ microbial culture chamber and the microbial enrichment and preservation device.

6. The deep-sea in-situ microbial culture and collection system as described in claim 5, characterized in that, The pump includes a peristaltic pump.

7. A method for in-situ cultivation and collection of deep-sea microorganisms, characterized in that, Deep-sea in-situ microbial culture and collection using the system described in any one of claims 1 to 6.

Images