Phytoplankton in-situ culture device and culture method

Abstract

The invention provides a phytoplankton in-situ culture device and a culture method, the phytoplankton in-situ culture device comprises: a culture cabin, the culture cabin is a cylinder structure with the top and the bottom open, the side wall of the cylinder is provided with a plurality of holes, each hole is covered with a screen, and an inner culture cabin can be arranged in the cylinder to form a double-layer culture cabin structure; the buoyancy platform comprises at least one floating body connected with an upper hook and is used for being connected with the top of the culture cabin through the upper hook and enabling the whole culture device to float at a specific position in the water body; and the gravity system comprises a weight connected with the bottom of the culture cabin and is used for keeping the culture device stable and enabling the culture cabin to be located at a preset water depth. The problems that in-situ culture of phytoplankton in the prior art, environment simulation is incomplete, water-sediment interface matter exchange is difficult, sampling is inconvenient, and experimental conditions are unstable can be effectively solved.

Description

Technical Field

[0001] This application relates to the field of microbial culture and aquatic ecological monitoring, and more specifically to an in-situ phytoplankton culture device and culture method. Background Technology

[0002] Phytoplankton are a class of tiny organisms that possess photosynthetic pigments, enabling them to perform photosynthesis and live a planktonic life. As an important component of aquatic ecosystems, phytoplankton are fundamental to energy flow and material cycling, and changes in their community structure can affect the stability of aquatic ecosystems. The growth, development, and distribution of phytoplankton are regulated by a variety of environmental factors, and the mechanisms involved are highly complex. Numerous studies have shown that physical factors such as temperature, light, and hydrodynamics; chemical factors such as nutrients, pH, and ions; and biological factors such as grazing pressure and interspecific competition are all driving factors for phytoplankton growth and development, and different phytoplankton species respond differently to these environmental factors. Therefore, exploring the different behaviors of various phytoplankton at different water layers can not only reveal the growth, development, and seasonal succession patterns of phytoplankton, clarify the spatial heterogeneity of phytoplankton community structure at horizontal and vertical scales, analyze the correlation between aquatic environmental factors and phytoplankton, and explore the factors and extent driving the spatiotemporal distribution characteristics of phytoplankton, but also elucidate the intrinsic mechanisms of phytoplankton community structure changes. This has a positive impact on biological research on phytoplankton species diversity and is also significant for understanding the processes of occurrence, development, succession, decline, and extinction involved in the development of aquatic microorganisms. Furthermore, dinoflagellates are an important component of phytoplankton in both freshwater and marine environments, exhibiting diverse morphological variations and being one of the algae that form red tides and algal blooms. Dinoflagellates possess the ability to live both planktonic and benthic lives, and their nutritional modes are diverse and complex, including autotrophic, heterotrophic, and mixed trophic types. Therefore, when exploring the mechanisms of dinoflagellate bloom outbreaks, persistence, and decline, as well as a series of driving forces for algal bloom succession, the mixed trophic mode of dinoflagellates should be considered. The mixed-trophic feeding behavior of dinoflagellates indicates a strong ability to utilize nutrients. They can obtain energy through photosynthesis and also engage in heterotrophic feeding by consuming cyanobacteria, green algae, diatoms, and other protozoa. This diverse nutritional approach makes the micro-food web structure of dinoflagellates more complex. However, current laboratory culture techniques have significant limitations in isolating and culturing certain dinoflagellates, and the selectivity of their feeding (such as cyanobacteria and green algae) remains unclear. Therefore, exploring the feeding behavior of mixed-trophic dinoflagellates is not only beneficial to the biological research on the diversity of aquatic microbial species, but also of great significance to understanding the processes involved in the occurrence, development, succession, decline, and extinction of algal blooms.

[0003] In the existing fields of microbial culture and aquatic ecological monitoring, research on the growth mechanism of phytoplankton mostly relies on indoor culture experiments. Although this experimental method reveals the growth characteristics of a single algal species to some extent, it has the following limitations because the laboratory environment cannot fully simulate the complexity of natural aquatic ecosystems: 1. Lack of species diversity: Laboratory culture can often only isolate or enrich some aquatic microorganisms, while ignoring those species that can only grow in natural environments or need to coexist with other microorganisms. This may result in research results that do not fully reflect the species composition of the real ecosystem.

[0004] 2. Environmental condition deviation: Indoor experiments cannot accurately replicate the real physicochemical conditions of water bodies, such as light, temperature, and hydrodynamics. In particular, the simulation of the important factor of light intensity changing with depth is insufficient, which makes the experimental results significantly different when explaining natural water phenomena.

[0005] 3. Neglecting the water-sediment interface: Most studies focus only on the surface of the water body, neglecting the study of the bottom layer and sediments. In particular, during the process of algal bloom extinction, the changes in the overlying water and sediments of the bottom sediment are equally important, but current technology makes it difficult to effectively observe and study this in situ.

[0006] 4. Operational inconvenience and cost issues: In situ culture experiments usually require control of individual environmental factors, which is relatively complex to operate. Furthermore, there is a lack of devices that are suitable for different water depths, easy to adjust, and reusable, which increases the cost and difficulty of the experiment.

[0007] 5. Limitations of short-term experiments: Existing in-situ culture devices often only allow for short-term experiments and cannot be used for long-term fixed-point observations, which limits in-depth research into the growth process of microorganisms, especially the seasonal succession patterns.

[0008] In summary, current cultivation methods and technologies have significant shortcomings in meeting the needs of different water depths, facilitating material exchange at the bottom and sediment interface, and adapting to long-term fixed-point monitoring. There is an urgent need to develop a new in-situ phytoplankton cultivation device to overcome these shortcomings, so as to more accurately study the vertical spatial heterogeneity of phytoplankton communities and the relationship between environmental factors, as well as to better study the feeding behavior of mixed-trophic dinoflagellates. Summary of the Invention

[0009] To address the aforementioned issues, this application presents an in-situ phytoplankton culture device and method. By optimizing the device structure, improving the in-situ nature of experimental conditions, and simplifying the operation process, a more comprehensive and precise study of phytoplankton growth and the feeding behavior of mixed-trophic dinoflagellates can be achieved.

[0010] The technical solution of this application is as follows: One aspect of this application provides an in-situ phytoplankton cultivation device, including a single-layer cultivation chamber, a buoyancy platform, and a gravity system.

[0011] The single-layer culture chamber is a cylindrical structure with openings at the top and bottom. The cylinder is made of a light-transmitting material, and the side wall of the cylinder has multiple openings. Each opening is covered with a screen block to allow the exchange of nutrients and water but prevent phytoplankton from passing through.

[0012] The buoyancy platform includes at least one float connected to an upper hook, which is used to connect to the top of the culture chamber via the upper hook, and to float the entire culture device at a predetermined water depth in the water, and to fix the shape of the entire culture device.

[0013] The gravity system includes a weight connected to the bottom of the single-layer culture chamber to maintain the stability of the culture device and position the single-layer culture chamber at a predetermined water depth.

[0014] This application also provides another in-situ phytoplankton culture device, including a double-layer culture chamber, a buoyancy platform and a gravity system.

[0015] The double-layered culture chamber consists of an inner culture chamber and an outer culture chamber. The outer culture chamber is a cylindrical structure with open top and bottom, made of a translucent material. Multiple openings are provided on the side walls of the cylinder, each covered with a screen block to allow nutrient and water exchange while preventing phytoplankton from passing through. The inner culture chamber is a screen cylinder with open top and bottom. The screen aperture of the inner culture chamber is 20-50 μm, used to accommodate larger target phytoplankton and some nutrients, while restricting the passage of target phytoplankton. The system allows for the movement of algae, but permits small algae (such as cyanobacteria <10μm and green algae <15μm) to freely enter and exit; the screen aperture on the outer culture chamber is 0.1-20μm, allowing the exchange of dissolved nutrients (such as nitrates and phosphates) with the water, but completely blocking the free entry and exit of natural water bodies and all planktonic algae in the culture device; the bottoms of the inner and outer culture chambers are connected by a circular screen, the aperture of which is consistent with that of the screen in the inner culture chamber, maintaining the nutrient gradient between the inner and outer layers and simulating the nutrient diffusion process in the natural environment.

[0016] The buoyancy platform includes at least one float connected to an upper hook, which is used to connect to the top of the double-layer culture chamber via the upper hook, and to make the entire culture device float in the water at a predetermined depth, and to fix the shape of the entire culture device. The gravity system includes a weight connected to the bottom of the double-layered culture chamber to maintain the stability of the culture device and keep the culture device at a predetermined water depth.

[0017] In one embodiment of this application, the openings on the side wall of the culture chamber are polygonal or circular openings, with the total opening area accounting for 30%-50% of the side wall surface area. The openings are arranged in an orthogonal matrix distribution manner, and the spacing between adjacent openings is 1.2-2 times the diameter of the opening.

[0018] In one embodiment of this application, the mesh size of the screen on the single-layer culture chamber is 0.1-20μm, which is determined in combination with the size of the target cultured algae. For example, Chlorella needs to be ≤5μm, while diatoms can be relaxed to 20μm, etc.

[0019] In one embodiment of this application, the culture chamber is made of PVC soft crystal plate material with high light transmittance, with a thickness of 0.5-5mm and a light transmittance of ≥90%.

[0020] In one embodiment of this application, the buoyancy platform comprises an upper float plate, a lower float plate, and a tubular column connecting the two. The height of the tubular column ranges from 30 to 100 cm to provide sufficient buoyancy. The tubular column adopts an annular cavity structure, with an inner diameter the same as the diameter of a single-layer culture chamber or an inner culture chamber, and an outer diameter the same as the diameter of an outer culture chamber. The outer diameter of the tubular column is 1.2-1.5 times the inner diameter, ensuring sufficient installation clearance through the area multiplication principle. The tubular column is made of PP polypropylene with a density of 0.9 g / cm³, providing approximately 200 kg of buoyancy.

[0021] In one embodiment of this application, the gravity system further includes a support platform connected to the bottom of the culture chamber via a lower hook, and the weight is connected to the support platform. Preferably, the weight is made of stone and is connected to the bottom of the culture chamber or the support platform via ropes.

[0022] Another aspect of this application provides a method for in-situ culture of phytoplankton, employing the above-mentioned single-layer culture chamber phytoplankton in-situ culture device, comprising the following steps: (1) The culture chamber is suspended on the buoyancy platform by the upper hook of the buoyancy platform, and the gravity system is connected to the bottom of the culture chamber by the lower hook of the support platform to form a stable vertical positioning structure.

[0023] (2) Slowly place the in-situ culture device into the water body at the predetermined location until it reaches the predetermined water depth, ensuring that the culture chamber remains in contact with both the surface layer and the bottom sediment layer. Contact with the surface layer ensures that the organisms (such as algae and phytoplankton) in the culture chamber receive natural light intensity, temperature, and gas exchange, simulating real photosynthetic conditions. At the same time, contact with the bottom sediment layer allows for benthic-water layer material exchange, reflecting the physical, chemical, and biological processes at the sediment-water interface, ensuring that the experiment accurately reflects the real functions of the benthic ecosystem.

[0024] (3) The in-situ culture device is left to stand in water for at least 24 hours to ensure that the natural exchange of water flow and nutrients inside and outside the culture chamber reaches a stable state.

[0025] (4) According to the cultivation objective, the target algae species that have been specially marked are slowly introduced into the cultivation chamber.

[0026] (5) At predetermined time intervals, water and mud samples are collected from the inside of the culture chamber at the same time point, and other monitoring activities are carried out to assess the growth status of phytoplankton and the impact of environmental factors on its growth.

[0027] This application also provides another method for in-situ phytoplankton culture to study the feeding behavior of mixed-trophic dinoflagellates, which uses an in-situ phytoplankton culture device with a double-layered culture chamber, and includes the following steps: (1) The double-layer culture chamber is suspended on the buoyancy platform by the upper hook of the buoyancy platform, and the gravity system is connected to the bottom of the culture chamber to form a stable vertical positioning structure.

[0028] (2) Slowly place the assembled in-situ culture device into the water body at the predetermined location until it reaches the predetermined water depth, ensuring that the culture chamber remains in contact with both the surface layer and the bottom sediment layer. In this way, the inner culture chamber, with its screen aperture designed to be 20-50μm, can effectively trap large algal communities and dissolved nutrients in the natural water body, restricting the target dinoflagellates from entering the outer culture chamber, but allowing small algae (such as cyanobacteria <10μm and green algae <15μm) to freely enter and exit, forming a semi-closed ecological retention effect. The bottom circular screen connecting the inner and outer culture chambers, with the same aperture as the inner screen, can also effectively prevent dinoflagellates from entering the outer culture chamber. At this time, the dinoflagellates are confined in the inner chamber and cannot escape, but can come into contact with small algae (such as cyanobacteria and green algae) that enter through the screen, while there are almost no large dinoflagellates in the outer culture chamber.

[0029] (3) The in-situ culture device is placed in water for at least 24 hours to ensure that the natural exchange of water flow and nutrients inside and outside the culture chamber reaches a stable state, the phytoplankton growth is stable, and the light reduction rate in the device is tested.

[0030] (4) According to the cultivation objective, the target dinoflagellates are slowly introduced into the inner culture chamber (starvation treatment to eliminate endogenous nutrient interference), and the target small algae species with special marking (such as cyanobacteria with added ¹³C) or small algae of different concentrations (such as curly anthocyanin) are placed in the outer culture chamber.

[0031] (5) Collect water and mud samples from the inner and outer culture chambers at the same time point within a predetermined time interval, and conduct other monitoring activities to assess the feeding behavior of mixed-trophic dinoflagellates. Compared with the prior art, this application has the following advantages and application benefits: 1. Realistic environmental simulation: The design of the culture device allows phytoplankton to grow in an environment close to nature, effectively avoiding the deviations that may occur under laboratory conditions, and the results are more reliable. Specifically, it is reflected in: (1) The screen on the side wall of the culture chamber can ensure the exchange of natural water and nutrients inside and outside, and the screen of a specific size allows phytoplankton of different sizes to selectively enter. (2) The staggered openings on the side wall of the culture chamber are covered by the screen, and water exchange can be carried out on all four sides, which can fully reflect the influence of nutrients and flow rate in the water on the growth of phytoplankton. (3) The double-layer culture chamber design ensures the exchange of natural water and nutrients inside and outside, and the inner culture chamber does not allow dinoflagellates to enter and exit but allows cyanobacteria and green algae to selectively enter, while the outer culture chamber does not allow phytoplankton inside and outside the device to freely enter and exit, which is suitable for the study of the feeding behavior of mixed-nutrient dinoflagellates. (4) The culture chamber uses high light transmittance material, which can ensure that phytoplankton receive sufficient light. (5) The culture chamber can be fixedly placed at a specific site in the river, and the experimental conditions have good in-situ properties. (6) The buoyancy platform and gravity system form an anchor system. The top and bottom of the cultivation device are open. The top of the cultivation device is in contact with the water surface and the bottom is in contact with the sediment at the bottom of the water, so as to realize the exchange of substances at the water-sediment interface, which is convenient for the release of algae seeds and for monitoring and sample collection.

[0032] 2. Simple and practical: The in-situ algae culture device of this application has a simple principle, making it easy to set up control and parallel experiments. It is convenient to operate, allows for better control of experimental variables, and facilitates the achievement of experimental objectives, demonstrating practicality and applicability. The assembly and underwater deployment of the device are relatively simple, reducing complex operating procedures and saving time and labor costs. It is easy to mass-produce and maintain, portable, easy to adjust, and reusable multiple times.

[0033] 3. Wide Applicability: Depending on the experimental purpose, the in-situ culture device of this application can also be used to study the autotrophic, heterotrophic, and mixed nutrient behaviors among different plankton, the effect of light intensity on plankton growth, the effect of different euphotic or mixed-layer depths on plankton growth, the effect of nutrient addition on plankton growth, and experiments on water-sediment interface material exchange, etc. Furthermore, the device is adaptable to freshwater lakes, rivers, and marine environments, expanding the geographical scope of phytoplankton research.

[0034] 4. Comprehensive data collection: Through regular sampling and monitoring, detailed information on phytoplankton growth can be collected, including factors such as nutrient absorption, light influence, and temperature changes, providing strong data support for ecosystem health assessment and restoration. Attached Figure Description

[0035] The accompanying drawings, which are provided to further illustrate this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application.

[0036] Figure 1 This is a three-dimensional schematic diagram of an in-situ culture device with a single-layer culture chamber provided in an embodiment of the present invention; Figure 2 This is a three-dimensional schematic diagram of a single-layer culture chamber provided in an embodiment of the present invention; Figure 3 This is a schematic cross-sectional view of a single-layer culture chamber provided in an embodiment of the present invention; Figure 4 This is a three-dimensional schematic diagram of the buoyancy platform provided in an embodiment of the present invention; Figure 5 This is a three-dimensional schematic diagram of the gravity system provided in an embodiment of the present invention.

[0037] Figure 6 This is a three-dimensional schematic diagram of an in-situ culture device with a double-layer culture chamber provided in an embodiment of the present invention; Figure 7 This is a three-dimensional schematic diagram of the inner and outer layers of the double-layer culture chamber provided in an embodiment of the present invention; Figure 8 This is a schematic diagram of the inner and outer cross-sections of the double-layer culture chamber provided in an embodiment of the present invention; Figure 9 This is a top view schematic diagram of the bottom circular screen of the double-layer culture chamber provided in an embodiment of the present invention.

[0038] The following are the labeling elements in the figure: 1. Upper floating plate, 2. Pipe column, 3. Lower floating plate, 4. Upper hook, 5. Lower hook, 6. Gravity support platform, 7. Rope, 8. Stones, 9. Screen block, 10. PVC crystal plate cylinder, 11. Outer culture chamber, 12. Screen cylinder, 13. Inner culture chamber, 14. Circular screen. Detailed Implementation

[0039] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0040] It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0041] In this application, unless otherwise stated, directional terms such as "upper," "lower," "top," and "bottom" are generally used in relation to the direction shown in the accompanying drawings, or in relation to the vertical, perpendicular, or gravitational direction of the component itself; similarly, for ease of understanding and description, "inner" and "outer" refer to the inner and outer contours of each component itself, but the above directional terms are not intended to limit this application. One embodiment of this application provides an in-situ phytoplankton culture device with a single-layer culture chamber, see [link to relevant documentation]. Figure 1 The device includes a single-layer culture chamber, which is a 120cm diameter PVC crystal plate cylinder 10 with openings at the top and bottom. Multiple openings are provided on the side walls of the cylinder, each covered with a 5μm sieve block 9 to allow nutrient and water exchange while preventing phytoplankton passage. A buoyancy platform is also included, comprising at least one float connected to an upper hook 4 for connecting to the top of the culture chamber and buoyant the entire device at a specific position in the water. A gravity system is also included, comprising at least one weight connected to the bottom of the culture chamber via a lower hook 5, to maintain the stability of the device, ensuring the culture chamber is in contact with the bottom of the water or at a predetermined depth. This embodiment, by designing a culture chamber with openings at the top and bottom, combined with a buoyancy platform and a gravity system, achieves stable positioning of the device in the water, ensuring natural exchange of substances at the water-sediment interface. It utilizes the sieve mesh size to control the free flow of nutrients and water, while effectively preventing the random migration of phytoplankton and maintaining the in-situ nature of the experimental conditions. The technology in this embodiment ensures the similarity of the internal and external environments of the cultivation device, promotes the study of phytoplankton growth under natural conditions, and improves the accuracy and reliability of the experiment. In other embodiments, the mesh size can be adjusted to accommodate phytoplankton of different sizes, addressing the in-situ cultivation needs of specific algal species.

[0042] In a further embodiment, see Figures 1-3The openings on the sidewall of the cultivation chamber are square, measuring 50cm x 50cm, and are arranged in an arithmetic, staggered pattern along the height of the sidewall. These openings are covered by 5μm pore size screen blocks 9, tightly sewn with nylon thread onto the outer PVC soft crystal plate of the cultivation chamber. This embodiment, by setting square openings on the sidewall of the cultivation chamber and using a staggered distribution design, increases the effective area for water and nutrient exchange between the inside and outside of the device. This design ensures the uniformity of the environment inside and outside the cultivation chamber, avoiding uneven local water flow or nutrient concentration, and providing a more realistic growth environment for phytoplankton. The technology in this embodiment optimizes the efficiency of water and nutrient exchange, promotes the uniform distribution and growth of phytoplankton, and improves the representativeness of the experimental results. In other embodiments, the shape and distribution pattern of the openings can be changed, such as circular, elliptical, or other geometric shapes, and a non-staggered distribution method can be used to adapt to different aquatic environments and experimental needs, solving the problem of in-situ phytoplankton cultivation under specific hydrological conditions.

[0043] In a further embodiment, refer to Figures 1 to 3 As shown, the cultivation chamber is made of highly transparent PVC soft crystal plate material, with a thickness of 0.39 mm. The height of the PVC soft crystal plate cylinder 10 can be flexibly selected according to the water depth. This embodiment uses highly transparent PVC soft crystal plate material, which provides the necessary conditions for phytoplankton photosynthesis, ensuring that the phytoplankton can receive sufficient natural light. At the same time, the choice of material flexibility and thickness balances light transmittance and structural strength, ensuring the stability of the device in water, as well as its portability and durability. Therefore, the technology in this embodiment ensures the normal growth of phytoplankton under natural light conditions, while the material's lightweight and thin characteristics make the device easy to transport and deploy, reducing experimental costs. In other embodiments, other highly transparent, lightweight, and durable materials, such as polycarbonate or special glass fiber composite materials, can be selected to address the need for in-situ phytoplankton cultivation under extreme light conditions or harsh environments.

[0044] In a further embodiment, refer to Figure 1 and Figure 4As shown, the upper float 1 and lower float 3 of the buoyancy platform are connected by a hollow tube column 2. The tube column 2 is a circular column with an inner diameter of 80cm, an outer diameter of 120cm, and a height of 50cm. The upper float 1, lower float 3, and tube column 2 are made of PP polypropylene material cut and bonded together. A hook 4 is installed on the bottom side wall of the lower float 3 of the buoyancy platform. This hook connects and suspends the PVC soft crystal plate cylinder 10 to the buoyancy platform. The buoyancy platform can fix the shape and water surface position of the entire culture device. This embodiment, by designing a hollow tube column to connect the upper and lower floats, forms a stable buoyancy structure, ensuring the vertical positioning of the culture device in the water and ensuring its stable suspension in the water. This avoids the influence of water flow or wind on the device's position, improving the controllability and accuracy of the experiment. The size design of the hollow tube column matches the culture chamber, ensuring sufficient buoyancy without excessively increasing the size and weight of the device, making the device easy to operate and position. In other embodiments, the height and outer diameter of the tubular column can be adjusted, and other types of floating materials, such as foam plastics or lightweight alloys, can be selected to adapt to experimental needs under different water depths and environmental conditions, thus solving the problem of in-situ culture of phytoplankton in specific water environments.

[0045] In a further embodiment, refer to Figure 1 and Figure 5 As shown, the gravity support platform 6 of the gravity system fixes the shape of the culture chamber and the experimental site via a lower hook 5. The weight in the gravity system is a granite block 8 with a perforation in the center, which is connected to the bottom of the gravity support platform 6 by a wear-resistant, waterproof, and corrosion-resistant nylon rope 7, allowing the entire culture device to contact the sediment at the bottom of the river. In this embodiment, the use of granite blocks as the gravity source ensures the stable sinking of the device in the water, while the wear-resistant, waterproof, and corrosion-resistant properties of the nylon rope ensure the reliability of the connection and the long-term use of the device. This ensures that the culture device can accurately contact the bottom of the water or reach a predetermined water depth, providing a basis for studying the material exchange between the bottom of the water body and the sediment interface. In other embodiments, other high-density materials, such as iron or lead blocks, and different types of rope materials, such as polyester rope or steel wire rope, can be selected to adapt to different water environments and experimental needs, solving the problem of in-situ phytoplankton culture under special water conditions.

[0046] This application also provides an embodiment of in-situ phytoplankton culture using the above-mentioned device, the culture method including the following steps: Step 1: The in-situ culture chamber of the in-situ culture device is set at a height according to the water depth. The culture chamber is suspended on the buoyancy platform through the upper hook of the buoyancy platform, and the gravity system is connected to the bottom of the culture chamber through the lower hook of the support platform to form a stable vertical positioning structure.

[0047] Step 2: Determine a specific location on the river surface and slowly lower the in-situ culture device into the water at the predetermined location, ensuring it reaches the desired depth and that the culture chamber remains in contact with both the surface and bottom sediment layers. A buoyancy platform and gravity system form an anchorage, and the entire device is placed at the specific location on the river. Water from the natural water body and phytoplankton enter the in-situ culture device.

[0048] Step 3: Place the device in the water for at least 24 hours to maintain the stability of the internal and external water flow and nutrient exchange rate, as well as the phytoplankton growth status, while testing the light reduction rate in the device.

[0049] Phytoplankton grow stably in the culture chamber, and water flow and nutrients can be stably exchanged through the square screen block 9. Due to differences in size, all phytoplankton in natural water bodies and culture devices cannot pass through the square screen block 9 of the culture chamber and cannot migrate freely within the culture chamber.

[0050] Step 4: According to the cultivation objective, slowly introduce the target algae species marked with ¹³C into the cultivation chamber 11.

[0051] For example, depending on the experimental purpose, target algae species such as Chlorella and Anabaena can be placed in the culture chamber 11 with ¹³C labeling. The in-situ culture device is stable, and the phytoplankton can move freely under natural conditions.

[0052] Step 5: As needed, collect water and mud samples from the culture chamber at the same time point within a predetermined time interval, and conduct other monitoring activities to assess the growth status of phytoplankton and the impact of environmental factors on its growth.

[0053] According to experimental requirements, the in-situ culture device can be placed in different water layers, and samples of sediment at the bottom of the gravity support platform 6 can be collected to gather system data for experimental analysis.

[0054] This embodiment, through a series of designed steps, ensures the correct deployment and experimental operation of the phytoplankton in-situ culture device, improving the standardization and reproducibility of the experiment. By slowly placing the device into the water and allowing it to stand for 24 hours, the internal and external environments of the device reach equilibrium, providing stable conditions for subsequent experiments, ensuring standardized experimental procedures, promoting the study of phytoplankton growth under natural conditions, and improving the accuracy and reliability of experimental results. In other embodiments, specific details of the experimental operation, such as the method of introducing algal species, the sampling time and frequency, can be adjusted to adapt to different experimental needs and solve the problems of in-situ culture of specific algal species and research on environmental factors.

[0055] In a further embodiment, the target algal species is a cyanobacterial or green algae labeled with a radioactive isotope. Radioactive isotope labeling technology provides a means for dynamic monitoring of algal species, distinguishing experimental algal species from algae in natural water bodies, and enabling the study of specific algal species behaviors. Therefore, the technology in this embodiment ensures the traceability of the experimental algal species, promotes in-depth research on algal growth and metabolic processes, and enhances the scientific value of the experimental results.

[0056] In a further embodiment, the slow descent into the water is performed by a diver to minimize disturbance to the water and bottom sediment. The diver's expertise and experience allow for precise control of the device's descent speed and direction, avoiding unnecessary agitation of the water flow and sediment. This provides a more natural and stable environment for the experiment, ensuring the in-situ nature of the experimental conditions, promoting research on phytoplankton growth in the natural environment, and improving the representativeness and reliability of the experimental results.

[0057] In a further embodiment, water quality multi-parameter analyzers, water samplers, and sediment samplers are used for sampling. The sampling frequency and sampling depth can be adjusted according to experimental needs. These devices can accurately measure key environmental factors such as water temperature, light intensity, and nutrient content, and collect water and sediment samples simultaneously. This allows for comprehensive monitoring of various parameters of the water body and sediments, ensuring the accuracy and completeness of experimental data and providing data support for analyzing the growth status and environmental impact of phytoplankton.

[0058] The above methods ensure the standardization and normalization of experimental operations, while minimizing external interference, providing an efficient and accurate tool for in-situ culture and environmental response research of phytoplankton.

[0059] In some of the following embodiments, an in-situ culture device with a double-layer culture chamber is further provided for studying the feeding behavior of mixed-trophic dinoflagellates.

[0060] See Figures 6-9This device features a double-layered culture chamber. Based on the aforementioned culture device, it incorporates a PVC crystal plate cylinder 10 as the outer culture chamber 11, and an additional screen cylinder 12 as the inner culture chamber 13. Specifically, the outer culture chamber 11, like the single-layered culture chamber in the previous embodiment, is a cylindrical cylinder with openings at the top and bottom, with a diameter of 120cm, made of 0.39mm thick PVC soft crystal plate material. The height of the cylinder can be flexibly selected according to the water depth. The cylindrical sidewall of the outer culture chamber 11 has 50cm x 50cm square openings arranged in a staggered, equally layered pattern, covered by nylon screen blocks 9 with a 5μm aperture, tightly sewn to the PVC soft crystal plate of the outer culture chamber with nylon thread. The added inner culture chamber 13 is also a cylindrical screen cylinder 12 with openings at the top and bottom, with a diameter of 80cm, made of wear-resistant, waterproof, and corrosion-resistant nylon screen material with a 50μm aperture. A 50μm circular screen 14 connects the bottom cylinders of the inner and outer culture chambers. The buoyancy platform and gravity system of the culture device can be the same as those of the single-layer culture chamber culture device described above.

[0061] The following examples illustrate an in-situ culture method for studying the predatory behavior of dinoflagellates against cyanobacteria and green algae, and the steps are as follows: Step 1: The in-situ culture chambers are set at a height according to the water depth. The inner and outer culture chambers are suspended on the buoyancy platform via the upper hooks of the buoyancy platform. The gravity system is suspended at the bottom of the inner and outer culture chambers to form a stable vertical positioning structure.

[0062] Step 2: Determine a specific location on the river surface and slowly lower the in-situ culture device into the water at the predetermined location, ensuring it reaches the desired depth. Minimize disturbance to the water flow and bottom sediment, guaranteeing contact between the culture chamber and both the surface and bottom sediment layers. A buoyancy platform and gravity system form an anchorage, and the entire device is placed at the specific river location. Water from the natural water body and phytoplankton enter the in-situ culture device.

[0063] Step 3: Place the culture device in water for at least one day to maintain the stability of the water flow inside and outside the in-situ culture device, the rate of nutrient exchange, and the growth status of phytoplankton, and test the light reduction rate in the device.

[0064] During this period, phytoplankton in the water grow stably in the inner and outer culture chambers, and water flow and nutrients can be stably exchanged through the inner culture chamber, the bottom screen, and the screen in the outer culture chamber. Due to the size differences of phytoplankton, larger dinoflagellates can only swim in the inner culture chamber 13 area, while smaller cyanobacteria and green algae can not only enter the inner culture chamber 11, but also enter the outer culture chamber 13 through the 50μm screen cylinder 12 of the inner culture chamber and the bottom circular screen 14. They can migrate freely in the inner and outer culture chambers 11 and 13, but cannot pass through the 5μm screen block 9 of the outer culture chamber.

[0065] Step 4: Based on the culture objective, slowly introduce the target dinoflagellate into the inner culture chamber, and slowly add cyanobacteria or green algae with ¹³C markers cultured indoors into the outer culture chamber. Small algae of different concentrations can also be introduced.

[0066] According to the experimental objective, the target dinoflagellates are slowly introduced into the inner culture chamber through the tube column 2 of the buoyancy platform. Blue-green algae or green algae with ¹³C labeling, or small algae such as Chlorella or Anabaena, are placed in the outer culture chamber 11. Once the in-situ culture device is stable, the dinoflagellates in the inner culture chamber can freely choose autotrophic or heterotrophic feeding modes under natural conditions.

[0067] Step 5: As needed, place water quality multi-parameter instrument, water sampler, mud sampler and other equipment into the inner and outer culture chambers at the same time intervals and slowly collect samples.

[0068] According to experimental requirements, the in-situ culture device can be sampled in different water layers in the inner and outer culture chambers, as well as the sediment at the bottom of the device, through the tubing of the buoyancy platform, and the system data can be collected for experimental analysis.

[0069] For example, monitoring changes in dinoflagellate biomass and the number of small algae in the inner chamber, as well as the nutrient concentration in the outer chamber, can demonstrate that if the dinoflagellate biomass increases in the inner chamber and the number of small algae decreases significantly, while the nutrient concentration in the outer chamber remains stable, it indicates that the dinoflagellates obtain nutrients by feeding on small algae (heterotrophic). To rule out autotrophic dinoflagellates, a black cloth with a light-blocking function can be placed on top of the cultivation device to block natural light and prevent the dinoflagellates from autotrophically consuming them through photosynthesis. If elemental analysis-isotope mass spectrometry shows a positive correlation between the dinoflagellate growth rate and the amount of ¹³C absorbed, and electron microscopy reveals cyanobacterial cell fragments present in the dinoflagellate phagocytic vesicles, it can be inferred that the dinoflagellates obtain nutrients through feeding and that a predator-prey relationship exists between them and cyanobacteria.

[0070] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0071] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A device for in-situ cultivation of phytoplankton, characterized in that, include: A single-layer culture chamber, wherein the single-layer culture chamber is a cylindrical structure with openings at the top and bottom. The cylinder is made of a light-transmitting material and has multiple openings on the side wall. Each opening is covered with a screen block to allow the exchange of nutrients and water but prevent phytoplankton from passing through. A buoyancy platform, comprising at least one float connected to an upper hook, for connecting to the top of the single-layer culture chamber via the upper hook, and for making the entire culture device float in the water at a predetermined depth, and for fixing the shape of the entire culture device; A gravity system, comprising a weight connected to the bottom of a single-layer culture chamber, for maintaining the stability of the culture device and positioning the single-layer culture chamber at a predetermined water depth.

2. A device for in-situ cultivation of phytoplankton, characterized in that, include: The double-layered culture chamber consists of an inner and an outer culture chamber. The outer culture chamber is a cylindrical structure with open top and bottom, made of a translucent material. Multiple openings are located on the side walls, each covered by a screen block to allow nutrient exchange with the water while preventing phytoplankton from passing through. The inner culture chamber is also a screen cylinder with open top and bottom. The screen aperture of the inner culture chamber is 20-50 μm, designed to accommodate larger target phytoplankton and some nutrients, restricting the movement of the target phytoplankton while allowing smaller algae to enter and exit freely. The screen block on the outer culture chamber has an aperture of 0.1-20 μm, allowing dissolved nutrients to exchange with the water but completely blocking the free entry and exit of natural water and all phytoplankton in the culture device. The bottoms of the inner and outer culture chambers are connected by a circular screen with the same aperture as the inner culture chamber, maintaining a nutrient gradient between the inner and outer layers and simulating the nutrient diffusion process in the natural environment. A buoyancy platform, comprising at least one float connected to an upper hook, for connecting to the top of the double-layered culture chamber via the upper hook, and for making the entire culture device float in the water at a predetermined depth, and for fixing the shape of the entire culture device; A gravity system, comprising a weight connected to the bottom of a double-layered culture chamber, is used to maintain the stability of the culture device and position the culture device at a predetermined water depth.

3. The in-situ phytoplankton culture device according to claim 1 or 2, characterized in that, The openings on the side wall of the culture chamber are polygonal or circular, with the total opening area accounting for 30%-50% of the side wall surface area. The openings are arranged in an orthogonal matrix distribution, and the spacing between adjacent openings is 1.2-2 times the diameter of the opening.

4. The in-situ phytoplankton culture device according to claim 3, characterized in that, The openings are arranged in equal layers and in a cross pattern along the height of the side wall of the culture chamber.

5. The in-situ phytoplankton culture device according to claim 1, characterized in that, The aperture of the screen block with the opening is 0.1-20 μm, determined in conjunction with the size of the target cultured algae.

6. The in-situ phytoplankton culture device according to claim 3, characterized in that, The light-transmitting material is PVC flexible crystal board material with a thickness of 0.5-5mm and a light transmittance of ≥90%.

7. The in-situ phytoplankton culture device according to claim 3, characterized in that, The buoyancy platform comprises an upper float plate, a lower float plate, and a connecting column. The height of the connecting column ranges from 30 to 100 cm to provide sufficient buoyancy. The connecting column adopts an annular cavity structure. The outer diameter of the connecting column is 1.2 to 1.5 times the inner diameter, ensuring sufficient installation clearance through the principle of area multiplication. The connecting column is made of PP polypropylene with a density of 0.9 g / cm³, which can provide approximately 200 kg of buoyancy.

8. The in-situ phytoplankton culture device according to claim 3, characterized in that, The gravity system also includes a support platform connected to the bottom of the culture chamber via a lower hook, and the weight is connected to the support platform.

9. A method for in-situ culture of phytoplankton, characterized in that, Using the phytoplankton in-situ culture apparatus according to any one of claims 1, 3-8, includes the following steps: (1) The single-layer culture chamber is suspended on the buoyancy platform by the upper hook, and the gravity system is connected to the bottom of the culture chamber by the lower hook to form a stable vertical positioning structure; (2) The in-situ culture device is slowly placed into the water body at the predetermined location so that the culture chamber reaches the predetermined water depth, ensuring that the culture chamber is in contact with the surface layer and bottom sediment layer of the water body. The single-layer culture chamber allows the exchange of nutrients and water but prevents phytoplankton from passing through, effectively intercepting algal communities and dissolved nutrients in natural water bodies, forming a closed ecological retention effect. (3) The in-situ culture device shall be left to stand in water for at least 24 hours to ensure that the natural exchange of nutrients and water inside and outside the culture chamber reaches a stable state; (4) According to the cultivation objective, the specially marked target algae species are slowly introduced into the single-layer culture chamber; (5) At the same time point within a predetermined time interval, water and mud samples are collected from the inside of the single-layer culture chamber, and other monitoring activities are carried out to assess the growth status of phytoplankton and the impact of environmental factors on its growth.

10. A method for in-situ culture of phytoplankton, used to study the feeding behavior of mixed-trophic dinoflagellates, characterized in that, Using the phytoplankton in-situ culture apparatus according to any one of claims 2 to 8, the following steps are included: (1) The double-layer culture chamber is suspended on the buoyancy platform by the upper hook, and the gravity system is connected to the bottom of the double-layer culture chamber to form a stable vertical positioning structure; (2) Slowly place the assembled in-situ culture device into the water body at the predetermined location so that the culture chamber reaches the predetermined water depth and ensures that the culture chamber is in contact with the surface and bottom sediment layers of the water body. In this way, the inner culture chamber effectively intercepts large algal communities and dissolved nutrients in the natural water body, restricting the target dinoflagellates from entering the outer culture chamber, but allowing small algae to enter and exit freely, forming a semi-closed ecological retention effect. The bottom circular screen connecting the inner and outer culture chambers effectively isolates the target dinoflagellates from entering the outer culture chamber. At this time, the target dinoflagellates are confined in the inner culture chamber and cannot escape, but can come into contact with small algae that enter through the screen, while there are almost no large dinoflagellates in the outer culture chamber. (3) The in-situ culture device is placed in water for at least 24 hours to ensure that the natural exchange of water and nutrients inside and outside the culture chamber reaches a stable state, the phytoplankton growth is stable, and the light reduction rate in the device is tested. (4) According to the cultivation objective, the target dinoflagellates are slowly introduced into the inner cultivation chamber, and the target small algae species with special markings or small algae of different concentrations are placed in the outer cultivation chamber. (5) Collect water and mud samples from the inner and outer culture chambers at the same time point within a predetermined time interval, and conduct other monitoring activities to assess the feeding behavior of mixed-trophic dinoflagellates.

11. The method for in-situ culture of phytoplankton according to claim 9 or 10, characterized in that, The target algal species or target small algal species are cyanobacteria or green algae with radioactive isotope labels.

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