Method for culturing beneficial algae by utilizing cyanobacterial bloom as resource

Abstract

This invention provides a method for resource-based cultivation of beneficial algae from cyanobacterial blooms, comprising: 1. Obtaining a cyanobacterial (Microcystis) bloom and concentrating it to a high concentration of chlorophyll a of 80–150 mg / L; 2. Continuously hydrodynamically disturbing this high-concentration cyanobacterial bloom, with the disturbance intensity preferably designed to prevent the bloom from turning black and emitting a foul odor; 3. Continuously disturbing for 20–60 days to obtain a cyanobacterial bloom that changes from green to light yellowish-green or whitish; 4. Diluting the color-changed cyanobacterial bloom with tap water, with the dilution concentration preferably between 0.5 and 2.3 mL / cm³. 2 5. Under normal temperature and low light conditions, beneficial algae such as diatoms and green algae are inoculated and cultured; 6. After 12-30 days of culture, the growth density of beneficial algae reaches its peak, with a cell density as high as 2.5 × 10⁻⁶. 6 ~5.6×10 7 cells / cm 2 This method can not only remove algal toxins, but also utilize cyanobacterial blooms to cultivate beneficial algae such as diatoms and green algae, providing a new approach for the control and resource utilization of cyanobacterial blooms.

Description

Technical Field

[0001] This invention relates to the field of resource utilization of harmful algae, and more particularly to the resource utilization of cyanobacterial blooms and methods for cultivating beneficial algae. Background Technology

[0002] Currently, cyanobacterial blooms mainly refer to blooms of the genus *Microcystis* within the phylum Cyanobacteria (abbreviated as cyanobacterial blooms). Cyanobacterial blooms are common and harmful algal blooms in eutrophic lakes, reservoirs, ponds, and other water bodies in my country. After occurring, cyanobacterial blooms tend to float on the water surface and, influenced by wind direction, tend to accumulate in large quantities downwind, often forming deposits resembling green paint. After accumulation, cyanobacterial blooms easily emit a foul odor during hot seasons, affecting the surrounding environment. Large-scale cyanobacterial blooms also consume oxygen in the water, causing nighttime hypoxia, producing algal toxins, disrupting the ecological balance, affecting drinking water and irrigation water, and impacting the yield and quality of aquaculture. These problems caused by cyanobacterial blooms seriously threaten the aquatic environment and water body functions. Therefore, the regulation and resource utilization of cyanobacterial blooms has always been an important research direction.

[0003] Compared to cyanobacteria, diatoms have a higher nutritional value. Diatoms are rich in siliceous shells and their nutritional characteristics include high protein content, moderate fat content, abundant vitamins and minerals, and rich unsaturated fatty acids. Diatoms are rich in high-quality protein, making them an ideal protein source, which plays an important role in improving the growth rate and weight gain of aquatic animals such as fish, shrimp, and shellfish. Diatoms contain a moderate amount of fat, which is not only an important source of energy but also a carrier for the storage and absorption of fat-soluble vitamins. Diatoms are rich in various vitamins and minerals, such as vitamin C, vitamin E, beta-carotene, calcium, iron, and zinc. These nutrients play an important role in the immunity, antioxidant capacity, bone development, and reproductive health of aquatic organisms. Diatoms contain abundant omega-3 unsaturated fatty acids, such as EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). These unsaturated fatty acids play an important regulatory role in the growth, immunity, and anti-inflammatory capabilities of aquatic organisms such as fish and shrimp. Therefore, diatoms are widely used in aquaculture as feed or additives, significantly improving the quality and yield of aquatic products. High-density diatom cultivation is an important research direction in aquaculture. High-density diatom cultivation is even more valuable given the current rising cost of formulated aquatic feeds.

[0004] Besides their crucial role in aquaculture, diatoms play important roles in many other areas. Diatoms are rich in fucoxanthin, a natural pigment belonging to the lutein class of carotenoids. Fucoxanthin ranges in color from pale yellow to brown, which is the main reason diatoms appear brown or dark brown. Fucoxanthin possesses anti-tumor, anti-inflammatory, antioxidant, weight-loss, and neuroprotective properties, and is widely used in pharmaceuticals, skincare products, and health supplements. Furthermore, diatoms have broad applications in bioremediation, chemical materials, nanotechnology, and diatomaceous earth, demonstrating extremely promising research and application prospects.

[0005] Numerous reports exist on the large-scale cultivation of diatoms in seawater. Currently, diatom cultivation technology primarily focuses on seawater diatom cultivation. For example, Shenzhen Zhaokai Bioengineering R&D Center Co., Ltd. has developed a large-scale open culture method and system for seawater diatoms, yielding diatoms with various applications. However, their diatom cultivation methods and technologies remain relatively lacking. Diatoms generally thrive better in nutrient-rich, low-light environments. Methods for the natural proliferation and cultivation of diatoms are less common. Other diatom cultivation technologies mainly utilize the ease with which diatoms attach and grow. By introducing diatom seed sources, diatoms are attached to a substrate and grown, serving as high-quality live feed for aquaculture. The commonly used method for attached diatom cultivation in aquaculture is the open culture method for shellfish feed. The inventors of this application have discovered a method for cultivating diatoms using Microcystis blooms. Its main characteristic is utilizing the substances and energy stored in cyanobacterial blooms to promote the succession of cyanobacterial blooms to diatom dominance under hydrodynamic disturbance conditions. The inventors have also provided a method for cultivating attached diatoms using aquatic plants during decay. These diatom cultivation methods facilitate the cultivation of high-density diatoms and promote the beneficial transformation of natural substances and the large-scale development and utilization of diatoms. To further shorten the time required for resource utilization of cyanobacterial blooms and improve the utilization rate and effectiveness of substances within these blooms, the applicant continues to optimize and innovate resource utilization methods for cyanobacterial blooms.

[0006] Besides diatoms, some species of green algae also possess outstanding nutritional value. For example, *Haematococcus pluvialis*, a freshwater single-celled green alga belonging to the Chlorophyta phylum, Volvoxales order, Haematococcidae family, and *Haematococcus pluvialis* genus, is another example. This algae accumulates large amounts of astaxanthin, giving it a red color, hence its other name, Haematococcus pluvialis. Astaxanthin is a natural pigment widely distributed in aquatic species such as salmon, shrimp, and crab. It has been found to have extremely strong antioxidant capabilities, being 1000 times that of vitamin E, 200 times that of tea polyphenols, 100 times that of α-tocopherol, 65 times that of vitamin C, and 17 times that of grape seed extract. It is typically 10 times higher than other carotenoids, such as β-carotene, canthaxanthin, zeaxanthin, and lutein. The internal structure of astaxanthin gives it strong antioxidant and anti-inflammatory activities, and since its first isolation from lobsters in 1938, it has been widely used in the feed industry. Currently, astaxanthin, along with canthaxanthin, is the most important and expensive pigment used in aquaculture for pigmentation in salmon, trout, and shrimp. It is also an essential component of ornamental fish and large ornamental fish feed. Furthermore, astaxanthin has a positive effect on egg yolk color and the skin and meat tissue of broiler chickens. Due to its strong antioxidant properties and other health benefits, astaxanthin is also used in nutritional supplements and cosmetics. In some countries, astaxanthin is also used as a food and beverage additive.

[0007] Haematococcus pluvialis is a single-celled green algae capable of synthesizing lipids and astaxanthin. Astaxanthin accounts for over 80% of its carotenoid secondary metabolites, with an overall astaxanthin accumulation level of 1%–3%, making it one of the main sources of natural astaxanthin. Numerous studies have elucidated the positive effects of Haematococcus pluvialis powder as an aquatic feed additive, such as improving the growth performance of rainbow trout and large yellow croaker. In crustaceans, Haematococcus pluvialis also shows excellent application prospects. Studies have found that it can increase the deposition levels of carotenoids in various tissues of Chinese mitten crab and red swamp crayfish, enhancing antioxidant properties; it can also improve the red swamp crayfish's resistance to hypoxia; and the addition of appropriate amounts of Haematococcus pluvialis powder can regulate the intestinal flora of red swamp crayfish, alleviating intestinal dysfunction and thus improving its growth performance. Haematococcus pluvialis has also been found to have a positive regulatory effect on the intestinal flora in whiteleg shrimp and Chinese mitten crab. Haematococcus pluvialis can accumulate high levels of astaxanthin under environmental stress conditions such as high salinity, high temperature, and high light.

[0008] Common culture media for freshwater algae include those commonly referred to as BG11, BBM, SE, and HUT (sourced from the Freshwater Algae Culture Bank of the Chinese Academy of Sciences: https: / / algae.ihb.ac.cn / MeSearch.aspx?type=00020001). These media are rich in various major and minor elements, meeting the growth requirements of algae. Furthermore, a single culture medium can often satisfy the growth needs of multiple algae species. A sufficient supply of nutrients is essential for rapid and abundant algal growth. The large-scale occurrence of cyanobacterial blooms indicates the accumulation of abundant nutrients necessary for algal growth; the nutrients released after the algae decompose under suitable conditions also become the basis for the growth of new algae.

[0009] Transforming cyanobacterial blooms into beneficial algae such as diatoms and green algae is one aspect of the resource utilization of cyanobacterial blooms, turning them from waste into treasure. It also provides an efficient culture medium for cultivating high-nutrient-value algae such as Haematococcus pluvialis (a member of the green algae phylum). Improving the purity and density of beneficial algae cells such as diatoms and green algae, reducing contaminated cyanobacterial cells, and increasing the cultivation efficiency of beneficial algae while reducing time and economic costs are important research directions in the resource utilization of cyanobacterial blooms. Summary of the Invention

[0010] The technical problem to be solved by this invention is to provide a method for cultivating beneficial algae by utilizing cyanobacterial blooms in a resource-efficient manner. This method can enrich the pathways of matter and energy transfer in the aquatic environment, enrich the cultivation methods of beneficial algae, enrich the theoretical research on algal ecology, and the cultivated beneficial algae also have many other benefits, such as providing high-quality live aquatic feed, purifying water quality, protecting the environment, developing nutritional products, developing drugs, and developing new energy sources.

[0011] The technical problem it aims to solve can be addressed through the following technical solutions.

[0012] A method for resource-based utilization of cyanobacterial blooms to cultivate beneficial algae, the method comprising the following steps:

[0013] S1. Obtain high concentrations of cyanobacterial (microcystis) blooms from lakes, ponds, reservoirs, and other water bodies where cyanobacterial blooms occur, concentrate the cyanobacterial blooms to obtain slurry-like cyanobacterial blooms, and control the chlorophyll a concentration of the cyanobacterial blooms to a high concentration of 80-150 mg / L.

[0014] S2. Accumulate high-concentration cyanobacterial blooms under normal temperature and low light conditions for 0-3 days;

[0015] S3. Continuously hydrodynamically disturb this high-concentration cyanobacterial bloom, with the disturbance intensity being appropriate to prevent the cyanobacterial bloom from turning black and smelling foul.

[0016] S4. At room temperature, continuous disturbance for 20 to 60 days will cause the green cyanobacterial bloom to turn white or light yellowish-green.

[0017] S5. Using a whitish or pale yellow-green cyanobacterial bloom stock solution, dilute it with tap water into the culture container. The dilution concentration of the whitish or pale yellow-green cyanobacterial bloom stock solution should be 0.5–2.3 mL / cm³. 2 The water depth should be between 5 and 100 cm.

[0018] S6. After allowing the diluted cyanobacterial bloom to stand for 1-3 days, some blooms will sink to the bottom, while others will float on the surface. For the floating blooms, inoculate them with beneficial algae, such as diatoms or green algae. The initial density of the inoculated beneficial algae should be 1.0 × 10⁻⁶. 3 cells / cm 2 above;

[0019] S7. After inoculating with beneficial algae, the algae were statically cultured under normal temperature and low light conditions. During the 7-15 day inoculation period, high-density growth of the inoculated algae appeared on both the floating algae and the cyanobacterial blooms at the bottom. The floating algae and the cyanobacterial blooms gradually sank to the bottom, and the beneficial algae gradually grew into high-concentration algal clusters on these sediments.

[0020] S8. During the total culture period after inoculation with beneficial algae, which is 12–30 days, the growth density of beneficial algae reaches its peak, with the cell density of beneficial algae on the sediment reaching as high as 2.7 × 10⁻⁶. 6 ~5.6×10 7 cells / cm 2 .

[0021] In step S1, the concentration of cyanobacterial blooms can be estimated by utilizing the buoyancy of the blooms without measuring the chlorophyll a concentration. This involves adding water to the high-concentration cyanobacterial blooms, stirring, and then letting it stand for 0.5–1 hour to obtain the cyanobacterial bloom accumulation layer floating on the water surface. This yields a high-concentration cyanobacterial bloom that meets the requirements, with a chlorophyll a concentration within the range of 80–150 mg / L.

[0022] In step S2, the normal temperature and low light conditions refer to the temperature conditions of the current season and the low light conditions with a shading rate of more than 70%, which can be completely dark conditions. The purpose is to promote the formation of a certain anaerobic fermentation process during the accumulation of high-concentration cyanobacterial blooms due to the large amount of respiration of cyanobacterial cells leading to oxygen deficiency.

[0023] In step S3, the disturbance is aeration disturbance, rotation or circulation agitation, in order to increase the dissolved oxygen level in the cyanobacterial bloom and prevent severe anaerobic decay and foul odor.

[0024] In step S4, the continuous disturbance of the high-concentration cyanobacterial bloom generates a lot of foam. The cyanobacterial bloom gradually changes from green to yellowish-green, and then gradually turns white. Given enough time, the cyanobacterial bloom becomes distinctly white, even turning off-white. At this point, the cyanobacterial cells in the bloom begin to decay, sometimes to the point where their round shape is no longer visible, while the cyanobacterial lamina remains relatively intact. The decaying cyanobacterial cells and cyanobacterial lamina remain aggregated, maintaining a fine granular state. The rate at which the green cyanobacterial bloom gradually turns white is related to water temperature. In summer, when the water temperature is high, the whitening speed is faster. Under summer daytime water temperatures of around 30℃, it can become noticeably white in about 20 days. Otherwise, the whitening speed is slower; green cyanobacterial blooms at daytime water temperatures of around 15-20℃ require nearly 60 days to show signs of whitening.

[0025] In step S5, when adding tap water to dilute the whitened cyanobacterial bloom, the degree of dilution is mainly related to the thickness of the whitened cyanobacterial bloom gathered on the water surface, and the water depth is controlled to meet the appropriate range of light and nutrient concentration in the water for algae growth.

[0026] In step S6, theoretically, the higher the initial density of beneficial algae inoculated, the faster the algae will reproduce.

[0027] In step S7, the cultivation process should preferably be carried out in the warm months of May to October, and the highest daytime cultivation water temperature should preferably be above 18°C.

[0028] In step S8, when the inoculated algae are diatoms, the color of the algal layer is brown or brownish-red; when the dominant algae inoculated are green algae, the color of the algal layer is green. If the color of Haematococcus pluvialis turns brownish-red or dark red, the color of the algal layer will also change from green to darker, or even turn brownish-red or dark red.

[0029] In the technical solution of this invention, three factors are key conditions for achieving the invention: high-concentration cyanobacterial blooms, continuous hydrodynamic disturbance until the cyanobacterial blooms turn white, and diluting the whitened cyanobacterial blooms with tap water at an appropriate ratio. Changes in any of these factors will affect the time and concentration required to cultivate beneficial algae. If the concentration of the cyanobacterial bloom during continuous hydrodynamic disturbance is unsuitable, it will affect the decay process of the cyanobacterial cells, making it difficult to obtain a clearly whitened cyanobacterial bloom—that is, a bloom where the cyanobacterial cells decay while the cyanobacterial colony gel remains relatively well preserved. If the concentration of the cyanobacterial bloom is too low, both the cyanobacterial cells and the cyanobacterial colony gel will decay, making it difficult to obtain a clear cyanobacterial gel. If the concentration of the cyanobacterial bloom is too high, the aeration and disturbance time will be longer, and it is easy for the cyanobacterial bloom to decay and turn brown, leading to bacterial dominance. After the cyanobacterial bloom turns white, the dilution level with tap water also needs to be carefully controlled. If the concentration of tap water is too low, the concentration of cyanobacterial blooms on the water surface will be too high, causing the cyanobacterial blooms to continue to rot, which is not conducive to the growth of inoculated algae. If the concentration of tap water is too high, the concentration of cyanobacterial blooms will be too low, and the concentration of beneficial algae inoculated will also be low.

[0030] The beneficial algae grown in this invention mainly float on the water surface and settle at the bottom of the container, unlike previous studies where they were attached to the inside of the container. Furthermore, as the cultivation time continues, some beneficial algae may become suspended in the water. The diatoms, green algae, and other beneficial algae grown in this invention use nutrients released from the decay of cyanobacterial blooms as their material basis, thus achieving the goal of utilizing cyanobacterial blooms as a resource for cultivating beneficial algae.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] 1. Under aerobic conditions created by hydrodynamic disturbance, cyanobacterial blooms decompose, which in particular promotes the decomposition of cyanobacterial cells while keeping the cyanobacterial colony's gel relatively intact. Since the main component of the cyanobacterial colony's gel is polysaccharide, this method effectively maintains the colony's gel and obtains cyanobacterial polysaccharides.

[0033] 2. It was clarified that the cyanobacterial colony, especially the dominant cyanobacterial polysaccharides, can be used for the efficient and rapid cultivation of beneficial algae such as diatoms and green algae, and in particular, it can be used to cultivate astaxanthin-producing Haematococcus pluvialis. This provides an efficient and high-quality culture medium for the large-scale cultivation of Haematococcus pluvialis.

[0034] 3. Compared with algae that grow on the inner wall of the container, beneficial algae that float on the water surface and grow at the bottom are easier to collect and concentrate, and are more conducive to further utilization after concentration.

[0035] 4. After the cyanobacterial bloom turns white, the cyanobacterial cells in the cyanobacterial colony have already undergone obvious decay, and the algal toxins produced by the cyanobacteria have also completely decayed and degraded within a few days. When cultivating beneficial algae such as diatoms and green algae, the cyanobacterial cells will not compete with each other, and there will be no algal toxin residues. The cell purity and density of the beneficial algae are both high, which is conducive to the further collection and utilization of these beneficial algae, such as for use as live feed in aquaculture and extraction of effective chemical substances.

[0036] 5. This method directly utilizes high-concentration cyanobacterial bloom slurry for resource utilization, providing a resource utilization method for the direct treatment of cyanobacterial bloom slurry collected from eutrophic lakes. Attached Figure Description

[0037] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: all algae photographs in the embodiments were taken under an optical microscope at 400x magnification, and were taken after the algae were diluted with an appropriate amount of pure water (not representing actual algae density).

[0038] Figures 1-1 to 1-30 This is a photograph of algae from Example 1.

[0039] Figures 2-1 to 2-6 This is a photograph of algae from Example 2.

[0040] Figures 3-1 to 3-10 This is a photograph of algae in Example 3.

[0041] Figures 4-1 to 4-8 This is a photograph of algae in Example 4. Detailed Implementation

[0042] Based on the current state of the technology, the inventors of this application have discovered a method for cultivating beneficial algae using cyanobacterial blooms in a resource-efficient manner. The main characteristic is that high-concentration cyanobacterial blooms decompose under aerobic conditions, resulting in a lighter color, particularly a whitish appearance. Specifically, the cyanobacterial cells within the bloom decompose before the algal bloom's gelatinous layer, which retains a relatively fine granular structure, providing an excellent culture medium for beneficial algae growth. Inoculating the aerobic, decomposed cyanobacterial bloom with beneficial algae, such as diatoms or green algae, allows for high-density growth on the whitish bloom layer. The gelatinous layer of the whitish cyanobacterial bloom, primarily composed of polysaccharides, is a crucial material basis for promoting the growth of beneficial algae.

[0043] The present invention will be further described and illustrated in detail below with reference to specific embodiments and accompanying drawings, with more specific implementation methods.

[0044] Example 1

[0045] Fresh cyanobacterial blooms (Microcystis aeruginosa) were collected from aquaculture ponds experiencing cyanobacterial (Microcystis) blooms during the warm month of June. The Microcystis aeruginosa colonies were observed in the fresh cyanobacterial blooms. Figures 1-1 to 1-4 The concentrated cyanobacterial bloom, expressed as a high concentration of chlorophyll a (Chl-a) at 100.0 mg / L, contained a Microcystis cell density of 3.1 × 10⁻⁶ cells. 11 The concentration of microcystin blooms was measured in cells / L, approximately 310 billion microcystin cells per liter of water. The resulting high-concentration cyanobacterial bloom had a volume of 35L. This high-concentration bloom was placed in a 60cm high, 80L white plastic tank and continuously aerated using air stones. The tank was placed in a glass greenhouse. The light conditions in the greenhouse were approximately 30% of the intensity of natural sunlight outdoors. No additional water was added during the continuous aeration process.

[0046] After 15 days of continuous aeration and disturbance, the fresh, high-density cyanobacterial bloom gradually lightened in color, turning whitish, from green to light yellowish-green, and produced a lot of foam, which overflowed the tank. After 30 days of continuous aeration and disturbance, the high-density cyanobacterial bloom became even lighter and whiter, and the amount of foam produced decreased. Microscopic examination of the cyanobacterial bloom at this point revealed that the Microcystis cells in the cyanobacterial community were significantly decayed and fewer in number, and the Microcystis cells were no longer fully formed, but the community still maintained a granular structure, meaning the colony's gelatinous coating remained intact. Figures 1-5 to 1-8 In this case, after the Microcystis cells in the cyanobacterial community decompose, the overall color of the cyanobacterial bloom becomes lighter and whiter, while the transparent community gel remains well preserved.

[0047] Then, the bleached cyanobacterial bloom was left to stand for 1 hour to obtain the cyanobacterial bloom layer floating on the water surface as the mother liquor. 0.18L, 0.36L, 0.54L, and 0.72L of these mother liquors were respectively transferred to colorless transparent glass tanks (30cm×30cm×30cm). Tap water was added to dilute the mixture to 23L. The concentration of the bleached cyanobacterial bloom in these tanks was 0.20mL / cm³, respectively. 2 0.40 mL / cm 2 0.60 mL / cm 2 and 0.80 mL / cm 2 During the cultivation process, it was found that the white cyanobacterial blooms in the water mainly sank to the bottom and floated on the surface, with the concentration primarily related to the water body area. Therefore, the unit for expressing the concentration of cyanobacterial blooms was converted to mL / cm³. 2 The organisms were cultured in a glass greenhouse under two conditions: with and without aeration. Specific treatment details are shown in Table 1.

[0048] Table 1: Treatments for algal cultivation using cyanobacterial blooms

[0049]

[0050] After adding tap water to each glass tank, the resulting cyanobacterial bloom was allowed to stand for 2 days. Then, rhomboid algae cells (diatoms) were inoculated into each glass tank at a density of 1.2 × 10⁻⁶. 3 cells / cm 2 During the cultivation process, different phenomena occurred in each treatment. In the four treatments without aeration and disturbance, some whitish blue-green algae particles floated on the water surface, while others settled at the bottom of the tank. Decaying whitish blue-green algae blooms were distributed in the water column, and the water column as a whole turned light yellowish-green. Furthermore, both the blue-green algae particles floating on the surface and those sinking to the bottom gradually turned brownish-brown.

[0051] On day 7, the brownish hue was most pronounced in the floating matter and sediment, especially in treatment III which was not aerated or disturbed. Microscopic observation revealed a very dense concentration of rhomboid algal cells. Figures 1-9 to 1-12 It also contained a small amount of green algae. The density of *Nyctaginosa* was as high as 1.2 × 10⁻⁶. 6 cells / cm 2 Meanwhile, rhomboid algae dominated the sediments from treatment III, interspersed with some green algae and filamentous cyanobacteria. Figures 1-13 to 1-16 This is very similar to the algae found in floating debris on the water surface. On day 7, some rhomboid algae grew in the cyanobacterial bloom cover on the surface of treatment IV, which was not aerated or disturbed. Figures 1-17 to 1-20 ).

[0052] On day 10, the surface floating matter in treatment III without aeration and disturbance evolved into a large amount of rhomboid algae. Figures 1-21 to 1-26 The cell density of *Nyctaginosa* reached 2.7 × 10⁻⁶. 6 cells / cm 2 Numerous rhomboid algae also grew in the floating debris and sediments of treatments I and II, with densities of rhomboid algae reaching 0.4 × 10⁻⁶ in treatments I and II, respectively. 6 cells / cm 2 and 1.5×10 6 cells / cm 2 The growth time of a large number of brownish-red rhomboid algae in the sediment and floating matter of treatment IV was 3 days later than that of treatment III, and the appearance of rhomboid algae was similar to that of treatment III. However, after the floating matter settled, the growth density of rhomboid algae cells in the sediment of treatment IV was higher, reaching as high as 1.7 × 10⁻⁶ on day 15. 7 cells / cm 2 .

[0053] On day 10, significant green algae growth was observed in all four tanks with aeration disturbance, especially in treatment VII, where the water turned a deep green color and a large amount of green algae was observed under a microscope. Figures 1-27 to 1-30 These green algae are mainly of the genus *Cryptocoryne*, with some filamentous blue-green algae mixed in.

[0054] This embodiment demonstrates that after appropriately diluting the decaying, whitish cyanobacterial bloom with tap water, diatoms can be cultured under static conditions, achieving a large-scale diatom growth. The suitable addition concentration for obtaining high-concentration diatom growth is in treatments III and IV, i.e., the concentration of added whitish cyanobacterial bloom is 0.60 mL / cm³. 2 and 0.80 mL / cm 2 However, aeration disturbance of diluted cyanobacterial blooms is not conducive to the dominance of diatoms, and green algae are more likely to dominate under aeration disturbance.

[0055] Example 2

[0056] The inventors' existing experimental research found that hydrodynamic disturbance cultivation of cyanobacterial blooms at suitable concentrations can achieve the succession to diatom dominance. However, if the concentration continues to increase to a certain level, the cyanobacterial blooms will rot and turn brown. If the concentration increases further, more types of changes will occur. In order to further study the degree of concentration required for cyanobacterial blooms to facilitate the whitening phenomenon under hydrodynamic disturbance and to prevent the cyanobacterial blooms from completely rotting and decomposing into dissolved nutrients under hydrodynamic disturbance, fresh cyanobacterial blooms floating on the water surface were collected from aquaculture ponds where cyanobacterial (Microcystis) blooms occurred in the hot month of July and concentrated to different degrees. The concentrated cyanobacterial (Microcystis) blooms were divided into five concentration gradients based on Chl-a concentration: 60 mg / L, 80 mg / L, 120 mg / L, 150 mg / L, and 170 mg / L. Each concentration of cyanobacterial (Microcystis) bloom had a volume of 25 L and was placed in five white circular plastic tanks, each 60 cm high and with a total volume of 80 L. Continuous aeration was achieved using air stones. The cell densities of Microcystis in the cyanobacterial (Microcystis) blooms with Chl-a concentrations of 60 mg / L, 80 mg / L, 120 mg / L, 150 mg / L, and 170 mg / L were 1.9 × 10⁻⁶. 11 cells / L, 2.5×10 11 cells / L, 3.7×10 11 cells / L, 4.7×10 11 cells / L and 5.3×10 11 cells / L. A white plastic bucket is placed in a glass greenhouse. No additional water is added during continuous aeration.

[0057] To obtain the desired concentration of cyanobacterial blooms quickly and easily, the inventors, based on the buoyancy of cyanobacterial blooms, discovered that after mixing the cyanobacterial blooms with water and letting them stand for one hour, they float to the surface, forming a clear stratification with the water. The Chl-a concentration in the cyanobacterial bloom slurry floating on the surface is exactly 100 mg / L. This constitutes a rapid and accurate method for obtaining high-concentration cyanobacterial blooms.

[0058] During aeration and disturbance, the fresh, high-density green cyanobacterial bloom gradually turned yellowish-green and produced a lot of foam, with the most foam produced between 10 and 20 days, overflowing the tank. After 20 days of continuous aeration and disturbance, the foam showed a color change. The foam color gradually deepened with increasing concentrations, especially at high concentrations of 150 mg / L and 170 mg / L, where the foam gradually turned brownish-orange, and the cyanobacterial bloom gradually changed from yellowish-green to brownish-red. In contrast, at lower Chl-a concentrations of 60 mg / L and 80 mg / L, the water color gradually became whitish. After 30 days of continuous aeration and disturbance, the cyanobacterial bloom colors at Chl-a concentrations of 60 mg / L, 80 mg / L, 120 mg / L, 150 mg / L, and 170 mg / L were, in order: light white, light white mixed with pale green, light white mixed with yellowish-green, light yellowish-green, brownish-yellow, and brownish-red. In the 170 mg / L treatment, the cyanobacterial community was almost entirely decomposed into brown bacteria, with no obvious community particles. Microscopic examination of the different colored cyanobacterial blooms in each treatment showed that they were basically the same as those in Example 1, except that the cyanobacterial community was relatively smaller in the 60 mg / L treatment. Representative cyanobacterial community decomposition in the 80 mg / L, 120 mg / L, and 150 mg / L treatments is shown in [reference needed]. Figures 2-1 to 2-6 .

[0059] This embodiment shows that the changes in the high-concentration cyanobacterial bloom obtained after concentration of cyanobacteria (Microcystis) bloom under aeration disturbance conditions are related to the concentration of the cyanobacterial bloom. It is speculated that aeration disturbance increases the dissolved oxygen concentration in the cyanobacterial bloom. Although the decay process of cyanobacterial bloom under aerobic conditions will result in the decay and damage of cyanobacterial cells, it is not easy to release odorous substances and it is easy to retain obvious colony gel, especially in the treatment where the Chl-a concentration in the cyanobacterial bloom is 80-150 mg / L, a relatively obvious cyanobacterial colony gel can be retained.

[0060] Example 3

[0061] In September, when the temperature was still relatively warm, diatoms were cultured using cyanobacterial blooms (Chl-a concentration 80 mg / L) from Example 2 that had turned white after 40 days of aeration and disturbance. Before cultivation, the whitened cyanobacterial blooms were allowed to stand for 0.5 hours, and the slurry of the whitened blooms floating on the surface was collected as a mother liquor. These mother liquors were diluted with tap water. The culture containers were 30cm × 30cm × 30cm colorless transparent glass tanks, filled with tap water to a volume of 8.5L, with a water depth of 10cm. The tanks were placed in a glass greenhouse for cultivation. The light conditions in the glass greenhouse were approximately 30% of the outdoor natural sunlight intensity. Six treatments were performed; details are shown in Table 2.

[0062] Table 2: Specific details of algae cultivation using cyanobacterial blooms.

[0063]

[0064] In the three treatment tanks with aeration and disturbance, the water appeared as a light white and turbid color. In the three treatment tanks without aeration, some of the whitish cyanobacterial bloom particles gradually floated to the surface, while others sank, forming light white layers on the surface and bottom of the water. All tanks were inoculated with *Rhizophora natans* at an initial density of 1.8 × 10⁻⁶. 3 cells / cm 2 .

[0065] On day 4, brownish spots appeared on the light-colored floating debris and sediment in treatment IV (non-aerated). Microscopic observation revealed these to be rhomboid algal cells. No significant changes were observed in other treatments. On day 6, the brownish patches on the floating debris and sediment in treatment IV (non-aerated) became more numerous and larger.

[0066] On day 9, the brownish patches on the surface and bottom of the non-aerated treatment IV water were more numerous and larger. Meanwhile, the foam on the surface of the three aerated treatments turned yellowish-green. No significant changes were observed in the other treatments.

[0067] On day 11, in the non-aerated Treatment IV, all floating debris on the water surface turned brown, and the sediment turned light brownish-yellow. Microscopic observation revealed that these brown, brownish-yellow, or brownish-red substances were all diatoms. Figures 3-1 to 3-6 The algae are primarily rhomboid, with cell lengths of approximately 30 μm and a maximum width of about 5 μm. The cell density of rhomboid algae in floating debris on the water surface reached 4.9 × 10⁻⁶. 6 cells / cm 2 Brownish spots also appeared on the surface of the non-aerated treatments V and VI. In the three aerated treatments, the foam on the surface turned yellowish-green, the water remained turbid, and the color was a light brownish-green. After 12 days, much of the floating debris in treatment VI sank, and the sediment gradually turned green, revealing the presence of green algae growth.

[0068] From day 13 to 16, the brownish-red substance in the floating matter and sediment of treatments IV and V (without aeration) reached its peak. Microscopic observation revealed a high concentration of *Nyctaginosa* algae, with *Nyctaginosa* cell densities reaching as high as 3.5 × 10⁻⁶ cells in the sediments of treatments IV and V. 7 cells / cm 2 and 5.6×10 7 cells / cm 2 In the three aeration treatments, the water color became more green. Microscopic observation revealed that these green substances were dominated by green algae, mainly *Cladophora*, *Euphorbia lathyris*, and a small amount of filamentous cyanobacteria. (See details...) Figures 3-7 to 3-10 .

[0069] From this example, it can be seen that the concentration of the mother liquor added to the whitened cyanobacterial bloom mother liquor is 1.11 mL / cm³. 2 2.22 mL / cm 2 When the concentration of diatoms is higher, a large amount of diatoms can be observed in both sediments and floating matter. However, when the concentration of cyanobacterial blooms is higher, diatoms are less likely to grow, and green algae are more likely to grow.

[0070] Example 4

[0071] In late September, when temperatures were relatively high, a milky-white cyanobacterial bloom of 80 mg / L Chl-a (as described in Example 2) was used to cultivate Haematococcus pluvialis. The Haematococcus pluvialis strain was obtained from the freshwater algae strain bank of the Institute of Hydrobiology, Chinese Academy of Sciences, and was further cultured until the Haematococcus pluvialis strain concentration reached approximately 1.0 × 10⁻⁶. 5 The concentration of the milky white cyanobacterial bloom was first allowed to stand for 0.5 hours, and then the milky white cyanobacterial bloom slurry floating on the surface was taken as the stock solution. These stock solutions were diluted with tap water. The culture containers were 500 mL Erlenmeyer flasks, each filled with tap water to a depth of 6 cm. The cultures were cultured at room temperature in the laboratory. The laboratory lighting conditions were approximately 2% of the outdoor natural sunlight intensity. Six treatments were performed, and the specific details are shown in Table 3. The concentration of the milky white cyanobacterial bloom was calculated using a volume of 400 mL (mL / cm³). 2 Each bottle was inoculated with 2 mL of Haematococcus pluvialis culture solution, with an initial inoculation density of 5.3 × 10⁻⁶. 3 cells / cm 2 Each bottle was not aerated; it was simply shaken once a day for about 20 seconds each time.

[0072] Table 3. Specific details of Haematococcus pluvialis cultivation using milky white cyanobacterial blooms.

[0073]

[0074]

[0075] For the first three days, there were no obvious changes in any of the bottles; the water was cloudy and milky white, with some milky white cyanobacterial blooms floating on the surface and settling at the bottom. Starting on the fourth day, the milky white cyanobacterial blooms that had settled at the bottom of each bottle gradually began to show a light green tinge. As time continued, most of the milky white cyanobacterial blooms sank to the bottom, and the water remained cloudy. The milky white cyanobacterial blooms gradually grew into flocculent structures, which gradually turned light green. By the 12th day of cultivation, the light green color was most pronounced in culture bottle IV. Therefore, the flocculent material from culture bottle IV was examined under a microscope and found to be Haematococcus pluvialis cells growing together with filamentous cyanobacteria. Figures 4-1 to 4-8 As cultivation continued, the amount of light green flocculent matter in each bottle gradually increased and settled loosely at the bottom.

[0076] On day 28, a large amount of green, filamentous culture material appeared in all culture flasks, especially in culture flask VI, where the amount was most abundant, and in culture flask I, the amount was least abundant. Therefore, the filamentous cultures in culture flasks I and VI were thoroughly mixed, and 50 mL was taken from each flask for cell counting; the remaining flasks were continued to be cultured. Calculations showed that the total cell density of Haematococcus pluvialis in the filamentous cultures of culture flasks I and VI on day 28 was 3.9 × 10⁻⁶. 5 cells / mL and 6.5×10 5 cells / mL, and the bottom area of ​​a 500mL Erlenmeyer flask is 63cm². 2 After conversion, the average cell density of Haematococcus pluvialis at the bottom surface is 2.5 × 10⁻⁶. 6 cells / cm 2 and 4.1×10 6 cells / cm 2 .

[0077] During continued cultivation, the amount of green filamentous culture material in each treatment bottle did not increase significantly compared to day 30. Therefore, it was inferred that the growth density of Haematococcus pluvialis reached its peak around day 30.

[0078] In comparison, the growth rate of the inoculated Haematococcus pluvialis was slower than that of the diatoms in Examples 1 and 3, and filamentous cyanobacteria grew between the Haematococcus pluvialis cells, exhibiting filamentous growth during the culture process, which affected the purity of Haematococcus pluvialis.

[0079] This example demonstrates that when the concentration of the whitening cyanobacterial bloom is 0.5–1.0 mL / cm³, the effect is achieved. 2When Haematococcus pluvialis is inoculated into it, Haematococcus pluvialis with filamentous growth can be obtained. That is, the aerobic conditions caused by hydrodynamic disturbance can be used to cultivate Haematococcus pluvialis after the algal bloom turns white.

Claims

1. A method for resource-based cultivation of beneficial algae from cyanobacterial blooms, characterized in that, The following processing steps are included: S1. Obtain high concentrations of cyanobacterial blooms from water bodies where cyanobacterial blooms occur, concentrate the cyanobacterial blooms to obtain slurry-like cyanobacterial blooms, and control the chlorophyll a concentration of the cyanobacterial blooms to a high concentration of 80-150 mg / L. S2. Accumulate high-concentration cyanobacterial blooms under normal temperature, low light or no light conditions to form anaerobic fermentation of cyanobacterial cells under hypoxic conditions. S3. Continuously hydrodynamically disturb this high-concentration cyanobacterial bloom, with the disturbance intensity being appropriate to prevent the cyanobacterial bloom from turning black and smelling foul. S4. At room temperature, continuous disturbance is carried out until the green cyanobacterial bloom turns white or light yellowish-green; the cyanobacterial gel is kept in a relatively fine granular state, serving as a culture medium for the growth of beneficial algae; the duration of continuous disturbance is 20 to 60 days. S5. Using a stock solution of cyanobacterial blooms that have turned whitish or pale yellowish-green, dilute it with water into the culture container. The concentration of the whitish or pale yellowish-green cyanobacterial bloom stock solution should be 0.5–2.3 mL / cm³. 2 The water depth is 5-100cm; S6. Allow the diluted cyanobacterial bloom to stand until some blooms sink to the bottom and others float on the surface. Inoculate the floating blooms with beneficial algae at an initial density of 1.0 × 10⁻⁶. 3 cells / cm 2 above; S7. After inoculating with beneficial algae, the algae were statically cultured under normal temperature and low light conditions. During this period, high-density growth of the inoculated algae appeared on both the floating algae and the cyanobacterial blooms at the bottom. The algae floating on the surface and the cyanobacterial blooms gradually sank to the bottom, and the beneficial algae gradually grew into high-concentration algal clusters on these sediments. S8. Continue culturing until the cell density of beneficial algae on the sediment reaches 2.7 × 10⁻⁶. 6 ~5.6×10 7 cells / cm 2 .

2. The method for resource utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 1, characterized in that, In step S1, the concentration of cyanobacterial blooms is estimated by utilizing the buoyancy of cyanobacterial blooms without measuring the concentration of chlorophyll a.

3. The method for resource utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 2, characterized in that, During the estimation, water is added to the high-concentration cyanobacterial bloom and stirred, then left to stand for 0.5 to 1 hour to obtain the cyanobacterial bloom accumulation layer floating on the water surface, thus obtaining a high-concentration cyanobacterial bloom that meets the chlorophyll a concentration requirements.

4. The method for resource utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 1, characterized in that, In step S2, the normal temperature and low light conditions refer to the temperature conditions of the current season and the low light conditions with a shading rate of more than 70%, while the no light condition refers to the completely dark conditions.

5. The method for resource utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 1, characterized in that, In step S2, the accumulation time is within 3 days; In step S3, the disturbance is aeration disturbance, rotation or circulation agitation. The disturbance increases the dissolved oxygen level in the cyanobacterial bloom, preventing severe anaerobic decay and foul odor. In step S6, the settling time is 1 to 3 days; In step S7, the inoculation and culture time is 7–15 days; In step S8, the total culture time after inoculating beneficial algae is 12 to 30 days.

6. The method for resource utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 1, characterized in that, In step S5, when diluting with water, the degree of dilution is mainly related to the thickness of the cyanobacterial bloom on the water surface, and the water depth is controlled to meet the appropriate range of light and nutrient concentration in the water for algal growth.

7. The method for resource utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 1, characterized in that, In step S6, the beneficial algae are diatoms or green algae. The higher the initial density of the inoculated beneficial algae, the faster the algae reproduce.

8. The method for resource utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 1, characterized in that, In step S7, the cultivation process is carried out in the warm months of May to October, with the daytime culture water temperature above 18°C.

9. A method for resource-based utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 1 or 7, characterized in that, In step S8, when the beneficial algae inoculated and grown are diatoms, the color of the algal layer is brown or brownish-red; when the beneficial algae inoculated and grown are green algae, the color of the algal layer is green; when the color of Haematococcus pluvialis turns brownish-red or dark red, the color of the algal layer will also change from green to darker, or even turn brownish-red or dark red.

10. A method for resource-based utilization of cyanobacterial blooms to cultivate beneficial algae according to claim 1, characterized in that, The cyanobacterial bloom is a microcystis bloom within the phylum Cyanobacteria; the water body is a lake, pond, or reservoir.

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