A method for treating a brown algae bloom by using gel microspheres and ultrasound and a method for regenerating the gel microspheres

Abstract

The present application relates to a kind of gel microspheres adsorption and ultrasonic combined management brown cystosphaera globosa red tide method and gel microspheres regeneration method in red tide control technical field, wherein the gel microspheres adsorption and ultrasonic combined management brown cystosphaera globosa red tide method, first, gel microspheres are prepared, then gel microspheres are loaded in filter bag and ultrasonic device is combined to manage the brown cystosphaera globosa red tide in water area.In addition, the regeneration method of gel microspheres is that the gel microspheres after being adsorbed and managed are recycled for regeneration and reuse, and the effect of removing brown cystosphaera globosa is still good.The management method of brown cystosphaera globosa red tide in the present application, by the adsorption of gel microspheres and the auxiliary effect of ultrasonic, not only can remove brown cystosphaera globosa cell in water body efficiently, but also avoid the release of intracellular hemolytic toxin;Meanwhile, it also has good elimination effect on phosphate in water body, avoids the recovery of red tide algal cell and reoccurrence.

Description

Technical Field

[0001] This invention relates to the field of red tide control technology, and in particular to a method for controlling spherical Phaeocystis red tides by combining gel microsphere adsorption and ultrasound, as well as a method for regenerating gel microspheres. Background Technology

[0002] Phaeocystis globosa red tides are a harmful algal bloom that has frequently occurred in many eutrophic coastal areas around the world in recent years. These red tides pose a significant threat to nearshore aquaculture, tourism, the safety of cooling sources for coastal power plants, and the health of ecosystems. Therefore, there is an urgent need to find an environmentally friendly treatment method that can both efficiently remove algae and purify water.

[0003] Currently known red tide control technologies include various approaches such as physical, chemical, and biological methods. Physical methods include common methods such as ultrasonication, flocculation, filtration, and adsorption. These methods are simple and do not cause secondary pollution, but the removal rate is unstable. Chemical methods eliminate red tide algae by adding algaecides and other chemical reagents. They are highly efficient and fast-acting, but can cause secondary pollution of the water body. Biological methods mainly involve cultivating organisms that specifically remove red tide algae, such as bacteria and viruses, but they suffer from slow effectiveness and limited results.

[0004] Ultrasonic control of red tide algae primarily relies on the action of sound energy on algal cells, inactivating them through mechanical or chemical oxidative damage. Due to its simplicity, cost-effectiveness, and high efficiency, ultrasonic technology has been widely proven effective in controlling various red tide algae species, including cyanobacteria, in laboratory studies and field applications. However, some limitations remain. For example, while ultrasound provides good immediate inhibition of red tide algae, incompletely inactivated algal cells are prone to recovery. Furthermore, ultrasonic mechanical damage to algal cells can lead to the release of intracellular toxins, thereby endangering the aquatic environment and other biological safety. Therefore, developing an environmentally friendly method that can efficiently kill red tide algae, prevent their recovery, and avoid harming aquatic health is crucial for fundamentally controlling *Phaeocystis globosa* red tides. Summary of the Invention

[0005] This invention addresses the problems existing in the current technology for controlling red tides caused by *Phaeocystis globosa* by providing a method for controlling red tides caused by *Phaeocystis globosa* through a combination of gel microsphere adsorption and ultrasound, thereby achieving efficient red tide control, prevention of recurrence, and prevention of secondary pollution of water bodies.

[0006] The objective of this invention is achieved by providing a method for the combined treatment of Phaeocystis spheroidis red tides using gel microsphere adsorption and ultrasound. The method is characterized by first preparing gel microspheres for adsorption treatment, and then placing the gel microspheres in a filter bag and using an ultrasound device to treat Phaeocystis spheroidis red tides in the water.

[0007] This invention presents a method for treating *Phaeocystis globosa* red tides using a combination of gel microsphere adsorption and ultrasound. Through the adsorption of gel microspheres and the assistance of ultrasound, not only are *Phaeocystis globosa* cells in the water efficiently removed, but the release of intracellular hemolytic toxins is also prevented. Simultaneously, it has a good effect on eliminating phosphate in the water, preventing the resurgence and re-emergence of red tide algae cells. Furthermore, the gel microspheres are placed inside filter bags during use, so the adsorption effect is not affected during water treatment. They are also easy to recycle and reuse after use, have no adverse effects on the water body, and can maintain water cleanliness.

[0008] To facilitate adsorption and treatment, the gel microspheres are prepared as follows: a mixed aqueous solution of sodium alginate and sodium humate is injected into a ferric chloride solution and solidified for 10-15 hours. The microspheres are then filtered out and calcined at 380-400 °C for at least 1 hour to obtain the gel microspheres. The gel microspheres obtained by this method exhibit good stability, strong algae removal ability, and environmental friendliness. They also demonstrate a good effect on eliminating phosphates in water, making them an effective material for multi-faceted red tide control.

[0009] Furthermore, the particle size of the gel microspheres is 0.5-1.0 mm, and the pore size of the filter bag is 8-10 μm.

[0010] Furthermore, the mass ratio of sodium alginate to sodium humate is 1:1, and the mass concentration of both sodium alginate and sodium humate in the mixed aqueous solution is 1-2%; the concentration of the ferric chloride solution is 0.05-0.1 M.

[0011] To further facilitate the thorough treatment of *Phaeocystis globosa*, gel microspheres are placed in filter bags and used in conjunction with ultrasonic devices to treat *Phaeocystis globosa* red tides in water bodies. The dosage of gel microspheres is 0.22-2.2 kg per cubic meter of water. The treatment is combined with ultrasonic treatment until the phosphate content in the water body is lower than 1 ug / L.

[0012] Furthermore, the frequency of the low-frequency ultrasonic treatment is 20-40 kHz.

[0013] Fe in the gel microspheres of the present invention 3+ This gel microsphere can rapidly catalyze ultrasonic cavitation to generate hydroxyl radicals, which not only improves the removal efficiency of *Phaeocystis globosa* algal cells but also degrades their intracellular hemolytic toxins. Secondly, the gel microsphere can continuously adsorb phosphates in the water, preventing any remaining *Phaeocystis globosa* from reviving and forming algal blooms. Tests showed that after treatment with the gel microsphere, the number of *Phaeocystis globosa* algal cells, chlorophyll a content, and algal toxin content all decreased significantly. Simultaneously, the gel microsphere can effectively prevent the revival of *Phaeocystis globosa* by adsorbing phosphates in the water.

[0014] Another objective of this invention is to provide a method for regenerating the above-mentioned gel microspheres, characterized in that the gel microspheres after combined treatment of Phaeocystis globosum red tide are placed in distilled water, treated at 0.2 MPa pressure and 121 °C for 20-30 min, and then dried at 60 °C to complete the regeneration of the gel microspheres.

[0015] After the gel microspheres of the present invention have been used for adsorption, they can still maintain good killing and anti-resurrection performance against *Phaeocystis globosa* after undergoing regeneration treatment under the above-mentioned pressure and temperature conditions. Attached Figure Description

[0016] Figure 1 The gel microspheres and gel microsphere filter bags prepared in Examples 1 and 2 are shown.

[0017] Figure 2 The results show the comparison of cell number, chlorophyll a, and hemolysin removal in Examples 1, 2, and Comparative Example 1 for Phaeocystis globosa.

[0018] Figure 3 Examples 1, 2 and Comparative Example 1 show the results of the recovery of cell number, chlorophyll a and hemolysin in *Phaeocystis globosa*, as well as the results of the removal of phosphate in the algal solution.

[0019] Figure 4 Examples 1, 2 and Comparative Example 1 show the results of cell number, chlorophyll a and hemolysin removal in *Phaeocystis globosa* for reuse after gel microsphere regeneration.

[0020] Figure 5 Examples 1, 2 and Comparative Example 1 show the results of the recovery of cell number, chlorophyll a and hemolysin in *Phaeocystis globosa*, as well as the results of the removal of phosphate in the algal solution, for reuse after gel microsphere regeneration. Detailed Implementation Example 1

[0021] 2 g of sodium alginate was weighed and dissolved in 200 mL of distilled water to obtain a 1 wt% mixed solution. Then, this mixed solution (200 mL) was dripped into 300 mL of 0.1 M ferric chloride solution using a syringe and solidified for 12 h. Finally, after calcination at 400 °C for 2 h, gel microspheres were prepared. 0.04 g of gel microspheres were weighed and placed into a small bag made of 10 μm sieve silk to prepare a gel microsphere filter bag. A camera image of the gel microspheres and gel microsphere filter bag prepared in this example is shown below. Figure 1 As shown.

[0022] The gel microsphere filter bag was placed in a solution of 25 mL of spherical *Phaeocystis jirovecii* algae (cell density 1×10⁻⁶). 4The sample was placed in glass test tubes (each containing 100 cells / mL) and 1‰ f / 2 culture medium was added. The samples were then subjected to low-frequency sonication. The sonication conditions were: sonication frequency 20 kHz, sonication intensity 180 W, and sonication time 8 min.

[0023] To examine the anti-recovery effect of *Phaeocystis globosa* treated with the above-mentioned combined gel microsphere adsorption and ultrasound, the *Phaeocystis globosa* solution treated with the above combined ultrasound was cultured under light for 7 days. The culture temperature was 25 ℃, the light intensity was 4000 Lux, and the light-dark ratio was 12 h:12 h. Then, its physiological activity after recovery was detected.

[0024] To further verify the regeneration and reuse performance of the gel microspheres after adsorption, the gel microsphere filter bag was removed from the *Phaeocystis spherica* algal solution, immersed in distilled water, digested under high temperature and high pressure (121 ℃, 0.2 MPa) for 20 min, and then dried at 60 ℃. The gel microsphere filter bag was then reintroduced into the *Phaeocystis spherica* algal solution from the initial test for secondary use, under the same test conditions, to examine its removal and anti-recovery effects on *Phaeocystis spherica*. Example 2

[0025] Example 2 differs from Example 1 only in that sodium humate is added during the preparation of the gel microspheres. 2 g of sodium alginate and 2 g of sodium humate were weighed and dissolved in 200 mL of distilled water to obtain a 1 wt% sodium alginate solution. This mixed solution (200 mL) was then dripped into a 0.1 M ferric chloride solution (300 mL) using a syringe and solidified for 12 h. Finally, after calcination at 400 °C for 2 h, gel microspheres were formed. 0.04 g of gel microspheres were weighed and placed into a small bag made of 10 μm sieve silk to form a gel microsphere filter bag. A photograph of the actual gel microspheres and gel microsphere filter bag prepared in this example is shown below. Figure 1 As shown, the difference between the gel microspheres in Example 1 and the gel microspheres is that sodium humate is added during the preparation process.

[0026] The gel microsphere filter bag was placed in a solution of 25 mL of spherical *Phaeocystis jirovecii* algae (cell density 1×10⁻⁶). 4 The sample was placed in glass test tubes (each containing 100 cells / mL) and 1‰ f / 2 culture medium was added. The samples were then subjected to low-frequency sonication. The sonication conditions were: sonication frequency 20 kHz, sonication intensity 180 W, and sonication time 8 min.

[0027] To test the anti-recovery effect of the *Phaeocystis globosum* treated with the above-described gel microsphere adsorption and ultrasonication in this embodiment, the treated *Phaeocystis globosum* algal solution was cultured under light for 7 days. The culture temperature was 25 ℃, the light intensity was 4000 Lux, and the light-dark ratio was 12 h:12 h.

[0028] To test the regeneration of the gel microspheres after use and verify their regeneration and reuse performance, the gel microsphere filter bags added to the *Phaeocystis spheroidae* algal solution were removed, placed in distilled water, digested under high temperature and high pressure (121 ℃, 0.2 MPa) for 20 min, and then dried at 60 ℃ to complete the regeneration process. These regenerated gel microsphere filter bags were then reintroduced into the *Phaeocystis spheroidae* algal solution for secondary use under the same testing conditions to examine their removal and anti-recovery effects on *Phaeocystis spheroidae*.

[0029] Comparative Example 1

[0030] This comparative example follows the process of Examples 1 and 2, except that no gel microsphere filter bags were added, and the *Phaeocystis globosum* algal solution was treated only with 20kHz low-frequency ultrasound. Under the same conditions, the cell number, chlorophyll a content, and hemolysin content of *Phaeocystis globosum* in the ultrasonically treated algal solution were measured, as well as the cell number, chlorophyll a content, hemolysin content, and phosphate content of *Phaeocystis globosum* after 7 days of recovery culture.

[0031] The removal effect of *Phaeocystis globosum* algal solutions from Examples 1-2 and Comparative Example 1 was measured. The cell number, chlorophyll a content, and hemolysin content of *Phaeocystis globosum* in the algal solutions were measured respectively. The measurement results are as follows: Figure 2 As shown.

[0032] like Figure 2 a shows the measurement results of the number of *Phaeocystis glomeratus* algal cells in Examples 1-2 and Comparative Example 1. This indicates that the gel microsphere filter bags in Examples 1 and 2, under ultrasound assistance, can eliminate *Phaeocystis glomeratus* algal cells more efficiently and quickly, achieving a removal rate of 50% in the first minute of the reaction. Furthermore, the removal rate at each measurement time is higher than that of Comparative Example 1, demonstrating the significant advantages of the algae removal method provided by this invention. Moreover, the gel microsphere filter bag prepared in Example 2 has a better removal effect on *Phaeocystis glomeratus* algal cells; no *Phaeocystis glomeratus* algal cells were observed by the 8th minute.

[0033] like Figure 2 b shows the chlorophyll a measurement result of *Phaeocystis globosa* in Examples 1-2 and Comparative Example 1 at 8 min. This also indicates that the gel microsphere filter bags in Examples 1 and 2 can more efficiently eliminate *Phaeocystis globosa* algal cells, and the chlorophyll a content in Example 2 is slightly lower than that in Example 1, indicating that the gel microsphere filter bag prepared in Example 2 has the best removal effect on *Phaeocystis globosa*.

[0034] like Figure 2c represents the measurement results of hemolytic toxins in the *Phaeocystis globosa* algal solutions of Examples 1-2 and Comparative Example 1 at 8 minutes. This also shows that the gel microsphere filter bags in Examples 1 and 2 can more efficiently reduce the content of hemolytic toxins in the water, and the inactivation effect of Example 2 is higher than that of Example 1. This indicates that the gel microsphere filter bag prepared in Example 2 has the best removal effect on *Phaeocystis globosa* cytotoxicants.

[0035] Measurements were performed on the recovery culture of *Phaeocystis globosum* algal solutions from Examples 1-2 and Comparative Example 1. The cell number, chlorophyll a content, hemolysin content, and phosphate content of *Phaeocystis globosum* in the algal solutions were measured. The measurement results are as follows: Figure 3 As shown.

[0036] like Figure 3 a shows the measurement results of the number of *Phaeocystis globosa* algal cells in Examples 1-2 and Comparative Example 1. This indicates that the *Phaeocystis globosa* cells in Comparative Example 1 recovered growth after 7 days of light cultivation. However, the *Phaeocystis globosa* cells in Examples 1 and 2 did not recover growth, and the cell number remained low. Therefore, both Examples 1 and 2 can inhibit the recovery of *Phaeocystis globosa* red tides, and Example 2 is more effective than Example 1.

[0037] like Figure 3 b shows the measurement results of chlorophyll a in *Phaeocystis globosa* in Examples 1-2 and Comparative Example 1. It also shows that the chlorophyll a content of *Phaeocystis globosa* in Comparative Example 1 recovered after 7 days of light cultivation. However, the chlorophyll a content of *Phaeocystis globosa* in Examples 1 and 2 remained low, indicating that both Examples 1 and 2 can inhibit the recovery of *Phaeocystis globosa* red tides, and that Example 2 is more effective than Example 1.

[0038] like Figure 3 c shows the measurement results of hemolysin in *Phaeocystis globosa* in Examples 1-2 and Comparative Example 1. This also indicates that the hemolysin content in *Phaeocystis globosa* in Comparative Example 1 recovered after 7 days of light cultivation. However, the hemolysin content in *Phaeocystis globosa* in Examples 1 and 2 was still significantly lower than that in Comparative Example 1. This suggests that both Examples 1 and 2 can inhibit the recovery of *Phaeocystis globosa* red tides, and that Example 2 is more effective than Example 1.

[0039] like Figure 3 d represents the measurement results of phosphate content in the algal solutions of Examples 1-2 and Comparative Example 1. The phosphate content in Examples 1 and 2 is significantly lower than that in Comparative Example 1, with Example 2 showing the lowest phosphate content. This indicates that both Examples 1 and 2 can prevent the resurgence of *Phaeocystis globosa* red tides by adsorbing phosphate in the water, and Example 2 is the most effective.

[0040] The removal efficiency of *Phaeocystis globulus* algal solutions from Examples 1-2 (for microsphere gel regeneration and reuse) and Comparative Example 1 was measured. The cell number, chlorophyll a content, and hemolysin content of *Phaeocystis globulus* in the algal solutions were measured. The measurement results are as follows: Figure 4 As shown.

[0041] like Figure 4 a shows the measurement results of the number of *Phaeocystis sphericalis* algal cells in Examples 1-2 and Comparative Example 1, which were reused for recycling. This indicates that the regenerated and reused gel microsphere filter bags can still efficiently eliminate *Phaeocystis sphericalis* algal cells. The number of algal cells in Example 2 is lower than in Example 1, and significantly lower than in Comparative Example 1. This demonstrates that the gel microsphere filter bag in Example 2 provided by the present invention has excellent recyclability and can be repeatedly used to eliminate *Phaeocystis sphericalis*.

[0042] like Figure 4 b shows the measurement results of chlorophyll a in *Phaeocystis globosa* in Examples 1-2 and Comparative Example 1, which were reused. This indicates that the recycled gel microsphere filter bags can still efficiently reduce the chlorophyll a content of *Phaeocystis globosa*. The chlorophyll a content in Example 2 is lower than that in Example 1, and significantly lower than that in Comparative Example 1. This demonstrates that the gel microsphere filter bags in Example 2 provided by this invention have excellent recyclability and can be repeatedly used to eliminate *Phaeocystis globosa*.

[0043] like Figure 4 c represents the measurement results of hemolysin from *Phaeocystis globosa* in Examples 1-2 and Comparative Example 1, which were reused. This shows that the regenerated and reused gel microsphere filter bags can still efficiently reduce the hemolysin content of *Phaeocystis globosa*. The hemolysin content in Example 2 was slightly lower than in Example 1, and significantly lower than in Comparative Example 1. This indicates that the gel microsphere filter bag in Example 2 provided by this invention has excellent recyclability and can be repeatedly used to eliminate *Phaeocystis globosa*.

[0044] Measurements were performed on the regenerated and reused *Phaeocystis globosum* algal solutions from Examples 1-2 and Comparative Example 1 to assess their recovery and culture. The cell number, chlorophyll a content, hemolysin content, and phosphate content of *Phaeocystis globosum* in the algal solutions were measured. The results are as follows: Figure 5 As shown.

[0045] like Figure 5 a shows the measurement results of the number of *Phaeocystis globosa* algal cells in Examples 1-2 and Comparative Example 1, which were reused. This indicates that in Comparative Example 1, after 7 days of light cultivation, the *Phaeocystis globosa* cells had recovered growth. However, the reused gel microsphere filter bag in Examples 1-2 still significantly inhibited the growth of *Phaeocystis globosa*, and the cell number remained low; in Example 2, almost no algal cells were visible. Therefore, the gel microsphere filter bag provided by this invention has excellent recyclability, and Example 2 is more effective than Example 1, both of which can inhibit the recovery of *Phaeocystis globosa* red tides.

[0046] like Figure 5 b shows the measurement results of chlorophyll a in *Phaeocystis globosa* from Examples 1-2 and Comparative Example 1, which were regenerated and reused. This indicates that in Comparative Example 1, after 7 days of light cultivation, the chlorophyll a content of *Phaeocystis globosa* had recovered to its initial level. However, the regenerated and reused gel microsphere filter bag in Examples 1-2 still significantly inhibited *Phaeocystis globosa*, and the chlorophyll a content remained low. Therefore, the gel microsphere filter bag provided by this invention has excellent recyclability, and Example 2 is more effective than Example 1, both of which can inhibit the recovery of *Phaeocystis globosa* red tides.

[0047] like Figure 5 c shows the measurement results of hemolysin in *Phaeocystis globosa* in Examples 1-2 and Comparative Example 1, which were reused. This indicates that in Comparative Example 1, the hemolysin in *Phaeocystis globosa* gradually recovered after 7 days of light cultivation. However, the reused gel microsphere filter bag in Examples 1-2 still significantly inhibited *Phaeocystis globosa*, and the hemolysin content remained low. Therefore, the gel microsphere filter bag provided by this invention has excellent recyclability, and Example 2 is more effective than Example 1, both inhibiting the recovery of *Phaeocystis globosa* red tides.

[0048] like Figure 5 Figure d shows the measurement results of phosphate in the algal solution in Examples 1-2 and Comparative Example 1, which were regenerated and reused. The phosphate content of the regenerated and reused gel microsphere filter bags in Examples 1 and 2 was significantly lower than that in Comparative Example 1, with the lowest phosphate content observed in Example 2. This indicates that the regenerated and reused gel microsphere filter bags still have good adsorption performance for phosphate and can prevent the resurgence of *Phaeocystis globosa* red tides by reducing phosphate in the water, with Example 2 showing the best effect.

[0049] In summary, this invention provides a method for controlling *Phaeocystis globosa* red tides using a combination of gel microsphere adsorption and ultrasound, as well as a method for regenerating the gel microspheres. This method is simple and easy to operate, effectively killing *Phaeocystis globosa* cells and degrading hemolytic toxins, while also exhibiting good adsorption of phosphates in the water, preventing the resurgence and re-emergence of red tide algae. Furthermore, the gel microspheres can be recycled and reused with good results. Therefore, this method for controlling *Phaeocystis globosa* red tides using a combination of gel microsphere adsorption and ultrasound is a simple, efficient, and environmentally friendly new method for red tide control.

Claims

1. A method for controlling red tides caused by *Phaeocystis globosum* using a combination of gel microsphere adsorption and ultrasound, characterized in that... First, gel microspheres for adsorption and treatment are prepared. The preparation method of the gel microspheres is as follows: a mixed aqueous solution of sodium alginate and sodium humate is injected into a ferric chloride solution and solidified for 10-15 h. The microspheres are filtered out and calcined at 380-400 ℃ for at least 1 h to obtain gel microspheres. Then, the gel microspheres are placed in a filter bag and placed in a spherical Phaeocystis algal solution, and culture medium is added. Low-frequency ultrasonic treatment combined with gel microsphere adsorption is used to treat the Phaeocystis algal red tide. The mass ratio of sodium alginate to sodium humate is 1:1, and the mass concentration of sodium alginate and sodium humate in the mixed aqueous solution is 1-2%. The concentration of the ferric chloride solution is 0.05-0.1M. The frequency of the low-frequency ultrasonic treatment is 20-40 kHz.

2. The method for controlling red tides caused by *Phaeocystis globulus* using a combination of gel microsphere adsorption and ultrasound according to claim 1, characterized in that... The gel microspheres have a particle size of 0.5-1.0 mm, and the filter bags have a pore size of 8-10 μm.

3. The method for controlling red tides caused by *Phaeocystis globulus* using a combination of gel microsphere adsorption and ultrasound according to claim 1, characterized in that... When using gel microspheres packed in filter bags and combined with an ultrasonic device to treat red tides of Phaeocystis globosa in water bodies, the dosage of gel microspheres is 0.22-2.2 kg per cubic meter of water. Low-frequency ultrasonic treatment is used until the phosphate content in the water body is lower than 1 ug / L.

4. A method for regenerating gel microspheres according to any one of claims 1-3, characterized in that, The combined-treatment gel microspheres were placed in distilled water and treated at 0.2 MPa pressure and 121 °C for 20-30 min. After being removed and dried at 60 °C, the gel microspheres were regenerated.

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