A method for inhibiting the growth of Microcystis aeruginosa by combining a low-voltage alternating electric field and allelochemical slow-release biochar.

Abstract

This invention provides a method for inhibiting the growth of *Microcystis aeruginosa* using a low-voltage alternating current electric field and allelochemical slow-release biochar, belonging to the field of algal bloom control. Specific steps: *Microcystis aeruginosa* algal solutions (both non-toxic and toxic) are pumped through a stainless steel mesh low-voltage alternating current electric field device using a peristaltic pump, with the voltage controlled at 2V for four cycles. Then, 50 mg / L of pyrogallic acid-coated corn cob biochar is added to the algal solution. After combined treatment, the inhibition rate can reach 70% or higher by day 7. Compared with traditional algal suppression methods, the low-voltage alternating current electric field increases cell membrane permeability, and when combined with pyrogallic acid-coated corn cob biochar, it has a higher algal suppression efficiency. This invention is simple to operate, inexpensive, does not affect the aesthetics of the water body, and effectively inhibits the growth of *Microcystis aeruginosa* in the water, showing good application prospects in controlling algal blooms in small landscape water bodies.

Description

Technical Field

[0001] This invention belongs to the field of algal bloom control, and particularly relates to a method for inhibiting the growth of Microcystis aeruginosa by using a low-voltage alternating electric field and allelochemical slow-release biochar. Background Technology

[0002] In recent years, with rapid industrial development and the continuous improvement of urban living standards, the amount of nutrient-rich wastewater generated by human activities has been increasing daily, causing considerable damage to the global water environment. In eutrophic water bodies, the large-scale growth and reproduction of microalgae can cause severe algal blooms. Microcystis aeruginosa is the most important cyanobacterial species causing algal blooms; its excessive reproduction covers the water surface, causing a decrease in dissolved oxygen, water quality deterioration, and mass mortality of aquatic organisms such as fish. Simultaneously, some Microcystis species release algal toxins, which can cause severe damage to aquatic organisms and may even accumulate in fish and shrimp or spread into drinking water, thus endangering human health. Therefore, controlling algal blooms remains one of the main tasks of water pollution control.

[0003] Currently, there are many common methods for suppressing algal blooms, mainly including mechanical harvesting, the addition of chemical agents, and biological predation. Among these, mechanical harvesting is energy-intensive and costly; the addition of chemical algaecides is simple to operate and has a fast removal efficiency, but it usually brings serious side effects to aquatic organisms and the ecosystem; biological predation for algae control technology is complex to manage, has a long treatment cycle, and poses a risk of ecosystem imbalance.

[0004] Plant-produced allelochemicals for algae control are attracting increasing attention due to their economic, environmentally friendly, and efficient characteristics. However, direct introduction of allelochemicals into water bodies can easily lead to excessively high local concentrations, affecting the growth of non-algae organisms and causing side effects on the ecological environment. Furthermore, their efficacy is relatively short-lived. Currently, emerging low-voltage AC electric field algae control technology enhances cell membrane permeability and uses electroporation to inactivate microalgae, offering advantages such as low cost and safety. However, algal cells can still regenerate. Addressing the issue of allelochemicals being easily decomposed and microalgae regrowth after low-voltage AC electric field treatment, this invention cleverly combines low-voltage AC electric field technology with allelochemical slow-release biochar algae control technology. By enhancing cell membrane permeability through a low-voltage AC electric field, algal cells absorb more allelochemicals to inhibit microalgae growth, thus achieving a better algae control effect and demonstrating broad application prospects. Summary of the Invention

[0005] This invention provides a technique for effectively inhibiting the growth of both non-toxic and toxic Microcystis aeruginosa by combining a low-voltage alternating electric field with slow-release biochar containing allelochemicals. The growth of Microcystis aeruginosa can be effectively inhibited by using a relatively small electric field strength and adding a trace amount of slow-release biochar containing allelochemicals.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] (1) Assembly of low-voltage AC electric field device

[0008] First, the stainless steel mesh electrodes were ultrasonically cleaned with methanol and acetone to remove surface impurities for 20–30 minutes. Then, the two stainless steel mesh electrodes, insulating gaskets, conductive sheets, and plexiglass electrode supports were assembled to form the electrode device. Subsequently, the electrode device was connected to a peristaltic pump using silicone tubing and a reducing connector to create a flowing low-voltage electric field device. During the experiment, a waveform generator was used to apply a voltage between the two electrodes.

[0009] (2) Preparation of pyrogallic acid corn cob biochar

[0010] First, prepare a 50% sulfuric acid solution. Weigh 5g of corn cob biochar material and add it to the solution. Then, use magnetic stirring at 500 rpm for 5 hours to ensure a complete reaction, removing impurities that readily react with acid from the surface and pores of the biochar. Next, take the stirred solution and filter it using a circulating water multi-purpose vacuum pump. Wash the filtered biochar several times with distilled water and dry it in an oven for 8 hours to obtain purified biochar. Then, take 5g of the purified corn cob biochar material and place it in a 50mL Erlenmeyer flask. Add 30mL of analytical grade pyrogallic acid and transfer the flask to a water bath constant temperature shaker for a shaking reaction at 50℃ and 120 rpm for 2 hours. After washing and filtration, dry in a drying oven to obtain pyrogallic acid-containing corn cob slow-release biochar.

[0011] (3) Joint processing

[0012] First, the stainless steel mesh electrode is ultrasonically cleaned to remove surface impurities for 20–30 minutes. The algal solution is then pumped through a stainless steel mesh low-voltage electric field reaction device using a peristaltic pump at a flow rate of 5 mL / min and a voltage of 0.5–2 V. Subsequently, 10–50 mg / L of pyrogallol corn cob biochar is added. The algal density is measured daily to calculate the algal inhibition rate.

[0013] The aforementioned Microcystis aeruginosa includes non-toxic Microcystis aeruginosa (FACHB-526) and toxic Microcystis aeruginosa (FACHB-905).

[0014] The stainless steel mesh electrode is a commercially available ordinary stainless steel mesh (60 mesh), which is ultrasonically cleaned with acetone and methanol to remove impurities. The preferred stainless steel mesh voltage is 2V and the process is repeated four times.

[0015] The pyrogallol corn cob biochar should be stored in a dry, well-ventilated glass petri dish.

[0016] The algal cell concentration is 1×10 6 The preferred concentration of pyrogallol corn cob biochar is 50 mg / L.

[0017] The culture time is 7 days.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention selects readily available and inexpensive stainless steel mesh as the electrode material, readily available corn cob biochar as the substrate material, and environmentally friendly pyrogallic acid as the allelochemical to prepare pyrogallic acid corn cob biochar. The low-voltage AC electric field of the stainless steel mesh and the pyrogallic acid corn cob biochar effectively inhibit the growth of *Microcystis aeruginosa*, with an inhibition effect superior to that achieved by treating with either the low-voltage AC electric field or the pyrogallic acid corn cob biochar alone. Applying this technology to algal blooms in small landscape water bodies not only does not affect the aesthetic appeal of the water body but also effectively inhibits the growth of *Microcystis aeruginosa*, representing an economical, environmentally friendly, and effective new approach to control algal blooms in small landscape water bodies, with promising application prospects. Attached Figure Description

[0020] Figure 1 The growth curves of non-toxic Microcystis aeruginosa under different treatment modes in Example 1 and Comparative Examples 1-2 are shown.

[0021] Figure 2 The graph shows the inhibition rate of different treatment modes on the growth of non-toxic Microcystis aeruginosa in Example 1 and Comparative Examples 1-2.

[0022] Figure 3 The growth curves of toxin-producing Microcystis aeruginosa under different treatment modes in Example 2 and Comparative Examples 3-4 are shown.

[0023] Figure 4 The graph shows the inhibition rate of different treatment modes on the growth of toxin-producing Microcystis aeruginosa in Example 2 and Comparative Examples 3-4. Detailed Implementation

[0024] This invention provides a method for inhibiting both non-toxic and toxic Microcystis aeruginosa using a combination of a low-voltage alternating electric field and allelochemical slow-release biochar, comprising the following steps:

[0025] (1) Cut a 60-mesh stainless steel mesh into pieces approximately 1cm x 1cm in size, and ultrasonically clean them with methanol and acetone for 20–30 min respectively. Then, at a fixed flow rate of 5 mL / min using a peristaltic pump, pump the *Microcystis aeruginosa* solution through the electrode reaction device. The voltage of the low-voltage AC electric field is set to 2V, and the frequency is set to 10. 6 Hz. The algal solution flows through the electrode reaction device and completes one electric field treatment. The treated algal solution is then passed through the electric field again and completes two electric field treatments, and so on.

[0026] (2) Purification of corn cob biochar

[0027] First, prepare a 50% sulfuric acid solution. Weigh out 5g of corn cob biochar material and add it to the solution. Then, use magnetic stirring at 500 rpm for 5 hours to ensure a complete reaction. This is to remove impurities that are easily reacted with acid from the surface and pores of the biochar. Next, take the stirred solution and filter it using a circulating water multi-purpose vacuum pump. Wash the filtered biochar several times with distilled water and then dry it in an oven for 8 hours to obtain purified biochar.

[0028] (3) Preparation of corn cob biochar composite pyrogallic acid algae-inhibiting material

[0029] Take 5g of purified corn cob biochar material and place it in a 50mL Erlenmeyer flask. Then add 30mL of analytical grade pyrogallol. Transfer the Erlenmeyer flask to a water bath constant temperature shaker for shaking reaction at 50℃, a rotation speed of 120r / min, and a preparation time of 2h. After washing and filtration, dry in a drying oven to obtain the allelochemical heavy composite material.

[0030] (4) The prepared pyrogallol corn cob biochar was added to the Microcystis aeruginosa algal solution obtained after four low-voltage AC electric field treatments. The algal density was measured daily and the algal inhibition rate was calculated.

[0031] In this invention, the *Microcystis aeruginosa* is preferably cultured in BG11 liquid medium to increase the microalgae density before inoculation. This invention does not impose any special limitations on the culture process before inoculation of *Microcystis aeruginosa*; conventional methods in the art can be used.

[0032] The technical solutions provided by this invention will be described in detail below with reference to specific examples, but they should not be construed as limiting the scope of protection of this invention.

[0033] Example 1

[0034] The stainless steel mesh was ultrasonically cleaned with methanol and acetone for 20 minutes each, then rinsed with distilled water. The two stainless steel mesh electrodes, insulating gaskets, conductive sheets, and plexiglass electrode holders were then assembled to form the electrode device. All parts of the electrode holder were bonded with silicone gaskets to ensure a sealed reactor. The electrode device was then connected to the peristaltic pump using silicone tubing and a reducer to create a low-voltage AC electric field. After reactor assembly, the initial inoculation density was 10-1. 6 Non-toxic Microcystis aeruginosa algal solution of 1 / mL was pumped through the reactor using a peristaltic pump at a fixed flow rate of 5 mL / min, with a 2V voltage applied between the two electrodes. After four cycles of electric field circulation, 50 mg / L of pyrogallol-coated corn cob biochar was added. The reaction period was set to 7 days, with algal density measured daily and inhibition rate calculated. Each experiment included three replicates.

[0035] Comparative Example 1

[0036] The stainless steel mesh was ultrasonically cleaned with methanol and acetone for 20 minutes each, then rinsed with distilled water. The two stainless steel mesh electrodes, insulating gaskets, conductive sheets, and plexiglass electrode holders were then assembled to form the electrode device. All parts of the electrode holder were bonded with silicone gaskets to ensure a sealed reactor. The electrode device was then connected to the peristaltic pump using silicone tubing and a reducer to create a low-voltage AC electric field. After reactor assembly, the initial inoculation density was 10-1. 6 Non-toxic Microcystis aeruginosa algal solution of 1 / mL was pumped through the reactor using a peristaltic pump at a fixed flow rate of 5 mL / min. A voltage of 2V was applied between the two electrodes for four cycles. The reaction period was set to 7 days, and the algal density was measured daily to calculate the algal inhibition rate. Each experiment included three replicates.

[0037] Comparative Example 2

[0038] Pyrogallol-containing corn cob biochar was added at a dosage of 50 mg / L to 100 mL of water with an initial algal density of 1×10⁻⁶. 6 In a solution of non-toxic Microcystis aeruginosa cells / mL, the growth of algal cells was recorded for seven consecutive days, the algal density was measured daily, and the algal inhibition rate was calculated. Each experiment included three parallel samples.

[0039] The results are as follows Figure 1 , 2 As shown, after 7 days of cultivation, the non-toxic Microcystis aeruginosa control group increased from an initial algal density of 1×10⁻⁶. 6 The number of cells / mL increased to 7.6 × 10⁻⁶ 6Cells / mL, low-voltage AC electric field, treatment with pyrogallic acid-coated corn cob biochar alone, and combined treatment with both all inhibited the growth of non-toxic Microcystis aeruginosa. After treatment with 50 mg / L pyrogallic acid-coated corn cob biochar alone and four cycles of treatment with a 2V electric field alone, the algal cell count increased to 3.8167 × 10⁶ cells / mL. 6 cells / mL and 3.6833×10 6 The combined treatment with both methods resulted in an algal density of 2.1333 × 10⁶ cells / mL, with inhibition rates of 49.78% and 51.54%, respectively. After 7 days of culture, the combined treatment yielded an algal density of 2.1333 × 10⁶ cells / mL. 6 The combined treatment group showed a concentration of 71.93% and an inhibition rate of 71.93%. Compared with the single treatment, the growth of non-toxic Microcystis aeruginosa was significantly inhibited.

[0040] Example 2

[0041] The stainless steel mesh was ultrasonically cleaned with methanol and acetone for 20 minutes each, then rinsed with distilled water. The two stainless steel mesh electrodes, insulating gaskets, conductive sheets, and plexiglass electrode holders were then assembled to form the electrode device. All parts of the electrode holder were bonded with silicone gaskets to ensure a sealed reactor. The electrode device was then connected to the peristaltic pump using silicone tubing and a reducer to create a low-voltage AC electric field. After reactor assembly, the initial inoculation density was 10-1. 6 Algal culture of *Microcystis aeruginosa* with a concentration of 1 / mL was pumped through the reactor using a peristaltic pump at a fixed flow rate of 5 mL / min, with a 2V voltage applied between the two electrodes. After four cycles of electric field circulation, 50 mg / L of pyrogallol-coated corn cob biochar was added. The reaction period was set to 7 days, with algal density measured daily and inhibition rate calculated. Each experiment included three replicates.

[0042] Comparative Example 3

[0043] The stainless steel mesh was ultrasonically cleaned with methanol and acetone for 20 minutes each, then rinsed with distilled water. The two stainless steel mesh electrodes, insulating gaskets, conductive sheets, and plexiglass electrode holders were then assembled to form the electrode device. All parts of the electrode holder were bonded with silicone gaskets to ensure a sealed reactor. The electrode device was then connected to the peristaltic pump using silicone tubing and a reducer to create a low-voltage AC electric field. After reactor assembly, the initial inoculation density was 10-1. 6 A solution of toxin-producing Microcystis aeruginosa at a concentration of 1 / mL was pumped through the reactor using a peristaltic pump at a fixed flow rate of 5 mL / min. A 2V voltage was applied between the two electrodes, and the mixture was cycled four times. The reaction period was set to 7 days, with algal density measured daily to calculate the algal inhibition rate. Each experiment included three replicates.

[0044] Comparative Example 4

[0045] Pyrogallol-containing corn cob biochar was added at a dosage of 50 mg / L to 100 mL of water with an initial algal density of 1×10⁻⁶. 6 In a solution of toxin-producing Microcystis aeruginosa containing 1 / mL of microcystis aeruginosa, the growth of algal cells was recorded for seven consecutive days, the algal density was measured daily, and the algal inhibition rate was calculated. Each experiment included three parallel samples.

[0046] The results are as follows Figure 3 , 4 As shown, after 7 days of cultivation, the toxin-producing Microcystis aeruginosa control group increased from an initial algal density of 1×10⁻⁶. 6 The number of cells / mL increased to 7.3667 × 10⁻⁶. 6 Cells / mL, low-voltage AC electric field, treatment with pyrogallic acid-coated corn cob biochar alone, and combined treatment with both all inhibited the growth of toxin-producing Microcystis aeruginosa. After treatment with 50 mg / L pyrogallic acid-coated corn cob biochar alone and four cycles of a 2V electric field alone, the algal cell count reached 3.7417 × 10⁶ cells / mL. 6 cells / mL and 3.8667×10 6 The combined treatment with both methods resulted in an algal density of 2.1667 × 10⁶ cells / mL, with inhibition rates of 49.21% and 47.51%, respectively. After 7 days of culture, the combined treatment yielded an algal density of 2.1667 × 10⁶ cells / mL. 6 The combined treatment group showed a concentration of 70.59% and an inhibition rate of 70.59%. Compared with the single treatment, the growth of toxin-producing Microcystis aeruginosa was significantly inhibited.

[0047] In summary, the combination of the stainless steel mesh low-voltage AC electric field and pyrogallol corn cob biochar of the present invention has a strong inhibitory effect on both non-toxic and toxic Microcystis aeruginosa, with inhibition rates of 71.93% and 70.59%, respectively. It has advantages such as being economical and environmentally friendly, having good treatment effect, and having a long action period.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for inhibiting the growth of Microcystis aeruginosa by combining a low-voltage alternating electric field and allelochemical slow-release biochar, characterized in that, Includes the following steps: (1) Purification of corn cob biochar; (2) Preparation of pyrogallol-containing corn cob biochar; (3) Set the initial algal density to 1×10 6 ~5×10 6 Microcystis aeruginosa algal solution of 1 / mL was pumped through a stainless steel mesh low-voltage AC electric field reaction device using a peristaltic pump, and then pyrogallol corn cob biochar was added to the algal solution; wherein, the Microcystis aeruginosa includes both toxic and non-toxic Microcystis aeruginosa.

2. The method for inhibiting the growth of Microcystis aeruginosa by combining a low-voltage alternating electric field and allelochemical slow-release biochar according to claim 1, characterized in that, The purification method of step (1) is as follows: prepare a sulfuric acid solution with a mass fraction concentration of 50%, add 5g of corn cob biochar material, stir and react at a stirring speed of 500r / min for 5h, filter the stirred solution, wash it several times with distilled water, and dry it for 8h to obtain purified biochar.

3. The method for inhibiting the growth of Microcystis aeruginosa by combining a low-voltage alternating electric field and allelochemical slow-release biochar according to claim 1, characterized in that, The method of step (2) is as follows: 5g of corn cob biochar purification material is mixed with 30mL of analytical grade pyrogallic acid and shaken in a water bath constant temperature shaker. The preparation temperature is 50℃, the preparation speed is 120r / min, and the preparation time is 2h. After washing, filtration and drying, pyrogallic acid corn cob slow-release biochar is obtained.

4. The method for inhibiting the growth of Microcystis aeruginosa by combining a low-voltage alternating electric field and allelochemical slow-release biochar according to claim 1, characterized in that, The low-voltage AC electric field reaction device is assembled from a stainless steel mesh electrode with a aperture of 60 mesh, a conductive sheet and an electrode support. The voltage is controlled between 0.5 and 2V, and the flow rate of the peristaltic pump is controlled at 5mL / min.

5. The method for inhibiting the growth of Microcystis aeruginosa by combining a low-voltage alternating electric field and allelochemical slow-release biochar according to claim 1, characterized in that, The concentration of the pyrogallol corn cob biochar is controlled at 10–50 mg / L.

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