A method for removing microcystis aeruginosa by using rose Bengal

Abstract

The present application belongs to the environmental governance technical field, and particularly relates to a method for removing Microcystis aeruginosa by using rose Bengal. Rose Bengal is added in an environment containing Microcystis aeruginosa; the final concentration of rose Bengal is 5 mg / L-10 mg / L, and Microcystis aeruginosa is removed under visible light. After adding rose Bengal, the highest algae density can remove 85.37% of Microcystis aeruginosa; at 168 h, the effect of stably inhibiting Microcystis aeruginosa is still maintained. The present application discloses a mild drug control mode by using the characteristics of high singlet oxygen yield of RB to target damage the photosynthetic system and core metabolic pathway of Microcystis aeruginosa, and discloses a three-damage mechanism of physiological and biochemical, photosynthetic system and metabolic network of RB photodynamic algae inhibition.

Description

Technical Field

[0001] This invention belongs to the field of environmental remediation technology, specifically relating to a method for removing Microcystis aeruginosa using Bengal rose red. Background Technology

[0002] Microcystis aeruginosa is a major algal species that causes cyanobacterial blooms in freshwater bodies. Its outbreaks can disrupt the aquatic ecological balance, release microcystin-releasing enzymes (MC-LR), threaten drinking water safety and human health, and cause huge economic losses.

[0003] Current cyanobacteria control technologies employ physical methods such as harvesting, ultrasonication, and membrane filtration, which are costly, time-consuming, and difficult to apply on a large scale. Further, chemical methods, such as algaecides like copper sulfate, potassium permanganate, and chlorine dioxide, can be used on a large scale at lower costs. However, these methods disrupt aquatic ecosystems, and KMnO4 can cause a sharp increase in extracellular algal toxins, posing safety risks. While H2O2 can degrade toxins, its sustained effectiveness is poor. In short, these methods indiscriminately kill aquatic organisms and easily cause secondary pollution. Further, biological methods, using microorganisms, algae-eating organisms, and aquatic plants to suppress algae, do not produce secondary pollution, but their stability is poor, making them unsuitable for emergency response.

[0004] Furthermore, while traditional photocatalytic methods offer good stability, such as the photocatalytic removal of Microcystis aeruginosa using TiO2 materials, they suffer from low efficiency in inhibiting Microcystis aeruginosa. Summary of the Invention

[0005] The present invention aims to provide the application of Bengal rose red in the removal of Microcystis aeruginosa, in order to solve the technical problem of low efficiency in inhibiting Microcystis aeruginosa in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for removing Microcystis aeruginosa using Bengal rose red involves adding Bengal rose red to an environment containing Microcystis aeruginosa, achieving a final concentration of 5 mg / L to 10 mg / L, and then removing Microcystis aeruginosa under visible light.

[0007] Furthermore, the final concentration of Bengal rose red added in the environment containing Microcystis aeruginosa is 7.5 mg / L.

[0008] Furthermore, the intensity of the visible light is 2000 lux to 4000 lux.

[0009] Furthermore, the illuminance of the visible light is 3500 lux.

[0010] Furthermore, the temperature of the environment containing Microcystis aeruginosa is 25℃~30℃.

[0011] Furthermore, the temperature of the environment containing Microcystis aeruginosa is 25°C.

[0012] Furthermore, the light-dark ratio of the environment containing Microcystis aeruginosa is 12h:12h~16h:8h.

[0013] The principle of this invention is as follows: Using Bengal rose red RB as a photosensitive algaecide, a large amount of singlet oxygen is generated under visible light irradiation. 1 O2 targets and disrupts the cell membrane structure, photosynthetic system, and core metabolic pathways of Microcystis aeruginosa, achieving highly efficient inactivation while gently controlling the release of algal toxins. It is suitable for the treatment of Microcystis aeruginosa blooms in eutrophic waters.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a method for removing Microcystis aeruginosa using Bengal rose red. Bengal rose red is added to an environment containing Microcystis aeruginosa to a final concentration of 5 mg / L to 10 mg / L, and Microcystis aeruginosa is removed under visible light. Compared with the algal density without Bengal rose red, the algal density with added Bengal rose red is reduced by 85.37%, which means that the inhibition efficiency of Microcystis aeruginosa is high. The inhibition effect of Microcystis aeruginosa is still stable after 168 hours.

[0015] (2) This invention utilizes the high singlet oxygen production of RB for the first time to target and damage the photosynthetic system and core metabolic pathway of Microcystis aeruginosa, and discloses a mild and controlled toxicity mechanism; it reveals the physiological and biochemical, photosynthetic system and metabolic network triple damage mechanism of RB photodynamic algae inhibition; as a typical type II photosensitizer, Bengal rose red RB is used for the first time to specifically remove Microcystis aeruginosa by RB photodynamic effect, and discloses the optimal action conditions, algae inhibition mechanism, algal toxin release control and metabolic disturbance law.

[0016] (3) This invention uses Bengal rose red (RB) as a photosensitive algaecide, overcoming the shortcomings of existing algaecide technologies such as high cost, secondary pollution, and uncontrolled toxin release; it provides a green, efficient, and low-cost method for removing Microcystis aeruginosa that can be applied in actual water bodies; it is green and environmentally friendly, with no heavy metal residues, good biocompatibility, and does not cause secondary pollution; it is efficient and low-cost, with low RB prices, low optimal concentration, and can be excited by visible light; it has strong targeting, specifically damaging the photosynthetic system and membrane structure of algal cells, and is friendly to non-target organisms; it has a synergistic effect of algae inhibition and toxin control, avoiding a sharp increase in extracellular toxins, and is suitable for emergency treatment of algal blooms; it is applicable to actual water bodies, has stable effects in natural water samples, and can be applied on a large scale.

[0017] (4) This invention clarifies the optimal conditions for the inhibition of Microcystis aeruginosa by Bengal rose red RB, including concentration, light and temperature; and achieves a synergistic effect of highly efficient algae inhibition and mild toxin control, reducing the risk of extracellular algal toxin release. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, instruments, etc. used are all commercially available. The following contents need to be specifically explained:

[0019] Rose Bengal (RB); Microcystis aeruginosa (Latin name: RB) Microcystis aeruginosa The genus FACHB905 is in the Freshwater Algae Culture Bank of the Chinese Academy of Sciences; it is also known as photodynamic algae inhibition and singlet oxygen. 1 O2; Reactive oxygen species (ROS); Microcystin-LR (MC-LR); Photosystem II (PSII); Chlorophyll fluorescence induction kinetics curve (OJIP); Malondialdehyde (MDA); Superoxide dismutase (SOD); Catalase (CAT); Adenosine triphosphate (ATP).

[0020] Example 1: A method for removing Microcystis aeruginosa using Bengal rose red, the specific steps of which are as follows: S1. Microcystis aeruginosa was cultured on BG-11 medium at 27°C, with a light intensity of 3000 lux and a light-dark ratio of 16 h:8 h until it reached the logarithmic growth phase.

[0021] S2. Dilute the algal solution cultured to the logarithmic growth phase with BG-11 medium solution until the absorbance is 0.1. Then add RB to the algal solution. Add RB to Microcystis aeruginosa to a final concentration of 5 mg / L. The light intensity is 3500 lux, the temperature is 25℃, and the light-dark ratio is 16h:8h.

[0022] Example 2: A method for removing Microcystis aeruginosa using Bengal rose red, the specific steps of which are as follows: S1. Microcystis aeruginosa was cultured using BG-11 medium at 27°C, light intensity of 3000 lux, and light-dark ratio of 16h:8h.

[0023] S2. Add RB to Microcystis aeruginosa to a final concentration of 7.5 mg / L, with a light intensity of 3500 lux, a temperature of 25℃, and a light-dark ratio of 16 h:8 h.

[0024] Example 3: A method for removing Microcystis aeruginosa using Bengal rose red, the specific steps of which are as follows: S1. Microcystis aeruginosa was cultured using BG-11 medium at 27°C, light intensity of 3000 lux, and light-dark ratio of 16h:8h.

[0025] S2. Add RB to Microcystis aeruginosa to a final concentration of 10 mg / L, with a light intensity of 3500 lux, a temperature of 25℃, and a light-dark ratio of 16 h:8 h.

[0026] Example 4: A method for removing Microcystis aeruginosa using Bengal rose red, the specific steps of which are as follows: S1. Microcystis aeruginosa was cultured using BG-11 medium at 27°C, light intensity of 3000 lux, and light-dark ratio of 13h:11h.

[0027] S2. Add RB to Microcystis aeruginosa to a final concentration of 10 mg / L, with a light intensity of 3500 lux, a temperature of 25℃, and a light-dark ratio of 12h:12h.

[0028] Example 5: A method for removing Microcystis aeruginosa using Bengal rose red, the specific steps of which are as follows: S1. Microcystis aeruginosa was cultured using BG-11 medium at 27°C, light intensity of 3000 lux, and light-dark ratio of 13h:11h.

[0029] S2. Add RB to Microcystis aeruginosa to a final concentration of 10 mg / L, with a light intensity of 3500 lux, a temperature of 24℃, and a light-dark ratio of 17 h:7 h.

[0030] Application Example 1: Verification using actual water samples Using lake water samples from Midong District, Urumqi, China as the actual water samples, Microcystis aeruginosa was artificially added. The absorbance of the lake water sample + algae solution in the actual water sample reached 0.1. RB was added to the actual water sample at a ratio of 1L:7.5mg.

[0031] Comparative Example 1: A method for removing Microcystis aeruginosa using KMnO4, the specific steps of which are as follows: Based on Example 1, the algal culture that had reached the logarithmic growth phase was diluted with BG-11 medium solution until the absorbance was 0.1. Then, KMnO4 was added to the algal culture with an absorbance of 0.1, resulting in a final concentration of 3 mg / L. The light intensity was 3500 lux, the temperature was 25°C, and the light-dark ratio was 16 h:8 h. Experiment 1: Detection of Microcystis aeruginosa concentration The number of *Microcystis aeruginosa* cells was counted using a hemocytometer under different absorbance values ​​at 680 nm. Based on the cell number and absorbance value, a regression equation was constructed for the quantitative analysis of *Microcystis aeruginosa* cell density: Y = 25.92X - 0.98, R0 2 =0.999.

[0032] The concentration of Microcystis aeruginosa after adding RB for 120 hours in Examples 1 to 3 was measured. Compared with the concentration of Microcystis aeruginosa cultured with BG-11 culture medium alone, the algal density of Microcystis aeruginosa with added RB decreased by 70.12%, 85.37%, and 83.15%, respectively.

[0033] When RB was added to the actual water body, the algae inhibition rate of Microcystis aeruginosa was 83.46% after 168 hours, and the algae inhibition effect remained stable in the real water sample after 168 hours.

[0034] Experiment 2 Oxidative Stress Detection Testing of *Microcystis aeruginosa* with added RB in Example 2 revealed oxidative stress damage, with SOD initially increasing and then decreasing, indicating a collapse of the antioxidant system; MDA significantly increased, indicating severe membrane damage; Na+... + / K + ATPase activity decreased by 90%.

[0035] Experiment 3 Detection of relevant indicators of the photosynthetic system The analysis of Microcystis aeruginosa after the addition of RB in Example 2 revealed that the photosynthetic system was disrupted, with chlorophyll a decreasing by 94.3%, Fv / Fm decreasing by 54.3%, PIabs decreasing by 56.9%, and PSII electron transport completely blocked.

[0036] Experiment 4 Structural observation of Microcystis aeruginosa The analysis of Microcystis aeruginosa after the addition of RB in Example 2 revealed that the structure of Microcystis aeruginosa was damaged, and the algal cells showed signs of depression, shrinkage, rupture and disintegration over time, with severe lipid peroxidation of the cell membrane.

[0037] Experiment 5 RB detection To assess the toxin control advantages of RB, in Example 2, the intracellular MCLR of Microcystis aeruginosa with RB added and in Comparative Example 1, the microcystis aeruginosa with KMnO4 added decreased by 80.8%, while the extracellular toxin level only increased slightly, far lower than that of the KMnO4 group, indicating no risk of toxin release. The microcystis aeruginosa with KMnO4 added is the KMnO4 group.

[0038] Experiment 6 Detection of Metabolic Indicators The metabolism of Microcystis aeruginosa after the addition of RB in Example 2 was tested, and metabolic disorders were found. Glycolysis, TCA cycle, pentose phosphate pathway, and amino acid / fatty acid / nucleotide metabolism were all inhibited, resulting in energy collapse of algal cells and inability to divide and repair.

[0039] Experiment 7: Mechanism of action of RB in inhibiting Microcystis aeruginosa 1 O2 is the main reactive oxygen species. - As a secondary factor, ·OH participates in the initial stage.

[0040] In summary, 7.5 mg / L RB is the economically optimal concentration for inhibiting *Microcystis aeruginosa*. The optimal light intensity is 2000 lux–4000 lux, with 3500 lux showing the best inhibitory effect. The optimal temperature is 25℃–30℃, with 25℃ being the optimal temperature for action. In actual water body verification, the inhibitory effect remained stable for 168 hours in real water samples. Adding 7.5 mg / L Bengal rose red to water containing *Microcystis aeruginosa*, adjusting the water temperature to 25℃, and continuously irradiating with 3500 lux of visible light for 96 hours resulted in highly efficient inactivation of algal cells. The efficacy verification indicators included cell density, cell morphology (SEM), antioxidant enzymes (SOD / CAT), MDA content, and Na+. + / K + ATPase, photosynthetic pigments, chlorophyll fluorescence (OJIP), intracellular and extracellular MCLR, intracellular and extracellular organic matter, reactive oxygen species quenching, and non-targeted metabolomics. Highly effective algae suppression: under conditions of 7.5 mg / L RB, 3500 lux, and 25℃, algal density decreased by 85.37% after 96 hours, and the actual algae suppression rate in water samples was 83.46% after 168 hours. Structural damage: algal cells showed indentation, shrinkage, rupture, and disintegration over time, with severe cell membrane lipid peroxidation. Oxidative stress damage: SOD initially increased and then decreased, indicating a collapse of the antioxidant system; MDA significantly increased, indicating severe membrane damage; Na... + / K + ATPase activity decreased by 90%. The photosynthetic system disintegrated, with chlorophyll a decreasing by 94.3%, Fv / Fm decreasing by 54.3%, PIabs decreasing by 56.9%, and PSII electron transport completely blocked. RB showed advantages in controlling toxins, reducing intracellular MCLR by 80.8%, while extracellular toxins only increased slightly, far lower than the KMnO4 group, with no risk of toxin burst release, but it can cause metabolic disorders in Microcystis aeruginosa; the mechanism of action is clear.

[0041] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for removing Microcystis aeruginosa using Bengal rose red, characterized in that, Add Bengal rose red to an environment containing Microcystis aeruginosa; make the final concentration of Bengal rose red 5 mg / L~10 mg / L, and remove Microcystis aeruginosa under visible light.

2. The method according to claim 1, characterized in that, The final concentration of Bengal rose red added in the environment containing Microcystis aeruginosa is 7.5 mg / L.

3. The method according to claim 1, characterized in that, The intensity of the visible light is 2000 lux to 4000 lux.

4. The method according to claim 3, characterized in that, The intensity of the visible light is 3500 lux.

5. The method according to claim 1, characterized in that, The temperature of the environment containing Microcystis aeruginosa was 24℃~30℃.

6. The method according to claim 5, characterized in that, The temperature of the environment containing Microcystis aeruginosa was 25°C.

7. The method according to claim 1, characterized in that, The light-dark ratio of the environment containing Microcystis aeruginosa was (12h:12h) to (17h:7h).

8. The method according to claim 1, characterized in that, The light-dark ratio of the environment containing Microcystis aeruginosa was 16h:8h.