CN114162884B

CN114162884B - Method for inhibiting harmful algae by using perylenequinone compounds

Info

Publication number: CN114162884B
Application number: CN202111362979.3A
Authority: CN
Inventors: 饶义剑; 袁振波; 刘美玲; 张艳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-11-17
Filing date: 2021-11-17
Publication date: 2023-04-28
Anticipated expiration: 2041-11-17
Also published as: CN114162884A

Abstract

The invention discloses a method for inhibiting harmful algae by using perylenequinone compounds, and belongs to the technical field of environmental application. The perylenequinone compound provided by the invention can obviously inhibit the growth of harmful algae under the irradiation of no light or visible light, so that the cell surface is not smooth and the cell morphology is incomplete; can destroy cell membrane, cell wall and thylakoid structure; degrading fatty acids in blue algae cell membranes; degrading chlorophyll a content therein; inhibit the growth of blue algae in the actual environment of the Taihu lake. The method of the invention uses perylenequinone compounds as the algae inhibitor for inhibiting blue algae in a broad spectrum, and has very wide application prospect.

Description

Method for inhibiting harmful algae by using perylenequinone compounds

Technical Field

The invention particularly relates to a method for inhibiting harmful algae by using perylenequinone compounds, and belongs to the technical field of treatment of pollutants in water.

Background

With the development of urban land of China, a large amount of nutrient substances required by organisms such as nitrogen, phosphorus and the like in industrial and agricultural wastewater enter a closed or semi-closed water body in a city, so that the water body is seriously eutrophicated, and algal bloom is initiated. Numerous studies have found that microcystis bloom is most prevalent in algal bloom. When bloom occurs, the excessively propagated algae cause anoxic death of other aquatic organisms, and secondary metabolites including organic substances are formed, so that the water body emits smell to different degrees. In addition, part of the water bloom dominant algae release a large amount of algae toxins into the water body during the metabolic process or after the algae body breaks. And algae toxins are extremely harmful to humans and animals. Therefore, how to effectively control the transitional propagation of algae which causes the dominant algae bloom is the primary task of algae bloom treatment.

According to the principle of controlling harmful algae, existing control methods can be classified into physical methods, chemical methods and biological methods. Physical methods, including mechanical salvage methods, ultrasonic methods and optical isolation methods, are time-consuming and labor-consuming and have high cost; the biological means mainly comprises that the microbial preparation inhibits the growth of blue algae, so that the stability is poor and the balance of ecological environment can be possibly damaged; chemical methods remain the most efficient. The commonly used chemical methods are largely classified into conventional oxidation methods and photocatalytic methods. The traditional oxidation method is easy to cause secondary pollution, is very expensive, toxic and harmful, however, the photocatalysis method has the advantages of easy availability, low cost, wide pollutant removal range, mild reaction condition, strong oxidation capability and the like, and has the potential of degrading pollutants. However, the existing natural photocatalyst is less, and the problems of high extraction cost and the like cause limited application. Therefore, it is particularly important to find an efficient, reliable, green, environment-friendly and economical algistat.

Disclosure of Invention

Aiming at the problem of lack of an economic and effective method for controlling harmful algae such as cyanobacteria bloom and the like, the invention provides application of a perylenequinone compound in inhibiting growth of the harmful algae.

The invention aims to provide application of perylenequinone compounds in inhibiting growth of harmful algae.

The invention aims at a method for inhibiting the growth of harmful algae, which is to add perylenequinone compounds as inhibitors into a water body containing the harmful algae for inhibition treatment.

In one embodiment of the present invention, the inhibition treatment may be performed in a non-illuminated or visible light environment.

In one embodiment of the invention, the light source of the visible light is a visible light lamp or sunlight with the power of 5-40W.

In one embodiment of the present invention, the visible light lamp includes a white light lamp, a violet light lamp, a blue light lamp, and a green light lamp. White light lamps are preferred.

In one embodiment of the present invention, visible light is specifically selected from: 5-32W white light lamp, 20W purple light lamp, 20W blue light lamp, 20W green light lamp, sunlight. Preferably 15-32W white light; further preferred are 23-32W white lamps.

In one embodiment of the invention, the perylenequinone compound is added in an amount of 1.25-20 mu M relative to the water body.

In one embodiment of the invention, the treatment time is 0-36 hours and is not 0.

In one embodiment of the invention, the perylenequinone compound comprises cercosporin, elsinochrome A, elsinochrome B, elsinochrome C, hypocrellin A, hypocrellin B, and tabasheer pigment A.

In one embodiment of the invention, cercosporin, elsinochrome A, elsinochrome BThe chemical formulas of the capsulorhein C, the hypocrellin A, the hypocrellin B and the tabasheer pigment A are respectively as follows: c (C) ₂₉ H ₂₆ O ₁₀ 、C ₃₀ H ₂₄ O ₁₀ 、C ₃₀ H ₂₆ O ₁₀ 、C ₃₀ H ₂₈ O ₁₀ 、C ₃₀ H ₂₆ O ₁₀ 、C ₃₀ H ₂₄ O ₉ 、C ₃₀ H ₂₆ O ₉ 、C ₃₀ H ₂₆ O ₁₀ The structural formula is as follows:

in one embodiment of the invention, the harmful algae includes any one or more of the following: microcystis aeruginosa (FACHB-905), anabaena (FACHB-82), aphanizomenon flos-aquae (FACHB-1040), oscillatoria (FACHB-528), phaeophyta albopictus (FACHB-920) and Taihu blue algae.

In one embodiment of the invention, the algae density in the body of water containing the detrimental algae is 1.6X10 ⁷ And each mL.

The invention also provides a harmful algae inhibitor, which comprises effective components and auxiliary materials; wherein the effective component is perylenequinone compound, and any one or more of the following components are selected: cercosporin, elsinochrome A, elsinochrome B, elsinochrome C, hypocrellin A, hypocrellin B, and tabasheer pigment A. The method comprises the steps of carrying out a first treatment on the surface of the The auxiliary materials comprise dispersing agents and/or regulating agents.

In one embodiment of the invention, the dispersant is selected from any one or more of the following: carrageenan, gelatin, alginic acid, hydroxypropyl cellulose, sodium dodecyl sulfonate, polyethylene glycol, polypropylene glycol, alkyl monoglycoside.

In one embodiment of the invention, the modulator is selected from any one or more of the following: sodium chloride, sodium nitrate, sodium sulfate, sodium gluconate, potassium chloride, potassium nitrate, and potassium sulfate.

The beneficial effects are that:

compared with the existing physical and biological methods for inhibiting the growth of harmful algae, the inhibition method of the invention is more economic and effective, has good algae inhibition effect, and the perylenequinone compounds such as CP and the like are widely distributed in natural pigments in nature, and has low cost and environmental friendliness when used as an algae inhibitor.

The method of the invention utilizes perylenequinone compounds to inhibit a plurality of harmful algae, and the reaction can be carried out under the illumination of visible light or natural sun only by adding the compounds in the reaction process.

The method of the invention utilizes perylenequinone compounds to inhibit the growth of various harmful algae, and can effectively degrade various harmful algae by only adding the algistat into the solution containing the harmful algae and properly carrying out illumination reaction under visible light, thereby being efficient and time-saving.

Drawings

FIG. 1 shows inhibition of 4 perylenequinones by 1.6X10 ⁷ Schematic diagram of growth change condition of microcystis aeruginosa in volume per mL

FIG. 2 is a schematic diagram showing the inhibition of growth changes of Microcystis aeruginosa by implementing different concentrations of CP.

FIG. 3 is a schematic diagram showing the effect of CP inhibition on growth variation of microcystis aeruginosa under different light intensities.

FIG. 4 is a schematic of a 6-well plate for various times to perform 20. Mu.M CP inhibition of microcystis aeruginosa.

FIG. 5 is a schematic diagram of an oil-microscopic image of a microcystis aeruginosa for various times with 20. Mu.M CP inhibition

Fig. 6 is a schematic diagram showing the variation of SEM structure of microcystis aeruginosa by implementing CP inhibition.

Fig. 7 is a schematic diagram showing the change of TEM structure of microcystis aeruginosa by implementing CP inhibition.

FIG. 8 is a schematic diagram showing the effect of CP inhibition on the change in fatty acid content of Microcystis aeruginosa.

FIG. 9 is a schematic diagram showing the inhibition of chlorophyll a of Microcystis aeruginosa by CP.

FIG. 10 is a schematic diagram showing the reaction of Taihu lake algae in sunlight.

FIG. 11 is a schematic diagram showing chlorophyll a changes in Taihu lake algae after CP inhibition.

FIG. 12 shows the measurement of the residual amount of CP in the reaction solution for outdoor solar light experiment.

Detailed Description

The following is provided by the present laboratory in connection with the use of cercosporin, elsinochrome C in the examples; the elsinochrome A, elsinochrome B, hypocrellin A, hypocrellin B and tabasheer pigment A are all purchased from the market, and the purity is 98%.

The quantum yields of singlet oxygen for the above several compounds are shown below:

all microcystis aeruginosa (FACHB-905), anabaena (FACHB-82), aphanizomenon flos-aquae (FACHB-1040), oscillatoria (FACHB-528) and Fusarium albolabrim (FACHB-920) are provided by aquatic organisms of the Chinese sciences, and are cultured by adopting a BG-11 culture medium, wherein the main components of the BG-11 culture medium are as follows: sodium nitrate: 1.5g/L; dipotassium hydrogen phosphate: 0.04g/L; magnesium sulfate heptahydrate: 0.075g/L, calcium chloride dihydrate: 0.036g/L; citric acid: 0.006g/L; ferric ammonium citrate: 0.006g/L; disodium edetate: 0.001g/L; sodium carbonate: 0.02g/L;1000 x trace elements: 1.0mL;1000 x trace element composition: h ₃ BO ₃ ：2.86g/L；MnCl ₂ ·4H ₂ O：1.81g/L；ZnSO ₄ ·7H ₂ O：0.222g/L；NaMoO ₄ ·2H ₂ O：0.39g/L；CuSO ₄ ·5H ₂ O：0.079g/L；Co(NO ₃ ) ₂ ·6H ₂ O：0.0494g/L；

The reaction device comprises: microcystis aeruginosa (FACHB-905), anabaena (FACHB-82), aphanizomenon flos-aquae (FACHB-1040), oscillatoria (FACHB-528) and Philippine seaweed (FACHB-920) are cultured in an illumination incubator (ZXSD-A1160), and have a light intensity of 3000LX, a light-dark time ratio of 12:12 and a temperature of 25deg.C; will be 1.6X10 ⁷ Microcystis aeruginosa seed per mL was seeded into 6-well plates, 4mL per well. Low speed stirring of 6-orifice plate on stirrerIn the middle, a visible light lamp was placed 15 cm from the 6-well plate, and experiments were performed.

The detection method comprises the following steps: the perylenequinone compounds have blood cell counting plate for survival rate of microcystis aeruginosa (FACHB-905), anabaena (FACHB-82), aphanizomenon flos-aquae (FACHB-1040), oscillatoria (FACHB-528) and Phaeophyllum alfa (FACHB-920). The counting of the counting plate is divided into 25 medium cells by a Shang Mai counting method from large square cells, each medium cell is divided into 16 small cells, namely 25 multiplied by 16, the counting formula is algae cell number/L=Nx5x10xdilution, and N is: total number of algae cells in five medium squares. EXAMPLE 1 inhibition of Microcystis aeruginosa (FACHB-905) by Cercosporin (CP)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M of Cercosporin (CP) and 3 duplicate wells were set up for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as follows:

survival (%) = (T _Time /C _Time ) 100% formula (1)

C, T is the control and CP treated algal cell densities, respectively.

As a result, as shown in FIG. 1, CP was reacted under a 23W white light for 36 hours, wherein 20. Mu.M CP inhibited microcystis aeruginosa cell viability by 5%.

EXAMPLE 2 inhibition of Tilletia (CP) on anabaena (FACHB-82)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

The results show that: after 36h, the cell viability of the anabaena inhibiting effect of 20. Mu.M CP was 23%.

EXAMPLE 3 inhibition of Courosporine (CP) on Phaeophyllum water bloom (FACHB-1040)

The results show that: after 36h, the cell viability of the 20. Mu.M CP effect on inhibition of Aphanizomenon flos-aquae was 21%.

EXAMPLE 4 inhibition of Oscillatorin (CP) against Oscillatoria (FACHB-528)

The results show that: after 36h, the cell viability of the inhibitory effect of 20. Mu.M CP on Oscillatoria was 12%.

EXAMPLE 5 inhibition of Fusarium Alcalis (FACHB-920) by Cercosporin (CP)

The results show that: the cell viability of 20. Mu.M CP inhibiting F.albopictus was 34%.

EXAMPLE 6 inhibition of Elsinon A (EA) on Microcystis aeruginosa (FACHB-905)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M EA and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

The results show that: the cell viability of 20 μm EA against microcystis aeruginosa was 15%.

EXAMPLE 7 inhibition of Elsinon B (EB) on Microcystis aeruginosa (FACHB-905)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M EB and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

The results showed that 20. Mu.M EB inhibited microcystis aeruginosa with a cell viability of 25%.

EXAMPLE 8 inhibition of Elsinon C (EC) on Microcystis aeruginosa (FACHB-905)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M EC and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

The results are shown in FIG. 1, and the cell viability of 20. Mu.M EC against Microcystis aeruginosa was 22% after 36h.

EXAMPLE 9 inhibition of Hypocrellin A (HA) on Microcystis aeruginosa (FACHB-905)

Modulating the cell density of algaeThe section is 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M HA and 3 duplicate wells were set up for each of the experimental group and control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

As a result, as shown in FIG. 1, after 36 hours, the cell viability of 20. Mu.M HA against Microcystis aeruginosa was 40%.

EXAMPLE 10 inhibition of Hypocrellin B (HB) on Microcystis aeruginosa (FACHB-905)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M HB and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

As a result, as shown in FIG. 1, after 36 hours, the cell viability of 20. Mu.M HB was 36% for Microcystis aeruginosa inhibition.

EXAMPLE 11 inhibition of Hypocrellin C (HC) on Microcystis aeruginosa (FACHB-905)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was charged with 20. Mu.M HC and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

The results are shown in FIG. 1, which shows that after 36h, the cell viability of 20. Mu.M HB against Microcystis aeruginosa is 28%.

EXAMPLE 12 inhibition of microcystis aeruginosa (FACHB-905) by tabasheer A (SA)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ individual/mL cellsInoculating the suspension into a 6-well plate, wherein each well is 4mL; the experimental group was added with 20 μm SA and 3 duplicate wells were set for the experimental group and the control well, respectively. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

The results showed that after 36h, 20. Mu.M SA inhibited microcystis aeruginosa with a cell viability of 18%.

As shown in examples 1-12 above, 20. Mu.M CP inhibited microcystis aeruginosa best.

EXAMPLE 13 inhibition of Microcystis aeruginosa (FACHB-905) by 1.25. Mu.M of Cercosporin (CP)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 1.25. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

As a result, as shown in FIG. 2, after 36 hours, the cell viability of 1.25. Mu.M CP against Microcystis aeruginosa was 72%.

EXAMPLE 14 inhibition of Microcystis aeruginosa (FACHB-905) by 2.5. Mu.M Cercosporin (CP)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 2.5. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

As a result, as shown in FIG. 2, the cell viability of 2.5. Mu.M CP against Microcystis aeruginosa was 56% after 36 hours.

EXAMPLE 15 inhibition of Microcystis aeruginosa (FACHB-905) by 5. Mu.M Cercosporin (CP)

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 5. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature and reacted under a 23W white light lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is as in formula (1) in example 1.

As a result, as shown in FIG. 2, after 36 hours, the cell viability of 5. Mu.M CP against Microcystis aeruginosa was 48%.

EXAMPLE 16 inhibition of Microcystis aeruginosa (FACHB-905) by 10 μM Cercosporin (CP)

As a result, as shown in FIG. 2, after 36 hours, the cell viability of the microcystis aeruginosa inhibition by 10. Mu.M CP was 28%.

EXAMPLE 17 inhibition of Microcystis aeruginosa (FACHB-905) by Cercosporin (CP) under 5W white light conditions

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature under 5W white light lamp reaction conditions. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is shown in the following example 1 formula (1).

As shown in FIG. 3, the cell viability of 20. Mu.M CP against Microcystis aeruginosa was 41% after 36 hours under 5W white light conditions.

EXAMPLE 18 inhibition of Microcystis aeruginosa (FACHB-905) by Cercosporin (CP) in a 15W white light reaction

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature under 15W white light lamp reaction conditions. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is shown in the following example 1 formula (1).

As shown in FIG. 3, the cell viability of 20. Mu.M CP against Microcystis aeruginosa was 13% after 36 hours under 15W white light conditions.

EXAMPLE 19 inhibition of Microcystis aeruginosa (FACHB-905) by Cercosporin (CP) in a 32W white light reaction

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed at room temperature under 32W white light lamp reaction conditions. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is shown in the following example 1 formula (1).

As shown in FIG. 3, the cell viability of 20. Mu.M CP against Microcystis aeruginosa after 36 hours under 32W white light conditions was: 5%.

EXAMPLE 20 inhibition of Microcystis aeruginosa (FACHB-905) by Cercosporin (CP) upon reaction under 20W blue light

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the concentration of experimental group CP was set at 20 μm and 3 duplicate wells were set for each of the experimental and control wells. The 6-well plate was left to react at room temperature under blue light conditions. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is shown in the following example 1 formula (1).

The results show that the cell viability of 20 μM CP against microcystis aeruginosa is 38% after 36 hours under 20W blue light conditions.

EXAMPLE 21 inhibition of Microcystis aeruginosa (FACHB-905) by Cercosporin (CP) upon reaction under 20W green light

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions of individual/mL, seeded in 6-well plates, 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was left at room temperature and reacted under a 20W green light. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is shown in the following example 1 formula (1).

The results showed that after 36h under green light conditions, the cell viability of 20. Mu.M CP against Microcystis aeruginosa was 45%. Example 22 inhibition of Microcystis aeruginosa (FACHB-905) by CP after reaction under 20W ultraviolet light

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was left at room temperature and reacted under a 20W ultraviolet lamp. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is shown in the following example 1 formula (1).

The results showed that after 36h of reaction under violet light, the cell viability of 20. Mu.M CP inhibiting Microcystis aeruginosa was 31%.

EXAMPLE 23 inhibition of Microcystis aeruginosa (FACHB-905) by CP in the absence of light

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions of individual/mL, seeded in 6-well plates, 4mL per well; the concentration of Cercosporin (CP) in the experimental group was set at 20 μm and 3 multiplex wells were set for each of the experimental group and the control well. The 6-well plate was left at room temperature and reacted without light. Cells were counted for 12h,24h, and 36h, respectively, and compared with control cells to determine survival. The survival rate calculation formula is shown in the following example 1 formula (1).

The results show that after 36 hours in the absence of illumination, the cell viability of 20 mu M CP against Microcystis aeruginosa is: 60%.

EXAMPLE 24 6 well plate diagram of 20. Mu.M CP inhibiting Microcystis aeruginosa

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-hole plate is placed in a white light lamp with the room temperature and the pressure of 23W for reaction, and the reaction solutions of 0h, 12h, 18h, 24h, 30h and 36h are taken for photographing respectively. As a result, as shown in FIG. 4, 20. Mu.M CP was reacted in a 23W white light lamp, and the liquid in 6 wells was changed from green to transparent over time.

The result shows that 20 mu M CP has inhibition effect on microcystis aeruginosa under the condition of 23W white light lamp.

EXAMPLE 25 oil-scoping image of Microcystis aeruginosa inhibited by 20 μM CP

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. And placing the 6-hole plate in a white light lamp with the room temperature and 23W for reaction, and taking reaction solutions of 0h, 12h, 18h, 24h, 30h and 36h for oil-level observation. As a result, as shown in FIG. 5, 20. Mu.M of CP was reacted under a 23W white light, and the microcystis aeruginosa changed from green to transparent and the number of cells in the visual field was varied little with the passage of time.

EXAMPLE 26 SEM test of 20. Mu.M CP inhibition of Microcystis aeruginosa

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-hole plate is placed in a white light lamp with the room temperature and the pressure of 23W for reaction, cells of a control group, 12h,24h and 36h are respectively taken, 6000 Xg are centrifuged for 10 minutes, PBS is used for washing for 3 times, the supernatant is discarded, thalli are obtained, 1mL of fixing solution is added, and the mixture is placed for 12 h. Pouring out the fixing solution, and slowing down the fixing solution by using 0.1M phosphoric acid with pH of 7.0Rinsing the sample for three times for 15min each time; dehydrating the sample with ethanol solutions of gradient concentration (including 30%,50%,70%,80%,90% and 95% concentration), each concentration being treated for 15min, and then 100% ethanol for 20min; critical point drying removes water from the sample. And after the ion sputtering is completed, a layer of uniform film is formed on the surface of the sample, the sample can be observed in a scanning electron microscope. As a result, as shown in FIG. 6, under the condition of 23W white light, 20. Mu.M of CP acting cells, the surface of the control cells was smooth and the cells were intact; the experimental group became not smooth and the cells became incomplete over time.

It can be seen that 20. Mu.M CP inhibits the effect of microcystis aeruginosa on cell morphology under 23W white light conditions.

EXAMPLE 27 TEM test of 20 μM CP inhibition of Microcystis aeruginosa

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed under a 23W white light lamp at room temperature for reaction. Taking cells of a control group, namely 12h,24h and 36h respectively, centrifuging 6000 Xg for 10 minutes, washing 3 times by PBS, discarding supernatant, and carrying out the following operations after obtaining thalli: placing into a 1.5mL centrifuge tube, adding 2.5% glutaraldehyde solution (fixing solution fills the centrifuge tube to completely submerge the sample in the fixing solution), standing at 4deg.C for preservation, and fixing for 12 hr without pouring the fixing solution. Pouring out the fixing solution, and rinsing the sample three times with 0.1M phosphate buffer solution with pH of 7.0 for 15min each time; fixing the sample with 1% osmium acid solution for 1-2h; carefully remove the osmium acid waste solution, rinse the sample three times with 0.1m phosphate buffer, ph 7.0, 15min each; dehydrating the sample with ethanol solutions of gradient concentration (including 30%,50%,70%,80%,90% and 95% concentration), each concentration being treated for 15min, and then 100% ethanol for 20min; finally, the mixture is transited to pure acetone for 20min. Treating the sample with a mixture of embedding medium and acetone (V/v=1/1) for 1h; treating the sample with a mixture of embedding medium and acetone (V/v=3/1) for 3h; treating the sample with pure embedding medium, and heating at 70deg.C overnight to obtain embedded productGood samples. Slicing the sample in an LEICA EMUC7 type ultrathin slicing machine to obtain 70-90nm slices, respectively dyeing the slices for 5-10min by a lead citrate solution and a 50% ethanol saturated solution of uranyl acetate, and airing the slices to observe in a transmission electron microscope.

The results are shown in FIG. 7, in which 20. Mu.M of CP-acting cells were under a 23W white light, the control cells were intact, the cytoplasm was uniform, the thylakoids were clearly visible, and the cell membranes and cell walls were intact; the experimental group became incomplete with time, uneven cytoplasm and had leakage, unclear thylakoids, incomplete cell membranes and cell walls.

EXAMPLE 28 influence of 20 μM CP on inhibition of fatty acid content of Microcystis aeruginosa

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed under a 23W white light lamp at room temperature for reaction. The cells were collected for 0h and 36h, centrifuged at 6000 Xg for 10min, washed 3 times with PBS, and lyophilized. About 5mg of lyophilized cells was added with 10mL of 5% methanol hydrochloride. Incubated at 90℃for 120 min in a sealed glass tube and heated in a water bath at 70℃for 2 h. The tube was cooled at room temperature for 30min, and 1mL of distilled water was added for vortexing. To extract Fatty Acid Methyl Esters (FAME), 2mL of hexane was added and subjected to strong vortexing. These tubes have two layers of material. The upper layer was transferred to a clean tube, blanketed with nitrogen and stored at-20 ℃ for later analysis.

As a result, as shown in FIG. 8, 9 fatty acids including saturated fatty acid component (C _12:0 ,C _14:0 ,C _15:0 ,C _16:0 ,C _17:0 ,C _18:0 ) And an unsaturated fatty acid component (C) _16:1 ,C _18:3 And C _18:1 ). C in the experimental group _12:0 、C _14:0 、C _15:0 、C _16:0 、C _17:0 C _18:1 And C _18:2 Decrease, and no C was detected in the experimental group _16:1 。

It can be seen that 20. Mu.M CP has a destructive effect on cell membranes under 23W white light conditions.

EXAMPLE 29 20 μM CP inhibits chlorophyll a status of Microcystis aeruginosa

The density of the cells of the algae was adjusted to 1.6X10 ⁷ Cell suspensions at each mL, seeded in 6-well plates at 4mL per well; the experimental group was added with 20. Mu.M CP and 3 duplicate wells were set for each of the experimental group and the control well. The 6-well plate was placed under a 23W white light lamp at room temperature for reaction. Taking 0h, 12h,24h and 36h of cells respectively, centrifuging the algae suspension at 4000rpm for 3-5 min, removing supernatant, obtaining thalli, adding 90% acetone overnight, placing the thalli in a refrigerator at 4 ℃ in a dark place, centrifuging the extract at 5000rpm and 4 ℃ for 10min by using a high-speed refrigerated centrifuge (Hitachi Co., japan) to collect supernatant, detecting absorbance at 630nm, 645nm, 663nm and 750nm of algae, and determining the content of chlorophyll a according to the following formula:

units mg.L ^-1 Wherein V is the volume of the algae liquid, the unit is the volume of the collected sample, the unit is L, and the unit is mL. In this experiment, v=10, v=5.

The results are shown in FIG. 9, where 20. Mu.M CP-affected cells were irradiated at 23W CFL, and the chlorophyll a content of the experimental group was lower and lower over time.

[ example 30 ]

2021, 7, 29, we collected fresh algae experiments in the vicinity of the Taihu new harbor bridge salvage station (31 ° '41'07s/120 ° 24'19' w) into three groups: control group, 7.5. Mu.M CP group, 20. Mu.M CP group, at light intensity of 14-25mW/cm ² The reaction time was 36h under solar conditions. As shown in fig. 10, the 7.5 μm group of algae changed from green to white over time, and the 20 μm group of algae changed from green to purple over time.

It can be seen that under solar radiation, CPs of 7.5. Mu.M and 20. Mu.M have an effect on Taihu lake algae.

Example 31

At 2021, 7 and 29, we collected fresh algae in the vicinity of the Taihu New harbor bridge salvage station (31 ° '41'07S/120 ° ' 24'19' W) and conducted further experiments. The experiments were divided into three groups: control group, 7.5. Mu.M CP group, 20. Mu.M CP group, reaction system was 2L. At a light intensity of 14-25mW/cm ² The reaction time was 36h under solar conditions. Samples of 0h, 6h, 12h, 18h, 24h, 30h, 36h were taken. Separating algae by suction filtration, adding 90% acetone overnight after obtaining thallus, placing in a refrigerator at 4deg.C in dark place, centrifuging the extractive solution at 4deg.C for 10min at 10000rmp rotation speed with high-speed refrigerated centrifuge (Hitachi Corp., japan) to collect supernatant, and calculating chlorophyll a content according to the formula shown in example 28 (2).

As a result, FIG. 11 shows that the chlorophyll a content of the control group and the experimental group at 36 hours was 1.466.+ -. 0.0763 mg.L, respectively ^-1 、0.078±0.007mg/L ^-1 And 0.025.+ -. 0.025mg/L ^-1 。

It can be seen that under solar radiation, 7.5. Mu.M and 20. Mu.M CPs have a good inhibition effect on Taihu lake algae.

Example 32 content of CP in reaction solution for solar light experiment

And (3) taking samples of 0h, 6h, 12h, 18h, 24h, 30h and 36h according to actual blue algae reaction. Separating algae by suction filtration to obtain liquid. The liquid was extracted with an equal volume of dichloromethane, the organic phase was rotary evaporated and redissolved with HPLC in methanol. The residual amount of CP was measured by Waters.

Table 1 liquid chromatograph operating parameters

As shown in FIG. 12, we examined the residual amount of CP in the reaction system. After 36 hours of reaction, no CPs were detected in the 7.5. Mu.M and 20. Mu.M CPs, indicating that CPs did not cause secondary pollution in the actual treatment of harmful algae.

Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Claims

1. The application of perylenequinone compounds in inhibiting the growth of harmful algae comprises cercosporin, elsinochrome A, elsinochrome B, elsinochrome C, hypocrellin A, hypocrellin B and hypocrellin A;

the chemical structural formulas of cercosporin, elsinochrome A, elsinochrome B, elsinochrome C, hypocrellin A, hypocrellin B and tabasheer pigment A are respectively as follows:

the harmful algae include any one or more of the following: microcystis aeruginosa, anabaena, aphanizomenon flos-aquae, oscillatoria, aphanizomenon alike, and Taihu blue algae.

2. A method for inhibiting the growth of harmful algae is characterized in that perylenequinone compounds are added into a water body containing the harmful algae for inhibition treatment under the irradiation of visible light or natural sun light; the perylenequinone compound comprises cercosporin, elsinochrome A, elsinochrome B, elsinochrome C, hypocrellin A, hypocrellin B and tabasheer pigment A;

3. The method of claim 2, wherein the visible light is derived from a 5-40W visible light lamp or sunlight.

4. The method of claim 2, wherein the perylenequinone compound is added in an amount of 1.25 to 20 μm relative to the water body.

5. The method of claim 2, wherein the treatment is for a time period of 0 to 36 hours and not 0.