Amino acid blue-green algae inhibitor and application thereof

Abstract

The invention discloses an amino acid blue-green algae inhibitor and application thereof, and belongs to the technical field of water body remediation. The amino acid blue-green algae inhibitor comprises levodopa, homoserine and threonine, and preferably, the concentration of the levodopa is 100 [mu] mol / L, the concentration of the homoserine is 160 [mu] mol / L, and the concentration of the threonine is 500 [mu] mol / L. Three amino acid substances, namely levodopa, homoserine and threonine, with high algal inhibition efficiency are screened by analyzing EC50 of amino acid substances with algal inhibition effects, and experiments prove that the three amino acid substances have extremely high blue-green algae killing activity, so that the amino acid blue-green algae inhibitor is obtained. And an efficient, convenient and safe way is provided for biological prevention and control of cyanobacterial bloom.

Description

Technical Field

[0001] The present invention belongs to the technical field of water body restoration, and particularly relates to an amino acid-based cyanobacteria inhibitor and its application. Background Art

[0002] Harmful cyanobacterial blooms (HCBs) have become a common phenomenon in global fresh waters. Due to the release of algal organic matter and cyanotoxins, the overgrowth of bloom-forming cyanobacteria poses a threat to both drinking water production and aquatic ecosystems. Among these bloom-forming cyanobacteria, Microcystis aeruginosa can produce microcystins and thus has a survival advantage, and it is the most common and widely distributed one. At present, the commonly used methods for inhibiting and killing Microcystis aeruginosa include physical methods, chemical methods and biological methods. Among them, the biological method has received extensive attention due to its advantages of high efficiency and environmental protection. In biological control, mainly algicidal treatment is carried out through co-cultivation of algicidal bacteria or the release of allelochemicals. And allelochemicals, due to their specificity and biodegradability, are regarded as an environmentally friendly method for controlling cyanobacterial blooms. Allelochemicals are generally divided into four categories: nitrogen-containing substances, polyphenols, fatty acids and terpenoids. Among them, nitrogen-containing substances have become good screening targets because of their rich variety, diverse action sites and easy availability. At present, the research on alkaloids, peptides and proteins has a relatively high use cost, while amino acid substances have the advantages of low price and easy availability.

[0003] At present, some amino acids with algicidal effects have been isolated from the metabolites of algicidal bacteria. L-lysine extracted from the anti-cyanobacteria substance secreted by Streptomyces phaeofaciens has a damaging effect on the cell wall of cyanobacterial cells. L-valine extracted from the fermentation broth of Streptomyces jiujiangensis JXJ 0074 has a stronger inhibitory effect than L-lysine, but Microcystis aeruginosa cells are more likely to return to normal growth state and are more likely to promote the release of microcystins. Systematic screening of twenty natural amino acids and low-cost amino acid analogs is conducive to obtaining low-cost, more efficient and safe amino acid substances, providing new ideas for allelochemicals to inhibit and kill harmful cyanobacterial blooms. Summary of the Invention

[0004] In view of this, the main object of the present invention is to provide an application of an amino acid composition in the biological control of cyanobacterial blooms. The amino acid composition includes levodopa, homoserine and threonine, and the above three amino acid substances with high efficiency in inhibiting and killing Microcystis aeruginosa are screened out from twenty natural amino acids and five low-cost amino acid analogs.

[0005] Another object of the present invention is to provide an amino acid-based cyanobacteria inhibitor, which includes levodopa, homoserine and threonine, and the three amino acid substances have the effect of efficiently inhibiting and killing Microcystis aeruginosa.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides an application of an amino acid composition in biological control of cyanobacterial blooms, and the amino acid composition includes levodopa, homoserine and threonine.

[0008] Preferably, the cyanobacterial bloom includes Microcystic aeruginosa.

[0009] Preferably, the amino acid composition acts on Microcystic aeruginosa in the logarithmic growth phase (2×10 6 cells / mL).

[0010] Preferably, the concentration of levodopa is 100 μmol / L.

[0011] Preferably, the concentration of homoserine is 160 μmol / L.

[0012] Preferably, the concentration of threonine is 500 μmol / L.

[0013] The screening method of the above amino acid substances for biological control of cyanobacterial blooms includes the following steps:

[0014] (1) Select twenty natural amino acids: tryptophan, lysine, phenylalanine, methionine, threonine, valine, leucine, isoleucine, cysteine, serine, glycine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, histidine and proline; and five low-cost amino acid analogues: levodopa, serotonin, γ-aminobutyric acid, homoserine and homoarginine;

[0015] (2) Using BG11 as a solvent, prepare an amino acid substance solution, or also include using dimethyl sulfoxide as a co-solvent;

[0016] (3) Primary screening stage: The concentration of different amino acid solutions is uniformly set at 300 μmol / L, treat Microcystic aeruginosa for 96 h, detect the absorbance at 680 nm, and calculate the inhibition rate according to the algal density corresponding to the absorbance;

[0017] (4) Re-screening stage: Set different concentration gradient solutions for the amino acid substances with algicidal effects, treat Microcystic aeruginosa for 96 h, detect the growth and photosynthetic activity of Microcystic aeruginosa, use the actual photosynthetic efficiency as a toxicity reference, calculate the EC50 of different amino acid substances, evaluate the inhibitory ability of the amino acid substances on Microcystic aeruginosa, and screen out the amino acid substances with high algicidal efficiency, that is, obtain; wherein the actual photosynthetic efficiency is measured by a phyto-PAM instrument.

[0018] The present invention also provides an amino acid - type cyanobacteria inhibitor, which includes L - dopa, homoserine, and threonine. These three amino acid substances have the effect of efficiently inhibiting and killing Microcystis aeruginosa.

[0019] Preferably, in the amino acid - type cyanobacteria inhibitor, the concentration of L - dopa is 100 μmol / L, the concentration of homoserine is 160 μmol / L, and the concentration of threonine is 500 μmol / L.

[0020] The present invention also provides the application of the amino acid - type cyanobacteria inhibitor in the biological control of cyanobacterial blooms.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: By analyzing the EC50 of amino acid substances with algicidal effects, the present invention screens out three amino acid substances with high algicidal efficiency, namely L - dopa, homoserine, and threonine, all of which have extremely strong cyanobactericidal activity, especially having a strong inhibitory effect on the growth and photosynthetic system of Microcystis aeruginosa. In addition, threonine is a natural amino acid with low cost and rapid reaction. It is an environmentally friendly and easy - to - use algicide. L - dopa and homoserine belong to tyrosine and serine analogs, which have the advantages of rapid reaction and low effective concentration in the process of inhibiting cyanobacteria. Combining them to prepare an amino acid - type cyanobacteria inhibitor is expected to provide an efficient, convenient, and safe prevention and control method for cyanobacterial bloom control. Description of the Drawings

[0022] Figure 1 It is the fitting curve of the algal density corresponding to the absorbance at a wavelength of 680 nm of Microcystis aeruginosa by 25 amino acid substances in the examples at 0, 24, 48, 72, and 96 h.

[0023] Figure 2 It is the influence of 25 amino acid substances in the examples on the absorbance at a wavelength of 680 nm of Microcystis aeruginosa at 0, 24, 48, 72, and 96 h, as a preliminary screening.

[0024] Figure 3 It is to detect the actual photosynthetic activity and chlorophyll content of total cells after the 12 amino acid substances with algicidal effects in the examples are divided into three concentrations of high, medium, and low (a, b, c) and detected at 0, 24, 48, 72, 96, 144, and 192 h respectively.

[0025] Figure 4 It is to set 6 concentration gradients for the 12 amino acid substances with algicidal effects in the examples and detect the inhibition rate of the actual photosynthetic efficiency of algal cells at 96 h respectively.

[0026] Figure 5For the three amino acids with the lowest EC50 selected in the examples, after treating the algal cells with the concentration at which the inhibition rate of actual photosynthetic efficiency is basically stable, the inhibition rate of actual photosynthetic efficiency (a) and the inhibition rate of cell density (b) of the algal cells are detected at 0, 24, 48, 72, and 96 h. Detailed implementation manners

[0027] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below in conjunction with the accompanying drawings and specific examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. It should be noted that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention shall fall within the protection scope of the present invention.

[0028] The Microcystis aeruginosa used in the following examples was purchased from the Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences and cultured to the logarithmic growth phase using BG11 medium.

[0029] Example 1 Primary screening of amino acid substances

[0030] Prepare an amino acid stock solution using BG11 as the solvent and add it to the Microcystis aeruginosa solution to make the initial concentration of algal cells 2×10 6 cells / mL and the substance concentration 300 μmol / L. Among them, 1‰ DMSO is added to the L-DOPA group for solubilization, and 3‰ DMSO is added to the Phe, Trp, and Ile groups for solubilization. The treated algal solution is placed in a full-automatic light incubator for cultivation. The cultivation conditions are as follows: temperature is 25°C, light intensity is 1500 Lux, and the light-dark ratio is 12 h:12 h. Samples are taken regularly every day to measure the absorbance of Microcystis aeruginosa at a wavelength of 680 nm, determine the correlation between algal density and absorbance (OD680), and obtain the algal density-absorbance standard curve: C = 12.889×OD 680 -0.0479, where C represents the algal cell density (×10 6 cells / mL), OD 680 represents the absorbance at a wavelength of 680 nm, and the R 2 of the fitted curve is 0.9995. The initial algal density during the experiment is 2×10 6 cells / mL, and OD 680 is 0.155 ± 0.003. From this, the algal density inhibition rate in different amino acid treatment groups is calculated ( Figure 1 , 2) As a result, it was found that 12 kinds of amino acids had algicidal activity. At the same time, amino acid substances that were relatively expensive, would turn green again in a short time, had a growth-promoting effect, and produced peculiar smells during the reaction process were excluded. The 12 kinds of amino acids included homoserine (Hse), L-dopa (L-DOPA), threonine (Thr), lysine (Lys), homoarginine (Har), histidine (His), tryptophan (Trp), phenylalanine (Phe), serine (Ser), glycine (Gly), isoleucine (Ile), and alanine (Ala).

[0031] Example 2 Rescreening of Amino Acid Substances

[0032] For the 12 kinds of amino acids screened in Example 1, three concentration gradients of high, medium, and low (a, b, c) (unit: μmol / L) were set respectively, and samples were taken on time to detect the chlorophyll content and actual photosynthetic efficiency. The results are as Figure 3 shown. It was found that the actual photosynthetic efficiency of algal cells in most high-concentration treatment groups dropped to zero, and the concentrations are shown in Table 1.

[0033] Table 1

[0034] Amino acid a b c Ala 3000 1500 1000 Ile 3000 1500 1000 Phe 3000 1500 1000 Gly 3000 1500 1000 Ser 3000 1500 1000 Trp 1500 750 100 Har 1000 500 50 His 750 250 100 Lys 750 250 100 Thr 750 250 100 L-DOPA 500 250 50 Hse 250 70 50

[0035] Example 3 Determination of EC50 of Twelve Kinds of Amino Acid Substances

[0036] Different amino acids were set to six concentration gradients (Table 2), and Microcystis aeruginosa was treated for 96 h. The actual photosynthetic efficiency of algal cells was detected, and the inhibition rate of actual photosynthetic efficiency was calculated. The results are as Figure 4 shown. After fitting the data, EC50 was calculated, and the best algicidal amino acids were determined to be L-DOPA, Hse, and Thr. The concentration gradients of various amino acid substances are shown in Table 2, with the unit of μmol / L.

[0037] Table 2

[0038] Ser Ile Gly Ala Phe Har 35000 25000 20000 20000 3750 2500 17500 12500 10000 10000 2500 1000 8750 6250 4000 4000 1250 500 3500 3750 2000 2000 625 100 1750 2500 1000 1000 250 50 875 1250 400 400 125 25 0 0 0 0 0 0 Trp His Lys Thr Hse L-DOPA 2500 2000 1500 700 500 200 1250 1000 750 350 250 100 250 400 300 175 100 50 125 200 150 87.5 50 25 62.5 100 75 35 25 10 25 40 30 8.75 10 2.5 0 0 0 0 0 0

[0039] Example 4 Application of Three Highly Effective Amino Acids

[0040] Select the amino acid substances with the smallest EC50 in Example 3, and use the concentrations at which the inhibition rate of actual photosynthetic efficiency is stable to treat Microcystis aeruginosa cells. The concentration of L-DOPA is 100 μmol / L, the concentration of Hse is 160 μmol / L, and the concentration of Thr is 500 μmol / L. The actual photosynthetic efficiency and algal cell density of algal cells are detected on time, and the actual photosynthetic efficiency and algicidal rate are calculated. Three parallel samples are set for both the control group and the experimental group, and the results are expressed in the form of mean ± standard deviation.

[0041] As Figure 5As shown, after treatment with three amino acid substances, the algal cells died rapidly at 48 h, the actual photosynthetic efficiency decreased significantly, and the algicidal rates reached up to 83.57% for L-DOPA, 81.11% for Hse, and 96.09% for Thr. The EC50 values of twelve amino acid substances with algicidal effects are shown in Table 3.

[0042] Table 3

[0043]

[0044]

[0045] The culture conditions of Microcystis aeruginosa were as follows: the temperature was 25 °C, the light intensity was 1500 Lux, the light-dark ratio was 12 h:12 h. It was cultured in an illumination incubator, and the conical flask was gently shaken every 12 h to keep the algal cells in a suspended state.

[0046] In summary, through the EC50 values of amino acid substances with algicidal effects, the present invention screened out three amino acid substances with the highest algicidal efficiency, namely L-DOPA, homoserine, and threonine. After treatment at concentrations of 100, 160, and 500 μmol / L for 96 h respectively, the algicidal rates exceeded 80%. The initial concentration of Microcystis aeruginosa was 2×10 6 cells / mL, indicating that the three amino acid substances have a strong inhibitory effect on the growth and photosynthetic system of Microcystis aeruginosa.

[0047] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An application of an amino acid composition in biological control of cyanobacteria blooms, the amino acid composition comprising levodopa, homoserine and threonine.

2. The use according to claim 1, characterized in that: The cyanobacterial blooms include Microcystis aeruginosa.

3. The use according to claim 1, characterized in that: The amino acid composition acts on Microcystis aeruginosa in the logarithmic growth phase.

4. The use according to claim 1, characterized in that: The concentration of levodopa was 100 μmol / L.

5. The use according to claim 1, characterized in that: The concentration of homoserine is 160 μmol / L.

6. The use according to claim 1, characterized in that: The concentration of threonine is 500 μmol / L.

7. A method for screening amino acid substances for biological control of cyanobacterial blooms, characterized in that: The steps include: (1) selecting twenty natural amino acids: tryptophan, lysine, phenylalanine, methionine, threonine, valine, leucine, isoleucine, cysteine, serine, glycine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, histidine and proline; and five low-priced amino acid analogs: levodopa, serotonin, gamma-aminobutyric acid, homoserine and homoarginine; (2) using BG11 as a solvent to prepare an amino acid substance solution, or further including using dimethyl sulfoxide as a co-solvent; (3) Preliminary screening: The concentration of different amino acid solutions was uniformly set to 300 μmol / L, and the Microcystis aeruginosa was treated for 96 h. The absorbance at 680 nm was detected, and the inhibition rate was calculated based on the algae density corresponding to the absorbance. (4) Rescreening stage: Different concentration gradient solutions were set for amino acid substances with algae inhibition effects, and Microcystis aeruginosa was treated for 96 h. The growth and photosynthetic activity of Microcystis aeruginosa were detected. The actual photosynthetic efficiency was used as a toxicity reference, and the EC50 of different amino acid substances was calculated. The inhibitory ability of the amino acid substances on Microcystis aeruginosa was evaluated, and the amino acid substances with high algae inhibition efficiency, such as L-DOPA, homoserine and threonine, were screened. The actual photosynthetic efficiency was determined using a phyto-PAM instrument.

8. An amino acid cyanobacteria inhibitor, characterized in that: It includes levodopa, homoserine and threonine.

9. The amino acid cyanobacteria inhibitor according to claim 8, characterized in that: The concentration of L-DOPA in the amino acid cyanobacteria inhibitor is 100 μmol / L, the concentration of homoserine is 160 μmol / L, and the concentration of threonine is 500 μmol / L.

10. Use of the amino acid cyanobacteria inhibitor according to claim 8 or 9 in biological control of cyanobacteria blooms.

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