Amino acid cyanobacteria inhibitor and use thereof
By screening out combinations of levodopa, homoserine, and threonine, an amino acid-based cyanobacterial inhibitor was prepared, which solved the problem of poor inhibition of Microcystis aeruginosa growth and photosynthetic system in existing technologies, and achieved efficient and low-cost control of cyanobacterial blooms.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI OCEAN UNIV
- Filing Date
- 2025-03-05
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies are insufficient to efficiently, cost-effectively, and safely inhibit the growth and photosynthetic system of Microcystis aeruginosa, especially the release of cyanobacterial toxins. Traditional amino acid-based substances are either too expensive or ineffective.
Three amino acid substances, namely levodopa, homoserine, and threonine, were screened out and used in combination to prepare amino acid-based cyanobacterial inhibitors at concentrations of 100 μmol/L, 160 μmol/L, and 500 μmol/L, which were used to inhibit the growth and photosynthetic system of Microcystis aeruginosa.
It achieves highly efficient inhibition of Microcystis aeruginosa, with an algae inhibition rate of over 80%, and is rapid and inexpensive, providing an environmentally friendly control method.
Smart Images
Figure CN120081491B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of water remediation technology, specifically relating to an amino acid-based cyanobacteria inhibitor and its application. Background Technology
[0002] Harmful cyanobacterial blooms (HCBs) have become a common phenomenon in freshwater worldwide. Due to the release of algal organic matter and cyanobacterial toxins, the excessive proliferation of cyanobacteria in blooms threatens both drinking water production and aquatic ecosystems. Among these blooming cyanobacteria, *Microcystis aeruginosa*, which produces microcystin and thus has a survival advantage, is the most common and widely distributed. Currently, commonly used methods for suppressing and controlling *Microcystis aeruginosa* include physical, chemical, and biological methods. Among these, biological methods have gained widespread attention due to their high efficiency and environmental friendliness. Biological control mainly involves co-culturing algae-inhibiting bacteria or administering allelochemicals. Allelochemicals, due to their specificity and biodegradability, are considered an environmentally friendly method for controlling cyanobacterial blooms. Allelochemicals are broadly classified into four categories: nitrogenous substances, polyphenols, fatty acids, and terpenes. Nitrogenous substances are a good selection target due to their abundance, diverse action sites, and ease of acquisition. Currently, alkaloids, peptides, and proteins, which are the most studied, are relatively expensive, while amino acids have the advantages of low price and easy availability.
[0003] Currently, studies have isolated some amino acids with algicidal effects from the metabolites of algicidal bacteria. L-lysine, extracted from the anti-cyanobacterial substance secreted by *Streptomyces phaeofaciens*, has been shown to damage the cell walls of cyanobacterial cells. L-valine extracted from the fermentation broth of *Streptomyces jiujiangensis* JXJ 0074 showed a stronger inhibitory effect than L-lysine, but *Microcystis* cells recovered to normal growth more easily and were more likely to release algal toxins. Systematic screening of twenty natural amino acids and low-cost amino acid analogs is beneficial for obtaining low-cost, more efficient, and safe amino acid substances, providing new ideas for allelochemical suppression of harmful cyanobacterial blooms. Summary of the Invention
[0004] Therefore, the main objective of this invention is to provide an application of an amino acid composition in the biological control of cyanobacterial blooms. This amino acid composition includes levodopa, homoserine, and threonine. The above three amino acid substances, which are highly effective at inhibiting and killing Microcystis aeruginosa, were selected from twenty natural amino acids and five low-priced amino acid analogs.
[0005] Another objective of this invention is to provide an amino acid-based cyanobacteria inhibitor, comprising levodopa, homoserine, and threonine, which are three amino acids that have a highly effective inhibitory effect on Microcystis aeruginosa.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] This invention provides an application of an amino acid composition in the biological control of cyanobacterial blooms, wherein 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 Microcystis aeruginosa in its logarithmic growth phase (2×10⁻⁶). 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 the threonine is 500 μmol / L.
[0013] The above-mentioned screening method for amino acid substances used in the biological control of cyanobacterial blooms includes the following steps:
[0014] (1) Twenty natural amino acids were selected: tryptophan, lysine, phenylalanine, methionine, threonine, valine, leucine, isoleucine, cysteine, serine, glycine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, histidine, and proline; as well as five low-priced amino acid analogs: levodopa, serotonin, γ-aminobutyric acid, homoserine, and homoarginine;
[0015] (2) Using BG11 as a solvent to prepare amino acid solutions, or including the use of dimethyl sulfoxide as a co-solvent;
[0016] (3) Initial screening stage: Different amino acid solutions were uniformly set to a concentration of 300 μmol / L, and Microcystis aeruginosa was treated for 96 h. The absorbance at 680 nm was detected, and the inhibition rate was calculated based on the algal density corresponding to the absorbance.
[0017] (4) Secondary screening stage: Different concentration gradient solutions of amino acid substances with algae-inhibiting effect were set up to treat Microcystis aeruginosa for 96 hours. The growth and photosynthetic activity of Microcystis aeruginosa were detected. The actual photosynthetic efficiency was used as a toxicity reference. The EC50 of different amino acid substances was calculated to evaluate the inhibitory ability of the amino acid substances on Microcystis aeruginosa. The amino acid substances with high algae-inhibiting efficiency were screened. The actual photosynthetic efficiency was measured by a phyto-PAM instrument.
[0018] The present invention also provides an amino acid-based cyanobacterial inhibitor, comprising levodopa, homoserine, and threonine, which have a highly effective inhibitory effect on Microcystis aeruginosa.
[0019] Preferably, in the amino acid-based cyanobacteria inhibitor, the concentration of levodopa 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-based cyanobacterial inhibitor in the biological control of cyanobacterial blooms.
[0021] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention screens three amino acids with high algae-inhibiting efficiency—L-DOPA, homoserine, and threonine—by analyzing the EC50 of amino acids with algae-inhibiting effects. All three exhibit extremely strong activity in killing cyanobacteria, particularly showing a strong inhibitory effect on the growth and photosynthetic system of *Microcystis aeruginosa*. Furthermore, threonine is a natural amino acid, inexpensive, and reacts rapidly; it is an environmentally friendly and easy-to-use algaecide. L-DOPA and homoserine are tyrosine and serine analogs, respectively, and have the advantages of rapid reaction and low effective concentration in the process of inhibiting cyanobacteria. Combining them to prepare amino acid-based cyanobacteria inhibitors holds promise for providing an efficient, convenient, and safe method for controlling cyanobacterial blooms. Attached Figure Description
[0022] Figure 1 The above are the algal density fitting curves corresponding to the absorbance of 25 amino acids at a wavelength of 680 nm for Microcystis aeruginosa at 0, 24, 48, 72, and 96 h, as shown in the examples.
[0023] Figure 2 The effects of 25 amino acid substances on the absorbance of Microcystis aeruginosa at a wavelength of 680 nm at 0, 24, 48, 72, and 96 h were studied in the examples as preliminary screening.
[0024] Figure 3 To investigate the actual photosynthetic activity and chlorophyll content of total cells after treatment with 12 amino acid substances that have an algae-inhibiting effect in the example, the concentrations were divided into high, medium, and low (a, b, c). The tests were conducted at 0, 24, 48, 72, 96, 144, and 192 h.
[0025] Figure 4 Six concentration gradients were set up for the 12 amino acid substances with algal inhibition effects in the examples, and the actual photosynthetic efficiency inhibition rate of algal cells was detected after 96 hours.
[0026] Figure 5The three amino acids with the lowest EC50 values selected in the examples were used to treat algal cells at concentrations where the actual photosynthetic efficiency inhibition rate was basically stable. The actual photosynthetic efficiency inhibition rate (a) and cell density inhibition rate (b) of the algal cells were then detected at 0, 24, 48, 72, and 96 h. Detailed Implementation
[0027] 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 with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.
[0028] The Microcystic aeruginosa used in the following examples was purchased from the Algal Culture Bank of the Institute of Hydrobiology, Chinese Academy of Sciences, and cultured in BG11 medium to the logarithmic growth phase.
[0029] Example 1: Preliminary screening of amino acid substances
[0030] An amino acid stock solution was prepared using BG11 as a solvent and added to the Microcystis aeruginosa solution to achieve an initial algal cell concentration of 2 × 10⁻⁶. 6 The concentration of the substance was 300 μmol / L, with 1‰ DMSO added to the L-DOPA group for dissolution and 3‰ DMSO added to the Phe, Trp, and Ile groups for dissolution. The treated algal solutions were cultured in an automated light incubator under the following conditions: temperature 25℃, light intensity 1500 Lux, light-dark ratio 12h:12h. The absorbance of *Microcystis aeruginosa* at 680 nm was measured daily, and the correlation between algal density and absorbance (OD680) was determined to obtain the algal density-absorbance standard curve: C = 12.889 × OD680. 680 -0.0479, C represents algal cell density (×10⁻¹⁰). 6 cells / mL), OD 680 The R-value of the fitted curve represents the absorbance at a wavelength of 680 nm. 2 The value was 0.9995, and the initial algal density during the experiment was 2 × 10⁻⁶. 6 cells / mL, OD 680 The value was 0.155 ± 0.003, from which the algal density inhibition rate in different amino acid treatment groups was calculated. Figure 1 , 2The results showed that 12 amino acids had anti-algae activity, while excluding amino acids that were expensive, would cause rapid regreening, would promote growth, or would produce an odor during the reaction. The 12 amino acids included homoserine (Hse), 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: Secondary screening of amino acid substances
[0032] The 12 amino acids screened in Example 1 were used to establish three concentration gradients (high, medium, and low, a, b, c) (unit: μmol / L), and samples were taken regularly to detect chlorophyll content and actual photosynthetic efficiency. The results are as follows: Figure 3 As shown in Table 1, the actual photosynthetic efficiency of algal cells in most of the high-concentration treatment groups was reduced to zero.
[0033] Table 1
[0034] amino acids 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 amino acids
[0036] Six concentration gradients of different amino acids were set up (Table 2) and Microcystis aeruginosa was treated for 96 h. The actual photosynthetic efficiency of the algal cells was detected, and the inhibition rate of actual photosynthetic efficiency was calculated. The results are as follows: Figure 4 As shown in Table 2, EC50 was calculated after fitting the data, and the optimal algae-inhibiting amino acids were determined to be L-DOPA, Hse, and Thr. The concentration gradients of various amino acids are shown in Table 2, with units 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 Efficient Amino Acids
[0040] The amino acid with the lowest EC50 in Example 3 was selected, and Microcystis aeruginosa cells were treated with the concentration at which the actual photosynthetic efficiency inhibition rate was stable. The concentrations were: L-DOPA 100 μmol / L, Hse 160 μmol / L, and Thr 500 μmol / L. The actual photosynthetic efficiency and algal density of the algal cells were measured regularly, and the actual photosynthetic efficiency and algal lysis rate were calculated. Three parallel samples were set for each of the control and experimental groups. The results are expressed as mean ± standard deviation.
[0041] like Figure 5As shown, after treatment with the three amino acid substances, the algal cells died rapidly at 48 h, and the actual photosynthetic efficiency was significantly reduced. The highest algal dissolution rate reached 83.57% for L-DOPA, 81.11% for Hse, and 96.09% for Thr. The EC50 of the twelve amino acid substances with algal inhibitory effects are shown in Table 3.
[0042] Table 3
[0043]
[0044]
[0045] The culture conditions for Microcystis aeruginosa were as follows: temperature 25℃, light intensity 1500 Lux, light-dark ratio 12h:12h, cultured in a light incubator, with the conical flask gently shaken every 12h to keep the algal cells in suspension.
[0046] In summary, this invention screened three amino acids with the highest algae-inhibiting efficiency—L-DOPA, homoserine, and threonine—using EC50 analysis. After treatment at concentrations of 100, 160, and 500 μmol / L for 96 hours, the algae inhibition rate exceeded 80%, with the initial concentration of *Microcystis aeruginosa* being 2 × 10⁻⁶. 6 The cells / mL indicates that the three amino acid compounds have a strong inhibitory effect on the growth and photosynthetic system of Microcystis aeruginosa.
[0047] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. The application of an amino acid composition in the biological control of cyanobacterial blooms, wherein the amino acid composition comprises levodopa, homoserine, and threonine.
2. The application according to claim 1, characterized in that, The cyanobacterial blooms include Microcystis aeruginosa.
3. The application according to claim 1, characterized in that, The amino acid composition acts on Microcystis aeruginosa in its logarithmic growth phase.
4. The application according to claim 1, characterized in that, The concentration of the levodopa was 100 μmol / L.
5. The application according to claim 1, characterized in that, The concentration of homoserine was 160 μmol / L.
6. The application according to claim 1, characterized in that, The concentration of threonine is 500 μmol / L.
7. A method for screening amino acid-based substances for the biological control of cyanobacterial blooms, characterized in that, Includes the following steps: (1) Twenty natural amino acids were selected: tryptophan, lysine, phenylalanine, methionine, threonine, valine, leucine, isoleucine, cysteine, serine, glycine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, histidine, and proline; as well as five low-priced amino acid analogs: levodopa, serotonin, γ-aminobutyric acid, homoserine, and homoarginine; (2) Using BG11 as a solvent to prepare amino acid solutions, or including the use of dimethyl sulfoxide as a co-solvent; (3) Initial screening stage: The concentration of different amino acid solutions was uniformly set to 300 μmol / L. Microcystis aeruginosa was treated for 96 h, and the absorbance at 680 nm was detected. The inhibition rate was calculated based on the algal density corresponding to the absorbance. (4) Secondary screening stage: Different concentration gradient solutions of amino acid substances with algae-inhibiting effect were set up to treat Microcystis aeruginosa for 96 hours. The growth and photosynthetic activity of Microcystis aeruginosa were detected. The actual photosynthetic efficiency was used as the toxicity reference. The EC50 of different amino acid substances was calculated to evaluate the inhibitory ability of the amino acid substances on Microcystis aeruginosa. The amino acid substances with high algae-inhibiting efficiency, such as L-DOPA, homoserine and threonine, were screened. The actual photosynthetic efficiency was measured by a phyto-PAM instrument.
8. An amino acid-based cyanobacteria inhibitor, characterized in that, It includes levodopa, homoserine, and threonine.
9. The amino acid-based cyanobacteria inhibitor according to claim 8, characterized in that, The amino acid-based cyanobacteria inhibitor contains 100 μmol / L of levodopa, 160 μmol / L of homoserine, and 500 μmol / L of threonine.
10. The application of the amino acid-based cyanobacterial inhibitor according to claim 8 or 9 in the biological control of cyanobacterial blooms.