Low-concentration H2O2 combined with bacteria as an algae inhibitor and its application

Abstract

This invention discloses a low-concentration H2O2 combined with bacteria algae-inhibiting agent and its application, belonging to the field of aquatic bloom pollution control technology. The algae-inhibiting agent comprises hydrogen peroxide, algae-inhibiting bacteria, and LB algae-inhibiting bacteria culture medium. This algae-inhibiting agent is used to inhibit the growth of Microcystis aeruginosa, comprising the following steps: Step 1, adding hydrogen peroxide to the water body at a concentration of 600–1000 nM; Step 2, 3–7 days after adding hydrogen peroxide, adding algae-inhibiting bacteria to the water body at a concentration of 300–500 ml / L, including LB algae-inhibiting bacteria culture medium by volume. The technical effects of this invention are: improved cyanobacteria inhibition rate, and environmentally friendly and rapid algae removal.

Description

Technical Field

[0001] This invention belongs to the field of algal bloom pollution control technology, specifically relating to a highly efficient algae-inhibiting agent composed of chemical reagents and bacteria, and the application of the algae-inhibiting agent. Background Technology

[0002] In response to the frequent and difficult-to-control cyanobacterial bloom pollution incidents, surface water algae removal mainly employs physical, biological, and chemical methods. Physical methods primarily include water diversion and replacement, sediment dredging, shading, clay flocculation and sedimentation, and ultrasonic methods. However, these methods are not widely adopted due to their lack of fundamental solutions, high costs, and complex operations.

[0003] Biological methods, including the use of large herbivorous fish or the interaction between microorganisms and microalgae, are used to control algal blooms. They have advantages such as low cost, no pollution, low energy consumption, and sustainability. However, biological methods are slow to take effect and are not suitable for the treatment of sudden algal blooms. In terms of using microorganisms to remove algae, the screening of algae-inhibiting microorganisms and how to improve the algae-inhibiting efficiency are challenges.

[0004] Chemical methods are currently the most widely used and fastest-acting treatments, including chemical reagent methods, electrochemical methods, and photocatalytic degradation methods. However, due to their side effects and high energy consumption, they cannot be used long-term. A common chemical reagent method involves adding hydrogen peroxide (H2O2) at high concentrations (>5 mg / L, i.e., 0.15 mmol / L). While this can provide a timely response to algal blooms, it can also be toxic to aquatic organisms in the water, thus damaging the ecosystem.

[0005] In addition, other microorganisms in the aquatic environment can provide protection for harmful algae against oxidative stress caused by high concentrations of hydrogen peroxide. According to the literature Weenink, EFJ, Interspecific protection against oxidative stress: green algae protect harmful cyanobacteria against hydrogen peroxide. ENVIRONMENTAL MICROBIOLOGY 2021, 23, (5), 2404-2419, Microcystis aeruginosa can resist hydrogen peroxide up to 15 mg / L under the protection of Chlorella. Even the intracellular lysates of Chlorella have a protective effect on Microcystis aeruginosa. Therefore, the use of chemical agents to remove algae not only has adverse effects on other aquatic organisms and the aquatic environment, but increasing the concentration of chemical agents does not necessarily achieve the effect of increasing the inhibition rate of harmful algae. This shows that using a single chemical agent often fails to achieve both algae suppression and ecological benefits.

[0006] Chemical agents have the function of inhibiting and killing microorganisms. According to the literature "Progress in the study of the sterilization mechanism of hydrogen peroxide", Yu Xiaofeng, Disinfection and Sterilization 1988, (03), 149-152, when the H2O2 concentration is 10mg / L, it can kill all bacteria after treatment at 25℃ for 1 hour. Hydrogen peroxide is an oxidant that can ionize the molecules or atoms of the bacterial cell wall, causing the lipid chains on the cell wall to break, thereby destroying the cell wall.

[0007] Therefore, chemical agents (H2O2) for algae removal and microbial bacteria for algae removal are two incompatible methods. When a high concentration of chemical agent (H2O2) is added to the water, microorganisms cannot survive and cannot exert their algae-removing effect. It is very difficult to use both chemical agents (H2O2) and microbial bacteria for algae removal, and it is difficult to achieve the ecological and environmental protection and algae removal effects of microbial bacteria. Summary of the Invention

[0008] To address the problems existing in current algal bloom control technologies, this invention aims to provide a low-concentration H2O2 combined with bacterial algae-inhibiting agent. This agent solves the challenge of using hydrogen peroxide and microbial bacteria for algae removal, improves the cyanobacteria inhibition rate, and combines the rapid action of chemical agents with the environmentally friendly benefits of microorganisms. This invention also provides applications of this algae-inhibiting agent.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0010] The present invention provides a low-concentration H2O2 combined with bacteria algaecide, comprising hydrogen peroxide, algaecide bacteria, and LB algaecide culture medium.

[0011] The algae-inhibiting bacteria are of the genus *Platydon*.

[0012] This invention also provides a low-concentration H2O2 combined with bacteria algaecide for inhibiting the growth of Microcystis aeruginosa, comprising the following steps:

[0013] Step 1: Add hydrogen peroxide to the water body, with the concentration of hydrogen peroxide in the water body being 600-1000 nM;

[0014] Step 2: 3-7 days after adding hydrogen peroxide, add algae-inhibiting bacteria to the water. The concentration of algae-inhibiting bacteria in the LB medium should not be less than 10. 8 The concentration of the bacteria in water is 300–500 ml / L, and the bacteria are added in LB culture medium.

[0015] Because different algae have varying tolerances to hydrogen peroxide, this invention selects a low concentration of hydrogen peroxide (600–1000 nM). This concentration is higher than the tolerance level of *Microcystis aeruginosa*, while still maintaining the survival of algae-inhibiting bacteria, thus solving the problem of incompatibility between H₂O₂ and microbial bacterial algae removal. This invention utilizes low-concentration hydrogen peroxide to disrupt the photosynthetic system and nucleosomes of cyanobacteria, thereby reducing the concentration of cyanobacteria in the water. It also utilizes algae-inhibiting bacteria to compete with cyanobacteria for nutrients or release algae-inhibiting active substances into the environment to inhibit or kill cyanobacteria, thereby improving the cyanobacteria inhibition rate.

[0016] Compared with the prior art, the technical advantages of the present invention are:

[0017] 1. Compared with existing single physical, biological, and chemical methods for inhibiting the growth of harmful algae, the combined algae-inhibiting agent provided by this invention is more economical, effective, and environmentally friendly, and the inhibition rate of cyanobacteria cells can reach more than 90%.

[0018] 2. The low-concentration hydrogen peroxide of the present invention will not harm non-target aquatic organisms. While inhibiting the growth of cyanobacteria, the low-concentration hydrogen peroxide does not cause algal cell rupture and death, and therefore will not cause a large amount of cyanobacterial toxins to be released into the extracellular environment.

[0019] 3. This invention has the advantages of rapid algae suppression by chemical agents, as well as the economical, safe, and aquatic ecological balance-maintaining characteristics of algae-suppressing bacteria, and has a long-term algae-suppressing effect. Attached Figure Description

[0020] The accompanying drawings of this invention are described below:

[0021] Figure 1The histogram shows the inhibition rate of H2O2 and *Pterygophyton floccosum* at different concentrations according to the present invention.

[0022] Figure 2 The histograms show the individual inhibition rates of H2O2 and *Pterygomyces lateralis* at different concentrations, representing comparative examples. Detailed Implementation

[0023] The present invention will now be described in detail:

[0024] The low concentration of H2O2 described in this patent application refers to a concentration of H2O2 in water bodies below 1100 nM (nM is an abbreviation for nmol / L, and μM is an abbreviation for μmol / L). Table 1 shows the concentration of H2O2 in natural water bodies.

[0025] Table 1. Hydrogen peroxide concentration measured in natural freshwater

[0026]

[0027] The low concentration H of the present invention O O2 combined with bacteria algaecide contains hydrogen peroxide, algaecide bacteria, and LB algaecide culture medium.

[0028] The concentration of hydrogen peroxide in the water is 600–1000 nM, and the concentration of algicidal bacteria in the LB medium is greater than 10. 8 The concentration of algae-inhibiting bacteria in water is 300–500 ml / L, based on the volume of LB medium.

[0029] The algae-inhibiting bacteria are of the genus *Platydon*.

[0030] Because different algae have varying tolerances to hydrogen peroxide, this invention selects a H2O2 concentration higher than that tolerated by *Microcystis aeruginosa*, while simultaneously maintaining the survival of the algae-inhibiting bacteria. This low-concentration H2O2... O O2 content in natural water is relatively high (see Table 1), which reduces H2 concentration. O According to the literature "Screening of Algicidal Bacteria and Effects of Algicidal Active Substances on the Physiological Activity of Microcystis aeruginosa", Chen Liting; Zuo Jun et al., Acta Hydrobiologica Sinica 2020, 44, (03), 638-646, algicidal bacteria can affect the photosynthetic system of microalgal cells, hinder electron transfer, inhibit their photosynthetic process, and cause oxidative damage to algal cells, destroying the integrity of algal cell membranes, thereby achieving algicidal effects. Therefore, the combined action of the chemical agent H2O2 and algicidal bacteria can maximize the inhibition rate of cyanobacteria.

[0031] LB medium was used as the culture medium for algae-inhibiting bacteria. The LB medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with the pH adjusted to 7.4. According to the literature "The Influence of Nutrients on the Growth of Microcystis aeruginosa and Algal Bacteria", Lü Ping, Li Huili et al., Environmental Science 2022, 43, (10), 4502-4510, LB medium can promote the growth of algae-inhibiting bacterial communities and has a certain inhibitory effect on algal cells. In implementation cases and engineering applications, LB medium and algae-inhibiting bacteria are poured into the water body at the same time, which will not aggravate eutrophication or cause other pollution. Preferably, the concentration of algae-inhibiting bacteria in the LB medium is not less than 10 g / L. 8 The algae-inhibiting bacteria concentration in the water is 300–500 ml / L, and LB algae-inhibiting bacteria culture medium is included when adding the algae-inhibiting bacteria.

[0032] The low concentration H of the present invention O O2 combined with bacteria to inhibit the growth of Microcystis aeruginosa includes the following steps:

[0033] Step 1: Add hydrogen peroxide to the water body, with the concentration of hydrogen peroxide in the water body being 600-1000 nM;

[0034] Step 2: 3-7 days after adding hydrogen peroxide, add algae-inhibiting bacteria to the water. The concentration of algae-inhibiting bacteria in the LB medium should be greater than 10. 8 The concentration of the bacteria in water is 300–500 ml / L, and the bacteria are added in LB culture medium.

[0035] The technical effects of the present invention are demonstrated below using examples and comparative examples:

[0036] The formula for calculating the inhibition rate of Microcystis aeruginosa growth in the examples and comparative examples is as follows:

[0037] IR (%) = (1 - N / N0) × 100

[0038] Wherein, IR represents the inhibition rate; N represents the number of algal cells in the experimental group; and N0 represents the number of algal cells in the control group.

[0039] Algal cell counts were determined by flow cytometry.

[0040] Control group:

[0041] Add the same volume of distilled water as the H2O2 and bacteria combined algaecide, keeping other conditions unchanged.

[0042] Example 1

[0043] The initial cell density was selected as 4×10⁻⁶. 5For Microcystis aeruginosa samples, 600 nmol / L hydrogen peroxide was first added to the water. Three days after adding hydrogen peroxide, 300 ml / L of a combination agent of Alternaria albopictus (based on LB medium volume) was added. Cells were counted at 3 days, 7 days, and 14 days and compared with the control group cells.

[0044] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this embodiment was 2.39 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The combined agent of 400 nmol / L hydrogen peroxide and 300 mL / L *Microcystis aeruginosa* showed an inhibition rate of 48% on the growth of *Microcystis aeruginosa*.

[0045] Example 2

[0046] The initial cell density was selected as 4×10⁻⁶. 5 For Microcystis aeruginosa samples of cells / mL, 800 nmol / L hydrogen peroxide was first added to the water. Five days after adding hydrogen peroxide, 300 ml / L of a combination agent of Alternaria albopictus (based on LB medium volume) was added. Cells were counted at 3d, 7d, and 14d and compared with the control group cells.

[0047] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this embodiment was 1.61 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The combined agent of 800 nmol / L hydrogen peroxide and 300 mL / L *Microcystis aeruginosa* showed a 65% inhibition rate on the growth of *Microcystis aeruginosa*.

[0048] Example 3

[0049] The initial cell density was selected as 4×10⁻⁶. 5 For Microcystis aeruginosa samples, 1000 nmol / L hydrogen peroxide was first added to the water. Seven days after the addition of hydrogen peroxide, 300 ml / L of a combination agent of Alternaria albopictus (based on the volume of LB medium) was added. Cells were counted at 3 days, 7 days, and 14 days and compared with the control group cells.

[0050] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this embodiment was 6.44 × 10⁻⁶. 4 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The combined agent of 1000 nmol / L hydrogen peroxide and 300 mL / L *Microcystis aeruginosa* showed an 86% inhibition rate on the growth of *Microcystis aeruginosa*.

[0051] Example 4

[0052] The initial cell density was selected as 4×10⁻⁶. 5 For Microcystis aeruginosa samples of cells / mL, 800 nmol / L hydrogen peroxide was first added to the water. Three days after adding hydrogen peroxide, 400 ml / L of a combination agent of Alternaria albopictus (based on LB medium volume) was added. Cells were counted at 3 days, 7 days, and 14 days and compared with the control group cells.

[0053] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this embodiment was 1.01 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The combined agent of 800 nmol / L hydrogen peroxide and 400 mL / L *Microcystis aeruginosa* showed a 78% inhibition rate on the growth of *Microcystis aeruginosa*.

[0054] Example 5

[0055] The initial cell density was selected as 4×10⁻⁶. 5 For Microcystis aeruginosa samples of cells / mL, 800 nmol / L hydrogen peroxide was first added to the water. Five days after adding hydrogen peroxide, 500 ml / L of a combination agent of Alternaria albopictus (based on LB medium volume) was added. Cells were counted at 3 days, 7 days, and 14 days and compared with the control group cells.

[0056] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this embodiment was 5.1 × 10⁻⁶. 4 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The combined agent of 800 nmol / L hydrogen peroxide and 500 mL / L *Microcystis aeruginosa* showed an 89% inhibition rate on the growth of *Microcystis aeruginosa*.

[0057] Example 6

[0058] The initial cell density was selected as 4×10⁻⁶. 5 For Microcystis aeruginosa samples, 1000 nmol / L hydrogen peroxide was first added to the water. Seven days after adding hydrogen peroxide, 500 ml / L of a combination agent of Alternaria albopictus (based on LB medium volume) was added. Cells were counted at 3, 7, and 14 days and compared with the control group cells.

[0059] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this embodiment was 3.6 × 10⁻⁶. 4 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5The combined agent of 1000 nmol / L hydrogen peroxide and 500 mL / L *Microcystis aeruginosa* showed a 92% inhibition rate on the growth of *Microcystis aeruginosa*.

[0060] Comparative Example 1

[0061] The initial cell density was selected as 4×10⁻⁶. 5 A sample of Microcystis aeruginosa with cells / mL was added to water at a concentration of 600 nmol / L. Cells were counted at 3, 7, and 14 days and compared with the control group.

[0062] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this comparative example was 3.54 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The concentration of hydrogen peroxide was 600 nmol / L, which means that the inhibition rate of 600 nmol / L hydrogen peroxide on the growth of Microcystis aeruginosa was 23%.

[0063] Comparative Example 2

[0064] The initial cell density was selected as 4×10⁻⁶. 5 A sample of Microcystis aeruginosa with cells / mL was added to water at a concentration of 800 nmol / L. Cells were counted at 3, 7, and 14 days and compared with the control group.

[0065] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this comparative example was 2.25 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The concentration of hydrogen peroxide was 800 nmol / L, which means that the inhibition rate of 800 nmol / L hydrogen peroxide on the growth of Microcystis aeruginosa was 51%.

[0066] Comparative Example 3

[0067] The initial cell density was selected as 4×10⁻⁶. 5 A sample of Microcystis aeruginosa with cells / mL was added to water at a concentration of 1000 nmol / L. Cells were counted at 3, 7, and 14 days and compared with the control group.

[0068] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this comparative example was 1.24 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The concentration of hydrogen peroxide was 1000 nmol / L, which means that the inhibition rate of 1000 nmol / L hydrogen peroxide on the growth of Microcystis aeruginosa was 73%.

[0069] Comparative Example 4

[0070] The initial cell density was selected as 4×10⁻⁶.5 A sample of Microcystis aeruginosa was prepared by adding 300 ml / L of Alternaria leucocephala in water (based on the volume of LB medium). Cells were counted at 3, 7, and 14 days and compared with the control group.

[0071] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this comparative example was 3.27 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The concentration of *Microcystis aeruginosa* was 300 ml / L, which showed a 29% inhibition rate against the growth of *Microcystis aeruginosa*.

[0072] Comparative Example 5

[0073] The initial cell density was selected as 4×10⁻⁶. 5 A sample of Microcystis aeruginosa was prepared by adding 400 ml / L of Alternaria leucocephala in water (based on the volume of LB medium). Cells were counted at 3, 7, and 14 days and compared with the control group.

[0074] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this comparative example was 2.71 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The concentration of *Microcystis aeruginosa* was 400 ml / mL, meaning that 400 ml / L of *Alternaria leucocephala* inhibited the growth of *Microcystis aeruginosa* by 41%.

[0075] Comparative Example 6

[0076] The initial cell density was selected as 4×10⁻⁶. 5 A sample of Microcystis aeruginosa was prepared by adding 500 ml / L of Alternaria leucocephala (based on the volume of LB medium) to the water. Cells were counted at 3, 7, and 14 days and compared with the control group.

[0077] The results showed that after 14 days, the cell density of *Microcystis aeruginosa* in this comparative example was 1.61 × 10⁻⁶. 5 The cell density of Microcystis aeruginosa in the control group was 4.6 × 10⁶ cells / mL. 5 The concentration of *Microcystis aeruginosa* was 500 ml / L, meaning that *Microcystis aeruginosa* inhibited the growth of *Microcystis aeruginosa* by 65%.

[0078] Based on the test results of the above embodiments and comparative examples, such as Figure 1 and Figure 2As shown, compared with the comparative example, the embodiments of the present invention have higher algae suppression efficiency, indicating that the treatment using a combination of low concentration H2O2 and algae-suppressing bacteria has a higher algae suppression rate than using H2O2 or algae-suppressing bacteria alone. Furthermore, the optimal ratio of the combined agents is: 1000 nmol / L hydrogen peroxide and 500 ml / L of *Pseudomonas albopictus* based on LB medium volume.

Claims

1. A method for inhibiting the growth of Microcystis aeruginosa using a low-concentration H2O2 combined with bacteria algaecide, wherein the algaecide comprises hydrogen peroxide, algaecide bacteria, and LB algaecide culture medium, characterized in that, Includes the following steps: Step 1: Add hydrogen peroxide to the water body, with the concentration of hydrogen peroxide in the water body being 600-1000 nM; Step 2: 3-7 days after adding hydrogen peroxide, add algae-inhibiting bacteria to the water. The concentration of algae-inhibiting bacteria in the LB medium should not be less than 10. 8 The concentration of the algae-inhibiting bacteria in water is 300-500 ml / L, and the algae-inhibiting bacteria are added with LB algae-inhibiting bacteria culture medium. The algae-inhibiting bacteria are of the genus *Platydon*.

2. The method for inhibiting the growth of Microcystis aeruginosa using a low-concentration H2O2 and bacterial combination algaecide according to claim 1, characterized in that: The LB anti-algae bacteria culture medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with the pH adjusted to 7.

4.

3. The method for inhibiting the growth of Microcystis aeruginosa using a low-concentration H2O2 and bacterial combination algaecide according to claim 2, characterized in that: The combination of hydrogen peroxide at a concentration of 1000 nM in the water and *Pterygodium lateralis* added to the water at a concentration of 500 ml / L based on the volume of LB medium.

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