An algicidal agent, its preparation method and application

Abstract

This invention discloses an algicidal agent, its preparation method, and its applications, relating to the fields of environmental microbiology and water treatment technology. The active ingredient of this algicidal agent includes extracellular metabolites of *Bacillus licheniformis*. This invention isolated an *Bacillus licheniformis* strain with algicidal capabilities. The extracellular metabolites of this strain are the core active ingredient, and experiments have confirmed that it has highly efficient algicidal effects against various common algae, including *Microcystis aeruginosa*, *Chlorella vulgaris*, and *Fragariae baumannii*, overcoming the technical bottleneck of narrow algicidal spectrum in single-strain formulations. Based on this *Bacillus licheniformis*, this invention developed a novel algicidal agent whose active ingredient is stable under conventional storage conditions, exhibiting both high efficiency and environmental friendliness. This invention provides stable and reliable technical support for algae control in complex aquatic environments and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology and water treatment technology, and in particular to an algicidal agent, its preparation method, and its application. Background Technology

[0002] Eutrophication-induced algal blooms are a prominent technical challenge in water treatment. Massive algal growth disrupts the material cycle and energy flow of aquatic ecosystems, leading to an imbalance in the aquatic ecosystem. Existing algal control technologies include physical methods such as harvesting and ultrasonic treatment, which are limited by high energy consumption, complex operation, and difficulty in treating large areas of water, and cannot fundamentally inhibit algal regeneration. Chemical methods achieve rapid algal control through the application of algicides, but these chemicals tend to remain in the water, causing secondary pollution. They may also alter the water's chemical properties, affecting the normal metabolism of aquatic organisms, and long-term use may induce drug resistance in algae, leading to a gradual decline in algal control effectiveness.

[0003] Microbial algicidal technology in biological methods has become a research focus in recent years due to its advantages such as strong environmental compatibility and high specificity. However, existing technologies still have significant technical bottlenecks. Single algicidal strains have a narrow algicidal spectrum, acting only against specific algal phyla, making it difficult to adapt to the complex community structure of multiple algae coexisting in actual water bodies. Some strains have low algicidal efficiency and long action cycles, failing to meet the technical requirements for rapid algae control during algal blooms. The environmental adaptability of these strains is weak; their survival and algicidal activity are easily affected by fluctuations in water temperature, changes in pH, or competition for nutrients, leading to unstable practical application results. Furthermore, live bacteria are prone to inactivation during production, processing, storage, and transportation, resulting in degradation of active ingredients, further limiting their large-scale application. Therefore, developing algicidal technologies with broad-spectrum algicidal activity, high efficiency, strong environmental adaptability, and excellent stability has become a key direction for overcoming the limitations of existing technologies. Summary of the Invention

[0004] The purpose of this invention is to provide an algicidal agent, its preparation method, and its application, to solve the problems existing in the prior art. This algicidal agent possesses highly efficient and broad-spectrum algicidal activity, as well as excellent stability, providing stable and reliable technical support for algae control in complex aquatic environments and showing broad application prospects.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of Bacillus licheniformis with algicidal ability (… Bacillus licheniformisThe Bacillus licheniformis described therein was deposited on December 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37050.

[0006] The present invention also provides the application of the above-mentioned Bacillus licheniformis in the preparation of algicidal agents.

[0007] The present invention also provides an algicidal agent, the active ingredient of which includes the extracellular metabolites of the above-mentioned Bacillus licheniformis.

[0008] Furthermore, the formulation of the algicidal agent is a liquid preparation or a lyophilized powder.

[0009] Furthermore, the lyophilized powder also includes a lyophilization protectant.

[0010] Furthermore, the freeze-drying protectant is trehalose, skim milk, or glycerin.

[0011] This invention also provides a method for preparing an algae-dissolving liquid formulation, comprising the following steps: The fermentation broth obtained by fermenting the above-mentioned Bacillus licheniformis is the algicidal liquid preparation.

[0012] This invention also provides a method for preparing an algae-soluble lyophilized powder, comprising the following steps: The above-mentioned Bacillus licheniformis was fermented to obtain a fermentation broth; After adding a freeze-drying protectant to the fermentation broth, the algae-soluble freeze-dried powder is obtained by freeze-drying.

[0013] The present invention also provides the application of the above-mentioned Bacillus licheniformis or algicidal agents in the removal of algae from water bodies.

[0014] The present invention also provides a method for removing algae from water, including the step of applying the above-mentioned Bacillus licheniformis or algicidal agent to the water to be treated.

[0015] The present invention discloses the following technical effects: This invention has isolated a strain of Bacillus licheniformis with algicidal capabilities. The extracellular metabolites of this strain are the core active ingredients. Experiments have confirmed that it has highly efficient algicidal effects on a variety of common algae, such as Microcystis aeruginosa, Chlorella vulgaris, and Hericium baumannii, breaking through the technical bottleneck of narrow algicidal spectrum of single strains.

[0016] This invention develops a novel algicidal agent based on Bacillus licheniformis. Its preparation process is simple and controllable, and both liquid and lyophilized powder formulations are suitable for different application scenarios. The lyophilized powder formulation, with the addition of a lyophilization protectant, effectively improves storage stability. Experiments show that this agent achieves excellent algicidal effects at addition levels of 10% (v / v) and above, and can efficiently remove algae at low, medium, and high initial concentrations. The agent prepared after 24 hours of expanded culture already achieves ideal algicidal effects, eliminating the need for extended culture periods and reducing production energy consumption. The active ingredients are stable under normal storage conditions and are heat-instantaneous proteins or peptides, posing no risk of chemical residues and combining high efficiency with environmental friendliness. This invention provides stable and reliable technical support for algae control in complex aquatic environments and has broad application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a colony morphology diagram of strain IMEE-BL-001; Figure 2 Phylogenetic tree analysis diagram of strain IMEE-BL-001; Figure 3 This is a diagram showing the results of the algae-dissolving experiment in Example 4; Figure 4 This is a statistical graph showing the chlorophyll a content of different groups at different times in Example 4; Figure 5 This is a statistical graph showing the chlorophyll a content of different groups at different times in Example 5; where Microcystis represents Microcystis aeruginosa and Herba salsa represents Herba salsa. Figure 6 This is a statistical graph showing the algae removal rate of different groups at different times in Example 5; where Microcystis represents Microcystis aeruginosa and Herba salsa represents Herba salsa. Figure 7 A statistical graph showing the chlorophyll a content of different groups at different times in Experiment Example 1; Figure 8 This is a statistical graph showing the algae removal rate of different groups at different times in Experiment Example 1; Figure 9 A statistical graph showing the chlorophyll a content of different groups at different times in Experiment Example 2; Figure 10 This is a statistical graph showing the algae removal rate of different groups at different times in Experiment Example 2; Figure 11 A statistical graph showing the chlorophyll a content of different groups at different times in Experiment Example 3; Figure 12 This is a statistical graph showing the algae removal rate of different groups at different times in Experiment Example 3. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 1. Strains Isolation A bacterial strain with algicidal ability was isolated from eutrophic water samples of Wuliangsuhai Lake and named IMEE-BL-001. Colony morphology is shown in [reference needed]. Figure 1 .

[0025] 2. Strain identification Phylogenetic analysis based on the 16S rRNA gene sequence was used to systematically identify the taxonomic position of strain IMEE-BL-001. First, the complete or near-complete 16S rRNA gene sequence of strain IMEE-BL-001 was amplified and used to construct a dataset along with sequences of representative closely related strains downloaded from the NCBI GenBank database. All reference sequences were derived from formally described species and had reliable accession numbers. After sequence alignment, phylogenetic analysis was performed on the dataset. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) method, which reconstructs evolutionary relationships between species by minimizing the sum of evolutionary distances and is suitable for taxonomic studies of conserved genes such as 16S rRNA. Evolutionary distances were calculated using the Kimura 2-parameter (K2P) model, which can distinguish between transitions and transversions, thus more accurately estimating evolutionary differences between nucleotide sequences. To ensure the reliability of phylogenetic inference, each branch of the tree underwent a bootstrap test (1000 replicates) 1000 times, and the resulting confidence scores (support rates) were annotated on the corresponding branch nodes to assess the stability of different branch topologies. During the phylogenetic analysis, all alignment sites containing gaps or missing data were completely removed using a complete deletion strategy. The final number of alignment sites used for analysis was 1412 bp, comprising 12 nucleotide sequences.

[0026] Phylogenetic results ( Figure 2 The results showed that strain IMEE-BL-001 clearly belongs to the genus Bacillus (Bacillus). Bacillus ), and with Bacillus licheniformis DSM 13 (NR_118996) A highly stable branch cluster was formed, with a support rate as high as 99%, indicating that the two have a very high phylogenetic relationship at the 16S rRNA gene level. Simultaneously, this branch and... Bacillus haynesii , Bacillus swezeyi These species together constitute a stable phylogenetic subgroup, with multiple key nodes showing support rates exceeding 95%, indicating high phylogenetic reliability. In contrast, IMEE-BL-001 and... Bacillus cytotoxicus , Bacillus mycoides as well as Bacillus pseudomycoides The species are located on different branches in the phylogenetic tree, with significantly increased evolutionary distances between them, and are clearly separated by nodes with high support, further ruling out the possibility that it belongs to the aforementioned species. Based on the combined information of phylogenetic topology, evolutionary distance, and branch support, it can be confirmed that strain IMEE-BL-001 is related to the aforementioned species at the 16S rRNA gene level. Bacillus licheniformisHaving a recent phylogenetic relationship supports its identification as Bacillus licheniformis ( Bacillus licheniformis ).

[0027] 3. Biological Preservation Bacillus licheniformis ( Bacillus licheniformis IMEE-BL-001 was deposited on December 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37050.

[0028] Example 2 bacterial strain: IMEE-BL-001 Culture medium: LB medium (components: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.0-7.2) Preparation method of algicidal agent: (1) Activation of strain: Pick a single colony from the plate and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL LB medium. Incubate in a constant temperature shaker at 37 °C and 180 rpm for 12 hours to obtain seed culture.

[0029] (2) Scale-up culture: Transfer the seed culture to fresh LB medium at an inoculation rate of 10% (v / v) and continue to culture under the same conditions (37℃, 180rpm) with shaking for 24 hours (at this time, the bacterial culture is in the late logarithmic growth stage, OD 600 =1.5-2.0), to obtain the fermentation broth.

[0030] (3) Collect the supernatant: Centrifuge the fermentation broth at 4℃ and 8000rpm for 10 minutes to obtain the supernatant.

[0031] (4) Sterilization filtration: The supernatant is sterilely filtered through a 0.22μm microporous membrane to obtain sterile filtrate, which is the algaecide, and stored at 4℃.

[0032] Tests showed that the active substances in this algicidal agent remained chemically stable and active under -20°C freezing and light-proof conditions; however, after high-temperature sterilization (121°C, 20 minutes), its algicidal activity was completely lost, indicating that it is one or more heat-instantaneous proteins or peptides. This characteristic clarifies its non-antibiotic small molecule nature.

[0033] Example 3 bacterial strain: IMEE-BL-001 Culture medium: LB medium (components: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.0-7.2) Preparation method of algicidal agent: (1) Activation of strain: Pick a single colony from the plate and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL LB medium. Incubate in a constant temperature shaker at 37 °C and 180 rpm for 12 hours to obtain seed culture.

[0034] (2) Scale-up culture: Transfer the seed culture to fresh LB medium at an inoculation rate of 10% (v / v) and continue to culture under the same conditions (37℃, 180rpm) with shaking for 24 hours (at this time, the bacterial culture is in the late logarithmic growth stage, OD 600 =1.5-2.0), to obtain the fermentation broth, which is the algicidal agent, and should be stored at 4℃.

[0035] Example 4 Following the method in Example 3, IMEE-BL-001 was activated and cultured to obtain a fermentation broth. A portion of the fermentation broth was centrifuged at 4°C and 8000 rpm for 10 minutes to obtain a supernatant, which was then aseptically filtered through a 0.22 μm microporous membrane to obtain a sterile filtrate. Another portion of the fermentation broth was centrifuged at 4°C and 8000 rpm for 10 minutes, the supernatant was discarded, and the bacterial cell precipitate was obtained. This precipitate was resuspended in an equal volume of sterile water to obtain a resuspension.

[0036] At a dosage of 10% (v / v), sterile LB medium, fermentation broth, sterile filtrate, and resuspension were added separately to the *Microcystis aeruginosa* algal solution (chlorophyll a content of 1.01 mg / L) and cultured continuously at 25°C for 5 days. The chlorophyll a content of the algal solution was determined using the hot ethanol method, and the algae removal rate was calculated. Algae removal rate = (Chlorophyll a content before treatment - Chlorophyll a content after treatment) / Chlorophyll a content before treatment × 100%.

[0037] The algae removal rates of each group are shown in Table 1 and Figures 3-4 As shown in the figure, the results indicate that, compared with the control group, both the fermentation broth and the sterile filtrate can effectively degrade chlorophyll a, while the resuspension is significantly less effective than the fermentation broth and the sterile filtrate.

[0038] Table 1. Algae removal rate of different active ingredients Example 5 The algicidal agent prepared in Example 2 was added at a dosage of 10% (v / v) to *Microcystis aeruginosa* algal solution (chlorophyll a content of 0.94 mg / L), *Chlorella vulgaris* algal solution (chlorophyll a content of 0.99 mg / L), and *Streptococcus baumannii* algal solution (chlorophyll a content of 1.09 mg / L), and cultured continuously at 37°C for 5 days. A control group was also set up, using an equal volume of sterile LB medium instead of the algicidal agent. The chlorophyll a content of the algal solution was determined using the hot ethanol method, and the algae removal rate was calculated, following the same method as in Example 3.

[0039] The statistical results of chlorophyll a content and algae removal rate of each group at different time points are shown in the figure. Figures 5-6 After 5 days of continuous treatment, the chlorophyll a content of *Microcystis aeruginosa* algal solution decreased from 0.94 mg / L to 0.07 mg / L, with an algae removal rate of 92.6%; the chlorophyll a content of *Chlorella vulgaris* algal solution decreased from 0.99 mg / L to 0.11 mg / L, with an algae removal rate of 88.9%; and the chlorophyll a content of *Gnaphalium baumannii* algal solution decreased from 1.09 mg / L to 0.18 mg / L, with an algae removal rate of 83.5%.

[0040] Experimental Example 1 The algicidal agent prepared in Example 2 was added to the *Microcystis aeruginosa* algal solution at dosages of 5%, 10%, and 15% (v / v), respectively, and cultured continuously at 37°C for 5 days. A control group was also set up, using an equal volume of sterile LB medium instead of the algicidal agent. The chlorophyll a content of the algal solution was determined using the hot ethanol method to calculate the algae removal rate, following the same method as in Example 3.

[0041] The statistical results of chlorophyll a content and algae removal rate of each group at different time points are shown in the figure. Figures 7-8 After 5 days of continuous treatment, the chlorophyll a content in the 5% (v / v) addition group decreased from 1.50 mg / L to 0.73 mg / L, with an algae removal rate of 51.3%; the chlorophyll a content in the 10% (v / v) addition group decreased from 1.50 mg / L to 0.18 mg / L, with an algae removal rate of 88.0%; and the chlorophyll a content in the 15% (v / v) addition group decreased from 1.53 mg / L to 0.21 mg / L, with an algae removal rate of 86.3%. This demonstrates that maintaining an addition level of 10% (v / v) or higher yields the best algae-dissolving effect.

[0042] Experimental Example 2 The extended culture time in Example 2 was adjusted to 1 day, 2 days, 3 days, 4 days, and 5 days to prepare different alginolytic agents. Then, each alginolytic agent was added to the *Microcystis aeruginosa* algal solution at a dosage of 10% (v / v) and cultured continuously at 37°C for 5 days. A control group was set up, using an equal volume of sterile LB medium instead of the alginolytic agent. The chlorophyll a content of the algal solution was determined using the hot ethanol method to calculate the algae removal rate, following the same method as in Example 3.

[0043] The statistical results of chlorophyll a content and algae removal rate of each group at different time points are shown in the figure. Figures 9-10 The results showed that the algae dissolution rate in each group was greater than 90%.

[0044] Experimental Example 3 The algicidal agent prepared in Example 2 was added to low-, medium-, and high-concentration Microcystis aeruginosa algal solutions at a dosage of 10% (v / v), and cultured continuously at 37°C for 5 days. A control group was also set up, using an equal volume of sterile LB medium instead of the algicidal agent. The chlorophyll a content of the algal solution was determined using the hot ethanol method to calculate the algae removal rate, following the same method as in Example 3.

[0045] The statistical results of chlorophyll a content and algae removal rate of each group at different time points are shown in the figure. Figures 11-12 The results showed that after 5 days of continuous treatment, the chlorophyll a content in the low concentration group decreased from 0.34 mg / L to 0.06 mg / L, with an algae removal rate of 82.4%; the chlorophyll a content in the medium concentration group decreased from 0.64 mg / L to 0.08 mg / L, with an algae removal rate of 87.5%; and the chlorophyll a content in the high concentration group decreased from 0.97 mg / L to 0.09 mg / L, with an algae removal rate of 90.7%. These results demonstrate that the algicidal agent prepared in this invention has a highly efficient algae-dissolving ability for different initial algae concentrations.

[0046] Example 6 bacterial strain: IMEE-BL-001 Culture medium: LB medium (components: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.0-7.2) Preparation method of algicidal agent: (1) Activation of strain: Pick a single colony from the plate and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL LB medium. Incubate in a constant temperature shaker at 37 °C and 180 rpm for 12 hours to obtain seed culture.

[0047] (2) Scale-up culture: Transfer the seed culture to fresh LB medium at an inoculation rate of 10% (v / v) and continue to culture under the same conditions (37℃, 180rpm) with shaking for 24 hours (at this time, the bacterial culture is in the late logarithmic growth stage, OD 600=1.5-2.0), to obtain the fermentation broth.

[0048] (3) Freeze-drying: Add 1 wt% trehalose to the fermentation broth (skim milk or other freeze-drying protectants such as glycerin can also be used), mix evenly and freeze-dry to obtain powdered algaecide, and store at 4℃.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus licheniformis with algicidal ability ( Bacillus licheniformis ), characterized in that, The Bacillus licheniformis was deposited on December 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37050.

2. The application of Bacillus licheniformis as described in claim 1 in the preparation of algicidal agents.

3. An algicidal agent, characterized in that, The active ingredients include the extracellular metabolites of Bacillus licheniformis as described in claim 1.

4. The algicidal agent according to claim 3, characterized in that, The algicidal agent is in the form of a liquid preparation or a lyophilized powder.

5. The algicidal agent according to claim 4, characterized in that, The lyophilized powder also includes a lyophilization protectant.

6. The algicidal agent according to claim 5, characterized in that, The freeze-drying protectant is trehalose, skim milk, or glycerin.

7. A method for preparing an algae-dissolving liquid formulation, characterized in that, Includes the following steps: The fermentation broth obtained by fermenting the Bacillus licheniformis according to claim 1 is the algicidal liquid preparation.

8. A method for preparing an algae-soluble lyophilized powder, characterized in that, Includes the following steps: Fermentation broth was obtained by fermenting the Bacillus licheniformis according to claim 1; After adding a freeze-drying protectant to the fermentation broth, the algae-soluble freeze-dried powder is obtained by freeze-drying.

9. The application of Bacillus licheniformis as described in claim 1 or the algicidal agent as described in any one of claims 3-6 in the removal of algae from water bodies.

10. A method for removing algae from water bodies, characterized in that, The method includes the step of applying the Bacillus licheniformis of claim 1 or the algicidal agent of any one of claims 3-6 to the water body to be treated.

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