Algicidal bacterium TH41 and application thereof in inhibiting cyanobacterial bloom

Abstract

The invention discloses algicidal bacteria TH41 and application thereof in inhibiting cyanobacterial bloom, and belongs to the technical field of biology. The algicidal bacterium TH41 is identified as bacillus pumilus TH41 and is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the algicidal bacterium TH41 is GDMCC No: 66948. The invention also provides an algicidal bacterial agent containing the algicidal bacteria TH41. The invention further provides application of the algicidal bacterium TH41 or the algicidal fungicide in inhibiting cyanobacterial bloom. The algicidal bacterium TH41 has a degradation effect on harmful blue-green algae, has an efficient blue-green algae inhibition effect, has biological safety on daphnia magna and zebra fish in an algae killing concentration range, is suitable for preventing and treating various blue-green algae such as microcystis aeruginosa, cuscuta larakii, pichia mold and anabaena pseudosciaena, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of environmental ecological microbiology, specifically relating to an algicidal bacterium TH41 and its application in inhibiting cyanobacterial blooms. Background Technology

[0002] Frequent cyanobacterial blooms in China's three major eutrophic lakes—Taihu Lake, Dianchi Lake, and Chaohu Lake—cause serious harm to human society, the economy, and the ecological environment, necessitating effective prevention and control. Cyanobacterial blooms encompass various types, with Microcystis blooms being the most common. Microcystis blooms, dominated by Microcystis, release microcystin, severely damaging aquatic ecosystems. In recent years, other cyanobacterial blooms have also frequently occurred. *Aureobasidium oosporum* often blooms in lakes in Israel, Australia, and other regions. Several countries, including Spain, Lebanon, and Italy, have suffered from *Aureobasidium oosporum* blooms, and there are increasingly signs of an outbreak in China. It can release large amounts of *Cyclophora* toxin. *Aureobasidium raspenicillium*, due to its high toxicity, explosiveness, and invasiveness, has become another harmful cyanobacterial species after Microcystis, attracting significant attention. Furthermore, the active range of *Aureobasidium raspenicillium* is gradually spreading from tropical to subtropical and temperate regions, and *Aureobasidium raspenicillium* blooms are becoming increasingly frequent in southern my country. Cyanobacterial blooms dominated by *Anabaena pseudocaryophylla* have occurred in Shanghai, Zhejiang, Guangdong, Hebei, Shandong, and other regions. During their growth, they produce large amounts of 2-methylisoborneol—a substance that, at extremely low doses (10 ng·L⁻¹)—[is mentioned in the original text]. -1 Substances that give water a muddy smell make the prevention and control of various harmful cyanobacteria urgent.

[0003] Currently, commonly used methods for controlling cyanobacteria both domestically and internationally mainly include physical, chemical, and biological methods. Physical methods are divided into engineering algae removal (such as sand removal, bottom sediment dredging, and dike interception) and direct algae removal (such as dredging, electromagnetic fields, ultrasound, and shading), which inhibit algae reproduction by removing algae or changing their growth environment. However, this method is costly, difficult to implement, and hard to apply on a large scale. Chemical methods are simple to operate and fast-acting, killing or flocculating algae by adding chemical agents, but they are prone to causing secondary pollution, damaging the aquatic ecosystem, and even exacerbating cyanobacteria recurrence. Biological methods utilize aquatic plants, animals, and microorganisms to inhibit cyanobacteria. Among them, microbial algae-suppressing technology is receiving increasing attention. By applying screened and domesticated algicidal microorganisms (such as bacteria, fungi, or viruses), the cell structure of cyanobacteria is destroyed, inhibiting their protein synthesis and metabolism, thereby achieving efficient algae control. It has the advantages of simple operation, low cost, environmental friendliness, and high safety, and is a promising green governance method.

[0004] In nature, fungi, phycophages, and algicidal bacteria play crucial roles in regulating algal community dynamics and promoting the resolution of algal blooms. Among them, algicidal bacteria, due to their diverse species, varied functions, environmental friendliness, and ease of cultivation and scale-up, show great application potential. Currently, most algicidal bacteria are strains isolated from algal bloom water bodies or sediments. Due to the diversity of microbial resources, the types and functions of algicidal bacteria vary in different algal bloom habitats, thus a large number of algicidal microbial resources remain to be explored and applied. Algicidal bacteria are widely distributed in various aquatic and soil microenvironments. Studies have shown that the natural resolution of algal blooms or red tides is often accompanied by significant changes in the abundance and activity of algicidal bacterial communities, suggesting their key role in algal bloom regulation. Screening for highly efficient algicidal microorganisms from algal bloom-affected water bodies has become an important approach to developing biocontrol agents.

[0005] In existing technologies, some algicidal microorganisms have been discovered, and a small number have been applied in actual production. For example, patent document CN112625952A discloses the first application of a strain of Bacillus subtilis subsp. stegal in cyanobacterial control. However, research on algicidal bacteria is mostly limited to the laboratory stage and often focuses on a single subject, lacking research on the inhibitory effects on some common toxic and harmful cyanobacteria blooms. Furthermore, the diversity of microbial resources still requires the exploration of more efficient algicidal bacteria. Therefore, isolating and screening highly efficient algicidal strains with broad-spectrum cyanobacterial activity, and systematically studying their algicidal characteristics and thresholds, is of great significance for advancing the application of biological control of cyanobacterial blooms. Summary of the Invention

[0006] The main objective of this invention is to provide an algicidal bacterium, TH41, which uses the algal bloom habitat of Meiliang Bay in Taihu Lake as a strain resource bank for highly efficient algicidal bacteria. By screening algicidal bacteria with highly efficient algicidal effects, it can effectively inhibit harmful cyanobacteria such as Microcystis aeruginosa, Acerola latae, Acerola oosporioides, and Anabaena spp., providing technical support for the control of various harmful cyanobacteria.

[0007] Another objective of this invention is to provide an algicidal agent containing the aforementioned algicidal bacterium TH41, which can effectively inhibit and kill a variety of harmful cyanobacteria—Microcystis aeruginosa, Spirodactylus raspe, Megia oosporioides, and Anabaena pseudocaryophyte.

[0008] Another object of the present invention is to provide the application of the algicidal bacterium TH41 or the algicidal agent in inhibiting cyanobacterial blooms.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides an algicidal bacterium TH41, which has been identified as a Bacillus thuringiensis TH41, deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:66948, deposit date 2025-09-10, and deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0011] Preferably, the algicidal bacterium TH41 was isolated from the algal bloom habitat of Taihu Lake.

[0012] In a second aspect, the present invention provides an algicidal agent comprising the aforementioned algicidal bacterium TH41.

[0013] Preferably, the algaecide is selected from at least one formulation of active bacterial powder, active emulsion, and active granules.

[0014] A third aspect of the present invention provides the application of the algicidal bacterium TH41 or the algicidal agent in inhibiting cyanobacterial blooms.

[0015] Preferably, the cyanobacteria include Microcystis aeruginosa, Spirodactylus rasti, Oomycetes malachite, and Anabaena pseudocaryophyte.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] I. This invention uses the Taihu Lake cyanobacterial bloom habitat as a resource bank of algicidal bacteria, and conducts low-cost cultivation and isolation of cyanobacterial microorganisms. It also screens out highly efficient algicidal bacteria through co-cultivation with various harmful cyanobacteria, which has the advantages of low cost, simplicity and safety.

[0018] Second, the algicidal bacterium TH41 of the present invention has a degradation effect on a variety of harmful cyanobacteria. With an inoculum of more than 3%, the actual algicidal efficiency against harmful cyanobacteria reaches more than 60% on the 7th day, and it has a highly efficient effect on inhibiting cyanobacteria.

[0019] Third, the algicidal bacterium TH41 of the present invention has biosafety against Daphnia magna and zebrafish within the algicidal concentration range, and can better exert its algicidal properties in natural water bodies. It is suitable for the prevention and control of various cyanobacteria such as Microcystis aeruginosa, Acerola latae, Acerola oosporioides, and Anabaena spp., and has broad application prospects. Attached Figure Description

[0020] Figure 1 The effect of the alginolytic bacterium TH41 on chlorophyll a treatment of Microcystis aeruginosa, Auricularia latifolia, Auricularia oosporioides, and Anabaena pseudoaquatica for 7 days was shown in the examples.

[0021] Figure 2 The algicidal rate of TH41 on Microcystis aeruginosa, Acerola latae, Mesquita oosporioides, and Anabaena pseudoacacia on day 7 in the examples is shown.

[0022] Figure 3 The algicidal rate of the TH41 inoculation concentration of the algicidal bacterium in the examples on the 7th day of co-culture of Microcystis aeruginosa, Auricularia latifolia, Auricularia oosporioides, and Anabaena pseudoacacia is shown.

[0023] Figure 4 The concentration of thiazolysin TH41 in the examples is the toxicity concentration of Daphnia magna.

[0024] Figure 5 The concentration of thiazolyl bacterium TH41 in the examples is the toxicity concentration of thiazolyl bacterium TH41 to zebrafish. Detailed Implementation

[0025] 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.

[0026] The following example screened a strain with highly efficient algicidal activity, identified as Bacillus aerius TH41, which is deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No:66948. The specific steps are as follows:

[0027] Step 1: Take cyanobacterial slurry from Taihu Lake blooms for cyanobacterial community isolation. The isolated cyanobacterial community is incubated upside down in LB solid medium at 37℃ for 24 hours to screen for culturable bacteria in the algal community. These bacteria are then cultured in LB liquid medium. The culture medium of these culturable bacteria is co-cultured with *Microcystis aeruginosa* for 7 days under the following conditions: 25℃, light intensity of 1500 Lux, and a light-dark cycle of 12h:12h (D:L). The initial density of *Microcystis aeruginosa* is 1.5 × 10⁻⁶. 6 cells / mL.

[0028] Step 2: Inoculate Bacillus aerius TH41 into LB liquid medium and culture it in a shaker at 37°C and 180 r / min until the logarithmic growth phase. Then, transfer it to fresh LB liquid medium at a volume fraction of 1% and culture it under the same conditions for 12 h until the logarithmic growth phase.

[0029] Step 3: Inoculate the Bacillus aerius TH41 strain cultured to the logarithmic growth phase into an initial algal density of 2 × 10⁻⁶ cells / year at a bacterial-to-algae ratio of 10%. 6Fresh Microcystis aeruginosa, Auricularia latifolia, Oospora micrantha, and Anabaena pseudocaryophyte were co-cultured in a light incubator at 25°C and 1500 Lux with a photoperiod of 12h:12h (D:L).

[0030] The LB liquid medium consists of 10g peptone, 5g yeast extract, 10g NaCl, 1L distilled water, and pH adjusted to around 7.0. The solid medium is the same as the LB liquid medium with 2% agar added.

[0031] Step 4: Inoculate fresh algae at a bacterial-to-algae volume ratio of 0.01% to 15% at a density of 2 × 10⁻⁶. 6 Four harmful cyanobacteria were cultured together for 7 days in a light incubator with four cell / mL algal solutions at 25℃, light intensity of 1500 Lux and light-dark cycle of 12h:12h (D:L).

[0032] Step 5: Conduct biosafety assessments of Bacillus aerius TH41 strain on Daphnia magna and zebrafish. Acute toxicity tests on Daphnia magna were conducted using five juvenile Daphnia magna born to the same mother within 6–12 hours in a 50 mL system, with bacterial concentration gradients of 0%, 0.02%, 0.05%, 0.5%, 5%, 10%, and 12%. Acute toxicity tests on zebrafish were conducted using eight zebrafish larvae in a 4 L system, with bacterial concentration gradients of 0%, 0.05%, 0.2%, 0.5%, 3%, 5%, 10%, and 12%, using water with the same aeration time.

[0033] Example 1

[0034] (1) Alginolytic effect of TH41 algaecide on four harmful cyanobacteria:

[0035] Preparation of harmful cyanobacteria solution: Weigh 1.7 g of BG-11 culture medium powder, place it in a beaker, add an appropriate amount of distilled water to dissolve it completely, then transfer it to a 1 L volumetric flask and dilute to the mark. Subsequently, autoclave at 121 ℃ for 20 min.

[0036] Activation of Alginate TH41: The strain of Alginate TH41 was inoculated into LB liquid medium and activated and expanded by shaking culture at 37 ℃ and 180 r / min.

[0037] The cyanobacterial cell density in the culture system was set at 2.0 × 10⁶ cells / year. 6Cells / mL were added to 10% bacterial culture medium, and a control group was set up with an equal volume of BG-11 medium. All experimental groups and control groups were cultured at 25℃, with a light-dark cycle of 12 h:12 h and a light intensity of 1500 Lux. The differences between the experimental and control groups were compared by periodically measuring chlorophyll a content.

[0038] To accurately describe the actual algicidal efficiency of the algicidal bacterium TH41, algal cell density was used as a reference index to calculate the actual algicidal efficiency: R (%) = [(N0−N) / N0] × 100%, where N0 and N represent the algal cell densities of the control group and the treatment group, respectively.

[0039] (2) Alginolytic effects of different inoculation concentrations of TH41 algaecide on four harmful cyanobacteria:

[0040] Set the cyanobacteria density to 2.0 × 10⁻⁶. 6 cells / mL, TH41 alginolytic bacteria were cultured to the logarithmic growth phase, and the colony concentration was controlled at 1×10⁻⁶. 8 CFU / mL, with bacterial-to-algae volume ratios (v / v) set as follows: 0%, 0.01%, 0.1%, 1%, 2%, 3%, 5%, 7%, 10%, 15%, and 3 replicates per group. Algal cell density was measured and algal dissolution rate was calculated.

[0041] (3) Safety test of Alginate TH41 on Daphnia magna and zebrafish larvae:

[0042] According to the national standard "Acute Activity Inhibition Test of Daphnia spp." (GB / T21830-2008), the static limit exposure method was used in the test. After large-scale culture of Daphnia spp., several large and healthy oviparous female Daphnia spp. were selected and cultured individually in beakers until parthenogenesis. The generation with the highest reproductive output was selected as the female Daphnia spp. and cultured separately. This process was repeated for more than 3 generations to obtain a pure strain of Daphnia spp. 48 hours before the test, oviparous female Daphnia spp. were selected from the pure strain and cultured separately to acclimatize to the environment. 24 hours before the test, the larvae that had already laid eggs were removed. 6 hours before the test, the female Daphnia spp. were removed, and the larvae laid within 6-24 hours were retained for the test. Because large daphnia are more sensitive, Bacillus inoculation rates were set at 0%, 0.005%, 0.02%, 0.05%, 0.2%, 0.5%, 5%, 10%, and 12%, with LB control groups at 5.09% and 10%. Each experimental group was supplemented with 50 mL of aerated water and 5 juvenile daphnia in a 100 mL beaker. Three replicates were set up for each concentration. The experiment lasted 48 hours. During the experiment, no feeding, aeration, or water changes were performed, and daphnia mortality was recorded.

[0043] According to the national standard "Acute Toxicity Testing of Chemicals in Fish" (GB / T27861-2011), this experiment used the static limit exposure method, selecting healthy, active zebrafish without disease or injury. The experimental system consisted of 8 zebrafish placed in 4 L of aerated water, with feeding stopped 24 hours prior to the experiment. Bacterial inoculum levels were set at 0%, 0.05%, 0.2%, 0.5%, 3%, 5%, 10%, and 12%, with a 5% LB medium control group included. Each group had three replicates, and the experimental period was 48 hours. During the experiment, zebrafish behavior and mortality were observed and recorded daily, and the pH and dissolved oxygen levels of the water were monitored.

[0044] The following are three examples of using the above-mentioned strains to treat four kinds of harmful cyanobacteria.

[0045] Example 2

[0046] The alginolytic bacterium TH41 was activated and cultured to the logarithmic growth phase, and then co-cultured with *Auricularia latifolia* and *Megakia oosporioides*, respectively, at a cell concentration of 2.0 × 10⁻⁶ cells / mL. 6 The inoculum size was 10% (cells / mL). Chlorophyll a content in the algal cells was monitored daily, and cell density was counted on day 7 of the experiment to calculate the algal lysis rate. Figure 1 and 2 As shown, inoculation with TH41 alginolytic bacteria significantly reduced the chlorophyll a content of *Microcystis aeruginosa* to 277.2 μg / L, while the chlorophyll a content of the control group was as high as 1939.6 μg / L; the chlorophyll a content of *Auricularia auricula-judae* in the experimental group decreased to 20.4 μg / L, while the chlorophyll a content of *Auricularia auricula-judae* in the control group reached 1927.4 μg / L; the chlorophyll a content of *Oomycetes molluscioides* in the experimental group decreased to 3.8 μg / L, while the chlorophyll a content of the control group reached 1626.5 μg / L; the chlorophyll a content of *Anabaena pseudocaryophylla* in the experimental group decreased to 0 μg / L, while the chlorophyll a content of the control group reached 1436.1 μg / L. Figure 1 Meanwhile, on day 7, the algicidal bacterium TH41 achieved algicidal rates of 96%, 99.9%, 100%, and 100% against Microcystis aeruginosa, Aster davidii, Mesquita oosporioides, and Anabaena pseudocaryophyte, respectively. Figure 2 ).

[0047] Example 3

[0048] The concentration of cyanobacteria cells in the unified culture system was set at 2.0 × 10⁻⁶. 6 cells / mL. Simultaneously, TH41 bacterial suspension pre-activated to the logarithmic growth phase (concentration 1×10⁻⁶ cells / mL) was added. 8The experiment was conducted with bacterial-algae volume ratios (V / V) of 0.01%, 0.1%, 1%, 2%, 3%, 5%, 7%, 10%, and 15%, with three replicates per group. The experimental period was 7 days. The algicidal effect of different inoculum concentrations was determined by measuring algal cell density. When the inoculum concentration of TH41 was ≥0.01%, it inhibited the growth of *Microcystis aeruginosa*. When the inoculum concentration was ≥3%, the algicidal rate was greater than 60% on day 7. For *Auricularia auricula-judae*, the inoculum concentration of TH41 ≥0.01% effectively inhibited its growth, especially when ≥1%, the algicidal rate reached over 85%. When the inoculum concentration of TH41 was ≥1%, the growth rate of *Oomycetes oosporioides* significantly decreased, and when the inoculum concentration reached 3%, the algicidal rate of TH41 against *Oomycetes oosporioides* reached 95%. For *Anabaena pseudocaryophylla*, its growth tolerance is stronger. When the inoculum amount of algicidal bacterium TH41 is ≥1%, the growth rate of *Anabaena pseudocaryophylla* slows down. However, when the inoculum amount of TH41 reaches 3%, the algicidal efficiency instantly reaches 96%. Figure 3 ).

[0049] Example 4

[0050] The alginolytic bacterium TH41 was cultured to the logarithmic growth phase, with inoculum concentrations of 0%, 0.005%, 0.02%, 0.05%, 0.2%, 0.5%, 5%, 10%, and 12%. Simultaneously, 5.09% and 10% LB control groups were added to 50 mL of *Daphnia magna* larvae, and mortality was observed in the larvae after 24 hours. Zebrafish were also tested with inoculum concentrations of 0%, 0.05%, 0.2%, 0.5%, 3%, 5%, 10%, and 12%, with zebrafish mortality observed every 24 hours. When the TH41 concentration was below 10%, the survival rate of *Daphnia magna* larvae exceeded 80%, demonstrating a certain level of safety. For zebrafish larvae, the survival rate was over 90% when the TH41 concentration was below 5%. Therefore, the safe concentration range for alginolytic bacterium TH41 is below 5%. Figure 4 and 5 ).

[0051] 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. An algicidal bacterium TH41, characterized in that, It was identified as a Bacillus aerius TH41, and is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:66948.

2. The algicidal bacterium TH41 according to claim 1, characterized in that, The algicidal bacterium TH41 was isolated from the algal bloom habitat of Taihu Lake.

3. An algicidal agent, characterized in that, It contains the algicidal bacterium TH41 as described in claim 1 or 2.

4. The algicidal agent according to claim 3, characterized in that, The algaecide is selected from at least one formulation of active bacterial powder, active emulsion, and active granules.

5. The application of the algicidal bacterium TH41 as described in claim 1 or 2, or the algicidal agent as described in claim 3 or 4, in inhibiting cyanobacterial blooms.

6. The application according to claim 5, characterized in that, The cyanobacteria include Microcystis aeruginosa, Spirodactylus rasti, Oomycetes malachite, and Anabaena pseudoaquae.

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