A Bacillus subtilis compound agent for controlling algae growth, its preparation and application

Abstract

This invention discloses a compound Bacillus agent for controlling algae growth, its preparation, and its application. The compound Bacillus agent comprises *Bacillus belyssus* AS5 strain, *Bacillus licheniformis* PNB3 strain, *Bacillus licheniformis* C1 strain, *Bacillus subtilis* JK2 strain, *Bacillus subtilis* GT5 strain, and *Bacillus amyloliquefaciens* YX01 strain. This compound Bacillus powder achieves an algae-dissolving rate of up to 85.75% against green algae and exhibits broad-spectrum algae-inhibiting activity, inhibiting algae growth in *Spirogyra*, *Nostoc*, *Hylocereus algae*, *Oscillatoria*, *Microcystis aeruginosa*, and *Anabaena*, but its algae-inhibiting activity against green algae is higher than that against other unicellular algae. This invention achieves excellent algae-dissolving effects at relatively low working concentrations, significantly reducing the actual working concentration of each strain and effectively lowering the cost of large-scale algae-dissolving applications.

Description

Technical Field

[0001] This invention relates to a microbial composition for inhibiting algae growth, and more particularly to an algae-controlling compound Bacillus agent and its preparation and application. Background Technology

[0002] Spirogyra, also known as green algae, is a common, harmful, multicellular filamentous algae found in aquaculture waters. It not only attaches to the cheeks and gills of shrimp and crabs, affecting their activity and feeding, but also impairs gas exchange, easily producing harmful gases such as hydrogen sulfide. Furthermore, after death, it decomposes, causing the pond bottom to turn black and smelly, producing toxic substances and leading to the death of sea cucumbers, shrimp, crabs, and other aquatic animals. Therefore, green algae pollution is an environmental problem that urgently needs to be addressed in aquaculture production.

[0003] Currently, microbial ecological control is expected to become an effective strategy to solve the problem of algae pollution in aquaculture production. For example, Bacillus subtilis A4 disclosed in Reference 1: Cao Haipeng, Gu Ying, Sun Miaomiao, et al. Safety analysis of Bacillus subtilis A4 for Chinese mitten crab farming [J]. Freshwater Fisheries, 2023, 53(05):104-112. and Bacillus subtilis GT5 disclosed in Reference 2: Teng Chenhao, Wang Huicong, Wang Youhong, et al. Isolation, identification and metabolomics characteristics of Bacillus subtilis [J]. Bulletin of Microbiology, 2025, 52(05):2156-2171. have been proven to have good inhibitory effects on algae. Chinese Patent CN122188868A discloses that the algae-dissolving rate of Bacillus belyi AS5 strain on algae reaches 87.04%.

[0004] Although some existing microorganisms have a high algae-dissolving rate, these algae-controlling microorganisms generally have a high working concentration. When used alone, the effective viable bacteria concentration in the water must reach 1×10⁻⁶. 7 Only concentrations above CFU / mL exhibit excellent algae-dissolving effects. In large-scale aquaculture production, the consumption of algae-controlling microorganisms is enormous, making large-scale application difficult. Therefore, how to utilize the combination of different microorganisms to significantly improve the algae-suppressing efficiency of microorganisms at low concentrations has become an urgent technical problem to be solved. Summary of the Invention

[0005] Objective of this invention: The objective of this invention is to provide a Bacillus-based compound agent for controlling algae growth, addressing the problems of low efficiency and high working concentration required for existing algae-controlling microorganisms. A second objective is to propose a method for preparing this compound agent, solving the problem of how to prepare it. A third objective is to propose the application of this compound agent in inhibiting algae growth, solving the problem of how to efficiently inhibit algae growth at low concentrations.

[0006] Technical solution: The present invention provides a compound Bacillus agent for controlling algae growth, comprising Bacillus belye AS5 strain, Bacillus licheniformis PNB3 strain, Bacillus licheniformis C1 strain, and Bacillus subtilis JK strain. Two strains, Bacillus subtilis GT5 strain and Bacillus amyloliquefaciens YX01 strain.

[0007] Preferably, the preservation number of the Bacillus belyssus AS5 strain is CCTCC No.: M 2026576; The Bacillus subtilis JK The preservation number of strain 2 is CCTCC No.: M 20221568; the preservation number of Bacillus subtilis GT5 strain is CCTCC No.: M 2023291; The preservation number of the Bacillus licheniformis C1 strain is CCTCC No.: M 2020680.

[0008] Both Bacillus licheniformis strain PNB3 and Bacillus amyloliquefaciens strain YX01 are existing strains disclosed in the references.

[0009] Preferably, the *Bacillus belyssus* AS5 strain, *Bacillus licheniformis* PNB3 strain, *Bacillus licheniformis* C1 strain, and *Bacillus subtilis* JK strain are... The ratio of viable counts of strain 2, Bacillus subtilis GT5 strain and Bacillus amyloliquefaciens YX01 strain was 5-9:70-80:50-60:20-30:10-15:10-15.

[0010] The second aspect of this invention discloses a method for preparing the above-mentioned anti-porphyria compound Bacillus agent, comprising the following steps: (1) Bacillus belysus AS5 strain, Bacillus licheniformis PNB3 strain, Bacillus licheniformis C1 strain, and Bacillus subtilis JK strain were cultured separately. Culture media of different strains were obtained from strains 2, Bacillus subtilis GT5 and Bacillus amyloliquefaciens YX01; (2) The culture broth of each strain was inoculated into Bacillus solid culture medium for fermentation. The fermented solid culture was dried and pulverized to obtain the bacterial powder of each strain. (3) Mix the bacterial powders of each strain in a certain proportion to obtain the moss-controlling compound Bacillus agent.

[0011] Preferably, in step (2), the AS5 strain, PNB3 strain, C1 strain, and JK strain... The effective viable cell concentration in the mycelial powder of strains 2, GT5, and YX01 was at least 1.40 × 10⁻⁶. 9 1.42×1010 1.49×10 10 5.30×10 9 2.30×10 9 4.00×10 9 CFU / g.

[0012] Furthermore, in step (3), PNB3 strain, C1 strain, and JK strain... The mass ratio of the mycelium powder of strains 2, GT5, YX01, and AS5 was 4-6:3-5:4-6:4-6:2-4:4-6.

[0013] The third aspect of this invention discloses the application of the above-mentioned moss-controlling compound Bacillus agent in inhibiting moss growth.

[0014] The application of the above-mentioned Bacillus subtilis compound agent to inhibit algae growth includes the following steps: The Bacillus spp. compound for controlling algae growth was added to the algal solution containing green algae at a final concentration of 0.01-10 mg / L and cultured using standard methods. Preferably, the concentration of chlorophyll a in the algal solution does not exceed 3 mg / L.

[0015] Preferably, the algal solution also contains at least one of Nostoc, Alternaria floscens, Oscillatoria, Microcystis aeruginosa, and Anabaena.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: This invention combines Bacillus belye and Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens to produce a synergistic effect, achieving excellent algae suppression at a low working concentration, significantly reducing the actual working concentration of each strain, and effectively reducing the cost of large-scale algae control applications.

[0017] Furthermore, this invention utilizes AS5 strain, PNB3 strain, C1 strain, and JK strain. The combination of strains 2, GT5, and YX01 significantly expanded the algicidal spectrum of the compound bacterial agent, inhibiting moss and various algae such as Nostoc, Alternaria floscens, Oscillatoria, Microcystis aeruginosa, and Anabaena. Furthermore, the algicidal rate against moss was higher than that against unicellular algae such as Nostoc, Alternaria floscens, Oscillatoria, Microcystis aeruginosa, and Anabaena. This indicates that the addition of strains such as Bacillus licheniformis, Bacillus subtilis, and Bacillus amyloliquefaciens significantly enhanced the inhibitory and killing ability of Bacillus belyss AS5 strain against moss, making the compound bacterial agent a promising candidate for practical production applications. Attached Figure Description

[0018] Figure 1 The algae-dissolving rate of the algae-controlling compound bacterial powder on different algae strains. Detailed Implementation The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0019] Example 1: The formulation and preparation method of a Bacillus subtilis compound agent for controlling algae growth are as follows: (1) Experimental materials: Bacillus belyssus AS5 strain (accession number CCTCC No.: M 2026576, this strain was deposited at the China Center for Type Culture Collection on April 2, 2026, and has been disclosed in Chinese Patent Publication No. CN122188868A), Bacillus subtilis JK 2 (deposited at the China Center for Type Culture Collection, accession number CCTCC No.: M 20221568, disclosed in patent publication number CN116042449A), Bacillus subtilis GT5 strain (accession number CCTCC No.: M 2023291, deposited at the China Center for Type Culture Collection on March 13, 2023, disclosed in patent publication number CN117004505A), Bacillus licheniformis C1 (accession number CCTCC No.: M 2020680, deposited at the China Center for Type Culture Collection, has been disclosed in patent publication number CN113088466A), Bacillus licheniformis PNB3 (disclosed in reference: Huang Xiaodong, Zhang Jian, Zhu Yuanhong, An Jian, Cao Haipeng. Isolation, identification and safety analysis of crab-derived Citrobacter freundii antagonistic bacteria PNB3 [J]. Journal of Shanghai Ocean University, 2021, 30(4): 653-663.) and Bacillus amyloliquefaciens YX01 (disclosed in reference: Cao Haipeng, Zhou Chengxiang, He Shan, et al. Isolation and safety analysis of Bacillus amyloliquefaciens with nitrite degradation activity [J]. Environmental Pollution and Control, 2013, 35(6):6.DOI:10.3969 / j.issn.1001-3865.2013.06.004.), as compound strains of algae-controlling microbial agents; Spirogyra ( Spirogyra sp.) FACHB-737, as a representative algae species of moss, and Nostoc ( Nostoc sp.) FACHB-1967, Alternaria flocculata ( Aphanizomenonflos-aquae )FACHB-116, Oscillatoria ( Oscillatoria sp.) FACHB-528, Microcystis aeruginosa ( Microcystisaeruginosa )FACHB-315, Anabaena ( Anabaena sp . )FACHB-82, purchased from the Freshwater Algae Culture Bank of the Chinese Academy of Sciences; Nutrient broth was purchased from Sinopharm Chemical Reagent Co., Ltd.; BG11 culture medium was prepared by our laboratory according to the reference (Li Xinzhou, Zhuang Sina. The effect of culture medium on Chlorella). Chlorella Effects of sp. KM-201305 on growth and oil production [J]. Chemical and Biological Engineering, 2019, 36(09): 35-38.) were prepared in-house; Bacillus liquid culture medium was prepared in our laboratory with reference to the literature (Cao Haipeng, He Shan, Wei Ruopeng, et al. A fermentation medium for Bacillus subtilis resistant to hydrolytic amyloliquefaciens: ZL201110346583.X [P]. 2013-04-24.); Solid fermentation medium was prepared in our laboratory with reference to the literature (Zhang Zhiyan, Xu Haiyan, Yang Junfang. Optimization of solid fermentation medium for Bacillus subtilis [J]. Feed Expo, 2005(9):34-36.). All the above culture media were autoclaved at 121℃ for 20 min and then naturally cooled before use.

[0020] (2) Preparation of culture medium for each strain Aseptic technique was used to process Bacillus belysus AS5 strain, Bacillus licheniformis PNB3 strain, Bacillus licheniformis C1 strain, and Bacillus subtilis JK strain. Two strains of Bacillus subtilis, Bacillus subtilis GT5, and Bacillus amyloliquefaciens YX01 were inoculated into their respective nutrient broths and cultured in a constant temperature shaker for 24 h under suitable conditions, resulting in effective viable cell counts of 6.03 × 10⁻⁶. 8 4.33×10 8 8.10×10 8 6.67×10 8 2.70×10 8 2.0×10 8 The culture medium contained CFU / mL. The effective viable count was determined by the dilution plating method (Jin Yuyang, Jia Yang, Xu Hanqing, et al. Isolation, identification and algicidal activity of marine algicidal bacterium Pseudoalteromonas sp. J75 [J]. Acta Microbiologica Sinica, 2025, 65(04):1542-1557.).

[0021] (3) Preparation of mycelium powder for each strain Bacillus belysinus AS5 strain, as well as Bacillus licheniformis PNB3 strain, Bacillus licheniformis C1 strain, and Bacillus subtilis JK strain were used. The cultures of strains 2, Bacillus subtilis GT5, and Bacillus amyloliquefaciens YX01 were inoculated at 25% into 250 mL of their respective corresponding Bacillus liquid culture medium and cultured under suitable conditions in a constant temperature shaker for 24 h to achieve an effective viable count of 2.74 × 10⁻⁶ cells / mL. 91.03×10 9 1.93×10 9 5.73×10 9 1.23×10 9 1.25×10 9 CFU / mL, then each culture solution was inoculated into a solid culture medium and fermented for 4 days in a constant temperature biochemical incubator at its suitable culture temperature. After that, it was dried and pulverized into powder under aseptic conditions at 45°C, so that *Bacillus belye* AS5 strain, *Bacillus licheniformis* PNB3 strain, *Bacillus licheniformis* C1 strain, and *Bacillus subtilis* JK strain could be used. The effective viable cell concentrations in the mycelial powders of strains 2, Bacillus subtilis GT5, and Bacillus amyloliquefaciens YX01 were 1.40 × 10⁻⁶. 9 1.42×10 10 1.49×10 10 5.30×10 9 2.30×10 9 4.00×10 9 CFU / g. The effective viable count was determined using the dilution plating method (Jin Yuyang, Jia Yang, Xu Hanqing, et al. Marine alginolytic bacteria *Pseudomonas aeruginosa*). Pseudoalteromonas Isolation, identification and algicidal activity of sp.)J75[J]. Acta Microbiologica Sinica, 2025, 65(4):1542-1557.).

[0022] (4) Optimization of the compound ratio of each bacterial powder Orthogonal experimental design was used to optimize the *Bacillus belyssus* AS5 strain, as well as *Bacillus licheniformis* PNB3, *Bacillus licheniformis* C1, and *Bacillus subtilis* JK strains. The compounding ratios of bacterial powders from strains 2, Bacillus subtilis GT5, and Bacillus amyloliquefaciens YX01, and the factor levels for the orthogonal experiment are shown in Table 1. Bacillus belyss AS5, Bacillus licheniformis PNB3, Bacillus licheniformis C1, and Bacillus subtilis JK were taken according to the compounding ratios in Table 1. Two strains of Bacillus subtilis, Bacillus subtilis GT5 strain, and Bacillus amyloliquefaciens YX01 strain were added to a total weight of 100.0 g. After stirring at 10 r / min for 30 min at room temperature, 0.1 g of the compound bacterial powder with different compound ratios was added to a centrifuge tube containing 9.9 mL of sterile water and mixed in a vortex mixer for 5 min until it was uniformly mixed to prepare a stock solution of 10000.0 mg / L. The stock solution was then diluted with sterile distilled water to a dilution of 10.0 mg / L. Following the method of Liu Jiamin et al. (Liu Jiamin, Zhou Zhijie, Huang Minsheng, et al. Comparative analysis of the control effect of three agents on Spirogyra[J]. Chemical World, 2024, 65(03): 163-170.), Spirogyra FACHB-737 was inoculated into BG11 medium to prepare an algal solution with an initial chlorophyll a concentration of 1.26 mg / L. Aseptically, 2 mL of each dilution was added to 18 mL of Spirogyra FACHB-737 algal solution (initial chlorophyll a concentration of 1.26 mg / L) to make the final dose 1.0 mg / L. Equal amounts of sterile water and single-strain bacterial powder were added as blank and positive controls, respectively. The mixture was incubated in a light incubator for 6 days, with shaking 3 times a day. The concentration of chlorophyll a in Spirogyra FACHB-737 (mg / L) was determined according to the spectrophotometric method for determination of chlorophyll a in water quality according to HJ 897-2017. The algal dissolution rate was calculated according to the formula in the literature (Lv Ping, Li Huili, Zheng Xiaoxu, et al. Effect of nutrient copper aluminum microcystis growth and algal bacteria. Environmental Science, 2022, 43(10): 4502-4510.): Algal dissolution rate = (chlorophyll a concentration of blank control group - chlorophyll a concentration of bacterial treatment group) / chlorophyll a concentration of blank control group × 100%. Each treatment was performed in triplicate. Experimental data are expressed as mean ± standard deviation (Mean ± SD) and were analyzed using SPSS 19.0. P <0.05 indicates a significant difference. The results are as follows: Table 1. Factor Level Table for Orthogonal Experiment

[0023] Table 2. Results of the orthogonal experiment

[0024] Note: Different letters in the same column indicate significant differences. P <0.05), the same letter in the same column indicates no significant difference ( P >0.05); K1 is the sum of the 1-level means; K2 is the sum of the 2-level means; K3 is the sum of the 3-level means; k1 is the average of the 1-level means; k2 is the average of the 2-level means; k3 is the average of the 3-level means; R is the range.

[0025] Table 3. Analysis of Variance of Orthogonal Experiments

[0026] Note: ** indicates P <0.01.

[0027] Table 4. Algae-dissolving rate of Spirogyra FACHB-737 by single bacterial powder

[0028] Note: Different letters indicate significant differences. P <0.05), the same letter indicates no significant difference ( P >0.05).

[0029] The experimental results (Table 2) show that the effects of the six single bacterial strains on the algae-controlling compound bacterial powder FACHB-737 were in the following order: PNB3 > JK. The order of influence of each factor is as follows: A: 5>2>4>1>3; B: 4>5>1>2>3; C: 5>2>3>4>1; D: 5>1>4>3>2; E: 3>4>5>2>1; F: 5>4>1>3>2. Analysis of variance (Table 3) shows that PNB3, C1, and JK... 2. The bacterial powders of GT5, YX01, and AS5 all had significant effects on the algae-dissolving rate of Spirogyra fachlenberg-737. Therefore, PNB3, C1, and JK... 2. The optimal mixing ratio of GT5, YX01, and AS5 bacterial powders is 5:4:5:5:3:5, with a total bacterial concentration of 8.2 × 10⁻⁶. 9 At this compound ratio, the algae-dissolving rate of Spirogyra fachlenberg-737 treated with the algae-controlling compound bacterial powder reached 85.75%, which is higher than the algae-dissolving rate of Spirogyra fachlenberg-737 prepared with the algae-controlling compound bacterial powder at each compound ratio in the orthogonal experiment, and significantly higher than that of PNB3 powder, C1 powder, and JK powder. The algae-dissolving rates of Spirogyra facsimile FACHB-737 were 45.45% higher than those of single bacterial powders such as 2-microbial powder, GT5-microbial powder, YX01-microbial powder, and AS5-microbial powder. P <0.05), 71.20% P <0.05), 65.26% P <0.05), 68.38% P <0.05), 57.03% P <0.05), 54.37% P <0.05) (Table 4).

[0030] According to the synergy effect evaluation formula: SR=E 实测 / E 理论 E 实测 E represents the actual algae dissolution rate. 理论 For the expected algae dissolution rate, Expected algae dissolution rate E 理论 =1-(1-0.4030)×(1-0.1455)×(1-0.2049)×(1-0.1737)×(1-0.2872)×(1-0.313 8)=1-0.597×0.8545×0.7951×0.8263×0.7128×0.6862=1-0.1639=0.8361; SR=E 实测 / E 理论 =0.8575 / 0.8361=1.03. Based on SR>1 indicating a synergistic effect, PNB3, C1, and JK are identified. 2. The single bacterial powders of GT5, YX01, and AS5 were compounded in a ratio of 5:4:5:5:3:5, which showed a synergistic effect on the algae-dissolving effect of Spirogyra FACHB-737.

[0031] Example 2: Analysis of the application effect of the optimal compound ratio of algae-controlling compound bacterial powder (1) Analysis of the effect of moss-controlling compound microbial powder on moss control Before the experiment, Spirogyra FACHB-737 algal solution with an initial chlorophyll a concentration of 1.33 mg / L was prepared using the same method as in Example 1. Aseptically, 0.1 g of the algae-controlling compound bacterial powder (PNB3, C1, JK) prepared by the above method was taken. 2. The bacterial powder mixture ratio of GT5, YX01, and AS5 strains was 5:4:5:5:3:5, with a total bacterial concentration of 8.2 × 10⁻⁶. 9Add the CFU / g of the solution to a centrifuge tube containing 9.9 mL of sterile water and vortex for 5 min to prepare a stock solution of 10000.0 mg / L. Then dilute with sterile distilled water to prepare dilutions of 100.0 mg / L, 50.0 mg / L, 10.0 mg / L, 5.0 mg / L, 1.0 mg / L, and 0.1 mg / L. Take 2 mL of each dilution and add it to 18 mL of Spirogyra FACHB-737 algal solution (initial chlorophyll a concentration of 1.33 mg / L) to achieve final doses of 10.0 mg / L, 5.0 mg / L, 1.0 mg / L, 0.5 mg / L, 0.1 mg / L, and 0.01 mg / L, respectively. Use an equal volume of sterile water as a blank control. Incubate in a light incubator for 6 days, shaking 3 times daily. Refer to HJ 897-2017 Water Quality Chlorophyll a Standard. The concentration of chlorophyll a in Spirogyra FACHB-737 was determined (mg / L) using the spectrophotometric method. The algal dissolution rate was calculated using the formula in the literature (Lv Ping, Li Huili, Zheng Xiaoxu, et al. Effects of nutrient-poisoned copper-aluminum on the growth of Microcystis aeruginosa and algal bacteria. Environmental Science, 2022, 43(10): 4502-4510). Each treatment was repeated in triplicate. Experimental data are expressed as mean ± standard deviation (Mean ± SD) and statistical analysis was performed using SPSS 19.0. P <0.05 indicates a significant difference. The results are shown in Table 5: The experimental results (Table 5) show that the algae-dissolving rates of Spirogyra FACHB-737 at final dosages of 0.01, 0.1, 0.5, 1.0, 5.0, and 10.0 mg / L were 37.36%, 45.88%, 51.80%, 85.75%, 57.19%, and 54.13%, respectively. However, unexpectedly, the algae-dissolving rate did not gradually increase with the increase of the dosage of the algae-dissolving compound bacterial powder. In fact, the algae-dissolving rate of Spirogyra FACHB-737 at 1 mg / L was higher than that at 5.0 mg / L and 10.0 mg / L. Table 5. Effect of different dosages of algae-controlling compound bacterial powder on the algae-dissolving rate of Spirogyra FACHB-737.

[0032] Note: Different letters in the same column indicate significant differences. P <0.05), the same letter in the same column indicates no significant difference ( P >0.05).

[0033] (2) Alginate spectrum analysis of algae-controlling compound bacterial powder Following the method of Liu Jiamin et al. (Liu Jiamin, Zhou Zhijie, Huang Minsheng, et al. Comparative analysis of the control effects of three agents on Spirogyra [J]. Chemical World, 2024, 65(03): 163-170.), Spirogyra FACHB-737, Nostoc fACHB-1967, Alternaria fACHB-1168, Oscillatoria facsimile fACHB-528, Microcystis aeruginosa fACHB-315, and Anabaena fACHB-82 were inoculated into BG11 medium to prepare initial chlorophyll a concentrations of 1.33 mg / L, 0.79 mg / L, 1.61 mg / L, 2.44 mg / L, 2.87 mg / L, and 1.38 mg / L, respectively. Algal solutions of Spirogyra facsimile FACHB-737, Nostoc facsimile FACHB-1967, Alternaria flosica facsimile FACHB-1168, Oscillatoria facsimile FACHB-528, Microcystis aeruginosa fACHB-315, and Anabaena facsimile FACHB-82 at mg / L.

[0034] Following the method of Zhao Tianyu et al. (Zhao Tianyu, Pan Chaoyang, Zhang Chaohui, et al. Study on preparation process of solid Bacillus subtilis inoculant [J]. Food Industry Technology, 2020, 41(19): 104-111, 120.), 0.1 g of the algae-controlling compound bacterial powder prepared by the optimal compound ratio selected by screening was aseptically taken and added to a centrifuge tube containing 9.9 mL of sterile water. The mixture was vortexed for 5 min to prepare a mother liquor of 10000.0 mg / L. The mother liquor was then diluted with sterile distilled water to a dilution of 10.0 mg / L. Two mL of the dilution were then added to chlorophyll a concentrations of 1.33 mg / L, 0.79 mg / L, 1.61 mg / L, 2.44 mg / L, 2.87 mg / L, and 1.38 mg / L, respectively. The following algae were cultured at a final concentration of 1.0 mg / L: Spirogyra facsimile F ... P <0.05 indicates a significant difference.

[0035] Experimental results are as follows Figure 1 As shown, Figure 1 Different letters in the middle indicate statistically significant differences ( P <0.05). Figure 1The results indicate that the algae-controlling compound bacterial powder has an algae-inhibiting effect on Spirogyra FACHB-737, Nostoc fACHB-1967, Alternaria fACHB-1168, Oscillatoria facsifolia fACHB-528, Microcystis aeruginosa fACHB-315, and Anabaena fACHB-82, and has broad-spectrum algae-inhibiting activity. Surprisingly, the algae-controlling compound bacterial powder showed a better algae-suppressing effect on Spirogyra than simpler unicellular algae such as Nostoc flagelliforme FACHB-1967, Alternaria floscens FACHB-1168, Oscillatoria FACHB-528, Microcystis aeruginosa FACHB-315, and Anabaena FACHB-82. The algae-dissolving rate of Spirogyra flagelliforme FACHB-737 was 51.93% higher than that of other unicellular algae such as Nostoc flagelliforme FACHB-1967, Alternaria floscens FACHB-1168, Oscillatoria FACHB-528, Microcystis aeruginosa FACHB-315, and Anabaena FACHB-82. P <0.05), 44.72% P <0.05), 56.92% P <0.05), 65.95% P <0.05), 55.17% P <0.05).

[0036] Comparative Example 1: Everything else is the same as in Example 1, except that: PNB3 strain powder was not added to the compound bacterial agent, and the proportions of the powders of the other strains were used in the optimal proportions shown in Example 2. The total bacterial concentration remained unchanged.

[0037] Comparative Example 2: Everything else is the same as in Example 1, except that: No C1 strain powder was added to the compound bacterial agent, and the proportions of the powders of the other strains were the optimal proportions shown in Example 2. The total bacterial concentration remained unchanged.

[0038] Comparative Example 3: Everything else is the same as in Example 1, except that: JK is not added to the compound microbial agent. The mycelial powder of strain 2 was used, and the mycelial powder of the remaining strains was mixed in the optimal ratio shown in Example 2. The total bacterial concentration remained unchanged.

[0039] Comparative Example 4: Everything else is the same as in Example 1, except that: GT5 strain powder was not added to the compound bacterial agent, and the proportions of the powders of the other strains were used in the optimal proportions described in Example 2. The total bacterial concentration remained unchanged.

[0040] Comparative Example 5: Everything else is the same as in Example 1, except that: YX01 strain powder was not added to the compound bacterial agent, and the proportions of the powders of the other strains were used in the optimal proportions described in Example 2. The total bacterial concentration remained unchanged.

[0041] Comparative Example 6: Everything else is the same as in Example 1, except that: AS5 strain powder was not added to the compound bacterial agent, and the proportions of the powders of the other strains were used in the optimal proportions described in Example 2. The total bacterial concentration remained unchanged.

[0042] Comparative Example 7: Everything else is the same as in Example 1, except that: Under the optimal compounding ratio, replace PNB3 strain with Bacillus licheniformis AQ19. 11 strains (CCTCC No.: M 2022012, published in Chinese Patent CN114717140A).

[0043] Comparative Example 8: Everything else is the same as in Example 1, except that: Under the optimal compound ratio, strain C1 was replaced with Bacillus cereus LY-3 (published in reference: An Jian, Chen Baiyao, Gong Qiben, et al. Study on the purification of aquaculture pond water by Bacillus cereus [C] / / Proceedings of the 2011 National Symposium on Marine Aquaculture. 2011:532-536.).

[0044] Comparative Example 9: Everything else is the same as in Example 1, except that: Under the optimal compounding ratio, JK 2 strains were replaced with Bacillus cereus LY-1 (published in the reference: An Jian, Fu Guanghui, Ruan Jiming, et al. Screening and characteristics of denitrifying phosphorus-removing bacteria [J]. Bulletin of Microbiology, 2012, 39(2):162-171.).

[0045] Comparative Example 10: Everything else is the same as in Example 1, except that: Under the optimal compounding ratio, the GT5 strain was replaced with the Bacillus subtilis A4 strain (published in the reference: Cao Haipeng, Gu Ying, Sun Miaomiao, et al. Safety analysis of Bacillus subtilis A4 for Chinese mitten crab farming [J]. Freshwater Fisheries, 2023, 53(05):104-112).

[0046] Comparative Example 11: Everything else is the same as in Example 1, except that: Under the optimal compounding ratio, strain YX01 was replaced with Bacillus amyloliquefaciens strain G1 (published in reference: Cao H., He S., Wei R., et al. Bacillus amyloliquefaciens G1: a potential antagonistic bacterium against eel-pathogenic Aeromonas hydrophila[J]. Evidence-Based Complementary and Alternative Medicine, 2011, 2011: 1-7).

[0047] Comparative Example 12: Everything else is the same as in Example 1, except that: Under the optimal compound ratio, the AS5 strain was replaced with Bacillus subtilis BYK00318-01-01 (published in the reference: An Jian, Fu Guanghui, Ruan Jiming, et al. Screening and characteristics of denitrifying phosphorus removal bacteria [J]. Bulletin of Microbiology, 2012, 39(2):162-171.).

[0048] The algae-dissolving rate of different compound bacterial agents on Spirogyra FACHB-737 was tested using the same method as in Example 2. The final concentration of each compound bacterial agent was 1 mg / L. The results are as follows: Table 6. Algae-dissolving rate of Spirogyra FACHB-737 by different compound bacterial agents

[0049] Table 6 shows that the algae-dissolving rates of the compound bacterial agents prepared in Comparative Examples 1-12 against Spirogyra FACHB-737 were significantly lower than those of the compound bacterial agent with the optimal compound ratio in this invention. Furthermore, the algae-dissolving rates of Comparative Examples 1-12 against Spirogyra FACHB-737 were all below 10%, indicating no practical application value. Specifically, Comparative Examples 1-6 demonstrate that the highly efficient algae-inhibiting effect of the compound bacterial agents against Spirogyra FACHB-737 depends on strains PNB3, C1, and JK. Simultaneous addition of strains 2, GT5, YX01, and AS5 was necessary; the absence of any one of these strains resulted in no effective algae-inhibiting effect. Comparative Examples 7-12 showed that the simultaneous addition of strains PNB3, C1, and JK... When strains 2, GT5, YX01, and AS5 were replaced with other strains, the resulting compound microbial agent still could not effectively inhibit moss at low working concentrations. Only when these specific strains were combined could a significant synergistic effect be produced, greatly improving the moss-inhibiting effect of the compound microbial agent.

Claims

1. A compound Bacillus inoculant for controlling algae growth, characterized in that, Includes Bacillus belyss AS5 strain, Bacillus licheniformis PNB3 strain, Bacillus licheniformis C1 strain, and Bacillus subtilis JK strain. Two strains, Bacillus subtilis GT5 strain and Bacillus amyloliquefaciens YX01 strain.

2. The Bacillus subtilis compound agent for controlling algae growth according to claim 1, characterized in that, The preservation number of the Bacillus belyssus AS5 strain is CCTCC No.: M 2026576; The Bacillus subtilis JK The preservation number of strain 2 is CCTCC No.: M 20221568; the preservation number of Bacillus subtilis GT5 strain is CCTCC No. M 2023291; The preservation number of the Bacillus licheniformis C1 strain is CCTCC No.:M 2020680.

3. The Bacillus subtilis compound agent for controlling algae growth according to claim 2, characterized in that, The Bacillus belyssus AS5 strain, Bacillus licheniformis PNB3 strain, Bacillus licheniformis C1 strain, and Bacillus subtilis JK strain are mentioned. The ratio of viable counts of strain 2, Bacillus subtilis GT5 strain and Bacillus amyloliquefaciens YX01 strain was 5-9:70-80:50-60:20-30:10-15:10-15.

4. The preparation method of the Bacillus subtilis compound agent for controlling algae growth according to claim 2, characterized in that, Includes the following steps: (1) Bacillus belysus AS5 strain, Bacillus licheniformis PNB3 strain, Bacillus licheniformis C1 strain, and Bacillus subtilis JK strain were cultured separately. Culture media of different strains were obtained from strains 2, Bacillus subtilis GT5 and Bacillus amyloliquefaciens YX01; (2) The culture broth of each strain was inoculated into Bacillus solid culture medium for fermentation. The fermented solid culture was dried and pulverized to obtain the bacterial powder of each strain. (3) Mix the bacterial powders of each strain in a certain proportion to obtain the moss-controlling compound Bacillus agent.

5. The preparation method of the Bacillus subtilis compound agent for controlling algae growth according to claim 4, characterized in that, In step (2), the AS5 strain, PNB3 strain, C1 strain, and JK strain... The effective viable cell concentration in the mycelial powder of strains 2, GT5, and YX01 was at least 1.40 × 10⁻⁶. 9 1.42×10 10 1.49×10 10 5.30×10 9 2.30×10 9 4.00×10 9 CFU / g.

6. The preparation method of the Bacillus subtilis compound inoculant according to claim 5, characterized in that, In step (3), PNB3 strain, C1 strain, and JK strain... The mass ratio of the mycelium powder of strains 2, GT5, YX01, and AS5 was 4-6:3-5:4-6:4-6:2-4:4-6.

7. The application of the Bacillus subtilis compound agent according to claim 1 in inhibiting moss growth.

8. The application according to claim 7, characterized in that, Includes the following steps: The Bacillus spp. compound for controlling algae growth was added to the algal solution containing green algae at a final concentration of 0.01-10 mg / L and cultured using standard methods.

9. The application according to claim 8, characterized in that, The concentration of chlorophyll a in the algal solution does not exceed 3 mg / L.

10. The application according to claim 8, characterized in that, The algal solution also contains at least one of the following: Nostoc, Alternaria floscens, Oscillatoria, Microcystis aeruginosa, and Anabaena.

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