A freeze-dried bacillus algae-lyticus agent, a preparation method thereof, and application thereof in algae inhibition

Abstract

The present invention discloses a freeze-dried algae-lytic bacteria agent, a preparation method thereof, and an application thereof in algae inhibition. The algae-lytic bacteria (Brevibacillus laterosporus) RZ1 is deposited with CCTCC NO: M2024725. The algae-lytic bacteria freeze-dried powder prepared by the present invention has a viable bacterial count of up to 7.27×10 10 cfu / g, the survival rate before and after freeze-drying can reach up to 171.62%, and the number of viable bacteria can be increased by 57.5% after storage at 4°C for one month. The strain and bacterial agent of the present invention can be added to water bodies before algae outbreaks to inhibit algae growth and reproduction and prevent algae outbreaks.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an algae-lytic Bacillus, a freeze-dried bacterial agent thereof, a preparation method thereof, and application thereof in inhibiting algae outbreaks. Background Art

[0002] Under the influence of human activities, increasing amounts of nutrients such as nitrogen and phosphorus are entering freshwater ecosystems such as rivers and lakes, causing eutrophication. Currently, cyanobacterial blooms have become the primary form of eutrophication in many rivers and lakes. High summer temperatures and excessive nutrient concentrations in water bodies can lead to explosive growth of harmful algae, such as cyanobacteria. These blooms, in turn, cause oxygen depletion, reduced water clarity, unpleasant odors, and even mass mortality in aquatic life, severely compromising water quality and ecological safety. Therefore, the development of efficient and sustainable algae control technologies is urgently needed.

[0003] Most current physical methods for algae removal, such as mechanical dredging and flotation, only treat the symptoms rather than the root cause and often require extensive engineering work. Chemical methods, such as chemical disinfection and flocculation sedimentation, are prone to secondary water pollution. In recent years, biological algae control, which can eliminate algae by fostering a healthy aquatic ecosystem or by introducing appropriate beneficial microorganisms, has demonstrated significant potential for combating harmful algae. Compared to traditional physical and chemical methods, these methods are safer, more economical, more ecologically sound, and more sustainable, and have therefore garnered increasing attention and research. Algae-lytic bacteria are a type of beneficial microorganism that can destroy algal cells and inhibit algal growth. They can also control algal blooms while reestablishing the ecological balance between bacteria and algae in the water, leading to their increasing application in algal bloom control. Traditional algal bloom control often involves emergency measures after an algal bloom occurs, which can cause irreversible damage to the aquatic ecosystem. However, algae-lytic microorganisms, due to their ability to maintain ecological balance, can be applied before an algal bloom occurs, making them more beneficial for ensuring a safer aquatic environment.

[0004] Prior art reports on algae-lytic bacteria include:

[0005] Patent specification with publication number CN114574409A discloses a strain of Pseudomonas aeruginosa, its fermentation products, and their application in algae lysis. The patented technology, Pseudomonas aeruginosa 630, has a deposit number of GDMCC No: 62328. Strain 630 has a certain algae-lysis effect on Raphidocelis subcapitata, Ulothrix sp., Chlorella pyrenoidosa, and Tetradesmus obliquus, indicating that it has a rich algae-lysis spectrum and has great application potential in biological agents for controlling algal blooms.

[0006] Patent specification with publication number CN114634895A discloses a strain of Bacillus cereus B1-XL001 with algae-lytic effect and its application. The Bacillus cereus B1-XL001 strain disclosed in the patented technology grows rapidly, has strong chemotaxis to algae, can attach to the outside of algal cells, and its secretions can destroy the cell walls, cell membranes, thylakoids and other structures of algal cells, inhibiting algal photosynthesis, causing irreversible damage to the algae and causing their death. It also has good stability, a wide range of temperature stability (-20 to 100°C), is resistant to strong acids, and repeated freezing and thawing of the bacterial liquid will not affect the algae-lytic effect, and there is no risk of recurrence of algal blooms. It can be prepared into an algae-lytic agent for controlling harmful algal blooms and has great application potential.

[0007] In the existing technology, algae-dissolving bacteria are mostly applied after an algae outbreak. If algae growth and reproduction can be inhibited by adding algae-dissolving bacteria before the algae outbreak, the occurrence of algae outbreak can be avoided, which has more practical application value. Summary of the Invention

[0008] One of the purposes of the present invention is to solve the problem of algae outbreaks such as blue algae in rivers and lakes by ecological means, by adding algae-dissolving bacteria to inhibit the growth and reproduction of algae in advance, thereby fundamentally preventing algae outbreaks.

[0009] In a first aspect, the present invention provides a strain of Brevibacillus laterosporus RZ1, with a deposit number of CCTCC NO: M2024725, which is a strain of Brevibacillus laterosporus with algae-lytic function.

[0010] The alginolytic Bacillus (Brevibacillus laterosporus) RZ1 was deposited in the China Center for Type Culture Collection (CCTCC) on April 19, 2024, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0011] In a second aspect, the present invention provides an alginolytic Bacillus agent, comprising the alginolytic Bacillus (Brevibacillus laterosporus) RZ1 described in the first aspect.

[0012] The Bacillus algae-lyticus bacterial agent may be a freeze-dried bacterial powder. Furthermore, the freeze-dried bacterial powder may be prepared by mixing raw materials including the fermentation culture of Bacillus algae-lyticus (Brevibacillus laterosporus) RZ1 and a freeze-protectant, followed by vacuum freeze-drying. The freeze-dried bacterial powder is preferably stored at -20 to 4°C.

[0013] The fermentation culture may be bacterial sludge obtained by centrifuging and removing the supernatant of the liquid fermentation culture of the Brevibacillus laterosporus RZ1.

[0014] The lyoprotectant preferably comprises skim milk powder, sucrose and mannitol in a mass ratio of 5:5 to 6:3.

[0015] The freeze-drying protective agent can be an aqueous solution, wherein the mass concentration of the effective protective component can be 13% to 14%. The effective protective component refers to skim milk powder, sucrose and mannitol.

[0016] The pH of the lyoprotectant may be 6.5 to 7.5, preferably 7. The pH adjustment may be achieved by adding a base such as sodium hydroxide.

[0017] The ratio of the volume of the lyoprotectant to the mass of the fermentation culture may be 2-2.5 mL:1 g, preferably 2 mL:1 g.

[0018] In a third aspect, the present invention provides a method for preparing the alginolytic Bacillus agent according to the second aspect, comprising the steps of:

[0019] (1) transferring the activated Bacillus laterosporus RZ1 to LB liquid culture medium and culturing at a constant temperature of 28 to 32° C. (e.g., 30° C.) to obtain a fermentation broth;

[0020] (2) centrifuging the fermentation liquid obtained in step (1) to collect the bacterial sludge;

[0021] (3) mixing the bacterial sludge obtained in step (2) with a freeze-drying protective agent, and resuspending the bacterial cells to obtain a bacterial suspension;

[0022] (4) The bacterial suspension obtained in step (3) is pre-frozen and then vacuum-freeze-dried to obtain freeze-dried bacterial powder.

[0023] In step (1), the activated Brevibacillus laterosporus RZ1 can be transferred to LB liquid culture medium at an inoculum volume of 0.1% to 1%.

[0024] In step (1), the rotation speed of the constant temperature shaking culture can be 150-180 rpm.

[0025] In step (1), the constant temperature shaking culture time can be 48 to 72 hours.

[0026] In step (2), the centrifugation temperature is preferably 4° C. Under 4° C., the survival rate of Brevibacillus laterosporus RZ1 is the highest.

[0027] In step (2), the centrifugal speed is preferably 8000 r / min.

[0028] In step (2), the centrifugation time is preferably 10 min.

[0029] The highest bacterial yield was achieved after the fermentation broth was centrifuged at 8000r / min for 10min.

[0030] In step (4), the pre-freezing temperature may be -20°C.

[0031] In step (4), the pre-freezing time may be 5 to 12 hours.

[0032] In a fourth aspect, the present invention provides use of the Brevibacillus laterosporus RZ1 described in the first aspect or the Brevibacillus laterosporus agent described in the second aspect in inhibiting the growth and reproduction of algae.

[0033] The Brevibacillus laterosporus RZ1 and the Brevibacillus laterosporus agent of the present invention can control the algae density and chlorophyll a concentration in water bodies within the threshold value before algae outbreak.

[0034] In a fifth aspect, the present invention provides a method for preventing algae outbreaks in water bodies, comprising: adding the Brevibacillus laterosporus RZ1 described in the first aspect and / or the Brevibacillus laterosporus agent described in the second aspect to the water body before the algae outbreak, inhibiting the growth and reproduction of algae and preventing the algae outbreak.

[0035] When the algae-lytic Bacillus agent is the freeze-dried powder mentioned above, it can be dissolved in water at a ratio of 1g:30-50mL before use, and activated for 30-60min. 3 The dosage is regularly added to the water body before the algae outbreak to suppress the algae outbreak.

[0036] In the use of the fourth aspect and the method of the fifth aspect, the algae include cyanobacteria such as Microcystis aeruginosa, Oscillatoria, and Planctomyces. The algae-lytic Bacillus (Brevibacillus laterosporus) RZ1 of the present invention has a broad-spectrum algae-inhibiting effect.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1) The present invention prepares the algae-dissolving bacteria into freeze-dried powder and optimizes its preparation process, thereby increasing the survival rate and storage time of the bacterial agent, which is beneficial for rapid response to algae outbreaks in practical applications. The viable cell count of the freeze-dried algae-dissolving bacteria powder prepared by the present invention can reach 7.27×10 10 cfu / g, the survival rate before and after freeze-drying can reach up to 171.62%, and the number of viable bacteria can be increased by 57.5% after storage at 4℃ for one month.

[0039] 2) In actual treatment of algae outbreaks in water bodies, microbial methods are often not well used because they are not as effective as physical and chemical methods. Although the water body after the algae outbreak is effectively treated in time, the aquatic ecosystem has suffered irreversible damage. However, the algae-lytic agent prepared by the present invention can be applied to water bodies showing signs of algae outbreak before the algae blooms, playing a role in preventing the large-scale growth of algae, maintaining the normal ecological balance of the water body, and achieving a balance between bacteria and algae. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a photo of the colony morphology of Brevibacillus laterosporus RZ1 growing on solid culture medium.

[0041] Figure 2 The figure shows the change of viable bacterial count of freeze-dried bacterial powder stored at different temperatures over storage time.

[0042] Figure 3 This is a diagram showing the treatment effect of freeze-dried bacterial powder on actual algal bloom water bodies, with the left side being the control group and the right side being the experimental group with added freeze-dried bacterial powder.

[0043] Figure 4 This is a graph showing the changes in chlorophyll a concentration in the freeze-dried bacterial powder in inhibiting algae outbreaks. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0045] Example 1

[0046] Isolation and identification of Brevibacillus laterosporus RZ1.

[0047] 1. Separation

[0048] (1) Water sample enrichment: Water samples from rivers during algae blooms were collected. After the water samples were enriched and cultured in LB medium, a large number of microbial strains were purified from them by the dilution coating method and streak separation method to serve as candidate strains for screening algae-lytic bacteria.

[0049] (2) High-throughput screening: After the purified strains were activated, they were co-cultured with Microcystis aeruginosa cultured to the logarithmic phase at a volume ratio of 10% in a 96-well plate. The plates were placed in a light incubator at a temperature of 25°C, a light intensity of 2000 lux, and a light-dark ratio of 14h:10h. The strains that caused the algae solution to turn yellow were screened out.

[0050] (3) Shake flask rescreening: The strain obtained from the initial screening was activated and added to 30 mL of Microcystis aeruginosa algae solution at a volume ratio of 5% for rescreening. The chlorophyll a concentration before and after incubation was measured, and the algae lysis rate was calculated. The strain with the highest algae lysis rate was the Bacillus algae lysis strain of the present invention.

[0051] The chlorophyll a concentration was determined using the 90% hot ethanol method, and the calculation formula was:

[0052]

[0053] Where Chl a is the concentration of chlorophyll a in the water sample (mg / L); D664, D647, D630, and D750 are the absorbances of the extract at 630 nm, 647 nm, 664 nm, and 750 nm, respectively; V e is the constant volume of the extract (mL); V s is the volume of water sample (mL).

[0054] The calculation formula of algae dissolution rate is:

[0055]

[0056] Where R a is the algae lysis rate (%); C0 is the initial chlorophyll a concentration (mg / L); C t is the chlorophyll a concentration after treatment (mg / L).

[0057] 2. Identification

[0058] (1) Colony morphology: The colonies are white and opaque in the initial stage of culture on solid culture medium. After prolonged culture time, the colonies age and become yellow with wrinkles on the surface and irregular edges, such as Figure 1 shown.

[0059] (2) Molecular identification: The algae-lytic bacteria species was identified by 16S rRNA gene sequencing. The sequencing results were compared with known sequences in GenBank by BLAST. The comparison analysis results showed that the strain was Brevibacillus laterosporus, and it was named Brevibacillus laterosporus RZ1. The bacteria was deposited in the China Center for Type Culture Collection (CCTCC) on April 19, 2024, with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the deposit number is CCTCC NO: M2024725. The 16S rRNA gene sequence of Brevibacillus laterosporus RZ1 is shown in SEQ ID NO: 1.

[0060] Example 2: Optimization of the centrifugal collection process of bacterial cells

[0061] A mother liquor of Bacillus laterosporus RZ1 was obtained by inoculating the culture medium with LB liquid medium from a plate for 24 hours. The mother liquor was then transferred to fresh LB liquid medium at a 1% inoculum rate and expanded for 48 hours in a constant temperature shaking incubator at 30°C and 180 rpm to obtain a large amount of Bacillus RZ1 fermentation liquid. The fermentation liquid was centrifuged at 4°C with the centrifugal speed set at 6000 r / min, 6500 r / min, 7000 r / min, 7500 r / min, 8000 r / min, and 8500 r / min, and the centrifugation time was 5 min, 10 min, and 15 min, respectively. The viable cell counts of the bacterial solution before centrifugation and the pellet after centrifugation were counted by the plate dilution coating method, and the bacterial cell yield under each centrifugation condition was calculated. The results are shown in Table 1 below. The bacterial cell yield was the highest at a centrifugal speed of 8000 r / min and a centrifugation time of 10 min, reaching 96.80%. Therefore, it was concluded that the optimal centrifugal conditions were a centrifugal speed of 8000 r / min and a centrifugal time of 10 min.

[0062] Table 1 Optimization results of bacterial centrifugation collection process

[0063]

[0064] Example 3: Optimization of compound freeze-drying protective agent formula

[0065] 1. Single factor screening of protective agents

[0066] Based on the properties and mechanisms of action of different protectants, a selection of lyoprotectants, including sodium glutamate, sucrose, mannitol, glucose, lactose, trehalose, skim milk powder, and maltodextrin, was selected. Bacillus RZ1 was seeded from a plate into LB liquid medium and cultured for 24 hours to obtain a stock solution. This stock solution was then transferred to fresh LB liquid medium at a 0.1% to 1% inoculum concentration. The culture was expanded for 48 to 72 hours in a constant temperature shaking incubator at 28-32°C and 150-180 rpm to obtain a large amount of Bacillus RZ1 fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 10 minutes at 4°C, and the supernatant was discarded to obtain a bacterial slurry. Solutions of equal mass concentrations of different protectants were prepared. A saline blank control was used as a blank control and mixed with the collected bacterial slurry at a ratio of 2 mL:1 g. The solution was pre-frozen in a -20°C refrigerator for 5-12 hours and then freeze-dried in a vacuum freeze dryer. After freeze-drying, freeze-dried bacterial powder was obtained. The freeze-dried bacterial powder was dissolved in an equal amount of sterile water, and the viable bacterial cells were counted before and after freeze-drying using the plate dilution spread method. The survival rate of the cells was calculated under the addition of different protectants. The top three protectants with the highest survival rates were screened: skim milk powder, sucrose, and mannitol. Based on this, single-factor experiments were conducted with different addition concentrations of the three screened protectants.

[0067] Bacillus RZ1 was inoculated from a plate into LB liquid medium and cultured for 24 hours to obtain a mother liquor. This mother liquor was then transferred to fresh LB liquid medium at a 1% inoculum rate and expanded for 72 hours in a constant-temperature shaking incubator at 30°C and 150 rpm to obtain a large amount of Bacillus RZ1 fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 10 minutes at 4°C, and the supernatant was discarded to obtain a bacterial slurry. Skim milk powder, sucrose, and mannitol were prepared to concentrations of 2%, 4%, 6%, 8%, and 10%, respectively. The collected bacterial slurry was mixed evenly at a ratio of 2 mL:1 g. The solution was pre-frozen in a -20°C refrigerator for 5 hours and then freeze-dried in a vacuum freeze dryer. Freeze-dried bacterial powder was obtained after completion of freeze-drying. The freeze-dried bacterial powder was dissolved in an equal amount of sterile water, and the viable bacterial counts before and after freeze-drying were counted using the plate dilution spread method to calculate the bacterial survival rate. The results are shown in Tables 2 to 4 below. According to the calculation results of bacterial survival rate, 6wt% skim milk powder, 6wt% sucrose and 4wt% mannitol were selected as the added concentrations after optimization in single-factor experiments.

[0068] Table 2 Single factor experimental results of skim milk powder

[0069]

[0070] Table 3 Results of single factor experiment on sucrose

[0071]

[0072] Table 4 Mannitol single factor experimental results

[0073]

[0074] 2. Orthogonal test of composite freeze-drying protective agent

[0075] Based on the results of the single-factor experiments with single-factor protectants, we selected the survival rate of bacteria before and after freeze-drying as the evaluation indicator. Using skim milk powder, sucrose, and mannitol as the experimental factors, we designed an orthogonal experiment with a 4-factor, 3-level composite freeze-drying protectant. The orthogonal experimental design is shown in Table 5, and the experimental results for each combination are shown in Table 6. The experimental results show that the optimal composite freeze-drying protectant combination was 5% skim milk powder, 5% sucrose, and 3% mannitol, achieving a bacterial survival rate of 141.72%. Range and variance analyses were performed on the experimental results, as shown in Tables 6 and 7. The range analysis results showed that the optimal level combination in the orthogonal experiment was 1, 2, 1, 2, that is, the optimal composite freeze-drying protectant combination was 5% skim milk powder, 6% sucrose, and 3% mannitol. Based on the R values ​​of each factor, the order of effect of the three protectants on survival was C (mannitol) > B (sucrose) > A (skim milk powder). Variance analysis also showed that mannitol and sucrose had significant effects on bacterial survival rate, while skim milk powder had no significant effect.

[0076] The two groups of optimal protective agent combinations obtained from the orthogonal experiment results and range analysis were further verified by experiments. The results are shown in Table 8 (the number of viable bacteria before freeze-drying was 2.27×10 8 CFU / mL), there was no significant difference between the two groups of protective agent combinations. Considering the cost, 5% skim milk powder, 5% sucrose, and 3% mannitol were selected as the best composite freeze-dried protective agent formula.

[0077] Table 5 Orthogonal experimental design of composite freeze-drying protective agent

[0078] level Skim milk powder A (%) Sucrose B (%) Mannitol C (%) 1 5 5 3 2 6 6 4 3 7 7 5

[0079] Table 6 Orthogonal analysis results of composite freeze-dried protective agents

[0080] combination Skim milk powder A Sucrose B Mannitol C D Survival rate (%) 1 1 1 1 1 141.72 2 1 2 2 2 89.51 3 1 3 3 3 65.89 4 2 1 3 2 55.63 5 2 2 1 3 135.87 6 2 3 2 1 59.38 7 3 1 2 3 80.13 8 3 2 3 1 83 9 3 3 1 2 113.58 K1 297.12 299.54 391.17 284.10 K2 280.88 308.38 221.08 288.72 K3 268.77 238.85 234.52 273.95 k1 99.04 99.85 130.39 94.70 k2 93.63 102.79 73.69 96.24 k3 89.59 79.62 78.17 91.32 R 9.45 23.18 56.70 4.92 Optimal level 1 2 1 2

[0081] Table 7 Orthogonal variance analysis of composite freeze-dried protective agents

[0082] Sources of Difference Sum of Squares of Deviations degrees of freedom mean square F-number P-value Significance Skim milk powder A 0.220 0.220 0.220 0.220 0.220 Sucrose B 0.038 0.038 0.038 0.038 0.038 * Mannitol C 0.006 0.006 0.006 0.006 0.006 ** Error D 38.058 2 19.029

[0083] Table 8 Further verification of the best protective agent combination

[0084] Portfolio Source Skim milk powder concentration (%) Sucrose concentration (%) Mannitol concentration (%) Survival rate (%) Experimental results 5 5 3 141.72 Range Analysis 5 6 3 140.42

[0085] 3. Optimization of the mixing ratio of protective agent and bacterial mud

[0086] Based on the optimized formula of the composite lyoprotectant, a protective agent solution prepared according to a concentration formula of 5wt% skim milk powder, 5wt% sucrose, and 3wt% mannitol was mixed with Bacillus RZ1 bacterial sludge at ratios of 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, and 3:1 (mL:g), respectively. After pre-freezing at -20°C for 12 hours, the mixture was placed in a vacuum freeze dryer for freeze drying. The number of viable bacteria before and after lyophilization was counted, and the bacterial survival rate was calculated. The results are shown in Table 9 below. Based on the calculated bacterial survival rate, the optimal mixing ratio of protective agent to bacterial sludge was determined to be 2mL:1g (v:w).

[0087] Table 9 Optimization of the mixing ratio of protective agent and bacterial mud

[0088] Protective agent and bacterial sludge ratio Survival rate (%) 0.5:1 93.57 1:1 110.53 1.5:1 113.97 2:1 141.72 2.5:1 126.96 3:1 131.03

[0089] 4. pH Optimization of Composite Lyoprotectant

[0090] Finally, the pH of the composite freeze-dried protective agent solution was optimized. After 5wt% skim milk powder, 5wt% sucrose, and 3wt% mannitol were prepared into a mixed solution (pH of about 6), the pH was adjusted to 6, 6.5, 7, 7.5, and 8, respectively. The solution was mixed evenly with the bacterial mud at a ratio of 2mL:1g (v:w). After pre-freezing for 12 hours, the solution was placed in a vacuum freeze dryer for freeze drying. The number of viable bacteria before and after freeze-drying was counted, and the survival rate of the bacteria was calculated. The results are shown in Table 10 below. According to the results of the bacterial survival rate calculation, the optimal pH of the composite freeze-dried protective agent was determined to be 7.

[0091] Table 10 pH optimization of composite freeze-drying protective agent

[0092]

[0093] Example 4

[0094] A method for preparing freeze-dried Bacillus powder, comprising:

[0095] Bacillus RZ1 was inoculated from a plate into an LB liquid culture medium and cultured for 24 hours to obtain a mother liquor, which was then transferred to a fresh LB liquid culture medium at an inoculum rate of 0.1% to 1%, and cultured in a constant temperature shaking incubator at a temperature of 28 to 32° C. and a rotation speed of 150 to 180 rpm for 48 to 72 hours to obtain a large amount of Bacillus RZ1 fermentation liquid.

[0096] The Bacillus RZ1 fermentation broth obtained by the expanded culture was centrifuged at 8000 r / min at 4° C. for 10 min, the supernatant was removed, the bacterial precipitate after centrifugation was collected, and washed twice with sterile water to obtain bacterial sludge.

[0097] Skimmed milk powder, sucrose and mannitol were mixed in distilled water at concentrations of 5 wt%, 5 wt% and 3 wt% respectively, the pH was adjusted to 7, and the mixture was sterilized at 121° C. for 20 min to obtain a composite freeze-drying protective agent.

[0098] The composite lyophilization protective agent and Bacillus RZ1 bacterial sludge were mixed at a ratio of 2 mL: 1 g (v:w) and the bacteria were resuspended to obtain a mixture of bacteria and lyophilization protective agent.

[0099] The mixture of bacteria and lyophilization protectant was placed in a -20°C refrigerator for pre-freezing for 5-12 hours, and then placed in a vacuum freeze dryer for freeze drying to obtain freeze-dried bacterial powder.

[0100] Example 5: Storage conditions and stability of freeze-dried bacterial powder

[0101] Freeze-dried bacterial agents were prepared according to the method of Example 4. After determining the viable bacterial count of the freeze-dried bacterial powder in its initial state, the freeze-dried bacterial powder was stored at room temperature, 4°C, and -20°C. Samples were accurately weighed every week, and the viable bacterial count was calculated after the bacterial powder was rehydrated. The change in the viable bacterial count of the freeze-dried bacterial powder stored at different temperatures over time was determined. The results are shown in FIG. Figure 2 As shown in Table 11, when freeze-dried bacterial powder was stored at 4°C, the number of viable bacteria could increase from 7.27×10 10 cfu / g steadily increased to 1.15×10 11 cfu / g, which increased by 57.5%, so 4℃ was selected as the optimal storage condition for freeze-dried bacterial powder.

[0102] Table 11 Changes in viable bacterial count (CFU / g) of freeze-dried bacterial powder at different temperatures with storage time

[0103]

[0104] Example 5: Treatment effect of freeze-dried bacterial powder on actual algal bloom water

[0105] Algae removal experiments were conducted on algae outbreak water bodies collected from actual rivers. The algae in the water included cyanobacteria such as Microcystis aeruginosa, Oscillatoria, and Phytosphaera. The initial chlorophyll a concentration of the algae solution was measured to be 0.368 mg / L. A freeze-dried bacterial agent was prepared according to the method in Example 4. The freeze-dried bacterial powder was dissolved in water at a ratio of 1g:50mL. After activation and recovery in a constant temperature shaker at 30℃ for 30 minutes, the mixture was diluted with 1g / mL water and then stirred at 1g / mL. 3 The dosage of was added to the algae solution, and sterile water was added to the control group. The chlorophyll a concentration was measured again after 48 hours. Figure 3 As shown, compared with the algae solution of the control group on the left, the transparency of the algae solution of the experimental group on the right increased after treatment, the algae turned yellow and sank to the bottom, and the chlorophyll a was reduced to 0.126 mg / L after measurement.

[0106] Example 6: Application of freeze-dried bacterial powder in suppressing algae outbreaks

[0107] Water from actual rivers was collected. The algae in the water included cyanobacteria such as Microcystis aeruginosa, Oscillatoria, and Thread-like algae. Nutrients were added exogenously to make the water eutrophic, and freeze-dried bacterial powder was added to conduct an experiment to inhibit algae outbreaks. 10L of water was added to a 20L bucket. The initial water quality of the experiment was: chlorophyll a: 1.36μg / L, total nitrogen (TN): 4.54mg / L, ammonia nitrogen: 3.305mg / L, and total phosphorus (TP): 0.505mg / L. Algae-lysing bacteria were added to the experimental group at 10 am every day. The freeze-dried bacterial powder was dissolved in water at a ratio of 1g:30mL. After activation and recovery in a constant temperature shaker at 30℃ for 30min, it was diluted at 0.1g / m 3 The dosage of 200mg was added to the algae solution, and sterile water was added to the control group. Water samples were taken every 2 days to monitor the water quality data. The monitoring results are as follows: Figure 4 As shown in the figure, the chlorophyll a concentration in the control group continued to increase to 35.2 μg / L, while the chlorophyll a concentration in the experimental group with freeze-dried bacterial powder remained stable and no algae bloom occurred.

[0108] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A strain of Bacillus alginolyticus ( Brevibacillus laterosporus ) RZ1, characterized in that, The deposit number is CCTCC NO: M2024725.

2. A Bacillus algae-lyticus agent, characterized in that: comprising the Bacillus alginolyticus according to claim 1 ( Brevibacillus laterosporus )RZ1.

3. The algae-lytic Bacillus agent according to claim 2, characterized in that The algae-lytic Bacillus agent is freeze-dried bacterial powder; The freeze-dried bacterial powder comprises the alginolytic Bacillus ( Brevibacillus laterosporus ) The fermentation culture of RZ1 is mixed with raw materials including a lyophilization protectant and then vacuum freeze-dried to prepare the product.

4. The algae-lytic Bacillus agent according to claim 3, characterized in that The fermentation culture is composed of the Bacillus alginolyticus ( Brevibacillus laterosporus ) RZ1 was cultured in liquid and then centrifuged and the supernatant was removed to obtain bacterial sludge.

5. The algae-lytic Bacillus agent according to claim 3, characterized in that The freeze-drying protective agent includes skim milk powder, sucrose and mannitol in a mass ratio of 5: (5-6):

3.

6. The Bacillus alginolyticus agent according to any one of claims 3 to 5, characterized in that The freeze-dried protective agent is an aqueous solution, wherein the mass concentration of the effective protective component is 13% to 14%; the effective protective component is skim milk powder, sucrose and mannitol; The pH of the lyophilization protectant is 6.5-7.5; The ratio of the volume of the lyoprotectant to the mass of the fermentation culture is 2-2.5 mL:1 g.

7. The algae-lytic Bacillus agent according to claim 6, characterized in that The pH of the lyoprotectant is 7.

8. The method for preparing the Bacillus alginolyticus agent according to any one of claims 2 to 7, characterized in that: Including steps: (1) The activated Bacillus algilyticus ( Brevibacillus laterosporus ) RZ1 was transferred to LB liquid medium and cultured at a constant temperature of 28-32°C with shaking to obtain fermentation broth; (2) centrifuging the fermentation liquid obtained in step (1) to collect the bacterial sludge; (3) mixing the bacterial sludge obtained in step (2) with a freeze-dried protective agent, and resuspending the bacterial cells to obtain a bacterial suspension; (4) The bacterial suspension obtained in step (3) is pre-frozen and then vacuum-freeze-dried to obtain freeze-dried bacterial powder.

9. The preparation method according to claim 8, characterized in that In step (1): The activated Bacillus alginolyticus ( Brevibacillus laterosporus ) RZ1 was transferred to LB liquid medium at an inoculum rate of 0.1% to 1%; The rotation speed of constant temperature shaking culture is 150~180 rpm; The constant temperature shaking culture time is 48~72 hours; In step (2): The centrifugation temperature was 4°C; The centrifugal speed is 8000 r / min; The centrifugation time was 10 min; In step (4): The pre-freezing temperature is -20℃; The pre-freezing time is 5~12 hours.

10. The Bacillus alginolyticus according to claim 1 ( Brevibacillus laterosporus ) Use of the Bacillus alginolyticus agent according to RZ1 or any one of claims 2 to 7 in inhibiting the growth and reproduction of algae, characterized in that: The algae are Microcystis aeruginosa, Oscillatoria and Planctomyces.

11. A method for preventing algae blooms in water, characterized in that: include: The alginolytic Bacillus sp. ( Brevibacillus laterosporus ) RZ1 and / or the Bacillus alginolyticus agent according to any one of claims 2 to 7 is added to the water body before the algae outbreak to inhibit the growth and reproduction of algae and prevent the algae outbreak; the algae are Microcystis aeruginosa, Oscillatoria and Planctomyces.

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