Microbial preparation obtained by fermenting bacillus pumilus S3 as well as preparation method and application of microbial preparation
By fermenting Bacillus pumilus S3 to prepare microbial preparations and utilizing its enzyme-producing and surfactant properties, the foam problem in aquaculture was solved, the foam was effectively eliminated and the water quality was improved, and secondary pollution from chemical synthetic agents was avoided.
Patent Information
- Application Number
- CN202510656446.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-21
- Publication Date
- 2025-09-12
AI Technical Summary
The foam problem in aquaculture leads to a decrease in dissolved oxygen in the water, affecting the survival of aquatic organisms. Existing chemical synthetic surfactants for eliminating foam pose a risk of secondary pollution.
Microbial preparations are prepared by fermentation with Bacillus pumilus S3 and made into liquid and solid dosage forms. Its extracellular protease and surfactant production properties are utilized to degrade foam organic matter and promote the growth of microalgae, thereby improving water quality.
It can effectively eliminate foam on the surface of water, promote the growth of microalgae, improve water quality, and avoid secondary pollution from chemical synthetic agents. It has the advantages of rapid reproduction and decomposition of organic matter, especially in low temperature seasons.
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Figure CN120624263A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a microbial preparation obtained by fermenting Bacillus pumilus S3, and a preparation method and application thereof. Background Art
[0002] Bacillus pumilus is a species of the genus Bacillus. It has been found in many extreme environments, and its spores are more common in soil than those of Bacillus subtilis. Bacillus pumilus secretes highly active enzymes such as cellulases, lipases, xylanases, and pectate lyases, which facilitate the degradation of macromolecules. It also produces antagonistic substances such as antibiotics and antimicrobial proteins, exhibiting a broad range of antimicrobial effects against a wide range of pathogens. Numerous studies have reported on Bacillus pumilus's antibacterial properties and its ability to degrade organic matter, ammonia nitrogen, and nitrite nitrogen in water.
[0003] Foam in aquaculture is often formed by organic particles such as leftover bait, feces, and algae secretions. These substances form a stable air-liquid interface film on the water surface, hindering oxygen entry into the water and causing a decrease in dissolved oxygen. This affects the breathing and survival of aquatic organisms, and sometimes overflows from the aquaculture pond, hindering aquaculture management. The development of microbial agents with biosurfactant properties is crucial for improving the foam problem in high-density aquaculture, eliminating the secondary pollution and food safety issues associated with using chemical synthetic surfactants to eliminate foam. Summary of the Invention
[0004] The present invention provides a microbial preparation obtained by fermenting Bacillus pumilus S3, as well as a preparation method and application thereof. After fermentation and drying, the Bacillus pumilus is made into liquid and solid microbial preparations. The microbial preparation can eliminate foam on the surface of aquaculture water bodies, and rapidly reproduce and occupy space in water bodies in low temperature seasons, decompose organic matter, promote the growth of microalgae, and play a role in assisting water fertilization and algae cultivation.
[0005] In order to achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions:
[0006] The present invention provides a microbial preparation obtained by fermenting Bacillus pumilus S3. The microbial preparation is prepared by fermenting Bacillus pumilus S3. The microbial preparation includes an S3 liquid microbial preparation and an S3 solid microbial preparation. The S3 liquid microbial preparation contains 2.0-3.0×10 10 CFU / g, the viable bacteria count of the S3 solid microbial preparation is 1.0×10 11 CFU / g~1.2×10 11 CFU / g.
[0007] Furthermore, the deposit number of the Bacillus pumilus S3 is CGMCC No.9161, and its 16S rRNA sequence is shown as SEQ ID No.1.
[0008] Furthermore, the Bacillus pumilus S3 is Gram-positive, forming colonies on nutrient agar plates with a diameter of approximately 2 mm, smooth edges, wrinkled centers, and a milky white color. The bacteria are rod-shaped, with oval, mesozoic spores, and exhibit strong antagonistic activity against Vibrio parahaemolyticus.
[0009] Furthermore, the Bacillus pumilus S3 has a growth-promoting effect on Chlorella.
[0010] Furthermore, the Bacillus pumilus S3 promotes the growth rate of Chlorella by more than 400%.
[0011] Furthermore, the ability of the strains obtained in the experiment to promote the growth of Chlorella is as follows: Bacillus pumilus S3 > Bacillus Velezii > Bacillus licheniformis > Bacillus subtilis subsp. subtilis > Bacillus subtilis deserticola.
[0012] Furthermore, the Bacillus pumilus S3 can adapt to growth at 15°C.
[0013] Furthermore, the growth ability of the strains obtained in the experiment at 15°C was as follows: Bacillus pumilus S3 > Bacillus velezensis > Bacillus licheniformis > Bacillus subtilis subsp. deserticola > Bacillus subtilis subsp. subtilis.
[0014] Furthermore, the Bacillus pumilus S3 has the ability to produce extracellular protease and has significant oil film removal ability.
[0015] The present invention also provides a method for preparing a microbial preparation obtained by fermentation of Bacillus pumilus S3, which specifically comprises the following steps:
[0016] (1) Seed solution preparation: Using Bacillus pumilus S3 as the strain, pick a single colony and culture it in a seed culture medium to obtain Bacillus pumilus S3 seed solution;
[0017] (2) Deep fermentation: inoculating the Bacillus pumilus S3 seed liquid into a fermentation medium for cultivation, and the resulting fermentation liquid is an S3 liquid microbial preparation;
[0018] (3) Drying and solidification: Light calcium carbonate is added to the Bacillus pumilus S3 seed liquid, and the mixture is stirred and spray-dried to obtain an S3 solid microbial preparation.
[0019] Furthermore, the components of the seed culture medium in step (1) include 2 g / L to 5 g / L glucose, 3 g / L to 6 g / L sucrose, 8 g / L to 10 g / L tryptone, 3 g / L to 6 g / L yeast extract, and 5 g / L to 11 g / L NaCl. The culture conditions are a temperature of 28°C to 32°C, a rotation speed of 180 r / min to 230 r / min, a pH of 7.0 to 8.0, and a culture time of 11 to 16 h.
[0020] Furthermore, after optimization, the seed culture medium components of step (1) include 3 g / L glucose, 5 g / L sucrose, 9 g / L tryptone, 5 g / L yeast extract powder, and 10 g / L NaCl, and the culture conditions are temperature 30°C, pH 7.5, rotation speed 220 r / min, and culture time 12 h.
[0021] Furthermore, the components of the fermentation medium in step (2) include 8 g / L to 12 g / L corn starch, 12 g / L to 17 g / L corn steep liquor powder, 10 g / L to 22 g / L soybean meal, 0.4 g / L to 0.6 g / L MgSO4, and 6 g / L to 10 g / L NaCl. The culture conditions are a temperature of 28°C to 32°C, a rotation speed of 180 r / min to 220 r / min, a pH of 7.0 to 8.0, and a culture time of 24 to 36 h.
[0022] Furthermore, after optimization, the components of the fermentation medium in step (2) include 10 g / L corn starch, 16 g / L corn steep liquor powder, 20 g / L soybean meal, 0.5 g / L MgSO4, and 8 g / L NaCl, and the culture conditions are 30°C, pH 7.2, 200 r / min, and the culture time is 26 h.
[0023] Furthermore, in step (2), the Bacillus pumilus S3 seed liquid is inoculated into the fermentation medium at an inoculation rate of 5%-15%.
[0024] Furthermore, after optimization, in step (2), the Bacillus pumilus S3 seed liquid was inoculated into the fermentation medium at an inoculum rate of 10%.
[0025] Furthermore, in step (3), the volume-to-mass ratio of the Bacillus pumilus seed solution to the light calcium carbonate is 8% to 12%.
[0026] Furthermore, the spray drying conditions in step (3) are as follows: an inlet air temperature of 145°C to 150°C and an outlet air temperature of 65°C to 70°C.
[0027] The present invention also provides the use of the microbial preparation in eliminating foam on the surface of aquaculture water and / or assisting in fertilizing water in low temperature seasons.
[0028] Furthermore, the microbial preparation for eliminating foam on the surface of aquaculture water is an S3 liquid microbial preparation, and the dosage range of the S3 liquid microbial preparation is 150-300 mL / m³.
[0029] Furthermore, the microbial preparation used to assist in fertilizing water in low temperature seasons is an S3 solid microbial preparation, and the dosage range of the S3 solid microbial preparation is 50~120 g / mu·m.
[0030] Furthermore, the conditions for auxiliary fertilizer and water in the low temperature season are spring temperature of 15~25℃ and water temperature of 15~18℃.
[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0032] 1. The present invention utilizes the characteristics of Bacillus pumilus S3 in producing high-quality proteases and surfactants. After liquid fermentation, it is used in factory farming. On the one hand, the surfactant produced by Bacillus pumilus S3 is an amphiphilic molecule that can significantly reduce the surface tension of water, thinning and breaking the liquid film, causing the foam to dissipate. On the other hand, Bacillus pumilus S3 can not only secrete surfactants, but also secrete large amounts of enzymes such as proteases and lipases, which directly degrade organic substances such as proteins and lipids in the foam, fundamentally eliminating the substrate for foam formation.
[0033] 2. Bacillus pumilus S3 has a small body and a fast reproduction speed, especially at a low temperature of 15℃, which has obvious reproduction advantages over other Bacillus. It can be used in aquaculture ponds in low temperature seasons such as spring and autumn. It can reproduce rapidly and occupy space in the water body, decompose organic matter, promote the growth of microalgae, and play a role in auxiliary fertilizer water cultivation of algae. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the colony image of Bacillus pumilus S3 on NA medium;
[0035] Figure 2 is the growth curve of Bacillus pumilus S3 at 15°C;
[0036] Figure 3 The turbidity of different strains cultured at 15°C for 24 hours;
[0037] Figure 4 The absorbance at 600 nm of different strains cultured at 15°C for 24 h;
[0038] Figure 5 It is the hydrolysis circle of Bacillus pumilus S3 protease;
[0039] Figure 6 It is the hydrolysis circle of Bacillus pumilus S3 lipase;
[0040] Figure 7 It is the hydrolysis circle of Bacillus pumilus S3 amylase;
[0041] Figure 8 The oil ring expelling ability of Bacillus pumilus S3 was compared with other strains;
[0042] Figure 9 The changes in foam before and after use of Bacillus pumilus S3 liquid microbial preparation;
[0043] Figure 10 The effect of Bacillus pumilus S3 on promoting the growth of Chlorella vulgaris;
[0044] Figure 11 This is the effect of using Bacillus pumilus S3 on water transparency. DETAILED DESCRIPTION
[0045] The technical solution of the present invention is further described in detail with reference to the following specific examples.
[0046] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.
[0047] Bacillus pumilus S3 originates from the shrimp intestine. Its 16S rRNA sequence was sequenced and aligned in Patent 2014106521303 (see SEQ ID No. 1), confirming it as Bacillus pumilus. This strain is Gram-positive and forms colonies approximately 2 mm in diameter on nutrient agar plates, with smooth edges and a wrinkled, milky white center. The bacteria are rod-shaped, with oval, mesozoic spores. It exhibits strong antagonistic activity against Vibrio parahaemolyticus. It was deposited with the China General Microbiology Center (CGMCC) on May 13, 2014, under the accession number CGMCC No. 9161.
[0048] Example 1: Low temperature growth advantage of Bacillus pumilus S3
[0049] Experiment 1: In a clean bench, use a sterile pipette to remove 0.1 mL of Bacillus pumilus S3 from a preserved glycerol tube and inoculate it into a test tube containing 5 mL of sterile LB liquid medium. Place it in a shaker at 37°C and 200 rpm for 12 hours to serve as a seed. Inoculate 3% of the inoculum into 100 mL of sterile LB liquid medium and shake it at 15°C and 200 rpm for 80 hours. Take appropriate samples every 8 hours, dilute them to an appropriate multiple, and measure the absorbance OD at 600 nm using a spectrophotometer. 600, control the absorbance between 0.1-0.6, and use LB liquid culture medium with the same dilution multiple as a control. 600 Draw the growth curve of S3 at 15℃ on the vertical axis. Draw the growth curves of Bacillus velez, Bacillus subtilis subsp. deserticola, Bacillus subtilis subsp. subtilis, and Bacillus licheniformis at 15℃ under the same conditions.
[0050] The LB liquid culture medium was prepared as follows: 10 g / L peptone, 5 g / L yeast powder, and 10 g / L sodium chloride.
[0051] The results showed that compared with other spore-forming strains, S3 grew fastest at 15°C and entered the logarithmic growth phase after 8 h, while other strains entered the logarithmic growth phase after 24 h. 600 The value is the highest, indicating that under the same conditions, S3 has the largest biomass compared with the other four Bacillus strains ( Figure 2 ).
[0052] Experiment 2: Pick a loopful of bacterial liquid from a glycerol tube of Bacillus subtilis subsp. deserticola, Bacillus pumilus S3, Bacillus velezensis, Bacillus subtilis subsp. subtilis, and Bacillus licheniformis, streak it on a nutrient agar plate, and incubate it upside down at 37°C in a 37°C incubator for 20-24 hours. Pick a single colony and inoculate it into a test tube containing 5 mL of LB liquid medium. Incubate it in a shaker at 30°C and 200 r / min for 17 hours. Under sterile conditions, take 2 mL of seed liquid and transfer it to a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium. Incubate it in a shaker at 15°C and 200 r / min. Figure 3 Observe the turbidity at different times, take photos and measure OD 600 . Combined Figure 4 The results showed that the turbidity and OD of Bacillus pumilus S3 600 It is significantly higher than other Bacillus, indicating that it has obvious growth advantages at 15℃.
[0053] Example 2: Enzyme production detection of Bacillus pumilus S3
[0054] Use a sterile toothpick to pick up a single S3 colony on the plate and spot it on casein medium, starch medium and fat medium respectively, and culture it in a constant temperature incubator at 37°C for 24 hours. Add an appropriate amount of acidic mercury reagent around the colony on the casein medium to determine the protease production ability, add Lugol's iodine solution to the starch medium and shake it to determine the amylase production ability. The lipase decomposition circle on the fat medium can be directly observed without the need for a color developer. Use a ruler to measure the size of the colony and the diameter of the transparent circle. The ratio of the transparent circle to the colony diameter is used to represent the enzyme activity. The larger the ratio, the greater the activity of the bacteria in producing the enzyme. Bacillus subtilis desert subspecies B2 was used as the control strain.
[0055] (1) Protease
[0056] Casein culture medium: 10g casein, 1g yeast extract, 20g agar, pH 7.0, dilute to 1 L with distilled water, and sterilize at 121°C for 30 minutes. The color developing solution is an acidic mercuric reagent: 15g HgCl2, 20mL concentrated HCl, and distilled water to 100mL. Note: If casein is difficult to dissolve, add some NaOH to make it alkaline. Heat magnetically and stir until completely dissolved, then adjust the pH to 7.0 with dilute hydrochloric acid.
[0057] (2) Amylase
[0058] Starch medium: 2g soluble starch, 10g peptone, 5g beef extract, 5g sodium chloride, 20g agar, pH 7.2, dilute to 1L with distilled water, and sterilize at 121°C for 30min. The color developing solution is Lugol's iodine solution: 1g iodine tablets, 2g potassium iodide, 300ml distilled water (Note: First dissolve the potassium iodide in a small amount of water, then dissolve the iodine tablets in the potassium iodide. Once the iodine is completely dissolved, add enough water). Notes: ① Soluble starch can be dissolved better by first using a small amount of cold water, stirring, and then adding it to boiling water. ② The color of the color developer disappears quickly, so measurements should be taken as soon as possible.
[0059] (3) Lipase
[0060] Lipid culture medium: 10g peptone, 5g yeast extract, 10g NaCl, 2mL tributyrin, 20g agar powder, 1L water, pH 7.5. Lipase hydrolysis zones can be directly observed without the need for a colorimetric reagent.
[0061] The results showed that Bacillus pumilus S3 has a good ability to produce extracellular protease ( Figure 5 ), the lipase production capacity is slightly higher than that of Bacillus subtilis deserticola B2 ( Figure 6 ), but does not produce amylase ( Figure 7 ), Table 1 shows the ratio of hydrolysis zone diameter to colony diameter.
[0062] Table 1 Ratio of 24h hydrolysis zone diameter to colony diameter
[0063]
[0064] Example 3: Evaluation of the oil film removal ability of Bacillus pumilus S3
[0065] A single colony of Bacillus pumilus S3 was picked and placed in the LB liquid medium described in Example 1. The culture was cultured at 30°C and 200 rpm for 24 h, and then centrifuged at 8000 rpm for 5 min. The supernatant was reserved for later use.
[0066] Take 1g of Sudan III dye solution and add it to 100mL of soybean oil. Stir thoroughly to dissolve Sudan III in the soybean oil. Take a 9cm disposable plastic dish and add 20mL of pure water. Pipette 1mL of red-dyed soybean oil and add it vertically to the surface of the water. Let it stand for 2 minutes so that the oil layer is evenly distributed on the surface of the culture dish. Take 20μL of fermentation supernatant and add it to the center of the oil film. If the strain fermentation liquid contains surfactant substances, the central oil film will be squeezed to the surrounding area to form an oil drainage ring. Judging from the oil drainage ring results, compared with ordinary strains, the fermentation supernatant of Bacillus pumilus S3 has a significant oil drainage ability ( Figure 8 ).
[0067] Example 4: Application of Bacillus pumilus S3 in promoting the growth of Chlorella
[0068] A loopful of culture suspension from each glycerol tube of Bacillus subtilis subsp. deserticola, Bacillus subtilis subsp. subtilis, Bacillus licheniformis, Bacillus pumilus S3, and Bacillus velezensis was streaked onto a nutrient agar plate and incubated upside down at 37°C for 20–24 hours. A single colony was transferred to an LB tube and incubated at 37°C and 200 rpm for 16 hours. Then, a 2% inoculum was inoculated into a 100-mL Erlenmeyer flask containing 50 mL of LB medium. Fermentation broths of the different strains were obtained and used for future use.
[0069] Chlorella was inoculated into BG11 medium diluted 6 times at a 4% inoculation rate and cultured until the algal cell density was about 1.3~1.5×10 6 cells / mL. Aliquot the algae solution into sterile test tubes, 10 mL per tube, and inoculate 1% fermentation broth from different strains. After 4 days of incubation in a light incubator, the cell density of the algae solution was determined using a hemocytometer, and the growth rate was calculated. Culture conditions: 12 hours of light at 30°C, 12 hours of darkness at 26°C, and a light intensity of 3000 lx. The BG11 medium composition includes: 1500 mg / L NaNO₃, 6 mg / L citric acid, 40 mg / L K₂HPO₄, 6 mg / L ammonium ferric citrate, 20 mg / L Na₂CO₃, and 51 mg / L A51.
[0070] Growth rate = (final cell density - initial cell density) / initial cell density × 100%.
[0071] The test results showed that the growth rates of Chlorella in the Bacillus subtilis subsp. deserticola, Bacillus subtilis subsp. subtilis, Bacillus licheniformis, Bacillus pumilus S3, and Bacillus velezensis groups were 255%, 285%, 289%, 419%, and 303%, respectively. The growth rate of Chlorella in the control group without probiotics was 81%. This shows that various probiotics in the diluted BG11 medium have a certain growth-promoting effect on the growth of Chlorella. In terms of growth rate, Bacillus pumilus S3 has the best effect ( Figure 10 ).
[0072] Example 5: Application of Bacillus pumilus S3 in eliminating water foam
[0073] The liquid fermentation of Bacillus pumilus S3 is carried out to produce the S3 liquid microbial preparation.
[0074] (1) Seed solution preparation: Using Bacillus pumilus S3 as the strain, single colonies were picked and incubated in a seed culture medium containing 3 g / L glucose, 5 g / L sucrose, 9 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. The culture was incubated at 30°C, pH 7.5, and 220 r / min for 12 h to obtain Bacillus pumilus S3 seed solution.
[0075] (2) Deep fermentation: The seed liquid of step (1) was inoculated at an inoculum rate of 10% into a fermentation medium containing 10 g / L corn starch, 16 g / L corn steep liquor, 20 g / L soybean meal, 0.5 g / L MgSO4, and 8 g / L NaCl. The culture was carried out at 30°C, pH 7.2, and 200 r / min for 26 h to obtain a fermentation liquid of Bacillus pumilus S3, which is the S3 liquid microbial preparation. The viable bacterial count detected by nutrient agar plate was 3.0×10 10 CFU / g.
[0076] Sprinkle 200 mL / m3 of Bacillus pumilus S3 liquid microbial preparation in the factory aquaculture cement pool. 3 , the foam can be observed to be significantly reduced on the same day, and the foam basically disappears within 24 hours ( Figure 9 ).
[0077] Example 6: Effect of Bacillus pumilus S3 in auxiliary pond low temperature fertilization water
[0078] The Bacillus pumilus S3 fermentation broth in this embodiment is the Bacillus pumilus S3 fermentation broth prepared in step (2) of Example 5.
[0079] Light calcium carbonate with a volume-to-mass ratio of 10% was added to the fermentation broth of Bacillus pumilus S3, stirred evenly, and then spray-dried with an inlet air temperature of 145°C to 150°C and an outlet air temperature of 65°C to 70°C. The viable bacterial count was 1.2×1011 CFU / g of Bacillus pumilus S3 solid microbial preparation. A low-temperature fertilization test was conducted in ponds under spring temperatures of 15-25°C and water temperatures of 15-18°C.
[0080] Nine ponds were selected and divided into three groups, with three ponds in each group. In test group 1, S3 solid microbial preparation was sprayed into the pond at a dosage of 100g / mu·m together with fertilizer and water-based organic matter. In test group 2, only fertilizer and water-based organic matter was added. Test group 3 was the control group, without S3 and fertilizer and water-based organic matter. The weather was clear during the test, and the Secchi transparency disk was used to measure the change in water transparency before and 48 hours after the test. The test results showed that the transparency of the three ponds to which S3 solid microbial preparation was added was further reduced than that of the ponds to which only fertilizer and water-based organic matter was added, indicating that the algae concentration was higher, and the S3 solid microbial preparation promoted the growth of algae in the pond ( Figure 11 ).
[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.
Claims
1. A microbial preparation obtained by fermentation of Bacillus pumilus S3, characterized in that: The microbial preparation is prepared by fermenting Bacillus pumilus S3. The microbial preparation includes S3 liquid microbial preparation and S3 solid microbial preparation. The S3 liquid microbial preparation contains 2.0-3.0×10 10 CFU / g, the viable bacteria count of the S3 solid microbial preparation is 1.0×10 11 CFU / g~1.2×10 11 CFU / g.
2. The microbial preparation obtained by fermentation of Bacillus pumilus S3 according to claim 1, characterized in that: The deposit number of the Bacillus pumilus S3 is CGMCC No.9161, and its 16S rRNA sequence is shown as SEQ ID No.
1.
3. The microbial preparation obtained by fermentation of Bacillus pumilus S3 according to claim 1, characterized in that: The Bacillus pumilus S3 has a growth-promoting effect on Chlorella.
4. The method for preparing a microbial preparation obtained by fermentation of Bacillus pumilus S3 according to claim 1, characterized in that: The preparation method specifically comprises the following steps: (1) Seed solution preparation: Using Bacillus pumilus S3 as the strain, pick a single colony and culture it in a seed culture medium to obtain Bacillus pumilus S3 seed solution; (2) Deep fermentation: inoculating the Bacillus pumilus S3 seed liquid into a fermentation medium for cultivation, and the resulting fermentation liquid is an S3 liquid microbial preparation; (3) Drying and solidification: Light calcium carbonate is added to the Bacillus pumilus S3 seed liquid, and the mixture is stirred and spray-dried to obtain an S3 solid microbial preparation.
5. The preparation method according to claim 4, characterized in that The components of the seed culture medium in step (1) include 2 g / L to 5 g / L of glucose, 3 g / L to 6 g / L of sucrose, 8 g / L to 10 g / L of tryptone, 3 g / L to 6 g / L of yeast extract powder, and 5 g / L to 11 g / L of NaCl. The culture conditions are a temperature of 28°C to 32°C, a rotation speed of 180 r / min to 230 r / min, a pH of 7.0 to 8.0, and a culture time of 11 to 16 h.
6. The preparation method according to claim 4, characterized in that The components of the fermentation medium in step (2) include 8 g / L to 12 g / L corn starch, 12 g / L to 17 g / L corn steep liquor powder, 10 g / L to 22 g / L soybean meal, 0.4 g / L to 0.6 g / L MgSO4, and 6 g / L to 10 g / L NaCl. The culture conditions are a temperature of 28°C to 32°C, a rotation speed of 180 r / min to 220 r / min, a pH of 7.0 to 8.0, and a culture time of 24 to 36 h.
7. The preparation method according to claim 4, characterized in that In the step (3), the volume mass ratio of the Bacillus pumilus seed liquid to the light calcium carbonate is 8% to 12%.
8. Use of the microbial preparation according to claim 1 for eliminating foam on the surface of aquaculture water and / or for assisting water fertilization in low temperature seasons.
9. The use according to claim 8, characterized in that The microbial preparation for eliminating foam on the surface of aquaculture water is an S3 liquid microbial preparation, and the dosage range of the S3 liquid microbial preparation is 150-300 mL / m³.
10. The use according to claim 8, characterized in that The microbial preparation used to assist in fertilizing water in low temperature seasons is an S3 solid microbial preparation, and the dosage range of the S3 solid microbial preparation is 50-120 g / mu·m.