Composite bacillus preparation for degrading nitrite and application thereof

Abstract

The invention discloses a composite bacillus preparation for degrading nitrite and application of the composite bacillus preparation, and belongs to the technical field of biology. The composite bacillus preparation provided by the invention comprises the following components: bacillus YHBLS-1 and bacillus licheniformis YHD2, the preservation number of the bacillus YHBLS-1 is CCTCC NO: M20251258, the preservation number of the bacillus licheniformis YHD2 is CCTCC NO: M 20251259, the two strains are respectively fermented to prepare bacterial powder of a single strain, then the bacterial powder is compounded to obtain the composite bacillus preparation, and the composite bacillus preparation can be used for preparing the composite bacillus preparation. Compared with a single strain, the composite bacillus preparation can efficiently degrade nitrite and is remarkable in effect, the concentration of the nitrite in the sewage is reduced to 0.15 mg / L from 2.12 mg / L before putting within 48 hours when the composite bacillus preparation is put into aquaculture sewage with the adding amount of 5g / m < 3 >, the effect is remarkable, and the nitrite can be effectively degraded. The method is of great significance to purification and improvement of nitrite pollution in an aquaculture water environment, and has an application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound Bacillus preparation for degrading nitrite and its application. Background Technology

[0002] With the continuous advancement of aquaculture technology, intensive aquaculture has developed rapidly, leading to year-on-year increases in aquaculture output. However, along with this increase in output, high-density intensive aquaculture has also significantly impacted the carrying capacity of the aquatic environment, resulting in a series of environmental problems. Especially in the later stages of aquaculture, the large-scale input of high-protein feed causes the accumulation of uneaten feed and excrement in the water, leading to an increase in nitrogenous organic matter. This hinders the nitrification process of nitrogen in the water, resulting in elevated levels of ammonia nitrogen and nitrite in the aquaculture water. However, due to the rapid conversion rate of ammonia nitrogen, the nitrite problem is the most prominent.

[0003] Nitrites can be absorbed by the gill epithelium of aquatic animals and enter the bloodstream through the gill filaments, affecting the blood's oxygen-carrying capacity and causing tissue hypoxia. This leads to respiratory distress, restlessness, and in severe cases, dysfunction of certain metabolic organs, physical decline, and increased susceptibility to disease and death. Furthermore, the accumulation of nitrites in water bodies can cause eutrophication, deterioration of water quality and bottom sediment, thereby disrupting the balance of the aquatic ecosystem. Therefore, timely and effective removal of nitrites from aquaculture water bodies is crucial for improving the economic benefits of the aquaculture industry, restoring the balance of the aquatic environment, and promoting the development of the aquaculture industry.

[0004] Currently, the main methods for treating nitrite in the industry include physical, chemical, and microbial methods. Physical methods primarily utilize substances with high adsorption capacity, such as activated carbon and zeolite powder, to adsorb nitrite, or involve directly replacing some water with healthy water. Chemical methods mainly involve applying inorganic fertilizers to the water to promote algae growth and thus purify the water, or using strong oxidants to degrade nitrite levels. However, physical and chemical methods are costly, time-consuming, and incomplete in degradation. Microbial methods, on the other hand, have become a research hotspot in recent years, offering advantages such as low cost, rapid processing, no pollution, and high safety. They can provide long-term, safe self-repair for aquaculture water, achieving a harmless transformation of nitrite and restoring nitrogen cycle balance.

[0005] Bacillus is ubiquitous in the environment and is one of the most commonly used probiotics in aquaculture. Due to its strong reproductive capacity, resilience, and efficient secretion of extracellular enzymes, it can rapidly multiply in the aquatic environment, degrading feed residues and metabolic waste in ponds, thereby improving aquaculture water quality. Currently, there are increasingly more microbial agents containing Bacillus, but they generally suffer from problems such as limited strain composition, low nitrite degradation concentration, and long degradation cycles. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a compound Bacillus preparation for degrading nitrite and its application. The compound Bacillus preparation can efficiently degrade nitrite, showing significantly better results than single strains, and can be widely used in aquaculture, with great market potential.

[0007] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides a compound Bacillus preparation comprising Bacillus YHBLS-1 and Bacillus licheniformis YHD2. The preservation number of Bacillus licheniformis YHBLS-1 is CCTCC NO: M20251258, and the preservation number of Bacillus licheniformis YHD2 is CCTCC NO: M 20251259.

[0008] Furthermore, the Bacillus belye Bacillus velezensis The 16S rDNA sequence of YHBLS-1 is shown in SEQ ID NO.1. Furthermore, the Bacillus licheniformis Bacillus licheniformis The 16S rDNA sequence of YHD2 is shown in SEQ ID NO.2. This invention also provides a method for preparing a compound Bacillus preparation, the preparation method specifically including the following operations: (1) Bacillus belyssus YHBLS-1 and Bacillus licheniformis YHD2 were activated and cultured in culture medium to obtain activated bacterial solutions of the corresponding strains; (2) Inoculate the activated bacterial solution into the fermenter and incubate at a constant temperature to obtain the fermentation broth of the corresponding strain; (3) Centrifuge the fermentation broth separately, discard the supernatant, and obtain the bacterial sludge precipitate of the corresponding strain; (4) The bacterial sludge sediment was washed with sterile water and resuspended. Heat-resistant protectant was added to each and mixed evenly. Then, it was spray-dried to obtain Bacillus vesiculosus YHBLS-1 bacterial powder and Bacillus licheniformis YHD2 bacterial powder. (5) Mix Bacillus beryl YHBLS-1 powder and Bacillus licheniformis YHD2 powder to prepare a compound Bacillus preparation.

[0009] Furthermore, in step (1), the culture medium is LB medium, and the culture time is 20-26 hours.

[0010] Furthermore, in step (2), the inoculation amount of activated bacterial solution is 3-8%; the constant temperature culture conditions are: temperature 35-40℃, time 25-30h, and fermentation tank rotation speed 100-150r / min.

[0011] Furthermore, the centrifugation operation in step (3) is specifically as follows: the rotation speed of the fermentation bacteria is 3500-4500 r / min, and the centrifugation time is 10-20 min.

[0012] Furthermore, the components and contents of the heat-resistant protective agent are 10-20 g / L of skim milk powder, 10-20 g / L of maltodextrin, and 10-20 g / L of glucose.

[0013] Furthermore, the mass ratio of the Bacillus vesiculosus YHBLS-1 powder and Bacillus licheniformis YHD2 powder is 1:0.5-2.

[0014] Furthermore, the viable count of the *Bacillus vesiculosus* YHBLS-1 powder or *Bacillus licheniformis* YHD2 powder is at least 10. 9 CFU / g.

[0015] This invention also provides the application of the aforementioned compound Bacillus preparation in improving nitrite pollution in aquaculture water environments.

[0016] Furthermore, the dosage of the compound Bacillus preparation used in the aquaculture environment is 3-10 g / m³. 3 .

[0017] Furthermore, the compound Bacillus preparation is fully diluted with actual aquaculture water and then evenly sprayed into the aquaculture environment.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a compound Bacillus preparation for degrading nitrite, which can be obtained by screening Bacillus belye. Bacillus velezensis YHBLS-1 and Bacillus licheniformis Bacillus licheniformis YHD2 is produced by spray drying after fermentation. It has a clear composition, is simple to prepare, and is convenient to use. It also efficiently degrades nitrite content in aquaculture water environments. Combined with practical application verification, after 48 hours of use of the compound Bacillus preparation, the nitrite concentration in the experimental group decreased from 2.12 mg / L before application to 0.15 mg / L. Compared with single strains, the compound Bacillus is more efficient and effective in degrading nitrite. At the same time, it does not pose any safety risks to aquatic products or environmental safety issues when treating nitrite in water bodies, and has broad application prospects in aquaculture wastewater treatment. Attached Figure Description

[0019] Figure 1 The degradation rates of 0.2 g / L NaNO2 by five strains at 24 h and 48 h were calculated. Figure 2 The colony characteristics of Bacillus belyssus YHBLS-1; Figure 3 The colony characteristics of Bacillus licheniformis YHD2; Figure 4 It is Bacillus baileyi YHBLS-1 bacterial powder; Figure 5 It is Bacillus licheniformis YHD2 bacterial powder; Figure 6 The degradation rates of 0.5 g / L NaNO2 by single Bacillus and combined Bacillus were measured at 24 h and 48 h. Figure 7 The effect of compound Bacillus preparation on the treatment of aquaculture wastewater. Detailed Implementation

[0020] To further illustrate the technical means and effects of this invention, the invention will be further described below in conjunction with the technical solutions, accompanying drawings, and application examples. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

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

[0022] Example 1: Isolation, screening, and identification of bacterial strains (1) Enrichment of strains Black mud was collected from the bottom of shrimp ponds in Tongzhou Bay, Rudong County, Nantong City using a mud collector. The mud was placed in sterile reagent bottles, sealed, and stored at low temperature before being transported back to the laboratory for bacterial enrichment. Under sterile conditions, 5g of sample was weighed from the black mud and added to 95mL of sterile water. The mixture was shaken at 37℃ and 200rpm for 30 minutes to ensure thorough mixing. 5mL of the mixed solution was added to 95mL of liquid tryptic soy broth (purchased from Haibo Biotechnology) and incubated at 37℃ and 200rpm for 24 hours. The previously inoculated culture solution was then inoculated into liquid tryptic soy broth at a 5% inoculum level, and this enrichment process was repeated three times.

[0023] (2) Separation and purification Dilute the enrichment culture medium sequentially to 10 with sterile water. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10-7 10 -8 8 gradients, each absorbing 10 -6 10 -7 10 -8 Three gradient bacterial suspensions (100 μL each) were spread onto solid tryptic soy agar plates and incubated at 37°C for 24–48 h. Each group was divided into three replicates. Single colonies with different morphologies capable of spore production were selected from the plates and streaked continuously onto fresh solid tryptic soy agar plates 3–5 times until purified Bacillus strains with essentially the same morphology were obtained.

[0024] (3) Strain screening The purified Bacillus strain was inoculated into LB medium and activated at 37℃ and 200 rpm for 24 h. Then, 5% of the activated bacterial solution was inoculated into selection medium supplemented with 0.2 g / L NaNO2 (purchased from MACKLIN). The control group was not inoculated with the strain. The culture was incubated at 37℃ and 200 rpm for 48 h. Samples were taken every 24 h during this period, and the nitrite concentration of different Bacillus bacterial solutions was determined using the naphthylethylenediamine hydrochloride method (referring to GB / T7493-1987, Determination of Nitrite Nitrogen in Water Quality). The nitrite degradation rate was calculated. Two Bacillus strains, C1 and C3, were screened out, demonstrating a degradation rate of over 90% for 0.2 g / L nitrite after 48 h. Figure 1 ).

[0025] Screening medium: 10g peptone, 10g NaCl, 5g yeast extract, 1L purified water, sterilized at 121℃ for 30min. After cooling, add 0.2g NaNO2.

[0026] (4) Strain identification Purified Bacillus strains C1 and C3 were inoculated onto LB broth for activation and culture. Bacterial DNA was extracted using a bacterial genome extraction kit (Beijing Tiangen), and its 16S rDNA sequence was amplified. Universal bacterial primers were used, with sequences 27F: 5'-AGAGTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction mixture (25 μL) contained 21 μL of PCR Mixture, 1 μL each of forward and reverse primers, and 2 μL of DNA template. The PCR amplification program was set as follows: 96 °C pre-denaturation for 5 min, 96 °C denaturation for 20 s, 57 °C annealing for 30 s, 72 °C extension for 30 s, 35 cycles; final extension at 72 °C for 10 min. The PCR products of strains C1 and C3 were sent to Qingdao Platinum Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of C1 is shown in SEQ ID NO.1, and the sequence of C3 is shown in SEQ ID NO.2.

[0027] The sequence determination results were compared and analyzed in the NCBI database, and phylogenetic analysis was performed using MEGA 12.0 software. The results showed that the screened bacterium C1 was named *Bacillus belyesense* YHBLS-1 (…). Figure 2 ), categorized and named Bacillus velezensis The selected bacterium C3 was named Bacillus licheniformis YHD2 ( Figure 3 Its classification is named Bacillus licheniformis .

[0028] (5) Preservation of strains The selected *Bacillus belyes* strain YHBLS-1 was deposited at the China Center for Type Culture Collection (CCCHC), Wuhan University, Wuhan, China, on June 4, 2025. Bacillus velezensis The accession number for YHBLS-1 is CCTCC NO: M 20251258.

[0029] The selected Bacillus licheniformis strain YHD2 was deposited at the China Center for Type Culture Collection (CCCHC), Wuhan University, Wuhan, China, on June 4, 2025. Bacillus licheniformis YHD2 has the accession number CCTCC NO: M 20251259.

[0030] Example 2: Preparation and efficacy testing of solid powder of compound probiotics Bacillus belyssus YHBLS-1 and Bacillus licheniformis YHD2 were activated and cultured in LB medium for 24 h. Then, the activated bacterial solutions were inoculated into fermenters at a 5% inoculum rate and cultured at 37℃ and 120 rpm for 26 h. The fermentation broth was centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the bacterial sludge precipitate was washed with sterile water and resuspended. A heat protectant (10 g / L skim milk powder, 10 g / L maltodextrin, 10 g / L glucose) was added and mixed thoroughly. The mixture was then spray-dried to obtain Bacillus belyssus YHBLS-1 bacterial powder. Figure 4 ) and Bacillus licheniformis YHD2 powder ( Figure 5 ).

[0031] Accurately weigh 1.00 g of spray-dried Bacillus belyeis YHBLS-1 and Bacillus licheniformis YHD2 bacterial powders, and add sterile water to a final volume of 10 mL. Viable cell counts were then performed using the dilution plate method. The results showed that the viable cell counts of Bacillus belyeis YHBLS-1 and Bacillus licheniformis YHD2 bacterial powders reached 1.75 × 10⁻⁶. 9 CFU / g and 1.12×10 9 CFU / g. A compound Bacillus preparation, a solid powder, was obtained by mixing Bacillus beryl YHBLS-1 and Bacillus licheniformis YHD2 in a 1:1 ratio.

[0032] Bacillus belyssus YHBLS-1 powder, Bacillus licheniformis YHD2 powder, and a compound Bacillus preparation were diluted 100-fold with sterile water. The diluted bacterial solutions were then inoculated into selection medium supplemented with 0.5 g / L NaNO2 at a 5% inoculum size and cultured at 37℃ and 200 rpm for 48 h. Samples were taken every 24 h, and the nitrite concentration of the bacterial solutions was determined using the naphthylethylenediamine hydrochloride method to compare the nitrite degradation effects of single and compound Bacillus preparations. The results showed that, with the same inoculum size, the compound Bacillus preparation exhibited better nitrite degradation than the single Bacillus preparation. Figure 6 ).

[0033] Example 3: Test on the treatment effect of compound Bacillus preparation on aquaculture wastewater An experiment was conducted using small-scale shrimp farming ponds in Tongzhou Bay, Rudong County, Nantong City, which were found to be contaminated with nitrite. The nitrite concentrations in the two selected ponds were 2.12 mg / L and 1.82 mg / L, respectively. The pond with a nitrite concentration of 2.12 mg / L was designated as the experimental group, and a compound Bacillus preparation was added to it at a dosage of 5 g / m³. 3For water bodies, the method of application is to dilute the bacterial agent with 10 times the amount of pond water and then evenly sprinkle it. A fishpond with a concentration of 1.82 mg / L served as the control group, receiving no treatment. After 48 hours, the nitrite concentration in the experimental group decreased from 2.12 mg / L before application to 0.15 mg / L, while the nitrite concentration in the control group increased from 1.82 mg / L to 2.06 mg / L. Figure 7 This indicates that the compound Bacillus preparation can promote the degradation of pollutants in water and ultimately achieve the purpose of water purification.

[0034] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A compound Bacillus preparation, characterized in that, The compound Bacillus preparation contains Bacillus YHBLS-1 and Bacillus licheniformis YHD2. The preservation number of Bacillus licheniformis YHBLS-1 is CCTCC NO: M 20251258, and the preservation number of Bacillus licheniformis YHD2 is CCTCC NO: M 20251259.

2. A method for preparing a compound Bacillus preparation, characterized in that the preparation method specifically includes the following operations: (1) Bacillus belyssus YHBLS-1 and Bacillus licheniformis YHD2 were activated and cultured in culture medium to obtain activated bacterial solutions of the corresponding strains; (2) Inoculate the activated bacterial solution into the fermenter and incubate at a constant temperature to obtain the fermentation broth of the corresponding strain; (3) Centrifuge the fermentation broth separately, discard the supernatant, and obtain the bacterial sludge precipitate of the corresponding strain; (4) The bacterial sludge sediment was washed with sterile water and resuspended. Heat-resistant protectant was added to each and mixed evenly. Then, it was spray-dried to obtain Bacillus vesiculosus YHBLS-1 bacterial powder and Bacillus licheniformis YHD2 bacterial powder. (5) Mix Bacillus beryl YHBLS-1 powder and Bacillus licheniformis YHD2 powder to prepare a compound Bacillus preparation.

3. The preparation method according to claim 2, characterized in that, The culture medium used in step (1) is LB medium, and the culture time is 20-26 hours.

4. The preparation method according to claim 2, characterized in that, In step (2), the inoculation amount of activated bacterial solution is 3-8%; the constant temperature culture conditions are: temperature 35-40℃, time 25-30h, and fermentation tank rotation speed 100-150r / min.

5. The preparation method according to claim 2, characterized in that, The heat-resistant protective agent consists of 10-20 g / L skim milk powder, 10-20 g / L maltodextrin, and 10-20 g / L glucose.

6. The preparation method according to claim 2, characterized in that, The mass ratio of the Bacillus beryl YHBLS-1 powder and Bacillus licheniformis YHD2 powder is 1:0.5-2.

7. The preparation method according to any one of claims 2-6, characterized in that, The viable count of the *Bacillus vesiculosus* YHBLS-1 powder is at least 1 × 10⁻⁶. 9 The CFU / g of Bacillus licheniformis YHD2 bacterial powder must have a viable count of at least 1×10⁻⁶. 9 CFU / g.

8. The application of the compound Bacillus preparation according to claim 1 in improving nitrite pollution in aquaculture water environments.

9. The application according to claim 8, characterized in that, The dosage of the compound Bacillus preparation used in the aquaculture environment is 3-10 g / m³. 3 .

10. The application according to claim 8, characterized in that, The compound Bacillus preparation is fully diluted in the actual aquaculture water and then evenly sprinkled into the aquaculture environment.

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