Bacillus proteolyticus B1, microbial inoculum and application of bacillus proteolyticus B1 and microbial inoculum in field of biological denitrification

Abstract

The invention belongs to the technical field of denitrifying microorganisms, and particularly relates to bacillus proteolyticus B1, a microbial agent and application of the bacillus proteolyticus B1 in the field of biological denitrification. Aiming at the problem of repairing the bottom mud of the water body, the invention designs that a denitrifying strain is screened from the bottom mud of the eutrophic water body, and the bacillus proteolyticus B1 is provided by qualitative and quantitative analysis on the denitrification capacity of the strain, and the strain has remarkable denitrification performance, can quickly degrade organic nitrogen and inorganic nitrogen in a culture environment, and can be applied to the field of remediation of the bottom mud of the water body. The research and development of microbial agents for biological denitrification are facilitated.

Description

Technical Field

[0001] This invention belongs to the field of denitrifying microbial technology, specifically relating to a strain of protein-hydrolyzing Bacillus B1, the inoculum of this strain, and its application in the field of biological denitrification. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] With rapid economic and social development, it is inevitable that some domestic wastewater will be discharged into rivers and lakes. Simultaneously, the decomposition of organic matter by numerous anaerobic microorganisms in the water releases irritating gases such as hydrogen sulfide and ammonia. Coupled with insufficient hydrodynamic conditions and poor flow in rivers and lakes, eutrophication and other problems arise. Sediments, as an important component of aquatic ecosystems, are crucial storage carriers of nutrients such as nitrogen and phosphorus, and also important mediators for the exchange of matter and energy cycling among aquatic plants and animals. When the input of exogenous pollutants increases, nutrients accumulate in sediments, potentially becoming the dominant factor in internal water pollution. Studies on eutrophication have found that the continuous release of nutrients such as nitrogen and phosphorus from sediments is a major obstacle to pollution control.

[0004] To address the aforementioned nitrogen pollution problem in water bodies, different remediation strategies can be adopted. Physical methods mainly involve river dredging, water diversion and replacement, and removal of some eutrophic sediments, but these methods require significant investment and involve substantial engineering work. Chemical methods primarily involve adding chemical reagents, which can rapidly reduce the concentration of dissolved nitrogen and phosphorus in the water or inhibit the release of pollutants from sediments; they are fast-acting but can easily cause adverse ecological impacts due to reagent residues. Currently, biological methods for water pollution remediation have become a research hotspot. These methods utilize microorganisms to treat nitrogen and phosphorus pollution in water bodies while simultaneously remediating river sediments, further enhancing the water remediation effect.

[0005] Adding bacteria directly to water bodies to treat pollutants is a relatively direct and efficient method of treatment and remediation. The added bacteria are typically genetically engineered or specific domesticated strains. Genetically engineered bacteria achieve specific treatment functions by altering the coding segments of existing genes within the bacteria. Domesticated bacteria are specific species collected from the natural environment, enriched, screened, and isolated. After verifying their treatment effectiveness, they are directly added to polluted water bodies as bacterial agents for reaction. Because native microorganisms in the environment have strong adaptability and the ability to stably degrade specific pollutants, and because the treatment and cultivation of native microorganisms is more convenient and faster than that of genetically engineered bacteria, and poses no potential safety hazards, the screening and isolation of native microorganisms in the environment has received widespread attention from researchers in recent years.

[0006] The advantages of screening denitrifying bacteria from sediment for treating nitrogen pollution and improving the aquatic environment are mainly reflected in its strong targeting, high environmental compatibility, and eco-friendliness. Since denitrifying bacteria are directly isolated from the target sediment, they are naturally adapted to the local sediment's physicochemical properties (such as pH, salinity, and pollutant composition) and microbial community environment. They can efficiently initiate denitrification without additional acclimatization, avoiding problems such as poor adaptability and failure to compete with native microorganisms that may be encountered with exogenous bacterial agents. By utilizing the screened denitrifying bacteria as a basis, novel microbial agents can be constructed that are more adaptable to the local environment and have higher treatment efficiency for nitrogen pollution in local sediment. This method reduces nitrates to nitrogen gas through bioconversion, completely eliminating nitrogen pollution and avoiding secondary pollution (such as residual agents or intermediate products) that may be caused by chemical treatments or sediment structure damage and ecological disturbance caused by physical dredging. Summary of the Invention

[0007] To address the problems of eutrophication in water bodies, the present invention aims to provide a microbial preparation with good denitrification activity. To achieve this objective, the present invention used sediment from a eutrophic riverbed in Jinan as the screening target, identified specific denitrifying strains, and conducted qualitative and quantitative analyses of the strains' denitrification capabilities, obtaining a Bacillus proteolyticus B1 strain with significant denitrification ability, whose NO2... - -N accumulation was 0.037 mg / L, while TN and NO3 levels reached their highest levels. - The removal rates of -N were 93.02% and 99.04%, respectively. This is expected to contribute to the development of microbial agents for the remediation of eutrophic pollution in rivers and provide technical guidance for in-situ remediation technologies for nitrogen and phosphorus polluted rivers.

[0008] Based on the above-mentioned technical effects, the present invention provides the following technical solution: In a first aspect, the present invention provides a strain of Bacillus proteolyticus (Bacillus) Bacillus proteolyticus B1, this strain was deposited on May 23, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC M 20251164.

[0009] The above-mentioned strain was isolated from the sediment of a eutrophic river in Jinan. Sequencing revealed its 16S rRNA sequence, as shown in SEQ ID NO:1. Sequence alignment was performed with that of a reference species. Bacillus proteolyticus The homology of MCCC 1A00365 reached 99.72%, and it was identified as Bacillus proteolyticus. Bacillus proteolyticus It was named Bacillus proteolyticus B1.

[0010] The morphological characteristics of the above-mentioned strains are as follows: Bacterial characteristics: The bacterial cells are straight rods with a slight curvature, and turn purple-red after staining, indicating that they are Gram-negative bacteria; Colony characteristics: Colonies are light yellow, round, moist, with a smooth surface and neat edges.

[0011] The optimal culture medium conditions for the above strains are as follows: Each 1000mL of water contains 4-6 g of yeast extract, 8-12 g of tryptone, 8-12 g of NaCl, and pH=7.0.

[0012] In a second aspect, the present invention provides a microbial agent comprising the *Bacillus proteolyticus* described in the first aspect (…). Bacillus proteolyticus B1 and / or its metabolites and extracts.

[0013] The metabolites refer to the chemical substances produced by the strain during its growth and reproduction, including primary metabolites and secondary metabolites.

[0014] The term "culture" refers to the collective name for viable bacterial strains and their growth vectors obtained through isolation, inoculation, and cultivation under specific culture conditions. These growth vectors include both solid and liquid culture media, and are typically pharmaceutically acceptable excipients or matrices.

[0015] Furthermore, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the source of the pesticide-acceptable excipients, and commercially available products are generally sufficient.

[0016] Furthermore, the matrix is ​​selected from porous materials (such as activated carbon, zeolite, sodium alginate gel), biomass carriers (such as straw), composite corrosion inhibitors (such as polyvinyl alcohol), or special carriers (such as sulfur / iron-based fillers).

[0017] The extract refers to a single or mixed component obtained by separating and concentrating bacteria and / or their growth carriers through physical and chemical methods.

[0018] In a third aspect, the present invention provides the application of the protein-hydrolyzing Bacillus B1 described in the first aspect and the bacterial agent described in the second aspect in the field of biological denitrification.

[0019] The biological denitrification field mentioned in the third aspect above aims to reduce the total nitrogen content in the environment to be treated through the action of microorganisms. The environment to be treated includes, but is not limited to, various aquatic environments, agricultural environments, or industrial scenarios.

[0020] The water environment includes, but is not limited to, industrial wastewater (such as wastewater from chemical plants or fertilizer plants), municipal wastewater, aquaculture wastewater, and natural water bodies (including water and sediment in eutrophic rivers).

[0021] The agricultural environment includes, but is not limited to, soils with excessively high ammonia nitrogen content and saline-alkali soils.

[0022] The industrial scenarios mentioned include high-nitrate wastewater generated from electronics / electroplating.

[0023] The specific methods of application mentioned above include, but are not limited to, any of the following: 1) Used in the preparation of biological denitrification agents; 2) Add the above-mentioned strains, inoculants, or biological agents from 1) to the environment to be treated; 3) Construct a bioreactor in the environment to be treated.

[0024] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a highly efficient denitrifying bacterium—Bacillus proteolyticus (B. proteolyticus). Bacillus proteolyticus B1 strains are effective at removing nitrate nitrogen in eutrophic river environments, exhibiting low nitrate accumulation and minimal growth requirements. Furthermore, these strains can efficiently degrade bottom sediment, effectively reducing sediment volume and organic matter content, thus preventing internal pollution of the water body. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 This is a comparison chart of the denitrification performance of the 25 strains in Example 1; Figure 2 This is a colony diagram of the Bacillus proteinophilus B1 strain in Example 1; Figure 3 This is an optical microscope image of Bacillus proteinophilus B1 in Example 1; Figure 4 This is a developmental tree diagram for the identification of Bacillus proteinophilus B1 in Example 1; The strain preservation information is as follows: Bacillus proteolyticus ( Bacillus proteolyticus strain B1 was deposited at the China Center for Type Culture Collection (CCTCC) on May 23, 2025. The address is: The biological accession number for Wuhan University, Wuhan, China is: CCTCC M 20251164 。 Detailed Implementation

[0027] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0029] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0030] The culture media used in the following examples have the following components: The bromothymol blue (BTB) medium comprises: 1.0 g KNO3, 1.0 g C6H5Na3O2, 1.0 g KH2PO4, 0.05 g FeSO4·7H2O, 0.2 g CaCl2, 1.0 g MgSO4·7H2O, 1 mL 1% bromothymol blue, 1000 mL distilled water, and pH=7.2.

[0031] The denitrification performance testing medium consisted of: CH3COONa 2.56 g, KNO3 0.361 g, MgSO4·7H2O 0.2 g, KH2PO4 1.0 g, K2HPO4 5.0 g, NaCl 0.5 g, trace element solution 1 mL, distilled water 1000 mL, pH=7.4; wherein the trace element solution comprised: EDTA 50 g, CaCl2 5.5 g, ZnSO4 2.2 g, MnCl2·4H2O 5.06 g, FeSO4·7H2O 5.0 g, (NH4)6·MO7O2·4H2O 1.1 g, CuSO4·5H2O 1.57 g, CoCl2·6H2O 1.61 g, distilled water 1000 mL, pH=7.0.

[0032] Example 1 In this embodiment, a strain of Bacillus proteolyticus (Bacillus) is provided. Bacillus proteolyticus B1, the screening method for the strain is as follows: I. Screening and Cultivation of Denitrifying Bacteria 1. Bacterial Enrichment: Weigh 10 g of bottom sediment sample and add it to a conical flask containing 90 mL of sterile water (with a few glass beads to disperse the sediment and ensure bacteria are fully suspended in the water). Incubate at 30 °C, 200 r / min for 3 h, and let stand for 1 h. Take 10 mL of the supernatant and inoculate it into a conical flask containing 90 mL of LB liquid medium. Incubate at 30 °C, 180 r / min for 24 h.

[0033] 2. Initial screening of bacterial strains: Take 1 mL of the enriched bacterial solution and mix it thoroughly in a test tube containing 9 mL of sterile water. Dilute stepwise to a concentration gradient of 10. -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 100 μL of each sample was spread onto bromothymol blue (BTB) solid medium, with each concentration repeated three times, and incubated at 30 °C for 2–3 days.

[0034] After observing the growth of single colonies on the plates, 25 colonies with a distinct blue halo were selected and streaked onto LB agar plates for purification. The plates were then incubated at 30°C until the colonies grew. This process was repeated 2-3 times. The purified cells were then streaked onto LB slant agar, numbered, and stored at 4°C. A total of 25 strains were obtained.

[0035] 3. Strain re-screening: A small amount of the purified strain was inoculated into LB liquid medium and cultured at 30°C and 180 r / min in a constant temperature shaking incubator for 10 h. The activated bacterial solution was centrifuged at 8000 rmp for 5 min, the supernatant was discarded, and the cells were washed with 0.75% sterile physiological saline, repeated twice. The concentrated bacterial cells were resuspended in sterile physiological saline to OD200. 600 The concentration was approximately 0.4% as the seed culture. 1 mL of the seed culture was inoculated into 100 mL of denitrification performance testing medium and cultured at 30°C and 180 r / min for 24 h. Three replicates were performed for each strain.

[0036] After the culture is completed, take 10 mL of bacterial culture, centrifuge at 8000 rpm for 5 min, and take the supernatant to determine the NO3 content in the culture medium. - -N, NO2 - Calculate the concentrations of -N and TN, and calculate NO3. - Removal rates of -N, TN, and NO2 - -N concentration. Selecting those with high TN and NO3- concentrations. - -N had the highest removal rate and NO2 - The strain with the lowest accumulation of -N.

[0037] II. Denitrification Performance Testing of Denitrifying Bacteria After purification, the 25 denitrifying strains obtained from the initial screening were tested for their denitrification performance. The supernatant was used to measure TN and NO3 after culture. - -N and NO2 - -N content, calculate TN and NO3. - -N removal rate and NO2 - -N concentration. Selecting those with high TN and NO3- concentrations. - -N had the highest removal rate and NO2 - The strain with the lowest -N accumulation. A comparison of the denitrification performance of the 25 bacterial strains is shown in Table 1. Figure 1 As shown.

[0038] Table 1. Comparison of denitrification performance of 25 bacterial strains Among them, strain 1 obtained lower NO2. - -N accumulation was 0.036 mg / L, while TN and NO3 levels reached their highest levels. - The removal rates of -N were 92.80% and 98.99%, respectively, making them the optimal denitrifying strains.

[0039] III. Identification of Denitrifying Strains 1. Morphological identification of strains: Observe the culture characteristics of the optimal denitrifying strains, mainly including the color, luster, shape, size, transparency, and edge shape of single colonies on solid culture media. Gram staining is performed on the above strains, and preliminary identification is carried out by observation under an optical microscope.

[0040] Observation of strain B1 revealed that the colonies were light yellow, round, with smooth edges, small and flat, as shown in the image. Figure 2 As shown.

[0041] Gram staining of this strain, observed under an optical microscope, revealed that strain B1 was a straight rod-shaped bacterium with a slight curve, and its purplish-red color after staining indicated it was a Gram-negative bacterium. Figure 3 As shown.

[0042] 2. 16S rRNA identification of the strain: 16S rRNA was identified from fresh agar slant culture medium containing the optimal denitrifying bacterium B1. The phylogenetic tree is shown below. Figure 4 As shown.

[0043] II. Degradation Effect of Bottom Sediment in Black and Odorous Water Bodies The sediment from a eutrophic river in Jinan was selected as the degradation target. Its TS content was 19.6%, VS content was 16.25%, and total nitrogen was 2.52 mg / g. The sediment was laid at the bottom of the reactor with a thickness of 5 cm, and covered with raw water from the same section of the river to a depth of 8 cm. The sediment was degraded at 30℃ and pH 7.0.

[0044] Strains numbered 1, 3, 9, and 25 were cultured to the logarithmic growth phase. Suspensions of these strains were inoculated at a ratio of 1% based on the sediment volume. The control group was inoculated with the same volume of culture medium. Samples were taken at the sediment-water interface on days 0, 2, 4, and 8 for analysis. The results are shown in Table 2 below. Table 2 Degradation effect of bottom sediment in water bodies According to Table 2, in the control group without the addition of exogenous microbial intervention, the degradation rate of TS (total dry matter) was 11.89% and the degradation rate of VS (organic dry matter) was 12.46% on day 8. Based on the results of the above denitrification performance test, strains numbered 1, 3, 9, and 25 had relatively good denitrification performance. However, there were significant differences in the degradation effects of these strains on the bottom sediment of the water body. Among them, strain number 1 had a TS degradation rate of 20.82% and a VS degradation rate of 23.63%, which significantly exceeded the other three strains.

[0045] The above verification results indicate that the protein-hydrolyzing Bacillus (Bacillus) provided by this invention Bacillus proteolyticus B1 has good denitrification and sediment degradation effects, and has good prospects for application in the treatment of black and odorous rivers.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of protein-hydrolyzing Bacillus ( Bacillus proteolyticus B1, this strain was deposited on May 23, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC M 20251164.

2. The protein-hydrolyzing Bacillus as described in claim 1 ( Bacillus proteolyticus B1, characterized in that, Its 16S rRNA sequence is shown in SEQ ID NO:

1.

3. The protein-hydrolyzing Bacillus as described in claim 1 ( Bacillus proteolyticus B1, characterized in that, The morphological characteristics of the strain are as follows: Bacterial characteristics: The bacterial cells are straight rods with a slight curvature, and turn purple-red after staining, indicating that they are Gram-negative bacteria; Colony characteristics: Colonies are light yellow, round, moist, with a smooth surface and neat edges.

4. The protein-hydrolyzing Bacillus as described in claim 1 ( Bacillus proteolyticus B1, characterized in that, The optimal culture medium conditions for this strain are as follows: Each 1000mL of water contains 4-6 g of yeast extract, 8-12 g of tryptone, 8-12 g of NaCl, and pH=7.

0.

5. A microbial agent, characterized in that, Includes the protein-hydrolyzing Bacillus as described in any one of claims 1-4 ( Bacillus proteolyticus Metabolites and extracts of B1 and / or bacteria.

6. The microbial agent as described in claim 5, characterized in that, The metabolites are chemical substances produced by the strain during its growth and reproduction, including primary metabolites and secondary metabolites; The culture refers to the collective term for active bacterial strains and their growth carriers obtained through isolation, inoculation and cultivation under certain culture conditions. The growth carriers include solid culture media and liquid culture media, which are pharmaceutically acceptable excipients or matrices. Furthermore, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents; Furthermore, the matrix is ​​selected from porous materials, biomass carriers, composite corrosion inhibitors, or special carriers; The extract refers to a single or mixed component obtained by separating and concentrating bacteria and / or their growth carriers through physical and chemical methods.

7. The protein-hydrolyzing Bacillus as described in any one of claims 1-4 ( Bacillus proteolyticus B1. The application of the bacterial agent described in claim 5 or 6 in the field of biological denitrification.

8. The application as described in claim 7, characterized in that, In the field of biological denitrification, the environment to be treated by the strains or agents includes, but is not limited to, various aquatic environments, agricultural environments, or industrial scenarios.

9. The application as described in claim 8, characterized in that, The water environment includes, but is not limited to, industrial wastewater, municipal wastewater, aquaculture wastewater, and natural water bodies; The agricultural environment includes, but is not limited to, soils with excessively high ammonia nitrogen content and saline-alkali soils; The industrial scenarios mentioned include, but are not limited to, high-nitrate wastewater generated from electronics / electroplating.

10. The application as described in claim 7, characterized in that, The specific methods of application include, but are not limited to, any of the following: 1) Used in the preparation of biological denitrification agents; 2) Add the above-mentioned strains, inoculants, or biological agents from 1) to the environment to be treated; 3) Construct a bioreactor in the environment to be treated.

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