A composite microbial agent, epiphyte biological filter material and application thereof in removal of inorganic nitrogen and inhibition of growth of harmful bacteria

Abstract

The application discloses a kind of composite microbial inoculant, attached fungus biological filter material and its application in removal inorganic nitrogen, inhibit the growth of harmful bacteria.It is contained Enterobacter cloacae, Baccillus velezensis and tobacco glutamic acid bacillus in the composite microbial inoculant, and the preservation number of Baccillus velezensis JYC1 is CGMCC No.26987.The attached fungus biological filter material includes filter material, and the filter material is loaded with the composite microbial inoculant.The composite microbial inoculant, attached fungus biological filter material of the application can not only efficiently remove ammonia nitrogen, nitrite and other inorganic nitrogen pollutants in water body, but also can significantly inhibit the proliferation of water aquaculture pathogenic bacteria, effectively reconstruct water microecological balance while purifying water quality, greatly improve the safety of cultured organisms.The composite microbial inoculant and attached fungus biological filter material of the application can be widely applied to aquaculture water body, and have important significance for promoting the protection and healthy aquaculture of aquaculture water body.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a composite microbial agent, a microbial filter material, and its application in removing inorganic nitrogen and inhibiting the growth of harmful bacteria. Background Technology

[0002] In aquaculture systems, biological treatment is a crucial step in ensuring water quality safety, typically accomplished by the microbial community attached to filter media and the biofilm it forms. Currently, two common methods are used to establish biological treatment systems: one is to add nutrients such as ammonia nitrogen to the system to enrich naturally occurring microorganisms capable of reducing ammonia nitrogen; the other is to directly add commercially available nitrifying bacteria products to accelerate system formation.

[0003] However, these practices often overlook a significant safety hazard—some microorganisms with highly efficient ammonia nitrogen reduction capabilities are also important pathogens or opportunistic pathogens of aquatic animals. For example, *Acinetobacter baumannii*, *Aeromonas hydrophila* strain gdHMN102, and *Klebsiella pneumoniae* all possess strong ammonia nitrogen and nitrite conversion capabilities and organic matter decomposition abilities, showing considerable application potential in water treatment. However, these strains are also zoonotic or opportunistic pathogens, posing a potential threat to the health of aquatic animals and humans.

[0004] For example, patent application CN108342339B discloses Klebsiella strains and their application in river sewage and rural domestic sewage containing ammonia nitrogen. Among them, Klebsiella strain KSND-3 has a fast growth rate, high cell yield, and low dissolved oxygen concentration requirement. Under the condition of using nitrate as the sole nitrogen source, the total nitrogen removal rate of 105 mg / L initial ammonia nitrogen reaches more than 78% within 48 hours, with no accumulation of nitrite nitrogen. However, this strain is a zoonotic bacterium, not only an important pathogen of community-acquired pneumonia and nosocomial endocarditis, but also a major pathogen causing gill rot in fish. In addition, the capsular polysaccharides it produces make this type of bacterium very easy to colonize on filter media and form biofilms, which also makes it highly resistant to drugs. For example, patent application CN202411728519.1 discloses an aerobic denitrifying bacterium with flocculent characteristics and its application. The aerobic denitrifying bacterium N-1 belongs to *Acinetobacter baumannii*. This strain can denitrify wastewater through aerobic denitrification and has a high denitrification capacity, with a total nitrogen removal rate reaching up to 76.89%. However, this bacterium is a zoonotic bacterium, a known pathogen that can cause acute, high-mortality infectious diseases in fish. It has multiple antibacterial properties and the ability to resist harsh environments, and it easily accumulates in low-temperature nitrification systems. As another example, patent application CN201810097594.0 discloses an aerobic denitrifying bacterium ZJ-17 and its application. ZJ-17 belongs to *Aeromonas hydrophila*. After activation and culture, this strain exhibits excellent denitrification performance and can be used in biological wastewater denitrification processes, achieving a nitrate removal rate of up to 100%. However, this bacterium is an extremely common and seriously harmful opportunistic pathogen in aquatic environments, causing "bacterial septicemia" that can lead to explosive mortality in various aquatic animals.

[0005] In summary, current practices in constructing aquaculture biochemical treatment systems often focus on the nitrogen removal efficiency of microorganisms, neglecting their potential pathogenic risks. Especially during natural acclimatization or enhanced inoculation processes, the accumulation of pathogenic or opportunistic pathogens is difficult to completely avoid, potentially posing a sustained threat to the health of farmed organisms.

[0006] Therefore, how to effectively control the introduction and proliferation of pathogenic microorganisms while ensuring efficient denitrification has become an important issue that urgently needs to be addressed in aquaculture water quality management. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a composite microbial agent, a microbial filter material, and its application in removing inorganic nitrogen and inhibiting the growth of harmful bacteria, so as to solve the problems in the prior art.

[0008] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0009] The first aspect of this invention protects a compound microbial agent comprising Enterobacter lutei, Bacillus nicotine, and Bacillus belyssus JYC1, wherein the Bacillus belyssus JYC1 has the accession number CGMCC No. 26987.

[0010] The second aspect of the present invention protects the method for preparing the composite microbial agent as described above, wherein Enterobacter lutei, Bacillus glutamate of tobacco and Bacillus belye JYC1 are inoculated into a culture medium and cultured to obtain the composite microbial agent.

[0011] A third aspect of the present invention protects a microbial filter media comprising a filter media loaded with the composite microbial agent as described above.

[0012] The fourth aspect of this invention protects the use of the composite microbial agent as described above or the bacterial-coated biological filter material as described above in at least one of the following:

[0013] A1) Removal of inorganic nitrogen;

[0014] A2) Prepare products that have removed inorganic nitrogen;

[0015] A3) Inhibits the growth of harmful bacteria;

[0016] A4) Prepare products that inhibit the growth of harmful bacteria.

[0017] The fifth aspect of the present invention protects a method for safely removing inorganic nitrogen from aquaculture water using microorganisms, comprising: treating the aquaculture water with the composite microbial agent as described above or the microbial filter material as described above.

[0018] As described above, the composite microbial agent, the bacterial-coated biological filter material of the present invention, and their application in removing inorganic nitrogen and inhibiting the growth of harmful bacteria have the following beneficial effects.

[0019] 1) The applicant's research found that *Enterobacter leukemiae*, *Glutamicinus tobacco*, and *Bacillus belyssus* JYC1, when used individually, had no inhibitory effect on harmful bacteria (the inhibition zone diameter was 0 mm for each). However, when the three strains were combined, the interaction between them significantly inhibited the accumulation and proliferation of *Saprolegnia*, *Aeromonas vesiculosus*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*. Furthermore, the inhibitory effect on harmful bacteria was further enhanced by adding *Bacillus amyloliquefaciens* (which also has no inhibitory effect on harmful bacteria when used alone) and / or *Bacillus belyssus* Mardel-KB01 to the three-strain combination system. These results indicate that the composite microbial agent of the present invention significantly broadens the antibacterial spectrum through the synergistic interaction between the strains, demonstrating good application potential.

[0020] 2) The applicant conducted a three-in-three combination study using denitrifying bacteria *Enterobacter leukemia*, *Bacillus amyloliquefaciens*, *Bacillus belyssus* JYC1, and *Bacillus nicotinibacillus* to evaluate the effectiveness of different strain combinations in removing inorganic nitrogen. The results showed that the composite microbial agent formed by *Enterobacter leukemia*, *Bacillus belyssus* JYC1, and *Bacillus nicotinibacillus* exhibited the best denitrification effect. Furthermore, the removal effect of inorganic nitrogen was further enhanced by adding *Bacillus amyloliquefaciens* and / or *Bacillus belyssus* Mardel-KB01 to the three-strain compound system. These results indicate that the composite microbial agent of the present invention significantly improves the removal rate of inorganic nitrogen through synergistic interactions among the strains, demonstrating good application potential.

[0021] 3) Compared with traditional denitrifying bacteria such as Acinetobacter baumannii, Aeromonas hydrophila, and Klebsiella pneumoniae, the strains used in the compound microbial agent of this application are all environmental isolates and are not common opportunistic pathogens in aquaculture. Existing literature and experimental evidence have not shown that the above-mentioned strains are pathogenic to aquatic animals or humans. Therefore, the compound microbial agent of this invention has good biosafety and poses no significant risk of pathogenicity to farmed aquatic animals.

[0022] 4) The strains in the compound microbial agent of this application do not have obvious antagonistic effects, exhibit good growth compatibility, and can coexist and synergistically function in water. Attached Figure Description

[0023] Figure 1 This is a photograph of the plate of strain SX-01 from Example 1 of the present invention.

[0024] Figure 2 The images show the inhibition zones of strain SX-01 from Example 1 of this invention against Saprolegnia, Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae.

[0025] Figure 3 This is a photograph of the plate of strain Mardel-KB01 from Example 2 of the present invention.

[0026] Figure 4 The images show the inhibition zones of strain Mardel-KB01 from Example 2 of this invention against Saprolegnia, Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae.

[0027] Figure 5 This is a photograph of the plate of strain JYC1 from Example 3 of the present invention.

[0028] Figure 6 The images show the inhibition zones of strain JYC1 in Example 3 of this invention against Saprolegnia, Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae.

[0029] Figure 7 The images show actual photographs of the inhibition zones of different combinations of compound bacteria fermentation broths against Saprolegnia in Example 4 of the present invention.

[0030] Figure 8A The images show the inhibition zones of different combinations of compound fermentation broths in Example 4 of this invention against Aeromonas versicolor, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae.

[0031] Figure 8B This is a photograph of a flat panel of the blank control group in Example 4 of the present invention.

[0032] Figure 9 The images show actual photos of TCBS plates in a koi breeding pond with and without the addition of compound microbial agents, as shown in Application Example 1 of this invention.

[0033] Figure 10 The images shown are actual photographs of TCBS flat plates in a fish tank treated with and without bio-filter media, as shown in Application Example 2 of the present invention.

[0034] Figure 11 These are photographs of the inhibition zones of strain CW-01 of the present invention against Aeromonas vernix, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae.

[0035] Figure 12 These are photographs of the inhibition zones of strain ZB of the present invention against Aeromonas vernix, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae. Detailed Implementation

[0036] In aquaculture biochemical treatment systems, some microorganisms with highly efficient ammonia nitrogen reduction capabilities (such as Acinetobacter baumannii, Aeromonas hydrophila, and Klebsiella pneumoniae) exhibit significant nitrogen removal efficiency, but they are also important opportunistic pathogens in aquatic animals, with some strains posing a potential zoonotic risk. For example, Klebsiella pneumoniae can cause gill rot in fish. These pathogenic microorganisms are difficult to completely avoid, and their accumulation process is often accompanied by increased drug resistance, thus posing a continuous threat to the health of farmed organisms. To address these issues, this invention provides a composite microbial agent and a biofilter material with attached bacteria, which not only possesses the ability to efficiently remove inorganic nitrogen but also effectively inhibits the accumulation and proliferation of harmful bacteria, thereby ensuring the biosafety and healthy operation of the aquaculture system.

[0037] The first aspect of this invention protects a compound microbial agent comprising Enterobacter lutei, Bacillus nicotine, and Bacillus belyssus JYC1, wherein the Bacillus belyssus JYC1 has the accession number CGMCC No. 26987.

[0038] The applicant's research found that *Enterobacter leukoi*, *Tobacco glutamate*, and *Bacillus belyssae* JYC1, when used individually, had no inhibitory effect on harmful bacteria. However, when the three were combined, the interactions between the strains significantly inhibited the accumulation and proliferation of *Saprolegnia spp.*, *Aeromonas veronii*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*.

[0039] In addition, the applicant conducted a three-in-three combination study using denitrifying bacteria Enterobacter leucis, Bacillus amyloliquefaciens, Bacillus belyss JYC1, and Bacillus nicotine glutamate to evaluate the effectiveness of different strain combinations in removing inorganic nitrogen. The results showed that the composite microbial agent formed from Enterobacter leucis, Bacillus belyss JYC1, and Bacillus nicotine glutamate exhibited the best denitrification effect.

[0040] In some embodiments, the *Bacillus belyssus* JYC1 was deposited on March 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26987. It was isolated from shrimp ponds and its morphological characteristics are: milky white, wrinkled, and moist. The 16S rDNA sequence includes the sequence shown in SEQ ID No. 3. This strain JYC1 can effectively remove inorganic nitrogen, but it has no inhibitory effect on harmful bacteria such as *Saprolegnia*, *Aeromonas velutipes*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*.

[0041] In some embodiments, the *Enterobacter ludwigii* strain includes, but is not limited to, *Enterobacter ludwigii* CW-01, which was deposited on April 17, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It was isolated from a wastewater treatment plant and its accession number is CGMCC No. 27126. This strain CW-01 can effectively remove inorganic nitrogen, but it has no inhibitory effect on harmful bacteria such as *Saprolegnia*, *Aeromonas vesiculosus*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae* (see [link to documentation]). Figure 11 ).

[0042] In some embodiments, the *Gluconacetobacter nicotianae* strain includes, but is not limited to, *Gluconacetobacter nicotianae* SX-01, which was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 33168. It was isolated from a wastewater treatment plant and its morphological characteristics are: yellow, moist surface, and no wrinkles. The 16S rDNA sequence includes the sequence shown in SEQ ID No. 1. This strain SX-01 can effectively remove inorganic nitrogen, but it has no inhibitory effect on harmful bacteria such as *Saprolegnia*, *Aeromonas vesiculosus*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*.

[0043] In some embodiments, the effective viable count of *Enterobacter leukemia* in the compound microbial agent is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, the effective viable count of the *Tobacco glutamate* bacteria is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, the effective viable count of the *Bacillus belyssus* JYC1 is at least 1 × 10⁻⁶ CFU / mL. 8 CFU / g or 1×10 8 CFU / mL. For example, the effective viable count of Enterobacter leukemia is 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, 4×10 8 CFU / g or 4×10 8 CFU / mL, 8×10 8 CFU / g or 8×10 8 CFU / mL, 2×10 9 CFU / g or 2×10 9 CFU / mL, 5×10 9 CFU / g or 5×10 9 CFU / mL, 8×10 9 CFU / g or 8×10 9 CFU / mL, 1×10 10 CFU / g or 1×10 10 CFU / mL, 5×10 10 CFU / g or 5×10 10 CFU / mL, 8×10 10 CFU / g or 8×10 10 CFU / mL, 1×10 11 CFU / g or 1×10 11CFU / mL, etc.; the effective viable count of *Bacillus glutamate* is at least 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, 4×10 8 CFU / g or 4×10 8 CFU / mL, 8×10 8 CFU / g or 8×10 8 CFU / mL, 2×10 9 CFU / g or 2×10 9 CFU / mL, 5×10 9 CFU / g or 5×10 9 CFU / mL, 8×10 9 CFU / g or 8×10 9 CFU / mL, 1×10 10 CFU / g or 1×10 10 CFU / mL, 5×10 10 CFU / g or 5×10 10 CFU / mL, 8×10 10 CFU / g or 8×10 10 CFU / mL, 1×10 11 CFU / g or 1×10 11 CFU / mL, etc.; the effective viable count of Bacillus vesiculosus JYC1 is at least 1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, 4×10 8 CFU / g or 4×10 8 CFU / mL, 8×10 8 CFU / g or 8×10 8 CFU / mL, 2×10 9 CFU / g or 2×10 9 CFU / mL, 5×10 9 CFU / g or 5×10 9 CFU / mL, 8×10 9 CFU / g or 8×10 9 CFU / mL, 1×10 10 CFU / g or 1×10 10 CFU / mL, 5×10 10 CFU / g or 5×10 10 CFU / mL, 8×10 10 CFU / g or 8×10 10 CFU / mL, 1×10 11 CFU / g or 1×10 11CFU / mL, etc.; those skilled in the art can select the appropriate value range based on actual needs.

[0044] In some embodiments, the CFU ratio of *Enterobacter leukoi*, *Bacillus nicotine*, and *Bacillus belyssus* JYC1 is (1 × 10⁻⁶). 8 ~1×10 11 ): (1×10 8 ~1×10 11 ): (1×10 8 ~1×10 11 Alternatively, 5×10 8 : 4×10 8 5×10 8 5×10 8 5×10 8 : 4×10 8 .

[0045] In some more specific embodiments, the compound microbial agent comprises Enterobacter leuciscus strain CW-01, Bacillus nicotineus strain SX-01, and Bacillus belyssus strain JYC1.

[0046] In some embodiments, the compound microbial agent further comprises one or more of Bacillus amyloliquefaciens and Bacillus belyss Mardel-KB01, wherein the Bacillus belyss Mardel-KB01 has the accession number CGMCCNo.37220.

[0047] In some embodiments, the *Bacillus velezensis* Mardel-KB01 strain was deposited on December 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37220. It was isolated from shrimp ponds and its morphological characteristics are: milky white, wrinkled, dry surface, and bristly edges. The 16S rDNA sequence includes the sequence shown in SEQ ID No. 2. However, this strain Mardel-KB01 has poor inorganic nitrogen removal efficiency but strong inhibitory effects against *Saprolegnia*, *Aeromonas velezensis*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*.

[0048] In some embodiments, the effective viable count of *Bacillus vesiculosus* Mardel-KB01 in the compound microbial agent is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, for example 1×10 8 CFU / g or 1×10 8 CFU / mL, 4×108 CFU / g or 4×10 8 CFU / mL, 8×10 8 CFU / g or 8×10 8 CFU / mL, 2×10 9 CFU / g or 2×10 9 CFU / mL, 5×10 9 CFU / g or 5×10 9 CFU / mL, 8×10 9 CFU / g or 8×10 9 CFU / mL, 1×10 10 CFU / g or 1×10 10 CFU / mL, 5×10 10 CFU / g or 5×10 10 CFU / mL, 8×10 10 CFU / g or 8×10 10 CFU / mL, 1×10 11 CFU / g or 1×10 11 CFU / mL, etc., those skilled in the art can select the range of values ​​according to actual needs.

[0049] In some specific embodiments, the compound microbial agent comprises *Enterobacter luteolyticus*, *Glutamoxifenobacterium tobaccosum*, *Bacillus belyssae* strain JYC1, and *Bacillus belyssae* strain Mardel-KB01. Specifically, it includes *Enterobacter luteolyticus* strain CW-01, *Glutamoxifenobacterium tobaccosum* strain SX-01, *Bacillus belyssae* strain JYC1, and *Bacillus belyssae* strain Mardel-KB01. The applicant has discovered that adding *Bacillus belyssae* strain Mardel-KB01 to *Enterobacter luteolyticus* strain CW-01, *Glutamoxifenobacterium tobaccosum* strain SX-01, and *Bacillus belyssae* strain JYC1 not only improves the inhibitory effect on *Saprolegnia*, but also enhances the inhibitory effect on four harmful bacteria: *Aeromonas verrucosa*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*. Furthermore, compared with the compound microbial agent formed by three strains of Enterobacter leuciscus CW-01, Bacillus nicotine glutamate SX-01 and Bacillus belysaeus JYC1, the removal rate of inorganic nitrogen was significantly improved after adding Bacillus belysaeus Mardel-KB01.

[0050] In some specific embodiments, the CFU ratio of Enterobacter leukoi, Bacillus nicotine, Bacillus belyssus JYC1, and Bacillus belyssus Mardel-KB01 is (1×10⁻⁶). 8 ~1×10 11 ): (1×10 8 ~1×1011 ): (1×10 8 ~1×10 11 ): (1×10 8 ~1×10 11 For example, it can be 3×10 8 3×10 8 : 4×10 8 : 4×10 8 It can also be 3×10 8 3×10 8 : 4×10 8 : 4×10 8 .

[0051] In some embodiments, the *Bacillus amyloliquefaciens* includes, but is not limited to, *Bacillus amyloliquefaciens* ZB, which was deposited on December 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29399. According to CN118406620B, the average diameter of its inhibition zone against *Saprolegnia* is 19.88 mm, while it has no inhibitory effect on *Aeromonas verrucosum*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae* (see...). Figure 12 At 3 days, strain ZB reduced the ammonia nitrogen concentration from 50 mg / L to 1.2 mg / L, removing 97.6% of the ammonia nitrogen; and reduced the nitrite concentration from 22 mg / L to 0.05 mg / L, removing 99.8% of the nitrite.

[0052] In some embodiments, the effective viable count of the *Bacillus amyloliquefaciens* in the compound microbial agent is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, for example 1×10 8 CFU / g or 1×10 8 CFU / mL, 4×10 8 CFU / g or 4×10 8 CFU / mL, 8×10 8 CFU / g or 8×10 8 CFU / mL, 2×10 9 CFU / g or 2×10 9 CFU / mL, 5×10 9 CFU / g or 5×10 9 CFU / mL, 8×10 9 CFU / g or 8×10 9 CFU / mL, 1×10 10 CFU / g or 1×1010 CFU / mL, 5×10 10 CFU / g or 5×10 10 CFU / mL, 8×10 10 CFU / g or 8×10 10 CFU / mL, 1×10 11 CFU / g or 1×10 11 CFU / mL, etc., those skilled in the art can select the range of values ​​according to actual needs.

[0053] In some specific embodiments, the CFU ratio of Enterobacter leukemiae, Bacillus nicotinibacillus, Bacillus belyceae JYC1, and Bacillus amyloliquefaciens is (1×10⁻⁶). 8 ~1×10 11 ): (1×10 8 ~1×10 11 ): (1×10 8 ~1×10 11 ): (1×10 8 ~1×10 11 For example, it can be 3×10 8 3×10 8 : 4×10 8 : 4×10 8 It can also be 3×10 8 3×10 8 : 4×10 8 : 4×10 8 .

[0054] In some more specific embodiments, the compound microbial agent comprises *Enterobacter leukemia*, *Bacillus tobaccomi*, *Bacillus belyssus*, and *Bacillus amyloliquefaciens*. Specifically, it includes *Enterobacter leukemia* strain CW-01, *Bacillus tobaccomi* strain SX-01, *Bacillus belyssus* strain JYC1, and *Bacillus amyloliquefaciens* strain ZB. The applicant has found that adding *Bacillus amyloliquefaciens* strain ZB to *Enterobacter leukemia* strain CW-01, *Bacillus tobaccomi* strain SX-01, and *Bacillus belyssus* strain JYC1 improves the inhibitory effect on *Saprolegnia*, and significantly enhances the inhibitory effect on four harmful bacteria: *Aeromonas vesiculosus*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*.

[0055] In some more specific embodiments, the composite microbial agent comprises *Enterobacter leukemia*, *Glutamicinus tobacco*, *Bacillus belyssae* strain JYC1, *Bacillus amyloliquefaciens* strain ZB, and *Bacillus belyssae* strain Mardel-KB01. Specifically, it includes *Enterobacter leukemia* strain CW-01, *Glutamicinus tobacco* strain SX-01, *Bacillus belyssae* strain JYC1, *Bacillus amyloliquefaciens* strain ZB, and *Bacillus belyssae* strain Mardel-KB01. The applicant found that, based on *Enterobacter leukemia* strain CW-01, *Glutamicinus tobacco* strain SX-01, and *Bacillus belyssae* strain JYC1, the simultaneous addition of *Bacillus amyloliquefaciens* strain ZB and *Bacillus belyssae* strain Mardel-KB01 resulted in the best inhibitory effect against *Saprolegnia*, *Aeromonas vesiculosus*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*. Simultaneously, the inorganic nitrogen removal rate was the highest.

[0056] In some specific embodiments, the CFU ratio of *Enterobacter leukoi*, *Bacillus nicotinica*, *Bacillus belyceae* JYC1, *Bacillus amyloliquefaciens*, and *Bacillus belyceae* Mardel-KB01 is (1×10⁻⁶). 8 ~1×10 11 ): (1×10 8 ~1×10 11 ): (1×10 8 ~1×10 11 ): (1×10 8 ~1×10 11 ): (1×10 8 ~1×10 11 For example, it can be 3×10 8 3×10 8 : 4×10 8 2×10 8 2×10 8 It can also be 3×10 8 3×10 8 : 4×10 8 2×10 8 2×10 8 .

[0057] In some embodiments, the formulation of the composite microbial agent can be of various forms, such as liquid, emulsion, suspension, or powder.

[0058] In some embodiments, the microbial agent further includes a carrier.

[0059] In some embodiments, the carrier comprises a solid carrier. The solid carrier includes mineral materials, plant materials, and / or polymeric compounds; the mineral material may be at least one selected from talc, maifanite, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one selected from milk powder, corn flour, soybean flour, rice husk powder, and starch; the polymeric compound may be polyvinyl alcohol and / or polyethylene glycol. Preferably, it is milk powder, specifically skim milk powder.

[0060] In some specific embodiments, the microbial agent may also contain surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc.

[0061] The second aspect of the present invention protects the method for preparing the composite microbial agent as described above, wherein Enterobacter lutei, Bacillus glutamate of tobacco and Bacillus belye JYC1 are inoculated into a culture medium and cultured to obtain the composite microbial agent.

[0062] In some embodiments, the culture medium includes, but is not limited to, MS medium, LB medium, PDA medium, Czapek's medium, and beef extract peptone medium.

[0063] In some specific embodiments, LB medium is used. The composition of the LB medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7-7.2.

[0064] In some embodiments, the culture temperature is 4–35°C, or it can be 15–35°C, or it can be 20–28°C, or it can be 25°C, 28°C, or 30°C.

[0065] In some embodiments, the culture time is 2 to 10 days, or it can be 2 to 5 days, or it can be 4 to 8 days, or it can be 7 to 10 days, or it can be 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0066] In some embodiments, inoculation may also include one or both of Bacillus amyloliquefaciens and Bacillus belyss Mardel-KB01.

[0067] In some embodiments, the Bacillus belyssima Mardel-KB01 is accessed under the number CGMCC No. 37220.

[0068] In some embodiments, the Bacillus amyloliquefaciens is registered under the CGMCC No. 29399.

[0069] In some embodiments, after culturing, a carrier is added to the fermentation broth; the carrier is selected from skim milk powder. The mass-to-volume ratio of the skim milk powder to the fermentation broth is (70-130) g:1 L, or it can be 70 g:1 L, 80 g:1 L, 90 g:1 L, 100 g:1 L, 1100 g:1 L, 120 g:1 L, or 130 g:1 L. Preferably, it is 100 g:1 L.

[0070] A third aspect of the present invention protects a microbial filter media comprising a filter media loaded with the composite microbial agent as described above.

[0071] The filter media serves as an immobilization carrier for the aforementioned composite microbial agent, providing a suitable attachment interface and growth space for the microorganisms within the agent. The microorganisms in the composite microbial agent attach to, proliferate on the filter media surface, and form a biofilm, thereby constructing a functional micro-ecosystem with inorganic nitrogen removal and antibacterial activity. This system removes ammonia nitrogen and nitrite from the water through nitrification, while simultaneously inhibiting the accumulation and proliferation of harmful bacteria through antagonistic effects.

[0072] In some embodiments, the filter material is selected from one or more of the following: spheres formed of filamentous fibers, bacterial houses, ceramic rings, porous glass rings, maifanite, and zeolite.

[0073] Ceramic rings are a type of biological filter media used in aquariums and filtration systems. Their microporous structure provides a breeding ground for various organisms in compound microbial agents, thereby helping to purify water.

[0074] Glass rings, also known as bio-rings or bacterial houses, are porous filter media made from talc powder through high-temperature firing. They are economical and efficient, and are commonly used in aquarium and aquaculture filtration systems.

[0075] The spheres formed from filamentous fibers can be spherical filter media made of chemical fibers such as polyester, polypropylene, or acrylic fibers. They can also be obtained by modifying chemical fibers such as polyester, polypropylene, or acrylic fibers.

[0076] In some embodiments, the spheres formed by the filamentous fibers have a diameter of 1-15cm, preferably 1cm, 2cm, 3cm, 5cm, 8cm, 10cm, 12cm, or 15cm, specifically 3.2cm.

[0077] In some embodiments, the filter material is immersed in a liquid composite microbial agent and then freeze-dried to obtain the microbial-coated biological filter material.

[0078] The fourth aspect of this invention protects the use of the composite microbial agent as described above or the bacterial-coated biological filter material as described above in at least one of the following:

[0079] A1) Removal of inorganic nitrogen;

[0080] A2) Prepare products that have removed inorganic nitrogen;

[0081] A3) Inhibits the growth of harmful bacteria;

[0082] A4) Prepare products that inhibit the growth of harmful bacteria.

[0083] In some embodiments, the harmful bacteria are selected from fungi and bacteria. The fungi include *Saprolegnia spp.*; the bacteria include one or more of *Aeromonas veronii*, *Aeromonas hydrophila*, *Vibrio alginolyticus*, and *Klebsiella pneumoniae*.

[0084] Water mold is a fungus that invades the surface wounds or dead eggs of aquatic animals through spores. The inner hyphae penetrate deep into the tissue to absorb nutrients, while the outer hyphae form a visible grayish-white cotton-like covering.

[0085] Aeromonas vera, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae are all bacteria. Aeromonas vera infects fish through wounds, and can also invade through the skin and gills, causing symptoms such as erythema, ulcers, organ bleeding, and severe ascites, leading to fish mortality. Aeromonas hydrophila primarily infects through the intestines, but can also infect injured or parasite-infected fish through the skin and gills, causing various diseases such as red fin disease, droplet disease, and print disease, resulting in mass fish mortality. Vibrio alginolyticus, as an opportunistic pathogen, is abundant in seawater and seafood, and when it reaches a certain quantity, it causes disease, infecting fish with symptoms such as skin bleeding, gill rot, and ascites, reducing growth rate and disease resistance. Klebsiella pneumoniae, as an opportunistic pathogen, multiplies rapidly and causes disease under conditions such as fish injury, water quality deterioration, and drastic water temperature changes, infecting fish and causing damage to organs such as the brain, liver, and kidneys, leading to mass mortality.

[0086] In some embodiments, the inorganic nitrogen includes one or more of ammonia nitrogen, nitrates, and nitrites.

[0087] In some embodiments, the inorganic nitrogen is present in the aquaculture water.

[0088] The fifth aspect of the present invention protects a method for safely removing inorganic nitrogen from aquaculture water using microorganisms, comprising: treating the aquaculture water with the composite microbial agent as described above or the microbial filter material as described above.

[0089] In some embodiments, the processing temperature is 4–35°C; it can also be 15–35°C, 20–28°C, or 25°C, 28°C, or 30°C.

[0090] In some embodiments, the inorganic nitrogen includes one or more of ammonia nitrogen, nitrate, and nitrite.

[0091] In some embodiments, based on the total mass of the water body, the amount of the composite microbial agent or the attached biological filter material added is 0.01g / T to 100g / T of water, or 0.01g / T to 0.5g / T of water, or 0.3g / T to 12g / T of water, or 10g / T to 40g / T of water, or 30g / T to 70g / T of water, or 60g / T to 100g / T of water, or the amount of water can be 0.05g / T of water.

[0092] This invention is the first to prepare a compound microbial agent by mixing Enterobacter leukemia, Bacillus belye, and Bacillus glutamate of tobacco. It can not only efficiently remove inorganic nitrogen pollutants such as ammonia nitrogen and nitrite from water, but also significantly inhibit the proliferation of harmful bacteria in aquaculture. In particular, it can simultaneously inhibit the proliferation of harmful bacteria such as Saprolegnia, Aeromonas vera, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae. While purifying water quality, it can effectively reconstruct the microecological balance of aquatic bodies and greatly improve the safety of aquatic organisms.

[0093] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0094] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0095] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0096] Unless otherwise specified, the raw materials used in the following embodiments and comparative examples of this application are all commercially available raw materials.

[0097] In the following embodiments of this application, the composition of LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7; LB solid plate: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 2% agar, pH 7-7.2.

[0098] Example 1: Isolation, identification, inorganic nitrogen removal, and antibacterial properties of *Bacillus glutamate* SX-01

[0099] In Example 1, *Bacillus nicotine* SX-01 was screened and isolated from wastewater from a sewage treatment plant, and its identification, inorganic nitrogen removal performance, and antibacterial performance were studied. These included the following:

[0100] 1.1 Isolation of strain SX-01

[0101] 1) 500 mL of wastewater was collected from a wastewater treatment plant in Shaoxing, Zhejiang Province using disposable sterile sampling bags. 5 mL of this sample was added to 200 mL of pre-sterilized culture medium A and incubated at 28°C on a shaker at a speed of 150 rpm for 7 days. Culture medium A consisted of: ammonium chloride 0.1 g / L, ferrous sulfate 0.02 g / L, dipotassium hydrogen phosphate 0.1 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.1 g / L, glucose 1 g / L, and pH 7 ± 0.2.

[0102] 2) After 7 days, the inoculum was transferred to fresh culture medium A at a rate of 5% (v / v) and cultured in a constant temperature shaker at 28°C for 7 days at a speed of 150 r / min.

[0103] 3) After culturing for another 7 days, 0.1 mL was spread onto agar plate A. After 5 purification cycles, a single strain was obtained and named strain SX-01. Agar plate A consisted of: ammonium chloride 0.1 g / L, ferrous sulfate 0.02 g / L, dipotassium hydrogen phosphate 0.1 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.1 g / L, glucose 1 g / L, and 2% agar.

[0104] The morphological characteristics of strain SX-01 on medium A plates were: yellow, moist surface, and without wrinkles, as shown in the image. Figure 1 As shown.

[0105] 1.2 Identification of strain SX-01

[0106] The purified strain SX-01 was sent to Shanghai Saiheng Biotechnology Co., Ltd. for 16S rDNA sequencing. The obtained sequence was compared with the database of the National Center for Bioinformatics (NCBI) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and it was found that the 16S rDNA sequence had 99.58% homology with the type strain Glutamicibacter nicotianae in GenBank.

[0107]

[0108] Based on morphological analysis and 16S rDNA analysis, this strain was identified as *Gluconacetobacter nicotianae* SX-01. The strain's taxonomic name is *Gluconacetobacter nicotianae* SX-01, its accession number is CGMCC No. 33168, and it was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0109] 1.3 Study on the inorganic nitrogen removal performance of strain SX-01

[0110] 1.3.1 Ammonia nitrogen removal performance test

[0111] Culture medium A: Ammonium chloride 0.1 g / L, ferrous sulfate 0.02 g / L, dipotassium hydrogen phosphate 0.1 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.1 g / L, glucose 1 g / L, pH 7 ± 0.2.

[0112] A single colony of strain SX-01 from Example 1 was inoculated into LB liquid medium and cultured for 1 day, resulting in a bacterial count of 7 × 10⁻⁶. 8 The concentration of CFU / mL was then inoculated into 150 mL of culture medium A at an inoculation rate of 0.1% (v / v), and cultured in a constant temperature shaker at 30°C with a shaker speed of 150 r / min. The ammonia nitrogen value was measured every 6 h.

[0113] One blank control group and three parallel experimental groups were set up (experimental group A1, experimental group A2, and experimental group A3).

[0114] Ammonia nitrogen determination: The Nessler's reagent spectrophotometric method was used. The principle is that mercuric iodide and potassium iodide in an alkaline solution react with ammonia to form a pale reddish-brown colloidal compound, which exhibits strong absorption over a wide wavelength range. The procedure was as follows: 5 mL of culture product was taken from each time point and added to a 50 mL colorimetric tube. The solution was diluted to the mark, and 1.0 mL of potassium tartrate solution and 1.5 mL of Nessler's reagent were added. After standing for 10 min, the absorbance was measured at 420 nm. For details, please refer to the Nessler's reagent spectrophotometric method (A) in "Methods for Monitoring and Analysis of Water and Wastewater (Fourth Edition)". The results are shown in Table 1.

[0115] Table 1

[0116]

[0117] As shown in Table 1, strain SX-01 has a high efficiency in removing ammonia nitrogen. In 24 hours, it can reduce the average concentration of ammonia nitrogen from 26.40 mg / L to 0.04 mg / L, removing 99.84% of the ammonia nitrogen.

[0118] 1.3.2 Nitrite Removal Performance Test

[0119] Culture medium B: Sodium nitrite 0.1 g / L, ferrous sulfate 0.02 g / L, dipotassium hydrogen phosphate 0.1 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.1 g / L, glucose 1 g / L, pH 7 ± 0.2.

[0120] A single colony of strain SX-01 from Example 1 was inoculated into LB liquid medium and cultured for 1 day, resulting in a bacterial count of 7 × 10⁻⁶. 8 The concentration of CFU / mL was then inoculated into 150 mL of culture medium B at an inoculation rate of 0.1% (v / v), and cultured in a constant temperature shaker at 30°C with a shaking speed of 150 r / min. The nitrite level was measured every 6 h.

[0121] One blank control group and three parallel experimental groups were set up (experimental group A1#, experimental group A2#, and experimental group A3#).

[0122] Nitrite determination: The N-(1-naphthyl)-ethylenediamine spectrophotometric method was used. The principle is that in a phosphoric acid medium at pH 1.8 ± 0.3, nitrite reacts with p-aminobenzenesulfonamide to form a diazonium salt, which then couples with N-(1-naphthyl)-ethylenediamine to form a red dye. Maximum absorption is observed at 540 nm. Results are shown in Table 2.

[0123] Table 2

[0124]

[0125] As shown in Table 2, strain SX-01 has a high efficiency in removing nitrite. In 24 hours, it can reduce the average concentration of nitrite from 21.40 mg / L to 0.31 mg / L, removing 98.55% of the nitrite.

[0126] 1.4 Study on the antibacterial properties of strain SX-01

[0127] 1.4.1 Test on Inhibition of Water Mold

[0128] 1) A single colony of the isolated strain SX-01 was inoculated onto an LB agar plate and activated in a 25°C incubator. The activated strain SX-01 was then inoculated into 100 mL of LB liquid medium and fermented at 25°C for 3 days. The bacterial concentration in the fermentation broth after fermentation was 8 × 10⁻⁶. 8 CFU / mL, for later use.

[0129] 2) Inoculate *Saprolegnia* onto PDA plates for activation. Colonies on PDA plates are white with long hyphae. Once the colonies have covered the entire plate, place it in a 4°C incubator as the inoculum for subsequent *Saprolegnia* inhibition experiments. PDA medium composition: 200g potato, 20g glucose, 1L purified water, natural pH, with 2% agar powder added when solid.

[0130] 3) Using a 0.6cm diameter punch, remove one piece of the *Saprolegnia* plate from step 2) and place it in the center of a blank PDA plate. Then, soak the SX-01 fermentation broth from step 1) in sterile antimicrobial susceptibility testing tablets for 2 hours. Afterward, place three sterile antimicrobial susceptibility testing tablets (arranged in a triangle on the PDA plate) around the *Saprolegnia* plate. Incubate for 2-3 days, repeating three times. Results are shown below. Figure 2 .

[0131] from Figure 2 The absence of a transparent zone indicates that strain SX-01 has no inhibitory effect on water mold.

[0132] 1.4.2 Performance test for inhibiting Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus and Klebsiella pneumoniae.

[0133] 1) A single colony of the isolated strain SX-01 was inoculated onto an LB agar plate and activated in a 25°C incubator. The activated strain SX-01 was then inoculated into 100 mL of LB liquid medium and fermented at 25°C for 3 days. The bacterial concentration in the fermentation broth after fermentation was 8 × 10⁻⁶. 8 CFU / mL, for later use.

[0134] 2) Aeromonas hydrophila gdHMN102, Aeromonas vesiculosus TCBS02, Vibrio alginolyticus WLSY01, and Klebsiella pneumoniae 7P-01 were inoculated onto LB solid plates for activation.

[0135] After activation, 200 mL of LB liquid fermentation broth for each of the four harmful bacteria was prepared for later use.

[0136] Aeromonas vilonis TCBS02, Aeromonas hydrophila gdHMN102, Vibrio alginolyticus WLSY01, and Klebsiella pneumoniae 7P-01 were isolated from fish with gill and flesh rot in a koi breeding pond in Shanghai and from water samples in the biochemical pond. They were identified by Shanghai Saiheng Biotechnology Co., Ltd.

[0137] 3) Take 0.1 mL of LB fermentation broth from each of the following bacteria (Step 2): Aeromonas vera TCBS02, Aeromonas hydrophila gdHMN102, Vibrio alginolyticus WLSY01, and Klebsiella pneumoniae 7P-01. Spread each broth onto an entire LB agar plate. Then place three sterile antimicrobial susceptibility testing tablets soaked in the SX-01 fermentation broth from Step 1) in the center of the plate (each tablet is soaked in the SX-01 fermentation broth for 20 min). Incubate for 2-3 days. Repeat for three groups.

[0138] Meanwhile, sterile drug sensitivity tablets soaked in LB liquid culture medium (each sterile drug sensitivity tablet soaked for 20 min) were used as a control group.

[0139] from Figure 2 It can be seen that no clear zone of inhibition was observed for any of the four pathogenic bacteria, proving that strain SX-01 has no inhibitory effect on Aeromonas hydrophila gdHMN102, Aeromonas vesiculosus TCBS02, Vibrio alginolyticus WLSY01, and Klebsiella pneumoniae 7P-01.

[0140] Example 2: Isolation, identification, inorganic nitrogen removal, and antibacterial properties of Bacillus belyssus Mardel-KB01

[0141] In Example 2, *Bacillus belye* Mardel-KB01 was screened and isolated from the water in an aquaculture pond, and its identification and antibacterial properties were studied. These included the following:

[0142] 2.1 Isolation of strain Mardel-KB01

[0143] 1) Collect 500 mL water samples from koi ponds in Suzhou using disposable sterile sampling bags. Add 5 mL of the sample to 100 mL of pre-sterilized LB liquid culture medium and place it in a constant temperature shaker at 28℃ for 3 days for enrichment culture. The shaker speed is 200 r / min.

[0144] 2) Take 1 mL of the enriched culture medium and dilute it in 9 mL of sterile pure water, then mix well to obtain a 10⁻⁶ solution. -1 Concentration mixture; take 10 -1 Dilute 1 mL of the concentration mixture with 9 mL of sterile pure water, mix well, and the resulting solution is 10. -2 Concentration mixture; dilute sequentially to 10 using the general method. -6 Concentration. From the minimum concentration of 10 -6 To begin, 0.1 mL of bacterial culture was pipetted into LB agar plates and spread evenly with a disposable spreader. The plates were then marked and inverted in a 25°C incubator to observe colony growth.

[0145] 3) Select single colonies with different morphology and color and good separation on LB solid plates, perform multiple streaking operations, purify for more than 5 generations, and finally screen out 1 strain, named strain Mardel-KB01.

[0146] The morphological characteristics of strain Mardel-KB01 on LB agar plates are: milky white, wrinkled, dry surface, with spiky edges, as shown. Figure 3 As shown.

[0147] 2.2 Identification of strain Mardel-KB01

[0148] The purified strain Mardel-KB01 was sent to Shanghai Saiheng Biotechnology Co., Ltd. for 16S rDNA sequencing. The obtained sequence was compared with the database of the National Center for Bioinformatics (NCBI) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and it was found that the 16S rDNA sequence had 99.52% homology with the type strain Bacillus velezensis in GenBank.

[0149]

[0150] Based on morphological analysis and 16S rDNA analysis, this strain was identified as *Bacillus velezensis* Mardel-KB01. The strain's taxonomic name is *Bacillus velezensis* Mardel-KB01, its accession number is CGMCC No. 37220, and it was deposited on December 16, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0151] 2.3 Study on the inorganic nitrogen removal performance of strain Mardel-KB01

[0152] 2.3.1 Ammonia nitrogen removal performance test

[0153] The same culture medium, culture method, and detection method as in step 1.3.1 of Example 1 were used, and the bacterial count was 7 × 10⁻⁶. 8 The ammonia nitrogen removal performance was tested using CFU / mL, and the ammonia nitrogen determination results are shown in Table 1. Three parallel experimental groups were set up (experimental group B1, experimental group B2, and experimental group B3).

[0154] As shown in Table 1, after 24 hours, strain Mardel-KB01 reduced the average concentration of ammonia nitrogen from 26.40 mg / L to 18.31 mg / L, removing 30.64% of the ammonia nitrogen. This indicates that strain Mardel-KB01 can remove ammonia nitrogen to some extent, but its ammonia nitrogen removal performance is relatively weak.

[0155] 2.3.2 Nitrite Removal Performance Test

[0156] The same culture medium, culture method, and detection method as in step 1.3.2 of Example 1 were used, and the bacterial count was 7 × 10⁻⁶. 8 The nitrite removal performance was tested using CFU / mL, and the nitrite determination results are shown in Table 2. Three parallel experimental groups were set up (experimental group B1#, experimental group B2#, and experimental group B3#).

[0157] Table 2 shows that after 24 hours, strain Mardel-KB01 reduced the average nitrite concentration from 21.4 mg / L to 18.21 mg / L, removing 14.82% of the nitrite. This indicates that strain Mardel-KB01 has relatively weak functionality in removing nitrite.

[0158] 2.4 Study on the antibacterial properties of strain Mardel-KB01

[0159] 2.4.1 Test on Inhibition of Water Mold

[0160] Using the same culture medium and culture method as in step 1.4.1 of Example 1, the bacterial strain content in the fermentation broth after fermentation culture was 7 × 10⁻⁶. 8 The CFU / mL concentration was used to detect its antibacterial effect against Saprolegnia, and the experimental results are shown below. Figure 4 Set up 3 parallel experimental groups (replication 1, replication 2, and replication 3).

[0161] from Figure 4 The presence of a large clear zone indicates that strain Mardel-KB01 has a strong inhibitory effect on Saprolegnia. The diameter of the inhibition zone is shown in Table 3.

[0162] Table 3

[0163]

[0164] As shown in Table 3, the average inhibition zone of strain Mardel-KB01 against Saprolegnia was 19.53 mm.

[0165] 2.4.2 Performance test for inhibiting Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus and Klebsiella pneumoniae.

[0166] Using the same culture medium and culture method as step 1.4.2 in Example 1, the bacterial strain content in the fermentation broth after fermentation culture was 7 × 10⁻⁶. 8 The antibacterial effect of CFU / mL on Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae was detected. The antibacterial results are shown in [Figure 1]. Figure 4 Set up 3 parallel experimental groups (repeat 1, repeat 2, repeat 3).

[0167] from Figure 4 It can be seen that all four pathogenic bacteria showed large clear zones, proving that the Mardel-KB01 strain has an inhibitory effect on Aeromonas hydrophila gdHMN102, Aeromonas vesiculosus TCBS02, Vibrio alginolyticus WLSY01, and Klebsiella pneumoniae 7P-01.

[0168] The diameter of the inhibition zone is shown in Table 4.

[0169] Table 4

[0170]

[0171] As shown in Table 4, the average inhibition zone diameters of strain Mardel-KB01 against Aeromonas villus TCBS02, Aeromonas hydrophila gdHMN102, Vibrio alginolyticus WLSY01, and Klebsiella pneumoniae 7P-01 were 13.03 mm, 14.29 mm, 13.34 mm, and 15.04 mm, respectively, indicating that strain Mardel-KB01 has a strong inhibitory effect on these four pathogens.

[0172] Example 3: Isolation, identification, inorganic nitrogen removal, and antibacterial properties of Bacillus belyssus JYC1

[0173] In Example 3, *Bacillus belye* JYC1 was screened and isolated from the water in an aquaculture pond, and its identification, inorganic nitrogen removal performance, and antibacterial performance were studied. These included the following:

[0174] 3.1 Isolation of strain JYC1

[0175] 1) A 500 mL water sample was collected from a shrimp pond in Dalian, Liaoning Province using a disposable sterile water sample collection bag. 5 mL of the sample was added to 100 mL of pre-sterilized LB liquid medium and placed in a constant temperature shaker at 30°C for 3 days for enrichment culture. The shaker speed was 200 r / min.

[0176] 2) Take 1 mL of the enriched culture medium and dilute it in 9 mL of sterile pure water, then mix well to obtain a 10⁻⁶ solution. -1 Concentration mixture; take 10 -1 Dilute 1 mL of the concentration mixture with 9 mL of sterile pure water, mix well, and the resulting solution is 10. -2 Concentration mixture; dilute sequentially to 10 using the general method. -6 Concentration. From the minimum concentration of 10 -6 To begin, 0.1 mL of bacterial culture was pipetted into LB agar plates and spread evenly with a disposable spreader. The plates were then marked and inverted in a 25°C incubator to observe colony growth.

[0177] 3) Select single colonies with different morphology and color and good separation on LB solid plates, perform multiple streaking operations, purify for more than 5 generations, and finally screen out 1 strain, which is named strain JYC1.

[0178] The morphological characteristics of strain JYC1 on LB agar plates are: milky white, wrinkled, and moist. Figure 5 As shown.

[0179] 3.2 Identification of strain JYC1

[0180] The purified strain JYC1 was sent to Shanghai Paisennong Gene Technology Co., Ltd. for 16S rDNA sequencing. The obtained sequence was compared with the database of the National Center for Biotechnology Information (NCBI) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and it was found that the 16S rDNA sequence had 99.72% homology with the type strain Bacillus velezensis in GenBank.

[0181]

[0182] Based on morphological analysis and 16S rDNA analysis, this strain was identified as *Bacillus velezensis*. The strain's taxonomic name is *Bacillus velezensis* JYC1, its accession number is CGMCC No. 26987, and it was deposited on March 31, 2023, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0183] 3.3 Study on the inorganic nitrogen removal performance of strain JYC1

[0184] 3.3.1 Ammonia nitrogen removal performance test

[0185] The difference between this method and step 1.3.1 in Example 1 lies in the composition of culture medium A, which is: ammonium chloride 0.2 g / L, ferrous sulfate 0.02 g / L, dipotassium hydrogen phosphate 0.1 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.1 g / L, glucose 2 g / L, pH 7 ± 0.2. The remaining culture and detection methods were used to test its ammonia nitrogen removal performance, and the bacterial count was 6 × 10⁻⁶. 8 The CFU / mL and ammonia nitrogen determination results are shown in Table 5. There are three parallel experimental groups (experimental group C1, experimental group C2, and experimental group C3).

[0186] Table 5

[0187]

[0188] As shown in Table 5, strain JYC1 has a high efficiency in removing ammonia nitrogen. In 24 hours, it can reduce the average concentration of ammonia nitrogen from 51.40 mg / L to 0.04 mg / L, removing 99.91% of the ammonia nitrogen.

[0189] 3.3.2 Nitrite Removal Performance Test

[0190] The same culture medium, culture method, and detection method as in step 1.3.2 of Example 1 were used, and the bacterial count was 6 × 10⁻⁶. 8 The CFU / mL concentration was used to test its nitrite removal performance, and the nitrite determination results are shown in Table 2. Three parallel experimental groups were established (group C1#, group C2#, and group C3#).

[0191] As shown in Table 2, strain JYC1 has a high efficiency in removing nitrite. In 24 hours, it can reduce the average concentration of nitrite from 21.40 mg / L to 0.04 mg / L, removing 99.80% of the nitrite.

[0192] 3.4 Study on the antibacterial properties of strain JYC1

[0193] 3.4.1 Test on inhibition of water mold

[0194] Using the same culture medium and method as step 1.4.1 in Example 1, the content of strain JYC1 in the fermentation broth after fermentation culture was 6 × 10⁻⁶. 8 The CFU / mL concentration was used to detect its antibacterial effect against Saprolegnia, and the experimental results are shown below. Figure 6 .

[0195] from Figure 6 The absence of a transparent zone indicates that strain JYC1 has no inhibitory effect on water mold.

[0196] 3.3.2 Performance test for inhibiting Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus and Klebsiella pneumoniae.

[0197] Using the same culture medium and method as step 1.4.2 in Example 1, the content of JYC1 strain in the fermentation broth after fermentation culture was 6 × 10⁻⁶. 8 The antibacterial effect of CFU / mL on Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae was detected. The antibacterial results are shown in [Figure 1]. Figure 6 .

[0198] from Figure 6 It can be seen that none of the four pathogenic bacteria showed a large clear zone, proving that strain JYC1 had no inhibitory effect on Aeromonas hydrophila gdHMN102, Aeromonas vesiculosus TCBS02, Vibrio alginolyticus WLSY01, and Klebsiella pneumoniae 7P-01.

[0199] Example 4: Preparation and Antibacterial Properties Study of Compound Microbial Agent

[0200] Example 4 describes the preparation of a composite microbial agent and its antibacterial properties. The steps include:

[0201] 4.1 Preparation of Compound Microbial Agents

[0202] Five bacterial strains were inoculated onto LB agar plates: Enterobacter leuciscus strain CW-01 (CGMCC No. 27126), Bacillus amyloliquefaciens strain ZB (CGMCC No. 29399), Bacillus glutamate from Example 1 (SX-01), Bacillus belyssus strain Mardel-KB01 from Example 2, and Bacillus belyssus strain JYC1 from Example 3. The plates were then activated in a 25°C incubator. Following the design in section 4.2, the activated strains were inoculated into 200 mL of LB liquid medium and cultured at 25°C for 3 days. The resulting compound bacterial solutions were prepared according to the bacterial concentration ratios, filtered through a 0.22 μm disposable syringe filter, and 200 mL of each solution was collected for later use.

[0203] 4.2 Study on the antibacterial properties of the compound bacterial solution

[0204] Divided into 4 groups, the specific groupings are as follows:

[0205] Group 1#: A compound bacterial culture of strain CW-01 + strain SX-01 + strain JYC1 + strain ZB. The concentration of strain CW-01 in the compound fermentation broth of Group 1# is 3 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 3×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 4×10⁻⁶. 8 The concentration of CFU / mL and strain ZB was 4 × 10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0206] Group 2#: A compound bacterial culture of strain CW-01 + strain SX-01 + strain JYC1 + strain Mardel-KB01. The concentration of strain CW-01 in the compound fermentation broth of Group 4# is 3 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 3×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 4×10⁻⁶. 8 The CFU / mL concentration of strain Mardel-KB01 was 4 × 10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0207] Group 3#: A compound bacterial culture of strain CW-01 + strain SX-01 + strain JYC1. The concentration of strain CW-01 in the compound fermentation broth of Group 3# is 5 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 4×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 5×10⁻⁶. 8 CFU / mL, total bacterial concentration 1.4×10 9 CFU / mL.

[0208] Group 4#: A compound bacterial culture consisting of strain CW-01 + strain SX-01 + strain JYC1 + strain ZB + strain Mardel-KB01. The concentration of strain CW-01 in the compound fermentation broth of Group 4# is 3 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 3×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 4×10⁻⁶. 8 The concentration of CFU / mL and strain ZB was 2×10⁻⁶.8 The concentration of CFU / mL and strain Mardel-KB01 was 2×10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0209] 4.2.1 Test on performance in inhibiting water mold

[0210] The antibacterial performance was tested using the same method as in step 1.4.1 of Example 1, and the results are shown below. Figure 7 .

[0211] from Figure 7 It can be seen that the PDA solid plates of the blank control group were covered with water mold and almost no inhibition zone was visible, while the compound bacterial solutions of groups 1#, 2#, 3# and 4# all showed clear inhibition zones against water mold.

[0212] The antibacterial effects of the compound bacterial solutions of Group 1#, Group 2#, Group 3# and Group 4# on Saprolegnia are shown in Table 6.

[0213] Table 6

[0214]

[0215] Table 6 shows that the average inhibition zones of groups 1#, 2#, 3#, and 4# against Saprolegnia were 20.09 mm, 20.08 mm, 6.98 mm, and 21.45 mm, respectively. Among them, the average inhibition zone of group 2#, after adding strain Mardel-KB01, increased by 187.68% compared to group 3#; the average inhibition zone of group 1#, after adding strain ZB, increased by 187.82% compared to group 3#; and the average inhibition zone of group 4#, after simultaneously adding strains Mardel-KB01 and ZB, increased by 207.31% compared to group 3#.

[0216] 4.2.2 Performance test for inhibiting Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus and Klebsiella pneumoniae.

[0217] The antibacterial performance was tested using the same method as in step 1.4.2 of Example 1, and the results are shown below. Figure 8A , 8B .

[0218] from Figure 8B It can be seen that the LB solid plates of the blank control group were covered with harmful bacteria, and the inhibition zone was almost invisible.

[0219] from Figure 8A It can be seen that the compound bacterial solutions of Group 1#, Group 2#, Group 3# and Group 4# all showed clear inhibition zones against the four harmful bacteria.

[0220] The antibacterial effects of the compound bacterial solutions of Group 1#, Group 2#, Group 3# and Group 4# on the four harmful bacterial strains are shown in Table 7.

[0221] Table 7

[0222]

[0223] Summary Table 7 and Figure 8A It can be seen that, for Aeromonas hydrophila TCBS02, compared with group 3#, the average inhibition zone of group 2# (17.05 mm) after adding strain Mardel-KB01 increased by 109.20% compared with group 3# (8.15 mm), the average inhibition zone of group 1# (13.18 mm) after adding strain ZB increased by 61.72% compared with group 3# (8.15 mm), and the average inhibition zone of group 4# (17.15 mm) after adding both strains Mardel-KB01 and ZB increased by 110.43% compared with group 3# (8.15 mm).

[0224] For Aeromonas villus gdHMN102, compared with group 3#, the average inhibition zone of group 2# (17.20 mm) after adding strain Mardel-KB01 increased by 142.94% compared with group 3# (7.08 mm); the average inhibition zone of group 1# (13.32 mm) after adding strain ZB increased by 77.14% compared with group 3# (7.08 mm); and the average inhibition zone of group 4# (17.32 mm) after adding both strains Mardel-KB01 and ZB increased by 144.63% compared with group 3# (7.08 mm).

[0225] For Vibrio alginolyticus WLSY01, compared with group 3#, the average inhibition zone of group 2# (15.33 mm) after adding strain Mardel-KB01 increased by 152.52% compared with group 3# (6.15 mm); the average inhibition zone of group 1# (13.79 mm) after adding strain ZB increased by 124.23% compared with group 3# (6.15 mm); and the average inhibition zone of group 4# (15.50 mm) after adding strains Mardel-KB01 and ZB was 152.03% of that of group 3# (6.15 mm).

[0226] For Klebsiella pneumoniae 7P-01, compared with group 3#, the average inhibition zone of group 2# (16.12 mm) after adding strain Mardel-KB01 increased by 62.99% compared with group 3# (9.89 mm). The average inhibition zone of group 1# (13.57 mm) after adding strain ZB increased by 37.21% compared with group 3# (9.89 mm). At the same time, the average inhibition zone of group 4# (16.25 mm) after adding strains Mardel-KB01 and ZB was 64.31% of that of group 3# (9.89 mm).

[0227] The compound bacterial solution of group 4# showed the strongest antibacterial effect against the four pathogenic bacteria, and subsequent application experiments were conducted using the compound bacterial solution of group 4#.

[0228] 4.3 Study on the Inorganic Nitrogen Removal Performance of Compound Bacteria

[0229] Divided into 7 groups, the specific groupings are as follows:

[0230] Group 1: A compound fermentation broth consisting of strain CW-01, strain ZB, and strain JYC1. The concentration of strain CW-01 in the compound fermentation broth of Group 1 was 5 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 5×10⁻⁶. 8 The concentration of CFU / mL and strain ZB was 4 × 10⁻⁶. 8 CFU / mL, total bacterial concentration 1.4×10 9 CFU / mL.

[0231] Group 2: A compound fermentation broth containing strains CW-01, ZB, and SX-01. The concentration of strain CW-01 in the compound fermentation broth of Group 2 was 5 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 5×10⁻⁶. 8 The bacterial concentration of strain ZB at CFU / mL was 4 × 10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0232] Group 3: A compound fermentation broth containing strains CW-01, JYC1, and SX-01. The concentration of strain CW-01 in the compound fermentation broth of Group 3 was 5 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 5×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 4×10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0233] Group 4: A compound fermentation broth containing strain ZB, strain JYC1, and strain SX-01. The concentration of strain SX-01 in the compound fermentation broth of Group 4 was 5 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 5×10⁻⁶. 8 The concentration of CFU / mL and strain ZB was 4 × 10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0234] Group 5: A compound bacterial culture of strain CW-01 + strain ZB + strain JYC1 + strain SX-01. The concentration of strain CW-01 in the compound fermentation broth of Group 5# is 3×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 3×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 4×10⁻⁶. 8 The concentration of CFU / mL and strain ZB was 4 × 10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0235] Group 6: A compound bacterial culture of strain CW-01 + strain Mardel-KB01 + strain JYC1 + strain SX-01. The concentration of strain CW-01 in the compound fermentation broth of Group 6 is 3 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 3×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 4×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain Mardel-KB01 is 4×10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0236] Group 7: A compound bacterial culture of strain CW-01 + strain ZB + strain JYC1 + strain SX-01 + strain Mardel-KB01. The concentration of strain CW-01 in the compound fermentation broth of Group 7# is 3 × 10⁻⁶. 8 CFU / mL, the bacterial concentration of strain SX-01 is 3×10⁻⁶. 8 CFU / mL, the bacterial concentration of strain JYC1 is 4×10⁻⁶. 8 The concentration of CFU / mL and strain ZB was 2×10⁻⁶. 8 The concentration of CFU / mL and strain Mardel-KB01 was 2×10⁻⁶. 8 CFU / mL, total bacterial concentration was 1.4 × 10⁻⁶. 9 CFU / mL.

[0237] Following the same steps as in Example 1, different combinations of fermentation broth were inoculated into 150 mL of culture medium A1 at an inoculation rate of 0.1% (v / v), and placed in a constant temperature shaker at 30°C with a shaker speed of 150 r / min. Ammonia nitrogen values ​​were measured every 6 hours. One blank control group and three parallel experimental groups were set up. The results are shown in Table 8-1 and Table 8-2.

[0238] The composition of culture medium A1 is as follows: ammonium chloride 0.2 g / L, ferrous sulfate 0.02 g / L, dipotassium hydrogen phosphate 0.1 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.1 g / L, glucose 2 g / L, pH 7 ± 0.2.

[0239] Table 8-1

[0240]

[0241] Table 8-2

[0242]

[0243] As shown in Tables 8-1 and 8-2, after 18 hours, the average concentration of ammonia nitrogen in groups 1-7 decreased from 52.40 mg / mL to 9.34 mg / mL, 7.93 mg / mL, 7.43 mg / mL, 8.43 mg / mL, 7.00 mg / mL, 6.31 mg / mL, and 5.78 mg / mL, respectively, representing reductions of 82.18%, 84.87%, 85.81%, 83.91%, 86.65%, 87.96%, and 88.99% compared to the initial concentration.

[0244] Compared with group 1, group 2, which uses strain SX-01 instead of strain JYC1, has a 3.28% higher ammonia nitrogen removal rate ((84.87-82.18) / 82.18), indicating that strain SX-01 is more effective than strain JYC1 in promoting ammonia nitrogen removal.

[0245] Compared with group 1, group 3, which uses strain SX-01 instead of strain ZB, showed a 4.42% increase in ammonia nitrogen removal rate, indicating that strain SX-01 is more effective than strain ZB in promoting ammonia nitrogen removal.

[0246] Compared with group 3, group 4, which uses strain ZB instead of strain CW-01, had a 2.21% lower ammonia nitrogen removal rate, indicating that strain CW-01 was more effective than strain ZB in promoting ammonia nitrogen removal.

[0247] Compared with group 3, the ammonia nitrogen removal rate of group 5 increased by 1% after adding strain ZB, indicating that adding strain ZB can promote the ammonia nitrogen removal effect of the compound bacterial solution based on strain CW-01+ strain JYC1+ strain SX-01.

[0248] Compared with group 3, the ammonia nitrogen removal rate of group 6 increased by 2.51% after adding strain Mardel-KB01, indicating that adding strain Mardel-KB01 can promote the ammonia nitrogen removal effect of the compound bacterial solution based on strain CW-01 + strain JYC1 + strain SX-01.

[0249] Based on group 3, strains ZB and Mardel-KB01 were added to form group 7. The ammonia nitrogen removal rate of group 7 was 3.71% higher than that of group 3.

[0250] Following the same steps as in Example 1, different combinations of fermentation broth were inoculated into 150 mL of culture medium B at an inoculation rate of 0.1% (v% / v%), placed in a constant temperature shaker at 30°C and 150 r / min, and cultured for 1 day. Nitrite levels were measured every 6 hours. One blank control group and three parallel experimental groups were set up. The results are shown in Tables 9-1 and 9-2.

[0251] Table 9-1

[0252]

[0253] Table 9-2

[0254]

[0255] As shown in Tables 9-1 and 9-2, after 18 hours, the average nitrite concentration in groups 1-7 decreased from 21.40 mg / mL to 4.25 mg / mL, 5.06 mg / mL, 4.06 mg / mL, 4.78 mg / mL, 3.46 mg / mL, 3.22 mg / mL, and 2.86 mg / mL, respectively, representing reductions of 75.47%, 75.42%, 81.03%, 77.66%, 83.93%, 84.95%, and 87.90% compared to the initial concentration.

[0256] Compared with group 1, group 2 was obtained by replacing strain JYC1 with strain SX-01, and the nitrite removal rate was comparable.

[0257] Compared with group 1, group 3 was obtained by replacing strain ZB with strain SX-01, and the nitrite removal rate increased by 7.37%, indicating that strain SX-01 is more effective than strain ZB in promoting nitrite removal.

[0258] Compared with group 3, group 4 was obtained by replacing strain CW-01 with strain ZB, and the nitrite level decreased by 4.16%, indicating that strain CW-01 was more effective than strain ZB in promoting nitrite removal.

[0259] Based on the results from groups 1 to 4, among the four strains CW-01, ZB, JYC1, and SX-01, group 3 (strain CW-01 + strain JYC1 + strain SX-01) showed the best effect in removing nitrite.

[0260] Compared with group 3, the nitrite removal rate of group 5 increased by 3.58% after adding strain ZB, indicating that adding strain ZB can promote the nitrite removal effect of the compound bacterial solution based on strain CW-01+ strain JYC1+ strain SX-01.

[0261] Compared with group 3, the nitrite removal rate of group 6 increased by 4.84% after adding strain Mardel-KB01, indicating that adding strain Mardel-KB01 can promote the nitrite removal effect of the compound bacterial solution based on strain CW-01 + strain JYC1 + strain SX-01.

[0262] Based on group 3, strains ZB and Mardel-KB01 were added to form group 7. The nitrite removal rate of group 7 was 8.48% higher than that of group 3.

[0263] In summary, considering the inorganic nitrogen removal and antibacterial properties, the compound microbial agent formed by strains CW-01+, ZB+, SX-01+, JYC1+, and Mardel-KB01 exhibits the best effect.

[0264] Application Example 1: Study on the antibacterial and inorganic nitrogen removal performance of compound microbial agents in aquaculture ponds.

[0265] In this application example 1, the antibacterial and inorganic nitrogen removal performance of the compound microbial agent (strain CW-01 + strain ZB + strain SX-01 + strain JYC1 + strain Mardel-KB01) from Example 4 was studied in an aquaculture pond. The compound microbial agent (strain CW-01 + strain ZB + strain SX-01 + strain JYC1 + strain Mardel-KB01) was freeze-dried into a lyophilized powder (i.e., the compound microbial agent) using a freeze-drying process (skim milk powder was added to the compound bacterial fermentation broth; the mass-to-volume ratio of skim milk powder to compound bacterial fermentation broth was 100g:1L, and then the lyophilized powder was obtained using a freeze-drying process).

[0266] The bacterial concentration of strain CW-01 in this lyophilized powder is 3.5 × 10⁻⁶. 10 CFU / g, the bacterial concentration of strain SX-01 is 2.5 × 10⁻⁶. 9 CFU / g, the bacterial concentration of strain JYC1 is 3×10⁻⁶. 9 The CFU / g concentration of strain ZB was 2.5 × 10⁻⁶. 9 The CFU / g concentration of strain Mardel-KB01 was 2.5 × 10⁻⁶. 9 CFU / g, total bacterial concentration was 4.55×10 10 CFU / g. Antibacterial properties and inorganic nitrogen removal properties include the following:

[0267] 5.1 Study on the antibacterial properties of compound microbial agents

[0268] In a koi breeding pond in Shanghai, fish exhibited gill and flesh rot. A 500mL water sample was collected using a disposable sterile sampling bag. The concentration of harmful bacteria in the water sample was determined using a TCBS plate (purchased from Beijing Luqiao Technology Co., Ltd.). The determined concentration of harmful bacteria was 4 × 10⁻⁶. 4 CFU / mL.

[0269] The compound microbial agent (strain CW-01 + strain ZB + strain SX-01 + strain JYC1 + strain Mardel-KB01) was added to the koi pond at a dosage of 0.1 g / ton of water, once daily. After 7 days, 500 mL of water was collected using a disposable sterile sampling bag, and the concentration of harmful bacteria was determined again using TCBS plates. The results are shown below. Figure 9 .

[0270] The method for determining bacterial concentration on TCBS plates is as follows: Samples are serially diluted 10-fold using sterile test tubes to create different dilutions. Then, 0.1 mL of the appropriate dilution is pipetted onto the surface of a TCBS plate. Using a sterile L-shaped spreader, the liquid is spread evenly and quickly until completely absorbed. The plate is then incubated at 28°C for 18 hours. Plates with colony counts between 30 and 300 CFU are selected for counting.

[0271] from Figure 9 It can be seen that after 7 days of biochemical treatment, the concentration of harmful bacteria in the koi breeding pond decreased significantly, by two orders of magnitude, from 10... 4 The order of magnitude (CFU / mL) decreased to 10 2 Order of magnitude (CFU / mL).

[0272] 5.2 Study on the Inorganic Nitrogen Removal Performance of Compound Microbial Agents

[0273] Artificially simulated ammonia nitrogen and nitrite wastewater was prepared by inoculating it with compound microbial agent at a dosage of 0.1g per ton of water, followed by aeration at room temperature for 24 hours, with ammonia nitrogen and nitrite data in the wastewater monitored every 6 hours.

[0274] The components of artificially simulated ammonia nitrogen wastewater were: ammonium chloride 0.2g, potassium dihydrogen phosphate 0.2g, sodium citrate 0.5g, glucose 0.5g, magnesium sulfate 0.1g, ferrous sulfate 0.02g, and water 1L. Sterilization was performed at 121℃ for 30min, with a pH of 7.0-7.2. One blank control group and three parallel experimental groups (group D1, group D2, and group D3) were set up. The components of artificially simulated nitrite wastewater were: sodium nitrite 0.1g, potassium dihydrogen phosphate 0.2g, sodium citrate 0.5g, glucose 0.5g, magnesium sulfate 0.1g, ferrous sulfate 0.02g, and water 1L. Sterilization was performed at 121℃ for 30min, with a pH of 7.0-7.2. One blank control group and three parallel experimental groups (group D1#, group D2#, and group D3#) were set up.

[0275] The determination of ammonia nitrogen and nitrite was performed in the same manner as in steps 1.3.1 and 1.3.2 of Example 1. The results are shown in Tables 10 and 11.

[0276] Table 10

[0277]

[0278] Table 11

[0279]

[0280] As shown in Tables 10 and 11, the compound microbial agent in group 4# effectively removed ammonia nitrogen and nitrite from the water. After 24 hours, the average concentration of ammonia nitrogen decreased from 51.4 mg / L to 0.04 mg / L, achieving a removal rate of 99.91%; the average concentration of nitrite decreased from 20.4 mg / L to 0 mg / L, achieving 100% removal of nitrite.

[0281] In summary, the compound microbial agent (strain CW-01 + strain ZB + strain SX-01 + strain JYC1 + strain Mardel-KB01) has good effects on water treatment and inhibition of harmful bacteria in aquaculture water.

[0282] Application Example 1: Preparation of microbial filter media, study on inorganic nitrogen removal and antibacterial properties.

[0283] In Example 5, the composite microbial agent (strain CW-01 + strain ZB + strain SX-01 + strain JYC1 + strain Mardel-KB01) from Example 4 was immobilized in filter media to form a microbially attached biological filter media. Its antibacterial properties and inorganic nitrogen removal performance were then studied. This included the following:

[0284] 6.1 Preparation of Bacterial Filter Media

[0285] Preparation of microbial-coated biological filter media: 135 spheres formed from 3.2cm diameter filamentous fibers (specifically, 5S Ma Dou filter media was used, but it was not loaded with any microorganisms). The spheres formed from the filamentous fibers were immersed in 1500 mL of the compound bacterial fermentation broth obtained in Group 4# (or Group 7) of Example 4 at room temperature (25℃) for 30 min, and then dried using a vacuum freeze dryer (Nanjing Yanwo Biotechnology Co., Ltd., model YWLG-50F) to form microbial-coated biological filter media.

[0286] Then, place the bio-filter media with bacteria in the middle layer of a three-layer drip box (17cm long x 7cm wide x 16.5cm high) for aquariums. Place the drip box on top of the aquarium and use a 40W water pump to create a circulating water system between the aquarium and the drip box. Water flows from the top layer of the drip box, through the middle layer, and then drains from the bottom. The aquarium (160cm long x 60cm wide x 55cm high) houses a 50cm long arowana, forming the complete aquarium ecosystem.

[0287] Experimental group: obtained by freeze-drying spheroids formed from filamentous fibers loaded with fermentation broth of compound bacteria.

[0288] Control group: spheres formed from filamentous fibers (without any microorganisms loaded).

[0289] 6.2 Study on the Inorganic Nitrogen Removal Performance of Bacterially Attached Biofilter Media

[0290] After the fish were introduced, the concentrations of ammonia nitrogen, nitrite, nitrate, and pH in the aquarium were measured at the same time every day for a total of 7 days. The results are shown in Table 12.

[0291] The determination of ammonia nitrogen and nitrite is the same as steps 1.3.1 and 1.3.2 in Example 1.

[0292] Nitrate determination: A UV spectrophotometer was used. The principle is to quantitatively determine nitrate nitrogen based on the absorption of nitrate ions at a wavelength of 220 nm. Dissolved organic matter also absorbs at 220 nm, while nitrate ions do not absorb at 275 nm. Therefore, another measurement was performed at 275 nm to correct the nitrate nitrogen value. The results are shown in Table 12.

[0293] Table 12

[0294]

[0295] Table 12 shows that, in terms of water quality, the levels of ammonia nitrogen, nitrite, and nitrate in the experimental group's aquarium remained relatively stable; while in the control group, ammonia nitrogen, nitrite, and nitrate accumulated to varying degrees, with levels of 0.45 mg / L, 0.35 mg / L, and 30.6 mg / L at 7 days, significantly higher than those in the experimental group. In summary, the filter media loaded with the compound microbial agent provides a better living environment, and the compound microbial agent can effectively colonize the filter media, establishing a biological system.

[0296] 6.3 Study on the antibacterial properties of bio-filter media with attached bacteria

[0297] On day 7, the concentration of harmful bacteria was determined using TCBS plate counting. The results are shown below. Figure 10 The number of harmful bacteria in the experimental group was less than 10. 4 (CFU / mL), the number of harmful bacteria in the control group was higher than 10. 4 (CFU / mL).

[0298] from Figure 10 It can be seen that, under the same dilution gradient of the water sample, the number of harmful bacteria in the aquarium water treated with the microbial filter media was significantly lower than that in the control group, proving that the microbial filter media can effectively antagonize harmful bacteria.

[0299] The compound microbial agent of this invention exhibits significant efficacy: on the one hand, it has a strong inhibitory effect on harmful bacteria such as Saprolegnia, Aeromonas vera, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae. For example, after applying the compound microbial agent containing Enterobacter leucis, Bacillus glutamate, Bacillus belyceae strain JYC1, Bacillus amyloliquefaciens strain ZB, and Bacillus belyceae strain Mardel-KB01 to aquaculture ponds where fish exhibit gill and flesh rot, the concentration of harmful bacteria decreased from 10... 4 The order of magnitude decreased to 10 2The concentration of ammonia nitrogen decreased by two orders of magnitude. On the other hand, it can efficiently remove inorganic nitrogen pollutants such as ammonia nitrogen and nitrite. For example, a composite microbial agent containing *Enterobacter leukemia*, *Glutamicinus tobacco*, *Bacillus belyssus* strain JYC1, *Bacillus amyloliquefaciens* strain ZB, and *Bacillus belyssus* strain Mardel-KB01, when treating artificially simulated high-concentration ammonia nitrogen and nitrite wastewater, reduced the average ammonia nitrogen concentration from 51.4 mg / L to 0.04 mg / L in 24 hours, removing 99.91% of the ammonia nitrogen; the average nitrite concentration decreased from 20.4 mg / L to 0 mg / L, achieving 100% nitrite removal. Furthermore, when the bio-filter material formed by the composite microbial agent is applied to aquaculture tanks, it can effectively remove inorganic nitrogen, preventing the accumulation of ammonia nitrogen, nitrite, and nitrate; while also significantly reducing the number of harmful bacteria. In summary, the composite microbial agent and the attached biological filter material of the present invention have shown significant effects in water purification and the inhibition of harmful bacteria and removal of inorganic nitrogen in aquaculture water, and have broad application prospects, especially suitable for water quality control and disease prevention in high-density aquaculture systems.

[0300] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A compound microbial agent, characterized in that, It contains Enterobacter luteolyticus, Bacillus nicotinibacillus, and Bacillus belyssus JYC1, wherein the preservation number of Bacillus belyssus JYC1 is CGMCC No. 26987, the preservation number of Enterobacter luteolyticus is CGMCC No. 27126, and the preservation number of Bacillus nicotinibacillus is CGMCC No. 33168.

2. The composite microbial agent as described in claim 1, characterized in that, In the compound microbial agent, the effective viable count of *Enterobacter leukemia* is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, the effective viable count of the *Tobacco glutamate* bacteria is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL, the effective viable count of the *Bacillus belyssus* JYC1 is at least 1 × 10⁻⁶ CFU / mL. 8 CFU / g or 1×10 8 CFU / mL; And / or, the compound microbial agent further comprises one or both of Bacillus amyloliquefaciens and Bacillus belyss Mardel-KB01, wherein the preservation number of Bacillus belyss Mardel-KB01 is CGMCC No. 37220.

3. The compound microbial agent as described in claim 2, characterized in that, The preservation number of the Bacillus amyloliquefaciens is CGMCC No. 29399; And / or, in the compound microbial agent, the effective viable count of the *Bacillus amyloliquefaciens* is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL; And / or, in the compound microbial agent, the effective viable count of the *Bacillus berberis* Mardel-KB01 is at least 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL.

4. The method for preparing the composite microbial agent according to any one of claims 1-3, characterized in that, Enterobacter luteoli, Bacillus nicotinib, and Bacillus belye JYC1 were inoculated into a culture medium and cultured to obtain the compound microbial agent; the preservation number of Bacillus belye JYC1 is CGMCC No. 26987, the preservation number of Enterobacter luteoli is CGMCC No. 27126, and the preservation number of Bacillus nicotinib is CGMCC No. 33168.

5. The preparation method according to claim 4, characterized in that, The culture temperature is 4–35°C; And / or, the culture time is 2 to 10 days; And / or, it also includes inoculation with one or both of Bacillus amyloliquefaciens and Bacillus belyss Mardel-KB01, wherein the Bacillus belyss Mardel-KB01 has the accession number CGMCC No. 37220.

6. A microbial filter media comprising a filter media loaded with a composite microbial agent as described in any one of claims 1-3.

7. The microbial filter material as described in claim 6, characterized in that, The filter media is selected from one or more of the following: spheres formed from filamentous fibers, bacterial houses, ceramic rings, porous glass rings, maifanite, and zeolite.

8. The application of the composite microbial agent as described in any one of claims 1-3 or the microbial-coated biological filter material as described in claim 6 or 7 in at least one of the following: A1) Removal of inorganic nitrogen; A2) Prepare products that have removed inorganic nitrogen; A3) Inhibits the growth of harmful bacteria, wherein the harmful bacteria are selected from one or more of the following: Saprolegnia, Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae; A4) Prepare a product that inhibits the growth of harmful bacteria, wherein the harmful bacteria are selected from one or more of Saprolegnia, Aeromonas verrucosa, Aeromonas hydrophila, Vibrio alginolyticus, and Klebsiella pneumoniae.

9. A method for safely removing inorganic nitrogen from aquaculture water using microorganisms, characterized in that, include: Aquaculture water bodies are treated using the composite microbial agent as described in any one of claims 1-3 or the microbial filter material as described in claim 6 or 7.

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