Application of nitrification and denitrification complex microbial inoculant in aquaculture

Abstract

The invention discloses an application of a nitrification and denitrification complex microbial inoculant in aquaculture. According to the present invention, the Acinetobacter lwoffii C25 and the Pseudomonas stutzeri C29 with nitrification and denitrification functions are obtained through directional screening, the C25 and the C29 are compounded to prepare the composite bacterial agent, and the bacterial agent can efficiently degrade NH4 < + >-N, NO2 <->-N and NO3 <->-N so as to achieve rapid and stable nitrogen removal, and is safe and harmless to fish bodies. The complex microbial inoculant disclosed by the invention has wide application prospects in the fields of denitrification of aquaculture pond water, treatment of aquaculture tail water or biological denitrification treatment of other nitrogen-containing sewage.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of a nitrification-denitrification compound bacterial agent in aquaculture. Background Technology

[0002] The aquatic environment is an essential survival factor for aquatic economic animals, and its quality directly affects the health of the fish. Aquaculture environments are often relatively closed, and the accumulation of large amounts of nitrogen in the water can easily exceed the tolerance range of aquatic animals, thus creating a stress effect. This stress not only leads to physiological dysfunction but also exacerbates the growth and spread of pathogenic microorganisms, thereby inducing large-scale disease outbreaks. Therefore, how to effectively mitigate the negative impact of nitrogen stress on farmed animals is a key issue that urgently needs to be addressed in the current aquaculture industry.

[0003] Utilizing microorganisms for nitrogen removal in aquatic environments has always been an important technological direction for improving aquaculture environments. Nitrifying and denitrifying bacteria can effectively degrade high concentrations of nitrogen, thereby alleviating the harmful effects of nitrogen stress on farmed animals. Compared with single strains, complex microorganisms have advantages such as functional diversity, high growth efficiency, and strong stability. Based on this, screening and isolating strains from aquaculture environments, constructing complex microbial communities with highly efficient nitrogen removal capabilities, and developing them into microbial preparations suitable for aquaculture water use are of great significance for the healthy breeding and disease control of aquatic animals. Summary of the Invention

[0004] This invention provides an application of a nitrifying and denitrifying compound bacterial agent in aquaculture, which includes Acinetobacter rumeni (… Acinetobacter lwoffii ) and Pseudomonas stearothermia ( Stutzerimonas stutzeri The compound microbial agent is composed of microorganisms that efficiently degrade NH4 through synergistic effects. + -N, NO2 - -N and NO3 - -N enables rapid nitrogen reduction, solving the problems of low degradation efficiency and poor stability in existing technologies that use a single strain for nitrogen removal.

[0005] The first objective of this invention is to provide a nitrification-denitrification compound microbial agent, said compound microbial agent being composed of Acinetobacter rumeni (… Acinetobacter lwoffii ) and Pseudomonas stearothermia ( Stutzerimonas stutzeri )composition.

[0006] Preferably, the Acinetobacter ruvidii is Acinetobacter ruvidii (… Acinetobacter lwoffii C25, the *Pseudomonas stearothermia* is *Pseudomonas stearothermia* (C25). Stutzerimonas stutzeri C29.

[0007] The Acinetobacter rumen ( Acinetobacter lwoffiiC25, which was deposited on December 12, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, postcode: 510070, accession number: GDMCC NO: 67476; the described *Pseudomonas stearothermia* ( Stutzerimonas stutzeri C29 was deposited on December 12, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67477.

[0008] Further preferably, the compound bacterial agent is prepared by compounding Acinetobacter rumeni C25 and Pseudomonas stearothermia C29 in a 1:1 ratio.

[0009] The second objective of this invention is to provide a method for preparing the above-mentioned nitrification-denitrification compound bacterial agent, comprising the following steps: inoculating Acinetobacter rumeni and Pseudomonas stenosum into a liquid culture medium and culturing them under shaking to obtain the corresponding bacterial solutions, and then mixing the two bacterial solutions to obtain the compound bacterial agent.

[0010] Preferably, the liquid culture medium is LB liquid culture medium.

[0011] Preferably, the shaking culture continues until the strain enters the logarithmic growth phase.

[0012] Preferably, the conditions for the shaking culture are: temperature 28℃ and rotation speed 200 rpm.

[0013] Preferably, the compound bacterial agent is prepared by mixing the two bacteria in a 1:1 ratio.

[0014] The third objective of this invention is to provide the application of the above-mentioned nitrification-denitrification composite bacterial agent in water purification.

[0015] Preferably, the application is the use of compound microbial agents in denitrification of aquaculture water. The compound microbial agents have denitrification capabilities and high-efficiency denitrification capacity.

[0016] Further preferably, the compound microbial agent degrades NH4 + -N, NO2 - -N and NO3 - -N achieves denitrification of water bodies.

[0017] The fourth objective of this invention is to provide a method for denitrification of water, which involves adding the aforementioned nitrification-denitrification composite bacterial agent to the water body to be treated, thereby degrading NH4. + -N, NO2 - -N and NO3 - -N achieves denitrification of water bodies.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention uses targeted screening to obtain Acinetobacter rumeni with nitrification and denitrification functions. Acinetobacter lwoffii C25 and Pseudomonas stearothermia ( Stutzerimonas stutzeri C29 was further compounded with C25 to form a composite microbial agent, which can efficiently degrade NH4. + -N, NO2 - -N and NO3 - -N achieves rapid and stable denitrification, and is safe and harmless to fish. Therefore, the compound bacterial agent of this invention has broad application prospects in the fields of denitrification of aquaculture ponds, treatment of aquaculture wastewater, and biological denitrification treatment of other nitrogen-containing wastewater.

[0020] Acinetobacter ruvidii ( Acinetobacter lwoffii C25 was deposited on December 12, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67476.

[0021] Pseudomonas stearothermia ( Stutzerimonas stutzeri C29 was deposited on December 12, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67477. Attached Figure Description

[0022] Figure 1 The images show the morphological structures of strains C25 and C29 in Example 1, including (A) the colony morphology of strain C25; (B) a Gram-stained microscopic image of strain C25; (C) the colony morphology of strain C29; and (D) a Gram-stained microscopic image of strain C29.

[0023] Figure 2 These are the growth curves of strains C25 and C29, and compound bacterial agent M6 in Example 3.

[0024] Figure 3 This describes the effect of adding strains C25 and C29 to the culture water in Example 4 on the survival rate of zebrafish.

[0025] Figure 4 This refers to the sensitivity of strains C25 and C29 in Example 4 to multiple antibiotics.

[0026] Figure 5 The conversion characteristics of ammonia nitrogen by the compound microbial agent M6 and single strains C25 and C29 in Example 5 are shown.

[0027] Figure 6 The conversion characteristics of nitrite by the compound microbial agent M6 and single strains C25 and C29 in Example 6 are shown.

[0028] Figure 7 The conversion characteristics of nitrate by the compound microbial agent M6 and single strains C25 and C29 in Example 7 are shown. Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] In the experiment, NH4 + -N, NO2 - -N, NO3 - The determination and analysis methods for -N are all based on the national standard determination methods. Among them, NH4 + The determination and analysis of NO2- were based on the "Water Quality - Determination of Ammonia Nitrogen - Nessler's Reagent Spectrophotometric Method (HJ 535-2009)"; - The determination and analysis of -N was performed according to the standard "Water Quality - Determination of Nitrite Nitrogen - Spectrophotometric Method (GB 7493-87)"; NO3 - The determination and analysis of -N were performed in accordance with the "Water Quality - Determination of Nitrate Nitrogen - Ultraviolet Spectrophotometry (HJ / T 346-2007)".

[0031] Example 1: Isolation, purification and identification of nitrifying and denitrifying bacteria

[0032] (1) Preparation of culture medium

[0033] LB medium (g / L): 10g tryptone, 5g yeast extract, 10g NaCl, water as solvent, pH = 7.0. If preparing a solid medium, add 15g agar.

[0034] Ammonia nitrogen medium (g / L) DMI: C4H4Na2O4·6H2O 5.62g, Vickers salt solution 50mL, (NH4)2SO4 0.47g, solvent is water, pH = 7.0.

[0035] Nitrite medium (g / L) DMⅡ: C4H4Na2O4·6H2O 5.62g, Vickers salt solution 50mL, NaNO2 0.49g, solvent is water, pH = 7.0.

[0036] Nitrate medium (g / L) DMⅢ: C4H4Na2O4·6H2O 5.62g, Vickers salt solution 50mL, KNO3 0.72g, solvent is water, pH = 7.0.

[0037] Giltay medium (g / L): 12.24 g Giltay powder, water as solvent, pH = 7.0. Add 15 g Agar if preparing a solid medium. Add 0.1% Agar if preparing a semi-solid medium.

[0038] Vickers salt solution (g / L): K2HPO4·3H2O 5.0g, MgSO4·7H2O 2.5g, NaCl 2.5g, MnSO4·4H2O 0.05g, FeSO4·7H2O 0.05g, solvent is water, pH = 7.0.

[0039] The above culture medium is prepared by mixing all components evenly and then sterilizing it with autoclave at 121℃ for 20 minutes.

[0040] (2) Isolation and purification of strains

[0041] Water was taken from sea bass farming ponds and diluted 10% with sterilized water. -1 Up to 10 -5 Different dilutions were spread onto LB agar plates and incubated at 28°C for 48 hours, with colony growth observed every 8-12 hours. For further purification, single colonies with different morphologies were selected and streaked onto fresh LB agar plates. The streaked plates were incubated at 28°C for 24 hours, and colony morphology was observed to ensure uniformity. Strains with uniform colony morphology were transferred to LB liquid medium and incubated with shaking at 200 rpm at 28°C. For purified strains obtained using ammonia nitrogen medium and Giltay agar, strains that grew on ammonia nitrogen medium and turned Giltay agar blue were selected.

[0042] (3) Strain morphology

[0043] The culture characteristics of the obtained strain are as follows: aerobic or facultative anaerobic bacteria; on LB solid medium, strain C25 colonies are pale yellow, with regular edges, smooth surface, raised, opaque, round colonies, slightly sticky, with no obvious odor; Gram-negative bacteria, coccobacilli, arranged in pairs or chains; its colony morphology and Gram staining microscopic examination are as follows. Figure 1 As shown in A and B; strain C29 colonies are pale yellow, with neat edges, wrinkled surface, slightly flattened, semi-transparent, irregular colonies, dry, with no obvious odor, Gram-negative, short rod-shaped, with slightly curved cells. Its colony morphology and Gram staining microscopic examination are as follows: Figure 1 As shown in C and D.

[0044] (4) Molecular identification of strains

[0045] The 16S rDNA of strains C25 and C29 was amplified using universal 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The purified PCR products were sequenced, and the results are as follows:

[0046] The 16S rDNA sequence of strain C25 is shown in SEQ ID No. 1:

[0047] The 16S rDNA sequence of strain C29 is shown in SEQ ID No. 2:

[0048] The 16S rDNA (SEQ ID No. 1) of strain C25 showed over 99% homology with *Acinetobacter rumenella*; the 16S rDNA (SEQ ID No. 2) of strain C29 showed over 99% homology with *Pseudomonas stearothermia*. Based on morphological and molecular biological analysis, strain C25 was identified as *Acinetobacter rumenella*, and strain C29 as *Pseudomonas stearothermia*.

[0049] The strain C25 was named Acinetobacter roux. Acinetobacter lwoffii C25 was deposited on December 12, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67476.

[0050] Strain C29 was named *Pseudomonas stearothermia*. Stutzerimonas stutzeri C29 was deposited on December 12, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67477.

[0051] Example 2 Physiological and biochemical characteristics of the strain

[0052] Acinetobacter rumeni C25 and Pseudomonas stearothermia C29 grew well at 20-28℃. Their physiological and biochemical characteristics are shown in Table 1 and Table 2.

[0053] Table 1. Physiological and biochemical test results of Acinetobacter rumenella C25

[0054] Table 2. Physiological and biochemical test results of Pseudomonas stearothermiae C29

[0055] Example 3 Construction of Compound Microbial Agent M6

[0056] Experimental methods:

[0057] Construction of compound microbial agent M6: C25 (OD25) in the logarithmic growth phase was used... 600 =0.1) bacterial solution and C29 (OD 600 =0.1) The bacterial solutions are mixed at a volume ratio of 1:1 to form compound bacterial agent M6.

[0058] Bacterial growth curve plotting: Equal amounts of single bacterial strains (C25, C29) and compound bacterial agent (M6) were inoculated into LB liquid medium, and the initial OD in the medium was adjusted. 600< 0.005, incubate at 220 rpm and 28℃ for 24 h with constant temperature shaking, and measure OD every 3 h. 600 .

[0059] Experimental results: The growth rate and quantity of the compound bacterial agent M6 were significantly higher than those of the single strain. (See...) Figure 2 The growth curves show that the combination of M6 bacterial agent is feasible and can be used for subsequent nitrogen compound conversion experiments.

[0060] Example 4 Safety of the strain

[0061] Experimental methods:

[0062] 1. Zebrafish Toxicity Experiment: Ninety zebrafish with a body length of 2.99±0.12cm and a weight of 0.14±0.02g were selected and divided into three groups. They were temporarily housed in aquariums at approximately 25℃ for two weeks, after which bacterial suspension was added to analyze the safety of the suspension on the fish. Ten fish were placed in each aquarium. Experimental setup: Strains C25 and C29 cultured on LB liquid medium were centrifuged and precipitated. The bacteria were resuspended in water used to house the zebrafish after aeration, and the resuspended bacterial suspension was evenly injected into the aquariums to achieve a bacterial concentration of 10⁻⁶. 6 Zebrafish were divided into two groups with cfu / mL, designated as treatment group C25 (3 replicates per group, n=3) and treatment group C29 (3 replicates per group, n=3). A Ctrl control group (n=3) was also included, meaning no other bacterial agents were added to this aquarium. The total water volume in each aquarium was kept constant. The experiment lasted 10 days. The survival rate of zebrafish in each aquarium was recorded daily.

[0063] 2. Antibiotic susceptibility test: C25 and C29 (OD600≈0.1) in the logarithmic growth phase were evenly spread on LB solid medium. After the solid medium was dried, the antibiotic susceptibility test strips were gently applied to the surface of the solid medium. The medium was incubated at 28℃ for 12-15 hours and the formation of inhibition zones was observed.

[0064] Experimental results: The results of the zebrafish toxicity test are shown below. Figure 3 After 10 days of culture in water supplemented with bacterial solution, zebrafish showed no significant difference compared to the control group (P > 0.05). Results of antibiotic sensitivity testing are shown below. Figure 4 Strains C25 and C29 are sensitive to a variety of antibiotics. The antibiotic sensitivity tablets 1-15 correspond to erythromycin, streptomycin, florfenicol, spectinomycin, norfloxacin, vancomycin, amikacin, ofloxacin, penicillin, tetracycline, kanamycin, ciprofloxacin, ampicillin, ceftriaxone, and clindamycin, respectively.

[0065] Example 5: Study on the conversion characteristics of ammonia nitrogen by compound microbial agent M6

[0066] Experimental method: The bacterial suspensions (C25, C29, and compound bacterial agent M6) in the logarithmic growth phase were centrifuged at 4000 rpm for 10 min to precipitate. The supernatant (culture medium and metabolites) was discarded, and the bacterial suspensions were resuspended in ammonia nitrogen medium to achieve an OD concentration of [missing value]. 600 =0.1. Following the principle of single-factor experiments, equal amounts of the three bacterial cultures were inoculated into ammonia nitrogen medium to achieve an initial OD value of 0.1. 600 <0.005, cultured at 200 rpm and 28℃ with constant temperature shaking. Collect samples at 0h, 6h, 12h, 24h, 36h, 48h, 60h and 72h after inoculation. Centrifuge and discard the precipitate. Determine the content of residual ammonia nitrogen in the culture medium according to the national standard detection method (HJ 535-2009).

[0067] Experimental results: see Figure 5 The conversion rate of ammonia nitrogen by compound microbial agent M6 was significantly higher than that of single strains. After 12 hours of inoculation, the conversion rate of ammonia nitrogen by single strains C25 and C29 was about 60%, while the conversion rate of ammonia nitrogen by compound microbial agent M6 reached more than 80%.

[0068] Example 6: Study on the conversion characteristics of compound bacterial agent M6 for nitrite nitrogen

[0069] Experimental method: The bacterial suspensions (C25, C29, and compound bacterial agent M6) in the logarithmic growth phase were centrifuged at 4000 rpm for 10 min to precipitate. The supernatant (culture medium and metabolites) was discarded, and the bacterial suspensions were resuspended in nitrite medium to achieve an OD concentration of [missing value]. 600 =0.1. Following the principle of single-factor experiments, equal amounts of the three bacterial cultures were inoculated into nitrite medium to adjust the initial OD value in the medium to 0.1. 600 < 0.005, cultured at 200 rpm and 28℃ with constant temperature shaking. Collect samples at 0h, 6h, 12h, 24h, 36h, 48h, 60h and 72h after bacterial inoculation. Centrifuge and discard the precipitate. Determine the content of residual nitrite in the culture medium according to the national standard test method (GB 7493-87).

[0070] Experimental results: see Figure 6 The conversion rate of nitrite by compound microbial agent M6 was significantly higher than that of single strains. At 12h, the conversion rate of nitrite by single strains C25 and C29 was less than 40%, while the conversion rate of nitrite by compound microbial agent M6 reached more than 65%.

[0071] Example 7 Study on the conversion characteristics of nitrate nitrogen by compound bacterial agent M6

[0072] Experimental method: The bacterial suspensions (C25, C29, and compound bacterial agent M6) in the logarithmic growth phase were centrifuged at 4000 rpm for 10 min to precipitate. The supernatant (culture medium and metabolites) was discarded, and the bacterial suspensions were resuspended in nitrate medium to achieve an OD concentration of [missing value]. 600 =0.1. Following the principle of single-factor experiments, equal volumes of the three bacterial cultures were inoculated into nitrate medium to achieve an initial OD value of 0.1. 600 < 0.005, cultured at 200 rpm and 28℃ with constant temperature shaking. Collect samples at 0h, 6h, 12h, 24h, 36h, 48h, 60h and 72h after bacterial inoculation. Centrifuge and discard the precipitate. Determine the content of residual nitrate in the culture medium according to the national standard test method (HJ / T 346-2007).

[0073] Experimental results: see Figure 7 The nitrate conversion rate of compound microbial agent M6 is higher than that of single strains. After 12 hours, the nitrate conversion rates of single strains C25 and C29 reached 27% and 23%, respectively, while the nitrate conversion rate of compound microbial agent M6 reached over 45%.

[0074] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A nitrification-denitrification composite microbial agent, characterized in that, The compound bacterial agent is composed of Acinetobacter rumeni (… Acinetobacter lwoffii ) and Pseudomonas stearothermia ( Stutzerimonas stutzeri )composition.

2. The compound microbial agent according to claim 1, characterized in that, The Acinetobacter ruvidii is Acinetobacter ruvidii ( Acinetobacter lwoffii C25, the *Pseudomonas stearothermia* is *Pseudomonas stearothermia* (C25). Stutzerimonas stutzeri The Acinetobacter rumeni C25 has the accession number GDMCC NO: 67476; the Pseudomonas stearothermiae C29 has the accession number GDMCC NO: 67477.

3. The compound microbial agent according to claim 2, characterized in that, The compound bacterial agent is prepared by mixing Acinetobacter rumeni C25 and Pseudomonas stearothermia C29 in a 1:1 ratio.

4. The method for preparing the compound microbial agent according to claim 1, 2 or 3, characterized in that, The process includes the following steps: Acinetobacter rumeni and Pseudomonas stenosum are inoculated into liquid culture medium and cultured by shaking to obtain the corresponding bacterial solutions, and then the two bacterial solutions are mixed to prepare a compound bacterial agent.

5. The method according to claim 4, characterized in that, The liquid culture medium is LB liquid culture medium.

6. The method according to claim 4, characterized in that, The shaking culture continued until the strain entered the logarithmic growth phase; the shaking culture conditions were: temperature 28℃, rotation speed 200 rpm.

7. The method according to claim 4, characterized in that, The two bacteria were mixed in a 1:1 ratio.

8. The application of the compound bacterial agent according to claim 1, 2 or 3 in water purification.

9. The application according to claim 8, characterized in that, The application is the use of compound microbial agents in denitrification of aquaculture water. Preferably, the compound microbial agents degrade NH4. + -N, NO2 - -N and NO3 - -N achieves denitrification of water bodies.

10. A method for denitrification of water, characterized in that, The compound bacterial agent described in claim 1, 2, or 3 is added to the water body to be treated to degrade NH4. + -N, NO2 - -N and NO3 - -N achieves denitrification of water bodies.

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