An Acinetobacter strain with water body nitrogen reduction function and production of signal molecule DSF quenching enzyme and its application

Abstract

The present invention discloses a strain of Acinetobacter having a water body nitrogen reduction function and producing a signal molecule DSF quenching enzyme and its application. The present invention belongs to the field of microbial degradation technology, and specifically relates to a strain of Acinetobacter having a water body nitrogen reduction function and producing a signal molecule DSF quenching enzyme and its application. The Acinetobacter provided by the present invention is Acinetobacter tandoii CGMCC No. 25032, and its registration number at the General Microbiological Center of the China Culture Collection Administration is CGMCC No. 25032. The Acinetobacter of the present invention has nitrification and aerobic denitrification capabilities, can efficiently degrade ammonia nitrogen and nitrite nitrogen in an aerobic aquaculture water environment, and the strain also has the ability to reduce quorum sensing signal molecules in water bodies, and is a potential strain for application in aquaculture.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial degradation, and particularly relates to an Acinetobacter strain having the function of reducing nitrogen in water and producing a signal molecule DSF quenching enzyme, and applications thereof. Background Art

[0002] In recent years, with the continuous expansion of aquaculture and the development of intensive farming methods, the aquaculture industry has developed rapidly. However, with the changes in farming methods, excessive feeding and inadequate management have led to a continuous deterioration of the aquaculture environment and water quality. Among them, excessive ammonia nitrogen and nitrite nitrogen are more serious hazards. Excessive levels can cause poisoning or even death in farmed animals, restricting the development of the aquaculture industry. Water quality restoration includes chemical remediation and biological remediation. Chemical remediation mainly involves adding chemicals to the water. This method is fast-acting and low-cost, but the maintenance time is short and it cannot continuously improve the water quality environment. Biological remediation mainly involves adding microbial agents with denitrification functions. It can effectively and continuously regulate the nitrogen balance of the water and improve the quality of aquaculture water. In recent years, studies have reported that strains such as Pseudomonas stutzeri, Bacillus, and Denitrifying Coccus have both nitrification and aerobic denitrification functions, which can reduce ammonia nitrogen and nitrite nitrogen in aquaculture water. However, different strains have different denitrification characteristics, resulting in differences in denitrification performance in different water bodies. Therefore, it is particularly important to isolate a strain with a wide adaptability and strong nitrogen reduction performance from the aquaculture environment. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to use bioremediation to reduce ammonia nitrogen and nitrite nitrogen in aquaculture water, effectively and continuously regulate the nitrogen balance of the water body, and improve the aquaculture water environment.

[0004] In order to solve the above problems, the present invention provides an Acinetobacter.

[0005] The Acinetobacter provided by the present invention is Acinetobacter tandoii CGMCC No. 25032, and its registration number in the General Microbiology Center of China Culture Collection Administration is CGMCC No. 25032.

[0006] The present invention also provides a bacterial agent, which contains the aforementioned Acinetobacter tankini and / or a metabolite of the Acinetobacter tankini.

[0007] The aforementioned culture of Acinetobacter is a substance obtained by culturing the aforementioned Acinetobacter tankii in a bacterial culture medium.

[0008] The active ingredient of the above-mentioned bacterial agent may be the above-mentioned Acinetobacter tankini and / or a metabolite of the above-mentioned Acinetobacter tankini. The active ingredient of the above-mentioned bacterial agent may also contain other biological components or non-biological components.

[0009] The above-mentioned microbial agent, in addition to the active ingredient, also contains a carrier. The carrier can be a biologically inert carrier commonly used in the field of bioremediation agents. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material, or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soy flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0010] The above-mentioned microbial agent can be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0011] As needed, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the above-mentioned bacterial agents.

[0012] In the above, the metabolite can be obtained from the fermentation liquid of the Acinetobacter tankii. The metabolite can be the sterile metabolite of the Acinetobacter tankii or the bacteria-containing metabolite of the Acinetobacter tankii. The sterile metabolite (sterile fermentation filtrate) of the Acinetobacter tankii can be specifically prepared according to the following method, and the Acinetobacter tankii is cultivated in a liquid culture medium, and the Acinetobacter tankii in the liquid culture (fermentation liquid) is filtered to obtain the sterile metabolite of the Acinetobacter tankii. The bacteria-containing metabolite of the Acinetobacter tankii can be specifically prepared according to the following method, and the Acinetobacter tankii is cultivated in a liquid fermentation medium, and the fermentation liquid is collected, and the fermentation liquid is the bacteria-containing metabolite of the Acinetobacter tankii.

[0013] The present invention also provides the use of the aforementioned Acinetobacter tankini, the bacterial agent, or the culture in at least one of the following:

[0014] (a1) Degradation of ammonia nitrogen;

[0015] (a2) preparing ammonia nitrogen degradation products;

[0016] (a3) degradation of nitrite nitrogen;

[0017] (a4) preparing a product for degrading nitrite nitrogen;

[0018] (a5) Reduce the ability of quorum sensing signal molecules in water.

[0019] The present invention also provides a method for preparing the above-mentioned bacterial agent.

[0020] The present invention provides a method for preparing the aforementioned bacterial agent, comprising the following steps: using the aforementioned Acinetobacter tankini as an active ingredient to obtain the bacterial agent.

[0021] The present invention also provides a bioremediation agent, which contains the aforementioned Acinetobacter tankini or the bacterial agent or the culture.

[0022] The present invention also provides a method for efficiently degrading ammonia nitrogen and nitrite nitrogen in a water environment, comprising the following steps: applying the aforementioned Acinetobacter tankii or the bacterial agent or the culture in the water environment, thereby degrading ammonia nitrogen and nitrite nitrogen in the water.

[0023] The Acinetobacter tankii isolated from the bottom mud of the shrimp aquaculture pond in the present invention has nitrification and aerobic denitrification capabilities, can efficiently degrade ammonia nitrogen and nitrite nitrogen in an aerobic aquaculture water environment, and has wide adaptability in terms of temperature, salinity, pH and carbon-nitrogen ratio. The strain also has the ability to produce the signal molecule DSF quenching enzyme, making it a potential strain for use in aquaculture.

[0024] Preservation Instructions

[0025] Species name: Acinetobacter tankii

[0026] Latin name: Acinetobacter tandoii

[0027] Strain ID: DD15

[0028] Depository: General Microbiology Center of China Culture Collection Administration

[0029] Abbreviation of depository institution: CGMCC

[0030] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0031] Date of deposit: June 9, 2022

[0032] CGMCC registration number: CGMCC No.25032 BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The colony morphology of strain DD15 on LB culture medium.

[0034] Figure 2 This is the phylogenetic tree of strain DD15 based on the 16S rRNA gene.

[0035] Figure 3 The growth characteristics of strain DD15 using different carbon sources.

[0036] Figure 4 is the denitrification ability of strain DD15.

[0037] Figure 5 The ammonia nitrogen degradation ability of strain DD15 under a. different C / N, b. temperature, c. salinity, d. pH, and e. inoculum size conditions.

[0038] Figure 6 The ability of strain DD15 to degrade nitrite nitrogen under a. different C / N ratios, b. temperature, c. salinity, d. pH, and e. inoculum size.

[0039] Figure 7 The strain DD15 produces the ability of quenching enzyme to degrade the signal molecule DSF.

[0040] Figure 8 This is the hemolytic detection experiment of strain DD15.

[0041] Figure 9 is the survival rate of zebrafish challenged with strain DD15. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0043] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0044] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0045] Example 1: Isolation, identification, and growth characteristics of bacterial strains

[0046] 1. Strain Isolation

[0047] Take 10g of pond mud from the shrimp farming pond and place it in a conical flask filled with 200mL of denitrification medium. Place it in a shaker, set the temperature to 30℃ and the speed to 180r / min for acclimation. One cycle is 3 days. After each cycle, let the culture medium in the bottle stand for a while, pour out the supernatant, and then add 200mL of fresh culture medium for acclimation. Acclimation is repeated twice. Take 1.00mL of the second acclimation solution and dilute it to 10-1 , 10 -3 , 10 -5 and 10 -7 The strain was then spread on a denitrification plate medium and cultured in a biochemical incubator at 30°C for 3 days. Single colonies with different morphologies were selected and streaked onto a denitrification solid medium using the plate streak method. The strain was then cultured under the same conditions for 3 days. Single colonies were then selected and streaked repeatedly until a pure strain was obtained.

[0048] Denitrification medium: CH3COONa 5.12 g, KNO3 0.722 g, MgSO4·7H2O 0.2 g, KH2PO4 1.0 g, K2HPO4 5.0 g, NaCl 0.5 g, trace element solution 1 mL, distilled water 999 mL, pH = 7.4.

[0049] Trace element solution: EDTA 50 g, CaCl2 5.5 g, ZnSO4 2.2 g, MnCl2·4H2O 5.06 g, FeSO4·7H2O 5.0 g, (NH4)6MO7O2·4H2O 1.1 g, CuSO4·5H2O 1.57 g, CoCl2·6H2O 1.61 g, distilled water 1000 mL, pH = 7.0.

[0050] 2. Strain identification

[0051] 1) Strain DD15 grows rapidly on LB solid plates, showing a light yellow color, with smooth colonies, neat edges, and a moist and translucent surface (e.g. Figure 1 shown).

[0052] 2) By sequencing the DD15 16S rRNA and comparing the sequence in NCBI, the strain had a similarity of 99.86% with Acinetobacter sp., accession number MW287273.1, and a phylogenetic tree was constructed based on the 16S rRNA gene. The results showed that the strain was most closely related to Acinetobacter tandoii (e.g. Figure 2 The strain DD15 was identified as Acinetobacter tandoii, abbreviated as Acinetobacter tandoii DD15.

[0053] The 16SrRNA identification results of Acinetobacter tankii DD15 are as follows:

[0054]

[0055] 3. Preservation of Acinetobacter tankii DD15 strain

[0056] Acinetobacter tankani DD15 was deposited with the China General Microbiological Culture Collection on June 9, 2022, under the accession number CGMCC No. 25032. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. It is hereafter referred to as Acinetobacter tankani CGMCC No. 25032 or strain DD15.

[0057] 4. Analysis of growth characteristics of strain DD15

[0058] Acinetobacter tankii DD15 was activated overnight in LB medium and inoculated into different carbon source medium at 1% addition rate. LB medium was used as the control group. Each experimental group had 3 parallels, cultured at 30°C and 200 rpm, and samples were taken every 2 hours to measure OD. 600 .

[0059] Culture medium: 2 g / L potassium hydrogen phosphate; 0.6 g / L magnesium sulfate; 10 g / L peptone; 2 g / L carbon source (carbon source types are: glucose; sucrose; maltose; lactose; soluble starch; molasses).

[0060] The experimental results are as follows Figure 3 As shown in the results, DD15 has a wide range of carbon energy utilization capabilities and can grow in a variety of carbon sources, among which molasses is the best carbon source for growth.

[0061] Example 2: Nitrogen reduction ability of strain DD15

[0062] 1. Determination of nitrogen reduction ability of strain DD15

[0063] The strain DD15 was inoculated into sterilized LB medium and cultured in a shaker at 30°C and 180 rpm for 24 h. The cells were collected by centrifugation at 12000 rpm for 2 min, dissolved in 0.01 M PBS (pH 7.2-7.4), and serially diluted to obtain a concentration of 10 8 CFU / mL of DD15 bacterial agent.

[0064] Then press 10 7 The inoculum size of CFU / mL was inoculated into Erlenmeyer flasks containing appropriate amounts of heterotrophic nitrification and aerobic denitrification culture medium, with an initial concentration of ammonia nitrogen and nitrite nitrogen of 20 mg / L. The culture was carried out at 30 °C and 120 r / min. Two parallel experiments were conducted. The same volume of culture medium was added as a control. The NH4 in the culture medium was measured at 6 h, 12 h, and 24 h. + -N, NO2 - -N content.

[0065] Nitrification medium: CH3COONa 1.024 g, (NH4)2SO4 0.0944 g, MgSO4·7H2O 0.2 g, KH2PO4 1.0 g, K2HPO4 5.0 g, NaCl 0.5 g, trace element solution 1 mL, distilled water 999 mL, pH = 7.4.

[0066] Denitrification medium: CH3COONa 1.024 g, NaNO2 0.0986 g, MgSO4·7H2O 0.2 g, KH2PO4 1.0 g, K2HPO4 5.0 g, NaCl 0.5 g, trace element solution 1 mL, distilled water 999 mL, pH = 7.4.

[0067] Trace element solution: EDTA 50g, CaCl2 5.5g, ZnSO4 2.2g, MnCl2·4H2O 5.06g, FeSO4·7H2O 5.0g, (NH4)6MO7O2·4H2O 1.1g, CuSO4·5H2O 1.57g, CoCl2·6H2O 1.61g, distilled water 1000mL, pH=7.0.

[0068] The experimental results are as follows Figure 4 As shown in the figure, the degradation rate of ammonia nitrogen and nitrite nitrogen of DD15 was 99% in 6 hours, which showed that DD15 had a high nitrogen reduction ability.

[0069] 2. Effect of different C / N ratios on nitrogen reduction capacity

[0070] According to the experimental method for determining the nitrogen reduction capacity in step 1, adjust the amount of carbon source added to the nitrification medium and the denitrification medium, with 3 parallels for each experimental group, so that C / N = 5, 10, 15, 20, and measure the concentrations of ammonia nitrogen and nitrite nitrogen after 3h, 6h, 9h, and 12h of cultivation.

[0071] The experimental results are as follows Figure 5 A and Figure 6As shown in Figure (a), strain DD15 achieved nitrogen reduction efficiencies ranging from 97% to 100% at initial carbon-nitrogen ratios (C / N) of 10, 15, and 20 after 6 h of cultivation. Ammonia degradation rates were 97.4%, 98.2%, and 98.2%, respectively, and nitrite degradation rates were 97.0%, 100%, and 100%, respectively. Reducing the C / N ratio to 5 significantly affected the nitrogen reduction rate of strain DD15, with significant reductions in ammonia and nitrite within 3 h of cultivation. However, no significant reductions were observed beyond 3 h of cultivation. Within 3 h, ammonia degradation rates were 48.4%, and nitrite degradation rates were 61.5%. These results indicate that the C / N ratio significantly influences the nitrogen reduction capacity of strain DD15. The C / N ratio range for efficient nitrogen reduction for strain DD15 is 10-20. At low C / N ratios (C / N = 5), strain DD15 exhibits nitrogen reduction capacity, but its efficiency is significantly reduced.

[0072] 3. Effect of different temperatures on nitrogen reduction capacity

[0073] According to the culture medium and experimental method for determining the nitrogen reduction ability in step 1, different culture temperatures were set at 15°C, 20°C, 25°C, 30°C, and 35°C. Each experiment was repeated three times, and the concentrations of ammonia nitrogen and nitrite nitrogen were measured after 3 h, 6 h, 9 h, and 12 h of culture.

[0074] The experimental results are as follows Figure 5 Zhongb and Figure 6 As shown in Figure b, DD15 achieved nitrogen reduction efficiencies ranging from 94.8% to 100% at 25°C, 30°C, and 35°C for 6 hours. Ammonia degradation rates were 96.8%, 99%, and 98.4%, respectively, and nitrite degradation rates were 94.8%, 99.9%, and 100%, respectively, within 6 hours. When the temperature dropped to 15°C and 20°C, DD15's nitrogen reduction rate slowed, with ammonia degradation rates reaching 34.6% and 39.9% after 6 hours and 92.3% and 99.2% after 12 hours, respectively. Nitrite degradation rates were 64.6% and 56.6% after 6 hours and 97.4% and 100% after 12 hours, respectively. These results indicate that although the nitrogen reduction rate slowed at temperatures between 15 and 20°C, strain DD15 maintained a high nitrogen reduction efficiency after 12 hours, demonstrating its adaptability to a wide range of temperatures (15-35°C).

[0075] 4. Effect of different salinities on nitrogen reduction capacity

[0076] According to the culture medium and experimental method for determining the nitrogen reduction ability in step 1, different culture salinities of 0‰, 5‰, 10‰, 20‰, and 30‰ were set during the culture, with 3 parallel experiments for each. The concentrations of ammonia nitrogen and nitrite nitrogen were measured after 3h, 6h, 9h, and 12h of culture, and the nitrogen reduction efficiency was calculated.

[0077] The experimental results are as follows Figure 5 Middle C and Figure 6 As shown in Figure c, at salinities of 0‰, 5‰, and 10‰, DD15 achieved a nitrogen reduction efficiency of 95.8%-99.9% after 6 hours of incubation. Ammonia degradation rates were 99.2%, 99.9%, and 97.8%, respectively, and nitrite degradation rates were 98.3%, 95.9%, and 96.9%, respectively. When salinity increased to 20‰ and 30‰, DD15's nitrogen reduction rate decreased. Ammonia degradation rates were 99.3% and 57.2% after 6 hours of incubation, and 99.5% and 98.2% after 12 hours. Nitrite degradation rates were 72.5% and 42.5% after 6 hours of incubation, and 98.8% and 93.9% after 12 hours of incubation, respectively. The results showed that although the nitrogen reduction rate decreased at a salinity of 20‰-30‰, it still maintained a high nitrogen reduction efficiency within 12 hours, and DD15 was able to adapt to a wide range of salinities (0‰-30‰).

[0078] 5. Effect of different pH on nitrogen reduction capacity

[0079] According to the experimental method for determining the nitrogen reduction capacity in step 1, set different pH values to 5, 6, 7, 8, and 9, and perform 3 parallel experiments for each experiment. Measure the concentrations of ammonia nitrogen and nitrite nitrogen after 3 h, 6 h, 9 h, and 12 h of incubation, and calculate the nitrogen reduction efficiency.

[0080] The experimental results are as follows Figure 5 Zhongd and Figure 6 As shown in (d), the nitrogen reduction efficiency of DD15 after 6 h of incubation at pH values of 7, 8, and 9 ranged from 85.2% to 99.9%. The ammonia degradation rates were 98.5%, 98.6%, and 99.0%, respectively, and the nitrite degradation rates were 99.9%, 99.9%, and 85.2%, respectively. At pH values of 5-6, the nitrogen reduction rate and efficiency of DD15 decreased. At pH 6, the ammonia degradation efficiency was 93.2% after 12 h of incubation, but at pH 5, the degradation efficiency was only 32.4%. At pH 6, the degradation efficiency of nitrite was 45.4%, and at pH 5, the degradation efficiency of ammonia was 53.7%. The results showed that the nitrogen reduction rate of DD15 under neutral to alkaline conditions (pH 7-9) was higher than that in acidic environment (pH 5-6), and the ammonia nitrogen degradation efficiency had a wider pH range, so DD15 could adapt to a wide pH range (pH 6-9).

[0081] 6. Effect of different inoculation amounts on nitrogen reduction capacity

[0082] According to the experimental method of nitrogen reduction capacity determination in step 1, different inoculation amounts were set 10 3 CFU / mL, 10 4 CFU / mL, 105 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, three parallels for each experiment, the concentrations of ammonia nitrogen and nitrite nitrogen at 3h, 6h, 9h, and 12h of culture were measured, and the nitrogen reduction efficiency was calculated.

[0083] The experimental results are as follows Figure 5 Zhongehe Figure 6 As shown in Figure e, DD15 has an inoculum size of 10 7 CFU / mL, the nitrogen reduction rate was the fastest. After 3 hours of culture, the ammonia nitrogen degradation rate was 96.9%, and the nitrite nitrogen degradation rate was 99.4%. With the decrease of the initial inoculum size, the nitrogen reduction rate of DD15 gradually decreased. 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL inoculation amount, the nitrogen reduction efficiency of ammonia nitrogen was 99.1%, 99.6%, 97.7%, and 92.3% after 12 hours of culture, and the nitrogen reduction efficiency of nitrite nitrogen was 100%, 97.3%, 80.5%, and 73.3% respectively. The results showed that at the minimum inoculation amount of 10 3 CFU / mL for 12 h, it still maintained a high nitrogen reduction efficiency, and DD15 could also have a high nitrogen reduction ability at a low inoculation amount.

[0084] Example 3: Study on the production of signal molecule DSF quenching enzyme by strains

[0085] 1. Determination of the production of signal molecule DSF quenching enzyme by strain DD15

[0086] The strain DD15 was activated overnight in LB liquid medium, and 2 μL of the bacterial solution was dropped onto the MM plate medium containing 5 mM DSF signal molecule. The plate was incubated at 30°C for 2 days, and the transparent zone produced by DSF degradation was observed. Figure 7 As shown, a clear transparent circle appeared in the DD15 colony, indicating that the bacteria had the ability to produce DSF signal molecule quenching enzyme.

[0087] MM inorganic salt culture medium (1L): K2HPO4: 10.5g; KH2PO4: 4.5g; (NH4)2SO4: 2.0g; MgSO4·7H2O: 0.2g; FeSO4: 0.005g; CaCl2: 0.01g; MnCl2: 0.002g.

[0088] 2. Determination of quenching enzyme activity produced by strain DD15

[0089] Strain DD15 was activated overnight and inoculated into LB liquid medium for 24 hours. The supernatant was centrifuged and filtered through a 0.22 μm filter. 100 μL of the supernatant was transferred to a 1.5 mL EP tube (an equal volume of the supernatant from the heat-inactivated culture was used as a control). 50 μL of a 20 mM sodium oleate stock solution and other reagents (1 M Tris–HCl pH 7.4, 0.5 M MgCl2, 10% Triton X-100, 1 M DTT, 0.1 M ATP) were added to prepare a 500 μL reaction system. A standard curve was generated using a gradient addition of 5 mM sodium oleate as the substrate. The reaction was incubated at 37°C in a water bath for 90 minutes. An equal volume of methanol was added to terminate the reaction, and the supernatant was filtered through a 0.22 μm filter for analysis.

[0090] The concentration of sodium oleate in the supernatant of the bacterial culture was determined using high-performance liquid chromatography (HPLC). The detection conditions were: a C18 column (4.6 mm × 250 mm, 5 μm), an organic phase of acetonitrile and an aqueous phase of 0.1% phosphoric acid (88:12), a flow rate of 1 mL / min, a column temperature of 30°C, a measurement wavelength of 203 nm, an injection volume of 20 μL, and an HPLC detection time of 20 min. Enzyme activity was defined as the amount of enzyme that converts 1 μmol of fatty acid to acyl-CoA per minute at 37°C.

[0091] According to the standard curve formula (Y = 1380112.8X - 15200.8, R 2 =0.99) and calculated the enzyme activity per liter of DD15 bacterial supernatant to be 2.8 U.

[0092] Example 4: Safety evaluation of bacterial strains

[0093] 1. Hemolytic test

[0094] The strain DD15 was activated by overnight culture, and 2 μL of bacterial solution and bacterial solution supernatant (filtered with 0.22 μm filter membrane) were spotted on 5% sheep blood plates and cultured at 30° C. for 24 h.

[0095] The results are as follows Figure 8 As shown, neither the DD15 bacterial suspension nor the supernatant produced a hemolytic zone and was non-hemolytic.

[0096] 2. Zebrafish Safety Assessment

[0097] (1) Preparation of bacterial agents: strain DD15 was cultured in LB medium for 20 h, centrifuged at 12,000 rpm for 2 min, and the supernatant was discarded. The bacterial pellet was washed twice with PBS and resuspended in PBS. The pellet was then serially diluted and plated for counting.

[0098] (2) Zebrafish preparation: Zebrafish were hatched for 4 days and placed in 6-well plates. Each well contained 10 mL of filtered water. Ten fish were placed in each well. The zebrafish were allowed to adapt to the conditions for 12 h before the experiment.

[0099] (3) Experimental group: On the fifth day of zebrafish hatching, DD15 bacterial agent was administered at 10 5 , 10 6 , 10 7 The amount of CFU / mL was added to 10mL of culture water, with 3 parallels in each group, and the mortality rate was recorded every 4 hours.

[0100] The results are as follows Figure 9 As shown in the figure, no zebrafish death was observed until 72 hours after the mortality statistics were collected. The DD15 bacterial agent had no effect on the mortality of zebrafish larvae.

[0101] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Acinetobacter tenella, characterized in that: The Acinetobacter tankii is Acinetobacter tankii ( Acinetobacter tandoii ) CGMCC No.25032, and its registration number in the General Microbiology Center of China Culture Collection Administration is CGMCC No.25032.

2. A bacterial agent, characterized in that: The bacterial agent contains the Acinetobacter tankini described in claim 1.

3. The culture of Acinetobacter tankini according to claim 1, which is obtained by culturing the Acinetobacter tankini according to claim 1 in a bacterial culture medium.

4. Use of the Acinetobacter tankini of claim 1, the bacterial agent of claim 2, or the culture of claim 3 in at least one of the following: (a1) Degradation of ammonia nitrogen; (a2) preparing products for degradation of ammonia nitrogen; (a3) degradation of nitrite nitrogen; (a4) preparing products for the degradation of nitrite nitrogen; (a5) Reduce the ability of quorum sensing signal molecules in water; The quorum sensing signal molecule is a DSF signal molecule.

5. A method for preparing the bacterial agent according to claim 2, comprising the following steps: using the Acinetobacter tankini described in claim 1 as an active ingredient to obtain the bacterial agent.

6. A bioremediation agent, characterized in that: The bioremediation agent contains the Acinetobacter tankini described in claim 1 or the bacterial agent described in claim 2 or the culture described in claim 3.

7. A method for efficiently degrading ammonia nitrogen and nitrite nitrogen in a water environment, comprising the steps of applying the Acinetobacter tankii of claim 1, the bacterial agent of claim 2, or the culture of claim 3 to the water environment, thereby degrading ammonia nitrogen and nitrite nitrogen in the water.

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