Strain suitable for efficient denitrification under florfenicol stress and application thereof

Abstract

The invention discloses a strain suitable for efficient denitrification under the stress of florfenicol and application of the strain. According to the present invention, the Acinetobacter calcoaceticus ND10 with characteristics of broad-spectrum antibiotic florfenicol resistance and good nitrogen removal ability is obtained through screening, and the Acinetobacter calcoaceticus ND10 can grow in the florfenicol-containing water body, and can further perform efficient nitrogen removal on the nitrogen element in the water body. The acinetobacter calcoaceticus ND10 has the potential of being developed into a pond culture water denitrification microbial agent, is expected to be applied to denitrification treatment of culture water, and has a good application prospect for solving the problem that nitrite ammonia nitrogen in florfenicol-containing water exceeds the standard.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain suitable for efficient nitrogen removal under florfenicol stress and application thereof. Background Art

[0002] In pond aquaculture water, when the content of ammonia nitrogen and nitrite is high, it will be toxic to fish, affect fish health and reduce aquaculture benefits. The main source of ammonia nitrogen is the metabolism of compound feed and aquatic organisms, among which free ammonia is the main factor causing aquatic organism toxicity. In high temperature seasons, on the one hand, the food intake of fish usually increases, the amount of feed added increases, the dissolved oxygen in the water will decrease, and the ammonia nitrogen content may increase; on the other hand, the light in summer is strong, the photosynthesis of phytoplankton in the water is strong, and the consumption of carbon dioxide will be more than other seasons. When the carbon dioxide in the water is consumed too much, the pH of the water will rise, thereby prompting the ammonia nitrogen to change from the form of ammonium ions to free ammonia, increasing the load of free ammonia in the water. Nitrite is an intermediate product in the process of ammonia conversion to nitrate. Its main hazard is to inhibit the oxygen carrying capacity of the blood, causing tissue hypoxia, and hypoxia and death when the fish body is in a metabolically active state and needs a lot of oxygen. In water bodies that have been in a low oxygen state for a long time, or in water bodies after heavy rain, the nitrite content is very easy to exceed the standard, causing fish death. Therefore, reducing the ammonia nitrogen and nitrite content in water is an important issue in aquaculture.

[0003] Compared with physical or chemical denitrification, biological denitrification is considered the most promising denitrification technology because it is environmentally friendly and has no secondary pollution. However, the effects of denitrification bacteria agents for aquaculture water on the market are uneven, and there are few products with obvious effects. This is probably related to the fact that the complexity of the aquaculture water environment is not taken into account.

[0004] The aquaculture pond system is a special type of water system because it must be artificially fed with a large amount of exogenous nitrogen, phosphorus and other nutrients, which changes the composition of nitrogen, phosphorus and other elements in the water system under natural conditions. Especially in recent years, with the increasing density of aquaculture, some veterinary antibiotics have been intermittently added to prevent the occurrence of fish diseases. Florfenicol is a commonly used antibiotic in aquaculture. Although it is mostly administered in the form of mixing with feed, after metabolism, a part of florfenicol is still excreted from the body with animal excrement, resulting in florfenicol residues in aquaculture water. Florfenicol is a broad-spectrum antibacterial drug. The residual florfenicol in the water often inhibits the activity of exogenously added bacterial agents, affecting the denitrification effect of denitrifying bacterial agents used in aquaculture water. Therefore, efficient denitrifying bacteria that can grow under the stress of florfenicol will have broad application prospects in the preparation of effective bacterial agents. Summary of the invention

[0005] The purpose of the present invention is to provide a strain suitable for efficient denitrification under florfenicol stress and its application. The strain belongs to a new species of bacteria of the genus Acinetobacter, can grow in water containing florfenicol and has efficient denitrification performance, so it is expected to be developed into a denitrification microbial agent for pond aquaculture water, thereby helping to solve the problems of excessive nitrite and ammonia nitrogen in water containing florfenicol.

[0006] In order to solve the above technical problems and achieve the above technical effects, the present invention is implemented through the following technical solutions:

[0007] The present invention obtains a Gram-negative bacterium which can grow in florfenicol-containing water and has high-efficiency denitrification performance by repeatedly screening sludge from a sewage treatment plant in Suzhou City, Jiangsu Province.

[0008] This strain was selected for morphological observation, biological characteristics research, taxonomic status determination, and growth medium optimization. 16SrDNA identification revealed that the strain belonged to a new species of the genus Acinetobacter sp. and was named Acinetobacter calcoaceticus ND10.

[0009] The calcoacetic acid Acinetobacter ND10 selected by the present invention can grow in water containing florfenicol and efficiently denitrify nitrogen in the water, so it has both the ability to resist the broad-spectrum antibiotic florfenicol and good denitrification ability.

[0010] The present invention provides a strain of Acinetobacter calcoaceticus ND10 suitable for efficient denitrification under florfenicol stress. The Acinetobacter calcoaceticus ND10 has been deposited in the China General Microbiological Culture Collection Center (CGMCC) on May 13, 2024, with a deposit number of CGMCC NO.30544, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] The 16SrDNA sequence of the calcoacetic acid Acinetobacter ND10 is shown in SEQ ID NO:1.

[0012] The present invention provides a microbial agent containing the above-mentioned Acinetobacter calcoaceticus ND10.

[0013] Furthermore, the microbial agent contains living cells of Acinetobacter calcoaceticus ND10 bacteria, dry bacteria of Acinetobacter calcoaceticus ND10 obtained by freeze-drying, immobilized cells of Acinetobacter calcoaceticus ND10, liquid agent of Acinetobacter calcoaceticus ND10, solid agent of Acinetobacter calcoaceticus ND10, or Acinetobacter calcoaceticus ND10 strains in any other form.

[0014] The present invention also provides the use of the above-mentioned Acinetobacter calcoaceticus ND10 or the above-mentioned microbial agent in the treatment of aquaculture water.

[0015] Furthermore, the application is to degrade nitrogen in aquaculture water containing 0.001-10 mg / L of florfenicol.

[0016] Furthermore, the application is to degrade ammonia nitrogen and nitrite in aquaculture water containing 0.001-10 mg / L of florfenicol.

[0017] Furthermore, the application is to first culture the calcoacetic acid Acinetobacter ND10, then collect the bacteria, and finally inoculate the collected calcoacetic acid Acinetobacter ND10 bacteria into the florfenicol-containing aquaculture water body, so that the calcoacetic acid Acinetobacter ND10 bacteria are cultured and propagated in the florfenicol-containing aquaculture water body while the ammonia nitrogen and nitrite nitrogen therein are effectively removed.

[0018] Furthermore, the culture is carried out at 30° C. and 120-160 rpm shaking conditions for 12-48 hours.

[0019] Furthermore, the bacteria are collected by centrifuging the cultured Acinetobacter calcoaceticus ND10 at 6000-8000 rpm for 8-10 minutes and discarding the supernatant.

[0020] Furthermore, the culture and propagation is carried out at 30°C and 120-160 rpm shaking conditions for 24-48 hours.

[0021] The beneficial effects of the present invention are:

[0022] The Acinetobacter calcoaceticus ND10 screened by the present invention can not only grow in water containing florfenicol, but also efficiently denitrify nitrogen in the water, has both the ability to resist the broad-spectrum antibiotic florfenicol and good denitrification ability, so the strain has the potential to be developed as a denitrification microbial agent for pond aquaculture water, and has good application prospects for solving the problems of excessive nitrite and ammonia nitrogen in water containing florfenicol.

[0023] Biomaterial Deposit

[0024] The Acinetobacter calcoaceticus ND10 of the present invention has been deposited in the China General Microbiological Culture Collection Center on May 13, 2024, with a taxonomic name of Acinetobactercalcoaceticus ND10, a deposit number of CGMCC NO.30544, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0027] Figure 1 The cell morphology of the Acinetobacter calcoaceticus ND10 screened by the present invention observed under an optical microscope;

[0028] Figure 2 A phylogenetic tree constructed from the 16SrDNA gene of Acinetobacter calcoaceticus ND10 screened by the present invention;

[0029] Figure 3 The denitrification performance test results of the Acinetobacter calcoaceticus ND10 screened by the present invention in a culture medium with nitrite nitrogen as the only nitrogen source;

[0030] Figure 4 The denitrification performance test results of the Acinetobacter calcoaceticus ND10 screened by the present invention in a culture medium with ammonia nitrogen as the only nitrogen source;

[0031] Figure 5 The denitrification performance test results of the Acinetobacter calcoaceticus ND10 screened in the present invention in a mixed nitrogen source medium;

[0032] Figure 6 The denitrification performance test results of the Acinetobacter calcoaceticus ND10 screened in the present invention under florfenicol stress conditions;

[0033] Figure 7 This is the test result of the maximum tolerance concentration of the calcoacetic acid Acinetobacter ND10 screened in the present invention in the culture medium with ammonia nitrogen as the only nitrogen source. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings so that the purpose, features and advantages of the invention can be more clearly understood. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0035] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0036] Unless the context requires otherwise, throughout the specification and claims, the word "comprise" and variations such as "include" and "have" should be construed in an open, inclusive sense, ie, should be interpreted as "including, but not limited to."

[0037] References throughout the specification to "one embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0038] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise.

[0039] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0040] The present invention has repeatedly screened the sludge from a sewage treatment plant in Suzhou, Jiangsu Province, and finally isolated a Gram-negative bacterium that can grow in water containing florfenicol and has high denitrification performance. The specific screening process is:

[0041] 10% of the sludge from the above-mentioned sewage treatment plant was inoculated into heterotrophic nitrification enrichment medium at 30°C and 150r / min shaking culture for 48h to enrich the target bacteria, and the culture was transferred twice in succession. Take 1mL of the enrichment culture solution for gradient dilution plate coating, culture at 30°C for 24h, pick a single colony, and test the ability to degrade ammonia nitrogen. After multiple rounds of separation and purification, 13 strains were obtained, numbered ND01~13. The heterotrophic nitrification ability of the above strains was tested, and the 10th strain (ND10) with better ammonia nitrogen degradation effect was determined for subsequent experiments.

[0042] First, this strain was selected for morphological observation. The morphological characteristics of the strain are as follows:

[0043] After culturing this strain on LB solid medium at 30°C for 24 hours, it grew into yellow-green round colonies with neat edges and a moist and smooth surface. Figure 1 As shown, under a microscope, the bacteria appear spherical after Gram staining.

[0044] Then the taxonomic status of the strain was determined, and the results of its species classification are as follows:

[0045] The effective sequence length of the 16S rDNA gene of this strain is 1402 bp, see Figure 2 As shown, through BLAST sequence homology comparison and construction of phylogenetic evolutionary tree, the strain was identified as a new species of Acinetobacter sp. and was named Acinetobacter calcoaceticus ND10.

[0046] The 16SrDNA sequence of the calcoacetic acid Acinetobacter ND10 is shown in SEQ ID NO:1.

[0047] SEQ ID NO: 1:

[0048]

[0049] Then, the biological characteristics of the Acinetobacter calcoaceticus ND10 were studied, and its physiological and biochemical characteristics are shown in Table 1:

[0050] Table 1 Results of physiological and biochemical identification of ND10

[0051]

[0052] ″+″-positive, ″-″-negative

[0053] The results of physiological and biochemical identification showed that the experimental results of the citric acid test, nitrate reduction test, glucose test and urea test of the calcoacetic acid Acinetobacter ND10 were all positive, and the experimental results of the esculin test, indigo matrix test, ONPG test, maltose test, xylose test, sucrose test and hydrogen sulfide test were all negative.

[0054] The calcoacetic acid Acinetobacter ND10 screened in the present invention was deposited in the China General Microbiological Culture Collection Center (CGMCC) on May 13, 2024, with a deposit number of CGMCCNO.30544, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0055] The present invention carried out the following nitrogen-containing water denitrification experiment on the screened Acinetobacter calcoaceticus ND10.

[0056] Example 1 Experiment on water bodies with nitrite as the only nitrogen source

[0057] A culture medium with nitrite nitrogen as the sole nitrogen source was prepared (Na2HPO4 7.9 g, KH2PO4 1.5 g, MgSO4·7H2O 0.1 g, trace elements 2 mL, NaNO2 100 mg, sodium succinate 9.4 g, pH = 7.0-7.5), and the initial nitrite nitrogen concentration was 21.69 mg / L.

[0058] Pick calcoacetic acid Acinetobacter ND10 from the slant and inoculate it into a 250 mL conical flask containing 50 mL LB medium (10 g tryptone, 5 g yeast powder, 5 g sodium chloride, 1 L distilled water, pH = 7.0), culture at 30 ° C, 120-160 rpm shaking conditions for 12-48 h, centrifuge at 6000-8000 rpm for 8-10 min (preferably culture at 30 ° C, 160 rpm shaking conditions for 12 h, centrifuge at 6000 rpm for 10 min), discard the supernatant and collect the bacteria.

[0059] Resuspend the bacteria in sterilized nitrite medium and transfer to a 150 mL conical flask containing 50 mL of medium with nitrite nitrogen as the sole nitrogen source. Culture and propagate at 30°C and 120-160 rpm for 24-48 h (preferably at 30°C and 160 rpm for 24 h). Take the culture medium and measure the OD value of the culture medium. 600 , and centrifuged at 8000rpm for 10min, and the supernatant was taken to determine TN and NO 2- -N content.

[0060] See also Figure 3 As shown, by measuring TN and NO 2- -N content, the results showed that in the culture medium with nitrite nitrogen as the only nitrogen source, the nitrite nitrogen removal rate was 100%.

[0061] Example 2 Denitrification experiment for water bodies with ammonia nitrogen as the only nitrogen source

[0062] A culture medium with ammonium sulfate as the sole nitrogen source (5 g CH3COONa, 50 mL Wei's salt solution, 0.5 g (NH4)2SO4, 1000 mL distilled water, pH = 7.5-8.0) was prepared, and the initial ammonia nitrogen concentration was 108.7 mg / L.

[0063] Pick calcoacetic acid Acinetobacter ND10 from the slant and inoculate it into a 250 mL conical flask containing 50 mL LB medium (10 g tryptone, 5 g yeast powder, 5 g sodium chloride, 1 L distilled water, pH = 7.0), culture at 30 ° C, 120-160 rpm shaking conditions for 12-48 h, centrifuge at 6000-8000 rpm for 8-10 min (preferably culture at 30 ° C, 160 rpm shaking conditions for 12 h, centrifuge at 6000 rpm for 10 min), discard the supernatant and collect the bacteria.

[0064] Resuspend the bacteria in sterilized ammonium sulfate medium and transfer them to a 150 mL conical flask containing 50 mL of medium with ammonium sulfate as the sole nitrogen source. Cultivate and propagate at 30°C and 120-160 rpm for 24-48 h (preferably at 30°C and 160 rpm for 24 h). Take the culture medium and measure the OD value of the culture medium. 600 , and centrifuged at 8000 rpm for 10 min, and the supernatant was taken to determine NO 3- -N, NO 2- -N, NH 4+ -N content.

[0065] See also Figure 4 As shown, by measuring NO 3- -N, NO 2- -N, NH 4+-N content, the results showed that in the water body where ammonia nitrogen was the only nitrogen source, the removal rate of ammonia nitrogen in 48 hours was 97.2%. There was almost no accumulation of nitrite during the experiment.

[0066] Example 3 Denitrification experiment on water bodies with mixed nitrogen sources

[0067] Prepare a mixed nitrogen source water culture medium (NaAc 281 mg, K2HPO4 2.83 mg, NH4Cl11.4 mg, NaNO316.6 mg, NaNO2 9.86 mg, MgSO4·7H2O 50.0 mg, CaCl2 20.0 mg, FeSO4·7H2O 40.0 mg, NaHCO340.0 mg, distilled water 1 L, pH = 7.0-7.5).

[0068] Pick calcoacetic acid Acinetobacter ND10 from the slant and inoculate it into a 250 mL conical flask containing 50 mL LB medium (10 g tryptone, 5 g yeast powder, 5 g sodium chloride, 1 L distilled water, pH = 7.0), culture at 30 ° C, 120-160 rpm shaking conditions for 12-48 h, centrifuge at 6000-8000 rpm for 8-10 min (preferably culture at 30 ° C, 160 rpm shaking conditions for 12 h, centrifuge at 6000 rpm for 10 min), discard the supernatant and collect the bacteria.

[0069] Resuspend the bacteria in sterilized mixed nitrogen source water and inoculate into a 150 mL conical flask containing 50 mL of mixed nitrogen source water culture medium. Cultivate and propagate at 30 ° C and 120-160 rpm shaking conditions for 24-48 h (preferably at 30 ° C and 160 rpm shaking conditions for 48 h). Take the culture solution and measure the OD of the culture solution. 600 , and centrifuged at 8000 rpm for 10 min, and the supernatant was taken to determine NO 3- -N, NO 2- -N, NH 4+ -N content.

[0070] See also Figure 5 As shown, by measuring NO 3- -N, NO 2- -N, NH 4+ -N content. The results showed that in water bodies with mixed nitrogen sources, the 48-hour removal rate of nitrate nitrogen was 96.8%, the 48-hour removal rate of nitrite nitrogen was 99.8%, and the 48-hour removal rate of ammonia nitrogen was 83.5%.

[0071] Example 4 Experiment on the effect of nitrogen degradation in simulated aquaculture tail water

[0072] Considering the presence of florfenicol as a conventional veterinary drug in water, simulated aquaculture tail water (SAEF) was prepared according to the following formula based on aquaculture practices:

[0073] NaAc 281mg, K2HPO4 2.83mg, NH4Cl 11.4mg, NaNO3 16.6mg, NaNO29.86mg, MgSO4·7H2O 50.0mg, CaCl2 20.0mg, FeSO4·7H2O 40.0mg, NaHCO340.0mg, florfenicol 10mg / L, distilled water 1L, pH=7.0~7.5.

[0074] Pick calcoacetic acid Acinetobacter ND10 from the slant and inoculate it into a 250 mL conical flask containing 50 mL LB medium (10 g tryptone, 5 g yeast powder, 5 g sodium chloride, 1 L distilled water, pH = 7.0), culture at 30 ° C, 120-160 rpm shaking conditions for 12-48 h, centrifuge at 6000-8000 rpm for 8-10 min (preferably culture at 30 ° C, 160 rpm shaking conditions for 12 h, centrifuge at 6000 rpm for 10 min), discard the supernatant and collect the bacteria.

[0075] The cells were resuspended with sterilized simulated aquaculture tail water and inoculated into a 150 mL conical flask containing 50 mL simulated aquaculture tail water. The cells were cultured and propagated at 30 ° C and 120-160 rpm for 24-48 h (preferably at 30 ° C and 160 rpm for 24 h). The culture fluid was taken to measure the OD value of the culture fluid. 600 , and centrifuged at 8000 rpm for 10 min, and the supernatant was taken to determine NO 3- -N, NO 2- -N, NH 4+ -N and TN content.

[0076] Table 2 Effect of ND10 on nitrogen degradation in simulated aquaculture tail water

[0077]

[0078] Note: SAEF contains simulated aquaculture tail water of florfenicol: SAE does not contain simulated aquaculture tail water of florfenicol

[0079] See also Figure 6 As shown in Table 2, by measuring NO 3- -N, NO 2- -N, NH 4+ -N and TN content. The results showed that in the simulated aquaculture tail water with the addition of 10 mg / L florfenicol, calcium-acetic acid Acinetobacter ND10 still showed a high denitrification efficiency.

[0080] The present invention also measured the maximum tolerance concentrations of ammonia nitrogen and nitrite nitrogen for the screened Acinetobacter calcoaceticus ND10. The specific process and results are as follows:

[0081] After activating and culturing the calcoacetic acid Acinetobacter ND10 strain for 12 h, the strain was inoculated into NH 4+ -N mass concentrations of 100mg / L, 500mg / L, 1000mg / L, 2000mg / L, 5000mg / L and 10000mg / L simulated aquaculture tail water (NaAc 281mg, K2HPO42.83mg, NaNO316.6mg, NaNO29.86mg, MgSO4·7H2O 50.0mg, CaCl220.0mg, FeSO4·7H2O40.0mg, NaHCO340.0mg, distilled water 1L, pH=7.0-7.5) were cultured in a 30℃ constant temperature shaker. Other variables were the same, and 3 parallels were set for each group. Samples were taken at 24h and 48h, the optical density of the bacterial solution was observed, and the NH in the culture solution was determined. 4+ -N content.

[0082] See also Figure 7 As shown, the results show that NH 4+ When the -N concentration reached 100 mg / L, the degradation rate of Acinetobacter calcoaceticus ND10 was still high after 48 h, while when NH 4+ When the -N concentration reached 5 g / L, the calcoacetic acid Acinetobacter ND10 still showed a significant degradation rate after 48 h. These results show that the calcoacetic acid Acinetobacter ND10 screened by the present invention can tolerate 5 g / L NH 4+ -N concentration.

[0083] After activating and culturing the ND10 strain of Acinetobacter calcoaceticus for 12 h, the strain was inoculated into NO 2 --N mass concentration of 100mg / L and 500mg / L simulated aquaculture tail water (NaAc 281mg, K2HPO4 2.83mg, NH4Cl 11.4mg, NaNO3 16.6mg, MgSO4·7H2O 50.0mg, CaCl2 20.0mg, FeSO4·7H2O 40.0mg, NaHCO3 40.0mg, distilled water 1L, pH = 7.0-7.5), 30℃ constant temperature shaker culture, other variables are the same, each group is set up 3 parallel. Sampling was carried out at 24h and 48h of culture, and the optical density of the bacterial liquid was observed and the NO in the culture liquid was determined 2 --N content.

[0084] As shown in Table 3, according to the observation of NO 2When the concentration of --N was 100 mg / L and 500 mg / L, the growth of Acinetobacter calcoaceticus ND10 was slow, but it also showed a certain degradation effect. These results show that the Acinetobacter calcoaceticus ND10 screened by the present invention can tolerate 500 mg / L NO 2 --N concentration.

[0085] Table 3 Results of maximum tolerance concentration of nitrite nitrogen

[0086]

[0087] The above experiments can fully prove that the calcium-acetic acid Acinetobacter ND10 selected by the present invention can not only grow in water containing florfenicol, but also efficiently denitrify nitrogen in the water. Therefore, it has the ability to resist the broad-spectrum antibiotic florfenicol and has good denitrification ability.

[0088] Based on the above properties, the calcium-acetic acid Acinetobacter ND10 selected in the present invention has the potential to be developed into a microbial agent, specifically, it is expected to be prepared into a microbial agent for denitrification of aquaculture water suitable for efficient denitrification under florfenicol stress.

[0089] Preferably, the aquaculture water denitrification microbial agent may contain living cells of Acinetobacter calcoaceticus ND10 bacteria, dry bacteria of Acinetobacter calcoaceticus ND10 obtained by freeze-drying, immobilized Acinetobacter calcoaceticus ND10 cells, liquid bacteria agent of Acinetobacter calcoaceticus ND10, solid bacteria agent of Acinetobacter calcoaceticus ND10, or Acinetobacter calcoaceticus ND10 strains in any other form.

[0090] In addition, the calcoacetic acid Acinetobacter ND10 selected by the present invention and the aquaculture water denitrification microbial agent prepared using the strain can be used in aquaculture water treatment, especially for degrading nitrogen (ammonia nitrogen and nitrite) in aquaculture water containing florfenicol, and has good application prospects for solving problems such as excessive nitrite and ammonia nitrogen in florfenicol-containing water.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Acinetobacter calcoaceticus ND10, characterized in that: The calcium-acetic acid Acinetobacter ND10 was deposited in the China General Microbiological Culture Collection Center on May 13, 2024, with the deposit number CGMCC NO.30544.

2. A microbial agent containing the calcoacetic acid Acinetobacter ND10 strain as claimed in claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial inoculum contains living cells of Acinetobacter calcoaceticus ND10 bacteria, dry bacteria of Acinetobacter calcoaceticus ND10 obtained by freeze drying, immobilized cells of Acinetobacter calcoaceticus ND10, liquid inoculum of Acinetobacter calcoaceticus ND10, solid inoculum of Acinetobacter calcoaceticus ND10, or Acinetobacter calcoaceticus ND10 strains in any other form.

4. Use of the calcoacetic acid Acinetobacter ND10 as claimed in claim 1 or the microbial agent as claimed in claim 2 in aquaculture water treatment.

5. The use according to claim 4, characterized in that: The application is to degrade nitrogen in aquaculture water containing 0.001-10 mg / L of florfenicol.

6. The use according to claim 5, characterized in that: The application is to degrade ammonia nitrogen and nitrite in aquaculture water containing 0.001-10 mg / L of florfenicol.

7. The use according to any one of claims 4 to 6, characterized in that: The application is to first culture the calcoacetic acid Acinetobacter ND10, then collect the bacteria, and finally inoculate the collected calcoacetic acid Acinetobacter ND10 bacteria into the florfenicol-containing aquaculture water body, so that the calcoacetic acid Acinetobacter ND10 bacteria are cultured and propagated in the florfenicol-containing aquaculture water body, while the ammonia nitrogen and nitrite contained therein are effectively removed.

8. The use according to claim 7, characterized in that: The culture is carried out at 30° C. and 120-160 rpm shaking conditions for 12-48 hours.

9. The use according to claim 7, characterized in that: The bacterial body is obtained by centrifuging the cultured Acinetobacter calcoaceticus ND10 at 6000-8000 rpm for 8-10 minutes and discarding the supernatant to obtain the collected bacterial body.

10. The use according to claim 7, characterized in that: The culture and propagation is carried out at 30° C. and 120-160 rpm shaking conditions for 24-48 hours.

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