Acinetobacter CN-D1 isolated strain and application thereof

Abstract

The invention discloses an acinetobacter CN-D1 isolated strain and application thereof, and belongs to the technical field of microorganisms. The invention provides an Acinetobacter sp. CN-D1 bacterial strain, and the preservation number of the bacterial strain is CCTCC (China Center for Type Culture Collection) NO: M 20242023. The acinetobacter CN-D1 separation strain provided by the invention is a synchronous nitrification and denitrification aerobic bacterium, and can remove substances such as total nitrogen, ammonia nitrogen, nitrite and the like in a water body at the same time; and meanwhile, the strain is high in environmental tolerance and can be applied to circulating aquaculture water and aquaculture tail water to play a biological denitrification role.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an Acinetobacter CN-D1 isolated strain and application thereof. Background Art

[0002] Recirculating Aquaculture System (RAS) is one of the main directions of aquaculture development at home and abroad in recent years. The high-density aquaculture model of circulating water integrates advanced concepts such as water treatment technology, high-density aquaculture, and intensive management. It is an environmentally friendly and sustainable aquaculture model. However, with the increase in aquaculture density, the deposition of a large amount of feces and leftover bait in the water body causes the accumulation of harmful substances such as ammonia nitrogen and nitrite nitrogen, leading to the deterioration of the aquaculture water body, which seriously affects the output and quality of aquaculture products. At the same time, a large amount of water needs to be changed to reduce the concentration of harmful nitrogen substances in the aquaculture water body. This operation not only wastes water resources, but also causes the surrounding natural seawater to be polluted by the discharge of tail water. Therefore, water purification in the recirculating aquaculture system has become an indispensable task.

[0003] Today, factory-based high-density farming has become a trend, among which commonly used water treatment technologies include air stripping, ion exchange adsorption, ozone oxidation treatment, electrodialysis treatment and biological denitrification. Biological denitrification technology has many advantages in wastewater treatment, such as environmental friendliness, economic efficiency, good treatment effect, simple operation, high flexibility and sustainability. Through reasonable system design and operation management, biological denitrification technology can effectively remove nitrogen compounds in wastewater and realize the protection and sustainable utilization of water resources.

[0004] Biological denitrification is a method of removing nitrogen compounds from wastewater through microbial metabolism, which mainly includes two steps: nitrification and denitrification. Among them, nitrification and denitrification bacteria, as a type of biological denitrification bacteria, are widely used in factories to treat aquaculture water. They are not only low-cost and highly efficient in denitrification, but also have no toxic residues, which can greatly improve the comprehensive benefits of aquaculture.

[0005] In the traditional biological denitrification process, the suitable environment and metabolic conditions for nitrifying and denitrifying bacteria are different. The process is complex and time-consuming, with high requirements for equipment and site. In addition, nitrifying bacteria are autotrophic bacteria with poor environmental tolerance, making it difficult to carry out efficient denitrification under different scenarios and water qualities. Summary of the invention

[0006] In view of the fact that nitrifying bacteria in the prior art have poor tolerance and cannot efficiently remove ammonia nitrogen, nitrite, nitrate and other substances in aquaculture tail water, the purpose of the present invention is to provide an isolated strain of Acinetobacter CN-D1 with high efficiency and simultaneous nitrification and denitrification.

[0007] The present invention provides an Acinetobacter sp. CN-D1 isolated strain. The Acinetobacter sp. CN-D1 isolated strain was deposited in the China Center for Type Culture Collection (Wuhan University, China) on September 23, 2024, with a deposit number of CCTCC NO: M 20242023.

[0008] Wherein, the Acinetobacter CN-D1 isolated strain has a nucleotide sequence as shown in SEQ ID No.1.

[0009] The colonies of the Acinetobacter CN-D1 isolated strain of the present invention are smooth and round, plump and convex, opaque, white, with neat edges, smooth, moist and shiny, and sticky and easy to pick up.

[0010] The present invention also provides a bacterial preparation, comprising the isolated strain of Acinetobacter CN-D1 as described above.

[0011] The bacterial preparation comprises the Acinetobacter CN-D1 isolated strain and a microbial culture, wherein the Acinetobacter CN-D1 isolated strain serves as an active ingredient of the bacterial preparation.

[0012] The present invention also provides a method for preparing the bacterial preparation as described above, characterized in that it comprises the following steps:

[0013] (1) inoculating the isolated strain of Acinetobacter CN-D1 into a seed culture medium for culturing to obtain a seed solution;

[0014] (2) adding the seed liquid to the seed culture medium for high-density fermentation and expansion to obtain the bacterial preparation.

[0015] Furthermore, the components of the seed culture medium in step (1) and step (2) are: 5 g glucose, 0.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4; 2 mL trace salt solution, 1% (v / v) yeast powder solution, 1% (v / v) peptone solution, and 1000 mL distilled water.

[0016] Furthermore, the pH of the seed culture medium in step (1) and step (2) is 7.0-7.2, the concentration of the yeast powder solution is 0.5 g / 10 mL, and the concentration of the peptone solution is 1 g / 10 mL.

[0017] Furthermore, in step (2), the volume ratio of the seed liquid to the seed culture medium is 1:100.

[0018] The present invention also provides an application of the bacterial preparation as described above in circulating aquaculture.

[0019] The present invention also provides an application of the bacterial preparation as described above in purifying aquaculture tail water.

[0020] The present invention provides an isolated strain of Acinetobacter CN-D1, which is a synchronous nitrifying and denitrifying aerobic bacterium and can simultaneously remove total nitrogen, ammonia nitrogen, nitrite and other substances in water bodies; at the same time, it has strong environmental tolerance and can be applied to circulating aquaculture water bodies and aquaculture tail water to play a role in biological denitrification. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a colony morphology diagram of the Acinetobacter CN-D1 isolated strain of the present invention;

[0022] Figure 2 is the phylogenetic tree in Example 2 of the present invention;

[0023] Figure 3 In Example 1 of the present invention, NH4 + -Bacterial growth curve when N is the single nitrogen source;

[0024] Figure 4 In Example 1 of the present invention, NH4 + -Denitrification effect diagram when N is the single nitrogen source;

[0025] Figure 5 In Example 1 of the present invention, NO2 - -Bacterial growth curve when N is the single nitrogen source;

[0026] Figure 6 In Example 1 of the present invention, NO2 - -Denitrification effect diagram when N is the single nitrogen source;

[0027] Figure 7 In Example 1 of the present invention, NO3 - -Bacterial growth curve when N is the single nitrogen source;

[0028] Figure 8 In Example 1 of the present invention, NO3 - -Denitrification effect diagram when N is the single nitrogen source;

[0029] Fig. 9 This is a diagram showing the denitrification effect of strain CN-D1 in Example 4 of the present invention in a circulating aquaculture water body;

[0030] Fig.10 This is a diagram showing the denitrification effect of strain CN-D1 in Example 5 of the present invention in aquaculture tail water. DETAILED DESCRIPTION

[0031] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] LB medium: peptone 10 g; yeast powder 5 g; NaCl 10 g; pH 7.0-7.2, distilled water 1000 mL.

[0033] Nitration medium: sodium acetate 5 g; (NH4)2SO4 0.235 g (50 mg / L); KH2PO4 0.5 g; Na2HPO4 0.5 g; MgSO4·7H2O 0.4 g; trace salt solution 2 ml; distilled water 1000 ml; pH 7.0-7.2, solid medium plus 2wt% agar.

[0034] Denitrification medium: sodium acetate 5 g; KNO3 0.36 g (50 mg / L); Na2HPO4 0.5 g, KH2PO4 0.5 g; MgSO4·7H2O 0.4 g; trace salt solution 2 ml; distilled water 1000 ml; pH 7.0-7.2, solid medium plus 2 wt% agar.

[0035] Trace salt solution: ethylenediaminetetraacetic acid (EDTA) 50 g, ZnSO4 2.2 g, CaCl2 5.5 g, CuSO4·5H2O1.57 g, MnCl2·4H2O 5.06 g, FeSO4·7H2O 5 g, CoCl2·6H2O 1.61 g, distilled water 1000 mL, pH 7.0-7.2.

[0036] Seed culture medium: 5 g glucose, 0.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4; 2 mL trace salt solution, 1% (v / v) yeast powder solution, 1% (v / v) peptone solution, 1000 mL distilled water, pH 7.0-7.2. Among them: yeast powder solution concentration: 0.5 g / 10 mL; peptone solution concentration: 1 g / 10 mL.

[0037] Solid medium was supplemented with 2 wt% agar.

[0038] Instruments: clean bench, biochemical incubator, UV-visible spectrophotometer, vertical high-pressure steam sterilizer, gas bath constant temperature oscillator, water quality rapid tester, etc.

[0039] Example 1

[0040] Isolation and screening of Acinetobacter CN-D1 strain:

[0041] (1) Enrichment culture: Take 1 mL of aquaculture water sample and add it to 100 mL of sterile pure water, shake and mix evenly to prepare bacterial suspension. Pipette 10 mL of bacterial suspension into 90 mL of nitrification medium and incubate at 30°C and 180 rpm. -1 Culture under the same conditions for 24 h, which is the first acclimation cycle. Take 10 mL of the previous culture and transfer it to the second nitrification medium, culture it under the same conditions, and repeat the acclimation process for 5 times.

[0042] (2) Isolation and purification: The domesticated and enriched bacterial solution was diluted to 10 -1 -10 -8 The different concentration gradient bacterial suspensions were streaked on LB solid plate medium to obtain single colonies.

[0043] (3) Preliminary screening: In order to screen out strains with good denitrification performance, the isolated and purified pure strains were first inoculated into 96-well culture plates of sterilized nitrification medium and denitrification medium, respectively, and placed in a shaker at 30°C and 180 r / min. -1 After shaking culture for 24 h under the conditions of 5% paraformaldehyde and 1% paraformaldehyde, nitrite colorimetric agent and ammonium salt colorimetric agent were added respectively, and the dominant strain was selected by observing the color depth.

[0044] (4) Rescreening and performance testing: The strains preliminarily screened were inoculated into nitrification medium and denitrification medium and incubated at 30°C and 180 r / min. -1 The seed solution was then inoculated into the nitrification medium and denitrification medium at a 1% (v / v) inoculation rate. Samples were taken every 24 hours to measure the total nitrogen, nitrate, nitrite, ammonia nitrogen, COD, and OD in the culture medium. 600 , selected strains with strong denitrification ability, and the strain with the best performance was named Acinetobacter sp. CN-D1. Subsequently, CN-D1 was inoculated into nitrification medium with (NH4)2SO4, KNO3 and NaNO2 as the only nitrogen source, with an initial nitrogen concentration of 20 mg / L. The experimental conditions were 30℃, 180r·min -1 , sampling every 24 hours, measuring the concentration of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, and the performance test results are as follows Figure 3-Figure 8 shown.

[0045] Acinetobacter sp. CN-D1 strain has good denitrification performance and has simultaneous nitrification and denitrification functions, such as Figure 3-Figure 4 As shown, NH4 + When -N is the only nitrogen source, OD 600The value reached the highest value after 36 h of cultivation and was greater than 1. Within 48 h, the ammonia nitrogen NH4 + -N removal rate reaches 93.9%, and there is no NO3 - -N, NO2 - -N accumulation. Figure 5-Figure 6 As shown, NO2 - When -N is the only nitrogen source, OD 600 The value reached the highest value after 24 h of cultivation, close to 1, and NO2 - The highest removal rate of -N reached 77.2%, and there was no NO3 - -N、NH4 + -N accumulation. Figure 7-Figure 8 As shown, NO3 - When -N is the only nitrogen source, OD 600 The value reached the highest value after 24 hours of cultivation, close to 1. Within 48 hours, NO3 - -N removal rate reaches 80.2%, and there is no NH4 + -N, NO2 - -N accumulation.

[0046] Example 2

[0047] Identification of Acinetobacter CN-D1 strain:

[0048] (1) Morphological characteristics

[0049] like Figure 1 As shown, after cultured on LB plates at 30°C for 12 hours, the colonies were smooth and round, 2-3 mm in diameter, plump and convex, opaque, white, with neat edges, smooth, moist and shiny, sticky and easy to pick up. The colonies increased in size with time, and after a few days, the diameter was ≥5 mm.

[0050] (2) Genetic identification of Acinetobacter CN-D1 strain

[0051] A single colony of Acinetobacter CN-D1 was selected for colony PCR, with primers 27F (5'-agagtttgatcctggctag -3') and 1495R (5'-CTACGGCTACCTTGT TACGA-3'), and the product was purified and sequenced. The sequence obtained was retrieved in GenBank by BLAST and compared, and the 16SrDNA sequence with high similarity to the sequence was screened out. The phylogenetic tree was constructed using MEGA7.0 to find the strain closest to its homology.

[0052] The 16SrDNA sequence of CN-D1 was determined, and the result was SEQ ID No.1:

[0053]

[0054] The results were compared with the 16S rDNA gene sequence of the model strain in the NCBI database. Figure 5 As shown, the salt-tolerant and efficient strain was identified as Acinetobacter proteolyticus, Gram-negative, and the developmental tree was as follows Figure 2 shown.

[0055] Example 3

[0056] Preparation of bacterial preparations:

[0057] The strain CN-D1 was inoculated into a 250 mL shake flask containing 100 mL of seed culture medium and cultured in a constant temperature shaker at 37°C and 180 r / min for 24 h to be used as seed solution. The seed solution was then transferred to the seed culture medium in the fermenter for high-density fermentation. During the expansion culture, the volume ratio of the seed solution to the culture medium in the fermenter was 1:100.

[0058] Example 4

[0059] Application of strain CN-D1 in circulating aquaculture water:

[0060] The bacterial agent was obtained according to the above bacterial preparation method, and added to the aquaculture water at a dosage of 1% (v / v), with a dosage frequency of once every 3 days. The indicators such as ammonia nitrogen and nitrite nitrogen in the water were recorded every day. The data for the first 6 days were when no bacterial agent was added, and the data from the seventh day onwards were after the addition of the bacterial agent. The comparative example was a commercially available denitrifying bacterial agent (aerobic denitrification bacteria produced by Zhuokai Water Diversion Company), and the dosage and addition method were the same as those of strain CN-D1.

[0061] like Fig. 9 As shown in the figure, the concentrations of ammonia nitrogen and nitrite nitrogen in the circulating aquaculture system before addition were 1.2-1.5 mg / L and 0.8-0.9 mg / L. After adding the commercially available denitrifying bacteria, the ammonia nitrogen and nitrite nitrogen in the water showed a downward trend. The stable concentrations of ammonia nitrogen and nitrite nitrogen were 0.8 mg / L and 0.6 mg / L, respectively, and the removal rates could reach 40.1% and 33.0%, respectively. After adding the CN-D1 bacteria, the concentrations of ammonia nitrogen and nitrite in the circulating aquaculture water body could reach about 0.4 mg / L and 0.3 mg / L, and the highest removal rates of ammonia nitrogen and nitrite nitrogen reached 70.8% and 69.2%. From the results, the CN-D1 strain showed good denitrification performance in the circulating aquaculture system. Compared with the denitrifying bacteria products on the market, the removal rates of ammonia nitrogen and nitrite nitrogen in the circulating aquaculture water body of the CN-D1 strain increased by 30.7% and 36.2%.

[0062] Example 5

[0063] Application of strain CN-D1 in aquaculture tail water:

[0064] The bacterial agent was obtained according to the above bacterial preparation method and added to the aquaculture tail water at a dosage of 1% (v / v). The frequency of addition was once every 3 days, and the total nitrogen, ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the water were recorded every day. Fig.10 As shown, the first seven days are data without adding bacterial agents, and the eighth day onwards are data after adding bacterial agents.

[0065] The results are as follows Fig.10 As shown in the figure, before adding bacteria, the concentrations of ammonia nitrogen, nitrite nitrogen, TN (total nitrogen) and nitrate nitrogen in the water were about 3.5 mg / L, 1.2 mg / L, 6.9 mg / L and 0.9 mg / L. After adding bacteria on the eighth day, it can be seen that the concentrations of ammonia nitrogen, nitrite, TN (total nitrogen) and nitrate nitrogen have dropped significantly. On the 10th day, the water quality began to stabilize. The concentrations of ammonia nitrogen, nitrite, TN (total nitrogen) and nitrate were 0.46 mg / L, 0.22 mg / L, 2.0 mg / L and 0.53 mg / L, and the removal rates reached 86.5%, 81.7%, 71.0% and 41%. According to the discharge standard of freshwater pond aquaculture water, the total nitrogen in the water body can reach the first-level discharge standard.

[0066] The present invention screens out a strain of highly efficient simultaneous nitrifying and denitrifying aerobic bacteria that can be applied to circulating aquaculture water bodies and aquaculture tail water, and exhibits good denitrification performance under different water quality conditions, such as in complex aquaculture water bodies and in highly polluted aquaculture tail water, and has strong environmental tolerance; it can efficiently degrade ammonia nitrogen, nitrite, nitrate and other harmful substances in the water body, thereby achieving the purpose of improving the water quality of the aquaculture water body and purifying the aquaculture tail water.

[0067] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An isolated strain of Acinetobacter sp. CN-D1, wherein: The Acinetobacter CN-D1 isolated strain was deposited in the China Center for Type Culture Collection on September 23, 2024, with the deposit number being CCTCC NO: M20242023.

2. The Acinetobacter CN-D1 isolated strain according to claim 1, characterized in that The Acinetobacter CN-D1 isolated strain has a nucleotide sequence as shown in SEQ ID No.

1.

3. The Acinetobacter CN-D1 isolated strain according to claim 1, characterized in that The colonies of the Acinetobacter CN-D1 isolated strain are smooth and round, plump and convex, opaque, white, with neat edges, smooth, moist and shiny, and sticky and easy to pick up.

4. A bacterial preparation, characterized in that It comprises the isolated strain of Acinetobacter CN-D1 as described in claim 1.

5. A method for preparing a bacterial preparation as claimed in claim 4, characterized in that: The following steps are involved: (1) inoculating the isolated strain of Acinetobacter CN-D1 into a seed culture medium for culturing to obtain a seed solution; (2) adding the seed liquid to the seed culture medium for high-density fermentation and expansion to obtain the bacterial preparation.

6. The method for preparing a bacterial preparation according to claim 5, characterized in that: The components of the seed culture medium in step (1) and step (2) are: 5 g glucose, 0.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4; 2 mL trace salt solution, 1% yeast powder solution, 1% peptone solution, and 1000 mL distilled water.

7. The method for preparing a bacterial preparation according to claim 6, characterized in that: The pH of the seed culture medium in step (1) and step (2) is 7.0-7.2, the concentration of the yeast powder solution is 0.5 g / 10 mL, and the concentration of the peptone solution is 1 g / 10 mL.

8. The method for preparing a bacterial preparation according to claim 7, characterized in that: In the step (2), the volume ratio of the seed liquid to the seed culture medium is 1:

100.

9. Use of the bacterial preparation as claimed in claim 4 in circulating aquaculture.

10. Use of the bacterial preparation as claimed in claim 4 in purifying aquaculture tail water.

Images