Acinetobacter sp. CN-D1 isolated strain and its application

Abstract

The present invention discloses an isolated Acinetobacter sp. CN-D1 strain and its application, belonging to the technical field of microbiology. The present invention provides an Acinetobacter sp. CN-D1 strain, wherein the preservation number of the strain is CCTCC NO: M 20242023. The isolated Acinetobacter sp. CN-D1 strain provided by the present invention is a synchronous nitrification and denitrification aerobic bacterium, which can simultaneously remove substances such as total nitrogen, ammonia nitrogen, and nitrite in water; at the same time, it has strong environmental tolerance and can be applied to circulating aquaculture water and aquaculture tail water to play a role in biological nitrogen removal.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an Acinetobacter sp. CN-D1 isolated strain and its application. Background Art

[0002] The recirculating aquaculture system (RAS) is one of the main directions of the development of the aquaculture industry at home and abroad in recent years. The recirculating high-density aquaculture mode integrates advanced concepts such as water treatment technology, high-density aquaculture, and intensive management, and is an environmentally friendly and sustainable aquaculture mode. However, with the increase in aquaculture density, the deposition of a large amount of feces and residual bait in the water body causes the accumulation of harmful substances such as ammonia nitrogen and nitrite nitrogen, resulting in the deterioration of the aquaculture water body, seriously affecting the yield 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 the discharge of tail water will cause pollution to the surrounding natural seawater. Therefore, the purification treatment of the water body in the recirculating aquaculture system has become an essential task.

[0003] Today, industrialized high-density aquaculture has become a trend. The 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 achieve the protection and sustainable utilization of water resources.

[0004] Biological denitrification is a method for removing nitrogen compounds in wastewater through the microbial metabolism process, mainly including two steps: nitrification and denitrification. Among them, nitrifying and denitrifying bacteria, as a type of biological denitrifying bacteria, are widely used in the treatment of aquaculture water in factories. They not only have low cost and high denitrification efficiency, but also have no toxic residues, which can greatly improve the comprehensive aquaculture benefits.

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

[0006] Aiming at the problems in the prior art that nitrifying bacteria have poor tolerance and cannot efficiently remove substances such as ammonia nitrogen, nitrite, and nitrate in aquaculture tail water, the purpose of the present invention is to provide an Acinetobacter sp. CN-D1 isolated strain with efficient synchronous nitrification and denitrification.

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

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

[0009] The colony of the isolated strain of Acinetobacter sp. CN-D1 described in the present invention is smooth, round, plump and raised, opaque, white in color, with a neat edge, smooth, moist and shiny, and sticky and easy to pick up.

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

[0011] The bacterial preparation includes the isolated strain of Acinetobacter sp. CN-D1 and a microbial culture. The isolated strain of Acinetobacter sp. CN-D1 is used as the active ingredient of the bacterial preparation.

[0012] The present invention also provides a preparation method of the bacterial preparation as described above, which is characterized by including the following steps:

[0013] (1) Inoculating the isolated strain of Acinetobacter sp. CN-D1 into a seed medium for culture to obtain a seed liquid;

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

[0015] Further, the components of the seed medium in the step (1) and the step (2) are: 5 g of glucose, 0.5 g of K 2 HPO 4 0.5 g of KH 2 PO 4 0.2 g of MgSO 4 ; 2 mL of trace salt solution, 1% (v / v) yeast powder solution, 1% (v / v) peptone solution, and 1000 mL of distilled water.

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

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

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

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

[0020] An Acinetobacter sp. CN-D1 isolated strain provided by the present invention is a synchronous nitrification and denitrification aerobic bacterium, which can simultaneously remove substances such as total nitrogen, ammonia nitrogen, and nitrite in water; at the same time, it has strong environmental tolerance and can be applied to recirculating aquaculture water bodies and aquaculture tail water to play a role in biological denitrification. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0023] Figure 3 It is a bacterial growth curve diagram in Example 1 of the present invention when NH 4 + -N is used as the sole nitrogen source;

[0024] Figure 4 It is a bacterial growth curve diagram in Example 1 of the present invention when NH 4 + -N is used as the sole nitrogen source;

[0025] Figure 5 It is a bacterial growth curve diagram in Example 1 of the present invention when NO 2 - -N is used as the sole nitrogen source;

[0026] Figure 6 It is a bacterial growth curve diagram in Example 1 of the present invention when NO 2 - -N is used as the sole nitrogen source;

[0027] Figure 7 It is a bacterial growth curve diagram in Example 1 of the present invention when NO 3 - -N is used as the sole nitrogen source;

[0028] Figure 8 It is a bacterial growth curve diagram in Example 1 of the present invention when NO 3 - -N is used as the sole nitrogen source;

[0029] Figure 9 It is a denitrification effect diagram of the strain CN-D1 in the recirculating aquaculture water body in Example 4 of the present invention;

[0030] Figure 10 It is the denitrification effect diagram of strain CN-D1 in Example 5 of the present invention in aquaculture tail water. Detailed implementation manners

[0031] To make the technical solutions of the present invention clearer, the present invention will be 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] Nitrification medium: sodium acetate 5 g; (NH 4 2 SO 4 0.235 g (50 mg / L); KH 2 PO 4 0.5 g; Na 2 HPO 4 0.5 g; MgSO 4 ·7H 2 O 0.4 g; trace salt solution 2 ml; distilled water 1000 ml; pH 7.0 - 7.2, add 2wt% agar to the solid medium.

[0034] Denitrification medium: sodium acetate 5 g; KNO 3 0.36 g (50 mg / L); Na 2 HPO 4 0.5 g, KH 2 PO 4 0.5 g; MgSO 4 ·7H 2 O 0.4 g; trace salt solution 2 ml; distilled water 1000 ml; pH 7.0 - 7.2, add 2 wt% agar to the solid medium.

[0035] Trace salt solution: ethylenediaminetetraacetic acid (EDTA) 50 g, ZnSO 4 2.2 g, CaCl 2 5.5 g, CuSO 4 ·5H 2 O1.57 g, MnCl 2 ·4H 2 O 5.06 g, FeSO 4 ·7H 2 O 5 g, CoCl 2 ·6H 2 ​1.61 g of O, 1000 mL of distilled water, pH 7.0 - 7.2.

[0036] Seed medium: 5 g of glucose, K 2 HPO 4 0.5 g, KH 2 PO 4 0.5 g, MgSO 4 0.2 g; 2 mL of trace salt solution, 1% (v / v) yeast extract solution, 1% (v / v) peptone solution, 1000 mL of distilled water, pH 7.0 - 7.2. Among them: the concentration of the yeast extract solution: 0.5 g / 10 mL; the concentration of the peptone solution: 1 g / 10 mL.

[0037] Add 2 wt% agar to the solid medium.

[0038] Instruments: laminar flow hood, biochemical incubator, UV-visible spectrophotometer, vertical autoclave, air bath thermostatic shaker, water quality rapid detector, etc.

[0039] Example 1

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

[0041] (1) Enrichment culture: Take 1 mL of aquaculture water sample into 100 mL of sterile pure water, shake and mix evenly to prepare a bacterial suspension. Pipette 10 mL of the bacterial suspension into 90 mL of nitrification medium, and culture at 30°C and 180 r·min -1 for 24 h, which is the first domestication cycle. Take 10 mL of the previous culture and transfer it to the nitrification medium of the second stage respectively, and culture under the same conditions, and so on, repeating the domestication 5 times.

[0042] (2) Isolation and purification: Dilute the domesticated and enriched bacterial liquid by gradient dilution method to different concentration gradients of bacterial suspension of 10 -1 -10 -8 , and streak culture on LB solid plate medium to obtain single colonies.

[0043] (3) Primary screening: In order to screen out strains with good denitrification performance, first inoculate the purified strains after isolation and purification into 96-well culture plates of sterilized nitrification medium and denitrification medium respectively, and place them in a shaker at 30°C and 180 r·min -1 for 24 h under the conditions of oscillation culture, then add nitrite chromogenic agent and ammonium salt chromogenic agent respectively, and observe the color depth to select the dominant strains.

[0044] (4) Re-screening and performance testing: Inoculate the preliminarily screened strains into nitrification medium and denitrification medium, and culture them at 30 °C and 180 r·min -1 for 24 h to obtain the seed liquid. Then, inoculate the seed liquid of each strain into nitrification medium and denitrification medium for culture at an inoculation amount of 1% (v / v). Take samples every 24 h to measure the total nitrogen, nitrate, nitrite, ammonia nitrogen, COD, and OD 600 in the medium, select the strains with stronger nitrogen removal ability, and complete the screening to obtain the strain with the best performance, named Acinetobacter sp. CN-D1. Subsequently, inoculate CN-D1 into nitrification media with (NH 4 ) 2 SO 4 , KNO 3 , and NaNO 2 as the sole nitrogen sources respectively. The initial nitrogen concentration is 20 mg / L for all. The experimental conditions are 30 °C and 180 r·min -1 . Take samples every 24 h to measure the ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen concentrations. The performance test results are as Figures 3 - 8 shown.

[0045] The Acinetobacter sp. CN-D1 strain has good nitrogen removal performance and has the function of simultaneous nitrification and denitrification. As Figures 3 - 4 shown, when using NH 4 + -N as the sole nitrogen source, the OD 600 value reaches the highest value after 36 h of culture and is greater than 1. The ammonia nitrogen NH 4 + -N removal rate reaches 93.9% within 48 h, and there is no accumulation of NO 3 - -N and NO 2 - -N. As Figures 5 - 6 shown, when using NO 2 - -N as the sole nitrogen source, the OD 600 value reaches the highest value after 24 h of culture, approaching 1. The highest removal rate of NO 2 - -N reaches 77.2%, and there is no accumulation of NO 3 - -N and NH 4 + -N. As Figures 7 - 8 shown, when using NO 3 - -N as the sole nitrogen source, the OD 600The value reached the highest value after 24 h of cultivation, approaching 1, and the NO 3 - -N removal rate reached 80.2%, and there was no accumulation of NH 4 + -N or NO 2 - -N.

[0046] Example 2

[0047] Identification of Acinetobacter sp. CN-D1 strain:

[0048] (1) Morphological characteristics

[0049] As Figure 1 shown, after culturing on an LB plate at 30 °C for 12 h, the colonies were smooth and round, with a diameter of 2 - 3 mm, plump and raised, opaque, white in color, with neat edges, smooth, moist and shiny, sticky and easy to pick up. The colonies increased in size over time and had a diameter ≥ 5 mm after several days.

[0050] (2) Gene identification of Acinetobacter sp. CN-D1 strain

[0051] Single colonies of Acinetobacter sp. CN-D1 were selected for colony PCR. The primers were 27F (5’-agagtttgatcctggctag -3’) and 1495R (5’-CTACGGCTACCTTGT TACGA-3’). The products were purified and sequenced. The sequences retrieved from GenBank through BLAST were aligned, and 16S rDNA sequences with higher similarity to this sequence were screened out. A phylogenetic tree was constructed using MEGA7.0 to find the strain with the closest homology.

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

[0053]

[0054] The obtained results were compared for homology with the 16S rDNA gene sequences of the type strains in the NCBI database. The results are as Figure 5 shown. It was identified that this salt-tolerant and highly efficient strain was Acinetobacter proteolyticus, Gram-negative. The phylogenetic tree is as Figure 2 shown.

[0055] Example 3

[0056] Preparation of the bacterial preparation:

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

[0058] Example 4

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

[0060] The bacterial agent was obtained according to the method of the above-mentioned bacterial preparation and added to the aquaculture water body at a dosage of 1% (v / v). The dosing frequency was once every 3 days. The indexes such as ammonia nitrogen and nitrite nitrogen in the water body were recorded every day. The data in the first 6 days were those before adding the bacterial agent, and the data from the seventh day were those after adding the bacterial agent. The control was a commercially available denitrifying bacterial agent (a special aerobic denitrifying bacterium produced by Zhuokai Water Conditioning Company). The dosage and dosing method were the same as those of strain CN-D1.

[0061] As Figure 9 shown, the concentrations of ammonia nitrogen and nitrite nitrogen in the circulating water aquaculture system before adding were 1.2 - 1.5 mg / L and 0.8 - 0.9 mg / L. After adding the commercially available denitrifying bacterial agent, the ammonia nitrogen and nitrite nitrogen in the water body 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 bacterial agent, the ammonia nitrogen and nitrite concentrations in the circulating water 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, strain CN-D1 showed good denitrification performance in the circulating water aquaculture system. Compared with the commercially available denitrifying bacterial agent products, the removal rates of ammonia nitrogen and nitrite nitrogen of strain CN-D1 in the circulating water aquaculture water body increased by 30.7% and 36.2%.

[0062] Example 5

[0063] Application of Strain CN-D1 in Aquaculture Tail Water:

[0064] The microbial agent was obtained according to the method of the above microbial preparation, and added to the aquaculture tail water at a dosage of 1% (v / v). The dosing frequency was once every 3 days, and indexes such as total nitrogen, ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the water body were recorded every day. The results are as Figure 10 shown. The data of the first seven days are those before adding the microbial agent, and the data from the eighth day are those after adding the microbial agent.

[0065] The results are as Figure 10 shown. Before adding the microbial agent, 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 respectively. Starting from the eighth day of adding the microbial agent, it can be seen that the concentrations of ammonia nitrogen, nitrite, TN (total nitrogen) and nitrate nitrogen decreased significantly. On the 10th day, the water quality began to stabilize. When stable, 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 respectively, and the removal rates reached 86.5%, 81.7%, 71.0% and 41%. According to the discharge standard of aquaculture water in freshwater ponds, the total nitrogen in the water body can reach the first-class discharge standard.

[0066] A highly efficient simultaneous nitrification and denitrification aerobic bacterium screened by the present invention, which can be applied to circulating aquaculture water bodies and aquaculture tail water, shows good denitrification performance under different water quality conditions, such as in complex aquaculture water bodies and highly polluted aquaculture tail water, and has strong environmental tolerance; it can efficiently degrade various harmful substances such as ammonia nitrogen, nitrite and nitrate in the water body, so as to achieve the purpose of improving the water quality of aquaculture water bodies and purifying aquaculture tail water.

[0067] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. An Acinetobacter species ( Acinetobacter sp.) CN-D1 isolated strain, among which, 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. A bacterial preparation, characterized in that: It comprises the isolated strain of Acinetobacter CN-D1 as described in claim 1.

3. A method for preparing a bacterial preparation as claimed in claim 2, 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.

4. The method for preparing a bacterial preparation according to claim 3, 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.

5. The method for preparing a bacterial preparation according to claim 4, 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.

6. The method for preparing a bacterial preparation according to claim 5, characterized in that: In the step (2), the volume ratio of the seed liquid to the seed culture medium is 1:

100.

7. Use of the bacterial preparation as claimed in claim 2 in circulating aquaculture.

8. Use of the bacterial preparation as claimed in claim 2 in purifying aquaculture tail water.

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