Acinetobacter calcoaceticus and application thereof

Abstract

The invention discloses acinetobacter calcoaceticus and application thereof. The preservation number of the acinetobacter calcoaceticus is CCTCC (China Center For Type Culture Collection) NO: M20232617. The acinetobacter calcoaceticus can be used for phosphorus accumulation and removal of sewage.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a strain of Acinetobacter calcoaceticus and application thereof. Background Art

[0002] With the development of rural economic construction and the improvement of people's living standards, regional environmental pollution has been increasing. Among them, the pollution problem caused by residents' daily water use is particularly important. Domestic sewage contains various harmful substances. If it is discharged directly into natural water bodies without treatment, it will have a serious impact on the environment and human health.

[0003] Phosphorus in domestic sewage is one of the key factors in the eutrophication of water bodies. Excessive phosphorus content in water bodies will cause eutrophication of water bodies, thereby endangering the environment and human health. The key to controlling eutrophication of water bodies is to reduce phosphorus emissions. Traditional phosphorus removal process research focuses more on physical phosphorus removal and chemical phosphorus removal, and less research on biological phosphorus removal. However, physical phosphorus removal technology has high requirements and cannot be applied on a large scale; chemical phosphorus removal requires the purchase of a large amount of reagents, has high operation and maintenance costs, and is prone to produce a large amount of sludge, leading to secondary pollution. Biological phosphorus removal technology has broad application prospects due to its safety, high efficiency, and economy.

[0004] Polyphosphate bacteria, also known as phosphate-absorbing bacteria, refers to a class of bacteria that can store polyphosphate and poly-β-hydroxybutyric acid in their bodies. Polyphosphate bacteria are a special type of facultative bacteria in traditional activated sludge processes. Under aerobic or anoxic conditions, they can absorb excessive amounts of phosphorus in sewage into their bodies, making the phosphorus content in their bodies several times higher than that in ordinary bacteria. Biological phosphorus removal mainly utilizes the phosphorus accumulation effect of polyphosphate bacteria.

[0005] When studying biological phosphorus removal to reduce the cost of wastewater phosphorus removal and reduce secondary pollution, the applicant isolated a strain of calcoacetic acid Acinetobacter from nature and found that it has the ability to accumulate phosphorus. Summary of the invention

[0006] In order to overcome the problems existing in the prior art, the present invention provides a strain of Acinetobacter calcoaceticus and application thereof.

[0007] To achieve the above object, the first aspect of the present invention provides a strain of Acinetobacter calcoaceticus, wherein the preservation number of Acinetobacter calcoaceticus is: CCTCC NO: M20232617.

[0008] The present invention also provides a bacterial agent, the active ingredient of which is the above-mentioned Acinetobacter calcoaceticus.

[0009] The third aspect of the present invention provides the use of the above-mentioned Acinetobacter calcoaceticus and bacterial agent in phosphorus removal.

[0010] Through the above technical solution, the present invention can at least achieve the following beneficial effects: The calcoacetic acid bacterium of the present invention can accumulate phosphorus under simple aerobic conditions, without the need for a complex and cumbersome aerobic / anaerobic sequencing batch process, with low use cost and no secondary pollution. The strain is cultured in NB culture solution for 48 hours under aerobic conditions and then transferred to simulated domestic sewage. After 96 hours, the total phosphorus concentration of the simulated domestic sewage supernatant drops from 5 mg / L to 0.83 mg / L, 1.1 mg / L, and 1.9 mg / L, with an average phosphorus removal rate of 74.47%, a high phosphorus removal rate, and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a phylogenetic tree of Acinetobacter calcoaceticus of the present invention; Figure 2 This is a diagram showing the growth of Acinetobacter calcoaceticus in a phosphorus-limited culture medium provided by the present invention; Figure 3 This is a diagram showing the growth of Acinetobacter calcoaceticus in a perphosphate culture medium provided by the present invention; Figure 4 It is a standard curve diagram of the phosphorus standard solution used in the present invention. Biological Deposit

[0012] The present invention provides acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ), classified and named: Calcium Acinetobacter FL1-2 Acinetobacter calcoaceticus FL1-2 was deposited in the China Center for Type Culture Collection on December 21, 2023, with the deposit number: CCTCC NO:M2023617, and the address of the deposit center is Wuhan University, Wuhan City, Hubei Province, China, Postal Code: 430072. DETAILED DESCRIPTION

[0013] Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Example

[0014] 1. Isolation and purification of strains During the research process, the inventor of the present invention isolated a bacteria strain from the soil of Baima Snow Mountain (4000m above sea level) in Deqin County, Diqing Prefecture, Yunnan Province by using the dilution spread plate method and the plate streak method.

[0015] 2. Identification of strains The total DNA of the strain was extracted and used as a template, and 16S rRNA was amplified using 27F (5'-AGAGTTTGATCCTGGCTCAG-3') as the upstream primer and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') as the downstream primer.

[0016] The amplified product was extracted and sequenced. The sequencing result is shown in SEQ ID NO.1.

[0017] SEQ ID NO.1:

[0018] The sequencing results were input into the NCBI website for data comparison, and the sequences related to the bacterial strain were found and the similarities between them were calculated. Finally, the phylogenetic tree was constructed using MEGA.11.0 software. The phylogenetic tree is shown in the attached figure. Figure 1 As shown. The strain isolated and obtained by the present invention was identified as Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ). Example

[0019] Identification of the ability of phosphate-accumulating bacteria 1. Identification of blue-white spots Under sterile conditions, inoculate the strain to be isolated with an inoculation loop into the BCIP phosphorus-limited solid medium and BCIP phosphorus-excessive medium after filtration and sterilization, record the corresponding number and inoculation time, and culture at 15°C for 3 days, and observe the changes of the inoculated strain regularly. The strains that turn blue on both the BCIP phosphorus-limited solid medium and the BCIP phosphorus-excessive solid medium are strains with the ability to accumulate phosphorus, which are the initial screening strains of this study (the blue-white spot identification can only determine that the screened strains have the ability to accumulate phosphorus, but the strength of the ability is not reflected, and the strength of the phosphorus accumulation needs to be further determined). The results of the blue-white spot identification are shown in Table 1 below. Pictures of the strains cultured for 48 hours are shown in the attached Figure 2 shown.

[0020] Table 1 Identification results of blue-white spots Blue time / h Phosphorus-limited medium Phosphorus-rich medium 24 - - 48 ＋ ＋ Note: + means turning blue; - means not turning blue; 2. Determination of the ability of strains to accumulate phosphorus The ammonium molybdate spectrophotometry method can be used to quantitatively verify the strain, and the phosphorus removal rate can be used to measure the phosphorus accumulation ability of Acinetobacter calcoaceticus.

[0021] Preparation of simulated domestic sewage liquid culture medium: peptone 5g / L, beef extract powder 1.5g / L, NaCl 2.5g / L, potassium dihydrogen phosphate 0.022g / L. (The starting concentration of total phosphorus in simulated domestic sewage is 5mg / L) Preparation of nutrient broth medium (NB): Weigh 18g of nutrient broth medium, add 1L of distilled water and mix well, and then put them into 250mL conical flasks, 200mL per bottle. After sterilization and cooling at 121℃, inoculate the polyphosphate bacteria stored at -80℃ into the conical flask, shake and culture at 15℃ and 180rpm for 48h to obtain the seed liquid, and then inoculate 6mL (3% (seed liquid / simulated wastewater liquid culture volume)) of seed liquid into each bottle of simulated domestic sewage liquid culture medium, do 3 replicates, shake and culture at 15℃ and 180rpm for 96h, measure the absorbance of the culture medium, and then convert the phosphorus concentration according to the absorbance and standard curve.

[0022] The polyphosphate effect is shown in Table 2 below.

[0023] Table 2 Phosphorus accumulation effect of strains serial number Initial phosphorus concentration Phosphorus concentration after 96 hours Phosphorus removal rate 1 5mg / L 0.83mg / L 83.4% 2 5mg / L 1.1mg / L 78% 3 5mg / L 1.9mg / L 62% 3. Drawing of standard curve: 10% Ascorbic Acid Solution: Dissolve 10 g of ascorbic acid in water and dilute to 100 mL.

[0024] Molybdate solution: Dissolve 13g of ammonium molybdate ((NH4)6Mo7·4H2O) in 100mL of water; dissolve 0.35g of potassium antimony oxide tartrate (K(SbO)C4H4O6·1 / 2H2O) in 100mL of water. While stirring continuously, slowly add the ammonium molybdate solution to 30mL (1+1) sulfuric acid, add the potassium antimony oxide tartrate solution and mix well.

[0025] Preparation of phosphorus standard stock solution: Weigh (0.2197±0.001) g of potassium dihydrogen phosphate dried at 110℃ for 2 hours and cooled in a desiccator, dissolve it in water and transfer it to a 1000ml volumetric flask, add about 800ml of water, add 5ml of sulfuric acid solution (1+1), dilute with water, make up to the mark and mix well. This standard solution contains 50µg phosphorus (in terms of P) per liter; Phosphorus standard working solution: Accurately pipette 10mL of the above phosphorus standard stock solution into a 250mL volumetric flask, dilute with water, make up to the mark and mix; this standard solution contains 2µg phosphorus per mL and is prepared on the day of use.

[0026] Preparation of standard curve: add 0, 0.5, 1, 3, 5, 10, 15 mL of phosphate standard solution (2 μg / mL) to a colorimetric tube (50 mL), and dilute to 50 mL with distilled water. The concentrations of the standard curve solutions are 0, 0.02, 0.04, 0.12, 0.2, 0.4, 0.6 μg / mL, respectively. The standard curve is as attached. Figure 3 shown.

[0027] Color development: Add 1 mL of 10% ascorbic acid solution to the colorimetric tube and mix well; after 30 seconds, add 2 mL of molybdate solution and mix well, and let stand for 15 minutes; Measurement: Use a micro-cuvette to measure the absorbance at a wavelength of 700nm with zero concentration solution as reference.

[0028] Sample determination: After centrifuging the simulated wastewater culture solution with bacterial solution at 12000rpm for 5min, 5mL was aspirated into a 50mL volumetric flask, and 1mL of 10% ascorbic acid solution was added to the volume. After 30s, 2mL of molybdate solution was added and mixed thoroughly. After standing for 15min, the absorbance was measured. Phosphorus removal rate calculation formula: Phosphorus removal rate = [(P0 -P i )∕ P0] ×100% (P0 is the total phosphorus content after culture without inoculation of calcoacetic acid Acinetobacter seed solution, P i is the total phosphorus content after inoculation with calcoacetic acid Acinetobacter seed solution).

[0029] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A strain of Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ), characterized in that: The deposit number of the Acinetobacter calcoaceticus is: CCTCC NO:M20232617.

2. A bacterial agent, characterized in that: The active ingredient of the bacterial agent is the calcoaceticus Acinetobacter described in claim 1.

3. The calcoacetic acid Acinetobacter as claimed in claim 1, and / or, the use of the bacterial agent as claimed in claim 2 in dephosphorization.

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