Acinetobacter B2-4, culture method thereof and application of acinetobacter B2-4 in degradation of polycyclic aromatic hydrocarbon

Abstract

The invention relates to the technical field of environmental remediation microorganisms, and discloses acinetobacter B2-4, a culture method thereof and application of the acinetobacter B2-4 in degradation of polycyclic aromatic hydrocarbon. The Acinetobacter sp. B2-4 is preserved in the China General Microbiological Culture Collection Center on March 3, 2025, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.33689. The Acinetobacter sp. B2-4 has the advantages that the Acinetobacter sp. The Acinetobacter sp. B2-4 disclosed by the invention is an anaerobic bacterium, and can utilize nitrate, iron, sulfate and the like to replace an electron acceptor in an anoxic environment to continuously degrade polycyclic aromatic hydrocarbon. After the strain is cultured for 45 days, the degradation rate of phenanthrene reaches 74.9%, and the degradation rate of pyrene reaches 56.2%.

Description

Technical Field

[0001] The present invention relates to Acinetobacter sp. B2-4, its cultivation method and application in degrading polycyclic aromatic hydrocarbons, belonging to the technical field of environmental remediation microorganisms (polycyclic aromatic hydrocarbon-degrading bacteria). Background Art

[0002] Polycyclic Aromatic Hydrocarbons (PAHs) are a class of persistent organic pollutants widely distributed in the environment. They not only have "carcinogenic, teratogenic and mutagenic" effects, but also have neurotoxicity, posing great hazards to the ecological environment and public health. Their main sources are the combustion of fossil fuels and industrial production processes, such as coke making from coal, oil extraction and refining, incomplete combustion of motor vehicle fuels, and forest fires. Due to the good stability and inertness of the structure of multiple benzene rings, and their natural attenuation rate is much lower than the input rate, PAHs cause persistent harm in the environment. PAHs can widely and persistently exist in various environments, such as the atmosphere, soil, water bodies and sediments. First, it can enter the atmosphere and exist in the atmospheric environment in the form of gas or combined with particulate matter, and then enter the ground and water bodies through sedimentation, and finally migrate and accumulate in the deep anaerobic environment, and the proportions of phenanthrene, fluoranthene and pyrene are the highest.

[0003] Although PAHs can be removed through physicochemical processes, biodegradation is still considered the main mechanism for detoxification. At present, the aerobic degradation of PAHs and related mechanisms have been widely studied. Although the aerobic degradation speed is fast and the efficiency is high, most of the polycyclic aromatic hydrocarbons in the actual environment will eventually accumulate in the anoxic environment due to their difficult-to-degrade characteristics. Due to the lack of oxygen as an electron acceptor, microorganisms can only use alternative electron acceptors such as nitrate, iron and sulfate. In addition, due to the high toxicity and low bioavailability of PAHs, their degradation usually requires the cooperative metabolism of multiple microorganisms to complete. Although the degradation of PAHs in the nitrate reduction system, iron reduction, sulfate reduction, and methanogenesis systems has been successively discovered since 1988, there is a severe lack of well-studied pure strains of anaerobic degradation of polycyclic aromatic hydrocarbons under these systems; moreover, the research on the anaerobic degradation pathways and mechanisms of PAHs still needs further study.

[0004] In view of this, it is still of great significance to isolate and screen strains that can efficiently degrade polycyclic aromatic hydrocarbons under anaerobic conditions, providing strain resources for the pollution remediation of polycyclic aromatic hydrocarbons in the environment. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a strain of Acinetobacter sp. B2-4, and also provides its cultivation method and application in degrading polycyclic aromatic hydrocarbons.

[0006] The technical solution of the present invention is as follows:

[0007] An Acinetobacter sp. strain B2-4, which was deposited on March 3, 2025 at the General Microbiological Center of the China National Microbial Culture Collection Center, with the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC No. 33689.

[0008] Preferably according to the present invention, the gene sequence of the 16S rDNA of the Acinetobacter sp. B2-4 is as shown in SEQ ID NO.1.

[0009] The cultivation method of the above-mentioned Acinetobacter sp. B2-4 includes the following steps:

[0010] Inoculate Acinetobacter B2-4 into LB liquid medium, and activate and culture it for 20-30 h under the conditions of 25-30 °C and 150-200 rpm, then centrifuge to collect the cells; then resuspend the cells and inoculate them into the inorganic salt medium, and continue to culture for 20-30 h under the conditions of 25-30 °C and 150-200 rpm to obtain an Acinetobacter sp. B2-4 bacterial liquid with OD600 = 0.8-1.2.

[0011] Preferably according to the present invention, the formula of the inorganic salt medium is: NaNO 3 1.5 g / L, K 2 HPO 4 4 g / L, KH 2 PO 4 6 g / L, MgSO 4 0.2 g / L, CaCl 2 0.02 g / L, FeCl 3 0.05 g / L, NH 4 Cl 1 g / L, trace element solution 1 mL, adjust the pH to 7.0;

[0012] Among them, the formula of the trace element solution is: EDTA 15 g / L, H 3 BO 3 ·0.014 g / L, MnCl 2 ·4H 2 O 0.99 g / L, CuSO 4 ·5H 2 O 0.25 g / L, ZnSO 4 ·7H 2 O 0.43 g / L, NiCl 2 ·6H 2O 0.19 g / L, Na 2 MoO 4 ·2H 2 O 0.22 g / L, CoCl 2 ·6H 2 O 0.24 g / L, NaSeO 4 ·10H 2 O 0.21 g / L.

[0013] The application of the above-mentioned Acinetobacter sp. B2-4 in degrading polycyclic aromatic hydrocarbons.

[0014] Preferably according to the present invention, the polycyclic aromatic hydrocarbons to be degraded are those in soil or sewage.

[0015] More preferably, the polycyclic aromatic hydrocarbons are phenanthrene or pyrene.

[0016] The application of the above-mentioned Acinetobacter sp. B2-4 in preparing a polycyclic aromatic hydrocarbon degrading bacterial agent.

[0017] A polycyclic aromatic hydrocarbon degrading bacterial agent, which uses the above-mentioned Acinetobacter sp. B2-4 as the main active ingredient.

[0018] The application of the above-mentioned Acinetobacter sp. B2-4 or the polycyclic aromatic hydrocarbon degrading bacterial agent in the bioremediation of polycyclic aromatic hydrocarbon-polluted environments.

[0019] Preferably according to the present invention, the application is to apply the above-mentioned Acinetobacter sp. B2-4 bacterial liquid or the polycyclic aromatic hydrocarbon degrading bacterial agent to an environment containing polycyclic aromatic hydrocarbons, and degrade the polycyclic aromatic hydrocarbons under anaerobic conditions.

[0020] For those aspects not detailed in the present invention, existing technologies can be adopted.

[0021] The technical features and beneficial effects of the present invention:

[0022] 1. In the present invention, a degrading strain B2-4 using polycyclic aromatic hydrocarbons as a carbon source is obtained through screening and isolation. According to the strain morphology, physiological characteristics, 16S rDNA gene sequencing analysis and phylogenetic analysis, the strain is identified as Acinetobacter sp., which has the function of degrading polycyclic aromatic hydrocarbons and can be used for the pollution remediation of polycyclic aromatic hydrocarbons, providing bacterial species resources for the pollution remediation of polycyclic aromatic hydrocarbons in the environment.

[0023] 2. The Acinetobacter sp. B2-4 of the present invention is an anaerobic bacterium that can utilize alternative electron acceptors such as nitrate, iron, and sulfate in an anoxic environment to continuously degrade polycyclic aromatic hydrocarbons. After 45 days of cultivation, the degradation rate of phenanthrene reached 74.9%, and the degradation rate of pyrene reached 56.2%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a morphological diagram of Acinetobacter sp. B2-4 cells under an electron microscope.

[0025] Figure 2 It is a morphological diagram of the colonies of Acinetobacter sp. B2-4 on a solid medium.

[0026] Figure 3 It is a phylogenetic tree analysis diagram of Acinetobacter sp. B2-4.

[0027] Figure 4 It is a degradation curve of phenanthrene with an initial concentration of 100 mg / L and a diagram of the growth of Acinetobacter sp. B2-4.

[0028] Figure 5 It is a degradation curve of pyrene with an initial concentration of 100 mg / L and a diagram of the growth of Acinetobacter sp. B2-4. DETAILED DESCRIPTION OF THE INVENTION

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Microbial source: Acinetobacter sp. B2-4. This strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 3, 2025. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 33689.

[0031] The formula of the inorganic salt medium is: NaNO 3 1.5 g / L, K 2 HPO 4 4 g / L, KH 2 PO 46 g / L, MgSO 4 0.2 g / L, CaCl 2 0.02 g / L, FeCl 3 0.05 g / L, NH 4 Cl 1 g / L, trace element solution 1 mL, adjust the pH to 7.0;

[0032] Among them, the formula of the trace element solution is: EDTA 15 g / L, H 3 BO 3 ·0.014 g / L, MnCl 2 ·4H 2 O 0.99 g / L, CuSO 4 ·5H 2 O 0.25 g / L, ZnSO 4 ·7H 2 O 0.43 g / L, NiCl 2 ·6H 2 O 0.19 g / L, Na 2 MoO 4 ·2H 2 O 0.22 g / L, CoCl 2 ·6H 2 O 0.24 g / L, NaSeO 4 ·10H 2 O 0.21 g / L.

[0033] The raw materials used in the examples are all conventional raw materials, and the equipment used is all conventional equipment, which can be obtained by purchasing in the market.

[0034] Example 1, Screening and Isolation of Acinetobacter sp. B2-4

[0035] 1. Sample Source

[0036] Samples were collected from the oil-containing soil of Shengli Oilfield in Dongying City, Shandong Province, and long-term acclimation was carried out using high-concentration phenanthrene or pyrene as the carbon source to obtain sludge with long-term acclimation to polycyclic aromatic hydrocarbons.

[0037] 2. Screening and Isolation of Strains

[0038] (1) Take 80 mL of the sludge sample with long-term acclimation to polycyclic aromatic hydrocarbons and place it in a 250 mL serum bottle, and add PBS buffer solution to ultrasonic clean it 3 times;

[0039] (2) Dilute the sludge sample after cleaning in step (1) with the inorganic salt medium to an OD 600 value of 0.5 - 1;

[0040] (3) Add acetone solutions of polycyclic aromatic hydrocarbons at gradient concentrations (0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L) to a sterile 3 mL anaerobic bottle. After the acetone has evaporated, add it to the sterilized inorganic salt medium;

[0041] (4) Inoculate the suspension in step (2) into the inorganic salt medium prepared in step (3) with polycyclic aromatic hydrocarbons as the sole carbon source at an inoculation amount of 10%. Incubate with constant shaking, and take samples at fixed intervals to test the microbial growth status of the culture solution;

[0042] (5) Gradient dilute the culture solution in the anaerobic vial with the best microbial growth with sterile normal saline. Take 100 μL of the bacterial suspensions of 10 -4 、10 -5 、10 -6 and 10 -7 respectively and spread them on the inorganic salt medium with polycyclic aromatic hydrocarbons as the sole carbon source. Incubate statically under anaerobic conditions at 30 °C;

[0043] (6) When colonies appear, pick single colonies with an inoculation loop and streak them on the plate, and streak twice; pick single colonies with an inoculation loop and transfer them to a serum bottle containing 50 ml of liquid medium. Incubate with shaking at 30 °C and 130 rpm for 24 h. Add glycerol and the bacterial solution in a ratio of 1:1 to the cryopreservation tube respectively. After mixing evenly, store it in a -80 °C ultra-low temperature refrigerator, and the strain number is B2-4.

[0044] Example 2. Identification of Acinetobacter sp. B2-4

[0045] 1. Stain the strain B2-4 obtained in Example 1 by Gram staining method, and observe the morphology of strain B2-4 using a field emission scanning electron microscope. The results are as shown in Figure 1 and Figure 2 .

[0046] It can be seen from Figure 1 and Figure 2 that the biological characteristics of this strain are: Gram-negative bacteria, cells are short rod-shaped, single, with a size of (0.6 μm - 0.8 μm) × (1.2 μm - 3 μm) (see Figure 1 ). And the colony surface is smooth, convex, moist, with a diameter of 2 - 3 mm (see Figure 2 ).

[0047] 2. Use the T5 Direct PCR Kit (Plant) bacterial genome extraction kit to extract the DNA of the strain B2-4 obtained in Example 1. The specific extraction method refers to the instruction manual of this kit. Use the primers 27F and 1492R to perform PCR amplification on the 16S rDNA gene of the strain B2-4, and sequence the amplified specific fragment.

[0048] The PCR primers use universal primers:

[0049] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.2),

[0050] 1492R: 5'-GGTTACCTTGTTACGACTTC-3' (SEQ ID NO.3).

[0051] The sequencing result is: the length of the 16S rDNA sequence of B2-4 is 1451 bp (shown in the specific SEQ ID NO.1). Compare this result on NCBI. After BLAST comparison, it is found that the strain B2-4 has the closest genetic relationship with Acinetobacter sp. Use the neighbor-joining method of MEGA7 to establish a phylogenetic tree, and the result is as Figure 3 shown.

[0052] According to the 16S rDNA sequence comparison result and combined with the biological characteristics of this strain, this strain is identified as Acinetobacter sp., named Acinetobacter sp. B2-4.

[0053] Example 3. Degradation of phenanthrene and pyrene by Acinetobacter sp. B2-4

[0054] 1. The preparation method of the Acinetobacter sp. B2-4 bacterial solution includes the following steps:

[0055] (1) Inoculate Acinetobacter sp. B2-4 into the LB liquid medium, activate and culture it at 30 °C and 150 rpm for 24 h, centrifuge, and collect the bacterial cells;

[0056] (2) After resuspending the bacterial cells in step (1), inoculate them into the inorganic salt medium, and continue to culture at 30 °C and 150 rpm for 24 h to obtain the Acinetobacter sp. B2-4 bacterial solution with OD600 = 1.

[0057] 2. Preparation of the degradation medium

[0058] Method for preparing phenanthrene degradation medium: Add 50 mL of inorganic salt medium into a 100 mL serum bottle, and then add an acetone solution of phenanthrene to make the concentration of phenanthrene in the inorganic salt medium 100 mg / L. Place it in a sterile operation box for 24 hours to volatilize acetone, and obtain the phenanthrene degradation medium.

[0059] Prepare a pyrene degradation medium according to the same method.

[0060] 3. Detection of polycyclic aromatic hydrocarbon degradation rate

[0061] According to the mass percentage of 5%, inoculate the Acinetobacter sp. B2-4 bacterial solution into the phenanthrene degradation medium, seal it with a butyl rubber stopper, and transfer it to a constant temperature shaking incubator. Cultivate it for 45 days under the conditions of 30 °C, 150 rpm, and a dark anaerobic environment. In addition, set three groups of parallel and sterile media as blank controls. Every week during cultivation, ultrasonically extract the culture vial with an equal volume of cyclohexane, and measure the content of phenanthrene in it by high performance liquid chromatography (HPLC), and calculate the degradation rate of phenanthrene. Detect the degradation rate of pyrene according to the same method. The results of the degradation rates of both phenanthrene and pyrene are as Figure 4 and Figure 5 shown.

[0062] It can be seen from Figure 4 and Figure 5 that under anaerobic conditions, the strain has a degradation effect on both phenanthrene and pyrene. After 45 days of cultivation, the degradation rate of phenanthrene by Acinetobacter sp. B2-4 is 74.9%; after 45 days of cultivation, the degradation rate of pyrene by Acinetobacter sp. B2-4 is 56.2%.

Claims

1. A strain of Acinetobacter B2-4, characterized in that The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 3, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.33689.

2. Acinetobacter B2-4 according to claim 1, characterized in that The gene sequence of 16S rDNA of the Acinetobacter B2-4 is shown in SEQ ID NO.

1.

3. The method for culturing Acinetobacter B2-4 according to claim 1, characterized in that: The steps include: The Acinetobacter B2-4 was inoculated into LB liquid culture medium, activated and cultured at 25-30°C and 150-200rpm for 20-30h, centrifuged, and the bacteria were collected; then the bacteria were resuspended and inoculated into inorganic salt culture medium, and cultured at 25-30°C and 150-200rpm for 20-30h to obtain an Acinetobacter B2-4 bacterial solution with OD600=0.8-1.

2.

4. The method for culturing Acinetobacter B2-4 according to claim 3, characterized in that: The formula of the inorganic salt culture medium is: NaNO3 1.5g / L, K2HPO4 4g / L, KH2PO4 6g / L, MgSO4 0.2g / L, CaCl2 0.02g / L, FeCl3 0.05g / L, NH4Cl 1g / L, trace element solution 1mL, adjusted to pH 7.0; Among them, the formula of trace element solution is: EDTA 15g / L, H3BO3·0.014g / L, MnCl2·4H2O 0.99g / L, CuSO4·5H2O 0.25g / L, ZnSO4·7H2O 0.43g / L, NiCl2·6H2O 0.19g / L, Na2MoO4·2H2O 0.22g / L, CoCl2·6H2O 0.24g / L, NaSeO4·10H2O 0.21g / L.

5. Use of the Acinetobacter B2-4 described in claim 1 in degrading polycyclic aromatic hydrocarbons.

6. The use of Acinetobacter B2-4 in the degradation of polycyclic aromatic hydrocarbons as claimed in claim 5, characterized in that: The degradation of polycyclic aromatic hydrocarbons is the degradation of polycyclic aromatic hydrocarbons in soil or sewage; the polycyclic aromatic hydrocarbons are phenanthrene or pyrene.

7. Use of the Acinetobacter B2-4 described in claim 1 in the preparation of a polycyclic aromatic hydrocarbons-degrading bacterial agent.

8. A polycyclic aromatic hydrocarbons-degrading bacterial agent, characterized in that: The bacterial agent uses the Acinetobacter B2-4 described in claim 1 as the main active ingredient.

9. Use of the Acinetobacter B2-4 described in claim 1 or the polycyclic aromatic hydrocarbons-degrading bacterial agent described in claim 8 in the bioremediation of an environment contaminated by polycyclic aromatic hydrocarbons, wherein the polycyclic aromatic hydrocarbons are degraded under anaerobic conditions by applying the bacterium to an environment containing polycyclic aromatic hydrocarbons.

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