Acinetobacter B2-4, its culture method and application in degradation of polycyclic aromatic hydrocarbons

Abstract

The present invention relates to the field of environmental remediation microbial technology, and discloses a strain of Acinetobacter B2‑4, its cultivation method, and its application in the degradation of polycyclic aromatic hydrocarbons. The Acinetobacter sp. B2‑4 was deposited at the General Microbiology Center of the China Culture Collection Administration on March 3, 2025, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33689. The Acinetobacter sp. B2‑4 of the present invention is an anaerobic bacterium that can use nitrate, iron, sulfate and other alternative electron acceptors 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%.

Description

Technical Field

[0001] The present invention relates to Acinetobacter B2-4, a culture method thereof, 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 exhibit not only "triggers," "drug poisoning," and "neurotoxic" effects but are also neurotoxic, posing significant risks to the ecological environment and public health. They primarily originate from fossil fuel combustion and industrial processes, such as coal-to-coke, oil extraction and refining, incomplete combustion of motor vehicle fuels, and forest fires. PAHs pose a persistent environmental hazard due to the stability and inertness of their multiple benzene ring structures, and their natural decay rate is much lower than their input rate. PAHs can persist widely in various environments, including the atmosphere, soil, water, and sediments. They first enter the atmosphere, remaining in the atmosphere in a gaseous state or bound to particulate matter. They then settle into the ground and water, ultimately migrating and accumulating in deep anaerobic environments, with phenanthrene, fluoranthene, and pyrene being the most prominent.

[0003] Although PAHs can be removed through physical and chemical processes, biodegradation is still considered the main mechanism of detoxification and detoxification. Currently, the aerobic degradation of PAHs and related mechanisms have been widely studied. Although aerobic degradation is fast and efficient, most PAHs in the actual environment will eventually accumulate in anoxic environments due to their difficult-to-degrade properties. Due to the lack of oxygen as an electron acceptor, microorganisms can only use nitrates, iron, and sulfates as alternative electron acceptors. In addition, due to the high toxicity and low bioavailability of PAHs, their degradation usually requires the coordinated metabolism of multiple microorganisms. Although PAHs degradation in nitrate reduction systems, iron reduction, sulfate reduction, and methanogenesis systems has been discovered since 1988, there is a lack of pure strains of PAHs anaerobic degradation that have been fully studied in 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 bacterial strains that can efficiently degrade PAHs under anaerobic conditions, so as to provide bacterial resources for the remediation of PAHs pollution in the environment. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides an Acinetobacter sp. B2-4, as well as a cultivation method and application thereof in the degradation of polycyclic aromatic hydrocarbons.

[0006] The technical solutions of the present invention are as follows:

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

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

[0009] The above-mentioned method for culturing Acinetobacter sp. B2-4 comprises the following steps:

[0010] Acinetobacter sp. B2-4 was inoculated into LB liquid culture medium, activated and cultured at 25-30°C and 150-200 rpm for 20-30 hours, centrifuged, and the bacteria were collected; the bacteria were then resuspended and inoculated into inorganic salt culture medium, and cultured at 25-30°C and 150-200 rpm for 20-30 hours to obtain an Acinetobacter sp. B2-4 bacterial solution with an OD600 of 0.8-1.2.

[0011] Preferably, according to the present invention, 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;

[0012] 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, Na2MoO4·2H2O0.22g / L, CoCl2·6H2O 0.24g / L, NaSeO4·10H2O 0.21g / L.

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

[0014] Preferably, according to the present invention, the degradation of polycyclic aromatic hydrocarbons is the degradation of polycyclic aromatic hydrocarbons in soil or sewage.

[0015] More preferably, the polycyclic aromatic hydrocarbon is phenanthrene or pyrene.

[0016] The application of the above-mentioned Acinetobacter sp. B2-4 in the preparation of polycyclic aromatic hydrocarbons degrading bacterial agents.

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

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

[0019] According to a preferred embodiment of the present invention, the application is to apply the above-mentioned Acinetobacter sp. B2-4 bacterial solution or PAH-degrading bacterial agent to an environment containing PAHs to degrade PAHs under anaerobic conditions.

[0020] Where the present invention is not exhaustive, existing technologies may be used.

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

[0022] 1. The present invention obtained a strain B2-4 that uses polycyclic aromatic hydrocarbons as a carbon source through screening and isolation. Based on the strain's morphology, physiological characteristics, 16S rDNA gene sequencing analysis and phylogenetic analysis, the strain was identified as Acinetobacter sp., which has the ability to degrade polycyclic aromatic hydrocarbons and can be used for the remediation of polycyclic aromatic hydrocarbons pollution, providing a strain resource for the remediation of polycyclic aromatic hydrocarbons pollution in the environment.

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

[0024] Figure 1 This is the cell morphology of Acinetobacter sp. B2-4 under an electron microscope.

[0025] Figure 2 This is the colony morphology of Acinetobacter sp. B2-4 on solid culture medium.

[0026] Figure 3This is the evolutionary tree analysis diagram of Acinetobacter sp. B2-4.

[0027] Figure 4 Figure 2 shows the degradation curve of Acinetobacter sp. B2-4 to phenanthrene with an initial concentration of 100 mg / L and the bacterial growth.

[0028] Figure 5 Figure 2 shows the degradation curve of Acinetobacter sp. B2-4 to pyrene with an initial concentration of 100 mg / L and the bacterial growth. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Microbial source: Acinetobacter sp. B2-4, the strain was deposited in the General Microbiology Center of China Culture Collection Administration on March 3, 2025, the deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33689.

[0031] 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;

[0032] 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, Na2MoO4·2H2O0.22g / L, CoCl2·6H2O 0.24g / L, NaSeO4·10H2O 0.21g / L.

[0033] The raw materials used in the examples are all conventional raw materials, and the equipment used are all conventional equipment, which can be purchased commercially.

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

[0035] 1. Sample source

[0036] Samples were collected from oil-bearing soil in Shengli Oilfield, Dongying City, Shandong Province, and were domesticated for a long time using high concentrations of phenanthrene or pyrene as carbon sources to obtain sludge domesticated with polycyclic aromatic hydrocarbons for a long time.

[0037] 2. Screening and Isolation of Strain

[0038] (1) Take 80 mL of PAHs-acclimated sludge sample and place it in a 250 mL serum bottle. Add PBS buffer and ultrasonically clean it three times.

[0039] (2) Dilute the sludge sample after cleaning in step (1) with inorganic salt culture medium to OD 600 The value is 0.5~1;

[0040] (3) Add gradient concentrations of PAH acetone solutions (0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L) to a sterile 3 mL anaerobic bottle. After the acetone evaporates, add it to the sterilized inorganic salt culture medium.

[0041] (4) The suspension in step (2) is inoculated at a rate of 10% into the inorganic salt culture medium prepared in step (3) with polycyclic aromatic hydrocarbons as the sole carbon source, cultured at a constant temperature with shaking, and samples are taken at fixed intervals to test the microbial growth status of the culture medium;

[0042] (5) Dilute the anaerobic culture solution in the vial with the best microbial growth with sterile saline, and take 10 -4 , 10 -5 , 10 -6 and 10 -7 100 μL of the bacterial suspension was applied to an inorganic salt culture medium with polycyclic aromatic hydrocarbons as the sole carbon source and cultured statically at 30°C under anaerobic conditions;

[0043] (6) When colonies appear, use an inoculating loop to pick a single colony and streak it on the plate. Transfer the streak twice. Use an inoculating loop to pick a single colony and transfer it to a serum bottle containing 50 ml of liquid culture medium. Incubate at 30°C and 130 rpm with shaking for 24 hours. Add glycerol and bacterial solution in a ratio of 1:1 to the frozen tube, mix well, and store in a -80°C ultra-low temperature freezer. The strain is numbered B2-4.

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

[0045] 1. The strain B2-4 obtained in Example 1 was stained with Gram staining, and the morphology of the strain B2-4 was observed using a field emission scanning electron microscope. The results are as follows: Figure 1 and Figure 2 shown.

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

[0047] 2. DNA from strain B2-4 obtained in Example 1 was extracted using the T5 Direct PCR Kit (Plant). The specific extraction method was described in the kit instructions. The 16S rDNA gene of strain B2-4 was amplified by PCR using primers 27F and 1492R, and the amplified specific fragment was sequenced.

[0048] PCR primers used are universal primers:

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

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

[0051] The sequencing results showed that the 16S rDNA sequence of B2-4 was 1451 bp long (specifically shown in SEQ ID NO. 1). Comparison of the results on NCBI revealed that strain B2-4 was most closely related to Acinetobacter sp. using the neighbor-joining method of MEGA7 to construct a phylogenetic tree. The results are as follows: Figure 3 shown.

[0052] Based on the 16S rDNA sequence comparison results and combined with the biological characteristics of the strain, the strain was identified as Acinetobacter sp. and named Acinetobacter sp. B2-4.

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

[0054] 1. A method for preparing an Acinetobacter sp. B2-4 bacterial solution, comprising the following steps:

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

[0056] (2) The cells obtained in step (1) were resuspended and inoculated into an inorganic salt culture medium, and cultured at 30°C and 150 rpm for 24 h to obtain an Acinetobacter sp. B2-4 bacterial solution with an OD600 of 1.

[0057] 2. Preparation of degradation medium

[0058] Preparation of a phenanthrene degradation medium: Add 50 mL of an inorganic salt medium to a 100 mL serum bottle, then add a phenanthrene acetone solution to a concentration of 100 mg / L. Place the medium in a sterile operating chamber for 24 hours to allow the acetone to evaporate, thereby obtaining the phenanthrene degradation medium.

[0059] The pyrene degradation medium was prepared according to the same method.

[0060] 3. PAH degradation rate detection

[0061] According to the mass percentage of 5%, Acinetobacter sp. B2-4 bacterial liquid was inoculated into the phenanthrene degradation culture medium, sealed with a butyl rubber stopper and moved to a constant temperature shaking incubator for 45 days at 30°C, 150 rpm and dark anaerobic environment. In addition, three groups of parallel and sterile culture media were set as blank controls. After each week of cultivation, the culture vials were ultrasonically extracted with equal volumes of cyclohexane, and the phenanthrene content was determined by high performance liquid chromatography (HPLC) to calculate the phenanthrene degradation rate. The degradation rate of pyrene was detected by the same method. The results of the degradation rates of phenanthrene and pyrene are as follows: Figure 4 and Figure 5 shown.

[0062] Depend on Figure 4 and Figure 5 The strain was found to be effective at degrading both phenanthrene and pyrene under anaerobic conditions. After 45 days of culture, Acinetobacter sp. B2-4 achieved a phenanthrene degradation rate of 74.9% and a pyrene degradation rate of 56.2%.

Claims

1. A strain of Acinetobacter that degrades polycyclic aromatic hydrocarbons under anaerobic conditions ( Acinetobacter sp. ) B2-4, characterized in that, The strain was deposited in the General Microbiology Center of China 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 as claimed in claim 1 ( Acinetobacter sp. ) B2-4, characterized in that, The gene sequence of 16S rDNA of the Acinetobacter B2-4 is shown in SEQ ID NO.

1.

3. Acinetobacter according to claim 1 ( Acinetobacter sp. ) The culture method of B2-4, characterized in that The method comprises the following steps: inoculating Acinetobacter B2-4 into LB liquid culture medium, activating and culturing the culture at 25-30°C and 150-200 rpm for 20-30 hours, centrifuging, and collecting the bacteria; then resuspending the bacteria and inoculating the culture into an inorganic salt culture medium, continuing to culture the culture at 25-30°C and 150-200 rpm for 20-30 hours, and obtaining an Acinetobacter B2-4 bacterial liquid with an OD600 of 0.8-1.

2.

4. Acinetobacter as claimed in claim 3 ( Acinetobacter sp. ) The culture method of B2-4, 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. The Acinetobacter according to claim 1 ( Acinetobacter sp. ) Application of B2-4 in degradation of polycyclic aromatic hydrocarbons, characterized in that, The degradation of polycyclic aromatic hydrocarbons is to degrade polycyclic aromatic hydrocarbons in soil or sewage under anaerobic conditions; the polycyclic aromatic hydrocarbons are phenanthrene or pyrene.

6. The Acinetobacter according to claim 1 ( Acinetobacter sp. ) Application of B2-4 in the preparation of a polycyclic aromatic hydrocarbon degradation bacterial agent, wherein the polycyclic aromatic hydrocarbon is phenanthrene or pyrene.

7. A polycyclic aromatic hydrocarbons-degrading bacterial agent, characterized in that: The bacterial agent is the Acinetobacter ( Acinetobacter sp. ) B2-4 is the main active ingredient, and the polycyclic aromatic hydrocarbon is phenanthrene or pyrene.

8. The Acinetobacter according to claim 1 ( Acinetobacter sp. ) Use of the polycyclic aromatic hydrocarbon-degrading bacterial agent described in B2-4 or claim 7 in the bioremediation of a polycyclic aromatic hydrocarbon-contaminated environment, wherein the polycyclic aromatic hydrocarbons are degraded under anaerobic conditions by application to an environment containing polycyclic aromatic hydrocarbons; the polycyclic aromatic hydrocarbons are phenanthrene or pyrene.

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