CN120060083B

CN120060083B - Acinetobacter B2-4, its culture method and application in degradation of polycyclic aromatic hydrocarbons

Info

Publication number: CN120060083B
Application number: CN202510512498.8A
Authority: CN
Inventors: 苏继新; 曹晓晴; 苏晓文; 牛启桂; 薛含含; 张易宁; 万瑜; 节龙飞; 麻琼艳
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2025-04-23
Filing date: 2025-04-23
Publication date: 2025-08-26
Anticipated expiration: 2045-04-23
Also published as: CN120060083A

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

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

Technical Field

The invention relates to acinetobacter B2-4, a culture method thereof and application thereof in degrading polycyclic aromatic hydrocarbon, belonging to the technical field of environmental repair microorganisms (polycyclic aromatic hydrocarbon degrading bacteria).

Background

Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons, PAHs) are a widely distributed class of persistent organic pollutants in the environment, not only having a "tri-induced" effect, but also neurotoxicity, and having great harm to the ecological environment and public health. It is mainly derived from fossil fuel combustion and industrial production processes, such as coal-to-coke, petroleum exploitation and refining, incomplete combustion of motor vehicle fuel, forest fire and the like. Because the structure of the benzene rings has better stability and inertness, and the natural decay rate is far lower than the input rate, the PAHs cause lasting harm in the environment. PAHs can be widely and permanently found in various environments such as the atmosphere, soil, water and sediments. Firstly, it can enter the atmosphere, exist in the atmosphere in a gaseous state or combined with particulate matters, then enter the ground and water body through sedimentation, finally migrate and accumulate in the deep anaerobic environment, and the phenanthrene, fluoranthene and pyrene are the highest in proportion.

Although PAHs can be removed by physicochemical processes, biodegradation is still considered to be the main mechanism of detoxification and detoxification. Currently, the aerobic degradation of PAHs and related mechanisms have been widely studied. Although the aerobic degradation speed is high and the efficiency is high, most of polycyclic aromatic hydrocarbons in the actual environment are accumulated in the anoxic environment finally due to the characteristic of difficult degradation, microorganisms can only utilize nitrate, iron, sulfate and the like to replace the electron acceptors due to the lack of oxygen as the electron acceptors, and in addition, due to the high toxicity of PAHs, the bioavailability is low, and the degradation is usually completed by the cooperative metabolism among various microorganisms. Although nitrate reduction systems, iron reduction, sulfate reduction, and PAHs degradation in methanogenic systems have been discovered successively since 1988, pure strains of polycyclic aromatic hydrocarbon anaerobic degradation have been well studied under these systems are very lacking, and further studies on the pathways and mechanisms of PAHs anaerobic degradation have been required.

In view of the above, the separation and screening of the strain capable of efficiently degrading the polycyclic aromatic hydrocarbon under the anaerobic condition has important significance, and strain resources are provided for the pollution repair of the polycyclic aromatic hydrocarbon in the environment.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides Acinetobacter (Acinetobacter sp.) B2-4, and simultaneously provides a culture method and application thereof in degrading polycyclic aromatic hydrocarbon.

The technical scheme of the invention is as follows:

Acinetobacter (Acinetobacter sp.) B2-4 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 3 months and 3 days, and the preservation address is number 1, no.3, and the preservation number is CGMCC No.33689 in the Korean region North Star of Beijing city.

According to a preferred embodiment of the present invention, the 16S rDNA of Acinetobacter (Acinetobacter sp.) B2-4 has a gene sequence shown in SEQ ID NO. 1.

The method for culturing Acinetobacter (Acinetobacter sp.) B2-4 comprises the following steps:

inoculating Acinetobacter B2-4 into an LB liquid culture medium, performing activation culture for 20-30 hours at 25-30 ℃ and 150-200 rpm, centrifuging, collecting thalli, then suspending the thalli again, inoculating into an inorganic salt culture medium, and continuously culturing for 20-30 hours at 25-30 ℃ and 150-200 rpm to obtain Acinetobacter (Acinetobacter sp.) B2-4 bacterial liquid with OD600 = 0.8-1.2.

According to the 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, microelement liquid 1mL, and the pH value is regulated to 7.0;

wherein the formula of the microelement liquid 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.

Application of Acinetobacter (Acinetobacter sp.) B2-4 in degrading polycyclic aromatic hydrocarbon is provided.

According to the invention, the degradation of the polycyclic aromatic hydrocarbon is the degradation of the polycyclic aromatic hydrocarbon in soil or sewage.

Further preferably, the polycyclic aromatic hydrocarbon is phenanthrene or pyrene.

Application of Acinetobacter (Acinetobacter sp.) B2-4 in preparing polycyclic aromatic hydrocarbon degrading bacterial agent.

A polycyclic aromatic hydrocarbon degrading bacterial agent comprises Acinetobacter (Acinetobacter sp.) B2-4 as main active ingredient.

The application of the Acinetobacter (Acinetobacter sp.) B2-4 or the polycyclic aromatic hydrocarbon degrading bacterial agent in the bioremediation of the polycyclic aromatic hydrocarbon polluted environment.

According to the invention, preferably, the application is that the Acinetobacter (Acinetobacter sp.) B2-4 bacterial liquid or the polycyclic aromatic hydrocarbon degrading bacterial agent is applied to an environment containing polycyclic aromatic hydrocarbon, and the polycyclic aromatic hydrocarbon is degraded under anaerobic conditions.

The present invention is not limited to the details of the prior art.

The invention has the technical characteristics and beneficial effects that:

1. according to the invention, the degrading strain B2-4 which takes the polycyclic aromatic hydrocarbon as a carbon source is obtained through screening and separation, and the strain is identified to be Acinetobacter sp according to strain morphology, physiological characteristics and 16S rDNA gene sequencing analysis and phylogenetic analysis, so that the degrading strain has the function of degrading the polycyclic aromatic hydrocarbon, can be used for pollution repair of the polycyclic aromatic hydrocarbon, and provides strain resources for pollution repair of the polycyclic aromatic hydrocarbon in the environment.

2. The Acinetobacter (Acinetobacter sp.) B2-4 is anaerobic bacteria, and can utilize nitrate, iron, sulfate and the like to replace electron acceptors in an anoxic environment to continuously degrade polycyclic aromatic hydrocarbon. After 45 days of culture, the degradation rate of phenanthrene reaches 74.9%, and the degradation rate of pyrene reaches 56.2%.

Drawings

FIG. 1 is a cell morphology of Acinetobacter (Acinetobacter sp.) B2-4 under electron microscopy.

FIG. 2 is a colony morphology of Acinetobacter (Acinetobacter sp.) B2-4 on solid medium.

FIG. 3 is a diagram of an analysis of a phylogenetic tree of Acinetobacter (Acinetobacter sp.) B2-4.

FIG. 4 is a graph showing degradation curves and bacterial growth of Acinetobacter (Acinetobacter sp.) B2-4 against phenanthrene having an initial concentration of 100 mg/L.

FIG. 5 is a graph showing degradation curves of Acinetobacter (Acinetobacter sp.) B2-4 against pyrene at an initial concentration of 100 mg/L and bacterial growth.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Microorganism source is Acinetobacter (Acinetobacter sp.) B2-4, the strain 2025 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 3 months, and the preservation address is number 3 of West Song No. 1, the Korean region North Star of Beijing, and the preservation number is CGMCC No.33689.

The formula of the inorganic salt culture medium is ：NaNO₃ 1.5g/L,K₂HPO₄ 4g/L,KH₂PO₄ 6g/L,MgSO₄0.2g/L,CaCl₂ 0.02g/L,FeCl₃ 0.05g/L,NH₄Cl 1g/L, microelement liquid 1mL, and the pH value is regulated to 7.0;

The raw materials used in the examples are all conventional raw materials, and the equipment used in the examples are all conventional equipment and are all commercially available.

EXAMPLE 1 screening separation of Acinetobacter (Acinetobacter sp.B2-4)

1. Sample source

Samples are collected from oil-containing soil of a victory oil field in eastern city of Shandong province, and long-term domestication is carried out by taking high-concentration phenanthrene or pyrene as a carbon source, so that sludge for long-term domestication of polycyclic aromatic hydrocarbon is obtained.

2. Screening and isolation of strains

(1) Placing a sludge sample subjected to long-term domestication of 80 mL polycyclic aromatic hydrocarbon in a 250 mL serum bottle, and adding PBS buffer solution for ultrasonic cleaning for 3 times;

(2) Diluting the sludge sample cleaned in the step (1) to an OD ₆₀₀ value of 0.5-1 by using an inorganic salt culture medium;

(3) Adding a gradient concentration polycyclic aromatic hydrocarbon acetone solution (0 mg/L, 50 mg/L, 100 mg/L, 200 mg/L) into a sterile 3mL anaerobic bottle, and adding the acetone into a sterilized inorganic salt culture medium after the acetone volatilizes;

(4) The suspension in the step (2) is inoculated into the inorganic salt culture medium which takes the polycyclic aromatic hydrocarbon as the only carbon source and is prepared in the step (3) in 10 percent of inoculum size, and the culture medium is subjected to constant temperature shaking culture, sampling and testing the microbial growth condition of the culture medium at fixed time intervals;

(5) Performing gradient dilution on an anaerobic small bottle culture solution with best microorganism growth by using sterile normal saline, respectively taking 100 mu L of bacterial suspensions of 10 ^-4、10^-5、10^-6 and 10 ^-7, coating the bacterial suspensions into an inorganic salt culture medium with polycyclic aromatic hydrocarbon as a unique carbon source, and performing stationary culture under the anaerobic condition at 30 ℃;

(6) When colonies appear, single colonies are selected by an inoculating loop, streaked on a plate, streaked twice in a transfer way, single colonies are selected by the inoculating loop, transferred into a serum bottle filled with a 50 ml liquid culture medium, and subjected to shake culture for 24 hours at 30 ℃ and 130 rpm. Glycerin and bacterial liquid with the ratio of 1:1 are respectively added into the freezing and storing tube, and the mixture is placed into an ultralow temperature refrigerator at-80 ℃ for storage after being uniformly mixed, and the bacterial strain number is B2-4.

EXAMPLE 2 identification of Acinetobacter (Acinetobacter sp.) B2-4

1. The strain B2-4 obtained in example 1 was stained by the gram staining method, and the morphology of the strain B2-4 was observed by using a field emission scanning electron microscope, and the results are shown in FIG. 1 and FIG. 2.

As can be seen from FIGS. 1 and 2, the strain is characterized in that the gram-negative bacteria and the cells are in the shape of short rods, and the size of each gram-negative bacteria is (0.6-0.8 μm) x (1.2-3 μm) (see FIG. 1). And the colony surface is smooth, convex and moist, and the diameter is 2-3 mm (see figure 2).

2. The DNA of the strain B2-4 obtained in example 1 was extracted using a T5 DIRECT PCR KIT (Plant) bacterial genome extraction kit, the specific extraction method was referred to the instructions of the kit, the 16S rDNA gene of the strain B2-4 was PCR amplified using 27F and 1492R primers, and the amplified specific fragment was sequenced.

The PCR primer adopts a universal primer:

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

1492R:5'-GGTTACCTTGTTACGACTTC-3'(SEQ ID NO.3)。

As a result of sequencing, the length of the 16S rDNA sequence of B2-4 was 1451 bp (specifically SEQ ID NO. 1). The results were aligned on NCBI, and after BLAST alignment, it was found that strain B2-4 was closest to Acinetobacter sp.

Based on the 16S rDNA sequence alignment and in combination with the biological properties of the strain, the strain was identified as Acinetobacter sp., designated Acinetobacter sp.) B2-4.

EXAMPLE 3 Acinetobacter (Acinetobacter sp.) B2-4 degradation of phenanthrene and pyrene

1. The preparation method of the Acinetobacter (Acinetobacter sp.) B2-4 bacterial liquid comprises the following steps:

(1) Inoculating Acinetobacter (Acinetobacter sp.) B2-4 into LB liquid culture medium, performing activation culture at 30deg.C and 150rpm for 24 hr, centrifuging, and collecting thallus;

(2) And (3) re-suspending the bacteria in the step (1) and inoculating the bacteria into an inorganic salt culture medium, and continuously culturing the bacteria for 24 hours at the temperature of 30 ℃ and the speed of 150rpm to obtain Acinetobacter (Acinetobacter sp.) B2-4 bacterial liquid with the OD600 of 1.

2. Preparation of degradation Medium

The preparation method of the phenanthrene degradation culture medium comprises the steps of adding 50mL of inorganic salt culture medium into a 100mL serum bottle, and then adding an acetone solution of phenanthrene to ensure that the concentration of phenanthrene in the inorganic salt culture medium is 100mg/L. Placing the strain in a sterile operation box for 24 hours to volatilize acetone, and obtaining the phenanthrene degradation culture medium.

And preparing the pyrene degradation culture medium according to the same method.

3. Polycyclic aromatic hydrocarbon degradation rate detection

According to the mass percentage of 5%, acinetobacter (Acinetobacter sp.) B2-4 bacterial liquid is inoculated into a phenanthrene degradation culture medium, a constant temperature shaking incubator is displaced after the bacteria liquid is sealed by a butyl rubber plug, and the bacteria liquid is cultured for 45 days in an anaerobic environment with the temperature of 30 ℃ and 150 rpm ℃. In addition, three parallel and sterile medium groups were used as blank. For each culture week, the culture vials were extracted with cyclohexane equivalent volume ultrasound and the phenanthrene content therein was determined by High Performance Liquid Chromatography (HPLC) to calculate the phenanthrene degradation rate. The degradation rate of pyrene was measured in the same manner, and the results of the degradation rates of both phenanthrene and pyrene are shown in fig. 4 and 5, respectively.

As can be seen from FIGS. 4 and 5, the strain has a degradation effect on both phenanthrene and pyrene under anaerobic conditions. After 45 days of culture, the degradation rate of Acinetobacter (Acinetobacter sp.) B2-4 to phenanthrene was 74.9%, and after 45 days of culture, the degradation rate of Acinetobacter (Acinetobacter sp.) B2-4 to pyrene was 56.2%.

Claims

1. An Acinetobacter sp. B2-4 that degrades polycyclic aromatic hydrocarbons under anaerobic conditions, characterized in that the strain was deposited in the General Microbiology Center of the China Culture Collection Administration on March 3, 2025, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 33689.

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

3. The method for culturing Acinetobacter sp. B2-4 according to claim 1, comprising the steps of: inoculating Acinetobacter sp. 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 cells; then resuspending the cells and inoculating them into an inorganic salt culture medium, and continuing to culture 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.

4. The method for culturing Acinetobacter sp. B2-4 according to claim 3, wherein the formula of the inorganic salt culture 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, and the pH is adjusted to 7.0;

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

5. Use of Acinetobacter sp. B2-4 according to claim 1 in the degradation of polycyclic aromatic hydrocarbons, characterized in that the degradation of polycyclic aromatic hydrocarbons is the degradation of polycyclic aromatic hydrocarbons in soil or sewage under anaerobic conditions; the polycyclic aromatic hydrocarbons are phenanthrene or pyrene.

6. Use of the Acinetobacter sp. B2-4 according to claim 1 in the preparation of a polycyclic aromatic hydrocarbon degrading bacterial agent, wherein the polycyclic aromatic hydrocarbon is phenanthrene or pyrene.

7. A polycyclic aromatic hydrocarbon-degrading bacterial agent, characterized in that the bacterial agent contains the Acinetobacter sp. B2-4 described in claim 1 as a main active ingredient, and the polycyclic aromatic hydrocarbon is phenanthrene or pyrene.

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