CN111748494B

CN111748494B - Nitrogen-fixing campylobacter for degrading polycyclic aromatic hydrocarbon and application thereof

Info

Publication number: CN111748494B
Application number: CN202010619449.1A
Authority: CN
Inventors: 黄玉屏; 张宏宏; 王黎明; 张晓昀
Original assignee: Hubei Environmental Restoration And Treatment Technology Research Co ltd; Wuhan University WHU
Current assignee: Hubei Environmental Restoration And Treatment Technology Research Co ltd; Wuhan University WHU
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2022-05-13
Anticipated expiration: 2040-06-30
Also published as: CN111748494A

Abstract

The invention belongs to the technical field of microbiology, and particularly relates to a campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons and application thereof, wherein the campylobacter azotobacter is named as campylobacter azotobacter (Azoarcus sp.) CE3, and is preserved in China center for type culture collection with the preservation number of CCTCC M2020003. The campylobacter azotoformans (Azoarcus sp.) CE3 has stable degradation performance, high tolerance concentration of polycyclic aromatic hydrocarbons and wide range of degradation substrates, not only can degrade tetracyclic fluoranthene and pyrene, but also can degrade low-ring phenanthrene and naphthalene, and can degrade high-ring benzo [3,4] pyrene, benzo [ a ] anthracene, benzo [ b ] fluoranthene, dibenzo [ a, h ] anthracene and other substrates, so the campylobacter azotoformans (Azoarcus sp.) CE3 can be used for degrading various polycyclic aromatic hydrocarbons.

Description

Nitrogen-fixing campylobacter for degrading polycyclic aromatic hydrocarbon and application thereof

Technical Field

The invention belongs to the technical field of microbiology, and particularly relates to a Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons and application thereof.

Background

Campylobacter azotobacter (Azoarcus) belongs to the kingdom of Bacteria (Bacteria), Proteobacteria (Proteobacteria), class B-Proteobacteria (Betaproteobacteria), order Rhodocyclales (Rhodocyclals), family Rhodocyclaceae (Rhodocyclaceae). The Campylobacter azotobacter is gram-negative bacteria, oxidase is positive, the cells contain poly-b-hydroxybutyrate particles, the cells are straight or slightly bent rod-shaped, and the content of G + C in DNA molecules is 62-68%. Campylobacter azotobacter can be classified into two groups, the first group is an endophytic bacterium capable of promoting the growth of a plant body, including Azoarcus communis SWub3, Azoarcus indegens VB32, Azoarcus sp.BH72, etc., which supply a nitrogen source to a host plant to promote the growth of a plant body through nitrogen fixation, and the second group is a bacterium capable of degrading organic contaminants, including Azoarcus evansii KB740, Azoarcus tolyticus Tol-4, Azoarcus tolyticus MF63, etc., which are present in petroleum-contaminated soil and water. However, some campylobacter azotoformans can survive not only independently by degrading aromatic compounds but also in plants, such as Azoarcus sp. Therefore, the Campylobacter azotoformans has important ecological significance.

Fluoranthene is a polycyclic aromatic hydrocarbon composed of four benzene rings, has a molecular structure similar to that of dibenzodioxin, dibenzofuran and dibenzothiophene, is a yellow-green needle-shaped crystal, and is a polycyclic aromatic hydrocarbon with high molecular weight and most abundant content in the environment. Pyrene is a tetracyclic polycyclic aromatic hydrocarbon with a symmetrical structure and is a colorless crystalline solid. Fluoranthene and pyrene, as high molecular weight polycyclic aromatic hydrocarbons, are not easily degraded by long-term distribution in various complex environments due to their high thermodynamic stability, low water solubility and low bioavailability. Therefore, they are generally used as model molecules of high molecular weight polycyclic aromatic hydrocarbons to study the degradation mechanism and toxicological properties of the high molecular weight polycyclic aromatic hydrocarbons.

Disclosure of Invention

One of the purposes of the invention is to provide the Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbon, which has stable degradation performance, high polycyclic aromatic hydrocarbon tolerance concentration and wide substrate degradation range.

The second purpose of the invention is to provide the application of the Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons.

The invention also aims to provide an application method of the Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons, which does not cause any harm to human health and public environment, has low requirements on experimental conditions and has relatively low cost.

The scheme adopted by the invention for realizing one purpose is as follows: a strain of Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons is named as Campylobacter azotobacter (Azoarcus sp.) CE3, and is preserved in China center for type culture Collection with the preservation number of CCTCC M2020003.

The second scheme adopted by the invention for achieving the purpose is as follows: the campylobacter azotobacter for degrading polycyclic aromatic hydrocarbon is applied to degrading polycyclic aromatic hydrocarbon, and the polycyclic aromatic hydrocarbon is at least one of fluoranthene, pyrene, benzo [3,4] pyrene, benzo [ a ] anthracene, benzo [ b ] fluoranthene, dibenzo [ a, h ] anthracene, phenanthrene and naphthalene.

The scheme adopted by the invention for realizing the third purpose is as follows: the method for applying the Campylobacter azotobacter for degrading the polycyclic aromatic hydrocarbon comprises the steps of taking the Campylobacter azotobacter as a spawn, adding the polycyclic aromatic hydrocarbon into a liquid inorganic salt culture medium as a unique carbon source, and culturing for 3-7 days to achieve the purpose of degrading the polycyclic aromatic hydrocarbon.

Preferably, the culture conditions are: the culture temperature is 25-40 ℃, the shaking culture rotation speed is 100-250rpm, and the culture time is 3-7 days.

The invention has the following advantages and beneficial effects: the campylobacter azotoformans (Azoarcus sp.) CE3 has stable degradation performance, high tolerance concentration of polycyclic aromatic hydrocarbons and wide range of degradation substrates, not only can degrade tetracyclic fluoranthene and pyrene, but also can degrade low-ring phenanthrene and naphthalene, and can degrade high-ring benzo [3,4] pyrene, benzo [ a ] anthracene, benzo [ b ] fluoranthene, dibenzo [ a, h ] anthracene and other substrates, so the campylobacter azotoformans (Azoarcus sp.) CE3 can be used for degrading various polycyclic aromatic hydrocarbons.

Compared with the common chemical method and physical method, the application method of the invention degrades the polycyclic aromatic hydrocarbon by using the Campylobacter azotoformans (Azoarcus sp.) CE3, does not cause any harm to the human health and the public environment, has lower requirements on experimental conditions and has lower cost.

In addition, the Campylobacter azotobacter sp CE3 can be permanently stored, and lays a foundation for future degradation mechanism research and further practical application.

Drawings

FIG. 1 is a phylogenetic tree of Campylobacter azotobacter CE 3;

FIG. 2 is a graph showing the degradation effect of Campylobacter azotobacter CE3 on fluoranthene with different concentrations in a single system;

FIG. 3 is a graph showing the degradation effect of Campylobacter azotobacter CE3 on pyrene of different concentrations in a single system;

FIG. 4 is a graph showing the degradation effect of Campylobacter azotobacter CE3 on a binary mixture of fluoranthene and pyrene in a mixed system.

Detailed Description

The following examples are provided to further illustrate the present invention for better understanding, but the present invention is not limited to the following examples.

Example 1 isolation, screening and identification of Campylobacter azotobacter (Azocarcus sp.) CE3

Campylobacter azotobacter (Azoarcus sp.) CE3 was isolated from a highly contaminated soil sample from east Steel, Huangshi, Hubei. Campylobacter azotobacter (Azoarcus sp.) CE3 was deposited at the China center for type culture Collection (deposition address: Wuhan university, China) at 1/2 of 2020, and was classified and named as Campylobacter azotobacter (Azoarcus sp.) CE3 with the deposition number of CCTCC M2020003.

Separation and screening of Campylobacter azotobacter (Azoarcus sp.) CE3

1. Enrichment and domestication: weighing 10g of soil sample, adding the soil sample into a 250mL triangular flask, adding 90mL of sterile water and a proper amount of glass beads, placing the triangular flask in a constant temperature shaking table at 30 ℃, carrying out shake culture for 3h, taking out and standing for 30 min. 10mL of the supernatant was added to 90mL of an inorganic salt medium containing fluoranthene, and the mixture was subjected to shaking culture at 30 ℃ and 180rpm in the dark. Taking out 10mL of culture solution from the triangular flask every 7 days, and transferring the culture solution into 90mL of fresh culture medium for subculture, and carrying out subculture for 4 times. The concentration gradient of enrichment domestication is as follows: 10mg/L, 20mg/L, 40mg/L, 60mg/L, 80 mg/L.

2. Separation and purification: fifth enrichment and domestication bacterial liquid dilution 10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸Taking 100 μ L of 10⁶、10⁷、10⁸The diluted solution was spread on a fluoranthene-containing inorganic salt plate, and the plate was incubated in a constant temperature incubator at 30 ℃ until a clear colony grew. Colonies with different colony morphologies were picked for numbering and streaked on inorganic salt plates with fluoranthene as the sole carbon source until pure single colonies were formed. The single colony after purification was inoculated in 5mL LB liquid medium and cultured to OD₆₀₀And (3) when the concentration reaches about 1, uniformly mixing 0.5mL of bacterial liquid with 0.5mL of 40% glycerol, and freezing and storing in a refrigerator at the temperature of minus 80 ℃.

3. Screening of high efficiency degradation bacterium CE3 (ultraviolet spectrophotometry)

(1) Drawing of fluoranthene standard curve

Arranging a standard product: weighing 10mg of fluoranthene solid powder, dissolving in 10mL of dichloromethane to prepare 1mg/mL of standard mother liquor, diluting the mother liquor with different concentrations (the concentration gradient is 1mg/L, 2mg/L, 3mg/L, 4mg/L, 5mg/L and 6mg/L), and storing 3 parts of each standard at-40 ℃.

Determining the maximum absorption wavelength: taking 3mL of fluoranthene standard solution with the concentration of 3mg/L, taking dichloromethane as a blank control, and carrying out full-wave-band scanning in a wave band range of 190-900 nm by using an MK3 type enzyme standard instrument. The fluoranthene has a maximum absorption wavelength of 237nm by analytical determination.

Drawing a standard curve: the absorbance of the standards at different concentrations was measured at 237nm with an ultraviolet spectrophotometer using dichloromethane as a blank control. And drawing a standard curve of the fluoranthene by taking the absorbance value as an ordinate and the concentration as an abscissa.

(2) Preparation of the bacterial suspension

500. mu.L of each strain was inoculated in 5mL of LB medium overnight for activation.

And centrifuging the activated bacterium liquid at 4000rpm for 10min to remove supernatant, and collecting thalli.

③ washing the bacteria obtained by centrifugation with sterile inorganic salt culture medium for 2 times, and resuspending the bacteria with the inorganic salt culture medium to obtain OD₆₀₀The suspension was stored at 4 ℃ until use.

(3) Preparation of degradation systems

Adding fluoranthene acetone solution into 50mL of inorganic salt culture medium, placing an triangular flask in a shaking table overnight, and oscillating to volatilize the acetone completely.

② inoculating 1mL of bacterial suspension into the culture medium, inoculating 3 bottles of each strain, setting a blank control without adding bacteria, and shake-culturing for 7 days in a triangular flask at 30 ℃ and 180 rpm.

(4) Extraction of fluoranthene in degradation system

Adding 25mL of dichloromethane into 50mL of culture solution, placing a triangular flask in a shaking table at normal temperature and oscillating at 180rpm for 30min, adding the mixed solution into a 250mL separating funnel, standing and layering for 15min, separating an organic phase and an aqueous phase, and continuously extracting the collected aqueous phase for 2 times according to the method. Finally, the organic phase was centrifuged at 8000rpm for 10min and the residual aqueous phase was aspirated off.

② combining organic phases obtained by 3 times of extraction, and measuring the total volume of the organic phases.

(5) And measuring the absorbance of the extracted sample by using an ultraviolet spectrophotometer, and calculating the degradation rate of each strain.

Screening by an ultraviolet spectrophotometry to obtain a fluoranthene high-efficiency degrading bacterium CE 3.

II, identifying 16S rDNA of efficient degrading bacterium CE3

And (3) selecting a single colony of the efficient degradation bacterium CE3 to perform colony PCR, wherein the amplification primers adopt universal primers 27F and 1492R identified by the 16S rDNA of the bacterium. The sequencing work of the PCR amplification products was performed by the Biotech company, Wuhan Tianyihui. Inputting the sequence information into NCBI website, and performing sequence homology comparison analysis with existing nucleic acid sequence in GenBank by Blast program. According to the sequence alignment results, a phylogenetic tree of the strain CE3 was constructed using MEGA 6.0 software by the Neighbor-Joining method.

The sequence alignment result shows that the 16S rDNA sequence of the strain CE3 has the highest homology of Azoarcus olericus DQS-4, and the nucleotide consistency is 99.64%. Phylogenetic trees showed that strain CE3 was associated with the Azoarcus strain. Therefore, the strain CE3 can be classified and named as Campylobacter azotobacter (Azoarcus sp.) CE 3. A phylogenetic tree of strain CE3 is shown in FIG. 1.

Example 2 research on degradation effect of Campylobacter azotobacter (Azoarcus sp.) CE3 on fluoranthene and pyrene at different concentrations

Establishment of method for detecting fluoranthene and pyrene in degradation system

According to the research, the fluoranthene and the pyrene are quantitatively detected by adopting a high performance liquid chromatography, standard liquid of the fluoranthene and the pyrene is used as an external standard, and the content of the fluoranthene and the content of the pyrene are directly calculated by comparison through a retention time method.

1. And (3) configuring a standard product: weighing and dissolving fluoranthene and pyrene solids in chromatographic pure methanol respectively to prepare 1mg/mL of standard mother liquor. 1mg/mL of the stock solution was diluted to prepare 1mg/L, 2.5mg/L, 5mg/L, 12.5mg/L, 25mg/L, 50mg/L and 100mg/L of standards at 3 concentrations.

2. And (3) performing on-machine analysis on a standard sample: the chromatographic column is

PAH column (5 μm, 250 mm. times.4.6 mm), mobile phase ratio methanol: the volume ratio of water is 9: 1, the detector is an ultraviolet detector, the detection temperature is room temperature, the set flow rate is 1mL/min, the sample volume is 30 muL, the detection wavelength of fluoranthene and pyrene is 254nm, and the analysis time is 30 min.

3. And drawing a standard curve of fluoranthene and pyrene by taking the peak area as an ordinate and the concentration as an abscissa.

Research on degradation effect of Campylobacter azotobacter (Azoarcus sp.) CE3 on fluoranthene and pyrene

A1.250 mL triangular flask was charged with 50mL of an inorganic salt medium for autoclaving, and a filter-sterilized acetone solution of fluoranthene and pyrene was added. Only one substrate of fluoranthene or pyrene is added into a single system, and the concentration gradient of fluoranthene or pyrene is 50mg/L, 100mg/L, 150mg/L, 200mg/L and 250 mg/L; a mixture of fluoranthene and pyrene is added into the mixing system, the total concentration gradient of the binary mixture is 50mg/L, 100mg/L, 150mg/L, 200mg/L and 250mg/L, wherein fluoranthene and pyrene respectively account for half of the total concentration.

2. 0.5mL of bacterial suspension was added at 1% inoculum size, 3 replicates per concentration and a blank without added bacteria. The flask was placed on a shaker and incubated at 30 ℃ and 180rpm in the dark for 7 days.

3. Residual fluoranthene and pyrene in the culture broth were extracted with equal volume of dichloromethane, and each sample was extracted 2 times. The combined extracted organic phases were rotary evaporated to complete the dichloromethane evaporation and finally 50mL of chromatographically pure methanol was added to the volume.

4. About 1mL of the sample was filtered through a 0.22 μm organic filter and stored at-40 ℃ for HPLC analysis.

The degradation effect of campylobacter azotoformans (Azoarcus sp.) CE3 on fluoranthene and pyrene is shown in fig. 2, fig. 3 and fig. 4. The results of the study showed that when the initial concentration of fluoranthene in the medium was 50mg/L, 100mg/L, 150mg/L, 200mg/L, 250mg/L, the degradation rates for fluoranthene were 25.50%, 50.76%, 43.94%, 8.26%, and 5.30%. When the initial concentration of pyrene in the culture medium is 50mg/L, 100mg/L, 150mg/L, 200mg/L and 250mg/L, the degradation rate of the corresponding pyrene is 41.57%, 65.26%, 53.09%, 30.74% and 26.10%. Research results also show that the strain CE3 has a good degradation effect on a binary mixture of fluoranthene and pyrene in a mixed system, the degradation rates of the strain CE3 on 25mg/L, 50mg/L, 75mg/L, 100mg/L and 125mg/L fluoranthene are 49.81%, 46.03%, 34.96%, 31.36% and 28.91% after being cultured for 7 days at 30 ℃ and 180rpm in a dark place, and the degradation rates of the strain CE3 on 25mg/L, 50mg/L, 75mg/L, 100mg/L and 125mg/L pyrene are 47.95%, 43.36%, 31.54%, 26.78% and 10.14%.

Example 3 investigation of the extent of utilization of other substrates by Campylobacter azotobacter (Azoarcus sp.) CE3

Adding acetone solutions of phenanthrene, naphthalene, benzene, ethylbenzene, benzo [ a ] pyrene, benzo [ a ] anthracene, benzo [ b ] fluoranthene, carbazole and dibenzo [ a, h ] anthracene with different concentrations and filter sterilization into a test tube filled with 5mL of an inorganic salt culture medium after autoclaving, wherein the specific concentrations are shown in Table 1, preparing 4 tubes for each substrate, inoculating bacterial suspension into the test tube according to 1% of inoculation amount, setting 3 parallel blank controls and 1 blank control without inoculation for each substrate, culturing at 30 ℃ and 180rpm for 7 days, and observing the growth condition of the strains. The result shows that the strain CE3 can degrade fluoranthene and pyrene, benzene, naphthalene and phenanthrene with low ring, and can also degrade benzo [3,4] pyrene, benzo [ a ] anthracene, benzo [ b ] fluoranthene and dibenzo [ a, h ] anthracene with high ring, which indicates that the strain CE3 has wide substrate utilization range.

TABLE 1 investigation of substrate utilization Range of Strain CE3

+: represents the ability to grow-: indicates that it is unable to grow

While the foregoing is directed to the preferred embodiment of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

1. A strain of Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons is characterized in that: the name of the campylobacter azotobacter is campylobacter azotobacterAzoarcussp.) CE3, wherein the Campylobacter azotoformans is preserved in China center for type culture Collection (CCTCC M2020003) in 2020 and at the time of 01-02 month.

2. The use of the Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons according to claim 1, wherein: the campylobacter azotobacter is applied to degrading polycyclic aromatic hydrocarbon, and the polycyclic aromatic hydrocarbon is at least one of fluoranthene, pyrene, benzo [3,4] pyrene, benzo [ a ] anthracene, benzo [ b ] fluoranthene, dibenzo [ a, h ] anthracene, phenanthrene and naphthalene.

3. The use of the Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons according to claim 2, wherein: the campylobacter azotoformans is taken as a growth bacterium, the polycyclic aromatic hydrocarbon is added into a liquid inorganic salt culture medium to serve as a unique carbon source, and the polycyclic aromatic hydrocarbon is cultured for 3-7 days to achieve the purpose of degrading the polycyclic aromatic hydrocarbon.

4. The use of Campylobacter azotobacter for degrading polycyclic aromatic hydrocarbons according to claim 3, wherein: the culture conditions are as follows: the culture temperature is 25-40 ℃, the shaking culture rotation speed is 100-250rpm, and the culture time is 3-7 days.