CN116042410A - Strain for simultaneously degrading polyethylene plastic and polycyclic aromatic hydrocarbon and application thereof - Google Patents

Abstract

The invention discloses a strain for simultaneously degrading polyethylene plastic and polycyclic aromatic hydrocarbon and application thereof, wherein the strain is preserved in the microorganism strain preservation center of Guangdong province in 2022, and the preservation number is GDMCC No:62761, the strain can degrade polyethylene plastics and polycyclic aromatic hydrocarbon simultaneously, and can be applied to the treatment and repair of polyethylene plastics-polycyclic aromatic hydrocarbon composite pollution environment.

Description

Strain for simultaneously degrading polyethylene plastic and polycyclic aromatic hydrocarbon and application thereof

Technical Field

The invention relates to the field of biological treatment of environmental pollutants, in particular to a strain for simultaneously degrading polyethylene plastics and polycyclic aromatic hydrocarbon and application thereof.

Background

The plastic and the products thereof have the advantages of low price, light weight, corrosion resistance and the like, and are widely applied to industrial production, medical industry and daily life, thereby producing a large amount of waste plastics. Polyethylene is the plastic type with the highest throughput and usage, and the detection rate in the environment is high. Polyethylene plastic can change physical and chemical properties of soil, destroy soil structure, influence migration of nutrients and moisture and prevent crops from growing. Polyethylene plastic can limit the movement of living organisms after being wound, and the living organisms can die; the plastic can abrade or block the feeding organs and digestive tracts after being ingested by organisms, reduce the feeding rate, induce toxic effects at the cellular and molecular level, and further influence the metabolism, growth and reproduction of the plastic. In addition, along with the acceleration of industrialization process, the consumption of fossil fuel is continuously increased, so that the pollution of Polycyclic Aromatic Hydrocarbons (PAHs) in the environment is more serious, and the polyethylene plastic has larger specific surface area and strong hydrophobicity, can be used as an adsorption carrier of the PAHs, forms composite pollution, seriously influences the ecological environment safety, and can be transferred along a food chain to threaten the health of human beings. Therefore, the method is very necessary to treat and repair the environment polluted by polyethylene plastic-PAHs.

Physical and chemical technologies are used to repair the environment polluted by polyethylene plastics/PAHs, such as coagulation-precipitation, filtration, photolysis, incineration and the like, to remove polyethylene plastics, and extraction, adsorption, leaching, oxidation and the like to remove PAHs. However, the physical/chemical repair technology has high investment cost, is easy to destroy the ecological environment and cause secondary pollution, and is difficult to remove coexisting polyethylene plastics and PAHs simultaneously. The microbial repairing technology has the advantages of low cost, good treatment effect, environmental protection and the like, has been developed as one of effective methods for removing pollutants in the environment, is characterized in that microorganisms are adsorbed on the surface of plastic, long chains of polyethylene plastic are broken into short chains by secreting hydrolase/oxidase and extracellular polymer, oligomers and monomers are formed, and finally the polyethylene plastic is decomposed into CO ₂ And H ₂ O. Microorganisms can utilize PAHs as a carbon source and energy source to convert PAHs into low-toxic or non-toxic compounds through their own metabolic activities. However, the current research only reports the removal of single polyethylene plastics or PAHs by microorganisms, and the existing strains can only separate the polyethylene plasticsCannot degrade PAHs, or can only degrade PAHs but cannot degrade polyethylene plastics, and strain resources capable of simultaneously and efficiently degrading polyethylene plastics and PAHs are lacking at present.

Disclosure of Invention

Aiming at the problems, the invention provides a strain for simultaneously degrading polyethylene plastic and polycyclic aromatic hydrocarbon and application thereof.

The aim of the invention is realized by adopting the following technical scheme:

the invention provides a nocardia strainNocardioidessp.) PE1W, accession No. GDMCC No:62761, deposited at the microorganism strain collection of Guangdong province in China at 9/2 of 2022, was addressed by the institute of microorganisms, sciences of Guangdong province, mirabilite, guangzhou, city.

The nocardia is [ ]Nocardioidessp.) domesticating, screening and purifying PE1W from the polluted soil, wherein the nocardia is obtained by the steps ofNocardioidessp.) the morphology of PE1W on the plate is shown in FIG. 1, the colonies are raised, milky white, and the edges are neat and smooth.

The strain is classified and identified by adopting 16S rDNA sequence analysis, DNA extraction of the strain PE1W is completed by entrusting the biological engineering (Shanghai) stock, and the sequence of 1478bp is obtained by carrying out 16S rDNA amplification sequencing by using 27F/1492R primer. The obtained PE1W sequence is subjected to homology comparison and analysis on a ribosome database http:// rdp.cme.msu.edu/index.jsp, and is compared with nocardia @Nocardioidessp.) up to 99%, the strain was assigned to nocardia.

The nocardia is [ ]Nocardioidessp.) the nucleotide sequence of the 16S rDNA of PE1W is shown in Seq ID No. 1.

Another object of the present application is to provide said NocardiaNocardioidessp.) PE1W in the degradation of plastics and/or polycyclic aromatic hydrocarbons.

Preferably, the plastic is polyethylene.

Preferably, the polycyclic aromatic hydrocarbon is phenanthrene and/or pyrene.

The beneficial effects of the invention are as follows:

the invention separates and screens to obtain a strainNocardia @ North AmericaNocardioidessp.) PE1W, can degrade polyethylene plastics and PAHs (phenanthrene and pyrene) simultaneously, can be applied to the treatment and repair of polyethylene plastics-PAHs composite pollution environments, has wide application prospect, and can effectively reduce the harm of polyethylene plastics and PAHs to ecological environment and human health, wherein the application objects comprise coking industrial sites, petroleum exploitation areas, water/soil around oil refinery areas, farmlands and the like.

Drawings

The invention will be further described with reference to the accompanying drawings, in which embodiments do not constitute any limitation of the invention, and other drawings can be obtained by one of ordinary skill in the art without inventive effort from the following drawings.

FIG. 1 is the morphology of strain PE1W on NB plates;

FIG. 2 shows the effect of removing PE1W from polyethylene plastics in a polyethylene plastics-PAHs composite pollution system;

FIG. 3 is a surface microform of polyethylene plastic (a: before treatment; b: control group; c: treatment group);

FIG. 4 shows the effect of PE1W in removing phenanthrene and pyrene from polyethylene plastic-PAHs composite pollution system.

Detailed Description

The invention will be further described with reference to the following examples.

Example 1

Isolation and identification of nocardia PE1W strain

1. Preparation of culture Medium and solution

Inorganic salt medium (MSM): 5mL of phosphate buffer solution (Na ₂ HPO ₄ ·12H ₂ O 33.4g/L，K ₂ HPO ₄ ·3H ₂ O 21.75 g/L，KH ₂ PO ₄ 8.5 g/L，NH ₄ Cl 5.0 g/L)；3mL MgSO ₄ Solution (22.5 g/L); 1mL FeCl ₃ Solution (0.25 g/L); 1mL CaCl ₂ Solution (36.4 g/L); 1mL trace element liquid (ZnSO) ₄ ·H ₂ O 42.8 mg/L，MnSO ₄ ·H ₂ O 39.9 mg/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O34.7 mg/L), constant volume to 1L.

PAHs stock solution: acetone is used as a solvent to prepare mother solutions of pyrene (2 g/L) and phenanthrene (10 g/L) respectively, and the mother solutions are placed in a refrigerator and stored at 4 ℃ for standby.

Nutrient broth agar solid medium (NB): 18 g nutrient broth, 18 g technical agar powder, distilled water 1L.

2. Domestication, screening and isolation and purification of strains

10g of contaminated soil was weighed and placed in a triangular flask containing MSM medium, polyethylene plastic (10 g/L) was added, and 60 d was cultured at 30℃with shaking at 140 rpm. Transferring 10 mL culture solution into MSM culture medium containing polyethylene plastic (10 g/L), phenanthrene (50 mg/L) and pyrene (10 mg/L), shake culturing 60 d, performing first domestication, repeating the steps, and performing domestication for 4 times;

and (3) absorbing 100 mu L of culture solution in the last domestication period, coating the culture solution on an NB culture medium, inversely buckling a flat plate, placing the flat plate in a constant temperature incubator at 30 ℃, after 2-3 d, taking out bacterial colonies with different forms on the flat plate, and carrying out streak purification to obtain pure bacterial strains. Inoculating pure strains into MSM culture medium (10 g/L polyethylene plastic, 50 mg/L phenanthrene and 10 mg/L pyrene) respectively, shake culturing to verify whether the strains have the capability of degrading polyethylene plastic and PAHs (phenanthrene and pyrene), selecting shake flasks with turbid culture solution, repeating the steps for 3 times, and purifying to ensure the purity and performance stability of the strains, and finally obtaining the strains capable of simultaneously degrading polyethylene plastic, phenanthrene and pyrene, wherein the number of the strains is PE1W;

the strain was classified and identified by 16S rDNA sequence analysis, and the strain was classified as Nocardia.

Example 2

Degradation Property of Nocardia PE1W Strain

Transferring the PE1W bacterial suspension into an MSM culture medium containing polyethylene plastic (10 g/L), phenanthrene (50 mg/L) and pyrene (10 mg/L) to obtain a treatment group; setting a control group without adding bacteria PE1W, setting three parallel groups for each treatment, carrying out shaking culture at 30 ℃ under 140 r/min for 60 d, centrifuging, collecting supernatant, adding methanol, carrying out ultrasonic treatment to completely dissolve phenanthrene and pyrene, transferring to a volumetric flask, fixing the volume by using the methanol, filtering by using a 0.22 mu m filter head, measuring the concentration of phenanthrene and pyrene by using a high performance liquid chromatograph, and calculating the removal rate of phenanthrene and pyrene.

Separating polyethylene plastic obtained by centrifugation from thalli, soaking the polyethylene plastic for more than 4 hours by using 75 percent ethanol, 95 percent ethanol and 100 percent ethanol in sequence, flushing the polyethylene plastic on an ultra-clean bench for 3 times by using sterile water, then sucking water by using filter paper, naturally airing, weighing, determining the degradation rate of the polyethylene plastic, and observing the microscopic characteristics of the polyethylene plastic by using a scanning electron microscope.

The reduction of the weight of the polyethylene plastic is an important index for judging the degradation effect of the polyethylene plastic, the degradation effect of PE1W on the polyethylene plastic in a polyethylene plastic-phenanthrene-pyrene composite pollution system is shown in a figure 2, after the strain PE1W is added, the degradation rate of the strain PE1W on the polyethylene plastic is gradually increased along with the extension of the culture time, the removal rate of the polyethylene plastic reaches 8.52% at 60 d, and the weight of the polyethylene plastic in a control group (without the strain) is hardly changed, so that the PE1W degrades the polyethylene plastic.

The surface morphology result of the polyethylene plastic observed by the scanning electron microscope is shown in figure 3, and the surface of the original polyethylene plastic (a) is smooth and complete. In the polyethylene plastic-phenanthrene-pyrene composite pollution system, after 60 d culture, the surface of the polyethylene plastic of the control group (b) is still smooth and flat, and the surface of the polyethylene plastic of the treatment group (c) is marked with large-area trace of microorganism corrosion, obvious holes and damages are shown, and the surface is very rough, which indicates that the bacteria PE1W can degrade the polyethylene plastic. Meanwhile, the bacteria PE1W has good degradation effect on phenanthrene and pyrene (see figure 4), and the removal rate of the phenanthrene and the pyrene reaches 74.96% and 66.95%, respectively. Therefore, nocardia PE1W has better degradation capability on PAHs (phenanthrene and pyrene) while degrading polyethylene plastic.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims (5)

1. Nocardia strainNocardioidessp.) PE1W, characterized by a deposit number GDMCC No:62761, at 2022, 9 and 2, was deposited with the collection of microorganism strains in Guangdong province, china.

2. Nocardia according to claim 1Nocardioidessp.) PE1W in the degradation of plastics and/or polycyclic aromatic hydrocarbons.

3. Use according to claim 2, characterized in that the plastic is polyethylene.

4. Use according to claim 2, characterized in that the polycyclic aromatic hydrocarbon is phenanthrene and/or pyrene.

5. Comprising nocardia of claim 1Nocardioidessp.) degrading bacterial agent of PE 1W.

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