A strain capable of simultaneously degrading polyethylene plastic and polycyclic aromatic hydrocarbons and its application

Abstract

The present invention discloses a strain capable of simultaneously degrading polyethylene plastic and polycyclic aromatic hydrocarbons and an application thereof. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center of China on September 2, 2022, with a deposit number of GDMCC No: 62761. The strain can simultaneously degrade polyethylene plastic and polycyclic aromatic hydrocarbons, and can be applied to the treatment and restoration of polyethylene plastic-polycyclic aromatic hydrocarbons composite pollution environment.

Description

Technical Field

[0001] The invention relates to the field of biological treatment of environmental pollutants, and in particular to a bacterial strain capable of simultaneously degrading polyethylene plastics and polycyclic aromatic hydrocarbons and application thereof. Background Art

[0002] Plastics and their products have the advantages of being cheap, light, and corrosion-resistant. They are widely used in industrial production, the medical industry, and daily life, which has resulted in a large amount of waste plastics. Polyethylene is the type of plastic with the highest production and usage, and has a high detection rate in the environment. Polyethylene plastics can change the physical and chemical properties of soil, destroy soil structure, affect the migration of nutrients and water, and hinder the growth of crops. Polyethylene plastics can restrict the movement of organisms and cause death after entanglement; after being ingested by organisms, plastics can wear or block the feeding organs and digestive tract, reduce the feeding rate, and induce toxic effects at the cellular and molecular levels, thereby affecting their metabolism, growth, and reproduction. In addition, with the acceleration of industrialization, the consumption of fossil fuels has continued to increase, leading to increasingly serious polycyclic aromatic hydrocarbons (PAHs) pollution in the environment. Polyethylene plastics have a large specific surface area and strong hydrophobicity, and can be used as an adsorption carrier for PAHs. The two form a composite pollution, which seriously affects the safety of the ecological environment and can also be transmitted along the food chain and threaten human health. Therefore, it is very necessary to control and repair the polyethylene plastic-PAHs composite pollution environment.

[0003] Physical and chemical techniques are used to repair the environment polluted by polyethylene plastics / PAHs. For example, coagulation-sedimentation, filtration, photolysis, incineration and other methods can remove polyethylene plastics, while extraction, adsorption, leaching, oxidation and other methods can remove PAHs. However, the investment cost of physical / chemical remediation technology is high, it is easy to damage the ecological environment and cause secondary pollution, and it is difficult to remove coexisting polyethylene plastics and PAHs at the same time. Microbial remediation technology has the advantages of low cost, good treatment effect, and green environmental protection. It has developed into one of the effective methods for removing pollutants in the environment. Microorganisms adsorb on the surface of plastics and break the long chains of polyethylene plastics into short chains by secreting hydrolases / oxidases and extracellular polymers, forming oligomers and monomers, and finally decomposing into CO2 and H2O. Microorganisms can use PAHs as a carbon source and energy source, and convert PAHs into low-toxic or non-toxic compounds through their own metabolic activities. However, current studies have only reported the removal of single polyethylene plastics or PAHs by microorganisms. Existing strains can only decompose polyethylene plastics but not PAHs, or can only degrade PAHs but not polyethylene plastics. There is currently a lack of bacterial strain resources that can simultaneously and efficiently degrade polyethylene plastics and PAHs. Summary of the invention

[0004] In view of the above problems, the present invention provides a bacterial strain capable of simultaneously degrading polyethylene plastic and polycyclic aromatic hydrocarbons and application thereof.

[0005] The purpose of the present invention is achieved by adopting the following technical solutions:

[0006] The present invention provides a Nocardia strain ( Nocardioides sp.) PE1W, with the deposit number GDMCC No: 62761, was deposited at the Guangdong Microbial Culture Collection Center, China on September 2, 2022, located at the Institute of Microbiology, Guangdong Academy of Sciences, Xianlie Middle Road, Guangzhou.

[0007] The Nocardia Nocardioides sp.) PE1W was domesticated, screened and purified from contaminated soil. The Nocardia sp. Nocardioides sp.) The morphology of PE1W on the plate can be found in Figure 1 The colonies are raised, milky white, with neat and smooth edges.

[0008] The strains were classified and identified by 16S rDNA sequence analysis. The DNA of PE1W was extracted by Sangon Biotech (Shanghai) Co., Ltd., and 16S rDNA was amplified and sequenced using primers 27F / 1492R to obtain a sequence of 1478 bp in length. The obtained PE1W sequence was compared with the ribosomal database http: / / rdp.cme.msu.edu / index.jsp for homology analysis. It was found that the PE1W sequence was similar to that of Nocardia sp. Nocardioides sp.) and classified the species as Nocardia spp.

[0009] The Nocardia Nocardioides The nucleotide sequence of 16S rDNA of Pseudomonas sp. PE1W is shown in Seq ID No: 1.

[0010] Another object of the present application is to provide the Nocardia ( Nocardioides sp.) PE1W in the degradation of plastics and / or polycyclic aromatic hydrocarbons.

[0011] Preferably, the plastic is polyethylene.

[0012] Preferably, the polycyclic aromatic hydrocarbons are phenanthrene and / or pyrene.

[0013] The beneficial effects of the present invention are:

[0014] The present invention isolates and screens a strain of Nocardia ( Nocardioidessp.) PE1W, which can degrade polyethylene plastics and PAHs (phenanthrene, pyrene) at the same time, can be used in the treatment and restoration of polyethylene plastic-PAHs composite pollution environment. The application objects include coking industrial sites, oil mining areas and water bodies / soil and farmland around refineries, etc. It can effectively reduce the harm of polyethylene plastics and PAHs to the ecological environment and human health, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative work.

[0016] Figure 1 This is the morphology of strain PE1W on NB plates;

[0017] Figure 2 It is the removal effect of bacteria PE1W on polyethylene plastic in the polyethylene plastic-PAHs composite pollution system;

[0018] Figure 3 is the surface microscopic morphology of polyethylene plastic (a: before treatment; b: control group; c: treatment group);

[0019] Figure 4 This is the removal effect of bacteria PE1W on phenanthrene and pyrene in the polyethylene plastic-PAHs composite pollution system. DETAILED DESCRIPTION

[0020] The present invention is further described in conjunction with the following examples.

[0021] Example 1

[0022] Isolation and Identification of Nocardia PE1W Strain

[0023] 1. Preparation of culture medium and solution

[0024] Mineral salt medium (MSM): 5 mL phosphate buffer solution (Na2HPO4·12H2O 33.4 g / L, K2HPO4·3H2O21.75 g / L, KH2PO4 8.5 g / L, NH4Cl 5.0 g / L); 3 mL MgSO4 solution (22.5 g / L); 1 mL FeCl3 solution (0.25 g / L); 1 mL CaCl2 solution (36.4 g / L); 1 mL trace element solution (ZnSO4·H2O 42.8 mg / L, MnSO4·H2O 39.9 mg / L, (NH4)6Mo7O 24 4H2O 34.7 mg / L), dilute to 1L.

[0025] PAHs stock solution: Use acetone as solvent to prepare the mother solutions of pyrene (2 g / L) and phenanthrene (10 g / L), respectively, and store them in a refrigerator at 4°C for future use.

[0026] Nutrient broth agar solid medium (NB): 18 g nutrient broth, 18 g technical agar powder, 1 L distilled water.

[0027] 2. Domestication, screening, isolation and purification of strains

[0028] Weigh 10g of contaminated soil, put it into a triangular flask containing MSM culture medium, add polyethylene plastic (10 g / L), and culture at 30℃ and 140 rpm for 60 days. Then take 10 mL of culture solution and transfer it to MSM culture medium containing polyethylene plastic (10 g / L), phenanthrene (50 mg / L), and pyrene (10 mg / L), and culture it for 60 days with shaking, completing the first domestication. Repeat this step and perform domestication 4 more times.

[0029] 100 μL of the culture solution from the last acclimation cycle was taken and spread on the NB medium. The plate was placed upside down in a 30 ℃ constant temperature incubator. After 2-3 days, when the colonies grew well, colonies of different forms were picked on the plate for streaking purification to obtain pure strains. The pure strains were inoculated into MSM medium (10 g / L polyethylene plastic, 50 mg / L phenanthrene, 10 mg / L pyrene) and cultured with shaking to verify whether they have the ability to degrade polyethylene plastic and PAHs (phenanthrene, pyrene). The shake flask with turbid culture solution was selected and the above steps were repeated for purification 3 times to ensure the purity of the strain and the stability of its performance. Finally, a strain capable of simultaneously degrading polyethylene plastic, phenanthrene and pyrene was obtained, which was numbered as PE1W;

[0030] The strain was identified by 16S rDNA sequence analysis and was classified as Nocardia.

[0031] Example 2

[0032] Degradation performance of Nocardia PE1W strain

[0033] The PE1W bacterial suspension was transferred into MSM medium containing polyethylene plastic (10 g / L), phenanthrene (50 mg / L), and pyrene (10 mg / L) as the treatment group; a control group without PE1W was set up, and three parallel cultures were set up for each treatment. The culture was shaken at 30 ℃ and 140 r / min for 60 d, and the supernatant was collected after centrifugation. Methanol was added and the phenanthrene and pyrene were completely dissolved by ultrasound. The supernatant was transferred to a volumetric flask and fixed to volume with methanol. The supernatant was filtered using a 0.22 µm filter head, and the concentrations of phenanthrene and pyrene were determined by high performance liquid chromatography, and the removal rates of phenanthrene and pyrene were calculated.

[0034] The polyethylene plastic and bacteria obtained by centrifugation were separated, and the polyethylene plastic was soaked in 75% ethanol, 95% ethanol and 100% ethanol for more than 4 hours, rinsed with sterile water on a clean bench for 3 times, dried with filter paper, dried naturally, and weighed to determine the degradation rate of the polyethylene plastic. The microscopic characteristics of the polyethylene plastic were observed using a scanning electron microscope.

[0035] The weight reduction of polyethylene plastic is an important indicator for judging its degradation effect. In the composite pollution system of polyethylene plastic-phenanthrene-pyrene, the degradation effect of PE1W on polyethylene plastic is shown in the attached figure. Figure 2 As shown in the figure, after adding strain PE1W, the degradation rate of polyethylene plastic by strain PE1W gradually increased with the extension of culture time. The removal rate of polyethylene plastic reached 8.52% at 60 days, while the weight of polyethylene plastic in the control group (without bacteria) remained almost unchanged, indicating that PE1W degraded polyethylene plastic.

[0036] The results of the surface morphology of polyethylene plastic observed by scanning electron microscopy are shown in the attached figure. Figure 3 As shown in the figure, the surface of the original polyethylene plastic (a) is smooth and intact. In the composite pollution system of polyethylene plastic-phenanthrene-pyrene, after 60 days of cultivation, the surface of the polyethylene plastic in the control group (b) remained smooth and flat, while the surface of the polyethylene plastic in the treatment group (c) showed large-scale traces of microbial erosion, with obvious holes and damage, and became very rough, which shows that the bacteria PE1W can degrade polyethylene plastic. At the same time, the bacteria PE1W has a good degradation effect on phenanthrene and pyrene (see Appendix Figure 4 ), the removal rates of phenanthrene and pyrene reached 74.96% and 66.95% respectively. It can be seen that Nocardia PE1W has a good degradation ability for PAHs (phenanthrene and pyrene) while degrading polyethylene plastics.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A strain of Nocardia Nocardioides sp.) PE1W, characterized in that The deposit number is GDMCC No: 62761, and it was deposited in the Guangdong Provincial Microbiological Culture Collection Center, China on September 2, 2022.

2. The Nocardia according to claim 1 ( Nocardioides sp.) Application of PE1W in the degradation of plastics and / or polycyclic aromatic hydrocarbons, wherein the plastic is polyethylene and the polycyclic aromatic hydrocarbons are phenanthrene and / or pyrene.

3. The Nocardia according to claim 1 ( Nocardioides sp.) PE1W degradation bacteria.

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