Screening And Identification Of PET Plastic-degrading Strains, Degradation Characteristics And Key Enzyme Genes Analysis

Polyethylene terephthalate?PET?is widely used in various commodities because of its good performance.However,due to its non-degradable properties,it has become the main source of"white pollution"of plastics,causing serious harm to nature and human life.At present,the main treatment methods of PET focus on incineration,landfill and chemical degradation,which are time-consuming,labor-intensive,and environment pollution.Therefore,biodegradation of PET has become a focus area that attracted researchers'attention.In this study,biological methods was applied in PET degradation,which it is expected to provide a certain theoretical basis for the treatment of"white pollution".The main contents of this study contain screening and identification of PET plastic-degraded strains,degradeation characteristics and key enzyme genes analysis.To this end,the main results are as follows:?1?Taking the PET plastic bottle debris from the WuXi Taohuashan landfill as the source sample of strains,two PET-degraded strains were successfully screened,JWG-G1 and JWG-G2.The fourier infrared spectroscopy analysis found that JWG-G2 had better damage capability of the ester bond functional groups on PET surface than that of JWG-G1,confirming that JWG-G2 had better degradation ability of PET.Strain JWG-G2 was identified as Micbacterium oleivorans,which belongs to genus Micbacterium.So,M.oleivorans JWG-G2 was used as the only degrading strain to further investigate its degradability and characteristics of PET.?2?Results of preliminary degradation showed that M.oleivorans JWG-G2 can grow on PET granules or films as the only carbon source.Products were detected in the processing solution by high performance liquid chromatography after M.oleivorans JWG-G2 treatment for 5 days,monohydroxyethyl terephthalate 1.39 mg·L^-1,terephalic acid 1.06 mg·L^-1 and bis?2-hydroxyethyl?terephthalate polymers 0.25 mg·L^-1 for PET granules;MHET 1.53 mg·L^-1,TPA 1.10 mg·L^-1 and BHET polymers 0.51 mg·L^-1 for PET film.The weight loss rates of PET granules and film were 1%and 1.25%,respectively.Their surface was more obvious rougher and erosion trace than that of control by scanning electron microscopy?SEM?analysis.Generally,degradability of M.oleivorans JWG-G2 on PET film was better than that of PET granules.?3?M.oleivorans JWG-G2 and T.fusca cutinase were combined to construct a mixed bacterial enzyme system to synergistic degrade PET film and BHET polymers for 15 h,whose degradation effect was superior to that of single bacteria and single enzyme treatment alone.For BHET polymers as substrate,its synergistic degradation efficiency was 122-167%;For PET film as substrate,the highest concentration of TPA and MHET was produced in mixed system that combination of 5m L(OD₆₀₀=1.0)strain suspension of M.oleivorans JWG-G2 and 3 mL(40 U·mL^-1)T.fusca cutinase.The synergistic degradation efficiency was 118%by TPA as evaluation standard,The synergistic degradation efficiency was 155%by MHET as evaluation standard,and the maximum loss weight rate increased by 1.3 times.SEM analysis revealed that the surface of PET-treated had obvious erosion and rough.?4?The second-generation high-throughput sequencing technology was used to complete the whole genome sequencing of M.oleivorans JWG-G2.The results showed that the genome length was3851210 bp,the base content of G+C was 70.71%,and the length of N50 was 260398 bp.Basis of results of gene sequencing,a genetic relationship tree was established by analysis of esterase,cutinase,lipase and reported PET degrading enzyme sequences.Several potential PET degradation genes was selected through database annotations,metabolic pathways,structural functions and previous data.Enzyme encoded by 19-8 gene could combined with the long-chain macromolecular substance,which break the ester bond therein to form a small molecule substance.Enzyme encoded by 4-138,5-212and 6-188 genes could combine and hydrolyze small molecular substances until completely degraded.Due to low activity and hydrolysis ability,it lead to excessive accumulation of intermediates,which is also consistent with the experimental results.