| In order to solve the problem that polyethylene plastic is difficult to degrade in the environment,a strain was selected to study the biodegradation of polyethylene plastics.After screening,purification,analysis of physiological and biochemical tests and 16 SrDNA sequence of the bacteria.The strain has inoculated with different molecular weight polyethylene(PE)powder(Mw=2000/5000/100000)and film(Mw > 100000)as the sole carbon source in the liquid medium,culture at 28?,180 rpm.Concentrate determination of cell and the extracellular protein content by spectrophotometric method in every 24 hours.After 60 days of culture,weight the residual of PE,the apparent of films were observed by SEM,measured mechanical property by tensile testing machine,measured the water contact angle and infrared spectrum of film.For further improve the degradation ability of the original strain to high molecular weight PE,the original strain was treated by mutation.By methods of UV irradiation,microwave(MW)thermal effect,LiCl,hydroxylamine hydrochloride(HH),acridine orange(AO)and UV-AO compound to mutant strain.Use sieve method of molecular weight grads which culture strains with the molecular weight as 2000/5000/100000 and PE film as sole carbon source to screened mutant strains.The growth status of mutant strains,extracellular protein content and film weight loss rate were determined,Select the strains with the best optimal capable of degrading PE,the strain VS the original strain under the same culture conditions(with PE membrane as the sole carbon source)were cultured for 60 days,determinate the mechanical properties of films.In order to find the key genes and metabolic pathways of degradable polyethylene,we culture the original strains with different carbon sources(group G: using soluble starch as the sole carbon source,W group: taking PE as the sole carbon source),the expression of genes was analyzed by RNA-sequence.Total RNA was extracted from the original strain,constructe cDNA library of G and W groups,sequencing,then mapping reference genome,contrast GO/COG/KEGG database,clustering and enrichment analysis.Compare the amount of expression of the two groups of genes.Finally,the differentially expressed genes were randomly selected to verify the expression of RT-PCR and the expression of the key genes in the mutant strains was determined.The results show that the isolates were belong to the genus of Actinomycesthe selected is Amycolatopsis orientalis.We named it ZEL-1.The strain in lower molecular weight of PE medium,the concentration higher,extracellular protein content higher,reducing sugar content higher,the weight loss rate of polyethylene higher,pH value of medium decrease faster.SEM images display that the surface of the films have a lot of degradation holes.Tensile strength of film decreased by 74.15%,the elongation decreased by 84.90%,the mechanical properties decreased significantly.The water contact angle from 78.58±0.10° to 41.25±0.16°,the hydrophilicity increased.The results of FTIR spectra showed that the ketone carbonyl and ester carbonyl groups were successively produced by the degradation of the films.Mutagenesis results show that UV irradiation of 50 s,MW heat 800Hz/120 s,0.02% AO,0.7%LiCl,0.6%HH were the best mutagenic dose in kinds of methods.The compound mutation use the stains which mutagenesis UV 50 s as start strains,and then was mutated by 0.02%AO.PE culture medium cell growth trend,in addition to HH group,the other groups were better than that of the original strain group.UV group of extracellular protein content than the original group increased 2.06 times,the MW group increased 1.24 times,AO group increased 2.70 times,HH group increased 1.60 times,the compound mutation group increase 3.30 times,averagely.The Situation of films weightlessness,HH group was lower than the original strain group,other groups were higher than the original strain group,compound mutation group maximum weight loss rate of films,2.28 times higher than the original group.So selection compound mutation strain UV-AO-1 strain as the excellent strains.Degradation of PE in UV-AO-1 group after 60 days,the water contact angle than the original strain was decreased by 2.5°.The tensile strength of 3.37 Mpa decreases,the elongation decreased by 39.62%.The results RNA-sequence showed that are were 12372(63.24%)non redundant Unigenes were obtained,of which 2095 Unigenes were enriched in the metabolic process group.Compared with the COG database,there are 3328 Unigenes and the gene of COG database have homology,Unigenes related to metabolic activity accounted for 50.57%.In this study,we found 423 differentially expressed genes in different treatment groups,compared with group G,have 293 genes were up-regulated in W group,have 130 genes were down-regulated,and the expression of the AlkB gene was the most significant.Compared with the KEGG Pathway database,there are 5776 transcripts in comparison to the KEGG database,there are 92 metabolic pathways appear difference,most of the expression in differentially genes up-regulated relate to carbon metabolism,amino acid synthesis,pyruvate metabolism,carbon metabolism,amino acid synthesis,pyruvate metabolism genes expression quantity.The expression of the differentially genes was significantly increased with acetyl-CoA synthesisand and fatty acid metabolism.The TCA cycle related genes were down regulated significantly.RT-PCR results which the up-down-type of the gene expression use RT-PCR verify are consistent with the results of gene expression use transcriptome sequencing.It is proved that the results of RNA-sequence are reliable.In summary,Amycolatopsis orientalis ZEL-1 can directly degrade the low,medium and high molecular weight PE plastics.The degradation performance of UV-AO-1 was better than that of the original strain.Under the condition of PE carbon source,the original strains metabolism tended to be constructed by carbon frame,which reduced the synthesis and metabolism of carbohydrate.We speculate that the degradation of polyethylene by strain ZEL-1 begins with the AlkB gene encoding an alkane monooxygenase,which breaks through the chain at the end of polyethylene,and accomplished by fatty acid ?/?oxidation to complete the PE inorganic mineralization.The differential gene expression of strain ZEL-1 degrading polyethylene reflect the difference of metabolic pathway caused by different carbon sources,which provides a theoretical basis for the further study of the mechanism of polyethylene degradation. |
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