Research On Degradation Of Phenolic Compounds By Strains M-1,W-1-1 And H-3-1 Isolated From Salt Marsh In Yancheng,Northern Jiangsu Province

Study on the biological properties and identification of M-1,W-1-1 and H-3-1 which were isolated from the salt marshes in the coastal area in Yancheng,northern Jiangsu was carried out in this paper.It reveals that all of the 3 strains are Gram-negative bacteria,without endospore structure and fexirubin pigment forming.Acetate,xylose,glucosamine,DNA broth,urea,glucose and gluconate are the common carbon sources that can be utilized for all of the 3 strains.Besides,each of the 3 strains utilizes some carbon sources which are specific to each of them.For example,M-1 has the ability to utilize aescin,ornithine,tartrate,hydrogen sulfide,arginine dihydrolase,malonate as carbon sources,while W-1-1 can use ornithine,sorbitol,tartrate,arginine dihydrolase,mannitol,malonate.For H-3-1,inositol,cellobiose,phenylalanine,mannitol,ribose are specific.M-I,W-1-1 and H-3-1 share the same optimum growth conditions.The optimum pH and temperature for growth were neutral and 30°C respectively.And 0%of sanlinity was optimal.The logarithmic grovwth phase of M-1 was 12-36h,W-1-1 was 18-42h,H-3-1 was 6-24h respectively.Finally,the results of 16S rDNA sequencing and phylogenetic analysis showed that M-1 was Acinetobacter sp.,W-1-1 was Brachybacterium sp.,and H-3-1 was identified as Neomicroccus sp..Another study on the ability of phenol degration,4-chlorophenol degradation and 4-nitrophenol degradation was carried out.H-3-1 can not degrade the 3 contaminants.M-1 and W-1-1 could degrade 100%of phenol in 48h,and M-1 showed the greater degradative capability of phenol.Both M-1 and W-1-1 possessed the capability of degrading 4-chlorophenol in 48h with low amplitude.But for 4-nitrophenol,M-1 could degrade a extremly small portion in 48h,and M-1 showed no ability of degradation.Taken together,M-1 and phenol were chosen as the reference strains and compound respectively for further investigation on the mechnism of phenol degradation.Proteomics research on the mechnism of phenol degradation of M-1 which combines iTRAQ and Label-free technology revealed that M-1 degrades phenol by ortho-cleavage pathway.Phenol was firstly transformed to catechol,then a ring opening reaction of catechol made the production of muconic acid with chain structure.Muconic acid was further transformed to mucoconate lactone.Finally,the product of succinyl coenzyme A and acetyl CoA were mineralized by TCA.Furthermore,KEGG pathway annotation was made for distinct proteins.There was a pathway of degradation of 4-chlorophenol found in the branch of chlorocyclohexane and chlorobenzene degradation pathway,which indicated that M-1 has the ability to degrade 4-chlorophenol.The comparative analysis of iTRAQ technology,sepectrum counting and LFQ intensity in screening and identifing distinct proteins revealed that distinct proteins could be found by each of the 3 methods and these proteins showed the apparent connection with phenol degradation by GO and KEGG annotation.But phenol hydroxylase and L-alanine-DL-glutamate epimerase were only screened by iTRAQ technology,which were significant proteins in the metabolism of phenol in microbe.In addition,the phenol hydroxylase gene was identified by PCR assay in M-1.