Isolation & Characterization Of Thiobencarb-degrading Strain S351 (Acidovorax Sp.) And The Researching Of Its Genetic Mechanism

One bacterial strain,which is capable of degrading thiobencarb,was isolated from the sludge collected from the wastewater pipe of a thiobencarb factory and named S351(GenBank Accession No:KR048280).This strain was identified as Acidovorax sp in accordance to its physiological&biochemical characteristic and the similarity analysis of its 16S rDNA sequence.The strain is gram-negative bacteria,the color of the colony is white,and it is not transparent,high viscosity,rounded,edge neat,bump.The colony is wet,not easy to pick.The shape of the thalli is short rod,surface crapy,it has a thick capsule,and observed no flagella,no spore production.The research on biological characteristics of strain S351 showed that,the optimal pH value for the growth of strain S351 was 7.0,the optimal temperature was 30?.The aeration was related positively to growth of this strain.The optimal osmotic pressure for the strain S351 was 0?10mg·L-1 NaCl.The optimal culture medium for the growth of strain S351 was glucose and fructose as carbon source.Strain S351 is able to degrade 99.6%of 100mg?L-1 thiobencarb in 36h.The optimal pH and temperature for the degradation is 7.0 and 30 ?,respectively.The degradation efficiency was related positively to initial inoculum size.When the initial inoculum size is more than 5%(containing 5%),the velocity is incrasing fast.By different substrate degradation experiments of strain S351,we found that S351 can utilize 4-Chlorobenzyl mercaptan and chlorobenzoic acid,4-hydroxybenzoic acid and so on,but it can't use of chlorobenzene,2,4,6-trichlorobenzene and benzyl mercaptan.A mutant strain was got,by streaking subculture on plates without thiobencarb many times,and named S3512.We got two whole genome framework map after whole genome sequencing of strain S351 and S3512.According to annotation of genome framework map and fcbABC gene downloaded from NCBI,primers are designed at conserved section.The degradation gene of chlorobenzoic acid was amplificated.The degradation pathway of thiobencarb studied in detail.According to results of experiments,and information given by genome framework map,A clear pathway of thiobencarb is determined.A few of suspected fragments was found according to comparison of two genome framework map.Two fragments,contig 138 and 180,was confirmed using PCR.Also,by PCR,we know the connection sequence is contig180-contig138.SEFA-PCR was done to detect the unknown fragments of this sequence,so far,a sequence about 9k long was got,and one end is clear,the other end need more research.Primitive research of crude enzyme was carried on,however,no obvious enzyme activity was observed,maybe it is because lacking one kind of coenzyme(cofactor).