Isolation And Characterization Of Highly Efficient BBP-degrading Bacteria From The River Sludge

N-butyl benzyl phthalate (BBP) is a phthalate ester (PAE) used extensively in the manufacture of plastics. It was found to exert estrogenic activities and proved to be reproductive toxicity in several studies. Due to the excellent plasticizing properties and gradually widespread use, it readily released into the environment and was threatening the human’health. Since the rates of photolysis and chemical hydrolysis of phthalate esters are very slow, biodegradation is considered to be one of the major routes for the environmental degradation of these pollutants in aquatic and terrestrial systems, such as sewage, soils, sediments, and surface waters. Studies showed that the biodegradation of PAEs was harder if the lateral chain was longer and more complicated. As an complicated aryl alkyl ester of 1,2-benzene dicarboxylic acid, BBP is hard to mineralize and constantly accumulating in natural environment. Since much less attention was paid to biodegradation of BBP, it will be meaningful to isolate more bacterium species with wide availability, high environmental endurance, or strong degradation capacity for it.In this paper, BBP was used as the sole carbon and energy source to screen and isolate microbes from river segments of Wuhan city and the surrounding area. We obtained 8 strains of BBP-degrading bacteria, and finally found one strain named HS-B1 that is relatively outstanding. Based on its morphological, physiological characteristics, 16S rDNA and gyrB gene sequence analysis, strain HS-B1 was preliminarily identified as a member of the genus Acinetobacter sp.. Shake flask experiments indicated the optimum pH,temperature for growth and BBP biodegradation by HS-B1 was found to be 8.0-9.0 and 30-35℃, respectively. BBP is an organic compound of high octanol/water partition coefficients and low water solubility. When using 0.10 mmol·L-1 non-ionic surfactant Tween-80 as co-solvent to increase the apparent solubility of BBP in water, HS-B1 was able to completely degrade 1000 mg·L-1 BBP within 48 hours, the degradation rate increased by 60%. According to the literatures on the aerobic biodegradation pathway of BBP, the BBP metabolites were detected by HPLC-UV and then identified as phthalate acid (PA) and benzoic acid (BA) by the retention time of the standard compound. In the end, the BBP degradation by Aci sp. HS-B1 was speculated:BBP was firstly hydrolyzed to PA and benzyl alcohol, then was further metabolized to BA and finally to produce CO2 and H2O. Diversity of degradable substrates also showed that HS-B1 can efficiently utilize many other phthalate esters such as dimethyl phthalate and diethyl phthalate. It revealed that the strain HS-B1 has special application potential in dealing with the pollution caused by phthalate esters.Catechol 1,2 dioxygenase (1,2-CTD) is a key enzyme in metabolism of aromatic compounds which catalyzes an intradiol cleavage reaction of catechol to form cis,cis-muconate. The conserved gene of catA was obtained by PCR from the total DNA of strain HS-B1. Analysis of the DNAStar software showed that catA gene organized as an operon and the size were 921bp which were predicted to encode peptides of 306 amino acids. According to homology analysis, the catA gene of Aci sp. strain HS-B1 was gather into a cluster with other strains of Acinetobacter sp. and had most high similarity with Aci baumannii AYE(99%). Then the catA gene was subcloned into pET-28a to construct the recombinant expression vector pET-catA by molecular cloning. The recombinant plasmid was transformed into E. coli BL21(DE3), after induced 9H by 0.05 mmol·L-1 IPTG at 18℃, a specific soluble protein band (about 37kDa) was observed in SDS-PAGE analysis.