Isolation,Identification And Characterization Of A PCBs-degrading Bacteria

Polychlorinated biphenyls(PCBs)are widely distributed and complex composition,PCBs are gradually chlorinated by biphenyl(BPH)under the action of metal catalysts,they are one of the 12 persistent organic pollutants to be priority controled and there are209 congeners and isomers for different chlorine substitution position.Because of the characteristics such as acid and alkali resistance,heat resistance and electrical insulation,it has been widely used as a flame retardant,heat carrier,and insulating oil in industry.PCBs are difficult to degrade,bioaccumulation,high toxicity and other environmental characteristics make it easy to enrich in the environment and organisms,causing harm to the environment and human health,so it is an urgent environmental problem to be solved.In recent years,bioremediation of polychlorinated biphenyls has been the focus and difficulty for scientists,it is the key to solve the problem that find aerobic bacteria which can efficiently degrade PCBs.In this experiment,two strains were isolated from estuary which have the ability to degrade polychlorinated biphenylsat.These two strains were named P-6 and P-6-5.After molecular biological identification and cell morphology identification,the two strains were identified as Pseudomonas and Ochrobactrum;environmental conditions were changed to explore the growth of degrading bacteria is affected by environmental factors;degradation bacteria utilized polychlorinated biphenyls and polychlorinated biphenyl congeners as the sole carbon and energy source in the degradation system,the degradation of PCBs was investigated;gas chromatographic analysis was used to infer the preferential attack of degrading bacteria on different chlorine substitution sites,the peak time of each PCB in the experiment was abtained and the degradation degree of 4-chlorobenzoate was verified.The main results of this study are as follows:(1)Two strains of PCBs degrading bacteria were isolated from the estuary utilizing biphenyl as sole carbon and energy source.P-6 is a short rod gram-negative bacteria,the sequence of P-6 gene was found to have 99.63%similarity with Ochrobactrum anthropi strain ATCC 49188~T(CP000758),they were thought to be the same bacteria;P-6-5 is an ellipsoidal gram-negative bacterium and the sequence of P-6-5 gene was found to have99.15%similarity with Pseudomonas oceani strain KX20~T(KT449732.1),they were thought to be the same bacteria.(2)Both salinity and pH can affect the growth of degrading bacteria,Ochrobactrum anthropi strain P-6 and Pseudomonas oceani strain P-6-5 can survive in the weakly acidic to weakly alkaline environment.The growth of strains P-6 and P-6-5 showed certain halophilic properties,the optimal salinity was 35 g/L.The two strains were isolated from estuary showed the characteristics of marine bacteria,it provied a good start to remove PCBs from the ocean in the future.(3)The degradation rates of Ochrobactrum anthropi strain P-6 and Pseudomonas oceani strain P-6-5 towrds 10 mg/L PCB3 were 92.4%and 95.3%,respectively.Induced by biphenyl and PCB3,PCBs degradation strains stimulate the production of enzymes and promote the ability of enzymes to bind to substrates.The ability to induced the growth of the degrading bacteria and to metabolize the substrate in this experiment was PCB3>BPH>LB.The degradation ability of bacteria is affected by the initial substrate concentration and there is an optimal substrate concentration range for the degradation.Within this concentration range of substrate,the degrading bacteria have the largest amount of substrate degradation.The substrate degradation rate of degrading bacteria was different towards different concentrations.The degradation speed of strain P-6 increased with the substrate concentration and the maximum degradation rate was 250?g/L·h.While the degradation speed of strain P-6-5 increased firstly and then decreased with increasing concentration and the maximum degradation rate was 344.8?g/L·h.(4)Ochrobactrum anthropi strain P-6 and Pseudomonas oceani strain P-6-5 exhibit a broad range of substrate applicability for polychlorinated biphenyl congeners.The two strains both have the ability to degrade up to four chlorinated polychlorinated biphenyls,especially for symmetrical coplanar PCBs with relatively high toxicity,such as2,2',4,4'-tetrachlorobiphenyl,2,2',5,5'-tetrachlorobiphenyl,2,2',6,6'-tetrachlorobiphenyl.In particularly,the degradation pattern of strain P-6 exhibits differences from other aerobic degrading bacteria,para-polychlorinated biphenyls was degraded preferentially.These finds have a great significance for practical soil remediation.(5)Both Ochrobactrum anthropi strain P-6 and Pseudomonas oceani strain P-6-5 can metabolize PCBs to 4-chlorobenzoic acid.Furthermore,strain P-6 can degrade4-chlorobenzoic acid,while strain P-6-5 has no obvious effect on the degradation of4-chlorobenzoic acid.It is speculated that the degradation pathway of the strain P-6-5 is similar to the BPH degradation pathway,the degradation of polychlorinated biphenyls is inhibited by the accumulation of the degradation product chlorobenzoic acid;Strain P-6may have new degradation pathways including the BPH pathway and the 4-chlorobenzoic acid degradation pathway.The above results indicate that Ochrobactrum anthropi strain P-6 and Pseudomonas oceani strain P-6-5 are two high-effective polychlorinated biphenyl-degrading bacteria which with the characteristics of both marine bacteria and terrestrial bacteria.It has a positive guide to remove contaminate from actual polluted soils and provide a basis for the degradation of polychlorinated biphenyls from the ocean.These data postulated that strain P-6 and P-6-5 have a great potential to be utilized in the bioremediation of PCBs pollution.