Isolation And Characterization Of A Novel Polychlorinated Biphenyl-degrading Bacterium

Polychlorinated biphenyls(PCBs),including 209 congeners and isomers,are a class of typical,complex,and ubiquitous persistent chlorinated pollutants in the environment.In recent 20 years,biodegradation of PCBs has captured great attention in the field of environmental science and technology.One of the promising solutions is to obtain microbial strains capable of removing more PCB congeners aerobically at a high biotransformation rate.In this paper,a new strain is isolated from a transformer-oil polluted site using PCB analogue as substrate.Molecular,physiological and biochemical assays identify it as a member of Enterobacter sp,named LY402.Further research has been done systematically on determination of the transformation potential of different PCB congeners in Aroclor by LY402 cells.Relevant factors that affect the degradation performance are also discussed.In addition,intermediate metabolites are identified by GC-MS;genes encoding responsible enzymes are analyzed mainly by PCR,with primers designed according to gene homology. The results are as below:(1)The resting cells could degrade 86.4%of Aroclor1242,60%of Aroclor1254,and 29.1% of Aroclor1260 at a concentration of 2 mg/L,respectively.When exposed to 100 mg/L Aroclor1242,LY402 cell growth did not seem to be significantly inhibited and still removes 60%of PCBs in six days,demonstrating its strong durabilitY against toxic contaminants.(2)After six days incubation of LY402 with 3 mg/L Aroclor(1242:1254:1260 = 1:1:1)in synthesis medium without carbon source,75%of PCBs was successfully degraded.Kinetic study shows that,among 91 chromatographic peaks,51 have disappeared,23 have reduced peak area in certain degree,and the remaining 10 have no significant changes.(3)The different number and positions of chlorine substituents widely affected the biotransformation rates of PCBs.It was found that di-～penta- chlorinated PCB congeners were able to be rapidly eliminated by LY402;whereas octachlorobiphenyls and nonachlorobiphenyls seemed not subject to microbial attack.Generally,the more the chlorine atoms PCBs possess,the lower the biotransformation rate is.Moreover,it was found that chlorinated positions greatly affect PCB biodegradation rate.Enterobacter sp.LY402 can attack both ortho- and para- substituted PCBs.It was supposed that para- substituted PCBs (4,4′and 3,3′,4,4′)pose negative effect on biodegradation.13 PCB congeners out of 112 have not been effectively degraded,12 of which contains 3,3′,4,4′- Chlorinated sites.Some of these highly toxic PCBs are coplanar or similar to coplanar congeners.Overall,compared to reported strains with high transformation rate,Enterobacter sp.LY402 is more competent and effective in transforming both ortho- andpara- chlorinated PCBs.(4)The metabolites study indicated that varied amount of chlorobenzoic acids were produced from differentially substituted PCBs,accompanied with significant dechlorination. Moreover,LY402 could continue to remove some chlorobenzoic acids.2-CBA and 4-CBA partially degraded,and 3-CBA completely eliminated.However,if more than one chlorine atoms located on the aromatic ring,the effect became negligible.(5)Constitution of the biphenyl-catabolic(bph)gene cluster,encoding enzymes for the degradation of PCBs,was analyzed in LY402.According to gene homology,primers of bph genes were designed.All the related genes were amplified by PCR and sequenced.The results showed that the entire bph gene cluster is located on an approximately 20 Kb plasmid in LY402 cells.Alignment with the published sequence through BLAST,it was found bph gene sequence was almost the same with those reported in Burkholderia xenovorans LB400 except one base mutation(C⁷⁰→T)which resulted in one amino acid replacement in small subunit of the terminal dioxygenase(Thr²⁴→Ile).However,the bph genes in LB400 locate on a 1.4 Mb plasmid.A definite explanation still lacks to explain the high similarity between two sets of PCB degradation genes,whose hosts show a far cry in phylogenesis.(6)Surfactants,as carbon sources,obviously promote the biodegradation of PCBs. Utilizing biosurfactant sucrose laurate and non-ionized Tween-80 as carbon sources,75.4% and 64.7%of 4 mg/L Aroclor 1242 were degraded in 12 h,which were respectively increased by 20.6%and 9.9%comparing with consuming biphenyl.Furthermore,the promoting effects of sucrose laurate on different Aroclors were measured.98.5%of Aroclor 1242,79.9%of Aroclor 1254 and 64.5%of Aroclor 1260 at the concentration of 2.0 mg/L were degraded after they were treated with 2.0 g/L sucrose laurate.The transformations were enhanced by12.2%,19.9%and 35.5%,respectively,when any carbon sources were not used.And the promoting effects were more notable for hexa- and hetpa-chlorobiphenyls.Therefore,sucrose laurate is expected to be applied as an accelerant to PCB contaminated soil.In conclusion,Enterobacter sp.LY402 is one of strains which show the highest rate as well as the most congeners of biodegradation to PCBs under aerobic condition.Kinetic study and mechanism exploration not only give deep insight into the microbial removal ability,but also pave the way for further genetic construction of recombinant strain and enhancement of bioremediation efficiency in persistent chlorinated complex contaminated sites.