The Isolation Of An Aerobic Diphenyl Ether And Lower Brominated Diphenyl Ether Degrading Bacterium And Characterization Of The Degradation Mechanism

Polybrominated diphenyl ethers (PBDEs) have been used as flame retardant for a long period of time. It was used in a lot of areas related to living and industry including electronic products, toys, clothing. PBDEs do not form any chemical bond with the plastic matrix, thus would easily be released into the environment. PBDEs have very low biodegradability, with half life in soil as long as 20 years. Once PBDEs enter into the environment, they would accumulate stably. Attributed to the hypophobicity of PBDEs, once they have entered into the bodies of animals, they would accumulate in tissues, and be enriched through food chain.Researches have shown PBDEs have toxicity against animals, the effects include neurodevelopmental toxicity, endocrine disruption, even carcinogenicity.Compared to the chemical remediation methods, bioremediation is more operable, more applicable for soil remediation. Among all biodegradation methods, anaerobic bacteria could remove the bromines from diphenyl ether backbone; aerobic bacteria can degrade diphenyl ether and its lower brominated derivatives into smaller carbohydrate. Since the aerobic bacteria are more suitable for in situ bioremediation, this work focused on discovering bioresource for degradation of diphenyl ether and lower brominated diphenyl ethers. We also carried out sophisticated studies around the mechanisms behind the degradation activity, which would be helpful for further improving this characteristic. Below listed are the advancements made in this work:1. Isolation and characterization of a diphenyl ether degrading bacterium.Using diphenyl ether as the substrate, a culture which could grow on diphenyl ether as sole carbon source and capable of degrading diphenyl ether was enriched from the sediment of a water treatment plant. From this enrichment, a gram negative bacterium was isolated from the enrichment culture, which was named Cupriavidus basilensis WS after identification. The strain could completely remove 1 g/L diphenyl ether within 6 days, and transform the substrate into its own biomass. During the diphenyl ether degradation process, no intermediate was detected. This strain could also degrade 4-bromodiphenyl ether and 4,4’-dibromodiphenyl ether. During the degradation process of 4-bromodiphenyl ether,4-bromophenol was detected as an intermediate.2. Identification of genes related to diphenyl ether degradation by transposon mutation of C. basilensis WS.By using the transposon mutagenesis mediated by mini-Tn5, a library containing thousands of different mutants was constructed. After selection,6 mutants which lost the ability to degrade diphenyl ether were obtained from the library. The genes interrupted by transposon were identified by using genome walking, which finally indicated the diphenyl ether degradation function was carried out by bph operon, especially the genes coding biphenyl 2,3-dioxygenase.3. Characterization of the functions of bph genes in the diphenyl ether degradation pathway.After finding the relationship between diphenyl ether degradation and bph operon, the functions of each candidate genes were studied. By using E. coli as platform, candidate genes were assembled into several sets, until the degradation pathway was reconstructed. Using the constructs, every intermediate product in the degradation pathway was identified. The result showed that BphA (BphAl+BphA2+BphA3+ BphA4), BphB and BphC acted sequentially to degrade diphenyl ether into phenol and PCA. Combining the previous analysis of diphenyl ether degradation products in C. basilensis WS, it is highly possible that this strain could mineralize diphenyl ether.4. Construction and application of a C. basilensis/E. coli shuttle vector based on the natural plasmid pWSAfter separating and sequencing of the natural plasmid pWS from C. basilensis WS, we identified the minimal replicon of pWS by construction of vectors with different parts from pWS. In the minimal replicon, all the materials except the coding region of rep is substitutable. Based on the minimal replicon, we constructed the C. basilensis/E. coli shuttle vector pCB5. Interestingly, the copy number of pCB5 in C. basilensis WS could be enhanced from 1-3 to 16 by manipulating the supply of Rep. On the basis of pCB5, two reporting vectors, pCB-lacZ and pDMP-lacZ were constructed and used to analyze the transcription of bph and dmp operons during diphenyl ether degradation.5. Development of a promoter prediction method based on sequence matchingThe method require a predicted promoter library obtained from genome using PWM, and a randomly constructed non-promoter library. Any sequence to be analyzed would be matched with each sequence from the former library and the later library. The possibility of the sequence to be a promoter will be determined by the difference of matching scores.