Isolation, Characterization And Degradation Pathway Of Polyaromatic Compound Degrading Bacteria

Polyaromatic compound has been produced and widely used in pharmaceutical, chemical and dyeing industries. Industrial wastewater containing polyaromatic compound such as naphthalene could lead to gross pollution of the environment. ZJ0273, propyl4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate, is a novel herbicide developed for oilseed crop in China.10%ZJ0273emulsifiable concentration and10%suspension concentration (the trade name is Youli) have been obtained registration of Ministry of Agriculture, China, and permitted to be used extensively for eliminating oil rape weed. However, Microbial degradation of ZJ0273, degradation pathway in aquatic system and microbial degradation proteome has not been reported until now. I n order to understand microbial behavior, biodegradation pathway and mechanism of ZJ0273in environment, biodegradation and bioremediation of ZJ0273contaminated water and soil need to be further studied. In this study, ZJ0273-degrading bacterium Amycolatopsis sp. M3-1and Naphthalene-degrading bacterium Pseudomonadaceae sp. Nai8were isolated from soil. Using traditional microbiological method, isotope tracer, modern biotechnology and modern instrument analysis, biodegrading characteristics, pathway and mechanism of ZJ0273in soils and water were studied. The degradation enzymes were identified by2-DE and MALDI-TOF-MS, gene engineering bacterium was constructed and it can degrade ZJ0273and naphthalene simultaneously. The results of this study will be will provide technical support and thoretical guidance for reasonable and safe use of ZJ0273, and it is very important for improving the quality of agricultural products, ecological environment and human health.The main results are as follows1. Five strains of naphthalene degrading bacteria and fifteen strains of ZJ0273degrading bacteria were screened, isolated and separated from soils by applying modern molecular biological technique with traditional microbiological method. The strain M3-1, Nai8and CY were identified as Amycolatopsis sp., Pseudomonadaceae sp. and Bacillus sp. by physiological, biochemical tests and16s rDNA sequence. The optimum ZJ0273degrading conditions of Amycolatopsis sp. M3-1were studied. The results showed that with condition of35°C, pH6.0,8%of inoculation and100mg/L of ZJ0273, the degrading ratio in25d was63.30%. The degradation ratio of ZJ0273by Amycolatopsis sp. M3-1was decreased with the increasing ZJ0273concentration,300mg/L of ZJ0273can strongly inhibit bio-activity of strain M3-1, which means that higher concentration of ZJ0273has higher toxicity to M3-1. And the optimum naphthalene degrading conditions of Pseudomonadaceae sp. Nai8was30℃, pH7.0,8%of inoculation and1000mg/L of naphthalene, the degrading ratio in100h was96.24%.2.Six metabolites (M1-M6) during the degradation of ZJ0273by Amycolatopsis sp. M3-1were identified by combination with multi-poa’tion14C-labeled compounds (B-ZJ0273and C-ZJ0273), chromatography, liquid scintillation spectrometer and LC-MS, a novel pathway of ZJ0273degradation by Amycolatopsis sp. M3-1was proposed based on the identified metabolites and their biodegradation courses.ZJ0273was initially hydrolyzed into M1(4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzaoicacid), then further oxidized into M3(2-(4,6-dimethoxypyimidin-2-yloxy) benzoic acid); M1also could undergo a carbonylation into M2(4-(2-(4,6-dimethoxypyimidin-2-yloxy) benzamido) benzoicacid), and then its C-N and C-0bonds were deaved to yield M3(2-(4,6-dimethoxypyimidin-2-yloxy) benzoicadd) and M4(4,6-dimethoxypyrimidin-2-ol), respectively. Moreover, another two new metabolites, M5(2-(4-hydroxy,6-methoxypyimidin-2-yloxy) benzoicadd) and M6(2,4-dihydroxy-pyrimidine) were found. M5was formed through de-methyl of M3, and then hydrolyzed into M6.In the natural environment, M4and M6could be used as carbon and energy source and bread down to CO2and H2O.3. ZJ0273could be degraded in the five sterile soil samples after inoculation of Amycolatopsis sp. M3-1. The residue of ZJ0273in S1, S4and S3was48.60%.23.32%and5.31%, respectively. Degradation rate of ZJ0273in S3and S4were higher than that of in add soil like S1, S2and S5. In the blank soil samples, the concentration of ZJ0273also decreased slowly, which showed that photodegradation, hydrolysis and mineralization in soil have certain effect to degrade ZJ0273. The residue of ZJ0273in unsterile S1, S2, S3, S4and S5after inoculation of Amycolatopsis sp.M3-1was33.50%,31.11%,31.52%,22.03%and4.22%. 4. To illuminate the mechanism of Z J0273-degrading pathway in bacteria, two-demensional protein SDS-PAGE electrophresis was used to identify variations in protein expression in ZJ0273degrading bacterium. The results showed that strain M3-1exposed to ZJ0273could lead to the6new proteins and the up-regulation of5proteins, and8proteins were indentified by MALDI-TOF-MS.5. Two auxotrophic mutant strains (Pro-and Asn-) were gain through ultraviolet mutagenesis method and chose to mark protoplast fusion.8fusants were obtained with genetic stability on the degradation of ZJ0273and naphthalene by protoplast fusion, the fusant named M N6could degrade ZJ0273and naphthalene simultaneously and has higher degradation rate of degrading ZJ0273and naphthalene. The degradation ratio could improve6.87%(20d) and18.01%(48h).