Isolation,Characterization And Degradation Characteristics Of A 3,5-dibromo-4-hydroxybenzoic Acid Degrading Strain

3,5-dibromo-4-hydroxybenzoic acid(DBHB),a relatively stable bromo-substituted aromatic compound,can cause irritation to eyes,skins and respiratory system.It is widely used for the production of many bromo-substituted aromatic drugs and is also the intermediate of some pesticides,imposing harmful effects to environments.Biodegradation by microorganisms is an efficient,cost-effective and environmental friendly means for the clean up of pollutants from environment,thus it’s considered as the most potential method to degrade DBHB in the environment.Besides,DBHB can be naturally produced in marine environment,and thus,study of the microbial degradation of DBHB is meaningful to elucidate the cycle of halogen in the marine environment.So,isolation of bacterial strains degrading DBHB,study of the growth and degradation characteristics of the isolate,and further analysis of the metabolic pathway of DBHB can deep our understandings of the degradation of bromo-substituted aromatic compounds and provide theoretical support to the bioremediation of bromo-substituted aromatic compound contaminated sites.A bacterial strain named H8,which was capable of degrading 3,5-dibromo-4-hydroxybenzoate,was isolated from a bromo-substituted aromatic compoundpolluted soil sample.It was able to grow with DBHB as its sole sources of carbon and energy,and completely mineralize 0.2 mM DBHB within 30 hours.The strain was identified as Pigmentiphaga sp.based on the phylogenetic analysis of its 16S rRNA gene sequence as well as its physiological-biochemical characteristics.The optimal carbon source and nitrogen source for strain H8 growth were sucrose and peptone,respectively.The optimum temperature for strain H8 growth was 37℃ and the optimum pH range was 6.0-7.0.Strain H8 could grow under the condition of 10-40 g/L NaCl,and the optimum NaCl concentration was 10 g/L.With the growing concentrations of NaCl,the growth of strain H8 was inhibited.Strain H8 was resistant to 50 mg/L chloramphenicol,50 mg/L streptomycin or 80 mg/L spectinomycin.When strain H8 degraded DBHB as the sole carbon source,about two equivalents of bromide ions were released during the process of DBHB degradation.In addition,strain H8 was able to degrade 3,5-dichloro-4-hydroxybenzoic acid,3-chloro-4-hydroxybenzoic acid and 3-bromo-4-hydroxybenzoic.With the condition of 30℃ and pH 7.0,the degradation rate of DBHB was the highest.Sodium acetate strongly promoted the degradation of DBHB;while organic nitrogen sources could dramatically enhance the degradation of DBHB.Strain H8 could degrade 3,5-dibromo-4-hydroxybenzoate in the condition of NaCl ranging from 1 g/L to 15 g/L.The optimum NaCl concentration was 1 g/L,and the degradation was inhibited when the NaCl concentration was too high.It was found that 0.2 mM of Co2+,Mn2+,Ni+ or Cu2+ could also inhibit the degradation,while 0.2 mM of Al3+,Ca2+,Zn2+,Mg2+,Fe3+ or Fe2+didn’t have significant effect on the degradation.In the crude enzyme reaction system,NADPH was the necessary cofactor for the enzyme activity,and the enzyme activity was the highest when NADPH and FAD were added simultaneously.The crude enzyme had enzyme activity during pH range of 6.0-10.0 and 4-50℃ with the highest enzymatic activity at pH 7.0-8.0 and 30-37℃.The DBHB degradation activity was inhibited when the pH was lower than 5.0.Compared to 30℃,the degradation rate of DBHB in crude enzyme was reduced by 60%at 4℃ and by 85%at 50℃.The crude enzyme activity was strongly inhibited by 1 mM Cu2+.The metabolic pathway of DBHB in strain H8 was also investigated.The metabolites,produced during the degradation of DBHB and CHB,were studied by resting cell experiment and high-performance liquid chromatography spectrum.The results showed that the metabolites were totally different with that of previous reports,indicating that there was a new debromination pathway and debromination mechanism in strain H8.By LC-MS/MS technique for detection and identification of metabolite,2,6-dibromo hydroquinone and 3-chloro-4,5-dihydroxy benzoic acid or 5-chloro-2,4-dihydroxy benzoic acid were identified as the metabolites of DBHB and CHB,respectively.The expression of genes involved in the degradation of DBHB in strain H8 was found to be induced by DBHB or CHB.According to the results,comparative proteomics were carried out to analyze and compare the differences of protein expression between different treatments.Combining these results with whole genome sequencing results and the metabolic pathway,some genes related with the degradation of DBHB were also speculated.