Isolation,Identification And Degradation Mechanism Of Bisphenol A And Triphenyl Phosphate Degrading Bacteria

Environmental endocrine disruptors(EEDs)which are widely used have caused serious environmental pollution problems.Bisphenol A(BPA)and triphenyl phosphate(TPHP)are broadly used in industrial production and human life.In recent years,the endocrine disrupting effects of BPA and TPHP have significantly threatened to the ecological environment and human health.How to effectively remove environmental endocrine disruptors from the environment has become a research hotspot.Microbial degradation of environmental pollutants for bioremediation has high efficiency,safty and low cost,and has been widely valued.Strain YC-JY1 which was able to degrade BPA and TPHP was isolated from contaminated river sludge polluted by electronic waste.Strain YC-JY1 was identified as Sphingobium yanoikuyae by colony morphology,bacterial morphology,Biolog analysis,phylogenetic analysis of 16 S rDNA sequences and housekeeping genes.The effect of different environmental conditions on the ability of YC-JY1 to degrade BPA was investigated using single factor analysis.The results indicated that strain YC-JY1 had good toleration with temperature 15-35 ?,pH5.5-8.0 and NaCl concentration ?0.4%,while glucose and culture speed have no significant effect on degradation efficiency.Metabolic intermediates of BPA were detected by high-performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry,and metabolic pathway was proposed.Strain YC-JY1 showed good application potential in promoting the biodegradation of BPA and TPHP for soil remediations.Genome of strain YC-JY1 was sequenced and the genome sequence was submitted to GenBank(Accession number CP047218).A circular genome(5.48 Mb,GC content 64.08%)and 5 plasmids(plasmid A,135.3 Kb,GC content 61.06%;plasma B,65 Kb,GC content 59.24%;Plasmid C,66.7 Kb,GC content 59.75%;plasma D,45.5 Kb,GC content 61.31%;plasma E,15.6 Kb,GC content 60.32%)were obtained.Total 5633 coding sequences(CDS),12 rRNA genes and 66 tRNA genes were obtained.Fourteen genes were proposed to be involved in the BPA degradation process of strain YC-JY1.Then the metabolic pathway of BPA degradation in strain YC-JY1 was proposed combining with the detection and analysis of metabolic intermediates.Cytochrome P450(CYP450)gene bisdB and ferredoxin gene bisdA were proposed to be involved in biodegradation of BPA in strain YC-JY1 according to genome sequence analysis.CYP450 inhibitor 1-aminobenzotriazole could significantly reduce the ability of strain YC-JY1 to degrade BPA,indicating that CYP450 may be involved in the degradation of BPA.Genes bisdA and bisdB were cloned and expressed.Escherichia coli BL21(pET28a-bisdAB)cells showed BPA degradation ability,demonstrating the BPA degradation ability of BisdAB.The bisdB gene knockout strain YCJY1?bisdB lost the ability to degrade BPA,indicating that BisdAB are important initiated catalytic proteins for BPA degradation in strain YC-JY1.To explore other genes related to the degradation of BPA and TPHP,a degradation-inactivating mutant library of strain YC-JY1 was constructed using the pUTmini-Tn5 plasmid.Five mutant strains that could not grow well using BPA or TPHP as the sole carbon source were screened.After analyzing the mutant sequences,it was predicted that the phosphomonophenylesterase gene phoP may participate in the degradation of TPHP in strain YC-JY1.The function of PhoP was verified by heterologous expression,showing that PhoP has the activity of hydrolyzing phenyl phosphate.