Isolation And Molecular Characterization Of Bisphenol A Degrading Bacteria

Bisphenol A?BPA?is an important organic chemical as an intermediate,final and inert ingredient in manufacturing of many important products like polycarbonate plastics,epoxy resins,flame retardants,food–drink packaging coating,and other.BPA is an endocrine disruptor compound that mimics the function of estrogen causing damage to reproductive organs.Bacterial degradation has been considered as a cost effective and eco-friendly method for BPA degradation compared with physical and chemical methods.This study aimed to isolate and identify bacterial strains capable to degrade and tolerate high concentrations of BPA,studying the factors affecting the degradation process and study the degradation mechanism of this strains.Two Gram negative bacterial strains?YC-AE1 and YC-AE2?were isolated from soil and identified as Pseudomonas putida and Achromobacter sp,respectively by using 16S rRNA gene sequence and BIOLOG identification system.These strains found to have a high capacity to degrade the endocrine disruptor BPA.Response surface methodology using central composite design was used to statistically optimize the environmental factors during BPA degradation by strain YC-AE1 and the results obtained by significant model were 7.2,30 ^oC and 2.5%for optimum initial pH,temperature and inoculum size,respectively.Prolonged incubation period with low NaCl concentration improve the biodegradation of BPA.Analysis of variance?ANOVA?showed high coefficient of determination,R² and Adj-R² which were 0.9979 and 0.9935,respectively.Substrate analysis found that,strain YC-AE1 could degrade a wide variety of BPA-related pollutants such as bisphenol B,bisphenol F,bisphenol S,Dibutyl phthalate,Diethylhexyl phthalate and Diethyl phthalate in varying proportion.Moreover,YC-AE1 mineralize it in a short time compared with reported strains?75%of BPA within40 hours?.One the other hand,one factor at time?OFAT?was used to optimize BPA degradation by strain YC-AE2 and the results obtained were sucrose,8 and 30 C for optimum co-substrate,pH and temperature.The addition of NaCl lead to decrease the degradation of BPA by YC-AE2 while,increasing of inoculum size improved it.The two strains showed high ability to degrade a wide range of BPA concentrations(0.5-1000 mg l^-1)with completely degradation for 500 mg/L,200 mg/L within72 hours for YC-AE1 and YC-AE2,respectively.Metabolic intermediates detected in this study by high performance liquid chromatography-mass spectroscopy?HPLC-MS?were identified as 4,4-dihydroxy-alpha-methylstilbene,p-hydroxybenzaldehyde,p-hydroxyacetophenone,4-hydroxyphenylacetate,4-hydroxyphenacyl alcohol,2,2-bis?4-hydroxyphenyl?-1-propanol,1,2-bis?4-hydroxyphenyl?-2-propanol and 2,2-bis?4-hydroxyphenyl?propanoate,for strain YC-AE1while,only three intermediates were detected during the degradation by YC-AE2 and it were p-hydroxyacetophenone,1,2-bis?4-hydroxyphenyl?-2-propanol and 4,4-dihydroxy-alpha-methylstilbene.We employed RNA sequencing to analyze the differentially expressed genes?DEGs?in YC-AE1strain during degradation of BPA.Total of 1229 genes were differentially expressed,including 725up-regulated and 504 down-regulated genes.Kyoto encyclopedia of genes and genomes?KEGG?enrichment analysis demonstrated that the pathway of microbial metabolism in diverse environments was significantly enriched among DEGs.Moreover,qRT-PCR for 10 genes involved in BPA degradation pathway was further confirmed.To delineate the molecular mechanism,we construct cytochrome P450(CYP₄₅₀)?bisdB?deficient strain?bisdB.The degradation capability of BPA was definitively abolished in?bisd compared with the wild type.Subsequently,CYP₄₅₀ inhibitor showed severe decreasing in the efficiency of YC-AE1 to degrade BPA.Further,Gene bisdB(CYP₄₅₀)and bisdAB(CYP₄₅₀50 and ferredoxin)were cloned in pET-32a and expressed in E.coli BL21?DE3?.Transformed Escherichia coli?E.coli?harboring pET-32a-bisdAB acquired the ability to degrade BPA?100 mg/L within 24 hours?.Taken together,our findings propose the molecular basis and the potential role of CYP₄₅₀ monooxygenase in BPA catabolism.The results of these studies suggested that Pseudomonas putida YC-AE1 and Achromobacter sp.YC-AE2 have a great and un-tapped biotechnological potential that could be utilized further in biotechnological uses especially in bioremediation of bisphenols.