Degradation/transformation Of Tetrabromobisphenol A (TBBPA) By Phanerochaete Chrysosporium And Proteomics Analysis

Tetrabromobisphenol A?TBBPA?is one of the most used brominated flame retardants,which is easily released into the environments and causes pollution.Due to its biotoxicity and bioaccumulation,tetrabromobisphenol A poses a certain threat to the safety and health of ecosystems and humans.Therefore,research on high-efficiency degradation technology of TBBPA has attracted much attention.Microbial remediation is considered to be an important means to remove toxic and harmful environmental pollutants.Among them,white rot fungi play an important role in the degradation of various organic pollutants due to its powerful enzyme system.Although previous researches proved the effectiveness of several kinds of fungi in removing TBBPA,more attention was focused on TBBPA conversion by laccase secreted by the fungi.There are few studies on the mechanism of direct biotransformation of TBBPA by fungi,and little is known about the molecular mechanism at proteome level in the process of fungal degradation of TBBPA.The objective of this study was to investigate the removal characteristics,degradation pathways,detoxification properties and degradation mechanism at proteome level.Besides,the feasibility of applying P.chrysosporium to the bioremediation of TBBPA in water/sediment system was also investigated.The detailed conclusions of this thesis are as follows:?1?The removal efficiency of TBBPA by P.chrysosporium was affected by various environmental factors.Under optimal conditions?pH 4⁵,inoculum size of 5%?V/V?,initial glucose concentration of 5 g/L,TBBPA concentration of 5 mg/L?,97.7%of initial TBBPA was removed after 3 days.Cd²⁺of low concentration?5 mg/L?did not significantly affect the removal efficiency of TBBPA by P.chrysosporium,but exerted significant inhibition at higher concentrations??20 mg/L?.The addition of cytochrome P450 enzyme inhibitor resulted in a significant decrease in the removal efficiency of TBBPA,indicating that the cytochrome P450enzyme involved in the degradation of TBBPA by P.chrysosporium.?2?Analysis by LC-Q-TOF-MS/MS demonstrated seven products of TBBPA,including tetrabromobisphenol A glycoside,tribromobisphenol A,tribromobisphenol A glycoside,monohydroxylated tetrabromobisphenol A,4-?2-hydroxy-isopropyl?-2,6-dibromophenol,4-?2-methoxy-isopropyl?-2,6-dibromophenol,andp-hydroxybenzoicacid.Four degradation/transformation pathways were speculated as debromination,glycosylation,oxidative hydroxylation and oxidative cleavage pathway.tetrabromobisphenol A glycoside,tribromobisphenol A glycoside and monohydroxylated tetrabromobisphenol A were being produced by glycosylation and oxidative hydroxylation,respectively,which were identified for the first time in fungal transformation of TBBPA.To clarify the detoxification properties of TBBPA transformed by P.chrysosporium,HepG2 was used as a model cell to investigate the cytotoxicity of different concentrations of TBBPA and its equivalent metabolic mixture.The results showed that TBBPA?10⁵0?mol/L?induced the accumulation of excess ROS in HepG2cells in a concentration-dependent manner,which caused mitochondrial damage and decreased mitochondrial membrane potential,and finally induced apoptosis of HepG2 cells.The TBBPA equivalent metabolic mixture exhibited lower impact on cellular ROS content,mitochondrial membrane potential,and apoptosis.TBBPA induced HepG2 cells to arrest at phase G0/G1,and the proportion of cells at phase S decreased.The disruption of the cell cycle by the TBBPA equivalent metabolic mixture was weaker than TBBPA.Current studies showed that the cytotoxicity of TBPPA on HepG2 is related to cell cycle regulation and mitochondrial pathway-mediated apoptosis.The metabolic mixture produced by P.chrysosporium degrading exhibited lower cytotoxicity.?3?The results of proteome quantitated by iTRAQ indicated that a total of 148 proteins were differentially expressed during the degradation of TBBPA by P.chrysosporium,90 of which were up-regulated and 58 were down-regulated.The results of protein functional analysis showed that the expression of two cytochrome P450 enzymes,three glutathione S-transferases,two UDP-glucosyltransferases,one O-methyltransferase and other oxidoreductases in P.chrysosporium was significantly up-regulated during TBBPA degradation,suggesting that they might be involved in the biodegradation of TBBPA in P.chrysosporium including hydroxylation,debromination,glycosylation,O-methylation and other oxidation reactions.In addition,the stress of TBBPA induced the up-regulation of phosphoglycerate kinase,glyceraldehyde-3-phosphate dehydrogenase,pyruvate decarboxylase,aspartate-tRNA ligase and serine/threonine-protein phosphatase 4 catalytic subunit in P.chrysosporium for more energy production and resistance to oxidative stress.The expression of various proteins involved in amino acid synthesis and catabolism was inhibited.?4?The results of bioremediation indicated that there may be microorganisms capable of degrading TBBPA in water/sediment system.The degradation efficiency of TBBPA was 13.91%in the unsterilized control group,and was significantly increased to 40.19%and 78.87%respectively in the bioaugmentation group and the bioaugmentation-biostimulation group.Both two treatments affected the microbial community and led to the enhancement of certain functional flora:As the result of bioaugmentation,Phanerochaete became the dominant fungus in water/sediment system,and Flavobacterium?0.3%⁴.4%?and Sporocytophaga?1.0%².8%?were found to be the dominant bacteria.After co-treatment with bioaugmentation and biostimulation,Pseudomonas proved to be the absolutely dominant bacterial genus?9.6%¹7.3%?,and the proportion of Sporocytophaga?1.0%⁵.5%?also increased.Based on previous studies,we speculated that Pseudomonas played an important role in the enhanced degradation of TBBPA,and Sporocytophaga and Flavobacterium possess the potential to degrade TBBPA.A variety of functional genes related to xenobiotic metabolism and microbial cell self-protection,including enoyl-CoA hydratase,3-oxoacyl-[acyl-carrier protein]reductase,glutathione S-transferase,methyl-accepting chemotaxis protein and ATP-binding cassette,were enriched by co-treatment with bioaugmentation and biostimulation.