Mechanisms Of The Phytodegradation Of Bisphenol A In Water By Submerged Macrophyte Ceratophyllum Demersum L.

As an important component of global aquatic ecosystems,wetland has great capability in water purification and toxic contaminants removing.During the process,aquatic plants associate with rhizosphere microbial play a key role by direct absorption,degradation and/or transformation.Bisphenol A(BPA),a typical endocrine disruptor,has been ubiquitously found in aquatic environments around the world,which can be detected in almost all types of aquatic ecosystems..Submerged macrophytes,as one of the important plant species and main primary producers in wetland ecosystem,have been proved to have functions in maintaining the diversity of wetland species,increasing water transparency,inhibiting algae growth and removing pollutants from water.However,few studies have been found on the BPA degradation abilities of submerged plants and on the contribution of plant andassociated epiphytic microorganisms.In addition,the responsible key enzymes,the functional genes involved and relating metabolic pathwayshave not been reported.Therefore,mechanisms in BPA degradation mechanism by submerged macrophytes were investigated here,from individual level,physiological level and molecular level,including BPA degradation abilitities of submerged macrophytes,contribution of plants and their epiphytic microorganisms,environmental influencing factors,the key enzymes and degradation products,and the related gene expression and metabolism pathways involved in BPA degradation process,and also the expression and validation of the functional genes of degrading enzyme.Therefore,this research substantially provides a theoretical basis for phytodegradation of toxic organic pollutants by macrophytes.The main results are summarized as follows:1.Compared the BPA removal rates of 9 kinds of common submerged macrophytes,it was found that the submerged macrophytes have efficient BPA removal abilities and the BPA removal rates were between 62.22%～100%in 12 d.The BPA accumulation rates in submerged macrophytes were between 0.1%～17.94%,indicating that BPA was mainly degraded by the macrophytes.Ceratophyllum demersum has the highest BPA degradation rate,and could removed 98.91%of total BPA at 12 d.BPA(5 mg/L)had no significant effect on the growth of submerged macrophytes(P>0.05).The contributions of the macrophytes and the associated epiphytic microorganisms to BPA removal were further detemined.Ceratophyllum demersum and Myriophyllum spicatum from lakes with diffrent water qualities showed no signifcant diffrences in BPA removal rates(P>0.05).Moreover,removal,inhibition or re-colonization of epiphytic microorganisms did not signifiantly change the BPA removal rates of macrophytes.These results indicated that submerged macrophytes,rather than epiphytic microorganisms,substantially contribute to the biodegradation of BPA in water.2.Effects of environmental factors including temperature,nutrient,initial BPA concentration and biomass,on BPA removal by submerged macrophytes were also tested.The results showed that environmental factors could significantly affect the BPA removal rate of submerged macrophytes.At 25℃ and 35℃ when TN and TP were 0.79 mg/L and 0.07 mg/L,respectively,BPA removal rates of C.demersum and M spicatum significantly increased than other nutrient and temperature conditions(P<0.05).BPA removal rates of the two plants were both highest(P<0.05),which reached 100%in 1d under the conditions that initial BPA concentration of was 1 mg/L and the plant biomass was 10 g/L.3.In order to determine the key degrading enzymes and metabolites of BPA by submerged macrophytes,C.demersum,which had the highest degradation ability of BPA,was selected for further study.Aseptic seedling of C.demersum without epiphytic microorganisms was prepared for comparation with wild seedlings with epiphytic microorganisms.The results showed that the BPA degradation ability of aseptic seedling of C.demersum was greatly higher than that of wild seedlings,which could reach 99%within 24h.Comparing the antioxidant enzymes activity of two types of C.demersum,the enzyme activities of peroxidase(POD)and Polyphenol oxidase(PPO)of aseptic seedling were accordingly significantly higher than that of wild seedlings.BPA degradation experiments by addition of antioxidant enzymes exctracted from C.demersum showed the necessity of H2O2 addition,suggesting POD may be the key enzyme.Purified POD which had greater enzyme activity had the greater rate of BPA degradation.In addition,the BPA metabolites of C.demersum and purified POD were the both 4-isopropenylphenol,determined by LC-MS/MS and GC-MS.Hence,it’s reasonable to deduce that POD is the unique key enzyme in BPA degradation by C.demersum.The toxicity of metabolites was also tested.The result showed that the metabolite of BPA degraded by POD of C.demersum showed no lethal toxicity to zebrafish and estrogen effect.4.In order to declare the molecular mechanism of BPA degradation by C.demersum,the gene expression level in the BPA degradation process of C.demersum was analyzed by RNA-Sequencing.The results showed that 996 differentially expressed unigenes were found in the process of BPA degradation by C.demersum,including that 687 of unigenes were significantly up-regulated and 309 of unigenes were significantly down-regulated.The results of GO function annotation indicated that the differently expressed genes were enriched into 93 GO terms,including 56 biological processes GO terms,1 cell components GO term and 36 molecular function GO terms.The enrichment results of the GO terms showed that after BPA treatment,C.demersum responded to the stress of BPA by regulating the expressions of the genes in GO terms such as oxidation-reduction process,defense response,response to oxidative stress,catalytic activity,oxidation-reduction enzyme activity and so on.The analysis of different expression genes of KEGG pathway classification revealed that differential genes were significantly enriched 15 pathways.The results showed that in order to respond to the oxidative stress caused by BPA,the related genes of Ca2+ and ABA signaling pathways were significantly up-regular expressed,and the related genes in the pathways such as activities of antioxidant enzymes,glutathione synthesis and metabolism,cell wall synthesis,energy metabolism,as well as amino acid metabolism were differently expressed.POD has been proved to be the key enzyme in BPA degradation by C.demersum,and its related genes were also significantly differently expressed,including 3 up-regulated unigenes and 3 down-regulated unigenes.5.Finally,the cDNA of peroxidase from C.demerum named CMPb was successfully cloned for the first time,which was involved in the degradation process of BPA.The full-length cDNA of CMPb was 1359 bp,and its open reading frame(ORF)was 972bp.The ORF encoded 323 amino acid residues with a predicted molecular weight of 35.2 kD,and an isoeletric point value of 8.87.Phylogenetic tree showed that CMPb had the closest evolutionary relationship with POD of Nelumb nucifera.NCBI conserved domain showed that the protein encoded by CMPb belonged to Class III of the plant heme-dependent peroxidase superfamily with a typical POD conserved regions and domains,including a heme-binding domain,an active binding region,a substrate binding region,and two Ca2+conserved binding regions.Prokaryotic expression results showed that efficient expression of CMPb protein could be realized after induced in E.coil BL21(DE3)for 4 h at 37℃.SDS-page analysis confirmed that the recombinant CMPb protein was about 49 kD,consistent with the predicted results and mainly existed as inclusion bodies.After dissolved by urea and purified through Ni column,the CMPb protein showed the peroxidase activity after renaturation,and it was found that CMPb protein had an efficient BPA degradation ability.The protein encoded by CMPb(100 U/mL)could degrade 79.52%of BPA at 30min(5mg/L).Based on all the results,submerged macrophytes especially C.demersum can efficiently degrade BPA from water,and plants rather than epiphytic microorganisms substantially contribute to the process.POD is the unique key enzyme in the degradation of BPA by C.demersum,and the main metabolite is 4-isopropenylphenol.The gene of peroxidase(CMPb),which has the ability of BPA degradation was obtained.The results of this study demonstrated that POD may be a key enzyme in the degradation process of toxic organic pollutants by macrophytes.