Study On The Mechanisms Of Biochar-enhanced Biodegradation Of Typical Polybrominated Diphenyl Ethers By Pseudomonas Plecoglossicida

Polybrominated diphenyl ethers（PBDEs）are a class of persistent organic pollutant that widely exist in e-waste-polluted areas.PBDEs are typically featured with high toxicity,environmental persistence,bioaccumulation,and susceptible to long-range transport through multiple environmental media,posing a significant threat to ecological security and human health.Biochar-enhanced microbial remediation has been proposed as a promising strategy to eliminate environmental pollutants.However,there has yet to fully understand the biotransformation mechanisms of PBDEs.Additionally,the pathways and mechanisms of biochar in affecting biodegradation have not been systematically studied.In this study,2,2’,4,4’-tetrabrominated diphenyl ether（BDE-47）was used as the representative contaminant of PBDEs.A novel BDE-47-degrading strain was isolated from an e-waste dismantling site.The biodegradation and adaption mechanisms of the strain to BDE-47were revealed through an integrative approach combining toxicological assessment,degradation product determination,metabolomic analysis,and transcriptomic analysis.Moreover,the effects of biochar as a biostimulator on the degradation of BDE-47 by the strain were studied.This study systemically investigated the interaction between biochar,the strain and BDE-47,and clarified the mechanism of biochar in affecting BDE-47biodegradation by the strain.Besides,a biochar-based microbial agent was prepared by immobilizing the strain on biochar.We studied the effectiveness of biochar-assisted microbial remediation on persistent organic pollutants and heavy metals co-contaminated soils,as well as the corresponding mechanisms of soil microbial regulation and response.The main research results are as follows:（1）A novel BDE-47-degrading strain,P.plecoglossicida,was isolated from an e-waste dismantling site.The effects of environmental factors on the degradation of BDE-47 by P.plecoglossicida were studied.The results showed that BDE-47 could be degraded efficiently by P.plecoglossicida under anaerobic and aerobic conditions,and the aerobic degradation efficiency was higher.The indigenous microorganisms and co-existing organic pollutants（PCBs and PAHs）in actual polluted water body would not interfere the degradation of BDE-47 by P.plecoglossicida,while the co-existing heavy metals significantly decreased the degradation efficiency.The results of pollutant degradation pathway analysis showed that the degradation of BDE-47 by P.plecoglossicida was mainly through the biological action of intracellular enzymes.The degradation products included two hydroxylated bromo diphenyl ether(C₁₂H₉Br O₂and C₁₂H₉Br O₃),three bromophenol/phenol products（C₆H₄Br₂O,C₆H₅Br O,and C₆H₆O）,two hydroxylated polybrominated dibenzofurans(C₁₂H₇Br O₂and C₁₂H₇Br O₃),and two ring-opening products（C₄H₄O₅and C₈H₈O₂）.（2）Toxicological assessment,transcriptomic analysis and metabolomics analysis were conducted to reveal the biological mechanism of bacterial strain adaptation and degradation to pollutants.The results showed that BDE-47 exposure produced excessive intracellular reactive oxygen species（ROS）,causing oxidative stress to P.plecoglossicida.The adaptation mechanism of P.plecoglossicida to BDE-47 was mainly to activate the antioxidant defense system to relieve the oxidative stress induced by BDE-47.However,the defense ability of P.plecoglossicida against BDE-47 was limited,and the high concentration of BDE-47 would result in the disturbance of amino acid and purine metabolism,and ultimately apoptosis.Transcriptomic analysis revealed gene expression variations of P.plecoglossicida involved in transmembrane transport,synthesis of oxidoreductase,and antioxidant response.Differential expression of genes related to the regulation of cell membrane components might be related to changes in the hydrophobicity and permeability of the cell membrane of P.plecoglossicida,which is beneficial for enhancing the affinity of the strain to BDE-47.Transport proteins on cell membranes of P.plecoglossicida,such as ATP-binding cassette（ABC）transporters and major facilitator superfamily（MFS）transporters might be involved in transportation of BDE-47.Various intracellular oxidoreductases,such as 2-haloacrylate reductase,NADH dehydrogenase,Cytochrome oxidases,as well as various monooxygenases and dioxygenase might be involved in BDE-47 debromination,hydroxylation,ring-cleavage,and ether bond breaking catabolic process.Active efflux of BDE-47 and its degradation products by efflux pump,ABC,and MFS transporters might contribute to the bacterial tolerance against them.（3）The effect of biochar on the degradation of BDE-47 by P.plecoglossicida was further studied.The results showed that biochar significantly promoted the degradation of BDE-47 at higher concentrations（>0.1 mg/L）by P.plecoglossicida.However,biochar addition had no significant effect on BDE-47 biodegradation,and even slightly reduced its biodegradation efficiency,when the initial concentration of BDE-47 was below 0.1 mg/L.This was due to the fact that pore-filling was the predominant mechanism for BDE-47adsorption on biochar at low concentration levels.And BDE-47 adsorbed in micropore of biochar was hard to desorbed,which was adverse to its degradation.The adsorption-desorption of BDE-47 on biochar was influenced by the physicochemical properties of biochar.Biochar with less aromatic carbon contents and micropore volumes had a weaker binding force with BDE-47,which was conducive to the desorption of BDE-47 from biochar,and thus promoted BDE-47 biodegradation.The enhancement of BDE-47 biodegradation caused by biochar was partly because the initial adsorption of BDE-47 by biochar alleviated the cytotoxicity of BDE-47 to the P.plecoglossicida,and then gradual desorption regulated its bioavailability.Another reason was that biochar acted as shelter and stimulated EPS secretion,protecting the bacteria against extreme conditions.Metabolomic analysis indicated that biochar promoted the expression of metabolites related to glyoxylate and dicarboxylate metabolism,citrate cycle,and serial amino acid metabolism,suggesting that biochar could improve the BDE-47 biodegradation via enhancing oxidative metabolism and energy supply of the bacterial cells.（4）The effectiveness of biochar-assisted microbial remediation on persistent organic pollutants and heavy metals co-contaminated soils was investigated.The results showed that biochar as a carrier was conducive to P.plecoglossicida colonization,and biochar-mediated immobilization of Cu and Pb alleviated the environmental stress,thereby prolonging the survival time of P.plecoglossicida in soil and maintained degradation activity to BDE-47.Besides,biochar facilitated stable-adsorbed and bound-residue formation of BDE-47,reducing the ecological risk of residual BDE-47.Besides,biochar-based microbial agent comprehensively improved environment by enhancing the soil microbial carbon metabolism and enzyme activities related to carbon,nitrogen,and phosphorus cycles.Our research also emphasized the influence of biochar on soil bacterial community assembly processes and co-existence patterns.In the presence of biochar,positive interactions in microbial ecological networks were more frequent,and higher network stability and more keystone taxa were observed（including potential degraders and ecologically beneficial bacteria）.Importantly,biochar-based microbial agent improved the structure,multifunction,and stability of the soil micro-ecosystem,contributing to the sustainable and safe ecological remediation of complex-polluted soils.In this study,the screening of new degrading strain was conducive to the construction of the resource library of PBDEs-degrading strain.And the above result deepened the understanding of the mechanisms of microbial degradation and transformation of PBDEs.Additionally,the influential mechanism of biochar on biodegradation of PBDEs illustrated in this study would provide a theoretical basis and strong support for the development and application of the removal technology of PBDEs and heavy metals co-contamination.