Regulation Mechanisms Of Biosynthesized Palladium Nanoparticles On Electron Transfer And Diversion In Pollutant Removal By Citrobacter Freundii

Microorganisms,as important drivers of geochemical element cycling,can directly link self-metabolism with the transformation and removal of pollutants in the environment via electron transfer systems.However,the low electron transfer rate greatly limits their practical application in the field of environmental remediation.Therefore,ascertaining microbial electron transfer pathways,and improving the electron transfer rate by means of the technologies both with environmental and economic friendliness,are currently important research directions.According to the above information,this study utilized the microbial metal-reduction capacity to construct a metal@bio combined system possessing cooperativity and complementarity,which successfully activated the capacities of microorganism to completely reduce Pt（Ⅳ）and degrade sulfamethoxazole（SMX）.Meanwhile,combined with multi-omics sequencing,the molecular mechanisms of Pd⁰ NPs on enhancing microbial electron transfer and inducing electron diversion were deeply explored.The main results were as follows:First,Citrobacter freundii JH with efficient Pd（Ⅱ）reducing ability was isolated from electroplating sludge.In addition,according to the results of whole-genome sequencing and functional annotation,multiple genetic information of C.freundii JH including protein functions,metabolic pathways,gene-encoded products were obtained.Accordingly,114encoding genes of NADH-ubiquinone oxidoreductase（Complex I）,F₀F₁-ATPase,cytochrome c（c-Cyts）,[Ni Fe]-hydrogenase 2/3,riboflavin synthase and other key functional proteins/enzymes were identified.Simultaneously,potential electron transfer pathways of C.freundii JH were predicted,including intracellular electron transfer（IET）coupled with energy metabolism,c-Cyts-based transmembrane electron transfer and flavin-or H₂-mediated extracellular electron transfer（EET）.Based on the high-efficiency Pd（Ⅱ）reducing ability of C.freundii JH,the Pd⁰ NPs@C.freundii JH combined system was constructed,meanwhile,the involved electron transfer pathways in Pd（Ⅱ）reduction and the internal reasons for the differences in Pd（Ⅱ）reducing ability of cells at different growth phases were explored.In detail,the results of inhibition experiments manifested that both logarithmic-phase（cells-log）and stationary-phase cells（cells-sta）exhibited a biphasic dose response of stimulation at the low dose and inhibition at the high dose when exposed to Pd（Ⅱ）,moreover,the inhibition effect of high Pd（Ⅱ）concentration for cellular growth was mainly due to the production of·OH.According to this dose response,the combined system was constructed with Pd（Ⅱ）exposure concentration of 10mg L^-1,which was able to maintain cellular activity to the greatest extent.Noteworthily,generated Pd⁰ NPs were uniformly distributed in the cytoplasm and cell membrane.Additionally,the results of FTIR analysis,dynamic monitoring of p H and electrochemical detection showed that C.freundii JH undergone ion-exchange with Pd（Ⅱ）through surface functional groups（hydroxyl and amino groups）,and delivered electrons to Pd（Ⅱ）with the assistances of c-Cyts and riboflavin.Furthermore,the results of kinetic analysis and protein denaturation showed that the reducing ability of cells-log was significantly higher than that of cells-sta(0.0543 vs.0.0216 L mg^-1 h^-1),which was mainly caused by intracellular active substances（non-protein）,while the metal binding sites,extracellular polymeric substances and functional enzymes had little influence.Based on the above,the synthesis mechanism of Pd⁰NPs was deduced.The Pd⁰ NPs@C.freundii JH combined system was subsequently used to reduce Pt（Ⅳ）,and the enhanced mechanism of Pd⁰ NPs on microbial electron transfer was clarified during this process.The kinetic and thermodynamic results indicated that Pt（Ⅳ）reduction was divided into two stages by Pt（Ⅱ）that was the only soluble intermediate product,namely,Reaction 1 of Pt（Ⅳ）reduction to Pt（Ⅱ）,and Reaction 2 of Pt（Ⅱ）reduction to Pt（0）.Notably,Reaction 2 was the rate-limited step in the entire Pt（Ⅳ）reduction process and existed a significant lag stage in comparison with Reaction 1.Moreover,when the Pd⁰ NPs loading of C.freundii JH was low,the activation energy was found to be ineffectively decreased,resulting in the failure of Reaction 2 to launch,while excess Pd⁰ NPs loading would cause surface passivation and a decreased reaction rate.The results of targeted inhibition experiments,key enzyme activity tests and electrochemical measurements demonstrated that Pd⁰ NPs intensified the electron transfer and energy metabolism of C.freundii JH by stimulating the secretion of flavins,strengthening the combination between flavins and c-Cyts,increasing the activities of formate dehydrogenase and hydrogenase（Hase）,and replacing the F₀ component of F₀F₁-ATPase.Furthermore,the linear regression analysis between the current responses of functional proteins,enzyme activities and reaction rate constants showed that Reaction 1 was mainly depended on free flavin-mediated EET,while Reaction 2 was closely related to the intracellular typical respiration long chain（L-chain）as well as Hase-based hydrogen-production short chain（S-chain）,here,the contribution of S-chain was as high as 71.7%～73.4%.These results proved that Pd⁰ NPs broke through the reduction bottleneck of Pt（Ⅳ）by enhancing the electron transfer of C.freundii JH.Finally,the Pd⁰NPs@C.freundii JH combined system was used to degrade SMX,and the molecular mechanism of Pd⁰ NPs inducing electron diversion was clarified according to the linear regression analysis and the difference analysis of gene expression.The corresponding results showed that under the induction of in-situ biosynthesized Pd⁰ NPs,the gene expression levels of Complex I,c-Cyts,formate dehydrogenase-N（FDH-N）,formate dehydrogenase-O（FDH-O）,[Ni Fe]-hydrogenase 2,c-Cyts synthase,menaquinone and flavin synthesis pathways were significantly up-regulated,the activity of related functional enzymes were improved and the dominant quinone mediator in electron transfer was converted,which strengthened the electron output of the respiratory chains,resulting in the activation of the capacity of C.freundii JH to degrade SMX.Furthermore,in the Pd⁰NPs@C.freundii JH combined system,the increased loading of Pd⁰ NPs drove electrons in bypassing Complex I to quinone pool and significantly reducing the NADH/NAD⁺ratio,which weakened the role of L-chain on electron shunting.Meanwhile,in response to the increased transmembrane proton gradient caused by NADH oxidation and proton backflow,Pd⁰ NPs was found to selectively induce the up-regulation of hyb A encoding[Ni Fe]-hydrogenase 2,hyc I encoding[Ni Fe]-hydrogenase 3,hydrogenase mature operons hyp ABC and fdn GI encoding FDH-N,thereby triggering the electron diversion from L-chain to S-chain,regulating the transmembrane proton gradient and alleviating the adverse effects of cytoplasmic acidification on C.freundii JH.The aforementioned results confirmed the roles of Pd⁰ NPs in IET and EET,quantified the contribution ratio of each transfer pathway,and clarified its molecular mechanism of enhancing electron transfer and inducing electron diversion,which broadens our understanding of microbial electron transfer behavior and provides a theoretical reference for solving water pollution.