Mediation Mechanisms Of Palladium Nanoparticles Synthesized In Situ By Bacillus Megaterium On Electron Transfer And Energy Metabolism

Due to the development of large scale breeding,the production and usage of antibiotics as well as the discharge of antibiotic wastewater significantly increased.The slow electron transfer rate of the biological treatment process resulted in the high residual of tetracyclines（TCs）and nitrate in the effluent of antibiotic wastewater,which has become ond of the main sources of TCs and nitrogen pollution in natural water.Thus,it was imminent to develop a feasible approach for improving the efficiency of denitrification and TCs biodegradation.Metal nanoparticles have been verified as a feasible strategy to improve the biological degradation efficiency by promoting electrons transfer and metabolic activity.Hence,in this work,Bacillus megaterium Y-4（B.megaterium）was employed to synthesize biogenic palladium nanoparticles（bio-Pd⁰）with high biocompatibility and catalytic activity,establishing a self-augmented biological system for denitrification and oxytetracycline（OTC）removal via the synergism of abiotic catalysis and biotic mediation of bio-Pd⁰.Meanwhile,the mediation mechanisms of bio-Pd⁰ on electrons transfer process and energy metabolism strategy were further investigated.The main results were as follows.Firstly,the dynamic process and formation mechanism of bio-Pd⁰ by B.megaterium were investigated.XRD,SEM and TEM results showed that B.megaterium could successfully synthesize bio-Pd⁰ in cytoplasm and periplasm as well as on the cell surface,which was a coupling process of biological reduction and autocatalytic reduction.Moreover,besides the contribution of extracellular enzymes and reductive functional groups on the cell surface to Pd（II）reduction,the bio-hydrogen generated via hydrogenase and the extracellular electron transfer mediated by NADH dehydrogenase also participated,and even dominated the biosynthesis of bio-Pd⁰.Particularly,electron donors could alter the biotic reduction pathway of Pd（II）via impacting NADH level and dehydrogenase activity,regulating the depositional site of bio-Pd⁰.With formate as the electron donor,bio-Pd⁰ was mainly synthesized in periplasm via bio-hydrogen,while more bio-Pd⁰ nanoparticles were synthesized extracellularly via extracellular electron respiration with lactate as the electron donor.In addition,it was proved that the direct electron transfer via c-Cyts and the indirect electron transfer via free flavins coexisted in gram-positive B.megaterium.Most importantly,the detection of DPV peak at-257 m V proved the involvement of one-electron EET via multiheme cytochrome-bound flavins in Gram-positive bacteria for the first time.Secondly,B.megaterium synthesizing bio-Pd⁰ in situ（bio-Pd@Cells）was employed to investigate the intracellular electron transfer（IET）mechanisms enhanced by metal nanoparticles in aerobic denitrification for the first time.Kinetic and thermodynamic results showed that the bio-Pd⁰ could significantly promote the removal of nitrate and nitrite by improving the affinity and decreasing activation energy.The results of enzymic activity measurement and the respiration chain inhibition experiment indicated that bio-Pd⁰ could facilitate the nitrate biotic reduction by improving the Fe-S center activity and serving as parallel H carriers to replace coenzyme Q to selectively increase the electron flux toward nitrate in IET,while promote the nitrite reduction by abiotic catalysis.Overall,this study expanded our understanding of the roles of bio-Pd⁰ on the aerobic denitrification process and provided an insight into the IET of Gram-positive strains.Thirdly,bio-Pd@Cells was used to establish a self-augmented biological system for oxytetracycline（OTC）removal.The results suggested that bio-Pd⁰could launch the extracellular OTC degradation by B.megaterium.Moreover,the catalytic hydrogenation via H₂rather than atomic H*was the main abiotic catalysis mechanism,while the activated biodegradation was attributed to extracellular enzymes,membrane-bound proteins and extracellular respiration.Particularly,bio-Pd⁰ NPs could establish a new electron transfer shortcut,promote direct electron transfer and one-electron reaction mediated via cytochrome-bound flavin,and accelerate electron hopping in EPS,thereby amplifying extracellular transfer flux and efficiency,which was the main reason for the activation of OTC biodegradation.In addition,the accelerated intracellular NADH regeneration and electron transfer could generate a larger proton motive force,boosting ATP generation,which further drove the energy-dependent efflux of OTC and glutathione to mitigate the toxicity of nanoparticles and OTC.Notably,hydrogenolysis and hydrodecyclization rather than oxidative cleavage were the main degradation pathways of OTC,avoiding the formation of more toxic hydroxylated and carboxylated intermediates.Overall,bio-Pd@Cells coupling of nanoparticles and biotechnology was an alternative to antibiotic degradation in wastewater.Finally,the extracellular p H was altered to investigate the regulation mechanism of TPG on OTC biodegradation by bio-Pd@Cells based on transmembrane electron transfer and energy metabolism.OTC degradation kinetics showed that the adsorption and absorption of OTC significantly decreased with the increase of p H due to the decreased TPG and OTC⁰ proportion（as reflected by the p H-dependent increased availability coefficient）,while OTC biodegradation rate showed a p H-dependent increase,indicating the involvement of unnormal intracellular biodegradation.Moreover,the cell fraction indicated that some biological processes relying on the intact respiration chain might participate and even dominate OTC biodegradation.Structural equation model results indicated that the activation of OTC biodegradation in bio-Pd@Cells was directly attributed to the dramatic increase of net outward proton flux due to the enhanced ETS activity as well as the improved STH activity mediating the substrate-level phosphorylation,but independent on ATPase-mediated energy metabolism.Combining with the results of respiratory chain inhibition experiment and electrochemical analysis,EET process relying on complex I and III dominated OTC biodegradation,and NADH-dependent EET efficiency via complex I and III with energy storage advantage was enhanced with the increase of p H,which was attributed to the increase of intracellular electron supply source and transmembrane electron output rate as well as the negative shift of onset potential and the decrease of electron transfer number due to the increase of bound flavin.These results perfected the extracellular respiratory mechanism of Gram-positive bacteria and provided new insights into the regulation and enhancement of extracellular respiratory process,which was expected to expand the application of electroactive microorganisms in biocatalysis and environmental remediation.