Molecular Mechanism And Regulation Methods For Extracellular Electron Transfer In Electrochemically Active Bacteria

Electrochemically active bacterias(EAB)are a class of microorganisms in nature that can undergo extracellular respiration,which is able to metabolize and transform environmental pollutants.Extracellular electron transfer(EET),a special talent owned by EAB,plays an indispensable role in this process.Taking advantage of such special ability,bioelectrochemical system(BES)could be constructed for pollutant degradation and energy transformation simultaneously.So far,studies on EET by molecular biology and theoretical calculation have been focusing on the composition and function of EET chain.The rate limiting step in BES and interaction and EET mechanism at the EAB/electron-acceptors interface remain unclear.However,the sluggish EET rate at the interface is the bottleneck,which restricts the practical applications of BES.This study aims at the key issues about the limited EET rate at the interface between outer membrane cytochrome c(OMCC)of EAB and extracellular electron acceptors.We explored the EET mechanism at the interface,proposed two new approaches to enhance EET process,i.e.,intracellular metabolism regulation and extracellular interface modification,and eventually improved the efficiency of pollutant degradation and energy conversion in BES.Main contents and results of this thesis are as follows:1.EET mechanism at the interface of OMCC OmcA and graphene oxide(GO).OmcA is one of the key OMCCs of a model EAB Shewanella,while GO is the precursor of graphene which could be used as anode material.An EET system was constructed by integrating the purified OmcA with GO.EET process at the interface was real-time monitored by the combination of electrochemical methods and multiple spectroscopies.Therefore,the in vitro interaction mechanism and direct electron transfer process were revealed.OmcA was confirmed to be at a high purity and active by SDS-PAGE and spectroscopy characterization.The OmcA was able to deliver electrons to GO with electrodes and chemical reductants as electron donors,which was evidenced by the protein film voltammetry and stopped-flow results.The EET reaction followed the Michaelis-Menten equation,showing its characteristics of enzyme-catalyzed reaction.The EET mechanism was revealed at the molecular level with the assistance of circular dichroism spectroscopy and two-dimensional IR correlation analysis.Hydrogen bond was formed between amino groups from OmcA and oxygen-containing groups from GO at the beginning,leading to a shorter distance and slight structural change of OmcA.Then,OmcA interacted with GO mainly via carbonyl groups,further altering the distance between the active site of OmcA and GO for electron transfer.These results are helpful for understanding the EET mechanism in pollutant-to-energy conversion,and also provide inspiration for designing highly efficient BES.2.EET mechanism at the interface of OmcA and nano-Fe2O3.EAB are also named as dissimilatory metal-reducing bacteria(DMRB)and capable of transferring electrons to iron and manganese oxides,in which the micro-environment of active site could be changed by the interaction between OMCCs and metal oxide,impacting the roles of EAB in bioremediation.In this case,hematite(?-Fe2O3),the most abundant mineral in soils,was chosen as the model mineral.IR spectral along with structural analysis reveal that ?-Fe2O3 exhibited excellent binding affinity with OmcA,accompanied with more exposed active site heme on the protein surface,leading to a higher reaction activity of OMCCs.Two-dimensional analysis indicates that peptide(Thr725-Pro726-Ser727)on protein surface could form a hydrogen bond with ?-Fe2O3,which could shorten the distance from Fe atom in heme to ?-Fe2O3.Further investigations with molecular dynamics simulations demonstrate the orientation of amino acid at the crystal face of ?-Fe2O3,structural information at the molecular level at the interface and effective electron transfer distance distribution from active center to ?-Fe2O3.Therefore,these results might be useful to understand the role of EAB in bioenvironmental remediation and bioenergy conversion with the synergistic effects of EAB-inorganic metal oxide.3.Regulation of EET in Shewanella by metabolic uncoupler 3,3',4',5-tetrachlorosalicylide(TCS).Intracellular metabolism of EAB for growth is essential in electricity generation.However,the impact of metabolic uncoupler in nature on the energy metabolism and EET process is poorly understood.When examining the impacts of TCS at different concentrations on the electricity production in microbial fuel cells and substrate utilization rate in anode chamber,we found that 50?g/l TCS could promote the substrate consumption by Shewanella and electricity generation.Thus,various schemes to enhance EET by low-concentration TCS were proposed,e.g.,accelerating electron and proton pumping by inhibition of proton motive force and replacing oxidative phosphorylation with substrate utilization.On the contrary,high-concentration TCS could reduce the activity of ATP synthase,resulting in a lower cell activity for electricity generation.These results suggest a possible regulation approach for BES.4.Regulation of EET by endogenous redox mediator riboflavin(RF)modified carbon electrode.Apart from tuning intracellular metabolism for EET regulation,interface modification is an effective way as well.With cyclic voltammetry RF was anchored on graphene surface,which not only improved the hydrophilia of electrodes,but also reduced the charge transfer resistance.In the operation of both microbial fuel cells and microbial electrolysis cells,more electricity was generated and the stability was improved after modification with RF.Electrochemical characterizations of biofilm and molecular simulation reveal that the electrochemical and physical properties of the electrode surface were altered by the immobilization of redox mediators.Redox mediators were found to function as a molecular bridge between OMCCs and inorganic electrodes.This not only accelerated the electron transfer,but also facilitated the biofilm attachment by improving the biocompatibility of electrode materials,ultimately promoted energy conversion efficiency.This result offers new possibilities for developing next-generation BES.