Study On Extracellular Electron Transport In Escherichia Coli Mediated By Endogenous Electron Shuttle

Electron transfer is an inherent feature of bacterial metabolism.Bacteria obtain energy for further growth and reproduction through electron transfer.Electron shuttles play an important role in the process of bacterial electron transfer.Therefore,mining the key endogenous electronic shuttles of bacteria can provide effective targets for inhibiting bacterial growth or killing bacteria.In this paper,Escherichia coli ATCC 25922 is used as the research object,and the electrochemical differential pulse voltammetry(DPV)method is used to explore the key extracellular electron shuttles of E.coli,combined with fluorescence spectroscopy,high performance liquid chromatography(HPLC),and chromatography-mass spectrometry(HPLC-MS-MS)analyzes it and conducts a preliminary exploration of its effect,intending to provide new ideas for exploring new inhibitory targets and mechanisms.The specific results are as follows:(1)A method for detecting the electrochemical activity of E.coli ATCC 25922 was established,and the method conditions were optimized..By cyclic voltammetry(CV)and differential pulse voltammetry(DPV),respectively,the E.coli bacteria and the culture supernatant after growth were analyzed,and it was found that E.coli can secrete electron shuttles for extracellular electron transfer.The apparent potential of the electronic shuttle is around-0.45 V.The conditions of the electrochemical detection method of E.coli extracellular electron shuttle were optimized,and the best test conditions were determined to be 60 mV amplitude and 0.1 s period.The i-t test results show that under a constant potential of 0.1 V,as the bacteria grow,the current response increases first and then decreases,indicating that the-0.45 V oxidation peak is closely related to the growth and metabolism of E.coli.(2)The existence of endogenous electronic shuttles on the surface of Escherichia coli was explored.The extracellular polymeric substances(EPS)wrapped around Escherichia coli was extracted,and the effect of different extraction conditions on cell viability was evaluated by colony plate count,TEM,and particle size determination.It was finally determined that heating at 45? for 5 h can extract more EPS.Thorough,and almost no damage to cells,it is the best extraction conditions.Finally,the differential pulse voltammetry test was performed on the E.coli after EPS removal.The results showed that the electron shuttles mainly existed on the surface of E.coli in a free state.(3)It is proved that the endogenous electron shuttle of E.coli is riboflavin.The oxidation peak potentials of riboflavin,FMN,and FAD measured by differential pulse voltammetry are all around-0.45 V,which is consistent with the electrical signals of E.coli culture medium and E.coli EPS.It is preliminarily inferred that the electron shuttle secreted by E.coli is Flavin substances.Afterwards,it was further proved that the main redox active substance in EPS is riboflavin through fluorescence spectroscopy,high performance liquid chromatography(HPLC),and high performance liquid chromatography mass spectrometry(HPLC-MS-MS).Finally,the flavin secretion during the growth and reproduction of Escherichia coli was studied by high performance liquid chromatography,and it was proved that riboflavin can promote the growth of Escherichia coli under anaerobic conditions and improve the electron transfer efficiency on the surface of Escherichia coli.