Design And Construction Of High Conductivity Biofilm Of Shewanella Oneidensis MR-1

The weak ability of biofilm formation and the low extracellular electron transport(EET)efficiency between electroactive microorganisms and electrode restrict the applications of microbial electrochemical technologies(METs).In this paper,the model electrogenic microorganism Shewanella oneidensis MR-1 was used as the research object.On the one hand,enhancing the extracellular electron transport capacity of S.oneidensis MR-1 by improving the biofilm formation and riboflavin synthesis with synthetic biology.On the other hand,enhancing the electron transfer efficiency between S.oneidensis MR-1 and electrodes and improving the conductivity of carbon cloth electrode by modifying the electrode surface with nano conductive materials,promoting the application of S.oneidensis in microbial fuel cell.The main research results are as follows:1.Enhancing the biofilm formation of S.oneidensis by gene knockout:We successfully constructed mutanted strains of Δ3171(2.1-folds increased in biofilm formation and 1.9-folds increased in output power density),ΔexeS(1.6-folds increased in biofilm formation)and Δ1942Δ3941(2.0-folds increased in output power density)that the biofilm formation ability and the extracellular electron transfer efficiency are improved by knocking out the genes of cell surface polysaccharide synthesis related genes(SO1860,SO3171),extracellular endonuclease genes(exeS,exeM)and c-di-GMP hydrolase genes(SO1942,SO3941,SO4711)in the wild-type S.oneidensis MR-1 by suicide plasmid and two-step homologous recombination.Based on that,we found the genes of SO3171,exeS,SO1942 and SO3941 have a greater influence on the biofilm formation and the extracellular electron transfer efficiency of S.oneidensis,and then we successfully constructed the multiple genes mutanted strain ΔPCC by knocking out these genes screened out.The biofilm formation capacity of the mutanted strain ΔPCC is 2.2-folds that of the wild type,and the maximum output power density reached 149.7± 3.5 mW/m~2,an increase of 2.2-folds.2.Enhancing the electron transfer of S.oneidensis by high-yielding riboflavin:To strengthen based on riboflavin mediated indirect electron transfer of the mutanted strainΔPCC,we enhanced the synthesis of riboflavin by introducing the exogenous riboflavin biosynthesis pathway.The riboflavin production of the ΔPCC/C5 is 113.4±4.3 mg/L,which is 6.5 times higher than the yield of WT.In addition,ΔPCC/C5 enabled a maximum electricity power density of 1335±65 mW/m~2 and maximum current density of 3621± 135 mA/m~2,which is 4.08-folds and 4.15-folds compared with the control strain,respectively.3.Enhancing the electron transfer of S.oneidensis with the modification of conductive materials:To improve the conductivity of the carbon cloth electrode and the electron transfer efficiency between the cell and the electrode,carbon nanotubes are used to modify the carbon cloth electrode.When using the modified carbon cloth as the anode and △PCC/C5 as a biocatalyst,the internal resistance of MFC reduced significantly,and the output power density increased significantly,reached 2662± 128 mW/m~2,and the maximum current density also reached 5211 ±236 mA/m~2,which is 8.1-folds and 6.0-folds compared with the control strain,respectively.The results showed that improving the extracellular electron transfer capacity of S.oneidensis by biological means or enhancing the electrical conductivity of electrodes could effectively improve the electron transfer efficiency between S.oneidensis and electrodes,which provided ideas and solutions for improving the extracellular electron transfer efficiency of other electroactive microorganisms.