Extracellular Electron Transfer Mechanism Of Shewanella Putrefaciens On Nanostructured Anodes

Microbial fuel cell(MFC) is a promising renewable power system that integrates microbial degradation and electrochemical energy harvest, which can directly convert the chemical energy of organic compounds to electricity by microbes as catalysts. In comparison to noble metal catalysts used in conventional fuel cells, the microbiological catalysts in MFCs are able to easily self-replicate and regenerate. In addition, a wide variety of microbes have rich and flexible metabolic pathways. Due to these merits, the microbiological catalysts could be theoretically capable of decomposing all organics even some inorganics for energy generation, thus becoming a sustainable technology to simultaneously degrade organic waste or environmental pollutants and produce clean electric energy and holding great promise in applications of wastewater treatment, environmental remediation and portable energy and so on. However, the current low power density, slow start-up and high cost of devices impede their commercial applications. In particular, the sluggish anode kinetics is mainly caused by the slow extracellular electron transfer from anode electroactive microbes(exoelectrogens) to electrodes and is generally considered as one of the main limiting factors for the low MFC performance. Nanostructured electrode materials with good biocompatibility can promote the interfacial electron transfer efficiency of MFC anode, thus providing a feasible route to address the discontented MFC performances for the time being. The effect of porous structure and surface physico-chemical property of nanostructured anode materials on the bacterial electron transfer efficiency as well as the electrochemical behavior of exoelectrogen itself occurring on the nanostructured interface, which play crucial roles in outstranding anode materials for high-performance MFCs, nevertheless, is still not fuly clear so far. In addition, the role of microbe catalyst in the MFCs performance is also important but it is not fully understang the enhancement mechanism of direct electrochemistry at a molecular biology level.Herein, three carbon based anode nanomaterials with flexible nanostructure and surface properties are designed and fabricated to promote the bioelectrocatalytic performance of MFC inoculated with Shewanella putrefaciens CN32(S. putrefaciens CN32) as an exoelectrogen in this study. The involved enhancement effect of these nanostructured anodes on extracellular electron transfer of S. putrefaciens CN32 are systematically studied. In addition, the export pathway of bio-electron from S. putrefaciens CN32 cells is investigated with the help of molecular biotechnology. Thus the extracellular electron transfer mechanism of MFC bio-anode is comprehensively researched from the exoelectrogen to nanostructured anode materials. The main research contents and results are as follows:1. The bioelectricity generation efficiency of S. putrefaciens CN32 strain used in this study is markedly higher than that of S.oneidensis MR-1(a typical model strain) in dual-chamber MFCs with conventional carbon cloth(CC) as an anode. The former is also superior to the latter in terms of utilizing flavins as electron shuttles to improve anode bioelectrocatalysis. A nanocrystal Ti O2/r GO nanocomposite is synthesized through a sol-gel process for Ti O2/GO hybrid followed by hydrothermal reduction of GO and used as an anode for promoting flavins secretion and enhancing bioelectrocatalytic efficiency in S. putrefaciens CN32 MFC. Ti O2 nanocrystals are uniformly deposited on the surface of r GO nanosheets, which not only effectively prevents the heavy agglomerations of GO sheets during the hydrothermal reduction to result in larger surface area and mesoporous volume, but also obviously increases the surface hydrophilicity of Ti O2/r GO nanocomposite. The Ti O2 nanocrystals and r GO sheets produce an unique strong synergistic effect on enhancing bioelectrocatalysis, in which the good hydrophilicity of Ti O2 nanocrystals promotes bacterial growth on r GO sheets, meanwhile the secretion of flavins from S. putrefaciens CN32 bioflim is stimulated in-situ by uniformly deposited Ti O2 nanocrystals for a high concentration of electron shuttle, and then the synergistic effect from conductive r GO sheets and hydrophilic Ti O2 accelerates the electron transfer process mediated by flavins between S. putrefaciens CN32 cells and the Ti O2/r GO anode. This unique synergistic effect significantly boosts the redox reaction of flavins by reduced the anode interface charge transfer resistance. The Ti O2/r GO anode delivers a maximum output power density of 540 m W m-2 in dual-chamber MFC fed with LB medium as the feedstock, which is 1.4-fold and 3.4-fold higher than that of r GO and plain CC anode, respectively.2. To investigate the effect of anode biofilm on the bioelectrocatalysis of S. putrefaciens CN32 MFC, a hierarchically porous carbon hybrid composed of carbon nanotube(CNT) and graphene, two most frequently used carbon nanomaterials for MFC anodes, is fabricated through one-step hydrothermal process followed by freeze-drying. MWCNT@r GO1:2 nanocomposite with the best porous structure is obtained by optimized the input ratio of multi-walled CNT(MWCNT) to GO, in which the appropriate amount of inserted 1D MWCNTs not only effectively prevent the aggregation of r GO sheets but also act as bridges for increasing polydirectional connection among 2D r GO sheets, resulting in 3D carbonaceous network structure with high specific surface area and high electron transfer efficiency. Consequently, compared to individual MWCNT and r GO, MWCNT@r GO1:2 nanocomposite not only increases capacitive current as well as redox peak current but also decreases interface charge transfer resistance. Due to the excellent 3D hierarchically porous structure of MWCNT@r GO1:2 nanocomposite and the inherent high biocompatibility of carbon materials, abundant S. putrefaciens CN32 cells grow on its surface even into its macropores to form a compact and dense biofilm. The obtained MWCNT@r GO1:2/biofilm hybrid anode delivers a maximum power output density of 789 m W m-2 in S. putrefaciens CN32 MFC, which is much higher than that of individual MWCNT and r GO, in particular, 6-folder higher than that of conventional carbon cloth. In addition, the generated current of MWCNT@r GO1:2/biofilm hybrid anode is mainly dominated by adherent bacteria in biofilm rather than planktonic bacteria in anolyte, indicating that anode biofilm plays a crucial role on the high current density generated in S. putrefaciens CN32 MFC. The tight electroactive biofilm provides a large amount of S. putrefaciens CN32 cells to generate a high concentration of local electron shuttles around MWCNT@r GO1:2 hybrid anode along with a short diffusion distance of electron shuttles from bacterial cells to electrodes, which greatly promote the direct electrochemistry of flavins, thus boosting the anode bioelectrocatalytic performance.3. Exploring microbe-available macroporous anode materials to improve the loading of microbial cells has attracted great inteerst from researchers for high-efficient biocatalysis. It is known that an MFC anode involves both bio- and electro-catalytic process, nevertheless, the effect of nanoporous structure on electrocatalysis, especially the direct electrochemistry of electron shuttles, has not been disclosed yet. A 3D hierarchically porous carbon nanofiber(CNF) aerogel is prepared from bacterial cellulose(a kind of biomass materials) by pyrolysis at high-temperature under inert atmosphere to address this issue. Due to its macroporous network structure of micron level and natural high biocompatibility, a large number of S. putrefaciens CN32 cells grow on the surface as well as into the inner macropores of CNF aerogels leading to a thick integrated biofilm, resulting in a very high loading of biocatalysts. The nanoporous structure(< 10 nm) is subtly tailored by varying pyrolytic temperature, it is found that the micropores gradually convert to mesopores with the increase of pyrolytic temperature, which gives rise to an increasing direct electrochemistry of flavins, indicating that the mesoporous structure is necessary for the high-efficient direct electrochemistry of electron shuttles. The unique enhancement mechanism of mesoporous structure on the direct electrochemistry of electron shuttles is proposed for the first time: mesoporous structure can greatly enlarge the avaliable surface area and provide large 3D pores for flavins to move in and out, moreover its curvature or kink surface could offer more opportunities to overcome the steric effect for directly contacting the two electroactive nitrogen atoms for effective surface absorption and to promote the two-electron transfer direct electrochemistry. The CNF1000 anode with rich mesopores delivers a maximum power output density of 1747 m W m-2 in S. putrefaciens CN32 MFC with a high plateau current density of 3.9 A m-2 under an external resistor of 1000 ?, 4-fold and 14-fold higher than that of CNT anode and bare CC anode, respectively. In addition, this CNF1000 anode shows good stability, repeatability and durability, thus holding great promise for practical industrial applications.4. In-frame deletions of mtr C, und A and mtr C/und A genes in S. putrefaciens CN32 is generated to study the roles of outer membrane c-type cytochrome Mtr C and Und A in its extracellular electron transfer process. There is no difference in terms of replication, growth activity and the secretory ability of flavins among wild-type and mutant strains with lactate as the sole carbon source and electron donor under aerobic conditions, indicating that deleting mtr C and und A genes have no observable influence on aerobic metabolism of S. putrefaciens CN32. By comparing the cell output voltages among wild-type and mutant strains discharged in dual-chamber MFCs, it is found that deletions of mtr C and/or und A genes reduce the bioelectricity generation of S. putrefaciens CN32. Moreover the negative effect of deleting mtr C gene is much larger than that of deleting und A gene, indicating the more important role of outer membrane c-cytochrome Mtr C in the extracellular electron transfer of S. putrefaciens CN32. Outer membrane c-type cytochrome Mtr C is demonstrated as a key terminal reductase of electron shuttles through real-time monitoring the concentration of endogenous flavins secreted from S. putrefaciens CN32 in MFC anolyte and comparing the effect of artificially added flavins on promoting bioelectricity generation. More importantly, the high-efficient bioelectrocatalysis of S. putrefaciens CN32 in MFC is executed by the fast direct electrochemistry of flavins between its outer membrane c-type cytochromes especially Mtr C and electrodes rather than the direct electron transfer through physical contact between them, this is further proved by comparing the decolorizing efficiency of methyl orange between the wild-type and ?mtr C mutant strain.In summary, the electron transfer mediated by endogenous flavins is the main pathway of bacterial extracellular electron transfer of S. putrefaciens CN32 on nanostructured anode interface in MFC, and the best anode nanomaterials should not only promote biofilm growth but also accelerate the direct electrochemistry of electron shuttles, thus achieving both high- efficient bio- and electro-catalytic process.