Modeling Studies Of Cytochromes And Their Applications In Mediating Bioenergy Storage And Microbial Fuel Cells

Cytochrome（C-type cytochrome）is an important component of microbial electron transferring,which not only exists inside microorganisms,but can also crosstalk with proteins and other medium on the extracellular membrane.Therefore,hemin protein has attracted widespread attention in the field of bioelectrochemistry.There have been many studies on the electron transfer of hemin proteins,and their working mechanism has been extensively investigated.Two electron transfer modes have been proposed:direct transfer and indirect transfer.Notably,the electron binding and dissociation process of hemin proteins has rarely been explored in practical applications.Demonstrating the electron binding characteristics of hemin proteins through energy storage is of great significance for expanding the application of functional materials of hemin proteins.In addition,electrodes modification enhances the electron transfer capability of hemin proteins between microorganisms and electrodes by increasing single functions,which greatly improves the performance of microbial fuel cells（MFCs）.However,electrode multi-functional modifications can provide a better working environment for electron transfer between electrodes and hemin proteins,such as increasing the biological load together with enhancing the electron transport capacity.Finally,based on the reversible electron transfer characteristics of hemin proteins,how to achieve the coexistence of electrically active microbial charge storage and self-discharge dual functions remains an urgent problem to be solved.To address the aforementioned issues,this study focuses on Shewanella oneidensis MR-1（S.oneidensis MR-1,S.O.）as an electrically active microorganism.Starting from modeling studies on cytochromes,artificial cytochrome complexes were constructed to mimic natural C-type cytochromes,and the charging/discharging capabilities of these cytochrome complexes were investigated.Furthermore,a dual-functional bioanode was constructed through electrode modification,aiming to increase the microbial load on the electrode while enhancing electron transport between the electrode and cytochromes.Additionally,a novel electrically active microbial gelatin platform was developed,capitalizing on the reversible electron transfer characteristics of cytochromes,to achieve a combination of energy storage and fuel cell functionalities.Specifically,the study includes the following aspects:1.Construction of artificial cytochrome complexes and investigation of energy storage capabilities.Inspired by the abundant involvement of pigment proteins in electron binding and detachment within electroactive microorganisms,we have constructed artificial pigment proteins to mimic the electrochemical properties of natural pigment proteins.We have conducted in vitro redox experiments on pigment protein complexes and developed green and flexible bioenergy storage devices based on these pigment proteins.Firstly,we used theoretical simulations to confirm the feasibility of the binding between natural proteins and hemin,an active molecule.After screening,bovine serum albumin（BSA）was preliminarily identified as a protein that can bind with the active molecule hemin and provide more uniform binding sites.Using BSA as a template,hemin molecules were introduced into the protein scaffold through hydrogen bonding and electrostatic interactions,creating artificial pigment protein complexes with hemin molecules resembling the active centers in natural pigment proteins.BSA modified with oxidized graphene（GO）was used as a control in parallel experiments.The experimental results show that iron（III）chloride hemin exhibits better dispersion in solution due to its supramolecular interaction with proteins.This feature directly affects the surface morphology and electrochemical behavior of protein-polymer materials.The artificially constructed pigment protein complexes exhibit excellent redox performance,with an energy storage capacity of up to 170.7 F·g^-1when used as pseudo-capacitors.2.Enhancement of electron transport between cytochromes and electrodes through non-metallic copolymers.To enhance the electron transfer efficiency between pigment proteins,electrodes,and electroactive microorganisms,we have constructed an anode material with dual functionality.This material not only enhances the adhesion of microorganisms but also improves the electron transfer efficiency of pigment proteins.Using a one-pot co-polymerization synthesis method,we created a positively charged conductive biofilm on the surface of a porous graphite felt（GF）.Co-polymerized poly-pyrrole supramolecular particles（PPy）were evenly distributed on the poly（1-vinyl-3-ethylimidazolium tetrafluoroborate）（P₁）membrane,compensating for the insulating properties of the cationic polymer coating itself.The PPy co-polymer material significantly increased the positive charge,affinity,and loading capacity of the bioanode and also improved the electron transfer efficiency of the microorganism’s surface pigment proteins.Using this bio-inorganic hybrid platform for constructing microbial fuel cells（MFCs）,we achieved a maximum power density of up to 7.02±0.35 W·m^-2,which is the highest reported to date using S.oneidensis MR-1 as a model microorganism.Additionally,this system can be extended to eukaryotic microorganisms,such as Saccharomyces cerevisiae（S.cerevisiae）,which reached a maximum power density of 13.82±2.91 W·m^-2,a 7.3-fold increase over the control group.This result suggests that this biohybrid platform is versatile in microbial fuel cells（MFCs）and can meet the electricity generation needs of different types of microorganisms.3.Preparation of dual-functional active microbial gelatin and its study on energy storage and self-powered generation.To construct a multifunctional device based on the electroactive microorganism S.oneidensis MR-1,linking fuel cells and charge storage requires a conductive scaffold material that interfaces with the microorganism’s pigment proteins without affecting the microorganism’s own metabolism.Gelatin,with its ability to melt at relatively low temperatures and be gelating at low temperatures,is employed for this purpose.Active microorganisms are introduced into the gelatin-based hydrogel scaffold.The amino acid chain residues in gelatin closely adhere to S.oneidensis MR-1,effectively protecting the fragile conductive protein chains on the surface of S.oneidensis MR-1,demonstrating excellent biocompatibility of the scaffold.During the construction of the porous scaffold,lithium ions（Li+）are introduced as electron carriers,enhancing the scaffold material’s electron binding and detachment capabilities,thereby improving the overall device’s electron transfer efficiency.Importantly,the porous scaffold constructed with gelatin almost does not interfere with the microorganism’s normal respiratory function and metabolism.With the aid of the rich amino acid chain network in the scaffold,electroactive microorganisms can store charges and self-generate electricity within the scaffold.The charge/discharge capacitance of this bio-inorganic hybrid hydrogel device can reach up to68 F·g^-1.Additionally,the self-discharge voltage output can reach a maximum of 0.67 V.Throughout the alternating charge/discharge and self-discharge operations,the level of active microorganisms is maintained close to normal levels.In summary,this dissertation has analyzed the electron transfer characteristics of pigment proteins.By leveraging the characteristics of redox reactions within electroactive microorganisms’pigment proteins and combining theoretical calculations,the feasibility of preparing artificial pigment-protein complexes for use in electrochemical energy storage devices has been validated.This approach reduces costs and simultaneously enhances preparation efficiency,broadening the research avenues for pigment proteins.Furthermore,based on the electron transfer mechanisms between conductive microorganisms and electrode materials,the design of dual-functional bioelectrodes has been optimized,resulting in a significant increase in power density.Finally,a scaffold system favorable for interactions between pigment proteins and gelatin has been designed.This system,while maintaining normal microbial metabolism,achieves efficient electron binding and detachment capabilities.Based on these achievements,the linkage between charge storage and microbial fuel cells has been established,resulting in the construction of a dual-functional device capable of energy storage and self-generation of electricity by active microorganisms.