Extracellular Electron Transfer Mechanisms Research Articles

Electro-stimulated anaerobic oxidation of methane with synergistic denitrification by adding AQS: Electron transfer mode and mechanism

Denitrifying anaerobic methane-oxidizing (DAMO) processes, which link anaerobic methane oxidation (AMO) and denitrification, have a promising prospect in anaerobic wastewater treatment. In bioelectrochemical systems (BES), DAMO consortium presented potent metabolic activity. However, the extracellular electron transfer (EET) in BES was poorly understood. This study investigated the EET mechanisms and modes of electron transport in BES dominated by anaerobic methanotrophic bacteria. In the bioreactors with the auxiliary voltage of 0.5 and 1.1 V, named EMN-0.5 and EMN-1.1, respectively, biological voltages of 0.198 and 0.329 V were generated with power densities of 0.6 and 1.20 mW/m2, after removing the voltage. High throughput and metagenome analyses demonstrated that main methanotrophs were DAMO bacteria and Methylocystis sp. The electroactive bacteria detected were Pseudomonas sp., Hypomicrobium sp., Thiobacillus sp, and Rhodococcus sp. The pil, cytochrome c, hdr, and he/fp genes related to EET were present on the electrode surfaces. Carbon 13 isotope tracing and chemicals analysis by GC-MS exhibited that methanol was an intermediate product released to extracellular environment and acted as the electronic carrier to drive the EET in methane oxidation. Extracellular electron transfer was achieved through the collaboration of DAMO bacteria, Methylocystis sp., and Pseudomonas sp. Anthraquinone 2-sulfonic acid ester (AQS) could improve the rate of electron transfer to the extracellular space, especially in the EMN-0.5 reaction system. This study provides a new understanding of AMO consortium metabolism in BES and may provide a scientific basis for developing methane control technology.

Direct extracellular electron transfer to an indium tin doped oxide electrode via heme redox reactions in Desulfovibrio ferrophilus IS5

Soluble redox mediators have been hypothesized to play a major role in cellular electron transportation to extracellular insoluble electron acceptors in sulfate-reducing bacteria (SRB) through a diffusion-based electron shuttle mechanism. Herein, in vivo electrochemistry shows that outer-membrane cytochromes (OMCs) directly mediated extracellular electron transfer (EET) in Desulfovibrio ferrophilus IS5. Anodic current production by IS5, coupled with lactate oxidation, was detected on indium-tin-doped oxide electrodes poised at +400 mV (vs. standard hydrogen electrode), which was the sole electron acceptor. Subsequent electrolyte replacement experiments with a fresh one showed that cells attached to the electrode contributed to 73% of current production by direct EET, while the electron shuttle mechanism may have contributed to the rest. Differential pulse voltammetry detected two redox species at E = −180 and 20 mV, with the former being most likely assignable to cell-surface cytochromes because nitric oxide decreased the peak current at −180 mV, as previously demonstrated with OMCs in iron-reducing bacteria. The EET onset potential at −200 mV further supports the notion that OMCs mediated EET in IS5, and that EET is thermodynamically favorable for reducing Fe(III) oxide. These results suggest that the direct EET mechanism regulates the reduction rate of insoluble substrates in SRB.

Moving towards the enhancement of extracellular electron transfer in electrogens

Electrogens are very common in nature and becoming a contemporary theme for research as they can be exploited for extracellular electron transfer. Extracellular electron transfer is the key mechanism behind bioelectricity generation and bioremediation of pollutants via microbes. Extracellular electron transfer mechanisms for electrogens other than Shewanella and Geobacter are less explored. An efficient extracellular electron transfer system is crucial for the sustainable future of bioelectrochemical systems. At present, the poor extracellular electron transfer efficiency remains a decisive factor in limiting the development of efficient bioelectrochemical systems. In this review article, the EET mechanisms in different electrogens (bacteria and yeast) have been focused. Apart from the well-known electron transfer mechanisms of Shewanella oneidensis and Geobacter metallireducens, a brief introduction of the EET pathway in Rhodopseudomonas palustris TIE-1, Sideroxydans lithotrophicus ES-1, Thermincola potens JR, Lysinibacillus varians GY32, Carboxydothermus ferrireducens, Enterococcus faecalis and Saccharomyces cerevisiae have been included. In addition to this, the article discusses the several approaches to anode modification and genetic engineering that may be used in order to increase the rate of extracellular electron transfer. In the side lines, this review includes the engagement of the electrogens for different applications followed by the future perspective of efficient extracellular electron transfer.

Extracellular electron transfer corrosion mechanism of two marine structural steels caused by nitrate reducing Halomonas titanicae

The extracellular electron transfer (EET) mechanism for corrosion of EH36 steel (with copper) and Q235 steel (without copper) caused by Halomonas titanicae was investigated. Due to copper cytotoxicity, the sessile H. titanicae cells on the EH36 surface was only 18% of that on Q235. This led to its lower EET rate, resulting in EH36 corrosion being 0.67 times of Q235 corrosion. The EET corrosion mechanism was further proven by the increased corrosion of the two steels after injection to the H. titanicae broth of 20 ppm (w/w) (in the broth) riboflavin, an electron mediator known to accelerate EET.

Enhanced purification of kitchen-oil wastewater driven synergistically by surface microelectric fields and microorganisms

The stable structure and toxic effect of refractory organic pollutants in wastewater lead to the problem of high energy consumption in water treatment technology. Herein, we propose a synergistic purification of refractory wastewater driven by microorganisms and surface microelectric fields (SMEF) over a dual-reaction-center (DRC) catalyst HCLL-S8-M prepared by an in situ growth method of carbon nitride on the Cu-Al2O3 surface. Characterization techniques demonstrate the successful construction of SMEF with strong electrostatic force over HCLL-S8-M based on cation-π interactions between metal copper ions and carbon nitride rings. With the catalyst as the core filler, an innovative fixed bed bioreactor is constructed to purify the actual kitchen-oil wastewater. The removal efficiency of the wastewater even with a very low biodegradability (BOD5/COD = 0.33) can reach 60% after passing through this bioreactor. An innovative reaction mechanism is revealed for the first time that under the condition of a small amount of biodegradable organic matter, the SMEF induces the enrichment of electric active microorganisms (Desulfobulbus and Geobacter) in the wastewater, accelerates the interspecies electron transfer of intertrophic metabolism with the biodegradable bacteria through the extracellular electron transfer mechanism such as cytochrome C and self-secreted electron shuttle. The electrons of the refractory organic pollutants adsorbed on the surface of the catalyst are delocalized by the SMEF, which can be directly utilized by microorganisms through EPS conduction. The SMEF generated by electron polarization can maximize the utilization of pollutants and microorganisms in wastewater and further enhance degradation without adding any external energy, which is of great significance to the development of water self-purification technology.

Accelerating anaerobic oxidation of methane coupled with extracellular electron transfer to electrodes via magnetite stimulating membrane-bound proteins of anaerobic methanotrophic (ANME) archaea/methanogens

Methane-dependent bioelectrochemcial systems (BESs) have been recently considered as a promising approach for mitigating methane emission, energy harvesting as well as water environment remediation. However, the mechanisms of extracellular electron transfer (EET) from anaerobic methanotrophic (ANME) archaea/methanogens to electrodes are still poorly understood, which limits the further improvement of methane oxidation and current generation. The study presented here showed that, in the presence of magnetite, anodic biofilms primarily comprised of ANME archaea/methanogens were highly conductive, compared to that without magnetite. Further examination with scanning electrochemical microscopy and electrochemical Fourier transform infrared spectra found that, anodic biofilms with magnetite were redox-based conductive, with a higher concentration of redox-active proteins displayed on their surface than that without magnetite. Metagenomic analysis showed that, the difference in the abundance of genes encoding the cytochrome c (CYC)-like proteins in the anodic biofilms with or without magnetite was not significant. However, magnetite specially increased the abundance of genes encoding the membrane-bound proteins, Rnf, and periplasmic heterodisulfide reductase, HdrABC, contained in ANME archaea/methanogens, providing a possibility that magnetite sped up methane oxidation as well as electron transfer from inner membrane to outer membrane in AOM coupled with EET to electrodes. As a result, the current density in methane-dependent BES with magnetite was increased by approximately 6–9 folds.

Mechanism and applications of bidirectional extracellular electron transfer of Shewanella.

Electrochemically active microorganisms (EAMs) play an important role in the fields of environment and energy. Shewanella is the most common EAM. Research into Shewanella contributes to a deeper comprehension of EAMs and expands practical applications. In this review, the outward and inward extracellular electron transfer (EET) mechanisms of Shewanella are summarized and the roles of riboflavin in outward and inward EET are compared. Then, four methods for the enhancement of EET performance are discussed, focusing on riboflavin, intracellular reducing force, biofilm formation and substrate spectrum, respectively. Finally, the applications of Shewanella in the environment are classified, and the restrictions are discussed. Potential solutions and promising prospects for Shewanella are also provided.

In Vivo Voltammetric Imaging of Metal Nanoparticle-Catalyzed Single-Cell Electron Transfer by Fermi Level-Responsive Graphene.

Metal nanomaterials can facilitate microbial extracellular electron transfer (EET) in the electrochemically active biofilm. However, the role of nanomaterials/bacteria interaction in this process is still unclear. Here, we reported the single-cell voltammetric imaging of Shewanella oneidensis MR-1 at the single-cell level to elucidate the metal-enhanced EET mechanism in vivo by the Fermi level-responsive graphene electrode. Quantified oxidation currents of ~20 fA were observed from single native cells and gold nanoparticle (AuNP)-coated cells in linear sweep voltammetry analysis. On the contrary, the oxidation potential was reduced by up to 100 mV after AuNP modification. It revealed the mechanism of AuNP-catalyzed direct EET decreasing the oxidation barrier between the outer membrane cytochromes and the electrode. Our method offered a promising strategy to understand the nanomaterials/bacteria interaction and guide the rational construction of EET-related microbial fuel cells.

Unveiling the Mechanism of Enhanced Extracellular Electron Transfer by Polarity Inversion of Bioelectrodes

Polarity inversion from the bioanode to the biocathode enables a rapid formation of efficient biofilms for microbial electrochemical reduction. Though this approach has been widely adopted for various applications, the mechanism behind the enhanced extracellular electron transfer (EET) during the polarity inversion process remains unclear. Therefore, in this study, the succession pattern and electron transfer mechanism of the inverted denitrifying biocathode were investigated by continuously monitoring the electrochemical properties, denitrification dynamics, and biofilm characteristics. The results indicated that the highest current density of the biocathode after inversion from bioanode was twice that of the steady current density, and the dominant flora of the electrode biofilm shifted from Geobacter sp. to denitrifying bacteria (e.g., Shinella and Comamonas). The reversal polarity induced a decreasing content of cytochrome C, while iron-containing compounds were found to be responsible for an enhanced EET within the denitrification process. This study provides a deep understanding of the EET mechanism behind the electrode polarity inversion and is of great significance for the future development of biocathode applications.

Screening of natural phenazine producers for electroactivity in bioelectrochemical systems.

Mediated extracellular electron transfer (EET) might be a great vehicle to connect microbial bioprocesses with electrochemical control in stirred-tank bioreactors. However, mediated electron transfer to date is not only much less efficient but also much less studied than microbial direct electron transfer to an anode. For example, despite the widespread capacity of pseudomonads to produce phenazine natural products, only Pseudomonas aeruginosa has been studied for its use of phenazines in bioelectrochemical applications. To provide a deeper understanding of the ecological potential for the bioelectrochemical exploitation of phenazines, we here investigated the potential electroactivity of over 100 putative diverse native phenazine producers and the performance within bioelectrochemical systems. Five species from the genera Pseudomonas, Streptomyces, Nocardiopsis, Brevibacterium and Burkholderia were identified as new electroactive bacteria. Electron discharge to the anode and electric current production correlated with the phenazine synthesis of Pseudomonas chlororaphis subsp. aurantiaca. Phenazine-1-carboxylic acid was the dominant molecule with a concentration of 86.1μg/ml mediating an anodic current of 15.1 μA/cm2 . On the other hand, Nocardiopsis chromatogenes used a wider range of phenazines at low concentrations and likely yet-unknown redox compounds to mediate EET, achieving an anodic current of 9.5μA/cm2 . Elucidating the energetic and metabolic usage of phenazines in these and other species might contribute to improving electron discharge and respiration. In the long run, this may enhance oxygen-limited bioproduction of value-added compounds based on mediated EET mechanisms.

Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34.

Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gfp and ΔpilAE::λPrgfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34.

Superior dimethyl disulfide degradation in a microbial fuel cell: Extracellular electron transfer and hybrid metabolism pathways

To enhance the biological degradation of volatile organic sulfur compounds, a microbial fuel cell (MFC) system with superior activity is developed for dimethyl disulfide (DMDS) degradation. The MFC achieves a removal efficiency near 100% within 6 h (initial concentration: 90 mg L−1) and a maximum biodegradation rate constant of 0.743 mM h−1. The DMDS removal load attains 2.684 mmol h−1 L−1, which is 6.18–2440 times the loads of conventional biodegradation processes reported. Meanwhile, the maximum power density output and corresponding current density output are 5.40 W m−3 and 40.6 A m−3, respectively. The main mechanism of extracellular electron transfer is classified as mediated electron transfer, supplemented by direct transfer. Furthermore, the mass balance analysis indicates that methanethiol, S0, S2−, SO42−, HCHO, and CO2 are the main intermediate and end products involved in the hybrid metabolism pathway of DMDS. Overall, these findings may offer basic information for bioelectrochemical degradation of DMDS and facilitate the application of MFC in waste gas treatment. Environmental implicationDimethyl disulfide (DMDS), which features poor solubility, odorous smell, and refractory property, is a typical pollutant emitted from the petrochemical industry. For the first time, we develop an MFC system for DMDS degradation. The superior DMDS removal load per unit reactor volume is 6.18–2440 times those of conventional biodegradation processes in literature. Both the electron transfer route and the hybrid metabolism pathway of DMDS are cleared in this work. Overall, these findings give an in-depth understanding of the bioelectrochemical DMDS degradation mechanism and provide an efficient alternative for DMDS removal.

Interspecies electron transfer between Geobacter and denitrifying bacteria for nitrogen removal in bioelectrochemical system

Groundwater nitrate pollution is a severe environmental problem causing global concern. Among others, autotrophic denitrification on polarized electrodes is one of the energy-efficient and sustainable solutions. However, the poor nitrogen-removal rate severely hinders the application of this process. Herein, biofilms consisted of the symbiotic growth of Geobacter and denitrifying bacteria with interspecies electron transfer ability were formed on carbon paper electrodes through periodic polarity inversion. The biofilm with an inversion period of 12 h obtained the highest cathode current density (2.19 A/m2 on average), and the total nitrogen removal rate reached 4.98 g/m2Ca/d, which was an order of magnitude higher than that of the control cathode (current density 0.13 A/m2, total nitrogen removal rate 0.52 g/m2Ca/d). The excellent performance of the biofilms was attributed to the symbiotic interactions between Geobacter and denitrifying bacteria, in which Geobacter transferred electrons from the electrodes to the denitrifying bacteria for denitrification via cytochrome c-like species. Such an electron transfer process was further verified by suppressing current generation and denitrification through dosing either electron transfer inhibitors or denitrifying inhibitors. This work reveals a new extracellular electron transfer mechanism underlying the autotrophic denitrification on the cathode electrode and provides an insight into the development of efficient biocathodes for treating nitrate pollution in groundwater.

Nanomaterials Facilitating Conversion Efficiency Strategies for Microbial CO2 Reduction.

Microbial electro- and photoelectrochemical CO2 reduction represents an opportunity to tackle the environmental demand for sustainable fuel production. Nanomaterials critically impact the electricity- and solar-driven microbial CO2 reduction processes. This minireview comprehensively summarizes the recent developments in the configuration and design of nanomaterials for enhancement of the bacterial adhesion and extracellular electron transfer (EET) processes, based on the modification technologies of improving chemical stability, electrochemical conductivity, biocompatibility, and surface area. Furthermore, the investigation of incorporating non-photosynthetic microorganisms using advanced light-harvesting nanostructured photoelectrodes for solar-to-chemical conversion, as well as the current understanding of EET mechanisms occurring at photosynthetic semiconductor nanomaterials-bacteria biohybrid interface is detailed. The crucial factors influencing the performance of microbial CO2 reduction systems and future perspectives are discussed to provide guidance for the realization of their large-scale application.

Two polarity reversal modes lead to different nitrate reduction pathways in bioelectrochemical systems

Polarity reversal is one of the effective strategies to rapidly start up denitrifying BESs，but the long-term performances of the denitrifying BESs operated under polarity reversal receive little attention. This study investigated the effects of periodic polarity reversal (PPR) and polarity reversal once only (PRO) on the long-term performances of denitrifying BESs. Repeatable oxidative and reductive currents were observed in the BESs obtained by PPR (PPR-BESs). The peak reductive currents of the PPR-BESs reached 0.95 A/m2, and nitrate was mainly removed by dissimilatory nitrate reduction to ammonium pathway with removal rates higher than 95 %. In contrast, the peak reductive currents of the BESs obtained by PRO (PRO-BESs) progressively decreased from 1.01 A/m2 to 0.12 A/m2. The nitrate removal rates of the PRO-BESs were <50 %, and the product of nitrate reduction turned to N2 instead of ammonium. 16S rDNA sequencing and metatranscriptomic analysis revealed that Geobacter capable of bidirectional extracellular electron transfer (EET) and Afipia capable of autotrophic growth were the dominant genera in the two types of BESs. Outer membrane cytochrome c and formate dehydrogenase were potentially involved in the cathodic electron uptake. These findings contribute to a better understanding of the EET mechanisms of electroautotrophic denitrifiers.

Enhancing Microbial Electron Transfer Through Synthetic Biology and Biohybrid Approaches: Part I

Traditional microbial synthesis of chemicals and fuels often rely on energy-rich feedstocks such as glucose, raising ethical concerns as they are directly competing with the food supply. Therefore, it is imperative to develop novel processes that rely on cheap, sustainable and abundant resources whilst providing carbon circularity. Microbial electrochemical technologies (MET) offer unique opportunities to facilitate the conversion of chemicals to electrical energy or vice versa, by harnessing the metabolic processes of bacteria to valorise a range of waste products, including greenhouse gases (GHGs). However, the strict growth and nutrient requirements of industrially relevant bacteria, combined with low efficiencies of native extracellular electron transfer (EET) mechanisms, reduce the potential for industrial scalability. In this two-part work, we review the most significant advancements in techniques aimed at improving and modulating the efficiency of microbial EET, giving an objective and balanced view of current controversies surrounding the physiology of microbial electron transfer, alongside the methods used to wire microbial redox centres with the electrodes of bioelectrochemical systems via conductive nanomaterials.

Electrochemical Characteristics of Shewanella loihica PV-4 on Reticulated Vitreous Carbon (RVC) with Different Potentials Applied.

The current output of an anodic bioelectrochemical system (BES) depends upon the extracellular electron transfer (EET) rate from electricigens to the electrodes. Thus, investigation of EET mechanisms between electricigens and solid electrodes is essential. Here, reticulated vitreous carbon (RVC) electrodes are used to increase the surface available for biofilm formation of the known electricigen Shewanella loihica PV-4, which is limited in conventional flat electrodes. S. loihica PV-4 utilizes flavin-mediated EET at potential lower than the outer membrane cytochromes (OMC), while at higher potential, both direct electron transfer (DET) and mediated electron transfer (MET) contribute to the current output. Results show that high electrode potential favors cell attachment on RVC, which enhances the current output. DET is the prevailing mechanism in early biofilm, while the contribution of MET to current output increased as the biofilm matured. Electrochemical analysis under starvation shows that the mediators could be confined in the biofilm. The morphology of biofilm shows bacteria distributed on the top layer of honeycomb structures, preferentially on the flat areas. This study provides insights into the EET pathways of S. loihica PV-4 on porous RVC electrodes at different biofilm ages and different set potential, which is important for the design of real-world BES.

Efficient bioelectricity generation and carbazole biodegradation using an electrochemically active bacterium Sphingobium yanoikuyae XLDN2-5

Carbazole and its derivatives are polycyclic aromatic heterocycles with unusual toxicity and mutagenicity. However, disposal of these polycyclic aromatic heterocycles remains a significant challenge. This study focused on efficient resource recovery from carbazole using an obligate aerobe, Sphingobium yanoikuyae XLDN2-5, in microbial fuel cells (MFCs). S. yanoikuyae XLDN2-5 successfully achieved carbazole degradation and simultaneously electricity generation in MFCs with a maximum power density of 496.8 mW m−2 and carbazole degradation rate of 100%. It is the first time that S. yanoikuyae XLDN2-5 was discovered as an electrochemically active bacterium with high extracellular electron transfer (EET) capability. Redox mediator analysis indicated that no self-produced redox mediators were found for S. yanoikuyae XLDN2-5 under analysis conditions, and the exogenous redox mediators used in this study did not promote its EET. The nanowires produced by S. yanoikuyae XLDN2-5 cells were found in the biofilm by morphology characterization and the growth process of the nanowires was consistent with the discharge process of the MFC. Conductivity determination further verified that the nanowires produced by S. yanoikuyae XLDN2-5 cells were electrically conductive. Based on these results, it is speculated that S. yanoikuyae XLDN2-5 may mainly utilize conductive nanowires produced by itself rather than redox mediators to meet the requirements of normal energy metabolism when it grows in the low dissolved oxygen zone of the anodic biofilm. These novel findings on the EET mechanism of S. yanoikuyae XLDN2-5 lay a foundation for further exploration of polycyclic aromatic heterocyclic pollutants treatment in electrochemical devices, which may create new biotechnology processes for these pollutants control.

Recent advances in electro-fermentation technology: A novel approach towards balanced fermentation

Biotransformation of organic substrates via acidogenic fermentation (AF) to high-value products such as C1-C6 carboxylic acids and alcohol serves as platform chemicals for various industrial applications. However, the AF technology suffers from low product titers due to thermodynamic constraints. Recent studies suggest that augmenting AF redox potential can regulate the metabolic pathway and provide seamless electron flow by lowering the activation energy barrier, thus positively influencing the substrate utilization rate, product yield, and speciation. Hence, the augmented AF system with an exogenous electricity supply is termed as electro-fermentation (EF), which has enormous potential to strengthen the fermentation technology domain. Therefore, this critical review systematically discusses the current understanding of EF with a special focus on the extracellular electron transfer mechanism of electroactive bacteria and provides perspectives and research gaps to further improve the technology for green chemical synthesis, sustainable waste management, and circular bio-economy.

Transcriptome Analysis to Identify Crucial Genes for Reinforcing Flavins-Mediated Extracellular Electron Transfer in Shewanella oneidensis.

Flavins serve as the electron mediators in Shewanella oneidensis, determining the extracellular electron transfer (EET) rate. Currently, metabolic engineering of flavins biosynthetic pathway has been studied for improving EET. However, the cellular response triggered by flavins that contribute to EET remains to be elucidated. In this study, the riboflavin-overproducing strain C5 (expressing the flavins synthetic genes in plasmid PYYDT) and the PYYDT strain (harboring the empty plasmid PYYDT) in the microbial fuel cells are applied for comparative transcriptomic analyses to investigate beneficial gene targets that could improve EET. From the differentially expressed genes, we select the significantly upregulated and downregulated genes for inverse engineering in S. oneidensis. The results show that overexpression of ahpC and ccpA, and inactivation of pubA, putB, and tonB are able to improve the EET capability. Combinatorial modulation of these five genes results in the recombinant strain CM4, achieving the maximum power density of 651.78 ± 124.60 mW/m2, 1.97 folds of the parental strain. These genes modulation is speculated to reduce the ROS damage and to promote cytochrome synthesis and heme accumulation, which coherently enhance EET. Our findings facilitate in-depth understanding of the mechanism of flavins-mediated EET and provide new insights in promoting EET of S. oneidensis for electricity generation.