Engineering Shewanella oneidensis-Carbon Felt Biohybrid Electrode Decorated with Bacterial Cellulose Aerogel-Electropolymerized Anthraquinone to Boost Energy and Chemicals Production

Abstract

Interfacial electron transfer between electroactive microorganisms (EAMs) and electrodes underlies a wide range of bio-electrochemical systems with diverse applications. However, the electron transfer rate at the biotic-electrode interface remains low due to high transmembrane and cell-electrode interfacial electron transfer resistance. Herein, a modular engineering strategy is adopted to construct a Shewanella oneidensis-carbon felt biohybrid electrode decorated with bacterial cellulose aerogel-electropolymerized anthraquinone to boost cell-electrode interfacial electron transfer. First, a heterologous riboflavin synthesis and secretion pathway is constructed to increase flavin-mediated transmembrane electron transfer. Second, outer membrane c-Cyts OmcF is screened and optimized via protein engineering strategy to accelerate contacted-based transmembrane electron transfer. Third, a S. oneidensis-carbon felt biohybrid electrode decorated with bacterial cellulose aerogel and electropolymerized anthraquinone is constructed to boost the interfacial electron transfer. As a result, the internal resistance decreased to 42 Ω, 480.8-fold lower than that of the wild-type (WT) S. oneidensis MR-1. The maximum power density reached 4286.6 ± 202.1 mW m^-2, 72.8-fold higher than that of WT. Lastly, the engineered biohybrid electrode exhibited superior abilities for bioelectricity harvest, Cr⁶⁺ reduction, and CO₂ reduction. This study showed that enhancing transmembrane and cell-electrode interfacial electron transfer is a promising way to increase the extracellular electron transfer of EAMs.

Keywords: CO2 reduction; Shewanella oneidensis; bioelectricity harvest; biohybrid electrode; interfacial electron transfer.

Figure 1

Modular engineering the S. oneidensis‐carbon felt biohybrid electrode decorated with bacterial cellulose aerogel‐electropolymerized anthraquinone to boost energy and chemicals production. A modular engineering strategy, including increasing flavins synthesis and secretion, reconstructing OM c‐Cyts conductive channel, and constructing bacterial cellulose‐based aerogel coated anthraquinone hybrid anode, was adopted to boost the cell‐electrode interfacial electron transfer rate. i) To address the limitations in the biosynthesis and secretion of endogenous electron shuttle flavins, a heterologous riboflavin synthesis and delivery pathway from B. subtilis and P. aeruginosa was designed to enhance flavin‐mediated transmembrane electron transfer. ii) To mitigate the issue of the physically insulative cytomembrane of S. oneidensis, outer membrane c‐type cytochromes OmcF from G. sulfurreducens were screened and optimized using protein engineering strategies to accelerate contact‐based transmembrane electron transfer. iii) To solve the problem of high electron transfer resistance at cell‐electrode interface, an efficient bacterial cellulose aerogel‐coated anthraquinone hybrid electrode was developed to enhance the loading of living cells within the electroactive biofilm on the anode, which boosted cell‐electrode interfacial electron transfer. To evaluate the catalytic performance of the engineered biohybrid electrode in the real‐world applications, bioelectricity harvest from thin stillage, reduction of Cr⁶⁺ to Cr³⁺, and electrosynthesis of CO₂ to formate were conducted in the dual‐chamber MFCs.

Figure 2

Enhancing flavins‐mediated transmembrane electron transfer by increasing flavins synthesis and secretion. a) Schematic of constructing a flavin biosynthesis pathway to accelerate flavin‐mediated indirect electron transfer, including heterologous expression of the gene cluster ribADHEC for flavin synthesis and pore protein OprF and Bfe for flavin transmembrane transport, as well as flavin‐mediated indirect electron transfer pathways. b) The flavin concentrations produced by the WT and recombinant strains (MC and MCO). c) Output power density curves of the WT and recombinant strains (MC and MCO). d) Current‐voltage curves of the WT and recombinant strains (MC and MCO). e) CLSM images of anode biofilms of the WT and recombinant strains (MC and MCO). f) Anode biomass of biofilm formed by the WT and recombinant strains (MC and MCO). g) Single‐cell output current of the WT and recombinant strains (MC and MCO). h) Nyquist plots of EIS spectra for anodes with the WT and recombinant strains (MC and MCO). i) EAC and EDC of the WT and recombinant strains (MC and MCO). Data were presented as mean ± SD (n = 3 biological replicates).

Figure 3

Accelerating contacted‐based transmembrane electron transfer by reconstructing OM c‐Cyts conductive channel. a) Schematic of the outer‐membrane c‐Cyts and riboflavin promoting transmembrane electron transfer from cell to anode. b) Amino acid sequences and mutation sites of the WT and mutant OmcF M1‐M6. c) Power densities of the mutant strains MCOF1‐6. d) The interaction between c‐Cyt OmcF M5 and riboflavin molecule. e) The 2D display of the interaction between the residues of OmcF M5 and RF molecule. f) Zeta potentials of strains MCOF and MCOF1‐6. g) Plasmids of the recombinant strains MCOF5 and CR1‐4 that included the assemblage of genes (ribADEHC, bfe, oprF, and omcF M5) and different RBSs (BBa_B0030, BBa_B0032, BBa_B0033, BBa_B0034, BBa_B0035, iGEM). h) Output power density curves of strains MCOF5 and CR1‐4. i) Single‐cell output current of the recombinant strains MCOF5 and CR1‐4. j) EIS analysis of the recombinant strains MCOF5 and CR1‐4. Data were presented as mean ± SD (n = 3 biological replicates).

Figure 4

Boosting cell‐electrode interfacial electron transfer by constructing S. oneidensis‐carbon felt biohybrid electrode decorated with bacterial cellulose aerogel‐electropolymerized anthraquinone. a) Schematic of interfacial electron transfer process after decoration of CNFA and AQ on the surface of the carbon felt anode. b) Power density curves of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). c) CV curves of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). d) Current output of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). e) EIS analysis of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). f) EAC and EDC of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). g) Electrochemically active surface area of the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). h) SEM images of colony morphology of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). i) CLSM images of colony morphology of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). j) Analysis of biomass of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). k) Single‐cell output current of strain CR1 equipped with the CF and decorated anodes (CF/CNFA and CF/CNFA@AQ). Data were presented as mean ± SD (n = 3 biological replicates).

Figure 5

Application of the constructed biohybrid electrode in real‐world scenarios for energy and chemicals production. a) Schematic diagram of electricity recovering from thin stillage using constructed BES. b) Power density curves of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control. c) The Coulombic efficiency and COD removal efficiency of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control in thin stillage. d) CLSM images of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control. e) Schematic diagram of Cr⁶⁺ degradation using constructed BES. f) The Cr⁶⁺ concentrations of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control in MFCs under anaerobic conditions. g) Cr⁶⁺ degradation kinetic constant of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control in MFCs. h) CLSM images of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control. i) Schematic diagram of CO₂ reduction to produce formate by electrosynthesis using the engineered biohybrid electrode. j) The formate concentrations of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control. k) The ATP concentrations and NADH/NAD⁺ ratio of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control. l) The Faraday efficiency of the engineered biohybrid electrode CF/CNFA@AQ‐CR1 and control. Data were presented as mean ± SD (n = 3 biological replicates).