A Microbial Cell Coating Based on a Conjugated Polyelectrolyte with Broad Reduction Potential Increases Inward and Outward Extracellular Electron Transfer

Bioelectrochemical systems hold the promise of enabling sustainable microbial‐mediated energy interconversion between electrical and chemical energy. Herein, it is demonstrated how a single conjugated polymer can be used to enhance bidirectional extracellular electron transfer through forming self‐assembled coatings on individual cells. Specifically, the n‐type conjugated polyelectrolyte p(cNDI‐gT2) exhibits a reduction potential window between −0.1 and −0.8 V (vs Ag/AgCl), thereby driving thermodynamically favored electron transfer in both directions across the abiotic‐biotic interface that involves the outer membrane cytochromes and flavins of Shewanella oneidensis MR‐1. Electrochemical tests show that injection from an external electrode into Shewanella oneidensis MR‐1 is enabled at negative potentials (−0.6 V), while electron extraction is possible at positive potentials (0.2 V). Relative to controls, the biohybrid shows a sixfold increase in biocurrent generation and a 35‐fold increase in current uptake for the bioelectrosynthesis of succinate from fumarate. This demonstrated abiotic‐biotic synergy provides new strategies for designing multifunctional biohybrids.


A Microbial Cell Coating Based on a Conjugated Polyelectrolyte with Broad Reduction Potential Increases Inward and Outward Extracellular Electron Transfer
Quek, Samantha R. McCuskey, Ricardo Javier Vázquez, Sarah J. Cox-Vázquez, and Guillermo C. Bazan* DOI: 10.1002/aelm.202300019 potential at the electrode controls the direction of electron flow. At more positive potentials typically found in microbial fuel cells, the electrode mediates outward extracellular electron transfer (EET) by coupling metabolic oxidation of organic substrates to electricity generation. [3] Meanwhile, the more negative potentials applied in microbial electrosynthesis platforms drive inward EET to promote metabolic reduction of substrates to high-value chemicals. [4] Practical implementation of BES technologies is challenged by conditions of poor interfacial contact(s) that limit the efficiency of EET on a per-cell basis and the total bacterial loading addressable from the electrode surface that contributes to overall EET. [5,6] Several strategies have been developed to address these challenges. For example, long-range EET has been facilitated by incorporating exogenous redox mediators, such as flavins, in dissolved or immobilized forms. [7,8] Another strategy involves modifying the electrode with conductive materials to maximize interfacial contact with bacteria. [9][10][11] An emerging biohybrid approach involves coating bacterial cells with conjugated polymers, which grants enhanced biofilm formation, increased cell viability, and decreased abiotic-biotic interfacial resistance. [12][13][14][15] This approach has been realized by either in situ polymerization or self-assembly on the cell surface. [12][13][14][15] To date, the biohybrid coating strategy usually employs p-type conjugated polymers and has focused predominantly on enhancing outward EET in the context of bioelectricity generation (Figure 1a, top). [12][13][14][15] In contrast, conjugated polymerbased cellular coatings that can mediate inward EET remain underexplored. Going one step further, demonstrating that a single polymer structure can be used to help mediate both processes would translate into versatile and multifunctional living biohybrid materials that can perform bioelectricity generation (outward EET) or microbial electrosynthesis (inward EET), depending on the tunable potential at the electrode (Figure 1a, bottom).
Conjugated polyelectrolytes (CPEs) are a class of conjugated polymers defined by a π-conjugated backbone bearing ionic pendant side chains. The latter can be designed for sufficient water-solubility and interfacing with biological systems. [16,17] Bioelectrochemical systems hold the promise of enabling sustainable microbial-mediated energy interconversion between electrical and chemical energy. Herein, it is demonstrated how a single conjugated polymer can be used to enhance bidirectional extracellular electron transfer through forming self-assembled coatings on individual cells. Specifically, the n-type conjugated polyelectrolyte p(cNDI-gT2) exhibits a reduction potential window between −0.1 and −0.8 V (vs Ag/AgCl), thereby driving thermodynamically favored electron transfer in both directions across the abiotic-biotic interface that involves the outer membrane cytochromes and flavins of Shewanella oneidensis MR-1. Electrochemical tests show that injection from an external electrode into Shewanella oneidensis MR-1 is enabled at negative potentials (−0.6 V), while electron extraction is possible at positive potentials (0.2 V). Relative to controls, the biohybrid shows a sixfold increase in biocurrent generation and a 35-fold increase in current uptake for the bioelectrosynthesis of succinate from fumarate. This demonstrated abiotic-biotic synergy provides new strategies for designing multifunctional biohybrids.

Introduction
Bioelectrochemical systems (BES) hold the promise of sustainable energy interconversion between electrical and chemical energy. [1] By interfacing electroactive bacteria with an external electrode, bacterial metabolism can be coupled to extracellular electrochemistry. This abiotic-biotic scheme exhibits merits of mild reaction conditions, high selectivity and activity, diverse catalytic functions, and self-regeneration. [2] Practical applications of this emerging technology are versatile-tunable www.advelectronicmat.de In this work, we employ an n-type CPE p(cNDI-gT2) with cationic quaternary ammonium side chains to coat the negativelycharged outer membrane of model electrogen S. oneidensis MR-1 ( Figure 1b) and enhance bidirectional EET. For EET to be thermodynamically favored in both directions, the reduction potential of the CPE must be higher than the redox potential of the bacteria for outward EET, and lower for inward EET. This enables the CPE to effectively function as both an electron acceptor and an electron donor at the CPE-bacteria interface, and requires a broad electrochemical response. Such features are indeed provided by the wide reduction potential of p(cNDI-gT2), which is spanned by two successive reduction peaks corresponding to polaron and bipolaron formation ( Figure 1c). Their midpoint potentials are −0.3 and −0.6 V, respectively (all potentials in this work are reported vs Ag/AgCl (3.5 m KCl)). Figure 1d compares the midpoint potentials of p(cNDI-gT2) to those associated with well-established redox processes in S. oneidensis MR-1-electron transfer via outer membrane cytochromes (−0.15 V), or in conjugation with flavins (−0.46, −0.35 V). [18] Based on these potential values, we reasoned that electrochemically n-doped p(cNDI-gT2) could drive inward EET in S. oneidensis MR-1, while its neutral form can be n-doped by S. oneidensis MR-1 for driving outward EET to the electrode. Furthermore, the conjugated backbone of p(cNDI-gT2) exhibits well-established high electrochemical stability and reversibility in aqueous electrolytes. [19][20][21] We report here that the resulting biohybrid indeed shows enhanced bidirectional EET, enabling more efficient bioelectricity generation when the electrode is poised at 0.2 V and selective microbial electrosynthesis of succinate from fumarate when poised at −0.6 V.

Results and Discussion
Biocompatibility is essential to constructing a viable biohybrid, therefore we first investigated to what extent p(cNDI-gT2) can be tolerated by S. oneidensis MR-1. Growth curves of S. oneidensis MR-1 in the presence and absence of p(cNDI-gT2) (20 µm) were found to be similar, suggesting no adverse effects of the coating on cell growth (Figure 2a). In addition, traditional agar plate colony forming unit counting was performed to determine cell viability after 24 h of incubation with p(cNDI-gT2).
These studies indicate biocompatibility up to concentrations of 100 µm ( Figure S1, Supporting Information). Next, the CPE cellular coating was characterized using microscopy and cellsurface charge measurements after incubating the bacteria (OD 600 = 0.22 at 30 °C) with [p(cNDI-gT2)] = 20 µm for 30 min. After incubation, color change of the cell pellet from orange to green (the color of p(cNDI-gT2)) preliminarily suggests the successful assembly of the biohybrid (Figure 2b,c). Figure 2d www.advelectronicmat.de shows a shift in zeta potentials of S. oneidensis MR-1 from −26.4 ± 0.9 to −17.3 ± 0.3 mV after incubating with p(cNDI-gT2), confirming electrostatic binding of the cationic CPE to the negatively-charged cell surface. Moreover, transmission electron microscopy (TEM) reveals the presence of p(cNDI-gT2) around the cells in the form of an irregular layer, which we will refer to as the coating (Figure 2e,f).
Fluorescence microscopy of SYTO 9-stained cultures show flocculation of the cells after being coated with p(cNDI-gT2), whereas the control cells form a sparse dispersion (Figure 2g,h). This may be from neutralization of repulsive negative charges on the cell surface by adsorbed p(cNDI-gT2), which favors cell aggregation. Furthermore, this propensity toward aggregation is potentially advantageous for establishing microcolonies essential for biofilm formation. [22] To establish that p(cNDI-gT2) can be reduced (n-doped) by extracellular electrons from S. oneidensis MR-1, absorption spectra were measured before and after the addition of S. oneidensis MR-1, see Figure 3a. After incubating the polymer with the cells for 1 h under anaerobic conditions, one observes the evolution of a broad peak at 550 nm and loss in the intensity of the peak at 800 nm. This change in absorption profile is consistent with previous spectroelectrochemical measurements on a neutral polymer with similar backbone and indicates the formation of an electron polaron in the polymer backbone, [19,20] i.e., reduction, confirming facile doping of p(cNDI-gT2) via outward EET from S. oneidensis MR-1. Moreover, the absorption profile does not change in the absence of S. oneidensis MR-1 under the same conditions ( Figure S2, Supporting Information).
The effects of the p(cNDI-gT2) cellular coating on bioelectricity generation were examined by using microbial threeelectrode electrochemical cells (M3Cs) with carbon felt as the working electrode and lactate as the electron donor. Biocurrent generation was measured by chronoamperometry (CA) at E = 0.2 V (Figure 3b), revealing a sixfold increase in steady-state current density (J SS ) for the biohybrid (34.8 ± 3.2 mA m −2 ) over the S. oneidensis MR-1 control (5.9 ± 1.3 mA m −2 ). This J SS for the biohybrid is slightly higher than that obtained in a previous report using the same M3C set-up but with a p-type CPE (27.5 ± 4.7 mA m −2 ). [14] Additionally, the abiotic p(cNDI-gT2) control produced negligible current, confirming that the current generated from the biohybrid is solely from S. oneidensis MR-1. The biocurrent generation from the biohybrid could be sustained for days and decreased with the depletion of lactate ( Figure S3, Supporting Information).
CV measurements were performed at t = 16 h under turnover conditions to obtain mechanistic insights. CV traces in Figure 3c reveal a larger catalytic current for the biohybrid than the S. oneidensis MR-1 control, consistent with the CA results. First-derivative plots of the CV traces (dJ/dE) in Figure 3d highlight relevant faradaic processes. The peaks at ≈−0.4 V are characteristic of flavin-based mediated electron transfer, while the peaks at potentials more positive than 0 V are attributed to membrane-protein direct electron transfer. [23,24] Of note is the additional peak at −0.26 V observed for the biohybrid but absent for the control. We attribute this catalytic current to the electrochemical dedoping of p(cNDI-gT2), as it matches the midpoint potential of polaron formation (−0.3 V). This observation

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implies that the biocurrent generation is mediated by repeated redox cycling of p(cNDI-gT2): n-doped by S. oneidensis MR-1 and dedoped by the electrode poised at 0.2 V (Figure 1a,d). Furthermore, the peak at −0.26 V coincides with the redox potential of cytochrome-bound flavins on the surface of S. oneidensis MR-1 (−0.35 V), which can provide a means of efficient direct electron transfer to n-dope the p(cNDI-gT2) cellular coating. [18,25] We next determined the Coulombic efficiency (CE), defined as the total charge collected by the electrode relative to the theoretical maximum charge that can be extracted from the consumed lactate. The total amount of consumed lactate was quantified via high-performance liquid chromatography (HPLC) analysis of the lactate concentration before and after CA (Table S1, Supporting Information). The calculation of CE assumes that S. oneidensis MR-1 generates four electrons for each equivalent of lactate oxidized to acetate. [26] The biohybrid has a CE of ≈84%, approximately twice that of the S. oneidensis MR-1 control with a CE of ≈40% (Table S1, Supporting Information), indicative of more efficient use of the substrate for biocurrent generation.
Biomass on the working electrode after CA was quantified by measuring total protein content. The biohybrid exhibits a ≈24% higher attached biomass than the control (Figure 3e; and  Table S1, Supporting Information). This is further corroborated by scanning electron microscopy (SEM) images of the working electrode ( Figure S4, Supporting Information) that show denser biofilms on the electrode surface for the biohybrid. Notably, SEM is unable to show the CPE coating as revealed by TEM (Figure 2f), possibly due to its lower resolution that fails to discern the subtle surface roughness of the coated cells or the pretreatment of samples that could have removed the CPE coatings. The increase in biomass of the biohybrid over the control is unable to completely account for the sixfold enhancement in biocurrent generation. Figure 3f reveals that the biohybrid indeed exhibits a higher current density per unit biomass relative to the control. We attribute this increased current per cell to the p(cNDI-gT2) coating effectively wiring-up the cellular surface to the electrode.
Having established enhanced outward EET in the biohybrid, we then probed its inward EET capabilities. The prototypical two-electron reduction of fumarate to succinate was chosen as the model reaction. [27] Current uptake was measured by CA at E = −0.6 V with fumarate added at the start. CA plots in Figure 4a show a 35-fold increase in J SS for the biohybrid (−66.2 ± 6.4 mA m −2 ) over the S. oneidensis MR-1 control (−1.9 ± 1.5 mA m −2 ). The abiotic p(cNDI-gT2) control showed negligible current uptake, confirming that the polymer does not contribute to any background cathodic current. The higher J SS in the biohybrid can also be observed in CV measurements (Figure 4b), which depict larger reductive catalytic currents for the biohybrid over the control. Additionally, the biohybrid could maintain substantial microbial electrosynthesis activity for several days ( Figure S5, Supporting Information). Figure 4c shows dJ/dE plots of the catalytic waves in Figure 4b to highlight the key faradaic processes involved in the electrode-driven fumarate reduction. The control presents a catalytic reduction wave at −0.42 V, consistent with reported potential values for fumarate reduction in S. oneidensis MR-1. [21,27] Differently, fumarate reduction in the biohybrid occurs with a lower overpotential than the control, at −0.35 V. Noteworthy, the potential at which fumarate reduction occurs in the biohybrid (−0.35 V) coincides with the potential for polaron formation in p(cNDI-gT2) (−0.3 V), in line with mediation of inward EET www.advelectronicmat.de by repeated redox cycling of p(cNDI-gT2): electrochemically n-doped by the electrode and dedoped by electron injection into S. oneidensis MR-1 (Figure 1a,d). A further boost in reductive catalytic current is observed in the biohybrid at lower potentials of −0.63 V, which matches the potential for bipolaron formation in p(cNDI-gT2) (−0.6 V). The combined results suggest that both polaronic and bipolaronic states of the p(cNDI-gT2) coating help to mediate inward EET.
The total amount of succinate produced from the reduction of fumarate was quantified via HPLC analysis of the succinate concentration before and after CA (Table S2, Supporting Information). Seen in Figure 4d, the biohybrid generated a significantly larger amount of succinate as compared to the S. oneidensis MR-1 control, agreeing with its higher J SS observed in CA (Figure 4a). Furthermore, the abiotic p(cNDI-gT2) and heat-killed biohybrid controls generated negligible amounts of succinate, confirming that bioelectrosynthesis of succinate is catalyzed by viable S. oneidensis MR-1 cells. Both the biohybrid and the S. oneidensis MR-1 control show high CE of close to 100% (Table S2, Supporting Information), reflecting the high selectivity and activity of the fumarate reductase enzyme that is coupled to the electrode. Quantification of the biomass on the working electrode at steady-state of microbial electrosynthesis reveals an ≈48% higher biomass for the biohybrid than the S. oneidensis MR-1 control (Figure 4e; and Table S2, Supporting Information). Figure S6 (Supporting Information) shows SEM images of the working electrodes. Similar to the outward EET experiments, the biohybrid exhibits a higher current density per unit biomass over the control (Figure 4f; and Table S2, Supporting Information), owing to the more efficient electron transfer at the abiotic-biotic interface mediated by p(cNDI-gT2).
In summary, we have demonstrated the first example of a single conjugated polymer cellular coating that can enhance bidirectional EET. This is underpinned by essential elements of molecular design, which include 1) cationic side chains for water-processibility and spontaneous electrostatic binding to cells, and 2) n-dopability of the conjugated backbone across a wide reduction potential window to thermodynamically drive inward and outward EET. As a result, the biohybrid exhibits improved performance over controls in bioelectricity generation and microbial electrosynthesis. Extensions of accelerated bidirectional EET opens opportunities to improve multifunctional bioelectrochemical systems that operate in both reductive and oxidative modes for emerging applications in sensing, wastewater treatment, and catalysis. [28][29][30] We also envision extending this biohybrid coating approach to other microbes with highvalue bioelectrochemical conversions.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.