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A Hierarchically Modified Graphite Cathode with Au Nanoislands, Cysteamine, and Au Nanocolloids for Increased Electricity-Assisted Production of Isobutanol by Engineered Shewanella oneidensis MR-1 Ju A La, Jong-Min Jeon, Byoung-In Sang, Yung-Hun Yang, and Eun Chul Cho ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09874 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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ACS Applied Materials & Interfaces

Revised MS, am-2017-09874n.R2

A Hierarchically Modified Graphite Cathode with Au Nanoislands, Cysteamine, and Au Nanocolloids for Increased Electricity-Assisted Production of Isobutanol by Engineered Shewanella oneidensis MR-1

Ju A La,† Jong-Min Jeon,‡ Byoung-In Sang,† Yung-Hun Yang,*, ‡ and Eun Chul Cho*,† †

Department of Chemical Engineering, Hanyang University, Seoul, 04763, South Korea

‡

Department of Biological Engineering, College of Engineering, Konkuk University, Seoul,

05030, South Korea

KEYWORDS: electrode surface nanostructures, isobutanol productivity of engineered Shewanella oneidensis MR-1, Au nanoislands, cysteamine, Au nanoparticles

ACS Paragon Plus Environment

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ABSTRACT: It is necessary to understand the surface structural effects of electrodes on the bioalcohol productivity of Shewanella oneidensis MR-1, but this research area has not been deeply explored. Here, we report that the electricity-assisted isobutanol productivity of Shewanella oneidensis MR-1::pJL23 can be enhanced by sequentially modifying a graphite felt (GF) surface with Au nanoislands (Au), cysteamine (NH2), and Au nanoparticles (Au NPs). After bacteria were incubated for 50 h with the unmodified GF under various electrode potentials (vs. Ag/AgCl), the bacterial isobutanol concentrations increased from 2.9 ± 1 mg/L under no electricity supply to a maximum of 5.9 ± 1 mg/L at −0.6 V. At this optimum electrode potential, the concentrations continued increasing to 9.1 ± 1 mg/L, 14 ± 2 mg/L, and 27 ± 2 mg/L when the GF electrodes were modified with Au, NH2-Au, and Au NP-NH2-Au, respectively. We further studied how each surface structure affected the bacterial adsorptions, current profiles, and biofilms’ electrochemical performances. In particular, these modifications induced the adsorption of elongated bacteria, with the amount dependent on the electrode structure. In the presence of electric supply, the amount of elongated bacteria further increased. We also found that the NH2-Au-GF and Au NP-NH2-Au-GF electrodes themselves could increase the concentrations to 11 ± 0.3 mg/L and 12 ± 2 mg/L, respectively, upon the bacterial incubation without electricity. Among the electrodes tested, the contribution of electricity to the bacterial isobutanol production was the greatest with the Au NP-NH2-Au-GF electrode. After 96 h of incubation, the concentration increased to 72 ± 2 mg/L, which was 4.7 and 3.7 times the previously reported values obtained without and with electricity, respectively.


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1. INTRODUCTION Electron transfer from a cathode to electrogenic bacteria is becoming an important issue in bioelectrochemical synthesis,1–8 as in the case of the electron transfer from bacteria to anode materials for the generation of electricity in microbial fuel cells.9–13 The electron supply to bacteria may be helpful in biochemical reduction reactions such as the transformation of CO2 into acetate14–16 or butyrate,17 or fatty acids into alcohols.18 In some cases, electrons have also been supplied to bacteria to obtain desired biochemical compounds,19,20 and to achieve a higher yield and productivity for the desired compound.21–24 From an environmental perspective, undesirable wastes (such as acids) can be converted into valuable fuels (alcohols). It has been known that many acetogenic bacteria effectively use electricity in CO2 reduction.14–16 Meanwhile, some other bio-electrochemical syntheses are known to require efficient metal-reducing moieties in the outer membrane of the bacteria, as in the case of Geobacter and Shewanella.3,4,25,26 Particularly in the latter case, Shewanella oneidensis MR-1 is known to actively exchange electrons between the electrodes and bacterial cells via several routes:27–31 i) electron transfer between the electrodes and cytochromes (MtrC and OmcA) in the outer membrane or conductive nanowires connected to the outer membrane of the periplasm and electrodes29–31 and ii) electron transfer through self-produced mediators such as riboflavin and flavin mononucleotide.27,28 Riboflavin and flavin mononucleotide are also reported to play some roles as cofactors in the direct electron transfer to MtrC and OmcA because they also exist in biofilms. To date, various factors have been extensively investigated to determine their possible influence on the electron exchange between Shewanella and electrodes, in relation to the electrochemical performance of anodes. It was reported that riboflavin solubilized in a culture medium affected the electron exchange between anodes and bacteria.27 The impact of riboflavin


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attached to the anode surface on the anodic electrochemical performance was also illustrated.32 Moreover, several studies showed that the composition, structure, and uniformity of a biofilm could be altered by the electrode potential,33 exogenous addition of a reagent,34 and introduction of positively charged anodes.35 In spite of these studies stressing the roles of anodes or a biofilm on the anode in the electron exchange with Shewanella, there is a lack of understanding about the effect of the cathode structure or biofilm structure on the production of biochemical compounds by S. oneidensis MR1. In particular, we are interested in the potentiality of S. oneidensis MR-1::pJL23 to increase the production of isobutanol, which is one of the attractive biofuels for replacing fossil fuels, by increasing reductive reactions via the electron supplement from the extracellular space.20 However, the bacterial isobutanol productivity has been very low, implying that the isobutanol synthetic pathway (Scheme 1A) is unfavorable. Meanwhile, Zhang et al.14 and Chen et al.15 recently suggested with Sporomusa ovata, one of the acetogenic bacteria, that the modification of the cathode surface with positively charged molecules or carbon nanotubes could enhance the ability of this bacterium to reduce CO2 into acetate. Zhang et al. also reported that some metallic (Au, Pt, Ni) coatings on the surface of electrodes also help to enhance the production of acetate. These studies suggested that modifying the electrode conditions could also improve the isobutanol productivity of S. oneidensis MR-1::pJL23. Nonetheless, unlike S. ovata, S. oneidensis MR-1::pJL23 possesses genetically-engineered non-natural biosynthetic genes to produce isobutanol (Scheme 1A), and it has another pathway to produce the other biochemical.27 As such case, it is unclear whether the change in the electrode potentials and structures can efficiently drive S. oneidensis MR-1::pJL23 to increase the isobutanol production. Moreover, it has not yet been definitely determined whether a change in the electrode structure itself could


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alter the bacterial biochemical productivity during an operation without electricity: if the electrode structures alter the bacterial adsorption patterns, these changes may greatly affect the performance of the bacteria.36–38 These two studies are essential to design an optimal electrode structure both to enhance the effect of electricity on the bacterial biochemical production and to increase the overall productivity.

Scheme 1. (A) A metabolic pathway of S. oneidensis MR-1::pJL23 for bacterial isobutanol synthesis. (B) A schematic illustrating the procedures for fabricating the hierarchical structure on a graphite felt (GF) electrode.

In this study, as shown in Scheme 1B, we modified a graphite felt (GF) surface sequentially with Au nanoislands (Au), cysteamine (NH2), and Au nanoparticles (NPs), not only to improve the isobutanol productivity of S. oneidensis MR-1::pJL23 but also to enhance the contribution of electricity to the bacterial bio-alcohol production during their incubation under an electricity supply. First, with the unmodified GF, we studied the effect of a working electrode potential on the bacterial adsorption, current profile, electrochemical performance of biofilms, and isobutanol concentration. Then, we determined the optimal electrode potential for this bacterium to increase


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the isobutanol production. Next, at the optimum electrode potential, we monitored how the sequential modification of GF with the previously mentioned nanomaterials influenced these characteristics and eventually the isobutanol concentration. In particular, we found a change in the electrode structure altered the amount and shape (e.g., elongated bacteria) of the bacteria on the electrodes, and the amount of adsorbed bacteria or degree of elongation was even altered when incubating the bacteria under the electricity supply. For this reason, we also investigated whether a change in the electrode’s surface nanostructure itself could affect the isobutanol concentration when the bacteria were incubated without electricity. More importantly, related to this issue, we further investigated the effect of the electrode structures on the electricity contribution to the bacterial isobutanol production. Last, the long-term stabilities of the modified electrodes (for 96 h) were investigated, and the isobutanol concentrations were compared with those in a previous report.20 From these studies, we suggested the Au NP-NH2-Au-GF electrode could not only make the bacteria produce isobutanol higher by 4.7 and 3.7 times than the previously reported values obtained without and with electricity, respectively, but also increase the effect of electricity on isobutanol concentrations.

2. EXPERIMENTAL SECTION 2.1. Materials. Gold(III) chloride trihydrate (HAuCl4·3H2O, 99.9%), cysteamine (≥98%), Nacetyl-D-glucosamine (≥95%), sodium pyruvate (≥99%), 50% sodium lactate, sodium phosphate monobasic dehydrate (≥99.9%), sodium phosphate dibasic dodecahydrate (≥99.9%), 10 v/v% phosphotungstic acid solution, and 50% glutaraldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trisodium citrate (99%) and ethanol (99.9%) were acquired from Fisher Scientific (USA), whereas M9 minimal salts 5× and yeast extract were acquired from Difco


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Laboratories (Franklin Lakes, NJ, USA). Graphite felt (GF), 20% Pt on carbon paper, Nafion cation exchange membranes (NR-212), and Ag/AgCl reference electrodes were purchased from Kureha (Japan), Naracelltech (Seoul, South Korea), DuPont (Germany), and BASi (Germany), respectively. 2.2. Synthesis of Au NPs. Au nanoparticles with a diameter of 15 ± 3 nm were synthesized by the conventional method. 2 mg of HAuCl4 was dissolved in 19 mL of deionized water. The solution was heated and equilibrated at 105 °C for 40 min. Then, 1.0 mL of a trisodium citrate aqueous solution (0.05 wt%) was added to the HAuCl4 solution. After a 30 min reaction, the Au nanoparticle dispersion was cooled to room temperature and stored at 4 °C before use. We purified the Au nanoparticles by centrifugation and redispersion in deionized water to modify the surface of NH2-Au-GF electrode. 2.3. Modification of GF electrode. GF served as the electrode material for improved isobutanol production. To fabricate the Au-GF, a 10 nm-thick Au layer was applied to both sides of the GF using E-beam evaporation (Daeki Hi-tech Co., Ltd., South Korea). To prepare the NH2-Au-GF, the Au-GF electrode was treated for 3 days at 4 °C with 2 mM cysteamine dissolved in ethanol, then washed with deionized water (18.2 MΩ cm) with stirring at 200 rpm for 30 min, which was repeated four times. Finally, to create the Au NP-NH2-Au-GF, the NH2Au-GF was immersed in an aqueous dispersion of 0.4 nM (based on particle number) Au NPs for 1 day at 30 °C. The concentration of Au NPs was quantified using inductively coupled mass spectroscopy (Perkin Elmer, USA). 2.4. Bacterial strains and media. S. oneidensis MR-1::pJL23 was used to produce isobutanol in a bioelectrochemical reactor. S. oneidensis MR-1::pJL23 has heterologous Ehrlich pathway genes, namely, kivD, which encodes ketoisovalerate decarboxylase, from Lactococcus lactis and


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adh, which encodes alcohol dehydrogenase, from Ralstonia eutropha.20 The pJL23 plasmid was obtained from Prof. Sinskey's lab at the Department of Biology, Massachusetts Institute of Technology. We used an optimized isobutanol production medium based on a method from the literature.20 A 1× M9 minimal medium was used, which contained 0.1 wt% yeast extract, 2 wt% N-acetyl-D-glucosamine, 1.5 wt% sodium pyruvate, and 2 wt% sodium lactate. Cells were grown in the optimized medium for 48 h at 30 °C with shaking at 200 rpm. 2.5. Isobutanol production in bioelectrochemical reactor. The bioelectrochemical Pyrex reactor was a slightly modified H-type reactor with dual chambers (300 mL each). The cathode and anode were a GF electrode (3 cm wide × 6 cm long × 0.5 cm thick) and an electrode consisting of 20% Pt on carbon paper (3 cm × 6 cm), respectively. The Ag/AgCl reference electrode was immersed in the cathode compartment. The anode and cathode compartments were separated by a Nafion NR-212 cation exchange membrane. Before the experiment, we sterilized all the glassware and reactors using an autoclave (Coretech, Korea). All the electrodes were sterilized on a clean bench using UV irradiation. In addition, the entire operation was performed on a clean bench to minimize the contamination. The cathode compartment was filled with the optimized medium with inoculated cells, and the anodic compartment was filled with 0.1 M sodium phosphate buffer (pH 7.0). The reactor was operated at various electrode potentials (vs. Ag/AgCl) created by a WMPG 1000 potentiostat/galvanostat (Wonatech Co. Ltd., South Korea). In all the experiments, the inoculum volumes were set at 2% for the total volume, and the initial cell number was 5 × 106 cells/mL, which was adjusted from optical density at 600 nm. In addition, we maintained microaerobic conditions20,39−41 because the electron respiration pathway of Shewanella is activated at low oxygen level, which results in higher yield. We maintained this condition under continuous magnetic stirring in the reactors with 200 rpm. The reactor


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temperature was maintained at 30 °C using heating tape (Daihan, Wonju, South Korea), and the temperature was monitored by means of a temperature sensor inserted into the anode compartment. 2.6. Gas chromatography. To analyze the growth of S. oneidensis MR-1::pJL23 in the previously described minimal medium, 200 µL of each culture sample was collected and analyzed at 595 nm after being put through a 96-well microplate washer (Tecan, Mannedorf, Switzerland). The isobutanol quantity was determined by gas chromatography using the following method. First, 1 mL of a bacterial culture was collected and mixed with 1 mL of chloroform. The organic phase (bottom) was collected using a pipette and transferred to clean borosilicate glass tubes containing Na2SO4. These samples were then injected into a gas chromatograph (Agilent, CA, USA) equipped with a fused silica capillary column (Supelco SPB5, 30 m × 0.32 mm, i.d. 0.25 µm film), with helium as the carrier gas. A 1 µL aliquot of the organic phase was injected using the autosampler. The inlet was maintained at 250 °C. The oven was held at 40 °C for 5 min, heated to 220 °C at 20 °C/min, and then held at 220 °C for 5 min. Peak detection was performed using a flame ionization detector, which was maintained at 250 °C. 2.7. Scanning electron microscopy (SEM). We observed the surface of electrode surfaces by using using a scanning electron microscope (S-4800, Hitachi, Germany). The biofilm-coated GF and biofilm-coated modified GF were fixed overnight at room temperature in 0.1 M phosphate buffer (pH 7.0) containing 2.5% glutaraldehyde. Then, the electrodes were stained with 25 mL of a 2 v/v% phosphotungstic acid solution. Next, the electrodes were dehydrated in a series of ethanol solutions (25%, 50%, 70%, and 100%) and dried under ambient conditions. The


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morphological features of the electrode surface were examined at an acceleration voltage of 15 kV.

3. RESULTS AND DISCUSSION 3.1. Effect of electrode potential of unmodified GF electrode on isobutanol production. S. oneidensis MR-1::pJL23 was genetically modified for the production of isobutanol via the introduction of the heterologous Ehrlich pathway genes of ketoisovalerate decarboxylase (kivD) and alcohol dehydrogenase (adh) into S. oneidensis MR-1 (Scheme 1A).20 For isobutanol production, the bacteria must possess the valine (from 2-acetolactate to 2-ketoisovalerate) and isobutanol (from isobutyraldehyde to isobutanol) synthetic pathways. N-acetyl-D-glucosamine and lactate can be oxidized to pyruvate and then converted to 2-acetolactate for these two routes. However, in the previous study, with the optimized composition of carbon sources (2 wt% Nacetyl-D-glucosamine, 2 wt% lactate, and 1.5 wt% pyruvate), the isobutanol concentration was very low: 10.3 mg/L.20 Even worse, in the present work, the bacteria produced only 2.9 ± 1 mg/L (n = 5) of isobutanol after 50 h of bacterial incubation at 30 °C under microaerobic conditions in a 300 mL H-type reactor containing a culture medium with identical composition (see EXPERIMENTAL SECTION). The concentration was enhanced to 8.9 ± 1 mg/L (n = 7) when we used a 20 mL reactor. Because the isobutanol production from this bacterium was sensitive to the supply of oxygen, the results suggested that controlled experimental conditions were required in these experiments. In addition, S. oneidensis MR-1 also has a metabolic pathway for the oxidation of lactate to pyruvate, acetyl-CoA, acetyl-phosphate, and finally acetate.27 Based on the experimental results and literature, it was thought that the metabolic pathway for isobutanol synthesis was unfavorable. Nevertheless, S. oneidensis MR-1 not only has diverse electron


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transport systems bearing the mtrCAB pathway with cytochrome C proteins on their membrane surfaces,42–44 but also various electron-exchange routes between the electrodes and bacteria.27–31 Therefore, we expected that an efficient supply of exogenous electrons from the electrodes could help increase the production of isobutanol. The present work mainly focused on understanding whether optimal electrode conditions (the electrode potential and surface structure) could influence the isobutanol production by bacteria, and eventually enhance their productivity.

Figure 1. (A–D) Effects of electrode potential on adsorption patterns of S. oneidensis MR1::pJL23 onto GF cathode: (A) no electricity, (B) −0.1 V, (C) −0.6 V, and (D) −0.9 V. (E) Bacterial cell numbers adsorbed on the surface of GF. For each average value and standard deviation, we analyzed at least 15 images (3 independent samples × 5 SEM images for each


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sample). (F) Isobutanol concentrations of the bacterial systems as a function of the electrode potential. All the data were obtained 50 h after the incubation of the bacteria with the electrode at various electrode potentials.

We first investigated the effect of the potential of a working electrode (vs. Ag/AgCl) consisting of unmodified GF on the concentration of isobutanol in the 300 mL H-type reactor. Figure 1A–D shows SEM images of the GF electrode after 50 h of exposure to S. oneidensis MR-1::pJL23 at various electrode potentials. In the comparison with the electrode incubated without electricity (Figure 1A), the amount of bacteria attached to the electrode seems to increase with the decreasing potential until −0.6 V, after which the amount appeared to decrease (Figure 1B–D). We also monitored the optical densities (OD at 600 nm) of the bacteriasuspended culture medium, and the values (n = 4) after 50 h were 1.08 ± 0.10, 1.19 ± 0.17, 1.21 ± 0.05, and 1.24 ± 0.20 under no electricity, −0.1 V, −0.6 V, and −0.9 V, respectively. Figure 1E and F shows plots of the densities of the bacteria attached to the electrodes and the isobutanol concentrations, respectively, as a function of the electrode potential. The number of bacteria attached to the unmodified GF electrode was quantified by directly counting the bacterial number from the SEM images. Both the cell density and isobutanol concentration reached maximum values at the electrode potential of −0.6 V. To understand Figure 1, the electrochemical performance of a bacteria-coated GF electrode was evaluated using cyclic voltammetry (CV) (Figure 2A–C). The bacteria-coated GF was obtained after the GF was incubated with the bacteria for 50 h without electricity. The CV measurement was conducted after replacing the culture medium containing bacteria (used for incubation) with a fresh culture medium (without bacteria) with a composition identical to that of the medium used for incubation. We detected two major oxidation peaks, at 0.07 and 0.37 V


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(Figure 2A, B), which may be associated with the direct electron transfer between the biofilm (riboflavin-rich) and electrodes.27,32 The oxidation peaks were more prominent than a reduction peak near 0.01 V (inset in Figure 2C), implying that the oxidation of the biofilm was more active than the reduction in the potential range of −0.1 to 0.4 V. Accordingly, positive currents were detected in the time-course current profiles (chronoamperogram, CA) observed during the bacterial incubation for 50 h at the electrode potential of −0.1 V (Figure 2D), implying that electrons were mostly transferred from the surroundings to the working electrode. The positive currents were also reported when a working electrode was exposed to an S. oneidensis MR-1 suspension at −0.3 V.32,35 Nevertheless, the isobutanol concentration was 4.7 ± 1 mg/L (n = 3), which was higher than the amount in the absence of electricity: 2.9 ± 1 mg/L (n = 5). We speculated that the small reduction near 0.01 V (electron transfer from the electrode to the biofilm) might have helped the bacteria to increase their isobutanol production.


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Figure 2. (A–C) Cyclic voltammograms of biofilm-coated GF electrode obtained after incubation of S. oneidensis MR-1::pJL23 for 50 h without electricity. (D) Chronoamperometry of GF at various electrode potentials during the electricity-assisted incubation of S. oneidensis MR1::pJL23 for 50 h.

Meanwhile, in the CV profile, we observed a major reduction peak at −0.40 to −0.65 V (Figure 2C), which was slightly lower than the oxidation/reduction potential associated with the riboflavin-mediated electron exchange between S. oneidensis MR-1 or Shewanella sp. MR-4 suspended in a culture medium and biofilm-coated electrodes (−0.45 to −0.4 V vs. Ag/AgCl).27,32 We also observed oxidation peaks at −0.52 and −0.40 V (inset in Figure 2B), but the magnitude of the current was much smaller than that of the current due to the reduction at −0.45 to −0.65 V. Accordingly, the operation at −0.6 V could drive the biofilm to accept electrons from the electrode for electrochemical reduction reactions, producing negative


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currents in the CA (Figure 2D; also see Figure S1, Supporting Information, SI), with average negative currents of −0.30 ± 0.06 µA/cm2 (n = 5). The isobutanol concentration increased from 4.7 ± 1 mg/L at −0.1 V to 5.9 ± 1 mg/L (n = 6). The number of bacterial cells attached was the highest at this potential. Finally, we did not observe any biofilm-associated electrochemical events at −0.9 and −1.2 V. According to Figure 2D, the average current increased to −2.62 µA/cm2 at −0.9 V and to −33.8 µA/cm2 at −1.2 V. The number of bacteria adsorbed on the GF electrode was higher at −0.9 V than the number without electricity. Nevertheless, the isobutanol concentrations were lower than the concentration without electricity, with values of 2.5 ± 2 mg/L (n = 2) at −0.9 V and 1.5 mg/L at −1.2 V. We speculated that the larger amounts of electrons supplied from the GF electrode at −0.9 and −1.2 V were used for undesired purposes such as a hydrogen evolution reaction.45−47 It is worth discussing that the overall shapes of CV profiles of the bacteria-coated electrodes obtained at various electrode potentials were more or less similar to those in Figure 2A (see Figure S2, SI). However, a close look at the data around −0.6 V in the CVs indicates that the reduction current densities of the bacteria-coated electrode were the largest when the electrode was obtained under the electrode potential of −0.6 V. Meanwhile, the oxidation currents around −0.6 V were not very different among the samples. Therefore, as reported in a previous literature,33 the characteristics of bacteria-coated electrodes might be influenced by the electrode potentials applied during the incubation of bacteria. However, a more systematic analysis of the chemical components of both the bacteria-coated electrode and culture medium is necessary to clarify the effect of the electrode potential on the characteristics of the bacteriacoated electrodes.


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Overall, an optimum electrode potential appears to exist for both enhanced bacterial adsorption and isobutanol production. Because the biofilm/bacteria on the electrode mostly participated in the above-mentioned electrochemical events,27,32 the enhanced bacterial adsorption may be helpful for more active electrochemical events. Nevertheless, the present results also suggest that an appropriate electrode potential for some electrochemical events should be accompanied for an increased isobutanol concentration. 3.2. Effect of modification of GF electrodes with Au nanoislands, cysteamine, and Au NPs on bacterial isobutanol production. Next, we modified the GF surface using various chemical components. We used an E-beam evaporator to deposit 10 nm-thick Au on the GF surface (Figure 3A). Thus, Au nanoislands smaller than 100 nm were densely formed on the GF. The Au-coated GF (hereafter: Au-GF) was further modified by means of Au-thiol chemistry with cysteamine to alter the adsorption behavior of the bacteria (Scheme 1B). We called this electrode “NH2-Au-GF.” These electrodes were incubated in the bacterial suspension at the electrode potential of −0.6 V for 50 h, and the isobutanol concentrations were compared. As shown in Figure 3B, the isobutanol concentrations can be ranked in the following order: NH2-Au-GF (14 ± 2 mg/L, n = 8) > Au-GF (9.1 ± 1 mg/L, n = 6) > unmodified GF (5.9 ± 1 mg/L, n = 6). Figure 3C shows the CAs of the three electrodes monitored during the incubation of the bacteria for 50 h at −0.6 V. The average reductive currents (n = 5) can be ranked in the same order: NH2-Au-GF (−0.49 ± 0.07 µA/cm2) > Au-GF (−0.36 ± 0.12 µA/cm2) > unmodified GF (−0.30 ± 0.06 µA/cm2). After the operation, we analyzed the CV results for the three electrodes that were coated with biofilms (Figure S3, SI). Although the shapes of the CV profiles were similar among the three electrodes, the total charges involved in the oxidation and reduction cycles increased from 4.51 ± 0.23 C for the unmodified GF to 8.91 ± 0.02 C and 8.00 ± 0.03 C for the Au-GF and


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NH2-Au-GF, respectively (Figure S4, SI). Moreover, as shown in Figure 3D, the reduction charges can be ranked in the following order: NH2-Au-GF (2.64 ± 0.06 C) > Au-GF (2.49 ± 0.24 C) > unmodified GF (0.84 ± 0.04 C). The ratio of the reduction to total charge increased from 0.19 for the unmodified GF to 0.28 for Au-GF and 0.33 for NH2-Au-GF.

Figure 3. (A) SEM image of GF after deposition of Au on it. (B) Effects of the electrode type on the isobutanol concentration produced by S. oneidensis MR-1::pJL23 after the electricity-assisted (−0.6 V) incubation of the bacteria for 50 h. (C) Chronoamperometry monitored during the electricity-assisted (−0.6 V) incubation of the bacteria with one of the three electrodes. (D) The average reductive charges (n = 3) estimated from the CV analysis for the three biofilm-coated electrodes (including Figure S3, SI) obtained after the electricity-assisted (−0.6 V) incubation of the bacteria for 50 h. The table in the inset shows the ratios of the reduction to the total charge for the three electrodes.


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We investigated the bacterial adsorption patterns on these electrodes. Figure 4 shows SEM images of the Au-GF electrode after 50 h of incubation in the aqueous suspension of S. oneidensis MR-1::pJL23 at −0.6 V. The results were compared with those of the unmodified GF (Figure 1C). Although the density of the bacteria does not look much different on this electrode (Figure 4A), high-magnification images (Figure 4B) show many elongated and filamentous bacterial cells connected to each other on the Au-GF electrode. In a very small region, we also observed elongated bacteria at −0.6 V on the unmodified GF (Figure S5A, SI). However, the lengths of the bacteria looked shorter than those shown in Figure 4B, and the bacteria were hardly detected in other areas. Instead, we occasionally observed bacteria with many nanowires when the bacteria were incubated at −0.6 V (Figure S5B), as in the case with no electricity (Figure S5C) and at −0.1 V (Figure S5D).


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Figure 4. (A) SEM image showing adsorption pattern of S. oneidensis MR-1::pJL23 on Au-GF electrode after electricity-assisted (−0.6 V) incubation of bacteria for 50 h. (B) A magnified SEM image showing elongated, wire-type bacteria.

We observed a number of interesting bacterial adsorption patterns (Figure 5) on the NH2-AuGF electrodes. The elongated bacteria were still found, but the thickness of the biofilm and the frequency of finding elongated bacteria seemed higher than with Au-GF (Figure 5A). The long bacteria overlapped with each other in a disorderly manner (Figure 5B). Another region showed an even thicker biofilm, inside of which filamentous bacteria were interwoven (Figure 5C) and constituted web-like structures (Figure 5D). We also observed a pile-up of filamentous bacteria (Figure 5E and F).

Figure 5. (A, C, E) SEM images showing adsorption patterns and (B, D, F) corresponding magnified images of S. oneidensis MR-1::pJL23 on sequentially modified GF with Au islands and cysteamine, after electricity-assisted (−0.6 V) incubation of bacteria for 50 h. Elongated and wire-type bacteria were (B, F) connected and attached to the electrode surface and (D) interwoven to form a biofilm.


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Elongated and filamentous bacterial cells have been reported for Desulfobulbaceae48 and S. oneidensis MR-1.49 It was also demonstrated that such elongated bacteria were effective at transporting electrons.48,49 According to Ref. 49, the elongated bacteria were found in the presence of cisplatin, and they showed enhanced electric current densities compared with the normal cells without any treatment. Meanwhile, our results might indicate that the change in the surface composition/nanomaterials of the GF electrode (Au and Au-NH2) could increase the population of elongated S. oneidensis MR-1::pJL23 on the electrode surfaces. To clarify this, we observed the bacteria adsorption patterns on the Au-GF (Figure 6A and B) and NH2-Au-GF electrodes (Figure 6C and D) after 50 h of incubation of the bacteria under the no electricity supply condition. In general, compared with Figure 1A (the unmodified GF electrode and no electricity), more elongated bacteria were found on the modified electrodes. The average size of the bacteria shown in Figure 1A was 0.56 µm in width × 1.50 µm in length. According to the literature, most Shewanella have a size of 0.66 µm × 1.89 µm.50 Meanwhile, the sizes in Figure 6B and D are 0.56 µm × 3.23 µm and 0.39 µm × 3.03 µm, respectively. These results indicate that the electrode surface composition could greatly affect the morphology of the bacteria. As for the electricity effect when using the Au-GF and NH2-Au-GF electrodes, the amounts of bacteria adsorbed on the electrode surfaces appeared to be much higher when incubating under an electricity supply at −0.6 V (Figures 4 and 5) compared to the case without electricity (Figure 6). In addition, the elongated bacteria looked longer when incubating under an electricity supply compared to the bacteria under no electricity.


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Figure 6. SEM images showing adsorption patterns of S. oneidensis MR-1::pJL23 on electrodes of (A, B) Au-GF and (C, D) NH2-Au-GF, after bacterial incubation for 50 h under no electricity supply.

To investigate the impact of elongated bacteria, we analyzed the CA and CV results shown in Figures 3C, 3D, S3, and S4. The CA results indicated that the Au-GF electrodes consumed a greater average current (−0.36 ± 0.12 µA/cm2), 20% more than the unmodified GF electrode (−0.30 ± 0.06 µA/cm2), although the bacterial density looked similar (Figure 1C vs. Figure 4A). Meanwhile, according to the CV analysis of the biofilm-coated Au-GF electrode, the total and reductive charges were significantly increased, by 1.98- and 2.96-fold, respectively, compared with the biofilm-coated GF electrode. Nonetheless, caution is needed before attributing this increase solely to the effect of the elongated bacteria because the contribution of conductive Au on the GF electrode must also be considered. On the other hand, the Au-GF and NH2-Au-GF electrodes had similar bacterial morphological features, but the density of elongated bacteria seemed to be greater on the NH2-Au-GF electrode. During the incubation of bacteria at −0.6 V, the average current consumed by NH2-Au-GF (−0.49 ± 0.07 µA/cm2) was 36% higher than that


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of the Au-GF electrode. According to the CV analysis of the biofilm-coated NH2-Au-GF electrode, the total charges (8.00 ± 0.03 C) were lower than the Au-GF (8.91 ± 0.02 C), but the reductive charge had slightly increased by 6% (2.64 ± 0.06 vs. 2.49 ± 0.24 C for Au-GF). As a result, the ratio of the reduction to total charge increased from 0.28 to 0.33, inferring that the number of elongated bacteria might be helpful for improving the reductive electrochemical performance.

Figure 7. (A) Schematic of hierarchical structure of GF, where GF was sequentially modified with Au islands, cysteamine, and Au NPs (coded as Au NP-NH2-Au-GF). (B) SEM image showing the morphology of the Au NP-NH2-Au-GF electrode. (C) Chronoamperometry during the electricity-assisted (−0.6 V vs. Ag/AgCl) incubation of the bacteria with Au NP-NH2-Au-GF and NH2-Au-GF electrodes. (D) The average reductive charges (n = 3) estimated by the CV analysis for the two biofilm-coated electrodes (including Figure S8, SI) obtained after the electricity-assisted (−0.6 V) incubation of the bacteria for 50 h. The table in the inset shows the ratios of the reduction to the total charge for the three electrodes. (E, F) Low- and (G) highmagnification SEM images showing the adsorption patterns on the Au NP-NH2-Au-GF after the


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electricity-assisted (−0.6 V) incubation of the bacteria for 50 h. Elongated, wire-type bacteria inter-connected with each other were still present in the biofilms.

Because the isobutanol concentration was still low, we further explored strategies to increase it. These strategies focused on developing structures both to maintain a high adsorption pattern for bacteria and to further increase the electrochemical efficiency at the bacteria–electrode interfaces. After numerous trials, we found that the introduction of Au NPs onto the surface of NH2-Au-GF could satisfy the two requirements (Figure 7A). Au NPs are known to exchange electrons with metal-containing enzymes and bacteria at their interfaces.51–55 In terms of catalysis, the electron transfer ability is generally increased with a decrease in the size of Au NPs.56 Thus, we selected tiny Au NPs (15 nm in diameter). The present Au NPs were stabilized with citrate ions (negatively charged) and attached to the surface of NH2-Au-GF (positively charged) by electrostatic attraction or a coordination reaction between an amine and Au. The Au NPs were partially deposited on the surface to maintain the high adsorption of bacteria to the electrode surfaces (Figure 7B, see also Figure S6 in SI). We called this electrode “Au NP-NH2Au-GF.” It should be noted that at −0.6 V, the isobutanol concentration approximately doubled from 14 ± 2 mg/L (n = 8) with NH2-Au-GF to 27 ± 2 mg/L (n = 9) with the Au NP-NH2-Au-GF. According to the CA (Figure 7C), the average consumed current (n = 5) was −0.53 ± 0.05 µA/cm2, which was slightly higher than that of the NH2-Au-GF electrode (−0.49 ± 0.07 µA/cm2). Instead, a large amount of current was consumed during the first 30 h of bacterial incubation, and multiple peaks were observed in the profile, indicating that several electrochemical events occurred at this electrode at −0.6 V. We analyzed the CV results (shown in Figure S7, SI) of the biofilm-coated NH2-Au-GF and Au NP-NH2-Au-GF electrodes (Figure 7D). The total charge decreased by 3% from 8.00 ± 0.03 to 7.74 ± 0.32 C. However, this was the result of a decrease in


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the oxidative charge (from 5.36 ± 0.49 to 4.99 ± 0.08 C). The reductive charges were slightly increased (from 2.64 ± 0.06 to 2.75 ± 0.23 C). Therefore, the large increase in the production of isobutanol might have been due to the increase in the ability of the electrode or biofilm-coated electrode to preferentially drive reductive electrochemical events at −0.6 V. Based on the SEM results for the Au NP-NH2-Au-GF electrode after 50 h of operation (Figure 7E–G), the morphology of the elongated bacteria was not very different from the morphology of the bacteria on the NH2-Au-GF electrode. We easily detected thick biofilms (Figure 7E and F), which resulted from the interweaving of the elongated bacteria (Figure 7G). In particular, the bacteria started forming a continuous film on top of the networks of filamentous bacteria. Therefore, the Au NP-NH2-Au-GF electrode not only induced the formation of a thick biofilm but also selectively facilitated the electron exchange, at a specific electrode potential, between the electrode and bacteria/biofilm for enhanced isobutanol production. We also compared Figure 7E–G with the morphology of the Au NP-NH2-Au-GF electrode under no electricity supply (Figure S8, SI). The morphology of the bacteria appeared to be less influenced by the electricity: the bacteria were also very elongated even when they were incubated without the electricity supply. Instead, the amount of bacteria looked influenced by the electricity. Putting Figures 1, 4–6, 7E–G, and S8 together, it seems that a change in the electrode structure could alter the morphology of S. oneidensis MR-1::pJL23, and the electricity supply could affect the amount of bacteria adsorbed on the electrode surface. Meanwhile, the effect of electricity on the morphology of S. oneidensis MR-1::pJL23 was dependent on the electrode structure.


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Since the SEM results indicated that the morphologies of the electrodes coated with bacteria were very different depending on the electrode type, we also could not rule out the possibility that the electrode structures themselves contributed to the enhancement of the isobutanol production: it has been known that the adsorption patterns can greatly affect the functioning of bacteria.36–38 Thus, we compared the bacterial isobutanol production values without electricity and at −0.6 V when the bacteria were incubated for 50 h with the unmodified GF, NH2-Au-GF, or Au NP-NH2-Au-GF (Figure 8A). Without electricity, the isobutanol concentrations increased from 2.9 ± 1 mg/L with the unmodified GF to 11 ± 0.3 mg/L (n = 2) for NH2-Au-GF and 12 ± 2 mg/L (n = 3) with the Au NP-NH2-Au-GF electrode. From the results, it is interesting to discuss the contribution of the electricity to the isobutanol production, as summarized in Figure 8B. The unmodified GF and NH2-Au-GF electrodes produced similar increases in the isobutanol concentration of 3 mg/L, respectively, when the bacteria were incubated at −0.6 V. In contrast, the effect of electricity was very significant with the Au NP-NH2-Au-GF: it increased production by 15 mg/L. Therefore, using Au NPs on the surface of the electrode may have effectively enhanced the contribution of electricity to the bacterial isobutanol productivity.


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Figure 8. (A) Comparison of isobutanol concentration produced by S. oneidensis MR-1::pJL23 with unmodified GF, NH2-Au-GF, and Au NP-NH2-Au-GF electrodes with (−0.6 V vs. Ag/AgCl) and without electricity. (B) The effect of electricity on the isobutanol concentration produced by the bacteria with the three electrodes. (C) A SEM image showing the morphology of the biofilm formed on the Au NP-NH2-Au-GF electrode. (D) A time-dependent profile of the isobutanol concentrations produced by the bacteria with the Au NP-NH2-Au-GF electrode during the electricity-assisted (−0.6 V) incubation of the bacteria for 96 h (n = 5).

We further monitored the isobutanol concentration profile during 96 h of bacterial incubation with the Au NP-NH2-Au-GF electrodes at −0.6 V. We noticed a thick and continuous biofilm (Figure 8C), in contrast to the images in Figure 7E and F. Figure 8D shows the profile of the isobutanol concentration with the bacterial incubation time. At 96 h, the amount reached 72 ± 2 mg/L (n = 5). Compared with the previous result obtained for 96 h of incubation without electricity (15.3 mg/L) and at −0.1 V with carbon electrodes (19.3 mg/L),20 the isobutanol concentration was enhanced 4.7- and 3.7-fold, respectively.


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It is worth discussing from Figure 8D that the slope of the profile increased after 72 h. We further investigated the profiles of the isobutanol concentrations with the NH2-Au-GF and AuGF electrodes over a period of 96 h (Figure S9A). For all the modified electrodes, the isobutanol concentrations were still increasing even when the incubation time was greater than 50 h, and the slopes increased around 50 h regardless of the electrode type. The profiles were different from those observed for 96 h of incubation without electricity and at −0.1 V with carbon electrodes.20 Regarding the adsorption patterns, we compared Figures 4 (Au-GF), 5 (NH2-Au-GF), and 6E–G (Au NP-NH2-Au-GF) with Figures 8C and S9B, C. More bacteria seemed to be adsorbed on the surfaces of the modified electrodes after 96 h of operation than the amount observed after 50 h (Figures 4 and 5). Therefore, we speculated that the different bacterial adsorption behavior or different amount of bacteria, as one of the reasons, could affect the productivity of the bacteria. However, to obtain a clearer explanation, further careful investigations should be investigated in more detail because there should be many factors involved in the results. Equally importantly, it is worth stressing that the concentrations after 96 h were 31 mg/L and 57 mg/L with the Au-GF and NH2-Au-GF, respectively. These values were lower than the concentration with the Au NP-NH2-Au-GF electrode, but still 1.6 and 2.9 times higher than the value at −0.1 V with the unmodified carbon electrode (19.3 mg/L).20 The average reduction currents during the incubation for 96 h were −0.59 ± 0.02 µA/cm2 (n = 5), −0.52 µA/cm2, and −0.40 µA/cm2 with the Au NPs-NH2-Au-GF, NH2-Au-GF, and Au-GF, respectively. The trends were in line with those obtained during the 50 h of incubation (−0.53 ± 0.05 µA/cm2 vs. −0.49 ± 0.07 µA/cm2 vs. −0.36 ± 0.12 µA/cm2). As for the biofilm formation (Figure S9B, C), the Au-GF and NH2-Au-GF electrodes were completely covered with the elongated bacteria after


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96 h of incubation. However, the film on the surface of the Au NPs-NH2-Au-GF electrode seemed to be much thicker than those on the Au-GF and NH2-Au-GF electrodes. Therefore, it could be said that the trends regarding the effects of the electrode structure on the isobutanol concentration, average current consumed, and bacterial adsorption pattern were unchanged even when the operation time was extended from 50 h to 96 h. Moreover, all the modified electrodes seemed to allowed bacteria to stably produce isobutanol even after a long period of incubation. Lastly, we calculated the coulombic efficiencies, the surface area based and volumetric production rates from the experimental results by referring to literatures (Table S1).57−59 For S. oneidensis MR-1::pJL23, the optimized Au NP-NH2-Au-GF electrode increased the coulombic efficiency from 25 % (the unmodified GF) to 66 % at 50 h operation, and further increased to 81 % at 96 h operation. In addition, we extended the operation time to 168 h with the Au NPNH2-Au-GF electrode (Figure S10, SI). The isobutanol concentration was also increased to 79 ± 1 mg/L (n = 2) at 120 h, but the concentrations were increased only slightly to 80 ± 0.5 mg/L (n = 2) and 81 ± 1 mg/L (n = 2) at 144 h and 168 h, respectively. Therefore, continual future efforts about improving the performance of present electrode are still required not only to increase the columbic efficiencies and thus isobutanol productivity with this bacterium, but also to maintain a high productivity of isobutanol for longer periods of time.

4. CONCLUSIONS In this study, we found that the isobutanol productivity of S. oneidensis MR-1::pJL23 could be enhanced by identifying the optimal electrode potential, and it was further improved when we sequentially modified the GF surface with Au nanoislands, cysteamine, and Au NPs. The change


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in the electrode surface structure altered both the amount and shape of the bacteria (e.g., elongated bacteria) adsorbed on the electrode, which were further altered when electricity was supplied. We also found that a change in the electrode’s nanostructure itself altered the bacterial isobutanol productivity when operating without electricity. The present optimized Au NP-NH2Au-GF electrode allowed S. oneidensis MR-1::pJL23 to enhance the production isobutanol for a long period of time, and it had effectively enhanced the contribution of electricity to the bacterial isobutanol productivity. In this sense, our study could be very useful to researchers who want to understand how the electrode surface structure affects the biofuel productivity of bacteria that can exchange electrons with electrodes via various routes, as S. oneidensis MR-1 does. There have been discussions about the cost-accounting of an electricity-supplied biochemical production system. A literature by Rabaey et al.60 reported in 2011 that the costs of the electrode materials, membranes, current collectors, and electricity supply should be overcome by a large increase in the productivity of biochemical compounds. Since then, among many efforts, an advance in the electrode fabrication technology made it possible to increase acetate production.14,15 The present optimized Au NP-NH2-Au-GF electrode also demonstrated the possibility that an electrode design can significantly enhance the bacterial productivity of isobutanol, which is one of the attractive biofuels for replacing fossil fuels.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI:. It contains CVs for the bacteria-coated unmodified GF obtained under various electrode potentials, CVs for the bacteria-coated Au-GF, NH2-Au-GF, and Au NP-NH2-Au-GF electrodes


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at −0.6 V, analysis of CV results, CA of the unmodified GF electrode monitored at −0.6 V, isobutanol concentration profiles during the electricity-assisted incubation of bacteria for 96 h with the three modified electrodes, an isobutanol concentration profile during the electricityassisted incubation of bacteria for 168 h with the Au NP-NH2-Au-GF electrodes, a table of calculation for Coulombic efficiencies, volumetric production rates, and surface based production rates and SEM images associated with the main text.

■ AUTHOR INFORMATION Corresponding Author *(Y. H. Yang) E-mail: [email protected] *(E. C. Cho) E-mail: [email protected] Author Contributions J.A L. and J.M.J. contributed equally to this work. Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENT The authors acknowledge the financial support from a KETEP grant (20133030000300) by the Ministry of Trade, Industry, and Energy of Korea. J.A L. and E.C.C. acknowledge the financial support from a research grant (NRF-2015R1A2A2A01007003) from the National Research Foundation of Korea (NRF).


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