Effect of Electrode Spacing on Electron Transfer and Conductivity of Geobacter Sulfurreducens Biofilms
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Bioelectrochemistry 131 (2020) 107395 Contents lists available at ScienceDirect Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem Effect of electrode spacing on electron transfer and conductivity of Geobacter sulfurreducens biofilms Panpan Liu a,b, Abdelrhman Mohamed b,PengLianga, Haluk Beyenal b,⁎ a State Key Joint Laboratory of Environment Simulation and Pollution Control School of Environment, Tsinghua University, Beijing 100084, PR China b The Gene and Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99163, USA article info abstract Article history: To understand electron transport in electrochemically active biofilms, it is necessary to elucidate the heteroge- Received 15 April 2019 neous electron transport across the biofilm/electrode interface and in the interior of G. sulfurreducens biofilms Received in revised form 12 September 2019 bridging gaps of varying widths. The conductivity of Geobacter sulfurreducens biofilm bridging nonconductive Accepted 13 September 2019 gaps with widths of 5 µm, 10 µm, 20 µm and 50 µm is investigated. Results of electrochemical gating measure- Available online 4 October 2019 ment show that biofilm conductivity peaks at the potential of −0.35 V vs. Ag/AgCl. The biofilm conductivity in- creases with gap width (10.4 ± 0.2 µS cm−1 in 5 µm gap, 13.3 ± 0.2 µS cm−1 in 10 µm gap, 16.7 ± 1.4 µS cm−1 in Keywords: −1 Electrochemically active biofilm 20 µm gap and 41.8 ± 2.02 µS cm in 50 µm gap). These results revealed that electron transfer in Exoelectrogens G. sulfurreducens biofilm is a redox-driven. In addition, higher biofilm conductivities and lower charge transfer Biofilm conductivity resistances are observed in all gaps under a turnover condition than in those under a non-turnover condition. Extracellular electron transfer Our results offer insights into the spatial heterogeneity of biofilm structure and extracellular electron transfer Microbial electrochemistry and spatial in electrochemically active biofilms. heterogeneity © 2019 Elsevier B.V. All rights reserved. 1. Introduction as a multicell EAB forms on an electrode, electrons produced by exoelectrogens far from the electrode surface must span the multi- Exoelectrogens are microorganisms that can oxidize organic mat- cell, layered biofilm. The diverse electrochemically active constitu- ter and use an inert electrode as an electron acceptor [1].Thistrait ents in an EAB make the EET in biofilm challenging to measure allows their use as live catalysts in bioelectrochemical systems, such directly and analyze over long distances. as microbial fuel cells, microbial desalination cells, microbial biosen- Long-distance electron transport in biofilms was previously pro- sors and microbial electrolysis cells [2–5].Ingeneral,exoelectrogens posed to be mediated by electrochemically active constituents including in a bioelectrochemical system colonize the electrode surface and outer membrane cytochrome, pili, and extracellular polymeric sub- form an electrochemically active biofilm (EAB) which catalyzes the stances [8–11]. As the adaptation to the local microenvironment and substrate consumption reaction and delivers the metabolic electrons the production of electrochemically active constituents by to the electrode. The efficiency of electron transfer in an EAB deter- exoelectrogens vary with the distance from the electrode surface, their mines the performance of the bioelectrochemical system [6].Deter- abundance is nonuniform throughout the biofilm [12]. As a result, mining the electron transport rates in biofilms can advance the EABs develop a heterogeneous spatial structure and the characteristics application of bioelectrochemical systems. It is well established of electron transport in biofilm are dependent on the distance from that extracellular electron transfer (EET) performed by model the electrode surface [13,14]. exoelectrogen Geobacter sulfurreducens is mediated by three differ- Biofilm conductivity was previously measured to indicate long- ent electrochemically active cellular components: outer membrane range electron transfer in biofilm [15,16]. Previous studies measured cytochromes, conductive pili and electron shuttles [7,8]. However, the biofilm conductance of mixed-culture and G. sulfurreducens biofilms using split electrodes with 50–µm to 1–mm nonconductive gaps in var- ious microbial fuel cells [17,18]. While these results emphasized that biofilms can conduct electrons spanning these nonconductive distances, ⁎ Corresponding author. they did not reveal whether the biofilm conductivity depended on the E-mail address: [email protected] (H. Beyenal). nonconductive gap width [8,17,19,20]. Moreover, there was a lack of https://doi.org/10.1016/j.bioelechem.2019.107395 1567-5394/© 2019 Elsevier B.V. All rights reserved. 2 P. Liu et al. / Bioelectrochemistry 131 (2020) 107395 comprehensive comparison of electron transport over different dis- reactor was operated in batch mode. The split gold electrode, an tances. Recent research found heterogeneous electron transport across Ag/AgCl electrode and a graphite rod were used as the working the biofilm is highly correlated with structure in G. sulfurreducens bio- electrode, reference electrode and counter electrode, respectively. film using the interdigitated array electrode [21]. The gap width of 5 We used one split electrode which included all the gaps as a single µm was too short to reveal the effect of distance on the electron trans- working electrode (Fig. S1). However, during the measurements, port in biofilm which had a thickness more than 50 µm. we only used the given gap distance. The growth medium did not To understand electron transport in EAB, it is necessary to elucidate contain any electron acceptor, and only 20 mM sodium acetate the heterogeneous electron transport across the biofilm/electrode inter- was used as the electron donor. The conductivity of the fresh face and in the interior of G. sulfurreducens biofilm by bridging gaps of growth medium was 5.11 mS cm−1. varying widths. We expect that this information will explain the depen- dence of electron transfer processes in biofilms on a variety of noncon- ductive gap widths. 2.3. Biofilm conductance measurement The goal of this research was to investigate the conductivity and electron transport of G. sulfurreducens biofilm by varying electrode spac- In the process of biofilm formation on the electrode, electrochem- ing. A split gold electrode with nonconductive gaps with widths of 5 µm, ical measurements to characterize the biofilms were performed using 10 µm, 20 µm and 50 µm was fabricated, and model exoelectrogen a potentiostat (Gamry Instruments Inc. G300™). Before each measure- G. sulfurreducens biofilm was grown on it until the turnover current ment, the working electrode was disconnected for 60 min to allow it reached a steady state and the biofilm bridged the nonconductive to reach a steady state. Cyclic voltammetry (CV) was conducted on gaps. Biofilm conductivities were measured across these gaps and re- the working electrode. The scan window ranged from −0.7 V vs. Ag/ lated to the gap widths. Bipotentiostat electrochemical gating measure- AgCl to +0.4 V vs. Ag/AgCl at a rate of 10 mV s−1.Thefirst derivative ments were performed to examine the change of biofilm conductivity of CV was obtained by plotting the slope of each CV point against the with the electrode potential. scan potential. To measure the conductance of the biofilm, a voltage difference (VSD, 0 mV, 25 mV, or 50 mV) was applied to the gaps. 2. Methods and materials For each voltage, a long period of 5 min was applied to allow the tran- sient current to decay. Current (ISD) was recorded every second over 2.1. Fabrication of the split gold electrode the 5–min period, and the conductance of the biofilm was calculated using the values ISD with VSD. For Electrochemical Impedance Spec- A split gold electrode with nonconductive gaps of 5 μm, 10 μm, 20 troscopy (EIS) measurement of the biofilm at the various gaps, one μmand50μm was designed to perform in situ measurement of biofilm side of the gap (source electrode) was used as the working electrode, conductivity. The electrode fabrication was adapted from a previous and the other side (drain electrode) was used as the reference and study [8]. Briefly, electrodes with different nonconductive gaps were counter electrode. The open circuit potential was used as the DC po- prepatterned on a Si/SiO2 wafer and then defined using electron-beam tential, and the AC potential was 10 mV rms. The frequency range lithography (EBL). A layer of 20 nm of Ti and 200–nm Au was spurted was from 100 mHz to 100,000 Hz with 10 points of data acquisition. onto the electrodes by evaporation. The whole electrode was 2 cm Zview software (Scribner Associates Inc., Southern Pines, NC, USA) long and about 5 cm wide (Fig. S1). Electrodes were wired with copper was used to analyze biofilm impedance obtained from EIS results. conductor by soldering them together with 60:40 tin/lead solder. The For the measurement of each gap, a period of 30 min was used to re- connecting joints were covered with silicone to prevent corrosion due cover the current production of the biofilm. to exposure of solder to growth medium. Before being placed into a re- actor, the split electrode was soaked in 3% acetone for 30 min, followed by rinsing with ethanol for 1 h to clean the surface, then rinsed with de- 2.4. Electrochemical gating measurements to estimate biofilm conductivity ionized water. at various applied gating potentials 2.2. G.