Kinetic, Electrochemical, and Microscopic Characterization of the Thermophilic, Anode-Respiring Bacterium <i>Thermincola ferriacetica</i>

<i>Thermincola ferriacetica</i> is a recently isolated thermophilic, dissimilatory Fe­(III)-reducing, Gram-positive bacterium with capability to generate electrical current via anode respiration. Our goals were to determine the maximum rates of anode respiration by <i>T. ferriacetica</i> and to perform a detailed microscopic and electrochemical characterization of the biofilm anode. <i>T. ferriacetica</i> DSM 14005 was grown at 60 °C on graphite-rod anodes poised at −0.06 V (vs) SHE in duplicate microbial electrolysis cells (MECs). The cultures grew rapidly until they achieved a sustained current density of 7–8 A m^–2 with only 10 mM bicarbonate buffer and an average Coulombic Efficiency (CE) of 93%. Cyclic voltammetry performed at maximum current density revealed a Nernst–Monod response with a half saturation potential (<i>E</i>_KA) of −0.127 V (vs) SHE. Confocal microscopy images revealed a thick layer of actively respiring cells of <i>T. ferriacetica</i> (∼38 μm), which is the first documentation for a gram positive anode respiring bacterium (ARB). Scanning electron microscopy showed a well-developed biofilm with a very dense network of extracellular appendages similar to <i>Geobacter</i> biofilms. The high current densities, a thick biofilm (∼38 μm) with multiple layers of active cells, and Nernst–Monod behavior support extracellular electron transfer (EET) through a solid conductive matrix – the first such observation for Gram-positive bacteria. Operating with a controlled anode potential enabled us to grow <i>T. ferriacetica</i> that can use a solid conductive matrix resulting in high current densities that are promising for MXC applications

Kinetic, Electrochemical, and Microscopic Characterization of the Thermophilic, Anode-Respiring Bacterium <i>Thermincola ferriacetica</i>

<i>Thermincola ferriacetica</i> is a recently isolated thermophilic, dissimilatory Fe­(III)-reducing, Gram-positive bacterium with capability to generate electrical current via anode respiration. Our goals were to determine the maximum rates of anode respiration by <i>T. ferriacetica</i> and to perform a detailed microscopic and electrochemical characterization of the biofilm anode. <i>T. ferriacetica</i> DSM 14005 was grown at 60 °C on graphite-rod anodes poised at −0.06 V (vs) SHE in duplicate microbial electrolysis cells (MECs). The cultures grew rapidly until they achieved a sustained current density of 7–8 A m^–2 with only 10 mM bicarbonate buffer and an average Coulombic Efficiency (CE) of 93%. Cyclic voltammetry performed at maximum current density revealed a Nernst–Monod response with a half saturation potential (<i>E</i>_KA) of −0.127 V (vs) SHE. Confocal microscopy images revealed a thick layer of actively respiring cells of <i>T. ferriacetica</i> (∼38 μm), which is the first documentation for a gram positive anode respiring bacterium (ARB). Scanning electron microscopy showed a well-developed biofilm with a very dense network of extracellular appendages similar to <i>Geobacter</i> biofilms. The high current densities, a thick biofilm (∼38 μm) with multiple layers of active cells, and Nernst–Monod behavior support extracellular electron transfer (EET) through a solid conductive matrix – the first such observation for Gram-positive bacteria. Operating with a controlled anode potential enabled us to grow <i>T. ferriacetica</i> that can use a solid conductive matrix resulting in high current densities that are promising for MXC applications

pH Dependency in Anode Biofilms of <i>Thermincola ferriacetica</i> Suggests a Proton-Dependent Electrochemical Response

Monitoring the electrochemical response of anode respiring bacteria (ARB) helps elucidate the fundamental processes of anode respiration and their rate limitations. Understanding these limitations provides insights on how ARB create the complex interfacing of biochemical metabolic processes with insoluble electron acceptors and electronics. In this study, anode biofilms of the thermophilic (60 °C) Gram-positive ARB <i>Thermincola ferriacetica</i> were studied to determine the presence of a proton-dependent electron transfer response. The effects of pH, the presence of an electron donor (acetate), and biofilm growth were varied to determine their influence on the electrochemical midpoint potential (<i>E</i>_KA) and formal redox potential (<i>E</i>°′) under nonturnover conditions. The <i>E</i>_KA and <i>E</i>°′ are associated with an enzymatic process within ARB’s metabolism that controls the rate and energetic state of their respiration. Results for all conditions indicate that pH was the major contributor to altering the energetics of <i>T. ferriacetica</i> anode biofilms. Electrochemical responses measured in the absence of an electron donor and with a minimal proton gradient within the anode biofilms resulted in a 48 ± 7 mV/pH unit shift in the <i>E</i>°′, suggesting a proton-dependent rate-limiting process. Given the limited energy available for anode respiration (<200 mV when using acetate as electron donor), our results provide a new perspective in understanding proton-transport limitations in ARB biofilms, one in which ARB are thermodynamically limited by pH gradients. Since the anode biofilms of all ARB that perform direct extracellular electron transfer (EET) investigated thus far exhibit an <i>n</i> = 1 Nernstian behavior, and because this behavior is affected by changes in pH, we hypothesize that the Nernstian response is associated with membrane proteins responsible for proton translocation. Finally, this study shows that the <i>E</i>_KA and <i>E</i>°′ are a function of pH within the physiological range of ARB, and thus, given the significant effect pH has on this parameter, we recommend reporting the <i>E</i>_KA and <i>E</i>°′ of ARB biofilms at a specific bulk pH

Characterization of Electrical Current-Generation Capabilities from Thermophilic Bacterium Thermoanaerobacter pseudethanolicus Using Xylose, Glucose, Cellobiose, or Acetate with Fixed Anode Potentials

<i>Thermoanaerobacter pseudethanolicus</i> 39E (ATCC 33223), a thermophilic, Fe­(III)-reducing, and fermentative bacterium, was evaluated for its ability to produce current from four electron donorsxylose, glucose, cellobiose, and acetatewith a fixed anode potential (+ 0.042 V vs SHE) in a microbial electrochemical cell (MXC). Under thermophilic conditions (60 °C), <i>T. pseudethanolicus</i> produced high current densities from xylose (5.8 ± 2.4 A m^–2), glucose (4.3 ± 1.9 A m^–2), and cellobiose (5.2 ± 1.6 A m^–2). It produced insignificant current when grown with acetate, but consumed the acetate produced from sugar fermentation to produce electrical current. Low-scan cyclic voltammetry (LSCV) revealed a sigmoidal response with a midpoint potential of −0.17 V vs SHE. Coulombic efficiency (CE) varied by electron donor, with xylose at 34.8% ± 0.7%, glucose at 65.3% ± 1.0%, and cellobiose at 27.7% ± 1.5%. Anode respiration was sustained over a pH range of 5.4–8.3, with higher current densities observed at higher pH values. Scanning electron microscopy showed a well-developed biofilm of <i>T. pseudethanolicus</i> on the anode, and confocal laser scanning microscopy demonstrated a maximum biofilm thickness (<i>L</i>_f) greater than ∼150 μm for the glucose-fed biofilm