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We investigated the role of green sulfur bacteria inlight-responsive electricity generation in microbial electrochemical cells (MXCs). We operated MXCs containing either monocultures or defined cocultures of previously enriched phototrophic Chlorobium and anode-respiring Geobacter under anaerobic conditions in the absence of electron donor. Monoculture control MXCs containing Geobacter or Chlorobium neither responded to light nor produced current, respectively. Instead, light-responsive current generation occurred only in coculture MXCs. Current increased above background levels only in the dark and declined slowly over 96 h. This pattern suggested that Chlorobium exhausted intracellular glycogen reserves via dark fermentation to supply an electron donor, presumably acetate, to Geobacter. With medium containing sulfide as the sole photosynthetic electron donor, current generation had a similar and reproducible negative light response. To investigate whether this metabolic interaction also occurred without an electrode, we performed coculture experiments in batch serum bottles. In this setup, sulfide served as the sole electron donor, whose oxidation by Chlorobium was required to provide S0 as the electron acceptor to Geobacter. Copies of Geobacter 16S rDNA increased approximately 14-fold in batch bottle cocultures containing sulfide compared to those lacking sulfide, and did not decline after termination of sulfide feeding. These results suggest that products of both photosynthesis and dark fermentation by Chlorobium were sufficient both to yield an electrochemical response by Geobacter biofilms, and to promote Geobacter growthin batch cocultures. Our work expands upon the fusion of MXCs with coculture techniques and reinforces the utility of microbial electrochemistry for sensitive, real-time monitoring of microbial interactions in which a metabolic intermediate can be converted to electrical current. Biotechnol. Bioeng. 2014;111: 223–231. © 2013 Wiley Periodicals, Inc.

Thermincola ferriacetica 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 T. ferriacetica and to perform a detailed microscopic and electrochemical characterization of the biofilm anode. T. ferriacetica 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 (EKA) of -0.127 V (vs) SHE. Confocal microscopy images revealed a thick layer of actively respiring cells of T. ferriacetica (~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 Geobacter 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 T. ferriacetica that can use a solid conductive matrix resulting in high current densities that are promising for MXC applications.

When anode-respiring bacteria (ARB) respire electrons to an anode in microbial electrochemical cells (MXCs), they harvest only a small amount of free energy. This means that ARB must have a high substrate-oxidation rate coupled with a high ratio of electrons used for respiration compared to total electrons removed by substrate utilization. It also means that they are especially susceptible to inhibition that slows anode respiration or lowers their biomass yield. Using several electrochemical techniques, we show that a relatively high total ammonium-nitrogen (TAN) concentration (2.2 g TAN/L) induced significant stress on the ARB biofilms, lowering their true yield and forcing the ARB to boost the ratio of electrons respired per electrons consumed from the substrate. In particular, a higher respiration rate, measured as current density (j), was associated with slower growth and a lower net yield, compared to an ARB biofilm grown with a lower ammonium concentration (0.2 g TAN/L). Further increases in influent TAN (to 3 and then to 4.4 g TAN/L) caused nearly complete inhibition of anode respiration. However, the ARB could recover from high-TAN inhibition after a shift of the MXC's feed to 0.2 g TAN/L. In summary, ARB biofilms were inhibited by a high TAN concentration, but could divert more electron flow toward anode respiration with modest inhibition and recover when severe inhibition was relieved. Biotechnol. Bioeng. 2017;114: 1151-1159. © 2017 Wiley Periodicals, Inc.

As interactive electronics become increasingly intimate and personal, the design of circuitry is correspondingly developing a more playful and creative aesthetic. Circuit sketching and design is a multidimensional activity which combines the arts, crafts, and engineering broadening participation of electronic creation to include makers of diverse backgrounds. In order to support this design ecology, we present Ellustrate, a digital design tool that enables the functional and aesthetic design of electronic circuits with multiple conductive and dielectric materials. Ellustrate guides users through the fabrication and debugging process, easing the task of practical circuit creation while supporting designers' aesthetic decisions throughout the circuit authoring workflow. In a formal user study, we demonstrate how Ellustrate enables a new electronic design conversation that combines electronics, materials, and visual aesthetic concerns.

A microbial peroxide producing cell (MPPC) for H2 O2 production at the cathode was systematically optimized with minimal energy input. First, the stability of H2 O2 was evaluated using different catholytes, membranes, and catalyst materials. On the basis of these results, a flat-plate MPPC fed continuously using 200 mm NaCl catholyte at a 4 h hydraulic retention time was designed and operated, producing H2 O2 for 18 days. H2 O2 concentration of 3.1 g L-1 H2 O2 with 1.1 Wh g-1 H2 O2 power input was achieved in the MPPC. The high H2 O2 concentration was a result of the optimum materials selected. The small energy input was largely the result of the 0.5 cm distance between the anode and cathode, which reduced ionic transport losses. However, >50 % of operational overpotentials were due to the 4.5-5 pH unit difference between the anode and cathode chambers. The results demonstrate that a MPPC can continuously produce H2 O2 at high concentration by selecting compatible materials and appropriate operating conditions.

Advances in digital fabrication (DF) technologies are making it easier to produce high-fidelity replicas of digital designs. However, this push-to-print paradigm limits the creative opportunities that arise from the process of "working through a material" which involves risk, uncertainty, and serendipitous discovery. We investigate how DF artifacts can function as static intermediary tools, which we term proxies, to support crafting practice. We focus on the wire-wrapping process where physical wire is bent into complex shapes and build DF fixtures to aid with construction and fabrication. We explore how these proxies can be generated to provide users with different levels-of-assistance and evaluate how these proxies affect the making process. We show that our proxies affect quality and speed and yield different making experiences between novice and expert craftspeople. We derive design principles to inform future proxy design and discuss how approaches such as ProxyPrint that are designed aware of the medium can create more engaging making tools that can embed tacit knowledge, encourage creativity, and sustain crafting practice.

LiveObjects approaches expressive object design from the lens of art theory utilizing theatricality, or the perception of an object having presence, to create a series of IoT data objects. This approach actuates materials and everyday objects to express social data and provokes new interactions between objects, viewers, and space. We showcase how such objects can form unique expressive personalities, expose relational data, and perturb environments to form information spaces.

Traditional HCI goals like efficiency and ease-of-use, while important, are not sufficient for digital technology to function as an expressive medium. This digital craftsmanship also requires diversity, risk, personal taste, mastery, and respect for materials. We discuss how digital tools can support expressive practices and highlight multiple strands of relevant HCI research. This workshop brings together tool developers, practitioners, ethnographers, and others engaged with digital technology as an expressive medium. It highlights digital craftsmanship as a distinct domain for HCI research and seeks to distill insights and best practices for the HCI community.

Microbial electrochemistry technologies (METs) take advantage of the connection of microorganisms with electrodes. In the classic case of a microbial anode, the maximization of current density produced is often the goal. But, current production is dependent on many transport processes occurring, which can be rate-limiting. These include the fluxes of electron donor and acceptor, the ionic flux, the acidity and alkalinity fluxes at anode and cathode respectively, the electron transport flux at the biofilm, and the reactant/product crossover flux. Associated with these fluxes are inherent concentration gradients that can affect performance. This critical review provides an analysis on how these transport processes have hindered the development of METs, and how MET designs have evolved as more knowledge of these transport limitations is gained. Finally, suggestions are provided on how to design MET systems taking into consideration critical transport processes that are intimately linked to the current produced.

The objective of this study was to employ microbial electrochemical cells (MXCs) to selectively enrich and examine anoxygenic photosynthetic bacteria for potential anaerobic respiration capabilities using electrodes. In the process, we designed a novel enrichment strategy that manipulated the poised anode potential, light, nitrogen availability, and media supply to promote growth of phototrophic bacteria while minimizing co-enrichment of non-phototrophic anode-respiring bacteria (ARB). This approach resulted in light-responsive electricity generation from fresh- and saltwater inocula. Under anoxic conditions, current showed a negative light response, suggesting that the enriched phototrophic consortia shifted between phototrophic and anaerobic respiratory metabolism. Molecular, physical, and electrochemical analyses elucidated that anode biofilms were dominated by green sulfur bacteria, and biofilms exhibited anode respiration kinetics indicative of non-mediated electron transfer, but kinetic parameters differed from values previously reported for non-phototrophic ARB. These results invite the utilization of MXCs as microbiological tools for exploring anaerobic respiratory capabilities among anoxygenic photosynthetic bacteria.

Cathodic limitation in microbial fuel cells (MFCs) is considered an important hurdle towards practical application as a bioenergy technology. The oxygen reduction reaction (ORR) needs to occur in MFCs under significantly different conditions compared to chemical fuel cells, including a neutral pH. The common reason cited for cathodic limitation is the difficulty in providing protons to the catalyst sites. Here, we show that it is not the availability of protons, but the transport of OH(-) from the catalyst layer to the bulk liquid that largely governs cathodic potential losses. OH(-) is a product of an ORR mechanism that has not been considered dominant before. The accumulation of OH(-) at the catalyst sites results in an increase in the local cathode pH, resulting in Nernstian concentration losses. For Pt-based gas-diffusion cathodes, using polarization curves developed in unbuffered and buffered solutions, we quantified this loss to be >0.3 V at a current density of 10 Am(-2) . We show that this loss can be partially overcome by replacing the Nafion binder used in the cathode catalyst layer with an anion-conducting binder and by providing additional buffer to the cathode catalyst directly in the form of CO(2) , which results in enhanced OH(-) transport. Our results provide a comprehensive analysis of cathodic limitations in MFCs and should allow researchers to develop and select materials for the construction of MFC cathodes and identify operational conditions that will help minimize Nernstian concentration losses due to pH gradients.

Geobacter spp. can form a biofilm that is more than 20 μm thick on an anode surface by utilizing the anode as a terminal respiratory electron acceptor. Just how microbes transport electrons through a thick biofilm and across the biofilm/anode interface, and what determines the upper limit to biofilm thickness and catalytic activity (i.e., current, the rate at which electrons are transferred to the anode), are fundamental questions attracting substantial attention. A significant body of experimental evidence suggests that electrons are transferred from individual cells through a network of cytochromes associated with cell outer membranes, within extracellular polymeric substances, and along pili. Here, we describe what is known about this extracellular electron transfer process, referred to as electron superexchange, and its proposed role in biofilm anode respiration. Superexchange is able to account for many different types of experimental results, as well as for the upper limit to biofilm thickness and catalytic activity that Geobacter biofilm anodes can achieve.

We evaluated the consumption of hydrogen gas at the anode of a microbial electrolysis cell (MEC) and characterized the significance of new interactions between anode respiring bacteria (ARB) and homo-acetogens. We demonstrated the significance of biofilm limitation for direct consumption of H2 over acetate by ARB, using the deep biofilm model. Selective inhibition of the major competing hydrogen sink at the biofilm anode, methanogenesis, resulted in significant increase in electron recovery as electric current (∼10–12 A/m2). The presence of acetate at high concentration in the anode compartment and detection of formate, a known intermediate of the acetyl-CoA pathway, provide evidence towards the role of homoacetogenic bacteria. We also assessed the activity of homoacetogens with reverse transcription quantitative PCR targeting formyltetrahydrofolate synthetase (FTHFS) transcripts, and observed a comparable decrease in the FTHFS transcript numbers with current density and acetate concentrations as we decreased the HRT below 4.5 h. The biofilm anode community was predominated by Deltaproteobacteria (70% of total readouts) along with a fraction of the homoacetogenic genus, Acetobacterium (4% of total readouts), established by pyrosequencing targeting the V6 region of the 16S rRNA. Homoacetogens seem to play a major role as syntrophic members of the biofilm anode community when electron recovery is high.

Sucralose, a chlorinated carbohydrate, is used as an artificial sweetener in more than 80 countries and in excess of 4,000 products. Thus far, minimal research has been done on the degradation and fate of sucralose in municipal wastewater treatment plants (WWTPs). We collected samples from WWTPs and surface waters in Arizona, United States. The average sucralose concentration of seven WWTP effluents was 2,800 ± 1,000 ng/L. Similarly, surface waters in Arizona contained sucralose at concentrations up to 300 ± 30 ng/L, which corroborates sucralose discharge from WWTPs into the environment. Biological degradation and chemical oxidation processes were evaluated to remove or transform sucralose under potential WWTP operation scenarios. Sucralose did not degrade in aerobic or anaerobic biological reactors, either metabolically or co-metabolically (in the presence of sucrose), after 42–62 days of experiments. Prolonged exposure to ultraviolet radiation did not oxidize sucralose significantly, and chlori...

Homo-acetogens in the anode of a microbial electrolysis cell (MEC) fed with H 2 as sole electron donor allowed current densities similar to acetate-fed biofilm anodes (∼10 A/m 2 ). Evidence for homo-acetogens included accumulation of acetate at high concentrations (up to 18 mM) in the anode compartment; detection of formate, a known intermediate during reductive acetogenesis by the acetyl-CoA pathway; and detection of formyl tetrahydrofolate synthetase (FTHFS) genes by quantitative real-time PCR. Current production and acetate accumulation increased in parallel in batch and continuous mode, while both values decreased simultaneously at short hydraulic retention times (1 h) in the anode compartment, which limited suspended homo-acetogens. Acetate produced by homo-acetogens accounted for about 88% of the current density of 10 A/m 2 , but the current density was sustained at 4 A/m 2 at short hydraulic retention time because of a robust partnership of homo-acetogens and anode respiring bacteria (ARB) in the biofilm anode.

Microbial electrochemical cells depend on the reaction by anode-respiring bacteria (ARB). The ARB reaction generates multiple e − and H + , which take diverging paths, creating a charge imbalance. An electric field must migrate ions to restore electrical neutrality. Here, the model proton condition in bioflim (PCBIOFILM) expands for evaluating the impact of migration on the biofilm anode: the expansion makes the proton condition (PC) work in tandem with the electrical-neutrality condition, which is a novel methodological advancement. The analysis with PCBIOFILM examines relevant scenarios of phosphate- and carbonate-buffered biofilm anodes using established parameters. The analysis demonstrates how: (1) the proton condition (PC) maintains electrical neutrality by achieving charge balance; (2) migration influences the biofilm anode more than non-ARB biofilms; (3) migration increases the overall current density, but by less than 15 percent; and (4) PCBIOFILM without migration accurately captures large-scale trends in biofilm anodes.

In microbial fuel cells and electrolysis cells (MXCs), anode-respiring bacteria (ARB) oxidize organic substrates to produce electrical current. In order to develop an electrical current, ARB must transfer electrons to a solid anode through extracellular electron transfer (EET). ARB use various EET mechanisms to transfer electrons to the anode, including direct contact through outer-membrane proteins, diffusion of soluble electron shuttles, and electron transport through solid components of the extracellular biofilm matrix. In this review, we perform a novel kinetic analysis of each EET mechanism by analyzing the results available in the literature. Our goal is to evaluate how well each EET mechanism can produce a high current density (> 10 A m(-2)) without a large anode potential loss (less than a few hundred millivolts), which are feasibility goals of MXCs. Direct contact of ARB to the anode cannot achieve high current densities due to the limited number of cells that can come in direct contact with the anode. Slow diffusive flux of electron shuttles at commonly observed concentrations limits current generation and results in high potential losses, as has been observed experimentally. Only electron transport through a solid conductive matrix can explain observations of high current densities and low anode potential losses. Thus, a study of the biological components that create a solid conductive matrix is of critical importance for understanding the function of ARB.

Anode-respiring bacteria (ARB) are able to transfer electrons contained in organic substrates to a solid electrode. The selection of ARB should depend on the anode potential, which determines the amount of energy available for bacterial growth and maintenance. In our study, we investigated how anode potential affected the microbial diversity of the biofilm community. We used a microbial electrolysis cell (MEC) containing four graphite electrodes, each at a different anode potential (E(anode) = -0.15, -0.09, +0.02, and +0.37 V vs SHE). We used wastewater-activated sludge as inoculum, acetate as substrate, and continuous-flow operation. The two electrodes at the lowest potentials showed a faster biofilm growth and produced the highest current densities, reaching up to 10.3 A/m(2) at the saturation of an amperometric curve; the electrode at the highest potential produced a maximum of 0.6 A/m(2). At low anode potentials, clone libraries showed a strong selection (92-99% of total clones) of an ARB that is 97% similar to G. sulfurreducens. At the highest anode potential, the ARB community was diverse. Cyclic voltammograms performed on each electrode suggest that the ARB grown at the lowest potentials carried out extracellular electron transport exclusively by conducting electrons through the extracellular biofilm matrix. This is supported by scanning electron micrographs showing putative bacterial nanowires and copious EPS at the lowest potentials. Non-ARB and ARB using electron shuttles in the diverse community for the highest anode potential may have insulated the ARB using a solid conductive matrix from the anode. Continuous-flow operation and the selective pressure due to low anode potentials selected for G. sulfurreducens, which are known to consume acetate efficiently and use a solid conductive matrix for electron transport.

The substrate-utilization rate of anode-respiring bacteria (ARB) directly correlates to the current density, one of the main factors in a microbial electrolysis/fuel cell. This study first evaluates the effects of donor-substrate diffusion and anode potential on the estimation of the half-maximum-rate concentration (K(s)) and the maximum specific substrate-utilization rate (q(max)) of a mixed culture biofilm in a microbial electrolysis cell oxidizing acetate. The intrinsic K(s) value is 119 g COD/m3, substrate diffusion has a significant impact on K(s) estimation, and the effect of the anode potential on K(s) is small. The intrinsic q(max) value is 22.3 g COD/g VS-d for an assumed biomass density of 50,000 g VS/m3 (q(max)X(f) = 1120 kg COD/m3-d). The maximum specific growth rate (micro(max)) is 3.2/d which is substantially faster than for acetate-utilizing methanogens and homoacetogens. Although the anode potential affects q(max), substrate diffusion has a negligible effect. The measured half-saturation anode potential (E(KA)) is very negative, -0.448 V (vs Ag/AgCl), and this low value minimizes anode-potential limitation on the current density and the substrate-utilization rate. Thus, the ARB selected in our biofilm anode were relatively fast growers able to take advantage of their low E(KA) value (-0.448 V).

With the goal of maximizing the H2-harvesting efficiency, we designed an upflow single-chamber microbial electrolysis cell (MEC) by placing the cathode on the top of the MEC and carried out a program to track the fate of H2 and electron equivalents in batch experiments. When the initial acetate concentration was 10 mM in batch-evaluation experiments lasting 32 h, the cathodic conversion efficiency (CCE) from coulombs (i.e., electron equivalents in current from the anode to the cathode) to H2 was 98 +/- 2%, the Coulombic efficiency (CE) was 60 +/- 1%, the H2 yield was 59 +/- 2%, and methane production was negligible. However, longer batch reaction time (approximately 7 days) associated with higher initial acetate concentrations (30 or 80 mM) led to significant H2 loss due to CH4 accumulation: up to 14 +/- 1% and 16 +/- 2% of the biogas at 30 and 80 mM of acetate, respectively. Quantitative PCR proved that no acetoclastic methanogens were present, but that hydrogenotrophic methanogens (i.e., Methanobacteriales) were present on both electrodes. The hydrogenotrophic methanogens decreased the CCE by diverting H2 generated at the cathode to CH4 in the upflow single-chamber MEC. In some experiments, the CE was greater than 100%. The cause was anode-respiring bacteria oxidizing H2 and producing current which recycled H2 between the cathode and the anodes, increasing CE to over 100%, but with a concomitant decline in CCE, despite negligible CH4 formation.

Anodes of biological fuel cells (BFCs) normally must operate at a near-neutral pH in the presence of various ionic species required for the function of the biological catalyst (e.g., substrate, nutrients, and buffers). These ionic species are in higher concentration than protons (H+) and hydroxides (OH-); slow transport of H+ and OH- equivalents between anode and cathode compartments can lead to a large pH gradient that can inhibit the function of biological components, decrease voltage efficiency in BFCs, or both. We evaluate the use of carbonate species as OH- carriers from the cathode to the anode compartment. This is achieved by adding CO2 to the influent air in the cathode. CO2 is an acid that combines with OH- in the cathode to produce bicarbonate and carbonate. These species can migrate to the anode compartment as OH- carriers at a rate much greater than can OH- itself when the pH is not extremely high in the cathode compartment We demonstrate this concept by feeding different air/CO2 mixtures to the cathode of a dual-chamber microbial fuel cell (MFC) fed with acetate as substrate. Our results show a 45% increase in power density (from 1.9 to 2.8 W/m2) by feeding air augmented with 2-10% CO2. The cell voltage increased by as much as 120 mV, indicating that the pH gradient decreased by as much as 2 pH units. Analysis of the anode effluent showed an average increase of 4.9 mM in total carbonate, indicating that mostly carbonate was transferred from the cathode compartment This process provides a simple way to minimize potential losses in BFCs due to pH gradients between anode and cathode compartments.

Coupling advanced oxidative pretreatment with subsequent biodegradation demonstrates potential for treating wastewaters containing biorecalcitrant and inhibitory organic constituents. However, advanced oxidation is indiscriminate, producing a range of products that can be too oxidized, unavailable for biodegradation, or toxic themselves. This problem could be overcome if advanced oxidation and biodegradation occurred together, an orientation called intimate coupling; then, biodegradable organics are removed as they are formed, focusing the chemical oxidant on the non-biodegradable fraction. Intimate coupling has seemed impossible because the conditions of advanced oxidation, for example, hydroxyl radicals and sometimes UV-light, are severely toxic to microorganisms. Here, we demonstrate that a novel photocatalytic circulating-bed biofilm reactor (PCBBR), which utilizes macro-porous carriers to protect biofilm from toxic reactants and UV light, achieves intimate coupling. We demonstrate the viability of the PCBBR system first with UV only and acetate, where the carriers grew biofilm and sustained acetate biodegradation despite continuous UV irradiation. Images obtained by scanning electron microscopy and confocal laser scanning microscopy show bacteria living behind the exposed surface of the cubes. Second, we used slurry-form Degussa P25 TiO2 to initiate photocatalysis of inhibitory 2,4,5-trichlorophenol (TCP) and acetate. With no bacterial carriers, photocatalysis and physical processes removed TCP and COD to 32% and 26% of their influent levels, but addition of biofilm carriers decreased residuals to 2% and 4%, respectively. Biodegradation alone could not remove TCP. Photomicrographs clearly show that biomass originally on the exterior of the carriers was oxidized (charred), but biofilm a short distance within the carriers was protected. Finally, we coated TiO2 directly onto the carrier surface, producing a hybrid photocatalytic-biological carrier. These carriers likewise demonstrated the concept of photocatalytic degradation of TCP coupled with biodegradation of acetate, but continued TCP degradation required augmentation with slurry-form TiO2.

Anode-respiring bacteria (ARB) are able to transfer electrons from reduced substrates to a solid electrode. Previously, we developed a biofilm model based on the Nernst-Monod equation to describe the anode potential losses of ARB that transfer electrons through a solid conductive matrix. In this work, we develop an experimental setup to demonstrate how well the Nernst-Monod equation is able to represent anode potential losses in an ARB biofilm. We performed low-scan cyclic voltammetry (LSCV) throughout the growth phase of an ARB biofilm on a graphite electrode growing on acetate in continuous mode. The (j)V response of 9 LSCVs corresponded well to the Nernst-Monod equation, and the half-saturation potential (E(KA)) was -0.425 +/- 0.002 V vs Ag/AgCl at 30 degrees C (-0.155 +/- 0.002 V vs SHE). Anode-potential losses from the potential of acetate reached approximately 0.225 V at current density saturation, and this loss was determined by our microbial community's E(KA) value. The LSCVs at high current densities showed no significant deviation from the Nernst-Monod ideal shape, indicating that the conductivity of the biofilm matrix (kappa(bio)) was high enough (> or = 0.5 mS/cm) that potential loss did not affect the performance of the biofilm anode. Our results confirm the applicability of the Nernst-Monod equation for a conductive biofilm anode and give insights of the processes that dominate anode potential losses in microbial fuel cells.

We established the first complete electron-equivalent balances in microbial fuel cells (MFCs) fed with non-fermentable (acetate) and fermentable (glucose) electron donors by experimentally quantifying current, biomass, residual organic compounds, H(2), and CH(4) gas. The comparison of the two donors allowed us to objectively evaluate the diversion of electron flow to non-electricity sinks for fermentable donors, leading to different behaviors in energy-conversion efficiency (ECE) and potential efficiency (PE). Electrical current was the most significant electron sink in both MFCs, being 71% and 49%, respectively, of the initial COD applied. Biomass and residual organic compounds, the second and third greatest sinks, respectively, were greater in the glucose-fed MFC than in the acetate-fed MFC. We detected methane gas only in the glucose-fed MFC, and this means that anode-respiring bacteria (ARB) could out-compete acetoclastic methanogens. The ECE was 42% with acetate, but was only 3% with glucose. The very low ECE for glucose was mostly due to a large increase of the anode potential, giving a PE of only 6%. Although the glucose-fed MFC had the higher biomass density on its anode, it had a very low current density, which supports the fact that the density of ARB was very low. This led to slow kinetics for electron transfer to the anode and accentuated loss due to the substrate-concentration gradient in the anode-biofilm. The large drop of PE with low current, probably caused by a low ARB density and electron (e(-)) donor concentration, resulted in a poor maximum power density (9.8mW/m(2)) with glucose. In contrast, PE reached 59% along with high current for acetate and the maximum power density was 360mW/m(2).

The biofilm of a microbial fuel cell (MFC) experiences biofilm-related (growth and mass transport) and electrochemical (electron conduction and charger-transfer) processes. We developed a dynamic, one-dimensional, multi-species model for the biofilm in three steps. First, we formulated the biofilm on the anode as a "biofilm anode" with the following two properties: (1) The biofilm has a conductive solid matrix characterized by the biofilm conductivity (kappa(bio)). (2) The biofilm matrix accepts electrons from biofilm bacteria and conducts the electrons to the anode. Second, we derived the Nernst-Monod expression to describe the rate of electron-donor (ED) oxidation. Third, we linked these components using the principles of mass balance and Ohm's law. We then solved the model to study dual limitation in biofilm by the ED concentration and local potential. Our model illustrates that kappa(bio) strongly influences the ED and current fluxes, the type of limitation in biofilm, and the biomass distribution. A larger kappa(bio) increases the ED and current fluxes, and, consequently, the ED mass-transfer resistance becomes significant. A significant gradient in ED concentration, local potential, or both can develop in the biofilm anode, and the biomass actively respires only where ED concentration and local potential are high. When kappa(bio) is relatively large (i.e., > or =10(-3) mS cm(-1)), active biomass can persist up to tens of micrometers away from the anode. Increases in biofilm thickness and accumulation of inert biomass accentuate dual limitation and reduce the current density. These limitations can be alleviated with increases in the specific detachment rate and biofilm density.

We determined the kinetic response of a community of anode-respiring bacteria oxidizing a mixture of the most common fermentation products: acetate, butyrate, propionate, ethanol, and hydrogen. We acclimated the community by performing three consecutive batch experiments in a microbial electrolytic cell (MEC) containing a mixture of the fermentation products. During the consecutive-batch experiments, the coulombic efficiency and start-up period improved with each step. We used the acclimated biofilm to start continuous experiments in an MEC, in which we controlled the anode potential using a potentiostat. During the continuous experiments, we tested each individual substrate at a range of anode potentials and substrate concentrations. Our results show low current densities for butyrate and hydrogen, but high current densities for propionate, acetate, and ethanol (maximum values are 1.6, 9.0, and 8.2 A/m(2), respectively). Acetate showed a high coulombic efficiency (86%) compared to ethanol and propionate (49 and 41%, respectively). High methane concentrations inside the MEC during ethanol experiments suggest that methanogenesis is one reason why the coulombic efficiency was lower than that of acetate. Our results provide kinetic parameters, such as the anode overpotential, the maximum current density, and the Monod half-saturation constant, that are needed for model development when using a mixture of fermentation products. When we provided no electron donor, we measured current due to endogenous decay of biomass (approximately 0.07 A/m(2)) and an open-cell potential (-0.54 V vs Ag/AgCl) associated with biomass components active in endogenous respiration.

Thermoanaerobacter pseudethanolicus 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), T. pseudethanolicus 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 T. pseudethanolicus on the anode, and confocal laser scanning microscopy demonstrated a maximum biofilm thickness (Lf) greater than ~150 μm for the glucose-fed biofilm.

Digital fabrication has enabled massive creativity in hobbyist communities and professional product design. These emerging technologies excel at realizing an arbitrary shape or form; however these objects are often rigid and lack the feel desired by designers. We aim to enable physical haptic design in passive 3D printed objects. This paper identifies two core areas for extending physical design into digital fabrication: designing the external and internal haptic characteristics of an object. We present HapticPrint as a pair of design tools to easily modify the feel of a 3D model. Our external tool maps textures and UI elements onto arbitrary shapes, and our internal tool modifies the internal geometry of models for novel compliance and weight characteristics. We demonstrate the value of HapticPrint with a range of applications that expand the aesthetics of feel, usability, and interactivity in 3D artifacts.

Thriving under alkaliphilic conditions, Geoalkalibacter ferrihydriticus (Glk. ferrihydriticus) provides new applications in treating alkaline waste streams as well as a possible new model organism for microbial electrochemistry. We investigated the electrochemical response of biofilms of the alkaliphilic anode-respiring bacterium (ARB) Glk. ferrihydriticus voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry. We observed there to be at least four dominant electron transfer pathways, with their contribution to the overall current produced dependent on the set anode potential. These pathways appear to be manifested at midpoint potentials of approximately -0.14 V, -0.2 V, -0.24 V, and -0.27 V vs standard hydrogen electrode. The individual contributions of the pathways change upon equilibration from a set anode potential to another anode potential. Additionally, the contribution of each pathway to the overall current produced is reversible when the anode potential is changed back to the original set potential. The pathways involved in anode respiration in Glk. ferrihydriticus biofilms follow a similar, but more complicated, pattern as compared to those in the model ARB, Geobacter sulfurreducens. This greater diversity of electron transport pathways in Glk. ferrihydriticus could be related to its wider metabolic capability (e.g., higher pH and larger set of possible substrates, among others).

A 3.19-Mbp draft genome of the Gram-positive thermophilic iron-reducing Firmicutes isolate from the Peptococcaceae family, Thermincola ferriacetica Z-0001, was assembled at ~100× coverage from 100-bp paired-end Illumina reads. The draft genome contains 3,274 predicted genes (3,187 protein coding genes) and putative multiheme c -type cytochromes.

Copyright © 2015 ACM. The creative promise of 3D digital fabrication tools is tremendous. However due to the wide range of tools and interfaces, a common static file format called STL is used for sharing designs. While customization tools add creative handles to these digital models, they are often constrained to pre-configured parameters limiting the creative potential of shared digital models. We introduce MetaMorphe, a novel digital fabrication framework that uses a common web-programming metaphor to enable users to easily transform static 3D models into re-formed, re-made, and re-imagined customized personal artifacts. We demonstrate the compatibility of Meta- Morphe with three well-established design interfaces, direction manipulation, scripted-CAD, and generative design. Through a user study with design experts, MetaMorphe reveals that decisions that physically produce bespoke artifacts or encode unique metadata actively affect perceptions of authorship, agency, and authenticity. We discuss how expressive model-building tools such as MetaMorphe enable a cultural shift in 3D design in terms of participation, personalization, and creativity.

We sequenced and annotated genomes of two haloalkaliphilic Deltaproteobacteria , Geoalkalibacter ferrihydriticus Z-0531 T (DSM 17813) and Geoalkalibacter subterraneus Red1 T (DSM 23483). During assembly, we discovered that the DSMZ stock culture of G. subterraneus was contaminated. We reisolated G. subterraneus in axenic culture and redeposited it in DSMZ and JCM.

Fluid User Interfaces (Fl.UIs) are liquid-based touch surfaces that use computer vision to detect and interpret a range of tactile user inputs. While Fl.UIs have less input resolution than digital touch screens, they provide an excellent low-cost solution for rapidly prototyping non-rectilinear screen designs as well as exploring novel surface interaction techniques. Fabricated on a laser cutter using low-cost materials, Fl.UIs use unique shape outlines to displace an internal colored liquid to regions-of-interest for a camera. This paper presents a set of software tools that help users rapidly design, fabricate and author interactions with Fl.UIs. The robust construction and an unpowered surface makes Fl.UIs well-suited for outdoor and public installations. Our Fl.UIs prototyping tool encourages these uncommon "screens" to emerge in complex environments (i.e. urban spaces, benches, tables, fountains, sidewalks).

In microbial electrochemical cells (MXCs), not only classic overpotentials known from other types of fuel cells are encountered, but also overpotentials associated with the metabolic processes and electron-transport pathways in bacteria are encountered. Of the many techniques that can be used to investigate the contributions of various processes to the total overpotential in MXCs, this chapter focuses on electrochemical impedance spectroscopy (EIS). This technique is an alternating current technique that involves applying sinusoidal voltage amplitude over a range of frequencies to investigate the processes that control the overall i–V response. The use of EIS allows differentiating these various processes, as they are often manifested at different frequencies. We discuss the principles and theory behind EIS, especially with respect to its use in MXCs, as well as important experimental methods and design parameters. We show that EIS is an important method that can be used to characterize Ohmic resistance, electrode processes, or an MXC as a whole.

The hydrogen-based membrane biofilm reactor (MBfR) is effective for reducing nitrate-N to N2 gas, but most wastewaters contain ammonium-N. Here, we document that an aerobic/anoxic MBfR system achieves nearly total N removal (

Water and energy are crucial aspects strongly connected when dealing with the problem of augmenting fresh water resources. Surprisingly, in the scientific and technological community there is a marked lack of attention to the combined research of water and energy issues. The present paper outlines a development-oriented object program in the said direction, oriented to the integrated analysis of power and desalination plants, which would hopefully fill the gap in having user-friendly software in the form of building blocks as an appropriate framework for the analysis, synthesis and design of either individual or integrated systems for energy and water. Such an approach would transcend other traditional approaches for process integration and obviously prove of great help not only to designers but also to researchers, educators and students. For illustrative purposes, in order to show the scope of the software being developed, an example is presented in which two different operation conditions of a dual-purpose power and desalination plant are compared. In particular, the effect of the throttling part of the steam extracted to the desalination unit is analyzed, which significantly increases the cost of water and electricity produced.

Butyrate is an important product of anaerobic fermentation; however, it is not directly used by characterized strains of the highly efficient anode respiring bacteria (ARB) Geobacter sulfurreducens in microbial electrochemical cells. By combining a butyrate-oxidizing community with a Geobacter rich culture, we generated a microbial community which outperformed many naturally derived communities found in the literature for current production from butyrate and rivaled the highest performing natural cultures in terms of current density (∼11 A/m2) and Coulombic efficiency (∼70%). Microbial community analyses support the shift in the microbial community from one lacking efficient ARB in the marine hydrothermal vent community to a community consisting of ∼80% Geobacter in the anode biofilm. This demonstrates the successful production and adaptation of a novel microbial culture for generating electrical current from butyrate with high current density and high Coulombic efficiency, by combining two mixed microbial cultures containing complementing biochemical pathways.

The development of microbial electrochemistry research toward technological applications has increased significantly in the past years, leading to many process configurations. This short review focuses on the need to identify and characterize the fundamental phenomena that control the performance of microbial electrochemical cells (MXCs). Specifically, it discusses the importance of recent efforts to discover and characterize novel microorganisms for MXC applications, as well as recent developments to understand transport limitations in MXCs. As we increase our understanding of how MXCs operate, it is imperative to continue modeling efforts in order to effectively predict their performance, design efficient MXC technologies, and implement them commercially. Thus, the success of MXC technologies largely depends on the path of identifying, understanding, and predicting fundamental phenomena that determine MXC performance.

Pre-fermentation of poorly biodegradable landfill leachate (BOD 5 /COD ratio of 0.32) was evaluated for enhanced current density ( j ), Coulombic efficiency (CE), Coulombic recovery (CR), and removal of organics (BOD 5 and COD) in a microbial electrolysis cell (MEC). During fermentation, the complex organic matter in the leachate was transformed to simple volatile fatty acids, particularly succinate and acetate in batch tests, but mostly acetate in semi-continuous fermentation. Carbohydrate had the highest degree of fermentation, followed by protein and lipids. j , CE, CR, and BOD 5 removal were much greater for an MEC fed with fermented leachate (23 A/m 3 or 16 mA/m 2 , 68%, 17.3%, and 83%, respectively) compared to raw leachate (2.5 A/m 3 or 1.7 mA/m 2 , 56%, 2.1%, and 5.6%, respectively). All differences support the value of pre-fermentation before an MEC for stabilization of BOD 5 and enhanced electron recovery as current when treating a recalcitrant wastewater like landfill leachate.

The discovery of Dehalococcoides mccartyi reducing perchloroethene and trichloroethene (TCE) to ethene was a key landmark for bioremediation applications at contaminated sites. D. mccartyi-containing cultures are typically grown in batch-fed reactors. On the other hand, continuous cultivation of these microorganisms has been described only at long hydraulic retention times (HRTs). We report the cultivation of a representative D. mccartyi-containing culture in continuous stirred-tank reactors (CSTRs) at a short, 3-d HRT, using TCE as the electron acceptor. We successfully operated 3-d HRT CSTRs for up to 120 days and observed sustained dechlorination of TCE at influent concentrations of 1 and 2 mM TCE to ≥ 97 % ethene, coupled to the production of 10(12) D. mccartyi cells Lculture (-1). These outcomes were possible in part by using a medium with low bicarbonate concentrations (5 mM) to minimize the excessive proliferation of microorganisms that use bicarbonate as an electron acceptor and compete with D. mccartyi for H2. The maximum conversion rates for the CSTR-produced culture were 0.13 ± 0.016, 0.06 ± 0.018, and 0.02 ± 0.007 mmol Cl(-) Lculture (-1) h(-1), respectively, for TCE, cis-dichloroethene, and vinyl chloride. The CSTR operation described here provides the fastest laboratory cultivation rate of high-cell density Dehalococcoides cultures reported in the literature to date. This cultivation method provides a fundamental scientific platform for potential future operations of such a system at larger scales.

Anode-respiring bacteria (ARB) generate electric current in microbial electrochemical cells (MXCs) by channeling electrons from the oxidation of organic substrates to an electrode. Production of high current densities by monocultures in MXCs has resulted almost exclusively from the activity of Geobacter sulfurreducens, a neutrophilic freshwater Fe(III)-reducing bacterium and the highest-current-producing member documented for the Geobacteraceae family of the Deltaproteobacteria. Here we report high current densities generated by haloalkaliphilic Geoalkalibacter spp., thus broadening the capability for high anode respiration rates by including other genera within the Geobacteraceae. In this study, acetate-fed pure cultures of two related Geoalkalibacter spp. produced current densities of 5.0 to 8.3 and 2.4 to 3.3 A m−2 under alkaline (pH 9.3) and saline (1.7% NaCl) conditions, respectively. Chronoamperometric studies of halophilic Glk. subterraneus DSM 23483 and alkaliphilic Glk. ferrihydriticus DSM 17813 suggested that cells performed long-range electron transfer through electrode-attached biofilms and not through soluble electron shuttles. Glk. ferrihydriticus also oxidized ethanol directly to produce current, with maximum current densities of 5.7 to 7.1 A m−2 and coulombic efficiencies of 84 to 95%. Cyclic voltammetry (CV) elicited a sigmoidal response with characteristic onset, midpoint, and saturation potentials, while CV performed in the absence of an electron donor suggested the involvement of redox molecules in the biofilm that were limited by diffusion. These results matched those previously reported for actively respiring Gb. sulfurreducens biofilms producing similar current densities (~5 to 9 A m−2)., IMPORTANCE This study establishes the highest current densities ever achieved by pure cultures of anode-respiring bacteria (ARB) under alkaline and saline conditions in microbial electrochemical cells (MXCs) and provides the first electrochemical characterization of the genus Geoalkalibacter. Production of high current densities among the Geobacteraceae is no longer exclusive to Geobacter sulfurreducens, suggesting greater versatility for this family in fundamental and applied microbial electrochemical cell (MXC) research than previously considered. Additionally, this work raises the possibility that different members of the Geobacteraceae have conserved molecular mechanisms governing respiratory extracellular electron transfer to electrodes. Thus, the capacity for high current generation may exist in other uncultivated members of this family. Advancement of MXC technology for practical uses must rely on an expanded suite of ARB capable of using different electron donors and producing high current densities under various conditions. Geoalkalibacter spp. can potentially broaden the practical capabilities of MXCs to include energy generation and waste treatment under expanded ranges of salinity and pH.

We investigated the role of green sulfur bacteria inlight-responsive electricity generation in microbial electrochemical cells (MXCs). We operated MXCs containing either monocultures or defined cocultures of previously enriched phototrophic Chlorobium and anode-respiring Geobacter under anaerobic conditions in the absence of electron donor. Monoculture control MXCs containing Geobacter or Chlorobium neither responded to light nor produced current, respectively. Instead, light-responsive current generation occurred only in coculture MXCs. Current increased above background levels only in the dark and declined slowly over 96 h. This pattern suggested that Chlorobium exhausted intracellular glycogen reserves via dark fermentation to supply an electron donor, presumably acetate, to Geobacter. With medium containing sulfide as the sole photosynthetic electron donor, current generation had a similar and reproducible negative light response. To investigate whether this metabolic interaction also occurred without an electrode, we performed coculture experiments in batch serum bottles. In this setup, sulfide served as the sole electron donor, whose oxidation by Chlorobium was required to provide S(0) as the electron acceptor to Geobacter. Copies of Geobacter 16S rDNA increased approximately 14-fold in batch bottle cocultures containing sulfide compared to those lacking sulfide, and did not decline after termination of sulfide feeding. These results suggest that products of both photosynthesis and dark fermentation by Chlorobium were sufficient both to yield an electrochemical response by Geobacter biofilms, and to promote Geobacter growthin batch cocultures. Our work expands upon the fusion of MXCs with coculture techniques and reinforces the utility of microbial electrochemistry for sensitive, real-time monitoring of microbial interactions in which a metabolic intermediate can be converted to electrical current.

Advances in data mining and knowledge discovery have transformed the way Web sites are designed. However, while visual presentation is an intrinsic part of the Web, traditional data mining techniques ignore render-time page structures and their attributes. This paper introduces design mining for the Web: using knowledge discovery techniques to understand design demographics, automate design curation, and support data-driven design tools. This idea is manifest in Webzeitgeist, a platform for large-scale design mining comprising a repository of over 100,000 Web pages and 100 million design elements. This paper describes the principles driving design mining, the implementation of the Webzeitgeist architecture, and the new class of data-driven design applications it enables.

Fermentation is a key process in many anaerobic environments. Varying the concentration of electron donor fed to a fermenting community is known to shift the distribution of products between hydrogen, fatty acids and alcohols. Work to date has focused mainly on the fermentation of glucose, and how the microbial community structure is affected has not been explored. We fed ethanol, lactate, glucose, sucrose or molasses at 100 me- eq. L−1, 200 me- eq. L−1 or 400 me- eq. L−1 to batch-fed cultures with fermenting, methanogenic communities. In communities fed high concentrations of electron donor, the fraction of electrons channeled to methane decreased, from 34% to 6%, while the fraction of electrons channeled to short chain fatty acids increased, from 52% to 82%, averaged across all electron donors. Ethanol-fed cultures did not produce propionate, but did show an increase in electrons directed to acetate as initial ethanol concentration increased. In glucose, sucrose, molasses and lactate-fed cultures, propionate accumulation co-occurred with known propionate producing organisms. Overall, microbial communities were determined by the substrate provided, rather than its initial concentration, indicating that a change in community function, rather than community structure, is responsible for shifts in the fermentation products produced.

Microbial fuel cells (MFCs) have been studied for over a decade for possible applications of generating electrical power from wastewater treatment. The power densities achieved so far – although increased by orders of magnitude since the first studies – are still well below those required to compete with other anaerobic treatment processes. We are exploring the use of MFCs for the production of hydrogen peroxide (H2O2). H2O2 is a powerful oxidant and disinfectant that can be used onsite at a treatment plant. Its production in MFCs is possible by allowing the 2-e- oxygen reduction reaction (ORR) to occur at the cathode. We have studied several aspects of the selection of cathode catalyst, design of the MFCs, and their operation, to optimize the rates and concentrations of H2O2 that can be achieved in a continuous-flow catholyte scheme. First, we determined the thickness of the Vulcan carbon catalyst necessary for the cathode to promote the 2-e- vs. the 4-e- ORR, as higher thicknesses tend to lead to the consumption of H2O2 within the catalyst layer. Next, we studied various anion exchange membranes (AEMs) for their compatibility with H2O2. Although high-conductivity AEMs are desirable to decrease Ohmic overpotentials, we had to select an AEM that has a relatively low conductivity, because of its higher compatibility with H2O2 that would allow long-term operation. We also optimized the catholyte composition and hydraulic retention time (HRT) within the MFCs to allow for H2O2 production at a high cathodic Coulombic efficiency, by avoiding its disintegration within the cathode chamber. Through these advances, we have been able to show consistent H2O2 production in MFCs fed with acetate as the electron donor on the anode to concentrations of >0.3%. These concentrations are significantly higher than those needed for many of the possible onsite applications of H2O2, for e.g. disinfection. Our results should pave the way for further development and scale-up of MFCs for the production of H2O2.

Anode-respiring bacteria (ARB) catalyze the complete oxidation of organic compounds (e.g. acetate, glucose) into electrical current and carbon dioxide. ARB naturally produce a biofilm at the electrode surface of up to 100 micrometers, where even cells on the outer part of the biofilm are participating in current production. The specific pathway by which ARB transport electrons intra- and extracellularly is still largely unknown. This is largely due to the fact that many ARB have redundant respiratory mechanisms, leading to a large set of possible proteins involved in these mechanisms. Our research utilizes a variety of electrochemical techniques in order to characterize electron transport responses from various ARB. Our goal is to provide insights into the possible mechanisms of electron transfer from the electrochemical responses of ARB biofilms. Through these experiments, we have observed a complex response to anode potential that allows ARB, such as G. sulfurreducens, to optimize their efficiency in electron transport. In G. sulfurreducens, two pathways were identified for anode respiration with midpoint potentials of – 0.155 ±0.005 and –0.095 ± 0.003 V versus SHE. G. sulfurreducens can shift between these two pathways depending on the anode potential in order to optimize its energy recovery from anode respiration. Pathway shifts were observed within 5-20 minutes of a shift in anode potential, suggesting that this microorganism has a potential-sensing mechanism. The fast shifts also confirm that the slow-scan cyclic voltammograms (~1 mV/s or slower) commonly performed in our field can be the result of transient responses by ARB, who can shift pathways within the experimental time. Multiple redox pathways have also been observed in thermophile T. ferriacetica and alkaliphile Geoalkalibacter ferrihydriticus, suggesting that most ARB can have multiple anode-respiration pathways. While the topic of electron transport is the focus of most ARB research, ionic transport is an important factor in determining rate-limiting and potential loss processes. ARB require near-neutral pH in the medium to grow, differing from chemical fuel cells commonly employed, which run under acidic or alkaline conditions. This pH requirement results in a major transport limitation, as H+ ions (now in mM range) should be transported from anode to cathode to achieve electron neutrality. In an MXC anode, H+ ions accumulate in the ARB biofilm, creating an acidification that limits current generation. This behavior has been observed with several ARB, including T. ferriacetica and Glk. ferrihydriticus. Through these experiments, we determined that the rate-limiting protein that is responsible for the potential response of ARB is a proton-coupled reaction. Experiments at different pH values result in shifts in the midpoint potential response of ARB, suggesting a proton coupled redox reaction. The specific stoichiometry of the reaction is difficult to elucidate since the H+ accumulation in the biofilm causes a pH gradient. Nonetheless, our studies provide insights into the characteristics of the rate-limiting proteins in anode respiration which are yet to be identified.