César I. Torres

A kinetic perspective on extracellular electron transfer by anode-respiring bacteria.

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.

Proton transport inside the biofilm limits electrical current generation by anode‐respiring bacteria

Anode‐respiring bacteria (ARB) in a biofilm anode carry out an oxidation half‐reaction of organic matter, producing an electrical current from renewable biomass, including wastes. At the same time, ARB produce protons, usually one proton for every electron. Our study shows how current density generated by an acclimated ARB biofilm was limited by proton transport out of the biofilm. We determined that, at high current densities, protons were mainly transported out of the biofilm by protonating the conjugate base of the buffer system; the maximum current generation was directly related to the transport of the buffer, mainly by diffusion, into and out of the biofilm. With non‐limiting acetate concentrations, the current density increased with higher buffer concentrations, going from 2.21 ± 0.02 A m−2 with 12.5‐mM phosphate buffer medium to 9.3 ± 0.4 A m−2 using a 100‐mM phosphate buffer at a constant anode potential of Eanode = −0.35 V versus Ag/AgCl. Increasing the concentration of sodium chloride in the medium (0–100 mM) increased current density by only 15%, indicating that ion migration was not as important as diffusion of phosphate inside the biofilm. The current density also varied strongly with medium pH as a result of the buffer speciation: The current density was 10.0 ± 0.8 A m−2 at pH 8, and the pH giving one‐half the maximum rate was 6.5. A j–V curve analysis using 100 mM phosphate buffer showed a maximum current density of 11.5 ± 0.9 A m−2 and half‐saturation potential of −0.414 V versus Ag/AgCl, a value that deviated only slightly from the standard acetate potential, resulting in small anode‐potential losses. We discuss the implications of the proton‐transport limitation in the field of microbial fuel cells and microbial electrolytic cells. Biotechnol. Bioeng. 2008;100: 872–881.

Selecting Anode-Respiring Bacteria Based on Anode Potential: Phylogenetic, Electrochemical, and Microscopic Characterization

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.

Syntrophic interactions among anode respiring bacteria (ARB) and Non‐ARB in a biofilm anode: electron balances

We demonstrate that the coulombic efficiency (CE) of a microbial electrolytic cell (MEC) fueled with a fermentable substrate, ethanol, depended on the interactions among anode respiring bacteria (ARB) and other groups of micro‐organisms, particularly fermenters and methanogens. When we allowed methanogenesis, we obtained a CE of 60%, and 26% of the electrons were lost as methane. The only methanogenic genus detected by quantitative real‐time PCR was the hydrogenotrophic genus, Methanobacteriales, which presumably consumed all the hydrogen produced during ethanol fermentation (∼30% of total electrons). We did not detect acetoclastic methanogenic genera, indicating that acetate‐oxidizing ARB out‐competed acetoclastic methanogens. Current production and methane formation increased in parallel, suggesting a syntrophic interaction between methanogens and acetate‐consuming ARB. When we inhibited methanogenesis with 50 mM 2‐bromoethane sulfonic acid (BES), the CE increased to 84%, and methane was not produced. With no methanogenesis, the electrons from hydrogen were converted to electrical current, either directly by the ARB or channeled to acetate through homo‐acetogenesis. This illustrates the key role of competition among the various H2 scavengers and that, when the hydrogen‐consuming methanogens were present, they out‐competed the other groups. These findings also demonstrate the importance of a three‐way syntrophic relationship among fermenters, acetate‐consuming ARB, and a H2 consumer during the utilization of a fermentable substrate. To obtain high coulombic efficiencies with fermentable substrates in a mixed population, methanogens must be suppressed to promote new interactions at the anode that ultimately channel the electrons from hydrogen to current. Biotechnol. Bioeng. 2009;103: 513–523.

On Electron Transport through Geobacter Biofilms

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.

Fate of H2 in an upflow single-chamber microbial electrolysis cell using a metal-catalyst-free cathode

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.

A μL-scale micromachined microbial fuel cell having high power density

We report a MEMS (Micro-Electro-Mechanical Systems)-based microbial fuel cell (MFC) that produces a high power density. The MFC features 4.5-μL anode/cathode chambers defined by 20-μm-thick photo-definable polydimethylsiloxane (PDMS) films. The MFC uses a Geobacter-enriched mixed bacterial culture, anode-respiring bacteria (ARB) that produces a conductive biofilm matrix. The MEMS MFC generated a maximum current density of 16,000 μA cm(-3) (33 μA cm(-2)) and power density of 2300 μW cm(-3) (4.7 μW cm(-2)), both of which are substantially greater than achieved by previous MEMS MFCs. The coulombic efficiency of the MEMS MFC was at least 31%, by far the highest value among reported MEMS MFCs. The performance improvements came from using highly efficient ARB, minimizing the impact of oxygen intrusion to the anode chamber, having a large specific surface area that led to low internal resistance.

Importance of OH− Transport from Cathodes in Microbial Fuel Cells

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.

Analysis of a microbial electrochemical cell using the proton condition in biofilm (PCBIOFILM) model.

Common to all microbial electrochemical cells (MXCs) are the anode-respiring bacteria (ARB), which transfer electrons to an anode and release protons that must transport out of the biofilm. Here, we develop a novel modeling platform, Proton Condition in BIOFILM (PCBIOFILM), with a structure geared towards mechanistically explaining: (1) how the ARB half reaction produces enough acid to inhibit the ARB by low pH; (2) how the diffusion of alkalinity carriers (phosphates and carbonates) control the pH gradients in the biofilm anode; (3) how increasing alkalinity attenuates pH gradients and increases current; and (4) why carbonates enable higher current density than phosphates. Analysis of literature data using PCBIOFILM supports the hypothesis that alkalinity limits the maximum current density for MXCs. An alkalinity criterion for eliminating low-pH limitation - 12 mgCaCO(3)/mg BOD--implies that a practical MXC can achieve a maximum current density with an effluent quality comparable to anaerobic digestion.

Hydrogen consumption in microbial electrochemical systems (MXCs): The role of homo-acetogenic bacteria

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 (1h) in the anode compartment, which limited suspended homo-acetogens. Acetate produced by homo-acetogens accounted for about 88% of the current density of 10A/m(2), but the current density was sustained at 4A/m(2) at short hydraulic retention time because of a robust partnership of homo-acetogens and anode respiring bacteria (ARB) in the biofilm anode.