Sudeep C. Popat

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.

Kinetic, Electrochemical, and Microscopic Characterization of the Thermophilic, Anode-Respiring Bacterium Thermincola ferriacetica

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.

Dynamic Potential-Dependent Electron Transport Pathway Shifts in Anode Biofilms of Geobacter sulfurreducens

Biofilms of the anode-respiring bacterium Geobacter sulfurreducens (G. sulfurreducens) demonstrate dynamic potential-dependent changes between two electron transport pathways that are used selectively depending on the anode potential. Electrochemical impedance spectroscopy (EIS) measurements suggest that these pathways (both n=1), with midpoint potentials of -0.155 (± 0.005) and -0.095 (± 0.003) V versus standard hydrogen electrode, are not additive within the biofilm, but are preferentially used depending on the anode potential. Potential step voltammetry and cyclic voltammetry (CV) confirmed rapid changes between the two pathways in minutes when the anode potential is changed. We confirm that the electrochemical response observed in a slow-scan-rate CV (∼1 mV s(-1) ) is often composed of at least the two pathways characterized. Thus, beyond understanding the electron transport pathways in G. sulfurreducens, this study also has implications on the interpretation of previously collected and future potential-dependent datasets.

Reductive dehalogenation of trichloroethene vapors in an anaerobic biotrickling filter.

Until now, it has not been possible to use biofiltration to treat trichloroethene (TCE) from waste gases generated by soil vapor extraction or dual-phase extraction at remediation sites because aerobic biodegradation of TCE is possible only via cometabolism, which is difficult to engineer on a large scale. This study looks at the possibility of conducting anaerobic gas-phase biotreatment of TCE vapors. The vision is that nitrogen sparging could be substituted for air sparging, resulting in TCE contaminated oxygen-free gas streams which require treatment A lab-scale anaerobic biotrickling filter inoculated with a mixed culture containing multiple Dehalococcoides strains was used for the proof of concept TCE vapors were removed via reductive dechlorination and converted to ethene, cis-1,2-dichloroethene (cis-DCE), and vinyl chloride (VC). Sodium lactate, a fermentable substrate, was provided to the reactor through the recirculating liquid as a source of hydrogen, the electron donor for Dehalococcoides strains. The biotrickling filter was able to remove >90% TCE at loadings of up to 4 g m(bed)(-3) h(-1) and sustained performance for over 200 days. The distribution of the intermediates of TCE biological reduction was found to be affected by the pH of the recirculating liquid. At pH 8.3, the primary accumulating productwas cis-DCE (approximately 92% of the TCE removed); while at pH 6.85-6.9, conversion to ethene, the intended end product, was 50-67% of the TCE removed. Kinetic determinations using batch biotrickling filter operation showed that VC reduction and not cis-DCE reduction was the sloweststep. Overall, the study shows that sustained anaerobic biotreatment of TCE vapors in biotrickling filters is possible.

Managing methanogens and homoacetogens to promote reductive dechlorination of trichloroethene with direct delivery of H2 in a membrane biofilm reactor

A study with H2‐based membrane biofilm reactors (MBfRs) was undertaken to examine the effectiveness of direct H2 delivery in ex‐situ reductive dechlorination of chlorinated ethenes. Trichloroethene (TCE) could be reductively dechlorinated to ethene with up to 95% efficiency as long as the pH‐increase effects of methanogens and homoacetogens were managed and dechlorinators were selected for during start‐up by creating H2 limitation. Based on quantitative PCR, the dominant bacterial groups in the biofilm at the end of reactor operation were Dehalococcoides, Geobacter, and homoacetogens. Pyrosequencing confirmed the dominance of the dechlorinators and identified Acetobacterium as the key homoacetogen. Homoacetogens outcompeted methanogens for bicarbonate, based on the effluent concentration of acetate, by suppressing methanogens during batch start‐up. This was corroborated by the methanogenesis functional gene mcrA, which was 1–2 orders of magnitude lower than the FTHFS functional gene for homoacetogens. Imaging of the MBfR fibers using scanning electron microscopy showed a distinct Dehalococcoides‐like morphology in the fiber biofilm. These results support that direct addition of H2 can allow for efficient and complete reductive dechlorination, and they shed light into how H2‐fed biofilms, when operated to manage methanogenic and homoacetogenic activity, can be used for ex‐situ bioremediation of chlorinated ethenes. Biotechnol. Bioeng. 2012;109: 2200–2210.

Critical transport rates that limit the performance of microbial electrochemistry technologies.

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.

Selective enrichment yields robust ethene-producing dechlorinating cultures from microcosms stalled at cis-dichloroethene.

Dehalococcoides mccartyi strains are of particular importance for bioremediation due to their unique capability of transforming perchloroethene (PCE) and trichloroethene (TCE) to non-toxic ethene, through the intermediates cis-dichloroethene (cis-DCE) and vinyl chloride (VC). Despite the widespread environmental distribution of Dehalococcoides, biostimulation sometimes fails to promote dechlorination beyond cis-DCE. In our study, microcosms established with garden soil and mangrove sediment also stalled at cis-DCE, albeit Dehalococcoides mccartyi containing the reductive dehalogenase genes tceA, vcrA and bvcA were detected in the soil/sediment inocula. Reductive dechlorination was not promoted beyond cis-DCE, even after multiple biostimulation events with fermentable substrates and a lengthy incubation. However, transfers from microcosms stalled at cis-DCE yielded dechlorination to ethene with subsequent enrichment cultures containing up to 109 Dehalococcoides mccartyi cells mL−1. Proteobacterial classes which dominated the soil/sediment communities became undetectable in the enrichments, and methanogenic activity drastically decreased after the transfers. We hypothesized that biostimulation of Dehalococcoides in the cis-DCE-stalled microcosms was impeded by other microbes present at higher abundances than Dehalococcoides and utilizing terminal electron acceptors from the soil/sediment, hence, outcompeting Dehalococcoides for H2. In support of this hypothesis, we show that garden soil and mangrove sediment microcosms bioaugmented with their respective cultures containing Dehalococcoides in high abundance were able to compete for H2 for reductive dechlorination from one biostimulation event and produced ethene with no obvious stall. Overall, our results provide an alternate explanation to consolidate conflicting observations on the ubiquity of Dehalococcoides mccartyi and occasional stalling of dechlorination at cis-DCE; thus, bringing a new perspective to better assess biological potential of different environments and to understand microbial interactions governing bioremediation.

Using electron balances and molecular techniques to assess trichoroethene-induced shifts to a dechlorinating microbial community

This study demonstrated the utility in correlating performance and community structure of a trichloroethene (TCE)‐dechlorinating microbial consortium; specifically dechlorinators, fermenters, homoacetogens, and methanogens. Two complementary approaches were applied: predicting trends in the microbial community structure based on an electron balance analysis and experimentally assessing the community structure via pyrosequencing and quantitative polymerase chain reaction (qPCR). Fill‐and‐draw reactors inoculated with the DehaloR⁁2 consortium were operated at five TCE‐pulsing rates between 14 and 168 µmol/10‐day‐SRT, amended with TCE every 2 days to give peak concentrations between 0.047 and 0.56 mM (6–74 ppm) and supplied lactate and methanol as sources of e‐ donor and carbon. The complementary approaches demonstrated the same trends: increasing abundance of Dehalococcoides and Geobacter and decreasing abundance of Firmicutes with increasing TCE pulsing rate, except for the highest pulsing rate. Based on qPCR, the abundance of Geobacter and Dehalococcoides decreased for the highest TCE pulsing rate, and pyrosequencing showed this same trend for the latter. This deviation suggested decoupling of Dehalococcoides growth from dechlorination. At pseudo steady‐state, methanogenesis was minimal for all TCE pulsing rates. Pyrosequencing and qPCR showed suppression of the homoacetogenic genera Acetobacterium at the two highest pulsing rates, and it was corroborated by a decreased production of acetate from lactate fermentation and increased propionate production. Suppression of Acetobacterium, which can provide growth factors to Dehalococcoides, may have contributed to the decoupling for the highest TCE‐pulsing rate. Biotechnol. Bioeng. 2012;109: 2230–2239.

Anode Biofilms of Geoalkalibacter ferrihydriticus Exhibit Electrochemical Signatures of Multiple Electron Transport Pathways.

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).

Kinetics and inhibition of reductive dechlorination of trichloroethene, cis-1,2-dichloroethene and vinyl chloride in a continuously fed anaerobic biofilm reactor.

Anaerobic bioreactors containing Dehalococcoides spp. can be effective for the treatment of trichloroethene (TCE) contamination. However, reductive dehalogenation of TCE often results in partial conversion to harmless ethene, and significant production of undesired cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC) is frequently observed. Here, a detailed modeling study was conducted focusing on the determination of biokinetic constants for the dechlorination of TCE and its reductive dechlorination intermediates cis-DCE and VC as well as any biokinetic inhibition that may exist between these compounds. Dechlorination data from an anaerobic biotrickling filter containing Dehalococcoides spp. fed with single compounds (TCE, cis-DCE, or VC) were fitted to the model to determine biokinetic constants. Experiments with multiple compounds were used to determine inhibition between the compounds. It was found that the Michaelis-Menten half-saturation constants for all compounds were higher than for cells grown in suspended cultures, indicating a lower enzyme affinity in biofilm cells. It was also observed that TCE competitively inhibited the dechlorination of cis-DCE and had a mild detrimental effect on the dechlorination of VC. Thus, careful selection of biotreatment conditions, possibly with the help of a model such as the one presented herein, is required to minimize the production of partially dechlorinated intermediates.