The aims of this project were to investigate a range of limitations affecting the electrical performance of bioelectrochemical systems (BES) and their use as analytical tools. The model cyanobacterium Synechocystis sp. PCC6803 was used to characterize light-driven BESs, or biophotovoltaic (BPV) devices. The phycobilisome (PBS) antenna size was altered to modify light absorption. At low to medium light intensities the optimum PBS antenna size was found to consist of one phycocyanin (PC) disc. Incorporating pulsed amplitude fluorescence (PAM) measurements into the BPV characterization allowed simultaneous comparison of photosynthetic efficiency to EET in Synechocystis. Non-photochemical quenching (NPQ) was investigated as a limiting factor in biophotovoltaic efficiency and was found to be reduced in the PBS antenna-truncated mutants. Fluorescence and electrochemical data were combined to develop a framework for quantifying the efficiency of light to bioelectricity conversion. This approach is a first step towards a more comprehensive and detailed set of analytical tools to monitor EET in direct relation to the underlying photosynthetic biology. A set of metabolic electron sinks were deleted to remove a selection of pathways that might compete with extracellular electron transfer (EET). The combined deletion of a bi-directional hydrogenase - HoxH, nitric oxide reductase - NorB, cytochrome-c oxidase - COX, bd-quinol oxidase - cyd, and the respiratory terminal oxidase - ARTO, roughly doubled light driven electron flux to EET. Deletion of nitrate reductase - NarB, and nitrite reductase - NirA, increased EET to a similar degree, but combination with the other knockouts compromised cell viability and did not increase output further. In addition to Synechocystis, the purple non-sulphur α-proteobacterium Rhodopseudomonas palustris CGA009 was used to test the effect of storage molecule synthesis knockout in a more industrially relevant organic carbon source driven BES, or microbial fuel cell (MFC). However, the removal of glycogen and poly-ß-hydroxybutyrate (PHB) did not have a significant effect on electrical output. Finally, the importance of electrode material and design for cell to anode connections in an MFC was investigated. EET from R. palustris was greatly enhanced using custom designed graphene and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) aerogels. Pristine graphene is also shown for the first time to be a viable, low cost alternative to platinum as a cathodic catalyst. Together, these results present a holistic view of major limitations on electrical output from BESs that may contribute to enhancing EET for power generation from MFCs in the long term, and optimization of BPV devices as reliable analytical tools in the short term.

Dans le cadre de ce projet initiant la nouvelle thématique de recherche sur les piles à combustible microbiennes (PCM) au LOCIE, nous tentons de répondre aux problématiques suivantes : Quelles stratégies d'intensification des transferts peuvent être mises en œuvre pour optimiser les efficacités de conversion chimiques et énergétiques des PCM ? Quels sont les moyens de caractérisation et de contrôle des phénomènes mécanistiques inhérents aux réactions bio-électro-chimiques à chaque électrodes ? Dans un premier temps, nous abordons le sujet à travers deux stratégies concrètes d'optimisation en terme d'architecture : l'utilisation d'électrodes poreuses en carbone vitreux réticulé (CVR) pour maximiser l'aire d'électrode active au sein d'un volume donné d'une part, et d'autre part, l'intégration multi-échelle via l'approche constructale, dont l'objectif est de minimiser la résistance à l'écoulement au sein du réacteur. Dans un second temps, nous conduisons une démarche fondamentale qui s'est attaché à identifier et caractériser les mécanismes électrochimiques, via l'évaluation de l'effet de facteurs d'ordre physico-chimiques (température, conductivité, pouvoir tampon et charge organique) et matériels (oxydation du CVR, catalyseur en platine sur la cathode, épaisseur de membrane, aire de cathode) sur le fonctionnement d'une PCM. Cette approche multifactorielle utilise la méthodologie des plans d'expérience via les tables de Tagushi. Des analyses par spectroscopie d'impédance électrochimique visent à apporter une vision complémentaire de notre système. L'analyse des spectres d'impédance des électrodes et du réacteur nous a permis de modéliser les mécanismes électrochimiques en jeu à travers des analogies électriques. / In this project we addressed the two following issues : what transfer intensification strategies can be set up to optimize the chemical and energetic yields in microbial fuel cells ? What are the most relevant methods to characterize and control the bio-electrochemical phenomenon that are taking place ? We first report two strategies regarding the reactor engineering that are (i) the use of reticulated vitreous carbon as high-surface area porous electrodes and (ii) the use of constructal approach as a multi-scale optimization for fluid distribution. Advantages and limits are discussed. In a second part, we address some basic research which aims at identifying and characterizing the electrochemical phenomenon occurring in our reactor and quantifying the effect of various physicochemical (temperature, conductivity, buffer and substrate concentration) and material factors (oxydized RVC, platinized air-cathode, membrane thickness, cathode surface area). This multifactorial analysis was performed using Tagushi experimental plans and electrochemical impedance spectroscopy (IES). IES was successfully used to simulate our electrodes and cell phenomenon based on electrical analogies using resistive and capacitive elements.

<p>Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O₂ to H₂O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the <i>aa</i>₃-type oxidases, to which cytochrome <i>c</i> oxidase (Cyt<i>c</i>O) belongs, which uses energy released in the O₂-reduction for proton pumping. The bacterial nitric oxide reductases (NORs) have been identified as divergent members of the HCuO-superfamily and are involved in the denitrification pathway where they catalyze the reduction of NO to NO₂. Although as exergonic as O₂-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the <i>cbb</i>₃-type oxidases are the closest relatives to the NORs and as such provide a link between the <i>aa</i>₃ oxidases and the NORs. The <i>cbb</i>₃ oxidases have been shown to pump protons with nearly the same efficiency as the <i>aa</i>₃ oxidases, despite low sequence similarity.</p><p>This thesis is focused on measurements of membrane potential generating reactions during catalysis in the Cyt<i>c</i>O and the <i>cbb</i>₃ oxidase from <i>Rhodobacter sphaeroides</i>, and the NOR from <i>Paracoccus</i> <i>denitrificans</i>, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in Cyt<i>c</i>O showed that only one proton is taken up and that no protons are pumped, at high pH. An additional kinetic phase was also detected at high pH that presumably originates to from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of Cyt<i>c</i>O. The measurements established that electrons and protons are taken up from the same side of the membrane in NOR. In addition, the directionality for proton uptake in <i>cbb</i>₃ oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O₂-reduction.</p>

Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O2 to H2O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the aa3-type oxidases, to which cytochrome c oxidase (CytcO) belongs, which uses energy released in the O2-reduction for proton pumping. The bacterial nitric oxide reductases (NORs) have been identified as divergent members of the HCuO-superfamily and are involved in the denitrification pathway where they catalyze the reduction of NO to NO2. Although as exergonic as O2-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the cbb3-type oxidases are the closest relatives to the NORs and as such provide a link between the aa3 oxidases and the NORs. The cbb3 oxidases have been shown to pump protons with nearly the same efficiency as the aa3 oxidases, despite low sequence similarity. This thesis is focused on measurements of membrane potential generating reactions during catalysis in the CytcO and the cbb3 oxidase from Rhodobacter sphaeroides, and the NOR from Paracoccus denitrificans, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in CytcO showed that only one proton is taken up and that no protons are pumped, at high pH. An additional kinetic phase was also detected at high pH that presumably originates to from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of CytcO. The measurements established that electrons and protons are taken up from the same side of the membrane in NOR. In addition, the directionality for proton uptake in cbb3 oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O2-reduction.