Depuis 2002 a émergé le concept de « catalyse électromicrobienne ». Cette même année, une équipe du LGC a démontré un phénomène de transfert d’électrons entre un biofilm aérobie marin et une cathode d’acier inoxydable. A partir de ces biofilms a été isolée une souche bactérienne, Algoriphagus yeomjeoni, pouvant former un biofilm électroactif monoespèce. Les objectifs de ce travail ont été de rechercher cette capacité à réduire du dioxygène chez des bactéries marines mais également chez une souche issue de l’industrie agroalimentaire Lactococcus lactis. La première partie de cette étude porte sur l’étude d’Algoriphagus yeomjeoni. Les essais électrochimiques ont eu lieu en eau de mer synthétique, dont on contrôle la composition. Aucune production de courant n’a pu être détectée même en repassant en eau de mer naturelle. La perte d’électroactivité de cette souche nous a amené à la deuxième partie de ce travail qui a été la recherche de nouveaux isolats bactériens électroactifs à partir d’un biofilm formé en milieu marin. La population microbienne de ce biofilm a été étudiée par pyroséquençage. Puis, quatre souches bactériennes ont pu être isolées et identifiées. Ces souches appartenant au genre Bacillus, Roseobacter, Pseudoalteromonas et Marinobacter ont toutes présentées des capacités de réduction du dioxygène à la cathode aussi bien en eau de mer naturelle qu’eau de mer synthétique. Enfin, des essais électrochimiques ont été réalisés avec Lactococcus lactis. Cette souche a présenté des capacités électrochimiques dans un compartiment anodique avec un record de performance de 400 mA.m-2. Et, pour la première fois, Lactococcus lactis a été capable de catalyser une réduction impliquant le dioxygène à une cathode avec une densité de courant maximale de 50 mA.m-2. / Since 2002 emerged the concept of "microbial electro-catalysis". That same year, a team from the Chemical Engineering Laboratory demonstrated a phenomenon of electron transfer between a marine aerobic biofilm and a stainless steel cathode. From these biofilms was isolated a bacterial strain Algoriphagus yeomjeoni which form a mono-species electroactive biofilm. The objectives of this work were to seek the ability to catalyze a reduction of oxygen amongst marine bacteria but also in a strain used in food industry Lactococcus lactis. The first part of this study focuses on the study of Algoriphagus yeomjeoni. Electrochemical tests were conduct in synthetic seawater, whose composition is controlled. No power generation could be detected even by returning in natural seawater. The loss of electroactivity of this strain led us to the second part of the work that has been looking for new electroactive bacterial isolates from a biofilm formed in a marine environment. This microbial population in this biofilm was studied by pyrosequencing. Then, four bacterial strains have been isolated and identified. These strains of the genus Bacillus, Roseobacter, Pseudoalteromonas and Marinobacter have all shown the ability to reduce the oxygen at the cathode in both natural and synthetic seawater. Finally, electrochemical tests were performed with, Lactococcus lactis. This strain showed electrochemical capacity in an anode compartment with a record performance up to 400 mA.m-2. Furthermore, and for the first time, Lactococcus lactis was able to catalyze the oxygen reduction involving a cathode with a maximum current density of 50 mA.m-2.

With the current interest in alternative methods of energy production and increased utilization of existing energy sources, microbial fuel cells have become an important field of research. Microbial fuel cells are devices which harvest electrons from microorganisms created by their enzymatic oxidation of complex carbon substrates or consumed by their reduction of chemical oxidants. Microbial fuel cells with photosynthetic biocathodes are of particular interest due to their ability to simultaneously produce electricity and hydrocarbons while reducing carbon dioxide. Most species of microorganisms including many bacteria and yeasts require exogenous electron transfer mediators in order to allow electron transfer with an electrode. While adding such chemicals is simple enough at a lab scale, problems arise with chemical costs and separation at a larger scale. The goal of this research was to develop electrodes composed of a robust material which will eliminate the need for added soluble electron mediators in a photosynthetic biocathode microbial fuel cell. Electrodes made from stainless steel 304L have been coated in a conductive polymer (polypyrrole) and an immobilized electron transfer mediator (methylene blue) and tested chemically for stability and in a microbial fuel cell environment for use in bioanodes and biocathodes. The use of these immobilized mediator in the photosynthetic biocathode increased the open circuit voltage of the cell from 0.17 V to 0.24 V and the short circuit current from 8 mA/m2 to 64 mA/m2 (normalized to the geometric surface area of the electrode) when compared to using the same mediator in solution. The opposite effect was seen when using the electrodes in a bioanode utilizing Saccharomyces cerevisiae. The open circuit voltage decreased from 0.37 V to 0.31 V and the short circuit current decreased from 94 mA/m2 to 24 mA/m2 when comparing the immobilized mediator to soluble mediators. The impact of the membrane and pH of the anode and cathode solutions were quantified and were found to have much less of an effect on the internal resistance than the microbial factors.

With the current interest in alternative methods of energy production and increased utilization of existing energy sources, microbial fuel cells have become an important field of research. Microbial fuel cells are devices which harvest electrons from microorganisms created by their enzymatic oxidation of complex carbon substrates or consumed by their reduction of chemical oxidants. Microbial fuel cells with photosynthetic biocathodes are of particular interest due to their ability to simultaneously produce electricity and hydrocarbons while reducing carbon dioxide. Most species of microorganisms including many bacteria and yeasts require exogenous electron transfer mediators in order to allow electron transfer with an electrode. While adding such chemicals is simple enough at a lab scale, problems arise with chemical costs and separation at a larger scale. The goal of this research was to develop electrodes composed of a robust material which will eliminate the need for added soluble electron mediators in a photosynthetic biocathode microbial fuel cell. Electrodes made from stainless steel 304L have been coated in a conductive polymer (polypyrrole) and an immobilized electron transfer mediator (methylene blue) and tested chemically for stability and in a microbial fuel cell environment for use in bioanodes and biocathodes. The use of these immobilized mediator in the photosynthetic biocathode increased the open circuit voltage of the cell from 0.17 V to 0.24 V and the short circuit current from 8 mA/m2 to 64 mA/m2 (normalized to the geometric surface area of the electrode) when compared to using the same mediator in solution. The opposite effect was seen when using the electrodes in a bioanode utilizing Saccharomyces cerevisiae. The open circuit voltage decreased from 0.37 V to 0.31 V and the short circuit current decreased from 94 mA/m2 to 24 mA/m2 when comparing the immobilized mediator to soluble mediators. The impact of the membrane and pH of the anode and cathode solutions were quantified and were found to have much less of an effect on the internal resistance than the microbial factors.

Microbial electrochemical technologies (MET) exploit the bioelectrocatalytic activity of microorganisms, with a main focus on waste-to-resource recovery. Electromethanogenesis, a type of MET, describes the process of CO2 reduction specifically to methane, catalyzed by methanogens that utilize the cathode directly as an electron donor or through H2 evolving from the cathode surface. Applications are mainly in the direction of bioelectrochemical power-to-gas, as well as biogas upgrading and carbon capture and utilization. As the cathode and its associated microbial consortia are key to the process, larger scale applications require improvements especially in terms of optimal operational parameters, cathode materials and the dynamics of the effect of electron transfer within the cathodic biofilm. The focus of this dissertation is to improve the understanding of the dynamics and function of methaneproducing biofilms grown on cathodes in electromethanogenic reactors in the presence of two different electron donors: the cathode and the H2 evolving from the cathode surface. The spatial homogeneity of the microbial communities across the area of the cathode was demonstrated, which is relevant for large scale applications where reproducibility is required for predictable engineered systems. Metagenomic and metatranscriptomic methods were applied to elucidate the short-term changes in the actively transcribed methanogenesis and central carbon assimilation pathways in response to varying the availability of electrons by changing the set cathode potential in a novel Methanobacterium species enriched from electromethanogenic biocathodes. Although changes in functional performance were evident with varying potential, no significant differential expression was observed and genes from the methanogenesis and carbon assimilation pathways were highly expressed throughout. Indium tin oxide (ITO) as a potentially hydrogen evolution reaction (HER) – inert cathode material was evaluated using the mixotrophic Methanosarcina barkeri in an attempt to develop a simplified material-science driven approach to future electron transfer studies. It was found to be electrochemically unstable under the tested conditions, losing its conductivity over time. Overall, the findings from these studies provide new knowledge on the effects of electron donor availability on the functional performance and the biocathode community dynamics. The understandings derived from the study are relevant to methanogenic processes and should aid in system scaleup design.

Microbial desalination cells (MDCs), a recent technological discovery, allow for simultaneous wastewater treatment and desalination of saline water with concurrent electricity production. The premise for MDC performance is based on the principles that bioelectrochemical (BES) systems convert wastewaters into treated effluents accompanied by electricity production and the ionic species migration (i.e. protons) within the system facilitates desalination. One major drawback with microbial desalination cells (MDCs) technology is its unsustainable cathode chamber where expensive catalysts and toxic chemicals are employed for electricity generation. Introducing biological cathodes may enhance the system performance in an environmentally-sustainable manner. This study describes the use of autothrophic microorganism such as algae and Anammox bacteria as sustainable biocatalyst/biocathode in MDCs. Their great potential for high valuable biomass production combined with wastewater treatment presents these systems as a viable option to replace expensive/unsustainable catalysts for oxygen production in MDCs. Since alga is a photosynthetic microorganism, the availability of light as well as the electron-donating anodic process may have significant effects on the biocathode performance. A series of experiments evaluating these effects proved that algae perform better under natural light/dark cycles and that higher COD concentrations do not necessarily improve the power density. Furthermore, three different process configurations of photosynthetic MDCs (using Chlorella vulgaris) were evaluated for their performance and energy generation potentials. Static (fed-batch, SPMDC), continuous flow (CFPMDC) and a photobioreactor MDC (PBMDC, resembling lagoon type PMDCs) were developed to study the impact of process design on wastewater treatment, electricity generation, nutrient removal, and biomass production and the results indicate that PMDCs can be configured with the aim of maximizing the energy recovery through either biomass production or bioelectricity production. In addition, the microbial community analysis of seven different samples from different parts of the anode chamber, disclosed considerable spatial diversity in microbial communities which is a critical factor in sustaining the operation of MDCs. This study provides the first proof of concept that anammox mechanism can be beneficial in enhancing the sustainability of microbial desalination cells to provide simultaneous removal of ammonium from wastewater and contribute in energy generation.

Partial nitritation combined with the anaerobic ammonium oxidation (Anammox) process offers a way of replacing the conventional nitrogen removal process of nitrification-denitrification, lowering the need for oxygen and chemical input, as well as reducing the production of sludge. However, as a by-product of the biochemical reaction driven by anammox bacteria, it produces nitrate-nitrogen (NO3- - N) (16-26% nitrogen removed), which is problematic. Microbial desalination cells (MDCs) are a promising technology capable of converting biodegradable organics into electricity (by electroactive bacteria), providing for simultaneous desalination, and wastewater treatment. Despite being a promising technology, MDCs have limitations. The first-proof of-concept of MDC was demonstrated using acetate as the organic source, expensive platinum as a catalyst, and ferricyanide as an electron acceptor in the cathode that makes MDC costly, environmentally unfriendly, and unsustainable. This research investigated the integration of the anammox and nitration processes in MDCs as a long-term biocatalyst/biocathode for sustainable and energy-efficient nitrogen removal and electricity generation. A series of experiments were designed and performed to evaluate the performance of the anammox process as a biocatalyst in MDCs. The results concluded that the anammox process can be used as a biocatalyst to accept electrons in MDCs producing 444 mW/m3 of power density and 84% of ammonium nitrogen removal. Furthermore, the concept of using a one-stage nitritation anammox process as a biocathode in MDC was evaluated and produced a maximum power output of 1007 mW/m3. Two configurations of anammox MDCs (anaerobic-anammox cathode MDC (AnAmmoxMDC) and nitritation-anammox cathode MDC (NiAmoxMDC) were compared with an air cathode MDC (CMDC), operated in fed-batch mode. The NiAmoxMDC showed better performance in terms of power production and nitrogen removal. The co-existence of aerobic ammonium oxidizing bacteria (AOB) and anammox bacteria in the same biocathode of single-stage NiAmoxMDC concluded the resource-efficient wastewater treatment. Furthermore, two-stage nitritation anammox as a biocathode in MDC was evaluated and proved to be energy-efficient bioelectrochemical wastewater treatment by producing 1500 mW/m3 (300 mW/m2) of maximum power output. This research provides the first proof of concept that nitritation-anammox biocathode can provide a sustainable and energy-efficient nitrogen removal along with desalination and bioelectricity generation.

Les piles à combustible microbiennes (PCMs) sont une technologie convertissant l’énergie chimique stockée dans la matière organique en énergie électrique à l’anode à l’aide de bactéries dites électroactives. A l'inverse des bioanodes, l’intérêt porté aux cathodes biologiques dans les PCMs est récent et ces dernières sont encore peu étudiées. Les objectifs de ces recherches ont donc été d’identifier et de décrire des bactéries ainsi que les mécanismes permettant de catalyser une réduction cathodique notamment celle de l’oxygène. La première partie des travaux portant sur la réalisation d’un criblage pour la formation de biocathodes a révélé l’inefficacité du métabolisme hétérotrophe pour la conception de biocathodes performantes. De ce fait, la suite des travaux a porté sur la bactérie chimiolithoautotrophe acidophile Acidithiobacillus ferrooxidans. Deux mécanismes de transfert d’électrons dépendant du fer ont été mis en évidence générant un courant maximal de -3,8 A.m-2. Nous avons également abordé les mécanismes de transfert d’électrons en milieu neutre chez une bactérie hétérotrophe, Pseudomonas brassicacearum NFM421 bien que peu performante en terme de production de courant. Cette dernière permet une catalyse indirecte de la réduction de l’oxygène à la cathode à pH neutre via un métabolite secondaire le 2,4-diacéthylphloroglucinol associé au fer, mettant encore une fois en lumière l’importance du fer dans le transfert d’électrons. Ce travail a également porté sur la potentielle application des biopiles dans des environnements anthropisés en vue de l’extraction de métaux et plus particulièrement de terre rares qui s’avèrent être une voie de recherche prometteuse. / Microbial fuel cells (MFCs) are devices that convert chemical energy contained in organic matter into electrical energy using electroactive bacteria that act at the anode. Currently, MFCs performances are limited by the use of abiotic cathode. The interest in biological cathode has recently started and less is known about bacterial diversity and mechanism that catalyze cathodic reduction. The aims of the research work are therefor to identify and describe potential bacteria and mechanism involved in such catalysis. The first part of the work is the realization of a screening that did not show conclusive results and might indicate that heterotrophic metabolism was not an efficient choice. Next, Acidithiobacillus ferrooxidans had been used and shown two extracellular electron transfer mechanism depending on iron. A maximum current intensity of -3,8 A.m-2 had been reached. To be close to operational condition of MFC, Pseudomonas brassicacearum NFM 421 has also be use and shown capacity to indirectly catalyze the oxygen reduction at a neutral pH using the 2,4-diacethylphloroglucinol, a secondary metabolite, associated to iron. However, current reach remained weak. Considering difficulty to build efficiant biocathode at a neutral pH, the end of this work had been focused on a new application of the MFC: metal and rare earth extraction from soil and contaminated site that appeared to be a great research opportunity to follow.

Este trabalho descreve a preparação e caracterização de biocátodos para biocélula a combustível Etanol e Metanol//O2 utilizando a enzima lacase (trametes versicolor) num sistema de transferência eletrônica mediada (TEM). Na primeira etapa do trabalho, os resultados de cinética enzimática com a enzima lacase em solução e imobilizada sobre tecido de carbono mostraram que os vários parâmetros experimentais (pH, temperatura, estabilidade) analisados devem ser considerados, a fim de se obter atividade máxima com os biocatalisadores. Além disso, em relação aos testes cinéticos e de estabilidade, pode-se inferir que o dendrímero PAMAM pode ser empregado como um bom agente imobilizante na preparação de bicátodos para biocélula a combustível enzimática. Na segunda etapa do trabalho, uma semibiocélula Etanol//O2 foi testada e os eletrocatalisadores testados foram o verde de metileno (VM) e o azul de meldola (AM). Os testes de potência mostraram a importância da presença do mediador ABTS e do eletrocatalisador (VM) para melhorar o desempenho do dispositivo. Na terceira etapa do trabalho, eletrodos com diferentes mediadores (ABTS, ferro porfirina, ferroceno, complexo de ósmio e complexo de rutênio) e com polipirrol eletropolimerizado na superfície do eletrodo foram testados numa semibiocélula Metanol//O2. Os testes de semibiocélula Etanol e Metanol//O2 com transferência eletrônica mediada mostraram que os biocátodos preparados com o dendrímero PAMAM e com os diferentes eletrocatalisadores e mediadores, se mostraram capazes de gerar densidades de potência competitivas em relação aos valores encontrados na literatura. / This work describes the preparation and characterization of biocathodes for Ethanol and Methanol//O2 biofuel cell using the enzyme laccase (trametes versicolor) enzyme and mediated electron transfer (MET). Investigation of the enzymatic kinetics of the enzyme laccase in solution and immobilized onto carbon platforms showed that the analyzed experimental parameters (pH, temperature, and stability) must be considered for maximum activity to be achieved. The kinetic and stability tests revealed that PAMAM dendrimers constitute very good immobilization agent to prepare biocathodes for enzymatic biofuel cell. The second part of this work, dealt with Ethanol//O2half-cell using methylene green (MG) ormeldola blue (MB) as electrocatalyst. The power test evidenced that it is important to have ABTS as mediator and an electrocatalyst, to ensure that the device performs better. The third part of this work evaluated electrodes with distinct mediators (ABTS, iron porphyrin, ferrocene, osmium complex, and ruthenium complex) and containing electropolymerized polypyrrole on their surface in a Methanol//O2half-cell. Ethanol and Methanol//O2 half-cell tests with mediated electron transfer showed that the biocathodes prepared with PAMAM dendrimers, electrocatalyst, and distinct mediators generated competitive power densities as compared with literature data.

Este trabalho descreve a preparação e caracterização de biocátodos para biocélula a combustível Etanol e Metanol//O2 utilizando a enzima lacase (trametes versicolor) num sistema de transferência eletrônica mediada (TEM). Na primeira etapa do trabalho, os resultados de cinética enzimática com a enzima lacase em solução e imobilizada sobre tecido de carbono mostraram que os vários parâmetros experimentais (pH, temperatura, estabilidade) analisados devem ser considerados, a fim de se obter atividade máxima com os biocatalisadores. Além disso, em relação aos testes cinéticos e de estabilidade, pode-se inferir que o dendrímero PAMAM pode ser empregado como um bom agente imobilizante na preparação de bicátodos para biocélula a combustível enzimática. Na segunda etapa do trabalho, uma semibiocélula Etanol//O2 foi testada e os eletrocatalisadores testados foram o verde de metileno (VM) e o azul de meldola (AM). Os testes de potência mostraram a importância da presença do mediador ABTS e do eletrocatalisador (VM) para melhorar o desempenho do dispositivo. Na terceira etapa do trabalho, eletrodos com diferentes mediadores (ABTS, ferro porfirina, ferroceno, complexo de ósmio e complexo de rutênio) e com polipirrol eletropolimerizado na superfície do eletrodo foram testados numa semibiocélula Metanol//O2. Os testes de semibiocélula Etanol e Metanol//O2 com transferência eletrônica mediada mostraram que os biocátodos preparados com o dendrímero PAMAM e com os diferentes eletrocatalisadores e mediadores, se mostraram capazes de gerar densidades de potência competitivas em relação aos valores encontrados na literatura. / This work describes the preparation and characterization of biocathodes for Ethanol and Methanol//O2 biofuel cell using the enzyme laccase (trametes versicolor) enzyme and mediated electron transfer (MET). Investigation of the enzymatic kinetics of the enzyme laccase in solution and immobilized onto carbon platforms showed that the analyzed experimental parameters (pH, temperature, and stability) must be considered for maximum activity to be achieved. The kinetic and stability tests revealed that PAMAM dendrimers constitute very good immobilization agent to prepare biocathodes for enzymatic biofuel cell. The second part of this work, dealt with Ethanol//O2half-cell using methylene green (MG) ormeldola blue (MB) as electrocatalyst. The power test evidenced that it is important to have ABTS as mediator and an electrocatalyst, to ensure that the device performs better. The third part of this work evaluated electrodes with distinct mediators (ABTS, iron porphyrin, ferrocene, osmium complex, and ruthenium complex) and containing electropolymerized polypyrrole on their surface in a Methanol//O2half-cell. Ethanol and Methanol//O2 half-cell tests with mediated electron transfer showed that the biocathodes prepared with PAMAM dendrimers, electrocatalyst, and distinct mediators generated competitive power densities as compared with literature data.

Las etapas de transferencia electrónica o transferencia de carga involucradas en reacciones electroquímicas juegan un papel muy importante en un gran número de procesos biológicos y bioquímicos. Hoy en día, el interés de la comunidad científica se centra en explorar y entender exhaustivamente la naturaleza biológica y química de los fenómenos bioelectroquímicos que ocurren en los seres vivos, con el objeto de mimetizarlos en el laboratorio. Los procesos bioelectrocatalíticos presentan un amplio abanico de aplicaciones dirigidas al: (i) desarrollo de biorreactores electroquímicos para la mitigación de las emisiones de gases de efecto invernadero, la eliminación de contaminantes presentes en aguas residuales y urbanas, o la síntesis de productos con alto valor añadido para la industria, (ii) el desarrollo de biopilas y biobaterías, y (iii) el desarrollo de (bio)sensores electroquímicos con fines analíticos. Sin embargo, la implantación en el mercado de dispositivos basados en procesos biocatalíticos aún se enfrenta a varios desafíos, como son la robustez, la estabilidad a largo plazo, la reproducibilidad y la rentabilidad de producción en términos de materiales y fabricación de los dispositivos electroquímicos. La motivación de esta tesis doctoral es la de enfrentarse a algunos de los desafíos con los que se encuentra hoy en día la bioelectrocatálisis, para ello esta tesis doctoral se centra, principalmente en el estudio de nuevos materiales y mejora de rutas y estrategias bioelectrocatalíticas, con la finalidad de desarrollar dispositivos electroquímicos con aplicaciones analíticas y en la obtención de productos de valor añadido. En primer lugar esta tesis doctoral recoge el estudio y desarrollo de (bio)sensores electroquímicos para la determinación de lactato, L-cisteína, peróxido de hidrógeno y pH en medios biológicos complejos, y en segundo lugar estudia la bioelectrosíntesis de ácido fórmico a través de la reducción bioelectroquímica de dióxido de carbono.