Hydrogen is re-emerging as a serious alternative to fossil fuels. It is a clean gas with high energy density and its combustion only generates water vapour. Nevertheless, the hydrogen industry has a significant carbon footprint since this gas is mostly derived from fossil fuels reforming processes. This project focusses on the development of sustainable alternatives to conventional hydrogen production, in which approaches based on dark fermentation (DF) using an inexpensive residue from the brewery industry as primary feedstock are presented. Firstly, a fungal pre-treatment (FT) was proposed to degrade a high-strength brewery waste slurry (BWS) to obtain an effluent with a lower concentration of chemical oxygen demand (COD) but rich in readily fermentable sugars for the ensuing DF, thus improving hydrogen yields (HY). Secondly, microbial electrolysis and fuel cells (MECs and MFCs) were proposed to assist DF, generating electricity in MFCs while improving HY by MECs. Coupling both microbial electrochemical technologies sequentially after DF did not show any advantage. However, promising results were obtained for electricity and hydrogen production when taking a single-staged approach. Treating BWS directly by MFCs produced 2.0 watts/g COD consumed, while the DF process assisted simultaneously by MECs (DF/MEC) produced 1.6 times more hydrogen than DF alone. An average HY of 2.32 ± 0.06 mol H₂/mol glucose was attained between both DF/MEC and DF after FT, hence approaching the theoretical value of 2.4 mol H₂/mol glucose, representing roughly a 50% improvement compared to DF alone. With an overall COD reduction above 76%, the DF after FT exhibited the highest energy conversion rate per substrate consumed (6.3 kJ/g COD). As valuable by-products obtained, up to 31 g/L of fungal biomass, which is appreciated in many state-of-the-art biomaterials applications, was produced by using BWS. While in the DF/MEC process, 18 g/L of butyric acid were generated, which is three times more than with DF alone. Butyric acid being the precursor to butanol and building block of biodegradable thermoplastics, this result is not without significance. The proposed approaches not only valorize BWS but also validate their economic and environmental attractiveness as promising alternative hydrogen production methods.

Wastewater treatment has developed as a mature technology over time. However, conventional wastewater treatment is a very energy-intensive process. Bioelectrochemical system (BES) is an emerging technology that can treat wastewater and also recover resources such as energy in the form of electricity/hydrogen gas and nutrients such as nitrogen and phosphorus compounds. Microbial electrolysis cell (MEC) is a type of BES that, in the presence of an additional voltage, can treat wastewater and generate hydrogen gas. This is a promising approach for wastewater treatment and value-added product generation, though it may not be sustainable in the long run, as it relies on fossil fuels to provide that additional energy. Thus, it is important to explore alternative renewable resources that can provide energy to power MEC. Waste heat is one such resource that has not been researched extensively, particularly at the low-temperature spectrum. This was utilized as a renewable resource by converting waste heat to electricity using a device called thermoelectric generator (TEG). TEG converted simulated waste heat from an anaerobic digester to power an MEC. The feasibility of TEG to act as a power source for an MEC was investigated and its performance compared to the external power source. Various cold sources were analyzed to characterize TEG performance. To explore this integrated TEG-MEC system further, a hydraulic connection was added between the two systems. Wastewater was used as a cold source for TEG and it was recirculated to the anode of the MEC. This system showed improved performance with both systems mutually benefitting each other. The operational parameters were analyzed for the optimization of the system. The integrated system could generate hydrogen at a rate of 0.36 ± 0.05 m3 m-3 d-1 for synthetic domestic wastewater treatment. For the practical application, it is necessary to estimate the cost and narrow the focus on the functions of the system. Techno-economic analysis was performed for MEC with cost estimation and net present value model to understand the economic viability of the technology. The application niche of the BES was described and directions for addressing the challenges towards a full-scale operation were discussed. The present system provides a sustainable method for wastewater treatment and resource recovery which can play an important role in human health, social and economic development and a strong ecosystem. / Doctor of Philosophy / An average person produces about 50-75 gallons of wastewater every day. In addition to the households, wastewater is generated from industries and agricultural practices. As the population increases, the quantity of wastewater production will inevitably increase. To keep our rivers and oceans clean and safe, it is essential to treat the wastewater before it is discharged to the water bodies. However, the conventional wastewater treatment is a very energy (and thus cost) intensive process. For low-income and developing parts of the world, it is difficult to adapt the technology everywhere in its present form. Furthermore, as the energy is provided mostly by fossil fuels, their limited reserves and harmful environmental effects make it critical to find alternative methods that can treat the wastewater at a much lower energy input. For a circular and sustainable economy, it is important to realize wastewater as a resource which can provide us energy, nutrients, and water, rather than discard it as a waste. Bioelectrochemical systems (BES) is an emerging technology that can simultaneously treat wastewater and recover resources in the form of electricity/hydrogen gas, and nitrogen and phosphorus compounds. Microbial electrolysis cell (MEC) is a type of BES that is used to treat wastewater and generate hydrogen gas. An additional voltage is supplied to the MEC for producing hydrogen. In the long run, this may not be sustainable as it relies on fossil fuels to provide that additional energy. Thus, it is important to explore alternative renewable resources that can provide energy to power MEC. Waste heat is a byproduct of many industrial processes and widely available. This was utilized as a renewable resource by converting waste heat to electricity using a device called thermoelectric generator (TEG). TEG converted simulated waste heat from an anaerobic digester to power an MEC. The mutual benefit for MEC and TEG was also explored by connecting the system electrically and hydraulically. Cost-estimation of the system was performed to understand the economic viability and functions of the system were developed. The present system provides a sustainable method for wastewater treatment and resource recovery which can play an important role in human health, social and economic development and a strong ecosystem.

L'hydrogène, qui constitue une solution alternative et durable à l’usage d’énergies fossiles, est produit essentiellement par reformage de combustibles fossiles (95%). Des filières de production plus soucieuses de l'environnement sont envisagées. Deux familles de technologies sont explorées: 1) par décomposition thermochimique ou électrochimique de l'eau et 2) à partir de différentes sources de biomasse. Parmi celles-ci, les cellules d'électrolyse microbienne ou «Microbial electrolysis cell (MEC)» permettent de produire de l'hydrogène par électrolyse de la matière organique. Une MEC consiste en une cathode classique qui assure la production d'hydrogène par la réduction électrochimique de l'eau, associée à une bioanode qui oxyde des substrats organiques en dioxyde de carbone. Ce processus d'oxydation n'est possible que grâce au développement sur l'anode d'un biofilm microbien électroactif qui joue le rôle d'électro-catalyseur. Par rapport aux procédés courants d'électrolyse de l'eau, une MEC requière un apport énergétique 5 à 10 fois plus faibles. En outre, les procédés « classiques » de production de bio-hydrogène par voie fermentaire en cultures mixtes convertissent des sucres avec des rendements limités à 2-3 moles d'hydrogène par mole d'hexose tout en coproduisant des acides organiques. Alimenté par de l'acétate, une MEC produit au maximum 3 moles d'hydrogène/mole d'acétate. Le couplage de la fermentation à un procédé d'électrolyse microbienne pourrait donc produire de 8 à 9 moles d'hydrogène/mole d'hexose, soit un grand pas vers la limite théorique de 12 moles d'hydrogène/mole d'hexose. L'objectif de cette thèse est d'analyser les liens entre la structure des communautés microbiennes dans les biofilms électroactifs et en fermentation, les individus qui les composent et les fonctions macroscopiques (électroactivité du biofilm, production d'hydrogène) qui leur sont associées dans des conditions permettant de réaliser le couplage des deux procédés. L'originalité de cette étude a été de travailler en milieu salin (30-35 gNaCl/L), favorable au transport de charges dans l'électrolyte de la MEC. Dans un premier temps, la faisabilité de la fermentation en conditions salines (3-75 gNaCl/L) a été démontrée en lien avec l'inhibition de la consommation de l'hydrogène produit et une forte prédominance d'une nouvelle souche de Vibrionaceae à des concentrations en sel supérieures à 58 gNaCl/L. D'autre part, la mise en œuvre de biofilms électroactifs dans des conditions compatibles avec la fermentation sombre a permis la sélection d'espèces dominantes dans les biofilms anodiques et présentant des propriétés électroactives très prometteuses (Geoalkalibacter subterraneus et Desulfuromonas acetoxidans) jusqu'à 8,5 A/m². En parallèle, la sélection microbienne opérée lors d'une méthode d'enrichissement utilisée pour sélectionner ces espèces à partir d'une source d'inoculum naturelle sur leur capacité à transférer leurs électrons à des oxydes de Fer(III) a été étudiée. Une baisse des performances électroactives du biofilm liée à une divergence de sélection microbienne dans ces deux techniques de sélection mène à limiter le nombre de cycle d'enrichissement sur Fer(III). Cependant, l'enrichissement sur Fer(III) reste une alternative efficace de pré-selection d'espèces électroactives qui permet une augmentation de rendement faradique de 30±4% à 99±8% par rapport au biofilm obtenu avec un inoculum non pré-acclimaté. Enfin, l'ajout d'espèces exogènes issues de la fermentation sombre sur le biofilm électroactif a révélé une baisse de l'électroactivité du biofilm se traduisant par une diminution de la densité de courant maximale produite. Cette baisse pourrait s'expliquer par à une diminution de la vitesse de transfert du substrat due à un épaississement apparent du biofilm. Cependant, un maintien de sa composition microbienne et de la quantité de biomasse laisse supposer une production d'exopolymères (EPS) dans le biofilm en situation de couplage. / Nowadays, alternative and sustainable solutions are proposed to avoid the use of fossil fuel. Hydrogen, which constitutes a promising energy vector, is essentially produced by fossil fuel reforming (95%). Environmentally friendly production systems have to be studied. Two main families of technologies are explored to produce hydrogen: 1) by thermochemical and electrochemical decomposition of water and 2) from different biomass sources. Among those last ones, microbial electrolysis cells (MEC) allow to produce hydrogen by electrolysis of organic matter. A MEC consists in a classical cathode, which provides hydrogen production by electrochemical reduction of water, associated to a bio-anode that oxidizes organic substrates into carbon dioxide. This process is only possible because of the anodic development of an electroactive microbial biofilm which constitutes an electrocatalyst. In comparison to classical water electrolysis process, a MEC requires 5 to 10 times less electrical energy and therefore reduces the energetic cost of produced hydrogen. Furthermore, classical process of dark fermentation in mixed cultures converts sugars (saccharose, glucose) to hydrogen with a limited yield of 2-3 moles of hydrogen per mole of hexose because of the coproduction of organic acids (mainly acetic and butyric acids). Fed with acetate, a MEC can produce up-to 3 moles of hydrogen per mole of acetate. Therefore, the association of these two processes could permit to produce 8 to 9 moles of hydrogen per mole of hexose, which represents a major step toward the theoretical limit of 12 moles of hydrogen per mole of hexose.Therefore, this work aims at analyzing the relationship between microbial community structures and compositions and the associated macroscopic functions (biofilm electroactive properties, hydrogen production potential) in electroactive biofilms and in dark fermentation in conditions allowing the coupling of the two processes. The originality of this study is to work in saline conditions (30-35 gNaCl/L), which favors the charges transfer in the MEC electrolyte.First of all, feasibility of dark fermentation in saline conditions (3-75 gNaCl/L) has been shown. This was linked to an inhibition of produced hydrogen consumption and the predominance of a new Vibrionaceae species at salt concentrations higher than 58 gNaCl/L. Secondly, electroactive biofilm growth in conditions compatibles to dark fermentation (pH 5.5-7 and fed with different organic acids) allowed to select dominant microbial species in anodic biofilms that present promising electroactive properties (Geoalkalibacter subterraneus and Desulfuromonas acetoxidans) with maximum current densities up to 8.5 A/m². In parallel, the microbial selection occurring during iron-reducing enrichment method used to select species from a natural inoculum source and based on their capacity to transfer electrons to iron oxydes (Fe(III)) has been studied. A decrease of electroactive performances of the biofilm linked to the divergence of microbial selection led to a limitation of the number of iron-enrichment steps. However, enrichment on Fe(III) presents an efficient alternative to pre-select electroactive species with an increase of coulombic efficiency from 30±4% to 99±8% in comparison with a biofilm obtained with a non-acclimated inoculum. Finally, the addition of exogenous bacteria from a dark fermenter on the electroactive biofilm revealed a decrease of electroactivity with a decrease of maximum current density produced. This diminution could be explained by a lower substrate transfer due to an apparent thickening of the biofilm. Nevertheless, the stability of microbial composition and of bacterial quantity on the anode suggests that a production of exopolymers (EPS) occurred.

La cellule d’électrolyse microbienne (CEM) permet la conversion de la biomasse en dihydrogène via un apport théorique en énergie électrique 10 fois moindre que celui de l’électrolyse de l’eau. Un tel procédé fonctionne via la technologie des bioanodes, qui permet la catalyse de l’oxydation de la biomasse en CO2 par l’intermédiaire d’un biofilm électro-actif. Les travaux exposés dans ce manuscrit de thèse ont pour but l’optimisation des performances de la bioanode, la compréhension des mécanismes de catalyse et la réalisation d’un prototype à échelle laboratoire. L’optimisation par l’étude de paramètres opératoires en montage 3 électrodes a montré que l’utilisation de sédiments de salins comme source de micro-organismes avec électrode en feutre de carbone polarisée à + 0,1 V / ECS à une température de 40°C permet la formation de bioanodes capables de débiter jusqu’à 85 A.m-2 pour une conductivité de 10,4 S.m-1. A 30°C, le pyroséquençage ADN a mis en lumière l’émergence des genres bactériens Desulfuromonas et Marinobacter. La conception et l’exploitation d’un modèle de voltammétrie cyclique a montré que le transport des électrons au sein du biofilm était environ 100 fois plus lent que le métabolisme bactérien. L’utilisation de la spectroscopie d’impédance électrochimique montre que la résistance au transfert de charge à l’interface électrode/solution baisse de 24 kΩ.cm2 à 64 Ω.cm2 lors de la formation du biofilm. Un taux de production maximum de 2,85 LH2.L-1.j-1 ainsi qu’une durée de vie de plus de 50 jours du procédé ont été obtenus lors de la conduite d’un prototype laboratoire de CEM. / Microbial electrolysis cell (MEC) is a recent and promising bioelectrochemical process that converts biomass onto hydrogen thanks to an amount of electrical energy 10 times smaller than for water electrolysis. The operation of the process is making possible by the bioanode technology which catalyses the biomass combustion onto CO2 through an electro-active biofilm. The purpose of the present work consists on the optimisation of the bioanode, the understanding of the catalysis mechanism and a scaling-up by the designing of a MEC prototype. Using a three-electrode device and sediment of salt marshes as inoculum, the study of the experimental parameters demonstrated that carbon felt poised at + 0.1 V /ECS at 40°C led to the formation of bioanode able to generate up to 85 A.m-2 at a solution conductivity of 10,4 S.m-1. For a temperature of 30°C, DNA pyrosequencing denoted the presence of the two bacterial genera Desulfuromonas and Marinobacter. The development and the exploitation of a cyclic voltammetric model showed that electron transfer within the biofilm ran almost 100 times slower than bacterial metabolism. Electrochemical impedance spectroscopy during biofilm formation revealed a decreasing of the charge transfer resistance at the electrode/solution interface from 24 kΩ.cm2 to 64 Ω.cm2. Designing and first experiments with a 6L CEM prototype led to a hydrogen production rate of 2.85 LH2.L-1.j-1 and a process life time of up to 50 days. Those performances were achieved in a reproducible way.

L’élimination des micropolluants organiques est devenue aujourd’hui un objectif de santé publique car leur toxicité et bioaccumulation au travers de la chaine trophique sont incontestables. Les hydrocarbures aromatiques polycycliques (HAP) et le nonylphénol (NP) présents en faible concentration dans l’eau usée sont peu éliminés par le traitement. Ces composés hydrophobes se retrouvent fortement sorbés à la matière organique des boues de station d’épuration. Les procédés de traitement de ces boues, comme la digestion anaérobie (DA) jouent un rôle central car ils constituent une des dernières barrières avant rejet vers l’environnement par épandage. La DA élimine les HAP et le NP mais les performances restent insatisfaisantes. L’objectif de cette thèse est d’améliorer les performances d’élimination des HAP et NP par la DA en utilisant l’électrolyse microbienne et l’ajout des matériaux conducteurs. Les résultats montrent que le graphite poreux permet de lever deux limites à la bioremédiation des HAP: le manque d'accepteurs d'électrons terminaux et la biodisponibilité limitée des HAP. En effet, le graphite poreux semble faciliter l'échange direct d'électrons avec la communauté syntrophique anaérobie ce qui améliore les performances d’hydrolyse de la matière et des contaminants associés, particulièrement, l’élimination de 12 HAP est accrue de 21 à 33 %. Pour le NP, les processus impliqués semblent être différents car aucune amélioration n’est observée quelles que soient les conditions.. L’addition du graphite non-poreux (avec une surface spécifique moindre) et du platine (avec une conductivité plus élevée) n’éliminait que deux HAP de faible poids moléculaire suggérant que la conductivité ne constitue pas un facteur majeur dans la dissipation des HAP. L’ajout du graphite poreux en plus grande quantité, par contre, n’a pas confirmé l’hypothèse qu’une augmentation de surface spécifique du matériau conducteur accroit également l’élimination des HAP. Lors de la DA assistée par différents matériaux, un enrichissement de méthanogènes hydrogénotrophs a été constaté qui pourra être à la racine des performances observées. Dans ce contexte, la composition microbienne de l’inoculum joue un rôle majeur dans l’ampleur d’une biodégradation des HAP. L'utilisation de matériaux conducteurs abordables tels que le graphite poreux et non-poreux pourrait présenter une stratégie de biodégradation alternative pour éliminer les HAP des boues, du sol ou des sédiments. / The elimination of organic micropollutants from the environment has become a public health goal today because of their toxicity and bioaccumulation through the trophic chain. Polycyclic aromatic hydrocarbons (PAHs) and nonylphenol (NP) are found at low concentrations in wastewaters. They are removed from wastewaters by sorption onto sewage sludge due to their hydrophobicity. Anaerobic digesiton (AD) that is used for sewage sludge treatmentThe treatments of these sludge, like anaerobic digestion (AD), therefore, plays a central role in reducing the micropollutant load before dissemination to the environment via sludge spreading. Removal performances of PAHs and NP are removed byunder AD are, yet, but limitedwith low performances. The aim of this PhD work is to enhance removalthese performances thanks to the use of two emerging techniques, microbial electrolysis and the addition of conductive materials. The results demonstrate that independent of the application of a potential porous graphite circumvents two limits of PAH bioremediation: the lack of terminal electron acceptors and PAHs’ limited bioavailability. Mediatorless electron exchange between the anaerobic syntrophic community and the conductive material presumably facilitates sludge hydrolysis which, in turn, leads to the enhanced bioavailability of PAHs and their subsequent biodegradation. The reductions of 12 PAHs were improved by 21 to 33% while NP was eliminated to the same extent in the digesters with and without conductive material. The addition of non-porous graphite (with less specific surface) and platinum (exhibiting higher conductivity) eliminated only two low molecular weight PAHs suggesting that conductivity is not a major factor in the dissipation of PAHs. The addition of porous graphite in larger quantities, however, did not support the hypothesis that an increase in specific surface area of the conductive material also enhances the removal of PAHs. During conductive material supplemented AD, an enrichment of hydrogenotrophic methanogens was ascertained which could be at the root of the removal performance. In this context, the microbial composition of the inoculum plays a major role in the extent of PAH biodegradation. The use of affordable conductive materials such as porous and non-porous graphite may present an alternative biodegradation strategy for removing PAHs from sludge, soil or sediments.

Notre étude porte sur la preuve de concept de contrôle électrochimique de la méthanogénèse, métabolisme clé de la digestion anaérobie et de la bioremédiation des eaux, en exploitant le principe des piles à combustible microbiennes. Une première partie bibliographique vise à décrire les mécanismes de la méthanogénèse dans le contexte de l'auto-épuration des eaux et de production naturelle de gaz à effet de serre (GES). Les technologies de pile à combustibles microbiennes y sont traitées. Une analyse critique des études sur le contrôle électrochimique de la méthanogénèse permet de dimensionner un montage expérimental dédié à la quantification des GES en cultures biologiques électro-stimulées. Sa conception, sa validation ainsi que les méthodes de mise en culture sont décrites dans une seconde partie. Une série de cultures préliminaires sur des boues digérées anaérobies de station d'épuration permettent d'identifier et fixer les paramètres expérimentaux. Dans une troisième partie, une étude expérimentale fait la preuve de concept de contrôle électrochimique de la méthanogénèse avec une diminution significative de 33% en CH4 (tension de +300 mV vs Ag/AgCl) par rapport à la méthanogénèse naturelle non stimulée. Toutefois, la stimulation contribue à multiplier par 10 la production de CO2. Ce constat amène la problématique supplémentaire d'impact sur l'effet de serre des cultures étudiées. Nous allons donc plus loin que l'objectif initial en nous intéressant à l'empreinte carbone générée par l'ensemble des GES. Le traitement électrochimique, outre la diminution du CH4 produit, permet de diminuer la contribution à l'effet de serre de 15% des cultures électro-stimulées / Our study focuses on the proof of concept of electrochemical control of methanogenesis, key metabolism of anaerobic digestion and water bioremediation, using the principle of microbial fuel cells. A first bibliographic section aims to describe the mechanisms of methanogenesis in the context of self-purification of water and natural production of greenhouse gases (GHG). Microbial fuel cell technologies are addressed. A critical analysis of the studies dealing with electrochemical control of methanogenesis makes it possible to size an experimental setup dedicated to quantification of GHGs in electro-stimulated biological cultures. Its design, validation and methods of cultivation are described in a second part. A series of preliminary cultures on anaerobic digested sewage sludge make it possible to identify and set the experimental parameters. In a third part, an experimental study proves the concept of electrochemical control of methanogenesis with a significant decrease of 33% in CH4 (voltage of +300 mV vs Ag/AgCl) compared to natural unstimulated methanogenesis. However, stimulation contributes to a 10-fold increase in CO2 production. This observation leads to the additional problem of impact on the greenhouse effect of the cultures studied. We go further than the initial objective by looking at the carbon footprint generated by all GHGs. The electrochemical treatment, in addition to the reduction of CH4 produced, makes it possible to reduce the contribution to the greenhouse effect of 15% of electro-stimulated cultures