La vinasse est le déchet liquide retiré de la base de colonnes de distillation de l'éthanol de canne à sucre à hauteur de 12 à 15 litres par litre d'alcool, ce qui entraîne une production estimée à environ 350 milliards de litres en 2012/2013 au Brésil. La vinasse a un pH faible et une forte demande chimique en oxygène, ce qui peut provoquer la désertification des terres, si elle est utilisée en excès comme amendement. En outre, une contamination des eaux souterraines liée aux épandages est observée dans certaines régions. L'aptitude de la vinasse à jouer le rôle de source d'éléments nutritifs pour la production de biohydrogène et d'acides gras volatils par des consortia microbiens anaérobies a été évaluée. Deux différents milieux à base de vinasse ont été proposés, un avec l’addition de jus de canne à sucre et l’autre avec l’addition de la mélasse comme source de carbone, et ont été comparés à un milieu supplémenté en saccharose. Des cultures bactériennes pures (4) et des consortia microbiens (7) ont été cultivées dans les milieux proposés et la production des acides gras volatils (AGV) et de biohydrogène ont été evalués. Le consortium LPBAH1, originaire d’un lac d’une ferme laitière et sélectionné pour la fermentation de la vinasse avec du jus de canne à sucre, conduit à un rendement en H2 de 7,14 molH2/molsucrose et à une teneur en hydrogène dans le biogaz d'env. 31% après optimisation. Par ailleurs, le processus optimisé en utilisant le consortium LPBAH2, originaire de fèces de chauves-souris frugivores, permet d'obtenir 3,66 molH2/molsucrose et 32,7% d'hydrogène dans le biogaz. Le processus proposé est d'une grande importance pour donner une destination plus rationnelle de la vinasse et d'élargir le bouquet énergétique brésilien en réduisant sa dépendance des combustibles fossiles. / Vinasse is the liquid waste removed from the base of sugarcane ethanol distillation columns at a ratio of 12-15 liters per liter of alcohol, resulting in an estimated production of approx. 350 billion liters in 2012/2013 in Brazil. Vinasse has a low pH and high chemical oxygen demand, which can cause land desertification when indiscriminately used as fertilizer. Also, underground water contamination is being observed in some regions. We evaluated the potential of vinasse as nutrient source for biohydrogen and volatile fatty acids production by means of anaerobic consortia. Two different vinasse-based media were proposed, using sugarcane juice or molasses as carbon source, and were compared to fermentation in a sucrosesupplemented medium. Pure cultures (4) and consortia (7) were cultured in the propose media and evaluated for volatile fatty acids (VFAs) and biohydrogen production. The consortium LPBAH1, originated from faeces of fruit bat, was selected for fermentation of vinasse supplemented with sugarcane juice and resulted in a higher H2 yield of 7.14 molH2/molsucrose and hydrogen content in biogas of approx. 31% after process optimization. Similarly, the optimized process using the consortium LPBAH2, originated from a lake of a dairy farm, resulted in 3.66 molH2/molsucrose and 32.7% hydrogen content in biogas. The proposed process is of great importance for giving a more rational destination to vinasse and expanding Brazilian energy matrix, reducing the dependence of fossil fuels.

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.

Les microorganismes photosynthétiques suscitent un intérêt biotechnologique important pour la production de dihydrogène. La cyanobactérie Synechocystis sp. PCC 6803 est capable d'initier une photoproduction d'hydrogène catalysée par une hydrogénase [NiFe] bidirectionnelle qui se présente sous la forme d'un complexe pentamérique (HoxEFUYH). Toutefois l'inhibition de cette enzyme par l'oxygène émis par le photosystème II rend cette photoproduction transitoire et constitue un verrou majeur au développement de tels procédés. L'exploitation de ces organismes impose une meilleure compréhension des bases moléculaires associées à la sensibilité de l'hydrogénase envers l'oxygène ainsi que des composantes limitant son activité de production d'H2, ce qui implique la connaissance détaillée des jeux d'interactions avec ses partenaires physiologiques NAD(P)+/NAD(P)H.Diverses substitutions d'acides aminés potentiellement impliqués dans la sensibilité de l'enzyme à l'O2 et situés au cœur du site actif (Ileu64, Leu107, Leu112) de la sous-unité catalytique HoxH ont été réalisées. Les résultats in vitro et in vivo indiquent une sensibilité envers l'O2 moindre chez le mutant I64M, qui présente une diffusion limitée et un biais vers l'activité de production d'H2.L'étude des interactions de mutants de délétion des gènes diaphorase hoxE et hoxF avec les cofacteurs NAD(P) a montré que NAD+/NADH semblent être les partenaires privilégiés de l'hydrogénase pour le transfert d'électrons, tandis que le NADPH a un effet activateur sur l'enzyme.Ces études apportent des éléments importants pour envisager une optimisation ciblée et maîtrisée pour la bioproduction d'H2. / Oxygenic photosynthetic organisms are a matter of great biotechnological interest for the production of dihydrogen using what seem to be infinite resources, water and solar energy. The cyanobacterium Synechocystis sp. PCC 6803 encodes a bidirectional [NiFe] hydrogenase consisting of a pentameric complex (HoxEFUYH) that allows it to carry H2 photoproduction. However, it is a transient process, mainly due to the oxygen sensitivity of hydrogenases, O2 being produced at PSII during photosynthesis. Future exploitation of these organisms in bioprocesses requires a better understanding of the molecular bases of O2 sensitivity of the hydrogenase and of the elements limiting H2 evolution which involves detailed knowledge of the interactions of the enzyme with its physiological partners NAD(P)+/NAD(P)H.Various mutants of the Synechocystis hydrogenase were created by genetic engineering, targeting specific amino acid residues (Ileu64, Leu107, Leu112) in the catalytic subunit HoxH identified as putative critical elements for O2 sensitivity. Results obtained in vitro and in vivo indicate that the substitution I64M slightly improves O2 tolerance and alters gas diffusion kinetics with a bias towards H2 production. Studying the interaction of diaphorase gene-deletion mutants hoxF and hoxE with partners NAD(P) showed that NAD+/NADH are the preferential electron acceptor/donor of the hydrogenase, while NADPH is more efficient for enzyme activation.These studies provide first insights on the determinants of the oxygen sensitivity of the hydrogenase of Synechocystis and its activation, which are critical elements to consider in targeted optimization for bioproduction of H2.

Orientador: Pedro Magalhães Sobrinho / Resumo: O Brasil tem uma grande capacidade para produzir biocombustíveis devido a sua extensa área territorial com grande produtividade de matéria-prima para este setor. O Governo Federal vem investindo bastante neste setor, pois além de ser estratégico economicamente, também tem grandes vantagens na luta contra o aquecimento global. Desde 2005, ano em que se iniciou a comercialização do biodiesel em caráter voluntário, até 2017 a produção cresceu de 736 m3/ano para 4291294 m3/ano. Isto se deu devido a política de aumento obrigatório da porcentagem de biodiesel misturado ao diesel de petróleo de forma gradativa até o nível de 10% (B10) em 2018. Neste trabalho propõe-se um sistema de cogeração utilizando um motor de combustão interna (queimando gás natural), um queimador suplementar e uma caldeira de recuperação para gerar energia elétrica e vapor superaquecido necessário para o processo de reforma a vapor do glicerol; comparando-o com outro sistema proposto por Galarza (2017) que utiliza microturbina à gás. Este sistema será estudado para uma planta capaz de produzir 17820 m3/ano de biodiesel operando 7920 h/ano. Foi escolhido um motor de combustão interna com potência de 200 kW e consumo de 42,9 kg/h, pois foi o motor com menor consumo, mas que mantêm o nível de temperatura dos gases de exaustão (471,8 ºC). Energeticamente o sistema se demonstrou viável, mas possui grande perda de calor na chaminé que poderia ser aproveitada, como na produção de água quente por exemplo. A análise ex... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Brazil has a large capacity to produce biofuels due to its extensive land area with high productivity of raw material for this sector. The Federal Government has been investing a lot in this sector, since besides being economically strategic, it also has great advantages in the fight against global warming. Since 2005, when the commercialization of biodiesel began on a voluntary basis, by 2017 production increased from 736 m3/year to 4291294 m3/year. This was due to the policy of mandatory increase of the percentage of biodiesel mixed with petroleum diesel gradually until the level of 10% (B10) in 2018. In this work, a cogeneration system is proposed using an internal combustion engine (burning natural gas), an additional burner and a recovery boiler to generate electrical energy and superheated steam necessary for the steam reforming process of glycerol; comparing it with another system proposed by Galarza (2017) that uses microturbine to gas. This system will be studied for a plant capable of producing 17820 m3/year of biodiesel operating at 7920 hours per year. An internal combustion engine with a power of 200 kW and a consumption of 42.9 kg/h was chosen, because it was the engine with the lowest consumption, but it maintains the level of temperature of exit (471.8 ºC). Energetically the system has proven viable, but it has great heat loss in the chimney that could be better used, as in the production of hot water for example. Exergetic analysis helps to understand the sys... (Complete abstract click electronic access below) / Mestre

Um reator anaeróbio com biomassa imobilizada e agitação mecânica foi operado em bateladas sequenciais com efluente sintético a base de sacarose visando à produção de biohidrogênio. O sistema foi inoculado com lodo proveniente de um reator anaeróbio metanogênico. Foram avaliados a produção de biohidrogênio, os rendimentos por carga aplicada e removida, a estabilidade e eficiência do reator quando submetido a diferentes cargas orgânicas volumétricas aplicada (COAV 9,0; 12,0 ;13,5; 18,0; 18,0 e 27,0 kgDQO/\'M POT.3\'.d), as quais foram modificadas em função da concentração afluente (3600 e 5400 mgDQO/L) e do tempo de ciclo (4, 3 e 2 h). O reator apresentou uma capacidade remoção da matéria orgânica (DQO) estável e próxima a um valor de 18%, e uma boa capacidade de conversão de carboidratos (sacarose) a qual permaneceu entre 83 e 97% ao longo da operação. Verificou-se uma diminuição do desempenho de remoção do reator com o aumento da carga orgânica aplicada e, além disso, valores crescentes de concentração afluente (e tempos de ciclo iguais) e tempos de ciclo menores (e concentrações afluente iguais) resultaram em eficiências menores de conversão. Houve predominância dos ácidos acético, butírico e propiônico com o aumento da carga orgânica, e de etanol em todas as condições. A maior concentração de biohidrogênio no biogás (24-25%) foi atingida nas condições com COAV de 12,0 e 13,5 kgDQO/\'M POT.3\'.d; a maior velocidade de produção diária (0,139 mol/d) foi atingida na condição com COAV de 18,0 kgDQO/\'M POT.3\'.d; e os maiores rendimentos de produção molares por carga aplicada e removida foram 2,83 e 3,04 mol \'H IND.2\'/kgSAC, respectivamente, na condição com COAV de 13,5 kgDQO/\'M POT.3\'.d. Não se verificou uma tendência de modificação do rendimento de biohidrogênio do reator em função da concentração afluente para tempos de ciclo iguais e do tempo de ciclo para concentrações afluente iguais, concluindo-se sobre a necessidade do estudo do comportamento do processo em função da carga orgânica aplicada e também das variáveis que definem a carga orgânica aplicada. / A mechanically stirred anaerobic sequencing batch reactor containing immobilized biomass treated sucrose-based synthetic wastewater to produce biohydrogen. The system was inoculated with sludge from an anaerobic methanogenic reactor. The following have been assessed: production of biohydrogen, yield per applied and removed load, reactor stability and efficiency under different applied volumetric organic loads applied (AVOL - 9.0, 12.0, 13.5, 18.0, 18. 0 and 27.0 kgDQO/\'M POT.3\'.d), which were modified according to the influent concentration (3600 and 5400 mgDQO/L) and cycle time (4, 3 and 2 h). The reactor\'s ability to remove organic matter (COD) remained stable and close to a value of 18%, and the system shows good ability to convert carbohydrates (sucrose) which remained between 83 and 97% during the operation. There was a decrease in removal performance of the reactor with increasing applied organic load, and furthermore, increasing influent concentration (at constant cycle length) and cycle lengths (at constant influent concentrations) resulted in lower conversion efficiencies. Under all conditions, as organic load increased there was a predominance of acetic, propionic and butyric acid, as well as ethanol. The highest concentration of bio-hydrogen in the biogas (24-25%) was achieved at conditions with AVOL of 12.0 and 13.5 kgDQO/\'M POT.3.d, the highest daily production rate (0.139 mol/d ) was achieved at AVOL of 18.0 kgDQO/\'M POT.3\'.d, and the highest production yields per removed and applied load were 2.83 and 3.04 mol \'H IND.2\'/kgSAC, respectively, at AVOL of 13.5 kgDQO/\'M POT.3\'.d. Biohydrogen yield showed no tendency to change with varying influent concentration at constant cycle length neither with varying cycle length at constant influent concentrations, indicating the need to study the behavior of the process as a function of applied organic load as well as of the variables which define the applied organic load.

A presente pesquisa teve como proposta avaliar o reator anaeróbio, operado de forma descontínua ou descontínua alimentada, contendo biomassa imobilizada em suporte inerte e com recirculação da fase líquida (AnSBBR) aplicado à produção de biohidrogênio a partir da codigestão de glicerina (efluente da produção de biodiesel) e soro de leite (efluente da produção de laticínios). A estabilidade, os índices de desempenho (referentes à produtividade e rendimento molar do hidrogênio) e o fator de conversão (entre biogás produzido e matéria orgânica consumida) foram analisados em função da composição afluente (porcentagem de cada substrato alimentado ao sistema), da variação da carga orgânica, do tempo de enchimento e da temperatura (20, 25, 30 e 35ºC). Os ensaios foram realizados em diferentes proporções dos substratos utilizando-se variadas cargas orgânicas volumétricas (10,3; 17,1 e 24,0 gDQO.L-1.d-1), as quais foram modificadas em função: (i) da concentração afluente (3, 5 e 7 gDQO.L-1) e (ii) do tempo de ciclo (4, 3 e 2 h, ou seja, 6, 8 e 12 ciclos diários). Também foram realizados ensaios para a produção de biometano a partir da codigestão proposta nesta pesquisa (com COAV de 7,6 gDQO.L-1.d-1) em diferentes proporções de mistura. Para a produção de biometano, a condição com 75% de soro e 25% de glicerina (base DQO) obteve os melhores resultados: produtividade molar de 101,8 molCH4.m-3.d-1 e rendimento por carga aplicada de 13,3 molCH4.kgDQO-1; o que representa um aumento de produtividade de cerca de 9% e 30% quando comparado com a digestão anaeróbia de soro e glicerina puros, respectivamente. A produção de metano no melhor ensaio aconteceu predominantemente pela rota hidrogenotrófica. Para a produção de biohidrogênio, a maior produtividade e rendimento do reator foram obtidas no ensaio operado com razão de mistura de 75% soro e 25% glicerina, com 7 gDQO.L-1 de concentração afluente, tempo de ciclo de 3 h e tempo de enchimento de 1,5 h (modo batelada alimentada - COAV de 23,9 kgDQO.m-3.d-1), a 30°C: foi obtida uma produtividade molar de 129,0 molH2.m-3.d-1 e rendimento de 5,4 molH2.kgDQO-1. Esses resultados representam um aumento de produtividade de 145% em relação a mono-digestão do soro na condição inicial, o que indica o benefício significativo da adição de glicerina ao afluente, provavelmente devido à sua capacidade tamponante, e a otimização das condições operacionais. A adição de glicerina e o aumento da COAV balancearam as rotas de produção de hidrogênio, sendo produzido de forma mais equilibrada pelas vias do ácido acético, butírico e valérico. A caracterização do consórcio microbiano desse ensaio indicou que a comunidade microbiana presente no AnSBBR foi dominada por Ethanoligenens e Megasphaera. / The current research evaluated an anaerobic reactor, operated in batch or fed-batch mode, containing immobilized biomass in inert support and with recirculation of the liquid phase (AnSBBR), applied to the production of biohydrogen co-digesting glycerin (effluent from biodiesel production process) and whey (effluent from dairy industry). Stability, performance (regarding productivity and molar hydrogen yield) and conversion factor (between biogas produced and organic matter consumed) were analyzed according to the percentage of each substrate fed to the system, organic loading rate, filling time and temperature (20, 25, 30 and 35ºC). Assays were carried out using different substrates proportions and organic loading rates (10.3; 17.1 and 24.0 gCOD.L-1.d-1), which have been modified in function of: (i) influent concentration (3, 5 and 7 gCOD.L-1) and (ii) cycle length (4, 3 and 2 h, i.e. 6, 8 and 12 cycles daily). Assays were also carried out aiming for biomethane production using the proposed co-digestion (with AVOL of 7.6 gDQO.L-1.d-1) with different proportions of substrate mixture. For biomethane production, the assay conducted with 75% whey and 25% glycerin (COD basis) obtained the best results: molar productivity of 101.8 molCH4.m-3.d-1 and yield per applied load of 13.3 molCH4. kgCOD-1; which is an increase in productivity of about 9% and 30% when compared with the anaerobic mono-digestion of whey and glycerin, respectively. Methane production in this assay came mainly from the hydrogenotrophic route. For biohydrogen production, the highest productivity and yield were achieved in the assay operated with 75% whey and 25% glycerin, with 7 gCOD.L-1 of influent concentration, 3 h of cycle time and filling time of 1.5 h (fed batch mode - AVOL of 23.9 kgCOD.m-3.d-1), at 30°C: a molar productivity of 129.0 molH2.m-3.d-1 and yield of 5.4 molH2.kgCOD-1 were obtained. These results represent a productivity increase of 145% in relation to whey mono-digestion at its initial condition, which indicates the significant benefit of glycerin addition to the influent, probably due to its buffering capacity, and improvement of operational conditions. The addition of glycerin and the increase in AVOL balanced the hydrogen production routes, since hydrogen was produced similarly by the acetic, butyric and valeric acid routes. The characterization of the microbial consortium of this assay indicated that the microbial community present in the AnSBBR was dominated by Ethanoligenens and Megasphaera.

A sustainable and low-cost system, namely a photo-bioelectrochemical system (photo-BES), based on the natural blueprint of photosynthetic microorganisms was studied. The aim of this research work is to improve the efficiency of electron transfer of the microorganisms for bioelectricity generation. The first strategy adopted was the evaluation of the exoelectrogenic activity of oxygenic photosynthetic cyanobaterium, Synechococcus elongatus PCC 7942, in biophotovoltaic (BPV) platforms through a comparative performance analysis of bioanode materials. The second approach involved improving the performance of anoxygenic photosynthetic bacterium, Rhodopseudomonas palustris ATCC® 17001™, by varying the ratio of nitrogen to carbon sources (N:C) to maximise both biohydrogen production and exoelectrogenesis for conversion into bioelectricity in photosynthetic microbial fuel cells (photoMFCs). A linear correlation was obtained between average surface roughness/surface area and maximum power density of ITO-coated and graphene/ITO-coated substrates. Graphene/ITO-coated PET bioanodes produced the highest maximum power output of 29±4 μW m-2 in a single chamber BPV device due to improved biofilm formation and improved electrochemical activity. XG Leaf®, also known as graphene paper, helped to bridge the shortcomings of carbon fibres in terms of wettability. The most hydrophilic, 240 μm thick graphene paper, produced the highest maximum power output of 393±20 μW m-2 in a membrane electrode assembly (MEA)-type BPV device, mainly due to reduced electrochemical polarisation. A proof of concept study compared the performance of screen-printed graphene onto a membrane separator against 3D-printed bioanodes coated with carbon nanotubes. One mm thick 3D-printed bioanode was better performing as its structures promoted a much denser biofilm with extensive fibrous extracellular matrix. Using a ratio of N:C=0.20 resulted in higher biohydrogen production and higher exoelectrogenic activity, generating a maximum power output of 361±157 mW m-2 and 2.39±0.13 mW m-2, respectively. This study provided additional insight in improving the electron transfer efficiency, which could be used to further optimise photo-BESs as part of future research and development for sustainable technologies.

Accelerated global warming plus an increasing need for energy is an equation not easily solved, thus new forms of sustainable energy production are urgently requested. In this context hydrogen production based on a cyanobacterial system offers an environmentally friendly alternative for energy capture and conversion. Cyanobacteria can produce hydrogen gas from sun light and water through the combination of photosystems and hydrogenases, and are suitable to cultivate in large scale. In the present thesis the maturation process of [NiFe]-hydrogenases is investigated with special focus on transcription of the accessory genes encoding proteins needed for assembly of the large and possibly also for the small hydrogenase subunit. The cyanobacteria used are two N2-fixing, filamentous, heterocystous strains; Nostoc sp. strain PCC 7120 and Nostoc punctiforme PCC 73102. For a biotechnological exploration of hydrogen production tools for regulatory purposes are important. The transcription factor CalA (cyanobacterial AbrB like) (Alr0946 in the genome) in Nostoc sp. strain PCC 7120 was found to be involved in hydrogen metabolism by regulating the transcription of the maturation protein HypC. Further the bidirectional hydrogenase activity was down-regulated in the presence of elevated levels of CalA, a result important to take into account when optimizing cyanobacteria for hydrogen production. CalA regulates at least 25 proteins in Nostoc sp. strain PCC 7120 and one of the down-regulated proteins was superoxide dismutase, FeSOD. The characterization of FeSOD shows that it has a specific and important function in the oxidative stress tolerance of Nostoc sp. stain PCC 7120. Since CalA is involved in regulation of both the hydrogen metabolism as well as stress responses these findings indicate that Alr0946 is an important transcription factor in Nostoc sp. strain PCC 7120 active on a global level in the cell. This thesis adds more knowledge concerning maturation and regulation of cyanobacterial hydrogenases which might be useful for future large scale hydrogen.

The pH is an important parameter in anaerobic reactors, may influence the rate of hydrogen production and inhibit the action of microorganisms hidrogenotróficos, it may affect the activity of hydrogenase as well as the route of metabolism. In this context, the present research aimed to study the best operating condition in relation to the pH factor in Anaerobic Fluidized Bed Reactor (RALF) for enhanced biological production of hydrogen from wastewater processing cassava (manipueira) plus supplements. The reactor used in laboratory scale possessed height of 190 cm and total volume 4192 cm3 net volume used was 2.7 L. It was used as support material for microbial adhesion, expanded clay having a diameter of 2.8 to 3.35 mm. For starting the reactor was used as the inoculum, an anaerobic lagoon sludge that was liquid swine waste, it has undergone a heat treatment so that there was a selection of microorganisms, resulting mainly in anaerobic hidrogenotróficos . It was used for room temperature operation of the reactor (25 to 30°C), and the hydraulic retention time (HRT) was applied 2 h. For substrate (cassava), took it a Chemical Oxygen Demand (COD) theoretical initial 4000 mg.L- 1. For this study we evaluated different pH values in the range 4.0 to 5.3 . In this sense, according to the results obtained can be said that the experiment was effective for biohydrogen production from cassava in RALF, observing an optimum pH of 4.9 with a production volume checked 0,31 L/h/L and 3.5 mol H2/mol yield glucose, a conversion rate of hydrogen in cassava 88%. The route of butyric acid fermentation is the most prevalent in this pH value. The percentages of metabolites soluble in this pH were: 4% acetic acid, 54% butyric acid, 4% propionic acid, 22% caproic acid and 16% ethanol. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O pH é um parâmetro fundamental em reatores anaeróbios, podendo influenciar na velocidade de produção de hidrogênio e inibir a ação de microrganismos hidrogenotróficos, pois pode afetar a atividade da hidrogenase, bem como na via de metabolismo. Neste contexto, a presente pesquisa objetivou estudar a melhor condição operacional em relação ao fator pH em Reator Anaeróbio de Leito Fluidificado (RALF) para uma maior produção biológica de hidrogênio a partir da água residuária do processamento da mandioca (a manipueira) acrescida de suplementos. O reator utilizado, em escala de laboratório, possuía altura de 190 cm e volume total 4192 cm3, o volume útil utilizado foi 2,7 L. Utilizou-se, como material suporte para adesão microbiana, a argila expandida com diâmetro de 2,8 à 3,35 mm. Para a partida do reator utilizou-se, como inóculo, lodo de uma lagoa anaeróbia que tratava resíduo líquido de suinocultura, o mesmo passou por um tratamento térmico para que houvesse uma seleção de microrganismos, resultando principalmente nos anaeróbios hidrogenotróficos. Utilizou-se a temperatura ambiente para a operação do reator (25 a 30 °C), e o Tempo de Detenção Hidráulica (TDH) aplicado foi de 2h. Para o substrato (manipueira), adotou-se uma Demanda Química de Oxigênio (DQO) teórica inicial de 4000 mg.L-1. Para este estudo foram avaliados diferentes valores de pH, na faixa de 4,0 a 5,3. Neste sentido, de acordo com os resultados verificados pode-se dizer que a realização do experimento foi eficiente para a produção de biohidrogênio a partir da manipueira em RALF, observando-se um pH ótimo de 4,9 com uma produção volumétrica verificada de 0,31 L/h/L e rendimento de 3,5 mol H2/mol glicose, a uma taxa de conversão de manipueira em hidrogênio de 88%. A rota fermentativa do ácido butírico foi a que predominou neste valor de pH. As percentagens dos metabólitos solúveis no pH de 4,9 foram: 4% de ácido acético, 54% de ácido butírico, 4% de ácido propiônico, 22% de ácido capróico e 16% de etanol.

Um reator anaeróbio com biomassa imobilizada e agitação mecânica foi operado em bateladas sequenciais com efluente sintético a base de sacarose visando à produção de biohidrogênio. O sistema foi inoculado com lodo proveniente de um reator anaeróbio metanogênico. Foram avaliados a produção de biohidrogênio, os rendimentos por carga aplicada e removida, a estabilidade e eficiência do reator quando submetido a diferentes cargas orgânicas volumétricas aplicada (COAV 9,0; 12,0 ;13,5; 18,0; 18,0 e 27,0 kgDQO/\'M POT.3\'.d), as quais foram modificadas em função da concentração afluente (3600 e 5400 mgDQO/L) e do tempo de ciclo (4, 3 e 2 h). O reator apresentou uma capacidade remoção da matéria orgânica (DQO) estável e próxima a um valor de 18%, e uma boa capacidade de conversão de carboidratos (sacarose) a qual permaneceu entre 83 e 97% ao longo da operação. Verificou-se uma diminuição do desempenho de remoção do reator com o aumento da carga orgânica aplicada e, além disso, valores crescentes de concentração afluente (e tempos de ciclo iguais) e tempos de ciclo menores (e concentrações afluente iguais) resultaram em eficiências menores de conversão. Houve predominância dos ácidos acético, butírico e propiônico com o aumento da carga orgânica, e de etanol em todas as condições. A maior concentração de biohidrogênio no biogás (24-25%) foi atingida nas condições com COAV de 12,0 e 13,5 kgDQO/\'M POT.3\'.d; a maior velocidade de produção diária (0,139 mol/d) foi atingida na condição com COAV de 18,0 kgDQO/\'M POT.3\'.d; e os maiores rendimentos de produção molares por carga aplicada e removida foram 2,83 e 3,04 mol \'H IND.2\'/kgSAC, respectivamente, na condição com COAV de 13,5 kgDQO/\'M POT.3\'.d. Não se verificou uma tendência de modificação do rendimento de biohidrogênio do reator em função da concentração afluente para tempos de ciclo iguais e do tempo de ciclo para concentrações afluente iguais, concluindo-se sobre a necessidade do estudo do comportamento do processo em função da carga orgânica aplicada e também das variáveis que definem a carga orgânica aplicada. / A mechanically stirred anaerobic sequencing batch reactor containing immobilized biomass treated sucrose-based synthetic wastewater to produce biohydrogen. The system was inoculated with sludge from an anaerobic methanogenic reactor. The following have been assessed: production of biohydrogen, yield per applied and removed load, reactor stability and efficiency under different applied volumetric organic loads applied (AVOL - 9.0, 12.0, 13.5, 18.0, 18. 0 and 27.0 kgDQO/\'M POT.3\'.d), which were modified according to the influent concentration (3600 and 5400 mgDQO/L) and cycle time (4, 3 and 2 h). The reactor\'s ability to remove organic matter (COD) remained stable and close to a value of 18%, and the system shows good ability to convert carbohydrates (sucrose) which remained between 83 and 97% during the operation. There was a decrease in removal performance of the reactor with increasing applied organic load, and furthermore, increasing influent concentration (at constant cycle length) and cycle lengths (at constant influent concentrations) resulted in lower conversion efficiencies. Under all conditions, as organic load increased there was a predominance of acetic, propionic and butyric acid, as well as ethanol. The highest concentration of bio-hydrogen in the biogas (24-25%) was achieved at conditions with AVOL of 12.0 and 13.5 kgDQO/\'M POT.3.d, the highest daily production rate (0.139 mol/d ) was achieved at AVOL of 18.0 kgDQO/\'M POT.3\'.d, and the highest production yields per removed and applied load were 2.83 and 3.04 mol \'H IND.2\'/kgSAC, respectively, at AVOL of 13.5 kgDQO/\'M POT.3\'.d. Biohydrogen yield showed no tendency to change with varying influent concentration at constant cycle length neither with varying cycle length at constant influent concentrations, indicating the need to study the behavior of the process as a function of applied organic load as well as of the variables which define the applied organic load.