Comparadas com células de mamífero, as células de inseto são mais fáceis de cultivar, não acumulam quantidade significativa de sub-produtos tóxicos e apresentam maiores rendimentos na expressão de proteínas heterólogas, porém apresentam uma menor capacidade de realizar modificações pós-traducionais. Células de inseto podem ser empregadas na produção in vitro de baculovírus, usados como pesticidas biológicos. As células Sf-9 estão entre as células de inseto com uso mais difundido. Entender o metabolismo destas células permitirá melhorias nos processos que as empregam, entretanto, ainda há relativamente pouca informação sobre o assunto. Considerando mais especificamente o uso dessas células para produção de baculovírus, também é necessário mais entendimento sobre o processo infectivo e parâmetros que o afetam. Este trabalho teve como objetivo estudar a influência da suplementação do meio de cultivo com diferentes aminoácidos no desenvolvimento das células Sf-9 e determinar a concentração de oxigênio dissolvido ideal para o cultivo destas células, visando elaborar uma metodologia de cultivo em biorreator otimizada e, paralelamente, estudar o processo de infecção de cultivos dessas células com o baculovírus Spodoptera frugiperda (SfMNPV) em diferentes escalas. Para estudar a influência que a adição de aminoácidos ao meio tem sobre o crescimento celular, células Sf-9 foram cultivadas em frascos schotts de 100 e 500 mL, com 20 mL do meio SF900III SFM (serum free medium) suplementados com cisteína, prolina, serina ou asparagina. Os resultados foram comparados com cultivos feitos sem suplementação (controles). A condição que apresentou o melhor resultado em frasco schott foi replicada em biorreator de 1 L de volume útil. Para estudar a influência do oxigênio dissolvido (O.D.) foram testados diferentes setpoints de O.D. em cultivos em biorreator. Em tais ensaios foram testadas as concentrações de O.D de 10%, 30% e 50% da saturação com o ar. Para o estudo do processo de infecção, foram realizadas infecções em frascos schotts de 500 mL, com 20 mL de cultivo, e em biorreator de 1 L. Também foram realizadas infecções em garrafas T-25 como forma de controle de virulência do inóculo viral e do vírus produzido. As principais variáveis analisadas foram µmáx, Xvmáx YX/Glc. Nos ensaios de influencia de O.D., analisou-se também qO2 e qCO2 e, nos ensaios de infecção, a porcentagem de células contendo poliedros. A suplementação com prolina foi prejudicial ao cultivo. A adição de asparagina não teve qualquer influência no desenvolvimento celular. Os resultados das adições de cisteína e serina não foram muito conclusivos, em alguns ensaios houve aumento de Xvmáx, já em outros não foi notado efeito significativo. Nos ensaios em biorreator, todos os valores de O.D. testados apresentaram resultados semelhantes, já a adição de cisteína ao meio em biorreator foi bastante maléfica ao crescimento celular. Os ensaios de infecção mostraram que células Sf-9 são bastante susceptíveis à infecção pelo baculovírus Spodoptera frugiperda e boas produtoras de poliedros virais, e que a vazão gasosa tem efeito negativo na concentração viral na fase líquida dos cultivos em biorreator (título viral). / Compared to mammalian cells, insect cells are easier to culture, do not accumulate significant amounts of toxic byproducts and are capable of higher heterologous protein yields, but have a lower ability to perform post-translational modifications. Insect cells may be employed in the in vitro production of baculoviruses, used as biological pesticides. Sf-9 cells are among the most used insect cell lines. Understanding the metabolism of these cells would allow improvements in the processes that employ them, however, Howeverreports on the metabolism and physiology of Sf-9 cells and insect cells in general are scarce. When considering the use of these cells for baculovirus production, it is also necessary more understanding about the infective process and parameters that can affect it. This work aimed to study the influence that the supplementation of the culture medium with different amino acids have on the development of Sf-9 cells and to determine the ideal dissolved oxygen concentration for the culture of these cells, aiming to elaborate an optimized bioreactor culture methodology and, in parallel, to study the infection process of these cells with the Spodoptera frugiperda baculovirus (SfMNPV) at different scales. To study the influence that the addition of amino acids to the medium has on cell growth, Sf-9 cells were cultured in 100 and 500mL schott flasks with 20mL SF900III SFM (serum free medium) supplemented with cysteine, proline, serine or asparagine. The results were compared with cultures without supplementation (controls). The condition that presented the best result in schott flasks was replicated in a 1L bioreactor. To study the influence of dissolved oxygen (O.D.), experiments with different values of O.D. were conducted at a 1L bioreactor. The O.D. tested were 10%, 30% and 50% of air saturation. To study the infection process, infections were carried out in 500 mL schotted flasks, with 20 mL of culture, and in a 1L bioreactor. Infections were also carried out in 25 cm² T-flasks as a form of virulence control of the viral inoculum and virus produced. The main variables analyzed were µmax, Xvmax YX/Glc. In the O.D. tests, qO2 and qCO2 were also analyzed and, in the infection assays, the percentage of cells containing polyhedra. Proline supplementation was detrimental to the culture. The addition of asparagine had no influence on cellular growth. The results of cysteine and serine additions were not very conclusive, in some studies there was an increase of Xvmax, while in others no significant effect was observed. In the bioreactor trials, all O.D. tested showed similar results and the addition of cysteine to the medium was quite harmful to cell growth. Infection assays showed that Sf-9 cells are quite susceptible to infection by the Spodoptera frugiperda baculovirus and good producers of viral polyhedra, and that the gas flow has a negative effect on the viral concentration in the liquid phase of the bioreactor cultures (viral titer).

A preocupação com a qualidade das águas está intensificando estudos com tecnologias avançadas para tratamento de efluentes. Uma tecnologia promissora no Brasil é a de biorreatores à membrana (MBR), pois há a possibilidade de trabalhar com altas taxas de aplicação e ainda pode obter um efluente tratado passível de reuso. Assim, o presente trabalho teve por objetivo estudar um MBR com módulo de membranas externo. Para tanto, foram estipulados os parâmetros operacionais em ensaios preliminares, tais como compactação da membrana, permeabilidade hidráulica e permeabilidade ao lodo ativado. Desta forma, efetuou-se a montagem do MBR, o qual foi inicialmente alimentado com um efluente sintético e posteriormente com efluente de curtume coletado pós-tratamento convencional. Operando com efluente sintético, obteve-se redução superior a 95% para DQO e turbidez. Com efluente de curtume pós-tratamento, obteve-se eficiência de 46%, 88%, 16%, 67% e 48% para DQO, DBO5, NTK, P-Total e cromo, respectivamente. Ademais, após 15 dias de operação, o sistema estabilizou com fluxo permeado elevado: 43 L/m².h. / The preoccupation with the quality of water resources is intensifying studies about advanced technologies for wastewater treatment. A promising technology in Brazil is the membrane bioreactors (MBR), due its capacity to operate with high application rates and may also obtains a treated effluent able to reuse. Thus, this work aimed to study a MBR with external membrane module. Firstly, it has been determined operational parameters by preliminary studies, namely: membrane compactation, hydraulic permeability and permeability to activated sludge. After that, the MBR was setting. Then, it was initially fed a synthetic wastewater and, then, with tannery wastewater collected after conventional treatment. Operating with synthetic wastewater, it was obtained a reduction of more than 95% for COD and turbidity. The operation with tannery wastewater post treatment resulted in efficiency of 46%, 88%, 16%, 67% and 48% for COD, BOD5, TKN, Total-P and chromium, respectively. Moreover, after 15 days of operation, the system has stabilized with high permeate flux: 43 L/m².h.

A utilização de inóculos líquidos na produção de Shiitake (Lentinula edodes (Berk.) Pegler) é uma tecnologia promissora para a industrialização do cultivo em sistema axênico, uma vez que permite a inoculação do micélio de forma rápida e bem distribuída, reduzindo riscos de contaminação e o período de incubação dos substratos. Neste trabalho, o cultivo submerso para a produção de micélio de Shiitake foi avaliado em um biorreator airlift de circulação externa, idealizado e projetado no grupo, utilizando o meio de cultura Mushroom Complete Medium adicionado de micronutrientes, nas condições de operação: taxas de aeração: 0.16, 0.20, e 0.24 vvm e tamanhos de inóculo (i.s.): 1.0 g.L-1 e 2.0 g.L-1. Na melhor condição de operação (0.16 vvm e 1.0 g.L-1 i.s.), um modelo matemático foi desenvolvido usando o software EMSO para descrever a cinética da cultura em ajuste aos dados experimentais. Foram atingidos 15,47 g.L-1 de biomassa total, após 10 dias de cultivo, 0,516 d-1 de taxa máxima de crescimento específico, fatores de rendimento Yx/s 1,06 g.g-1, YP/X 0,485 (g.L-1).g-1, YP/S 0,140 (g.L-1).g-1 e produtividade PX 0,061 (g.L-1).h-1. A predição do modelo demonstrou ser um parâmetro confiável para estudos de aumento de escala já que descreveu bons resultados preditivos para biomassa em relação aos produtos do metabolismo (CO2 e síntese de H+ pela acidificação do meio) e ao consumo de nutrientes (O2 e glicose). A seleção e a avaliação de linhagens no cultivo também são fatores a serem considerados para obtenção de boas produtividades e, deste modo, o desempenho produtivo de cinco linhagens de Shiitake foi avaliado em cultivo axênico, utilizando um planejamento composto central (CCD) para avaliar as variáveis independentes: tempo de incubação I(t), e percentual de farelo de trigo em relação a serragem Eucalypitus saligna (representada pela relação carbono/nitrogênio (RC/N)), tendo a eficiência biológica (BE) e o peso unitário dos cogumelos como resultados de destino . Os substratos utilizados tiveram sua composição físico-química determinada e as taxas de suplementação do substrato foram otimizadas, não só para o rendimento em cultivo, mas também para o crescimento vegetativo do micélio. Os melhores resultados de BE em todas as linhagens foram obtidos em cultivos com RC/N 57,62/1, com indução dos primórdios em 130 dias de I(t). Os cogumelos colhidos nesta condição de produção tiveram sua composição centesimal determinada. Atividades enzimáticas amilolíticas e celulolíticas, β-glicosidase e proteases específicas foram realizadas em ensaios de colonização do substrato. Três linhagens foram selecionadas para o isolamento de esporos e hibridização intraespecífica. As linhagens híbridas foram comparadas com as parentais em termos de rendimento e síntese das enzimas extracelulares. Os resultados deste trabalho demonstraram que a linhagem cultivada tem um efeito considerável sobre o rendimento, sobre o peso unitário e sobre a síntese de enzimas extracelulares produzidas pelo fungo, tanto pelas linhagens híbridas, como pelas parentais. / The use of liquid inoculant production of Shiitake (Lentinula edodes (Berk.) Pegler) is a promising technology for the industrialization farming of this fungus in axenic system because it allows the mycelium inoculation in a quick and well distributed way, thus reducing the risks of contamination and the incubation time of cultures. In this work, the submerged cultivation for the production of Shiitake mycelium was evaluated in an airlift bioreactor of external circulation, conceived and designed in the group, using the culture medium Mushroom Complete Medium added of micronutrients, under the operating conditions: rates of aeration: 0.16, 0.20, and 0.24 vvm and inoculum sizes (i.s.): 1.0 g. L-1 and 2.0 g. L-1. Under the best operating condition (0.16 vvm and 1.0 g L-1 i.s.), a mathematical model was developed using the EMSO software to describe the kinetics of culture fittnes to experimental data. It was possible to obtain 15.47 g L-1 of total biomass, after 10 days of cultivation, a maximum specific growth rate of 0.516 d-1, yields of Yx/s 1,06 g.g-1, YP/X 0,485 (g.L-1).g-1, YP/S 0,140 (g.L-1).g-1 and PX (productivity) 0.061 (g.L-1).h-1. The prediction of the model has proven to be a reliable parameter for studies for scaling up because it was predictive for biomass considering the products of metabolism (CO2 and H+ synthesis by acidification of the medium) and nutrient consumption (O2 and glucose). The selection and evaluation of strains in cultivation are also factors to be considered to obtain good productivity and, therefore, the productive performance of five strains of Shiitake cultivated under axenic system, was carried out using a central composition design (CCD) to evaluate the independent variables: incubation time I(t), and percentage of wheat bran in relation to sawdust of Eucalypitus saligna (represented by the carbon/nitrogen ratio (RC/N)), and having the biological efficiency (BE) and the unit weight of the mushrooms as target results. The best results for BE for all the strains were obtained in cultures with RC/N 57.62/1, with primordia induced at 130 days of I(t). The substrates used had their physico-chemical composition determined and the rates of substrate supplementation have been optimized, not only for the cultivation yields, but also for vegetative growth of the mycelium. The mushrooms harvested in this condition of production had their centesimal composition determined. Amilolytic, cellulolytic, β-glucosidase and specific proteases activities were performed in tests of colonization of the substrate. Three strains were selected for the isolation of spores and intraspecific hybridization was carried out. Hybrid strains were compared with the parental strains in terms of yields and synthesis of extracellular enzymes. The results of this study showed that strain has a considerable effect on both the yield and the unit weight of mushrooms.

A bactéria Zymomonas mobilis atraiu considerável interesse nas últimas décadas devido ao seu metabolismo único e a suas eficientes características fermentativas na produção de etanol através de açúcares simples. Além disso, dependendo do substrato utilizado outros produtos podem ser obtidos como ácido lático, ácido acético, ácido fórmico, acetona, levana, e sorbitol. Na literatura, a Z. mobilis tem sido proposta como microrganismo mais promissor que a convencional levedura Sacharomyces cerevisiae para a produção industrial de etanol. Na fermentação em modo contínuo o microrganismo apresenta oscilações (i.e., bifurcações Hopf) em baixas taxas de diluição (Df < 0,1/h). Diversos modelos têm sido propostos para descrever a dinâmica oscilatória do cultivo contínuo de Z. mobilis. Entre tais está o modelo de Jöbses et al. (1986) que foi ajustado experimentalmente em baixas taxas de diluição (Df < 0,1/h) e concentrações médias de substrato alimentado (Cso = 150 kg/m³). Recentemente, o modelo foi extrapolado por Elnashaine et al. (2006) que encontrou uma região operacional muito mais rentável a altas taxas de diluição (Df < 2,0/h) e concentração de substrato (Cso = 200 kg/m³). Embora o modelo de Jöbses não tenha sido validado nesta região, nossa contribuição assumirá que esta extrapolação é aceitável e então uma estratégia de controle foi proposta para manter o sistema trabalhando nesta região operacional. Para uma aplicação industrial bem sucedida da Z. mobilis, é necessário uma estratégia de controle eficiente e simples. Esse trabalho analisa o problema de controle e otimização de um biorreator contínuo de biosíntese de etanol pela bactéria modelado por Jöbses et al. (1986). Esse sistema apresenta multiplicidade de equilíbrios em determinadas condições operacionais. A idéia é manter o processo próximo à região de maior produtividade, localizada nas proximidades de um conjunto de bifurcações sela (onde o sistema torna-se instável). Baseado em uma análise sistemática da controlabilidade do sistema usando o índicie não-linear RPN percebe-se que é possível controlar o processo usando um controlador linear. Finalmente o trabalho aborda algumas características importantes no sistema de controle como a utilização de uma transformada nas ações do controlador com vistas a manter o biorreator no ótimo operacional. / Zymomonas mobilis has attracted considerable interest over the past decades pursuant to its unique metabolism and ability to rapidly and efficiently produce ethanol from simple sugars. In addition to ethanol depending on the substrate other fermentation products can occur, such as lactic acid, acetic acid, formic acid, acetone, and sorbitol. In the literature, Zymomonas mobilis has been proposed as a more promising microorganism than conventional yeast Saccharomyces cerevisiae for industrial production of ethanol. The major drawback of this microorganism is that it exhibits sustained oscillations (i.e., Hopf bifurcation) for low dilution rates (i.e., ,Df <=0.1 h-1) when grown in continuous mode. This leads to decreased ethanol productivity and less efficient use of available substrate. Various models have been proposed to describe the oscillatory dynamics of continuous Zymomonas mobilis cultures. One of them is the Jöbses et al. (1986) model that was fitted to experimental data with low dilution rate (i.e.,Df <= 0.1 h-1) and middle inlet substrate concentration (i.e., Cso~=150 kg/m³). Later, it was extrapolated outside of this operating region by Elnashaie et al. (2006), who have found a much more profitable operating region at higher dilution rates (Df~= 2.0 h -¹) and inlet concentrations (Cso~= 200 kg/m³). Notwithstanding the Jöbses's models has not been validated at this region, our contribution will assume that this extrapolation is acceptable and we will propose a control strategy to maintain the system working at this more profitable operating region. For a successful application of any industrial Z. mobilis facility, it is necessary to have an efficient and simple control strategy. This work analyzes the control and optimization problem of a continuous Z. mobilis bioreactor modeled by Jöbses et al. (1986). This system has steady state multiplicity in part of the operating range. The idea is to maintain the process close to the manifold border where is achievable the highest ethanol production. Based on a systematically analysis of the operational controllability using the nonlinear RPN indices it is identified that the process can be controlled using a linear controller. Finally in this work is proposed a variable transformation that makes easy to maintain the bioreactor close to the optimum.

Le sélénium et le tellurium partagent des propriétés chimiques communes et appartiennent à la colonne des éléments chalcogènes de la classification périodique des éléments. Ces métalloïdes ont des propriétés physico-chimiques remarquables et ils ont été utilisés dans un grand nombre d'applications dans le domaine des hautes technologies (électronique, semi-conducteurs, alliages). Ces éléments, qui se retrouvent généralement sous formes d'oxyanions, sont extrêmement solubles dans l'eau et présentent une forte toxicité. Leur libération dans l'environnement est donc d'un enjeu capital. Différentes méthodes physico-chimiques ont été développées pour la récupération de ces metalloïdes, en particulier pour le sélénium. Néanmoins, ces méthodes requièrent un équipement lourd et couteux et ne sont pas très recommandables sur le plan écologique. Le traitement biologique est donc une bonne alternative pour la récupération de Se et de Te provenant des effluents pollués. Cette approche réside dans la bioréduction des différents oxyanions sous formes métalliques. Ceux-ci sont moins toxiques et d'intérêts commerciales notables surtout lorsqu'ils se présentent sous forme nanométrique. L'utilisation de micro-champignons comme microorganismes catalyseur de la réduction de Se et de Te a été démontrée dans cette étude. La réactivité du champignon responsable de la pourriture blanche, Phanerochaete chrysosporium en présence de sélénite et de tellurite a été évaluée, ainsi que son application potentielle pour le traitement des eaux contaminées et la production de nanoparticules. La présence de Se et de Te a une influence importante sur la croissance et la morphologie du champignon. Il s'avère que P. chrysosporium est très sensible à la présence de sélénites. La synthèse de Se° et de Te° sous forme de nanoparticules piégées dans la biomasse fongique a été observée, ainsi que la formation de nano-composites Se-Te lorsque le champignon était cultivé simultanément en présence des deux métalloïdes. L'usage potentiel de biofilm fongiques pour le traitement des effluents semi-acides (pH 4.5) contenant du Se et du Te a été suggéré. De plus, le traitement en mode continu de sélénite dans un réacteur à biofilm fongique granulaire a été évalué. Le réacteur a montré un rendement d'élimination du sélénium en régime permanent de 70% pour differentes conditions opératoires. Celui-ci s'est montré efficace pendant une période supérieure à 35 jours. La bonne sédimentation du biofilm granulaire facilite la séparation du sélénium de l'effluent traité. L'utilisation du biofilm granulaire contenant du sélénium élémentaire comme bio-sorbant a également été étudiée. Cet adsorbant hybride s'est montré prometteur pour l'immobilisation du zinc présent dans les effluents semi-acides. La plupart des recherches effectuées se sont focalisées sur l'utilisation des biofilms granulaires. Toutefois, la croissance du champignon suite à l'exposition à des concentrations différentes de sélénites a également été étudiée. Des micro-électrodes à oxygène et un microscope confocal à balayage laser ont été utilisées pour évaluer l'effet du sélénium sur la structure des biofilms fongiques. Quel que soit le mode de croissance de P. chrysosporium, le mécanisme de réduction du sélénite semble être toujours le même tout en menant à la formation de sélénium élémentaire. Cependant, l'architecture des biofilms et l'activité en oxygène sont influencées par la présence de sélénium / Selenium (Se) and tellurium (Te) are particular elements, they are part of the chalcogens (VI-A group of the periodic table) and share common properties. These metalloids are of commercial interest due to their physicochemical properties, and they have been used in a broad range of applications in advanced technologies. The water soluble oxyanions of these elements (i.e., selenite, selenate, tellurite and tellurate) exhibit high toxicities, thus their release in the environment is of great concern. Different physicochemical methods have been developed for the removal of these metalloids, mainly for selenium. However, these methods require specialized equipment, high costs and they are not ecofriendly. The biological treatment is a green alternative to remove Se and Te from polluted effluents. This remediation technology consists on the microbial reduction of Se and Te oxyanions in wastewater to their elemental forms (Se0 and Te0), which are less toxic, and when synthesized in the nano-size range, they can be of commercial value due to their enhanced properties. The use of fungi as potential Se- and Te-reducing organisms was demonstrated in this study. Response of the model white-rot fungus, Phanerochaete chrysosporium, to the presence of selenite and tellurite was evaluated, as well as their potential application in wastewater treatment and production of nanoparticles. The presence of Se and Te had a clear influence on the growth and morphology of the fungus. P. chrysosporium was found to be more sensitive to selenite. Synthesis of Se0 and Te0 nanoparticles entrapped in the fungal biomass was observed, as well as the formation of unique Se-Te nanocomposites when the fungus was cultivated concurrently in the presence of Se and Te. Potential use of fungal pellets for the removal of Se and Te from semi-acidic effluents (pH 4.5) was suggested. Moreover, the continuous removal of selenite in a fungal pelleted reactor was evaluated. The reactor showed to efficiently remove selenium at steady-state conditions (~70%), and it demonstrated to be flexible and adaptable to different operational conditions. The reactor operated efficiently over a period of 35 days. Good settleability of the fungal pellets facilitated the separation of the selenium from the treated effluent. The use of elemental selenium immobilized fungal pellets as novel biosorbent material was also explored. This hybrid sorbent was promising for the removal of zinc from semi-acidic effluents. The presence of selenium in the fungal biomass enhanced the sorption efficiency of zinc, compared to Se-free fungal pellets. Most of the research conducted in this study was focused on the use of fungal pellets. However, the response of the fungus to selenite in a different kind of growth was also evaluated. Microsensors and confocal imaging were used to evaluate the effects of selenium on fungal biofilms. Regardless of the kind of fungal growth, P. chrysosporium seems to follow a similar selenite reduction mechanism, leading to the formation of Se0. Architecture of the biofilm and oxygen activity were influenced by the presence of selenium

La production de protéines recombinantes, d’anticorps, de vaccins, … est de plus en plus réalisée par culture de cellules animales. Le bioréacteur classiquement utilisé en industrie pour réaliser ces cultures est le bioréacteur à cuve agitée. Ce bioréacteur présente un volume important ce qui rend difficile le développement d’un bioréacteur à usage unique dans l’optique de réduire les risques de contaminations entre deux cultures consécutives. L’intensification du procédé et le développement de bioréacteur à usage unique sont donc deux défis intéressants dans la réalisation de cultures cellulaires à l’échelle industrielle. Un bioréacteur particulièrement prometteur pour l’intensification de culture cellulaire est le bioréacteur lit-fixe. Dans cette thèse, nous étudions le bioréacteur lit-fixe à usage unique iCELLis développé par Artelis S.A.<p>Le lit-fixe du bioréacteur iCELLis est composé d’un empilement de porteurs maintenu entre deux grilles perforées. Ces porteurs sont utilisés comme support par les cellules au cours de la culture. Le bioréacteur est équipé d’un système de transfert gaz-liquide par film tombant afin d’oxygéner le milieu de culture en continu. Une pompe centrifuge plongée dans un bac d’immersion assure la circulation du milieu de culture à travers l’ensemble du bioréacteur. Les cultures se déroulent en trois phases :une phase d’adhérence des cellules aux porteurs du lit-fixe, une phase de croissance cellulaire et une phase de production.<p>Les avantages d’un bioréacteur lit-fixe sont nombreux :une concentration cellulaire élevée impliquant une productivité élevée, un petit volume de bioréacteur, une faible exposition des cellules aux contraintes de cisaillement, Les bioréacteurs lits-fixes présentent cependant certains inconvénients qui freinent leur développement à l’échelle industrielle. Le lit-fixe se présente comme un réacteur piston ce qui implique l’apparition de gradients de concentrations de cellule et d’espèces extracellulaires (nutriments et produits) le long du lit-fixe. L’intérieur du lit-fixe est également difficilement accessible au cours de la culture. Le suivi des concentrations de cellules et d’espèces dans cette zone est donc problématique.<p>Une modélisation globale du bioréacteur lit-fixe nous permet de mieux comprendre les différents phénomènes qui prennent place dans le bioréacteur. Grâce à cette modélisation, nous sommes donc capables d’identifier les phénomènes clés contrôlant le procédé et ainsi fournir des pistes de travail pour l’optimisation et la montée en échelle du bioréacteur, ceci sur base de critères rationnels.<p>Nous choisissons de développer un modèle à compartiments du bioréacteur lit-fixe. Dans ce type de modèle, le bioréacteur est représenté par un réseau de compartiments interconnectés. Nous définissons trois compartiments :un premier pour la pompe, un deuxième pour les cellules et le lit-fixe, et un troisième pour le système de transfert gaz-liquide.<p>Pour le premier compartiment, nous souhaitons caractériser divers paramètres identifiés comme pertinents pour une sélection adéquate de la pompe. Dans cette thèse, nous présentons une méthode pour caractériser ces paramètres pour une pompe de référence (celle du bioréacteur iCELLis) et pour une pompe en similitude géométrique à la pompe de référence (dans le but d’étudier la montée en échelle).<p>La pompe de référence est étudiée numériquement (grâce aux logiciels Gambit 2.4 et Fluent 6.3) et expérimentalement. Nous mettons en évidence les liens entre les paramètres de la pompe déterminés numériquement et ceux déterminés expérimentalement. Ces liens définissent notre modèle. En intégrant au modèle les résultats de la simulation numérique de l’écoulement du milieu de culture dans le bac d’immersion contenant la pompe en similitude géométrique à la pompe de référence, nous déterminons entièrement les paramètres recherchés de la seconde pompe sans avoir recours à un prototype. Ceci permet donc de tester différentes échelles avant de choisir la version finale de la seconde pompe.<p>Le deuxième compartiment du modèle caractérise les cellules et le lit-fixe. La sélection de certains paramètres opératoires dépend du métabolisme cellulaire. Nous souhaitons développer un outil de surveillance en ligne de l’évolution des concentrations de certaines espèces extracellulaires sur base de la connaissance de la concentration cellulaire dans le bioréacteur. Cet outil est développé sur base d’un modèle structuré du métabolisme des cellules animales. Dans un tel modèle, nous établissons des bilans de matière sur les espèces extra- et intracellulaires en considérant les voies métaboliques intracellulaires. Un paramètre requis pour l’emploi de cet outil est la connaissance de la concentration cellulaire au cours de la culture. Or, la surveillance de cette concentration est l’un d’un problème évoqué dans les bioréacteurs lits-fixes. Nous développons donc un modèle ségrégé de culture cellulaire en bioréacteur lit-fixe. Dans ce modèle, nous considérons l’entièreté du lit-fixe. Le modèle comprend différentes populations de cellules :les cellules en suspension dans le milieu au début de la culture et les cellules adhérentes au lit-fixe. Le modèle inclut une distribution spatiale de la concentration d’espèces extracellulaires dans le lit-fixe. Par conséquent, le modèle rapporte les gradients potentiels de concentration de cellules et d’espèces extracellulaires dans le lit-fixe.<p>Le troisième compartiment du modèle du bioréacteur caractérise le système de transfert gaz-liquide. L’oxygénation est très souvent un paramètre clé dans la conception d’un bioréacteur. Dans le bioréacteur iCELLis, le système de transfert gaz-liquide est un film liquide tombant turbulent. Dans cette thèse, nous proposons une méthode pour caractériser le transfert d’oxygène à travers ce type de film tombant. Notre méthode, basée sur une approche numérique (grâce à Gambit 2.4 et Fluent 6.3), est scindée en deux parties. Premièrement, nous calculons la forme de l’interface gaz-liquide. Une simulation de l'écoulement est réalisée avec le modèle Volume of Fluid (VOF). A partir de cette simulation, la forme de l'interface est traquée. Deuxièmement, la forme de l'interface est générée dans un nouveau domaine de calcul afin de simuler le transfert d’oxygène. Grâce à cette seconde simulation, le coefficient de transfert d’oxygène de la phase gazeuse vers le milieu de culture est déterminé. Grâce à notre méthode, nous caractérisons ce coefficient pour différentes conditions opératoires. Nous étudions notamment l’influence du débit et de la température du milieu de culture sur le coefficient de transfert d’oxygène. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

Solid state fermentation (SSF) refers to the microbial fermentation, which takes place in the absence or near absence of free water, thus being close to the natural environment to which the selected microorganisms, especially fungi, are naturally adapted. The current status of SSF research globally was discussed in terms of articles publication. This was followed by discussion of the advantages of SSF and the reason for interest in SSF as a notable bioprocessing technology to be investigated and compared to submerged fermentation (SmF) for the production of various added-value products. SSF also proved to be a potential technology to treat solid waste produced from food and agricultural industry and to provide environmental benefits with solid waste treatment. A summary was made of the attempts at using modern SSF technology for future biorefineries for the production of chemicals. Many works were carried out in the Satake Centre for Grain Process Engineering (SCGPE), University of Manchester, to prove the strategy of using SSF for the production of hydrolysate rich in nutrients for sequel microbial fermentation with or without adding any commercial nutrients. The research findings presented in this thesis are based on a series of SSF experiments carried out on systems varying in complexity from simple petri dishes to our own design of bioreactor systems. They were conducted to assess a solution for biomass estimation, enzymes production, and successful mass and heat transfer. A proper technique for inoculum transfer prior to the start of the fermentation process was developed. In SSF, estimation of biomass presents difficulties as generally the fungal mycelium penetrates deep and remains attached with the solid substrate particles. Although many promising methods are available, the evaluation of microbial growth in SSF may sometimes become laborious, impractical and inaccurate. Essentially, this remains another critical issue for monitoring growth. In these studies, measurement of colour changes during SSF are presented as one of the potential techniques that can be used to describe growth, complementary to monitoring metabolic activity measurement, such as CER, OUR and heat evolution, which is directly related to growth. For the growth of Aspergillus awamori and Aspergillus oryzae on wheat bran, soybean hulls and rapeseed meal, it was confirmed that colour production was directly proportional to fungal growth. This colourimetric technique was also proved to be a feasible approach for fungal biomass estimation in SmF. This new approach is an important complementation to the existing techniques especially for basic studies. The key finding is that the colourimetric technique demonstrated and provided information of higher quality than that obtained by visual observation or spores counting. The effect of aeration arrangements on moisture content, oxygen (O2), mass and heat transfer during SSF was investigated. A. awamori and A. oryzae were cultivated on wheat bran in newly designed four tray solid state bioreactor (SSB) systems. The new tray SSB systems were: (1) single circular tray SSB, (2) multi-stacked circular tray SSB, (3) Single rectangular tray SSB and (4) multi-square tray SSB. The purpose was to study the effect, on heat and water transfer, of operating variables, fermentation on the perforated base tray and internal moist air circulation under natural and forced aeration. Temperature, O2 and carbon dioxide were measured continuously on-line. Enzyme activity, moisture content and biomass were also measured. The results suggest that the air arrangements examined have a remarkable effect on the quantity of biomass produced using measurement of spores and enzymes production. The strategy presented in these studies allowed quantitative evaluation of the effect of forced internal moist air circulation on the removal of metabolic heat. With the proposed strategy, it was possible to maintain the bed temperatures at the optimum level for growth. However, the effect on moisture content was very different for the two fungi. It was found that the ability of A. oryzae to retain moisture was much higher than that of A. awamori. This is possibly due to the higher levels of chitin in A. oryzae. Greater spores and enzymes (glucoamylase, xylanase and cellulase) production was observed for A. awamori in multi-stacked circular tray and multi-square tray SSB systems compared to the conventional petri dishes and the other two systems. A. oryzae was excellent in producing protease in the same bioreactors. A direct technique of establishing a correlation between fungal growth and CER, OUR, heat evolved was proven successful in this work. The information obtained from CER and OUR led to the estimation of respiratory quotient (RQ). RQ describes the state of the fungal population in the tray SSB and gives an indication of fungal metabolic behaviour. RQ values < 1 were obtained from 38 experiments using four tray SSB systems for the two fungi. A kinetic model based on CO2 evolution instead of biomass concentration was examined in order to simplify the required experiments for kinetic model development. A Gompertz model was used to fit the integrated CO2 data and predict the quantity of CO2 evolution in all experiments. A correlation was found between the heat evolution and CER. The performances of tray SSB systems can be improved by constructing them as multi-trays. The multi-tray systems improved the mass transfer considerably compared with single tray systems. In addition, the multi-tray systems allowed precise measurement of the gradients of CO2, enzymes, spores and fungal biomass. In addition, the air arrangements using moistened air were successful in maintaining moisture content, adequate O2 supply and control of temperature, and hence, increased the productivity of both fungi. Overall A. awamori and A. oryzae have their own ability and performance to degrade and utilise the complex compositions contained in the solid substrate and fermentation conditions may lead to possible comparisons. In addition, multi-stacked circular tray and multi-square tray SSB systems demonstrated an excellent system for further investigations of mass transfer and possibly for large scale operation, though considerable optimisation work remains to be done, for example the height/diameter ratio and total number of trays should be optimised.

For decades, mainstream domestic wastewater treatment has relied on activated sludge processes to remove organic matter, and on biological nutrient removal systems like the A2/O process to remove nutrients. Recently, membrane filtration was also added to the realm of possible technologies for domestic wastewater treatment, with aerobic membrane bioreactors (MBRs) becoming increasingly popular, especially for decentralized, and small to medium scale applications. However, the aerobic activated sludge and MBR processes, which are often combined with biological nutrient removal processes, have high energy costs associated with supplying oxygen to the process, and end up converting the organic matter into CO2 and high amounts of microbial biomass, instead of more useful byproducts. In order to remedy the aforementioned shortcomings of the aerobic processes, anaerobic wastewater treatment has been a focus of research, with anaerobic baffled reactors (ABRs) and anaerobic membrane bioreactors (AnMBRs) having shown promise for achieving acceptable organic matter removal performance, along with potential to be energy neutral or positive through biogas production. In addition, phototrophic technologies, such as algal photobioreactors, have recently been shown to be able to remove nutrients from waste streams, while at the same time having the potential to be used as feedstock to produce biofuels. In this dissertation, a novel concentrically-baffled reactor (CBR) was designed that has the potential to reduce heat loss by transfering more of the heat between reactor zones than traditional baffled reactor designs, which will increase energy efficiency for heated systems. A prototype CBR was operated abiotically under varying hydraulic retention times (HRTs) from 4 h to 24 h, and achieved over 90% removal of total suspended solids (TSS) for all HRTs tested with feed particle sizes below 1.7 mm. In parallel with the baffled reactor research, phototrophic membrane bioreactors (PMBRs) were tested with low aeration conditions to decrease their energy demand, which resulted in nitrification-dominated systems. A phototrophic technology was developed for increasing the pH of waste streams to potentially aid pH-sensitive nutrient recovery processes. Phototrophic pH increase from 6.42±0.13 to 8.87±0.06 was achieved using batch reactors, and an increase of pH from 6.73 to 8.61 was recorded during a continuous reactor trial. Finally, the CBR was combined with a post-CBR membrane filtration process, and two PMBRs treating the effluent and permeate streams from the CBR in order to achieve complete organic matter and nutrient removal. The combined systems were tested both for high strength-high HRT and low strength-low HRT scenarios. Using the combined CBR-PMBR system, over 90% TN and TP removal were possible for 10 d HRT operation at high-strength feed conditions, with post-CBR membrane filtration. COD removal over 90% was possible for both high-strength and low-strength scenarios under all conditions tested.

The bioreactor landfill is an emerging concept for solid waste management that has gained significant attention in the last decade. This technology employs specific operational practices to enhance the microbial decomposition processes in landfills. However, the unsupervised management and lack of operational guidelines for the bioreactor landfill, specifically leachate manipulation and recirculation processes, usually results in less than optimal system performance. Therefore, these limitations have led to the development of SMART (Sensor-based Monitoring and Remote-control Technology), an expert control system that utilizes real-time monitoring of key system parameters in the management of bioreactor landfills. SMART replaces conventional open-loop control with a feedback control system that aids the human operator in making decisions and managing complex control issues. The target from this control system is to provide optimum conditions for the biodegradation of the refuse, and also, to enhance the performance of the bioreactor in terms of biogas generation. SMART includes multiple cascading logic controllers and mathematical calculations through which the quantity and quality of the recirculated solution are determined. The expert system computes the required quantities of leachate, buffer, supplemental water, and nutritional amendments in order to provide the bioreactor landfill microbial consortia with their optimum growth requirements. Soft computational methods, particularly fuzzy logic, were incorporated in the logic controllers of SMART so as to accommodate the uncertainty, complexity, and nonlinearity of the bioreactor landfill processes. Fuzzy logic was used to solve complex operational issues in the control program of SMART including: (1) identify the current operational phase of the bioreactor landfill based on quantifiable parameters of the leachate generated and biogas produced, (2) evaluate the toxicological status of the leachate based on certain parameters that directly contribute to or indirectly indicates bacterial inhibition, and (3) predict biogas generation rates based on the operational phase, leachate recirculation, and sludge addition. The later fuzzy logic model was upgraded to a hybrid model that employed the learning algorithm of artificial neural networks to optimize the model parameters. SMART was applied to a pilot-scale bioreactor landfill prototype that incorporated the hardware components (sensors, communication devices, and control elements) and the software components (user interface and control program) of the system. During a one-year monitoring period, the feasibility and effectiveness of the SMART system were evaluated in terms of multiple leachate, biogas, and waste parameters. In addition, leachate heating was evaluated as a potential temperature control tool in bioreactor landfills. The pilot-scale implementation of SMART demonstrated the applicability of the system. SMART led to a significant improvement in the overall performance of the BL in terms of methane production and leachate stabilization. Temperature control via recirculation of heated leachate achieved high degradation rates of organic matter and improved the methanogenic activity.

Ce mémoire a été préparé dans le cadre du projet européen ERUDESP. Il décrit en détail les études qui ont été réalisées dans ce travail. Un bioréacteur sous forme d’une cellule bioélectrochimique à flux a été développé dans le but de servir pour la bioélectrosynthèse enantiopure. Le mémoire est consacré au design de cellules, screenings de différents médiateurs, au test de différentes réactions électroorganiques et électroenzymatiques à la fois dans une cellule électrochimique classique et dans une cellule électrochimique à flux. Un thème central de ce mémoire représente la synthèse d’électrodes macroporeuses tri-dimensionnelles. Grâce à cela, la surface active de l’électrode est augmentée de manière significative. Pour la préparation de ces électrodes une approche de template a été suivie. Des particules de polystyrène monodisperses ont été synthétisées de différentes manières et sont utilisées pour la synthèse des cristaux colloïdaux qui constituent les templates. Comme procédure de préparation contrôlée de ces templates, la technique de Langmuir-Blodgett et la méthode d’évaporation contrôlée, suivi par l’électrodéposition des métaux et des oxydes métalliques sont appliquées. Les diamètres des pores des matériaux macroporeux résultants sont parfaitement contrôlables par le diamètre des particules utilisées. La méthode de Langmuir-Blodgett a été étendue et appliquée pour la première fois à l’utilisation de particules de polystyrène. Le dépôt est réalisé dans les interstices des cristaux colloïdaux préparés préalablement, suivi par la dissolution des particules. De plus, des électrodes poreuses avec une grande surface de 6 x 6 cm2 ont été préparées. Les matériaux obtenus montrent une très bonne interconnéctivité avec une porosité ouverte et une surface active fortement augmentée ce qui se traduit électrochimiquement en une augmentation significative de la puissance du signal. Les matériaux poreux représentent un bénéfice non-seulement pour la (bio)électrosynthèse mais aussi dans le cadre de la (bio)électroanalyse. Dans ce mémoire, d’électrodes poreuses d’oxyde de ruthénium pour l’oxydation direct de NADH avec une surtension significativement améliorée ont été élaborée. La méthode de l’agrandissement de la surface est également appliquée et une meilleure densité de courant a été obtenue. Basé sur le projet ERUDESP, les méthodes apprises pour créer des électrodes poreuses à base de cristaux colloïdales ont dans la suite aussi été appliquées à d’autres domaines d’investigation. L’évolution méthodique de la technique de Langmuir-Blodgett a été utilisée pour le développement d’un système d’électrode renouvelable. Dans ce système, la surface peut être renouvelée sur commande par application d’un potentiel fixe (effet click).Les électrodes de l’oxyde de ruthénium ont non seulement été étudié dans le cadre du projet ERUDESP, mais la miniaturisation de ce matériau poreux et stable sous forme de microélectrodes a permis d’étudier une application comme capteur pH chimiquement et mécaniquement stable avec un meilleur ratio signal sur bruit. Dans ce cas le bruit thermique est diminué grâce à la porosité de l’électrode. Grâce à la technicité acquise par rapport à la synthèse des microélectrodes poreuses, des microélectrodes implantables pour les prothèses de main ont été aussi modifiées avec une couche macroporeuse pour augmenter la surface active et diminuer l’impédance de transition.Nous avons également exploré des couches multicatalyseurs macroporeuses de platine et nickel pour effectuer la génération d’hydrogène in-situ et l’hydrogénation simultanée dans un seul système catalytique.Comme dernière possibilité pour une structuration de surface contrôlée, des îlots de platine d’une étendue nanométrique furent examinées et biofonctionnalisées, ce qui résulte également en une augmentation significatif de la densité de courant. / The present work has been prepared within the framework of the European project ERUDESP and describes the research that has been carried out during this work. A bioreactor as a bioelectrochemical flow-cell was designed and realized with the goal to serve for enantiopure bioelectrosynthesis. The work deals with the cell design and screening of different mediators in a batch-cell and multi-cells, the development of different electroorganic and electroenzymatic reactions in an electrochemical batch- and flow-cell. With respect to the flow-cell, the upscaling of electrochemical reactions was carried out in the present work not only for electroorganic but also for electroenzymatic reactions with regard to the final application. A main focus of the present work represents the synthesis of three-dimensional macroporous electrodes in order to increase significantly the active surface. These macroporous structures were obtained by using the template approach. For the preparation of the templates monodisperse polystyrene particles were synthesized in different ways, and then used for the preparation of colloidal crystals serving as templates. As controlled assembly procedures, the Langmuir-Blodgett technique and the controlled evaporation method with subsequent electrodeposition of metals and metal oxides were chosen. With the present process the pore diameter could be exactly controlled by the diameter of the used particles. The approach of the Langmuir-Blodgett technique has been extended and optimized. For the first time, the Langmuir-Blodgett technique could be used with polystyrene particles. The deposition took place in the interspaces of the prepared colloidal crystals and is followed by the dissolving of the particles. Furthermore, the size of the porous electrodes could be upscaled (6 x 6 cm2). The obtained materials showed a very good interconnectivity with an open porosity and a highly increased active surface, which led to an increased electrochemical signal. The prepared porous materials represent a great benefit not only for (bio)electrosynthesis but also in the field of (bio)electroanalysis. In the framework of this work, the use of porous ruthenium oxide electrodes for direct oxidation of NADH with a significantly improved overvoltage was studied. Also in this context the increase of the surface led to an improved current density. Based on the ERUDESP project, the studied techniques for preparing porous electrodes with colloidal crystals were used for further scientific studies. The new variant of the Langmuir-Blodgett technique has also been used for the elaboration of a renewable electrode system where the surface can be simply renewed by applying a positive potential to the porous multilayers (click effect). The porous ruthenium oxide electrodes have not only been studied with respect to the ERUDESP project, but it was also possible to miniaturize this stable porous material as microelectrodes and use them as chemically and mechanically stable pH sensor with an improved signal to noise ratio. In this case the thermal noise decreased due to the porosity of the electrode. Due to the acquired expertise in the field of the preparation of porous microelectrodes, implantable microelectrodes for hand prosthesis were modified with a porous layer on the surface for increasing the active surface and decreasing their impedance.In addition, macroporous multicatalyst layers of platinum and nickel were synthesized for the simultaneous in-situ generation of hydrogen and hydrogenation reaction in the same catalyst system.As a final example for controlled surface structuring, nanoscale platinum islands were in detail examined and biofunctionalized. This led also to a significant increase of the current density.