O reator anaeróbio em batelada seqüencial é constituído por um frasco de vidro cilíndrico de volume total de cinco litros, envolvido por uma camisa de vidro, por onde escoa a água aquecida, permitindo a operação em temperatura controlada. A biomassa encontra-se imobilizada em partículas cúbicas de espuma de poliuretano (densidade aparente de 23 kg/\'M POT.3\', as quais estão colocadas em um cesto adaptado dentro do frasco cilíndrico. A mistura é promovida por três impelidores de 3,0 cm de diâmetro, distanciados 4,0 cm um do outro, situados ao longo do eixo vertical no centro do reator. O desempenho dessa nova configuração de reator anaeróbio foi avaliado sob diferentes condições os efeitos de transferência de massa nas fases sólida e líquida. Todos os ensaios foram efetuados à temperatura de 30 ± 1 grau Celsius. Cada batelada compreende três etapas: alimentação, reação e descarga. Para avaliar os efeitos da transferência de massa na fase sólida foram feitos quatro ensaios utilizando-se partículas cúbicas de espumas de poliuretano com tamanhos de 0,5 cm; 1,0 cm; 2,0 cm e 3,0 cm de lado, com impelidor tipo hélice e intensidade de agitação de 500 rpm, determinada através de um ensaio preliminar. Para avaliar os efeitos da transferência de massa nas fases sólida e líquida foram feitos experimentos com quatro tipos de impelidores: hélice, turbina plana, turbina inclinada e turbina curva, com intensidades de agitação na faixa de 100 rpm a 1100 rpm. Também foram realizados ensaios hidrodinâmicos para verificar o tempo de mistura e ensaio para verificar a condição de anaerobiose no sistema. A água residuária utilizada em todos os ensaios foi sintética com concentração de 530 ± 37 mg DQO/L. Em todas as condições estudadas o reator apresentou boa eficiência de remoção da matéria orgânica, em torno de 87%. A concentração efluente de ácidos voláteis totais manteve-se em 13 ± 9 mg HAc/L, alcalinidade a bicarbonato de 223 ± 14 mg Ca\'CO IND.3\'/L e pH entre 6,7 e 7,2. A transferência de massa na fase sólida não foi a etapa limitante na conversão da matéria orgânica, quando partículas de 0,5 cm a 2,0 cm de aresta foram usadas no reator anaeróbio em batelada seqüencial. A resistência à transferência de massa na fase sólida somente influenciou a taxa global de reação, quando foram usados tamanhos de partículas cúbicas de 3,0 cm de aresta. A resistência à transferência de massa na fase líquida não foi somente afetada pela intensidade de agitação, mas também pela eficiência da mistura obtida por cada tipo de impelidor. A mistura do líquido dentro do reator obtida pelo impelidor turbina plana foi a mais eficiente. O uso deste tipo de impelidor resultou em menores consumos de energia e ótimo desempenho do reator com baixas taxas de agitação. Os resultados deste estudo permitiram concluir que esta nova configuração não permite a manutenção de condição de anaerobiose estrita no meio, principalmente quando altas intensidades de agitação foram aplicadas e as limitações da eficiência do processo, neste sistema, estão relacionadas principalmente as resistências à transferência de massa do que restrições cinéticas bioquímicas. / The bench-scale anaerobic sequencing batch reactor consisted of a cylindrical glass flask with a total capacity of 5 liters. The reactor was surrounded by a water jacket that allowed the operation to proceed at a constant temperature throughout the experiment. The biomass was immobilized in 5-mm cubic particles of polyurethane foam (apparent density of 23 kg/\'M POT.3\') placed in a basket inside the cylindrical flask. The mixing was provided by three mechanical impellers with diameters of 3 cm, placed 4 cm apart along a vertical axis, at the center of the reactor. All experiments were conducted at the temperature of 30 Celsius degrees. Each batch consisted of three steps: feed, react and liquid withdrawal. The performance of this new reactor configuration was evaluated under different conditions of solid and liquid-phase mass transfer. In order to evaluate the effects of the solid-phase mass transfer, four experiments were carried out with cubical polyurethane foam particles of 0.5 cm, 1.0 cm, 2.0, cm and 3.0 cm side, and with propeller impellers rotating at 500 rpm, achieved by preliminary experiment. The effects of the liquid-phase mass transfer were evaluated through four experiments with four types of impellers: propeller, flat-blade, pitched-blade and curved-blade turbines, at agitation rates from 100 rpm and 1100 rpm. A hydrodynamic test was also carried out in order to verify the mixing time, energy consumption and occurrence of strict anaerobic activity in system. A low-strength synthetic substrate was used in all the experiments with a mean chemical oxygen demand (COD) of 530 ± 37 mg DQO/L. The influence of the solid and liquid-phase mass transfer on the reactor\'s performance was assessed by measuring COD temporal profiles along batch cycles. In all conditions studied the reactor achieved good efficiency, with mean removal of organic matter (COD) of 87%. The effluent mean TVA concentration was 13 ± 9 mg HAc/L, bicarbonate alkalinity was 223 ± 14 mg Ca\'CO IND.3\'/L and the pH values ranged from 6,7 e 7,2. The solid-phase mass transfer was not the limiting step in the organic matter conversion when 0.5 to 2.0-cm side bioparticles were used in the anaerobic sequencing batch reactor. Solid-phase mass transfer resistance only influenced the overall reaction rate when 3.0-cm cubic bioparticles were used. The liquid-phase mass transfer resistance was affected both by agitation and by efficiency of mixture provided by each type of impeller. Among the impellers assayed, the flat-blade one was the most efficient in providing the required mixing conditions. The use of this type impeller resulted in small energy consumption and excellent performance of the reactor with low agitation rate (N = 300 rpm). The results of this study also indicated that this new configuration did not provide conditions for the establishment of strict anaerobic conditions, mainly when high agitation rates were used. Anaerobic process efficiency limitations in this system were mainly related to mass transfer resistances rather than biochemical kinetic restrictions.

O reator anaeróbio em batelada seqüencial é constituído por um frasco de vidro cilíndrico de volume total de cinco litros, envolvido por uma camisa de vidro, por onde escoa a água aquecida, permitindo a operação em temperatura controlada. A biomassa encontra-se imobilizada em partículas cúbicas de espuma de poliuretano (densidade aparente de 23 kg/\'M POT.3\', as quais estão colocadas em um cesto adaptado dentro do frasco cilíndrico. A mistura é promovida por três impelidores de 3,0 cm de diâmetro, distanciados 4,0 cm um do outro, situados ao longo do eixo vertical no centro do reator. O desempenho dessa nova configuração de reator anaeróbio foi avaliado sob diferentes condições os efeitos de transferência de massa nas fases sólida e líquida. Todos os ensaios foram efetuados à temperatura de 30 ± 1 grau Celsius. Cada batelada compreende três etapas: alimentação, reação e descarga. Para avaliar os efeitos da transferência de massa na fase sólida foram feitos quatro ensaios utilizando-se partículas cúbicas de espumas de poliuretano com tamanhos de 0,5 cm; 1,0 cm; 2,0 cm e 3,0 cm de lado, com impelidor tipo hélice e intensidade de agitação de 500 rpm, determinada através de um ensaio preliminar. Para avaliar os efeitos da transferência de massa nas fases sólida e líquida foram feitos experimentos com quatro tipos de impelidores: hélice, turbina plana, turbina inclinada e turbina curva, com intensidades de agitação na faixa de 100 rpm a 1100 rpm. Também foram realizados ensaios hidrodinâmicos para verificar o tempo de mistura e ensaio para verificar a condição de anaerobiose no sistema. A água residuária utilizada em todos os ensaios foi sintética com concentração de 530 ± 37 mg DQO/L. Em todas as condições estudadas o reator apresentou boa eficiência de remoção da matéria orgânica, em torno de 87%. A concentração efluente de ácidos voláteis totais manteve-se em 13 ± 9 mg HAc/L, alcalinidade a bicarbonato de 223 ± 14 mg Ca\'CO IND.3\'/L e pH entre 6,7 e 7,2. A transferência de massa na fase sólida não foi a etapa limitante na conversão da matéria orgânica, quando partículas de 0,5 cm a 2,0 cm de aresta foram usadas no reator anaeróbio em batelada seqüencial. A resistência à transferência de massa na fase sólida somente influenciou a taxa global de reação, quando foram usados tamanhos de partículas cúbicas de 3,0 cm de aresta. A resistência à transferência de massa na fase líquida não foi somente afetada pela intensidade de agitação, mas também pela eficiência da mistura obtida por cada tipo de impelidor. A mistura do líquido dentro do reator obtida pelo impelidor turbina plana foi a mais eficiente. O uso deste tipo de impelidor resultou em menores consumos de energia e ótimo desempenho do reator com baixas taxas de agitação. Os resultados deste estudo permitiram concluir que esta nova configuração não permite a manutenção de condição de anaerobiose estrita no meio, principalmente quando altas intensidades de agitação foram aplicadas e as limitações da eficiência do processo, neste sistema, estão relacionadas principalmente as resistências à transferência de massa do que restrições cinéticas bioquímicas. / The bench-scale anaerobic sequencing batch reactor consisted of a cylindrical glass flask with a total capacity of 5 liters. The reactor was surrounded by a water jacket that allowed the operation to proceed at a constant temperature throughout the experiment. The biomass was immobilized in 5-mm cubic particles of polyurethane foam (apparent density of 23 kg/\'M POT.3\') placed in a basket inside the cylindrical flask. The mixing was provided by three mechanical impellers with diameters of 3 cm, placed 4 cm apart along a vertical axis, at the center of the reactor. All experiments were conducted at the temperature of 30 Celsius degrees. Each batch consisted of three steps: feed, react and liquid withdrawal. The performance of this new reactor configuration was evaluated under different conditions of solid and liquid-phase mass transfer. In order to evaluate the effects of the solid-phase mass transfer, four experiments were carried out with cubical polyurethane foam particles of 0.5 cm, 1.0 cm, 2.0, cm and 3.0 cm side, and with propeller impellers rotating at 500 rpm, achieved by preliminary experiment. The effects of the liquid-phase mass transfer were evaluated through four experiments with four types of impellers: propeller, flat-blade, pitched-blade and curved-blade turbines, at agitation rates from 100 rpm and 1100 rpm. A hydrodynamic test was also carried out in order to verify the mixing time, energy consumption and occurrence of strict anaerobic activity in system. A low-strength synthetic substrate was used in all the experiments with a mean chemical oxygen demand (COD) of 530 ± 37 mg DQO/L. The influence of the solid and liquid-phase mass transfer on the reactor\'s performance was assessed by measuring COD temporal profiles along batch cycles. In all conditions studied the reactor achieved good efficiency, with mean removal of organic matter (COD) of 87%. The effluent mean TVA concentration was 13 ± 9 mg HAc/L, bicarbonate alkalinity was 223 ± 14 mg Ca\'CO IND.3\'/L and the pH values ranged from 6,7 e 7,2. The solid-phase mass transfer was not the limiting step in the organic matter conversion when 0.5 to 2.0-cm side bioparticles were used in the anaerobic sequencing batch reactor. Solid-phase mass transfer resistance only influenced the overall reaction rate when 3.0-cm cubic bioparticles were used. The liquid-phase mass transfer resistance was affected both by agitation and by efficiency of mixture provided by each type of impeller. Among the impellers assayed, the flat-blade one was the most efficient in providing the required mixing conditions. The use of this type impeller resulted in small energy consumption and excellent performance of the reactor with low agitation rate (N = 300 rpm). The results of this study also indicated that this new configuration did not provide conditions for the establishment of strict anaerobic conditions, mainly when high agitation rates were used. Anaerobic process efficiency limitations in this system were mainly related to mass transfer resistances rather than biochemical kinetic restrictions.

La réponse globale de cellules animales à des contraintes hydrodynamiques lors de leur culture en suspension dans des réacteurs agités a été étudiée grâce à une approche intégrative couplant les outils du génie biochimique à ceux de la mécanique des fluides numérique. En premier lieu, la description de l’hydrodynamique moyenne et locale de deux systèmes de culture agités de laboratoire, spinner et bioréacteur, a été réalisée. Puis, l'étude des cinétiques macroscopiques de cellules CHO cultivées en suspension, en milieu sans sérum et sans protéine, a été réalisée avec différentes vitesses d’agitation, pour évaluer l'impact de l'agitation sur les vitesses de croissance et de mort cellulaires, ainsi que de consommation des substrats et de production des métabolites et de l'interféron-gamma recombinant. Des caractérisations supplémentaires des cellules (apoptose, protéines intracellulaires) et de l'interféron ont également été réalisées. Les effets de l'intensification de l'agitation ont été représentés avec plusieurs corrélations globales reliant : (i) en milieu contenant du pluronic, l'intégrale des cellules viables au nombre de Reynolds, et la proportion de cellules lysées à la valeur moyenne de l'énergie de dissipation, <[epsilon]? (ii) en milieu sans pluronic, les vitesses spécifiques de croissance et de mort cellulaires à <[epsilon]. De plus, l'analyse par CFD de la distribution spatio-temporelle des contraintes indique que la lyse cellulaire, observée dans le réacteur aux conditions extrêmes d'agitation, serait plutôt liée à des valeurs locales très élevées de [epsilon], ainsi qu’à la fréquence d'exposition des cellules dans ces zones énergétiques. Un modèle hydro-cinétique original, couplant l’hydrodynamique locale aux cinétiques cellulaires de croissance et de mort, et basé sur l’intermittence de la turbulence permet la prédiction de la lyse massive observée en réacteur sous certaines conditions. Pour confirmer le fait que les effets liés à l'intensification de l'agitation sont bien le résultat d'une augmentation des contraintes hydrodynamiques, et non d'une amélioration du transfert d'oxygène, ce dernier a été mesuré et modélisé par couplage avec une simulation numérique de type Volume Of Fluid , concluant en une absence de limitation d'oxygène. Enfin, la conception, le dimensionnement et la caractérisation hydrodynamique d'un réacteur innovant de type Couette-Taylor, sont proposées pour la mise en œuvre de cultures perfusées dans un environnement hydrodynamique mieux contrôlé / The global response of animal cells to hydrodynamic stress when cultivated in suspension in stirred tank reactors was studied. To do this, an integrative approach coupling biochemical engineering and fluid mechanics tools were used. First, the description of the global and local hydrodynamics of two bench-scale agitated reactors, a spinner flask and a bioreactor, was carried out. Then, macroscopic kinetics of CHO cells cultivated in a serum and protein-free medium were obtained at various agitation rates, in order to evaluate the impact of agitation on cellular growth and death, as well as substrates consumption and metabolites and recombining IFN-[gamma] production. IFN-[gamma] and cells physiological state were more precisely characterised by glycosylation, apoptosis state and intracellular proteins measurements. The effects of the agitation increase were represented by several global correlations that related: (i) in a medium containing Pluronic F68, the Integral of the Viable Cells Density to the Reynolds number, and the proportion of lysed cells with the average value of energy dissipation rate <[epsilon]? (ii) in a medium without pluronic, specific cell growth and death rates to <[epsilon]. Moreover, CFD analysis of the stress distribution indicated that the cellular lysis observed in the bioreactor at the highest agitation rate, would be related to very high local values of [epsilon], and to the exposure frequency of the cells in these energetic zones. An original hydro-kinetic model based on the intermittency of turbulence and coupling the local hydrodynamics with cell growth and death kinetics, allowed the prediction of the massive cell lysis observed in the bioreactor under some mixing conditions. To decouple shear stress effects from oxygen transfer improvement, the oxygen transfer coefficient was experimentally measured and modelled using a Volume Of Fluid numerical simulation. Our results indicated the absence of an oxygen limitation, which confirmed that this cell response resulted from the hydrodynamic stress increase alone. Lastly, an innovative continuous and perfused Couette-Taylor reactor, allowing a better-controlled hydrodynamic environment was designed and sized. Its hydrodynamic description was carried out using CFD calculations