Cette thèse porte sur l’analyse mathématique de différents modèles de la digestion anaérobie. Dans la première partie, nous étudions un modèle à quatre étapes avec dégradation enzymatique du substrat (matière organique) qui peut être sous forme solide. Nous étudions l’effet de l’hydrolyse sur le comportement du processus de la digestion anaérobie et de la production du biogaz (méthane et hydrogène). Nous considèrons, dans un premier modèle, que l’hydrolyse se fait d’une manière enzymatique, alors que dans un second, nous supposons qu’elle est réalisée par un compartiment microbien. Les modèles considérés incluent l’inhibition de croissance des bactéries acétogènes, méthanogènes hydrogénétrophes et acétoclastes par plu- sieurs substrats. Pour étudier l’effet de ces inhibitions en présence de l’étape de l’hydrolyse, nous étudions dans un premier temps un modèle sans inhibition. Nous déterminons les équilibres et nous donnons des conditions nécessaires et suffisantes pour leur stabilité. L’existence et la stabilité des équilibres sont illustrées avec des diagrammes opératoires. Nous montrons que le modèle avec hydrolyse enzymatique change la production du méthane et d’hydrogène. En outre, l’introduction du com- partiment hydrolytique microbien donne de nouveaux équilibres et affecte les régions de stabilité. Nous prouvons que la production de biogaz est maximale en un seul point d’équilibre selon les paramètres opératoires et nous déterminons le taux maxi- mal de biogaz produit, dans chaque cas. Dans la deuxième partie, nous nous sommes intéressés à un modèle à deux étapes décrivant les phases de l’acétogénèse et de la méthanogénèse hydrogénotrophe. Le modèle représente une relation de syntrophie entre deux espèces microbiennes (les bactéries acétogènes et méthanogènes hydro- génotrophes), avec deux substrats à l’entrée (l’acide gras volatile et l’hydrogène), incluant les termes de mortalité et l’inhibition de croissance des bactéries acéto- gènes par un excès d’hydrogène dans le système. L’analyse de l’existence et de la stabilité des équilibres du modèle donne naissance à un nouvel équilibre qui peut être stable selon les paramètres opératoires du système. En utilisant les diagrammes opératoires, on remarque que, quelle que soit la région de l’espace considérée, il existe un seul équilibre localement exponentiellement stable. Cette étude est géné- ralisée dans le cas où la croissance des bactéries méthanogènes hydrogénotrophes est inhibée. Ce modèle donne naissance à deux équilibres strictement positifs et une bistabilité. Nous illustrons, en utilisant les diagrammes opératoires l’effet de cette inhibition sur la réduction des régions de coexistence et l’émergence de régions de bistabilité. / This PhD thesis focuses on the mathematical analysis of different anaerobic digestion (AD) models. In a first part, we study a 4-step model with enzymatic degradation of the substrate (organic matter) that can partly be under a solid form. We investigate the effects of hydrolysis on the behavior of the AD process and the production of biogas (namely, the methane and the hydrogen). We consider, in a first model, that the microbial enzymatic activity is constant, then we take into consideration an explicit hydrolytic microbial compartment for the substrate biodegradation. The considered models include the inhibition of acetogens, hydroge- notrophic methanogens and acetoclastic methanogens growth bacteria. To examine the effects of these inhibitions in presence of a hydrolysis step, we first study an inhibition-free model. We determine the steady states and give sufficient and neces- sary conditions for their stability. The existence and stability of the steady states are illustrated by operating diagrams. We prove that modeling the hydrolysis phase by a constant enzymatic activity affects the production of methane and hydrogen. Furthermore, introducing the hydrolytic microbial compartment yields new steady states and affects the stability regions. We prove that the biogas production occurs at only one of the steady states according to the operating parameters and state variables and we determine the maximal rate of biogas produced, in each case. In the second part, we are interested in a reduced and simplified model of the AD pro- cess. We focus on the acetogenesis and hydrogenetrophic methanogenesis phases. The model describes a syntrophic relationship between two microbial species (the acetogenic bacteria and the hydrogenetrophic methanogenic bacteria) with two in- put substrates (the fatty acids and the hydrogen) including both decay terms and inhibition of the acetogenic bacteria growth by an excess of hydrogen in the sys- tem. The existence and stability analysis of the steady states of the model points out the existence of a new equilibrium point which can be stable according to the operating parameters of the system. By means of operating diagrams, we show that, whatever the region of space considered, there exists only one locally exponentially stable steady state. This study is generalized to the case where the growth of the hydrogenetrophic methanogens bacteria is inhibited. This model exhibits a rich be- havior with the existence of two positive steady states and bistability. We illustrate by means of operating diagrams the effect of this inhibition on the reduction of the coexistence region and the emergence of a bistability region.

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No. of bitstreams: 1 DissJRFN.pdf: 1477527 bytes, checksum: cb09da4bfdd14f1e2c076f57d365b7a1 (MD5) Previous issue date: 2017-02-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / In recent years, the application of attenuated Salmonella spp. has been investigated for development of several biotechnological products, mainly vaccines. However, the implementation of industrial processes to obtain these products depends on the development of strategies for this microorganism high-cell density cultures (HCDC). One of the HCDC’s greatest challenges is overcoming Salmonella’s metabolic limitations, as it presents a high organic acids production (mainly acetic acid) that inhibits biomass formation. In this context, this work proposes two approaches to deal with this problem and implement Salmonella’s HCDC: studying the cultivation of S. typhimurium using glycerol as carbon source to reduce the generation of organic acids; studying the cultivation of a recombinant strain of S. typhimurium expressing the enzyme acetyl-CoA synthetase (ACS) from E. coli, for improved acetate assimilation). Initially, cultures were grown in agitated flasks in minimal media for two carbon sources (glucose or glycerol) for the wild-type and the recombinant strain. After the preliminary experiments, the recombinant strain was cultivated in bioreactor operated in batch mode with minimal medium formulated with glycerol, glucose or acetic acid as carbon source, to evaluate acetate production and assimilation. The wild-type strain was cultivated in a continuous-mode bioreactor on minimal medium with glycerol at D=0.10; 0.17 and 0.22 h-1 to evaluate the S. typhimurium glycerol metabolism. During the cultivations, samples were collected and analyzed by high-performance liquid chromatography to quantify the production of organic acids and substrate consumption. To quantify the concentration of biomass, optical density (600 nm) readings of culture broth and dry cell weight measurements were performed. Agitated flasks and batch cultivations results indicated that the acetate production is reduced in medium with glycerol for both strains, and that the genetically modified cells present a lower acetate accumulation phenotype compared to the wild-type. Continuous cultures of the wild-type strain showed no acetate accumulation for a 0.1 h-1 dilution rate. At rates of 0.17 h-1 and 0.22 h-1 acetate accumulation was observed, but acetate flux was 2-fold lower than the flux reached in chemostat with glucose-formulated medium. Simulations were performed with the STM_v1.0 model using as input data the glycerol and oxygen fluxes estimated from the experimental results. Good predictions were obtained for the biomass, CO2 and acetate fluxes at the higher dilution rates. The results suggest that fed-batch culture using glycerol as carbon source along with an exponential feed to maintain a 0.1 h-1 specific growth rate as a promising strategy to obtain high cellular concentrations of wild-type S. typhimurium. The efficient acetate uptake observed for the recombinant S. typhimurium cells may allow higher values of specific growth rate to be used for this strain, resulting in a higher productivity of biomass. / Nos últimos anos, a aplicação de linhagens atenuadas de Salmonella spp. vem sendo amplamente investigada para o desenvolvimento de diversos produtos biotecnológicos, principalmente vacinas. No entanto, a implementação de processos industriais para a obtenção destes produtos depende do desenvolvimento de estratégias para o cultivo em alta densidade celular (CADC) deste microrganismo. Para isso, um dos grandes desafios a ser superado se refere às limitações metabólicas da Salmonella, uma vez que esta apresenta elevada produção de ácidos orgânicos (principalmente ácido acético) que inibem a formação de biomassa. Neste contexto, este trabalho propõe duas abordagens para lidar com este problema e implementar CADC de Salmonella: estudar o crescimento de Salmonella typhimurium em glicerol avaliando seu metabolismo como uma fonte de carbono menos propensa à geração de ácidos orgânicos; e estudar a produção destes ácidos por uma cepa de S. typhimurium geneticamente modificada para diminuir o acúmulo de acetato (super-expressão do gene acs, de Escherichia coli, que codifica a enzima acetil-CoA sintetase (ACS) responsável pela assimilação de acetato). Inicialmente foram realizados cultivos em frascos agitados em meio mínimo para duas fontes de carbono (glicose ou glicerol), para a cepa selvagem e para a cepa recombinante. Após os experimentos preliminares, foram realizados cultivos em biorreator operado em modo batelada com a cepa modificada em meio mínimo formulado com glicerol, glicose ou ácido acético, como fonte de carbono, a fim de avaliar a produção e a assimilação de acetato. Foram realizados cultivos em biorreator em modo contínuo com a cepa selvagem em meio mínimo com glicerol com D=0,10; 0,17 e 0,22 h-1 para avaliar o metabolismo do glicerol pela S. typhimurium. Ao longo dos cultivos foram coletadas amostras do caldo e analisadas por cromatografia líquida de alta eficiência para quantificar a produção de ácidos orgânicos e o consumo de substrato. Para quantificar a biomassa produzida, foram realizadas medidas de densidade ótica (600 nm) e de massa seca. Os resultados dos cultivos em frascos agitados e das bateladas indicam que, para ambas as cepas, a produção de acetato é reduzida em meio formulado com glicerol, e que as células modificadas geneticamente apresentam um fenótipo de menor acúmulo de acetato comparadas à linhagem selvagem. Os cultivos contínuos realizados com a cepa selvagem mostraram que não houve acúmulo de acetato para a taxa de diluição de 0,1 h-1. Já nas taxas de 0,17 h-1 e 0,22 h-1, apesar de haver acúmulo, o fluxo de produção de acetato foi cerca de 2 vezes menor que o observado em quimiostato em meio formulado com glicose. Foram realizadas simulações com o modelo STM_v1.0, tendo como dados de entrada os fluxos de glicerol e de oxigênio estimados a partir dos dados experimentais. O modelo descreveu bem os fluxos de biomassa, CO2 e acetato para as taxas de diluição mais altas. Os resultados obtidos sugerem o cultivo em batelada alimentada com glicerol como fonte de carbono, e alimentação exponencial definida para manter a velocidade específica de crescimento em 0,1 h-1, como uma estratégia promissora para obter altas concentrações celulares de S. typhimurium selvagem. Para a S. typhimurium recombinante, devido à sua eficiente assimilação de acetato, valores ainda maiores de velocidade específica de crescimento poderiam ser impostos, com elevado aumento de produtividade em biomassa.

Focalisé sur le groupe phytoplanctonique des diatomées, ce travail de thèse a permis d'étudier l'impact du réchauffement climatique et de l'acidification des océans afin d'appréhender la réponse de ce taxon au changement climatique actuel. L'augmentation de la pCO2 atmosphérique depuis la première révolution industrielle est à l'origine de l'augmentation de la concentration en carbone inorganique dissous (DIC) dans l'océan de surface, et donc d'une acidification des océans à laquelle s'ajoute un réchauffement de l'océan de surface conduisant finalement à l'extension des zones oligotrophes très stratifiées et pauvres en sels nutritifs. Différentes approches in situ et au laboratoire ont donc été utilisées lors de ce travail de thèse afin de mieux comprendre la place des diatomées dans une province oligotrophe et d'apprécier ensuite leur réponse à un réchauffement et une acidification dans un «chémostat oligotrophe», limité en phosphate. / Specifically orientated on the diatoms' phytoplanktonic group, this work tryed to understand the impact of warming and ocean acidification on diatoms and aimed at understand the taxon's response to the ongoing climate change. Amospheric pCO2 increase since the first industrial revolution lead to the augmentation of dissoveld inorganic carbon (DIC) concentration in the surface ocean, and thus to the ocean acidification, accompanied by an ocean surface warming leading finally to the extension of oligotrophic areas well stratified and nutrient depleted. Different in situ and lab techniques were used in order to better understand the diatom role in oligotrophic system and their response to warming and acidification in an oligotrophic chemostat, limited by phosphate.

Estudou-se a influência do fosfato na multiplicação de Saccharomyces cerevisiae em uma fermentação contínua em mini-fermentador. Determinou-se a massa seca, a concentração dos substratos, a velocidade específica de consumo dos substratos, a velocidade específica de formação de gás carbônico, velocidade específica de consumo de oxigênio e o quociente respiratório bem como, o fósforo intracelular em diversas vazões específicas de alimentação, em cultivo contínuo de levedura de panificação, em condições de substratos limitantes. Controlando-se a quantidade de fosfato no meio de alimentação de tal modo que o fosfato residual no meio de fermentação mal pudesse ser detectado, o valor da vazão específica de alimentação crítica era aparentemente aumentado de 0,23 h-1 para 0,32 h-1. Isto sugere uma possível influência do fosfato nas funções anaeróbicas e aeróbicas da levedura de panificação. / The influence of phosphate in a continuous culture was studied using mini-fermentor on the Saccharomyces cerevisiae multiplication. Dry matter, substrate concentration, specific substrate comsumption, specific carbon dioxide release, specific oxygen uptake rates and respiration quotient , as well as phosphorous content of the cells were measured in dependence on the dilutionrate. In continuous culture glicose-limited, of baker\'s yeast if the supply of phosphorous were restricted to a extent that residual phosphate in the medium could hardly be observed, the value of critical dilution rate was apparently enhanced from 0,23 h-1 to 0,32 h-1. This observation suggests a possible mediation by phosphate between anaerobic and aerobic functions of the baker\'s yeast.

As células de Drosophila melanogaster (S2) têm sido utilizadas como sistemas de expressão de proteínas recombinantes. Neste trabalho foi utilizada uma linhagem S2 geneticamente modificada com vetores de expressão para a produção da glicoproteína do vírus da raiva (GPV). O principal objetivo deste trabalho foi avaliar o comportamento destas células cultivadas em processo contínuo, visando-se manter elevadas concentrações celulares. Para os ensaios contínuos, utilizou-se meio livre de soro fetal bovino SF 900 II em um reator Biostat B, com 500 mL de volume útil e controle de temperatura (28ºC), oxigênio dissolvido (30% da saturação com ar), frequência de agitação (90 rpm) e monitoramento do pH. Verificou-se o comportamento do metabolismo celular em diferentes vazões específicas de alimentação (0,8 dia-1, 0,5 dia-1 e 0,2 dia-1) através parâmetros como fatores de conversão e variáveis como concentração celular máxima, concentração residual de glicose e glutamina, dentre outras. Ainda, avaliou-se a influência de aminoácidos, tais como, glutamina, asparagina, prolina, serina e cisteína suplementados no meio de alimentação, sob a concentração celular alcançada no estado estacionário. Diferentes vazões específicas de alimentação - em estado estacionário - resultaram em concentrações celulares próximas entre si. A adição de glutamina (1,7 g/L) no meio de alimentação não contribuiu para o aumento na concentração celular, indicando que este aminoácido não limitou o processo de crescimento celular. Uma observação similar ocorreu quando o meio SF 900 II foi suplementado com asparagina, prolina, serina e cisteína. Porém, a adição de cisteína (0,3 g/L) isoladamente no meio de alimentação resultou em um aumento de 12% na concentração celular quando comparada ao meio SF 900 II puro. Assim, pode-se concluir que a cisteína limitava o crescimento celular. Verificou-se ainda que a célula não apresentou grande variabilidade nos diferentes ensaios, sob mesma vazão específica de alimentação. Isso indica que processo contínuo constituiria um método viável para a compreensão do metabolismo desta célula. / Drosophila melanogasters cells (S2) have been used as expression systems for recombinant proteins. This study uses a genetically modified S2 line with expression vectors for production of rabies virus glycoprotein (RVPG). The main objective was to evaluate the growth trend of S2 cells in a continuous process, aiming to maintain high cell concentrations. In order to set the continuous culture, the experiments used serum-free medium SF 900 II in a Biostat B reactor, with working volume of 500 mL and temperature controlled at 28 º C, dissolved oxygen at 30% air saturation, agitation speed at 90 rpm, and pH monitoring. Cellular metabolism behavior was observed under different dilution rates (0.8 day-1, 0.5 day-1, and 0.2 day-1) through parameters such as yield factors, in addition to variables such as maximum cell concentration, residual concentration of glucose and glutamine, among others. Yet, this work evaluates the influence of amino acids such as glutamine, asparagine, proline, serine and cysteine supplemented in the feed, over cellular concentration value reached in the steady state. Different dilution rates decreasing (under steady state) resulted in cell concentrations quite simillar. The addition of glutamine (1.7 g/L) in the feed did not contribute to the increase of cell concentration, which indicates that this amino acid did not limit cell growth process. A similar observation occurred when SF 900 II medium was supplemented with asparagine, proline, serine and cysteine. However, the cysteine addition (0.3 g/L) alone in the feed resulted in a 12% increase in cell concentration, compared to pure SF 900 II. Thus, it is possible to conclude that cysteine limited cell growth. It was also found that the cell did not show great variability in the various tests under the same dilution rate. This indicates that chemostat culture would be a viable method for understanding the metabolism of this cell.

<p>There are many types of bioreactors used for producing bacteria populations in commercial, medical and research applications. </p><p>This report presents a systematic discussion of some of the most important models corresponding to the well known reproduction kinetics such as the Michaelis-Menten kinetics, competitive substrate inhibition and competitive product inhibition. We propose a modification of a known model, analyze it in the same manner as known models and discuss the most popular types of bioreactors and ways of controlling them. </p><p>This work summarises much of the known results and may serve as an aid in attempts to design new models.</p>

There are many types of bioreactors used for producing bacteria populations in commercial, medical and research applications. This report presents a systematic discussion of some of the most important models corresponding to the well known reproduction kinetics such as the Michaelis-Menten kinetics, competitive substrate inhibition and competitive product inhibition. We propose a modification of a known model, analyze it in the same manner as known models and discuss the most popular types of bioreactors and ways of controlling them. This work summarises much of the known results and may serve as an aid in attempts to design new models.

As células de Drosophila melanogaster (S2) têm sido utilizadas como sistemas de expressão de proteínas recombinantes. Neste trabalho foi utilizada uma linhagem S2 geneticamente modificada com vetores de expressão para a produção da glicoproteína do vírus da raiva (GPV). O principal objetivo deste trabalho foi avaliar o comportamento destas células cultivadas em processo contínuo, visando-se manter elevadas concentrações celulares. Para os ensaios contínuos, utilizou-se meio livre de soro fetal bovino SF 900 II em um reator Biostat B, com 500 mL de volume útil e controle de temperatura (28ºC), oxigênio dissolvido (30% da saturação com ar), frequência de agitação (90 rpm) e monitoramento do pH. Verificou-se o comportamento do metabolismo celular em diferentes vazões específicas de alimentação (0,8 dia-1, 0,5 dia-1 e 0,2 dia-1) através parâmetros como fatores de conversão e variáveis como concentração celular máxima, concentração residual de glicose e glutamina, dentre outras. Ainda, avaliou-se a influência de aminoácidos, tais como, glutamina, asparagina, prolina, serina e cisteína suplementados no meio de alimentação, sob a concentração celular alcançada no estado estacionário. Diferentes vazões específicas de alimentação - em estado estacionário - resultaram em concentrações celulares próximas entre si. A adição de glutamina (1,7 g/L) no meio de alimentação não contribuiu para o aumento na concentração celular, indicando que este aminoácido não limitou o processo de crescimento celular. Uma observação similar ocorreu quando o meio SF 900 II foi suplementado com asparagina, prolina, serina e cisteína. Porém, a adição de cisteína (0,3 g/L) isoladamente no meio de alimentação resultou em um aumento de 12% na concentração celular quando comparada ao meio SF 900 II puro. Assim, pode-se concluir que a cisteína limitava o crescimento celular. Verificou-se ainda que a célula não apresentou grande variabilidade nos diferentes ensaios, sob mesma vazão específica de alimentação. Isso indica que processo contínuo constituiria um método viável para a compreensão do metabolismo desta célula. / Drosophila melanogasters cells (S2) have been used as expression systems for recombinant proteins. This study uses a genetically modified S2 line with expression vectors for production of rabies virus glycoprotein (RVPG). The main objective was to evaluate the growth trend of S2 cells in a continuous process, aiming to maintain high cell concentrations. In order to set the continuous culture, the experiments used serum-free medium SF 900 II in a Biostat B reactor, with working volume of 500 mL and temperature controlled at 28 º C, dissolved oxygen at 30% air saturation, agitation speed at 90 rpm, and pH monitoring. Cellular metabolism behavior was observed under different dilution rates (0.8 day-1, 0.5 day-1, and 0.2 day-1) through parameters such as yield factors, in addition to variables such as maximum cell concentration, residual concentration of glucose and glutamine, among others. Yet, this work evaluates the influence of amino acids such as glutamine, asparagine, proline, serine and cysteine supplemented in the feed, over cellular concentration value reached in the steady state. Different dilution rates decreasing (under steady state) resulted in cell concentrations quite simillar. The addition of glutamine (1.7 g/L) in the feed did not contribute to the increase of cell concentration, which indicates that this amino acid did not limit cell growth process. A similar observation occurred when SF 900 II medium was supplemented with asparagine, proline, serine and cysteine. However, the cysteine addition (0.3 g/L) alone in the feed resulted in a 12% increase in cell concentration, compared to pure SF 900 II. Thus, it is possible to conclude that cysteine limited cell growth. It was also found that the cell did not show great variability in the various tests under the same dilution rate. This indicates that chemostat culture would be a viable method for understanding the metabolism of this cell.

No description available.

Les bioréacteurs de grandes dimensions, en raison de phénomènes de transfert limitant, sont le siège d’hétérogénéités se traduisant par des gradients locaux de concentration et température. Les microorganismes circulant au sein de ces bioréacteurs subissent donc des fluctuations environnementales qui peuvent affecter leur comportement aux niveaux métaboliques et/ou moléculaires. La réponse microbienne est fonction de la nature, de l’intensité, de la fréquence et de la durée de la perturbation. L’objectif de ce travail est l’étude quantitative de l’impact de l’intensité, la fréquence et l’amplitude d’un stress nutritionnel sur le comportement dynamique d’Escherichia coli, à savoir des ajouts pulsés de glucose lors de cultures continues en régime permanent. Un effort particulier est consacré au développement et à la validation des outils expérimentaux indispensables pour une caractérisation rigoureuse des dynamiques de réponses transitoires sur des échelles de temps allant de secondes à quelques minutes. Pour permettre le suivi in situ et en temps réel des changements métaboliques et moléculaires, une souche bioluminescente est mise en œuvre. Les réponses transitoires sont caractérisées par les vitesses spécifiques, les rendements, les profils d’induction transcriptionnelle, les temps caractéristiques. Selon les différents scenarii réalisés, l’ajustement du métabolisme face aux hétérogénéités de substrat est quantifié selon des échelles de temps aux niveaux macroscopiques et/ou moléculaires ; ces résultats originaux contribuent ainsi à l’implémentation des connaissances sur les interactions dynamiques entre les phénomènes biologiques et les phénomènes physiques ; l’enjeu réside à terme en l’amélioration des processus d’optimisation et d’extrapolation des bioprocédés par l’identification et la quantification des dynamiques des phénomènes limitants / Ineffective mixing entailing heterogeneity issues within industrial bioreactors have been reported to affect microbial metabolisms at cellular and/or molecular levels. Substrate gradients inside large-scale bioreactors are common environmental fluctuations that microorganisms would have to encouter along with the bioprocess. Depending on intensity, frequency and duration of those fluctuations, microorganisms may respond in a different manner. The objective of this work is to study the impact of intensity, frequency and amplitude of glucose perturbations on the dynamics of Escherichia coli responses. An E. coli bioluminescent strain is used for in situ and real-time monitoring of both metabolic and transcriptional changes. For this purpose, short-term glucose excess was simulated, using pulse-based experiments into glucose-limited chemostat cultures. In addition, an important effort is devoted to the development and validation of technical and mathematical tools in order to acquire quantitative and kinetic data on time scales from seconds to minutes. The transient responses are characterized, using specific rates, yields, transcriptional induction profiles and characteristic response times, and are compared in the different defined perturbation scenarios. The results reflected the fact that short-term heterogeneities of substrate affect both cell metabolism and regulation at macroscopic and/or molecular levels. Quantitative understandings of the dynamics during transient responses to environmental perturbations can thus shed light on the bioprocess optimization