As características do processo industrial brasileiro para a produção do bioetanol tornam as destilarias susceptíveis a presença de microrganismos contaminantes que ocasionam na queda de rendimento de produção. Dentre as etapas do processo, tem-se a fermentação alcoólica, que consiste na metabolização de açúcares pela cepa de levedura selecionada da espécie Saccharomyces cerevisiae produzindo o etanol. Nesta etapa ocorre a contaminação por leveduras do gênero Saccharomyces, leveduras não Saccharomyces e bactérias. As bactérias mais encontradas são as pertencentes ao grupo das bactérias láticas (LAB), que por utilizarem diferentes vias para metabolizar os açúcares, são classificadas em heterofermentativa obrigatória, homofermentativa obrigatória e heterofermentativa facultativa. Entre os gêneros deste grupo, os Lactobacillus são os mais comuns devido à sua habilidade em tolerar altas concentrações de etanol e açúcares, altas temperaturas e baixo pH. As espécies L. fermentum e L. plantarum foram relatadas em diversos trabalhos como entre as espécies mais encontradas contaminando esse ambiente. Os Lactobacillus contaminantes estão em constante interação com a cepa de levedura que por consequência, tem sua eficiência fermentativa reduzida. Este trabalho teve por objetivo analisar os metabólitos produzidos durante a fermentação alcoólica industrial e que podem ser utilizados pelas bactérias contaminantes, obtendo assim, condições para serem competitivas e persistentes no processo. Para isso, foram utilizadas duas linhagens, isoladas de destilaria e identificadas como L. fermentum (I3a) com metabolismo heterofermentativo obrigatório e L. plantarum (I4a) com metabolismo heterofermentativo facultativo, apresentando nas condições estudadas um metabolismo homofermentativo. Ambas as linhagens foram submetidas ao crescimento na presença do glicerol, malato e piruvato, que são metabólitos produzidos e excretados pela levedura e o manitol produzido e excretado pela bactéria heterofermentativa obrigatória. Foi observado que o metabólito manitol é uma eficiente fonte de carbono para ambas as linhagens proporcionando crescimento mesmo sem a presença de açúcares. Além disso, a combinação entre glicose, frutose, manitol e malato foi capaz de aumentar o crescimento das linhagens. Já a presença do piruvato, apresentou estímulo de crescimento para a linhagem heterofermentativa. Em relação ao consumo, as linhagens foram capazes de metabolizar o manitol, malato e piruvato, entretanto, não apresentaram consumo do glicerol. Com isso, ambas as linhagens são beneficiadas pelo metabolismo da levedura e ainda a heterofermentativa é capaz de reabsorver o manitol quando os açúcares fermentescíveis são esgotados e de disponibilizar o metabólito para uso da homofermentativa. / The characteristics of the Brazilian industrial process to produce bioethanol make the distilleries susceptible to the presence of contaminating microorganisms that cause in the fall of production yield. Among the stages of the process, we have the alcoholic fermentation, which consists in the metabolization of sugars by the yeast strain selected from the Saccharomyces cerevisiae species producing the ethanol. At this stage contamination occurs by yeasts of the genus Saccharomyces, yeasts not Saccharomyces and bacteria. The most commonly found bacteria are those belonging to the group of lactic bacteria (LAB), which, because they use different routes to metabolize sugars, are classified as obligate heterofermentative, obligate homofermentative and facultative heterofermentative. Among the genera of this group, Lactobacillus are the most common because of their ability to tolerate high concentrations of ethanol and sugars, high temperatures and low pH. The species L. fermentum and L. plantarum have been reported in several studies as among the most frequent species contaminating this environment. Lactobacillus contaminants are in constant interaction with the yeast strain which consequently has its fermentative efficiency reduced. This work aimed to analyze the metabolites produced during industrial alcoholic fermentation and that can be used by the contaminating bacteria, thus obtaining conditions to be competitive and persistent in the process. For this, two strains were used, isolated from distillery and identified as L. fermentum (I3a) with obligate heterofermentative metabolism and L. plantarum (I4a) with facultative heterofermentative metabolism, presenting a homofermentative metabolism under the conditions studied. Both strains were submitted to growth in the presence of glycerol, malate and pyruvate, which are metabolites produced and excreted by yeast and mannitol produced and excreted by the obligate heterofermentative bacterium. It was observed that the metabolite mannitol is an efficient source of carbon for both strains providing growth even without the presence of sugars. In addition, the combination of glucose, fructose, mannitol and malate was able to increase strains growth. However, the presence of pyruvate presented growth stimulus for the heterofermentative strain. In relation to consumption, the strains were able to metabolize mannitol, malate and pyruvate, however, they did not present glycerol consumption. Thus, both strains are benefited by yeast metabolism and the heterofermentative can reabsorb mannitol when the fermentable sugars are depleted and to make the metabolite available for homofermentative use.

Le procédé de transformation des racines de manioc en gari reste la plupart du temps artisanal et dépendant du savoir-faire des opératrices. Étant constitué d’une suite d’opérations unitaires (râpage, fermentation/pressage, tamisage et cuisson), il est nécessaire de déterminer les opérations-clefs déterminantes de la qualité du produit. Ainsi, dans ce travail, le procédé a été analysé dans sa continuité au travers du suivi de réponses biochimiques, granulométriques et hydro-texturales du produit durant sa transformation. L’objectif du travail de thèse a été dans un premier temps de développer des outils et méthodes permettant le suivi des états physiques et biochimiques du produit tout au long de sa transformation à l’échelle laboratoire. Cette démarche a permis de dégager certains mécanismes contribuant à la transformation du produit. Il a été mis en évidence le drainage d’une partie du liquide intra-cellulaire lors du pressage ainsi que deux mécanismes d’agglomérations successifs lors de l’élaboration du gari. Les résultats et les mécanismes déduits des observations réalisées à l’échelle laboratoire ont par la suite été confrontés à ceux obtenus sur le terrain (Bénin) dans des conditions moins maîtrisées et sous l’effet d’un changement d’échelle. L’opération de cuisson, à la fois très contraignante et ressortant comme une opération unitaire clef a été, par la suite, plus particulièrement étudiée. Pour cela, le comportement (choix et gestes) de deux opératrices a été enregistré de façon à mettre en évidence le rôle de l’opératrice lors de la cuisson. L’ensemble des résultats montrent que la qualité des garis repose en grande partie sur le savoir faire de l’opératrice. Il ressort cependant que les critères de qualité du gari qui guide l’opératrice lors de la cuisson et la conduisent à ajuster son comportement sont, bien sûr, ceux qui lui sont accessibles (couleur, taille, …), mais ne sont pas suffisants pour atteindre teneurs en composés cyanogénique résiduels recommandées par le Codex Alimentarius de la FAO. / The cassava process of converting cassava roots into gari remains mostly artisanal and depends on the know-how of the operators. As it consists of a series of unit operations (grating, fermentation/pressing, sieving and cooking), it is necessary to determine the key operations that determine the quality of the product. Thus, in this work, the process was analyzed in its continuity through the monitoring of biochemical, granule size and hydro-textural responses of the product during processing. The objective of the thesis work was initially to develop tools and methods to monitor the physical and biochemical states of the product throughout its processing on a laboratory scale. This approach has made it possible to identify some of the mechanisms that contribute to the elaboration of the product. It was found that part of the intracellular liquid drained during pressing and two successive agglomeration mechanisms were identified during the preparation of the gari. The results and mechanisms derived from laboratory scale observations were then compared with those obtained in the field (Benin) under less controlled conditions and scaling effect. The cooking operation, which is both very constraining and emerging as a key unitary operation, was then more particularly studied. For this purpose, the performance (choice and gestures) of two operators was recorded in order to highlight the role of the operator during the cooking process. All the results show that the gari quality is largely based on the operator's know-how. However, it appears that the product quality criteria that guide the operator during cooking lead her to adjust her practices are, of course, those that are accessible to her (colour, granule size, ...), but are not enough to reach the levels of residual cyanogenic compounds recommended by the FAO Codex Alimentarius.

Ces dernières années ont vu un grand développement des biotechnologies blanches et de l'ingénierie métabolique avec l'objectif de remplacer les procédés de synthèse de molécules d’intérêt de l’industrie chimique classique par des voies de synthèse biologique. Dans ce contexte, les réactions anaplérotiques, qui produisent les acides dicarboxyliques, sont particulièrement intéressantes puisqu'au delà de la production de ces molécules d’intérêt elles permettent une fixation nette de carbone, réduisant ainsi l’impact environnemental des procédés. Ce travail de thèse a donc porté sur l'élaboration d'une stratégie d'ingénierie métabolique faisant appel à des réactions de fixation de carbone inorganique chez la levure pour la production d'acide malique, une molécule plateforme ayant de nombreuses applications industrielles. La levure Saccharomyces cerevisiae a été choisie comme hôte pour sa commodité d’utilisation dans les procédés industriels et ses nombreux outils génétiques. L'approche développée repose sur la mise en place d'une voie de production d'acide malique par surexpression de la phosphonéolpyruvate carboxylase d'Escherichia coli (PEPC), de la malate déshydrogénase peroxysomale de S. cerevisiae relocalisée dans le cytosol (MDH) et du transporteur d'acides dicarboxyliques de Schizosaccharomyces pombe. La souche de levure recombinante obtenue a été caractérisée lors d'essais en fioles, en présence notamment de carbonate de calcium pour assurer un apport de carbone inorganique. Ces essais ont permis de mettre en évidence un effet stimulant de l'apport de carbone inorganique sur la production de malate et d'obtenir des concentrations de malate de l'ordre de 2,5 g/L à partir de 50 g/L de glucose, pour un rendement maximal de 0,046 gramme de malate par gramme de glucose. Des essais en bioréacteur de 5 L en présence d'air ou d'air enrichi à 5% de CO2 ont montré un effet positif de l'apport de carbone inorganique sous forme de dioxyde de carbone sur la production de malate. La concentration maximale de malate obtenue est de 1,46 g/L à partir de 50 g/L de glucose, soit un rendement de 0,029 gramme de malate par gramme de glucose. Des souches intermédiaires exprimant la PEPC et la MDH obtenues pour la production de malate ont également été caractérisées pour la production d'éthanol, car elles semblaient présenter une augmentation du rendement de production d'éthanol par effet transhydrogénase par rapport à la souche sauvage. Les essais n'ont cependant pas permis de confirmer cette augmentation de rendement. / White biotechnologies have been developing quickly during the last decades, aiming at replacing chemical syntheses by biological processes for the industrial production of target compounds. In this context, the implementation of anaplerotic reactions in the metabolism is of great interest, since those reactions allow both production of dicarboxylic acids and direct fixation of inorganic carbon. This work is about the development of a metabolic engineering strategy using inorganic carbon fixation reactions to produce malic acid, a compound with various industrial applications. The yeast Saccharomyces cerevisiae was chosen as a host for its convenient use in industrial processes and the availability of genetic tools. The approach developed to produce malic acid is based on the overexpression of Escherichia coli phosphoenolpyruvate carboxylase (PEPC), S. cerevisiae peroxysomale malate dehydrogenase relocated in the cytosol (MDH) and Schizosaccharomyces pombe dicarboxylic acid carrier. A recombinant yeast strain expressing those three genes was obtained and characterised in shake-flasks experiments, involving more specifically calcium carbonate as an inorganic carbon source. Those experiments showed an enhancement of the malate production in the presence of calcium carbonate and allowed to obtain a concentration of 2.5 g/L from 50 g/L glucose, for a maximal yield of 0.046 gram malate per gram glucose. Fermentation experiments were performed in a 5 L bioreactor in the presence of air or 5% CO2 enriched air; they confirmed the positive effect of inorganic carbon addition as CO2 on malate production. A malate concentration of 1.46 g/L from 50 g/L glucose was obtained, giving a yield of 0.029 gram malate per gram glucose. Intermediate recombinant strains expressing PEPC and MDH were also characterised, for ethanol production, as they seemed to give increased ethanol yields, probably due to a transhydrogenase effect. Shake flasks and bioreactors experiments did unfortunately not confirm the yield improvement.

Le vin est un milieu peu propice à la croissance de la levure mais il est néanmoins possible de la faire croître sur base de vin enrichit en nutriments et dilué pour diminuer la concentration en éthanol. En vue de l’élaboration des vins effervescents par une seconde fermentation, produire la levure Saccharomyces cerevisiae dans ces conditions est indispensable pour l’acclimater mais il s’agit d’un enjeu complexe qui doit prendre en compte de nombreux paramètres physico-chimiques mais aussi économiques. En effet, les paramètres opératoires peuvent induire des conditions de croissance pouvant affecter le développement de la levure. Seule la levure S. cerevisiae (Fizz+) a été utilisée car elle est spécialement sélectionnée pour cette seconde fermentation en vase clos. Le principal enjeu était donc d’obtenir une bonne adaptation de la levure à croître dans un milieu hydro-alcoolique, conditions contraignantes pour elle, mais aussi d’obtenir une production maximale.Nous avons tout d’abord étudié en fioles Erlenmeyer (250 mL) l’influence de divers paramètres : conditions physico-chimiques, concentrations en nutriments, concentration minimale en levure sèche active nécessaire à une bonne activité ainsi que son temps de réhydratation.Dans un deuxième temps, nous avons effectué des propagations en mode batch dans un bioréacteur (5 L) pour valider les conclusions réalisées à la suite de l’étude en Erlenmeyer et ainsi étudier l’influence de différentes aérations sur la production de S. cerevisiae. Les données obtenues ont servi de base pour comparer les améliorations apportées par le procédé développé en mode fed-batch. Les concentrations en levures obtenues suite à l’optimisation des conditions du milieu de culture en cinq litres sont supérieures d’un facteur cinq à celles obtenues dans la pratique en cave.Ensuite l’étude s’est concentrée sur le développement d’un nouveau procédé d’alimentation en nutriments pour cultiver S. cerevisiae en métabolisme respiratoire dans des cuves réalisées par la société partenaire du projet, OEno Concept. La nouveauté réside dans la façon de réguler la température de la culture qui se fait simultanément à l’apport des nutriments suite au dégagement de chaleur lors de la croissance de S. cerevisiae. Un brevet a été déposé sur cette technologie. Ce nouveau procédé a permis une augmentation de la productivité cellulaire, d’un facteur supérieur à quatre, car il a permis aux levures de s’adapter à cet environnement stressant et a favorisé l’oxydation du glucose au détriment de la fermentation. / Wine is an aggressive/stressful growth medium; it is depleted of micronutrients, rich in ethanol and very poor in assimilable nitrogen. Despite all these difficulties, it is possible to grow yeast in a medium largely based on wine by diluting the ethanol concentration and enriching the medium with micronutrients, a carbon source and assimilable nitrogen. It is, desirable to propagate Saccharomyces cerevisiae in such environment in order to produce a culture of yeast adapted to a second fermentation of alcoholic beverages. Production of microorganism in wine growing environment, is a complex issue that must take into account many, physicochemical and economic parameters. Indeed, the operating parameters can affect the development of yeast in a bioreactor. Therefore, it is important to know the most influential parameters on growth. The strain S. cerevisiae (Fizz+), a commercial strain that has been selected for the second fermentation in bottles, was used during this project. The propagation process served to increase the amount of yeast as well as to adapt the yeast to grow in an alcoholic environment. We first studied in shake-flasks cultures various physicochemical conditions such as nutrients concentration, the rehydration time and the minimum concentration of active dry yeast necessary for good yeast activity.In a second step, we performed batch fermentations in bioreactors (5 L) to confirm the conclusions from the shake-flask cultures and additionally to study the influence of aeration on S. cerevisiae production. The data obtained served as a basis for performing fed-batch cultures. The yeast concentrations obtained as a result of the optimization of the conditions of the culture medium in five liters were five times greater than those obtained in actual industrial production processes. The next step was to develop an automated fed-batch culture to grow S. cerevisiae respiratively in partnership with the industrial partner of the project, OEno Concept. The novelty of the process is the way in which the growth medium feed-rate is linked to the heat produced by the growing S. cerevisiae.This research has allowed an increase in cell productivity, by a factor greater than four, thanks to the novel process in stressful growth environment promoting respiration with regard to fermentation.

L'étude et le développement des procédés de fabrication de levures immobilisées en vue de la réalisation de fermentations de vins a débuté au milieu des années 80. Malgré les bénéfices potentiels que cette technologie pouvait apporter pour le secteur œnologique, peu de procédés d'immobilisation ont réussi à dépasser l'échelle laboratoire ou pilote et ceux qui sont arrivés à l'échelle industrielle n'ont pas eu le succès désiré pour des questions d'ordre technique ou économique. Le premier objectif de ce travail concerne la mise au point du procédé industriel en insistant sur les aspects les plus sensibles, et qui comme tels ont exigé des études complémentaires. Le deuxième objectif de ce travail vise à caractériser du point de vue cinétique et lorsque possible sensoriel, les fermentations avec les levures immobilisées pour la production de vins effervescents et pour la désacidification biologique de moûts. Le troisième et dernier objectif de ce travail consiste à évaluer l'utilisation de levures immobilisées pour la réalisation de la fermentation alcoolique en continu de moût. Pour cela on a fait appel à des fermenteurs continus à lit fixe et à lit fluidisé. / The study and development of yeast immobilization processes for wine fermentations started in the mid 80’s. Even though this technology could be of great benefit for the oenological sector very few process left the laboratory or pilot scale and those which arrived to industrial scale didn’t have the ambitioned success due to technical or economical constraints. The first goal of this work was to develop an industrial process for yeast immobilisation with emphasis on the most sensitive aspects which required further studies. The second objective of this work was to characterise the fermentation kinetics of immobilised yeasts cells during the production sparkling wines and during the deacidification of grape must. Whenever possible the wines produced were also characterised from a sensorial point of view. The third and last goal was to evaluate the use of immobilised yeast cells for continuous fermentation of grape must. For that we have used continuous fixed bed and fluidized bed fermenters.

Glucose isomerase (GI) catalyzes the conversion of D-glucose to D-fructose in vitro. It is one of the bulkiest commercial enzymes, essential for the mass production of high-fructose corn syrup (HFCS) and crystalline fructose. / In this study, the effects of nitrogen sources, carbon sources, expression vectors, host strains, bacterial (Vitreoscilla) hemoglobin, selective pressure, plasmid stability and fermentation process on the GI production were investigated. The results showed that E. coli could express cloned thermostable GI at high expression level. E. coli transformed with the recombinant plasmid P-lac-GI gave the best result in term of total GI production and expression level. Corn steep liquor could be used as a cheap alternative nitrogen source for what was in LB medium. The concentration of glucose affected the expression level of GI significantly. Replacement of the ampicillin resistance gene by kanamycin resistance gene improved the plasmid stability leading to high productivity of GI in fed-batch fermentation. A suicide system could further improve the plasmid stability resulting in a high productivity of GI. A feeding strategy for fed-batch fermentation with the optimized parameters was developed to result in the production of up to 3g/L recombinant GI, which constituted 50% of the total soluble proteins. The total yield was 5-fold higher than that from flask experiments and 7-fold higher than the highest ever recorded. The expression level was also 100% higher than it was in other reports. / Liu Zhaoming. / "August 2005." / Advisers: J. Wang; W. P. Fong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3780. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 129-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Orientador: Eleni Gomes / Coorientador: Gustavo O. Bonilla-Rodriguez / Banca: Heizir Ferreira de Castro / Banca: Ernandes Benedito Pereira / Banca: Humberto Márcio Santos Milagre / Banca: Teresa Cristina Zangirolami / Resumo: As enzimas são catalisadores muito eficientes e de grande interesse na aplicação industrial. As lipases (E.C. 3.1.1.3) são serina-hidrolases que agem na hidrólise, esterificação e transesterificação de acilgliceróis de cadeia longa. Lipases microbianas têm sido amplamente usadas devido à sua especificidade. Na transesterificação moléculas de triacilglicerol reagem com um álcool na presença de um catalisador formando uma mistura de glicerol e ésteres de ácidos graxos. O biodiesel, definido como ésteres metílicos ou etílicos, tem atraído crescente interesse como uma fonte de energia renovável, substituindo o diesel produzido a partir de combustíveis fósseis. O presente trabalho tem como objetivo principal efetuar a prospecção de fungos termofílicos que apresentem produção significativa de lipase e, concomitantemente, atividade transesterificante. As linhagens foram selecionadas por detecção de atividade lipolítica em placas de ágar contendo Rodamina B, por fermentação submersa (FSm) e fermentação em estado sólido (FES). Foram testadas linhagens de fungos termofílicos da coleção do laboratório de Bioquímica e Microbiologia Aplicada, sendo Thermomucor indicae-seudaticae N31, Rhizomucor pusillus Myceliophtora sp F2.1.4, Myceliophtora sp M7.7, F2.1.1, F2.1.3 e Thermomyces lanuginosus TO-05 os que mostraram um maior potencial hidrolítico. Estudos adicionais avaliaram a produção de lipase através da modificação da fonte de componentes nutricionais e algumas propriedades físicas na produção de lipase por T. indicae-seudaricae N31 em FSm, e por R. pusillus e T. indicae-seudaticae N31 em FES. Os estudos dos processos fermentativos foram bem sucedidos, havendo um aumento de 16 vezes na produção de lipase de R. pusillus e de 36 vezes na lipase de T indicae-seudaticae... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Enzymes are efficient catalysts and interesting for industrial applications. Lipases (EC 3.1.1.3) constitute a group of serine hydrolases that catalyze the hydrolysis, esterification and transesterification reactions of long chain acylglycerols. Microbial lipases have been widely used for biotechnological applications due to their specificity. In transesterification, molecule of a tryacylglicerol react with an alcohol in the presence of catalyst, producing a mixture of fatty esters and glycerol, Biodiesel, defined as methyl or ethyl fatty esters and has attracted considerable attention as a renewable source of energy, in substitution of fossil fuels. The main goal of the present work is the screening of thermophilic fungi that present outstanding lipase production and in parallel able to perform transesterification reaction. Strains were screened for lipase activity on agar plates containing Rhodamine B, for submerged fermentation (SmF) and solid state fermentation (SSF). The tested thermophilic strains were from the collections of the Biochemistry and Applied Microbiology Laboratory, where Thermomucor indicae-seudancae N31, Rhizomucor pusillus Myceliophtora sp F2.1.4, Myceliophtora sp M7.7, F2.1.1, F2.1.3 and Thermomyces lanuginosus TO-O5 had the highest lipase production. Additional studies attempted to improve lipase production by nutrient source modifications and physicals conditions in FmS by T. indicae-seudaticae N31 and in FSS by T. indicae-seudaticae N31 and R. pusillus. The fermentations studies were successful, with a 16 fold enhancement in lipase yield compared to the initial medim from lipase R. pusillus and 36 fold for the lipase T. indicae-seudaticae N31, both in FES. The lipase from T. indicae-seudaticae N31 cultured in SSF and SmF, exhibited maximum lipolytic activity at 40°C and stability for the pH range from 4 to 8. The enzyme produced by FmS presented maximum activity... (Complete abstract click electronic access below) / Doutor

La fermentation sombre est un procédé utilisant des déchets organiques dont le passage à l'échelle pilote est limité par les rendements de production d'hydrogène trop faibles ainsi que par l'utilisation des sous-produits. Cette étude a pour premier objectif d'étudier l'effet du pH, de la combinaison du pH et de la concentration en substrat, du prétraitement du substrat et de l'adaptation de l'inoculum sur la fermentation sombre de trois types de déchet différents. Il a notamment été montré que la biodégradabilité des substrats joue un rôle majeur dans le choix des paramètres opérationnels utilisés pour optimiser la production d'hydrogène. De plus, la faisabilité et la stabilité à long terme de la production d'hydrogène par le procédé de fermentation sombre ont été mises en évidence en utilisant des déchets agroalimentaires et du petit lait dans deux réacteurs thermophiliques fonctionnant en mode semi-continu. En particulier, il a été discuté l'influence de la charge organique (OLR), du temps de rétention hydraulique (HRT) et de l'addition de co-substrats (fumier de buffle) comme source d'alcalinité. Cette étude a montré que la combinaison de ces trois paramètres peut jouer un rôle important sur le pH et la stabilité de la production d'hydrogène. De plus, les sous-produits de la fermentation sombre ont été utilisés pour produire de l'hydrogène via la photo-fermentation, alors que les déchets générés par le couplage de la fermentation sombre et de la photo-fermentation ont été valorisés pour la production de méthane par digestion anaérobie. Ce concept de bioraffinerie basé sur la conversion en trois étapes des déchets agroalimentaires augmente le rendement énergétique global du procédé. Par ailleurs, il a été montré le potentiel important du procédé de photo-fermentation pour la production de polyhydroxybutyrate (polymère), parallèlement à celle d'hydrogène. De même, l'utilisation de la fermentation par voie sèche dans une bioraffinerie concept apparaît prometteuse vis à vis de la production de bioénergie et de molécules telles que les acides organiques et les alcools / Low biohydrogen (H2) yields and use of process by-products from dark fermentation (DF) of waste biomass is limiting its scaled-up application. This study aims to investigate the effects of culture pH, combination of substrate concentration and culture pH, pre-treatment of substrate and inoculum adaptation in H2 yields during the DF of three different wastes biomass. The study showed that the biodegradability of the substrates is important for the selection and application of optimum operational parameters aimed at enhancing H2 production. Moreover, long-term operational feasibility and stability of dark fermentative H2 production was demostrated using food waste and cheese whey in two semi-continuous thermophilic DF reactors. The effect of organic loading rates (OLRs), hydraulic retention times (HRTs) and co-substrates (buffalo manure) addition as a source of alkalinity on culture pH and H2 production stability was discussed. The study showed that combination of OLR, HRT and co-substrate addition could play an important role in the culture pH and stability of H2 production. Furthermore, the by-products of DF process was utilized for H2 production via photo fermentation (PF), while the waste stream generated from coupling of DF and PF processes was converted to methane in anaerobic digestion (AD). The three-step conversion of food waste in a biorefinery concept increased the total energy yields. Moreover, PF also showed a good potential for concomitant production of H2 and polyhydroxybutyrate (biopolymer). Likewise, dry fermentation could be promising to a biorefinery concept based on waste biomass for the production of bioenergy and biochemicals (organic acids and alcohols)

A viabilidade da produção em escala industrial de produtos biotecnológicos de interesse comercial e terapêutico, como os fármacos, depende significativamente das técnicas de separação e purificação utilizadas. A aplicação do sistema de duas fases aquosas (SDFA) é proposta como alternativa para a purificação, pois permite a separação e análise de biomoléculas, de modo que estas não percam sua atividade ou propriedades desejadas. Esta técnica é interessante para a purificação em larga escala, pois permite partição seletiva, com potencial de obtenção de altos rendimentos, além de apresentar boa relação custo-benefício. O presente trabalho estudou a purificação por extração líquido-líquido do ácido clavulânico em SDFA utilizando um novo sistema polimérico aquoso, formado pelos polímeros polietileno glicol (PEG) e ácido poliacrílico (APA). Foram estudadas diferentes composições do sistema polimérico aquoso PEG/APA, empregando diferentes massas molares e concentrações para o PEG e utilizando a massa molar 8000g/mol para o APA. Com base nas informações obtidas o melhor ponto de extração para o ácido clavulânico na presença de Na2SO4 foi definido como MPEG=400 g/mol, CPEG=17,5% (m/m) e CNaPA=22,5% (m/m) com K= 19,14, ηT=91,21%, BM=101,69 e R=0,45. Enquanto que na presença de NaCl, o melhor ponto encontrado foi: MPEG=400 g/mol, CPEG=35% (m/m) e CNaPA=10% (m/m) com K=11,96 ηT=80,04%, BM=90,18 e R=0,66. No trabalho será avaliada, também, a influência da temperatura, pH e força iônica nesse sistema. Estabeleceram-se os melhores parâmetros de separação do ácido clavulânico presente em meio fermentado produzido por Streptomyces clavuligerus utilizando a metodologia de fermentação extrativa com SDFA PEG/APA. O efeito do ácido clavulânico no diagrama de fases do sistema PEG-APA, bem como sua partição na forma pura e na presença de homogeneizado celular, foi estudado principalmente através da determinação do coeficiente de partição e recuperação do respectivo fármaco. / The viability of industrial scale production of commercial and therapeutical biotechnological products, such as drugs, is significantly dependent on the separation and purification techniques applied. The use of two-aqueous phase systems (ATPS) is proposed as an alternative to purification because it allows the separation and analysis of biomolecules, so that they do not lose their activities or desired properties. This technique is interesting for large scale purification because it allows selective partition with high potential yield and good cost/benefit ratio. The present work studied the purification of clavulanic acid (CA) by liquid-liquid extraction in ATPS applying a new aqueous polymeric system composed of two polymers, namely polyethylene-glicol (PEG) and sodium polyacrylate (NaPA). Different compositions of the aqueous polymeric system (PEG/PAA) were utilized, employing different PEG molar masses (MPEG) and concentrations (CPEG) and a molar mass of PAA of 8000 g/mol. In the light of the results obtained, the best conditions for clavulanic acid extraction, in the presence of Na2SO4, were MPEG = 400 g/mol, CPEG = 17.5% (m/m) and CNaPA = 22.5% (m/m), which allowed obtaining a partition coefficient (K) of 19.14, a yield in the top phase (ηT) of 91.21%, a mass balance (MB) of 101.69 and a volume ratio (R) of 0.45. On the other hand, in the presence of NaCl, the best results (K = 11.96, ηT = 80.04%, MB = 90.18 and R = 0.66) were found at: MPEG = 400 g/mol, CPEG = 35% m/m and CNaPA = 10% m/m. The effect of clavulanic acid in the PEG-PAA system phase diagram and its partition either in its pure form or in the cell homogenate were studied mainly through both the determination of the partition coefficient and the recovery of the drug selected for this study.

Liu, Haijing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 112-116). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of contents --- p.vi / List of figures --- p.xii / List of tables --- p.xv / List of abbreviations --- p.xvi / Chapter / Chapter 1 --- Introduction / Chapter 1.1 --- Fermentation --- p.1 / Chapter 1.1.1 --- Biopharmaceutical production --- p.1 / Chapter 1.1.2 --- History of fermentation --- p.1 / Chapter 1.1.3 --- Fermentation Technology --- p.2 / Chapter 1.1.3.1 --- Fermentation process --- p.2 / Chapter 1.1.3.2 --- Fermenter --- p.4 / Chapter "1,1.3.3" --- Mode of fermentation process --- p.5 / Chapter 1.1.3.4 --- Bacterial growth --- p.6 / Chapter 1.1.4 --- High cell density fermentation for plasmid DNA production --- p.7 / Chapter 1.1.4.1 --- Porcine growth hormone releasing factor (pGRF) --- p.8 / Chapter 1.1.4.2 --- H5N1 avian influenza viruses DNA vaccine --- p.9 / Chapter 1.1.4.3 --- Fed-batch high-cell-density fermentation --- p.9 / Chapter 1.1.5 --- Fermentation process monitoring --- p.11 / Chapter 1.2 --- Near-infrared spectroscopy (NIRS) --- p.12 / Chapter 1.2.1 --- Basic near-infrared spectroscopy --- p.13 / Chapter 1.2.1.1 --- Rationale of near-infrared spectroscopy --- p.13 / Chapter 1.2.1.2 --- NIR spectra acquisition --- p.16 / Chapter 1.2.1.3 --- Interpretation of NIR spectra --- p.18 / Chapter 1.2.2 --- Multivariate calibration --- p.20 / Chapter 1.2.2.1 --- Why multivariate calibration --- p.22 / Chapter 1.2.2.2 --- The problem of collinearity --- p.25 / Chapter 1.2.2.3 --- Spectral range selection --- p.26 / Chapter 1.2.2.4 --- Signal optimization --- p.26 / Chapter 1.2.2.5 --- Spectral pretreatment --- p.27 / Chapter 1.2.2.6 --- Parameter selection --- p.28 / Chapter 1.2.3 --- Applications of NIRS in high cell density E. coli fermentation --- p.31 / Chapter 1.2.4 --- Adaptive calibration strategy --- p.33 / Chapter 1.3 --- Aims of study --- p.34 / Chapter 2 --- Materials and methods / Chapter 2.1 --- High cell density fermentation of plasmid DNA --- p.35 / Chapter 2.1.1 --- Fermentation system --- p.35 / Chapter 2.1.2 --- pGRF fermentation --- p.38 / Chapter 2.1.2.1 --- Prepare seed flask --- p.38 / Chapter 2.1.2.2 --- Reagents for bacterial culture by fermenter --- p.38 / Chapter 2.1.2.2.1 --- LB based complex fermentation medium --- p.38 / Chapter 2.1.2.2.2 --- Batch fermentation medium --- p.39 / Chapter 2.1.2.2.3 --- Feeding medium-1 --- p.41 / Chapter 2.1.2.2.4 --- Base feed --- p.41 / Chapter 2.1.2.3 --- Fermentation methods --- p.42 / Chapter 2.1.2.3.1 --- Fermenter set-up --- p.42 / Chapter 2.1.2.3.2 --- Inoculate seed culture into the fermenter --- p.43 / Chapter 2.1.2.3.3 --- Transfer the feeding medium --- p.44 / Chapter 2.1.2.3.4 --- Heat induction --- p.44 / Chapter 2.1.2.3.5 --- Fermentation harvest --- p.44 / Chapter 2.1.2.4 --- Scale-up fermentation and large scale production 45 / Chapter 2.1.3 --- H5N1 fermentation --- p.45 / Chapter 2.1.4 --- Reference analytical testing --- p.45 / Chapter 2.1.4.1 --- Optical density --- p.45 / Chapter 2.1.4.2 --- Cell dry weight --- p.45 / Chapter 2.1.4.3 --- Specific plasmid DNA yield --- p.46 / Chapter 2.1.4.3.1 --- Plasmid DNA isolation --- p.46 / Chapter 2.1.4.3.2 --- Plasmid DNA measurement --- p.46 / Chapter 2.1.4.3.3 --- Plasmid DNA identification --- p.46 / Chapter 2.1.4.4 --- Analysis of glycerol and acetate --- p.46 / Chapter 2.1.4.4.1 --- Standard samples --- p.46 / Chapter 2.1.4.4.2 --- Enzymatic test kits --- p.47 / Chapter 2.1.4.4.3 --- Automatic biochemistry analyzer --- p.47 / Chapter 2.1.5 --- Summary of all fermentation batches --- p.48 / Chapter 2.2 --- Full factorial design and semi-synthetic samples --- p.49 / Chapter 2.2.1 --- Adaptive calibration samples --- p.49 / Chapter 2.2.1.1 --- Matrix 1 calibration samples --- p.49 / Chapter 2.2.1.2 --- Matrix 2 calibration samples --- p.50 / Chapter 2.2.1.3 --- Matrix 3 calibration samples --- p.50 / Chapter 2.2.2 --- Summary of all samples --- p.51 / Chapter 2.3 --- NIR sample presentation and spectral acquisition --- p.52 / Chapter 2.3.1 --- NIR spectrophotometers --- p.52 / Chapter 2.3.2 --- Cuvettes for transmittance spectral acquisition --- p.53 / Chapter 2.3.3 --- Bottles for reflectance spectral acquisition --- p.54 / Chapter 2.3.4 --- Spectral acquisition --- p.55 / Chapter 2.3.4.1 --- Transmittance --- p.55 / Chapter 2.3.4.2 --- Reflectance --- p.55 / Chapter 2.4 --- Multivariate calibration and validation --- p.56 / Chapter 2.4.1 --- Spectral preprocessing --- p.56 / Chapter 2.4.2 --- Multivariate calibration --- p.57 / Chapter 2.4.3 --- Model validation --- p.57 / Chapter 3 --- Results and discussion / Chapter 3.1 --- Sample presentation and NIR spectrum --- p.59 / Chapter 3.1.1 --- Transmission measurement --- p.60 / Chapter 3.1.2 --- Reflectance measurement --- p.63 / Chapter 3.1.3 --- Spectral responses and pre-treatment method --- p.64 / Chapter 3.1.4 --- Design of experiments for calibration sample preparation --- p.67 / Chapter 3.1.5 --- Summary --- p.68 / Chapter 3.2 --- Adaptive calibration --- p.69 / Chapter 3.2.1 --- Selection of Multivariate calibration model --- p.70 / Chapter 3.2.1.1 --- Matrix 1 calibration models --- p.70 / Chapter 3.2.1.2 --- Matrix 2 calibration models --- p.74 / Chapter 3.2.1.3 --- Matrix 3 calibration models --- p.76 / Chapter 3.2.1.4 --- Summary --- p.78 / Chapter 3.2.2 --- Model validation --- p.79 / Chapter 3.2.2.1 --- Performance of Reference analytical methods --- p.79 / Chapter 3.2.2.1.1 --- Enzymatic test kits --- p.79 / Chapter 3.2.2.1.2 --- Automatic biochemistry anaylyzer (Bioprofile) --- p.81 / Chapter 3.2.2.1.3 --- Summary --- p.83 / Chapter 3.2.2.2 --- Model validation using external test samples --- p.83 / Chapter 3.2.2.2.1 --- Matrix 1 models --- p.83 / Chapter 3.2.2.2.2 --- Matrix 2 models --- p.87 / Chapter 3.2.2.2.3 --- Matrix 3 models --- p.89 / Chapter 3.2.2.2.4 --- Overall NIR measurement errors --- p.90 / Chapter 3.2.2.2.5 --- Summary --- p.91 / Chapter 3.3 --- Use of calibrated NIRS in at-line monitoring and control of fermentation --- p.93 / Chapter 3.3.1 --- Analysis of small-scale fermentation --- p.94 / Chapter 3.3.1.1 --- pGRF plasmid DNA production --- p.94 / Chapter 3.3.1.2 --- H5N1 plasmid DNA production --- p.95 / Chapter 3.3.1.3 --- Summary --- p.97 / Chapter 3.3.2 --- Analysis of large scale fermentation --- p.97 / Chapter 3.3.2.1 --- 30 L clinical production of H5N1 plasmid DNA --- p.97 / Chapter 3.3.2.2 --- 80 L scale-up production of H5N1 plasmid DNA --- p.99 / Chapter 3.3.2.3 --- Summary --- p.100 / Chapter 3.3.3 --- Effective control of fermentation production using at-line NIR analysis --- p.101 / Chapter 3.3.3.1 --- At-line monitoring of Batch 11 --- p.101 / Chapter 3.3.3.2 --- At-line monitoring of Batch 12 X --- p.102 / Chapter 3.3.3.3 --- Summary --- p.104 / Chapter 3.4 --- General discussion and conclusion --- p.105 / Chapter 3.5 --- Future prospects --- p.108 / References --- p.112 / Appendix 1 --- p.117 / Appendix 2 --- p.118 / Appendix 3 --- p.123 / Appendix 4 --- p.131 / Appendix 5 --- p.134