Estudou-se o processo descontínuo alimentado de fermentação alcoólica, utilizando-se mosto de melaço de cana-de-açúcar e Saccharomyces cerevisiae na forma de fermento prensado. Foram analisados a influência da vazão de alimentação exponencialmente decrescente e do tempo de enchimento do fermentador no comportamento do sistema, considerando os seguintes parâmetros: 1. produtividade em etanol e em células, 2. rendimento em etanol e 3. razão de crescimento celular. / The fed-batch ethanol fermentation of sugar-cane blackstrap molasses by the action of Saccharomyces cerevisiae (pressed yeast) was studied. The influence of exponencialy decreasing feeding rates and of the fermentor filling up time on the system behavior was analysed considering the following parameters: 1. ethanol and cell productivities, 2. ethanol yield and 3. cell growth ratio.

O presente projeto avaliou um reator anaeróbio operado em batelada e batelada alimentada seqüenciais (ASBR), em ciclos de 8 horas, utilizando biomassa granulada e agitação mecânica em um draft-tube, alimentado com água residuária sintética (500 mgDQO/L), contendo sulfato em diferentes relações DQO/[\'SO IND.4\' POT.2-\']. Em todos os ensaios o reator apresentou uma operação estável, produzindo alcalinidade e com concentração de ácidos voláteis totais em níveis adequados. Para os tempos de alimentação de 10 min, 3 h e 6 h, respectivamente, as eficiências de remoção de sulfato foram de 30, 72 e 72% nas operações nas quais o reator foi alimentado com uma relação DQO/[\'SO IND.4\'POT.2-\'] de 1,34. Nos ensaios nos quais o reator foi alimentado na relação DQO/[\'SO IND.4\'POT. 2-\'] de 0,67, as eficiências para a redução de sulfato foram de 25, 58 e 55%, respectivamente. Na operação com relação DQO/[\'SO IND.4\'POT.2-\'] de 0,34, as eficiências para redução de sulfato foram de 23, 37 e 27%, respectivamente. Desta maneira, pode-se concluir que as operações em batelada alimentada favoreceram a remoção de sulfato, enquanto foi observado que nas operações em batelada a remoção de matéria orgânica atingiu melhores eficiências. / This present work evaluate an anaerobic sequencing batch reactor (ASBR), fed in batch and fed-batch, and cycles of 8 hours, using granulated biomass and mechanical stirring in a draft-tube, fed with synthetic wastewater (500 mgCOD/L), enriched with sulfate in some COD/[\'SO IND.4\'POT.2-\'] relations. In all operations the reactor showed a stable operation, producing alkalinity and maintaining the volatile acids in adequate levels. Considering the fed periods of 10 min, 3 h and 6 h, respectively, the removal efficiencies of the sulfate was 30, 72 e 72%, in the operations when the reactor was fed with a COD/[\'SO IND.4\'POT.2-\'] relation of 1,34. In the essays when the reactor was fed in COD/[\'SO IND.4\'POT.2-\'] relation of 0,67, the efficiencies of the sulfate reduction was 25, 58 e 55%, respectively. When the reactor was operated with COD/[\'SO IND.4\'POT.2-\'] relation of 0,34, the efficiencies of sulfate reduction 23, 37 e 27%, respectively. Thus, is possible to conclude that the operations in fed-batch increased the efficiency of sulfate removal, at what time was observed that in batch operations the organic matter removal attained improved efficiencies.

Abstract Small-scale cultivation methods are a necessity for the development of new biotechnological processes. The most common method for submerged microbial cultivation is a shake flask used with a batch operation protocol. Well plate cultivation formats have also increased their importance, due to the need to utilize high-throughput cultivations for efficient product development. However, batch cultivation is often not the optimal method for obtaining high cell densities and good product quality, due to unlimited microbial growth. The aim of this dissertation was to improve small-scale microbial cultivations for microbial growth and product formation. Hydrolytic enzymes were utilized to relieve nutrient limitation by hydrolysis of proteins in lactic acid bacteria cultures to improve lactic acid production from dairy side products. Hydrolytic enzymes were also utilized in the enzymatic release of glucose from starch to create a fed-batch-like cultivation system applicable on small scale. The wireless sensor system developed was applied in shake flask cultivations to monitor oxygen and pH levels. Enzymatic polymer processing was applicable for small-scale cultivations. Lactic acid production by Lactobacillus salivarius ssp. salicinius was enhanced four-fold when the proteins were hydrolyzed either by proteases or by proteolytic microbes. The fed-batch-mimicking controlled glucose feeding and growth control were obtained by means of the simultaneous enzymatic hydrolysis of starch-polymer during cultivation. Controlled growth, higher cell densities, decreased side product formation and increased amount of soluble protein product were obtained in Escherichia coli cultivations. When this method was applied to the cultivation and recombinant protein production of the methylotrophic yeast Pichia pastoris, higher cell densities and higher amounts of active protein were obtained. The glucose concentration remained low enough to avoid the substrate repression of the alcohol oxidase promoter. The fed-batch method is suitable for high-throughput cultivations since the method can be utilized in well plate formats without external feeding devices. The method can be utilized in the development of new biotechnological products, especially when the production system is sensitive to growth conditions, and growth control is preferred. / Tiivistelmä Pienen mittakaavan mikrobikasvatusmenetelmiä tarvitaan kehitettäessä uusia bioteknologisia prosesseja. Tavallisin menetelmä mikrobien liuoksessa tapahtuvaan kasvatukseen on panostyyppisesti tehtävä sekoituspullokasvatus. Kuoppalevykasvatukset ovat myös tulleet entistä tärkeämmiksi, koska tuotekehityksen tehostamiseksi on tarvetta käyttää high-throughput-menetelmiä. Tavoiteltaessa korkeita mikrobisolutiheyksiä ja tuotteen hyvää laatua, panostyyppinen kasvatus ei ole usein paras vaihtoehto, johtuen mikrobien rajoittamattomasta kasvusta. Tämän työn tarkoituksena oli parantaa mikrobien kasvua ja tuotteen muodostusta pienen mittakaavan kasvatuksissa. Meijeriteollisuuden sivutuotteiden proteiineja pilkottiin entsyymien avulla, jotta maitohappobakteerit pystyivät hyödyntämään proteiinit tehokkaammin ja tuottamaan enemmän maitohappoa. Hydrolyyttisiä entsyymejä hyödynnettiin myös glukoosin vapauttamiseen tärkkelyksestä, jolloin saatiin luotua pieneen mittakaavaan sopiva panossyöttötyyppinen kasvatusmenetelmä. Työn aikana kehitettyä langatonta mittausjärjestelmää hyödynnettiin sekoituspullokasvatuksissa happipitoisuuden ja pH:n seurantaan. Entsymaattinen polymeerien käsittely oli soveltuva menetelmä pienen mittakaavan kasvatuksiin. Maitohapon tuotto Lactobacillus salivarius ssp. salicinius -mikrobilla nelinkertaistui, kun ravinneproteiinit pilkottiin joko proteaasien tai proteolyyttisten mikrobien avulla. Panossyöttömenetelmää muistuttava hallittu glukoosin syöttö ja mikrobin kasvun hallinta saavutettiin pilkkomalla tärkkelystä glukoosiksi kasvatuksen aikana. Escherichia coli kasvatuksissa saavutettiin hallittu solumäärän kasvu, korkeammat solutiheydet, vähentynyt sivutuotteiden muodostus ja suurempi liukoisen tuoteproteiinin määrä. Tätä menetelmää sovellettiin myös vierasproteiinin tuottoon metylotrofisella Pichia pastoris -hiivalla, jolloin saavutettiin korkeammat solutiheydet ja suurempi aktiivisen tuoteproteiinin määrä. Glukoosin määrä kasvatusliuoksessa pysyi riittävän alhaisena, jotta se ei repressoinut hiivan alkoholioksidaasi-promoottoria. Panossyöttömenetelmä on sopiva high-throughput-mikrobikasvatuksiin, koska sitä voidaan käyttää kuoppalevyillä ilman syöttölaitteita. Menetelmää voidaan hyödyntää uusien bioteknisten tuotteiden kehittämisessä erityisesti silloin, kun tuottoisäntä on herkkä kasvuolosuhteiden suhteen ja mikrobin kasvua halutaan hallita.

The word cybernetics has its roots in the Greek word \kybernetes" or \steers-man" and was coined by Norbert Wiener in 1948 to describe \the science of control and communication, in the animal and the machine". The discipline focuses on the way various complex systems (animals/machines) steer towards/maintain their goals utilizing information, models and control actions in the face of various disturbances. For a given animal/machine, cybernetics considers all the possible behaviors that the animal/machine can exhibit and then enquires about the constraints that result in a particular behavior. The thesis focuses on the application of principles of cybernetics to the modeling and optimization of bioreactors and lies at the interface of systems engineering and biology. Specifically, it lies at the interface of control theory and the growth behavior exhibited by microorganisms. The hypothesis of the present work is that the principles and tools of control theory can give novel insights into the growth behavior of microorganisms and that the growth behavior exhibited by microorganisms can in turn provide insights for the development of principles and tools of control theory. Mathematical models for the growth of microorganisms such as stoichiometric, optimal and cybernetic assume that microorganisms have evolved to become optimal with respect to certain cellular goals or objectives. Typical cellular goals used in the literature are the maximization of instantaneous/short term objectives such biomass yield, instantaneous growth rate, instantaneous ATP production rate etc. Since microorganisms live in a dynamic world, it is expected that the microorganisms have evolved towards maximizing long term goals. In the literature, it is often assumed that the maximization of a short term cellular goal results in the maximization of the long term cellular goal. However, in the systems engineering literature, it has long been recognized that the maximization of a short term goal does not necessarily result in the maximization of the long term goal. For example, maximization of product production in a fed-batch bioreactor involves two separate phases: a first phase in which the growth of microorganisms is maximized and a second phase in which the production of product is maximized. An analogous situation arises when the bacterium E. coli passes through the digestive tract of mammals wherein it first encounters the sugar lactose in the proximal portions and the sugar maltose in the distal portions. Mitchell et al. (2009) have experimentally shown that when E. coli encounters the sugar lactose, it expresses the genes of maltose operons anticipatorily which reduces its growth rate on lactose. This regulatory strategy of E. coli has been termed asymmetric anticipatory regulation (AAR) and is shown to be beneficial for long term cellular fitness by Mitchell et al. (2009). The cybernetic modeling framework for the growth of microorganisms, developed by Ramakrishna and co-workers, is extended in the present thesis for modeling the AAR strategy of E. coli. The developed model accurately captures the experimental observations of the AAR phenomenon, reveals the inherent advantages of the cybernetic modeling framework over other frameworks in explaining the AAR phenomenon, while at the same time suggesting a scope for the generalization of the cybernetic framework. As cybernetics is interested in all the possible behaviors that a machine (which is, in the present case, microorganism) can exhibit, a rigorous analysis of the optimal dynamic growth behavior of microorganisms under various constraints is carried out next using the methods of optimal control theory. An optimal control problem is formulated using a generalized version of the unstructured Monod model with the objective of maximization of cellular concentration at a fixed final time. Optimal control analysis of the above problem reveals that the long term objective of maximization of cellular concentration at a final time is equivalent to maximization of instantaneous growth rate for the growth of microorganisms under various constraints in a two substrate batch environment. In addition, reformulation of the above optimal control problem together with its necessary conditions of optimality reveals the existence of generalized governing dynamic equations of the structured cybernetic modeling framework. The dynamic behavior of the generalized equations of the cybernetic modeling framework is analyzed further to gain insights into the growth of microorganisms. For growth of microorganisms on a single growth limiting carbon substrate, the analysis reveals that the cybernetic model exhibits linear growth behavior, similar to that of the unstructured Contois model at high cellular concentrations, under appropriate constraints. During the growth of microorganisms on multiple substitutable substrates, the analysis reveals the existence of simple correlations that quantitatively predict the mixed substrate maximum specific growth rate from single substrate maximum specific growth rates during simultaneous consumption of the substrates in several cases. Further analysis of the cybernetic model of the growth of S. cerevisiae on the mixture of glucose and galactose reveals that S. cerevisiae exhibits sub-optimal dynamic growth with a long diauxic lag phase and suggests the possibility for S. cerevisiae to grow optimally with a significantly reduced diauxic lag period. Since cybernetics is interested in understanding the constraints under which a particular machine (microorganism) exhibits a particular behavior, a methodology is then developed for inferring the internal constraints experienced by the microorganisms from experimental data. The methodology is used for inferring the internal constraints experienced by E. coli during its growth on the mixture of glycerol and lactose. An interesting question in the study of the growth behavior of microorganisms concerns the objective that the microorganisms optimize. Several studies aim to determine these cellular objectives experimentally. A similar question that is relevant to the optimization of fed-batch bioreactors is \what are the objectives that are to be optimized by the feed flow rate in various time intervals for the optimization of a final objective?" It was mentioned previously that the maximization of product production in a fed-batch bioreactor involves maximization of growth of microorganisms first and the maximization of product production later. However, such guidelines can only be stated for relatively simple bioreactor optimization problems and no such guidelines exist for sufficiently complex problems. For complex problems, the answer to the above question requires the formulation and solution of a genetic programming problem which can be quite challenging. An alternative numerical solution methodology is developed in the present thesis to address the above question. The solution methodology involves the specification of bioreactor objectives in terms of the bioreactor trajectory in the state space of substrate concentration-volume. The equivalent control law of the sliding mode control technique is used for finding the inlet feed ow rate that tracks the bioreactor trajectory accurately. The search for the best bioreactor trajectory is carried out using the stochastic search technique genetic algorithm. The effectiveness of the developed solution methodology in determining the optimal bioreactor trajectory is demonstrated using three challenging bioreactor optimization problems.

The recombinant product is secreted to the periplasm in many processes where E. coli is used as host. One drawback with secretion is the undesired leakage of the periplasmic products to the medium. The aim of this work was to find strategies to influence the periplasmic retention of recombinant products. We have focused on the role of the specific growth rate, a parameter that is usually controlled in industrial bioprocesses. The hypothesis was that the stability of the outer membrane in E. coli is gained from a certain combination of specific phospholipids and fatty acids on one side and the amount and specificity of the outer membrane proteins on the other side, and that the specific growth rate influences this structure and therefore can be used to control the periplasmic retention. We found that is possible to control the periplasmic retention by the growth rate. The leakage of the product increased as the growth rate increased. It was however also found that a higher growth rate resulted in increased productivity. This resulted in equal amounts of product inside the cells regardless of growth rate. We also showed that the growth rate influenced the outer membrane composition with respect to OmpF and LamB while OmpA was largely unaffected. The total amount of outer membrane proteins decreased as the growth rate increased. There were further reductions in outer membrane protein accumulation when the recombinant product was secreted to the periplasm. The lowered amount of outer membrane proteins may have contributed to the reduced ability for the cell to retain the product in the periplasm. The traditional way to control the growth rate is through a feed of substrate in a fed-batch process. In this work we used strains with a set of mutations in the phosphotransferase system (PTS) with a reduced uptake rate of glucose to investigate if these strains could be used for growth rate control in batch cultivations without the use of fed-batch control equipment. The hypothesis was that the lowering of the growth rate on cell level would result in the establishment of fed-batch similar conditions. This study showed that it is possible to control the growth rate in batch cultivations by using mutant strains with a decreased level of substrate uptake rate. The mutants also produced equivalent amounts of acetic acid as the wild type did in fed-batch cultivation with the same growth rate. The oxygen consumption rates were also comparable. A higher cell density was reached with one of the mutants than with the wild type in batch cultivations. It is possible to control the growth rate by the use of the mutants in small-scale batch cultivations without fed-batch control equipment. / QC 20101108

La biomasse lignocellulosique comprend les sous-produits agricoles et industriels pouvant être utilisés comme matière première dans des bioprocédés variés destinés à produire des molécules d'intérêt énergétique ou chimique. Ces ressources lignocellulosiques, peuvent notamment être fournies par l'industrie papetière qui est particulièrement adaptée pour les bio-raffineries modernes car elle est en capacité de produire en grande quantité un substrat ayant une faible teneur en lignine et sans composés inhibiteurs. La bagasse de canne à sucre est également un substrat prometteur par sa composition chimique simple et son abondance dans les pays tropicaux. Lors de l'utilisation de ces substrats, l'hydrolyse enzymatique constitue une étape cruciale permettant la transformation des fibres de cellulose en une source de carbone fermentescible. Si les aspects biochimiques de cette étape d'hydrolyse font l'objet de nombreuses recherches et de développements, les réactions sous haute teneur en matière sèche font apparaître des limitations physiques qui sont beaucoup moins étudiées et analysées mais constituent des verrous scientifiques et technologiques qui freinent actuellement l'utilisation de cette ressource abondante et durable. Ce travail s'inscrit dans ce contexte et propose l'étude de cette étape d'hydrolyse enzymatique de la lignocellulose en s'intéressant conjointement aux aspects biochimiques et physiques de façon à aller vers une compréhension et une maîtrise des transferts (de masse, de chaleur) dans les réactions à forte concentration en substrat. La stratégie adoptée a consisté à réaliser et analyser des réactions d'hydrolyse sous différentes conditions opératoires en travaillant dans un premier temps sur des concentrations intermédiaires (suspension semi-diluée), c'est-à-dire en introduisant, mais de façon limitée, les complexités dues aux interactions entre particules/fibres de lignocellulose. Les résultats obtenus sont ensuite utilisés pour élaborer une stratégie adaptée aux fortes concentrations. Les aspects physiques analysés sont essentiellement le comportement rhéologique du milieu réactionnel ainsi que la morpho-granulométrie des objets en suspension. Différentes métrologies, tant in-situ que ex-situ, ont été mises en œuvre et apportent des résultats complémentaires. Les études ont été menées sur un substrat de référence, le papier Whatman, et deux substrats à vocation industrielle: la pâte à papier et la bagasse de canne à sucre. La stratégie d'étude porte sur les aspects suivants: (i) le suivi de l'évolution des comportements rhéologiques et des propriétés morphologiques des suspensions au cours de l'hydrolyse, (ii) l'étude des mécanismes d'hydrolyse lors de la dégradation des substrats, (iii) l'étude de l'impact de la composition et de la structure des substrats sur les cinétiques de solubilisation et d'hydrolyse, (iv) la quantification de la contribution des différentes activités enzymatiques, seules ou en mélange par une approche physique multi-échelle et (v) le contrôle et l'optimisation des conditions d'alimentation dans un procédé discontinu alimenté (fed-batch) afin d'atteindre des conditions de milieu concentré. Les chapitres 1 et 2 de ce document sont consacrés à une étude bibliographique du sujet et à la présentation des matériels et méthodes mis en œuvre. Le troisième chapitre présente les résultats obtenus et leur analyse. Il est constitué de trois sections: tout d'abord une étude des propriétés des différents enzymes ou cocktail d'enzymes utilisés, des substrats retenus et des suspensions avec, notamment, la détermination des régimes semi-dilués et concentrés. Ensuite sont présentées et analysées les hydrolyses effectuées en milieu semi-dilué. Les mécanismes d'hydrolyse (fragmentation, solubilisation, hydratation et séparation des agglomérats) sont étudiés pour diverses concentrations et divers enzymes/cocktails. Enfin les résultats en milieu concentré sont présentés dans une dernière section. / Lignocellulosic biomass consists of several agriculture and industrial by-products that can be used as raw material for several bioprocesses to obtain range of products. Among lignocellulosic sources, the pulp & paper industry is appropriated for modern bio-refining thank to pulp with low lignin content and free of inhibitory compounds. Besides, sugarcane bagasse is a very promising feedstock because of its simple chemical composition and its abundancy especially in tropical countries. In the bioconversion of lignocellulose, enzymatic hydrolysis is a crucial step that allows the transformation of cellulosic and hemicellulosic fibers into fermentable carbon sources. The lack of knowledge about physical limitations and hydrolysis mechanisms, especially at high dry matter content, stands as the main barrier which forbids the scale-up of bio-refinery processes. Thus, the efficient and sustainable use of lignocellulosic resources is currently a major challenge and need to be investigated. In this context, this PhD focused on the enzymatic hydrolysis of lignocellulose by both physical and biochemical approaches. The strategy consisted in carrying out and in analyzing the hydrolysis reactions under different operating conditions with semi-dilute suspensions. Then, obtained results were used to develop a hydrolysis strategy for concentrated suspensions. Different methodologies, in- and ex-situ analyses, were implemented and provided complementary results. From physical approach, analyses consisted in rheological behavior of suspensions as well as the morpho-granulometry of particles. The study was carried out on a reference substrate, Whatman paper, and on two industrial substrates, paper pulp and sugarcane bagasse. The strategy aimed to investigate different stakes: (i) evolution of rheological behaviors and the morphological properties of suspensions, (ii) hydrolysis mechanisms during the degradation of substrates, (iii) impact of substrate composition and structure on solubilization and hydrolysis kinetics, (iv) quantification of the contribution of single enzyme and enzyme mixture activities by multi-scale physical approaches and (v) control and optimization of feeding parameters for fed-batch process in order to access to concentrated suspension. Chapters 1 and 2 of this document are devoted to a research bibliographic and presentation of materials and methods. The third chapter presents obtained results and discussion in three sections. The first one is a study of the properties of different enzymes and substrates, in particular, the determination of semi-dilute and concentrated regime. Subsequently the enzymatic hydrolysis at semi-dilute regime is presented to highlight the hydrolysis mechanisms (fragmentation, solubilization, solvation and agglomerate separation) in relationship with enzyme mixtures and dosages. Finally, results in concentrated regime are discussed in the final section.

L'acide succinique est une molécule linéaire, bi-fonctionnelle qui possède de nombreuses applications alimentaires, chimiques et pharmaceutiques etc. Les connaissances de la régulation des voies métaboliques d’organismes d’intérêt industriel, le génie génétique et le génie des procédés ont permis à des microorganismes recombinants (C. glutamicum) de produire jusqu'à 100 g.L-1 de succinate avec des rendements intéressants. C. glutamicum est largement connu comme l'un des meilleurs producteurs industriels de nombreux acides aminés (glutamate, lysine etc.). Cependant, des études de C. glutamicum ont démontré sa capacité à produire plusieurs acides organiques (succinate, lactate, acétate, etc.). Au cours de ce travail, nous avons supprimé le gène ldhA de C. glutamicum en utilisant le plasmide pk19mobsacBΔldhA. Nous avons démontré que la délétion de ce gène n’avait pas d’incidence sur la capacité de croissance de la bactérie. Par ailleurs, nous avons étudié les effets de l’oxygénation sur la réponse physiologique de C. glutamicum 2262ΔldhA à travers des expériences de cultures en fioles en verre lisses en imposant différentes conditions de kLa. Les résultats ont montré que des faibles kLa (<33 h-1) favorisaient la production d’acides organiques tandis que les kLa élevés amélioraient surtout l’accumulation de la biomasse. Nous avons également mis en œuvre un procédé de production très efficace avec une phase de transition aérobiose-anaérobiose basée sur la régulation de la concentration en oxygène dissous. Avec ce procédé, 327 mM de succinate avec un rendement de 0,94 mole par mole de glucose ont pu être produits avec le mutant ΔldhA. En outre, nous avons vérifié l’efficacité de ce nouveau procédé en l’appliquant à la souche sauvage qui normalement produit 10 fois plus de lactate que de succinate. Ces résultats ont montré une production de 793 mM (94 g.L-1) de succinate et 785 mM (71 g.L-1) de lactate. Ils soulignent ainsi, l'importance de la phase de transition aérobiose-anaérobiose lors des procédés de production de succinate par des bactéries aérobie facultatif. Enfin, des expériences en système bi-étagé ont montré que C. glutamicum 2262 pouvait s’adapter très facilement aux gradients et hétérogénéités en oxygène dissous dans les cultures à grande échelle / Succinic acid is a linear and bi-functional molecule that has several practical applications including food, chemical and pharmaceutical industries. Thanks to increased knowledge on metabolism and pathway regulation of industrially relevant organisms, to the development of performant genetic tools and process engineering, recombinants strains (Escherichia coli, Corynebacterium glutamicum etc.) have been reported to be able to produce up to 100 g.L-1 with interesting yields (> 1.5 mole per mole glucose). C. glutamicum is well known as one of the best industrial producers of numerous amino acids (glutamate, lysine etc.). However, recent studies of C. glutamicum revealed its capability to produce several organic acids (succinate, lactate, acetate, etc.). In this work, we have deleted the ldhA gene of C. glutamicum by using a plasmid vector pk19mobsacBΔldhA. We demonstrated that the mutant and the wild type presented similar growth kinetics with maximal growth rate of about 0.7 h-1. We studied also the effects of oxygenation on C. glutamicum 2262 ΔldhA through cultures at different kLa and it appeared that lower kLa (<33 h-1) favored organic acids production wile higher favored bacterial growth. Furthermore, we designed a tri-phasic process with transition phase by regulation of dissolved oxygen concentration which resulted in the production of 327 mM of succinic acid with a yield of 0.94 mole per mole glucose. The application of the designed process to C. glutamicum 2262 wild type that normally produces lactate with a lactate to succinate production ratio up to 13.3 mol.mol-1, resulted in succinate concentration up to 793 mM (94 g.L-1) and 785 mM (71 g.L-1) of lactate. The succinate production yield was 1.1 mole per mole glucose and acetate production was negligible. These results underlined the importance of aerobic to anaerobic transition in succinate production processes of facultative aerobes and the necessity to engineer not only the microorganism but also the process. Finally, scale-down study have demonstrated the robustness of C. glutamicum against the oxygen gradients in bioreactor

A produção da cianobactéria Arthrospira (Spirulina) platensis é considerada de interesse nas indústrias de alimentos, farmacêuticas e químicas. Diferentes fontes de nitrogênio alternativas têm sido sugeridas na literatura para seu cultivo, incluindo uréia e sais de amônio, visando à diminuição no custo do meio de cultivo. A perda de amônia por degasificação ocorre quando utilizados tanques abertos, justificando dessa forma o uso da associação de fontes de nitrato e de amônia em fotobiorreatores fechados. O nitrato de amônio (NH4NO3) contorna estas condições, proporcionando ao cultivo uma fonte de nitrogênio prontamente assimilável (amônio) e outra de reserva (nitrato), no mesmo composto. Neste trabalho verificou-se, pelo uso de um planejamento fatorial 22 com configuração de estrela, a influência da intensidade luminosa (I) e da adição de diferentes concentrações de NH4NO3 (mM), no crescimento e composição da biomassa de A. platensis em cultivos realizados em fotobiorreator tubular por processo descontínuo alimentado, utilizando um tempo de 6 dias alimentação para a fonte de nitrogênio. Parâmetros cinéticos de crescimento, como concentração celular máxima (Xm), produtividade em células (PX) e o fator de conversão de nitrogênio em células (YX/N), bem como o teor de proteínas e lipídios na biomassa foram avaliados. Através do uso da regressão multivariável para a otimização das condições experimentais, foram obtidos valores de Xm e de PX correspondentes a 4710 mg L-1 e 478,9 mg L-1d-1, respectivamente, pelo emprego de I = 148 &#181;mol fótons m-2 s-1 e a adição de 9,7 mM de NH4NO3. O maior valor de YX/N registrado foi 8,1 mg mg-1, sendo superior aos obtidos com nitrato de sódio (0,80 mg mg-1). Os teores de proteína e de lipídios alcançaram valores de até 63,2 e 17,3 %, respectivamente. O emprego de NH4NO3 conduziu à redução dos custos de produção, sendo gastos em média R$ 15,97 da fonte de nitrogênio por quilograma de células, valor inferior se comparado com outras fontes usadas no cultivo de A. platensis como NaNO3 (R$ 59,10 por quilograma de células). O uso do NH4NO3 em fotobiorreator tubular mostrou-se promissor para o cultivo de A. platensis, ao proporcionar um aumento na produção de biomassa e diminuir a perda de amônia por degasificação. / The production of the cyanobacterium Arthrospira (Spirulina) platensis is considered of interest to the food, pharmaceutical and chemical industries. Alternative nitrogen sources have been suggested in literature, including urea and ammonium salts, in order to reduce the cost of cultivation medium. Ammonia off-gassing occurs when open ponds are used, thus justifying the utilization of nitrate and ammonia sources association. Ammonium nitrate (NH4NO3) attend this condition, providing the cultivation with a nitrogen source readily assimilated (ammonium) and with one of reserve (nitrate), in the same molecule. In this work, it was verified, using a 22 factorial design with star configuration, the influence of light intensity (I) and the addition of different concentrations of NH4NO3 (mM) on the growth and biomass composition of S. platensis in cultures carried out in tubular photobioreactor by fed-batch process, using a 6-day feeding time of the nitrogen source. Kinetic growth parameters, such as maximum cell concentration (Xm), cell productivity (Px) and yield of nitrogen to biomass (YX/N), as well the biomass content of protein and lipids, were evaluated. Through the use of regression analysis for the optimization of experimental conditions, values of Xm and PX such as 4710 mg L-1 and 478.9 mg L-1d-1, respectively, were obtained by the use of I = 148 &#181;mol photons m-2 s-1 and the addition of 9.7 mM of NH4NO3. The highest value of YX/N obtained was 8.1 mg mg-1, higher than that provided by the use of sodium nitrate (0.80 mg mg-1). The levels of protein and lipid achieved up to 63.2 and 17.3 %, respectively. The use of NH4NO3 led to the reduction of culture medium costs, in which R$ 15.97 (Real/Brazil) were spent on nitrogen source per kilogram of cells, value that is lower if compared to other common sources used for A. platensis growth, such as NaNO3 (R$ 59.10 per kilogram of cells, Real/Brazil). The use of NH4NO3 in tubular photobioreactor proved to be promising for the cultivation of A. platensis, leading to an increase in biomass production and diminishing the ammonia lost by off-gassing.

A Arthrospira (Spirulina) platensis, cianobactéria fotoautotrófica, tem sido cultivada para a produção de biomassa, apresentando em sua constituição proteínas, ácidos graxos poliinsaturados, pigmentos, minerais, vitaminas e aminoácidos. O uso de fontes de nitrogênio de baixo custo, como o sulfato de amônio, pode contribuir para a viabilização da produção de A. platensis. Neste trabalho foram verificados a influência de diferentes intensidades luminosas e protocolos de alimentação utilizando o sulfato de amônio, em cultivos realizados com reatores tubulares por processo descontínuo alimentado. As variáveis dependentes selecionadas foram concentração celular máxima (Xm), produtividade em células (Px), fator de conversão de nitrogênio em células (YX/N), bem como teores de proteínas e lipídios da biomassa. Foram obtidos excelentes resultados em cultivos realizados aplicando-se um protocolo de alimentação de sulfato de amônio, considerando biomassa seca de A. platensis com teor de 7 % de nitrogênio, com valor médio de Xm igual a 11414 mg.L-1. Este protocolo de adição de sulfato de amônio com intensidade luminosa de 240 µmol fótons. m-2.s-1 levou à melhor combinação de obtenção de Xm e Px, com valores finais de 12200 mg.L-1 e 1686 mg.L-1.d-1, respectivamente. Adicionalmente, nesse protocolo de adição de sulfato de amônio, os valores de concentração celular máxima foram da mesma ordem de grandeza que aqueles onde se utilizou nitrato de sódio, evidenciando, assim, a possibilidade do uso do sulfato de amônio como uma fonte de nitrogênio alternativa para o cultivo da A. platensis. / Arthrospira (Spirulina) platensis, a photoautotrophic cyanobacterium, has been cultivated for the production of biomass, and it is constituted by proteins, polyunsaturated fatty acids, chlorophyll, minerals, vitamins and amino acids. The use of low cost nitrogen sources, such as ammonium sulphate can contribute to the A. platensis production viability. It was verified in this work the influence of different light intensities and nutrient feeding protocols in fed-batch cultures developed in tubular reactors, where ammonium sulphate was used as a nitrogen source. The dependent variables were the maximum cell concentration (Xm), cell productivity (Px), nitrogen-to-cell conversion factor (YX/N), as well as proteins and lipids contents of the biomass. Excellent results were obtained when ammonium sulphate protocols were studied, considering A. platensis dry-weight with nitrogen levels of 7%, obtaining an average value of Xm corresponding to 11414 mg L-1. It was also obtained with this protocol, with light intensity of 240 µmol photons. m-2.s-1, the best combination to high values of Xm and Px, corresponding to 12200 mg.L-1 e 1686 mg.L-1.d-1, respectively. Moreover, in this protocol of ammonium sulphate addition, it was obtained values of the maximum cell concentration as high as the ones obtained with sodium nitrate as nitrogen source. This fact highlighted the possibility of using this alternative nitrogen source for the A. platensis cultivation.

Este trabalho tem como finalidade estudar e estabelecer alguns parâmetros no processo fermentativo descontínuo alimentado de uma levedura recombinante Saccharomyces cerevisiae W303-181, visando aumentar a obtenção da enzima glicose-6-fosfato desidrogenase (G6PDH). Foram feitas a padronização e otimização do preparo de inóculo de S. cerevisiae recombinante. Foram três as condições estudadas. Reduziu-se o tempo de preparo do inóculo de 114 h, da primeira condição, para e 64 e 48 h para a segunda e terceira condições, respectivamente. Essas duas últimas condições mostraram-se adequados para dar continuidade com os processos fermentativos. Foi feito um estudo de otimização da concentração de micronutrientes (adenina, histidina, triptofano e uracila) no meio de cultivo usando a metodologia de superfície de resposta. Concluiu-se que, ao empregar o meio de cultivo cujas concentrações dos micronutrientes tenham sido otimizadas, a atividade específica de G6PDH atingiu 7927 U/L, 3,2 vezes maior que para o meio controle. Estudou-se a influência da constante de tempo (K), na síntese de G6PDH em processo descontínuo alimentado com vazão de alimentação exponencialmente crescente e decrescente, utilizando meio de cultivo otimizado e não otimizado. Os valores estudados de K para vazão de alimentação exponencialmente crescente foram 0,2, 0,3, 0,5, 0,7 e 0,8 h-1 e, para a decrescente, foram 0,2, 0,5 e 0,8 h-1. Dentre os cultivos com vazão exponencialmente crescente e com meio de cultivo não otimizado, encontrou-se para K=0,2 h-1 uma atividade enzimática (558 U/L) 4,1 vezes maior que para a levedura selvagem. Dentre os cultivos nas vazões exponencialmente crescente e decrescente, encontrou-se para a vazão crescente e K=0,2 h-1 os maiores níveis de produção de G6PDH (847 U/L). Foram estudados a influência da concentração inicial de glicose e o tempo de alimentação do processo descontínuo alimentado com vazão de alimentação exponencialmente crescente e K=0,2h-1. Verificou-se que a concentração inicial de glicose não favoreceu a formação de biomassa ou a produção de enzimas. Contudo, determina e as máximas velocidades específicas de crescimento celular e de produção de G6PDH, com as maiores velocidades correspondendo às menores concentrações iniciais. Por fim, foi estudada a influência da concentração de leucina na síntese da G6PDH e verificou-se que teores de leucina entre 0-240 mg/L não influenciaram o crescimento celular ou a produção de enzima nos cultivos estudados. / The purpose of this work is to study and to establish some parameters in the fed-batch process of a recombinant strain of Saccharomyces cerevisiae, aiming to increase the production the enzyme glucose-6-phosphate dehydrogenase (G6PDH). The recombinant S. cerevisiae inoculum preparation was standardized and optimized. Tree methods were studied. The inoculum preparation time was reduced to 114 h, from first method, to 64 and 48 h to second and third methods, respectively. These two last methods were adequate in order to proceed with the fermentation process. It was evaluated the best micronutrients (adenine, histidine, tryptophan and uracil) concentration in the cultivation medium to produce G6PDH, using a response surface methodology. We concluded that using cultivation medium witch optimized micronutrients concentration, the G6PDH activity reach 7927 U/L, 3.2 fold higher than to not optimized medium. The influence of time constant (K) on the G6PDH synthesis was studied at fed-batch bioreactor under exponentially increasing and decreasing feeding rates, using optimized medium cultivation and not optimized medium. The values for K at increasing rates were 0.2, 0.3, 0.5, 0.7 and 0.8 h-1, and for decreasing rates were 0.2, 0.5 and 0.8 h-1. Among K values for exponentially increasing rates with not optimized cultivation medium, K=02 h-1 shows higher G6PDH production (558U/L), 4.1 fold higher than wild yeast. Among cultivations proceeded with exponentially increasing and decreasing feeding rates and using a optimized medium, increasing rate and k=0.2 h-1 shows the higher G6PDH production (847 U/L) too. The initial glucose concentration and feeding time was studied at fed-batch bioreactor under exponentially increasing feeding rate with K=0.2 h-1. It was verified that initial glucose concentration do not favored mass or G6PDH production. Although, it determines the maximum G6PDH production and the maximum growth rates, with higher rates at lowest initial glucose concentration. At the end, the influence of leucin at G6PDH production was evaluated. It was verified that concentrations values between 0-240 mg/L did not showed influence at cell growth or G6PDH production, at the studied cultivations.