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The exponential development of biotechnology has lead to a quasi unlimited number of potential products going from biopolymers to vaccines. Cell culture has therefore evolved from the simple cell growth outside its natural environment to its use to produce molecules that they do not naturally produce. This rapid development could not be continued without new control and supervising tools as well as a good process understanding. This requirement involves however a large diversity and a better accessibility of process measurements. In this framework, software sensors show numerous potentialities. The objective of a software sensor is indeed to provide an estimation of the system state variables and particularly those which are not obtained through in situ hardware sensors or laborious and expensive analysis. In this context, This work attempts to join the knowledge of increasing bioprocess complexity and diversity and the time scale of process developments and favours systematic modelling methodology, its flexibility and the speed of development. In the field of state observation, an important modelling constraint is the one induced by the selection of the state to estimate and the available measurements. Another important constraint is the model quality. The central axe of this work is to provide solutions in order to reduce the weight of these constraints to software sensors development. On this purpose, we propose four solutions to four main questions that may arise. The first two ones concern modelling uncertainties. 1."How to develop a software sensor using measurements easily available on pilot scale bioreactor?" The proposed solution is a static software sensor using an artificial neural network. Following this modelling methodology we developed static software sensors for the biomass and ethanol concentrations in a pilot scale S. cerevisae cell culture using the measurement of titrating base quantity, agitation rate and CO₂ concentration in the exhaust gas. 2."How to obtain a reaction scheme and a kinetic model to develop a dynamic observation model?". The proposed solution is to combine three elements: a systematic methodology to generate, identify and select the possible reaction schemes, a general kinetic model and a systematic identification procedure where the last step is particularly dedicated to the identification of observation models. Combining these methodologies allowed us to develop a software sensor for the concentrations of an allergen produced by an animal cell culture using the discrete measurement of glucose, glutamine and ammonium concentrations (which are also estimated in continuous time by the software sensors). The two other questions are dealing with kinetic model uncertainty. 3 "How to correct kinetic model parameters while keeping the system observability?". We consider the possibility to correct some model parameters during the process observation. We propose indeed an adaptive observer based on the theory of the most likely initial conditions observer and exploiting the information from the asymptotic observer. This algorithm allows to jointly estimate the state and some kinetic model parameters. 4 "How to avoid any state observer selection that requires an a priori knowledge on the model quality?". Answering this question lead us to the development of hybrid state observers. The general principle of a hybrid observer is to automatically evaluate the model quality and to select the appropriate state observer. In this work we focus on kinetic model quality and propose hybrid observers that evolves between the state observation from an exponential observer (free convergence rate tuning but model error sensitivity) and the one provided by an asymptotic observer (no kinetic model requirement but a convergence rate depending on the dilution rate). Two strategies are investigated in order to evaluate the model quality and to induce the state observation evolution. Each of them have been validated on two simulated cultures (microbial and animal cells) and one real industrial one (B. subtilis). ∙ In a first strategy, the hybrid observer is based on the determination of a parameter that drives the state estimation from the one obtained with an exponential observer (exponential observation) when the model is of good quality to the one provided by an asymptotic observer (asymptotic observation) when a kinetic model error is detected. The evaluation of this driving parameter is made either with an a priori defined function or is coupled to the identification of the initial conditions in a most likely initial conditions observer. ∙ In another strategy, the hybrid observer is based on a statistical test that compares the state estimations provided by an exponential and an asymptotic observer and corrects the state estimation according to it./ Le rapide développement des biotechnologies permet actuellement d'envisager un nombre quasi illimité de produits potentiels allant du biopolymère au vaccin. La culture cellulaire a dès lors évolué de la simple croissance de cellules en dehors de leur environnement naturel à son exploitation pour la production de molécules qu'elles ne produisent pas naturellement. Un tel développement ne peut se poursuivre sans l'utilisation de nouvelles technologies de contrôle et de supervision ainsi q'une bonne compréhension et maîtrise du biprocédé. Cette exigence nécessite cependant une meilleure accessibilité et une plus grande variabilité des mesures des différentes variables de ce procédé. Dans ce contexte, les capteurs logiciels présentent de nombreuses potentialités. L'objectif d'un capteur logiciel est en effet de fournir une estimation des états d'un système et particulièrement de ceux qui ne sont pas mesurés par des capteurs physiquement installés sur le système ou par de longues et coûteuses analyses. Cet objectif peut être obtenu en combinant un modèle du système avec certaines mesures physiques au sein d'un algorithme d'observation d'état. Dans ce domaine de l'observation des bioprocédés, ce travail tente de considérer, à la fois, l'augmentation de la complexité et de la diversité des bioprocédés et l'exigence d'un développement rapide en favorisant le caractère systématique, flexible et rapide des méthodes proposées. Dans le cadre de l'observation des bioprocédés, une importante contrainte de modélisation est induite par la sélection des états à estimer et des mesures disponibles pour cette estimation. Une seconde contrainte est la qualité du modèle. L'axe central de ce travail est de fournir certaines solutions afin de réduire le poids de ces contraintes dans le développement de capteurs logiciels. Pour ce faire, nous proposons quatre réponses à quatre questions qui peuvent survenir lors de ce développement. Les deux premières questions concernent l'incertitude de modélisation. Quant aux deux questions suivantes, elles concernent l'incertitude du modèle lui-même. 1."Comment développer un capteur logiciel exploitant des mesures facilement disponibles sur un bioréacteur pilote?". La réponse que nous apportons à cette question est le développement d'un capteur logiciel statique basé sur un réseau de neurones artificiels. Cette structure nous a permis de développer des capteurs logiciels de concentrations en biomasse et éthanol au sein d'une culture de S. cerevisae utilisant les mesures en ligne de quantité de base titrante, de vitesse d'agitation et de concentration en CO₂ dans le gaz sortant du réacteur. 2."Comment obtenir un schéma réactionnel et un modèle cinétique pour l'identification d'un modèle dynamique d'observation". Afin de répondre à cette question, nous proposons de combiner trois éléments: une méthode de génération systématique de schémas réactionnels, une structure générale de modèle cinétique et une méthode d'identification dont la dernière étape est particulièrement dédiée à l'identification de modèles d'observation. La combinaison de ces éléments nous a permis de développer un capteur logiciel permettant l'estimation continue de la concentration en un allergène produit par une culture de cellules animales en utilisant des mesures échantillonnées de glucose, glutamine et ammonium (qui sont elles aussi estimées en continu par le capteur logiciel). 3."Comment corriger certains paramètres cinétiques tout en maintenant l'observabilité du système?". Nous considérons ici la possibilité de corriger certains paramètres du modèle cinétique durant le procédé de culture. Nous proposons, en effet, un observateur d'état adaptatif exploitant la théorie de l'observateur par identification des conditions initiales les plus vraisemblables et l'information fournie par un observateur asymptotique. L'algorithme proposé permet ainsi de fournir une estimation conjointe de l'état et de certains paramètres cinétiques. 4."Comment éviter la sélection d'un observateur d'état nécessitant une connaissance, a priori, de la qualité du modèle?". La dernière contribution de ce travail concerne le développement d'observateurs d'état hybrides. Le principe général d'un observateur hybride est d'évaluer automatiquement la qualité du modèle et de sélectionner l'observateur d'état approprié. Au sein de ce travail nous considérons la qualité du modèle cinétique et proposons des observateurs d'état hybrides évoluant entre un observateur dit exponentiel (libre ajustement de la vitesse de convergence mais forte sensibilité aux erreurs de mesures) et un observateur asymptotique (ne nécessite aucun modèle cinétique mais présente une vitesse de convergence dépendante du taux de dilution). Afin de réaliser cette évaluation et d'induire l'évolution de l'observation d'état entre ces deux extrémités, deux stratégies sont proposées. Chacune d'elle est illustrée sur deux cultures simulées (une croissance bactérienne et une culture de cellules animales) et une culture réelle de B. subtilis. ∙ Une première stratégie est basée sur la détermination d'un paramètre de pondération entre l'observation fournie par un observateur exponentiel et un observateur asymptotique. L'évaluation de ce paramètre peut être obtenue soit au moyen d'une fonction définie a priori soit par une identification conjointe aux conditions initiales d'un observateur par identification des conditions initiales les plus vraisemblables. ∙ Une seconde stratégie est basée sur une comparaison statistique entre les observations fournies par les deux types d'observateurs. Le résultat de cette comparaison, lorsqu'il indique une incohérence entre les deux observateurs d'état, est alors utilisé pour corriger l'estimation fournie par l'observateur exponentiel.

Em caso de envenenamento ofídico, o tratamento no Brasil hoje é realizado pela administração de soros geralmente produzidos por equinos, que apresentam eficácia limitada: são úteis para os efeitos sistêmicos, mas não inibem efetivamente a evolução dos danos locais, podem causar reações adversas e apresentam alto custo de produção. De acordo com a Organização Mundial da Saúde (OMS), trata-se de uma doença negligenciada pelas autoridades científicas mundiais. O presente projeto, em parceria com o Instituto de Pesquisas em Patologias Tropicais da Fundação Oswaldo Cruz - Rondônia, propõe a produção por Escherichia coli de fragmentos de anticorpos de cadeia pesada de camelídeos, denominados VHH, contra as toxinas do veneno de Bothrops jararacussu, utilizando biorreator. Neste trabalho há interesse em produzir VHH, através da otimização do crescimento desta E. coli. A cinética do crescimento bacteriano foi realizada em shaker orbital sob diferentes condições, variando tamanho do frasco, rotação do shaker, composição do meio de cultura e concentração de substrato; e em biorreatores, alternando meios de cultura e modo de operação do reator (descontínuo e descontínuo alimentado), alterando a vazão de alimentação (linear e exponencial) O processo cinético é fortemente limitado pela formação de acetato, por condições auxotróficas da célula e pela transferência de oxigênio. Nos ensaios em frascos agitados, uma melhor condição de crescimento foi obtida utilizando frascos de 1 L, sob rotação a 270 rpm e 5,0 g/L de glicose. Nos ensaios em reator, quando operados em batelada obtiveram-se cerca 5,5 g/L de células finais, contra 9,3 g/L de células em batelada alimentada com vazão constante. Um maior crescimento foi ainda obtido em um reator de 2 L em regime de batelada alimentada exponencialmente. O biorreator varia a agitação do meio e mantém um nível pré-definido de oxigênio dissolvido, evitando a limitação de oxigênio e controlando a oferta de glicose para o crescimento celular. Neste processo, atingimos 25,6 g/L de células e 0,35 g/L de proteína total após purificação, utilizando meio M9 suplementado. / Nowadays in Brazil, the treatment for snakebite poisoning is carried out by the administration of horse sera, which have limited effectiveness: they are useful for systemic effects, but do not effectively inhibit local damage, cause adverse reactions, and present high production costs. According to the WHO, this disease is neglected by the world`s scientific authorities. This project, in partnership with the Institute for Research in Tropical Diseases of Oswaldo Cruz Foundation - Rondonia, proposes the production of heavy chain antibody fragments from camelids, called VHH, using Escherichia coli, to be used against the toxins from the Bothrops jararacussu poison, using a bioreactor. This work is interested in producing VHH through the use of E. coli. The kinetics of bacterial growth were performed in orbital shaker under different conditions, varying vial size, shaker rotation, composition of the culture medium and substrate concentration; and bioreactors, alternating culture media and operation mode of reactor (batch and fed-batch), changing feeding rate (linear and exponential). The kinetic process is statically bound to acetate formation, auxotrophic conditions of the cell and oxygen transfer. In assays in shaken flasks, an output of 270 rpm and 5.0 g/L of glucose. In reactor runs, when operated in a batch 5.5 g/L of final cells were obtained, against 9.3 g/L of final cells in fed-batch with constant flowrate. A larger value was obtained in an exponentially fed-batch reactor of 2 L. The bioreactor varies the agitation of oxygen and controls glucose addition for cell growth. In this process, 25.6 g/L cells and 0.35 g/L total protein after purification were reached, using supplemented M9 medium.

Catalysts are utilized in 80% of all chemical synthesis operations. The industrial catalysts primarily used in oxidation reactions are highly polluting and expensive metal catalysts. Enzymes and whole cell biocatalysts are used to a lesser extent. Nowadays, several industrial sectors are developing bio-based technologies to reduce the high costs and environmental impact of traditional chemical processes. However, these applications are limited by the challenge of developing economically competitive biologically based systems. The key for adopting these sustainable advancements is the development of novel process designs, which assure robustness, simplicity, and sustainabile operations compatible with the current development of chemical reactions. In this regard, filamentous fungi may be considered good biocatalysts due to their natural biodiversity and their broad heterogeneous enzymatic pattern. The great selectivity of fungal catalysis is now well recognized for the production of commercially valuable steroids in the pharmaceutical industry. Although this inherent capacity is mainly used for functionalization of unactivated carbons, it can be further exploited for the oxidiation of heteroatoms, such as sulfur. Focusing on the oxidation of sulfur compounds, the widely used industrial processes are produced by an organometallic catalyst. This PhD project aims to overcome low substrate conversion and enzymatic expression by proving that exposure of cells to insecticides and hydrocarbons increases cell's oxidative capacity expressed as higher substrate conversion and CYP450 content. This study is focused in the application of pest management strategies, designed to enhance the biopesticide's efficacy, to induce and improve Beauveria bassiana oxidation. B. bassiana has a very flexible metabolism and is widely used as a biocontrol agent. It can metabolize hexadecane as a sole carbon source. In addition, it shows a synergistic effect over pest control efficacy when it is applied with low pesticides (carbaryl and/or imidacloprid) concentrations. A biocatalytic system was optimized to increase the conversion of organosulfur compounds under different fermentation conditions. Phenothiazine was used as our model substrate. Phenothiazine conversion was followed by GC-MS and HPLC. By NMR and MS fragmentation pattern product, phenothiazine metabolites were identified as (R)-hydroxyl metabolites (63% enatiomeric excess) and sulfoxide, the latter being the main metabolite. Phenothiazine conversions with growing cells resulted in 65±1.4% conversion with initial phenothiazine concentration of 500 ppm and final 325 ppm after 7 days. The highest conversion, 74±1 % was achieved with resting cells at the lowest cell concentration, 0.78 mg cell dry weight (cdw) /mL. Furthermore, the use of insecticides as inducers was an effective way to increase phenothiazine conversion from 47% to 64±3%. The major enzymes involved in catalysis of xenobiotic are heme-binding monooxygenases, in particular cytochrome P450. Heme positive proteins were identified by an SDS benzidine assay as well as the content of CYP450 by the CO difference spectrum. The P450 enzymes content was 12.3±1 pmol/µg protein for hexadecane adapted cells and 8.1± 1 pmol/µg protein for insecticides, respectively. The heme-positive proteins were characterized by MALDI-ToF and their peptide mass fingerprint compared to the available sequences on the SwissProt/Universal Protein Resource catalog of information on proteins (UniProtKB). Hemoproteins were found, including a cluster of catalase-peroxidase, alkane hydroxylase, and chloroperoxidase. The results from this project helped bridge the progress from agricultural biotechnology strain development into industrial biotechnology biocatalyst improvement. The success of this project helps us expand B. bassiana's catalysis and make it a better candidate for industrial biocatalysis.

Face à la montée de la prise de conscience des enjeux écologiques actuels, la recherche se tourne vers le développement des bioprocédés pour développer de nouvelles solutions aux problèmes environnementaux. Cette thèse porte sur l’étude de la faisabilité d’un procédé de capture de CO2 à partir de la culture de la microalgue Chlorella vulgaris en photobioréacteur continu. Ce travail a permis d’identifier l’algue C. vulgaris comme une candidate prometteuse pour cette application. En effet C. vulgaris présente une capacité de production de biomasse et de fixation de CO2 très intéressante pour cette application. Les études menées lors de ce travail de thèse ont également permis de mettre à jour les interactions complexes entre les cellules algales et le CO2 présent à de fortes concentrations. De même, elles ont apporté un approfondissement à la compréhension des verrous existants pour le développement d’un procédé de captage du CO2 et de la nécessité de prendre en compte tous les paramètres de culture (lumière, concentration en nitrate). A partir des études menées, il a été possible de proposer un modèle pour la croissance de C. vulgaris en photobioréacteur continu. Bien que de futures études soient encore nécessaires pour être en mesure de parfaitement modéliser le comportement de l’algue lors de cultures en photobioréacteur, ce modèle présente une bonne corrélation avec les expérimentations. Enfin une étude de pré-dimensionnement a été proposée qui a permis de mettre en lumière les nombreux points d’interrogations encore existants avant d’être en mesure d’adapter le procédé de laboratoire à une échelle industrielle / Faced with the growing awareness of environmental issues, the research turns to the development of bioprocesses to develop new solutions to environmental problems. This thesis concerns the study of the feasibility of a process for CO2 capture from the culture of the microalgae Chlorella vulgaris in a continuous photobioreactor. This work has identified the algae C. vulgaris as a promising candidate for this application. Indeed C. vulgaris has a capacity of biomass production and CO2 biofixation very interesting for this application. Studies in this thesis allowed us to update the complex interactions between the algal cells and high CO2 concentrations. Also they have provided a deeper understanding of existing locks for the development of a process for CO2 capture and the need to take into account all the parameters of culture (light, nitrate concentration). A model for the growth of C. vulgaris in continuous photobioreactor has been proposed. This model shows good correlation with experiments; although future studies are still needed to be able to fully simulate the behaviour of algae in photobioreactor cultures. Finally a study of pre-design has been proposed allowing highlighting the many questions that still exist before being able to adapt the laboratory process to an industrial scale.

Em caso de envenenamento ofídico, o tratamento no Brasil hoje é realizado pela administração de soros geralmente produzidos por equinos, que apresentam eficácia limitada: são úteis para os efeitos sistêmicos, mas não inibem efetivamente a evolução dos danos locais, podem causar reações adversas e apresentam alto custo de produção. De acordo com a Organização Mundial da Saúde (OMS), trata-se de uma doença negligenciada pelas autoridades científicas mundiais. O presente projeto, em parceria com o Instituto de Pesquisas em Patologias Tropicais da Fundação Oswaldo Cruz - Rondônia, propõe a produção por Escherichia coli de fragmentos de anticorpos de cadeia pesada de camelídeos, denominados VHH, contra as toxinas do veneno de Bothrops jararacussu, utilizando biorreator. Neste trabalho há interesse em produzir VHH, através da otimização do crescimento desta E. coli. A cinética do crescimento bacteriano foi realizada em shaker orbital sob diferentes condições, variando tamanho do frasco, rotação do shaker, composição do meio de cultura e concentração de substrato; e em biorreatores, alternando meios de cultura e modo de operação do reator (descontínuo e descontínuo alimentado), alterando a vazão de alimentação (linear e exponencial) O processo cinético é fortemente limitado pela formação de acetato, por condições auxotróficas da célula e pela transferência de oxigênio. Nos ensaios em frascos agitados, uma melhor condição de crescimento foi obtida utilizando frascos de 1 L, sob rotação a 270 rpm e 5,0 g/L de glicose. Nos ensaios em reator, quando operados em batelada obtiveram-se cerca 5,5 g/L de células finais, contra 9,3 g/L de células em batelada alimentada com vazão constante. Um maior crescimento foi ainda obtido em um reator de 2 L em regime de batelada alimentada exponencialmente. O biorreator varia a agitação do meio e mantém um nível pré-definido de oxigênio dissolvido, evitando a limitação de oxigênio e controlando a oferta de glicose para o crescimento celular. Neste processo, atingimos 25,6 g/L de células e 0,35 g/L de proteína total após purificação, utilizando meio M9 suplementado. / Nowadays in Brazil, the treatment for snakebite poisoning is carried out by the administration of horse sera, which have limited effectiveness: they are useful for systemic effects, but do not effectively inhibit local damage, cause adverse reactions, and present high production costs. According to the WHO, this disease is neglected by the world`s scientific authorities. This project, in partnership with the Institute for Research in Tropical Diseases of Oswaldo Cruz Foundation - Rondonia, proposes the production of heavy chain antibody fragments from camelids, called VHH, using Escherichia coli, to be used against the toxins from the Bothrops jararacussu poison, using a bioreactor. This work is interested in producing VHH through the use of E. coli. The kinetics of bacterial growth were performed in orbital shaker under different conditions, varying vial size, shaker rotation, composition of the culture medium and substrate concentration; and bioreactors, alternating culture media and operation mode of reactor (batch and fed-batch), changing feeding rate (linear and exponential). The kinetic process is statically bound to acetate formation, auxotrophic conditions of the cell and oxygen transfer. In assays in shaken flasks, an output of 270 rpm and 5.0 g/L of glucose. In reactor runs, when operated in a batch 5.5 g/L of final cells were obtained, against 9.3 g/L of final cells in fed-batch with constant flowrate. A larger value was obtained in an exponentially fed-batch reactor of 2 L. The bioreactor varies the agitation of oxygen and controls glucose addition for cell growth. In this process, 25.6 g/L cells and 0.35 g/L total protein after purification were reached, using supplemented M9 medium.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Cellulases are enzyme complex composed of endoglucanases, exoglucanases and β-glucosidases with several biotechnological applications. However, their production cost is a major obstacle for its industrial application. About 40% of the total cellulase production cost is related to the culture medium used for the microorganism growth. In this context, efficient processes for cellulolytic enzyme production are of technical and economical interest. Thus, the present study aimed to optimize the production of cellulases by Melanoporia sp. using coconut shell powder as substrate in submerged fermentation. The influence of pH and temperature on the enzyme activity was evaluated by univariate experimental design. Then, the composition of the culture medium was sequentially optimized through Plaket -Burman followed by Central Composite experimental designs. The fermentation under optimized conditions was subsequently conducted in bioreactor to evaluate the influen ce of pH control and aeration on enzyme production. The stability of the enzyme was evaluated for 6 and 8 months at 4 ÂC and - 20 ÂC, respectively. The ability of the enzyme to hydrolyze coconut shell powder was evaluated at 65 ÂC and 80 ÂC using the crude enzyme extract produced by Melanoporia sp. The enzyme activity was determined by the quantification of reducing sugars using DNS method at pH 5.5 and 80 ÂC (optimum conditions). The composition of the culture medium which provided the highest enzyme yield was: 5 g/L of coconut shell; 15 g/L lactose; 3% tween 80; 1 g/L of KH2PO4 and 0.05 g/L FeSO4; pH 6.5 at 30ÂC for 72 hours. For batch enzyme production, the cult ure medium using non-delignified substrate, with pH controlled at 6.5, without aeration resulted in an increase of 90% in enzyme activity compared to the fermentation in a rotatory shaker. Under these conditions, the maximal enzyme production was obtained after 24 hours of fermentation. The crude enzyme extract produced by Melanoporia sp. was able to hydrolyze cellulose (coconut shell powder) efficiently, presenting industrial potential for the degradation of lignocellulosic residues. Unlike most of the cellulases produced by Trichoderma species, the strain reported as one of the best producers, the microorganism was capable of producing cellulases efficiently without the need of substrate pretreatment. Another feature of this enzyme complex is its high stability in the crude broth at-20ÂC e 4 ÂC / Celulases sÃo um complexo enzimÃtico constituÃdo por endoglucanases, exoglucanases e β-glicosidases com diversas aplicaÃÃes biotecnolÃgicas. No entanto, o elevado custo de produÃÃo dessas enzimas à o principal obstÃculo para sua aplicaÃÃo industrial. Estima-se que cerca de 40% do custo total de produÃÃo de celulases esteja relacionado ao meio de cultura utilizado para o crescimento do micro-organismo. Nesse contexto, à de fundamental importÃncia o desenvolvimento de processos para a produÃÃo de enzimas do complexo celulolÃtico que se mostrem tÃcnico e economicamente viÃveis. Diante do exposto, o presente estudo teve como objetivo avaliar a produÃÃo de celulases produzidas por Melanoporia sp. utilizando o pà da casca de coco como substrato em fermentaÃÃo submersa. A influÃncia dos parÃmetros pH e temperatura na determinaÃÃo da atividade da enzima foi avaliada atravÃs de planejamento experimental univariado. Em seguida, a composiÃÃo do meio de cultura foi otimizada atravÃs dos planejamentos experimentais Plaket-Burman e Composto Central. A fermentaÃÃo em condiÃÃes otimizadas foi posteriormente conduzida em fermentador para avaliar a influÃncia do controle de pH e oxigÃnio na produÃÃo da enzima. A estabilidade da enzima foi avaliada por 6 e 8 meses nas temperaturas de 4 ÂC e -20 ÂC, respectivamente. A capacidade das enzimas em hidrolisar o pà da casca do coco foi avaliada nas temperaturas de 65 ÂC e 80 ÂC utilizando o extrato enzimÃtico bruto produzido por Melanoporia sp. A atividade da enzima foi determinada atravÃs da quantificaÃÃo de aÃÃcares redutores pelo mÃtodo de DNS. O pH e a temperatura de determinaÃÃo da atividade enzimÃtica foram pH 5,5 e 80 ÂC, respectivamente. A composiÃÃo do meio de cultura que proporcionou o maior rendimento de produÃÃo da enzima foi: 5 g/L de casca de coco; 15 g/L de lactose; 3% de tween 80; 1 g/L de KH2PO4 e 0,05 g/L de FeSO4; pH 6,5 a 30 ÂC em 72 horas. Para a produÃÃo da enzima em fermentador, o meio de cultura utilizando substrato nÃo deslignificado, com controle do pH em 6,5, sem aeraÃÃo proporcionou um aumento de 90% na atividade da enzima, comparado à fermentaÃÃo em shaker. Nessas condiÃÃes, a mÃxima produÃÃo da enzima foi obtida apÃs 24 horas de fermentaÃÃo. O extrato enzimÃtico bruto produzido por Melanoporia sp. exibiu capacidade de hidrolisar celulose presente na casca de coco com eficiÃncia, apresentando potencial industrial para a degradaÃÃo de resÃduos lignocelulÃsicos. Diferentemente da maior parte das celulases produzidas por espÃcies de Trichoderma, micro-organismo reportado como bom produtor de enzimas celulolÃticas, o micro-organismo utilizado neste trabalho à capaz de produzir celulases de forma eficiente, sem necessidade de prÃ-tratamento do substrato. Outra caracterÃstica diferencial desta enzima à sua elevada estabilidade nas temperaturas de -20 ÂC e 4 ÂC no caldo bruto.

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2008. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-24T17:43:54Z No. of bitstreams: 1 dissertao marta.pdf: 2219216 bytes, checksum: dabe85cdb78a95658dc970cbd0e37ce1 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-11-09T13:16:33Z (GMT) No. of bitstreams: 1 dissertao marta.pdf: 2219216 bytes, checksum: dabe85cdb78a95658dc970cbd0e37ce1 (MD5) / Made available in DSpace on 2012-11-09T13:16:33Z (GMT). No. of bitstreams: 1 dissertao marta.pdf: 2219216 bytes, checksum: dabe85cdb78a95658dc970cbd0e37ce1 (MD5) Previous issue date: 2008 / Os biossurfactantes são moléculas anfipáticas produzidas por microrganismos, que reduzem tensões superficiais e interfaciais e possuem propriedades de emulsificação. Apresentam vantagens com relação aos surfactantes químicos como a biodegradabilidade e baixa toxicidade, podendo ser aplicados na indústria de alimentos, farmacêutica, cosmética, na biorremediação e na recuperação de petróleo. Os processos biotecnológicos freqüentemente são limitados pelo investimento de capital. A utilização de resíduos agroindustriais para a produção de biossurfactante é importante do ponto de vista econômico, já que a matéria-prima representa grande parte dos custos de obtenção deste bioproduto. Os objetivos do presente trabalho foram selecionar bactérias com potencial para produzir biossurfactante e estudar a produção por bactéria e fungo em diferentes biorreatores, através de cultivos em estado sólido e submerso utilizando resíduos agroindustriais. O trabalho foi dividido em três etapas: 1) seleção de bactérias produtoras de biossurfactante e estudo da cinética dos processos fermentativos, 2) produção de biossurfactantes bacteriano e fúngico em diferentes biorreatores através de cultivo em estado sólido e 3) produção de biossurfactantes bacteriano e fúngico em diferentes biorreatores através de cultivo submerso. Na primeira etapa foram utilizadas 4 culturas de bactérias: cultura pura de Corynebacterium aquaticum, cultura mista contendo Corynebacterium aquaticum e Bacillus sp., cultura mista contendo Corynebacterium sp., Bacillus cereus e Bacillus mycoides e cultura pura de Bacillus subtilis. A cultura pura de Corynebacterium aquaticum foi a que apresentou menor tensão superficial (28,8 mN.m-1) e, em geral, maiores valores para os parâmetros cinéticos, sendo selecionada como produtora de biossurfactante. Resultados da segunda etapa mostraram que as menores tensões superficiais foram encontradas no cultivo de Corynebacterium aquaticum em biorreator de coluna (33,1 mN.m-1) e frasco Erlenmeyer (31,3 mN.m-1). A menor tensão interfacial foi 6,2 mN.m-1, obtida em cultivo bacteriano em biorreator de coluna. Os cultivos com o fungo Aspergillus fumigatus em biorreator de coluna e frasco Erlenmeyer apresentaram maiores atividades emulsificantes óleo em água (291,8 UE e 327,9 UE, respectivamente). A atividade emulsificante água em óleo foi incrementada nos cultivos de Aspergillus fumigatus e pela utilização do biorreator de coluna, atingindo 60,4 UE. Na terceira etapa verificou-se que as menores tensões superficiais foram encontradas no cultivo de Corynebacterium aquaticum em biorreator de mistura (28,6 mN.m-1) e frasco Erlenmeyer (29,1 mN.m-1). As menores tensões interfaciais foram obtidas nos cultivos bacterianos (3,0 mN.m-1). A maior atividade emulsificante óleo em água foi 232,0 UE, obtida no cultivo com a bactéria Corynebacterium aquaticum em biorreator de mistura. Os cultivos bacterianos em biorreator de mistura e frasco Erlenmeyer apresentaram maiores atividades emulsificantes água em óleo (37,9 UE e 36,1 UE, respectivamente). Logo, o biossurfactante produzido pela bactéria foi capaz de reduzir tensões e promover a formação de emulsões tanto em meio sólido quanto em meio líquido, enquanto o fungo mostrou-se apto a produzir biossurfactante com alta capacidade emulsificante em meio sólido. Constatou-se a viabilidade de produção de biossurfactante a partir de resíduos agroindustriais, o que torna o processo mais econômico, obtendo-se um bioproduto de valor agregado a partir de substratos de baixo custo, além de contribuir com o meio ambiente pela diminuição da poluição e desequilíbrio gerados pela emissão desses resíduos. / Biosurfactants are amphipathic molecules produced by microorganisms that reduce surface and interfacial tensions and possess emulsification properties. They present advantages with relationship to the chemical surfactants as the biodegradability and low toxicity, and can be applied in the food, pharmaceutical and cosmetic industry, in the bioremediation and in the recovery of petroleum. The biotechnology processes are frequently limited by the capital investment. The use of agroindustrial wastes for the biosurfactant production is important from the economical point of view, since the raw material represents great part of the costs in the obtaining of this bioproduct. The purposes of this study were to select bacteria with potential to produce biosurfactant and to study the production by bacteria and fungus in different bioreactors, through solid-state and submerged cultivations using agroindustrial wastes. The work was divided in three stages: 1) selection of bacteria for biosurfactant production and fermentation process kinetics study, 2) bacterial and fungic biosurfactants production in different bioreactors through solid-state cultivation and 3) bacterial and fungic biosurfactants production in different bioreactors through submerged cultivation. In the first stage 4 bacterial strains were used: pure culture of Corynebacterium aquaticum, mixed culture containing Corynebacterium aquaticum and Bacillus sp., mixed culture containing Corynebacterium sp., Bacillus cereus and Bacillus mycoides and pure culture of Bacillus subtilis. The pure culture of Corynebacterium aquaticum presented the smallest surface tension (28.8 mN.m-1) and, in general, larger kinetic parameters, being selected as biosurfactant producer. Results of the second stage showed that the smallest surface tensions were found in the cultivation of Corynebacterium aquaticum in column bioreactor (33.1 mN.m-1) and Erlenmeyer flask (31.3 mN.m-1). The smallest interfacial tension was 6.2 mN.m-1, obtained in bacterial cultivation in column bioreactor. The cultivations with the fungus Aspergillus fumigatus in column bioreactor and Erlenmeyer flask presented larger emulsifying activities oil in water (291.8 UE and 327.9 UE, respectively). The emulsifying activity water in oil was increased in the cultivations of Aspergillus fumigatus and for the use of the column bioreactor, reaching 60.4 UE. In the third stage it was verified that the smallest surface tensions were found in the cultivation of Corynebacterium aquaticum in mixture bioreactor (28.6 mN.m-1) and Erlenmeyer flask (29.1 mN.m-1). The smallest interfacial tensions were obtained in the bacterial cultivations (3.0 mN.m-1). The largest emulsifying activity oil in water was 232.0 UE, obtained in the cultivation with the bacteria Corynebacterium aquaticum in mixture bioreactor. The bacterial cultivations in mixture bioreactor and Erlenmeyer flask presented larger emulsifying activities water in oil (37.9 UE and 36.1 UE, respectively). Therefore, the biosurfactant produced by the bacteria was able to reduce tensions and to promote emulsions formation in solid and liquid medium, while the fungus was able to produce biosurfactant with high emulsifying capacity in solid medium. The viability of biosurfactant production from agroindustrial wastes was verified, what makes the process more economical, with the obtaining of a value joined bioproduct from low cost substrates, besides contributing with the environment for the decrease of the pollution and unbalance generated by the emission of those residues.

Användning av etanol som drivmedel och en efterfråga på gröna kemikalier driver utvecklingen av bioetanol framåt. Etanolpiloten, SEKAB, i Örnsköldsvik är en av få anläggningar i världen med kompetens och kunskap att producera bioetanol baserat på lignocellulosa. På senare tid har det dock uppstått problem vid etanolframställningen på grund av att en del jästodlingar blivit kontaminerade av bakterier vilket lett till ett sämre utbyte av biomassa och etanol. Det huvudsakliga syftet med detta examensarbete var att ta reda på orsaken till dessa misslyckade jästodlingar.   Examensarbetet delades upp i två huvudsakliga problemområden. Förutom orsaken till de kontaminerade odlingarna studerades även funktionen hos en ny jäststam, Saccaromyces cerevisiae torrjäst, i syfte att undersöka om det finns bättre alternativ till den jäststam som används i etanopiloten i nuläget.   En specialstudie av rengöringen av odlingstankar och ledningar i etanolpiloten utfördes i syfte att kartlägga var i utrustningen som infektionsrisken är som störst. Försöken påvisade att det huvudsakliga problemet kan lokaliseras till den största jästodlingstanken. Där befinner sig jästen under en längre tid i en miljö som är gynnsam för tillväxt av både jäst och bakterier. En annan orsak till de infekterade odlingarna är att rengöringen av utrustningen inte har skett på rätt sätt, samt att temperaturen hos tvättkemikalierna har varit för låg. En viktig slutsats är därför att bättre rutiner vid hanteringen av jästodlingsutrustningen samt att större noggrannhet i samband med rengöringen bör eftersträvas.   En bidragande orsak till de infekterade odlingarna kan också härröra från uppodlingsprocessen av ympjäst som i dagens läge sker på laboratorium. Genom att använda en stam av S. cerevisiae som köps in i frystorkad form kan flera steg i jästodlingsprocessen elimineras. Det både förkortar odlingsprocessen och minskar infektionsrisken. S. cerevisiae torrjäst undersöktes både i laboratorium och i etanolpiloten. Tre olika odlingsskalor användes, skakflaskor (250 ml), labfermentorer (3 l) och pilotskala (10m3). Försöken påvisar höga utbyten av både biomassa och etanol. För att kunna hålla nere produktionskostnaderna för etanolframställningen är det viktigt att jästen som används går att odla på det hydrolysat som produceras vid förbehandlingen av råvaran. Försök i pilotskala visar på lovande resultat vid uppodling av S. cerevisiae torrjäst när hela 70 % av sockerkällan kommer från hydrolysat. Ytterligare utvärdering och optimering av odlingsprocessen samt en ekonomisk jämförelse mellan de tillgängliga jäststammarna krävs dock innan S. cerevisiae torrjäst eventuellt kan användas kontinuerligt i pilotskala.