Ces travaux visaient à étudier la possibilité de réduire la production de glycérol chezSaccharomyces cerevisiae, afin d’améliorer le rendement éthanol, tout en préservant les capacités decroissance et de production des levures. La production minimale de glycérol nécessaire à la croissancea été déterminée à l'aide d'un modèle de calcul des flux métaboliques. Des souches présentant uneactivité des enzymes de la voie de production du glycérol modulée, afin de s'approcher au plus près del'activité minimale nécessaire estimée in silico, ont été utilisées.Cette stratégie d’ajustement de l’activité de la voie de synthèse du glycérol a permis, encondition aérobie, de réduire de 88 % le rendement glycérol et d'améliorer le rendement éthanol de4,7 % sans modifier la tolérance des mutants à l'éthanol, mais au détriment de la vitesse spécifique decroissance, légèrement réduite. En condition anaérobie, une diminution de 61 % du rendementglycérol et une amélioration de 7 % du rendement éthanol ont pu être obtenues, mais au détriment dela vitesse spécifique de croissance,qui subit une sévère diminution, et de la tolérance à l'éthanol,qui estréduite.L'analyse fine des résultats, grâce à un modèle métabolique, a permis de mettre en évidence,chez les souches mutantes, un besoin accru en énergie, interprété comme la traduction d'une plusgrande difficulté à gérer le stress du procédé et une réorganisation du métabolisme oxydo-réductif,interprétée comme l'impact de la réduction du glycérol sur les voies de réoxydation du cofacteurNADH dans les cellules.Ces résultats ont permis de valider la pertinence de la stratégie de réajustement des fluxmétaboliques, assistée par modélisation stoechiométrique pour l'amélioration des souches, mais aussid'accroître la compréhension du rôle physiologique du glycérol et son intégration au métabolismecellulaire. / This work aimed to assess the possibility of reducing Saccharomyces cerevisiae's glycerolproduction, in order to improve ethanol yield, without altering the abilities of yeasts to grow andproduce ethanol. Minimum glycerol production required for growth was found, thanks to a metabolicflux calculation model. Strains showing a fine tuned activity in the glycerol synthesis pathway enzymeswere used, to get close to the minimum activity established in silico.This fine tuning strategy lead, in aerobiosis, to a 88 % glycerol yield decrease together with a4.7 % ethanol yield increase, with no reduction of mutants'ethanol tolerance, but there is a slightdecrease of the growth rate. In anaerobiosis, a 61 % glycerol yield decrease, together with a 7 %ethanol yield increase were obtained, but mutant strains suffered of a sharp growth rate reduction anda decrease in their ethanol tolerance.A close analysis of the results, with the help of a metabolic model, highlighted both an increaseof mutants' energy requirements, interpreted as an increased difficulty to cope with osmotic stress,and a reorganisation of their oxydo-reductive metabolism, interpreted as glycerol reduction's impacton the NADH cofactor reoxydation pathway.These results validated the relevance of metabolic fine-tuning, assisted with in silicostoichiometric model for strains improvement and they increased the understanding of the integrationof glycerol in cell metabolism as well as its physiological role.

Durant les cultures cellulaires en bioréacteur, la physiologie des micro-organismes et les paramètres physico-chimiques (alimentations en gaz et en substrat, agitation, température, pH, pression) interagissent très fortement. La spécificité des bioréactions microbiennes, en relation avec les couplages irréductibles entre les transferts de chaleur, de matière et de quantité de mouvement, réside dans la complexité (milieu triphasique) et la dynamique (bioréaction autocatalysé) de ces systèmes. L’objectif de ce travail est de progresser dans la compréhension et le contrôle dynamique des intéractions entre les aspects biologiques et les aspects physiques à différentes échelles (macro, micro et moléculaire) pour conduire la réaction biologique vers l’objectif défini (production de biomasse, de métabolites intra ou extra cellulaires, …) et l’optimiser. Les cellules (concentration, forme, dimension, physiologie, …) affectent fortement les propriétés physico-chimiques des moûts et par conséquent, les performances des bioprocédés (vitesses spécifiques, rendements, productivité). Le comportement rhéologique particulier du moût est souvent utilisé pour comprendre l’impact de la biomasse microbienne sur le rendement et les performances du bioprocédé.Dans ce travail, des cultures axéniques, définies comme des cultures pures de microorganismes unicellulaires procaryote et eucaryote, sont considérées. Notre approche s’appuie sur des mesures physiques et physico-chimiques en ligne et hors ligne réalisées sur un bioréacteur instrumenté, mesures qui sont mises en place de façon à respecter les conditions imposées par les contraintes biologiques propres aux microorganismes et à la stratégie de culture choisie. Des cultures d’Escherichia coli et d’Yarrowia. lipolytica, à taux de croissance controlé par l’apport de substrat, ont été réalisées dans une gamme de concentration allant de 0.1 à 100 g l-1. Le bilan qui peut être dressé pour ce travail, tant sur les aspects scientifiques que technologiques, est le suivant :- conception et réalisation d’un outil d’investigation original construit sur la base d’un bioréacteur (20 l) et pourvu d’une boucle de recirculation instrumentée pour la mesure,- identification hydrodynamique (courbes de frottement) de conduites calibrées permettant la viscosimétrie en ligne durant une culture cellulaire, - conception, développement et validation d’un code, LoCoPREL, permettant simultanément le contrôle de la culture cellulaire suivant une stratégie définie, la gestion de séquences de débit dans la boucle de dérivation et l’acquisition des données issues de l’instrumentation spécifique employée,- comparaison des mesures réalisées en ligne à débit constant ou selon des séquences de débit,- mise en évidence du comportement non newtonien des moûts et d’écarts entre les mesures en ligne et hors ligne,- analyse des mesures physiques réalisées en ligne et hors ligne, en lien avec les performances de la culture / During cell cultures in bioreactors, micro-organism physiology closely interacts with physico-chemical parameters such as gas and feed flowrates, mixing, temperature, pH, pressure. The specificity of microbial bioreactions in relation with irreductible couplings between heat and mass transfers and fluid mechanics, led into complex (three-phase medium) and dynamic (auto-biocatalytics reaction) systems. Our scientific approach aims to investigate, understand and control dynamic interactions between physical and biological systems at different scales (macro, micro and molecular) for molecules, strains and/or bioprocess innovation. Cells (concentration, shape, dimension, physiology…) strongly affect physico-chemical properties of broth. Then the modification of these characteristics interacts with bioprocess performances (specific rates, yields…) with an improvement or, more frequently, a decrease of yields. Among these properties, rheological behaviour is a strategy widely used to understand the impact of cells and the derivation of bioprocess performances.In this manuscript, axenic cultures, defined as cultures of a pure and unicellular Prokaryote and Eukaryote microorganisms in bioreactors, are considered. Our approach is based on physical and physico-chemical on-line and off-line measurements in respect with accurate and stringent conditions imposed by cell culture strategy. Escherichia coli and Yarrowia lipolytica cultures were investigated with a control of growth rate by carbon feed in the range from 0.1 up to 100 g l-1. Our scientific and technical actions and results led:- to design and realize an original pilot based on a bioreactor (20 l) with a derivation loop including a specific on-line rheometric device as well as additional physical and biological measurements,- to identify, from a hydrodynamic standpoint, the generalized friction curves of calibrated ducts enabling on-line viscosimetry during cell cultures,- to conceive and validate a homemade software, named LoCoPREL, enabling simultaneously to control cell cultures under defined strategy and to manage flow sequences within the derivation loop,- to discuss and compare on-line physical measurements under constant flow rate and various sequence strategy related to investigated shear-rates,- to highlight about the non-newtonian rheological behaviour of broths and the gap between on-line and off-line measurements,- to analyse on-line and off-line physical measurements in the light of biological performances during fed-batch cultures (mass balance, specific rate, yield).

Rhodotorula glutinis (Rhodosporidium toruloides) est une levure oléagineuse dont les fortes capacités d’accumulation lipidique à partir de glucose comme source carbonée ont fait de la souche un modèle d’étude. La capacité de cette levure à utiliser le glycérol ou le xylose en simple ou co-substrat avec le glucose est toutefois encore peu explorée. De l’analyse des travaux antérieurs il a été possible de dégager les verrous scientifiques qui nécessitent une amélioration des connaissances du comportement physiologique de cette levure pour la conversion des substrats cités. Des stratégies expérimentales adaptées à la quantification rationnelle des dynamiques de Rhodotorula glutinis en conditions de croissance et accumulation lipidique à partir de xylose et de glycérol en simple ou co-substrats avec le glucose ont été développées. Des résultats originaux ont été obtenus dont :- la mise en évidence des potentialités de co-consommation des substrats xylose et glucose ou glycérol et glucose sans accumulation de substrat ni production de métabolites en conditions contrôlées des flux de substrats. Il a été possible de déterminer la vitesse spécifique maximale de consommation du carbone de la souche qui diminue lorsque la part de xylose ou glycérol augmente dans l’apport de carbone total.- la quantification de la dynamique de croissance sur xylose et glycérol pur en terme de taux de croissance et de rendement : sur xylose µmax= 0.034h-1 et RS/X= 0.28 Cmolx.Cmolxylose-1; sur glycérol µmax=0.04h-1 RS/X=0.31Cmolx.Cmolglycérol-1.- la quantification des vitesses spécifiques et rendements de production de lipides à partir de xylose ou de glycérol en simple ou co-substrat avec du glucose : 20%xylose-80%glucose : qp=0.065CmolTAG.Cmolbiomasse.h-1, RS/P=0.3CmoleTAG.Cmolesubstrat-1 100%xylose : qp=0.035065CmolTAG.Cmolbiomasse.h-1, RS/P=0.31CmoleTAG.Cmolesubstrat-1, 25% glycérol-75%glucose : qp=0.07065CmolTAG.Cmolbiomasse.h-1, RS/P=0.25CmoleTAG.Cmolesubstrat-1 , 100% glycérol : qp=0.03065CmolTAG.Cmolbiomasse.h-1, RS/P= 0.29CmoleTAG.Cmolesubstrat-1.- L’impact de la nature des substrats sur le profil lipidique de Rhodotorula glutinis demeure léger : il apparait que le xylose entraîne une surproduction de C16:0 et C18:3et le glycérol favorise l’accumulation de C18:0 / Rhodotorula glutinis (Rhodosporidium toruloides) is an oleaginous yeast. The micro-organism has demonstrated high lipid accumulation when utilizing glucose as a substrate, and has become a model for oil production. Glycerol and xylose are interesting as substrates for production of oil from renewable resources, but the capacity of R. glutinis to utilize glycerol and xylose as substrates has not been characterized well. Fermentation strategies were designed to quantify growth and lipid accumulation dynamics of R. glutinis when utilizing glycerol and xylose - either as pure substrates, or as co-substrates with glucose. Several original results have been found, including: - Co-consumption of xylose or glycerol along with glucose was observed, without carbon substrate accumulation or byproduct formation, when the carbon feed rate was carefully controlled. The specific carbon consumption rate decreases when the proportion of the second substrate (glycerol or xylose) increases in the feed, relative to glucose. - Growth capacities were characterized on pure xylose and pure glycerol in terms of growth rate and carbon yields: on xylose μmax= 0.034h-1 and RS/X= 0.28 Cmolx.Cmolxylose-1; on glycerol μmax=0.04h-1 RS/X=0.31Cmolx.Cmolglycerol-1. - specific production rate of lipid production and substrate to product carbon conversion yields from xylose or glycerol as single or cosubstrate with glucose were determinated: 20%xylose-80%glucose : qp=0.065CmolTAG.Cmolbiomasse.h-1, RS/P=0.3CmoleTAG.Cmolesubstrat-1 100%xylose : qp=0.035065CmolTAG.Cmolbiomasse.h-1, RS/P=0.31CmoleTAG.Cmolesubstrat-1, 25% glycerol-75%glucose : qp=0.07065CmolTAG.Cmolbiomasse.h-1, RS/P=0.25CmoleTAG.Cmolesubstrat-1 , 100% glycerol : qp=0.03065CmolTAG.Cmolbiomasse.h-1, RS/P= 0.29CmoleTAG.Cmolesubstrat-1. - Substrate diversification slightly impacts Rhodotorula glutinis´s lipid profile: xylose leads to an overproduction of C16:0 and C18:3 and glycerol increases C18:0 accumulation

L’accumulation de lipides chez une espèce levurienne Yarrowia lipolytica souche sauvage a été caractérisée par l’analyse dynamique et systémique des différents états métaboliques identifiés lors des cultures sous conditions environnementales parfaitement maitrisées, à hautes densités cellulaires selon deux stratégies bien distinctes. En premier lieu sur substrat osidique avec le phosphore comme élément inducteur de l’accumulation de lipides, stratégie originale pour déclencher l’accumulation de lipides chez cette souche. Et deuxièmement sur co-susbtrats glucose et oléate et sans aucune limitation nutritionnelle.Ces stratégies de conduites ont permis de dégager les points suivants :- La limitation phosphore déclenche une accumulation en lipides mais aussi en polysaccharides de réserves mobilisables mais non transitoire contrairement à la limitation azote.- La teneur en phosphore de la biomasse catalytique est très variable. De ce fait, le taux de croissance de la biomasse catalytique n’est pas contrôlable par le débit en phosphore.- Le phosphore joue un rôle dans la régulation de l’entrée de glucose dans la cellule, et permet d’éviter la production de citrate lorsque les voies de production de biomasse et de lipides sont en débordement sur une large gamme de rapport C/P (de 0 à 8000 Cmole.mole-1).- La capacité maximale d’accumulation en réserves carbonées chez Y. lipolytica wT est identique quelle que soit la méthode d’accumulation (limitation azote, limitation phosphore, co-substrats glucose / oléate) et est égale à 0,5 Cmole/CmoleX-1. Il existe donc un phénomène de régulation de la levure encore inconnu et limitant l’accumulation en réserves carbonées chez cette souche.Ces résultats ont permis d’identifier des points clés dans l’accumulation en réserves carbonées de cette espèce levurienne et de proposer un mode de conduite original faisant l’objet d’un dépôt de brevet / Lipid accumulation by the yeast Yarrowia lipolytica wT was characterized by dynamic and systemic analysis of different metabolic states in a microbial culture under fully controlled environmental conditions with high cell concentration and under two different strategies:Glucose as the substrate and phosphorus limitation as an inducer of lipid accumulation, an original strategy for lipid accumulation in Y. lipolytica wT.A co-substrate strategy with glucose and oleic acid and without any nutritional limitation.These strategies allowed showing the following points:- Phosphorus limitation triggers a lipid accumulation and a non-transient accumulation of reserve polysaccharide that can be consumed by biomass when necessary, contrary to nitrogen limitation- Phosphorus rate in catalytic biomass shows great variations. Catalytic growth rate cannot be governed by phosphorus input. - Phosphorus has a role in regulating cellular glucose uptake and allows avoiding citric acid production due to overflow of carbon input over a large range of C/P ratios (0 to 8000 Cmol.mol-1)- Maximum capacity of reserve carbon accumulation in Y. lipolytica wT is similar for any culture strategy tested (under nitrogen limitation, phosphorus limitation or with glucose and oleic acid co-substrates) and is equal to 0,5 Cmol/CmolX-1. There is an unknown phenomenon of carbon regulation limiting reserve carbon accumulation in Y. lipolytica wT. Results allowed identifying key points in reserve carbon accumulation in this particular yeast strain and suggesting an original process, claim of a patent

Thesis (MScEng)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Biosurfactants are microbially produced molecules that show excellent surface-active properties. Bacillus subtilis ATCC 21332 produces the biosurfactant, surfactin, which exhibits antimicrobial activity against bacteria as well as fungi. Although antimicrobial activity has been exhibited by a number of bacterially produced biosurfactants, notably the rhamnolipid from the pathogen Pseudomonas aeruginosa, the GRAS status B. subtilis makes the use of this organism preferable for large scale bioprocesses. The objectives of this study were to: (1) evaluate the effect of different nutrient conditions on growth and surfactin production; (2) evaluate the growth of B. subtilis ATCC 21332 and associated surfactin production on a hydrocarbon substrate; (3) evaluate the antimicrobial activity of surfactin against Mycobacterium aurum, and (4) to establish whether active growth of B. subtilis ATCC 21332 and associated surfactin production can be extended during fed-batch culture. B. subtilis ATCC 21332 was grown on low-nitrate; phosphate-limited and nutrient rich media with glucose as substrate during shake flask culture. Nitrate, phosphate, glucose and surfactin were quantified by HPLC analyses and growth via CDW and optical density measurements. Growth and surfactinproduction were further evaluated during shake flask cultureon a hydrocarbon substratereplacing the glucose in the nutrient rich medium with an equivalent amount of n-hexadecane. The antimicrobial activity was quantified by growth inhibition of M. aurum. Bioreactor batch and fed-batch studies were conducted to evaluate growth and surfactin production under controlled conditions. The fed-batch experiments included four constant dilution rate (D=0.40h-1; D=0.15h-1; D=0.10h-1 and D=0.05h-1) and two constant feed rate (F=0.40L/h and F=0.125L/h) fed-batch strategies. The nutrient rich medium was used for these experiments and also as the feed medium for fed-batch experiments. A CDW of 12.6 g/L was achieved in the nutrient rich medium during shake flask culture and was 2.5- and 1.6-fold higher than that achieved in the phosphate-limited medium and the lownitrate medium respectively. A surfactin concentration of 652 mg/L was achieved in the nutrient rich medium, while a maximum surfactin concentration of 730 mg/L was achieved in the phosphate-limited medium. A surfactin concentration of only 172 mg/L was achieved in the low-nitrate medium. Subsequently, growth and surfactin production were evaluated on n-hexadecane as sole carbon source. After inoculation, the CDW did not increase over a period of 119 h, which indicated that B. subtilis ATCC 21332 was unable to utilize n-hexadecane for growth and surfactin production. The maximum CDW (27 g/L) and maximum surfactin concentration (1737 mg/L) achieved in the bioreactor batch experiments were 2.1- and 2.6-fold higher respectively than that achieved in the nutrient rich medium during shake flask experiments. These results served as a benchmark for further fed-batch experiments. During the fed-batch phase of the D=0.40h-1 experiment, the biomass further increasedby 9 g/h, which was 3.5-, 3.1- and 5.3-fold higher compared to the fed-batch phases of the D=0.15h-1, D=0.10h-1 and D=0.05h-1 experiments respectively. Similarly, the biomass increased by 10.7 g/h during the fed-batch phase of the F=0.40L/h experiment, which was 4.6-fold higher than that of the F=0.125L/h experiment. The average rate of surfactin production was 633 mg/h during the fed-batch phase of the D=0.40h-1 experiment, 29.4-, 5.4- and 34.2-fold higher compared to the fed-batch phases of the D=0.15h-1, D=0.10h-1 and D=0.05h-1 experiments respectively. Analogously, the average rate of surfactin production (544 mg/h) of the F=0.40L/h experiment was 9.4 fold higher than that of the F=0.125L/h experiment. The antimicrobial assay showed that surfactin inhibits M. aurum growth. An inhibition zone diamater of 4mm was measured at a surfactin concentration of 208 mg/L, which linearly increased to 24mm at a surfactin concentration of 1662 mg/L. High feed flow rate strategies achieved higher rates of biomass increase and surfactin production and will thus decrease the production time required for large scale surfactin production.The antimicrobial activity of surfactin against M. aurum indicates that this biosurfactant has the potential to be used against M. tuberculosis, and as such has the potential to be used in the medical industry to reduce the spread of this, and other deadly diseases. / AFRIKAANSE OPSOMMING: Biosurfaktante is oppervlak-aktiewe molekules wat deur sekere mikro-organismes geproduseer word. Bacillus subtilis ATCC 21332produseer ‘n biosurfaktant genaamd surfactin, wat antimikrobiese eienskappe toon teen bakterieë sowel as fungi.Menige bakterieël geproduseerde biosurfaktante toon antimikrobiese eienskappe, vernaam die rhamnolipied van die patogeen Pseudomonas aeruginosa, maar die algemene veiligheids-status van B. subtilis gee voorkeur aan hierdie organisme vir grootskaalse bioprosesse. Die doelwitte van hierdie studie was: (1) om die effek van verskillende medium samestellings (in terme van voedingstowwe) ten opsigte van bakteriële seldigtheid en surfactin-produksie te evalueer; (2) om die bakteriële seldigtheid van B. subtilis ATCC 21332 en geassosieerde surfactin produksie vanaf ‘n alkaan-substraat te evalueer; (3) om die antimikrobiese aktiwiteit van surfactin teen Mycobacterium aurum te evalueer; (4) om vas te stel of die aktiewe groei van B. subtilis ATCC 21332 en geassosieerde surfactin-produksie gedurende voer-lot kultuur verleng kan word. B. subtilis ATCC 21332 was op lae-nitraat; fosfaat-beperkte en voedingstofryk-media met glukose as substraat in skudflesse gekultiveer. Nitraat, fosfaat, glukose en surfactin was deur hoëdruk vloeistofchromatografie gekwantifiseer en die seldigtheid deur middel van seldroëmassa en optiese digtheid metings. Verder was die groei van B. subtilis, en geassosieerde surfactin produksie, vanaf ‘n alkaan-substraat in skudflesse ge-evalueer deur die glukose in die voedingstofryke medium met ‘n ekwivalente hoeveelheid van n-heksadekaan te vervang. Die antimikrobiese aktiwiteit van surfactin was deur die geїnhibeerde groei van M. aurum gekwantifiseer. Bioreaktor lot en voer-lot studies was uitgevoer om die groei en surfactin produksie onder beheerde toestande te evalueer. Die voer-lot eksperimente het vier konstante verdunningstempos (D=0.40h-1; D=0.15h-1; D=0.10h-1 en D=0.05h-1) en twee konstante voertempos (F=0.40L/h and F=0.125L/h) ingesluit. Die voedingstofryke medium was vir hierdie eksperimente en ook as die voermedium vir dievoer-lot eksperimente gebruik. ‘n Seldigtheid van 12.6 g/L is bereik gedurende skudfleskultuur in die voedingstofryk-media en was 2.5- en 1.6-voud hoër as die seldigthede wat in die fosfaat-beperkte en lae-nitraat media bereik is. ‘n Surfactin konsentrasie van 652 mg/L is bereik in die voedingstofryke medium, terwyl ‘n maksimum surfactin konsentrasie van 730 mg/L in die fosfaat-beperkte medium bereik is. ‘n Surfactin konsentrasie van slegs 172 mg/L is in die lae-nitraat medium bereik.Hierna was bakteriële seldigtheid en surfactin produksie geuvalueer met slegs n-heksadekaan as die enigste koolstof bron. Die bakteriële seldigtheid het geen verandering getoon na inokulasie nie, wat aangedui het dat B. subtilis ATCC 21332 nie die vermoë beskik om n-heksadekaan vir groei en surfactin produksie te gebruik nie. Die maksimum seldigtheid (27 g/L) en maksimum surfactin konsentrasie (1737 mg/L) bereik in die bioreaktor lot eksperimente was 2.1- en 2.6-voud hoër onderskeidelik as dit bereik in die voedingstofryke medium gedurende skudfles eksperimente. Hierdie resultate dien as ‘n basis vir verdere voer-lot eksperimente. Gedurende die voer-lot fase van die D=0.40h-1 het die biomassa verder verhoog teen 9 g/h, wat 3.5-, 3.1- en 5.3-voud hoër was as dit van die D=0.15h-1, D=0.10h-1 en D=0.05h-1 eksperimente onderskeidelik. Die biomassa het soortgelyk tydens die voer-lot fase van die F=0.40L/h eksperiment teen 10.7 g/h verhoog, wat 4.6-voud hoër was as dit van die F=0.125L/h eksperiment. Die gemiddelde tempo van surfactin produksie was 633 mg/h gedurende die voer-lot fase van die D=0.40h-1 eksperiment, 29.4-, 5.4- en 34.2-voud hoër vergeleke met die voer-lot fases van die D=0.15h-1, D=0.10h-1en D=0.05h-1 eksperimente onderskeidelik. Die gemiddelde tempo van surfactin produksie (544 mg/L) was soortgelyk 9.4-voud hoër gedurende die voer-lot fase van die F=0.40L/h eksperimente, vergeleke met die die F=0.125L/h eksperiment. Die antimikrobiese toetse van surfactin teen M. aurum het positief getoets, wat aandui dat surfactin die groei van hierdie organisme inhibeer. ‘n Inhibisie sone deursnee van 4mm was gemeet teen ‘n surfactin konsentrasie van 208 mg/L, wat lineêr verhoog het tot 24 mm teen ‘n surfactin konsentrasie van 1662 mg/L. Hoë voertempo strategieë het hoër biomassa verhogingstempos en surfactin produksie tempos getoon en sal dus die produksietyd aansienlik verkort tydens grootskaalse surfactin produksie. Die antimikrobiese aktiwiteit van surfactin teen M. aurum toon dat hierdie biosurfaktant die vermoë het om gebruik te word teen M. tuberculosis. Daarom het surfactin die potensiaal om gebruik te word in die mediese industrie om die verspreiding van Tuberkulose, en ander dodelike patogene, te voorkom.

O presente trabalho teve como objetivo o estudo do processo fermentativo de produção de retamicina, em biorreatores de bancada, utilizando-se a linhagem mutante Streptomyces olindensis So20, visando a obtenção de elevadas quantidades deste antibiótico, em processo descontínuo alimentado (fed-batch) e contínuo, bem como a implementação de técnicas de análise de imagens para a caracterização morfológica do microrganismo durante os cultivos. Os ensaios descontínuos alimentados visaram o controle da velocidade específica de crescimento durante a alimentação em três valores distintos (0,03 1/h, 0,10 1/h e 0,17 1/h), por meio do emprego de vazões exponenciais de alimentação. Foram realizados três conjuntos de ensaios descontínuos alimentados. No primeiro conjunto, foi verificada a influência da concentração de nutrientes utilizada na alimentação, mantendo-se a velocidade específica de crescimento em 0,03 1/h, durante a alimentação. De uma maneira geral, o aumento da concentração de nutrientes na alimentação, levou a um aumento na produção do antibiótico. No segundo conjunto de ensaios, foi estudada a influência da velocidade específica de crescimento sobre a produção de retamicina, empregando-se como alimentação o meio R5 modificado com apenas a concentração de glicose quadruplicada. Nesse conjunto de ensaios, ocorreu limitação de outros nutrientes, o que prejudicou o controle da velocidade específica de crescimento durante a alimentação. No entanto, essa limitação nutricional mostrou-se importante para a produção do antibiótico. No terceiro conjunto de ensaios, foi novamente estudada a influência da velocidade específica de crescimento sobre a produção de retamicina, empregando-se como alimentação o meio R5 modificado, agora com as concentrações de todos os nutrientes quadruplicadas, o que evitou limitações nutricionais e permitiu o controle da velocidade específica de crescimento durante a alimentação. Estes ensaios mostraram que o processo de produção de retamicina é favorecido pela manutenção de baixas velocidades específicas de crescimento, por ser este um metabólito secundário. A maior produção de retamicina ocorreu quando a velocidade específica de crescimento foi fixada em 0,03 1/h, durante a alimentação. Nos ensaios contínuos realizados, foi estudada a influência da vazão específica de alimentação (D) e, conseqüentemente, da velocidade específica de crescimento (µx) sobre a produção do antibiótico retamicina. Foram, realizados quatro ensaios contínuos, variando-se D entre 0,03 1/h e 0,30 1/h. A máxima produção de retamicina (tanto em termos de concentração, como em termos de produtividade e produção específica em retamicina total) ocorreu para D = 0,05 1/h. O aumento de D levou a uma diminuição da produção do antibiótico, a qual cessou para D = 0,30 1/h, mostrando um comportamento típico de um metabólito secundário. Foram utilizadas técnicas de análise de imagens na caracterização morfológica do microrganismo. O cisalhamento mostrou-se um importante fator no rompimento de pellets e na formação de clumps e hifas livres, principalmente nos ensaios contínuos. Em geral, a maior produção do antibiótico esteve associada a altas porcentagens de clumps no meio de cultivo. / The objective of the present work was to study retamycin production in fed-batch and continuous cultivations of Streptomyces olindensis So20, a mutant strain, in order to obtain high antibiotic concentrations and analyse microorganism morphology employing image analysis techniques. The fed-batch runs were performed in order to control the specific growth rate in three different values, during feed (0.03 1/h, 0.10 1/h, and 0.17 1/h), employing exponential feed rates. Three sets of fed-batch runs were carried-out. The first set, feed composition was varied, controlling the specific growth rate in a low value (0.03 1/h) during feed. In general, higher nutrient concentrations in the feed led to higher antibiotic production. In the second set of fed-batch runs, the control of the specific growth rate in three different values during feed was studied. The feed was composed by R5 Modified medium, with four-fold the glucose concentration only, which led to nutrient limitation during runs. This nutrient limitation prejudiced the specific growth rate control, but led to high antibiotic production. This fact showed that nutrient limitation is an important factor in retamycin production. In the third set of fed-batch runs, the control of the specific growth rate in three different values during feed was studied. In these runs, the feed was composed by R5 Modified medium, with four-fold all the nutrient concentrations, which avoided nutrient limitation, during runs. In these runs, the control of the specific growth rate during feed was possible. Results showed that retamycin production is favored by the maintenance of low specific growth rates, since it is a secondary metabolite. Higher antibiotic production was achieved controlling the specific growth rate in 0.03 1/h, during feed. In the continuous runs, the influence of the dilution rate (D), and consequently, of the specific growth rate (µx) on the retamycin production was studied. Four continuous runs were performed, varying D in the range from 0.03 1/h to 0.30 1/h. The highest antibiotic production (in terms of retamycin concentration, retamycin productivity and retamycin specific production) was obtained at D = 0.05 1/h. The increase in the dilution rate led to lower antibiotic production, which ceased at D = 0.30 1/h, showing a typical behavior of a secondary metabolite. Image analysis was used to assess the morphological characteristics of the microorganism. Shear showed to be an important factor in pellet disruption and in clump and free filaments formation, mainly in the continuous runs. In general, higher antibiotic production was obtained with the growth mainly in the form of clumps.

O presente trabalho teve como objetivo avaliar a influência da intensidade de agitação e tipo de impelidor, bem como a estratégia de alimentação, em reator anaeróbio operado em batelada seqüencial, escala piloto (\'da ordem de\' 1 \'M POT.3\'), com agitação mecânica e em duas configurações: uma com biomassa granulada (ASBR) e outra com biomassa imobilizada em suporte inerte de espuma de poliuretano (ASBBR), aplicadas ao tratamento de esgoto doméstico. No estudo da intensidade de agitação e tipo de impelidor, para cada reator foram avaliadas três tipos de impelidores (turbina de pás planas, turbina de pás inclinadas 45º e hélice) associados a duas intensidades de agitação (40 e 80 rpm), obtendo uma combinação de 6 (seis) condições experimentais. A combinação da intensidade de agitação e tipo de impelidor que apresentou melhor desempenho no processo foi utilizado no estudo da estratégia de alimentação, na qual foi avaliada as seguintes condições: batelada alimentada durante 25%, 50%, 75% e 100% do ciclo. Os resultados obtidos permitiram concluir que: no ASBBR o aumento da intensidade de agitação de 40 rpm para 80 rpm permitiu uma melhoria nos fluxos de transferência de massa e, portanto, aumentou a velocidade de consumo de substrato; no ASBR o aumento da intensidade de agitação de 40 rpm para 80 rpm proporcinou uma desestabilização do sistema, provavelmente por causa da ruptura dos grânulos provocada pela maior agitação; os sistemas operados com impelidor do tipo hélice apresentaram vantagens, tais como: melhor eficiência de remoção de sólidos, maior valor da constante cinética de primeira ordem (melhor fluxo de transferência de massa e conseqüentemente maior consumo de substrato) e maior produção de alcalinidade, ou seja, maior estabilidade para o sistema; tanto o sistema ASBR como o ASBBR quando operados nas condições de batelada típica, batelada alimentada durante 50% e 75% do ciclo apresentaram melhores desempenhos no processo de tratamento, mostrando a flexibilidade operacional dos reatores anaeróbios operados em bateladas seqüenciais; e comparando os sistemas ASBBR e ASBR verificou-se que estes apresentaram comportamentos similares em todas as condições de operação de batelada alimentada avaliadas, não sendo possível, estatisticamente apontar um sistema com melhor desempenho. / The objective of this work was to assess the effect of the stirring speed, the type of impeller and the feed strategy in a mechanically stirred pilot-scale (\'da ordem de\' 1 \'M POT.3\') anaerobic sequencing batch reactor to two configurations: a containing granulated biomass (ASBR) and the other containing immobilized biomass (AnSBBR). Domestic wastewater was treated in 8-h cycles. Three impeller types (turbine with six-flat blades, turbine with six 45º-inclined blades and helix with three blades) were assessed at two different stirring speeds (40 and 80 rpm), totaling six experimental conditions. The stirring speed and the impeller that resulted in the best combination was used in work of the feed strategy . The reactors were operated at room temperature at four different feed strategies (fed batch during 25%, 50% and 75% of the cycle, and conventional fed-batch). The results allowed conclude that: in the AnSBBR increasing the stirring speed from 40 rpm to 80 rpm showed to improve mass transfer, with consequent increase in substrate consumption; in the ASBR increasing the stirring speed from 40 rpm to 80 rpm showed desestabilization in system, because of the disruption caused in the granules witth greater agitation; operation with the helix impeller showed some advantages over the turbine impellers, such as: improved efficiency in solids removal, higher value of the first order kinetic constant and higher alkalinity production; both for the ASBR as for the ASBBR the best performance in wastewater treatment was obtained when the reactors were operated at conventional batch, fed-batch during 50% and 75% of the cycle; no significant difference in performance was observed among these three conditions. Despite poor performance of the conventional fed-batch and fed-batch during 25% of the cycle compared to the other conditions, both these conditions presented operational stability. Hence, the anaerobic sequencing batch reactors presented operational flexibility as far as feed strategy is concerned.

A Cianobactéria Spirulina platensis representa uma fonte de proteínas e ácidos graxos que a tornam importante como suplemento alimentar. A fonte de nitrogênio é um nutriente que exerce influência em seu metabolismo. Neste trabalho, observou-se o crescimento de S. platensis e a composição da biomassa obtida, com a utilização de cloreto de amônio como fonte de nitrogênio por processo descontínuo alimentado. Com a utilização de planejamento fatorial, foi realizado o estudo do tempo de alimentação do cloreto de amônio e da intensidade luminosa no cultivo da S. platensis. Os resultados foram avaliados com auxílio da metodologia de superfície de resposta. Menores teores de proteínas e lipídios na biomassa final foram encontrados nos cultivos submetidos a maiores intensidades luminosas. A condição ótima calculada, obtida pela análise estatística, para a obtenção da concentração celular máxima (Xm) e fator de conversão de nitrogênio em células (YX/N) foi encontrada no cultivo sob intensidade luminosa de 13 klux e tempo de alimentação de 17,2 dias. Nessas condições foram obtidos Xm igual 1771 ± 2,3 mg/L e YX/N igual a 5,7 ± 0,17 mg/mg, 3,4% e 4,0% menores que os valores máximos estimados pelo modelo matemático, respectivamente. Maiores produtividades foram obtidas nos cultivos submetidos a maiores intensidades luminosas e menores tempos de alimentação. / Cyanobacterium Spirulina platensis represents a protein and fatty acid source that becomes it important as food supplement. The nitrogen source is a nutrient that influences in its metabolism. In this work, it was observed the S. platensis growth and biomass composition using ammonium chloride as nitrogen source in fed-batch process. Using factorial experimental design, it was carried out the study of the ammonium chloride feeding time and light intensity in the S. platensis culture. The results were evaluated by response surface methodology (RSM). Lower protein and lipids contents were found in cell cultures cultivated at higher light intensity. The predictive optimal condition, obtained by statistic analysis, for maximum cellular concentration (Xm) and nitrogen-cell conversion factor (YX/N) was obtained in the culture grown at 13 klux and feeding time of 17.2 days. In these conditions, Xm of 1771 ± 2.3 mg/L and YX/N of 5.7 ± 0.17 mg/mg were obtained. These values are 3.4 % and 4.0 % lower than that ones predicted by mathematical model, respectively. Higher productivities were observed in the culture grown at higher intensity light and lower feeding time.

O reator em batelada seqüencial com biomassa em suspensão foi submetido a concentrações de N-amoniacal de 125, 250 e 500 mg N/L e em condições de oxigênio dissolvido (OD) no reator de 2 mg 'O IND.2'/L, em ciclos de 2h/2h de anóxico/aeróbio. Em todas as fases, o reator foi operado como batelada alimentada. Na condição de 125 mg N/L obteve-se eficiência de remoção de 87% de N, tendo o nitrato sido o principal produto da nitrificação. Na condição de 250 mg N/L, obteve-se eficiência de remoção de N de 84%, com predominância de nitrito como principal produto da nitrificação e com ocorrência de nitrificação e desnitrificação simultânea durante os dois primeiros ciclos aeróbios. Na condição de 500 mg N/L, as condições de concentração de OD de 2 mg 'O IND.2'/L e aeração intermitente a cada 2h não foram suficientes para promover a remoção total de nitrogênio amoniacal. Foram feitas alterações: ciclos de 2h anóxico e 9h aeróbios, com concentração média de 2,8 mg 'O IND.2'/L, que resultaram em eficiências de remoção de N de 94%, com predominância de nitrito. Foram isoladas cepas desnitrificantes com similaridade de 97% para Thauera mechernichensis e Thauera sp. 27 nas condições operacionais de 125 e 250 mg N/L e de 99% para Ochrobactrum anthropi e Ochrobactrum tritici, na condição operacional de 500 mg N/L. O longo tempo de operação resultou na diminuição da população de bactérias oxidantes de nitrito, podendo ter sido uma das causas que contribuiu para que se criassem condições que levariam à nitrificação via nitrito na concentração de 500 mg N/L. O sucesso na prevenção da inibição do processo por amônia livre foi atribuído à adoção das condições operacionais do reator, que foi operado sob aeração intermitente e batelada alimentada. / The sequential batch reactor with suspended bioma was subjected to ammonium concentrations of 125, 250 and 500 mg N/L, oxygen dissolved (OD) concentrations of 2 mg 'O IND.2'/L, 2h/2h of anoxic/aerobic steps. The reactor was operated under fed-batch feeding. At the operational condition of 125 mg N/L, the mean nitrogen removal efficiency of 87% was obtained and nitrate was the main nitrification product. At operational condition of 250 mg N/L, was gotten nitrogen removal efficiencies of 84% and nitrite was the predominant form of nitrogen and simultaneous nitrification and denitrification occurred during the two first aerobic steps of the cycle. At 500 mg N/L, the operating conditions imposed to the reactor (OD concentrations of 2 mg 'O IND.2'/L and intermittent aeration of 2h) did not lead to complete nitrification during the aerated steps, thus affecting nitrogen removal. The conditions were altered increasing the aerated steps to 9 hours, with OD concentration of 2,8 mg 'O IND.2'/L, and keeping the duration of the anoxic steps in 2 hours. Under such conditions, the mean nitrogen removal efficiency attained 94% and nitrite was the predominant oxidized nitrogen specie. It was isolated denitrifiers with similarity of 97% for Thauera mechernichensis and Thauera sp. 27 at the operational conditions of 125 and 250 mg N/L and of 99% for Ochrobactrum anthropi and Ochrobactrum tritici, at operational condition of 500 mg N/L. The long term operation resulted in the decrease of nitrite oxidizers populations and this was probably the main factor contributing for the creation of conditions for the partial nitrification via nitrite to prevail during the operation at ammonium concentration of 500 mg N/L. The success in preventing free ammonia inhibition was attributed to the adoption of intermittent aeration and fed batch operation.

A toxina tetânica é uma proteína sintetizada pelo bacilo Clostridium tetani que após destoxificação através da ação do formol, continua apresentando propriedades antigênicas e imunogênicas, obtendo a denominação toxóide tetânico. A síntese dessa proteína ocorre quando esse bacilo encontra-se na sua forma vegetativa e em meio de cultura específico relativamente complexo contendo glicose e peptonas. O efeito simultâneo de diferentes níveis de glicose (Go) e N-Z Case TT® (NZo) como fontes de carbono e nitrogênio, respectivamente, na produção de toxina tetânica foi investigada nesta primeira parte do trabalho em cultivo estático por meio de planejamento fatorial em estrela com cinco níveis e avaliado por metodologia de superfície de resposta, com a finalidade de otimização do processo. O valor mais alto de toxina tetânica encontrado, correspondente a Go = 9,7 g/L e NZo = 43,5 g/L, foi 79% maior que aqueles obtidos em condições padrões de cultivo (Go = 8,0 g/L e NZo = 25,0 g/L). Também foram realizados cultivos de C. tetani utilizando o processo descontínuo alimentado com diferentes protocolos para a correção da concentração de glicose no meio de cultivo ao longo do tempo em diferentes concentrações iniciais de N-Z Case TT®. Dois grupos de ensaios foram executados: a) experimentos realizados com a correção da concentração de glicose para 3,0 g/L nos instantes 16, 56 e 88 h e b) experimentos com correção inicial da concentração de glicose para 3,0 g/L e após esta cair para 1-1,5 g/L. O primeiro protocolo de correção da concentração de glicose e NZo = 50,0 g/L foram as melhores condições para obtenção de toxina tetânica. Nestas condições, o título de toxina tetânica foi 300% maior que aqueles obtidos em cultivos padrão. / The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivations of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (Go) and N-Z Case TT® (NZo) as carbon and nitrogen sources, respectively, on the production of tetanus toxin, have been investigated in this work in static cultivations by means of a five-levels star-shaped experimental design and evaluated by Response Surface Methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin, achieved at Go = 9.7 g/L and NZo = 43.5 g/L, was 79% higher than that obtained with standard cultivations (Go = 8.0 g/L and NZo = 25.0 g/L). Also, there were carried out cultivations of C. tetani using fed-batch process at different protocols to correct the glucose concentration in the cultivation medium along the time at different initial N-Z Case TT® concentrations (NZo). Two series of runs were performed: a) experiments with the correction of the glucose concentration to 3.0 g/L in the times 16, 56 and 88 hours and b) experiments with initial correction of the glucose concentration to 3.0 g/L and after it to drop to 1-1,5 g/L. The former protocol to correct the glucose concentration and NZo = 50.0 g/L were the best condition to obtain tetanus toxin. In these conditions, the yield of tetanus toxin was 300% higher than that obtained with standard cultivations.