Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

Arthrospira (Spirulina) platensis vem sendo cultivada para a produção de biomassa pelos altos conteúdos de proteína, ácidos graxos poliinsaturados e vitaminas. O nitrogênio é um nutriente que exerce influência em seu metabolismo, e o uso de fontes nitrogenadas de baixo custo pode contribuir para a viabilização da produção de A. platensis. Foi verificado o crescimento do microrganismo empregando uréia como fonte de nitrogênio por meio de processo contínuo, visando evitar níveis inibitórios de amônia no meio. Verificou-se a influência da vazão específica de alimentação (D, 0,04 a 0,44 dia-1) utilizando duas concentrações de uréia (N0) de 0,5 e 5 mM no meio de alimentação. Para N0=0,5mM, a maior produtividade em células (PX) obtida foi 201,5 mg.L-1.dia-1com D=0,44d-1; nessas condições a concentração celular em regime permanente (XP) foi 458 mg.L-1, obtendo-se uma biomassa com um teor de proteínas de 27,2%. Para N0=5mM, o máximo valor de PX encontrado foi 148,2 mg.L-1.dia-1, com D=0,12d-1; nessa vazão de alimentação, o XP foi 1235 mg.L-1 e o teor protéico na biomassa (71,9%) o mais alto encontrado. / Arthrospira platensis is being cultivated for biomass production because of its high content of proteins, polyunsaturated fatty acids and vitamins. Nitrogen is a nutrient which has a big influence in its metabolism, and the use of low cost sources of this element could contribute for the feasibility of A. platensis production. It was verified the growth of the microorganism using urea as a nitrogen source by a continuous process, to avoid ammonia inhibitory levels into the culture. It was evaluated the influence of the specific dilution rate (D, 0.04 to 0.44 day-1) in the culture applying two urea concentrations (N0) 0.5 and 5mM in the feeding medium. For N0=0,5mM, the highest cell productivity (PX) obtained was 201.5 mg.L-1.day-1using D=0.44d-1; under these conditions the cell concentration in steady state (XP) was 458 mg.L-1, obtaining a biomass with a protein content of 27.2%. For N0=5mM, the highest PX value was 148.2 mg.L-1.day-1, with D=0.12 day-1; under this dilution rate, the XP was 1235 mg.L-1 and its protein content (71.9%) was the highest achieved.

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

Arthrospira (Spirulina) platensis vem sendo cultivada para a produção de biomassa pelos altos conteúdos de proteína, ácidos graxos poliinsaturados e vitaminas. O nitrogênio é um nutriente que exerce influência em seu metabolismo, e o uso de fontes nitrogenadas de baixo custo pode contribuir para a viabilização da produção de A. platensis. Foi verificado o crescimento do microrganismo empregando uréia como fonte de nitrogênio por meio de processo contínuo, visando evitar níveis inibitórios de amônia no meio. Verificou-se a influência da vazão específica de alimentação (D, 0,04 a 0,44 dia-1) utilizando duas concentrações de uréia (N0) de 0,5 e 5 mM no meio de alimentação. Para N0=0,5mM, a maior produtividade em células (PX) obtida foi 201,5 mg.L-1.dia-1com D=0,44d-1; nessas condições a concentração celular em regime permanente (XP) foi 458 mg.L-1, obtendo-se uma biomassa com um teor de proteínas de 27,2%. Para N0=5mM, o máximo valor de PX encontrado foi 148,2 mg.L-1.dia-1, com D=0,12d-1; nessa vazão de alimentação, o XP foi 1235 mg.L-1 e o teor protéico na biomassa (71,9%) o mais alto encontrado. / Arthrospira platensis is being cultivated for biomass production because of its high content of proteins, polyunsaturated fatty acids and vitamins. Nitrogen is a nutrient which has a big influence in its metabolism, and the use of low cost sources of this element could contribute for the feasibility of A. platensis production. It was verified the growth of the microorganism using urea as a nitrogen source by a continuous process, to avoid ammonia inhibitory levels into the culture. It was evaluated the influence of the specific dilution rate (D, 0.04 to 0.44 day-1) in the culture applying two urea concentrations (N0) 0.5 and 5mM in the feeding medium. For N0=0,5mM, the highest cell productivity (PX) obtained was 201.5 mg.L-1.day-1using D=0.44d-1; under these conditions the cell concentration in steady state (XP) was 458 mg.L-1, obtaining a biomass with a protein content of 27.2%. For N0=5mM, the highest PX value was 148.2 mg.L-1.day-1, with D=0.12 day-1; under this dilution rate, the XP was 1235 mg.L-1 and its protein content (71.9%) was the highest achieved.

Depuis la découverte des cheminées hydrothermales, de multiples travaux ont été menés afin d’en étudier la diversité microbienne. Les inventaires moléculaires réalisés ont ainsi mis en évidence une grande diversité d’espèces qui contraste avec la faible proportion (1 %) d’espèces isolées par approche culturale. Une nouvelle approche d’immobilisation cellulaire par inclusion dans une matrice de polymères (gellane et xanthane) a ainsi été développée pour permettre l’étude de ces communautés thermophiles anaérobies marines. Le système, basé sur la formation d’une émulsion entre une solution de polymères inoculée et de l’huile, permet le piégeage de cellules dans des billes de gel de 1 à 2 mm de diamètre. Les conditions optimales d’immobilisation ont été obtenues pour une émulsion réalisée à 80 °C sous agitation (150 tr/min) à partir d’une solution de gellane (2,5 %) et de xanthane (0,25 %) avec 12 g/L de NaCl et 4 g/L de citrate de sodium, bullée à l’azote et réduit au Na2S avant inoculation. Les billes ont montré une bonne résistance mécanique après 5 semaines d’incubation à des pH compris entre 5,4 et 8, des températures allant jusqu’à 90 °C et des concentrations en NaCl et soufre allant jusqu’à respectivement 80 et 5 g/L. Des cultures en batch de Thermosipho sp. AT1272 et Thermococcus kodakarensis KOD1 immobilisées ont permis d’obtenir des concentrations allant jusqu’à 107 cellules/g de billes et 108 cellules/mL de fraction liquide. Une culture en continu réalisée en bioréacteur gas-lift pendant 41 jours à partir d’une communauté synthétique immobilisée composée de 8 souches (hyper)thermophiles a démontré la capacité de l’immobilisation cellulaire à protéger les cellules face à un stress oxique et à les maintenir (3 des 8 souches) dans le bioréacteur jusqu’à ce que les conditions de culture soient propices à leur croissance. La réactivité de la communauté immobilisée face aux changements environnementaux (température) a également été démontrée. Enfin, la culture en continu réalisée pendant 64 jours d’un échantillon immobilisé de diffuseur du site Rainbow a permis la croissance de plusieurs espèces bactériennes et archéennes (Oceanithermus sp., Thermococcus sp.) dont une partie n’a été détectée que dans les billes (Sulfurimonas sp., Nitratifractor sp., Vibrio sp.) par clonage-séquençage. L’ensemble de ces résultats ont permis de valider l’utilisation d’un protocole d’immobilisation par inclusion dans une matrice de polymères pour l’étude des communautés hydrothermales, de leur diversité et de leur dynamique. / Since the discovery of hydrothermal vents, multiple studies have been conducted in order to study microbial diversity. Molecular inventories realized have thus demonstrated a great diversity of species that contrasts with the low proportion (1%) of species isolated by culture approach. A new cell immobilization approach by inclusion in a polymer matrix (gellan and xanthan) has been developed for the study of these thermophilic anaerobic marine communities. The system, based on the formation of an emulsion between an inoculated polymer solution and oil, allows the entrapment of cells in gel beads with a diameter between 1 and 2 mm. The optimal immobilization conditions were obtained for emulsion performed at 80 °C with stirring (150 rpm) with a polymer solution composed of gellan (2.5%) and xanthan (0.25%) with 12 g/L of NaCl and 4 g/L of sodium citrate, bubbled with nitrogen and reduced with Na2S before inoculation. The beads showed a good mechanical stability after a 5-week incubation at pH between 5.4 and 8, temperatures up to 90 °C and NaCl and sulfur concentrations up to respectively 80 and 5 g/L. Batch cultures of immobilized Thermosipho sp. AT1272 and Thermococcus kodakarensis KOD1 yielded concentrations up to 107 cells/g of beads and 108 cells/mL of liquid fraction. A continuous culture performed in a gas-lift bioreactor for 41 days of an immobilized synthetic community composed of 8 (hyper)thermophilic strains demonstrated the capacity of cell immobilization to protect cells from oxique stress and to maintain them (3 of 8 strains) in the bioreactor until having suitable culture conditions for their growth. The reactivity of the immobilized community to environmental change (temperature) was also demonstrated. Finally, the continuous culture performed for 64 days of an immobilized diffuser sample from Rainbow site allowed the growth of several bacterial and archaeal species (Oceanithermus sp., Thermococcus sp.), part of which was detected only in the beads (Sulfurimonas sp., Nitratifractor sp., Vibrio sp.) by cloning-sequencing. All these results have validated the use of an immobilization protocol by inclusion in a polymer matrix for the study of hydrothermal communities, of their diversity and their dynamics.

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

Ce travail de thèse vise à mieux comprendre l'effet de l'environnement buccal sur le comportement d'une bactérie orale probiotique, Streptococcus salivarius K12. La croissance et la maintenance de S. salivarius K12 ont tout d'abord été caractérisées dans une salive artificielle complémentée (CAS) conçue pour l'étude. Dans ce milieu, cette bactérie démontre un taux de croissance élevé et un temps de latence court, mais elle ne produit pas de bactériocines actives. La survie de S. salivarius K12 en phase stationnaire est, en revanche, affectée dans le milieu CAS. Ce phénomène est expliqué par une synthèse moindre des protéines impliquées dans le métabolisme énergétique, dont celui du glycogène. Toutefois, malgré une sensibilité accrue en phase stationnaire, le milieu CAS permet la croissance et la maintenance de S. salivarius K12. Les effets de plusieurs facteurs environnementaux spécifiques de la bouche, sur S. salivarius K12, ont été déterminés en milieu CAS. Ainsi, l'apport de saccharose, conduit à une dégradation de la viabilité. Des enzymes, ajoutées à leur concentration physiologique, affectent également les cellules bactériennes. Le lysozyme accroît la mortalité de S. salivarius par son action sur la paroi bactérienne. La peroxidase améliore sa viabilité en diminuant le potentiel redox du milieu. Le rôle clé du potentiel redox sur S. salivarius K12 est confirmé par l'impact négatif de l'injection d'air enrichi à 5% de CO2, qui accroît le potentiel redox. Enfin, l'amylase a démontré un rôle à la fois positif (augmentation de la biomasse) et négatif (diminution du taux de croissance). En conséquence, les études impliquant des bactéries orales se doivent de prendre en compte ces facteurs environnementaux influant sur les l'état physiologique bactérien. La mise en place de cultures continues respectant les variations de flux salivaire et permettant l'apport périodique de nutriments, tout en combinant l'ensemble des conditions environnementales identifiées précédemment, a permis de simuler la dynamique des conditions buccales. Les résultats démontrent que S. salivarius K12 est bien adapté à ces conditions de culture. Les cellules sont capables de se maintenir à un niveau de cultivabilité constant, malgré la carence nutritionnelle et le lessivage auxquels elles sont soumises. Certains mécanismes moléculaires expliquant cette adaptation ont été caractérisés : activation des voies d'utilisation de sources de carbone alternatives, stockage de l'énergie, augmentation de la compétence génétique naturelle. Finalement, ces travaux ont permis d'identifier certains mécanismes permettant à Streptococcus salivarius K12 de s'adapter à l'environnement buccal, grâce à la mise en place de méthodes d'étude in vitro du comportement des bactéries orales. / This thesis aims to better understand the effect of oral environmental conditions on the behaviorof the probiotic bacteria Streptococcus salivarius K12. Growth and maintenance of S. salivarius K12 have been characterized in a complemented artificial saliva (CAS), designed for this study. In this medium, S. salivarius demonstrated highspecific growth rate and low lag time, but it did not produce active bacteriocins. However, the survival of S. salivarius K12 during stationary phase was affected during fermentation in CAS medium. This was mainly explained by a reduced synthesis of proteins involved in energy and glycogen metabolisms. Thus, despite an increased sensitivity in stationary phase, the "complemented artificial saliva" allowed the growth and maintenance of S. salivarius K12. The effects of several environmental oral factors on S. salivarius K12 were determined in complemented artificial saliva. Adding sucrose decreased cellular viability. Enzymes added to their physiological concentration also affected the bacteria. Lysozyme increased S. salivarius mortality by acting on cellular wall. Peroxidase enhanced viability, by reducing the redox potential. The key role of redox potential on S. salivarius K12 was confirmed by the negative impact of the injection of air containing 5% CO2, which increased redox potential. The amylase demonstrated both positive (biomass increase) and negative roles (reduced growth rate). Consequently, studies involving oral bacteria must integrate these environmental factors that affected the bacterial physiological state. Continuous cultures, taking into account the variations in salivary flow and the periodical supply of nutrients, and combining all environmental conditions previously identified, allowed simulating oral dynamic conditions. From our results, a good adaptation of S. salivarius K12 took place in these culture conditions. Cells were able maintaining a constant level of cultivability despite nutritional starvation and wash out. Some molecular mechanisms explaining this bacterial adaptation have been characterized: activation of alternative carbon sources pathways, energy storage, and increase of natural genetic competence. Finally, this work made it possible identifying some mechanisms used by Streptococcussalivarius K12 to adapt itself to the oral environment, through the establishment of in vitro methods for studying the behavior of oral bacteria.

Μελετήθηκε ο μεταβολισμός της γλυκερόλης στη ζύμη Yarrowia lipolytica ACA–DC 50109 με έμφαση στη μετατροπή της σε λιπίδια και κιτρικό οξύ, μεταβολικά προϊόντα που παρουσιάζουν ιδιαίτερο ενδιαφέρον για τη βιοτεχνολογία. Σε καλλιέργειες που πραγματοποιήθηκαν σε βιοαντιδραστήρα διαλείποντος έργου, επί πολλαπλώς περιοριστικού μέσου, διαπιστώθηκε η ύπαρξη τριών διακριτών φάσεων αύξησης που χαρακτηρίζονται από ιδιαίτερα μορφολογικά και βιοχημικά χαρακτηριστικά: η φάση βιοσύνθεσης κυτταρικής μάζας (κατά την οποία συντέθηκαν 4–4,5 g/l βιομάζας), η ελαιογόνος φάση (κατά την οποία πραγματοποιήθηκε συσσώρευση λιπιδίων 20–22% wt/wt επί ξηρής βιομάζας, 90% wt/wt των οποίων ήταν ουδέτερα) και η φάση παραγωγής κιτρικού οξέος (κατά την οποία εκκρίθηκαν στο περιβάλλον της αύξησης 14–30 g/l κιτρικού οξέος). Κατά τη διάρκεια των ανωτέρω φάσεων η ζύμη διήλθε από διάφορα μορφολογικά στάδια: μικρού μήκους αληθή μυκήλια και ψευδομυκήλια που κυριάρχησαν των κυττάρων ζύμης κατά τη φάση βιοσύνθεσης κυτταρικής μάζας, ευμεγέθη κύτταρα κατά τη φάση της ελαιογένεσης και μικρού μεγέθους κύτταρα ζύμης κατά τη φάση παραγωγής κιτρικού οξέος. Η γλυκερόλη διαπερνά την κυτταροπλασματική μεμβράνη με διευκολυνόμενη διάχυση και καταβολίζεται μέσω των αντιδράσεων της κινάσης της γλυκερόλης – GK και της NAD+ εξαρτώμενης αφυδρογονάσης της 3–P–γλυκερόλης. Την υψηλή ενεργότητα της NAD+ εξαρτώμενης ισοκιτρικής αφυδρογονάσης (NAD+–ICDH) κατά τη διάρκεια της φάσης βιοσύνθεσης κυτταρικής μάζας διαδέχθηκε σημαντική πτώση της ενεργότητάς της, επάγοντας τη λιπογένεση. Απρόσμενη αποδόμηση των αποθεματικών (ουδέτερων) λιπιδίων και σημαντική βιοσύνθεση γλυκολιπιδίων, σφιγγολιπιδίων και φωσφολιπιδίων – Ρ παρατηρήθηκε κατά τη διάρκεια της φάσης παραγωγής κιτρικού οξέος, φάση κατά την οποία η ενεργότητα της GK είχε μειωθεί σημαντικά ενώ η ενεργότητα της NAD+–ICDH είχε σχεδόν μηδενιστεί. Το ελαϊκό οξύ ήταν το κυριότερο λιπαρό οξύ ενώ η φωσφατιδυλχολίνη – PC το κύριο Ρ. Σε συνεχές σύστημα καλλιέργειας επί θρεπτικού υλικού περιοριστικού σε άζωτο, βιοσυντέθηκαν περιορισμένες μόνο ποσότητες λιπιδίων (~10% wt/wt, επί της ξηρής βιομάζας), γεγονός που μπορεί αποδοθεί στο ότι δεν υπήρχε μια περιοχή του ειδικού ρυθμού αραίωσης (D, h–1) στην οποία τα ένζυμα – κλειδιά που εμπλέκονται στη λιπογένεση (όπως η ΑΤΡ:κιτρική λυάση – ATP:CL και το μηλικό ένζυμο – ME) να παρουσιάζουν συγχρόνως υψηλές ενεργότητες, ενώ η ενεργότητα της NAD+–ICDH μειώθηκε, όχι όμως σημαντικά, στους χαμηλούς D. Η ενεργότητα της ATP:CL χαρακτηρίστηκε από υψηλές τιμές (60–300 Units/mg DW) σε D 0,033 h–1 ενώ οι μέγιστες τιμές ενεργότητας του ME (650 Units/mg DW) εμφανίστηκαν σε D=0,104 h–1. Τα λιπίδια της ζύμης ήταν περισσότερο ακόρεστα σε ενδιάμεσες τιμές D. Σε όλους τους D η φωσφατιδυλαιθανολαμίνη – PE, η φωσφατιδυλινοσιτόλη – PI και η PC αντιπροσωπεύουν τις κυριότερες κλάσεις των Ρ. Όσον αφορά τη μορφολογία της ζύμης, βρέθηκε ότι σε D<0,055 h–1 επικρατούσαν αληθή μυκήλια και ψευδομυκήλια ενώ σε D 0,055 h–1 παρατηρήθηκαν μόνο κύτταρα ζύμης. Σε πειράματα που πραγματοποιήθηκαν επί θρεπτικού υλικού περιοριστικού σε άζωτο, σε D=0,026 h–1, σε διαφορετικές συγκεντρώσεις διαλυμένου οξυγόνου – DO παρατηρήθηκε αυξημένο ποσοστό του κλάσματος των Ρ επί των ολικών λιπιδίων στις ακραίες σε τιμές DO ( 70% και 7%). Ανεξάρτητα των τιμών DO η PC ήταν η κλάση με το μεγαλύτερο ποσοστό, ακολουθούμενη από την PI και PE. Ειδικότερα το ποσοστό της ΡΕ παρουσιάστηκε ιδιαίτερα αυξημένο σε ενδιάμεσες τιμές DO (20% και 30%). Σε DΟ 50% επικρατούσαν αληθή μυκήλια και ψευδομυκήλια ενώ σε DΟ 50% εμφανίστηκαν στην καλλιέργεια περισσότερα κύτταρα ζύμης. Σε πειράματα που πραγματοποιήθηκαν σε D=0,026 h–1 βρέθηκε ότι ο περιορισμός της αύξησης από ιχνοστοιχεία όπως το μαγνήσιο και το ασβέστιο τα οποία εμπλέκονται σε πολλαπλές κυτταρικές λειτουργίες, είχαν δυσμενή επίδραση στη φυσιολογία της ζύμης, ωστόσο η σύσταση των λιπιδίων σε λιπαρά οξέα δεν επηρεάστηκε από τη φύση του περιοριστικού για την αύξηση παράγοντα. Η παρούσα διδακτορική διατριβή φιλοδοξεί να συμβάλει στη μελέτη της φυσιολογίας των ελαιογόνων μικροοργανισμών και στη χρήση της γλυκερόλης ως υποστρώματος σε μελλοντικές βιοτεχνολογικές εφαρμογές. / In this thesis the metabolism of glycerol in Yarrowia lipolytica ACA–DC 50109, with emphasis on glycerol conversion into value–added biotechnological products, such as single cell oils and citric acid, was studied. The growth of Y. lipolytica was studied in bioreactor batch cultures in multiple limited medium and three distinct phases were identified during growth cycle. In each phase, yeast cells were characterized by specific morphological and biochemical features: biomass formation phase (in which 4–4.5 g/l of biomass were synthesized), lipogenic phase (in which 20–22% lipids wt/wt in dry weight were accumulated in biomass, containing 90% wt/wt neutral lipids) and citric acid production phase (in which 14–30 g/l of citric acid were secreted in the growth environment). Distinct cellular forms of Y. lipolytica were developed during the above phases: in biomass formation phase short true mycelia and pseudo–mycelia were predominant while a few yeast–like cells were observed, in lipogenic phase large obese cells were predominant and in citric acid production phase cells size was diminished. Glycerol passes into the microbial cell by facilitated diffusion. Y. lipolytica successfully converts glycerol via phosphorylation pathway, in which glycerol kinase (GK) and glycerol–3–P–dehydrogenase are implicated. Though high activity of NAD+ dependent isocitric dehydrogenase (NAD+–ICDH) was detected during biomass formation phase, this activity was significantly decreased afterwards inducing lipogenesis. Surprisingly, storage (neutral) lipid turnover and synthesis of glycolipids, sphingolipids and phospholipids – Ρ simultaneously occurred with citric acid production, and happened when GK activity was considerably reduced and NAD+–ICDH activity was minimised. Oleic acid was the major fatty acid in all lipid fractions and phosphatidylcholine – PC was the main Ρ. In continuous culture in nitrogen limited medium Y. lipolytica accumulated low quantities of lipids (~10% w/w, in dry weight), maybe due to the fact that there was not a region of specific dilution rate (D, h–1) in which the key–enzymes that are implicated in lipogenesis (i.e. ΑΤΡ:citrate lyase – ATP:CL and malic enzyme – ME) presented simultaneously high activity while NAD+–ICDH activity was insignificantly decreased in low D. ATP:CL presented high activity (60–300 Units/mg DW) in D 0,033 h–1 while ME presented maximum activity (650 Units/mg DW) in D=0,104 h–1. Lipids were more unsaturated in intermediate D values while phosphatidylethanolamine – PE, phosphatidylinositol – PI and PC are the main Ρ classes. As far as the morphology is concerned, in D<0,055 h–1 short true mycelia and pseudo–mycelia were predominant in culture medium while in D 0,055 h–1 only yeast cells were observed. In experiments performed in nitrogen limited medium in D=0,026 h–1 in different dissolved oxygen – DO concentrations, it was found that in extreme DO values ( 70% and 7%) the percentage of P was increased. Independently the DO concentration PC was the main class followed by PI and PE. The morphology of Y. lipolytica was influenced by the different concentration of DO and it was observed that in DΟ 50% short true mycelia and pseudo–mycelia were predominant in culture medium while in DΟ 50% more yeast cells were appeared. In experiments performed in D=0,026 h–1, it was found that the absence of micronutrients from the growth medium, i.e. magnesium and calcium that are implicated in multiple cellular functions, had severe effects in yeast physiology, while the fatty acid composition of cellular lipids was not affected by the nature of the growth limiting factor. The present thesis aspires to contribute in the study of oleaginous microorganisms’ physiology and in use of glycerol as substrate in future biotechnological applications.

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科学研究費補助金 研究種目:一般研究(C) 課題番号:05650796 研究代表者:山根 恒夫 研究期間:1993-1994年度