Potentialités de production de Poly-Hydroxy-Alcanoates (PHA) chez Cupriavidus necator sur substrats de type acides gras volatifs (études cinétiques et métaboliques.)

L accumulation de biopolymère de réserve (PolyHydroxyAlcanoates ou PHA) par la souche Cupriavidus necator, à partir de substrats de type acides gras volatils (acide butyrique, acide propionique et acide acétique) a été étudiée. Elle est induite par une limitation phosphore. Les performances atteintes lors des cultures se situent parmi les meilleures de la littérature pour ce type de substrat : jusqu à 66 g.L-1 de biomasse totale avec un pourcentage d accumulation massique de 88% en PHB PolyHydroxyButyrate- ou en PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- comportant jusqu à 52% de motifs d HV.Pour chaque source carbonée, une caractérisation cinétique et stœchiométrique de la souche a été réalisée en l absence d effets inhibiteurs dus aux substrats acides grâce à des cultures de type Fed-Batch avec des apports non limitants et non inhibiteurs en carbone. Il a été dégagé :- un taux de croissance maximal de la souche de 0,33 h-1 pour les trois acides étudiés- une relation entre vitesse spécifique de production de PHA et taux de croissance fixée par la disponibilité et les flux de production de NADPH2 avec un découplage inverse pour les taux de croissance supérieurs à 0,05 h-1 et un couplage partiel pour les taux de croissance inférieurs- un optimum de 0,35 Cmole.Cmole-1.h-1, associé à un taux de croissance de l ordre de 0,05 h-1.- une amélioration de la production de PHB en termes de vitesses spécifiques mais également en termes de rendements si une faible croissance résiduelle est maintenueLa réponse de la souche à un excès de substrat acide a été caractérisée via l étude de régimes transitoires induits par des pulses sur des cultures continues préalablement stabilisées en régime permanent. Il a été montré qu en excès de phosphore, face à un brusque excès de substrat, la souche est incapable d adapter rapidement son taux de croissance. L excès est donc dirigé vers la production de PHA dont les voies sont plus rapidement mobilisables. En conditions limitantes de phosphore, le substrat excédentaire est utilisé pour la production de PHA. L inhibition par les acides se traduit par une diminution des capacités de biosynthèse de la biomasse et des PHA entrainant une réduction de l assimilation du carbone puis une diminution des rendements de conversion. D autre part la sensibilité d un système continu à un excès de substrat dépend du point de fonctionnement choisi : plus il est optimal en termes de vitesse, moins le système est robuste. L acide propionique est très inhibiteur comparé aux autres acides étudiés (dès 3-4 mM contre 30-40 mM). Il n agit pas simplement via une accumulation excessive dans le cytoplasme mais il exerce également une inhibition spécifique des voies métaboliques.Un antagonisme entre les substrats (acide acétique et butyrique) a été constaté et expliqué grâce à une analyse des flux métaboliques. L acide acétique est assimilé préférentiellement pour produire la biomasse, l énergie et les cofacteurs nécessaires à la production de PHA, alors que l acide butyrique est utilisé pour la synthèse de PHB. La proportion maximale d acide acétique admise dans l alimentation en fonction des conditions fixées en régime permanent est calculée et peut être limitée à 40% du carbone.Enfin il a été déterminé que si une croissance résiduelle est assurée grâce à un apport en phosphore, le pourcentage maximal d HV dans le polymère dépend du taux d acide propionique dans l alimentation et ne peux dépasser 33 +- 5% sur acide propionique pur. Par contre, si aucune croissance résiduelle n est assurée, il est possible de convertir l acide propionique en motifs d HV uniquementReserve Biopolymer (PolyHydroxyAlkanoates or PHA) accumulation by the strain Cupriavidus necator, from Volatile Fatty Acids (VFA, like butyric acid, propionic acid and acetic acid) was investigated. This production is induced by a phosphorus limitation. For this type of substrates, performances reached during cultures are among the best listed in the literature: up to 66 g.L-1 of total biomass with 88% (w/w) of PHB PolyHydroxyButyrate- or PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- with a HV content up to 52 Mole%.For each carbon source, kinetic and stoechiometric characterization has been carried out thanks to Fed-Batch cultures with non-limiting and non-inhibitory carbon feed. It has been established:- a maximal growth rate of 0,33 h-1 for the three acid investigated- a relationship between specific PHA production rate and growth rate which is set by the availability and production flux of NADPH2. For growth rate above 0,05 h-1, there is an inverse coupling. For growth rate under 0,05 h-1, there is a partial coupling.- an optimum of 0,35 Cmole.Cmole-1.h-1 is associated with a growth rate of 0,05 h-1.- if a low residual growth rate is maintained, an improvement of PHB production is recorded in terms of specific production rate and yieldsThe response of the strain to an excess of acid substrate was characterized through the investigation of transient state induced by pulsed addition of substrate during continuous cultures stabilized in steady state. It was shown that in excess of phosphorus, when there is a substrate excess, the strain is unable to quickly adapt its growth rate, so the excess is directed to PHA production whose ways seem to be more easily mobilized. Under phosphorus limitation, an excess of substrate is used for PHA production. Acid inhibition results in a decrease in biomass and PHA production capacity which leads to a decrease in carbon assimilation and conversion yields. The sensitivity of a continuous system to an excess of substrate depends on the chosen operating point: the more it is optimal in terms of specific production rate, the less the system is robust. Propionic acid is highly inhibitory compared to the other acids studied (from 3-4 mM versus 30-40 mM). It does not act only via an excessive accumulation in the cytoplasm but also exerts a specific inhibition of metabolic pathways.An antagonism between substrates (acetic and butyric acid) has been established and explained thanks to the Metabolic Flux Analysis. Acetic acid is preferentially used to produce biomass, energy and cofactors for PHA synthesis, whereas butyric acid is used to product PHB. According to the conditions set during steady state, maximal content of acetic acid admitted in the feed can be calculated. It can be limited to 40% of the carbon in the feed.Finally if a growth rate is maintained thanks to a phosphorus supply, the maximal HV content in polymer is function of propionic acid in the feed and cannot exceed 33 +- 5 Mole% on pure propionic acid. Conversely, if there is no residual growth, a total conversion of propionic acid into HV is allowedTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Growth of the extremophilic Deinococcus geothermalis DSM 11302 using co-substrate fed-batch culture

Deinococcus geothermalis metabolism has been scarcely studied to date, although new developments on its utilization for bioremediation have been carried out. So, largescale production of this strain and a better understanding of its physiology are required. A fed-batch experiment was conducted to achieve a high cell density non-limiting culture of D. geothermalis DSM 11302. A co-substrate nutritional strategy using glucose and yeast extract was carried out in a 20-L bioreactor in order to maintain a non-limited growth at a maximal growth rate of 1 h−1 at 45 °C. Substrate supplies were adjusted by monitoring online culture parameters and physiological data (dissolved oxygen, gas analyses, respiratory quotient, biomass concentration). The results showed that yeast extract could serve as both carbon and nitrogen sources, although glucose and ammonia were consumed too. Yeast extract carbon-specific uptake rate reached a value 4.5 times higher than glucose carbon-specific uptake rate. Cell concentration of 9.6 g L−1 dry cell weight corresponding to 99 g of biomass was obtained using glucose and yeast extract as carbon and nitrogen sources

Quantitative Characterization of the Growth of Deinococcus geothermalis DSM-11302: Effect of Inoculum Size, Growth Medium and Culture Conditions

Due to their remarkable resistance to extreme conditions, Deinococcaceae strains are of great interest to biotechnological prospects. However, the physiology of the extremophile strain Deinococcus geothermalis has scarcely been studied and is not well understood. The physiological behaviour was then studied in well-controlled conditions in flask and bioreactor cultures. The growth of D. geothermalis type strains was compared. Among the strains tested, the strain from the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen DSM) DSM-11302 was found to give the highest biomass concentration and growth rate: in a complex medium with glucose, the growth rate reached 0.75 h−1 at 45 °C. Yeast extract concentration in the medium had significant constitutive and catalytic effects. Furthermore, the results showed that the physiological descriptors were not affected by the inoculum preparation steps. A batch culture of D. geothermalis DSM-11302 on defined medium was carried out: cells grew exponentially with a maximal growth rate of 0.28 h−1 and D. geothermalis DSM-11302 biomass reached 1.4 g•L −1 in 20 h. Then, 1.4 gDryCellWeight of biomass (X) was obtained from 5.6 g glucose (Glc) consumed as carbon source, corresponding to a yield of 0.3 CmolX•CmolGlc−1 ; cell specific oxygen uptake and carbon dioxide production rates reached 216 and 226 mmol.CmolX −1•h −1 , respectively, and the respiratory quotient (QR) value varied from 1.1 to 1.7. This is the first time that kinetic parameters and yields are reported for D. geothermalis DSM-11302 grown on a mineral medium in well-controlled batch cultur

Transcriptomic Analyses during the Transition from Biomass Production to Lipid Accumulation in the Oleaginous Yeast Yarrowia lipolytica

We previously developed a fermentation protocol for lipid accumulation in the oleaginous yeast Y. lipolytica. This process was used to perform transcriptomic time-course analyses to explore gene expression in Y. lipolytica during the transition from biomass production to lipid accumulation. In this experiment, a biomass concentration of 54.6 gCDW/l, with 0.18 g/gCDW lipid was obtained in ca. 32 h, with low citric acid production. A transcriptomic profiling was performed on 11 samples throughout the fermentation. Through statistical analyses, 569 genes were highlighted as differentially expressed at one point during the time course of the experiment. These genes were classified into 9 clusters, according to their expression profiles. The combination of macroscopic and transcriptomic profiles highlighted 4 major steps in the culture: (i) a growth phase, (ii) a transition phase, (iii) an early lipid accumulation phase, characterized by an increase in nitrogen metabolism, together with strong repression of protein production and activity; (iv) a late lipid accumulation phase, characterized by the rerouting of carbon fluxes within cells. This study explores the potential of Y. lipolytica as an alternative oil producer, by identifying, at the transcriptomic level, the genes potentially involved in the metabolism of oleaginous species

Experimental and statistical analysis of nutritional requirements for the growth of the extremophile Deinococcus geothermalis DSM 11300

Few studies concerning the nutritional requirements of Deinococcus geothermalis DSM 11300 have been conducted to date. Three defined media compositions have been published for the growth of this strain but they were found to be inadequate to achieve growth without limitation. Furthermore, growth curves, biomass concentration and growth rates were generally not available. Analysis in Principal Components was used in this work to compare and consequently to highlight the main compounds which differ between published chemically defined media. When available, biomass concentration, and/or growth rate were superimposed to the PCA analysis. The formulations of the media were collected from existing literature; media compositions designed for the growth of several strains of Deinococcaceae or Micrococcaceae were included. The results showed that a defined medium adapted from Holland et al. (Appl Microbiol Biotechnol 72:1074–1082, 2006) was the best basal medium and was chosen for further studies. A growth rate of 0.03 h-1 and a final OD600nm of 0.55 were obtained, but the growth was linear. Then, the effects of several medium components on oxygen uptake and biomass production by Deinococcus geothermalis DSM 11300 were studied using a respirometry-based method, to search for the nutritional limitation. The results revealed that the whole yeast extract in the medium with glucose is necessary to obtain a non-limiting growth of Deinococcus geothermalis DSM 11300 at a maximum growth rate of 0.64 h-1 at 45 °C

Homéostasie cellulaire chez Saccharomyces cerevisiae (Etude des effets des acides faibles)

TOULOUSE-INSA (315552106) / SudocSudocFranceF

Description de la production de spiramycine par Streptomyces ambofaciens (modélisation métabolique, simulation et capteur logiciel)

TOULOUSE-INSA (315552106) / SudocSudocFranceF

Contrôle de processus dynamiques par systèmes multi-agents adaptatifs (application au contrôle de bioprocédés)

Cette thèse a pour cadre le contrôle auto-adaptatif de procédés biologiques et son objectif est de permettre l'application de ce contrôle à une grande variété de problèmes distincts en évitant la phase usuelle de leur modélisation. Les bioprocédés sont des systèmes complexes, hautement dynamiques et parvenir à les contrôler en vue d'obtenir une production définie se révèle une tâche difficile. De plus, les incertitudes liées aux mesures et le manque de connaissances des réactions biologiques précises se déroulant à l'intérieur même du bioprocédé, font que les méthodes usuelles mises en œuvre pour contrôler un bioprocédé particulier doivent être largement re-calibrées dès qu'il s'agit d'adapter ce contrôle à un bioprocédé différent. L'apport de cette thèse est de proposer une approche informatique de cette problématique, centrée sur l'utilisation de Systèmes Multi-Agents Adaptatifs (AMAS). Les propriétés auto-organisatrices de tels systèmes, ainsi que leur conception centrée sur les comportements locaux, permettent d'appréhender la complexité des procédés biologiques et de fournir un système apte à leur contrôle sans nécessiter d'informations détaillées sur ceux-ci, tout en étant capable de s'adapter à leurs dynamiques. Deux systèmes multi-agents adaptatifs distincts, répondant chacun à un ensemble de contraintes différentes, ont permis d'étudier la faisabilité et l'apport de cette approche au domaine du contrôle de procédés. L'aboutissement est un modèle AMAS générique qui est associé à chaque variable contrôlable du procédé afin d'apprendre et de déterminer les actions de contrôle pour atteindre les objectifs définis par l'utilisateur. Les agents observent en temps réel l'évolution des variables du procédé pour en extraire les informations nécessaires à son contrôle. Le système de contrôle établit alors une contextualisation des contrôles à appliquer qui devient indépendante de la connaissance du bioprocédé. Ce contrôle auto-adaptatif a été évalué sur un ensemble de problèmes de contrôle de systèmes dynamiques, notamment celui d'un bioprocédé simulé, et les résultats obtenus sont analysés dans ce mémoireThis work aims at creating a self-adaptive approach to control bioprocesses while still being generic enough to be applied on a wide range of problems without relying on their modeling. Bioprocesses are complex and highly dynamic systems, and controlling them to reach a user-defined objective is a difficult task. Furthermore, the limited amount of available measures and the lack of a precise biological knowledge of what is happening inside the bioprocess lead usual control approaches to be recalibrated before being applied on a different bioprocess. The benefit of this work is a computer-based approach relying on the Adaptive Multi-Agent Systems (AMAS) theory. The objectives of adaptability and genericity are reached thanks to the self-organization of such systems and their design focusing on the local behavior of the agents, dealing with the complexity of such a problem without needing an extensive amount of information on the system to control.Two distinct multi-agent systems, defined by a specific set of constraints, were created to study the feasibility and benefits of such an approach. The outcome is a generic AMAS model, associated with each controllable variable, which learns the actions to apply in order to fulfill user-defined objectives. Agents observe in real-time the evolution of the variables and extract the information needed to control the target system. The AMAS is then able to contextualize the control without modeling the behavior of the system to control itself.This self-adaptive control was evaluated on a set of problems involving the control of dynamic systems, such as bioprocesses, and the associated results are analyzedTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Biologie systémique et intégrative pour l'amélioration de l'accumulation et de la sélectivité des acides gras accumulés dans les espèces levuriennes.

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 brevetLipid 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 patentTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Quantification of the transient and long-term response of Saccharomyces cerevisiae to carbon dioxide stresses of various intensities

Carbon dioxide (CO2) is a major compound of microbial metabolism both as a substrate and as a product but high dissolved carbon dioxide concentrations have long been known to affect yeast physiology. This work investigates the energetic aspects of the transient and long-term responses of yeast chemostat cultures submitted to different step-increases of the dissolved CO2 concentrations. It was demonstrated that a CO2 shift-up triggered a transient metabolic response characterized by an increase of the specific respiration rate (up to +37%), the mobilization of storage sugars, excretion of ethanol and acetate (up to 50 mg L-1) and by the uptake of potassium (+30 mM) and magnesium (+20 mM) ions. The energy generation by the yeast metabolism temporarily increased and a transient decrease in the biomass synthesis was observed. This short-term response was not dependent of the amplitude of the shift-up and could not be explained by pH homeostasis only. Moreover, on the long-run CO2 created an energy drain that was proportional to its concentration as the limp value decreased from 12.7 gx mole(ATP)(-1) to 9.57 gx mole(ATP)(-1) when the dissolved CO2 concentiation was increased from 1.4 mM to 17.09 mM. Succinate excretion was also observed for CO2 enriched cultures and its production rate was found proportional with increasing CO2 concentrations. Finally, we pointed out that yeast cells may adapt to high CO2 concentrations in the long-run. This work illustrates the regulation of physiologic and metabolic homeostasis of yeast cells when facing CO2 stress