L'objectif de cette thèse est l'étude du métabolisme de Corynebacterium glutamicum, et de ses potentialités, au cours de procédés aéro-anaérobies. Après une première phase avec apport d'oxygène pour permettre la croissance bactérienne, une phase anaérobie est induite par arrêt de l'aération et réduction de la vitesse d'agitation. Dans ces conditions, le lactate est le principal métabolite produit. La synthèse de ce dernier a été améliorée en jouant, essentiellement, sur le moment de la transition entre les 2 phases. C. glutamicum 2262 peut ainsi produire 27 g/l de lactate en mode discontinu et 55 g/l en mode semi-continu, suite à un arrêt de l'aération lorsque la concentration en biomasse est d'environ 2,6 g/l. Afin d'exploiter la voie de synthèse d'acide lactique chez C. glutamicum pour la production d'éthanol, les gènes PDC et ADH de Zymomonas mobilis ont été exprimés sous le contrôle du promoteur ldhA endogène de C. glutamicum 2262 et d'une souche de C. glutamicum 2262 sans ldhA. Bien que les productivités en éthanol de ces souches aient été relativement faibles, la suppression de ldhA a entraîné des augmentations de la concentration en éthanol d'environ 15 fois. Une stratégie similaire a été utilisée pour la production d'itaconate. Comme dans le cas de l'éthanol, la concentration en itaconate obtenue est demeurée très faible malgré des essais d'amélioration du procédé de mise en oeuvre de la souche productrice d'itaconate / The objective of this work is the study of Corynebacterium glutamicum metabolism, and of its potentialities, during an aero-anaerobic process. After a first phase during which the oxygen was supplied to favor the bacterial growth, the anaerobic phase was induced by the stopping of the oxygen supply and the decreasing of the agitation speed. In these culture conditions, lactate was the main metabolite produced. The production of this organic acid has been increased by modifying the transition time between the aerobic and the anaerobic phases. C. glutamicum 2262 was able to produce up to 27 g/l lactate during a batch process and up to 55 g/l during a fed batch process. To exploit the lactic acid synthesis pathway of C. glutamicum for ethanol production, the PDC and ADH genes from Zymomonas mobilis were expressed under the control of the endogenous promoter of ldhA, in the wild-type strain and in a ldhA-disrupted strain of C. glutamicum 2262. Although the ethanol productivities of these engineered strains were relatively low, the depletion of ldhA resulted in the increases of ethanol final concentration up to 15 times. A similar strategy was applied for the production of itaconate. As previously for the ethanol production, the final concentration of itaconate remained very low despite of some modifications of the process

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13248号 / 論農博第2873号 / 新制||農||1070(附属図書館) / 学位論文||H31||N5172(農学部図書室) / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

An indigenous mixed culture of bacteria collected from a Wastewater Treatment Plant (Brits, North West Province, South Africa), biocatalytically reduced Cr(VI) in the presence of As(III). Both the reduced chromium (Cr(III)) and the oxidised arsenic (As(V)) readily form amorphous hydroxides that can be easily separated or precipitated from the aqueous phase as part of the treatment process. Treatment of Cr(VI) and As(III) before disposal of wastewater is critical since both compounds are known to be carcinogenic and mutagenic at very low concentrations, and acutely toxic at high concentrations. Batch experiments were conducted to evaluate the rate of Cr(VI) reduction under anaerobic condition in the presence of its co-contaminant As(III) typically found in the groundwater and mining effluent. Results showed near complete Cr(VI) reduction under initial Cr(VI) concentrations up to 70 mg/L in a batch amended with 20 mg/L As(III). However, increasing Cr(VI) concentrations up to 100 mg/L resulted in the inhibition of Cr(VI) reduction activity. Further investigation was conducted in a batch reactor amended with 70 mg/L Cr(VI) concentration at different As(III) concentrations ranging from 5-70 mg/L to evaluate the effect of varying As(III) concentration on Cr(VI) reduction efficiency. Results showed that Cr(VI) reduction efficiency increased as As(III) concentrations increased from 5-40 mg/L. However, further increase in As(III) concentration up to 50 mg/L resulted in incomplete Cr(VI) reduction and decrease in Cr(VI) reduction efficiency. These results suggest that the rate of Cr(VI) reduction depends on the redox reaction of As(III) and As(V) with Cr(VI). Moreover, the inhibitory effect observed at high Cr(VI) and As(III) concentration may also be attributed to the dual toxicity effect of Cr(VI) and As(III) on microbial cell. From the above batch kinetic studies lethal concentration of Cr(VI) and As(III) for these strains was evaluated and established. Initial evaluation of the bacteria using 16S rRNA partial sequence method showed that cells in the mixed culture comprised predominantly of the Gram-positive species: Staphylococcus sp., Enterobacter sp., and Bacillus sp. The biokinetic parameters of these strains were estimated using a non-competitive inhibition model with a computer programme for simulation of the Aquatic System “AQUASIM 2.0”. Microbial reduction of Cr(VI) in the presence of As(III) was further investigated in continuous-flow bioreactors (biofilm reactor) under varying Cr(VI) loading rates. The reactor achieved Cr(VI) removal efficiency of more than 96 % in the first three phases of continuous operation at lower Cr(VI) concentration ranging from 20-50 mg/L. However, 20 % decrease in Cr(VI) removal efficiency was observed as Cr(VI) concentration increase up to 100 mg/L. The reactor was able to recover from Cr(VI) and As(III) overloading phase after establishing the resilient nature of the microorganism. Similarly to the batch reactor studies the overall performance of the reactor also demonstrated that the presence of As(III) greatly enhance Cr(VI) reduction in a bioreactor. This was evident by near complete removal of Cr(VI) concentration up to 50 mg/L. The basic mass balance expressions on Cr(VI) along with the non-competitive inhibition model were used to estimate the biokinetic parameters in the continuous flow bioreactor system. Cr(VI) reduction efficiency along the longitudinal column was also evaluated in this study. Results showed that Cr(VI) efficiency increased as Cr(VI) concentration travels along the longitudinal column. Other important factors such as oxygen and pH during biological Cr(VI) reduction in the presence of As(III) oxidation were also evaluated. / Dissertation (MEng)--University of Pretoria, 2014. / tm2015 / Chemical Engineering / MEng / Unrestricted