36 research outputs found

    Improvements in Fermentative Hydrogen Production through Physiological Manipulation and Metabolic Engineering

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    La production biologique d'hydrogĂšne (H2) reprĂ©sente une technologie possible pour la production Ă  grande Ă©chelle durable de H2 nĂ©cessaire pour l'Ă©conomie future de l'hydrogĂšne. Cependant, l'obstacle majeur Ă  l'Ă©laboration d'un processus pratique a Ă©tĂ© la faiblesse des rendements qui sont obtenus, gĂ©nĂ©ralement autour de 25%, bien en sous des rendements pouvant ĂȘtre atteints pour la production de biocarburants Ă  partir d'autres processus. L'objectif de cette thĂšse Ă©tait de tenter d'amĂ©liorer la production d'H2 par la manipulation physiologique et le gĂ©nie mĂ©tabolique. Une hypothĂšse qui a Ă©tĂ© Ă©tudiĂ©e Ă©tait que la production d'H2 pourrait ĂȘtre amĂ©liorĂ©e et rendue plus Ă©conomique en utilisant un procĂ©dĂ© de fermentation microaĂ©robie sombre car cela pourrait fournir la puissance supplĂ©mentaire nĂ©cessaire pour une conversion plus complĂšte du substrat et donc une production plus grande d'H2 sans l'aide de l'Ă©nergie lumineuse. Les concentrations optimales d’O2 pour la production de H2 microaĂ©robie ont Ă©tĂ© examinĂ©es ainsi que l'impact des sources de carbone et d'azote sur le processus. La recherche prĂ©sentĂ©e ici a dĂ©montrĂ© la capacitĂ© de Rhodobacter capsulatus JP91 hup- (un mutant dĂ©ficient d’absorption-hydrogĂ©nase) de produire de l'H2 sous condition microaĂ©robie sombre avec une limitation dans des quantitĂ©s d’O2 et d'azote fixĂ©. D'autres travaux devraient ĂȘtre entrepris pour augmenter les rendements d'H2 en utilisant cette technologie. De plus, un processus de photofermentation a Ă©tĂ© crĂ©Ă© pour amĂ©liorer le rendement d’H2 Ă  partir du glucose Ă  l'aide de R. capsulatus JP91 hup- soit en mode non renouvelĂ© (batch) et / ou en conditions de culture en continu. Certains dĂ©fis techniques ont Ă©tĂ© surmontĂ©s en mettant en place des conditions adĂ©quates de fonctionnement pour un rendement accru d'H2. Un rendement maximal de 3,3 mols de H2/ mol de glucose a Ă©tĂ© trouvĂ© pour les cultures en batch tandis que pour les cultures en continu, il Ă©tait de 10,3 mols H2/ mol de glucose, beaucoup plus Ă©levĂ© que celui rapportĂ© antĂ©rieurement et proche de la valeur maximale thĂ©orique de 12 mols H2/ mol de glucose. Dans les cultures en batch l'efficacitĂ© maximale de conversion d’énergie lumineuse Ă©tait de 0,7% alors qu'elle Ă©tait de 1,34% dans les cultures en continu avec un rendement de conversion maximum de la valeur de chauffage du glucose de 91,14%. Diverses autres approches pour l'augmentation des rendements des processus de photofermentation sont proposĂ©es. Les rĂ©sultats globaux indiquent qu'un processus photofermentatif efficace de production d'H2 Ă  partir du glucose en une seule Ă©tape avec des cultures en continu dans des photobiorĂ©acteurs pourrait ĂȘtre dĂ©veloppĂ© ce qui serait un processus beaucoup plus prometteur que les processus en deux Ă©tapes ou avec les co-cultures Ă©tudiĂ©s antĂ©rieurĂ©ment. En outre, l'expression hĂ©tĂ©rologue d’hydrogenase a Ă©tĂ© utilisĂ©e comme une stratĂ©gie d'ingĂ©nierie mĂ©tabolique afin d'amĂ©liorer la production d'H2 par fermentation. La capacitĂ© d'exprimer une hydrogĂ©nase d'une espĂšce avec des gĂšnes de maturation d'une autre espĂšce a Ă©tĂ© examinĂ©e. Une stratĂ©gie a dĂ©montrĂ© que la protĂ©ine HydA orpheline de R. rubrum est fonctionnelle et active lorsque co-exprimĂ©e chez Escherichia coli avec HydE, HydF et HydG provenant d'organisme diffĂ©rent. La co-expression des gĂšnes [FeFe]-hydrogĂ©nase structurels et de maturation dans des micro-organismes qui n'ont pas une [FeFe]-hydrogĂ©nase indigĂšne peut entraĂźner le succĂšs dans l'assemblage et la biosynthĂšse d'hydrogĂ©nase active. Toutefois, d'autres facteurs peuvent ĂȘtre nĂ©cessaires pour obtenir des rendements considĂ©rablement augmentĂ©s en protĂ©ines ainsi que l'activitĂ© spĂ©cifique des hydrogĂ©nases recombinantes. Une autre stratĂ©gie a consistĂ© Ă  surexprimer une [FeFe]-hydrogĂ©nase trĂšs active dans une souche hĂŽte de E. coli. L'expression d'une hydrogĂ©nase qui peut interagir directement avec le NADPH est souhaitable car cela, plutĂŽt que de la ferrĂ©doxine rĂ©duite, est naturellement produit par le mĂ©tabolisme. Toutefois, la maturation de ce type d'hydrogĂ©nase chez E. coli n'a pas Ă©tĂ© rapportĂ©e auparavant. L'opĂ©ron hnd (hndA, B, C, D) de Desulfovibrio fructosovorans code pour une [FeFe]-hydrogĂ©nase NADP-dĂ©pendante, a Ă©tĂ© exprimĂ© dans diffĂ©rentes souches d’E. coli avec les gĂšnes de maturation hydE, hydF et hydG de Clostridium acetobutylicum. L'activitĂ© de l'hydrogĂ©nase a Ă©tĂ© dĂ©tectĂ©e in vitro, donc une NADP-dĂ©pendante [FeFe]-hydrogĂ©nase multimĂ©rique active a Ă©tĂ© exprimĂ©e avec succĂšs chez E. coli pour la premiĂšre fois. Les recherches futures pourraient conduire Ă  l'expression de cette enzyme chez les souches de E. coli qui produisent plus de NADPH, ouvrant la voie Ă  une augmentation des rendements d'hydrogĂšne via la voie des pentoses phosphates.Biological hydrogen (H2) production represents a possible technology for the large scale sustainable production of H2 needed for a future hydrogen economy. However, the major obstacle to developing a practical process has been the low yields that are obtained, typically around 25%, well below those achievable for the production of other biofuels from the same feedstock. The goal of this thesis was to improve H2 production through physiological manipulation and metabolic engineering. One investigated hypothesis was that H2 production could be improved and made more economical by using a microaerobic dark fermentation process since this could provide the extra reducing power required for driving substrate conversion to completion and hence more H2 production might be obtained without using light energy. The optimal O2 concentrations for microaerobic H2 production were examined as well as the impact of carbon and nitrogen sources on the process. The research reported here proved the capability of Rhodobacter capsulatus JP91 hup- (an uptake-hydrogenase deficient mutant) to produce H2 under microaerobic dark conditions with limiting amounts of O2 and fixed nitrogen. Further work should be undertaken to increase H2 yields using this technology. In addition, a photofermentation process was established to improve H2 yield from glucose using R. capsulatus JP91 hup- strain either in batch and/or continuous culture conditions. Some technical challenges in establishing the proper operational conditions for increased H2 yield were overcome. A maximum yield of 3.3 mols of H2/ mol of glucose was found for batch cultures whereas in continous cultures it was 10.3 mols H2/ mol glucose, much higher than previously reported and close to the theoretical maximum value of 12 mols H2/ mol glucose. In batch cultures the maximum light conversion efficiency was 0.7% whereas it was 1.34% in continuous cultures with a maximum conversion efficiency of the heating value of glucose of 91.14%. Various approaches to further increasing yields in photofermentation processes are proposed. The overall results suggest that an efficient single stage photofermentative H2 production process from glucose using continuous cultures in photobioreactors could be developed which would be a much more promising alternative process to the previously studied two stage photofermentation or co-culture approaches. Furthermore, the heterologous expression of hydrogenases was used as a metabolic engineering strategy to improve fermentative H2 production. The capability of expressing a hydrogenase from one species with the maturation genes from another was examined. One strategy demonstrated that the orphan hydA of R. rubrum is functional and active when co-expressed in E. coli with hydE, hydF and hydG from different organisms. Co-expression of the [FeFe]-hydrogenase structural and maturation genes in microorganisms that lack a native [FeFe]-hydrogenase can successfully result in the assembly and biosynthesis of active hydrogenases. However, other factors may be required for significantly increased protein yields and hence the specific activity of the recombinant hydrogenases. Another strategy was to overexpress one of the highly active [FeFe]-hydrogenases in a suitable E. coli host strain. Expression of a hydrogenase that can directly interact with NADPH is desirable as this, rather than reduced ferredoxin, is naturally produced by its metabolism. However, the successful maturation of this type of hydrogenase in E. coli had not been previously reported. The Desulfovibrio fructosovorans hnd operon (hndA, B, C, and D genes), encoding a NADP-dependent [FeFe]-hydrogenase, was expressed in various E. coli strains with the maturation genes hydE, hydF and hydG of Clostridium acetobutylicum. Hydrogenase activities were detected in vitro, thus a multi-subunit NADP-dependent [FeFe]-active hydrogenase was successfully expressed and matured in E. coli for the first time. Future research could lead to the expression of this hydrogenase in E. coli host strains that overproduce NADPH, setting the stage for increased hydrogen yields via the pentose phosphate pathway

    Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd)

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    Background: Biologically, hydrogen (H-2) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H-2 production yield is unsatisfactorily low as < 4 mol H-2/ mol glucose. To address this challenge, simultaneous production of H-2 and ethanol has been suggested. Co-production of ethanol andH(2) requires enhanced formation of NAD(P) H during catabolism of glucose, which can be accomplished by diversion of glycolytic flux from the Embden-Meyerh-of-Parnas (EMP) pathway to the pentose-phosphate (PP) pathway in Escherichia coli. However, the disruption of pgi (phosphoglucose isomerase) for complete diversion of carbon flux to the PP pathway made E. coli unable to grow on glucose under anaerobic condition. Results: Here, we demonstrate that, when glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), two major enzymes of the PP pathway, are homologously overexpressed, E. coli.pgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of Delta hycA,Delta hyaAB,Delta hybBC,Delta ldhA, and Delta frdAB, the recombinant.pgi mutant could produce 1.69 mol H-2 and 1.50 mol ethanol from 1 mol glucose. However, acetate was produced at 0.18 mol mol(-1) glucose, indicating that some carbon is metabolized through the Entner-Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologous zwf and gnd from Leuconostoc mesenteroides and Gluconobacter oxydans, respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62 mol mol(-1) glucose along with 1.74 mol H-2 mol(-1) glucose, which are close to the theoretically maximal yields, 1.67 mol mol(-1) each for ethanol andH(2). However, the attempt to delete the ED pathway in the.pgi mutant to operate the PP pathway as the sole glycolytic route, was unsuccessful. Conclusions: By deletion of pgi and overexpression of heterologous zwf and gnd in E. coli Delta hycA Delta hyaAB Delta hybBC Delta ldhA Delta frdAB, two important biofuels, ethanol andH(2), could be successfully co-produced at high yields close to their theoretical maximums. The strains developed in this study should be applicable for the production of other biofuels and biochemicals, which requires supply of excessive reducing power under anaerobic conditions

    Photofermentative Hydrogen Production

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    There are many biological paths to hydrogen production, each with potential advantages, but also with its own challenges to implementation. The nonsulfur photosynthetic bacteria use a process termed as photofermentation to harness solar energy for the close to stoichiometric conversion of various carbon substrates to hydrogen, releasing carbon dioxide. These organisms can potentially use various feedstocks, but are particularly adept at the light-driven production of hydrogen from organic acids. Thus they are ideal candidates for two-stage or coculture systems, which derive additional hydrogen from the effluents of dark fermentations or organic acid-rich agricultural and industrial waste streams. The possible waste streams, as well as the metabolic and enzymatic properties, underlying photofermentation are reviewed. Recent progress, including the use of immobilized systems and metabolic engineering, is highlighted
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