Production of butyric acid and hydrogen by metabolically engineered mutants of Clostridium tyrobutyricum

Butyric acid has many applications in chemical, food and pharmaceutical industries. The production of butyric acid by fermentation has become an increasingly attractive alternative to current petroleum-based chemical synthesis. Clostridium tyrobutyricum is an anaerobic bacterium producing butyric acid, acetic acid, hydrogen and carbon dioxide as its main products. Hydrogen, as an energy byproduct, can add value to the fermentation process. The goal of this project was to develop novel bioprocess to produce butyric acid and hydrogen economically by Clostridial mutants.; Conventional fermentation technologies for butyric acid and hydrogen production are limited by low reactor productivity, product concentration and yield. In this project, novel engineered mutants of C. tyrobutyricum were created by gene manipulation and cell adaptation. Fermentation process was also optimized using immobilizing cells in the fibrous-bed bioreactor (FBB) to enhance butyric acid and hydrogen production.; First, metabolic engineered mutants with knocked-out acetate formation pathway were created and characterized. Gene inactivation technology was used to delete the genes of phosphotransacetylase (PTA) and acetate kinase (AK), two key enzymes in the acetate-producing pathway of C. tyrobutyricum , through homologous recombination. The metabolic engineered mutants were characterized by Southern hybridization, enzyme assay, protein expression and metabolites production. The enzyme assays showed that PTA and AK activities in the pta-deleted mutant (PPTA-Em) were reduced by 44% and 91%, respectively, whereas AK activity in the ack-deleted mutant (PAK-Em) decreased by 50%. Meanwhile, the activity of BK in PPTA-Em increased by 44%, and hydrogenase activity in PAK-Em increased by 40%. The SDS-PAGE showed that the expression of the proteins around 32 kDa and 70 kDa had significant changes in the mutants. Two dimensional protein electrophoresis gels showed that both PTA and AK were deleted from mutants. Butyric acid tolerances were improved significantly in the mutants, indicating high butyric acid productivity.; The free cell fermentation by PPTA-Em produced 40 g/L of butyric acid using glucose with lower specific cell growth rate, lower acetate yield (0.058 g/g), higher butyric acid yield (0.38 g/g), butyrate productivity (0.63 g/L · h), and consequent higher selectivity of butyrate over acetate as compared with that of wild type. In order to improve butyric acid and hydrogen production, a novel fibrous-bed bioreactor (FBB) was applied to immobilize the metabolically engineered mutants. The immobilization fermentation using PPTA-Em showed that butyric acid concentration was improved to 50 g/L with higher butyric acid yield compared with that of free cell fermentation. The effect of sugar sources and cell adaptation on the fermentation was also studied. (Abstract shortened by UMI.)...