Production of butyric acid and hydrogen by metabolically engineered mutants of Clostridium tyrobutyricum
The main goal of this research was to develop an economical process for butyric acid and hydrogen production by Clostridium tyrobutyricum. First, metabolically engineered mutants, PPTA-Em and PAK-Em with knocked-out pta and ack genes, were characterized by Southern hybridization, enzyme assay, protein expression, and final product tolerance analysis. The original pta and ack genes were inactivated in the mutants, and the activities of PTA, AK, and hydrogenase changed greatly. SDS-PAGE and 2D electrophoresis showed that both PTA and AK were deleted from mutants. Butyric acid tolerance was also significantly improved in these mutants. Second, free cell fermentation by PPTA-Em from glucose showed that butyric acid concentration reached about 40 g/L with yield of 0.38 g/g and higher productivity 0.63 g/L?h. The immobilization fermentation using fibrous-bed bioreactor (FBB) produced butyric acid at a concentration of 50 g/L. It was found that different sugar sources affected fermentation, protein expression, and metabolic flux. Third, a higher butyric acid yield (0.42 g/g) and final concentration (42 g/L) was obtained with PAK-Em from free cell fermentation. Hydrogen production by PAK-Em also increased significantly with yield of 2.61 and hydrogen/carbon dioxide ratio of 1.44. Fourth, through adaptation in the FBB fibrous matrix, the highest butyric acid concentration of 81 g/L was obtained at pH 6.3 by PAK-Em with yield of ~0.45 g/g. A mutant that produced even more hydrogen, with hydrogen/carbon dioxide ratio of 2.69 and with very fast growth rate, was also discovered from the FBB adaptation. Different pHs (from pH 5.0 to pH 7.0) and sugar sources (glucose, xylose, and fructose) were applied to FBB fermentation to better understand the metabolic mechanism. Metabolic shift analysis proved that both PTA-AK metabolic pathways were blocked by the gene manipulation. Finally, shotgun DNA microarray is being applied to globally study bacterial genomic function, and to analyze and identify the key genes and proteins present in C. tyrobutyricum under different environmental conditions. The novel metabolically engineered mutants and the FBB application are important to the development of an economical bioprocess for butyric acid and hydrogen production from biomass by C. tyrobutyricm.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:fermentation metabolic engineering butyric acid hydrogen
Date of Publication:01/01/2005