Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants

Clostridium tyrobutyricum produces butyric acid, acetic acid, hydrogen and carbon dioxide as its main fermentation products. In this work, mutants with inactivated pta gene, encoding phosphotransacetylase (PTA) and ack gene, encoding acetate kinase (AK), were studied for their potential to improve butyric acid production in the fermentation. PTA and AK are two key enzymes in the acetate-producing pathway. 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 butyrate kinase (BK) in PPTA-Em increased by 44% and hydrogenase activity in PAK-Em increased by 40%. As compared with the wild type, the specific growth rate of the mutants decreased by 32% (from 0.28 to 0.19 h−1) because of the impaired PTA-AK pathway. Meanwhile, butyric acid production by these mutants was improved greatly, with higher butyric acid yield (>0.4 g/g versus 0.34 g/g) and final concentration (43 g/L versus 29 g/L), which also indicated that the mutants had better tolerance to butyric acid inhibition. However, acetate production in the mutants was not significantly reduced even though more butyrate was produced from glucose, suggesting the existence of additional acetate forming pathway in C. tyrobutyricum. Also, hydrogen production by PAK-Em mutant increased significantly, with higher hydrogen yield (2.61 mol/mol glucose versus 1.35 mol/mol glucose) and H2/CO2 ratio (1.43 versus 1.08). The SDS-PAGE also showed significantly different expression levels of proteins with molecular mass around 32 and 70 kDa. These results suggested that integrational mutagenesis resulted in global metabolic shift and phenotypic changes, which also improved production of butyric acid and hydrogen from glucose in the fermentation.