Enhanced Butyric Acid and Hydrogen Production by the Mutants of Clostridium tyrobutyricum

Clostridium tyrobutyricum is a gram-positive, rod-shaped, spore-forming, obligate anaerobic bacterium that produces butyric acid, acetic acid, hydrogen and carbon dioxide as its main fermentation products from various carbohydrates including glucose and xylose (Wu and Yang, 2003). Butyric acid is used to synthesize butyryl polymers in the chemical industry and to enhance butter-like note in food flavors in the food industry. Esters of butyrate are used as additives for increasing fruit fragrance and as aromatic compounds for production of perfumes. Butyric acid, as one of the short-chain fatty acids generated by anaerobic fermentation of dietary substrates, is known to have therapeutic effects on colorectal cancer and hemoglobinopathies (Willims et al., 2003). The production of butyric acid from renewable resources has become an increasingly attractive alternative to the current petroleum-based chemical synthesis because of public concerns on the environmental pollution caused by the petrochemical industry and consumer’s preference to bio-based natural ingredients for foods, cosmetics and pharmaceuticals. Recently, we have demonstrated that butyric acid can be better produced by C. tyrobutyricum immobilized in a fibrous-bed bioreactor (Zhu and Yang, 2003; Yang, 1996). However, for economical production of butyric acid from biomass, it is desirable to further improve the fermentation process in its final product yield and concentration. Hydrogen, with a high energy content per unit weight (141.86 kJ/g or 61,000 Btu/lb), can be used as a clean fuel and easily converted to electricity by fuel cells. Hydrogen is thus considered as the most promising future fuel if its production cost can be greatly reduced. Hydrogen can be generated in several ways, including catalytic fuel reforming and electrolysis of water. However, the present chemical routes of hydrogen production are energy intensive and expensive. On the other hand, biological production of hydrogen, either by photosynthesis or by fermentation, can be operated at ambient temperature and pressure (Momirlan and Veziroglu, 1999; Das and Veziroglu, 2001). Hydrogen, as an energy byproduct from the butyric acid fermentation, can add value to the fermentation process. The goal of this project was to develop mutants with knocked-out acetate formation pathway to enhance the butyrate and hydrogen production from renewable resources. In this work, the partial pta or ack gene from C. tyrobutyricum was cloned and sequenced. Gene inactivation by integrational plasmid was then carried out to develop a pta or ack-deleted mutants. The enzyme activities of the mutants were examined to understand how the gene manipulation worked. Finally, their ability to produce butyric acid and hydrogen from glucose fermentation was evaluated and characterized in this paper.