Quantification of acetone-butanol-ethanol(ABE) fermentation in clostridium acetobutylicum using systems biology approach

Abstract

Clostridium acetobutylicum exhibits a two-step metabolic pathway where substrates are first converted to organic acids accompanied by a decrease in pH. The acids are then assimilated to organic solvents. The transition from the acid-producing (acidogenesis) to the solvent-producing phase (solventogenesis) is controlled by integration of a number of cellular and environmental cues, whose precise mode of action are not well understood. In this study, a series of batch experiments were performed to understand the impact of extracellular cues in regulating the dynamics of acidogenesis and solventogenesis. It is demonstrated that the two phases operate independently of each other and the growth phase of the cell, i.e. the cues controlling a phase are not linked to the status of the other phase or the growth phase of the cell. Kinetic experiments demonstrated that there exist two previously uncharacterized negative feedback loops controlling the amounts of acids produced in the acidogenesis phase. In the next investigation of this work, we have used Elementary Mode Analysis (EMA) to quantify fluxes of metabolic network of C. acetobutylicum under different physical and chemical conditions. Biochemical reactions and their stoichiometry based metabolic model helped to reveal that, in response to external stresses, the organism invokes Elementary Modes which couple acidogenesis and solventogenesis. This coupling leads to the organism exhibiting physiological characteristics of both, acidogenesis and solventogenesis at the same time. Significantly, this coupling was not invoked during any ‘‘unstressed’’ conditions tested in this study. Overall, this work highlights the flexibility in C. acetobutylicum to modulate its metabolism to enhance chances of survival under harsh conditions. During this thesis work, we have also developed a pH based phenomenological model of fermentative production of butanol from glucose using C. acetobutylicum in batch system. The model relates the dynamics of growth and metabolite production with the extracellular pH of the media and was validated using data from a number of fermentation experiments, and was found to be successful in predicting the fermentation dynamics in C. acetobutylicum. Further, the proposed model can be extended to represent the dynamics of butanol production in other fermentation system like fed-batch or continuous fermentation using single and multi-sugars.