With the rising concerns of energy crisis, biofuel production becomes a very popular topic. As a possible replacement to transportation fuel, butanol attracted more attention because of its similar properties to automobile gasoline. Its low hygroscopicity and low vapor pressure may prevent increase in moisture content in butanol and make butanol a safe and environmentally friendly biofuel. Butanol can be produced by clostridia through acetone-butanol-ethanol (ABE) fermentation. However, this practice was not economically feasible with low titer, yield and productivity. The objectives of this study were to develop enhanced butanol production from ABE fermentation in continuous FBB by mutants of C. beijerinckii, and to explore the possibility of producing butanol by metabolic engineered hosts.
A stoichiometric model was developed for the analysis of the metabolic pathway in ABE fermentation. The model helped process development by predicting the maximum butanol yield based on mass and energy balance at steady state in continuous process. In the stoichiometric analysis, the uptake of butyric acid was shown to benefit butanol production by reducing required energy to produce butanol.
Two mutants of C. beijerinckii were cultured in continuous fermentation with butyric acid as a supplement in the medium under different conditions. At a high dilution rate of 1.88 h-1, asporogenic C. beijerinckii ATCC 55025 in the fibrous bed bioreactor (FBB) was able to utilize glucose and butyrate and steadily give a high butanol productivity of 17.29 g/(L*h), which was 170 folds higher than that in conventional batch fermentation. A new strain isolated from C. beijerinckii ATCC 55025 in repeated-batch culture in the FBB, C. beijerinckii JB200, was able to increase butanol titer to 12 g/L in continuous fermentation. In continuous fermentation, fibrous bed bioreactor (FBB) was used to help cell immobilization at high cell density. C. beijerinckii mutants were able to be retained at high cell density (>100 g dried weight/L) and steadily produce butanol without contamination or degeneration using both glucose and butyric acid.
Besides solvent producing clostridia, metabolically engineered Clostridium tyrobutyricum could actively produce butanol over six repeated batches in the FBB using glucose and/or xylose. In batches with only xylose, more butanol was produced at 7.12 g/L with a higher yield with the slower cell growth that made the foreign enzyme competitive to the native enzyme for butyryl CoA.
A pilot scale FBB was constructed for the evaluation of the feasibility of large scale butanol production in the FBB. Two batch fermentations were operated and the results of ABE fermentation were similar to those at lab scale. ABE fermentation at pilot scale was able to produce butanol at ~9.1 g/L with media containing glucose and butyric acid.
In summary, continuous butanol production with improved productivity from ABE fermentation with supplement of butyric acid as additional carbon source in the FBB was demonstrated. More studies are necessary to improve butanol. At pilot scale, the FBB produced butanol with a result that was comparable to that at lab scale. The pilot scale FBB can be further operated under different modes for process optimization.