(47b) Consolidated Process for Biobutanol Production from Cellulose By Two Step Fermentation of Different Clostridia Strains

n-Butanol is an important industrial chemical and also has been proposed as an alternative biofuel superior to ethanol. Biological production of n-butanol is promising because it can use renewable biomass as feedstock and is environmentally friendly. Conventional biorefinery of lignocellulosic biomass needs separate processes, including feedstock pretreatment, cellulase/hemicellulase production, biomass enzymatic hydrolysis, sugar fermentation, and product recovery, which is not cost-effective due to the complex process and subsequently high equipment capital requirement. Consolidated process only use one tank for enzyme production, biomass hydrolysis, and sugar fermentation, which will greatly simplify the process and reduce the equipment investment. In our research, Clostridium cellulovorans, a natural cellulose/hemicelluloses utilizing, but non-butanol producing bacterium, was metabolically engineered for n-butanol production from cellulose by overexpressing enzymes in the butanol-producing pathway. The obtained mutants could produce above 2 g/L n-butanol from cellulose. However, the n-butanol productivity was very low and n-butanol titer was hard to be further improved, which could be attributed to the poor specific cellulose hydrolysis activity of the enzyme system from Clostridium cellulovorans, as well as limited amount of the secreted enzymes under the circumstances of metabolic burden and butanol stress. To unblock this bottleneck, a novel consolidated process was developed by applying another clostridia strain, Clostridium thermocellum, with naturally high cellulose hydrolyzing activity. There two clostridia strains have similar nutrient requirement and could grow in the same cellulose medium. Due to the difference in optimal growth temperature (55^oC vs. 37^oC), a two step fermentation was necessary. The first step of fermentation under 55^oC is a dedicated enzyme production phase by Clostridium thermocellum, in which maximal cellulose hydrolyzing activity of the whole culture system is the target of process optimization. The second step under 37^oC could be believed as a simultaneous enzymatic hydrolysis and n-butanol production phase by Clostridium cellulovorans mutant strain. By combining the advantages of these two clostridia strains, this novel consolidated process showed promising potential for cost-effective biobutanol production from lignocellulosic biomass.