Scale-up of Production and In-Situ Extraction of Saccharomyces-derived Bisabolene, an Advanced Biofuel Precursor

Engineering microbial biochemical pathways is a promising approach for production of advanced and sustainable biofuels. Through enzyme screening and metabolic engineering, Joint BioEnergy Institute (JBEI) invented a pathway for production of bisabolene, which is a precursor of bisabolane, an alternative to D2 diesel fuel.  Advanced Biofuels Process Demonstration Unit (ABPDU) in collaboration with JBEI performed process development at 2-L scale and scaled-up the process to 20-L. ABPDU studied the effect of product inhibition, overlay solvent (decane and oleyl alcohol) toxicity, and dissolved oxygen concentration during the production of bisabolene with engineered Saccharomyces cerevisiae. From our results, we found that the fed-batch mode of fermentation improved biomass yield and bisabolene titer from 1.2 g/L to 4 g/L, which is notably higher than results observed in previous lab scale research. When we used oleyl alcohol, a water-immiscible organic solvent for in-situ product recovery, we were able to reduce product inhibition and boost yield. Production at this scale provides some clarity on the path forward for this technology by answering some questions and raising more, particularly around viable recovery strategies during scale-up. In this presentation, we will discuss what we learned during the development of a commercially relevant fed-batch fermentation process for bisabolene and the demonstration of this technology beyond laboratory scale.