Converting biomass into biofuels through hybrid processes that integrate thermochemical and biochemical steps has the potential to reduce processing costs, but is understudied. This proposed research seeks to improve the capacity of microorganisms to produce biofuels using substrates derived from the fast pyrolysis of cellulosic biomass. The fast pyrolysis of biomass, which involves rapidly heating the material in the absence of oxygen and in presence of catalysts, yields a complex mixture called bio-oil. Fermentation of bio-oil is complicated by the fact that in addition to useful substrates, it also contains many inhibitory (contaminant) compounds, including furans and phenols.
The overall goals of this proposed research are to increase the tolerance of two model microorganisms, Escherichia coli (ethanol) and Chlamydomonas reinhardtii (lipids for biodiesel), to the inhibitory contaminants found in bio-oil, and to improve the capacity of these organisms to ferment bio-oil fractions into biofuel. Metabolic evolution will be used to increase the robustness of these model microorganisms to the contaminants, largely because the bio-oil is complex and the mechanisms of inhibition are not known. The research plan has three objectives. The first objective is to use metabolic evolution to increase the tolerance of ethanol-producing E. coli to the inhibitory contaminants in the sugar-rich bio-oil fraction. The anhydrosugar levoglucosan is the most abundant substrate in the sugar-rich bio-oil fraction, but it is metabolized by most microorganisms. Therefore, an ethanol producing strain of E. coli will be engineered to utilize levoglucosan as a carbon and energy source. The second objective is use metabolic evolution to utilize the acetate-rich fraction of bio-oil by a heterotrophic, lipid-producing strain of C. reinhardti, and then increase the tolerance of C. reinhardtii to the inhibitory contaminants as well as acetate itself with the acetate-rich bio-oil fraction. The third objective is to reverse engineer the contaminant-tolerant E. coli strain developed under objective 1 through analysis of genome sequence and transcriptome data in order to identify the important mutations that enable contaminant tolerance.
The proposed education activities will train two graduate students from the disciplines of chemical engineering and food science, and engage three undergraduates to assist with the proposed research. Core concepts from the research topic will be introduced into three courses at Iowa State University (ISU): a sophomore-level Material and Energy Balances course, a graduate-level Metabolic Engineering, course, and a graduate-level course on the Thermochemical Processing of Biomass. With respect to K-12 outreach, the PI will develop a half-day module on bio-renewable chemicals for the ISU Science Bound program, a Saturday program that targets underrepresented middle school students. Students will have the opportunity to see an operating bioreactor and discuss how bio-renewable chemicals relate to their everyday life. Students will then play a web-based game that introduces the concept of metabolic evolution using the Biology in Motion Lab.