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The objective of this project was to engineer novel Clostridium strains to produce n-butanol from low-cost lignocellulosic biomass and gases (CO<
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and H<
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). Butanol is an important industrial solvent and potentially a superior fuel that can fit the existing fuel infrastructure and replace gasoline in auto engines without modification. This project focused on metabolic engineering of cellulolytic Clostridium cellulovorans for directly converting cellulose to n-butanol and carboxydotrophic Clostridium carboxidivorans to convert CO<
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and H<
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to butanol and ethanol. These clostridial strains were engineered using synthetic biology, gene overexpression and gene knockout strategies to produce n-butanol as the main product from lignocellulose and gaseous substrates. With these metabolically engineered strains, high-titer and high-yield n-butanol production from lignocellulosic biomass were achieved with minimal CO<
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released to the environment in a sustainable and co-cultured consolidated bioprocess (CBP) integrated with in situ butanol separation by gas stripping. A well-to-pump life cycle analysis showed that the integrated biobutanol production process could reduce CO<
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and greenhouse gas (GHG) emissions by ~75% compared to traditional chemical process. Moreover, the production of cellulosic butanol from CBP would not require enzymatic hydrolysis, an expensive step in current biorefinery using lignocellulosic biomass. The successful development of CBP using engineered clostridia for n-butanol production from lignocellulosic biomass and CO<
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can meet the BETO 2022 cost target of $3/gallon gasoline equivalent (gge) for advanced biofuels. With the higher energy content, butanol will be cheaper than ethanol and similar to gasoline at $2.50/gal in terms of energy cost ($ per MJ). With the reduced production cost and product value, biobutanol from lignocellulose and CO<
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can one day become a major liquid transportation biofuel, replacing bioethanol and gasoline.<
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Strain development via metabolic engineering and fermentation process optimization, assisted by omic analysis of the mutation and process effects, helped us to achieve our goal of producing n-butanol and ethanol (biofuel) from lignocellulosic biomass at a cost of $3/gge (gallon gasoline equivalent) or less. The co-fermentation technology using both cellulose and CO<
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/H<
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for biofuel production can greatly increase product yield from the biomass feedstock while also reduce GHG emissions, and is novel and advantageous compared to existing ethanol and acetone-butanol-ethanol fermentation technologies. The same metabolic engineering and process approaches can also be applied to other microorganisms to produce bio-based chemicals from renewable resources. Overall, the CBP technology using the novel engineered clostridia is unique and innovative, and complements well with the Biochemical Conversion technologies in the BETO Multi-Year Program Plan (MYPP), which largely focuses on the hydrolysis of cellulosic biomass and utilization of the hydrolysates for ethanol production.<
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