This STTR Phase II project will develop novel engineered Clostridia strains for fermentation and economically produce butanol as a biofuel from sugars derived from starchy and lignocellulosic biomass. The conventional acetone-butanol-ethanol (ABE) fermentation has low butanol yield (<25%), butanol concentration (<16 g/L), and reactor productivity (<0.5 g/L?h) due to a strong butanol inhibition, and the fermentation process is difficult to improve due to the complicated metabolic pathways and gene regulation involved in the production microorganisms, mainly Clostridium acetobutylicum.
The broader impact/commercial potential of the project is to produce butanol as a biofuel from sugars derived from starchy and lignocellulosic biomass. Biobutanol has great value as an alternative transportation fuel. There is a huge potential commercial and societal impact in improving yields and reducing costs of butanol production. The research and other activity proposed could lead directly to a marketable product and process and leads to several enabling technologies, including better manipulation of C. tyrobutylicum, further demonstration of strain improvements using the FBB, and others.
This STTR project was to develop novel engineered Clostridia strains for fermentation to economically produce butanol as a biofuel from sugars derived from starchy and lignocellulosic biomass. Butanol is an important industrial solvent and potentially a better transportation fuel than ethanol. Recent rising oil prices and limited petroleum resources have generated a high interest in the production of biobutanol by anaerobic Clostridial fermentation. However, the conventional acetone-butanol-ethanol (ABE) fermentation has low butanol yield (<0.25 g/g), butanol concentration (<15 g/L), and reactor productivity (<0.5 g/L·h) due to a strong butanol inhibition, and the fermentation process is difficult to improve due to the complicated metabolic pathways and gene regulation involved in the production microorganisms, mainly Clostridium acetobutylicum. To develop a novel high-butanol producer, a high-butyrate producing Clostridia mutant with inactivated ack (acetate kinase) and ptb (phosphotransbutyrate) was cloned with an alcohol/aldehyde dehydrogenase gene (aad). The mutants were further adapted in a fibrous bed bioreactor to attain a high butanol tolerance. Further metabolic engineering and process development were carried out, and the feasibility and advantages of producing butanol from glucose and xylose present in lignocellulosic biomass hydrolysates using the engineered mutants were demonstrated. The new fermentation process cans double the butanol yield and concentration, thus reducing the product cost to an economically competitive level for fuel application. In summary, the mutant strains can produce n-butanol at a high titer (>15-20 g/L) and s high yield (>0.3-0.35 g/g) from glucose and other sugars present in the lignocellulosic biomass hydrolysates. With online butanol recovery by gas stripping, more than 50 g/L of butanol can be continuously produced in the fermentation, and the final butanol product from the gas stripping process has a high concentration of more than 600 g/L in the condensate. The process is thus highly efficient and can be used to economically produce n-butanol from biomass. Currently, butanol is almost exclusively produced via petrochemical routes. Its uses include industrial applications in solvent, rubber monomers and brake fluids. Butanol has also been shown to be a good alternative transportation fuel. Biobutanol will have a great potential to compete with ethanol as a transportation fuel when its production cost is reduced by using advanced fermentation technologies such as metabolically engineered butanol-tolerant mutants. By increasing the butanol yield from glucose and xylose from the current low of <25 % (w/w) to ~35%, the economics of biobutanol can be greatly improved. With the engineered mutants, the productivity and butanol product concentration can also be improved by at least 100%. Overall, the biobutanol product cost can be reduced to less than $2 per gallon using low-cost lignocellulosic biomass. This technology thus can provide an economical and better biofuel than ethanol. The generation of value-added products such as butanol from industrial waste streams and low-cost biomass feedstock can greatly enhance the economic viability of the biorefinery industry. The developed butanol fermentation technology would satisfy the public interest, especially in providing a safe, renewable energy, protecting natural resources and the environment, and enhancing economic opportunity and quality of life. There will be job creation throughout the commercial development and manufacturing phases. This project also trained several postdoctoral and Ph.D. students, and provided research opportunities for 3 undergraduate and 3 high school students.
