As the price of crude oil continues to reach record highs, biorefining becomes a necessary option for renewable fuels and chemical production. However, robust and versatile microorganisms for bioconversion that can utilize multiple feedstocks are currently lacking. Better engineered microorganisms designed to function within an integrated biorefining system are required to significantly reduce petroleum utilization and develop a diverse portfolio of biomass-based processes that can generate comparable alternatives for a wide range of petroleum products.
Utilization of biodiesel-derived crude glycerol for the production of butanol represents a tangible approach to improving the sustainability of the biofuels industry in general and is critical for the growth and stability of the biodiesel industry in particular. Furthermore, utilizing crude glycerol as an alternative to corn-derived feedstocks will provide much-needed diversity and flexibility to an emerging biorefining industry limited by the price and availability of corn. Butanol is a key biorefinery platform chemical and has significant advantages as a renewable transportation fuel. The potential to generate higher yields using an essentially no-cost feedstock strengthens the technical and economic feasibility for industrial-scale butanol fermentation.
Intellectual Merit
The goal of this research is to quantify metabolism and tolerance in Clostridium pasteurianum and to engineer this bacterium to improve the value-added conversion of biodiesel-derived crude glycerol using anaerobic fermentation. This project will focus on fundamental research to understand and improve the metabolic pathways that control glycerol utilization and substrate formation from both extracellular and intracellular approaches. The proposed research has three objectives. The first objective is to elucidate the cellular response of C. pasteurianum to both substrate and product toxicity. The second objective is to evaluate cell membrane structure and stability in response to increasing concentrations of substrate impurities and butanol. And the third objective is to metabolically engineer C. pasteurianum to increase butanol production.
The research is innovative because it proposes an alternative pathway to glycerol fermentation, where metabolic engineering approaches are used to knock down the genes in the reductive pathway as opposed to up-regulation of genes in the oxidative pathway. The research is potentially transformative because it opens up new approaches for metabolic engineering of microorganisms to convert crude glycerol byproducts from biorefinery operations to liquid transportation fuels.
Broader Impacts
This research project provides opportunities to educate students and other educators about alternative energy, renewable resources, and biorefining. The education plan will develop the technical and laboratory expertise for graduate and undergraduate students to prepare them for careers in the biofuels industry, which is currently experiencing substantial growth. Various outreach activities are planned, including the development of a seminars and demonstrations to educate K-12 students, teachers, and community members about biofuels production and utilization. Education efforts will also focus on the recruitment of students from underrepresented groups through activities that will target the inclusion of these students. These educational and outreach activities will stimulate broad interest in science and engineering, and inspire K-12 students to pursue higher education and careers in engineering and energy industries. The proposed outreach activities also include a web based wiki site and summer exchange program that will facilitate collaboration among graduate and undergraduate students in the two research groups.
The production of biodiesel increased steadily over the past decade, reaching 315 million gallons of biodiesel produced in 2010. However, for every 100 pounds of biodiesel, there are 10 pounds of crude glycerol produced as well. Unfortunately, there is no use for this glycerol, as the massive amount of impurities prohibits its use in traditional glycerol applications. Hence, there is a growing "glycerol-glut" that severely impedes biodiesel production. Research performed under this project sought novel approaches to convert this waste product into a useful and value-added new product (butanol). Research accomplished under this project yielded 3 major outcomes: This project is situated in the area of sustainable fuels. A new process was developed in which crude glycerol, as obtained from the biodiesel producers, was converted into butanol by microbial fermentation, that is, a waste product (crude glycerol) was converted into a value-added product (butanol). Butanol is a solvent and fuel additive, and as a fuel additive it is more efficient than the currently used ethanol, due to its higher carbon content, better miscibility with petroleum and gasoline products and lower vapor pressure. It is important to note that the microorganism used here, Clostridium pasteurianum, does not need any additional food/carbon sources besides crude glycerol. It can produce 0.3 pounds of butanol for each pound of glycerol consumed. While it has been known that this microorganism is capable of converting glycerol to butanol, very little is known about intracellular chemical processes that lead to the production of butanol. Hence, this research project concentrated on elucidating the fermentation process on a molecular level. Butanol, the product of this fermentation, is actually toxic to the cells. Our research contributed new insights into how and why the cells still produce butanol. We contributed new knowledge on how the microorganisms cope with this toxicity that is, how they adjust their membrane composition to "fortify" against the detrimental effects that butanol, and alcohols in general, have on bacterial cell membranes. New insights into the biophysics of cellular membranes were garnered. These results are of tremendous importance for future work that will focus on genetically engineering this microorganism to further enhance its butanol-producing (solventogenic) capacity. 3. Three graduate and six undergraduate students were trained under this research project. This, in our opinion, it is the most noble outcome of academic research. Society benefits from the initial research investment as a new generation of highly trained engineers enters the work force and continues to drive engineering and development. This project is situated in a STEM area, and more than 50% of the undergraduate students who participated in the project throughout the period of performance were female students.
