- Angela Zhu
- Steven Chang
- Samantha Alvarez
- Team Red
Title of Proposed Project
20.109(F12) Pre-Proposal: Engineering Alkyl Hydroperoxide Reductase Subunit C in E. coli to Extend the Oxidative Stability of Biodiesel Fuel
Biodiesel fuel is both a non-toxic and renewable resource, making it a promising alternative to the more common petroleum-derived diesel fuel. Current research focuses on synthesizing biodiesel from bacteria but does not address the poor oxidative stability of the biodiesel, which lowers its shelf life to a six month span. The proposed research would address this problem by engineering pre-existing biodiesel-producing bacteria to secrete antioxidative protein in order to prolong the oxidative stability of the biodiesel and therefore make it useable for a longer period of time.
Diesel fuel is a non-renewable resource made from a limited supply of fossil fuels, which is drawing attention towards more renewable biofuels like biodiesel . Biodiesel is primarily composed of methyl esters, which are unsaturated fatty acid chains that are prone to oxidation via the free radical mechanism. It can be found in vegetable oils, animal fats, and even in used frying oil. The reason why biodiesel is currently not very commercially accepted, however, is because it is expensive and has poor oxidative stability when exposed to atmospheric oxygen when it is being stored . This degradation of fuel quality can affect properties like kinematic viscosity, acid value, and peroxide value . Treating the fatty derivatives with oxidation inhibitors, or antioxidants, prevents this premature degradation . A previous study by Tang et al. found that the addition of synthetic antioxidants into biodiesel improves oxidative stability. However, these are synthetically produced through complex chemical processes in laboratories. Our group's goal is to create a more self-sustaining process by using bacteria to add naturally-occuring antioxidants to biodiesel over time. By genetically engineering a biofuel-tolerant strain of E. coli to produce these antioxidants, the bacteria eliminate the need for a complex and expensive synthetic chemical process. This would result in a continuous supply of antioxidants for the biodiesel, which in the long run can be more cost-efficient because it prolongs the useability and stability of the biodiesel. By producing higher quality and lower cost biodiesel, commercial acceptance of biofuels will grow and lead to widespread use of an effective and environmentally friendly fuel source. Eventually, this can relieve the current over-dependence on nonrenewable fossil fuels.
 Dunn, Robert. O. "Effect of antioxidants on the oxidative stability of methyl soyate (biodiesel)." Fuel Processing Technology 86.10 (2005): 1071-1085.
 Knothe, Gerhard. "Some aspects of biodiesel oxidative stability." Fuel Processing Technology 88.7 (2007): 669-677.
 Meng, Xin and Jianming, et al. Yang. "Biodiesel production from oleaginous microorganism." Renewable Energy 34.1 (2009): 1-5.
 Tang, Haiying and Rhet C. et al. De Guzman. "The oxidative stability of biodiesel:Effects of FAME composition and antioxidant." Lipid Technology 20.11 (2008): 1-5.
Biofuel as a source of renewable and green energy has great potential in reducing the need for fossil fuels. Currently, however, biodiesel that is newly produced or recycled from used oils can only be stored up to 6 months before auto-oxidation, the cause of rancidification, occurs. After that, exposure to oxygen causes the unsaturated methyl esters, the major chemical component of biodiesel, to undergo oxidation via a free radical process. This chain reaction resulting from initiation and propagation of lipid peroxyl radicals forms long polymers, which can clog fuel filters and makes the biodiesel unusable. It is plausible, therefore, to prolong the usability of stored biodiesel by preventing or at least minimizing the free radical oxidation of the methyl esters with the use of antioxidants. Our group seeks to develop a strain of E. coli that can continuously produce and secrete an antioxidant protein into the biodiesel that will prevent the oxidation of its chemical components, creating higher quality biodiesel.
The two main components of this research design are: one, to genetically engineer a biodiesel tolerating E. coli strain that secretes the antioxidant protein Alkyl Hydroperoxide Reductase Subunit C (AhpC); and two, to test the ability of the secreted AhpC to increase oxidative stability in biodiesel samples. The AhpC genetic sequence from Helicobacter pylori will be spliced and inserted into a plasmid with specific restriction enzymes. A protein secretion system that will allow AhpC to translocate across the inner and outer membranes of E. coli will also be inserted into the plasmid. The plasmid will then be transformed into a modified E. coli that is already able to withstand surviving in a biodiesel environment, and E. coli that successfully take in our plasmid will be isolated via a genetic screen. Furthermore, the robustness of AhpC secretion in E. coli will be analyzed based on the expression level of the AhpC gene and successful trafficking of the protein through its secretory pathways. The effectiveness of AhpC in inhibiting lipid oxidation will be analyzed by performing an AhpC to methyl ester concentration assay quantifying the relative concentration of methyl esters and their undesired lipid radical oxidation products. If AhpC is able to act as an effective antioxidant in the biodiesel, the next objective will be to increase output of AhpC by improving E. coli viability in a biodiesel environment and increase AhpC output from E. coli. Future studies include scale-up production of the engineered E. coli to produce AhpC levels that can effective prevent oxidation of biodiesel stored in an industrial-sized fuel tank.
Dunn, Robert. O. "Effect of antioxidants on the oxidative stability of methyl soyate (biodiesel)." Fuel Processing Technology 86.10 (2005): 1071-1085.
Knothe, Gerhard. "Some aspects of biodiesel oxidative stability." Fuel Processing Technology 88.7 (2007): 669-677.
Meng, Xin and Jianming, et al. Yang. "Biodiesel production from oleaginous microorganism." Renewable Energy 34.1 (2009): 1-5.
Tang, Haiying and Rhet C. et al. De Guzman. "The oxidative stability of biodiesel:Effects of FAME composition and antioxidant." Lipid Technology 20.11 (2008): 1-5.