Groundwater Remediation through Environmental Biotechnology: Transgenic Phytoremediation of Methyl Tertiary Butyl EtherEPA Grant Number: U916152
Title: Groundwater Remediation through Environmental Biotechnology: Transgenic Phytoremediation of Methyl Tertiary Butyl Ether
Investigators: Skinner, Kristin M.
Institution: Oregon State University
EPA Project Officer: Jones, Brandon
Project Period: January 1, 2003 through January 1, 2006
Project Amount: $105,983
RFA: STAR Graduate Fellowships (2003) Recipients Lists
Research Category: Fellowship - Environmental Engineering , Academic Fellowships , Engineering and Environmental Chemistry
The objectives of this research project are to design and characterize a transgenic phytoremediation system for the attenuation of the fuel additive methyl tertiary-butyl ether (MtBE) and other recalcitrant environmental pollutants from contaminated groundwater.
A filamentous fungus, Graphium sp. cometabolizes MtBE. A cytochrome P450 propane monooxygenase (PMO) is thought to be responsible for this activity in Graphium sp. The gene encoding this protein will be heterologously expressed in an alternate fungus to demonstrate function and to determine the substrate range of the cytochrome P450 PMO. Once proof of function is established, the cytochrome P450 PMO will be transformed into plants using established Agrobacterium tumefaciens transformation techniques. Kanamycin-resistant tobacco plants will be transferred to soil and grown in a growth chamber. Reverse transcriptase-polymerase chain reaction will be used to determine if the cytochrome P450 PMO gene is transcriptionally active. Transformed tobacco plants that demonstrate high levels of expression will be evaluated further in subsequent microcosm tests. Although it ultimately will be necessary to tranform the cytochrome P450 PMO into poplar hybrids, a plant that is practical for phytoremediation, proof of concept and modeling experiments will be conducted initially in tobacco. Transgenic and null vector individuals will be grown under greenhouse conditions that mimic the presence of groundwater. Aqueous concentrations of MtBE will be monitored with a gas chromatograph to determine specific kinetic parameters including rates of reaction and the limiting concentration at which MtBE can be taken up and transformed. Similar assays with radiolabeled MtBE will establish the metabolic fate of MtBE and MtBE oxidation products in transgenic plants. This work is expected to generate an innovative and inexpensive system for the removal of recalcitrant chemicals from contaminated groundwater.