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Determination of Limiting Factors in Fungal BioremediationEPA Grant Number: U915026
Title: Determination of Limiting Factors in Fungal Bioremediation
Investigators: Cross, Deanna S.
Institution: Texas A & M University
EPA Project Officer: Edwards, Jason
Project Period: January 1, 1996 through January 1, 1999
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1996) RFA Text | Recipients Lists
Research Category: Fellowship - Plant Pathology , Academic Fellowships , Ecological Indicators/Assessment/Restoration
The objective goal of this research project is to better understand factors important for developing viable fungal bioremediation strains using Trichoderma virens and organophosphate hydrolase (OPH) as a model system. Several factors identified for analysis and optimization were,: (1) growth and survival of fungal propagules in soil,; (2) survival in the presence of concentrated organophosphate levels,; (3) effective reduction of organophosphate compounds,; and (4) effectively remediating complex pesticide mixtures in concert with other strains.
Growth in soil of a strain, of T. virens (GvT6), that has been was modified with the bacterial gene (opd) encoding OPH was determined. GvT6 and its near isogenic parent (29-8) were grown in soil microcosms on the surface of a submerged membrane. The membrane was destructively sampled at 2, 3, 4, 6, and 8 days after inoculation. The fungal strands were stained and digitized. The digitized images were enhanced and analyzed using Image Pro Plus v.3.0. The number of pixels representing fungal biomass on each membrane was calculated, and the total growth for each day was recorded. This information was used to compare the growth of the parent with the growth of the transformant to determine if the transformed organism grows differently than the parent. The growth is being modeled using a critical exponential model. Gene expression and growth in the presence of contaminant were was tested with four different strains, which differ in the amount of OPH produced and in the transformation system used to incorporate OPH into the genome. Strains will be grown over a range of temperatures and enzyme activity in the culture supernatant will be determined using paraoxon as the substrate. The activity will be measured in units per mg of total protein and units per mg of fungal biomass. The strains will then be tested for growth in the presence of different levels of methyl parathion in vitro. The strains performing best in vitro will be further tested for growth and degradation of high levels of methyl parathion in soil. To investigate the ability of different strains of T. virens to work in consortia, several forms of opd encoding OPH with different metal binding potentials in the active site, were placed into T. virens. Enzyme profiles of the transformants will be characterized as above. The strains will be challenged individually with demetons, methyl parathion, and paraoxon alone and in combination. The strains will be placed in all combinations and challenged with the compounds to determine if there are any synergistic affects of strain combinations on degradation.