Water Resources Research Act Program

Details for Project ID 2009DE153B

Development of an Assay to Monitor the Activity of Fungal Enzymes in Soil

Institute: Delaware
Year Established: 2009 Start Date: 2009-03-01 End Date: 2010-02-28
Total Federal Funds: $3,500 Total Non-Federal Funds: $7,000

Principal Investigators: Anastasia Chirnside, Brian Jayne

Project Summary: White rot fungi (WRF), specifically Phanerochaete chrysosporium, are able to produce non-stereo selective enzymes that oxidize toxic environmental pollutants. Consequently, this has brought the fungi great attention in the field of environmental remediation. Studies have shown that the white rot fungus is able to degrade pesticides (Bumpus and Aust, 1987; Yadav and Reddy, 1993; Kenedy et al., 1990), polyaromatic hydrocarbons (Carberry, 1990; Mileski et al., 1988; Reddy and Gold, 2000), polychlorinated biphenyls (Thomas et al., 1992), and most halogenated aromatics. The WRF use a secondary growth phase in order to completely mineralize lignin, a complex polymer. This ligninolytic activity initiates the production of the non-specific, non-stereo selective extracellular enzymes: lignin peroxidases (LiP), manganese-dependent peroxidases (MnP) lactases, laccases, a hydrogen peroxide-generating system, other enzymes and co-factors (Barr and Aust, 1994; Maloney et al., 2001; Reedy and Mathew, 2001; and Wu et al., 1996). Activation of Lip and/or MnP results in formation of a porphyrin cation radical that can oxidize compounds, two times, before the enzyme returns to its resting state. The creation of these cation radicals with high redox potentials and large substrate ranges is what makes the fungus able to oxidize a variety of compounds (Hammel, 1995). Immobilizing P. chrysosporium in a packed-bed bioreactor (PBR) resulted in an increase of LiP production and successful degradation of pesticides and other recalcitrant compounds found in industrial wastewaters. Past research has investigated the ability of the fungal enzymes, LiP and MnP, to degrade pesticides and other compounds present in wastewater and the herbicides, atrazine and alachlor, in contaminated soil (Chirnside et al., 2005). Although past studies have successfully measured the percentage of compound degradation, there still remain difficulties with measuring the enzyme activity after application to the soil due to interacting compounds. The goal of this research is to develop an assay and method to monitor the activity of LiP and MnP within the soil over time. Current methods of enzyme extraction lead to inhibition of typical assays due to interfering compounds extracted with the enzymes. An extensive literature search of soil enzyme extraction methods led to the selection of several possible extraction techniques that could be modified to overcome these difficulties. The objective of this research is to develop an accurate assay to monitor the activity of LiP and MnP within the soil during degradation experiments. Specifically we want to define how long the enzymes stay active in the soil during degradation studies.