Year of Publication

2014

Degree Name

Master of Science in Biosystems and Agricultural Engineering (MSBiosyAgE)

Document Type

Master's Thesis

College

Agriculture; Engineering

Department

Biosystems and Agricultural Engineering

First Advisor

Dr. Sue Nokes

Abstract

Scaling biological pretreatment from the bench scale to the production scale may be more economical if unsterilized feedstock are used, however these allow for microbial competition from contaminates. An accurate and rapid method for identifying the desired biological pretreatment organism is necessary to confirm the presence of the desired organism when contaminates are morphologically similar to the target organism. Traditional methods, such as visual identification, sequencing, and selective plating can be time consuming and are sometimes still inconclusive. Based on methods described in the literature, plasmid DNA containing the marker genes gus (�-glucuronidase), LacZ, and gfp (green fluorescence protein) incorporated into the lignin-degrading basidiomycete Phanerochaete chrysosporium would result in a rapid genetic test for the desired organism. The presence of these genes can be confirmed either through an X-Gluc (cyclohexylammonia salt), X-Gal histochemical assay or observing the gfp’s fluorescence by a specially equipped confocal microscope. Each reporter systems will allow for rapid, reliable identification of the target species. This study will report on the success of the transformation methods in creating a transformed fungus to be used in the context of a large-scale fermentation operation.

Additionally, a novel in-harvest lignocellulose feedstock biological pretreatment inoculation trial was performed comparing lignolytic performance between fungal inoculum application techniques. Optimization of carbohydrate availability for enhanced saccharification was determined by analyzing glucose release by treated and non-treated unsterilized switchgrass. This study also focused on identifying parameters to enhance saccharification efficacy at the farm-scale.

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