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ARS Home » Midwest Area » Ames, Iowa » Corn Insects and Crop Genetics Research » Research » Publications at this Location » Publication #215167

Title: Development of a Fluorescence-based Method for Monitoring Glucose Catabolism and its Potential use in a Biomass Hydrolysis Assay

item RAMAN, D - ISU
item Scott, Marvin

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2008
Publication Date: 11/19/2008
Citation: Haney, L.J., Lamkey, K.R., Kirkpatrick, K., Coors, J.G., Lorenz, A.J., Raman, D.R., Anex, R.P., Scott, M.P. 2008. Development of a Fluorescence-based Method for Monitoring Glucose Catabolism and its Potential use in a Biomass Hydrolysis Assay. Biotechnology for Biofuels. 1:17.

Interpretive Summary: Increasing our domestic liquid fuel production will increase national security by reducing our dependance on foreign oil. One potenital feedstock for domestic liquid fuel production is crop residue biomass such as corn stover. Harvesting and storage strategies and genetic differences in varieties impact the amount of liquid fuel that can be produced from a given amount of biomass. We developed a method to predict the suitabiliy of different batches of corn stover for fermentation-based liquid fuel production and use it to show that different varieties differ in their suitabilities for this use. Our method is based on a biosensor that gives real-time measurements of the level of sugars produced in a hydrolysis reaction. This biosensor may be useful for optimization of processes used for biofuel production. Our method of corn stover analysis will allow breeders to develop varieties that yield more biofuel per land area. This will increase our domestic liquid fuel production and benefit the environment by reducing the amount of land required to meet our energy needs.

Technical Abstract: Availability and low cost of lignocellulosic biomass has caused tremendous interest in the fermentation of lignocellulosic-derived sugars for the production of liquid fuels. The economic feasibility of lignocellulosic biofuels can be improved by evaluating the fermentation potential of different feedstocks. During conversion, pretreated biomass is combined with hydrolytic enzymes that convert polymeric sugars into monomers. Such hydrolysis is feedback-inhibited by sugar products, limiting the extent and rate of the reaction. This feedback inhibition can be overcome by removal of sugar products in the reaction, as is done in simultaneous saccharification and fermentation (SSF). The objective of this study was to develop a sugar-consuming biosensor to monitor hydrolytic reactions and to demonstrate its application in monitoring corn stover hydrolysis. The biosensor is based on Escherichia coli strain CA8404, modified to produce green fluorescent protein (GFP), and is capable of catabolizing both five- and six-carbon sugars. After several biosensor characterization experiments, we found that growth rate was proportional to GFP-fluorescence, and total growth and growth rate depend upon how much sugar is present at inoculation. This biosensor has a dynamic range of 0.100-1.600 mg glucose/mL and can accurately measure sugar mixtures where 50-80% of the total sugar is glucose, the remainder xylose. We also demonstrated that stovers can be differentiated based on sugar yields in enzymatic hydrolysis reactions. It was possible to monitor the course of enzymatic hydrolysis in real-time. This biosensor could be used in screening methods to characterize hydrolysis of feedstocks or to evaluate the performance of hydrolytic systems.