Development of a fluorescence-based method for monitoring glucose catabolism and its potential use in a biomass hydrolysis assay
Lisa J Haney1, James G Coors2, Aaron J Lorenz2, D Raj Raman3, Robert P Anex3 and M Paul Scott4
1Syngenta Seeds Inc, Bloomington, IL, USA
2Department of Agronomy, University of Wisconsin-Madison, Madison, WI, USA
3Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA, USA
4Corn Insects and Crop Genetics Research Unit, ARS, USDA, USA
Biotechnology for Biofuels 2008,
1:17. An Open Access article.
The availability and low cost of lignocellulosic biomass has caused
tremendous interest in the bioconversion of this feedstock into liquid
fuels. One measure of the economic viability of the bioconversion
process is the ease with which a particular feedstock is hydrolyzed and
fermented. Because monitoring the analytes in hydrolysis and
fermentation experiments is time consuming, the objective of this study
was to develop a rapid fluorescence-based method to monitor sugar
production during biomass hydrolysis, and to demonstrate its
application in monitoring corn stover hydrolysis.
Hydrolytic enzymes were used in conjunction with Escherichia coli strain
CA8404 (a hexose and pentose-consuming strain), modified to produce
green fluorescent protein (GFP). The combination of hydrolytic enzymes
and a sugar-consuming organism minimizes feedback inhibition of the
hydrolytic enzymes. We observed that culture growth rate as measured by
change in culture turbidity is proportional to GFP fluorescence and
total growth and growth rate depends upon how much sugar is present at
inoculation. Furthermore, it was possible to monitor the course of
enzymatic hydrolysis in near real-time, though there are
instrumentation challenges in doing this.
We found that instantaneous fluorescence is proportional to the
bacterial growth rate. As growth rate is limited by the availability of
sugar, the integral of fluorescence is proportional to the amount of
sugar consumed by the microbe. We demonstrate that corn stover
varieties can be differentiated based on sugar yields in enzymatic
hydrolysis reactions using post-hydrolysis fluorescence measurements.
Also, it may be possible to monitor fluorescence in real-time during
hydrolysis to compare different hydrolysis protocols.