Background: 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. Results: 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. Conclusion: 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.
Bibliographical noteFunding Information:
LJH was supported by the Iowa State University Agronomy Department Endowment. AJL was supported by the USDA-DOE grant 'Integrated Feedstock Supply Systems for Corn Stover Biomass', NRCS 68-3A75-4-137. The funding organizations had no role in study design, collection, analysis, or interpretation of data, writing of the manuscript or in the decision to submit the manuscript for publication. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. This paper is a joint contribution from the Corn Insects and Crop Genetics Research Unit, USDA-ARS, and project no. 3781 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011.