Miniaturized, high-throughput hydrolysis assay for comparison of cellulase performance at high solids content of biomass substrates

Lignocellulosic biomass is a renewable feedstock that can be converted to fuels and chemicals through simple sugar intermediates.  Significant advances have been made in reducing the cost of cellulases for conversion of pretreated biomass substrates to fermentable sugars; additional efforts are currently underway to further improve cellulase performance and reduce the cost of enzymes.  Here we introduce a novel method for high-throughput screening of cellulase activity at high solids content of commercially relevant biomass substrates in microtiter plate format.  The assay requires minimal processing of pretreated biomass substrates for hydrolysis, and can be used under a variety of conditions, including pH and temperatures.  The amount of protein required for microtiter high solids reactions is significantly reduced compared to larger scale assays.  With dilute acid-pretreated corn stover (NREL, Golden, CO) as a model substrate and using this method, we demonstrate the effects of temperature, pH, solids loading and relative cellulase composition on saccharification, and show the correlation to a larger scale, high-solids assay benchmark.  We further demonstrate that the assay can be used on a variety of substrates, with CV values between 1% and 5%.  The assay represents a useful tool for identifying new and improved enzymes for biomass hydrolysis, enzyme mix optimization and assessment of the digestibility of biomass substrates.