Role of enzyme blend composition, biomass particle size and solids loading on pretreated biomass saccharification efficiency

One of the limitations of screening enzyme combinations using microplate based assays (Gao et al. 2010, Bioresour Technol 101, 8:2770) is that these are typically carried out using milled biomass (< 500 mms) at low solids loading (< 1%, w/v). Biomass particle size and solids loading can both influence the enzyme set necessary for efficient hydrolysis. In this study, three different harvest varieties of AFEX pretreated corn stover were hydrolyzed at 0.6-18% solids loading using multiple combinations of cellulases, hemicellulases and other accessory enzymes for extended hydrolysis time periods. Three particle size ranges were tested (< 100 mm, < 500 mm, >1 mm). The crude glycosyl hydrolase mixtures were also analyzed using a high-throughput proteomics approach to identify enzyme activities necessary for efficient hydrolysis. We show that proteomics based spectral counting approach is a reasonably accurate and rapid analytical technique that can be used to determine protein composition of complex glycosyl hydrolase mixtures that also correlates with the specific activity of individual enzymes present within the mixture. We also find that both particle size and solids loading can significantly impact the optimal combination of hydrolytic enzymes necessary for efficient hydrolysis. In summary, we found that optimal cellulase-hemicellulase mixtures can achieve near-theoretical glucan and xylan yields at greater than fourfold lower total protein loading than using individual commercial enzyme blends alone.