Sugar loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolysis

Authors: XUE, SAISI - Michigan State University; UPPUGUNDLA, NIRMAL - Michigan State University; Bowman, Michael; CAVALIER, DAVID - Michigan State University; DA COUSTA SOUSA, LEONARDO - Michigan State University; DALE, BRUCE - Michigan State University; BALAN, VENKATESH - Michigan State University

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/5/2015
Publication Date: 11/26/2015
Publication URL: http://handle.nal.usda.gov/10113/62507
Citation: Xue, S., Uppugundla, N., Bowman, M.J., Cavalier, D., Da Cousta Sousa, L., Dale, B.E., Balan, V. 2015. Sugar loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolysis. Biotechnology for Biofuels. 8:195. doi: 10.1186/s13068-015-0378-9.

Interpretive Summary: High sugar concentrations are required in biomass fermentations to achieve high ethanol titers thereby reducing the energy required to distill the ethanol from fermentation broths; however, high solids loadings (e.g. 25% solid loading) required to achieve high sugar concentrations can interfere with the efficiency of the bioconversion process at a variety of levels. Due to high solids loading, an accumulation of unhydrolyzed oligosaccharides occurs during enzymatic hydrolysis of cellulosic biomass reducing biofuel yields and increases processing costs for a cellulosic biorefinery. These oligosaccharides accumulate to 18-25% of the total soluble sugars in the hydrolysate and represent a yield loss and can inhibit commercial hydrolytic enzymes hindering economical biofuel production from cellulosic biomass. Little is known about the features of these oligomers that make them recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gram quantities facilitating recalcitrance and enzyme inhibition studies. Such studies can clarify: the reasons for oligosaccharide accumulation; contribute to development of strategies to convert oligosaccharides into fermentable sugars to provide higher biofuel yields; and potentially reduce enzyme loadings.

Technical Abstract: Oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, ammonia fiber expansion: AFEX and extractive ammonia: EA). The methodology for large-scale separation of recalcitrant oligosaccharides from 25% solids-loading AFEX-corn stover hydrolysate (ACH) using charcoal fractionation and size exclusion chromatography is reported here for the first time. Oligosaccharides with higher degree of polymerization (DP) are recalcitrant towards commercial enzyme mixtures (Ctec2, Htec2 and Multifect pectinase (MP)) compared to lower DP oligosaccharides. Enzyme inhibition studies using isolated substrates (Avicel and xylan) show that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes.