title: Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis.
abstract: BACKGROUND
  Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18-25 % of the total soluble sugars in the hydrolysate and 12-18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7-9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production from cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature.
  
  
  RESULTS
  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, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEX-corn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes.
  
  
  CONCLUSIONS
  The carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme.
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Biomass Conversion Research Lab (BCRL), Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910 USA.
authors.forename: Saisi
authors.identifier: 0000-0002-1973-0666
authors.initials: S
authors.lastname: Xue
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Biomass Conversion Research Lab (BCRL), Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910 USA.
authors.forename: Nirmal
authors.initials: N
authors.lastname: Uppugundla
authors.affiliation: USDA, Agricultural Research Service, National Center for Agricultural Utilization Research, Bioenergy Research Unit, Peoria, IL 61604 USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bowman
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Biomass Conversion Research Lab (BCRL), Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910 USA ; DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 USA.
authors.forename: David
authors.initials: D
authors.lastname: Cavalier
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Biomass Conversion Research Lab (BCRL), Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910 USA.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Da Costa Sousa
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Biomass Conversion Research Lab (BCRL), Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910 USA.
authors.forename: Bruce
authors.initials: B
authors.lastname: E Dale
authors.affiliation: DOE Great Lakes Bioenergy Research Center, Biomass Conversion Research Lab (BCRL), Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910 USA.
authors.forename: Venkatesh
authors.initials: V
authors.lastname: Balan
keywords: AFEX-CS hydrolysate; Charcoal fractionation; Commercial enzymes; Enzyme inhibition; High solids-loading; Recalcitrant oligosaccharides; Size exclusion chromatography