Fundamental understanding of the recalcitrant cell wall present in hydrolysate at different stages of enzyme hydrolysis

Ammonia fiber expansion (AFEX^TM) pretreatment has been proven to improve the enzymatic digestibility of plant cell walls. Although complex mixtures of enzymes have been identified to effectively break down and generate simple sugars from pretreated lignocellulosic biomass, there is a consistent decrease in enzymatic activity as a function of hydrolysis time. This decrease in enzymatic activity cause the accumulation of unhydrolyzed oligosaccharides (both water soluble and insoluble) and polysaccharides that cannot be further hydrolyzed to fermentable sugars. The cause for such loss of enzymatic activity may be related to missing enzyme activities for breaking down specific linkages present in the plant cell wall, or by product inhibition resulting from the accumulation of soluble sugars. Fundamental understanding of the nature of these polysaccharides and oligosaccharides at different stages of enzyme hydrolysis can lead to the identification of specific enzyme activities and, eventually, missing enzyme activities in commercial cocktails to break down AFEX pretreated biomass.

In this study, AFEX pretreated corn stover was hydrolyzed at 20% solids loading using optimized commercial enzyme cocktails. The resulting unhydrolyzed solids (UHS) and soluble oligossacharides were analyzed for sugar composition and used to determine the respective glycome profiling, where specific linkages that tend to accumulate during enzymatic hydrolysis were characterized. For better understanding of the hydrolysis process, this analysis was performed for various time points during the course of reaction. This work will help us to identify the limitations of current commercial enzyme cocktails for deconstructing AFEX pretreated corn stover.