The success of an economically feasible and environmentally sustainable lignocellulosic biorefinery has been largely impeded because of the native recalcitrance of lignocellulosic plant cell walls to thermochemical treatment (e.g. chemical pretreatment) and biochemical processing (e.g. enzymatic hydrolysis). Several leading pretreatment technologies, including Ammonia Fiber Expansion, Extractive Ammonia, Ionic Liquid, Steam Explosion and Dilute Acid, significantly help enhance the enzyme digestibility of pretreated biomass. Comprehensive enzyme cocktails have also been developed to maximize the biomass hydrolysis to fermentable sugars. However, due the complex nature of plant cell wall, inefficient sugar conversion remains to be a universal problem for various pretreatment technologies. The mechanism of recalcitrant oligosaccharides accumulation is poorly understood. The goal of this dissertation is to understand the limiting factors that contribute to unconverted carbohydrates in bio-based chemical industry, and provide information for the rational design of enzyme cocktail, fermentation and pretreatment process adjustments.We first proposed the methodology for large-scale purification of oligosaccharides using charcoal fractionation and size exclusion chromatography. The carbohydrate composition of recalcitrant oligosaccharides, their mass and molecular weight distribution profiles were determined. Enzyme activity assay revealed that sugar inhibition and the lack of enzyme activity in commercial enzyme mixtures are major reasons for inefficient monomeric sugar conversion.A novel glycome profiling method (Elisa screening), combined with biotinylation derivatization, was used as a rapid, high-throughput tool to identify recalcitrant non-cellulosic glycan structures for small-molecule oligosaccharides and insoluble polysaccharides. 4-O-methyl-D-glucuronic acid substituted xylan and pectic-arabinogalactan were found to be the most abundant epitopes recognized by monoclonal antibodies in liquid hydrolysate and unhydrolysed solids. These structures were further validated by MALDI-TOF and TMS composition analysis. Based on the epitope information, novel accessory enzymes were supplemented into existing enzyme cocktails with the required activities for achieving complete sugar deconstruction.An artificial, chemically-defined Synthetic Hydrolysate was used to study the inhibitory effect of water-soluble components of crude lignin stream produced extractive ammonia pretreatment (EA) on yeast fermentation. Key inhibitory compounds that were identified using LC-MS and GC-MS included p-coumaroylamide, feruloylamide and p-coumaroylglycerol. Chemical genomics was employed to show the fingerprints of genes deletion response to inhibitors. The different sensitive/resistant genes clusters confirmed the variability and similarity of inhibitors in water-soluble components with real hydrolysate.
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