Facing inexorable growth of global population and energy consumption, lignocellulosic biomass has taken a more central role in global energy strategy by providing sustainable sources for both liquid fuels and chemicals without competing with food and feed supplies. To establish a large scale, reliable, and economic lignocellulosic industry, biomass recalcitrance must be well understood and overcome. Currently cutting edge biological conversion research focuses mainly on three aspects: genetic modification of plant cell wall structure to reduce biomass recalcitrance, pretreatment of biomass as a critical prerequisite to achieve high sugar yields, and consolidated processing of enzymes and microorganisms to lower deconstruction costs. More importantly, however, synergistic concert of these three approaches is critical to develop practical solutions for this challenge. Thus, interactions and impacts among cell wall structural characteristics, pretreatment, and biological deconstruction are vital to understand.

In light of this, six technical studies towards three main objectives were pursued in this thesis. First, an integrated chromatographic method for xylooligosaccharides determination and a high throughput system using dilute alkali conditions were developed as characterization tools to facilitate understanding how pretreatment affects cell wall polysaccharide deconstruction. Next, two fundamental studies on lignin deposition during pretreatment and reduced methylation of xylan side-chains, respectively, were employed to identify important recalcitrant aspects of lignin and hemicellulose on biomass digestion. Finally, systematic research was undertaken to develop Agave as a low recalcitrant, drought resistant biofuels feedstock for semi-arid lands. The findings of this thesis provide fundamental insights towards understanding interactions of lignin and hemicellulose, the two primary barriers to biological conversion, for more effective biomass deconstruction and better energy crops design.