A SYSTEMATIC UNDERSTANDING OF BIOMASS RECALCITRANCE FROM FIBER, FIBRIL AND MOLECULAR LEVELS
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Deep understanding of "biomass recalcitrance" is a key to the development of cost effective pretreatment and discovery of highly efficient polysaccharide degrading enzymes. This thesis presents a systematic investigation of biomass recalcitrance across plant cell wall structures from fiber, fibril and molecular levels (Figure below). To achieve this goal, a set of biomass reference substrates with controlled levels of physico-chemical properties were prepared, and a number of analytical techniques to investigate specific substrate characteristics were developed.The effects of fiber size, fiber swelling, and resulting surface area changes on biomass substrate digestibility were investigated by using reference substrates (Chapter Two). Results showed that fiber size changes have negligible influence while swelling changes have significant influence on enzymatic hydrolysis efficiency. At the fibril level, the interactions between different cell wall components, cellulose, hemicellulose and lignin and their effects on biomass recalcitrance were revealed. X-ray photoelectron spectroscopy was established to quantify the amount of lignin on biomass substrate surface. Apart from its hindrance effect, xylan was found to enhance fibril swelling and thus generate more accessible surface area to facilitate enzyme and substrate interactions. Surface lignin has a direct impact on enzyme adsorption kinetics and hydrolysis rate (Chapter Three). Cellulose crystallinity is the one of the major factor contributing to biomass recalcitrance from molecular level. In this thesis, refined X-ray diffraction was developed to monitor changes in the crystallite structure of NCC (Chapter Four). Results provide strong evidence to demonstrate that the decrystallization of cellulose does not involve a "swelling component" to disrupt hydrogen bond in cellulose crystallite which has been a main hypothesis to explain cellulose hydrolysis mechanism. Our research support that cellulose decrystallization proceed through delamination mechanism which can be enhanced by putative oxidative enzymes (Chapter Five).Finally, reference substrates have been applied to evaluate the performance of commercial cellulase as well as cellulase mutants toward biomass hydrolysis (Chapter Six). It is apparent that biomass references substrates and the methodology established by this thesis can provide an effective and practical approach to identify highly efficient plant cell-wall degradation enzyme to overcome biomass recalcitrance.