| Summary: | The production of biofuels from non-edible plant biomass has been necessitated by the concern for the environmental consequences of fossil fuel use and the tightening of supply and demand for liquid fuels. In contrast to first generation biofuels which rely on crops used for food supplies, second generation biofuels, derived from lignin-containing feedstocks, completely eliminate the competition for food. The major challenges associated with second generation biofuels are both technical and economic. Due to the recalcitrant nature of the raw biomass materials to further biological conversion, their structural degradation often requires severe and costly pretreatment processes such as heat, physical and chemical treatments to disturb and fractionate the biomass. Significant research effort has been devoted to understanding the recalcitrant nature and to accelerate the commercialization process of second generation biofuels. In this thesis, three pretreatment methods that belong to different categories have been investigated to understand their impacts on cellulose and/or lignocellulose and the subsequent hydrolysis steps.
Physicochemical pretreatments, such as steam explosion, have been identified as one of the most effective and cost-efficient pretreatment methods for lignocelluosic materials. In Chapter 2, SO2-catalyzed steam explosion will be discussed and the effect of pretreatment severity on the substrate characteristics and degradation efficiency is also elucidated. Although the crystallinity index (CrI) of cellulose decreases as the severity increases, significant non-specific degradation and low yield of cellulose was observed at high severity. A new method for cellulose CrI determination has been developed with least squares curve fitting and validated with mechanically mixed cellulose samples. Biological pretreatment is another pathway through which the biomass structure can be modified to obtain a more amenable state for enzymatic degradation. Cellulose-binding domain (CBD) originated from Trichoderma reesei Cel7A (i.e. Tr cellobiohydrolase I) has been discovered as a potential biological pretreatment agent which is capable of modifying cellulose crystal structure. An extensive study on the protein engineering, expression, purification and functionalities of Cel7A CBDs was carried out (Chapter 3). The target mutations were identified with a computational protein engineering method involving principal component analysis (PCA). Due to the lack of catalytic activity and high throughput screening method, the library size was limited to nine. The wild-type and mutated CBDs were compared for their adsorption behavior and decrystallization effect on cellulose. Resulting saccharification efficiency after CBD pretreatment were studied and a possible explanation for the rate enhancement was proposed.
In addition to physicochemical and biological pretreatment methods, chemical pretreatment is also a commonly employed method to overcome the recalcitrance of lignocellulosic materials. The most widely studied include dilute acid, alkaline, and organosolv processes. Inspired by the rapidly growing green solvent ionic liquid (IL) researches in biomass pretreatment, substituted imidazoles have been investigated in this thesis to assess their potential as pretreatment agents for lignocelluloses (Chapter 4). 1-Methylimidazole (MI), a precursor to some ILs, has been determined to be the most promising agent for lignocellulose pretreatment due to its exceptional delignification and cellulose expansion efficiency. The chemical recovery and MI process development will also be discussed in Chapter 4.
In order to understand pretreatment effect, a semi-quantitative assay utilizing low molecular weight direct dyes and cellulases to estimate the accessibility and pore size distribution has been developed for application on pure cellulose substrates in Chapter 5. Finally, main conclusions as well as future perspectives for this work will be discussed in Chapter 6.
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