While no one particular pretreatment process can presently be viewed as the “ideal” ap­proach for all feedstocks or for all process circumstances, a well-accepted list of the desired properties of an ideal pretreatment process has been generated (3). Such an ideal pretreat­ment process:

• Produces a highly digestible pretreated solid

• Does not significantly degrade pentoses

• Does not significantly inhibit subsequent fermentation steps

• Requires little or no size reduction of biomass feedstock

• Can work in reactors of reasonable size and moderate cost

• Produces no solid-waste residues

• Has a high degree of simplicity

• Is effective at low moisture content

14.2 Physicochemical properties of pretreated biomass believed to affect cellulose digestibility

A significant effort has been expended, aimed at increasing our understanding of the factors that are the most critical in controlling the susceptibility of cellulosic substrates to enzymatic hydrolysis. A wide variety of physical and chemical properties of pretreated biomass and the cellulose remaining after pretreatment have been studied and reviewed (4-7). Changes in the physicochemical properties of pretreated lignocellulosics have been correlated with the enzymatic hydrolysis of the cellulose. There are general conclusions that have come out of these reviews that should be considered when trying to identify the critical properties in pretreated biomass. As stated by Coughlan (4), “There is considerable disagreement in the literature regarding the relative importance of the various factors that render cellulose so recalcitrant to hydrolysis.” Mansfield et al. (5) cautions that “when contemplating these characteristics (structural and physicochemical features of the substrate) and identifying potential contributing factors or limitations (to enzymatic hydrolysis) care must be taken to consider some undisputable principles: (i) all samples of insoluble cellulose (both native and pretreated) are structurally non-uniform; (ii) the pretreatment method and conditions can effectively alter the structure of the original cellulose; (iii) native cellulose contains inherent regions of highly ordered and disordered molecular polymers (i. e., crystalline and amorphous regions); (iv) considerable attention must be paid to the anatomical and structural ‘levels’ of organization (i. e., microfibril, fibril, or fiber) which is being modified or characterized during hydrolysis…”

A key factor for successful enzymatic conversion of biomass to fermentable sugars is the accessibility of the [3(1—>4) glycosidic bonds in cellulose to cellulase enzymes. Pretreatment regimes must be designed to remove substrate-specific barriers to cellulases to improve cel­lulose digestion. The precise nature of the obstacles encountered by cellulases in the complex biomass ultra-structure remains ambiguous. The effect of pretreatment is typically evalu­ated on the basis of improved enzyme digestibility and downstream ethanol production. The link between changes in cell wall chemistry/structure and cellulase digestibility is ultimately dependent on improved access to the cellulose microfibril. Accurate and direct assessment

of changes in enzyme accessibility is challenging primarily due to the complexities of both the cellulase system and the biomass. (8).

Some of the factors that could influence the rate of enzymatic hydrolysis of cellulose in pretreated lignocellulosic feedstocks are cellulose crystallinity, degree of cellulose poly­merization, feedstock particle size, the lignin barrier (content and distribution), substrate available surface area (pore volume), and cell wall thickness (coarseness). In addition, irre­versible binding of enzymes onto lignin is also influenced by the nature of the substrate (6, 7). Typical physicochemical properties of biomass obtained from the pretreatments used by the CAFI group are shown in Table 14.1. Zhang and Lynd (9) have attempted to take this a step further by building functional models of cellulose hydrolysis that incorporate substrate features in addition to concentration and the activities of multiple cellulase components.