There have been only limited studies assessing the significance of initial cellulose crystallinity and DP of lignocellulosic substrates with regard to subsequent substrate hydrolysis by cellulases; however, the importance of these factors has been the subject of considerable debate [113,114]. It has been suggested that amorphous cellulose is hydrolyzed, initially resulting in an accumulation of crystalline cellulose rendering the substrate increasingly recalcitrant as the hydrolysis progresses [113,114]. Most studies that have established a correlation between crystallinity and hydrolysis have utilized substrates of relatively pure cellulose, which most likely do not represent the heterogeneous lignocellulosic substrate encountered during the hydrolysis of substrates pretreated for bioconversion [115-117]. Furthermore, to demon­strate the effect of crystallinity on hydrolysis, these studies frequently utilize physical treatments such as ball milling [116] or gamma irradiation [118] to alter the initial substrate crystallinity, which can also result in increases in specific surface area. As a result, in previous work both crystallinity and spe­cific surface area of pure cellulose substrates have been combined into models predicting the rate and extent of hydrolysis [119]. As for crystallinity, it is dif­ficult to assess the effects of DP exclusively, since altered DP can be associated with crystallinity or accessible surface area. Nevertheless, there have been a few studies investigating the effects of the crystallinity and DP of chemical pulps on their hydrolysis by cellulases.

Employing unbeaten, beaten and recycled softwood pulps as substrates to assess various substrate characteristics that influence the enzymatic hydro­lysis of cellulose, Nahzad et al. [105] showed that although the pulps pos­sessed similar crystallinity, the beaten pulp hydrolyzed more readily than the unbeaten pulp without any appreciable changes in crystallinity occur­ring during the hydrolysis. Similar results have been found by Ramos et al. during the treatment of fully bleached eucalyptus Kraft pulp [104] and by Mansfield et al. during combined cellulase-xylanase treatment of Douglas — fir Kraft pulps [99]. Nahzad et al. [105] also showed that the initial DP of the pulps did not play a role in affecting subsequent hydrolysis; however, the DP was decreased by 2/3 during the hydrolysis period and the polydispersi — ties of all the hydrolyzed pulps were quite similar. Mansfield et al. [99] did not observe appreciable changes in cellulose DP during hydrolysis, however, it should be noted that they employed low cellulase loadings to impart sub­tle modifications to the pulp fiber. It is evident from the literature presented here that attributing the ease of enzymatic digestibility of a given substrate to initial crystallinity or DP is a dubious task, compared to studies that have tied the ease of hydrolysis of substrates to their initial surface area. However, pore volume determinations require a significant investment in time to ob­tain reproducible results. Also, it is likely that the pores in a lignocellulosic substrate will have irregular shapes, thus affecting the accuracy and preci­sion of the measurement [120]. Another drawback is that the method does not measure the areas in pores that are larger than the size of the probe, which would provide the easiest access for cellulases. Investigations into the sub­strate physical factors that affect hydrolysis should be aided by the continual evolution of analytical techniques such as thermoporosimetry [121] and high resolution fiber quality analysis [122], which may be capable of dealing with the diversity of pretreated lignocellulosic substrates produced for subsequent hydrolysis and fermentation in the bioconversion process.

6

Conclusions

In this review we suggested that, although the properties of the cellulase en­zyme complex has a significant effect on how effectively a lignocellulosic material will be hydrolyzed, it is the biomass pretreatment and the intrin­sic structure/composition of the substrate itself that are primarily responsible for its subsequent hydrolysis by cellulases. It is apparent that in sequential series of events, the conditions employed in the chosen pretreatment will af­fect various substrate characteristics, which in turn govern the susceptibility of the substrate to hydrolysis by cellulases and the subsequent fermentation of the released sugars. Choosing the appropriate pretreatment for a particu­lar biomass feedstock is frequently a compromise between minimizing the degradation of the hemicellulose and cellulose components while maximizing the ease of hydrolysis of the cellulosic substrate. The digestibility of pretreated lignocellulosic substrates is further complicated by the lignin-hemicellulose matrix in which cellulose is tightly embedded. Pretreatment conditions can be tailored to create either solid or solid/liquid substrates with varying levels of cellulose, hemicellulose and lignin. It is apparent that lignin affects enzy­matic hydrolysis by blocking cellulose and by chemical interactions facilitated by its hydrophobic surface properties and various functional groups. The role of hemicellulose is less obvious although there is good evidence to support the action of hemicellulose as a barrier restricting access to cellulases. In the past, many investigators have attributed the enhanced enzymatic hydrolysis performance of a particular pretreatment to changes in the proportion of the lignin, hemicellulose and cellulose in the substrate. However, it is important to advance this conclusion one step further as it is likely that decreases in lignin and hemicellulose content that occur as a result of pretreatment also affect the physical properties of the cellulosic component, such as its crys­tallinity, the degree of polymerization and the surface area of the substrate accessible to cellulases.

Various studies conducted with different cellulase systems and a range of cellulosic substrates all indicate that it is ultimately the “accessibility” of the cellulose fraction to the enzyme system that determines how fast (reaction rate) and how far (% conversion) the hydrolysis reaction can proceed [112]. In work conducted with either wood pulps or substrates pretreated for bio­conversion we and other groups have shown that accessibility is a property that describes the static environment encountered by the cellulase complex when it is combined with the substrate, and its action is governed by the in­trinsic pore size distribution, degree of swelling and other gross and detailed substrate characteristics. As enzymatic hydrolysis commences, the situation becomes dynamic, as substrate attributes begin to change due to cellulose hydrolysis and the hydrolysis rate decreases. Some workers have reported de­creases in accessible surface area as hydrolysis proceeds without appreciable changes in crystallinity [99,105,123], while others have reported decreases in crystallinity and increases in accessible surface area during hydrolysis [124]. The discrepancy in results regarding the decrease in the hydrolysis rate of pretreated substrates can most likely be attributed to variations in the sub­strates being studied and the techniques used for measurement of substrate properties. Furthermore, as cellulose is hydrolyzed, the lignin and hemicellu — lose that accumulate in the hydrolysis residue can potentially restrict access to cellulases and decrease the hydrolysis rate. Therefore, pretreatments should aim to produce a readily hydrolyzable substrate by increasing accessibility to cellulases and limiting the negative effects of hemicellulose and lignin on hydrolysis, while maximizing the total carbohydrate recovery.

However, it is important to recognize that studies which try to optimize pretreatment (as assessed by product recovery) need to be performed in parallel with measurements of key substrate characteristics in order to as­sociate specific aspects of pretreatment to substrate attributes that facilitate subsequent hydrolysis by cellulases. This emphasizes the significance of the pretreatment since the effectiveness of pretreatment affects both the up­stream selection of biomass, the efficiency of recovery of the overall cellulose, hemicellulose and lignin components, and the chemical and morphological characteristics of the resulting cellulosic component, which governs down­stream hydrolysis and fermentation.

Adv Biochem Engin/Biotechnol (2007) 108: 95-120 DOI 10.1007/10_2007_066 © Springer-Verlag Berlin Heidelberg Published online: 27 June 2007

Progress and Challenges