Pretreatment methods such as solvent extraction (organosolv pulping) [57] or ammonia fiber explosion treatment (AFEX) [58] either modify or remove lignin, while a large proportion of lignin remains intact in the solid phase after SO2-catalyzed steam pretreatment [59] or dilute acid pretreatment [60]. Since lignin is intertwined amongst cellulose and hemicellulose, varying the pretreatment method or conditions employed to improve cellulose or hemi — cellulose yields will undoubtedly affect the lignin content [57,59]. For ex­ample, it has been shown that the amount of lignin in the solid fraction of the substrate increases as the severity of SP is increased. Based on 13 severity fac­tors used during SO2-catalyzed SP of corn fiber, it was shown that the amount of lignin in the solid phase increased as the severity of the pretreatment was raised [59]. The lignin was most likely concentrated in the solid fraction due to the solubilization and degradation of the carbohydrates as the severity was raised [59]. It should be noted that the Klason method that is commonly used to estimate the lignin content of the pretreated substrate can result in arti­ficially high values for lignin, as sugar degradation products and entrapped low molecular weight phenolics can also be measured as “lignin”. For ex­ample, it was shown that the Klason lignin contents of steam-pretreated aspen at a series of severities ranged from 6-30%. However, the supposedly corres­ponding methoxyl groups in the samples were only in the 0.8-7.7% range. This possible elevation of lignin values should be taken into consideration when determining Klason lignin content of a steam-pretreated substrate [61]. Realizing that it is difficult to reduce the amount of substrate lignin by fine — tuning SP process conditions, various researchers have explored methods of “post-treatment” to reduce the lignin content of steam-pretreated substrates.

In the past [62-64], we have tried to enhance the removal of lignin from steam-pretreated substrates, and consequentially increase the rate and ex­tent of hydrolysis by cellulases, by applying several chemical post-treatments (Table 1). Oxygen-alkali and hydrogen peroxide post-treatments removed similar amounts of lignin and thus improved hydrolysis. However, of note, we also showed [64] that the removal of only 7% of the lignin from a steam — pretreated Douglas-fir substrate using a cold NaOH treatment resulted in a 30% improvement in hydrolysis yields, indicating that, in addition to the amount of lignin, the location of lignin is also an important factor affect­ing hydrolysis. Palonen et al. [65] have applied an alternative delignification method employing laccase enzymes in combination with mediators to steam — pretreated softwood, resulting in a slight release of aromatics into the system (delignification was not measured) and a corresponding 21% increase in hydrolysis yield. These researchers also showed that the oxidation of lignin surfaces by the application of laccases in the absence of mediators, as shown by others with pulp fibers [66,67], also resulted in a 13% improvement in hydrolysis yield. These results suggest that the modification of lignin surfaces may also play a role in reducing its inhibitory effect on hydrolysis, perhaps affecting non-productive binding of cellulases to lignin. Since most of the studies have been concerned with altering lignin content by affecting the pretreatment conditions or applying post-treatments, there have only been

limited studies linking the various chemical structures in lignin to changes in hydrolytic activity of cellulases.

3.2