*3.4. EH of the Fiber Fraction with and without Alkaline Extraction*

The enzymatic hydrolysis of steam-refined fibers was compared with and without alkaline extraction. To enable a detailed comparison all carbohydrate yields were calculated on the theoretically available carbohydrates in the used fibers, detected after two-stage acidic hydrolysis (Figure 9). Therefore, the detected monomeric carbohydrates were concerned as a percentage based on the carbohydrate content in the fibers after an EH with Cellic® CTec2.

**Figure 9.** Effect of severity and alkaline extraction on the enzymatic hydrolysis (EH) of the fibers with Cellic® CTec2. The EH yields are calculated based on the theoretically available carbohydrates in the used fibers.

It is obvious that without extraction of the lignin, the yields after EH increases strongly with increasing steaming severity. After alkaline lignin extraction, there is no more positive effect of severity. There are slightly higher yields at severities lower than 3.5 and yields surpass constant 100%. This is because available carbohydrates in the fiber fraction were determined with a two-step acid hydrolysis. In this process, the carbohydrate content can be underestimated due to the secondary degradation of

carbohydrates in the analytical method. This degradation will be more pronounced at severities below around 4 because more hemicelluloses and amorphous cellulose are present in the fiber. Hydrolysis with enzymes is more selective to carbohydrates and results in fewer degradation products. Therefore, higher yields are explainable by the high efficiency of EH. It can be concluded that after steam refining and alkaline extraction, all carbohydrates are available for the EH.

The described improvement of the EH yields by steam refining is an undisputed fact and pretreatments are often regarded as indispensable prerequisites for the economic implementation of such processes [6]. There are several parameters influencing the EH. Important characteristics for the impact on the EH are, e.g., the ratio of amorphous and crystalline regions, the degree of polymerization (DP) of the sample, the moisture content, available surface areas, the lignin content or the pore size of the samples [52]. Higher yields after EH are reported for wet substrate in contrast to dried biomass [53], as also found in this study (data not shown here). However, the effect of lignin removal is controversially discussed in the literature. Some authors complained about studies on wood having a reduced enzymatic digestibility after lignin extraction due to collapsing pores [54–56]. Schwalb et al. [57] reported no influence of the alkali extraction, whereas Excoffier et al. [58] and Schütt et al. [11] presented higher yields of the EH after lignin removal. Ishizawa et al. [25] report that a nearly complete delignification with acidified sodium chlorite below 5% lignin content decreases the yields after EH strongly. The authors report further that the influence of partial lignin and xylan extraction improves the EH. However, the xylan removal is suggested to be more significant for the improvement of the EH yields than lignin removal [25].

Regarding the discussions in the literature, a positive influence of the partial lignin removal on the EH results could also be stated in the present study for corn stover. Furthermore, the higher impact at severities below 4 and the fading influence at severities above 4 (Figure 10a,b) could be confirmed in accordance with Schütt et al. [11]. For more findings of the overall process efficiency, the glucose (Figure 10a) and xylose yield (Figure 10b) based on raw material were calculated for the process variants with and without lignin extraction prior to EH. Taking the yields after steam refining into account a strong influence of the extraction is apparent at severities below 4. This effect is disappearing at severities >4. After that point, no significant difference can be determined for the two process variants. Similar findings were reported for the steam refining of poplar by Schütt et al. [11]. The described influence at severities lower than 4 is also visible for the xylan yields. However, the influence is not that strong as for the glucose yields.

**Figure 10.** Glucose yields (**a**) and xylose yields (**b**) after EH based on raw material for the process variants with and without alkaline extraction prior to EH.
