*3.3. Enzymatic Hydrolysis*

The results included in this section provide a quantitative assessment of the susceptibility of selected delignified and autohydrolyzed-delignified solids towards enzymatic hydrolysis. The composition of the solids is listed in Table 5. The experimental plan included acid-catalyzed organosolv treatments (assays 18–19), and experiments in ethanol/NaOH media (runs 13–14).


**Table 5.** Substrates and composition of solids employed as substrates for enzymatic hydrolysis. Data expressed as average values ± standard deviations.

Both AS and the solids obtained under the conditions listed in Table 5 presented different behavior as hydrolysis substrates. Figure 1a shows the cellulose conversions into glucose achieved at selected reaction times. The highest cellulose conversion (74.2%, corresponding to a volumetric glucose concentration of 28.52 g/L) was reached when the solids from exp. 18 (HS subjected to acid organosolv delignification) were hydrolyzed for 96 h. In comparison, the solid from exp. 19 (AS delignified under the same conditions) was poorly hydrolyzed (conversion below 15%, or 5.62 g glucose/L). HS delignified with aqueous NaOH/ethanol under the conditions of exp. 13 showed higher hydrolysis conversion (52.5%) than the one achieved using AS delignified under the same conditions (exp. 14). Despite the low conversions reached in the experiments performed with autohydrolyzed or autohydrolyzed-delignified solids (obtained in exp. 14 and 19), the highest enzymatic susceptibility was observed for the substrate from exp. 14, carried out in aqueous NaOH/ethanol media. In comparative terms, the susceptibility of the diverse substrates to hydrolysis varied as follows: AS < acid-catalyzed-organosolv AS (exp. 19) < aqueous NaOH-organosolv AS (exp. 14) < aqueous NaOH-organosolv HS (exp. 13) < acid-catalysed organosolv HS (exp. 18).

Literature data reports that the lignin content affects the enzymatic hydrolysis, since cellulases may bound to lignin irreversibly [40]. In our study, the solid from exp. 18 (which showed the highest conversion into glucose) presented the highest cellulose/lignin ratio (1.86). In comparison, the solid from exp. 19 (cellulose/lignin ratio, 1.36) reached considerably lower conversion into glucose than the solids from exp. 13 and 14 (which presented cellulose/lignin ratios of 0.68 and 0.87, respectively). Çöpür et al. [27] reported the behavior of different pretreatment techniques on regard to their efficiencies for the subsequent enzymatic hydrolysis of glucan into glucose from hazelnut husks. The highest glucan to lignin ratio of pretreated solids was used as criteria for the subsequent saccharification. Then, they reported a glucan to lignin ratio of 0.99 when HS were treated with combined steam explosion-NaOH delignification, and a high conversion of glucan into glucose (74.7%) in enzymatic hydrolysis performed at LSR 20. Ho¸sgün and Bozan [20] compared the enzymatic susceptibility of HS treated by different methods (acid, alkali, and steam) and found that the maximum glucose recovery (58.7%) corresponded to samples pretreated with NaOH. Similar results were reported with alkali delignified HS when solids were enzymatically hydrolyzed at higher LSR and ESR than were used in this work [28].

Hallac et al. [43] reported that the relationship between the lignin content of pretreated *Buddleja davidii* samples and their enzymatic susceptibility was not proportional. Huijgen et al. [41] compared the digestibility of delignified and prehydrolyzed-delignified wheat straw, and concluded that prehydrolysis prior to organosolv improved the enzymatic cellulose digestibility, despite the low lignin removal in delignification. Yang and Pan [44] discussed some aspects of the complexity of lignin effects on the enzymatic hydrolysis: a) high lignin content does not necessarily predict poor enzymatic digestibility; b) not all lignins have the same inhibitory effect on enzymatic hydrolysis of cellulose, since lignin from different sources had varying inhibitory effects, greatly related to lignin structures and properties; c) not all the lignin in the same substrate has the same inhibitory effect. On the basis of these observations, they studied the inhibitory effect of lignin with varied physicochemical properties from different biomass sources on the enzymatic hydrolysis of cellulose, and concluded that the lignin inhibition to enzymatic hydrolysis of cellulose was related to the hydrophobicity or the phenolic hydroxyl groups of lignin.

The data in Figure 1b show that the residual xylan in the hydrolysis substrates was extensively hydrolyzed into xylose. For example, in exp. 18, 80% of xylan was converted into xylose, which reached 7.03 g/L. Sun et al. [30] reported that some accessory enzymes in the enzymatic hydrolysis of natural lignocellulosic substrates favored an efficient conversion, and underlined the role of the high specific xylanase activity of the complex Cellic CTec2.

**Figure 1.** Effect of the enzymatic hydrolysis in the conversion of: (**a**) cellulose to glucose; (**b**) xylan to xylose, of spent solids from HS and AS processed under selected conditions of alkaline-organosolv, acid-catalyzed organosolv, and AS.

### **4. Conclusions**

This study deals with the integral fractionation of HS, based on selected delignification methods (in alkaline, alkaline-organosolv, organosolv or acid-catalyzed organosolv media). Both HS and autohydrolyzed HS (AS) were used as substrates for delignification.

HS treated in acid-catalyzed organosolv media reached the highest delignification degree (65.3%), enabling the production of valuable hemicellulose-derived products (about half of them in the form of oligosaccharides), with limited cellulose losses from the solid phase. Alternatively, autohydrolysis led to the partial solubilization of hemicelluloses (mainly as oligosaccharides), and the subsequent acid-catalyzed organosolv of AS resulted in the simultaneous solubilization of the remaining hemicelluloses (87.7%) and lignin (47.9%).

The enzymatic hydrolysis of solids from delignification or autohydrolysis-delignification treatments confirmed that acid-catalyzed organosolv of HS provided the best substrate for enzymatic hydrolysis (74.2% cellulose conversion into glucose, with a volumetric concentration of 28.52 g glucose/L).

Then, the strategy approaches reported in this work can be considered as "conventional" but promising alternatives for the integral fractionation of HS in the scope of biorefinery.

**Author Contributions:** S.R. designed the research and discussed the data. S.R., L.L., A.M. and C.V. performed experiments and analyzed the data. S.R. and J.C.P. prepared the original draft. All authors discussed the data. All authors have read and agreed to the published version of the manuscript.

**Funding:** S.R. contract was supported by Ministerio de Ciencia, Innovación y Universidades (IJC2018-037665-I).

**Acknowledgments:** S.R. thanks Ministerio de Ciencia, Innovación y Universidades for her "Juan de la Cierva" contract (IJC2018-037665-I).

**Conflicts of Interest:** The authors declare no conflict of interest.
