**3. Results and Discussion**

Unpretreated agricultural residues are resistant to cellulolytic enzymes and characterized by a low reducing sugar yield in hydrolysis with a mixture of *P. verruculosum* B151 cellulase complex and F10 β-glucosidase. The wheat straw convertibility was 12%. For the sugar beet pulp, it was 20%, for oat husks it was 5%, for sunflower peels it was 3%, for corn stalks it was 10%, and for bagasse it was 17%. Only the soy husk convertibility was relatively high, at 31% (Table 2).

**Table 2.** Convertibility of different cellulose-containing materials in hydrolysis by a mixture of *P. verruculosum* B151 cellulase complex and F10 β-glucosidase.



**Table 2.** *Cont*.

Mechanical pretreatments such as fine ball milling or extruding have resulted in increasing the convertibility of these substrates 1.35–3.75 fold. The data of enzymatic hydrolysis obtained for mechanically pretreated materials have shown that the ball milling (which gives an average particle size of less than 20 µm) of wheat straw and bagasse results in increased convertibility to 45% and 42%, respectively. The ball milling of sunflower peels leads to a very limited improvement in convertibility at 7%, which indicates that they are practically not digestible by cellulolytic enzymes. Sugar beet pulp pretreatment by extrusion has also shown a limited improvement in convertibility (to 27% only).

The wheat straw convertibility after delignification with hot alkaline solution increased 4.6-fold (up to 55%).

Steam explosion pretreatment with different additives (H2SO4, Ca(OH)2) demonstrated that wheat straw, out husks, soy bean husks, corn stalks, and bagasse were easy to hydrolyze with enzymes, and this pretreatment enhanced the convertibility up to 69–75%, 76%, 58%, 36–55%, and 34–41%, respectively. Supplementation with H2SO<sup>4</sup> and Ca(OH)<sup>2</sup> has shown an opposite result for different materials: calcium hydroxide is preferable for bagasse pretreatment (7% higher sugar yield), while a corn stalk pretreatment required sulfuric acid (19% higher sugar yield). Wheat straw steam pretreatment required no additives.

The enzymatic hydrolysis of unpretreated food-industry waste has shown that this kind of cellulose-containing material is far from being a potential source of simple sugars for biotechnology. Thus, the convertibility of brewing waste, destarched corn, and wheat bran was very low, at 10%, 12%, and 14%, respectively. The convertibility of wet and dry distillers grains hydrolysis was slightly higher, at 18% wet and 16% dry, respectively.

Pulp and paper production is a large-tonnage and streamlined industry. The range of products in this area is very wide. They differ in the raw materials (hardwood or softwood, others) used and the way they are produced (wood cooking, bleaching). Creating an integrated biorefinery plant around existing pulp and paper mills would enhance their marketability by efficient converting all biomass components into value-added products [32]. The Kraft pulping process is a promising pretreatment technology for biocatalytic conversion of cellulose and hemicelluloses to glucose and other monosaccharides [33,34]. The convertibility of newer dry as well as dried kraft fibers representing by bleached and unbleached soft wood and hardwood pulp was evaluated (Table 1). The highest convertibility was demonstrated by wet bleached softwood pulp, at 78%; the reason for the high convertibility is the almost complete removal of lignin by the Kraft process (a remaining lignin content was 2–3% [33]). The convertibility of wet bleached hardwood pulp was 58%; this is lower compared with bleached softwood pulp because of the xylan influence [33]. Unbleached wet softwood and hardwood pulp had approximately a 1.1 times lower convertibility compared with similar types of bleached pulp; the decrease in convertibility of unbleached pulp is explained by the higher lignin content.

The drying and subsequent hornification of all kraft pulps types had a significant effect on convertibility as it reduces swelling and the cellulose fiber accessibility [35] and causes a collapse in the pore structure [36]. The convertibility was reduced by 1.3–1.4 times compared to wet pulp.

The recycling and utilization of wood industry wastes and forestry residues is crucial for wood processing and environmental security. In total, five types of wood species were included in this study. The convertibility of pine, larch, aspen, and hevea sawdust was low and found to be in the range of 4–8% (Table 1). Mechanical pretreatment (dry fine ball-milling that results in an average particle size less than 20 µm) has resulted in a significant increase in the convertibility of pine wood and aspen wood to up to 45–50%; in the case of larch, it is up to 22%. The increase in convertibility was due to defibrillation and reduction in the crystallinity of fibers and increasing surface area related to reducing particle size [35]. Despite its high efficiency, fine ball-milling has serious disadvantages, such as being energy consuming and difficulties in scaling up [37]. In view of the rising energy prices and power intensity, fine milling is not economically reasonable [38].

To counteract these disadvantages, less intensive milling processes can be combined with chemical and physicochemical pretreatments such as dilute acid and organosolv pretreatments. We have studied the convertibility of aspen wood subjected to pretreatment by different water and organic solutions of mineral acids. Relatively low temperatures of pretreatment process were selected to prevent the unfavorable degradation of carbohydrates and inhibitors formation.

Pretreatment of aspen wood by dilute acids: Five samples were obtained using dilute sulfuric acid pretreatment of aspen wood particles (200–300 µm). The results demonstrate a linear correlation between acid concentration and substrate convertibility (Table 3). There was no difference found (58.4% convertibility) for samples processed with 12.7% and 8.7% sulfuric acid, which could mean that the maximum available polysaccharides for enzymatic hydrolysis due to the solubilization of hemicelluloses are limited. Further reduction in the acid concentration to 4.4% and 1.8% results in the convertibility decreasing to 44.2% and 42.9%, respectively. Further reduction in the acid concentration to 0.9% provides a convertibility of 41.6%, which is just 1.4 times lower than the result obtained by 12.7% acid. Such a reduction in chemical consumption can be economically feasible even with a lower biomass convertibility.


**Table 3.** Convertibility of aspen wood pretreated using different types of acid-containing solutions at elevated temperatures.

Enhancing the hydrolysis of lignocellulose biomass for the efficient conversion of cellulose and hemicellulose by pretreatment using nitric acid has not been highly studied compared to sulfuric acid. To estimate the influence of nitric acid on the convertibility of 200–300 µm aspen wood in severe and mild conditions, another series of experiments was carried out.

The experimental results demonstrate that the reducing sugars yields after a relatively mild pretreatment at 100–130 ◦C, 5–6 at, and 1–4.8% nitric acid were purely comparable, at about 60% (Table 3). Maximum convertibility was achieved with a subsequent elevation of gas pressure. The aspen wood convertibility enhanced significantly from 60.6% to 78.7% as the pressure increased from 9 to 18–22, while the temperature and acid concentration remained constant (125 ◦C, 4.8%). These nitric acid

concentration and pressure values found in this study are optimal for pretreatment, since increasing the temperature up to 160 ◦C results in a convertibility below 50% for acid concentrations of 0.3–0.7%. There was no additional effect on the reducing sugars yield when the pressure was raised from 18 to 22 at.

It could be concluded that aspen wood pretreatment with dilute nitric acid (convertibility 78.7% at an acid concentration of 4.8%) is more efficient than with dilute sulfuric acid (convertibility 58.7% at an acid concentration of 12.7%) but requires more complex equipment.

Organosolve pretreatment of aspen wood: The results obtained in this study demonstrate that dilute acid pretreatment is very effective. However, after such pretreatment lignin solubilized poorly [39], even though the hemicellulose matrix is being dissolved. Thus, the next panel of experiments was aimed to discover best conditions for fractionation and recovery of lignin, cellulose, and hemicelluloses. All the experiments were conducted at constant temperature 140 ◦C for the same time 1 h, but at different concentrations of organic solvents (ethanol and *n*-buthanol) and sulfuric acid (0.18–0.54%) as a catalyst.

The data display that 50% (*v*/*v*) alcohol–water mixtures have the same efficiency of 36–37% with 0.36% acid. Using a 0.54% acid concentration, 65% *n*-butanol is more preferable than 65% ethanol, ensuring a convertibility of 58.7% and 48.3%, respectively (Table 3). Organosolve pretreatment with 80% alcohol results in a lower convertibility (43.9% and 54.1% for ethanol and *n*-butanol respectively), but *n*-butanol still provided a better enzymatic digestibility. This likely could be explained as the swelling of cellulose fibers decreased as the ethanol concentration increased [40].

During organosolve pretreatment with *n*-butanol, three fractions were obtained: black liquor containing dissolved lignin, hemicellulose-enriched liquid fraction, and cellulose-containing solid fraction [41]. This spatial separation also could explain better biomass hydrolysability after *n*-butanol pretreatment. Previously [42], it has been found that the swelling of cellulose in organic solvent strongly depends on the species of organic solvents—the solvent basicity, the molar volume, and the hydrogen bonding capability—thus, *n*-butanol is more significant in two-component mixtures. This was shown in experiments with alcohol concentrations of 25% EtOH + 25% BtOH (convertibility 54%) and 40% EtOH + 10% BtOH (convertibility 51%). However, in general, more concentrated *n*-butanol single-component mixtures are preferable to two-component mixtures.

Decreasing acid concentrations from 0.36% to 0.18% resulted in a lower convertibility using the same mixture composition (20% EtOH + 40% BtOH)—52% and 46%, respectively. At an acid concentration of 0.18%, this mixture composition has no effect on the convertibility.

These results indicate that alcohol-water mixtures allow using less concentrated acids, while biomass components could be fractionated and alcohols could be recirculated. In this study, the optimal conditions for pretreatment were found: 140 ◦C, 1 h, 0.54% sulfuric acid, 65% *n*-butanol. These conditions lead to a 7.4-fold increase in the convertibility or aspen wood (59% compared to 8% of untreated substrate).
