Hydrolysis of HPAC-pretreated pine
Table 1 summarizes the rates of pretreatment and enzymatic hydrolysis depending on lignocellulosic biomass. The differences among them were attributed to three factors that were reported to be responsible for the delay in enzymatic hydrolysis of lignocellulosic biomasses: (1) the structural complexity and solidity of lignin, (2) the structural recalcitrance of cellulose caused by the interaction strength between the β-1,4-glucose chain bundles in cellulose microfibrils, and (3) the inhibition of cellulases by glucose, cellobioses, and xylo-oligomers during enzymatic hydrolysis.[28, 35–40] HPAC pretreatment of pine wood showed high efficiencies in lignin removal and cellulose fiber swelling, which resulted in an 85% hydrolysis rate after 3 h and complete hydrolysis after 9 h with 3 FPU mL-1 of cellulase and xylanase in 1% substrate.[21, 28] These observations indicated that there was no structural recalcitrance of cellulose fibers. Additionally, the tracheid structures from HPAC-pretreated pine wood (pit, window-like pits, and lumen diameter) provided a wider accessible surface for cellulases and increased the hydrolysis rate compared to hardwood fibers.[28]
Hemicellulose consisting of galactoglucomannan and arabinoglucuronoxylan coats the microfibril units composed of cellulose chains.[41, 42] The trunks of the Korea red pine (P. densiflora ) used in this study are composed of 41.9% cellulose, 14.9% galactomannan, and 6.4% xylan (Rahmini et al., 2019).[43] A cellulase solution obtained fromT. reesei was reported to contain several hemicellulases, including one mannanase and six xylanases. Mannanase (Man5A, 53.6 kDa) hydrolyzes galactoglucomannan, which accounts for 60%–70% of the hemicellulose in softwood. Endo-xylanases cleave the β-1,4-xylosidic bonds within the xylan structures including arabinomethylglucuronoxylan and methylglucuronoxylan, which account for 13%–30% and 5%–15% of the hemicellulose, respectively.[44] Among the aforementioned endo-xylanases, XYNIV (55 kDa) is remarkably effective in the treatment of soluble beechwood, whereas the activity of hemicellulase in the cellulase solution was very low, suggesting that additional xylanases are required to enhance the hydrolysis rate. The xylanase solution from Thermomyces lanuginosus containing β-xylanase (Xyn11A, 23kDa) and β-xylosidase (GH43, 38.1 kDa) is considered a surrogate of the small molecular weight xylanases XYN1 and XYN2 of T. reesei. Previous studies have reported that additional xylanase enhanced the hydrolysis rates through a synergistic interaction with mannanase (Varnail et al., 2011).[45]
The open structural cellulose surface of HPAC-pretreated pine can be saturated by hydrolytic enzymes such as Cel7A (cellobiohydrolase I) and Cel6A (cellobiohydrolase II) and accounts for 68–78% of the secretome of T. reesei Rut-C30.[46] Moreover, these enzymes release cellobiose from solid cellulose fibers, which is a strong inhibitor of Cel7A, while glucose inhibits Cel6A (cellobiohydrolase II) and β-glucosidase.[39]It means that end-product inhibition can be a dominant retardation factor throughout the enzymatic saccharification process. In Fig. 3, the addition of the xylanase cocktail and β-glucosidases of A. niger into a 5–10 FPU cellulase g biomass-1 showed remarkable enhancement of the hydrolysis rate, increasing it from 39.0–42.1% to 84.0–87.5% for 12 h. HPAC pretreatment on softwood provided rapid saccharification and a small dose of hydrolysis enzymes, which begins to overcome the aforementioned major challenges and reduce the costs of the enzymes and energy supplied throughout the bioconversion process of the lignocellulosic biomass.[47]
The occurrence of the strong end-product inhibition when scaling-up the conditions caused retardation of enzymatic saccharification and contribute to the loss of fermentable sugars, predominantly due to the remaining solid fraction and the existence of oligomers or un-identified sugars (Table 2), compared to the results in Fig. 3. It is inferred that the insufficient dosages of the high cost of β-glucosidase were responsible for the retardation during the large scale hydrolysis. Preparation of low-cost and highly efficient β-glucosidase on cellobiose is key for rapid and economical saccharification.