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.