HPAC Pretreatment Facilitates Enzymatic Hydrolysis of Softwood


 Background: Woody plants with high glucose content are alternative bioresources for the production of biofuels and biochemicals. Various pretreatment methods may be used to reduce the effects of retardation factors such as lignin interference and cellulose structural recalcitrance on the degradation of the lignocellulose material of woody plants. Results: Here, a hydrogen peroxide-acetic acid (HPAC) pretreatment was used to reduce the lignin content of several types of woody plants, and the effect of the cellulose structural recalcitrance on the enzymatic hydrolysis was analyzed. The cellulose structural recalcitrance and the degradation patterns of the wood fibers in the xylem tissues of Quercus acutissima (hardwood) resulted in greater retardation in the enzymatic saccharification than those in the tracheids of Pinus densiflora (softwood). In addition to the HPAC pretreatment, application of supplementary enzymes (7.5 FPU cellulose for 24 hours) further increased the hydrolysis rate of P. densiflora from 61.42% to 91.94% whereas the same effect was not observed for Q. acutissima. Also, it was observed that endoxylanase synergism significantly affects the hydrolysis of P. densiflora. However, this synergistic effect was lower for other supplementary enzymes. The maximum concentration of the reducing sugars produced from 10% softwood was 89.17 g L-1 in 36 hours of hydrolysis with 15 FPU cellulase and other supplementary enzymes. Approximately 80 mg mL-1 of reducing sugars was produced with the addition of 7.5 FPU cellulase and other supplementary enzymes after 36hours, achieving rapid saccharification. Conclusion: HPAC pretreatment thus removed the interference of lignin, reduced structural recalcitrance of cellulose in the P. densiflora, and thereby enabled rapid saccharification of the woody plants including a high concentration of insoluble substrates with only low amounts of cellulase. HPAC pretreatment may thus be a viable as an alternative for the cost-efficient production of biofuels or biochemicals from softwood plant tissues.

in turn assemble to form primary and secondary tracheid cell walls and wood bers in the xylem tissues of softwood or hardwood [4]. This structural complexity of lignocellulosic biomass hinders enzymatic degradation performed by microbes or fungi. Therefore, various pretreatment methods involving the usage of dilute acids, steam, organosolv, or sodium sul te have been developed in the past to improve the e ciency of enzymatic sacchari cation for lignocellulose degradation.
The molecular mechanism of enzymatic hydrolysis of softwood kraft pulp bers and the relationship between ber fragmentation and dislocation sites have been evaluated previously [5]. Supramolecular interactions at cellulase dislocation sites for softwood tracheids have also been found [6,7]. In addition, the cellulose binding module CBM44 (high a nity to the amorphous region of cellulose) was found to bind to the dislocation sites of hardwood-derived dissolving pulp at a greater level than CBM2a (high a nity to crystalline cellulose). Furthermore, ber fragmentation level with endoglucanase Cel5A was higher than those with xylanase XYN10A, the cellobiohydrolase Cel7A, and swollenin [3].
Hardwoods or softwoods are two types of woody plants. In the context of biofuel production from lignocellulosic biomass, pine trees and poplar aspens are representative softwood and hardwood species, respectively. Hardwoods are composed of wood bers, tracheids, ray parenchyma cells, and vessel elements in the sap and heart wood [8], whereas softwoods are comprised of tracheids, ray parenchyma cells, and resin canals. Wood bers (62.4%) and tracheids (91.8%) are the major components of hardwood (e.g., oak), and softwood, (especially pine trees), respectively [9]. These xylem components also constitute major physically limitations that reduce hydrolysis e ciency. Analysis of tracheid and wood ber hydrolysis patterns at both micro and macromolecular levels is therefore required for a better understanding of the lignocellulose bioconversion processes.
The e ciency of enzymatic hydrolysis of woody plants depends on the choice of the pretreatment method and its intensity. For example, steam explosion was shown to increase the effectiveness of sacchari cation of corn stover more than those of poplar and lodgepole pine, whereas the organosolv technique showed higher sacchari cation e ciency with poplar than with pine or corn stover [10]. Dilute acid was found to be more effective for poplar and eucalyptus rather than spruce [1]. Sul te pretreatment also led to over 90% sacchari cation with both soft and hardwoods. Hence, enzymatic hydrolysis e ciency clearly depends on the pretreatment method as well as lignocellulosic biomass type.
Three properties or components of wood materials act as "retardation factors" that limit or determine pretreatment and sacchari cation e ciencies: (1) the cuticle and epicuticular waxes on plant epidermal tissues, such as those found in corn stover, wheat, and rice straws, (2) ligni cation degrees of xylem tissues, (3) the structural heterogeneity and complexity of cell-wall constituents such as micro brils and matrix polymers [11]. The deligni cation and mechanical delamination of lignocellulosic biomass were shown to be useful methods to study structural recalcitrance of lignocellulosic biomass at micro-and macro bril levels [12,13]. Hydrogen peroxide-acetic acid (HPAC) pretreatment was previously reported to reduce lignin content of rice straw, pine wood, and oak wood by 85.12%, 98.08% and 97.61%, respectively [12]. Hence, deligni ed rice straw was shown to yield lower hydrolysis e ciency than deligni ed pine or oak wood. Considering the presence of cuticle and epicuticular waxes on epidermal tissues of rice straw, these components may negatively affect the rice straw deligni cation process and limit the e ciency of cellulase. Further comparisons of enzymatic hydrolysis patterns of HPAC-deligni ed hardwoods and softwoods can be performed via assessments of the structural heterogeneity and complexity of cell-wall constituents.
Here, hydrolysis pattern of xylem tissues from HPAC-pretreated hard-and softwoods of various wood plant types and cellulose recalcitrance in tracheids and wood ber of these materials were analyzed to assist the development of rapid hydrolysis methods for economically feasible production of biofuels.

Results And Discussion
Change in recalcitrance during enzymatic hydrolysis Various pretreatment methods such as dilute acid addition, steam explosion, organosolv, and sul te pretreatment have been used to improve enzymatic hydrolysis e ciency of woody biomass [1]. Steam explosion was found to be more advantageous for enzymatic hydrolysis of hardwoods rather than softwoods with reported conversion rates of 65-83% (hardwoods) and 21-9% (softwoods). Similar to steam explosion, dilute acid pretreatment produced readily hydrolysable cellulose bers from hardwoods, and was shown to yield enzymatic conversion rates of around 80% for hardwood eucalyptus , and 40% and 20-70% for softwood spruce and red pine, respectively [1,14]. Organosolv and sul te pretreatments have been reported to achieve high conversion rates (over 90%) for both hardwoods and softwoods [1,15]. These results suggest that each pretreatment method is suitable for a particular type of lignocellulosic biomass in terms of reducing the lignin interference and the structural recalcitrance of cellulose. Therefore, these methods should be selectively utilized to enhance enzymatic sacchari cation of woody plants.
HPAC pretreatment has been found to delignify woody substrates e ciently, as evidenced by 98.08% and 97.61% reduction in acid-insoluble lignin content from pine and oak, respectively. Moreover, swelling of xylem tissues has been observed as well [12]. Substrate concentration affects the enzymatic conversion rate and greatly contributes to end-product inhibition and cellulose recalcitrance. Here, initial hydrolysis rates of HPAC-pretreated softwoods and hardwoods were investigated at low substrate concentration to minimize end-product inhibition. HPAC pretreatment was performed on several softwoods (Larix kaempferi, Pinus. rigida, Cryptomeria japonica, Pinus densi ora, Pinus koraiensis, and Chamaecyparis obtuse) and hardwoods (Liriodendron tulipifera L., Quercus acuta Thunb, Camellia japonica, Mallotus japonica, Castanopsis sieboldii Hatus, Quercus acutissima, and Populus deltoids), followed by hydrolysis using 7.5 FPU cellulose at 50 °C for 3 h. Finally, the initial rates were calculated (Fig. 1). In contrast to previous results from steam explosion and dilute acid pretreatments [1], HPAC-pretreated softwoods degraded faster than the hardwoods. This was especially evident from data for P. densi ora and C. japonica. Q. Thunb and Q. acutissima yielded the highest and lowest hydrolysis rates among all hardwoods, respectively. P. densi ora (softwood) and Q. acutissima (hardwood) showed different hydrolysis rates, and were thus included in further experiments to evaluate rate-limiting factors related to the structural recalcitrance of cellulose at both macro-and micro bril levels during enzymatic hydrolysis. P. densi ora and Q. acutissima are also representative softwood and hardwood species from forests in Korea, respectively.

Analysis of structural recalcitrance
The variation in the initial hydrolysis rates implies variation in the structural recalcitrance of wood plants as well. Hydrolysis was therefore repeated multiple times (re-hydrolysis) for P. densi ora and Q. acutissima to further study the variation in the initial hydrolysis rates. One round of re-hydrolysis included the hydrolysis followed by washing. Re-hydrolysis was carried out several times until the reducing sugars released from the substrates were no longer detected.
The initial hydrolysis rate of HPAC-pretreated substrates showed an overall steep increase until the third re-hydrolysis round. This indicates that as hydrolysis proceeded, the effect of structural recalcitrance become apparent, particularly in comparison to commercial substrates such as lter paper or avicel ( Fig. 2A). This result is also consistent with a previous report on the gradual increase in crystallinity of pre-hydrolyzed lodgepole pine [16]. The hydrolysis of P. densi ora was completed at the end of the 6th round, whereas that of Q. acutissima proceeded until the 9th round. However, undigested solid celluloses residues were present even after 14th round for avicel and lter paper. These results indicate that structural properties of cellulose from each biomass type determine the enzymatic hydrolysis rate.
Xylem tissues including tracheids, ray parenchyma cells, and wood bers of different softwood and hardwood types were previously suggested to include different cellulose structures and degrees of resistance to cellulases that retard sacchari cation to varying degrees. However, this hypothesis has not been con rmed so far. The analysis of xylem tissues may enable an estimation of the effect of cellulose structural on enzymatic hydrolysis. Hence, ray parenchyma cells, wood bers, and tracheids from P. densi ora and Q. acutissima were isolated and their hydrolysis rates were determined. Tracheids of P. densi ora were separated into either early and late tracheids, where the xylem tissues of Q. acutissima were separated into ray parenchyma cells and wood bers (including medullary rays and ber tracheids). Figure 2B indicated that ray parenchyma cells yielded the highest hydrolysis rates in the early stages of hydrolysis, and the concentration of reducing sugars later approached a steady level. No further increase in reducing sugar concentration was detected when the incubation time was prolonged to 24 h. Late tracheids and wood ber showed the strongest recalcitrance at the early stage and late stages (after 4 h) of hydrolysis, respectively.

Softwood and hardwood degradation patterns
Fiber cutting (fragmentation) mechanism can be observed at the macromolecular level during the enzymatic hydrolysis of lignocellulosic biomass [5,7]. Fiber cutting was found to level off with shorter ber lengths between 130 and 220 µm during the early stage, the length of which varies depending on pretreatment conditions and the chemical composition of the substrate [17].
Here, P. densi ora and Q. acutissima were subjected to HPAC pretreatment and subsequent hydrolysis to investigate ber cutting mechanism. The initial average length of the tracheids from P. densi ora was 1239.06 ± 301.68 µm (Fig. 3), which includes tracheids fragmented during pretreatment or preparation. The lengths of major tracheid fragments ranged between 900 and 1600 µm. Initial ber fragments (ranging between 500 and 2100 µm in length) were enzymatically hydrolyzed for 3 or 6 h. The ratios of the length of resulting fragments with respect to the average length ranged between 1/4 and 1/8, and the amount of these fragments accounted for 64.63% of all fragments after 3 h hydrolysis. After 6 h, this ration ranged between 1/8 and 1/20, and fragments within this size range accounted for 73.53% of all fragments. For Q. acutissima, the initial average length of wood bers was 515.9 µm, and lengths ranged between 200 and 850 µm. The ratios of lengths of resulting fragments with respect to the average length ranged between 1/4 and 1/8 as well, and the amount of these fragments accounted for 66.55% of all fragments after 3 h. Similar to values found for P. densi ora, this ration ranged between 1/8 and 1/20 after 6 h. and fragments within this size range accounted for 74.32% of all fragments.
The ber cutting and fragmentation patterns were thus insu cient to explain why faster overall hydrolysis rates were observed for P. densi ora than Q. acutissima. Therefore, tracheids and wood bers of P. densi ora and Q. acutissima were comparatively evaluated. For this purpose, the surface of the fragments was monitored for 24 h after cellulase treatment (Fig. 4). The widths of tracheids ranged between 20 and 40 µm, and cell walls included pits and window-like pits. The diameter of lumen of P. densi ora was wider than that of wood bers from Q. acutissima. Cellulase-tracheid binding pro les in softwood were previously shown to include high levels endoglucanase (EGV from Humicola insolens, GH 45) and cellobiohydrolase (CBH1 from T. reesei, GH 7) in the lumen (Hideayat et al., 2015). These characteristics of tracheids allow cellulase to easily approach cellulose bers on the surface and lumen. Window-like pits were found to be targets for cellulase attacks during the initial stage. The tracheid bodies also cracked and spread out in the form of irregular mosaic shapes, whereas the wood bers of Q. acutissima were longitudinally cracked by cellulase. Deconstruction of the ber cutting ends and a peeling-or erosion-like effect on the surface of the wood bers were also observed. The shortened bers remained intact during the prolonged 24-h incubation. The remaining fragments at the late stage included more recalcitrant cellulose bers that were resisted cellulase action, implying that the primary wall or S 1 layer of wood ber was responsible for this result [18]. These results may thus explain the retardation of Q. acutissima degradation compared to that of P. densi ora.

Rapid hydrolyzation of softwood
Enzymatic hydrolysis e ciency depends on the pretreatment method and utilized source of biomass. Hydrolysis e ciencies of eucalyptus and cedar that were pretreated with NaOH were found to be approximately 70% and 80% in 24 h, respectively [19]. Spruce that was pretreated via steam explosion was found to be hydrolyzed at 29% e ciency with 96 h [20]. Dilute acid pretreatment was also shown to increase enzymatic hydrolysis e ciency in hardwoods [1,14,21]. Organosolv-pretreated poplar cellulose was found to be converted to glucose at approximately 85% e ciency for 48 h, and the cellulose of mixed softwoods (spruce, pine, and Douglas r) showed conversion e ciencies of around 98% [22,23].
Here, HPAC-pretreated softwood P. densi ora and hardwood Q. acutissima were rapidly hydrolyzed at low substrate concentrations (1%), resulting in a yield of 90 ~ 100% at 12 h depending on the dosage of cellulase (7.5-30 FPU, data not shown). Hydrolysis patterns of these substrates at the macromolecular level were also different from each other. Based on the results, P. densi ora was chosen to achieve rapid sacchari cation at a highly insoluble substrate, instead of Q. acutissima which showed greater recalcitrance during the late stage of hydrolysis [18].
The hemicellulose network within softwood is known to hinder assess of cellulase composed to cellulose bers during enzymatic hydrolysis. The two main components of softwood hemicellulose are galactoglucomannan and arabinoglucuronoxylan [24]. The chemical composition of hemicellulose of Pinus radiata was reported to include xylose at 19% and mnannose at 37% [25]. On the contrary, Korean red pine (P. densi ora) trunk was found to include cellulose, xylan and galactomannan at rates of 41.9%, 6.4%, and 14.9%, respectively [26].
In order to investigate the possible utility of xylanase addition to HPAC-pretreated P. densi ora, micro bril surfaces of tracheids were examined, and found to be covered by hemicellulose or other unspeci ed materials (Fig. 5). Extracellular enzymes of T. reesei are known to include 68-78% cellobiohydrolases, 10-15% endoglucanases and others enzymes such as beta-glucosidase, xylanases, and accessory enzymes [27,28]. T. reesei produces XYN I and XYN II as two main xylanases types in addition to XYN III, and XYN IV. However, zymogram analysis showed that a single 55 kDa band corresponding to XYN IV showed enzymatic activity on birchwood and beechwood xylans [29,30]. Tenkanen et al. (2012) previously reported that XYN IV showed a typical exo-action on linear β-1,4-xylooligosaccharides [30], and low activity on xylo-biose, -triose, and -tetraose. Xylooligomers were also reported to strongly inhibit the activity of Cel7A (cellobiohydrolase I) by binding to the active site of this enzyme [31,32]. Moreover, the synergistic action of xylanase and mannanase was shown to improve the total hydrolysis e ciency of softwoods [33]. Here, mannanase activity was also observed in enzymes produced by T. reesei RUT C30 as well (Fig. 5B). Xylanase, supplementation educed enzymatic inhibition and physical hindrance on micro bril surfaces during hydrolysis of the HPAC-pretreated P. densi ora. Addition of xylanase into 3 FPU cellulase increased the hydrolysis e ciency of 1% HPAC-pretreated P. densi ora, from 22-85% for 3 h pretreatment and complete hydrolysis was observed at 9 h (Fig. 5C) High substrate concentrations are required to obtain high concentrations of fermentable sugars, and thus produce bioethanol e ciently. However, hydrolysis of high concentration of insoluble substrates was hindered by end-product inhibition that strongly reduced the hydrolysis rate ( Supplementary Fig. 2). Therefore, supplementary enzymes such as xylanase, lytic polysaccharide monooxygenases (LPMO), and beta-glucosidase were added to cellulase cocktail solutions (7.5 or 15 FPU cellulase) to reduce endproduct inhibition and the structural recalcitrance of the substrates (Fig. 6). Xylanase in combination with 7.5 and 15 FPU cellulase increased 24 h hydrolysis e ciency from 61.42-91.94% and 104.41%, respectively. No enhancement was found when GtLPMOs was added, while beta-glucosidase addition led to 102.69% and 109.60% 24 h e ciency with xylanase and 7 and 15 FPU cellulase solution, respectively. In contrast, the synergistic effect of the supplemented enzymes, especially xylanase, was lower on HPAC-pretreated Q. acutissima (Fig. 6B), even though this plant contains a higher proportion of xylose as its main hemicellulose component [12].
Overall, these results indicate that hemicellulose causes a retardation of the enzymatic hydrolysis of HPAC-pretreated softwood, while structural recalcitrance of cellulose primarily delays hardwood hydrolysis.
With concentration of insoluble substrates above 5-10%, the hydrolysis rate is negatively affected by ine cient agitation due to higher viscosity, which acts as a strong retardation factor, and leads to more severe end-product inhibition. Here, the initial concentration of P. densi ora, which has an insoluble substrate concentration of 5%, was hydrolyzed using 7.5-30 FPU cellulase and other accessory enzymes such as xylanase, GtLPMOs, and beta-glucosidase (Fig. 7). After 12 h, insoluble substrate was added to the reaction mixture to obtain a nal insoluble substrate concentration of 10%. The maximum concentration of the reducing sugars was found to be 89.17 g L − 1 at 36 h with 15 FPU cellulase and accessory enzymes, reaching 74.68% of the theoretical maximum rate. Hence, an economically feasible hydrolysis process was achieved using 7.5 FPU cellulase and other accessory enzymes, which is a remarkable result compared to that with 30 FPU cellulase only.
In summary, HPAC pretreatment of softwood e ciently reduced lignin interference and cellulose structural recalcitrance. Only a low amount of cellulase was required for the production of a high concentration of fermentable sugars within a short reaction time. HPAC is therefore an advantageous pretreatment for the economical production of biofuels or biochemicals.

Conclusions
The structural complexity of woody plants results in recalcitrance and prevents e cient enzymatic hydrolysis, and thereby leads to long reaction times for the production of high amounts of fermentable sugars. The level of recalcitrance throughout enzymatic hydrolysis is determined by pretreatment conditions and biomass type. Here, the HPAC pretreatment facilitated the removal of lignin from both softwood and hardwood biomass types. Moreover, this pretreatment method led to a more e cient reduction of cellulose structural recalcitrance of the softwood P. densi ora. Rapid enzymatic conversion was achieved with even low cellulase doses and high concentrations of woody plants. The methods described here may enable the economically feasible production of biofuels and biochemicals. (KobioTech, South Korea) was operated at 400 × g and 28 ºC for 8 days. Cellulase expression was induced by the addition of 1% (w/v) avicel. The air ow rate and pH adjustment were auto-regulated to 1vvm and pH 4.8, respectively.

Preparation of deligni ed bers and xylem tissues fraction
Pine (P. densi ora, diameter: 13 cm) and oak (Q. acutissima, diameter: 15 cm) trees were used to prepare the substrates that were used in all process of enzymatic sacchari cation processes. The wood was cut to 0.2 cm (width) × 0.3 cm (height) × 4 cm (length) pieces. A hydrogen peroxide (H 2 O 2 ) and acetic acid (CH 3 COOH; HPAC) solution was prepared in a 1:1 ratio and 1 L of the solution was added to 100 g lignocellulosic biomass [12]. The mixture was incubated in a water bath at 80 °C for 2 h and then washed several times to remove the solution. The deligni ed samples were squeezed and freeze-dried. The substrates were used to conduct enzymatic sacchari cation and to separate xylem tissues from one another.
Deligni ed pine and oak wood, avicel, and lter paper were used to measure change of in the strength of cellulose ber recalcitrance during enzymatic hydrolysis. Each sample (1%) was hydrolyzed with 10 FPU cellulase g − 1 biomass in 0.1 mol L − 1 citrate buffer (pH 0.5) at 50 °C for 1 h. The reducing sugar concentration in the supernatant was measured, and the pellet fraction was washed for subsequent hydrolysis. The pellet was then hydrolyzed under the same conditions as previously described. This was considered to be one subsequent hydrolysis cycle. Six to 14 subsequent hydrolysis cycles were performed depending on the substrate. Recalcitrance rates were determined by measuring the initial rate of each round.
The bers, tracheids, medullary rays, and ray parenchyma cells of HPAC-pretreated oak were separated by ltration with 60 and 100 mesh (S1020, Sigma-Aldrich). HPAC-pretreated pine tracheids were separated, and the tissue fractions (0.5%, g/v) were hydrolyzed in 0.1 M citrate buffer (pH 5.0) containing 15 FPU cellulase at 50 °C for 6 h. Nelson-Somogyi (NS) and DNS assays were used to measure the concentration of the reducing sugars [29].

Analysis of wood hydrolysis pattern
Tracheids or wood bers (2%, g/v) from the pine and oak were hydrolyzed with 7.5 FPU cellulase in 1 mL of 0.1 M citrate buffer (pH 5.0) at 50 °C. The hydrolysis patterns were monitored and the lengths of tracheids or bers were measured via microscopy (Eclipse TE2000-U, Nikon; Olympus BX41).
Tracheids were stained with congo-red and were placed on glass slides, a rectangle wall was made with nail enamel, and 0.1 M citrate buffer (pH 5.0) containing 1.5 FPU cellulase was added. The glass slides were sealed with a cover glass and nail enamel and placed in a small batch incubator and incubated at 50 °C. The tracheids were observed for 24 h. The same process was employed for hardwood bers.

Scanning electron microscopy (SEM)
The surface morphology of tracheids from HPAC-pretreated pine was observed using a JSM-7500 F (Jeol, Japan) eld-emission scanning electron microscope with a beam voltage of 3 kV. Brie y, the sample was dehydrated with a graded ethanol series and freeze-dried. After being sputter-coated with osmium, the external surface of the sample was observed.

Zymogram analysis of mannanase
The mannanase activity in cellulase produced by T. reesei RUT C30 was analyzed by using a modi ed method described by [35]. The cellulase (3, 6 µg) was loaded on SDS-PAGE with galactomannan. After electrophoresis, the gel was incubated in refolding buffer (20 mM citrate buffer pH 5.0, 0.1 mM CaCl 2 , 1% triton X-100) at room temperature for 30 min, and washed with 20 mM citrate buffer (pH 5.0). The reaction of the enzyme was performed at 37 °C for appropriate time. The gel was neutralized in phosphate buffer (pH 7.0) and stained with Congo red. Destaining was conducted with 1 M NaCl solution until a white band appeared. Citrate buffer (pH 3 ~ 5) was added to turn the red background to dark blue for better clarity.
Enzymatic hydrolysis of P. densi ora The sacchari cation e ciency of different concentrations of pretreated pine was analyzed. One per cent (w/v) of HPAC-pine was hydrolyzed with 7.5 FPU cellulase, 5 units of xylanase, and 10 µg of AnBgls in 1 mL of 0.1 M citrate buffer (pH 5.0) at 50 °C for 24 h. This amount of time was su cient to degrade the solid fraction of the substrate. The supernatant was subsequently incubated until the cellobiose was completely digested to glucose in the solution. The reducing sugar concentration was measured by DNS assay. A total of 12.01 mg mL − 1 of reducing sugars from 1% HPAC-pretreated pine was assigned as the standard concentration. The theoretical concentration of reducing sugars from each concentration of HPAC-pine was calculated by multiplying the standard concentration by each concentration. One unit of xylanase was de ned as the amount of xylanase that produced 5 mg mL − 1 of reducing sugars from 1% beechwood xylan during 10 min at 50 °C. A total of 40 µg mL − 1 AnBgls was determined to be the enzyme amount that completely hydrolyzed 20 mM cellobiose to glucose during 10 min at 50 °C.
To achieve the rapid sacchari cation of HPAC-pine, 1% (w/v) HPAC-pretreated pine was incubated with 1.5 ~ 3 FPU cellulase or cocktail solution of 1.5 ~ 3 FPU cellulase with 5 units of xylanase (T. longibrachiatum, Sigma) in 1 mL of 0.1 M citrate buffer (pH 5.0) at 50 °C for 3 h. The enzymatic hydrolysis of 5 or 10% of HPAC-pretreated pine was also determined in 1 mL of 0. LDS performed experiments and analysis of data with LYG who produced and supplied all of hydrolytic enzymes, and drafted the manuscript. LDS and LYG contributed equally. CEJ carried out microscopic analysis and SYH prepared HPAC-pretreated pine and oak wood. BHJ designed the project, critically analyzed the data, and improved the manuscript. All authors read and approved the nal manuscript. Figure 1 Comparison of initial hydrolysis rate of various woody plants. The HPAC-pretreated substrates (1%, w/v) were hydrolyzed using 7.5 FPU cellulase g biomass -1 at 50 °C for 3 h. Comparison of the change in recalcitrance during enzymatic hydrolysis. (A) Each re-hydrolysis round consisted of hydrolysis with 7.5 FPU cellulase at 50 °C for 1 h followed by three washing rounds with distilled water. The hydrolysis of P. densi ora was complete at the end of 6th round, and the hydrolysis of Q. acutissima was complete at the end of 9th round. Filter paper and avicel released reducing sugars even at the end of 14th round. (B) The enzymatic degradation of wood bers and ray parenchyma cells from Q. acutissima and tracheids (early and late tracheids) from P. densi ora are compared.     (xylanase, GtLPMOs, and beta-glucosidase). The initial concentration (5%) was hydrolyzed for 12 h, and additional substrate was added to obtain a total concentration of 10 %, as indicated by the arrow. The most economically feasible enzyme dose was found to be 7.5 FPU cellulase at 10% of the substrate when incubated with accessory enzymes.

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