Biomass and Bioenergy 158 (2022) 106377

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Biomass and Bioenergy

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Combined acetic acid and enzymatic hydrolysis for xylooligosaccharides

and monosaccharides production from poplar
Wenjun Ying a, Xin Fang a, Yong Xu a, b, Junhua Zhang a, b, c, *
a
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing,
210037, China
b
Key Laboratory of Forestry Genetics & Biotechnology (Nanjing Forestry University), Ministry of Education, Nanjing, 210037, China
c
College of Forestry, Northwest A&F University, Yangling, 712100, China

A R T I C L E I N F O A B S T R A C T

Keywords: Efficient production of xylooligosaccharides (XOS) from lignocellulosic biomass without preliminary xylan
Poplar isolation is challenging. Previously, organic acid has been used to produce XOS from lignocelluloses without
Acetic acid hydrolysis xylan isolation. However, the knowledge about XOS production from the residual xylan in acid-hydrolyzed
Delignification
lignocelluloses is still unclear. In this study, acetic acid (AC) hydrolysis of poplar was used to produce XOS,
Xylanase hydrolysis
Xylooligosaccharides
then AC-hydrolyzed poplar was delignified by hydrogen peroxide-acetic acid (HPAC) solution. The AC-HPAC-
pretreated poplar was sequentially hydrolyzed by xylanase and cellulase to produce XOS and mono­
saccharides. The results showed that the XOS yield of poplar after AC hydrolysis was 31.6% (based on the xylan
in raw poplar). A high lignin removal (94.8%) of AC-hydrolyzed poplar was obtained by HPAC pretreatment. The
highest XOS yield based on residual xylan in AC-HPAC-pretreated poplar by xylanase was 17.5%, and finally a
total XOS yield of 36.5% based on xylan in raw poplar was achieved. The highest glucose yield was 88.0% with
20 FPU/g dry mass cellulase and 1.0 mg/mL Tween 80. Finally, 65.1 g of XOS was obtained from 1,000 g of
poplar through this combined process including AC and xylanase hydrolysis, increased by 2.2 times compared
with that from xylanase hydrolysis without AC hydrolysis. This work greatly improves the XOS production from
poplar and provides a novel strategy to efficiently transform poplar carbohydrates into XOS and
monosaccharides.

1. Introduction
Now-a-days, due to traditional fossil energy causing serious energy
and environmental problem, it is particularly important to find a
renewable energy source. Lignocellulosic feedstocks as renewable re­
sources are rich in carbohydrates, which can be utilized to produce high-
valued platform chemical compounds through biorefinery [1]. The
optimized utilization strategy of hemicellulose is producing the xyloo­
ligosaccharides (XOS) [2], which contain 2–6 xylose units. It has been
reported that XOS can promote the proliferation of intestinal probiotics
and are beneficial to human health [3].
In the traditional method, xylan is first extracted from lignocellulosic
biomass by alkali, followed by autohydrolysis or enzymatic hydrolysis to
produce XOS [4,5]. Lian et al. [6] reported that 100 g of alkali-extracted
xylan could yield 21.4 g of XOS through autohydrolysis at 180 ◦ C for 40
min. This process is low-cost and environmentally friendly, but the
operation of which is complex. Recently, the single step of dilute organic
acid hydrolysis for XOS production from lignocellulosic biomass is
developed, which is more convenient than the two-step methods as
mentioned above [7,8]. It has been reported that the highest XOS yield
(36.0%) is obtained through adopting 5% (w/v) acetic acid (AC) to
pretreat poplar at 170 ◦ C for 30 min [4], with a high yield (26.1%) of
by-product xylose. Dilute acid hydrolysis is a promising method to
produce XOS from lignocellulosic biomass [9]. Nevertheless, there is still
a large amount of xylan (10%–60%) retains in the acid-hydrolyzed solid
residue, which can be further utilized [10]. Endo-xylanase hydrolysis is
a widely employed method for XOS production. Due to the mild reaction
condition and the absence of β-xylosidase, the hydrolysate from xyla­
nase hydrolysis has less by-products like xylose [11,12]. Hao et al. [11]
reported that 19.8% of XOS yield with a low ratio of xylose/XOS (0.14)

* Corresponding author. Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing
Forestry University, Nanjing, 210037, China.
E-mail address: junhuazhang@njfu.edu.cn (J. Zhang).

https://doi.org/10.1016/j.biombioe.2022.106377
Received 30 August 2021; Received in revised form 23 January 2022; Accepted 30 January 2022
Available online 12 February 2022
0961-9534/© 2022 Elsevier Ltd. All rights reserved.
W. Ying et al.
was obtained from hydrogen peroxide-acetic acid (HPAC)-pretreated
poplar by xylanase hydrolysis. In conclusion, dilute organic acid and
xylanase hydrolysis both show great potential to produce XOS from
lignocellulosic biomass. After dilute acetic acid hydrolysis of lignocel­
lulose, the knowledge of the XOS production from the residual xylan in
acid-hydrolyzed substrate is still unclear.
In this study, AC hydrolysis was first used to produce XOS and the
AC-hydrolyzed poplar was delignified through HPAC pretreatment.
Then, xylanase hydrolysis was further employed to produce XOS from
AC-HPAC-pretreated poplar and the final solid residue was utilized to
produce monosaccharides by cellulase hydrolysis. The solid loading,
enzyme loading and hydrolysis time were optimized for XOS production
from AC-HPAC-pretreated poplar by xylanase hydrolysis. The role of
surfactants in the monosaccharides production by cellulase from the
final solid was also investigated. The purpose of this work was to
develop a novel strategy of combined organic acid and xylanase hy­
drolysis to maximize the production of XOS from poplar.
2. Materials and methods
2.1. Raw materials and enzymes
Poplar sawdust was provided by a farm in Suqian
(33◦ 58′ 15.95′′ N,118◦ 16′ 43.55′′ E), Jiangsu Province, China. It was
smashed and collected the portion that passed through the 60-mesh
screen (≤0.25 mm). Chemical reagents used in the experiment were
provided by Nanjing Chemical Reagent Co. Ltd (Nanjing, China).
Cellulase (Cellic CTec2) and xylanase (X2753, Pentopan Mono BG)
were both purchased from Novozymes A/S, Bagsværd, Denmark.
Cellulase has an activity of 120.2 FPU/mL [13] and a protein content of
170.8 mg/mL [14]. Xylanase has an enzyme activity of 1667 nkat/mL.
2.2. Pretreatment
Poplar was first hydrolyzed by acetic acid (5%, w/v) at 170 ◦ C for 30
min [15] and the solid residue was named AC-hydrolyzed poplar.
The HPAC reagent was prepared according to Ying et al. [16]. Poplar
and AC-hydrolyzed poplar (30 g) were, respectively, pretreated by 300
mL 75% (v/v) HPAC solution and 100 mM H2SO4 in Erlenmeyer flask
(500 mL) at 80 ◦ C. After 2 h of reaction, the solid was separated and
washed with deionized water to neutral for subsequent structural
characterization and enzymatic hydrolysis experiments.
2.3. Enzymatic hydrolysis
Substrates were incubated in 50 mM sodium citrate buffer at 50 ◦ C
and 150 rpm for xylanase hydrolysis using a water bath shaker (SHA-B,
Guohua Industry & Trade Group Co., Ltd, China). The effects of solid
concentration (2%–10%, w/v), enzyme dosage (1,000–10,000 nkat/g
dry mass (DM) and hydrolysis time (24–96 h) on XOS yields were
investigated. Each experiment was conducted two times.
After xylanase hydrolysis, the final solid residue was used for
monosaccharides production by cellulase hydrolysis in 50 mM sodium
citrate buffer (pH 5.0) at 50 ◦ C and 150 rpm using a water bath shaker
(SHA-B, Guohua Industry & Trade Group Co., Ltd, China). The solid
concentration was 2% (w/v) and the enzyme dosage was 5–20 FPU/g
DM with the addition of 1 g/L of Tween 80 or PEG 4000.
2
Biomass and Bioenergy 158 (2022) 106377
2.4. Characterization of solid residue
2.4.1. SEM analysis
Surface morphology of experimental sample was observed by SEM
(Quanta 200, FEI, Holland). The sample was gold-plated before imaging
to enhance its conductivity. The working voltage of the instrument was
25 kV.
2.4.2. XRD analysis
Crystallinity index (CrI) was measured by Rigaku D/Ultima Ⅳ in­
strument (Rigaku Corporation, Japan) with a copper target as an X-ray
source. The scanning speed was 5◦ per min from 5◦ to 50◦ , and the value
of CrI was calculated as follows [17]:
CrI (%) = [(I002 − Iam ) / I002 ] × 100 (1)
where, I002 and Iam are maximum (around 22.5◦ ) and minimum (around
18◦ ) diffraction peak intensity, respectively.
2.4.3. XPS analysis
Sample surface was analyzed by X-ray photoelectron spectrometer
(AXIS Ultra DLD, England) according to Kumar et al. [18]. The carbon
spectrum was fitted into three carbon peaks using XPS peak 4.0 soft­
ware. C1 stands for carbon atom single bonded to a carbon or hydrogen
(C–C, C–H, 284.6 eV). C2 represents carbon atom single bonded to an
oxygen (C–O, 286.6 eV). C3 means carbon atom double bonded to one
oxygen or single bonded to two oxygens (C–– O, O–C–O, 288.8 eV).
2.4.4. Determination of cellulose accessibility
Direct red dye (DR 28) adsorption was used to measure the cellulose
accessibility to enzymes [15]. A gradient ascending DR 28 solution
(0.00, 0.05, 0.10, 1.00, 2.00, 3.00 and 4.00 g/L) was blended with 1%
(w/v) sample at 150 rpm and 50 ◦ C for 24 h. After incubation, the
specific measurement and calculation methods were used according to
Ying et al. [16].
2.5. Analysis method
Chemical compositions of all samples were determined according to
the NREL Analytical Procedure [19].
Concentrations of monosaccharides and by-products were quantified
by HPLC (Agilent 1260 series, Germany) with an Aminex HPX-87H
column, using 5 mM sulfuric acid as mobile phase [20]. The concen­
tration of XOS (DP2-6) was detected by HPAEC (Dionex ICS-3000, USA)
with a CarboPac™ PA200 column, using sodium acetate and sodium
hydroxide as mobile phases [21].
Contents of aromatic products in the pretreatment liquors were
measured by RP-HPLC (Agilent 1100 series, Germany) with a Zorbax
Eclipse XDB-C18 column. The mobile phases were 1.5% (v/v) aqueous
acetic acid and pure acetonitrile, respectively [22].
2.6. Calculations
The calculating formula for solids recovery was showed as follows:
( / )
Recovery (%) = mafter moriginal × 100 (2)
where, mafter and moriginal is the dry weight of solid residue after and
before treatment (g).
The removal of chemical components including glucan, xylan and
W. Ying et al. Biomass and Bioenergy 158 (2022) 106377

Table 1
Chemical compositions of poplar before and after pretreatment.
Samples Glucan (%) Xylan (%) Acid insoluble lignin (%) Acid soluble lignin (%) Solid recovery (%) Removal (%)

Glucan Xylan Lignin

None 40.0 ± 0.9 17.8 ± 0.6 25.7 ± 0.6 2.3 ± 0.5 / / / /

AC-hydrolyzed poplar 51.4 ± 0.1 11.1 ± 1.1 24.6 ± 2.3 1.7 ± 0.4 74.7 4.1 53.4 30.0
HPAC-pretreated poplar 70.2 ± 0.8 16.3 ± 0.1 0.9 ± 0.0 0.8 ± 0.0 52.5 5.7 52.0 97.0
AC-HPAC-pretreated poplar 82.1 ± 0.2 11.5 ± 0.3 2.1 ± 0.1 0.9 ± 0.0 59.4 4.8 31.4 94.8

None: non-pretreated poplar.

AC-hydrolyzed poplar: Poplar hydrolyzed by 5% (w/v) acetic acid solution at 170 ◦ C for 30 min.
HPAC-pretreated poplar: Poplar pretreated by 75% (v/v) HPAC solution at 80 ◦ C for 2 h.
AC-HPAC-pretreated poplar: Poplar pretreated by AC and HPAC pretreatments.

3.2. Delignification of non-pretreated and AC-hydrolyzed poplar

Table 2
XRD, XPS and maximum DR 28 adsorption analysis of non-, AC-, HPAC- and AC-
3.2.1. Chemical compositions
HPAC-pretreated poplar.
As shown in Table 1, raw poplar contained 40.0% glucan, 17.8%
Samples O/C C1 (%) C2 (%) C3 (%) CrI Γmax/DR xylan and 28.0% lignin. After AC hydrolysis, 53.4% of xylan was
(%) 28 (mg/
g)
removed from raw poplar and AC-hydrolyzed poplar contained 51.4%
glucan, 11.1% xylan and 26.3% lignin. After AC hydrolysis, the residual
None 0.34 49.3 40.4 10.3 48.8 240.5
lignin in poplar samples limited the enzymatic digestibility of poplar
± 0.0 ± 1.2 ± 0.8 ± 1.0 ± 0.9
AC-hydrolyzed 0.35 34.1 38.0 27.9 61.2 360.4 carbohydrates through physical barrier and non-productive adsorption
poplar ± 0.0 ± 0.4 ± 0.3 ± 0.8 ± 1.4 toward enzyme protein [15,25]. Thus, an effective lignin-removal pre­
HPAC- 0.39 34.2 50.2 15.6 65.4 702.1 treatment is indispensable. Selective delignification can increase the
pretreated ± 0.0 ± 0.3 ± 0.9 ± 0.7 ± 1.6 opportunity of cellulase to bind to cellulose and reduce the invalid
poplar
AC-HPAC- 0.43 29.1 55.2 15.7 73.8 542.3
adsorption of substrate toward enzymes, which can promote carbohy­
pretreated ± 0.0 ± 0.6 ± 0.6 ± 0.8 ± 1.8 drates degradation of lignocellulosic biomass [7,16,25,26]. After HPAC
poplar pretreatment, 97.0% of lignin was removed from poplar and 94.3% of
glucan was retained (Table 1), resulting in the relative contents of
glucan and xylan in HPAC-pretreated poplar were increased to 70.2%
lignin was calculated as follows:
and 16.3%, respectively. It was found that 94.8% of lignin was removed
[( )/ ]
Removal (%) = mnon− treatment − mafter treatment mnon− treatment × 100 (3) from AC-hydrolyzed poplar by HPAC pretreatment, resulting in a low
lignin content (3.0%) in AC-HPAC-pretreated poplar. High carbohydrate
The XOS and monosaccharides yields in hydrolysates were calcu­ retention was also observed (95.2% glucan and 68.6% xylan) with a
lated as follows: relative glucan content of 82.1% and a relative xylan content of 11.5% in

XOS yield (%) = [(Mass of XOS in hydrolysate) / Mass of xylan in substrate] × 100 (4)

Glucose yield (%) = [(Mass of glucose in hydrolysate × 0.9) / Mass of glucan in substrate] × 100 (5)

Xylose yield (%) = [(Mass of xylose in hydrolysate × 0.88) / Mass of xylan in substrate] × 100 (6)

AC-HPAC-pretreated poplar, which was similar with the results of Wen

et al. [7]. A high proportion of carbohydrates was beneficial to the hy­
drolysis process [27,28], and the residual xylan could be further
3. Results and discussion degraded into high-valued XOS by xylanase as performed later.

3.1. Production of XOS by AC hydrolysis 3.2.2. Surficial and physical properties

Acetic acid hydrolysis is an efficient method to degrade hemi­
celluloses into XOS [15,23]. In our previous report [7], AC catalysis of
poplar was conducted by 0–10% AC solution at 170 ◦ C for 10–50 min
and the optimized conditions for XOS production were 5% of AC solu­
tion at 170 ◦ C for 30 min. In this work, raw poplar was also catalyzed by
5% of AC solution at 170 ◦ C for 30 min and the results show that the XOS
yield was 31.6% (based on the xylan in raw poplar), which was close to
the data (37.3%) in other literature [24].
To intuitively understand the surficial and physical properties of
each sample after treatments, the surface morphology observed by SEM
is shown in supplementary material. After AC hydrolysis, the poplar
surface was rough and uneven. The color of AC-hydrolyzed poplar
became darker while that of HPAC-pretreated poplar and AC-HPAC-
pretreated poplar became bright white due to the bleaching ability of
HPAC pretreatment. It was observed that the smooth fiber surface dis­
appeared and the dense structure showed a loose and porous state after
HPAC pretreatment.
The measurement of cellulose accessibility by the direct red dye
adsorption method made the porosity of materials visual (Table 2). The
accessibilities of poplar and AC-hydrolyzed poplar were 240.4 and
360.4 mg/g substrate, respectively, indicating that AC hydrolysis

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W. Ying et al. Biomass and Bioenergy 158 (2022) 106377

Table 3
Analysis of by-products in pretreatment liquors from poplar after HPAC and AC-HPAC pretreatments.
Samples Xylose (g/L) Formic acid (g/L) HMF (g/L) Vanillin acid (mg/L) Syringic acid (mg/L) p-Coumaric acid (mg/L)

HPAC-pretreated poplar 0.9 ± 0.1 3.5 ± 0.2 0.0 ± 0.0 78.9 ± 0.8 20.7 ± 0.7 38.9 ± 1.1
AC-HPAC-pretreated poplar 0.9 ± 0.1 3.7 ± 0.3 – 90.2 ± 1.2 13.0 ± 1.0 35.6 ± 1.7

Fig. 1. Production of XOS from HPAC-pretreated (A, B, C) or AC-HPAC-pretreated poplar (D, E, F) by 1,000, 5000 and 10,000 nkat/g DM of xylanase at 50 ◦ C for 24,
48, 72 and 96 h. The experiment substrate concentrations were 2%, 5% and 10%.

weakened the encapsulation effect on cellulose by removing xylan [30].
After HPAC treatment, the cellulose accessibilities of HPAC-pretreated
and AC-HPAC-pretreated poplar were 702.1 and 542.3 mg/g sub­
strate, respectively, which were increased by 192% and 50.5%
compared with raw poplar and AC-hydrolyzed poplar. It has been re­
ported that the removal of lignin results in increasing accessibility,
which is beneficial to improve cellulose digestibility [29]. Furthermore,
it was noticed that HPAC-pretreated poplar had higher accessibility than
AC-HPAC-pretreated poplar (Table 2), although AC-HPAC-pretreated
poplar underwent a two-step pretreatment and had lower hemicellu­
lose and lignin contents (Table 1). The results might be attributed to the
aggregation of cellulose caused by excessive delignification, resulting in
reduced cellulose accessibility of raw materials [30].
The crystallinity index (CrI) is usually used as an indicator to eval­
uate enzymatic hydrolysis efficiency of lignocelluloses, and the crys­
tallinity of raw material is greatly affected by the pretreatment [31,32].
As shown in Table 2, due to a lot of hemicelluloses being degraded by AC
hydrolysis, the CrI of poplar was increased from 48.8% to 61.2%, which
was consistent with the trend of the result in Wen et al. [7]. Similarly,
the CrIs of non-pretreated poplar and AC-hydrolyzed poplar were
increased to 65.4% and 73.8% respectively after HPAC pretreatment
(Table 2).

4
W. Ying et al.
Fig. 2. Yields of X1-X6 in the hydrolysates of HPAC-, AC-HPAC-pretreated
poplar. X1-X6 represents xylose, xylobiose, xylotriose, xylotetraose, xylo­
pentaose and xylohexaose, respectively.
XPS is used to analyze the chemical state of the surface of samples.
Higher O/C ratios reflect higher carbohydrates contents, and lower O/C
ratios indicate higher lignin content in samples [18]. The level of lignin
and extractives on sample surface can be represented by C1 value [33],
and C2 and C3 values represent cellulose and hemicellulose contents
[34], respectively. The O/C values of non-pretreated and AC-hydrolyzed
Fig. 3. Monosaccharides yields of HPAC-pretreated poplar after xylanase hydrolysis (A, B) and AC-HPAC-pretreated poplar after xylanase hydrolysis (C, D) by 5–20
FPU/g DM of cellulase (CTec2) at 50 ◦ C and pH 5.0 for 72 h.
5
Biomass and Bioenergy 158 (2022) 106377
poplar were increased from 0.34 and 0.35 to 0.39 (HPAC-pretreated
poplar) and 0.43 (AC-HPAC-pretreated poplar) (Table 2), respectively,
meaning that the relative contents of carbohydrates were increased after
HPAC pretreatment. The C1 values in non-pretreated and
AC-hydrolyzed poplar were 49.3% and 34.1% respectively, which were
higher than those in HPAC-pretreated (34.2%) and AC-HPAC-pretreated
poplar (29.1%), indicating that the lignin was removed by HPAC pre­
treatment. The above results were consistent with the data of chemical
components (Table 1).
3.2.3. Compositions of pretreatment liquid
The liquid compositions of non-pretreated and AC-hydrolyzed poplar
after HPAC pretreatment are shown in Table 3. Only 0.9 g/L xylose was
found in the liquid phase, which meant that fewer carbohydrates were
lost during HPAC pretreatment. The main organic acid in liquid phase
was formic acid (about 3.5 g/L). The hydroxymethylfurfural and furfural
were not detected in HPAC pretreatment liquid phase, indicating that
HPAC pretreatment reduced the accumulation of HMF and furfural in
the pretreatment liquor [35]. The lignin degradation products mainly
included vanillin acid, p-coumaric acid, and syringic acid, of which
amounts were 78.9–90.2, 13.0–20.7, and 35.6–38.9 mg/L, respectively.
It has been reported that after dilute sulfuric acid pretreatment of
bamboo residues (10%, w/v), the lignin degradation products in the
hydrolysate mainly consist of syringaldehyde (285.4 mg/L) and vanillin
(31.5 mg/L), and other products like syringic acid, ferulic acid and
W. Ying et al. Biomass and Bioenergy 158 (2022) 106377

Fig. 4. The glucose (A) and xylose (B) yields of HPAC-pretreated (after xylanase hydrolysis) and AC-HPAC-pretreated poplar (after xylanase hydrolysis) by 5–20
FPU/g DM of CTec2 and 1.0 mg/mL Tween 80 or PEG 4000 at 50 ◦ C and pH 5.0 for 72 h.

p-coumaric acid are less than 20 mg/L [36]. These lignin degradation
products can be separated and used as important chemicals in medicine,
materials and food fields [37].
3.3. Optimization of XOS production from AC-hydrolyzed poplar by
xylanase
After the HPAC pretreatment of non-pretreated and AC-hydrolyzed
poplar, three important factors including xylanase dosage (1,000,
5,000, 10,000 nkat/g DM), hydrolysis time (24, 48, 72, 96 h) and sub­
strate concentration (2%, 5%, 10%) were chosen to optimize XOS pro­
duction from those poplar substrates (Fig. 1). According to Fig. 1A and
D, as the xylanase dosage was increased from 1,000 to 10,000 nkat/g
DM, the XOS yields of HPAC-pretreated and AC-HPAC-pretreated poplar
were increased from 17.8% and 9.7% to 24.2% and 17.5% (based on the
xylan in HPAC-pretreated poplar and AC-HPAC-pretreated poplar),
respectively. Fig. 1B and E shows that the highest XOS yields of HPAC-
pretreated and AC-HPAC-pretreated poplar were obtained at 72 h while

6
W. Ying et al.
Fig. 5. Mass balance of XOS and monosaccharides production from HPAC-pretreated and AC-HPAC-pretreated poplar by xylanase and cellulase.
the 96 h of hydrolysis time was not beneficial to improve XOS yields.
This was because XOS was further degraded by the trace amount of
β-xylosidase in xylanase preparations [38]. Fig. 1C and F shows that the
XOS yields of HPAC-pretreated and AC-HPAC-pretreated poplar were
both decreased from 24.2% and 17.5% to 14.9% and 13.7%, respec­
tively, with substrate concentration being increased from 2% to 10%.
Previous literatures has reported that the accumulated xylan degrada­
tion products during enzymatic hydrolysis exhibit great inhibition to
xylanase activity, resulting in the decreased XOS yield with increasing
solid concentration [39,40]. Besides, the ratio of xylose/XOS was
increased from 0.19 and 0.24 to 0.23 and 0.33, respectively, with the
substrate concentration, meaning that the undesired xylose was pro­
duced (Fig. 1C and F). In conclusion, the highest XOS yields obtained
from HPAC-pretreated and AC-HPAC-pretreated poplar were 24.2% and
17.5%, respectively, and the xylose/XOS ratios were 0.19 and 0.24,
7
Biomass and Bioenergy 158 (2022) 106377
respectively, with 10,000 nkat/g DM of xylanase dosage and 2% of solid
concentration for 72 h.
Fig. 2 shows the xylose and X2–X6 yields in the hydrolysates of
HPAC-, AC-HPAC-pretreated poplar by xylanase. The xylose yield of
HPAC- and AC-HPAC-pretreated poplar were 4.1% and 3.8%, respec­
tively. The X2–X4 were the main components of XOS, accounting for
92.8% and 99.4% of total XOS in the hydrolysates of HPAC- and AC-
HPAC-pretreated poplar, respectively. Under the same hydrolysis con­
ditions of 10,000 nkat/g DM of xylanase dosage and 2% of solid con­
centration for 72 h, the XOS yields of non-pretreated and AC-hydrolyzed
poplar were just 0.6% and 4.9%, respectively (data not shown). In
addition, it was found that the XOS yield of AC-HPAC-pretreated poplar
(17.5%) was lower than that (24.2%) of HPAC-pretreated poplar. This
was because a large amount of xylan in poplar which was easily hy­
drolyzed (31.6%) had been hydrolyzed during the previous AC
W. Ying et al.
hydrolysis.
3.4. Production of monosaccharides from pretreated poplar by cellulase
3.4.1. From HPAC- and AC-HPAC-pretreated poplar after xylanase
hydrolysis
Xylanase hydrolyzed residue of HPAC- and AC-HPAC-pretreated
poplar were hydrolyzed by cellulase (Cellic CTec2) with enzyme dos­
ages of 5–20 FPU/g DM for monosaccharides production. Fig. 3A and B
shows that the glucose and xylose yields of non-pretreated poplar were
just 17.0% and 6.0% at the highest enzyme dosage (20 FPU/g DM),
respectively. The glucose and xylose yields of HPAC-pretreated poplar
were remarkably increased by 4.0–4.2 and 6.9–9.3 times, respectively,
as the enzyme dosage was increased from 5 to 20 FPU/g DM. After 72 h
of hydrolysis with 20 FPU/g DM of cellulase dosage, the glucose and
xylose yields of HPAC-pretreated poplar were 84.3% and 47.5%,
respectively. These results were close to the data in previous literature
[25], in which the maximum glucose yield of peracetic acid-pretreated
yellow poplar (100 mM H2SO4, 120 ◦ C, 30 min) was 84.3% at 5
FPU/g glucan.
Despite a lot of hemicellulose had been degraded by AC hydrolysis,
the glucose and xylose yields of AC-hydrolyzed poplar were both 27.0%
at 20 FPU/g DM (Fig. 3C and D). The unsatisfying enzymatic hydrolysis
efficiency of AC-hydrolyzed poplar has also been reported by Lai et al.
[16]. HPAC pretreatment has been reported to remove 92.7% of lignin in
AC-hydrolyzed poplar and the glucose yield of final material with
cellulase dosage of 7.2 mg/g DM is 86.6% [7]. Although the hydrolysis
efficiency of AC-HPAC-pretreated poplar was increased by 94.3%–
154.3% with the increased cellulase dosage in this work, the glucose
yield was still low (62.3%) at 20 FPU/g DM. Above results meant that
the AC-HPAC-pretreated poplar could not be efficiently hydrolyzed
despite the high content of cellulose (82.1%). The low glucose yield
could be attributed to the excessive delignification, which caused the
cellulose aggregation and consequently reduced the cellulose accessi­
bility [41].
3.4.2. Effects of Tween 80 and PEG 4000
The role of surfactants in enzymatic hydrolysis is to improve the
digestibility of lignocelluloses by mitigating the nonproductive adsorp­
tion of lignin to enzymes and improving the stability of enzymes [20,
42]. The effects of surfactants (1 g/L), Tween 80 and PEG 4000, on
monosaccharides production at different enzyme dosages were investi­
gated. Fig. 4 shows that the glucose yields of non-pretreated poplar and
AC-hydrolyzed poplar were still low (<35%) with surfactants added.
The surfactants promoted the enzymatic hydrolysis efficiency of
HPAC-and AC-HPAC-pretreated poplar to some extent at varied enzyme
dosages. When the cellulase dosage was 20 FPU/g DM, the addition of
Tween 80 (1 g/L) increased the glucose yields of HPAC-and AC-H­
PAC-pretreated poplar from 84.3% and 62.3% to 99.4% and 88.0%,
respectively. Previous literature has reported many promoting mecha­
nisms of surfactants to enzymatic hydrolysis, such as preventing cellu­
lase from being denatured by heat, shear force shock and pH value
during enzymatic hydrolysis [43]. Interestingly, it was found that sur­
factants performed a greater improvement in digestibility of
AC-HPAC-pretreated poplar than that of HPAC-pretreated poplar. A
possible reason was that surfactants could promote the dispersal of
aggregated cellulose in water, resulting in the increased accessibility of
cellulase to cellulose.
3.5. Mass balance
The mass balance of the combined process for XOS and mono­
saccharides production from poplar is shown in Fig. 5. After AC hy­
drolysis, 56.2 g of XOS and 24.3 g of xylose were produced from 1,000 g
of poplar. After HPAC pretreatment (75% HPAC solution, 100 mM
H2SO4, 80 ◦ C, 2 h), 444.1 g of AC-HPAC-pretreated poplar consisting of
8
Biomass and Bioenergy 158 (2022) 106377
364.7 g of glucan, 50.8 g of xylan and 13.0 g of lignin was recovered.
Subsequently, 8.9 g of XOS and 2.2 g of xylose were obtained from AC-
HPAC-pretreated poplar by xylanase hydrolysis (10,000 nkat/g DM, 72
h). Finally, 320.9 g of glucose and 30.4 g of xylose were produced from
the final solid residue by cellulase hydrolysis. In conclusion, 65.1 g of
XOS and 377.8 g of monosaccharides were released from 1,000 g of
poplar. It has been reported that 1,000 g of poplar pretreated by 60%
HPAC solution at 60 ◦ C for 2 h released 17.0 g of XOS and 296.0 g of
glucose by the combined cellulase and xylanase hydrolysis [11], which
were lower than the XOS and glucose yields reported in this work. For
comparison, after HPAC pretreatment and two-step enzymatic hydro­
lysis, 20.5 g of XOS and 419.1 g of monosaccharides were recovered
from 1,000 g of poplar. Obviously, the XOS yield (36.5%, based on the
xylan in raw poplar) from the combined AC and xylanase hydrolysis
process was increased by 2.2 times compared with that (11.5%, based on
the xylan in raw poplar) from the xylanase catalysis without AC
hydrolysis.
Yang et al. (2021) produced XOS with a yield of 42.7% from HPAC-
delignified poplar by lactic acid hydrolysis [44], and Hao et al. (2020)
produced XOS with a yield of 19.8% from HPAC-delignified poplar by
xylanase hydrolysis [11]. The above researches claim that dilute organic
acid and xylanase hydrolysis are efficient to extract XOS from ligno­
cellulosic biomass and a delignification process is necessary before
xylanase hydrolysis. This work proposed an idea that the combined
dilute organic acid and xylanase hydrolysis process was conducted to
produce XOS from poplar. The final XOS yield of poplar was 36.5%,
which was higher than those from AC hydrolysis (21.7%) and lactic acid
hydrolysis (30.3%) of raw poplar directly [26,44] but lower than that
from lactic acid hydrolysis (42.7%) of delignified poplar [44]. However,
AC is cheaper than lactic acid, thus has a greater application potential. In
the future, other xylan-rich lignocelluloses such as sugarcane bagasse
and corncob could be pretreated by this combined process to efficiently
produce XOS and monosaccharides. To further increase XOS and
monosaccharides yields in this combined process, the following re­
searches will focus on further optimizing the conditions of HPAC pre­
treatment, xylanase hydrolysis and cellulase hydrolysis.
4. Conclusions
In this work, the combined AC and xylanase hydrolysis was suc­
cessfully carried out for maximum XOS production. Especially, AC hy­
drolysis yielded 31.6% of XOS (based on the xylan in raw poplar), and
HPAC pretreatment removed 94.8% of lignin in AC-hydrolyzed poplar.
After xylanase hydrolysis, 17.5% of XOS was produced from AC-HPAC-
pretreated poplar (based on the xylan in AC-HPAC-pretreated poplar).
The total XOS yield of the combined process was increased by 2.2 times
compared with that of xylanase catalysis. The glucose yield of final solid
residue reached to 88.0%. This work proposed a novel strategy for
efficient preparation of XOS and monosaccharides from poplar by
organic acid hydrolysis and two-step enzymatic hydrolysis.
Acknowledgments
This work was greatly supported by the National Natural Science
Foundation of China (Grant No. 32171731) and the Starting Research
Fund from the Nanjing Forestry University, China (Grant No.
163030103).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biombioe.2022.106377.
W. Ying et al. Biomass and Bioenergy 158 (2022) 106377

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