Bioresource Technology 313 (2020) 123675

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Using low carbon footprint high-pressure carbon dioxide in bioconversion of T

aspen branch waste for sustainable bioethanol production
Yingji Wua,1, Shengbo Gea,1, Changlei Xiaa,b,1, Liping Caia, Changtong Meia, Christian Sonnec,
Young-Kwon Parkd, Young-Min Kime, Wei-Hsin Chenf,g,h, Jo-Shu Changg,i, Su Shiung Lamj,k,b,
⁎

a
Co-Innovation Center of Efficient Processing and Utilization of Forestry Resources, College of Materials Science and Engineering, Nanjing Forestry University, Nanjing,
Jiangsu 210037, China
b
Anhui Juke Graphene Technology Co., Ltd., Bozhou, Anhui 233600, China
c
Aarhus University, Department of Bioscience, Arctic Research Centre (ARC), Frederiksborgvej 399, PO Box 358, DK-4000 Roskilde, Denmark
d
School of Environmental Engineering, University of Seoul, Seoul 02504, Republic of Korea
e
Department of Environmental Engineering, Daegu University, Gyeongsan 38453, Republic of Korea
f
Department of Aeronautics and Astronautics, National Cheng Kung University, Tainan 701, Taiwan
g
Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung 407, Taiwan
h
Department of Mechanical Engineering, National Chin-Yi University of Technology, Taichung 411, Taiwan
i
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
j
Pyrolysis Technology Research Group, Institute of Tropical Aquaculture and Fisheries (AKUATROP), Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu,
Malaysia
k
Henan Province Engineering Research Center for Biomass Value-Added Products, Henan Agricultural University, Zhengzhou, Henan 450002, China

GRAPHICAL ABSTRACT
Enzymatic hydrolysis yield (%)

120
AW-190°C
Regular hot water AW-190°C-68atmCO2
pretreatment
80 AW-190°C-68atmN2

Aspen wood (AW) AW-190 C

High-pressure CO2 hot water 40
pretreatment

(I)
0
Cellulose
(II) Gas releasing 0 24 48 72
Hemicellulose

High-pressure CO2 gas Incubation time (h)

Lignin AW-190 C-68atmCO2

ARTICLE INFO ABSTRACT

Keywords: An innovative approach was developed by incorporating high-pressure CO2 into the separate hydrolysis-fer-
Separate hydrolysis-fermentation (SHF) mentation of aspen leftover branches, aiming to enhance the bioethanol production efficiency. The high-pressure
Biomass CO2 significantly increased the 72-h enzymatic hydrolysis yield of converting aspen into glucose from 53.8% to
Carbon dioxide (CO2) 82.9%. The hydrolysis process was performed with low enzyme loading (10 FPU g−1 glucan) with the aim of
Yeast fermentation
reducing the cost of fuel bioethanol production. The ethanol yield from fermentation of the hydrolyzed glucose
Biofuel
using yeast (Saccharomyces cerevisiae) was 8.7 g L–1, showing increment of 10% compared with the glucose
control. Techno-economic analysis indicated that the energy consumption of fuel bioethanol production from
aspen branch chips was reduced by 35% and the production cost was cut 44% to 0.615 USD L–1, when 68 atm

⁎
Corresponding author.
E-mail address: lam@umt.edu.my (S.S. Lam).
1
These authors contributed equally to the manuscript.

https://doi.org/10.1016/j.biortech.2020.123675
Received 11 May 2020; Received in revised form 9 June 2020; Accepted 9 June 2020
Available online 13 June 2020
0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
Y. Wu, et al.
CO2 was introduced into the process. These results furtherly emphasized the low carbon footprint of this sus-
tainable energy production approach.
1. Introduction
Driven by the environmental impacts and unsustainability of using
fossil fuels, second-generation biofuels, e.g., bioethanol, biodiesel, and
biomethane, are widely investigated for their potential to alter the
status of global energy consumption (Ragauskas et al., 2006). Those
biofuels are quite applicable to substitute fossil fuels because they have
similar properties as conventional fuels (Kesharwani et al., 2019). This
makes them easier to be applied to conventional fossil-fuel engines. In
2018, the proportion of the biomass-derived fuels was approximately
5% in all energy resources used in the United States, and 46% of the
biomass fuels was mainly in the form of bioethanol (Ge et al., 2020).
Furtherly, as alternative fuels, global warming could also be hugely
relieved by counting the life cycle effects (Zhao et al., 2019). Separate
hydrolysis-fermentation (SHF) is one of the current approaches for
converting biomass into biofuels, including two stages, i.e., sacchar-
ification and fermentation (Yang et al., 2014). The advantage of SHF is
mainly on its easiness to separately optimize hydrolysis to prepare high-
concentration sugar broth for subsequent fermentation (Maslova et al.,
2019). However, bioethanol is primarily derived from sugar and starch
in the current stage, which substantially conflicts with the food supply
chains and thereby affecting the food prices (Weng et al., 2019).
To avoid competition with food supplies, other hydrocarbon feed-
stocks, for instance, lignocellulosic biomass, are investigated and de-
veloped to be used as biofuel production resources (Cordella et al.,
2012). Those lignocellulosic biomasses could be forestry materials,
agricultural residues, industrial wastes, etc., containing abundant
polysaccharides, for instance, cellulose (Ewulonu et al., 2019). After
being hydrolyzed, the released sugar (e.g., glucose) could be the pro-
mising feedstock used in the biofuel production industries (Yang et al.,
2019b), as a replacement of food crops, e.g., corn, sugarcane, etc.
However, the recalcitrance of lignocellulosic materials to biological
conversion is very serious, which increases the difficulty of cellulose
conversion into sugars (Shafiei et al., 2015). The compact structures in
the plant cells, where the cells of biomass are stacked together (called
middle lamella) due to the high lignin content, which will result in the
enzyme used for hydrolysis was inhibited from accessing the cellulose,
are undesirable for the cell decomposition. Thus, there was a need to
pretreat the lignocellulosic material for increasing the enzyme acces-
sibility to maximize the enzymatic hydrolysis of cellulose (Karimi et al.,
2013).
To upgrade lignocellulosic biomass for producing enzymatic hy-
drolysis glucose, various pretreatment approaches were explored, e.g.,
biological (Sharma et al., 2019), hot water (Xu et al., 2019), organic
solvent (Zhang et al., 2016), dilute acid (Jensen et al., 2010), Fenton
chemical (Yang et al., 2019a), etc. As a chemical-free method, hot water
pretreatment is preferred, especially for industrial production (Karimi
et al., 2013). However, the industrialization of hot water pretreatment
is limited by its unsatisfactory efficiency in eliminating the inhibition
from the residue biomass, for instance, high-residue hemicellulose
biomass (Karimi et al., 2013). Being a fast-growing wood, Aspen (Po-
pulus tremuloides) trees are widely planted as economic forest in North
America. The high cellulose content of aspen wood (AW) is a desired
resource for sugar production (Jagtap et al., 2013). Pretreatments, for
instance, dilute acid (Jensen et al., 2010), have been applied to AW for
enhancing the cellulose hydrozability by enzymes, however, the en-
vironmental concerns from the use of chemicals are obvious. A hot
water pretreatment is an ideal approach with no additional chemicals.
However, the hemicellulose removal from aspen in the hot water pre-
treatment is not efficient, for instance, the reduction of hemicellulose in
2
Bioresource Technology 313 (2020) 123675
the solid residue was only approximately 50% after subjecting to 1 h of
autohydrolysis (Mittal et al., 2009).
The CO2-assisted and CO2-catalyzed processes become attractive
due to their chemical-free nature (Park and Lee, 2020). To enhance the
hot water pretreatment efficiency without raising environmental con-
cerns, high-pressure CO2 was introduced into the process, which could
promote hemicellulose degradation without using chemicals. It was
contributed by the formation of carbonic acids that could help to hy-
drolyze the hemicellulose (Zheng et al., 1998). The effectiveness of
high-pressure CO2 can be highly correlated with pretreatment tem-
perature and pressure. As a chemical-free method, the use of high-
pressure CO2 in hot water pretreatment is hypothesized to be able to
help to break down cell wall structures and lead to more glucose pro-
duction without causing environmental burden. High-pressure CO2 gas
is used as an agent in the physical forming process, which could fab-
ricate foam with high surface area (Kuang et al., 2017). It is also
thought that the high-pressure CO2 may also increase the surface area
in the pretreated substances, enhancing the enzyme adsorption and
thereby promoting enzymatic hydrolysis stage. Furthermore, the utili-
zation of CO2 will not generate any toxic residue in the saccharine
broth, which could be beneficial to the fermentation stage for biofuel
production. In the economic aspect, the increased glucose yield is
predicted to reduce the production cost of bioethanol (Toor et al.,
2020).
To promote the bioconversion of waste aspen branch chips into
bioethanol, high-pressure CO2 was introduced into the SHF process to
improve pretreatment efficiency by combining hot-water pretreatment
with high-pressure CO2 application. The enzymatic hydrolysis results
were utilized to examine the pretreatment parameters. The mechanism
of the high-pressure CO2 hot water pretreatment was illustrated. Yeast
fermentation was used to produce bioethanol from the hydrolyzed
glucose. Furthermore, the energy consumption and techno-economic
investigation of the SHF processes were performed and compared. This
high-pressure CO2 process is hypothesized to show improved perfor-
mance on refining enzymatic feedstock compared to regular hot water
pretreatment, and less environmental impact and reagent cost than
chemical assisted pretreatments. Those beneficial features are expected
to enhance the feasibility of using the high-pressure CO2 process for
ethanol production.
2. Materials and methods
Aspen (Populus tremuloides) leftover branches were collected from
timber cutting and chopped into chips with an approximate size of
1 × 1 × 0.2 cm3 (length × width × thickness), and naturally dried.
The biomass chemical compositions were determined by following
National Renewable Energy Laboratory (NREL) standard (Sluiter et al.,
2008). The chemicals, including microcrystalline cellulose (Avicel® PH-
101, ~50 μm particle size), citric acid monohydrate (≥99%), sodium
citrate dihydrate (≥99%), sodium chloride (≥99.5%), acetic acid
(≥99%), D-glucose (≥99.5%), and ethanol (≥99.5%) were obtained
from Sigma-Aldrich, USA. The buffer solution (50 mM, pH = 4.8) was
prepared from citric acid, sodium citrate and deionized (DI) water.
Novozym® 22C cellulase was employed for experiments of enzymatic
hydrolysis. Cellulase C2730 was used for the Langmuir adsorption
studies. A commercial strain, Baker’s yeast (Saccharomyces cerevisiae)
was utilized in the yeast fermentation to produce bioethanol.
Y. Wu, et al.
2.1. Hot water pretreatments
A 2-L pressure reactor was employed for hot water pretreatment.
Briefly, aspen branch chips (100 g) were soaked in DI water (1 L) for
approximately 12 h, and subsequently poured into the reactor. The
pretreatments were carried out under various temperatures, i.e., 150,
170, and 190 °C, respectively. After 1-h of dwelling at various pre-
treatment temperatures, the reactor was quenched to less than 35 °C by
tap water. The suspension was filtrated (200-mesh sieve) and DI water
washed for three cycles. The solid substance was collected and kept in a
refrigerator (4 °C). The resulted substances were named AW-150 °C,
AW-170 °C, and AW-190 °C, respectively, corresponding to their pre-
treatment temperatures. Triplicate experiments were performed for
each pretreatment in this work. The chemical compositions of the
pretreated samples were investigated through the NREL procedure
(Sluiter et al., 2008), and then converted to calculate the contents of
cellulose, hemicellulose and lignin.
2.2. High-pressure CO2 hot water pretreatments
An innovative approach combining high-pressure carbon dioxide
(CO2) and hot water pretreatment was developed and tested on waste
aspen branch chips. The experiments were performed in a 2-litre her-
metical reactor connected with an external CO2 gas tanker. Similar to
that performed by regular hot water pretreatment at 190 °C, the soaked
aspen branch chips were subjected for 1 h cooking at 190 °C. During the
pretreatments, the pressures in the reactor were kept at 34 or 68 atm
(500 or 1000 psi) by introducing high-pressure CO2 gas, and the re-
sulting solid substances were named as AW-190 °C-34atmCO2 and AW-
190 °C-68atmCO2, respectively. All other procedures were the same as
those conducted in the regular hot water pretreatment.
2.3. High-pressure N2 hot water pretreatment
As a control variant and comparison to the high-pressure CO2 pre-
treatment, high-pressure N2 hot water pretreatment was also carried
out. The N2 is an inert gas that does not react with water, thus con-
sidering a suitable control variant for examination of the effect of
pressure on the pretreatment. All the procedures were the same as those
for producing AW-190 °C-68atmCO2 substance from aspen branch
chips, except with high-pressure N2 as the introduced gas, i.e., N2 at
1000 psi (68 atm). The treated substance was named as AW-190 °C-
68atmN2.
2.4. Enzymatic hydrolysis
The enzyme activity of Novozym 22C cellulase was determined in
terms of NREL standard (Sluiter et al., 2008), which was 110 FPU g−1.
By using Novozym 22C cellulase, the enzymatic hydrolysis was per-
formed in 250-mL flasks. The detail procedures were followed ac-
cording to a previously reported method (Sun et al., 2016). Briefly, the
substance with 2% glucan (w/v) was added to each 250-mL flask, fol-
lowed by 50 mL freshly prepared citrate buffer (pH = 4.8) and 10 FPU
enzymes (equivalent to 10 FPU g−1 glucan). All the flasks were set up
on an incubator shaker and the enzymatic hydrolysis of the substances
was performed under 150 rpm of orbital rotation and 50 °C of en-
vironmental temperature.
For analysis by Agilent 1260 (USA) high-performance liquid chro-
matography (HPLC) (Aminex HPX-87P column), small amounts (0.5 mL
each time) of samples were taken at various incubation time durations
(i.e., 0, 3, 6, 12, 24, 48, and 72 h, respectively) for determining the
cellulose and glucose concentrations. The cellulose and glucose stan-
dard solutions were prepared in the lab using Avicel (pure cellulose)
and D-glucose (≥99.5%), respectively. Divided by the theoretical glu-
cose availability in each substance, the determined glucose concentra-
tion in the flask was used to calculate the hydrolysis yield.
3
Bioresource Technology 313 (2020) 123675
2.5. Mercury intrusion
A Micromeritics Autopore IV 9500 with a 3-mL penetrometer
(0.387IV and 0.412SV) was employed for mercury intrusion analysis.
The pressure range from 0.5 to 33,000 psi was used for data collection.
Prior to the tests, the substances were freeze-dried for approximately
48 h by a freeze dryer (Christ Alpha 1–2 LD plus, Germany).
2.6. Langmuir adsorption
Langmuir adsorption tests were conducted to investigate the en-
zyme adsorption on the substances (Tu et al., 2009). Cellulase C2730
enzyme (low β-glucosidase) was used for the tests. The protein content
of the enzyme suspension was 41 mg mL−1. Briefly, the substance was
mixed into sodium citrate buffer for preparing samples with various
enzyme amounts, i.e., 0.1, 0.25, 0.5, 1 and 2 mg mL−1, respectively.
The prepared samples were then placed in an incubation shaker with
150 rpm of orbital rotation and at 50 °C of environmental temperature.
After equilibrium (approximately 12 h), the free enzyme concentrations
were obtained. Being subtracted from the initial concentrations, the
adsorbed enzyme concentrations were calculated. The correlation be-
tween free and adsorbed enzyme concentrations was established as
follows:
max KC
=
(1 + KC ) (1)
where ‘Γ’ = adsorbed enzyme concentration; ‘Γmax’ = maximum en-
zyme concentration (i.e., enzymes fully covered the surface of sub-
stance); ‘K’ = Langmuir constant; and ‘C’ = free enzyme concentration.
The distribution coefficient (R) can be defined:
R= (2)
max K
2.7. Yeast fermentation to produce bioethanol
The commercially available Fleischmann’s yeast (Saccharomyces
cerevisiae) was employed for the fermentation experiments. The dried
yeast was pre-cultured in yeast extract peptone dextrose medium (YPD)
at 30 °C for 12 h. The grown strains were subsequently harvested,
concentrated by centrifuging, sterile DI-water washed, and inoculated
into 125-mL serum bottles. The inoculation amount of yeast was kept
2.0 g L−1 for each bottle, the initial glucose concentration was adjusted
to 20 g L−1 by adding pure glucose, and liquid (pH = 6.0) volume was
60 mL, respectively. The batch fermentation was then conducted in an
incubation shaker with 150 rpm of orbital rotation and 30 °C of en-
vironmental temperature. Sampling 0.5 mL mixture at various in-
cubation time durations (0, 1, 3, 6, 9, 12, 24, and 36 h, respectively)
was used for detecting the glucose and bioethanol concentrations by
HPLC method. The ethanol standard solutions were prepared in the lab
using ethanol (≥99.5%).
3. Results and discussion
3.1. Chemical compositions of the substances
The chemical compositions of the substances (i.e., untreated AW,
AW-150 °C, AW-170 °C, AW-190 °C, AW-190 °C-34atmCO2, and AW-
190 °C-68atmCO2) are listed in Table 1. The mass fractions of the main
compounds of the untreated AW including glucan, xylan, and lignin
(total of acid-soluble lignin and Klason lignin) were analyzed to be
45.0%, 16.3%, and 26.8%, respectively, which were closed to other
reports (Liu and Zhu, 2010; Wang et al., 2018). The glucan fraction was
gradually increased to 48.9% (AW-150 °C), 53.0% (AW-170 °C), 56.2%
(AW-190 °C) with the increase of hot water temperature, and furtherly
increased to 60.0% (AW-190 °C-34atmCO2) and 61.5% (AW-190 °C-
68atmCO2) by introducing high-pressure CO2 gas. Similar to the glucan,
Y. Wu, et al. Bioresource Technology 313 (2020) 123675

Table 1
Chemical composition of the untreated, regular hot water (150, 170 and 190 °C) pretreated, and high-pressure CO2 (34 and 68 atm) pretreated aspen wood.
Composition[a] (%) Klason lignin Acid-soluble lignin Glucan Xylan Galactan Arabinan Mannan Total

[b] [e]
Untreated AW 24.6 ± 0.3 2.1 ± 0.0 45.0 ± 0.6 16.3 ± 0.3 1.6 ± 0.0 1.1 ± 0.1 2.2 ± 0.0 92.9
AW-150 °C[c] 29.1 ± 1.0 2.9 ± 0.2 48.9 ± 2.6 9.6 ± 0.7 0.7 ± 0.0 0.9 ± 0.0 1.7 ± 0.1 93.7
AW-170 °C 29.5 ± 1.5 3.0 ± 0.3 53.0 ± 2.3 8.4 ± 1.3 0.5 ± 0.0 0.6 ± 0.0 1.1 ± 0.0 96.0
AW-190 °C 30.3 ± 1.2 2.8 ± 0.2 56.2 ± 3.5 5.6 ± 1.0 0.5 ± 0.1 0.4 ± 0.1 1.0 ± 0.1 96.8
AW-190 °C-34atmCO2[d] 32.4 ± 1.8 2.0 ± 0.3 60.0 ± 2.3 1.8 ± 0.1 0.4 ± 0.0 0.5 ± 0.2 0.6 ± 0.0 97.8
AW-190 °C-68atmCO2 31.7 ± 1.9 2.0 ± 0.3 61.5 ± 2.5 1.9 ± 0.4 0.3 ± 0.0 0.3 ± 0.1 0.2 ± 0.0 97.7

Notes: [a] Determined in terms of the NREL standard procedure (Sluiter et al., 2008); [b] AW = aspen wood; [c] AW-150 °C = hot water (150 °C) pretreated aspen
wood; [d] AW-190 °C-34atmCO2 = hot water (190 °C) pretreated aspen wood in the presence of 34 atm CO2 gas; [e] Mean values of replicates (n = 3) are reported
with ± standard deviations.

the lignin fractions in the substances were increased, i.e., from un- (AW-170 °C) and 5.0% (AW-190 °C) after regular hot water pretreat-
treated AW at 26.8% to treated AWs at 32.0% (AW-150 °C), 32.5% ments, and to 1.8% (AW-190 °C-34atmCO2) and 1.6% (AW-190 °C-
(AW-170 °C), 33.2% (AW-190 °C), 34.5% (AW-190 °C-34atmCO2), and 68atmCO2) after high-pressure CO2 hot water pretreatments. The results
33.6% (AW-190 °C-68atmCO2), respectively. The increase of glucan and revealed that hot water pretreatments could dramatically reduce the
lignin fraction was probably derived by the dramatical reduction of hemicellulose in the substance but have fewer effects on the cellulose and
xylan contents in the substances (Table 1). The hot water pretreatments lignin components (Tian et al., 2019). Additionally, further decomposi-
broke down the xylan content from 16.3% (untreated AW) to 9.6% tion of the hemicellulose by high-pressure CO2 gas might benefit the
(AW-150 °C), 8.4% (AW-170 °C), and 5.6% (AW-190 °C), respectively. subsequent enzymatic hydrolysis of cellulosic substances (Tian et al.,
The addition of CO2 gas to the pretreatments could furtherly reduce the 2019). High-pressure CO2 was likely to have increased the formation of
xylan content to 1.8% (AW-190 °C-34atmCO2) and 1.9% (AW-190 °C- carbonic acid, which might promote the hydrolysis of hemicellulose
68atmCO2), respectively. A similar phenomenon was illustrated by Tian (Morais et al., 2014).
et al. (2019), which was mainly due to the various decomposition rates
of glucan (mainly in cellulose), lignin and xylan (mainly in hemi-
3.2. Parameters of the pretreatments with high-pressure CO2
cellulose), because hemicellulose is much easier to be hydrolyzed into
small molecules in the hot water pretreatment compared to cellulose
The enzymatic hydrolysis of the substances was performed (Fig. 2a).
and lignin.
Compared to the untreated AW (3.4%), the hydrolysis yields at 72 h of
The saccharide contents in the substances were converted from their
the hot water pretreated AWs were dramatically increased, i.e., 36.2%
chemical compositions (Table 1), i.e., 43.6% cellulose and 19.7%
for AW-150 °C, 44.5% for AW-170 °C, and 53.8% for AW-190 °C. The
hemicellulose for untreated AW, 47.4% and 11.8% for AW-150 °C, 51.3%
hot water pretreatment with higher temperatures could produce the
and 10.1% for AW-170 °C, 54.4% and 6.8% for AW-190 °C, 58.2% and
substance with higher enzymatic hydrolysability, which was inversely
2.6% for AW-190 °C-34atmCO2, and 59.6% and 2.4% for AW-190 °C-
correlated with their hemicellulose contents (Table 1), i.e., less hemi-
68atmCO2 (Fig. 1a). By incorporating the solid yields of the treated AWs
cellulose in the substance was beneficial to the hydrolysis yield
(i.e., 85.9% for AW-150 °C, 78.6% for AW-170 °C, 73.1% for AW-190 °C,
(Ngamsirisomsakul et al., 2019). Those improvements were probably
68.1% for AW-190 °C-34atmCO2, and 66.7% for AW-190 °C-68atmCO2)
derived by the reduction of hemicellulose that resulted in the micro-
with their component compositions (Fig. 1a), the component yields from
structure damage of the substance, and thereby increasing the enzyme
the raw AWs were calculated (Fig. 1b). It was shown that the cellulose
accessibility onto the cellulose surface (Garrote et al., 1999). However,
yields of the treated AWs were very close (39.6 – 40.7%), which were
the hydrolysis yields of the hot water pretreated AWs were still much
slightly lower than that of untreated AW (43.6%). The lignin yields of the
lower than that of the Avicel control (70.4%), which was probably
treated AWs were also very close to each other but presenting slight
owing to the relatively high remaining hemicellulose that reduced the
decrease with the increase of pretreatment temperature and CO2 pres-
cellulose accessibility (Bansal et al., 2012). It was indicated that the hot
sure, i.e., 27.5% for AW-150 °C, 25.5% for AW-170 °C, 24.3% for AW-
water pretreatment may not be enough to pretreat the AW for preparing
190 °C, 23.5% for AW-190 °C-34atmCO2, and 22.4% for AW-190 °C-
highly hydrolysable substrates.
68atmCO2. In comparison, the decrease of hemicellulose yields were very
To improve the pretreatment, dilute acid was an attractive method
significant from 19.7% (untreated AW) to 10.2% (AW-150 °C), 8.0%
to be used in the preparation of enzymatic hydrolysis substances, since

Fig. 1. Component compositions of the untreated,

(a) Others Hemicellulose
(b) Others Hemicellulose
based on the resulted biomass (%)

Cellulose regular hot water (150, 170 and 190 °C) pretreated,
Lignin Cellulose Lignin
and high-pressure CO2 (34 and 68 atm) hot water
based on the raw AW (%)
Component composition
Component composition

100 100 pretreated aspen wood from 100 g raw aspen wood,
based on the resulted biomass (a) and raw material
80 80
(b). Mean values of replicates (n = 3) are reported
60 60 with standard deviations given as error bars.

40 40

20 20

0 0
C C C 2 2

AW 0° 0° O O
8a O 2

0°
A W 0°C

C
A W 0°C
AW AW

C C
0°

5 7 9
C
C

ed -1 -1 -1 tm tm
7

A W °C 19
5

tm
m
ed

t a a
-1
-1

ea
t

AW AW AW 34 68
-

90 4a
at

r - -
nt
re

-3

-6

°C °C
nt

°C

U 0 0
U

19 19
90

- -
-1

-1

AW AW
AW

4
Y. Wu, et al. Bioresource Technology 313 (2020) 123675

Fig. 2. Enzymatic hydrolysis results of (a) Avicel

(a) (b) control, untreated and hot water (150, 170 and
120 120
Enzymatic hydrolysis yield

Enzymatic hydrolysis yield

Avicel Avicel AW-190°C 190 °C) pretreated aspen wood, (b) high-pressure
AW-150°C AW-190°C-34atmCO2
CO2 (34 and 68 atm) pretreated aspen wood, and (c)
AW-170°C AW-190°C-68atmCO2
80 AW-190°C 80 high-pressure N2 (68 atm) pretreated aspen wood.
Untreated AW Mean values of replicates (n = 3) are reported with
(%)

(%)
standard deviations given as error bars.
40 40

0 0
0 24 48 72 0 24 48 72
Incubation time (h) Incubation time (h)

(c)
120
Enzymatic hydrolysis yield

AW-190°C
AW-190°C-68atmCO2
80 AW-190°C-68atmN2
(%)

40

0
0 24 48 72
Incubation time (h)

the hemicellulose could be easily hydrolyzed in the acidic condition,
and in turn enhancing the enzyme accessibility to the cellulose surfaces
(Pu et al., 2013). The acidic condition was formed by dissolving high-
pressure CO2 into water, which might promote the hemicellulose de-
composition (Morais et al., 2014). In terms of the enzymatic hydrolysis
results from hot water pretreated AWs (Fig. 2a), the optimized hot
water processing temperature at 190 °C was chosen for the high-pres-
sure CO2 hot water pretreatments. The enzymatic hydrolysis curves of
the Avicel and treated AWs (i.e., AW-190 °C, AW-190 °C-34atmCO2,
and AW-190 °C-68atmCO2) are presented in Fig. 2b. The 72-h hydro-
lysis yields of AW-190 °C-34atmCO2 and AW-190 °C-68atmCO2 were
77.6% and 82.9%, respectively, which were dramatically increased
from 53.8% for AW-190 °C. The highest 72-h hydrolysis yield of AW-
190 °C-68atmCO2 exceeded 80%, indicating that the hydrolysis reac-
tion was almost completed for the substance after being treated at
190 °C combined with 68 atm CO2 (Santa-Maria and Jeoh, 2010). The
increment of the hydrolysis yield of 68 atm CO2 treated AW (AW-
190 °C-68atmCO2) could be 54%, compared to the regular hot water
treated AW (AW-190 °C).
3.3. Beneficial mechanism behind incorporation of high-pressure CO2 into
hot water pretreatment
The mechanism behind this approach was investigated and dual
beneficial effects can be derived. The dual effects of high-pressure CO2
on the hot water pretreatment of AW are illustrated in Fig. 3. Compared

Regular hot water pretreatment

Aspen wood (AW) Hot water pretreated AW

High-pressure CO2 hot water pretreatment

(I)
CO2 + H2O H+ + HCO3-
Cellulose
(II) Gas releasing
Hemicellulose

High-pressure CO2 gas

Lignin High-pressure CO2 hot
water pretreated AW

Fig. 3. Illustration of the dual-effects mechanism of high-pressure CO2 in the hot water pretreatment of aspen wood.

5
Y. Wu, et al.
to the regular hot water pretreatment, the high-pressure CO2 dramati-
cally enhanced the hydrolysability of the pretreated substance from
53.8% to 82.9% (Fig. 2b). As shown in Fig. 3, the dual effects of the
high-pressure CO2 were likely to have contributed to the increased
hydrolysability. Firstly, the introduction of high-pressure CO2 aided in
decomposing the hemicellulose from the treated substance, i.e., the
hemicellulose content of AW-190 °C (5.0%) was reduced to 1.6% after
introduction of 68 atm CO2 (Fig. 1b). Secondly, the high-pressure
condition enlarged the surface area of the treated substance and in turn
contributed to improved enzyme adsorption. Both the effects increased
the access of enzymes to the cellulose molecules, leading to enhanced
hydrolysability of the treated substance. It has been reported that the
CO2 dissolved in the aqueous solution could form carbonic acid to help
reduce the hemicellulose in the substance (Morais et al., 2014), which
could promote the subsequent enzymatic hydrolysis. Apart from the
effect as an acid source, a novel mechanism of high-pressure CO2 on the
pretreatment was firstly proposed and verified in this study. It was
suggested that the release of pressurized CO2 might increase the pore
area and porosity of the substance, leading to adsorption of more en-
zymes onto the substance and thereby enhancing the enzymatic hy-
drolysis process.
The hemicellulose contents of AW-190 °C, AW-190 °C-34atmCO2
and AW-190 °C-68atmCO2 were 6.8%, 2.6% and 2.4%, respectively
(Fig. 1a). It was revealed that the hemicellulose content was reduced by
61% and 65% after the application of high-pressure CO2 (34 and
68 atm, respectively). The slight difference of the residual hemi-
cellulose content between AW-190 °C-34atmCO2 and AW-190 °C-
68atmCO2 was probably attributed to the lower pH at higher CO2
pressure (Peng et al., 2013). The decomposition of hemicellulose by
CO2 was commonly reported (Morais et al., 2014), and the explanation
could be the formation of carbonic acid from CO2 and water, promoting
hemicellulose hydrolysis in the substance. The lower hemicellulose
content in the substance dramatically contributed to the enzymatic
hydrolysis of the corresponding substance (Bansal et al., 2012), e.g.,
53.8% of 72-h hydrolysis yield for AW-190 °C, 77.6% for AW-190 °C-
34atmCO2, and 82.9% AW-190 °C-68atmCO2 (Fig. 2b).
Fig. 4. Cumulative pore area (a) and pore distribution (b) of the regular hot water (190 °C) and high-pressure CO2 (34 and 68 atm) pretreated aspen wood. Cellulase
adsorption isotherms (c) on Avicel, the regular and high-pressure CO2 (34 and 68 atm) hot water (190 °C) pretreated aspen wood.
6
Bioresource Technology 313 (2020) 123675
To further confirm the contribution of acidic condition to the hy-
drolysis, the hot water (190 °C) pretreatment with 68 atm N2 for AW
(AW-190 °C-68atmN2) was performed. The enzymatic hydrolysis curves
of AW-190 °C-68atmCO2 and AW-190 °C-68atmN2 are compared in
Fig. 2c. Obviously, the 72-h hydrolysis yield of AW-190 °C-68atmCO2
(82.9%) was higher than that of AW-190 °C-68atmN2 (61.2%), proving
that the acidic condition generated by CO2 could promote the hydro-
lysis of pretreated substance. However, the hydrolysis yield at 72 h of
AW-190 °C-68atmN2 (61.2%) was higher than that of AW-190 °C
(53.8%), indicating that the high-pressure environment itself also
contributed to preparing pretreated substance with better enzymatic
hydrolysability.
Inspired by the physical foaming process using high-pressure gas
that could expand the pores in the substance (Hamidinejad et al., 2018),
the pores in the AW-190 °C-34atmCO2 and AW-190 °C-68atmCO2
substances might be expanded by the high-pressure CO2. The porosities
and pore distributions of AW-190 °C, AW-190 °C-34atmCO2 and AW-
190 °C-68atmCO2 were determined by mercury intrusion porosimetry.
The cumulative pore areas and pore distributions of the substances are
shown in Fig. 4a–b, and the corresponding parameters are presented in
Table 2. The total pore areas and porosities were increased from 4.3 m2
g−1 and 46.3% (AW-190 °C) to 5.3 m2 g−1 and 51.4% (AW-190 °C-
34atmCO2) after adding 34 atm CO2 to the pretreatment, and the values
were further increased to 5.5 m2 g−1 and 53.6% (AW-190 °C-
68atmCO2) when the pretreatment CO2 pressure was 68 atm. Further-
more, the increases of average pore diameter in the substance were
more significant, i.e., from 2.2 µm (AW-190 °C) to 4.5 µm (AW-190 °C-
34atmCO2) and 4.8 µm (AW-190 °C-68atmCO2). The results were per-
fectly matched with the assumption that high-pressure CO2 would ex-
pand the pores in the substance for a higher pore area and porosity
(Fig. 3).
Langmuir adsorption tests of the substances were performed to
evaluate their capabilities on enzyme adsorption (Fig. 4c and Table 3).
Commonly, the higher Γmax of the substance could result in higher
hydrolysis yield (Lai et al., 2015). The distribution coefficient (R) in-
creased with the raising of CO2 pressure from 0 to 68 atm, indicating
Y. Wu, et al. Bioresource Technology 313 (2020) 123675

Table 2 those obtained from SHF using different pretreatment conditions in the
Mercury intrusion porosimetry results of the regular hot water and high-pres- literatures. The ethanol production was calculated incorporating pre-
sure CO2 pretreated aspen wood. treatment, hydrolysis and fermentation yields, reflecting the overall
Substance Total pore area Porosity Average pore diameter efficiency of the SHF process. Compared to the literatures, the ethanol
production using high-pressure CO2 in this work exhibited higher value
(m2 g−1) (%) (µm) (0.157 g g−1 dry biomass) compared to those using other pretreatment
AS-190 °C[a] 4.3 46.3 2.2
conditions in the literature: 0.126 g g−1 dry biomass using 4 vol%
AS-190 °C-34 atm CO2[b] 5.3 51.4 4.5
AS-190 °C-68 atm CO2 5.5 53.6 4.8 H2SO4 (Zhu et al., 2011), 0.084–0.144 g g−1 dry biomass using dilute
acid (Abedinifar et al., 2009; Jutakridsada et al., 2019; Slathia et al.,
Notes: [a] AW-190 °C = aspen wood after hot water (190 °C) pretreatment; 2020), 0.135 g g−1 dry biomass using H2SO4 and H2O2 (Kaushal et al.,
[b]
AW-190 °C-34atmCO2 = aspen wood after hot water (190 °C) pretreatment 2016). This is probably contributed by the high cellulose retention in
in the presence of 34 atm CO2 gas. the pretreatment stage and the subsequent high hydrolysis yield when
using high-pressure CO2 hot water pretreatment.
Table 3
Langmuir adsorption parameters of Avicel and the pretreated aspen wood.
Substance Γmax[c] K[d] R[e] 3.5. Energy consumption and techno-economic analysis

(mg g−1) (mL mg−1) (L g−1) The ethanol production from waste aspen branch chips can be de-
Avicel 53.7 5.52 0.296
scribed by three major steps, namely: pretreatment of the raw aspen for
AW-190 °C [a] 19.3 0.64 0.012
AW-190 °C-34atmCO2 [b]
28.0 0.79 0.022 upgrading the feedstock, SHF of the feedstock for ethanol production,
AW-190 °C-68atmCO2 39.2 0.81 0.032 followed by distillation and dehydration to produce concentrated
ethanol. The major energy consumption was from the stages of pre-
Notes: [a] AW-190 °C = aspen wood after hot water (190 °C) pretreatment; treatment, distillation and dehydration (Piccolo and Bezzo, 2009).
[b]
AW-190 °C-34atmCO2 = aspen wood after hot water (190 °C) pretreatment Piccolo et al reported that the energy consumption in the pretreatment,
in the presence of 34 atm CO2 gas; [c–e] definition in Eqs. (1) and (2).
distillation and dehydration processes were approximately 4.7 and
3.5 MJ L–1 ethanol, respectively, which were estimated from the com-
that the cellulose surface accessibility for the enzymes was gradually
mercial SHF process with 80% hydrolysis yield and 92% ethanol yield
enhanced, which resulted in the higher hydrolysis yields (Fig. 2b). The
(Piccolo and Bezzo, 2009). For comparison, the energy consumptions of
increased CO2 pressure slightly enhanced the Langmuir constant (K)
SHF process for AW-190 °C and AW-190 °C-68atmCO2 samples were
value, indicating the improvement of affinity between enzymes and
calculated (Table 4). The calculations were conducted by considering
lignin (Lai et al., 2015). These results were perfectly matched with the
the hydrolysis and ethanol yields and assuming the same condition as
assumption from mercury intrusion porosimetry tests (Fig. 4a–b and
reported by Piccolo and Bezzo (2009). It was found that the major
Table 2), i.e., the higher porosity and larger pore area of the substance
energy consumption of SHF using 68 atm CO2 approach was 8.6 MJ L–1
would absorb more enzymes (Fig. 4c and Table 3).
ethanol, which was reduced by 35% compared that without adding CO2
It could be concluded that the addition of high-pressure CO2 is
(13.2 MJ L–1 ethanol) (Table 4).
beneficial to producing hydrolysis substance with desirable properties
Economic comparison of SHF process before and after introducing
from AW (Fig. 3). The acidic condition from the formed carbonic acid
68 atm CO2 was carried out by counting the material and energy costs
would greatly remove hemicellulose from the substance, and the high-
(Table 5). The consumption of enzyme (approximately 44%) was con-
pressure environment could create a larger pore area, benefiting to
sidered as a major cost (Khajeeram and Unrean, 2017). The price of the
adsorbing enzymes. The combined effects contributed to the enzymatic
enzyme was assumed to be 0.9 USD kg−1 (Pratto et al., 2020). Ac-
hydrolysis of the pretreated AWs (Fig. 2b).
cordingly, the material cost was estimated in terms of the use of en-
zymes in the hydrolysis stage. The energy used in this study was as-
3.4. Bioethanol production from hydrolyzed glucose sumed from the electricity consumed, which was estimated to be 80.8
USD per MWh (Longati et al., 2018). Besides, 10% of the cost was added
The yeast fermentation using Saccharomyces cerevisiae was con- to the total for representing the other expenditures (Pratto et al., 2020).
ducted (Ewanick et al., 2007), to evaluate the performance of the glu- As a result, the total cost of bioethanol from SHF with 68 atm CO2 was
cose-rich mixture from the enzymatic hydrolysis of AW-190 °C- estimated to be approximately 0.615 USD L–1 ethanol, which was
68atmCO2 (namely hydrolyzed glucose). The glucose and ethanol
curves showed similar trends for the glucose control and the hydrolyzed
glucose (Fig. 5), representing as good fermentability of the hydrolyzed
glucose for yeast. However, the glucose consumption time was a lightly
prolonged of the hydrolyzed glucose, i.e., 12 – 24 h, which was ap-
proximately 12 h for glucose control (Fig. 5). The ethanol production of
the hydrolyzed glucose was simultaneously delayed, but the yield (8.7 g
L–1) increased 10%, which was 7.9 g L–1 for glucose control. The initial
fermentation rate of the hydrolyzed glucose (3.1 and 1.0 g L–1 of glu-
cose consumption and ethanol production, respectively) was lower than
that of glucose control (4.3 and 1.4 g L–1) (Fig. 5), probably due to the
weak inhibitors in the hydrolyzed glucose solution. Wahlbom and
Hahn-Hägerdal (2002) reported that moderate weak inhibitors could
inhibit the initial fermentation but improve the final ethanol yield.
Another potential reason could be the availability of other carbon re-
sources in the hydrolyzed glucose which increased its final ethanol
yield through yeast fermentation, compared to the glucose control Fig. 5. Yeast fermentation results of the hydrolyzed glucose and glucose con-
(Zhang et al., 2018). trol, i.e., glucose consumption and ethanol concentration. Mean values of re-
A comparison of ethanol production in this work was made with plicates (n = 3) are reported with standard deviations given as error bars.

7
Y. Wu, et al.
Table 4
Comparison of energy consumption of bioethanol production from different approaches.
Pretreatment approach Hydrolysis yield Ethanol yield Pretreatment energy
(%) (%) consumption (MJ L–1 ethanol)
Piccolo and Bezzo (2009) 80 92 4.7
190 °C (this study) 53.8 84.8 7.6
190 °C with 68 atm CO2 82.9 84.8 4.9
(this study)
Table 5
Production cost (USD L–1 ethanol) estimation of SHF for bioethanol production.
Pretreatment approach Material Energy Other expenditures[a]
190 °C 0.698 0.297 0.099
190 °C with 68 atm CO2 0.367 0.192 0.056
Note: [a] Other expenditures represent the costs from minor energy use ex-
cluding pretreatment, distillation and dehydration, operation, etc., which was
assumed to be 10% of Material and Energy in the table (Piccolo and Bezzo,
2009).
reduced by 44% compared to that without CO2 (1.094 USD L–1 ethanol)
(Table 5). The cost of bioethanol production (0.615 USD L–1 ethanol)
was comparable with other reports, for instance, 0.623 USD L–1 ethanol
from sugarcane (Pratto et al., 2020), 0.634 USD m−1 ethanol from
sugarcane bagasse (Khajeeram and Unrean, 2017). Compared with the
ethanol price in United States, i.e., 0.618 USD L–1 at Dec 30th, 2019
(data from globalpetrolprices.com), the production cost (0.615 USD L–1
ethanol) estimated is yet to be competitive. It is envisaged that opti-
mization of the processes will break down the cost furtherly, for in-
stance, the production cost could bring down to 0.599 USD L–1 ethanol
if the ethanol yield raised from 84.8% to 92% in the fermentation stage.
4. Conclusions
High-pressure CO2 was successfully applied to the SHF process,
which significantly improved the glucose yield from enzymatic hydro-
lysis of aspen by 54% and the subsequent ethanol production from yeast
fermentation by 10%. The high-pressure CO2 generated acidic condi-
tion for reducing the hemicellulose content and simultaneously ex-
panded the pores in the treated substance, enhancing the enzyme ac-
cessibility to cellulose in the substance for improved SHF process. The
study of energy consumption and techno-economic analysis indicated
that the utilization of high-pressure CO2 could reduce the bioethanol
production cost. Further optimization was required to increase its
competence with the price of commercial ethanol.
CRediT authorship contribution statement
Yingji Wu: Conceptualization, Data curation, Funding acquisition,
Project administration, Resources, Supervision, Validation, Writing -
original draft. Shengbo Ge: Data curation, Formal analysis,
Investigation, Writing - original draft, Writing - review & editing.
Changlei Xia: Data curation, Formal analysis, Investigation,
Methodology, Writing - review & editing. Liping Cai:
Conceptualization, Writing - review & editing. Changtong Mei:
Conceptualization, Project administration, Supervision, Writing - re-
view & editing. Christian Sonne: Writing - review & editing. Young-
Kwon Park: Writing - review & editing. Young-Min Kim: Writing -
review & editing. Wei-Hsin Chen: Writing - review & editing. Jo-Shu
Chang: Writing - review & editing. Su Shiung Lam: Conceptualization,
Data curation, Funding acquisition, Project administration, Resources,
Supervision, Validation, Writing - review & editing.
Bioresource Technology 313 (2020) 123675
Distillation and dehydration energy Total energy consumption (MJ
consumption (MJ L–1 ethanol) L–1 ethanol)
3.5 8.2
5.6 13.2
3.7 8.6
Declaration of Competing Interest
Total The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
1.094 ence the work reported in this paper.
0.615
Acknowledgments
This authors acknowledge financial support from the National
Natural Science Foundation of China (31901372), the Natural Science
Foundation of Jiangsu Province (BK20190758), and the China
Postdoctoral Science Foundation (2019M661851). The authors also
thank to the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD), the Golden Goose Research
Grant (GGRG) (Vot 55191) by Universiti Malaysia Terengganu, and the
Analysis and Test Center of Nanjing Forestry University.
References
Abedinifar, S., Karimi, K., Khanahmadi, M., Taherzadeh, M.J., 2009. Ethanol production
by Mucor indicus and Rhizopus oryzae from rice straw by separate hydrolysis and
fermentation. Biomass Bioenergy 33 (5), 828–833.
Bansal, P., Vowell, B.J., Hall, M., Realff, M.J., Lee, J.H., Bommarius, A.S., 2012.
Elucidation of cellulose accessibility, hydrolysability and reactivity as the major
limitations in the enzymatic hydrolysis of cellulose. Bioresour. Technol. 107,
243–250.
Cordella, M., Torri, C., Adamiano, A., Fabbri, D., Barontini, F., Cozzani, V., 2012. Bio-oils
from biomass slow pyrolysis: a chemical and toxicological screening. J. Hazard.
Mater. 231, 26–35.
Ewanick, S.M., Bura, R., Saddler, J.N., 2007. Acid-catalyzed steam pretreatment of lod-
gepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol.
Biotechnol. Bioeng. 98 (4), 737–746.
Ewulonu, C.M., Liu, X., Wu, M., Yong, H., 2019. Lignin-containing cellulose nanoma-
terials: a promising new nanomaterial for numerous applications. J. Bioresour.
Bioprod. 4 (1), 3–10.
Garrote, G., Dominguez, H., Parajo, J., 1999. Hydrothermal processing of lignocellulosic
materials. Eur. J. Wood Wood Prod. 57 (3), 191–202.
Ge, S., Wu, Y., Peng, W., Xia, C., Mei, C., Cai, L., Shi, S.Q., Sonne, C., Lam, S.S., Tsang,
Y.F., 2020. High-pressure CO2 hydrothermal pretreatment of peanut shells for en-
zymatic hydrolysis conversion into glucose. Chem. Eng. J. 385, 123949.
Hamidinejad, M., Zhao, B., Zandieh, A., Moghimian, N., Filleter, T., Park, C.B., 2018.
Enhanced electrical and electromagnetic interference shielding properties of
polymer-graphene nanoplatelet composites fabricated via supercritical-fluid treat-
ment and physical foaming. ACS Appl. Mater. Interfaces 10 (36), 30752–30761.
Jagtap, S.S., Dhiman, S.S., Kim, T.-S., Li, J., Lee, J.-K., Kang, Y.C., 2013. Enzymatic hy-
drolysis of aspen biomass into fermentable sugars by using lignocellulases from
Armillaria gemina. Bioresour. Technol. 133, 307–314.
Jensen, J.R., Morinelly, J.E., Gossen, K.R., Brodeur-Campbell, M.J., Shonnard, D.R., 2010.
Effects of dilute acid pretreatment conditions on enzymatic hydrolysis monomer and
oligomer sugar yields for aspen, balsam, and switchgrass. Bioresour. Technol. 101
(7), 2317–2325.
Jutakridsada, P., Saengprachatanarug, K., Kasemsiri, P., Hiziroglu, S., Kamwilaisak, K.,
Chindaprasirt, P., 2019. Bioconversion of saccharum officinarum leaves for ethanol
production using separate hydrolysis and fermentation processes. Waste Biomass
Valorization 10 (4), 817–825.
Karimi, K., Shafiei, M., Kumar, R., 2013. Progress in physical and chemical pretreatment
of lignocellulosic biomass. In: Gupta, V., Tuohy, M. (Eds.), Biofuel Technologies.
Springer, Berlin, Heidelberg, pp. 53–96.
Kaushal, R., Sharma, N., Dogra, V., 2016. Molecular characterization of glycosyl hydro-
lases of Trichoderma harzianum WF5-A potential strain isolated from decaying wood
and their application in bioconversion of poplar wood to ethanol under separate
hydrolysis and fermentation. Biomass Bioenergy 85, 243–251.
Kesharwani, R., Sun, Z.Y., Dagli, C., Xiong, H.Y., 2019. Moving second generation biofuel
manufacturing forward: Investigating economic viability and environmental sus-
tainability considering two. strategies for supply chain restructuring. Appl. Energy
242, 1467–1496.
Khajeeram, S., Unrean, P., 2017. Techno-economic assessment of high-solid simultaneous
8
Y. Wu, et al.
saccharification and fermentation and economic impacts of yeast consortium and on-
site enzyme production technologies. Energy 122, 194–203.
Kuang, T.R., Chen, F., Chang, L.Q., Zhao, Y.N., Fu, D.J., Gong, X., Peng, X.F., 2017. Facile
preparation of open-cellular porous poly (L-lactic acid) scaffold by supercritical
carbon dioxide foaming. for potential tissue engineering applications. Chem. Eng. J.
307, 1017–1025.
Lai, C., Tu, M., Yong, Q., Yu, S., 2015. Disparate roles of solvent extractable lignin and
residual bulk lignin in enzymatic hydrolysis of pretreated sweetgum. RSC Adv. 5
(119), 97966–97974.
Liu, H., Zhu, J., 2010. Eliminating inhibition of enzymatic hydrolysis by lignosulfonate in
unwashed sulfite-pretreated aspen using metal salts. Bioresour. Technol. 101 (23),
9120–9127.
Longati, A.A., Lino, A.R.A., Giordano, R.C., Furlan, F.F., Cruz, A.J.G., 2018. Defining
research & development process targets through retro-technoeconomic analysis: the
sugarcane biorefinery case. Bioresour. Technol. 263, 1–9.
Maslova, O., Stepanov, N., Senko, O., Efremenko, E., 2019. Production of various organic
acids from different renewable sources by immobilized cells in the regimes of sepa-
rate hydrolysis and fermentation (SHF) and simultaneous saccharification and fer-
mentation (SFF). Bioresour. Technol. 272, 1–9.
Mittal, A., Scott, G.M., Amidon, T.E., Kiemle, D.J., Stipanovic, A.J., 2009. Quantitative
analysis of sugars in wood hydrolyzates with H-1 NMR during the autohydrolysis of
hardwoods. Bioresour. Technol. 100 (24), 6398–6406.
Morais, A.R., Mata, A.C., Bogel-Lukasik, R., 2014. Integrated conversion of agroindustrial
residue with high pressure CO 2 within the biorefinery concept. Green Chem. 16 (9),
4312–4322.
Ngamsirisomsakul, M., Reungsang, A., Liao, Q., Kongkeitkajorn, M.B., 2019. Enhanced
bio-ethanol production from Chlorella sp. biomass by hydrothermal pretreatment and
enzymatic hydrolysis. Renew. Energy 141, 482–492.
Park, C., Lee, J., 2020. Recent achievements in CO2-assisted and CO2-catalyzed biomass
conversion reactions. Green Chem. 22 (9), 2628–2642.
Peng, C., Crawshaw, J.P., Maitland, G.C., Trusler, J.M., Vega-Maza, D., 2013. The pH of
CO2-saturated water at temperatures between 308 K and 423 K at pressures up to 15
MPa. J. Supercritical Fluids 82, 129–137.
Piccolo, C., Bezzo, F., 2009. A techno-economic comparison between two technologies for
bioethanol production from lignocellulose. Biomass Bioenergy 33 (3), 478–491.
Pratto, B., Dos Santos-Rocha, M.S.R., Longati, A.A., de Sousa Junior, R., Cruz, A.J.G.,
2020. Experimental optimization and techno-economic analysis of bioethanol pro-
duction by simultaneous saccharification and fermentation process using sugarcane
straw. Bioresour. Technol. 297, 122494.
Pu, Y., Hu, F., Huang, F., Davison, B.H., Ragauskas, A.J., 2013. Assessing the molecular
structure basis for biomass recalcitrance during dilute acid and hydrothermal pre-
treatments. Biotechnol. Biofuels 6 (1), 15.
Ragauskas, A.J., Williams, C.K., Davison, B.H., Britovsek, G., Cairney, J., Eckert, C.A.,
Frederick, W.J., Hallett, J.P., Leak, D.J., Liotta, C.L., Mielenz, J.R., Murphy, R.,
Templer, R., Tschaplinski, T., 2006. The path forward for biofuels and biomaterials.
Science 311 (5760), 484–489.
Santa-Maria, M., Jeoh, T., 2010. Molecular-scale investigations of cellulose micro-
structure during enzymatic hydrolysis. Biomacromolecules 11 (8), 2000–2007.
Shafiei, M., Kumar, R., Karimi, K., 2015. Pretreatment of lignocellulosic biomass. In: K.
Karimi (Ed.), Lignocellulose-Based Bioproducts. Biofuel and Biorefinery
Technologies, vol. 1, Springer, Cham, pp. 85–154.
Sharma, H.K., Xu, C.B., Qin, W.S., 2019. Biological pretreatment of lignocellulosic bio-
mass for biofuels and bioproducts: an overview. Waste Biomass Valorization 10 (2),
235–251.
Bioresource Technology 313 (2020) 123675
Slathia, P.S., Raina, N., Kiran, A., Kour, R., Bhagat, D., Sharma, P., 2020. Dilute acid
pretreatment of pine needles of Pinus roxburghii by response surface methodology for
bioethanol production by separate hydrolysis and fermentation. Biomass Convers.
Biorefin. 10 (1), 95–106.
Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008.
Determination of structural carbohydrates and lignin in biomass. Lab. Anal.
Procedure 1617, 1–16.
Sun, S., Huang, Y., Sun, R., Tu, M., 2016. The strong association of condensed phenolic
moieties in isolated lignins with their inhibition of enzymatic hydrolysis. Green
Chem. 18 (15), 4276–4286.
Tian, D., Shen, F., Yang, G., Deng, S., Long, L., He, J., Zhang, J., Huang, C., Luo, L., 2019.
Liquid hot water extraction followed by mechanical extrusion as a chemical-free
pretreatment approach for cellulosic ethanol production from rigid hardwood. Fuel
252, 589–597.
Toor, M., Kumar, S.S., Malyan, S.K., Bishnoi, N.R., Mathimani, T., Rajendran, K.,
Pugazhendhi, A., 2020. An overview on bioethanol production from lignocellulosic
feedstocks. Chemosphere 242, 125080.
Tu, M., Pan, X., Saddler, J.N., 2009. Adsorption of cellulase on cellulolytic enzyme lignin
from lodgepole pine. J. Agric. Food. Chem. 57 (17), 7771–7778.
Wahlbom, C.F., Hahn-Hägerdal, B., 2002. Furfural, 5-hydroxymethyl furfural, and acetoin
act as external electron acceptors during anaerobic fermentation of xylose in re-
combinant Saccharomyces cerevisiae. Biotechnol. Bioeng. 78 (2), 172–178.
Wang, Z., Winestrand, S., Gillgren, T., Jönsson, L.J., 2018. Chemical and structural factors
influencing enzymatic saccharification of wood from aspen, birch and spruce.
Biomass Bioenergy 109, 125–134.
Weng, Y., Chang, S., Cai, W., Wang, C., 2019. Exploring the impacts of biofuel expansion
on land use change and food security based on a land explicit CGE model: a case
study of China. Appl. Energy 236, 514–525.
Xu, C., Wang, H., Li, X., Zhou, W., Wang, C., Wang, S., 2019. Explosion characteristics of a
pyrolysis biofuel derived from rice husk. J. Hazard. Mater. 369, 324–333.
Yang, J., Tu, M., Xia, C., Keller, B., Huang, Y., Sun, F.F., 2019a. Effect of fenton pre-
treatment on C1 and C6 oxidation of cellulose and its enzymatic hydrolyzability. ACS
Sustain. Chem. Eng. 7 (7), 7071–7079.
Yang, J., Xu, H., Jiang, J., Zhang, N., Xie, J., Wei, M., Zhao, J., 2019b. Production of
itaconic acid through microbiological fermentation of inexpensive materials. J.
Bioresour. Bioprod. 4 (3), 135–142.
Yang, T.C., Kumaran, J., Amartey, S., Maki, M., Li, X., Lu, F., Qin, W., 2014. Biofuels and
bioproducts produced through microbial conversion of biomass. In: Bioenergy
Research: Advances and Applications, Elsevier, pp. 71–93.
Zhang, K., Pei, Z.J., Wang, D.H., 2016. Organic solvent pretreatment of lignocellulosic
biomass for biofuels and biochemicals: a review. Bioresour. Technol. 199, 21–33.
Zhang, Y., Xia, C., Lu, M., Tu, M., 2018. Effect of overliming and activated carbon de-
toxification on inhibitors removal and butanol fermentation of poplar pre-
hydrolysates. Biotechnol. Biofuels 11.
Zhao, Y., Damgaard, A., Xu, Y.J., Liu, S., Christensen, T.H., 2019. Bioethanol from corn
stover - global warming footprint of alternative biotechnologies. Appl. Energy 247,
237–253.
Zheng, Y., Lin, H.M., Tsao, G.T., 1998. Pretreatment for cellulose hydrolysis by carbon
dioxide explosion. Biotechnol. Prog. 14 (6), 890–896.
Zhu, Z.-S., Li, X.-H., Zheng, Q.-M., Zhang, Z., Yu, Y., Wang, J.-F., Liang, S.-Z., Zhu, M.-J.,
2011. Bioconversion of a mixture of paper sludge and extraction liquor from water
prehydrolysis of eucalyptus chips to ethanol using separate hydrolysis and fermen-
tation. Bioresources 6 (4), 5012–5026.