Industrial Crops and Products 76 (2015) 638646

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Industrial Crops and Products

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Effect and characterization of sodium lignosulfonate on alkali

pretreatment for enhancing enzymatic saccharication of corn stover
Huanfei Xu a,b,1 , Guang Yu c,1 , Xindong Mu a , Chunyan Zhang d , Paul DeRoussel e , Chao Liu a
, Bin Li a, , Haisong Wang c,
a
CAS Key Laboratory of Bio-Based Material, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong
266101, Peoples Republic of China
b
College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, Peoples Republic of China
c
CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101,
Peoples Republic of China
d
Materials, Process and Delivery, Procter and Gamble Singapore Innovation Center, Procter and Gamble Co., 138648, Singapore
e
Procter and Gamble Manufacturing, Procter and Gamble Co., Mason, OH 45040, United States

a r t i c l e i n f o a b s t r a c t

Article history: A modied alkali pretreatment (MAP) process was established in both lab-scale and pilot-scale, based
Received 13 March 2015 on the on-line production and utilization of lignosulfonate for improving enzymatic saccharication of
Received in revised form 7 July 2015 corn stover. Sodium lignosulfonate (SLS) produced by directly sulfonating lignin in spent liquor separated
Accepted 26 July 2015
from alkali pretreatment was reused in the next cycle of alkali pretreatment. The results showed that, by
using 11 wt% NaOH, 2 wt% SLS and 0.1 wt% anthraquinone (AQ) in MAP, delignication rate and nal total
Keywords:
sugar yields could reach about 78% and 80% respectively, which were about 6% and 9% higher compared
Alkali pretreatment
to the blank conventional NaOH pretreatment under the same conditions. In addition, sulfonation degree
Sodium lignosulfonate (SLS)
Corn stover of SLS had linearly impact on the nal total sugar yields.
Lignin 2015 Elsevier B.V. All rights reserved.
Enzymatic saccharication

1. Introduction sugars by changing the submicroscopic structure and chemical

Nowadays, both from the viewpoint of energy and environment,
a great deal of effort has been making to reduce the dependence
of depleting fossil resources in many countries around the world,
and more and more attentions have been paid on the develop-
ment of biofuel production via the conversion of lignocellulosic
biomass (e.g. corn stover) (Agbor et al., 2011; Singh et al., 2014).
For lignocellulosic biomass conversion, enzymatic saccharication
is an environmentally friendly process to produce fermentable
sugars, but the efciency of enzymatic saccharication is highly
limited by the natural recalcitrance of lignocelluloses (e.g. the pres-
ence, content, structure and distribution of lignin, the content of
hemicelluloses and acetyl groups, the crystallinity and degree of
polymerization of cellulose, etc.) (Leu and Zhu, 2013). The role of
pretreatment is to signicantly ameliorate the enzyme accessibil-
ity for the subsequent hydrolysis of carbohydrates to fermentable
Corresponding author. Fax: +86 532 80662724.
Corresponding author.
E-mail addresses: libin@qibebt.ac.cn (B. Li), wanghs@qibebt.ac.cn (H. Wang).
1
These authors contributed equally to this work.
compositions of biomass (Ding et al., 2012). Thus, developing cost-
effective pretreatment is of vital importance for lignocellulosic
biomass conversion.
Pretreatment can be classied as physical pretreatment,
biological pretreatment, chemical pretreatment and combined pre-
treatment approaches (e.g. physicochemical pretreatment) (Agbor
et al., 2011). Each pretreatment process has advantages and dis-
advantages. For instance, there is no chemical added in liquid hot
water pretreatment, but the nal sugar yields is not high mainly
due to the re-adsorption of the extracted hemicelluloses on cellu-
lose and the large amount of remaining lignin (Kabel et al., 2007).
However, most recently, Ruiz et al. (2013) reported that the liquid
hot water pretreatment caused re-localization of lignin on the sur-
face of the lignocellulosic material, producing a good performance
in the enzymatic hydrolysis. This indicated that the structure and
distribution of lignin also had impact on enzyme accessibility to
substrates. Organosolv pretreatment can efciently dissolve lignin
and gain the unaltered and puried lignin, but the processing of
organic solvent is expensive (Ostovareh et al., 2015). Compared to
liquid hot water pretreatment and dilute acid pretreatment, alkali
pretreatment can efciently remove lignin by breaking the cross-
linking bonds between lignin and carbohydrates, achieve high nal

http://dx.doi.org/10.1016/j.indcrop.2015.07.057
0926-6690/ 2015 Elsevier B.V. All rights reserved.
 H. Xu et al. / Industrial Crops and Products 76 (2015) 638646
total sugar yields, and use the existing and mature pulping equip-
ment as well as the chemical recovery and waste water treatment
system well developed in pulping industry (Chen et al., 2013).
Thus, alkali pretreatment is considered as one of the promising pre-
treatment technologies. However, there were also some issues of
alkali pretreatment: (1) Typically, about 30% of hemicelluloses were
degraded and dissolved in spent liquor during conventional alkali
pretreatment, leading to some waste of fermentable sugars (Asghar
et al., 2015; Zhang et al., 2014a,b). (2) The efciency of enzymatic
saccharication achieved by conventional alkali pretreatment was
limited. For instance, after enzymatic hydrolysis by using cellulase
of 20 FPU/g-substrate, the nal total sugar yields of corn stover
pretreated by 10 wt% sodium hydroxide was only 61.37% (Xu et al.,
2014). (3) The spent liquor containing much alkali and lignin has
not been fully and efciently utilized. In paper mills, lignin as a
waste product usually goes to alkali recovery system to be burned to
generate power and electricity, as well as recover chemicals. Most
recently, the more efcient utilization of lignin have been recog-
nized as being critically important to the economic viability for the
integrated biorenery (Ragauskas et al., 2014).
To address these issues mentioned above, many efforts have
been made. Firstly, adding suitable reagents like anthraquinone
(AQ) during alkali pretreatment could stabilize carbohydrate and
promote lignin removal (Von Schenck et al., 2013), but AQ was
not soluble in water, which may cause inhomogeneous treat-
ment of substrates particularly for the cooking with short heat up
time and shorter cooking time (Hart and Rudie, 2014); Although
the active form of AQ is the anthrahydroquinone (AHQ) which
is highly soluble in water, the well penetration of AQ in the ini-
tial stage of pretreatment is still important. The surfactant with
high hydrophilicity could promote chemical penetration and dis-
solution of lignin during pretreatment (Cao and Aita, 2013), but
surfactant was usually expensive and can also bring some bubble
issues in pretreatment. Secondly, mechanical rening after chem-
ical pretreatment could usually be conducted to further improve
the enzyme accessibility by increasing the porosity of substrate and
reducing cellulose crystallinity (Xu et al., 2014; Jones et al., 2014).
Lastly, spent liquor reuse could be carried out to save alkali and
reduce water usage. Lignin derived from spent liquor of pulping
or pretreatment could be sulfonated to produce lignosulfonates.
Sodium lignosulfonate (SLS) could be used to enhance the enzy-
matic saccharication of lignocellulosic biomass because SLS as
a surfactant could block the nonspecic binding of cellulase by
bound lignin on the solid substrates (Wang et al., 2013). It was
also reported that the SLS with low molecular weight and high
sulfonation degree could enhance enzymatic cellulose sacchari-
cation (Lou et al., 2014), and the structure of lignin in substrate had
clear impact on the performance of SLS during enzymatic hydrolysis
(Zhou et al., 2013).
However, there was no report on the utilization of SLS products
in pretreatment. SLS as an inexpensive surfactant was renewable,
biodegradable and environmentally benign. It was expected that
the addition of SLS during pretreatment could also be benecial
for chemical penetration and lignin removal. By this way, part of
lignin (spent liquor) could be recovered and reused in pretreatment
system. To verify this hypothesis, in the present work the alkali
pretreatment was modied by adding AQ and SLS, to improve the
effectiveness of pretreatment. After the modied alkali pretreat-
ment (MAP), post mechanical rening was performed to further
increase the enzymatic digestibility of substrate. Firstly, the com-
mercial SLS products were used, and secondly, the SLS produced by
direct sulfonation of spent liquor separated from MAP was added
in the following pretreatment process and the spent liquor reuse
was investigated as well. At last, the MAP process was established
at pilot scale.
639
2. Materials and methods
2.1. Raw material and chemicals
Lignocellulosic biomass (corn stover) was collected from the
village of Qingdao, China. The corn stover was air dried and
screened to remove the fraction less than 0.25 mm, and then
the screened corn stover was stored in plastic bags for mois-
ture balance. The chemical compositions of raw corn stover
are given in Table 1. Enzymes including -glucosidase, cellu-
lase and xylanase were obtained from SigmaAldrich China Inc.,
and used as received. As measured according to the IUPAC stan-
dards (Ghose, 1987), the activities of the enzymes were 741 IU/mL
(-glucosidase, Novozyme 188), 52 FPU/mL (Cellulase, Celluclast
1.5 L), and 365 IU/g (Xylanase), respectively. Commercial sodium
lignosulphonate (CSLS) was obtained from Aladdin. All other chem-
icals were bought from Sinopharm Chemical Reagent Co. Ltd. and
used without any further purication.
2.2. MAP of corn stover
A cooking digester (PL1-00, Xianyang TEST Equipment Co., Ltd.,
Xianyang, China) was used for pretreatment experiments. The
cooking digester had four small cooking tubes. For each small tube
(size 1 L), 50 g corn stover (oven dried) was added for pretreat-
ment tests. The temperature was elevated from room temperature
to 120 C in 30 min (heat up time), and the heating duration was
40 min at 120 C. The cooking digester was rotated with the speed
of 1 rpm. After pretreatment, the small tubes were immediately
cooled down with tap water and the total cool down time was about
10 min. The samples were washed to neutral pH and stored at 4 C
for further test. For spent liquor reuse study, 50 mL spent liquor
separated after pretreatment was added in the next cycle of pre-
treatment using the same cooking conditions. With regards to the
scaling-up study, pretreatment was carried out in the same digester
(15 L) without the use of small tubes under the same conditions as
described above.
2.3. Rening
The pretreated and washed corn stover was rened in a lab-scale
PFI mill (PL11-00, Xianyang TEST Equipment Co., Ltd., Xianyang,
China) at 10 wt% rening consistency with the revolution number
of 4000 and the rening gap of 0.24 mm. The rening treatment of
pretreated corn stover at pilot scale was performed by the use of a
pressurized disc rener with throughput of 100 kg/h. The rening
conditions are listed in Table 2.
2.4. Enzymatic hydrolysis
At lab scale, the enzymatic hydrolysis of the pretreated and
rened samples was carried out at 2% (w/v) substrate consistency
in a 25 mL small bottle (50 C, pH 4.8, 48 h). The addition of the -
glucosidase and cellulase were 9 IU/g-glucan and 36 FPU/g-glucan,
respectively. The nal hydrolyzate was ltered and stored at 4 C
for further analyses. For the pilot scale study, the enzymatic hydrol-
ysis was conducted at the solid loading of 8-9% with the addition
of 36 FPU cellulase/g-glucan, 9 IU -glucosidase/g-glucan, and 9 U
xylanase/g-glucan (50 C, pH 4.8, 48 h).
2.5. Analytical of pretreatment and enzymatic hydrolysis
The compositions of corn stover were tested using the National
Renewable Energy Laboratory (NREL) procedure (Sluiter et al.,
2008). The high performance liquid chromatography (HPLC)
(Model 1200, Agilent, USA) was used to test the enzymatic
640 H. Xu et al. / Industrial Crops and Products 76 (2015) 638646

Table 1
Composition of the raw material and pretreated corn stover.

Sample Glucan (%) Xylan (%) Arabinan (%) ASLa (%) AILb (%) Extractivesc (%) Ash (%)

Raw corn stover 31.22 0.53 17.66 0.09 1.91 0.02 0.85 0.01 14.20 0.23 22.61 0.07 6.89 0.18
NaOH pretreated 57.11 0.47 25.33 0.13 2.15 0.03 0.74 0.02 6.89 0.37 ND ND
NaOH AQ pretreated 56.50 0.36 24.88 0.27 2.18 0.04 0.76 0.03 6.03 0.49 ND ND
NaOH CSLS pretreated 56.24 0.09 25.18 0.08 2.18 0.01 0.76 0.02 6.31 0.12 ND ND
NaOH AQ CSLS pretreated 56.24 0.01 25.11 0.06 2.19 0.07 0.69 0.01 5.25 0.36 ND ND
a
Acid soluble lignin.
b
Acid insoluble lignin.
c
Hot water soluble and ethanol soluble material. ND: not detected.

Table 2
Rening conditions and comparable results of PFI and disc rening.

Substrate Rotational speed, rpm Rening gap, mm Rening time, min Steam pressure, MPa Final total sugar yields, %a

Control (MAP treated) 72.91 2.96c

MAP + PFI reningb 1460 0.24 3 0 78.96 1.32c
MAP + disc reningb 2980 0.20 0.25 0.4 79.40 1.72c
a
Enzymatic hydrolysis conditions: 36 FPU cellulase/g-glucan, 9 IU -glucosidase/g-glucan, 9 U xylanase/g-glucan, 50 C for 48 h, three enzymes were added together in
the beginning of enzymatic hydrolysis. The solid loading of enzymatic hydrolysis at pilot scale was 8 9%.
b
The mechanical rening consistency was 10%.
c
At least three replicates for each test were conducted.

hydrolyzate. The column was a Bio-Rad Aminex HPX-87H column,
and the mobile phase was the 0.005 M sulfuric acid. The calibration
was performed with the standard sugar solutions. The effectiveness
of alkaline pretreatment and enzymatic hydrolysis was evaluated
by the variables as follows:
exchange method (Yu et al., 2013a,b). Briey, the solution of SLS
product passed through an anion exchange resin rst to remove
inorganic acid, and then passed through a cation exchange resin.
In this case, the SLS was converted into lignosulfonic acid. Sub-

Rsolid (%) = (Pretreatedbiomass(g)/Originalbiomass(g)) 100% (1)

Rglucan/xylan (%) = (Rsolid P glucan/xylanofpretreatedbiomass )/P glucan/xylanofrawbiomass 100%(2)

Dlignin (%) = 1(Rsolid P ligninofpretreatedbiomass )/P ligninofrawbiomass 100%(3)

E glucan (%) = (Dglucoseinhydrolyzate 0.9/Dglucaninpretreatedbiomass ) 100%(4)

E xylan (%) = (Dxyloseinhydrolyzate 0.88/Dxylaninpretreatedbiomass ) 100%(5)

Wherein, P was the percent of the corresponding component con-

tent (wt.%), and D was the oven dry weight of sugar (g), Rsolid
was the percentage of solid recovery, Rglucan/xylan was the recovery
rate of glucan or xylan, Dlignin was the delignication rate, Eglucan
was the enzymatic hydrolysis percentage of glucan, Exylan was the
enzymatic hydrolysis percentage of xylan. Final total sugar (glu-
can + xylan) yields were calculated as the percentage of the total
sugar in enzymatic hydrolyzate divided by the original total sugar
in the corresponding raw material. All tests including pretreatment,
enzymatic hydrolysis, and component analyses were carried out in
duplicate and the average plus standard deviation was reported.
The statistical analyses were performed by Microsoft Excel 2013.
2.6. Preparation of SLS
SLS products were prepared by direct sulfonation of spent liquor
separated from alkali pretreatment. The sulfonation reaction was
carried out at 95 C for a certain reaction time (varying from 3 h
to 6 h) to obtain different SLS products with different sulfonation
degree. The dosage of sodium sulte ranged from 10 wt% to 30 wt%
was based on the dry weight of lignin in spent liquor. The agitation
speed for sulfonation reaction was 800 rpm. Upon completion, the
prepared SLS solution was cooled and stored at 4 C for further
study.
2.7. Characterization
2.7.1. Sulfonation degree test of SLS
The sulfonation degree tests of SLS products were performed
following the previously reported procedure based on the ion
sequently, the obtained solution was titrated by 0.04 M NaOH
standard solution, and detected by a conductivity meter (DDS-307,
Jingke, Shanghai, China). The sulfonation degree of the SLS products
was calculated by the following formula,
Sulfonationdegree(mmol/g) = (N NaOH V NaOH )/DSLS (6)
Where NNaOH was the concentration of the standard NaOH solution
(M), VNaOH was the volume of standard NaOH solution (mL), and
DSLS was the dry weight of SLS product (g).
2.7.2. FTIR analysis
The FTIR spectra of the corn stover, lignin, SLS product were
determined on a FTIR spectrometer (Nicolet 6700, Thermo Fisher
Scientic Inc.). The samples were prepared by KBr pellet (the ratio of
the samples to the KBr was 1:100). The resolution of the spectra was
4 cm1 in the range of 400-4000 cm1 , and 32 scans were carried
out for each sample.
2.7.3. SEM analysis
The SEM analyses of the samples were conducted by a scanning
electron microscopy (S-4800, Hitachi, Japan). The freeze-dried sam-
ples were coated with platinum and treated with the accelerating
voltages from 3.0 kV to 8.0 kV.
2.7.4. XRD analysis
The freeze-dried corn stover samples were tested by X-ray
diffractometer (D8 ADVANCE, Bruker Co., Germany) to get the crys-
tallinity index. The scanning speed was 0.5 per min with the varied
 H. Xu et al. / Industrial Crops and Products 76 (2015) 638646
scattering angle (2) (from 10 to 80 ), and the Ni-ltered Cu K
radiation was generated at 80 mA and 40 kV. The crystallinity index
(CrI) of corn stover was calculated by the owing formula according
to the empirical method (Segal et al., 1959):
CrI = (I 002 -I am )/I 002 100%
Wherein, I002 was the maximum of the diffraction intensity
(the 0 0 2 plane), and Iam was the minimum diffraction intensity
(between 1 0 1 and 0 0 2 peaks).
3. Results and discussion
In this study, corn stover was broken into pieces by a cutting
machine and screened to obtain the fraction with the suitable par-
ticle size (0.25 mm). Then, the screened corn stover was treated
by MAP in a cooking reactor. After pretreatment, the spent liquor
and solid residue (pretreated corn stover) were separated. The pre-
treated corn stover was then washed by tap water and subjected
to mechanical rening treatment (PFI or disc rening). Subse-
quently, the rened corn stover went to enzymatic hydrolysis to
produce fermentable sugars. Meanwhile, part of spent liquor could
be directly reused for MAP, and part of spent liquor could be used
for the production of SLS via direct on-line sulfonation. The pro-
duced SLS was then applied in the next cycle of MAP process. When
the solid content of spent liquor was relatively high after multi-
reuse, the spent liquor could go to chemical recovery system to
recover alkali and generate electric power and steam. Yet, in the
present work, the chemical recovery, energy recovery and waste
water treatment were not included. To evaluate the effectiveness
of MAP, enzymatic hydrolysis with 2% solid loading was conducted
at lab-scale, while 8-9% solid loading for enzymatic hydrolysis was
adopted at pilot-scale.
3.1. Effect of MAP of corn stover by using commercial sodium
lignosulfonate
It has been known that surfactants contain both hydrophilic
and hydrophobic properties. They could promote the removal of
hydrophobic compounds by reducing the surface tension between
two liquid phases, and modify the surface and structure of biomass,
making it more hydrophilic (Qing et al., 2010). To investigate
whether SLS could be used in alkali pretreatment and improve
the pretreatment effectiveness, commercial sodium lignosulfonate
(CSLS) was rstly applied at lab-scale experiment. The impact of
CSLS loading on the nal total sugar yields after pretreatment and
enzymatic hydrolysis is shown in Fig. 1. As can be seen, for the
one without addition of AQ, the nal total sugar yields increased
with the increase of CSLS loading (from 0 to 6%, based on the dry
weight of raw corn stover), particularly for the samples with the
supplement of post rening (PFI rening). For instance, about 7%
improvement (p < 0.01) of nal total sugar yields could be achieved
when the CSLS loading increased from 0 to 4% (deep blue line in
Fig. 1). This was probably because CSLS as a surfactant could facil-
itate the removal of lignin in pretreatment. On the other hand, AQ
could stabilize carbohydrates (particularly for xylan) in alkaline
pretreatment (by oxidizing the reducing end groups to stable car-
boxyl groups) and break down lignin molecule (Sixta, 2006). Thus,
sugar recovery and lignin removal could be improved. Meanwhile,
the improved lignin removal could promote enzymatic hydrolysis.
Although more xylan retained after pretreatment was not bene-
cial for cellulose digestibility, post PFI rening could compensate
this, thus leading to the signicant improvement (p < 0.01) of nal
total sugar yields (green line in Fig. 1). PFI rening could enlarge the
benets obtained from CSLS due to the further modications of ber
(Xu et al., 2014). However, the nal total sugar yields decreased
when CSLS loading was over 2%. This was probably due to the
641
(7)
Fig. 1. Effect of CSLS dosage on the nal total sugar yields.
Pretreatment conditions: Alkali charge 11 wt%, 40 min maintenance at maximum
temperature of 120 C, liquid to solid ratio 6, AQ dosage 0.1 wt%; PFI rening: 4000
revolutions, beating gap 0.24 mm, rening consistency 10%.
fact that the improvement in biomass enzymatic digestibility by
mechanical rening was dependent upon the severity of chemical
pretreatment (Leu and Zhu, 2013; Zhang et al., 2014a,b). High CSLS
loading (>2%) might lead to severer chemical pretreatment, thus
lowering the effectiveness of PFI rening. Therefore, the suitable
CSLS loading was 2% based on the results obtained.
The impact of AQ loading on the nal total sugar yields was also
investigated, and the results showed that the suitable AQ loading
would be 0.1% (based on the dry weight of raw corn stover), which
should be enough to get a high nal total sugar yields (over 80%).
The addition of extremely small amounts of AQ resulted in both
an enhanced delignication and an improvement of yield (due to
carbohydrate stabilization). According to the highly simplied con-
cept, the quinone was initially reduced by the carbohydrates to the
hydroquinone, which in turn reduces lignin, whereby the quinone
was regenerated (Liu et al., 2013). Thus, AQ worked as a catalyst
in the MAP process. It was known that the reaction mechanism of
AQ in the pretreatment was the oxidation reduction reaction in the
quinone ring of AQ (Sixta, 2006). AQ oxidized the aldehyde end of
the carbonhydrates to the carboxyl group, which could prevent the
peeling action and degradation. In this process, AQ was reduced
to AHQ, which could be ionized into the ions of AHQ in alkaline
conditions. The AHQ ions could react with the methylene quinone
structure of the lignin macromolecules, which provided the elec-
tronic to lignin molecule and made the aryl ether bonds cleave, thus
leading to the degradation of the lignin macromolecules (Falk et al.,
1984; Hart and Rudie, 2014).
More detailed results regarding the effectiveness of MAP pre-
treatment (NaOH + AQ + CSLS) with post PFI rening can be seen
in Fig. 2. With the addition of 0.1% AQ, recovery rate of glucan
(Rglucan ), recovery rate of xylan (Rxylan ), and delignication rate
(Dlignin ) were improved by about 4% (p = 0.06), 3% (p = 0.11), and
2% (p = 0.37), respectively, and the corresponding solid recovery
(Rsolid ) and nal total sugar yields were increased by about 5%
and 6% (p < 0.05), respectively, compared with the control. For the
samples with the supplement of 0.1% AQ and 2% CSLS, the effective-
ness of pretreatment was further enhanced, and the improvement
of Rxylan , and Dlignin was about 5% (p < 0.05) and 6% (p < 0.01),
respectively, in comparison with the control. This was probably
because SLS could promote the penetration and distribution of AQ
in pretreatment (Hart and Rudie, 2014). It was reported that the
combination of AQ with penetrants could improve the transport of
642 H. Xu et al. / Industrial Crops and Products 76 (2015) 638646
Fig. 2. Effect of different pretreatment on enzymatic hydrolysis.
Pretreatment conditions: Alkali charge 11 wt%, AQ dosage 0.1 wt%, CSLS charge 2
wt%, 40 min maintenance at maximum temperature of 120 C, liquid to solid ratio
6; PFI rening: 4000 revolutions, rening gap 0.24 mm, rening consistency 10%. a:
the effectiveness of different alkali pretreatment; b: the effect of alkali pretreatment
on enzymatic hydrolysis.
AQ (Hart and Ransdell, 2008). After the post PFI rening, the nal
total sugar yields could reach about 80.5%, which was about 10%
higher (p < 0.01) compared to NaOH pretreatment under the same
conditions. Also, with the supplement of 0.1% AQ and 2% CSLS, the
alkali charge could decrease about 3%. In addition, based on our
study (effect of pretreatment temperature and cooking duration
on nal total sugar yields was not shown), 120 C for 40 min cook-
ing for MAP was sufcient to obtain a higher nal total sugar yields.
Higher temperature or longer cooking time may cause more degra-
dation of carbohydrates (especially for xylan), thus lowering the
nal total sugar yields. It has been reported that random hydrol-
ysis of carbohydrate take place when the temperature was higher
than 140 C in alkaline conditions (Sixta, 2006). Compared to hot
water pretreatment (150230 C) (Wang et al., 2015), dilute acid
pretreatment (150190 C) (Mafe et al., 2014) and SPORL method
(150180 C) (Zhang et al., 2014a,b), the lower temperature of MAP
could reduce the process cost of pretreatment.
The characterization of pretreated corn stover was carried out
by FTIR, XRD and SEM analyses. In the FTIR spectra of untreated and
Fig. 3. FTIR spectra of untreated and pretreated corn stover.
a: raw corn stover; b: the samples pretreated by NaOH pretreatment; c: the samples
pretreated by the modied alkaline pretreatment. Pretreatment was performed at
120 C for 40 min with the liquid to solid ratio of 6; the dosages of NaOH, AQ and
CSLS were 11 wt%, 0.1 wt%, and 2 wt%, respectively.
Fig. 4. XRD spectra of untreated and pretreated corn stover.
a: raw corn stover; b: the samples pretreated by NaOH pretreatment; c: the samples
pretreated by the MAP pretreatment. Pretreatment was performed at 120 C for
40 min with the liquid to solid ratio of 6; the dosages of NaOH, AQ and CSLS were
11 wt%, 0.1 wt%, and 2 wt%, respectively.
pretreated corn stover (Fig. 3), the broad absorption band located
from 3400 to 3500 cm1 was due to the stretching of OH groups
and the absorption at 2900 cm1 was assigned to CH2 groups (Li
et al., 2013). The decreased intensity of the peaks at 1740 cm1
(C O in hemicellulose), 1627 cm1 (C C in lignin), 1510 cm1 (C C
in lignin), and 795 cm1 (lignin), as well as the increased inten-
sity of the peaks at 1158 cm1 (C O C stretching in cellulose)
and 896 cm1 (-glucosides, in cellulose) indicated the removal
of lignin and some hemicelluloses after pretreatment (Mou et al.,
2014). Also, compared to raw corn stover the decrease of the peak at
465 cm1 (Si O) for the pretreated samples was due to the removal
of ash.
CrI was considered as a crucial factor which can inuence the
enzymatic saccharication of lignocellulosic biomass (Van Dyk and
Pletschke, 2012). The CrI of raw corn stover was 0.43, while the CrI
of pretreated corn stover was higher (Fig. 4). The increase of corn
 H. Xu et al. / Industrial Crops and Products 76 (2015) 638646
Fig. 5. Effect of sulfonation degree of SLS on nal total sugar yields.
Pretreatment conditions: Alkali charge 11 wt%, AQ charge 0.1 wt%, SLS/CSLS dosage
2 wt%, 40 min maintenance at maximum temperature of 120 C, liquid to solid ratio
was 5; PFI rening: 4000 revolutions, rening gap 0.24 mm, rening consistency
10%.
stover CrI after pretreatment was mainly because of the removal
of amorphous components (e.g. lignin and hemicelluloses) (Mou
et al., 2014), which was in consistence with FTIR results (Fig. 3).
The CrI of NaOH pretreated corn stover (0.51) was similar with
the CrI of MAP pretreated corn stover (0.52). This was probably
because despite the more lignin was removed by MAP, more car-
bohydrates (e.g. hemicelluloses) were retained compared to NaOH
pretreatment (Fig. 2a).
SEM analyses could provide detailed information with respect to
the morphology changes of biomass before and after pretreatment.
More compact and smooth surface could be observed from Fig. S1a
for raw corn stover. After NaOH pretreatment, the ber surface of
corn stover became coarser and looser due to the removal of lignin,
ash and some hemicelluloses (Fig. S1b). Much coarser and looser
surface of the corn stover could be obtained after MAP, and the
porosity was increased as well (Fig. S1c). This was the consequence
of the addition of AQ and CSLS, which promoted the lignin removal.
3.2. Effect of MAP by using the SLS produced from spent liquor
In order to check if the SLS products produced by lignin derived
from the spent liquor of MAP had the similar performance as CSLS,
more experiments were conducted. The preparation of SLS prod-
ucts and the determination of sulfonation degree were carried out
according to the procedure reported previously (Yu et al., 2013a,b).
The impact of sulfonation degree of different SLS products on
MAP effectiveness is shown in Fig. 5. It was seen that the nal
total sugar yields were linearly linked to the sulfonation degree
of SLS both for the unrened corn stover and the PFI rened corn
stover. This was probably due to the fact that the higher the sul-
fonation degree the higher hydrophilicity of SLS products could
be obtained. This was also in agreement with the previous nd-
ing that surfactant with high hydrophile-lipophile balance (HLB)
values were more effective to extract hydrophobic degradation
products from lignin (Kurakake et al., 1994). Fig. 5 also indicates
that the sulfonation degree of SLS products should be no less than
1.28 mmol/g to achieve a high nal total sugar yield based on the
results obtained, and the nal total sugar yield could reach about
80% when the SLS with sulfonation degree of 1.28 mmol/g was used
in MAP (with PFI rening). This result was comparable with that of
CSLS (Figs. 1 and 2), verifying that the SLS produced by direct sul-
643
Fig. 6. Final total sugar yield as the function of enzymatic hydrolysis time.
fonation of spent liquor separated from MAP could be reused in the
following MAP process.
The nal total sugar yield as the function of enzymatic hydrolysis
time is displayed in Fig. 6. As can be seen, the enzymatic hydrol-
ysis efciency was much higher in the rst 24 h compared to the
longer hydrolysis duration, and the nal total sugar yields for all
the samples were basically steady after 48 h hydrolysis. This was
mainly due to the feedback inhibition of high concentration of fer-
mentable sugars in hydrolysate (Jing et al., 2009). Similar results
were also reported previously (Yu et al., 2013a,b; Mou et al., 2014).
On the other hand, Fig. 6 shows that MAP could gain higher nal
total sugar in comparison with NaOH pretreatment, and PFI rening
could obviously enhance enzymatic saccharication. These results
were in agreement with the data presented in Figs. 1 and 2.
In the FTIR spectra of CSLS, spray-dried lignin products and SLS
prepared by the direct sulfonation of spent liquor (Fig. 7), the peaks
at 1218 cm1 and 1268 cm1 were associated with C O vibration
in guaiacyl ring, and the peak at 1315 cm1 was responsible for
C O vibration in syringyl ring, while the peaks between 1502 cm1
and 1600 cm1 were related to the aromatic skeletal vibration in
lignin structure (Liu et al., 2014). The clear peak at 11551245 cm1
in CSLS and SLS products linked to S O stretching, which proved
the existing of SO3 H groups in the products (Lambert et al., 1987).
Yet, there was no corresponding peak for the spray-dried lignin,
indicating that no sulfuric groups existed in the lignin separated
from the spent liquor of MAP.
3.3. Spend liquor reuse in the MAP process
The properties of spent liquor of MAP are given in Table 3, which
presents that the pH of spent liquor was similar when the liquid to
solid (L/S) ratio for pretreatment was 5 or 6, but the concentration
of total titratable alkali (TTA) and the solid content were higher
when the L/S ratio was 5. The spent liquor with low solid content
could be reused as cooking liquor. In this study, the investigation
of spent liquor reuse was conducted for the pretreatment with the
L/S ratio of 5.
The impact of spent liquor reuse on nal total sugar yields and
the properties of spent liquor after reuse are given in Table 4. The
nal total sugar yields were slightly decreased after the spent liquor
was reused once. Yet, only 75% nal total sugar yields could be
obtained after the spent liquor was reused twice. The decrease of
nal total sugar yields by reusing spent liquor was probably due
to the increase of solid content in spent liquor, resulting in the
644 H. Xu et al. / Industrial Crops and Products 76 (2015) 638646

Table 3
Properties of spent liquor of MAPa .

Chemical combination (liquid to solid ratio) pH value of spent liquor Total titratable alkali, (g/L) Solid content of spent liquor, (%)

NaOH (5) 11.98 0.02 1.81 0.05 9.56 0.04

NaOH+AQ+SLS5 (5) 11.12 0.09 1.84 0.08 9.62 0.06
NaOH (6) 11.87 0.05 1.01 0.09 8.63 0.23
NaOH+AQ+SLS5 (6) 11.11 0.07 0.77 0.11 8.86 0.07
a
Pretreatment conditions: alkali charge 11 wt%, AQ charge 0.1 wt%, SLS (1.30 mmol/g) dosage 2%, 30 min heating-up, 40 min maintenance at maximum temperature of
120 C; PFI rening: 4000 revolutions, rening gap 0.24 mm, rening consistency 10%.

Table 4
Properties of spent liquor and the effect of spent liquor reuse on nal total sugar yieldsa .

Chemical combination (reuse time of spent liquor) Properties of spent liquor Solid recovery after MAP, % Final total sugar yield, %

pH value Solid content, % TTA, % Without rening With rening

NaOH (0) 11.29 0.09 9.70 0.11 1.94 0.03 56.17 0.19 65.07 0.23 70.05 0.55
NaOH (1) 11.03 0.08 11.49 0.09 2.13 0.07 57.03 0.16 65.13 0.09 71.12 0.07
NaOH (2) 11.85 0.11 10.33 0.06 2.82 0.06 56.83 0.09 63.33 1.03 67.59 0.69
NaOH (3) 11.09 0.09 9.89 0.05 2.01 0.16 58.15 0.07 62.81 0.49 67.52 0.63
NaOH SLS AQ (0) 11.77 0.02 8.88 0.06 2.08 0.11 60.70 0.23 72.53 0.68 78.47 1.18
NaOH SLS AQ (1) 11.83 0.06 10.83 0.14 2.43 0.09 60.78 0.22 71.78 0.89 77.82 0.96
NaOH SLS AQ (2) 12.39 0.07 11.98 0.11 3.30 0.08 59.23 0.36 70.93 0.66 75.64 0.76
NaOH SLS AQ (3) 11.65 0.13 10.35 0.06 2.11 0.09 59.26 0.11 67.55 0.87 71.20 0.48
a
Pretreatment conditions: alkali charge 11 wt%, AQ charge 0.1 wt%, SLS/CSLS dosage 2 wt%, 40 min maintenance at maximum temperature of 120 C, liquid to solid ratio
was 5; PFI rening: 4000 revolutions, rening gap 0.24 mm, rening consistency 10%. The sulfonation degree of SLS was (1.30 mmol/g).

Therefore, the application of disc rener at pilot scale was more

Fig. 7. FTIR spectra of lignin, CSLS and SLS.
a: the CSLS; b: the lignin separated from the spent liquor of the modied alkaline
pretreatment via spray-dry method; c: SLS prepared by the lignin (the pink line) via
sulfonation modication. The sulfonation degree of SLS was 1.30 mmol/g.
less removal of lignin. Therefore, based on the results obtained, the
spent liquor of MAP pretreatment could only be reused twice to
maintain a relatively high nal total sugar yields. The reuse of spent
liquor separated from MAP process could reduce alkaline chemical
consumption and water usage, thus lowering the overall process
cost.
3.4. Pilot scale test and mass balance of MAP process
The scaling-up study of MAP process was carried out in our pilot
plant by using a disc rener. The conditions of PFI rening and
disc rening as well as the comparison of nal total sugar yields
are listed in Table 2. As can be seen, MAP plus disc rening and
MAP plus PFI rening could yield similar results. However, it was
reported that the energy consumption of PFI rener was typically
10 times higher than that of industrial disc rener (Kerekes, 2005).
cost-effective.
Fig. 8 shows the overall mass balance diagram of MAP process
based on the pilot test. 1 kg corn stover was crushed by a cutting
machine and then screened to obtain the fraction with the particle
size larger than 0.25 mm. The mass loss was 0.27% during crushing
and screening. Subsequently, the screened corn stover was sub-
jected to the cooking reactor with the addition of NaOH, SLS and
AQ. When the dosages of NaOH, SLS and AQ were 11 wt%, 2 wt% and
0.1 wt%, respectively, about 54.5% solid could be recovered after
MAP (120 C for 40 min with the L/S ratio of 5), separation of spent
liquor and washing. Then, the washed substrate went to disc rener
to be further modied. Ideally, there was no mass loss during ren-
ing. The throughput and pressure of the disc rener were 100 kg/h
and 0.4 MPa, respectively. After rening, the substrate was blown
out, and then directly went to enzymatic hydrolysis with the addi-
tion of cellulase, -glucosidase and xylanse. The small amount of
xylanse could promote the hydrolysis of xylan, thus further enhanc-
ing the hydrolysis of glucan (Leu and Zhu, 2013). The solid loading
of enzymatic hydrolysis at pilot test was 89%. Upon completion
of enzymatic hydrolysis, 306.1 g glucose and 122.4 g xylose could
be obtained. The corresponding nal total sugar yields were 79.4%
(based on the sugars in raw corn stover), which was about 9% higher
compared to the conventional NaOH pretreatment (Table 2). In
addition, part of spent liquor could be directly sulfonated to pro-
duce SLS and the prepared SLS could be used in the next cycle of
MAP. In this case, part of spent liquor could be reused and recy-
cled in the system of MAP process. It was noted that unlike other
surfactants, the use of SLS could not cause bubble issues during
pretreatment. The remaining spent liquor could be subjected to
chemical recovery system to recover chemicals and generate power
and steam. Another option would be that the spent liquor could
also be spray-dried to produce lignin products, which could be fur-
ther modied to produce other value-added products like aromatic
chemicals or phenol-formaldehyde resins (Doherty et al., 2011;
Rahimi et al., 2014).
Based on the above results and discussion, it could be sum-
marized that: (1) MAP process could be conducted at relatively
low temperature (120 C) and achieve a high nal total sugar yield
(80%); (2) On the basis of the on-line sulfonation and utilization
 H. Xu et al. / Industrial Crops and Products 76 (2015) 638646
Fig. 8. The overall mass balance diagram of the MAP process based on the pilot test.
of lignin, part of spent liquor can be more efciently recycled and
reused in pretreatment system; (3) MAP process could also be inte-
grated in the alkali-based pulp mills and use the mature chemical
recovery and waste water treatment system to further lower the
capital cost and bring more benets for the mills. Therefore, MAP
process could be considered as a promising technology, although
further optimization of the overall process was needed for cost
reduction (e.g. the optimization of enzymatic hydrolysis at high
solid loading).
4. Conclusions
Sodium lignosulfonate (SLS) was produced by direct sulfonation
of spent liquor of alkali pretreatment. The produced SLS could be
reused as a surfactant to promote chemical penetration and lignin
removal in the modied alkali pretreatment (MAP) process, thus
boosting the enzymatic saccharication of pretreated corn stover.
After NaOH pretreatment with 2% SLS and 0.1% AQ, about 78% of
645
lignin was removed and the nal total sugar yields could reach 80%.
Based on the on-line sulfonation and utilization of SLS, part of spent
liquor could be recycled and reused more efciently in MAP, which
could bring more economic and environmental benets.
Acknowledgements
The nancial support of this research was mainly from research
funding of Procter and Gamble Co. This work was also partially sup-
ported by the National Natural Science Foundation of China (No.
21206184, No. 31370582, No. 21306216, and No. 31470609), the
Qingdao Institute of Bioenergy and Bioprocess Technology Direc-
tor Innovation Foundation for Young Scientists (No. Y37205110C),
Shandong Provincial Natural Science Foundation for Distinguished
Young Scholar (China) (No. JQ201305), as well as the National High
Technology Research and Development Program (863 program)
of China (No. 2012AA022301).
646 H. Xu et al. / Industrial Crops and Products 76 (2015) 638646
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.indcrop.2015.07.
057
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