Bioresource Technology 233 (2017) 284–290

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

Mild alkaline presoaking and organosolv pretreatment of corn stover

and their impacts on corn stover composition, structure, and digestibility
Qing Qing, Linlin Zhou, Qi Guo, Xiaohang Gao, Yan Zhang, Yucai He, Yue Zhang ⇑
Department of Biochemical Engineering, College of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou 213164, Jiangsu, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Mild alkaline presoaking and

organosolv pretreatment of CS were
evaluated.

The maximum total sugar yield
achieved was 98.6% with a moderate
enzyme loading.

Large amounts of lignin and
hemicelluloses were removed after
pretreatment.

The SSA and PV significantly enlarged
after pretreatment.

The CrI of the pretreated residue
increased but the cellulose was highly
digestible.

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

Article history: An efficient strategy was developed in current work for biochemical conversion of carbohydrates of corn
Received 11 January 2017 stover into monosaccharides. Corn stover was first presoaked in mild alkaline solution (1% Na2S) under
Received in revised form 10 February 2017 40 °C for 4 h, after which about 35.3% of the lignin was successfully removed while the specific surface
Accepted 23 February 2017
area was notably enlarged. Then the presoaked solids were subjected to organosolv pretreatment that
Available online 27 February 2017
employed 20% methanol with an addition of 0.2% HCl as catalyst at 160 °C for 20 min, and the maximum
total sugar yield of the pretreated corn stover achieved was 98.6%. The intact structure of corn stover was
Keywords:
disrupted by this two-step process, which resulted in a porous but crystalline structure of the regener-
Alkaline presoaking
Organosolv pretreatment
ated solids that were mainly composed of cellulose. The enlarged specific surface area and increased
Corn stover accessibility made the regenerated solids highly digestible by a moderate enzyme loading.
Saccharification Ó 2017 Elsevier Ltd. All rights reserved.
Digestibility

1. Introduction with this process is significantly greater than the first generation
biofuels due to the complex association of cellulose-lignin-
Production of biofuels and value-added bio-based chemicals hemicelluloses in native lignocellulosic biomass, making it difficult
from lignocellulosic biomass has become a substitute in pursuit to break down and offering limited accessibility to enzymes and
of renewable surrogate to fossil sources. Lignocellulosic biomass microorganisms (Auxenfans et al., 2014; Perez-Pimienta et al.,
is an abundant, low-cost, and promising source that could be con- 2013). In this context, an adequate pretreatment step is essential
verted to fermentable sugars through biochemical or thermochem- to mitigate the complexity of biomass structure and to facilitate
ical process (Kim et al., 2015). However, the challenge associated the accessibility of enzymes to cellulose surface (Kumar and
Wyman, 2009; Toquero and Bolado, 2014). Existing efforts are
devoted to development of a technically and economically feasible
⇑ Corresponding author.
pretreatment method that fulfill this objective, and a variety of
E-mail address: [email protected] (Y. Zhang).

http://dx.doi.org/10.1016/j.biortech.2017.02.106
0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
 Q. Qing et al. / Bioresource Technology 233 (2017) 284–290 285

chemical, physical, physicochemical, and biological pretreatment nauer–Emmett–Teller method (BET), Scanning electron micro-
methods were therefore established and intensively studied. Each scopy (SEM), Fourier transform infrared spectroscopy (FTIR), and
method has different impacts on the composition and structure X-ray diffraction (XRD) in order to elucidate the impacts of differ-
characteristics of the lignocellulosic material, and possesses perva- ent pretreatment processes on composition, structure, and surface
sive impacts on all the other steps of the biomass conversion pro- characteristics of corn stover.
cess, in terms of enzymatic hydrolysis, fermentation, product
separation, and waste water treatment (Yang and Wyman, 2008).
Among existing pretreatment techniques, alkaline pretreat- 2. Materials and methods
ments under high severity conditions are more effective for lignin
and hemicelluloses removal, while minor cellulose solubilization 2.1. Materials
occurs concomitantly. In comparison, alkaline presoaking pro-
cesses, such as aqueous ammonia or dilute NaOH presoaking, are Corn stover (CS) was obtained from nearby farm in the city of
usually applied under milder pretreatment conditions, thus pre- Changzhou, Jiangsu, China. After CS was milled to a size smaller
serve larger portion of carbohydrate, especially hemicelluloses than 3 mm, it was washed with deionized water and dried at
(Gupta and Lee, 2010). However, these alkaline presoaking pre- 85 °C until constant weight. The dry CS was stored in sealed plastic
treatments have been proven to be only effective in treatment of sample bags at 20 °C for future use. Accellerase 1500 (96 FPU/mL)
low lignin content feedstock and require high recovery cost. was generously provided by Genencor (Wuxi, Jiangsu Province,
Recently, alkalic salts has been employed to increase the efficiency China), while Novozyme 188 (066K0676603 ± 5 CBU/mL,
of alkaline pretreatments (Kim et al., 2014), with the advantages of 68 ± 5 mg protein/mL) was purchased from Sigma (St. Louis, MO,
less corrosion and recyclable. Compared to other pretreatment USA) for b-glucosidase supplementation. Sodium sulfide (Na2S)
methods, alkali salt pretreatment shows obvious saponification (purity  98.0%) and methanol (purity  99.5%) were purchased
of uronic bonds which link lignin to hemicelluloses (Reilly et al., from Changzhou Runyou Commercial and Trading Co. Ltd.
2015), partial removal of hemicelluloses (including acetyl groups (Changzhou, China). All other chemicals used were from commer-
and uronic acids), and swelling of cellulose structure, resulting in cial source and of reagent grade.
a substantial increase of the fiber surface area (Asgher et al.,
2013; Carvalho et al., 2016; Mendes et al., 2015). However, an 2.2. Pretreatment
effective alkalic salt pretreatment usually requires a pretreatment
temperature higher than 100 °C, besides a high alkali concentra- 2.2.1. Presoaking of CS by Na2S solution
tion (4%) and a corrosion-resistant reactor (Geng et al., 2014; The CS was presoaked in Na2S solution under different condi-
Gu et al., 2012; Kim et al., 2014). Apply of alkali salts in presoaking tions that optimized by orthogonal experimental design. The
process under mild conditions has yet been reported. impacts of crucial variables including Na2S concentration of 0.5–
Organosolv pretreatment is another promising technology that 1% (w/w), reaction temperature of 30–50 °C, presoaking residence
has attracted considerable attention in past decades (Obama et al., time of 3–5 h, and solid-to-liquid ratio of 1:10 to 1:20 (g:L) were
2012; Zhang et al., 2016a,b). Compared with other pretreatments, investigated. After presoaking, the CS solid was filtrated and
organosolv pretreatment has some particular advantages: (1) the washed several times by distilled water until neutral. The collected
high-quality lignin isolated from organosolv pretreatment can be solids were used for organosolv pretreatment in the second step or
used for the synthesis of several co-products (Hage et al., 2011; subjected to enzymatic hydrolysis and other analyses.
Pan et al., 2006) and (2) the organic solvents are easy to recover
by distillation and can be recycled (Zhang et al., 2016a). Most of
2.2.2. Organosolv pretreatment of CS
the organic solvents reported are bulk commodity chemicals, such
The presoaked solid residue from the first step was then pre-
as ethanol, methanol, acetone, glycerol, or solvents that used for
treated by organic solvent with addition of extremely dilute acid.
biphasic fraction systems, such as methyl isobutyl ketone (MIBK)
The impacts of different types of organic solvent, such as methanol,
and tetrahydrofuran (THF). These solvents are cost effective com-
ethanol, ethylene glycol, glycerol, acetone, and toluene were eval-
pared to other cellulose dissolving solvents, such as ionic liquids.
uated in this study. Dilute acid, including hydrochloric acid, sulfu-
However, the non-catalyst organosolv pretreatment process usu-
ric acid, phosphoric acid, acetic acid, oxalic acid, maleic acid, citric
ally requires high reaction temperature (160–220 °C) and high
acid, and formic acid were also screened. The organosolv pretreat-
organic solvent concentration (60%) (Chen et al., 2015; Novo
ment was conducted in a high-temperature and high-pressure
et al., 2011) in order to achieve self-acidification by the cleavage
stainless steel reactor with a total volume of 100 mL (Zhenjiang
of a-aryl ether and arylglycerol-b-aryl ether bonds (Zhang et al.,
Dantu Universal Electrical Equipment, China). The influences of
2016a). Using of dilute acid as catalyst can reduce the high reaction
operating parameters, such as organic solvent concentration
temperature which was considered as a necessity for the organo-
(0–50 w/v%), acid concentration (0–0.25 w/v%), pretreatment tem-
solv pretreatment, and facilitates the decomposition of hemicellu-
perature (120–170 °C), and residence time (10–60 min) were eval-
lose that leads to efficient enzymatic hydrolysis (Sookyeong et al.,
uated. After pretreatment, the reactor was quenched in an iced
2016).
water bath and then the solid residue was separated and washed
In order to achieve a high sugar recovery from lignocelluloses
until neutral.
and reduce the formation of inhibitory compounds, lots of pre-
treatment methods that combine two-stage pretreatment have
been explored (Liu et al., 2016; Min et al., 2015). In present work, 2.3. Compositional analysis
dilute alkali salt solution was chose to presoak the lignocellulosic
biomass before organosolv pretreatment to disrupt the cross- The chemical compositions of CS were determined using a two-
linked bonds between lignin and carbohydrates, partially remove step acid hydrolysis of standard protocol by National Renewable
lignin, and to make cellulose swollen. Afterwards, methanol pre- Energy Laboratory (Sluiter et al., 2011). Monomer sugars were
treatment with the addition of very dilute hydrochloride acid quantified by HPLC (high performance liquid chromatography)
was applied to the presoaked solid residue to dissolve lignin, method as described below. The carbohydrates and lignin contents
hydrolyze hemicelluloses, and make the cellulose residue highly in raw corn stover determined were 37.1% of cellulose, 20.9% of
digestible. The pretreated solid residues were characterized by Bru hemicellulose, 13.5% of lignin, and 1.3% of ash.
286 Q. Qing et al. / Bioresource Technology 233 (2017) 284–290

2.4. Enzymatic hydrolysis 3. Results and discussion

Tested CS samples were mixed with acetate buffer (pH 4.8, 3.1. 1Impacts of Na2S presoaking conditions on solid recovery and
0.05 M) containing 40 lL tetracycline hydrochloride (10 mg/mL) digestibility
in a 50 mL Erlenmeyer flask and the total solid loading was con-
trolled at 1% (w/v) according to the NREL Laboratory Analytical An orthogonal experiment was designed to investigate the
Procedure (Selig et al., 2008). Cellulase (30 FPU/g substrate) and impacts of Na2S presoaking (step 1) conditions on the solid recov-
b-glucosidase were added into the mixture. The flask was incu- ery and enzymatic digestibility of the recovered solids. Compara-
bated at 50 °C and 160 rpm for 72 h in a shaking incubator. All tively low presoaking temperatures from 30 to 50 °C were
samples were run in triplicates and an average value of the parallel evaluated with a moderate sodium sulfide loading of 0.5–1% under
experiments was shown. three different solid to liquid ratios. The orthogonal experiment
results are listed and compared in Table 1. Compared with raw
2.5. Sugar analysis corn stover (Entry 1), the total sugar yields and glucose yields of
the presoaked corn stover were obviously changed and most of
The concentrations of monomer sugars in the liquid samples the tested entries showed 10–20% of increment. The R values for
were analyzed by an HPLC. Liquid samples together with calibra- each of the factors were then calculated based on the total sugar
tion sugar standards were run on a Waters Alliance HPLC system yields of each factors and levels. It can be found that the influences
(Model 2695, Waters Corporation, Milford, MA) employing an Ami- of these three factors on the total sugar yield were in the order of
nex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) and a A > D > B > C according to the R values. Obviously, the concentra-
refractive index detector (Waters 2414). The glucose and total tion of Na2S plays the most important role to the pretreatment per-
sugar yields were calculated as following: formance in terms of the total sugar yield, which brought the
maximum R value of 9.94. Based on the experiment result analysis,
a Glucose released  0:9 the optimum Na2S presoaking conditions determined are 1% Na2S
Glucose yield ð%Þ ¼  100%
CS dry weight presoaking at 40 °C for 4 h with a solid to liquid ratio of 1:20, under
which conditions, the maximum total sugar and glucose yields
a C6 sugar released  0:9 þ C5 sugar released  0:88 were 42.54% and 24.80%, respectively. In addition, based on the
Total sugar yield ð%Þ ¼ compositional analysis of the Na2S presoaked solids, a considerable
CS dry weight
 100% part of lignin (35.31%) had been removed from the solid residue,
while there was just 22.05% of the hemicelluloses removal simul-
taneously (Table 2). In addition, the reusability of the spent Na2S
b C6 sugar released  0:9 þ C5 sugar released  0:88
Total sugar yield ð%Þ ¼ solution was investigated in this study (Table S1). The recycled
Original carbohydrates in CS
Na2S liquor was reused without further processing for 5 times.
 100%
The results indicated a stable reusability and recyclability of this
pretreatment liquor.
a
Based on the regenerated biomass weight.
b
Based on the total sugar weight in regenerated biomass.
3.2. Impacts of different organosolv systems on pretreatment of CS

2.6. Characterization of CS
To further improve the digestibility of CS, various organic sol-
vents with the addition of very dilute organic or mineral acid as
The surface morphological features of CS before and after pre-
catalysts were firstly applied to the presoaked solid residue (step
treatment were recorded by a scanning electron microscope
2) and their impacts on CS digestibility were evaluated as shown
(SEM, Model # JSM-6360LA, JEOL, Japan) operated at 15 kV. Fourier
in Fig. 1. Compared with the Na2S presoaked CS (Entry 11 in Table 1,
transform infrared spectroscopy (FTIR) was performed using a
designed as control sample), the total sugar and glucose yields of
Nicolet PROTÉGÉ 460 FT-IR Spectrometer (Nicolet, USA). The spec-
the organosolv pretreated solids remarkably enhanced. For metha-
tra were recorded between 4000 and 500 cm1. Samples were
nol, ethanol, and acetone, the digestibility of the pretreated CS
ground and mixed with the spectroscopic grade KBr then pressed
increased 39.8%, 29.5%, and 28.3%, respectively, compared to the
in a standard device to produce diameter pellets. Specific surface
control sample. The comparatively smaller molecule size of metha-
area (SSA) was calculated using the Brunauer–Emmett–Teller
nol, which is important to the interactions between solvent and
(BET) method on ASAP 2020M instrument from Micrometritics
substrate in pretreatment (Zhang et al., 2016b), might be the rea-
which was equipped with N2 adsorption/desorption isotherms.
son that led to a higher digestibility of the methanol pretreated
The total volume of pores (PV) was measured by single point
CS. In addition, methanol pretreatment was successfully applied
adsorption with P/P0 at 0.950. The pore size (PS) was estimated
in the field of pulping in order to produce high quality cellulose
by adsorption average pore diameter (4V/A by BET) (Qing et al.,
fiber, whereas only limited data was found for methanol pretreat-
2016). Powder X-ray diffractometry (PXRD) was used to examine
ment for hydrolysis and fermentation. Therefore, even though con-
changes of the crystallinity of different samples. PXRD spectra
sidering the toxicity and inflammability, methanol was chosen as
were recorded by using a D/max 2500 PC diffractometer with Cu
the pretreatment solvent in the organosolv system discussed in
Ka radiation (Rigaku Corporation, Tokyo, Japan). It was operated
this study.
at a voltage of 60 kV and a current of 300 mA. The 2h range was
In addition, exogenous organic or inorganic acid was added to
from 5° to 40° in steps of 0.02°. Crystallinity index (CrI) was calcu-
promote the delignification process and hydrolysis of hemicellu-
lated as following:
loses, as a supplementation to the acetic acids from acetyl groups
I002  Iam released from hemicelluloses hydrolysis. The impacts of different
CrIð%Þ ¼  100% dilute acid types on the acid-catalyzed organosolv pretreatment
I002
performances were illustrated in Fig. 1b. Among the eight different
where I002 is the intensity of the peak at near 21.1°, and Iam is the organic and inorganic acids evaluated, the CS by the hydrochlo-
intensity of the peak at near 15.7°. ric acid catalyzed pretreatment exhibited a better enzymatic
 Q. Qing et al. / Bioresource Technology 233 (2017) 284–290 287

Table 1
Solid recovery and digestibility of CS presoaked by sodium sulfide under different conditions.

Entry Na2S % Temperature °C Time h Solid:Liquid w:v Step 1

A B C D Solid recovery% Total sugara % Glucosea %
1 0 0 0 0 100 20.98 12.60
2 0.5 30 3 1:10 67.33 19.69 13.19
3 0.5 40 4 1:15 65.33 34.53 19.49
4 0.5 50 5 1:20 62.67 29.31 17.09
5 0.75 30 4 1:20 63.67 36.69 20.46
6 0.75 40 5 1:10 65.67 35.49 19.61
7 0.75 50 3 1:15 62.67 36.26 21.19
8 1 30 5 1:15 63.00 38.55 22.37
9 1 40 3 1:20 60.67 40.68 23.61
10 1 50 4 1:10 63.33 34.12 20.65
11 (Selected) 1 40 4 1:15 61.50 42.54 24.80
a
Total sugar yields and glucose yields were calculated based on the regenerated biomass weight.

Table 2
Impacts of different pretreatment strategies on CS composition, crystallinity, SSA, and digestibility.

Samples Cellulose Hemicellulose Lignin RS CrI CrI/ SSA PVb PSb HRb DLb TSa,b
% % % % % Cellulose m2/g cm3/g nm % % %
Untreated-CS 37.11 20.90 13.51 100 37.6 1.01 0.032 0.0009 – – – 35.89
Step 1 45.96 26.49 14.20 61.5 41.7 0.92 2.941 0.0016 2.1999 22.05 35.31 58.71
Step2 65.25 7.40 20.52 42.0 40.3 0.61 6.605 0.0108 6.5350 85.16 36.22 67.15
Step 1–Step 2 75.06 7.68 15.41 47.0 48.8 0.65 6.228 0.0080 5.1365 89.40 67.03 98.66
a
Total sugar yields were calculated based on the total carbohydrate in CS.
b
PV-Pore volume, PS-Pore size, HR-Hemicellulose removal, DL-Delignification, TS-Total sugar.

hydrolysis performance than the other seven groups. The total

a Toluene Glucose sugar and glucose yields of the methanol-HCl pretreated CS
increased 16.8% and 38.8%, respectively, compared with the control
Total sugar
sample which was presoaked CS pretreated by methanol only.
Acetone
Furthermore, all the three inorganic acids showed better catalytic
performances than the five organic acids screened in this study.
Glycerol
The total sugar yields of all the five organic acids had no substantial
enhancement compared to the methanol only pretreated CS. How-
Ethylene glycol ever, the glucose yields of these samples were promoted, indicat-
ing an increased cellobiose to glucose conversion by the acidified
Ethanol pretreatment solvents.

Methanol
3.3. Optimization of organosolv pretreatment conditions
0 20 40 60 80
The effects of methanol concentration, hydrochloric acid con-
Relative increment,% centration, pretreatment temperature, and pretreatment duration
on the organosolv pretreatment were elucidated in Figs. 2 and 3.
b Formic acid Glucose The total sugar and glucose yields obtained in the enzymatic
hydrolysis process were used as the indices to determine the opti-
Citric acid Total sugar
mum conditions. Impacts of various methanol concentrations were
determined at 140 °C with addition of 0.1% HCl and results were
Maleic acid
shown as relative changes compared to the CS pretreated under
Oxalic acid identical conditions without methanol (Fig. 2a). The addition of
20% methanol was found to be the most suitable dosage compared
Acetic acid
to other concentrations. The total sugar and glucose yields
Phosphoric acid increased 11.4% and 8.7%, respectively, compared to the methanol
blank reference. The delignification effect showed a corresponding
Sulfuric acid maximum value of 20.5% under this methanol addition dosage.
Hydrochloric acid With the increase of methanol concentration beyond 20%, the total
sugar and glucose yields obviously decreased. In addition, the
0 10 20 30 40 50 delignification also has a certain relationship with the methanol
Relative increment,% concentration, which increased first and then leveled off after
20% methanol addition.
Fig. 1. Effects of different organic solvents (a) and acid catalysts (b) on organosolv The organosolv pretreatment could be also significantly affected
pretreatment. Pretreatment condition: CS pretreated by 20 w/w% organic solvents
with 0.1% acid catalyst at 140 °C for 40 min. Values were calculated as relative
by acid catalyst concentration, due to the role of hydrolytic agent
increment compared to the control sample (a: CS from step 1; b:CS pretreated that interrupts the linkages in the lignin-carbohydrate complex
without acid catalyst). plays by acid catalyst. Evident enhancements of the total sugar
288 Q. Qing et al. / Bioresource Technology 233 (2017) 284–290

a 15 Total sugar Glucose

60
Relative percentage content,%

Lignin Hemicellulose 50
12

40

Removal, %
9
30
6
20

3
10

0 0
10 20 30 40 50
Methanol concentration, w/v

b 120 Total sugar Glucose

120
Relative percentage content,%

Lignin Hemicellulose
100 100
Removeal, %
80 80

60 60

40 40

20 20

0 0
0.05 0.10 0.15 0.20 0.25
HCl consentration,w/v Fig. 3. Impacts of pretreatment temperature and duration. (a) CS pretreated by
organic solvent system in the temperatures range of 120–170 °C for 40 min; (b) CS
Fig. 2. Impacts of methanol and HCl concentrations on pretreatment. (a) CS
pretreated by organic solvent system at 160 °C for different time (10–60 min).
pretreated at 140 °C for 40 min with different concentrations of methanol and 0.1%
HCl. (b) CS pretreated at 140 °C for 40 min with different concentrations of HCl and
20% methanol. Total sugar and glucose yields were shown as a relative change
compared to the CS pretreated under identical conditions without methanol (a) or 0.2% HCl were determined as the optimized organosolv pretreat-
without HCl (b). ment conditions.

yields, glucose yields and lignin and hemicelluloses removals were 3.4. Impacts of Na2S presoaking and organosolv pretreatment on
observed when the HCl concentration was increased from 0.05% to enzymatic digestibility
0.20%, while no further increase occurred from 0.20% to 0.25%
(Fig. 2b). The total sugar and glucose yields increased 47.8% and To comprehensively evaluate the impacts of step 1 and step 2
108.5%, respectively, with the addition of 0.2% HCl acid as catalyst pretreatments on the digestibility of the regenerated cellulose,
compared to non-catalyzed organosolv pretreatment. The hemicel- the enzymatic hydrolysis performances of different samples were
luloses removal showed a similar pattern as the sugar yields, indi- compared as shown in Fig. 4. The total sugar yields of Step1-
cating a strong hemicellulose decomposition effect by the Step2-CS were improved significantly compared to the UN-CS or
exogenous acid catalyst. CS pretreated by others strategies, which showed a maximum
Furthermore, impacts of the pretreatment temperature (120– sugar yield of 98.6% at 72 h with an enzyme dosage of 30 FPU/g
170 °C) and duration (10–60 min) involved in the organic solvent substrate. The total sugar yields for Step1-0.2% HCl-CS and Step1-
system were also investigated (Fig. 3). The pretreated CS exhibited 20% Methanol-CS were 83.5% and 81.1%, respectively, which were
a gradually increased digestibility in the temperature range of 45–47% higher than the UN-CS. In comparison, the digestibility of
120–160 °C, and the total sugar yield slightly decreased at pre- CS pretreated by only one step (step 1 or step 2) was considerably
treatment temperature higher than 170 °C, primarily due to the lower, which could be due to the limited removal of hemicelluloses
further degradation of carbohydrates to furfural or 5-HMF. In addi- and lignin. Considering the composition changes shown in Table 2,
tion, both of the hemicelluloses and lignin removal increased with these results also indicated that lignin removal may have a more
the raise of temperature, suggesting a promoted bond cleavage in profound impact on cellulose digestibility than hemicelluloses
the carbohydrate-lignin complex at higher reaction temperature. removal. The enhanced enzymatic digestibility of the Step1-
In comparison, the pretreatment duration had a minor impact on Step2-CS was tallied to the compositional change implied in
both of the CS digestibility and composition change as reflected Table 2, which indicates a large portion of lignin and hemicellulose
in Fig. 3b. Under the pretreatment temperature of 160 °C, all of has been removed after the two-stage pretreatment.
the six pretreatment time evaluated in this study had hemicellu-
loses removal higher than 90% and lignin removal from 37% to 3.5. Effect of pretreatment on CS composition and structure
49%, as well as total sugar yields around 80%. With the objective
to maximize the total sugar yield and reduce pretreatment sever- To reveal the impacts of Na2S presoaking and methanol-HCl
ity, 160 °C and 20 min with the addition of 20% methanol and pretreatment on the surface morphology, chemical bonding, and
 Q. Qing et al. / Bioresource Technology 233 (2017) 284–290 289

the aromatic ring, and 1465 cm1 associated with asymmetric

bending in C-H3 (Salehi et al., 2013), indicating that most of the lig-
nin were removed from the solid residue after pretreatment. These
results were in accordance with those of the composition analysis
shown in Table 2. In addition, the signal at approximately
899 cm1 was attributed to b-1,4-glycosidic linkages between the
sugar units in cellulose (Lu et al., 2014), the increase of which indi-
cates an increase of the cellulose portion in the solids.
The XRD profiles of different samples were recorded and their
crystallinity indexes were also shown in Table 2. The CrI was
increased from 37.6% up to 48.8% by different pretreatment strate-
gies. However, the ratio of CrI to the cellulose content showed an
opposite trend, decreasing from 1.01 to 0.65, which suggests that
while the crystallinity index is increased due to the removal of
the amorphous structure, the crystallinity of cellulose itself was
also decreased as supported by previously reported considerations
(Driemeier et al., 2011; Kim et al., 2003; Pihlajaniemi et al., 2016;
Singh et al., 2015). The XRD results indicated that the proposed
Fig. 4. The enzymatic hydrolysis performances of CS pretreated by different two-step pretreatment effectively remove most of the amorphous
methods. Pretreatment conditions were the optimal conditions determined in this components in CS, resulted in increased cellulose proportion and
study. Step1-20% methanol and Step1-0.2% HCl refer to CS pretreated by step 1 then improved enzymatic hydrolysis by reducing non-productive bind-
subjected to 0.2% HCl or 20% methanol pretreatment at 160 °C for 20 min.
ing of cellulases.

4. Conclusion
crystalline structure of the pretreated corn stover, the characteris-
tics of the untreated and pretreated CS were recorded and com- An efficient pretreatment strategy that employs Na2S presoak-
pared by means of SEM, FTIR, XRD, and BET analyses. ing and methanol-HCl pretreatment was proposed and systemati-
Surface morphology of the UN-CS and the pretreated CS were cally investigated in this study. The mild alkaline presoaking step
recorded by SEM. The UN-CS shows an ordered, smooth, and con- successfully removed 35.3% of lignin and enhanced the SSA and
tiguous surface structure without pores, preventing the access of PV of the presoaked solids, making more lignin and hemicelluloses
cellulase to cellulose fibrils. The tissue surface and cell wall were vulnerable in the methanol-HCl pretreatment step. The regener-
altered after the Na2S presoaking, which were perforated and par- ated cellulose-rich solids from the organosolv pretreatment step
tially disrupted in this step. Moreover, after the methanol-HCl pre- became much more susceptible to enzymatic hydrolysis and
treatment, coarse surface and porous areas were very evident. The exhibited an outstanding digestibility of 98.6% with a moderate
cellulose microfibrils were separated from the initial connected enzyme loading. The impacts of the combined pretreatment on
structure and a much more fragmental structure was displayed. the chemical composition and structure of CS were also better
In accordance with the morphological observations, the SSA and understood.
porosity of the pretreated CS increased after both step 1 and step
2 as shown in Table 2. After the Na2S presoaking, the SSA was Acknowledgements
increased dramatically from 0.032 to 2.941 m2/g, and the pore vol-
ume was increased from 0.0009 to 0.0016 cm3/g. The increase of This work was financially supported by the funds from Jiangsu
the SSA and pore volume could be resulted from the swollen effect Natural Science Funds through the contract Number of
of presoaking and partial removal of lignin and hemicelluloses. BK20140258, Changzhou Sci &Tech Program through the grant
Furthermore, the organosolv pretreatment of step 2 indicated a Number of CE20145053, and Natural and Science Research Pro-
much more significant effect on SSA and PV. After the methanol- gram of Jiangsu Universities through the contract Number of
HCl pretreatment, the SSA dramatically increased to 6.228 m2/g 15KJB530002. We also acknowledge the Laboratory of Cellulosic
and the PV was five times larger than the presoaked CS. Biofuel, Changzhou University for providing the facilities and
FTIR analyses of Un-CS, step 1 only and two steps pretreated CS equipments used in this research.
were performed to investigate the chemical changes before and
after pretreatment. The wide band at 3400 cm1 was associated
Appendix A. Supplementary data
with stretching of O–H hydrogen bonds of cellulose and the band
at 2900 cm1 was attributed to C–H stretching vibrations of cellu-
Supplementary data associated with this article can be found, in
lose/lignin (Chen et al., 2015). Absorption of both peaks were
the online version, at http://dx.doi.org/10.1016/j.biortech.2017.02.
enhanced after pretreatment indicating the increase of cellulose
106.
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