Bioresource Technology 101 (2010) 6994–6999

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

Xylose and cellulose fractionation from corncob with three different strategies
and separate fermentation of them to bioethanol
Yefu Chen *, Boyu Dong, Weijun Qin, Dongguang Xiao
Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, PR China
Tianjin Industrial Microbiology Key laboratory, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China

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

Article history: To the aim of efficient utilization of both of xylose and cellulose, a laboratory xylose/cellulose fraction-
Received 18 January 2010 ation and separate fermentation (XCFSF) bioethanol process was performed. Three xylose/cellulose frac-
Received in revised form 28 March 2010 tionation strategies: (A) dilute sulfur acid hydrolysis and detoxification, (B) lime pretreatment and
Accepted 30 March 2010
xylanase hydrolysis, (C) bio-treatment with Phanerochaete chrysosporium and xylanase hydrolysis were
Available online 18 April 2010
applied to corn cobs. As a result, the maximum xylose yields obtained from A, B and C fractionation meth-
ods were 78.47%, 57.84% and 42.54%, respectively, and 96.81%, 92.14% and 80.34% of cellulose were pre-
Keywords:
served in the corresponding solid residues. The xylose dissolved in acid and enzymatic hydrolysates was
Xylose
Cellulose
fermented to ethanol by Candida shahatae and the cellulose remaining in solid residues was converted to
Bioethanol ethanol by simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae. Finally,
Pretreatment for A, B, C fractionation methods, 70.40%, 52.87%, 39.22% of hemicellulose and 89.77%, 84.30%, 71.90%
Xylanase hydrolysis of cellulose in corn cobs was converted to ethanol, respectively.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction In our opinion, XCFSF process for ethanol production contains

D-Xylose is the second most abundant sugar in lignocellulosic
materials and its efficient conversion is one of the prerequisites
for lignocellulosic ethanol industrialization (Hahn-Hägerdal et al.,
2007). In the presence of glucose, xylose could not be converted
into ethanol at an acceptable efficiency for industrial level because
of preferential glucose utilization (Yomano et al., 2009; Panchal
et al., 1988). Efforts have been taken to explore the regulatory
mechanism of preferential glucose utilization, but it is very com-
plex and no clear solution has been achieved yet (Matsushika
et al., 2009).
Since xylose in the C5 and C6 sugars mixture is difficult to be
converted into ethanol, it is a good option to separate xylose from
cellulose and ferment xylose and cellulose to ethanol separately.
This leads to another lignocellulosic ethanol production strategy
which we called here XCFSF process.
Lignocellulose fractionation is not a novel idea. Previously,
some strategies (Ibrahim and Glasser, 1999; Kim and Lee, 2005,
2006; Hongzhang and Liying, 2007; Persson et al., 2009) have been
adopted to different lignocellulosic materials to fractionate the
three main components (hemicellulose, cellulose and lignin) for
high value-added materials production.
* Corresponding author. Tel.: +86 22 60601396; fax: +86 22 60602298.
E-mail address: yfchen@tust.edu.cn (Y. Chen).
the following steps: pretreatment, hydrolysis of hemicellulose,
hemicellulose hydrolyzate/cellulosic residues fractionation, xylose
ethanol fermentation and cellulosic residues ethanol fermentation.
Lignin has significantly negative effect on the hydrolysis of hemi-
cellulose and cellulose (Hendriks and Zeeman, 2009). For efficient
hemicellulose and cellulose hydrolysis, certain level of pretreat-
ment for decomposing the lignin is necessary in XCFSF process.
Hemicellulose can be easily hydrolyzed into dissolved sugars
(mainly xylose) with enzyme or chemicals, and then separated
from cellulosic residues by proper liquid–solid separation method.
Cellulose remaining in residues can be easily converted into etha-
nol by successful glucose-fermenting yeast Sacchromyces cerevisiae
after enzymatic hydrolysis to glucose. For xylose fermentation,
some xylose-fermenting yeasts, such as Pichia stipitis, Candida
shahatae, and Pachysolen tannophilus, could be used. These yeasts
possess relatively high xylose fermentation efficiency, and have
the potential for further engineering (Jeffries et al., 2007).
In this paper, we performed a laboratory XCFSF bioethanol pro-
cess using corn cobs as raw materials. Three leading pretreatment
methods, dilute acid hydrolysis, lime pretreatment, and bio-degra-
dation, were applied in xylose/cellulose fractionation. The hemicel-
lulose remaining in pretreated corn cobs were hydrolyzed to xylose
with commercial xylanase. Xylose-containing hydrolysate was fer-
mented to ethanol by C. shahatae. The cellulose remaining in solid
residues was converted to ethanol employing simultaneous sac-
charification and fermentation (SSF) method with S. cerevisiae.

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.03.132
 Y. Chen et al. / Bioresource Technology 101 (2010) 6994–6999
2. Methods
2.1. Raw material
Corn cob samples were obtained from local farmer in the sub-
urb of Tianjin, China, and grinded to particle size of 1–2 mm with
a Straw stalk knife mill (Shandong, China). It contained 40.67% cel-
lulose, 31.1% hemicellulose and 11.7% lignin and 4.43% ash. The
processed substrate was washed thoroughly and dried overnight
at 60 °C.
Commercial cellulase (XWS-G-1) from Trichoderma reesei
(10 FPU/mg), xylanase (JT-G-M) from Aspergillus niger (18 U/mg)
were purchased from Noao Sci & Tech Development Co., Ltd., Tian-
jin, China. The rest of the chemicals and media components were
purchased locally.
2.2. Microorganisms and seed culture
Candida shehatae CICC1766 and Phanerochaete chrysosporium
CICC40719 were obtained from China Center of Industrial Culture
Collection. Saccharomyces cerevisiae TCCC34074, an osmo-, ther-
mo-tolerant and high ethanol-producing yeast, was from our col-
lection (TCCC, Tianjin University of Science and Technology’s
Center of Culture Collection).
Liquid inoculum of C. shehatae was prepared in sequential pro-
cess. First, inoculating C. shehatae to the culture medium (g/L): xy-
lose, 20.0; yeast extract, 10.0; malt extract, 3.0; peptone, 20.0 and
maintaining at 28 °C for 24 h on an orbital shaker agitated at
120 rpm, hereafter, inoculating the liquid seed of C. shehatae to
the same culture medium and maintaining in same condition.
S. cerevisiae liquid inoculum was grown in the medium contain-
ing (g/L): glucose, 40.0; yeast extract, 3.0; peptone, 5.0; (NH4)2SO4,
2.0 at 30 °C. The other detailed approaches are as same as above.
Liquid inoculum of P. chrysosporium was prepared in a culture
medium of pH 5.0 containing (g/L): glucose, 20; MgSO4
7H2O,
0.5; KH2PO4, 0.5; (NH4)2SO4, 0.1 at temperature 30 °C for 72 h on
an orbital shaker agitated at 120 rpm.
2.3. Dilute acid pretreatment
The dilute sulfuric acid pretreatment of corn cob was optimized
by employing a L9 (34) orthogonal design with factors of tempera-
tures (100, 110, 120 °C), treatment time (100, 120, 140 min), acid
concentrations (0.5%, 1.0%, 2.0%, v/v) and substrate concentrations
(7.5%, 10.0%, 12.5%, w/v). The process was carried out in 250 mL
Erlenmeyer flask, using an autoclave. The acid hydrolysate was
recovered by filtering the contents through double-layered muslin
cloth. After washing with tap water till neutral pH, the remaining
solid residue was dried overnight at 60 °C till constant weight
and used for compositions analysis and further experiments.
2.4. Detoxification of acid hydrolysate
The corncob acid hydrolysate produced was concentrated 4-fold
in a 4-dm3 evaporator at 70 ± 5 °C under vacuum condition (0.07–
0.08 MPa). The overliming process was performed at temperature
80 °C by adding dried Ca(OH)2 till the pH reached 10.0, with con-
stant stirring for 5 min by an electric stirrer. After overliming, the
hydrolysate was neutralized with concentrated H3PO4 and centri-
fuged to remove the precipitate formed during process. The con-
centrated or overlimed hydrolysate was further detoxified by
adsorption with activated charcoal, ion-exchange resin adsorption,
or macroporous adsorbent resin. Adsorption of activated charcoal
was carried out with activated charcoal (Feiyang Company, Chon-
gqing, China) in a consistency of 1% (w/v) with constant stirring
6995
(120 rpm) in a rotatory shaker at room temperature for 1 h. The
adsorption processes could also be carried out by treating with
ion-exchange resin 201  7 (anionic) (Lianxing Company, Tianjin,
China) or macroporous adsorbent resin NKA II (Nankai Resin Com-
pany, Tianjin, China) in a consistency of 3% (w/v) with constant
stirring (120 rpm) in a rotatory shaker at temperature 50 °C for 1 h.
The concentrated hydrolysate was subjected to six detoxifica-
tion procedures as the following adsorption processes: (A) over-
liming and subsequently adsorption on the ion-exchange resin
201  7; (B) overliming followed by adsorption on macroporous
resin NKA II; (C) overliming followed by adsorption on activated
charcoal; (D) concentration and subsequently adsorption on the
ion-exchange resin 201  7; (E) concentration followed by adsorp-
tion on macroporous resin NKA II; (F) concentration followed by
adsorption on activated charcoal. The sugar syrup was recovered
through vacuum filtration for components analyse.
2.5. Lime pretreatment
The lime pretreatment was carried out in the presence of water,
applying a consistency of 10% (w/v) with constant stirring
(120 rpm) in a rotatory shaker. To reach non-oxidative and oxida-
tive conditions, the process was performed in a 250 mL Erlenmeyer
flask sealed by mono-layer gauze and rubber stopper, respectively.
The reaction temperature was 55 °C and the pretreatment time
was four weeks according to the reported results (Kim and Holtz-
apple, 2005). The calcium hydroxide loadings in dry weight were
varied from 0.02 to 0.20 g/g corn cob with an interval of 0.01 g to
evaluate the role of lime in deligninfication. After the pretreat-
ment, the residue was moved out of the system by filtration and
washed with tap water till neutral pH. After cooling down to ambi-
ent temperature, the remaining samples were then collected for
various analyses.
2.6. Bio-degradation of corn cob by P. chrysosporium
An orthogonal experiment was performed to optimize the pre-
treatment by P. chrysosporium for degradation of lignin. The corn
cob in dry weight 10 g was taken as the substrate in orthogonal
experiments, and the processes were conducted at substrate con-
centration of 50.0% (w/v). The medium consists of (g/L)
MgSO4
7H2O, 1.6; KH2PO4, 2.0; FeSO4
7H2O, 0.004; NaCl, 0.4;
ZnSO4, 0.007. The L9 (34) orthogonal table was chosen, the lignio-
lytic conditions (high carbon low nitrogen) (Kirk, 1984) were de-
signed as follows: glucose loadings (0.2, 0.3, 0.4 g/10 g substrate),
(NH4)2SO4 loadings (0.001, 0.002, 0.003 g/10 g substrate), MnSO4
loadings (0.001, 0.003, 0.005 g/10 g substrate) and inoculum size
(5%, 10%, 15%). The substrate was adjusted to pH 5.5 and auto-
claved at 121 °C for 15 min sequentially before inoculation. The
bio-degradation temperature is 30 °C and the pretreatment time
was 20 days. After processing, the delignified corn cob was sub-
jected to filtration and washed several times with tap water till
the filtrate reached neutral pH. The remaining residues were dried
overnight at 60 °C till constant weight and collected for various
component analyses.
2.7. Enzymatic hydrolysis of the lime treated and bio-degradated
corncobs by xylanase
The enzymatic saccharification of hemicellulose in dried residue
after lime treatment or bio-degradation was performed by shaking
gently (120 rpm) at 50 °C in 250 mL Erlenmeyer flask after adjust-
ing the pH to 5.3 with H3PO4. The substrate content for reaction
was 10.0% (w/v) and Tween-80 as surfactant (1.0%, v/v) was used.
Before enzyme loading, slurry was acclimated by incubating at
50 °C on a rotatory shaker (ZHWY-211B, Tocan Scientific, Shanghai,
6996 Y. Chen et al. / Bioresource Technology 101 (2010) 6994–6999
China) at 120 rpm for 30 min. Xylanase loading ranges from 100 to
900 U/g substrate with a regular interval of 100 U/g substrate spe-
cially. Samples (1 mL) of the entire xylanase loading were taken
from the reaction mixture periodically during incubation, and
boiled for 10 min to terminate the reaction and stored at 20 °C
before xylose analysis.
2.8. Fermentation of acid and enzymatic hydrolysates by C. shehatae
The hydrolysates were further supplemented with additional
nutrients (g/L) (NH4)2SO4, 1; KH2PO4, 2.0; MgSO4
7H2O, 0.5; yeast
extract, 1.5; CaCl2
2H2O, 0.1; FeCl3
2H2O, 0.1; ZnSO4
7H2O, 0.001
and the pH adjusted to 4.5. The corncobs hydrolysate and the
nutrients were autoclaved separately and combined after steriliza-
tion. Fermentation medium was inoculated with 10% (v/v) cultures
of C. shehatae. Unless otherwise stated, all treated hydrolysates
were fermented at 28 °C, in 250 mL Erlenmeyer flask having
100 mL medium shaking at 160 rpm for 72 h. The optimization
experiments were performed for single factor in terms of ethanol
yield. The factors and their levels were designed as follows: tem-
perature (28, 30, 32 °C) and constant stirring (120, 160, 200 rpm).
Liquid samples were taken with 24 h as a regular interval and di-
luted several times for ethanol determination. All experiments
were performed in triplicate.
2.9. Simultaneous saccharification and fermentation (SSF) by
S. cerevisiae
SSF was performed according to reference (Krishna et al., 2001).
The medium composition consists of (g/L) (NH4)2SO4, 2.0; KH2PO4,
5.0; MgSO4
7H2O, 0.4; CaCl2, 0.2; yeast extract, 2.0. The pH of sub-
strate was adjusted to 4.8 and autoclaved for 15 min at 121 °C or-
derly before adding enzymes and inoculum. The inoculum size was
10% (v/v) of the SSF medium. Unless otherwise stated, all SSF pro-
cesses were carried out statically with substrate concentration of
10% (w/v) at 35 °C in incubator for 96 h and the cellulase loading
is 20 FPU/g substrate. The optimization experiments were per-
formed for single factor in terms of ethanol yield. The factors and
their levels were designed as follows: substrate concentration
(8.0%, 10.0%, 12.0%, w/v), temperature (30, 35, 40 °C) and cellulase
loading (20, 30 and 40 FPU/g substrate). Samples were withdrawn
with 24 h as a regular interval and the solid substrate was removed
by centrifugation. The supernatant was used for ethanol determi-
nation, and cellulose content of the remaining solid substrate
was measured. All experiments were performed in triplicate.
2.10. Analytical methods
The compositions (cellulose, hemicellulose and lignin) of the
dry materials were determined according to the method reported
(Wang and Qi, 1987).
Xylan content of hydrolysate was measured by transforming
pentosans with HCl to furfural which was collected in the distillate
and determined colorimetrically with orcinol FeCl3.
The amount of acetic acid, furfural and 5-hydroxymethyl-furfu-
ral (HMF) were determined simultaneously with a RP-HPLC (Agi-
lent Technology 1100 series system equipped with UV detector).
Quantitative assessments of three inhibitory compounds were per-
formed on a Hypersil ODS-2 (5 lm, 250  4.6 mm) column with
0.1‰ (v/v) H2SO4:CH3OH = 95:5 as the mobile phase at a flow rate
of 1 mL/min. The wavelengths for detection of three compounds
were changed to their maximal UV absorption wavelengths (acetic
acid, 210 nm; furfural, 275 nm; HMF, 284 nm). The phenolic com-
pounds acting as representative for a type of inhibiting mixture
with phenyl could be estimated by UV spectrophotometry at
280 nm (Shimadzu, Kyoto, Japan). Ethanol was estimated by gas
chromatography with an elite-wax (cross-bond-polyethylene gly-
col) column (30.0 m  0.25 mm) at oven temperature 85 °C and
flame ionization detector (FID) at 200 °C. Nitrogen with a flow rate
of 0.5 mL/min was used as carrier gas.
3. Results and discussion
3.1. Pretreatment
Different pretreatment methods have been developed during
the past a few decades for effective enzymatic hydrolysis of ligno-
cellulosic materials (Chandra et al., 2007). In our study, three strat-
egies were adopted to fractionate xylose and cellulose. The first
one was to treat corn cob with dilute sulfur acid and hydrolyze
the hemicellulose into dissolved sugar directly. Second one was
to pretreat corncob with lime to increase enzymatic accessibility
and then hydrolyze hemicellulose into sugars by xylanase. The
third one was to bio-delignify with P. chrysosporium at first and
then hydrolyze hemicellulose into sugars by xylanase.
3.1.1. Dilute sulfur acid hydrolysis
Dilute acid hydrolysis has been successfully developed for pre-
treatment of lignocellulosic materials (Sun and Cheng, 2002).
Treatment with dilute acid at moderate temperatures could effec-
tively hydrolyze and recover most of the hemicellulose as dis-
solved sugars (Wyman et al., 2005). We first performed dilute
H2SO4 treatment to corn cob for xylose and cellulose fractionation
purpose.
Basing on our previous study, we selected temperature, treat-
ment time, H2SO4 concentration, and solid to liquid ratio as the
determinant factors to hemicellulose hydrolysis efficiency and de-
signed a L9 (34) orthogonal experiment to optimize the hydrolysis
condition. The optimum condition obtained from orthogonal
experiment were temperature 120 °C, acid concentration 1.0% (v/
v), reaction time 2 h and solid to liquid ratio 10% (w/v). When corn
cob was treated under this condition, 84.6% of hemicellulose in the
corncobs was dissolved into hydrolysate, the maximum xylose
concentration (29.84 g/L) was obtained and the xylose yield is
84.43%. Lignin (31.90%) was removed during acid hydrolysis. Cellu-
lose in corncobs were stable under this hydrolysis condition com-
pared with hemicellulose and lignin, only 3.10% were lost (Table 1).
3.1.2. Lime pretreatment for delignification
Using alkaline chemicals to remove lignin to improve cellulose
digestibility has been known for years. Sodium hydroxide and
other bases are too expensive and too difficult to recover to make
them viable for producing fuels. Pretreatment with lime is a low-
cost alternative for lignin removal (Wyman et al., 2005). The com-
bined action of alkali and oxygen could solubilize most portions of
the lignin, which made even recalcitrant biomass digestible (Chang
et al., 2001).
Table 1
Removals of components of corncobs by different treatments.
Removal (%)a
Hemicellulose Cellulose Lignin
Acid hydrolysis 85.60 3.10 31.90
Lime pretreatment 30.60 1.30 81.20
Xylanase hydrolysis to the lime 59.47 6.63 9.80
pretreated residues
Bio-pretreatment 20.80 18.50 42.70
Xylanase hydrolysis to bio-pretreated 42.90 1.00 5.13
residues
a
The initial hemicellulose, cellulose and lignin contents of the corn cob are
considered as 100.
 Y. Chen et al. / Bioresource Technology 101 (2010) 6994–6999 6997

We carried out two form of lime pretreatment on a rotatory
shaker, one with air (oxidative lime pretreatment), and one with-
out air (non-oxidative lime pretreatment). In the lower range of
lime consumption (60.07 g/g corn cob), lignin was removed up
to approximate 43% rapidly and easily and oxygen is not essential.
But for more lignin removal, lime over-loading alone did not work
under non-oxidative condition and the air (oxygen) much be pro-
vided at the same time. As shown in Fig. 1, during non-oxidative
lime treatment, the maximum lignin degradation is 43.2% when
specific lime loading was up to 0.08 g Ca(OH)2/g corn cob. On the
other hand, during oxidative pretreatment, the delignification
tended to increase as specific lime loading increased, up to 0.16 g
Ca(OH)2/g corn cob and then the lignin removal did not increase
even if more lime was supplied. The maximum lignin removal
was achieved as 81.5% during oxidative pretreatment with lime
loading 0.16 g Ca(OH)2/g corn cob at 55 °C for four weeks. Never-
theless about 31% of hemicellulose was removed together with lig-
nin (Table 1). Cellulose was stable during lime treatment and only
1.30% was decomposed.
3.1.3. Bio-degradation by P. chrysosporium to remove lignin
Biological pretreatment is considered to be environmentally
friendly and energy saving as it is performed at low temperature
and without chemicals (Galbe and Zacchi, 2007). P. chrysosporium,
one kind of white-rot fungi which could use lignin as a sole carbon
and energy source, has been most widely studied for lignocellu-
losic material pretreatment purpose (Sánchez, 2009).
Co-metabolizable carbon source, and additional nitrogen source
are essential to P. chrysosporium for lignin degradation. High carbon
to nitrogen ratio was employed as ligninolytic conditions in our
study based on the reported result that lignin enzymes was pro-
duced in response to nitrogen starvation (Keyser et al., 1978). In
addition, the amount of MnSO4 loaded in corncob substrate was
optimized because manganese peroxidases (MnP, EC1.11.1.13)
were described as main lignases in P. chrysosporium (Gold et al.,
2000). The amount of liquid inoculum was selected as the fourth
factors for a L9 (34) orthogonal experiment. The results of orthogo-
nal experiment indicate that optimum condition for bio-degrada-
tion are glucose 0.2 g/10 g substrate, (NH4)2SO4 0.003 g/10 g
substrate, MnSO4 0.005 g/10 g substrate and amount of inoculum
10% (v/v). When the corn cob was treated under this optimized
condition, the maximum delignification rate was 42.7% (Table 1).
But 20.8% of hemicellulose and 18.5% of cellulose were degradated
together with lignin.
The removal rate of different components in corncob with
above three treatments is presented in Table 1. Acid hydrolysis
was most efficient for hemicellulose hydrolysis purpose. During di-
lute treatment, only 3.10% of cellulose was decomposed and lost,
and quite amount of lignin (31.90%) was degraded and removed.
As to the aspect of lignin removal, lime pretreatment was most
effective obviously, most part (81.20%) of lignin was removed with
lime treatment and almost all of cellulose was preserved at the
same time. But parts of hemicellulose (30.60%) are degraded and
lost. Lignin removal by bio-degradation was relatively lower com-
pared with lime pretreatment, only 42.70% of lignin were de-
graded, and quiet amounts of cellulose and hemicellulose were
degraded by P. chrysosporium at the same time.
3.2. Preparation of fermentable xylose hydrolate
Acid hydrolysis resulted in the release of some kinds of inhibi-
tory compounds such as phenolics, acetic acid, HMF and furfural.
These chemicals are toxic to xylose-fermenting yeast at certain le-
vel and must be removed from hemicellulose hydrolate before fer-
mentation (Palmqvist and Hahn-Hägerdal, 2000). For lime and
biological pretreated materials, we hydrolyzed the hemicellulose
into sugars using xylanase. Enzymatic hydrolysis did not produce
the above toxic compounds and the hydrolyzate obtained from
enzymatic hydrolysis step can be used as fermentation substrate
directly without detoxification.
3.2.1. Detoxification of acid hydrolysate
The hydrolysate obtained under above optimized acid hydroly-
sis conditions was found to contain furfural (0.74 g/L), HMF
(0.08 g/L), acetic acid (2.23 g/L) and phenolic compounds (2.33 g/
L), and the xylose-fermenting strain C. shehatae CICC1766 could
not ferment it even extra nutrients were supplied. Consequently,
we performed six different detoxification procedures (see details
in Section 2) to the dilute acid hydrolysate to select an optimal
one for toxic compounds removing and the results were presented
in Table 2.
The results showed that all the six detoxification procedures
were effective in removing acetic acid, furfural and HMF. After
detoxification, the concentrations of all those toxic compounds
were lower than the reported levels causing inhibition of microor-
ganism metabolism (Wilson et al., 1989; Roberto et al., 1991). Nev-
ertheless, as synergistic effects of the toxic compounds could occur
even when they are present in low concentrations, it would be nec-
essary to remove these compounds completely from the hydroly-
sate. The detoxification procedure B, based on the use of
overliming followed by adsorption of macroporous resin NKA II,
was efficient for this purpose. It almost resulted in the highest
removals of HMF (100%), furfural (97.43%), acetic acid (97.39%)
and phenolic compounds (97.88%). The xylose loss during proce-
dure B is 7.04%, which is acceptable for xylose recovery purpose.
After acid hydrolysis and detoxification, 2.774 g xylose was ob-
tained from 10 g corncob which corresponded to 78.47% of theo-
retical yield (Table 3).

Table 2
Detoxification results.

Concentration in hydrolysate (g/L)

Xylose Acetic acid Furfural HMF Phenolics
Acid hydrolysate 29.84 2.23 0.74 0.08 2.33
A 27.82 0.019 0.045 0.006 0.415
0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.11 0.12 0.13 0.14 0.15 0.16 0.17 0.18 0.19 0.2 B 27.74 0.058 0.019 0 0.049
C 26.24 0.056 0.027 0.003 0.362
D 28.94 0.361 0.069 0.025 0.651
E 28.88 0.148 0.03 0.013 0.256
Fig. 1. Effects of lime loading on lignin removals in non-oxidative and oxidative F 27.55 0.415 0.026 0.037 0.492
condition.
6998 Y. Chen et al. / Bioresource Technology 101 (2010) 6994–6999

Table 3
Fermentation profiles of xylose in the hydrolysate using C. shehatae.

Xylose output Xylose yield Ethanol Ethanol output Ethanol yield Hemicellulose conversation
(g)a (%)b concentration (g/L) (g)a (g/g) rate (%)c
Acid hydrolysis and detoxification 2.774 78.49 9.16 1.145 0.421 70.40
Lime pretreatment and hydrolyzed with 2.032 57.84 8.45 0.860 0.429 52.87
xylanase
Bio-degradation and hydrolyzed with 1.509 42.54 6.65 0.638 0.427 39.22
xylanase
a
From 10 g raw corncobs.
b
Xylose yield = (xylose obtained (g)  0.88)/(raw material (10 g)  hemicellulose contents)  100%.
c
Hemicellulose conversion rate = ethanol output (g)/(raw material (10 g)  hemicellulose contents  theoretical yield of ethanol to hemicellulose (0.523 g/g))  100%.

3.2.2. Enzymatic hydrolysis of hemicellulose after lime treatment and
bio-degradation
In this section, we hydrolyzed the hemicellulose in the lime
treated and bio-treated residues into xylose using commercial
xylanase, which with optimal pH 5.3 and optimal temperature
50 °C, under their optimized conditions. For lime treated substrate,
the maximum xylose yield (57.84%) was achieved at a dose of
xylanase 500 U/g substrate after 48 h and the xylose concentration
in hydrolysate is 25.08 g/L. But for the biodegraded substrate, the
maximum xylose yield (42.54%) was obtained at increased enzyme
dose (xylanase 800 U/g substrate) and elongated reaction time
(60 h), and the xylose concentration in hydrolysate (19.60 g/L)
was also lower than lime treated sample. The removal of hemicel-
lulose relative to total hemicellulose content in corn cob material
were 59.47% and 42.90% corresponding to lime treatment and
bio-degradation materials (Table 1).
As compared to lime pretreated corncob, the hydrolysis of
hemicellulose in the bio-pretreated corncob needed more enzyme
and more time. Moreover, the xylose yield and xylose concentra-
tion of the hydrolyzate obtained from bio-pretreated corncob were
also lower than lime pretreated corncob. All these results maybe
due to the more lignin remained in bio-degradated materials. From
these results, we can see the importance of pretreatment for XCFSF
purpose.
3.3. Xylose fermentation
Conversion of the xylose from hemicellulose in hydrolysate to
ethanol can greatly improve overall yield of cellulosic ethanol pro-
duction and reduce the cost of unit amount ethanol. The fermenta-
tion with C. shehatae was then performed as described in methods.
The same optimized fermentation conditions including tempera-
ture (28 °C) and constant stirring (160 rpm) were used to all the
three kinds of xylose-containing hydrolysates obtained from differ-
ent treatments. Ethanol (9.16 g/L) was produced from the acidic
hydrolysate with a yield of 0.421 g/g after 96 h. For enzymatic
hydrolysates employed lime treatment and bio-degradation for
72 and 48 h, 8.45 and 6.65 g/L ethanol were obtained with yields
of 0.429 and 0.427 g/g (in Table 3), respectively. Ethanol yield of
acidic hydrolysate after detoxification was a little less than the
yields of other two enzymatic hydrolysates, this might be caused
by the synergistic inhibitory effect of the toxic chemicals remained
in the hydrolysate after detoxification.
the components of acid hydrolysis residue (AHR), lime pretreat-
ment and xylanase hydrolyzed residues (LPXHR) and biological
pretreatment and xylanase hydrolyzed residues (BPXHR) varied
in wide range (Table 4), so the optimized SSF process parameters
for each residues, except pH and temperature, were quiet different.
pH 4.8, the optimum pH for cellulase, was found to be appropriate
for fermentation by S. cerevisiae AY-15. Therefore, pH 4.8 was
adopted in all the SSF processes. In SSF, a compromise between
the optimal temperatures for cellulase and for yeast should be con-
sidered. After a series of experiments, 35 °C was chosen in all the
SSF processes. The contents of cellulose in AHR, LPXHR and BPXHR
are 72.1%, 59.65% and 54.75%, as shown in Table 4, correspondingly
the optimal enzyme loadings were 30, 20 and 24 FPU/g. The opti-
mal enzyme loading for BPXHR was more than that for LPXHR
abnormally, it suggested that residual lignin blocked the action
of cellulase. The optimal substrate concentrations for SSF of AHR,
LPXHR and BPXHR were 10% (w/v), 10% (w/v) and 12% (w/v),
respectively. The fermentation time was 120, 96 and 96 h for
AHR, LPXHR and BPXHR. Under the optimal conditions as described
above, 37.85, 30.96 and 33.3 g/L ethanol were produced from AHR,
LPXHR and BPXHR with the yields of 0.538, 0.522 and 0.510 g/g
cellulose, respectively (Table 5).
For dilute acid pretreatment process, 1.145 and 2.070 g ethanol
was produced from hemicellulose and cellulose in 10 g corncobs.
According to the theoretical yield (ethanol/component) of
0.523 g/g (ethanol/hemicellulose) and 0.567 g/g (ethanol/cellu-
lose), 70.40% and 89.77% of hemicellulose and cellulose in raw
material were biotransformed to ethanol, respectively, as shown
in Tables 3 and 5. For lime treatment process, 0.860 and 1.944 g
ethanol were produced from hemicellulose and cellulose in 10 g
corn cob. That meant 52.87% and 84.30% of hemicellulose and cel-
lulose in raw material were converted to ethanol. For bio-degrada-
tion process, 0.638 and 1.658 g ethanol were produced from
hemicellulose and cellulose in 10 g corn cob material, that meant
39.22% and 71.90% of hemicellulose and cellulose in raw material
were fermented to ethanol product, respectively.
Totally, 3.215, 2.804 and 2.296 g ethanol were produced from
10 g corn cob material employed acid hydrolysis, lime treatment
and bio-degradation process. The utilization of usable components
employed lime treatment and bio-degradation process were in a
Table 4
Components of the residues obtained after different treatments.

Hemicellulose Cellulose Lignin

3.4. SSF of cellulosic residues into ethanol
(%) (%) (%)
Original corn cob 31.10 40.67 11.70
The cellulose in lignocellulosic material is the great contributor
Acid hydrolysis 8.20 72.10 14.57
to ethanol. To avoid end-product inhibition caused by accumula- Lime treated 26.49 49.30 2.70
tion of glucose during cellulase hydrolysis and to enhance ethanol Lime treated and hydrolyzed by 4.91 59.65 1.68
yield, SSF process was applied for cellulosic residues ethanol fer- xylanase
mentation. In SSF process, the presence of yeast together with cel- Biodegraded 32.10 43.20 8.74
Biodegraded and hydrolyzed by 18.86 54.75 10.21
lulase reduced the accumulation of glucose, thereby increased
xylanase
saccharification rate and ethanol yield (Saha et al., 2005). Because
 Y. Chen et al. / Bioresource Technology 101 (2010) 6994–6999
Table 5
SSF profile of cellulose in residues.
Cellulose preserved in Cellulose
residues (g)a preserving rateb (%)
After acid hydrolysis 3.94 96.81
After lime treatment and 3.75 92.14
xylanase hydrolysis
After bio-degradation and 3.27 80.34
xylanase hydrolysis
a
From 10 g raw corncobs.
b
To total cellulose in initial corncobs.
c
Cellulose conversion rate = ethanol produced from cellulose (g)/(raw material (10 g)  cellulose contents  theoretical yield of ethanol to cellulose (0.567 g/g))  100%.
poor way compared with acid hydrolysis process, that attributed to
the lost of hemicellulose and cellulose during the course of deligni-
fication by two treatments.
4. Conclusions
In this study, we applied three leading pretreatment methods
for XCFSF purpose. Dilute acid hydrolysis was most effective for xy-
lose recovery, but with inhibitors generation. Lime pretreatment
was found very effective to remove lignin, but quite amount of
hemicellulose were lost at the same time. Biodelignification with
P. chrysosporium was friendly to environment. However, lots of
hemicellulose and cellulose were consumed during the processing.
Based on analysis of data, the contribution to ethanol production
from hemicellulose was not as significant as that from cellulose,
but it is still crucial to boost the industrialization of bioethanol.
Acknowledgements
The authors thank Dr. Baojun Xu and Dr. Jianhua Yan for the li-
vely discussion of and valuable comments on the manuscript.
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