Bioresource Technology 101 (2010) 7679–7682

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

Short Communication

Production of xylooligosaccharides from the steam explosion liquor

of corncobs coupled with enzymatic hydrolysis using a thermostable xylanase
Chao Teng a, Qiaojuan Yan b, Zhengqiang Jiang a,*, Guangsen Fan a, Bo Shi c
a
Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
b
Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, China
c
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

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

Article history: The production of xylooligosaccharides (XOs) from corncobs was studied using a two-stage process based
Received 16 January 2010 on a steam explosion pretreatment followed by enzymatic hydrolysis. Corncobs with different chip sizes
Received in revised form 2 May 2010 were subjected to steam explosion under different experimental conditions of temperature and time,
Accepted 4 May 2010
namely 188–204 °C for 2.5–7.5 min. The results indicate that corncobs were optimally steam exploded
at 196 °C for 5 min, resulting in hemicellulose recovery of 22.8%. Especially, corncobs with large chip sizes
(100 mm) during steam explosion pretreatment were suitable to produce XOs. Furthermore, a thermo-
Keywords:
stable xylanase from Paecilomyces themophila J18 was used to hydrolyze steam explosion liquor of corn-
Corncobs
Enzymatic hydrolysis
cobs (SELC) for the production of XOs. A maximum XOs yield of 28.6 g XOs/100 g xylan in corncobs was
Steam explosion achieved and XOs syrup contained more than 90% of xylobiose and xylotriose when the hydrolysis was
Steam explosion liquor of corncobs (SELC) carried out under the optimized conditions (pH 7.0, 70 °C, 7.5 U mL 1 and 2.5 h). These results suggest
Thermostable xylanase that the process might be effective in production of XOs for industrial applications.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction degradation of xylans to XOs by dilute solutions of mineral acids,

Lignocelluloses as abundant and renewable raw materials are
potential feedstocks for industrial utilization. Pretreatment of
lignocelluloses with saturated steam (i.e. steam explosion) or hot
water (autohydrolysis), or with addition of small amounts of acids,
has been widely studied to weaken the lignocellulosic structure
(Chen and Liu, 2007; Cara et al., 2008). Studies on the steam explo-
sion of lignocelluloses have mainly focused on lignin fraction, and
the cellulose fraction, as in the case of prehydrolysis pulping and
lately as a possible pretreatment before enzyme saccharification
of celluloses (Chen and Liu, 2007; Cara et al., 2008; Wang et al.,
2009). So far, the amount of work done with regard to the utiliza-
tion of hemicellulose fraction using steam explosion is limited
(Akpinar et al., 2007).
Xylooligosaccharides (XOs) have been produced for use as a
valuable food sweetener or as an additive with many functional-
ities. The production of XOs from xylan rich lignocelluloses has
been investigated by several ways such as direct enzymatic treat-
ments, enzymatic hydrolysis of the isolated xylans and hydrolytic
* Corresponding author. Address: China Agricultural University, College of Food
Science and Nutritional Engineering, Department of Biotechnology, P.O. Box 294,
No.17 Qinghua Donglu, Haidian District, Beijing 100083, China. Tel.: +86 10
62737689; fax: +86 10 82388508.
E-mail address: zhqjiang@cau.edu.cn (Z. Jiang).
steam or water (Yang et al., 2007; Tan et al., 2008). Hydrothermal
reaction (autohydrolysis) is an effective method to degrade the
hemicellulose from lignocelluloses to XOs. However, this process
is time consuming (usually requires 0.5–3.0 h) and produces XOs
which have wide range of DP 2-20 (Parajó et al., 2004; Makishima
et al., 2009). Generally, the commercial XOs contain mainly xylobi-
ose (DP 2) to xylotetraose (DP 4). The thermophilic fungus Paecilo-
myces thermophila J18 has been proved to be a good producer
(18 580 U g 1) of xylanase in solid-state fermentation (SSF). The
xylanase has an optimal temperature of 75–80 °C and exhibits
good hydrolysis patterns of xylans which is suitable for production
of XOs (Li et al., 2006; Yang et al., 2006).
Corncobs show great potential as a renewable raw material for
producing XOs. There are some reports on the production of XOs
from corncobs using different methods (Parajó et al., 2004; Tan
et al., 2008). However, no previous reports could be found that de-
scribe the production of XOs from corncobs using the steam explo-
sion pretreatment followed by enzymatic hydrolysis using
thermostable xylanases (Makishima et al., 2009). Therefore, the
main objective of this study was to develop a new and efficient
process for the production of XOs from corncobs. We have investi-
gated a new type of steam explosion method for pretreatment of
corncobs and optimized enzymatic hydrolysis conditions using a
thermostable xylanase from P. themophila. The process was proved
to be effective in producing XOs from corncobs.

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.05.004
7680 C. Teng et al. / Bioresource Technology 101 (2010) 7679–7682
2. Methods
2.1. Materials
Corncobs obtained locally were chopped and milled into differ-
ent fractions (chip sizes): larger than 100 mm, smaller than 100,
1.49, and 0.75 mm. Birchwood xylan was from Sigma Chemical
Company (St. Louis, MO). All other chemicals used were of reagent
grade obtained from standard sources.
2.2. Steam explosion and preparation of steam explosion liquor of
corncob (SELC)
Corncobs with 35.7% xylan, 5.5% moisture and 3.1% ash were
provided by Henan Taixing Co. Ltd. (Henan Province, China). Corn-
cobs were soaked in water overnight and then the excess liquid
was removed by centrifugation. Steam explosion was performed
in a pilot-scale equipment with 30-L reaction vessel designed by
the company according to the patent of Li (Li, 2007). The reactor
was filled with 20 kg (dry matter) of feedstock per batch, and
was then heated to the desired temperatures, directly with satu-
rated steam. The severity parameter (R0) was used to map the
destruction, degradation, and depolymerization of corncobs. R0
was calculated from the following relation: R0 = t exp(T 100/
14.75), where T is the temperature (°C), and t is the time (min).
The experimental conditions used in the present study for corn-
cobs were 2.5–7.5 min and 188–204 °C, which correspond to val-
ues of log R0 2.99–3.94. After steam-explosion, all the exploded
samples were recovered and extracted with 200 L of tap water
for 1 h at 60 °C, and then filtered to remove the water-insoluble
fraction to obtain the steam explosion liquor of corncob (SELC).
The content of xylose, XOs, total sugars and reducing sugars was
analyzed in SELC.
2.3. Production and purification of a thermostable xylanase
The production and purification of a thermostable xylanase
from P. thermophila J18 was carried out under the conditions pre-
viously described (Li et al., 2006). Xylanase activity was assayed
according to the previous report (Li et al., 2006). The amount of
reducing sugar liberated was determined by DNS method using xy-
lose as the standard.
2.4. Enzymatic hydrolysis of SELC by the xylanase
In order to produce XOs with suitable DP, 1000 mL of SELC was
further hydrolysed by the thermostable xylanase. After adjusting
pH to 7.0, the xylanase was added to SELC. The influence of differ-
ent conditions such as enzyme concentration (2–12 U mL 1) and
hydrolysis period (0–4 h) on the enzymatic hydrolysis yield was
studied at 70 °C. A 10 mL sample was taken at regular intervals
and heated to 100 °C for 5 min to inactivate enzyme. The formation
of XOs was monitored using high-performance liquid chromatog-
raphy (HPLC).
2.5. Analytical methods
Total sugars (TS) were determined according to the Orcinol–HCl
method with xylose as the standard. Reducing sugars (RS) were
determined by DNS (dinitrosalicylic acid) method with xylose as
the standard. The content of xylose and XOs was analyzed using
LC-10AD HPLC (Shimadzu, Japan), equipped with an InterWAX
(KS-802) column (DM) and a RID-10A refractive index detector.
The column was maintained at 80 °C and eluted with deionized
water at a flow rate of 0.6 mL min 1. The XOs yield (w/w) was cal-
culated using the equation: Yield (w/w) = XOs (g L 1)  Volume
(L)/xylan in raw corncobs (g), where XOs = X2 + X3 + X4 + X5 (X2,
xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose).
The content of X2 and X3 in XOs syrup was (X2 + X3)/XOs. The per-
cent yield of TS was calculated using the following equation: Hemi-
cellulose recovery (%) = TS yield (%) = TS in SELC (g L 1)  Volume
(L)  100/raw corncobs (g).
3. Results and discussion
3.1. Optimum conditions of steam explosion pretreatment
Steam explosion conditions were optimized to obtain maxi-
mum recovery of hemicellulose and to minimize the xylose con-
tent in SELC (data not shown). TS in SELC ranged from 16.1 to
21.6 g L 1 with the increment of temperatures from 188 to
204 °C when steam exploded for 5 min. The xylose content in SELC
at 188 and 196 °C was 15.4% and 21.4%, respectively whereas at
204 °C, the xylose content was greater than 40%. This indicates that
temperature is a critical factor in controlling the content of xylose.
Besides, TS increased from 18.7 to 20.9 g L 1 with the increment of
residence time from 2.5 to 7.5 min when steam explosion was car-
ried out at 196 °C. However, the lowest xylose content (21.4%) was
obtained when residence time was 5.0 min.
In order to reduce energy consumption, the effect of chip sizes
on the steam explosion pretreatment of corncobs was further eval-
uated (data not shown). The yields of hemicelluloses increased
from 22.3% to 28.4% and TS increased from 18.6 to 23.6 g L 1 with
decrease in chip sizes while DP decreased from 2.5 to 1.8. This indi-
cates that corncobs with smaller chip sizes significantly promoted
the solubilization of hemicellulose in corncobs during steam explo-
sion. However, chip sizes of 100 mm are considered to be good, in
which a low DP (1.9) with a hemicellulose recovery of 22.8% was
achieved.
The most important operational conditions in steam explosion
pretreatment are time, temperature and chip size. Results also
indicate that increasing steam temperature results in higher solu-
bilization of hemicellulose. Steam explosion pretreatment is gener-
ally performed at temperatures above 200 °C or using higher
severity parameters (Cara et al., 2008). However, pretreatment of
increased severity lead to increased solubilization of hemicellulose
and decomposition of XOs into xylose. Thus, a severe treatment is
not necessary to control substantial xylose content (less than 30%)
as shown in this study. The severity parameter adopted was mild
[log (R0) = 3.53], obtained with a steam temperature of 196 °C
and a residence time of 5 min. The mass ratio of steam to corncobs
was 0.17 kg steam/kg corncobs under this condition.
Chip sizes smaller than 100 mm did not have a significant influ-
ence on DP in this study. Prior to steam explosion, chip sizes are
usually reduced greatly, which requires significant amount of en-
ergy (Ballesteros et al., 2002). Corncobs are milled to rather small
chip sizes (<10 mm) for autohydrolysis of corncobs to produce
XOs (Ballesteros et al., 2002; Yuan et al., 2004). Taking into consid-
eration, the amount of compromise between energy consumption
and TS, as well as DP, corncobs with large chip sizes (100 mm)
exploded at 196 °C for 5 min are suitable to prepare SELC.
3.2. Morphological study
The raw and steam exploded corncobs under different condi-
tions were observed using a KYKY-2800 scanning electron
microscope operating at 20 kV, after sputter coating with gold–
palladium (data not shown). The raw corncobs exhibited a rigid
and compact surface structure and highly ordered fibrils. After
steam explosion, the surface was no longer smooth and covered
 C. Teng et al. / Bioresource Technology 101 (2010) 7679–7682
10
Sugar concentration (g L-1 )
8
0
0 0.5 1 1.5 2 2.5 3 3.5 4
Incubation time (h)
(a)
Fig. 1. Time course for production of XOs from hydrolysis of SELC by the thermostable xylanase (a) and TLC analysis of hydrolysis products (b). Symbols: XOs (N), xylobiose
(d) and xylotriose (j). Data represents the mean ± S.D. of three replicates. The hydrolysis conditions were as follows: xylanase, 7.5 U mL 1 (pH 7.0) at 70 °C for 4.0 h. Lanes
Xn, authentic xylose to xylopentose from top to bottom. Time of incubation (h or min) and xylooligosaccharides are indicated.
with grooves and ridges indicative of the organization of the
microfibrils within the fibers. The fibers of corncobs shattered with
some breaks across the fibers. Increasing the temperature or resi-
dence time resulted in muddy appearance. Most of the lignin–sac-
charidic complex in corncobs is lignin–xylan complex. SEM
confirmed that steam explosion is an effective method for causing
the breakdown of the lignin network polymer in corncobs. Simi-
larly, the fibers of steam explosion pretreated samples appear to
be distorted according to the previous results of SEM (Chen and
Liu, 2007; Wang et al., 2009).
3.3. Hydrolysis of SELC by the thermostable xylanase
Since previous study shows that the thermostable xylanase
from P. thermophila hydrolysed xylans to produce mainly xylobiose
and xylotriose (Li et al., 2006), the enzyme was used for production
of XOs. The effects of enzyme concentration and hydrolysis period
on the hydrolysis of SELC were carried out at 70 °C and pH 7.0 (data
not shown). XOs and xylobiose increased up to 8.3 and 4.3 g L 1,
respectively with the increment of enzyme concentration, whereas
increased slightly from 7.5 to 10.0 U mL 1. However, xylotriose in
SELC decreased with the increment of enzyme concentration.
Fig. 1 illustrates the time course for production of XOs from
hydrolysis of SELC by the thermostable xylanase. The accumulation
of xylobiose and xylotriose increased rapidly during the initial
0.5 h. After 0.5 h incubation, xylobiose increased slowly while
xylotriose decreased. XOs concentration reached 8.5 g L 1 by
hydrolysing SELC with 7.5 U mL 1 xylanase for 2.5 h, resulting in
a yield of 28.6% XOs from xylan in corncobs. The main hydrolysis
products of SELC were xylobiose and xylotriose, which contain
2.3 g L 1 xylose, 1.45 g L 1 arabinose, 0.3 g L 1 acetic acid,
0.26 g L 1 formic acid and 0.22 g L 1 furfural. The concentration
of xylobiose and xylotriose was 3.1 g L 1 which increased to
8.2 g L 1 after 2.5 h. Furthermore, XOs were mostly composed of
xylobiose and xylotriose (the content was more than 90%).
The conventional enzymatic hydrolysis in the production of XOs
is a time consuming process possibly due to use of mesophilic
xylanases having temperature optima of 40–60 °C (Yuan et al.,
2004; Akpinar et al., 2007; Yang et al., 2007). As compared to the
above mentioned results, the hydrolysis period in this study was
short (2.5 h) due to the use of thermostable xylanase from P. ther-
mophila. These results suggest that the thermostable xylanase is
7681
(b)
effective in hydrolysing SELC and shows a great potential in pro-
duction of XOs.
4. Conclusions
A process consisting of steam explosion pretreatment and
hydrolysis of SELC by the thermostable xylanase for the production
of XOs has been developed. Corncobs with larger chip sizes
(100 mm) exploded at 196 °C for 5 min were suitable for hydro-
lysis by the thermostable xylanase to produce XOs mainly with the
preferred DP. Finally, the total yield of XOs based on xylan in corn-
cobs was 28.6% and the syrup contained more than 90% of xylobi-
ose and xylotriose. The process appears to be a promising
technique for practical production of XOs.
Acknowledgements
This work is supported by the National Key Technology R&D
Program of China (No. 2006BAD07A08) and the National High
Technology Research and Development Program of China (863 Pro-
gram, No. 2006AA10Z337).
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