Accepted Manuscript

Enhanced Enzymatic Hydrolysis of Rapeseed Straw by Popping Pretreatment

for Bioethanol Production
Seung Gon Wi, Byung Yeoup Chung, Yoon Gyo Lee, Duck Joo Yang, Hyeung-
Jong Bae
PII: S0960-8524(11)00216-1
DOI: 10.1016/j.biortech.2011.02.031
Reference: BITE 8149
To appear in: Bioresource Technology
Received Date: 21 September 2010
Revised Date: 5 February 2011
Accepted Date: 6 February 2011
Please cite this article as: Wi, S.G., Chung, B.Y., Lee, Y.G., Yang, D.J., Bae, H-J., Enhanced Enzymatic Hydrolysis
of Rapeseed Straw by Popping Pretreatment for Bioethanol Production, Bioresource Technology (2011), doi:
10.1016/j.biortech.2011.02.031
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1

Enhanced Enzymatic Hydrolysis of Rapeseed Straw by Popping Pretreatment for
Bioethanol Production

Author list: Seung Gon Wi
a
, Byung Yeoup Chung
b
, Yoon Gyo Lee
c
, Duck Joo Yang
d
,
Hyeung-Jong Bae
a,c,e,
*

a
Bio-energy Research Institute, Chonnam National University, Gwangju 500-757,
Republic of Korea
b
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute,
Jeongeup 580-185, Korea
c
Department of Forest Products and Technology (BK21 Program), Chonnam National
University, Gwangju 500-757, Republic of Korea
d
Dept of Chemistry and The AG MacDiarmid nanotech Institute, The University of Texas
at Dallas, TX 75080
e
Department of Bioenergy Science and Technology, Chonnam National University,
Gwangju 500-757, Republic of Korea

* Corresponding author. Address: Department of Forest Products and Technology,
Chonnam National University, Gwangju 500-757, Republic of Korea; Tel.: +82 62 530
2097; fax: +82 62 530 0029.
E-mail address: baehj@chonnam.ac.kr (H.-J. Bae)

Short running title: popping pretreatment of rapeseed straw


2

ABSTRACT
The objective of this study was to find a pretreatment process that enhances enzymatic
conversion of biomass to sugars. Rapeseed straw was pretreated by two processes: a wet
process involving wet milling plus a popping treatment, and a dry process involving
popping plus dry milling. The effects of the pretreatments were studied both in terms of
structural and compositional changes and change in susceptibility to enzymatic hydrolysis.
After application of the wet and dry processes, the amounts of cellulose and xylose in the
straw were 3738% and 1415%, respectively, compared to 31% and 12% in untreated
counterparts. In enzymatic hydrolysis performance, the wet process presented the best
glucose yield, with a 93.1% conversion, while the dry process yielded 69.6%, and the un-
pretreated process yielded < 20%. Electron microscopic studies of the straw also showed a
relative increase in susceptibility to enzymatic hydrolysis with pretreatment.


Keywords: bioethanol, enzymatic hydrolysis, popping pretreatment, rapeseed straw

3

1. Introduction
Crude oil is a major resource that has been drawn on to meet increased energy demands
(Greene et al., 2004). However, high oil prices and public concerns over greenhouse gas
emissions have spurred interest in finding alternative fuel sources that could replace or
alleviate demand for crude oil, particularly for automotive liquid fuels (Keshwani and
Cheng, 2009).
Lignocellulosic biomass, defined as all natural vegetable and tree matter containing
carbohydrate compounds as main components, has great potential as an annually
renewable energy resource and has attracted much interest as a raw material for the
production of bioethanol (Alvira et al., 2009; Kumar, 2009). Production of bioethanol from
lignocellulosic biomass is a well-known process by which sugars are fermented into
ethanol using yeast. However, lignocellulosic biomass is made of sugar polymers, which
are not as easily saccharified and fermented (Chandra et al., 2007; Wi et al., 2009).
Processes used to produce ethanol efficiently from biomass include (i) pretreatment to
make the biomass readily hydrolyzable, (ii) enzymatic hydrolysis to convert cellulose and
hemicelluloses components to their sugar monomers, and (iii) fermentation of sugar
monomers to ethanol.
Enzymatic saccharification is considered a more promising technology than other
saccharification methods. While biomass pretreatment is not required for acid-catalyzed
saccharification, this method still has some disadvantages in terms of cost competitiveness
and environmental impacts. To provide efficient enzymatic degradation of lignocellulose,
the cellulosic fibers of the raw material must be rendered accessible to the enzymes. The
efficiency and effectiveness of cell wall saccharification are affected by many factors,
including feedstock characteristics, pretreatment technology, and hydrolysis conditions
such as use of enzyme mixtures and type (Mansfield et al., 1999). To achieve the highest

4

saccharification rate with a given feedstock, a pretreatment must render the fiber readily
degraded by the enzymes, recognizing that pretreatment must overcome the main factors
governing the ease of lignocellulose breakdown to fermentable monosaccharides, namely,
pore size (Chandra et al., 2007), cellulose crystallinity (Chang and Holtzapple, 2000), and
lignin removal (Mansfield et al., 1999).
Various substrate pretreatment processes have been used to alter the structure of
cellulose biomass, including biological, physical, and chemical processes or a combination
of these processes (Sun and Cheng, 2002). Diverse pretreatment methods have been
reported for various biomasses, making these biomasses potentially useful for industrial
applications. Many methods have reported high sugar yields, above 90% of the theoretical
yield for lignocellulosic biomasses such as woods, grasses, and corn (Daz et al., 2010;
Kim et al., 2006; Kim and Holtzapple, 2006; Liu and Wyman, 2005; Liu et al., 2009; Prez
et al., 2008; Teymouri et al., 2004; Wang et al., 2010).
Biological pretreatment utilizes wood-degrading fungi to modify the chemical
composition of lignocellulosic biomass, but requires careful control of growth conditions
and large amounts of space (Taniguchi et al., 2005; Zhang et al., 2007). Physical
pretreatment, such as hammer and ball milling, can procure smaller feedstock particles that
are more amenable to enzymatic hydrolysis. However, neither of these pretreatment
methods is considered commercially attractive at present. In chemical pretreatment, the
pulping process is already used commercially and is more effective for biomass containing
low lignin, but chemical processes in general significantly solubilize hemicelluloses and
have high negative environmental impact compared to biological and physical
pretreatments (Alvira et al., 2009).
Rapeseed straw, an agricultural waste product and a bio-oil extracted substrate, is a
lignocellulosic material that is abundant and inexpensive in European and Asian countries

5

(Karaosmanoglu et al., 1999). Previously studied chemical pretreatments of straw include
a high-pressure and hot water pretreatment (Daz et al., 2010), a phosphoric acid-acetone
pretreatment (Li et al., 2009), and a dilute sulfuric acid pretreatment (Jeong et al., 2010; Lu
et al., 2009). However, existing chemical methods are both expensive and environmentally
undesirable because of solvent recycling issues and corrosion and pollution from waste.
Conversely, biological and physical pretreatments are more environmentally friendly as
they do not require solvents and use chemicals with little or no generation of hazardous
waste.
Using a new approach, we have successfully developed a popping pretreatment method
that gives very high glucose yield from rapeseed straw treated with commercial enzymes.
This method employs a reactor that requires a short thermal reaction time without using
chemicals.

2. Methods
2.1. Substrates
Rapeseed straw was obtained from a field in Mooan, South Korea, after being harvested
for oil and air dried at ambient temperature to equilibrium moisture content. The dried
rapeseed straw was then cut into approximately 2-cm lengths and stored for pretreatment.

2.2. Popping pretreatment of rapeseed straw
Figure 1 illustrates the overall pretreatment of rapeseed straw applied in this work. For the
dry process, 100 g (dry weight, DW) of rapeseed straw was soaked in tap water for 1 day
at room temperature and administered the popping pretreatment. Popping pretreatment was
performed in a laboratory-scale cast iron cylindrical reactor with a total volume of 3 L, a
gas heater, a hatch, and a mechanical rotator (Fig. 2). The reactor was heated at a rate of

6

between 15 and 20C min
1
. When the temperature and pressure inside the reactor reached
220C and 21 kg f cm
1
, respectively, the sample was rapidly exposed to one atmospheric
pressure through a hatch attached to the reactor. After the popping pretreatment, popped
samples were ground for size reduction (particle size: 251422 m) in a Willy mill fitted
with stainless steel blades. For the wet process, 100 g (DW) of rapeseed straw were
fiberized in a single rotating disk atmospheric refiner and dehydrated in a centrifugal
dehydrator (moisture content: 7075%). Popping pretreatment was then conducted under
the same conditions as described above.

2.3. Enzyme assays
The commercial enzymes used for this study were cellulose (C8546, Sigma-Aldrich, St
Louis, MO, USA) from Trichoderma reesei and xylanase (X2753, Sigma) from
Thermomyces lanuginosus produced by submerged fermentation of a genetically modified
Aspergillus oryzae microorganism.
The activities of FPA, CMCase, avicelase, and xylanase from both enzymes were
measured versus Whatman filter paper (1%, w/v), CMC-Na (1%, w/v), avicel (1%, w/v)
and birch wood xylan (1.0%, w/v), respectively (Wood and Bhat, 1988). The reducing
sugar released was measured with the dinitrosalicylic acid (DNS) method. The activity of
-glucosidase was determined by measuring p-nitrophenyl from p-nitrophenyl
glucopyranoside (Kwon et al., 1992). One unit of -glucosidase was defined as the amount
of enzyme required to release 1 mol of p-nitrophenyl per minute under the assay
conditions. The activities of both cellulase and xylanase using filter paper, CMC, avicel,
xylan, and pNPG as the substrates are presented in Table 1.

2.4. Enzymatic hydrolysis of pretreated rapeseed straw

7

Enzymatic saccharification was conducted at 1% DM (w/v) initial substrate loading in a
conical tube (50 ml). A sample of pretreated rapeseed straw was soaked in sodium acetate
buffer (pH 5) with sodium azide as an antibiotic (0.2%) to prevent microbial
contamination. Enzymatic hydrolysis was performed at 37C with enzyme loading of 8
FPU cellulase g
1
biomass and 300 U xylanase g
1
biomass for 24 h. The hydrolytic
reaction was followed by measuring the carbohydrates in the hydrolyzates with a DNS
assay (Miller, 1959) or with high performance liquid chromatography (HPLC, Waters,
USA)

2.5. Chemical composition
The chemical composition (holocellulose, Klason lignin, organic solvent extractives, and
ash) of raw and pretreated rapeseed straw was determined using TAPPI Standard Methods
(1992). Quantitative and qualitative analyses of monosaccharide in the raw and pretreated
sample were conducted using a gas chromatograph. A two-step acid hydrolysis was
performed to quantify sugar polymers in the raw material and the sample after
pretreatment. The first hydrolysis step was performed at 30C for 60 min with H
2
SO
4

(72%), followed by dilution with water to give 4% sulfuric acid. The second hydrolysis
step was performed at 121C for 60 min. Myo-inositol was added as an internal standard
and the solution was neutralized with ammonia solution. An aliquot was reduced using 2%
sodium tetrahydroborate and the excess sodium tetrahydroborate was decomposed with
acetic acid. Alditol was acetylated with methylimidazole as a catalyst, followed by acetic
anhydride, and then extracted with dichloromethane. The samples were analyzed with a
chromatograph (CP-9100, Chrompack, Netherlands) equipped with a DB-225 capillary
column (30 m 0.25 mm ID, 0.25 m film thickness, J&W Scientific, CA, USA) and a
flame ionization detector. The operating conditions were as follows: detector temperature

8

250C, injector temperature 220C, with oven temperature programmed to rise from
100C (1.5 min) to 220C at 5C min
1
. Compounds were determined by comparing
retention times with those of standard compounds (Sigma).

2.6. HPLC analysis of carbohydrates in liquid phase
Samples for reducing-sugar analysis were taken from the reaction mixture at intervals of 6
h during the first 12 h and then at intervals of 12 h until reactions were complete. When the
concentrations of reducing sugars reached a plateau, the glucose and xylose contents in the
hydrolyzate were also determined by HPLC using a column (300 7.8 mm, Rezex RPM-
monosaccharide, Phenomenex, CA, USA) at 85C; distilled water was used as an eluent at
a flow rate of 0.6 mL min
1
. A refractive detector was used for the reducing-sugar analysis.

2.7. Crystallinity of rapeseed straw
The crystallinity of rapeseed straw before and after pretreatment was measured by X-ray
diffraction using a diffractometer with Cu K radiation at 40 kV and 30 mA (X'Pert PRO
MPD, XPANalytical, Netherlands). The samples were scanned and the intensity recorded
in a 2 range from 10

to 30

. The crystallinity of each sample was expressed in terms of a

crystallinity index (CrI) using the following equation (Segal et al., 1959): CrI = (I
002

I
am
)/I
002
100, where I
002
is the overall intensity of the peak at 2 at about 22 and I
am
is the
intensity of the baseline at 2 at about 18.

2.8. Surface characterization of rapeseed straw
Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to
study each substrate sample before and after the popping pretreatment. Each sample was
dehydrated in a graded ethanol series and then freeze-dried. Each sample was then

9

mounted on a stub and sputter-coated with gold prior to imaging with a field emission
scanning electron microscope (FE-SEM; JSM-7500F, Jeol, Japan) using 3 kV accelerating
voltage. The surface morphology of the rapeseed straw fibers was examined using AFM in
the tapping mode. Single fibers were mounted on magnetic holders using sticky tabs. AFM
measurements were made using a commercial AutoProbe CP system (XE-100, Park
System Inc., South Korea). A standard silicon tip was used and measurements were
recorded in the non-contact mode under ambient conditions. The morphology of rapeseed
straw in both pretreatment conditions was also imaged using a transmission electron
microscope (TEM; JEM 1010, Jeol, Japan) on an ultrathin section stained with 1% KMnO
4

solution and mounted on an uncoated nickel grid.

3. Results and Discussion
3.1. Chemical composition
Table 2 lists the chemical compositions of the prepared samples, such as organic solvent
extractives, ash, carbohydrates (glucose, xylose, arabinose, galactose, rhamnose, and
mannose), and Klason lignin. Untreated raw rapeseed straw contained 59.3% holocellulose,
16.5% Klason lignin, and 6.3% ash content, and its glucose and xylose concentrations
represented 31.7% and 12.5%, respectively, of the total sugars analyzed by GC. The major
glucose yield from cellulose increased, while the mannose yield from hemicellulose
significantly decreased after the popping pretreatment in both processes. In addition, the
xylose yield slightly increased, suggesting 4-O methylglucuronoxylan, a major component
in hemicelluloses (Preston et al., 2003), as a xylose source. After the treatment, the yields
of rhamnose, arabinose, and galactose were slightly lower than those of the control sample.

3.2. Enzymatic hydrolysis of pretreated rapeseed straw

10

The kinetic data of three samples prepared from the wet and dry processes, including the
control, are shown in Fig. 2A. After 24 h of hydrolysis with the mixture of cellulose and
xylanase, the highest concentration of reducing sugar reached was 6.0 01 g L
1
by the
wet process and 2.9 0.4 g L
1
by the dry process. In both cases, sugar release was
stopped after approximately 24 h. In contrast, only a negligible concentration of reducing
sugar (0.7 01 g L
1
) was produced in un-pretreated rapeseed straw. These results suggest
that the combined popping with wet-milling pretreatment was a key factor in obtaining
high reducing-sugar yields from rapeseed straw.
After 24 h of hydrolysis, the HPLC results showed that the highest levels of detectable
glucose and xylose were found with the wet process (Fig. 2B). GC results for the chemical
composition of rapeseed straw residues between pretreated and post-enzymatic hydrolysis
also showed that the wet process presented the best conversion to glucose (Table 3). Thus,
overall, the wet milling combined with popping pretreatment process yielded the highest
sugar contents from enzymatic hydrolysis.

3.3. X-ray diffraction analysis
The crystallinity of cellulose, representing its accessible surface area protected by lignin
and hemicellulose, is believed to have a significant effect on enzymatic saccharification of
glucan (Zhang and Lynd, 2004). X-ray diffraction analysis results showed that the wet
process yielded straw residues of lower crystallinity compared to residues from the dry
process (data not shown). The lignocellulose complex includes cellulose, hemicellulose,
and lignin. The crystallinity of pretreated material decreased, implying not only the
breakdown of the crystalline cellulose region, but also an increase in the amorphous
regions, hemicellulose and lignin, which occur in high-energy treatment processes such as
steaming (Mosier et al., 2005). Therefore, our result suggests that the wet process

11

developed in this study was very effective in reducing cellulose crystallinity. Our wet
milling-popping process, on the other hand, may have caused thermal damage to cell walls,
disrupting hemicellulose and lignin and leading to deformation of crystallites.

3.4. Surface morphology
Since direct physical contact between enzymes and the treated substrate is required for
hydrolysis, the amount of surface area available for such contact is of primary importance
to the reaction rate (Lee et al., 2010). Thus, we were highly interested in examining the
physical changes in the biomass, as enzymatic hydrolysis on rapeseed straw was
significantly increased by the popping treatment. For this purpose, SEM, AFM, and TEM
were used to gather information on the effect of the popping pretreatment and enzymatic
hydrolysis on the ultra-structure and possible disruption of the cell wall. The sample
pretreatment affected the shape and size of the fibers considerably, as revealed by SEM
and AFM examinations of the substrates (Supplementary Fig. S1A-C). The rapeseed straw
fibers were shattered after the popping process, with some breaks observed across the
fibers, accompanied by disruptions in surface morphology of rapeseed straw
(Supplementary Fig. S1D-I). The removal of non-cellulosic materials by the popping
process resulted in rougher surfaces and better microfibril exposure, thus enhancing
enzymatic hydrolysis. These observations were similar to those reported by Lee et al.
(2010), who investigated the fibrillation of woody biomass using a batch-type kneader
with twin-screw elements.

3.4. Surface morphology
Cell wall ultra-structure before and after enzymatic hydrolysis as examined by FE-SEM
and TEM (Supplementary Fig. S2) showed that the microfibril in cell walls before

12

hydrolysis exhibited intact dense structure, but was heavily degraded and disconnected
after hydrolysis (Supplementary Fig. S2B). TEM showed similar drastic changes in cell
wall integrity after enzymatic hydrolysis (Supplementary Fig. S2D), at which point the
dense cell walls were disintegrated by hydrolysis into less-dense walls with holes,
suggesting that most of the cellulose was hydrolyzed by glucose digestibility (93%)
(Supplementary Fig. S2D).

3.5. Overall mass balance
An overall mass balance diagram describing the process stages from pretreatment to
enzymatic hydrolysis was created (Fig. 4). Rapeseed straw was mechanically defibrated to
reduce its size using a single-disk refiner and 100 g (DW, M.C. 70%) of fiber was popped
at 220C. For this process, temperature and pressure were gradually increased using a
propane gas burner. The pretreated sample was recovered and enzymatic hydrolysis was
performed on 1 g (DW) of recovered solids with an enzyme loading of 8 FPU cellulose
and 300 U xylanase at 37C for 24 h. Enzymatic hydrolysis yielded 25.1 g of glucose and
8.7 g of xylose per 100 g of raw material. The overall mass balance showed approximately
93.1% of cellulose in the pretreated biomass. These results indicate that the cost of
biomass ethanol can be significantly reduced by high sugar yields, obtained by low
enzyme loadings without chemical additions.

4. Conclusions
Two processes were studied to improve enzymatic conversion of biomass to sugars. After
application of wet and dry processes, the amounts of cellulose and xylose from the straw
were 37.2% and 15.2%, and 38.0% and 14.1%, respectively, compared to 31.7% and
12.5% for untreated substrates. The best enzymatic hydrolysis performance was from the

13

wet process, which yielded a 93.1% conversion of cellulose to glucose compared to the dry
(69.6%) and un-pretreated processes (< 20%). From these results, we conclude that the wet
process is preferable. Our conclusions were supported by microscopy and by chemical
analysis of structural and compositional changes in pretreated and enzymatically
hydrolyzed substrates.

Acknowledgements
This work was supported by Priority Research Centers Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (Project No. 2010-0020141) to H.-J. Bae and from Nuclear R&D program
through the National Research Foundation of Korea funded by the ministry of Education,
Science and Technology. YGL is grateful for the BK21 program provided by the Ministry
of Education.

Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version.





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Figure Captions
Fig. 1. Schematic diagram of pretreatment process.
Fig. 2. Diagram of laboratory-scale popping machine.
Fig. 3. Time course of changes in glucose concentration by the DNS method in

18

experiments with a mixture of cellulase and xylanase (A) and changes in glucose and
xylose concentration by HPLC analysis after 24 h enzymatic hydrolysis (B).
Fig. 4. Overall mass balance of rapeseed straw using the wet process and enzymatic
hydrolysis.

Supplementary Fig. S1. FE-SEM (A-F) and AFM (G-I) images of rapeseed straw powder
pretreated with popping. Untreated rapeseed straw (A, D, and G), rapeseed straw
pretreated with dry (B, E and H) and wet (C, F and I) processing. Bar: 100 nm.
Supplementary Fig. S2. FE-SEM (A and C) and TEM (B and D) images of popping
pretreated rapeseed straw before (A and B) and after (C and D) enzymatic hydrolysis. Bar:
1 m.

19




20




21




23




24


Table 1
Specific activities of both commercial cellulase and xylanase used in this experiment.
(U mg
-1
protein) Filter paper CMC Avicel Xylan p-NPG
Cellulase
0.2
15.4 1.4 4.3 123.6
Xylanase
-
0.8 - 7.1 36.9
Mean values of three determinations.


25


Table 2
Changes in percent composition of components of rapeseed straw depending on each
process. Organic solvent extractives, holocellulose, Klason lignin, and ash were analyzed
by the TAPPI standard method and monosaccharide was analyzed using GC. Components:
Ara (arabinose), Man (mannose), Gal (galactose), Glu (glucose), HL (holocellulose), KL
(Klason lignin), OSE (organic solvent extractives), Rham (rhamnose), Xyl (xylose).
HL (% of dry matter)
OSE
Rham Ara Xyl Man Gal Glu Total
KL Ash
59.3
Control 0.8
0.6 1.0 12.5 9.7 1.5 31.7 56.99
16.5 6.3
57.6
Dry
process
4.4
0.4 0.4 14.1 1.9
***
1.0 38.0
**
55.79
19.5 3.2
61.6
Wet
process
4.3
0.4 0.5 15.2 4.2
***
1.1 37.2
*
58.54
16.2 2.1
Mean values of three determinations.
***; P<0.001, **; P<0.01, *; P<0.05




26


Table 3
Chemical composition of residues of rapeseed straw before and after enzymatic hydrolysis
by GC. D; sample after dry process, Cel; cellulase, W; sample after wet process, Xyl;
xylanase. Components: Ara; arabinose, Man; mannose, Gal; galactose, Glu; glucose,
Rham; rhamnose, Xyl; xylose.
Rham Ara Xyl Man Gal Glu Total
D 0.2 0.5 12.1 2.0 0.8 44.5 60.1
D: Cel+Xyl 0.1 0.1 2.2 1.0 0.3 16.4 20.1
W 0.2 0.1 8.6 2.1 0.4 48.5 59.9
W: Cel+Xyl 0.1 0.2 3.3 1.3 0.5 4.2 9.7
Mean values of three determinations.