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, *

aBio-energy Research Institute, Chonnam National University, Gwangju 500-757,

Republic of Korea

bAdvanced Radiation Technology Institute, Korea Atomic Energy Research Institute,

Jeongeup 580-185, Korea

cDepartment of Forest Products and Technology (BK21 Program), Chonnam National

University, Gwangju 500-757, Republic of Korea

dDept of Chemistry and The AG MacDiarmid nanotech Institute, The University of Texas

at Dallas, TX 75080

eDepartment 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

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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 37–38% and 14–15%, 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

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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

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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 (Díaz et al., 2010;

Kim et al., 2006; Kim and Holtzapple, 2006; Liu and Wyman, 2005; Liu et al., 2009; Pérez

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 (Díaz 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 20°C min–1. When the temperature and pressure inside the reactor reached

220°C 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: 251–422 µm) 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: 70–75%). 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 37°C 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 30°C for 60 min with H2SO4

(72%), followed by dilution with water to give 4% sulfuric acid. The second hydrolysis

step was performed at 121°C 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

250°C, injector temperature 220°C, with oven temperature programmed to rise from

100°C (1.5 min) to 220°C at 5°C 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 85°C; 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 = (I002 –

Iam)/I002 ×100, where I002 is the overall intensity of the peak at 2� at about 22° and Iam 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% KMnO4

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 ± 0·1 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 ± 0·1 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 220°C. 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 37°C 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 µm.

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20

21

23

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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.