Improved enzymatic hydrolysis yield of rice straw using electron beam irradiation pretreatment

Bioresource Technology 100 (2009) 1285–1290

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Improved enzymatic hydrolysis yield of rice straw using electron beam irradiation pretreatment Jin Seop Bak a, Ja Kyong Ko a, Young Hwan Han c, Byung Cheol Lee c, In-Geol Choi b, Kyoung Heon Kim a,b,* a

Brain Korea 21 School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea c Quantum Optics Research Division, Korea Atomic Energy Research Institute, Daejeon 305-353, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 4 July 2008 Received in revised form 4 September 2008 Accepted 4 September 2008 Available online 17 October 2008 Keywords: Electron beam irradiation Lignocellulose Biomass Pretreatment Biofuel

a b s t r a c t Rice straw was irradiated using an electron beam at currents and then hydrolyzed with cellulase and bglucosidase to produce glucose. The pretreatment by electron beam irradiation (EBI) was found to significantly increase the enzyme digestibility of rice straw. Specifically, when rice straw that was pretreated by EBI at 80 kGy at 0.12 mA and 1 MeV was hydrolyzed with 60 FPU of cellulase and 30 CBU of b-glucosidase, the glucose yield after 132 h of hydrolysis was 52.1% of theoretical maximum. This value was significantly higher than the 22.6% that was obtained when untreated rice straw was used. In addition, SEM analysis of pretreated rice straw revealed that EBI caused apparent damage to the surface of the rice straw. Furthermore, EBI pretreatment was found to increase the crystalline portion of the rice straw. Finally, the crystallinity and enzyme digestibility were found to be strongly correlated between rice straw samples that were pretreated by EBI under different conditions. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Cellulose is the most abundant renewable biomass on earth (Klemm et al., 2005). In addition, the high price of oil and global warming have emphasized the importance of biofuels such as bioethanol (Farrell et al., 2006). Therefore, the production of biofuels from lignocellulosic resources has become an important area in the development of alternative energy sources (Lynd et al., 2008; Orts et al., 2008; Waltz, 2008). Recently, the selection of raw materials for biofuel production has become a critical issue due to the conflict between food and energy resources for fuel production (Cassman and Liska, 2007). Accordingly, non-food resources are now being considered as feedstocks for biofuel production. For example, the US Department of Energy set a goal to replace 30% of the current gasoline used in the US with cellulosic ethanol by 2030 (Sherwood, 2006). To achieve such an aggressive goal, significant improvement in the production cost of cellulosic biofuels is required. Lignocellulose, which is a complex of cellulose, hemicelluloses and lignin, only renders approximately 20% of its theoretical glucose yield upon subjection to enzymatic hydrolysis due to its recal-

* Corresponding author. Address: Brain Korea 21 School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea. Tel.: +82 2 3290 3028; fax: +82 2 925 1970. E-mail address: [email protected] (K.H. Kim). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.09.010

citrance (Kim et al., 2006; Koullas et al., 1992). Therefore, lignocellulose needs to be pretreated to enable the cellulose to be more accessible to cellulolytic enzymes. Accordingly, many physicochemical pretreatment processes for lignocellulose have been evaluated in the last few decades, including a variety of physical and chemical pretreatments (Gharpuwy et al., 1983; Jeoh et al., 2007; Lee and Kim, 1983). These studies have led to the development of highly effective thermochemical processes, such as acid or alkaline pretreatment, which enable 80–90% of the theoretical enzyme digestibility of cellulase to be attained (Kim et al., 2002; Merino and Cherry, 2007; Zhao et al., 2008). However, these thermochemical pretreatment processes often result in the generation of byproducts such as furfural, hydroxymethylfurfural (HMF) and acetic acid, which significantly inhibit enzymatic hydrolysis and fermentation (Haagensen et al., 2008). Therefore, it is necessary to develop enzymes and microorganism that are resistant to such inhibitory substances or to employ additional steps to remove the inhibitors. However, both of these methods poses further cost burdens on the production of biofuel from lignocellulose. Therefore, pretreatment and enzymatic hydrolysis steps to achieve fermentable sugar are currently known to have much more room for reducing processing cost than other processes (Lynd et al., 2008). The milling process, which is a physical pretreatment method known to increase the surface area of biomass, was studied during the early stages of lignccellulose pretreatment. However, the high energy consumption and low effectiveness of this method

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prevented its application (Gharpuwy et al., 1983; Lee and Kim, 1983). Alternatively, c-ray (Beardmorel et al., 1980; Khan et al., 2006) and electron beam irradiations (Kamakura and Kaetsu, 1978; Khan, 1986) were considered as physical pretreatment processes. Because these methods do not involve the use of extreme temperatures, the generation of inhibitory substances produced during acid or alkali pretreatment can be either avoided or minimized. For example, when electron beam irradiation (EBI) was applied to lignocellulosic feedstocks such as rice straw, sawdust and softwood spruce (Kamakura and Kaetsu, 1978; Khan, 1986), it was found to increase the cellulase digestibility of the biomass (Khan, 1986). However, when compared to the vast amount of information available regarding thermochemical pretreatment, there is very little information available regarding the effectiveness and mechanisms involved in EBI pretreatment. Therefore, this study was conducted to verify the effectiveness and feasibility of EBI as a lignocellulose biomass pretreatment method for the enhanced enzymatic hydrolysis of cellulose for biofuel production. To accomplish this, rice straw was pretreated with EBI, after which its impact was evaluated based on the enzymatic digestibility (with cellulase and b-glucosidase) and various physical characteristics of the pretreated rice straw.

creases, which results in an increase in electric current. Therefore, both the electric current in mA and time were regarded as the same parameter. In this experiment, the current used ranged from 0.03 to 0.24 mA and the irradiation lasted from 4 to 36 min depending on the current used. 2.4. Analysis of rice straw The composition the rice straw and the pretreated rice straw and the concentration of inhibitory compounds such as acetic acid, furfural and HMF were analyzed following the Standard Biomass Analytical Procedures of National Renewable Energy Laboratory (NREL, 2008). Analysis revealed that the rice straw contained 35.7% glucan, 3.7% mannan, 2.5% galactan, 10.8% xylan, 3.3% arabinan and 19.7% lignin on a dry weight basis. For the analysis of sugar from biomass, an HPLC (Agilent 1100, Agilent Technologies, Waldbronn, Germany) equipped with a Shodex SP-0810 column (Pb2+ form; Showa Denko, Tokyo, Japan) and a refractive index detector (Agilent Technologies) was used with a mobile phase of HPLC-grade water at a flow rate of 0.5 ml/min at 80 °C. Inhibitors were quantified by the HPLC with an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) and a refractive index detector, and 0.01 N H2SO4 was run at 0.6 ml/min at 55 °C.

2. Methods

2.5. Enzyme digestibility test

2.1. Biomass feedstock

To evaluate the increase in the enzyme digestibility of the rice straw following EBI pretreatment, untreated and pretreated rice straw samples were hydrolyzed using Celluclast 1.5L and b-glucosidase in triplicate following the NREL standard procedure (NREL, 2008). The hydrolysis reaction was conducted using unwashed pretreated rice straw with 5, 15 and 60 FPU of cellulase and 30 CBU of b-glucosidase per g of glucan in 0.05 M sodium citrate buffer at pH 4.7 and 50 °C. Samples that were pretreated by EBI were then used in an enzyme digestibility test without washing. Samples were then collected at certain time intervals and then filtered using a 0.2 lm syringe filter (PVDF syringe filter; Whatman). The filtrate was then analyzed by HPLC using the Shodex SP-0810 column. The enzyme digestibility was expressed as the percentage of the theoretical maximum amount of glucose obtained from the biomass substrate, as shown in Eq. (1).

Rice straw harvested from Korea University Farm (Deokso, Korea) in 2006 was air-dried at ambient temperature. The dried rice straw was then milled using a cutting mill (MF 10, IKA, Staufen, Germany), sieved using two sieves with mesh sizes of 425 and 710 lm (Chung Gye Sang Gong Sa, Seoul, Korea), and then dried in a vacuum drying oven (SH-45S, BioFree, Seoul, Korea) at 45 °C for 5 days. The resulting solid content of the milled and dried rice straw measured at 105 °C was 96.6% (w/w). The rice straw samples were then packed in a polystyrene bag under vacuum until EBI pretreatment. Avicel (Sigma–Aldrich, St. Louis, MO, USA) and filter paper (Whatman No. 1, Whatman, Brentford, UK) were used as pure cellulose in this study. 2.2. Enzymes The enzymes used for the saccharification of pretreated rice straw, untreated rice straw and Avicel were cellulase (Celluclast 1.5L, Sigma) and b-glucosidase from Aspergillus niger (Novozyme 188, Sigma). The enzymatic activity of the cellulase and b-glucosidase as received from the manufactures were determined to be 84 filter paper units (FPU) ml1 and 963 cellobiase units (CBU) ml1, respectively. 2.3. Electron beam irradiation Milled dry rice straw was irradiated with accelerated electrons, which were driven in pairs through bridge couplers from 1 to 10 kW klystrons by a linear electron accelerator that had the capacity to produce electron beams of up to 0.24 mA and 2 MeV (Korea Atomic Energy Research Institute, Daejeon, Korea). For the irradiation of the rice straw samples, electrons generated by an electron gun were accelerated at a high frequency and sprayed through an electromagnet to a plate where the sample was placed. The EBI pretreatment was applied uniformly over the sample area on a plate. The irradiation dose was determined based on the sum of an integrated charge, and the doses used were 7.6, 20, 50, 80 and 90 kGy, where 1 Gy is equivalent to 100 rad. The strengths were 1, 1.5 and 2 MeV. As time increases, the number of electrons in-

Glucose yield ð%theoretical maximumÞ ¼

g of glucose by HPLC  100 g of glucan  1:1

ð1Þ

2.6. Analysis by X-ray diffractometry and scanning electron microscopy The crystallinity index (CrI) of the pretreated rice straw samples was determined using a powder X-ray diffractometer (Bruker D5005, Karlsruhe, Germany). The operating conditions were 40 kV, 40 mA, an angular range of from 100 to 168, and a multi-layer mirror for high intensity was gained by parallel beam. The diffraction spectra were collected using the h2h method (Fan et al., 1980; Kim et al., 2003; Segal et al., 1959). Briefly, samples were scanned in duplicate at 1° min1 from 2h = 3–40° with a step size of 0.02°. Radiation (Cu Ka) is 1.5406 Å. The amount of water contained in the sample was characterized by X-ray, where the maximum intensity of water was at 2h = 28°. The CrI was calculated by Eq. (2) using the intensities of crystalline region at 2h = 22–22.5° and the amorphous region at 2h = 18°, respectively.

Crystallinity index ðCrIÞ ¼ where I : Intensity:

Icrystalline  Iamorphous  100 Icrystalline ð2Þ

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Scanning electron microscopy (SEM) was conducted to analyze microstructural changes on EBI-pretreated rice straw samples. Prior to analysis by SEM, rice straw samples were dried in a vacuum drying-oven at 45 °C for 5 days, after which they were coated with gold-palladium. Photomicrographs of the samples were then taken using a scanning electron microscope (Hitachi S-4700, Tokyo, Japan) at a voltage of 10 kV. 3. Results and discussion 3.1. Effect of EBI current After the rice straw samples were pretreated by EBI at various currents at fixed doses and strengths, they were hydrolyzed by the addition of cellulase at a concentration of 60 FPU per g of glucan for 120 h. Fig. 1 shows the yields of glucose from glucan (calculated based on% theoretical maximum as in Eq. (1) in samples that were taken at the specified reaction time intervals. As the hydrolysis reaction time increased, the accumulated glucose yield, which indicates the enzymatic digestibility of lignocellulose, increased. The pure cellulose with moderate crystallinity, Avicel, showed the highest glucose yield, which was 88.5% after 120 h of hydrolysis. However, untreated rice straw exhibited a glucose yield of only 22.6% after 120 h of hydrolysis. Regarding the pretreated rice straw by EBI, the glucose yield was significantly higher than that of the untreated rice straw. When currents were used from 0.03 to 0.12 mA, both the production rate of glucose and the extent of the reaction increased as the EBI current increased. The glucose yields after 120 h of hydrolysis were 52.1% and 52.2% from the pretreated rice straw at 0.12 and 0.24 mA, respectively. Therefore, the increase of current from 0.12 to 0.24 mA did not result in a significant increase of glucose yield. This finding indicates that when a fixed dose and strength of 80 kGy and 1 MeV are used, an optimal current for the effective pretreatment of rice straw exists. The overall enzymatic digestibilites of the EBI-pretreated rice straw, shown as glucose yields, were generally lower than those obtained from lignocellulose pretreated by conventional physicochemical or chemical methods such dilute-acid (Kim et al., 2002; Merino and Cherry, 2007), ammonia fiber explosion (AFEX) (Teymouri et al., 2005), soaking in aqueous ammonia (SAA) (Kim

60

40

20

0

0

20

Fig. 2 shows the results of varying the dose of EBI on the pretreatment of rice straw when the MeV and mA were both fixed at 1. Although there was no correlation between irradiation dose and enzyme digestibility, a dose of 80 kGy produced the highest glucose yield at each reaction time. Following 132 h of hydrolysis, a higher glucose yield (52.1%) was obtained when a dose of 80 kGy was used in comparison with those at lower doses such as 7.6, 20 and 50 kGy. Probably, lower doses were too weak to bring about significant changes in the glucose yield. However, treatment with a dose of 90 kGy resulted in a decreased glucose yield, most likely because the components of the lignocellulose decomposed at the higher dose (Kamakura and Kaetsu, 1978). Pretreatment at 7.6 kGy resulted in the second highest glucose yield. Irradiation with doses of 20, 50 and 90 kGy produced similar glucose yields that were all lower than those observed when samples were irradiated with 80 and 7.6 kGy. In a study conducted to evaluate pretreatment by EBI, approximately 30–80% of the theoretical maximum reducing sugar yield and 14–37% of the theoretical maximum glucose yield were obtained after 26 h of enzymatic hydrolysis conducted using rice straw that had been pretreated in water by EBI at 2 MeV, 2 mA and a dose of 0.1–5 MGy (Kamakura and Kaetsu, 1978). In addition, in that study, the irradiation was run for up to 250 min at 5 MGy depending upon the irradiation dose. In another study that evaluated the effect of EBI pretreatment of spruce wood, a glucose yield of 40% on the basis of the input materials was achieved after 72 h of enzymatic hydrolysis following EBI pretreatment at 2 MGy (Khan, 1986). In both studies, the enzyme digestibility increased as the dose of EBI increased in the range of 0–5 MGy. 3.3. X-ray diffractometry and crystallinity index Fig. 3 shows the X-ray diffractograms of rice straw untreated or pretreated by EBI and Avicel. The intensities of the amorphous and

100

Avicel Untreated 0.03 mA 0.06 mA 0.12 mA 0.24 mA

80

3.2. Effect of dose of EBI

Glucose yield (% theoretical maximum)

Glucose yield (% theoretical maximum)

100

and Lee, 2005), ammonia recycle percolation (ARP) (Kim et al., 2006) and alkaline pretreatments (Merino and Cherry, 2007; Zhao et al., 2008), which exhibited 71–99%, 83–98%, 80–98%, 86–95% and 30–78%, respectively.

40

60

80

100

120

140

Hydrolysis time (h) Fig. 1. Effect of current of electron beam irradiation (at 1 MeV and 80 kGy) on the enzyme digestibility of unwashed pretreated rice straw. The enzymatic hydrolysis was conducted using 60 FPU of cellulase (Celluclast 1.5L) and 30 CBU of bglucosidase (Novozyme 188) per g of glucan at pH 4.8, 50 °C and 150 rpm. Data shown represent the mean ± standard deviation of experiments that were conducted in triplicate.

80

12 h 24 h 48 h 132 h

60

40

20

0

Untreated

7.6

20

50

80

90

Dose of irradiation (kGy) Fig. 2. Effect of dose of electron beam irradiation (at 1 MeV and 0.12 mA) on the enzyme digestibility of unwashed pretreated rice straw. The enzymatic hydrolysis was conducted using 60 FPU of cellulase (Celluclast 1.5L) and 30 CBU of bglucosidase (Novozyme 188) per g of glucan at pH 4.8, 50 °C and 150 rpm. Experimental data presented represent the mean ± standard deviation of experiments conducted in triplicate.

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60

1600 Untreated EBI-1-90 EBI-1-80 Avicel

Intensity

1200

Y= - 0.9131 X + 60.0265 R2 = 0.5107

55

Glucose yield (% theoretical maximum)

1400

1000 800 600 400

50 45 40 35 30 25

200 0 10

20

30

40

2θ

20 10

15

20

25

30

35

40

Crystallinity index (CrI)

Fig. 3. X-ray diffraction diagram of untreated rice straw, rice straw that was pretreated by EBI and Avicel. Pretreatment by EBI was conducted at 0.12 mA and 1 MeV and at 80 or 90 kGy. The intensities of the amorphous region at 2h = 18° and the crystalline region at 2h = 22–22.5° were used to calculate the crystallinity index (CrI).

Fig. 4. Correlation of the crystallinity index (CrI) with the enzyme digestibility of rice straw pretreated by EBI under different conditions. EBI was conducted at 0.12 mA, at 1, 1.5 and 2 MeV and at 7.6, 20, 50, 80 and 90 kGy. Enzymatic hydrolysis was conducted using 60 FPU of cellulase (Celluclast 1.5L) and 30 CBU of b-glucosidase (Novozyme 188) per g of glucan at pH 4.8, 50 °C and 150 rpm.

crystalline regions were measured at 2h = 18° and 2h = 22–22.5°, respectively. In lignocellulose, hemicellulose and lignin are considered to be amorphous components while cellulose is considered to be the crystalline component (Gharpuwy et al., 1983; Jeoh et al., 2007). Following EBI pretreatment at both 80 and 90 kGy, the intensity of the amorphous region of rice straw decreased slightly; however, the intensity of the crystalline region increased compared to those of the amorphous and crystalline regions in the untreated rice straw, respectively. In general, the crystalline portion of native cellulose is 70% (Fan et al., 1987). In the present study, the crystalline portion of the untreated rice straw was determined to be 54.5%, but this value slightly increased to 58.0% and 57.2% after pretreatment by EBI at 80 and 90 kGy (at 0.12 mA and 1 MeV), respectively. This likely occurred due to exposure of the crystalline portion of the rice straw in response to pretreatment. Based on these results, it is probable that the cellulose portion, which is crystalline in nature, became more exposed than the amorphous portion due to the effects of EBI. In the lignocellulose complex structure, cellulose microfibrils are protectively surrounded by hemicellulose and lignin. Therefore, the EBI may have disrupted the hemicellulose and lignin, which may have opened up the cellulose. The relationship between crystallinity and enzyme digestibility by cellulase has been the subject of many previous studies (Fan et al., 1987; Gharpuwy et al., 1983; Kim et al., 2003; Puri, 1984; Sasaki et al., 1979). Fig. 4 shows the correlation of the crystallinity index of rice straw that has been pretreated under various conditions by EBI with the extent of cellulose hydrolysis. The range of the CrI of the pretreated rice straw was found to be 10–40. In addition, the CrI of micrystalline cellulose, Avicel, was determined to be 78–80 in this study, which agrees well with the CrI of microcrystalline cellulose reported in a previously conducted study (e.g., 88) (Fan et al., 1987). In this study, as the CrI of rice straw decreased, its enzyme digestibility increased with a correlation coefficient slightly greater than 0.5. In addition, the majority of previously conducted studies report that the crystallinity index of cellulose or lignocellulose is inversely proportional to the extent of hydrolysis of cellulose (Gharpuwy et al., 1983; Sasaki et al., 1979), even though some studies have found cellulose crystallinity and enzyme digestibility to be irrelevant (Puri, 1984). Furthermore, as evidence of the negative correlation between crystallinity and digestibility, the gradual increase in the crystallinity of cellulose that occurred

during enzymatic hydrolysis indicates that the amorphous fraction of cellulose is preferentially hydrolyzed by cellulase (Bertran and Dale, 1985; Jeoh et al., 2007). 3.4. Scanning electron microscopy SEM revealed ultrastructral changes in rice straw following EBI pretreatment. When compared to the structure of untreated rice straw, which had a continuous, even and smooth flat surface, the EBI-pretreated rice straw had a rugged, unsmooth and broken face. In addition, the internal surface of the lignocellulose was more exposed, possibly due to bombardment with electrons during EBI. 3.5. Compositional change of pretreated rice straw Table 1 presents the changes in the mass of each primary biomass component in rice straw following pretreatment by EBI under several different conditions. The change in total mass following EBI pretreatment was negligible to within the error range, regardless of pretreatment conditions. Among the three major biomass components, xylan and lignin exhibited higher losses of mass (5–12% and 1–7%, respectively) following pretreatment at all operating conditions of EBI. In addition, the glucan mass decreased by 1–3% following EBI pretreatment. It is generally known that a cellulose microfibril formed by elementary fibrils cemented with hemicellulose is surrounded by a protective layer of hemicellulose and lignin complex (Fan et al., 1987). Therefore, the hemicellulose and lignin layers may have had a greater chance of being hit by electrons during EBI. These consequences may have appeared as higher losses of xylan and lignin, as shown in Table 1. Acid or alkali pretreatment of lignocellulose primarily removes hemicellulose or lignin, respectively (Esteghlalian et al., 2001; Kim et al., 2003, 2006; Kim and Lee, 2005; Sun and Cheng, 2002; Zhao et al., 2008). In this study, the amounts of removed hemicellulose (e.g., xylan) and lignin were similar or slightly higher than that of glucan, but the extent of removal of such lignocellulose components was much lower than occurs following physicochemical pretreatment using acid or alkali. For example, as shown in Table 1, the formation of monomeric sugars, which are predominantly produced from hemicellulose during dilute-acid pretreatment (Kim et al., 2002), was almost negligible in response to EBI treatment (0.6– 1.3% theoretical maximum). In addition, pretreatment using

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J.S. Bak et al. / Bioresource Technology 100 (2009) 1285–1290 Table 1 Analytical results of biomass components of rice straw following EBI pretreatment Conditions of EBI pretreatment

Loss of glucan (w/w, %)a

Loss of xylan (w/w, %)b

Loss of lignin (w/w, %)c

Loss of total mass (w/w, %)d

Formation of monomeric sugars (% theoretical yield)e

HMF (w/w, %)f

Furfural (w/w, %)g

Acetic acid (w/w, %)h

1 Mev, 80 kGy at 0.12 mA 1 Mev, 90 kGy at 0.12 mA 2 Mev, 80 kGy at 0.12 mA

2.31 ± 0.71 2.05 ± 1.27 1.23 ± 1.57

12.09 ± 7.35 8.92 ± 1.87 6.11 ± 2.03

7.65 ± 0.13 2.06 ± 0.49 1.56 ± 1.07

0.02 ± 0.01 0.01 ± 0.00 0.01 ± 0.00

1.25 ± 0.09 0.99 ± 0.07 0.64 ± 0.05

N.D.i N.D.i N.D.i

N.D.i 0.019 ± 0.010 N.D.i

Tracej Tracej Tracej

a

Determined as

g of untreated glucan-g of treated glucan g of initial weight of glucan

b

Determined as

g of untreated xylan-g of treated xylan g of initial weight of xylan

 100.  100.

 100.

c

Determined as

g of untreated lignin-g of treated lignin g of initial weight of lignin

d

Determined as

g of untreated RS-g of treated RS g of initial weight of RS

e

Determined as

g of monomeric sugars of untreated RS-g of monomeric sugars of pretreated RS g of total monomeric sugars of RS

f

Determined as

g of HMF of treated RS g of initial weight of RS

g

Determined as

g of furfural of treated RS g of initial weight of RS

h

Determined as

g of aceticacid of treated RS g of initial weight of RS

j

 100.

 100.  100.  100.

N.D.-not detected. Trace-smaller than 0.002% (w/w).

ammonia generally results in the removal of 50–78% and 70-85% of the initial content of lignin present in corn stover following pretreatment by SAA (Kim and Lee, 2005) or ARP (Kim et al., 2006), respectively. However, the loss of lignin in the present study was only 1–8%. In addition, EBI pretreatment without the use of chemicals and water exerted somewhat different compositional changes compared to conventional physicochemical pretreatment using acid or alkali. Furfural and HMF are generally considered to be the primary inhibitors generated by high temperature (Boussaid et al., 1999) or acid pretreatment (Lohmeier-Vogel et al., 1998; Roberto et al., 2003). However, some carboxlylic acids that are known to be produced through saponification during alkaline pretreatment may also cause inhibition during fermentation (Sun and Cheng, 2002). These inhibitors could act as obstacles to either the enzymatic hydrolysis or ethanol fermentation. Generally, inhibitors generated from acidic or alkaline pretreatment processes are more toxic to yeast or cellulase, respectively, during ethanol production from lignocellulose (Haagensen et al., 2008). In enzymatic hydrolysis, unwashed acid-pretreated corn stover was found to have lower cellulose conversion (approximately 30% of theoretical maximum in glucose yield) than washed acid-pretreated corn stover (Merino and Cherry, 2007). Therefore, washing pretreated biomass or an inhibitor removal step could be an essential in preventing a lower conversion yield from pretreated biomass. In this study, the generation of such inhibitory substances was either negligible or not detected (Table 1) although the enzyme digestibility of the EBIpretreated rice straw was generally lower than those of lignocellulose pretreated by the conventional physicochemical or thermochemical methods. 3.6. Effect of enzyme loading Fig. 5 shows the effect of the loading size of cellulase on the hydrolysis yield of glucose from untreated and EBI pretreated rice straw, as well as that of the Avicel. As the loading of cellulase increased while the b-glucosidase concentration was maintained at a constant level, glucose yield increased, regardless of biomass types. Additionally, when cellulase loading was increased from 5 to 60 FPU per g of glucan, the glucose yield after 132 h increased from 34.7% to 52.1%; however, that of untreated rice straw increased from 19.2% to only 24.5%. This finding implies that lignocellulose that has not been properly pretreated cannot be efficiently hydrolyzed, even when a high concentration of cellulose is used. The glucose yields of Avicel after 132 h showed a relatively small increase (from 81.3% to 91.3%) as the concentration of cellu-

100

Glucose yield (% theoretical maximum)

i

 100.

80

Untreated (72 h) Untreated (132 h) EBI 1-80 (72 h) EBI 1-80 (132 h) Avicel (72 h) Avicel (132 h)

60

40

20

0

5 FPU + 30 CBU

15 FPU + 30 CBU

60 FPU + 30 CBU

Enzyme loading per g glucan Fig. 5. Effect of enzyme loading on the enzymatic hydrolysis of rice straw and Avicel. Rice straw samples were untreated or pretreated by EBI at 0.12 mA, 80 kGy and 1 MeV (i.e., EBI 1–80). Enzymatic hydrolysis was then conducted using 60 FPU of cellulase (Celluclast 1.5L) and 30 CBU of b-glucosidase (Novozyme 188) per g of glucan at pH 4.8, 50 °C and 150 rpm. Results shown represent the mean ± standard deviation of experiments conducted in triplicate.

lase increased from 5 to 60 FPU per g of glucan, respectively. Therefore, pure cellulose containing no protective barriers, such as layers of hemicellulose and lignin, is readily hydrolyzed by cellulase, regardless of the enzyme dosage. Taken together, these results indicate that proper physical or physicochemical pretreatments are necessary for effective enzymatic cellulose hydrolysis as well as for reducing the amount of cellulase required. 4. Conclusion Rice straw was irradiated to increase the enzyme digestibility by cellulase using an electron beam at a current of 0.03–0.24 mA, a dose of 7.6–90 kGy and 1–2 MeV. Enzymatic hydrolysis of untreated rice straw with 60 FPU of cellulase and 30 CBU of b-glucosidase, resulted in a yield of 5.1% and 22.6% of theoretical maximum after hydrolysis for 24 and 132 h, respectively. However, enzymatic hydrolysis of rice straw that was pretreated by EBI at 80 kGy at 0.12 mA and 1 MeV under the same conditions produced a glucose yield of 43.1% and 52.1% after hydrolysis for 24 and 132 h, respectively. Both SEM and X-ray diffraction analysis revealed that physical changes in the rice straw were likely a result

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