Enhancement of enzymatic digestibility of Miscanthus by electron beam irradiation and chemical combined treatments for bioethanol production

Chemical Engineering Journal 275 (2015) 227–234

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Enhancement of enzymatic digestibility of Miscanthus by electron beam irradiation and chemical combined treatments for bioethanol production Soo Jeong Yang a,1, Hah Young Yoo a,1, Han Suk Choi a, Ja Hyun Lee a, Chulhwan Park b,⇑, Seung Wook Kim a,⇑ a b

Department of Chemical and Biological Engineering, Korea University, 5 Ga, Anam-dong, Sungbuk-Gu, Seoul 136-701, Republic of Korea Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-dong, Nowon-Gu, Seoul 139-701, Republic of Korea

h i g h l i g h t s  Korean Miscanthus was pretreated by electron beam irradiation (EBI).  The optimal irradiation was determined as 500 kGy of total exposed energy at 7.4 mA and 1 MeV.  The thermo-chemical treatments combined with EBI was performed.  Alkali + EBI combined treatment shows the highest glucose conversion (87.97%).  The ethanol yield was about 96.8% by hydrolysate from pretreated Miscanthus.

a r t i c l e

i n f o

Article history: Received 30 January 2015 Received in revised form 8 April 2015 Accepted 10 April 2015 Available online 17 April 2015 Keywords: Lignocellulosic biomass Pretreatment Electron beam irradiation Enzymatic hydrolysis Bioethanol

a b s t r a c t In this study, Korean Miscanthus sinensis was pretreated by electron beam irradiation (EBI) and then enzymatically hydrolyzed for fermentable sugar production. The total exposed energy (kGy) on Miscanthus which is considered as a significant factor in the pretreatment was investigated to obtain the optimal condition. As a result, total dose was determined as 500 kGy at 7.4 mA and 1 MeV and glucose conversion was about 1.26-fold enhanced compared with control (none treatment). To enhance the enzymatic digestibility, the thermo-chemical treatments combined with EBI was performed by sulfuric acid and aqueous ammonia. The result indicates that aqueous ammonia with EBI treatment shows the highest glucose conversion (87.97%) and 32.3% of biomass to glucose recovery which is 2.4-fold enhanced than control. Finally, the hydrolysate from pretreated Miscanthus was utilized to ethanol production by Saccharomyces cerevisiae K35 and the ethanol yield was about 96.8%. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction As a non-edible biomass, Miscanthus is an attractive energy crop because it has high contents of cellulose and hemi-cellulose with low cultivation cost and is available in anywhere. It is important that Miscanthus can be sustainably supplied in light of the high demand for biofuel production [1]. Biomass utilization of biofuels and biochemicals has received much attention over the recent decades due to the high price of petroleum and the accompanying global warming [2]. Lignocellulose which consists of cellulose and hemi-cellulose that are tightly bound with lignin has been limited to a direct

⇑ Corresponding authors. Tel.: +82 2 940 5173; fax: +82 2 912 5173 (C. Park). Tel.: +82 2 3290 3300; fax: +82 2 926 6102 (S.W. Kim). E-mail addresses: [email protected] (C. Park), [email protected] (S.W. Kim). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.cej.2015.04.056 1385-8947/Ó 2015 Elsevier B.V. All rights reserved.

feedstock of bioconversion, because of its rigid and hard-to-degrade cell wall structure. Therefore, pretreatments are required in the beginning of utilization of lignocellulosic biomass to increase recovery of fermentable sugar in hydrolysis [3–5]. The pretreatment technology has been developed for various utilizations of lignocellulosic biomass. Firstly, the collected biomass is ground to decrease particle size by crushing, milling, and homogenizing. Then, appropriate chemical pretreatments can be employed. Generally, acid and alkaline reagents have been widely used in chemical pretreatments, which selectively remove the barriers of lignocellulosic biomass such as hemi-cellulose and lignin. Therefore, the accessibility of enzyme to biomass could be enhanced. However, over-degradation of products, large amount of energy input, occurrence of secondary pollution and toxic chemical are still major problem in chemical pretreatments. To minimize those negative effects with removal of barrier

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compounds, the optimization of operating conditions is greatly required in this process [6,7]. Recently, high-energy irradiation treatments, such as electron beams, proton beams, microwaves, and c-rays, have been used for pretreatment of biomass by changing their properties. The basic principle of irradiation pretreatment is that chemical bonds of biomass are initiated and cleaved using ions produced by radiation, similar to the chemical reaction. Table 1 shows the summary of previous studies that include the various types of energy radiation and dosages on cellulosic biomass [8–17]. The dose is defined as the amount of absorbed energy to matter per unit mass and the dose of 1 kGy is the absorption energy of 1 J/g. The first research related irradiation pretreatment of cellulosic biomass was reported by Aoki, in which the crystallinity degree and cellulose strength of the biomass were decreased by increasing dose of c-ray [8]. Also, the high energy irradiation was utilized in many studies for enhancement of glucose yield from biomass [9–16]. The studies found that the physical properties of biomass such as degree of crystallinity, surface area, porosity and pore diameter were changed by high energy radiation. Gryczka et al., reported the changes in the morphology of willow plant fibers caused by a high energy electron beam [17]. Effect of electron beam irradiation on pure cellulose (Avicel PH101) was reported by Driscoll et al., and the result shows that the molecular weight and crystallinity of cellulose were reduced and its available surface area was increased by increasing dosage of radiation [18]. This improved accessibility of cellulose to enzymes on crystalline regions directly increases the rate of hydrolysis and the conversion of sugar by the irradiation, which increases the feasibility of biomass as a raw material in bioethanol production. The irradiation treatments have many advantages that produces less inhibitory compounds, short radiation time, not required a neutralization step and heat control. Therefore, many studies reported the potential applications in viscose industry [19], pulp and paper industry [20] and lignocellulose-based biorefineries [12–16]. In this study, electron beam irradiation (EBI) was applied to Miscanthus treatment for enhancement of enzymatic digestibility. The operating conditions of EBI were determined based on glucose conversion. The enzyme loading tests were performed by cellulases. The effect of chemical treatments combined with EBI was investigated for enhancement of glucose conversion and the results were analyzed by various methods (solid composition analysis, XRD, and SEM). Finally, bioethanol production was carried out by Saccharomyces cerevisiae K35 from hydrolysates of pretreated Miscanthus. 2. Materials and methods 2.1. Materials Sulfuric acid (H2SO4) and aqueous ammonia (NH3H2O) were utilized as chemical reagents for pretreatment. All chemical reagents were purchased from Dea-jung Chemical, Korea. Commercial enzymes, CelluclastÒ 1.5 L (synonym: 1,4-(1,3:1,4)b-D-Glucan 4-glucano-hydrolase; cellulase from Trichoderma reesei, Cat. No. C2730, Lot No. 077K0737) and Cellobiase (synonym: Novozyme 188; cellobiase from Aspergillus niger, Cat. No. C6105, Lot No. 011M2020) were purchased from Sigma–Aldrich, USA. AvicelÒ PH-101 (Sigma–Aldrich, USA) and Whatman No. 1 filter paper (Whatman, UK) were used as a-cellulose in this study. 2.2. Biomass preparations and thermo-chemical treatments Korean Miscanthus sinensis used in this study was collected from Honam area of Korea. The raw materials were air-dried at 40 °C for 3 days using a dry oven and milled by a cutting mill. Different

particle sizes of the materials were prepared by using two types of sieve (US standard sieves, mesh size of 300 and 425 lm), packed in air-tight polystyrene bag, and stored at room temperature. In combined treatment, the prepared Miscanthus was thermochemically pretreated by using acid or alkaline reagents before EBI treatment. Acid pretreatment was performed at 121 °C of temperature and 1% of sulfuric acid with 1:10 of solid–liquid ratio (w/ w) for 1 h [21]. Also, the alkaline pretreatment was performed by soaking in aqueous ammonia (SAA) method and the reaction conditions were as follow: 60 °C of temperature and 15% of aqueous ammonia with 1:10 of solid–liquid ratio (w/w), 150 rpm in shaking incubator for 24 h [22]. After chemical treatments, the samples were washed by deionized water for neutralization and then were dried in a vacuum oven for 2 days. The dried samples were packed in air-tight polystyrene bag and then utilized to EBI combined treatment. 2.3. Treatment of electron beam irradiation The prepared samples were irradiated on a conveyor belt using an industrial scale linear electron beam accelerator (ELV-8, EBTech, Daejeon, Korea), which can produce electron beams with an energy of 1.0–2.5 MeV and 50 mA maximum current. The electrons were generated by an electron gun accelerated at a high frequency and sprayed. The electron beam was uniformly irradiated on the samples which were packed in polystyrene bags (200  200 mm). The irradiation dose per pass (dose rate) was determined based on current and conveyor speed. Total irradiated dose of sample was achieved by regulating the number of passes. Under 10 kGy of dose per pass, the samples were exposed 10, 30, and 50 times of passes to achieve 100, 300, and 500 kGy of total dose exposure, respectively. 2.4. Enzymatic hydrolysis Enzymatic hydrolysis was carried out to investigate the effect of EBI treatment on glucose conversion and hydrolysis rate. The treated sample was hydrolyzed by using a commercial enzyme mixture, which consist of CelluclastÒ 1.5 L (loading of 30 FPU/g-glucan) and Cellobiase (loading of 60 CBU/g-glucan) in 0.05 M sodium citrate buffer at pH 4.8 and 50 °C, for 48 h in a shaking incubator (150 rpm) according to the NREL standard procedure [23]. One unit of filter paper (FPU) and cellobiase (CBU) activity was defined as the amount of enzyme that releases 1 lmol of glucose per min under standard assay conditions. All hydrolyses were performed in triplicate to indicate standard deviation. Samples were pretreated and collected at certain time intervals during hydrolysis. Enzymatic hydrolysis was stopped by heating the sample of reaction mixture at 100 °C for 5 min and then the sample was centrifuged to obtain the transparent supernatant. Finally, the supernatant was filtrated using a 0.2 lm syringe filter (hiPTFE membrane, G1320, GENIE, Taiwan), and analyzed by HPLC. Experimental data is presented as mean ± standard deviation of triplicate measurements. The enzymatic hydrolysate from treated Miscanthus was utilized as a carbon source of the main medium for fermentation process. The hydrolysate was filtered by GF/C filter (Whatman, UK) and then concentrated by using a rotary evaporator at 60 °C. Finally, glucose concentration of the hydrolysate was about 30 g/L. 2.5. Bioethanol production Bioethanol production using hydrolysates from Miscanthus was performed for evaluation of fermentability by S. cerevisiae K35 [24,25]. The strain was cultured on Difco™ YM (Yeast Mold) broth medium at 30 °C and 150 rpm for 24 h; the subculture was performed on Difco™ YM agar plate at 30 °C for a week. The culture

229

c

d

500

Electron beam Electron beam Proton beam Electron beam Electron beam Electron beamc Electron beamd Electron beam

Electron beam

aim of the study is to investigate the changes in physical properties of wood and cellulose induced by the irradiation of gamma rays. electron beam was irradiated on waste newspaper for improvement of acid hydrolysis; the glucose yield was enhanced about 1.4-fold. aim of the study is to determine the changes in the morphology of willow plant fibers caused by a high energy electron beam. molecular weight of MCC (Avicel PH101) was reduced from 82 kDa (0 kGy) to 5 kDa (100 kGy) and 2 kDa (1000 kGy). The available surface area was increased from 274 m2/g (0 kGy) to 318 m2/g (1000 kGy).

cellulase (Celluclast 1.5 L) + 60 CBU Cellobiase (Novo 188)

(Celluclast 1.5 L) + b-glucosidase (Novozym 342) (Celluclast 1.5 L) + 30 CBU b-glucosidase (Novo 188) (Celluclast 1.5 L) + 15 CBU b-glucosidase (Novo 188) (T. reesei) (T. reesei) + b-glucosidase (Novo 188) cellulase cellulase cellulase cellulase cellulase

20 FPU 60 FPU 60 FPU 30 FPU 35 FPU – – 30 FPU

1000 2000 450 80 15 250 1000

250–1000 500–2000 150–450 7.6–90 1–25 75–250 250–1000 25–300 10–1000 100–500

30–35 FPU cellulase (T. reesei)

10 100 5–50 10–100 Electron beam Electron beam

c-ray

The The The The a

b

[11] 1987

2008 [12] 2009 [13] 2011 [14] 2012 [15] 2014 [16] 2014 [17] 2009 [18] Current study

[9] [10]

– 21 16 90 90 35 52 70 37 21 – – 42 46 88 – 14 10 52 45 29 30 28 20 5 – – 33 38 71

1979 1984

– – –

b

Hinoki wood (Chamaecyparis obtusa) Hoonoki wood (Magnolia obovata) Waste newspapers Sawdust Chaff Newsprint Pulp and paper mill wastes Industrial hemp (Cannabis sativa L.) Rice straw Rice straw Hybrid napier grass with bajra Switch grass Willow plants (Salix viminalis L.) Microcrystalline cellulose (MCC) Miscanthus sinensis Acid treated Miscanthus Alkali treated Miscanthus

3.12–312

– 1% cellulase (Onozuka R-10)

After Control Range

Determined a

Biomass Irradiation type

Table 1 Summary of various irradiations and dosages on different biomass.

Irradiation dosages (kGy)

Enzyme loadings (Unit/g-biomass)

Glucose conversion (%)

Year

1977

[8]

Ref.

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broth was centrifuged at 12,000 rpm for 15 min. The cell pellet was washed using saline, suspended, and inoculated (10%, v/v) into the main medium. The composition of main medium was as follows: 5 g/L of yeast extract, 5 g/L of peptone, 1 g/L of MgSO4 and 1 g/L of K2HPO4, and the hydrolysate from Miscanthus (about 30 g of glucose with mixed sugars). The main culture for bioethanol production was performed in a 250 mL Erlenmeyer flask containing 50 mL medium at 30 °C and 150 rpm for 24 h. The initial pH and the optical density (OD) of the fermentation process were 5.7 and 0.4, respectively. Cell growth was monitored by measuring the optical density (OD) at 600 nm with a UV–vis spectrophotometer (UV mini-1240, Shimadzu, Japan). Experimental data are presented as mean ± standard deviation of triplicate measurements. The theoretical ethanol yield from glucose was calculated using the following equation (1):

 Ethanol yield ð%Þ ¼

 g of ethanol released  2  100 g of glucose consumed

ð1Þ

where 2 is the conversion factor of glucose to ethanol. 2.6. Analytical methods 2.6.1. Detection of solid compositions The solid composition of biomass was analyzed by standard procedure established by NREL [26]. For a two-step acid hydrolysis, the biomass was firstly soaked with sulfuric acid (72%, w/w) at 30 °C for 2 h, and the solution was diluted with distilled water to a concentration of 4% acid. Then, the reaction mixture was autoclaved at 121 °C for 1 h. After cooling, the reaction mixture was neutralized by calcium carbonate, and the transparent supernatant was obtained by filtration with a 0.2 lm syringe filter (hiPTFE membrane, G1320, GENIE, Taiwan). Finally, the filtrate was analyzed using HPLC. Cellulose content in the biomass was measured by the Updegraff method using Avicel (Sigma–Aldrich, USA) [27]. 2.6.2. X-ray diffraction (XRD) and crystallinity index (CrI) To measure the CrI of the biomass samples, the XRD analysis was performed using a X-ray diffractometer (Rigaku Model D/ MAX-2500V/PC, Tokyo, Japan). The operational voltage and current were maintained at 40 kV and 40 mA, respectively. Radiation (Cu Ka) wavelength was 1.5406 Å. The diffraction spectra were collected by the h–2h method [28,29]. The scan range was 5–35° with a step size of 0.01°. The CrI was calculated from the intensities of amorphous region (2h = 18°) and crystalline region (2h = 22°) by using the following equation (2):

Crystallinity index ðCrI; %Þ ¼

  Icrystalline  Iamorphous  100 Icrystalline

ð2Þ

where I is the intensity. 2.6.3. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) analysis was performed to evaluate the effect of EBI treatment by analyzing the change of microstructure on surface of biomass samples. The samples for SEM examination were prepared according to a standard procedure, fixed onto adhesive carbon tape on an aluminum stub and coated with a thin layer of platinum. The surface morphology of the samples was studied using a scanning electron microscope (Hitachi S-4700, Tokyo, Japan) at an accelerating voltage of 15 kV. 2.6.4. High performance liquid chromatography (HPLC) In order to analyze the concentrations of ethanol and sugars from biomass, HPLC was employed with an AminexÒ HPX-87H ion exclusion column (300 mm  7.8 mm, Bio-Rad, USA) and a refractive index detector (RID-10A, Shimadzu, Japan) using a

S.J. Yang et al. / Chemical Engineering Journal 275 (2015) 227–234

mobile phase of 0.005 N H2SO4 at flow rate of 0.8 mL/min. The temperature of the column and detector was kept at 50 °C, and the injection volume was 20 lL. The enzymatic digestibility was expressed as the percentage of the glucose conversion and calculated by Eq. (3):

Glucose conversion ð%; Enzymatic digestibilityÞ   g of glucose released  100 ¼ g of glucan  1:1

ð3Þ

where 1.1 is the conversion factor of glucan to glucose.

40 15 CBU/g-biomass 30 CBU/g-biomass 60 CBU/g-biomass

Glucose conversion (%)

230

30

20

10

3. Results and discussion 3.1. Effect of enzyme loading 0

3.2. Effect of electron beam irradiation treatment The pretreatment of Miscanthus was performed by electron beam at various irradiation doses with fixed current of 7.4 mA and beam energy of 1 MeV. By the fundamental experiments, the conditions of electron accelerator per pass were determined as the conveyor speed of 10 m/min with a dose of 10 kGy, respectively (data not shown). The effect of irradiation doses of electron beam on glucose conversion was shown in Fig. 2. The conversion of samples was analyzed by enzymatic hydrolysis which is performed under the previously determined conditions. The hydrolysis was performed during 48 h, but the results indicate that the reactions were almost finished at 24 h. The enzymatic digestibility of Miscanthus shows slightly enhanced in all treatment group and their conversions at 48 h were as follows: 35.23%, 38.15%, and 41.83% with a dose of 100, 300, and 500 kGy during the EBI

15

30

60

Enzyme loading (FPU/g-biomass) Fig. 1. The enzyme loading tests. Enzymatic hydrolysis of untreated Miscanthus was performed by using cellulase with cellobiase at pH 4.8 and 50 °C for 48 h.

Control (untreated) EBI-100 EBI-300 EBI-500

50

Glucose conversion (%)

Enzyme concentration is one of the main factors that affect the conversion and initial rate of enzymatic hydrolysis of cellulose. The increasing of cellulases dosage in the process can enhance the yield with reaction rate of hydrolysis, but also increase the cost of whole process. Therefore, determination of reasonable loadings of enzyme is necessary for economical process based on the optimal reaction conditions (temperature, pH, shaking speed and substrate concentration). The Korean Miscanthus was chosen as a substrate and the composition (41.3% glucan, 25.6% xylan, 1.4% galactan, 1.7% arabinan, 20.2% lignin, 2.4% ash and 7.4% others) was determined by NREL LAP procedure based on its dry weight [26]. Fig. 1 shows the effect of enzyme loading on glucose conversion of untreated Miscanthus. The loading range of commercial cellulase with cellobiase was 15, 30, 60 units per gram of biomass. When 15 CBU/g-biomass of cellobiase was used in addition, the glucose conversion was 8.5%, 15.5% and 16.6% at 15, 30 and 60 FPU/g-biomass of cellulase, respectively. Similarly, in case of 30 CBU/g-biomass, the glucose conversion was increased about 2-fold when using 30 and 60 FPU/g-biomass of cellulase compared with 15 FPU/g-biomass. Especially, with 60 CBU/g-biomass, the highest glucose conversion was achieved in each case and the result shows 25.5%, 33.2% and 34.8% at 15, 30 and 60 FPU/g-biomass, respectively. This indicates that the glucose conversion significantly depends on the loading of cellobiase. Therefore, cellobiase is a major factor during this enzymatic hydrolysis and 60 CBU/g-biomass of cellobiase was determined as a reasonable concentration. Meanwhile, over 30 FPU/g-biomass of cellulase loading, the glucose conversion was not significantly influenced at all concentrations of cellobiase. Therefore, the loading concentration of cellulase mixture was finally determined to be 30 FPU/g-biomass of cellulase with 60 CBU/g-biomass of cellobiase and all enzymatic hydrolysis of current study was performed by this condition.

40

30

20

10

0 0

3

6

12

24

48

Hydrolysis time (h) Fig. 2. Effect of EBI treatment on glucose conversion of Miscanthus. EBI-100, EBI300, and EBI-500 indicate total exposed energy at 100, 300, and 500 kGy, respectively. Enzymatic hydrolysis was performed by 30 FPU of cellulase with 60 CBU of cellobiase per g-biomass at pH 4.8 and 50 °C for 48 h.

treatment, respectively. This implies that the glucose conversion was enhanced depending on irradiation dose, and it should be a major factor on electron beam treatment. Therefore, 500 kGy of irradiation dose was finally determined as optimal condition in current study. The optimized conditions of EBI treatments with different biomass were compared with this study (Table 1). Many studies have been focused on the degradation of lignocellulosic biomass by various dose of EBI. Shin et al., used industrial hemp as a feedstock; the determined condition was 450 kGy of total dose [12]. Bak et al., determined 80 kGy of total dose for rice straw treatment, which shows about 1.77-fold enhancement of glucose conversion [13]. Karthika et al., reported that 37% of glucose conversion from hybrid napier grass with bajra under 250 kGy of total dose [15]. Sundar et al., reported that 1000 kGy of total dose was determined as an optimal conditions for treatment of switch grass. About 3.89-fold enhanced conversion was shown, but enzymatic digestibility indicates 21% [16]. In this regard, a biochemical composition of feedstock causes different glucose conversions (enzymatic digestibility).

S.J. Yang et al. / Chemical Engineering Journal 275 (2015) 227–234

1600

Avicel EBI-treated Untreated

1400

22°

Intensity

1200 1000 18° 800 600 400 200 0 10

15

20

25

30

2θ (degree) Fig. 3. The results of X-ray diffractogram of Avicel, untreated and EBI-treated Miscanthus. The EBI treatment was carried out at 1 MeV, 7.4 mA, and 500 kGy of EBI dose. The intensities of the amorphous region at 2h = 18° and the crystalline region at 2h = 22° were used to calculate the crystallinity index (CrI).

After treatment, the surface change of Miscanthus was analyzed by XRD and SEM. Many researches reported the relationship between crystallinity index and enzyme digestibility [14,28,29]. In lignocellulosic biomass, cellulose is considered as the crystalline region, whereas hemi-cellulose and lignin are considered as the amorphous regions. It is possible to determine cellulose content to evaluate pretreatment for recovery of sugars by measuring the crystalline region using XRD. The results of XRD analysis (Fig. 3) indicated the intensity of amorphous and crystalline regions of Miscanthus compared with that of Avicel as a standard. The intensities of amorphous and crystalline regions were measured at 2h = 18° and 22° [28], which was also used to calculate the crystallinity index (CrI). The CrI of the untreated Miscanthus was 59.72%, and this value slightly increased to 66.67% after EBI treatment at 500 kGy based on that of Avicel as pure cellulose was 83.33%. This result indicates that exposure of the crystalline region of Miscanthus was increased by EBI treatment. In other words, EBI

treatment could increase the exposure of crystalline region (cellulose portion) by removal of the amorphous region (lignin and hemi-cellulose portion). Increasing the CrI value resulted in increased surface area of the substrate which in turn enhanced the enzyme digestibility. In addition, cellulose is cemented and covered by lignin with hemi-cellulose. Lignin, as a physical barrier, limits enzyme access to cellulose and consequently decreases its degradability. Hemi-cellulose has an amorphous structure with side chains of short chain polymers which also reduces the accessibility of enzymes to the cellulose fibers. Therefore, removal of both lignin and hemi-cellulose are often reported in pretreatment studies to improve enzymatic hydrolysis using lignocelluloses. It is possible that EBI treatment disrupts the hemicelluloses and lignin to recover the cellulose [10]. SEM image analysis shows the effects of pretreatment, which was performed to observe the surface change of Miscanthus. The images at 250 or 3000 magnification were shown in Fig. 4. The morphological changes were clearly distinguished before and after the EBI treatment, which shows the obvious effect of EBI treatment on lignocellulosic biomass. Untreated Miscanthus showed a flat and smooth surface (Fig. 4a). Whereas, the cleavages on the fibers were shown in the EBI-treated sample (Fig. 4b). Therefore, the rough surface of treated Miscanthus could be evidence of the effect of EBI treatment, which also contribute to improvement of enzyme accessibility with large surface area. 3.3. Effect of combined treatments The combined treatment of Miscanthus was performed by chemical pretreatments with acid or alkaline reagents before electron beam irradiation. In acid pretreatment, dilute sulfuric acid was utilized and the reaction was carried out with the reference conditions which is reported by Sun et al., and Kim et al., [21,30]. As a result, the glucose conversion and solid recovery shows 41.19% and 61.18%, respectively. Aqueous ammonia with soaking method was utilized for alkaline pretreatment. Kim et al., reported the optimal conditions of soaking in aqueous ammonia (SAA) pretreatment, and the major conditions such as reaction temperature, time and concentration of catalyst were determined [22,31]. By the

(B) EBI-500

3,000 ×

250 ×

(A) Untreated Miscanthus

231

Fig. 4. The representative SEM images of untreated Miscanthus as control (A), EBI-treated Miscanthus before hydrolysis (B). The EBI treatment was carried out at 1 MeV, 7.4 mA, and 500 kGy of EBI dose.

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Table 2 The solid composition and glucose conversion of Miscanthus after combined treatments. Combined treatments

Glucose conversion (%)e

Solid composition (wt%) d

Chemical

EBI (kGy)

Glucan

XMGA

Nonea

Nonea 100 300 500

41.34 ± 1.56 40.67 ± 2.06 39.42 ± 1.74 38.67 ± 0.53

28.66 ± 0.24 28.34 ± 0.08 28.28 ± 2.44 26.82 ± 0.27

33.14 ± 0.94 35.23 ± 0.22 38.15 ± 0.51 41.83 ± 1.17

Acidb (Dilute sulfuric acid)

Nonea 100 300 500

62.42 ± 2.94 59.19 ± 0.38 57.09 ± 0.35 56.84 ± 1.07

9.69 ± 0.44 9.54 ± 0.01 9.15 ± 0.65 8.80 ± 0.03

38.06 ± 3.39 41.19 ± 0.92 42.33 ± 0.43 46.30 ± 0.21

Alkalic (Soaking in aqueous ammonia, SAA)

Nonea 100 300 500

55.38 ± 0.53 54.07 ± 0.66 52.88 ± 0.50 51.34 ± 1.97

34.58 ± 1.01 33.85 ± 0.79 32.69 ± 0.83 31.66 ± 0.10

71.25 ± 0.98 75.67 ± 4.07 78.56 ± 0.49 87.96 ± 3.27

a

None: it means the control group of experiment (untreated). Acid: the treatment of dilute sulfuric acid was performed in autoclave at 121 °C and 1% sulfuric acid with 1:10 of solid–liquid ratio (w/w) for 1 h. Alkali: the treatment of soaking in aqueous ammonia was performed in shaking incubator at 60 °C and 15% aqueous ammonia with 1:10 of solid–liquid ratio (w/w), 150 rpm for 24 h. d XMGA: summation of xylan, mannan, galactan and arabinan content in the sample. b

c

e

Glucose conversion (%, Enzymatic digestibility): it is determined as

g of glucose released g of glucan1:1

 100.

Table 3 Summary of mass balance during pretreatment and enzymatic hydrolysis on Miscanthus. Pretreatments

Solid recovery (%)a

Glucan composition (%)b

Glucose conversion (%)c

Biomass to glucose recovery (%)d

None (Untreated) EBI (500 kGy) Acid Acid + EBI (500 kGy) Alkali Alkali + EBI (500 kGy)

100 99.33 61.18 60.77 73.53 73.04

41.34 38.67 62.42 56.84 55.38 51.34

33.14 41.83 38.06 46.30 71.25 87.97

13.70 16.07 14.53 15.99 29.01 32.99

g of weight after treatment g of initial weight

a

Solid recovery: determined as

b

Glucan composition: determined as

c

Glucose conversion: determined as

d

Biomass to glucose recovery: determined as

 100.

g of glucan weight in sample  g of weight of sample g of glucose released  100. g of glucan1:1

100.

g of weight after treatment g of initial weight

weight in sample glucose released  g ofgglucan  g ofg of  100. of weight of sample glucan1:1

treatment, glucose conversion of 71.25% and solid recovery of 73.53% were achieved, respectively. A synergetic effect of thermo-chemical with EBI treatment of Miscanthus was investigated and the analytical results are shown in Table 2. ‘XMGA’ indicates the summation of xylan, mannan, galactan and arabinan content. The results showed that the contents of glucan (cellulose) and XMGA (hemi-cellulose) were decreased with all types of treatments by increasing exposure dose. These results suggest that EBI could lead to scission of the lignocellulosic polymer chain and degradation of cellulose [18]. Charlesby, also showed that radiation caused the molecular weight of cellulose to decrease with increasing irradiation dose. To improve the recovery of fermentable sugar, it is reported that EBI treatment was carried out to increase the porosity and accessibility of enzyme during hydrolysis when lignin and hemicellulose were removed by pretreatment [32]. Compared to the EBI treatment only, the glucose conversion of combined (acid or alkali with EBI) treatment shows great potential for application in pretreatment process. The glucose conversion in alkali + EBI treated samples was significantly increased than only EBI treated samples under all dose of irradiation. The highest glucose conversion (87.97%) was obtained by alkali + EBI 500, and that shows 2.1-fold increase compared with only EBI 500 (41.83%) treatment. In acid + EBI treated samples, a large amount of the hemicellulose was leached and removed from the biomass during the acid pretreatment. Therefore, the content of hemicellulose was

greatly reduced, and the content of cellulose was relatively increased. Arabinan, a type of hemicellulose and polymer of arabinose, was not detected in samples with only acid pretreatment, but was detected at about 2% of the total composition of samples in other pretreatment. A previous study have revealed that hydrolysis of hemicelluloses were enhanced by EBI treatment [12,13]. When samples were exposed to 500 kGy of EBI, glucose conversion was 41.83% (EBI 500 kGy) and 46.30% (acid + EBI 500 kGy) after hydrolysis, respectively. This slightly enhanced effect might be due to both acid and EBI treatment removing the similar component (hemicelluloses). In this view, a synergetic effect between acid + EBI and alkali + EBI treatment was confirmed, and the result indicates that alkali + EBI treatment was more effective on enzymatic digestibility. It seems that the highly selective degradation of lignin was achieved by aqueous ammonia, then the breakage of structural bonds which includes cellulose, hemicellulose and lignin complex in the biomass was occurred by high energy electron beam irradiation. Overall mass balance of various pretreatments is shown in Table 3. In the solid recovery, EBI treatment shows the highest recovery (99.33%) and solid losses are not came out, whereas chemical pretreatments cause the solid losses more than 30% by the reaction. The pretreatment of Miscanthus by dilute sulfuric acid was not effective on glucose conversion (enzymatic digestibility) with solid recovery. It seems that the reaction under current conditions was not suitable for Miscanthus. Kim et al., reported that the selectivity among competing reactions is controlled by the

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Glucose Ethanol

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0

24

Fermentation time (h) Fig. 5. Profiles on growth, glucose consumption and ethanol production of Saccharomyces cerevisiae K35 by using Miscanthus hydrolysate. The hydrolysate was obtained by enzymatic hydrolysis at pH 4.8 and 50 °C for 48 h and then concentrated by using a rotary evaporator at 60 °C. The fermentation was performed at 30 °C and 150 rpm for 24 h.

reagent and reaction conditions [24]. Therefore, optimization of the reaction conditions should be required in further study because current study has not focused on the chemical treatment. The composition of glucan in each sample was enhanced about 15–20% by both chemical treatments, whereas the sample from EBI treatment has decreased due to the degradation of cellulose structure by high energy radiation. The biomass to glucose recovery (BtG) was evaluated by the fundamental data (solid recovery, glucan composition and glucose conversion). The control group (none treatment) was found to be 13.7% of BtG. This indicates that about 137 g of glucose should be recovered, if 1 kg of raw biomass is loading in the process. The BtG was enhanced in all the cases of treatments. Especially, 32.3% of BtG was obtained by alkali + EBI combined treatment which is 2.4-fold enhanced compared to control group, whereas only EBI and acid + EBI treatment were found similar BtG (about 16%). Therefore, alkali + EBI combined treatment has a strong effect in glucose recovery from Miscanthus, and the results would be useful for process development or design. 3.4. Bioethanol production by hydrolysates from Miscanthus The pretreated Miscanthus was enzymatically hydrolyzed in a 2 L Erlenmeyer flask at the determined conditions. The theoretical maximum glucose conversion is 51.34 g when 100 g of Miscanthus is loading in the process. The hydrolysate contains about 30 g/L of glucose and the nutrient sources were added (0.5% of yeast extract, 0.5% of peptone, 0.1% of MgSO4 and 0.1% of K2HPO4). The fermentation was conducted using the hydrolysate for bioethanol production by S. cerevisiae K35. The fermentation was performed at 30 °C, 150 rpm and the cultivation profiles are shown in Fig. 5. The initial glucose concentration was about 28.7 g/L and it was almost consumed after 18 h. The cells rapidly grew after an initial 3 h; as a result ethanol production increased and pH decreased. The maximum concentration of ethanol was obtained at 18 h (11.58 g/L) and the ethanol yield was about 96.8%. The high ethanol yield was obtained by hydrolysate from pretreated Miscanthus, and the result shows that inhibitors are negligible during fermentation and there were no negative effect on yeast fermentation. 4. Conclusions Korean Miscanthus was chosen as a feedstock for bioethanol production, and 30 FPU/g-biomass of cellulase with 60 CBU/g-biomass of cellobiase were determined as optimal enzyme loading.

Total irradiation dose of electron beam was considered as significant factor for pretreatment and 500 kGy of irradiation dose at 7.4 mA and 1 MeV was determined by glucose conversion (enzymatic digestibility) which is enhanced about 1.26-fold compared with control (none treatment). In synergetic effect of pretreatment, alkali + EBI combined treatment was more effective than acid + EBI, and the results shows that 32.3% of biomass to glucose recovery which is 2.4-fold enhanced compared with control. It indicates that about 323 g of glucose should be recovered, if 1000 g of Miscanthus is loading in the process. The hydrolysate from pretreated Miscanthus was utilized as carbon sources for bioethanol production by S. cerevisiae K35. As a result, about 11.6 g/L of ethanol was obtained when 28.7 g/L of glucose was initially added. It indicates about 96.8% of ethanol yield and the result shows that inhibitors are negligible during fermentation. Acknowledgements This research was supported by the Advanced Biomass R&D Center (ABC-2011-0031360) of the Global Frontier Project funded by the Ministry of Science, ICT and Future Planning of Korea and the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (No. 2014R1A2A2A01007321) and Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2012M2B2A4029962). References [1] A.E. Farrell, R.J. Plevin, B.T. Turner, A.D. Jones, M. O’Hare, D.M. Kammen, Ethanol can contribute to energy and environmental goals, Science 311 (2006) 506–508. [2] C.H. Chou, Miscanthus plants used as an alternative biofuel material: the basic studies on ecology and molecular evolution, Renew. Energy 34 (2009) 1908– 1912. [3] M.H. Han, Y. Kim, Y.R. Kim, B.W. Chung, G.W. Choi, Bioethanol production from optimized pretreatment of cassava stem, Korean J. Chem. Eng. 28 (1) (2011) 119–125. [4] R. Kataria, R. Ruhal, R. Babu, S. Ghosh, Saccharification of alkali treated biomass of Kans grass contributes higher sugar in contrast to acid treated biomass, Chem. Eng. J. 230 (2013) 36–47. [5] S. Imman, J. Arnthong, V. Burapatana, V. Champreda, N. Laosiripojana, Influence of alkaline catalyst addition on compressed liquid hot water pretreatment of rice straw, Chem. Eng. J. (2014), http://dx.doi.org/10.1016/ j.cej.2014.12.032. [6] X. Erdocia, R. Prado, M.A. Corcuera, J. Labidi, Effect of different organosolv treatments on the structure and properties of olive tree pruning lignin, J. Ind. Eng. Chem. 20 (2014) 1103–1108.

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