Ethanol-based organosolv treatment with trace hydrochloric acid improves the enzymatic digestibility of Japanese cypress (Chamaecyparis obtusa) by exposing nanofibers on the surface

Bioresource Technology 132 (2013) 64–70

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Ethanol-based organosolv treatment with trace hydrochloric acid improves the enzymatic digestibility of Japanese cypress (Chamaecyparis obtusa) by exposing nanofibers on the surface Akihiro Hideno a,⇑, Ayato Kawashima b, Takashi Endo c, Katsuhisa Honda b, Masatoshi Morita b a

Senior Research Fellow Center, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan Biomass Refinery Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 737-0046, Japan b c

h i g h l i g h t s " Alcohol organosolv treatment with 0.4% HCl improved the enzymatic digestibility. " Ethanol-based organosolv treatment with 0.4% HCl exposed cellulose nanofibers. " Accellerase1500 was suitable for hydrolysis of organosolv treated softwood.

a r t i c l e

i n f o

Article history: Received 31 July 2012 Received in revised form 7 January 2013 Accepted 8 January 2013 Available online 18 January 2013 Keywords: Ethanol-based organosolv treatment Trace hydrochloric acid Japanese cypress Enzymatic hydrolysis Cellulose nanofiber

a b s t r a c t The effects of adding trace acids in ethanol based organosolv treatment were investigated to increase the enzymatic digestibility of Japanese cypress. A high glucose yield (60%) in the enzymatic hydrolysis was obtained by treating the sample at 170 °C for 45 min in 50% ethanol liquor containing 0.4% hydrochloric acid. Moreover, the enzymatic digestibility of the treated sample was improved to 70% by changing the enzyme from acremonium cellulase to Accellerase1500. Field emission scanning electron microscopy revealed the presence of lignin droplets and partial cellulose nanofibers on the surface of the treated sample. Simultaneous saccharification and fermentation of the treated samples using thermotolerant yeast (Kluyveromyces marxianus NBRC1777) was tested. A high ethanol concentration (22.1 g/L) was achieved using the EtOH50/W50/HCl0.4-treated sample compared with samples from other treatments. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Biorefinery processes such as the production of bio-ethanol production and chemicals from lignocellulose, have attracted significant attention worldwide (Pan et al., 2005). In particular, the production of bioethanol from lignocellulose biomass has been widely investigated because it does not compete with the production of food and fodder (Hägerdala et al., 2006). The Japanese cypress (JC; Chamaecyparis obtusa) is known for its high quality lumber and is used for constructing traditional Japanese buildings, such as the Horyji Temple, one of the oldest wooden buildings (1,300 years old) in the world (Ohtsuki et al., 2011). In 2007, the annual production of JC was 65,000 m3 however, 70% of JC was wasted because of its inferior quality. This ⇑ Corresponding author. Tel.: +81 089 946 9771; fax: +81 089 946 9980. E-mail address: [email protected] (A. Hideno). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.048

wasted JC has potential as a biomass resource. However, utilizing this wood in biorefineries is not easy because it is highly recalcitrant and rot resistant. Therefore there have been very few reports regarding the utilization of JC. Pretreatment and saccharification are the key processes in the establishment of bio-refineries, because fermentable sugars obtained after pretreatment and saccharification can be used for the production of not only bioethanol but also other chemicals (Hendriks and Zeeman, 2008; Sun and Cheng, 2002). Sugar yields and the amount of enzymes required for saccharification depend on the types of pretreatments and the properties of the material. Various pretreatments have been investigated, including (1) physical pretreatments such as ball milling (Mosier et al., 2005), hummer milling (Hideno et al., 2007), and disk milling (Hideno et al., 2009) (2) physicochemical treatments such as steam explosion, ammonia fiber explosion (Holtzapple et al., 1992), and hot-compressed water treatment (Yu et al., 2010) (3) chemical

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pretreatments such as organosolv treatment, sulfuric acid treatment (Esteghlalian et al., 1997) and lime treatment (J. Park et al., 2010; S. Park et al., 2010) and (4) biological treatments such as using white-rot fungi (Itoh et al., 2003). Organosolv pretreatment is a pretreatment technique for achieving highly effective enzymatic hydrolysis. In this technique, lignin and hemicellulose are extracted from lignocellulose biomass using organic solvents (e.g., methanol, ethanol, ethylene glycol, glycerol, acetic acid, formic acid, phenol, and dioxane) or their aqueous solutions (Zhao et al., 2009) with an acid catalyst. Teramoto et al. (2008) pretreated eucalyptus with a mixed solvent containing ethanol (EtOH; low boiling point), water (W), and acetic acid (AA) instead of sulfuric acid, with approximately 100% conversion yields. Sun and Chen reported that atmospheric aqueous glycerol (high boiling point) autocatalytic organosolv pretreatment enhanced the enzymatic hydrolysis of wheat straw (Sun and Chen, 2008). Hallac et al. reported a high efficiency of lignin and hemicelluloses removal, reduction in the degree of polymerization, decrease in the amount of crystalline cellulose, and increase in the digestibility of biomass following ethanol organosolv pretreatment (Hallac et al., 2010). In general, the increase in enzymatic digestibility correlates with an increase in the energy consumption or environmental load of the pretreatment. Desirable pretreatments are those that enhance the enzymatic digestibility of lignocellulosic biomass with low-to-intermediate energy consumption or environmental load. Inoue et al. (2008) reported that combined pretreatment without chemicals consisting of ball milling (BM) and hot-compressed water (HCW) treatment in mild conditions enhanced the enzymatic digestibility of eucalyptus. Recently, Hideno et al. (2012) reported that the combination of organosolv treatment with acetic acid and short-duration ball milling was effective for the enzymatic hydrolysis of JC. However, ball milling consumes significant amounts of energy (Hideno et al., 2009; Yanagida et al., 2009), and should ideally be fully omitted. Mineral acid is a good catalyst in organosolv treatment for the dissolution of hemicelluloses and lignin (Sun and Cheng, 2002). Organosolv treatment is a hydrothermal treatment; its energy consumption is nearly equal to that of hot-compressed water treatment, and greatly lower than that of ball milling. In previous reports, organosolv treatments based on ethanol solvent with acetic acid and sulfuric acid were carried out (Teramoto et al., 2008; Pan et al., 2005). However, the effects of hydrochloric acid, especially in trace amounts, on the ethanol organosolv treatment of soft wood are unknown. To cut off the BM process while maintaining high enzymatic digestibility and understand the reaction behavior of cellulose, which contribute increasing enzymatic digestibility, in the organosolv treatment process, the effects of trace amounts of mineral acids (sulfuric acid and hydrochloric acid) on alcohol organosolv treatment for the enzymatic hydrolysis of JC were investigated in this study. It was found that high glucose yields were obtained using ethanol-organosolv treatment with trace amounts of hydrochloric acid instead of short-duration BM. The physicochemical properties (crystallinity and surface structure at the nanometer scale) of treated samples were elucidated to investigate the causes of high enzymatic digestibility. Moreover, simultaneous saccharification and fermentation of treated samples using thermotolerant yeast were performed.

2. Methods 2.1. Japanese cypress (JC) Sample wood chips were electrically sieved to collect chips ranging in size from 125 to 500 lm. The monomeric sugars and lignin contents of JC were analyzed by the TAPPI Test Method T249

(Tappi, 1992) as described later, and were as follows (in terms of dry weight): glucose 449 mg, xylose 44 mg, mannose 105 mg per g-JC, acid-insoluble lignin (Klason lignin) 31.9%, and acid soluble lignin 1.1%. 2.2. Organosolv treatments with trace acid The samples were subjected to alcohol organosolv treatment using a portable reactor (TPR-1 type, Taiatsu Grass Co., Tokyo, Japan) consisting of SUS-316 (Swagelok Co., Hyogo, Japan) and equipped with a glass-inner cylinder and a band heater. The working volume of this reactor is 120–500 mL. Approximately 15 g of the raw sample and 80 mL of reaction solvents were added into the inner cylinder, mixed using a stirring paddle, and placed in the reactor. Air in the headspace was purged and replaced by nitrogen. The initial pressure was set at 0.2 MPa, and the cylinder was heated to the required temperature for the required time using the band heater. The mixed reaction solvents were selected on the basis of the preliminary results: a mixture of ethanol (EtOH), methanol (MeOH), ethylene glycol (EG), water (W), acetic acid (AA), sulfuric acid (SA), and hydrochloric acid (HCl). A detailed description of treatment conditions and solid recoveries after the reaction are shown in Table 1. The numbers after solvent abbreviations show the blend ratios. For example, EtOH75/W25/AA1 denotes the mixed solvent blended as ratio EtOH:W:AA = 75:25:1. After the reaction, the reactor was cooled to room temperature using a fan. Heated samples were transferred, washed, and filtered using approximately 400 ml of ethanol. The washed residues were vacuum dried at 55 °C for 20–21 h, and the dry weights were measured to calculate the percentage of solid recovery (%) using Eq. (1).

Solid recovery ð%Þ ¼ ðdry weight of residue= original dry weight of sampleÞ  100 Some samples (each approximately 1.8 g) were ground at 400 rpm for 10 min using a planetary ball mill (P-6, Fritsch Co., Idar-Oberstein, Germany) with approximately 90 g of alumina balls (diameter 5 mm). 2.3. Enzymatic hydrolysis of cooked samples using commercial cellulase enzymes Acremonium cellulase (Meiji Seika Co., Tokyo, Japan) and Accellerase 1500 (ACC1500; Genencor Co. Rochester, NY, USA) were mainly used for enzymatic hydrolyses following the method of Inoue et al. (2008). The specific activities of filter paperase (FPase), xylanase, carboxymethylcellulase (CMCase), b-glucosidase, and b-xylosidase in acremonium cellulase and Accellease1500 were 0.57, 61.5, 7.1, 1.7, and 9.3 U/mg protein (acremonium cellulase), and 0.57, 20.3, 9.2, 3.0, and 29.9 U/mg protein (Accellease1500), respectively. Approximately 6 g of the treated sample and 60 mL of the enzyme solution (containing 0.24 g-protein) were added into 100 mL Erlenmeyer flasks and incubated on the orbital shaker (150 rpm) at 45 °C for 72 h. For small-scale treatment, the pretreated samples (dry weight: 0.05 ± 0.01 g) and 1.0 mL of the enzyme cocktail in 50 mM acetate buffer (pH 5.0) were placed in 2 mL tubes and incubated at 45 °C for 72 h with agitation using a rotator. Both small- and large-scale enzymatic hydrolyses were performed depending on the purpose of each experiment. The large-scale enzymatic hydrolyses (in flasks) were performed to obtain the time course of sugar yields and assess the differences in the rate of sugar release between samples subjected to different pretreatments. Those on a small scale (in tubes) were performed in the end-point assay to assess the effect of the type of enzyme on sugar yield, and to select the suitable enzyme for hydrolysis of the treated samples. After hydrolysis, the sample was

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Table 1 Conditions of organosolv treatments used and solid recoveries obtained in this study. Solvents

Acids (%)

Time (min)

Temp. (°C)

Max pressure (Mpa)

Solid recovery (%)

EtOH75/W25/AA1 EG75/W25/AA1 EtOH50/W50/SA0.05 MeOH50/W50/HCl0.4 EtOH50/W50/HCl0.4

1 1 0.05 0.4 0.4

30 30 60 45 45

140 170 200 170 170

0.9 0.5 2.4–2.5 1.5 1.5

97.00 91.45 65.02 51.61 47.73

AA: acetic acid; SA: sulfuric acid; HCl: hydrochloric acid; EtOH: ethanol; EG: ethylene glycol; MeOH: methanol. The numbers after abbreviations of solvents denote the blend ratio of solvents and acids.

1

ð2Þ

where a denotes the amount of monomeric sugars produced (mg), and b is the amount of monomeric sugars component in the original residue. All enzymatic hydrolyses were performed in duplicate or triplicate. 2.4. Field emission scanning electron microscopy (FE-SEM) Raw JC and treated samples with trace acid were treated by an osmium tetraoxide to introduce conducting properties, and observed by FE-SEM (S-4800, Hitachi High-Technologies Co., Tokyo, Japan) at 1.4 kV to investigate the morphological alterations to the surface. 2.5. X-ray diffraction analysis Approximately 100 mg of sample was packed and smoothed using a hydraulic system pump. X-ray diffraction (XRD) analyses were performed using a Rigaku RINT-TTR3 X-ray diffractometer (Rigaku Co., Tokyo, Japan) with Cu Ka radiation at 50 kV and 300 mA. Samples were scanned over the range of 2 h = 2–60° at a rate of 2°/min. The crystallinity index (CrI) was calculated using Eq. (3) based on the method of Segal et al. (1959).

Crystallinity Index ð%Þ ¼ ½ðI002  Iam Þ=I002   100

2.7. Analysis of sugar composition and acid-insoluble lignin The monomeric sugar and acid-insoluble lignin in raw JC and pretreated samples were analyzed by TAPPI Test Method T249 (Tappi, 1992). Samples of approximately 0.5 g were crushed in a mortar with 10 mL of 72% sulfuric acid for 1 h at room temperature. The mixture was diluted to 11.8% sulfuric acid with water, autoclaved at 121 °C for 10 min, and neutralized to pH 5.0 using 5 N sodium hydroxide. The residual material was cooled, washed using 100 mL of hot water, and filtered through a glass filter using an aspirator. The solids were dried to a constant weight at 105 °C, and the dry weight of the solids was measured as acid-insoluble lignin (Klason lignin) content. The neutralized solution was centrifuged at 10,000 rpm for 5 min, 2-deoxyglucose was added to the supernatant as an internal standard, and the mixture was filtered through a 0.45 lm filter. The neutral sugars (glucose, xylose, galactose, and mannose) in the filtrate were analyzed using an HPLC (Dionex, Sunnyvale, CA) equipped with an ion-exchange CarboPac PA1 column, and a pulsed amperometric detector at room temperature. Deionized water was used for elution at a flow rate of 1 mL/ min. To wash the column, 0.3 N NaOH, 0.1 N KOH, and 1.5 N Na2CO3 were used. 3. Results and discussions 3.1. Composition of treated samples The sugar and Klason lignin content of the residues is shown in Fig. 1. A previous report (Hideno et al., 2012) presented data of Raw 800

ð3Þ

where I002 is the intensity at about 2h = 22.5°, and Iam is the intensity at 2h = 18.7°. 2.6. Simultaneous saccharification and fermentation (SSF) of treated samples using thermotolerant yeast SSF was performed using the pretreated sample (materials), ACC1500 enzyme, and Kluyveromyces marxianus NBRC1777, a thermotolerant yeast. Approximately 6 g of the treated sample was added into 60 mL of the medium in a 100 mL-Erlenmeyer flask. The flask was sterilized by autoclaving at 121 °C for 20 min and cooled naturally to room temperature. The filter-sterilized enzyme solution (40 mg protein/g substrate) and a preculture of K. marxianus NBRC1777 were added to the flask. The flask culture was incubated on a rotary shaker at 150 rpm at 43 °C. In total, 500 lL of sample was taken at each time point (3, 6, 12, 24, 48, and 72 h) to measure ethanol and sugar levels. The ethanol and sugar concentrations in the sample were measured using an HPLC (JASCO, Japan) instrument equipped with an Aminex HPX-87H column

Sugar content (mg-sugar/g-residue)

Sugar yield ð%Þ ¼ 100ab

(BioRad). The column oven was set at 60 °C. Samples were eluted at 1 mL/min with 10 mM H2SO4.

50

700 600 500

40

30

400 300 200

20

Klason lignin (%)

centrifuged (20,000g for 10 min), and the supernatant was diluted and applied to a high-performance liquid chromatography (HPLC, Jasco Co., Tokyo, Japan) instrument equipped with a HPX-87P column (7.8 mm ID 30 cm, Bio Rad, Hercules, CA, USA) for sugar analysis. The column oven was set at 80 °C, and samples were eluted at 1 mL/min with water. The enzymatic digestibility was represented by the sugar yield (%), which was calculated using Eq. (2) given below:

10

100 0

0

Fig. 1. Monomeric sugar components and Klason lignin content in treated samples. White, gray and black boxes denote contents of glucose, mannose and xylose content, respectively. Black triangles denote contents of Klason lignin content (%).

67

3.2. Enzymatic hydrolysis of treated samples The results of enzymatic hydrolyses of treated samples are shown in Fig. 2. A previous report (Hideno et al., 2012) has shown sugar yields in the enzymatic hydrolysis of JC subjected to a combination of organosolv treatment and BM. These data were obtained using the end-point assay. In this section, the time course of enzymatic hydrolyses are shown, and these data of ball-milled samples are shown for comparison. The highest glucose concentration was obtained from the EtOH50/W50/HCl0.4-treated sample (approximately 40 g/L), and was 2 times that of samples treated with a combination of ball milling and acetic acid (EtOH75/W25/ AA1 and EG75/W25/AA1) for 120 h (Fig. 2a). The saccharification speeds of the EtOH50/W50/SA0.05 and MeOH50/W50/HCl0.4 samples were slower than those of the ball-milled samples. Raw JC was hardly hydrolyzed. On the other hand, the glucose yields of ballmilled samples were higher than that of the EtOH50/W50/HCl0.4 sample until 72 h. However, these yields were surpassed by those of the EtOH50/W50/HCl0.4 sample (Fig. 2b). Interestingly, the glucose yields of ball-milled samples increased rapidly and then plateaued. On the other hand, the glucose yields of organosolvtrace acid-treated samples gradually increased and continued to do so. These differences are likely due to the physico-chemical properties of the treated residues. A difference was also observed between methanol-based solvent and ethanol-based solvents. There are only a few reports regarding organosolv treatment with trace hydrochloric acid. In all probability, this is the first report that ethanol and trace hydrochloric acid were the suitable mixture of solvent and catalyst, in organosolv treatment for enhancing the enzymatic digestibility of woody biomass.

(a)

50 40 30 20 10

(b)

60

Glucose yields (% )

0

50 40 30 20 10 0 0

24

48

72

96

120

Time (h) Fig. 2. Enzymatic hydrolysis of treated samples by acremonium cellulase (a) Changes in glucose concentration (g/L) over 120 h and (b) time course of glucose yield based on the constituent glucose content in treated sample over 120 h. White circles, black triangles, white diamonds, white boxes, gray boxes, and black diamonds represent the enzymatic hydrolysis of raw JC, EtOH50/W50/SA0.05, MeOH50/W50/HCl0.4, EtOH75/W25/AA1_B10, EG75/W25/AA1_B10 and EtOH50/ W50/HCl0.4, respectively.

HCl0.4), although slightly improved that of ball-milled samples (EtOH75/W25/AA1_B10 and EG75/W25/AA1_B10, Fig. 3) and raw samples (Raw JC). The glucose yield from EtOH50/W50/HCl0.4 using ACC1500 eventually reached approximately 70%, greater than that from ball-milled samples during 72 h of enzymatic hydrolysis. For the enzymatic hydrolyses of organosolv treated JC (soft wood), ACC1500 is more suitable than acremonium cellulase.

80 70 Glucose yields (% )

JC and samples subjected to alcohol organosolv treatment with acetic acid (EtOH/W25/AA1 and EG75/W25/AA1). However, these samples were obtained by using a reactor operating on a different scale and were used as references for comparison in this study. In the EtOH75 or EG75/W25/AA1-treated samples, the sugar content was similar to that of raw JC, although the xylose content slightly decreased. While, treatment with sulfuric acid (EtOH50/W50/ SA0.05) caused the elimination of xylose, a decrease in mannose, and a relative increase in glucose. Lavarack et al. (2002) reported that hydrochloric acid was found to be a less active catalyst for the degradation of xylose compared to sulfuric acid. However, xylose and mannose derived from hemicelluloses disappeared completely in both samples treated with hydrochloric acid (MeOH50/ W50/HCl0.4 and EtOH50/W50/HCl0.4), and these comprised the only glucose content derived from cellulose despite using trace hydrochloric acid (0.4%, equal to approximately 0.1 N). These results indicate that hydrochloric acid is an active catalyst for the degradation of mannose and xylose derived from hemicelluloses in JC. The Klason lignin contents of the EtOH50/W50/SA0.05 and EtOH50/W50/HCl0.4-treated samples were lower than those of other samples. A comparison of the use of MeOH and EtOH indicated that the lignin solubilities obtained using solvents based on EtOH were higher than those using MeOH solvents. Organosolv treatment resulted in the delignification of the lignocellulose biomass by the cleavage of lignin-carbohydrate and lignin–lignin bonds, and the solubilization of lignin by the organic solvents (McDonough, 1993; Sarkanen, 1990).

Glucose conc. (g/ L )

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60 50 40 30 20 10 0

3.3. Comparison of acremonium cellulase and ACC1500 To increase glucose yield in the enzymatic hydrolysis of organosolv-treated samples, ACC1500 was applied instead of acremonium cellulase. ACC1500 greatly improved the glucose yields of organosolv-treated samples (MeOH50/W50/HCl0.4 and EtOH50/W50/

Fig. 3. Comparison of acremonium cellulase and Accellerase1500 in enzymatic hydrolysis of treated JC for 72 h. White and black boxes denote the results of using acremonium cellulase and Accellerase1500, respectively.

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The specific activities of b-glucosidase and cellobiohydrolase in ACC1500 were higher than those in acremonium cellulase although filter paperase activity is the same in both. The main reason for the high glucose yield using ACC1500 would be that cellobiohydrolase and b-glucosidase are essential for the hydrolysis of the samples treated by organosolv with hydrochloric acid, as well as hydrolysis of cellulose. 3.4. XRD-analysis of treated samples Gentle peaks were observed in the ball-milled samples in X-ray diffraction spectra of the treated samples. On the other hand, the peaks of the organosolv-acid treated samples were sharper than that of raw JC. The CrI values calculated from these spectra were 60.2% (a: Raw JC), 33.3% (b: EtOH75/W25/AA1_B10), 34.1% (c: EG75/W25/AA1_B10), 78.5% (d: MeOH50/W50/HCl0.4), and 80.9% (e: EtOH50/W50/HCl0.4), respectively. The CrI-values of ballmilled samples were lower than that of raw JC. On the other hand, the CrI-values of organosolv-acid treated samples were slightly higher than that of raw JC. It was found that hydrothermal treatments, such as hot-compressed water (HCW) treatment and organosolv treatment, increased sugar yield in the enzymatic hydrolysis although the CrI values of the treated samples were high. In ball-milled samples, there is a positive correlation between the decrease in CrI and the increase in enzymatic digestibility. However, CrI alone may not be adequate to explain the increase in enzymatic digestibility, because enzymatic cellulose hydrolysis is complicated (J. Park et al., 2010; S. Park et al., 2010). Especially, the results indicate that CrI is not suitable for the explanation of improving enzymatic hydrolysis of ‘‘boiled’’ samples such as HCW-treated samples (Hideno et al., 2009) and organosolv-treated samples (this study). The CrI values of these ‘‘boiled’’ samples were slightly higher than those of the raw materials, although the enzymatic digestibilities of these samples were greatly improved. Organosolv/acid-treated samples had high crystallinities compared with ball-milled samples although the enzymatic digestibilities of organosolv-acid treated samples were higher than those of ballmilled samples. This would be because amorphous hemicelluloses and lignin were eluted away and highly crystalline cellulose was exposed on the surface of organosolv-acid treated samples. This phenomenon has been generally observed and reported (Zhao et al., 2009). The activities of cellobiohydrolase and b-glucosidase would be required for the enzymatic hydrolysis of samples with high crystallinities such as the EtOH50/W50/HCl0.4-treated sample. 3.5. Field emission scanning electron microscopy (FE-SEM) To elucidate the reason for the increase in the enzymatic digestibility of organosolv-acid treated samples, observation of the solid surface was carried out using FE-SEM. The surface of raw JC was very smooth and had no pores. On the other hand, there were many droplets (100 nm–5 lm) on the surface of organosolv-acid treated samples. These droplets are likely dissolved and denatured lignin, because the hemicelluloses content of these samples was fully lost (Fig. 1) and there have been reports of lignin droplets on the surface of thermochemically treated samples (Selig et al., 2007). Some broken lignin droplets were observed on the surface of the MeOH50/ W50/HCl0.4 samples, although these broken droplets were hardly seen on the surface of the EtOH50/W50/HCl0.4 samples. The strength and chemical composition of these lignin droplets may be different with different solvents (MeOH vs. EtOH). Selig et al. reported that redeposition of the lignin droplets has a negative effect on the enzymatic saccharification (Selig et al., 2007). The results and related reports (Selig et al., 2007) indicate that the lignin droplets on the surface of the MeOH50/W50/HCl0.4 sample were par-

tially broken, and these droplets covering the surface resulted in a decrease in enzymatic activity. Li et al. (2012a,b); (SFBC abstract) reported that these lignin droplets have strong negative effects on enzymatic processing system, and that increasing the amount of cellulases loaded or increasing the hydrolysis time would improve the enzymatic digestibility of such samples. Igarashi et al. (2009) directly showed using high-speed atomic force microscopy that Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules unidirectionally slid on the surface of crystalline cellulose. Moreover, Igarashi et al. (2011) showed that TrCel 7A molecules reached a collective halt analogous to a traffic jam on the bulky crystalline cellulose surface. The lignin droplets that were observed on the surface of MeOH50/W50/HCl0.4 samples by FE-SEM could cause such traffic jams of cellulase molecules. On the surface of the MeOH50/ W50/HCl0.4-treated sample, there were many nanometer-scale pores and microfibrils on the back (e). On the other hand, directly exposed cellulose nanofibers were dotted on the surface of EtOH50/ W50/HCl0.4-treated sample (f). These exposed cellulose nanofibers would contribute to the high crystallinity and enzymatic digestibility of the sample. These results indicate that the generation of such nanoscale structures on the surface of the treated biomass depends on the solvents in the organosolv treatment, and is closely related to the enzymatic digestibility of the sample. Hydrochloric acid is a good catalyst for delignification and hemicellulose degradation. Zhao et al. (2009) reported that hemicellulose and lignin are solubilized, while almost all the cellulose content remains insoluble. An increase in delignification results in an increase in enzymatic digestibility. Obama et al. (2012) reported that a good correlation was observed between the delignification of cellulosic pulp samples and their enzymatic digestibilities. H. Li et al. (2012) and Z. Li et al. (2012) reported that the removal of hemicelluloses and lignin increased the susceptiability of cellulose to cellulases, and that the content of hemicelluloses and lignin affected the enzymatic digestibility of the treated sample. However, ball-milled samples that retain almost all of their hemicellulose and lignin content have high enzymatic digestibility. This is usually explained as being due to decreasing CrI. The CrI of ball-milled samples is low, and the sugar yield increases. However, organosolv/acid-treated samples had high CrI values although their enzymatic digestibility was higher than that of ball-milled samples (Fig. 2). Moreover, the extent of delignification in organosolv-acid samples was approximately 26%, and more than 70% of lignin remained (Fig. 1). This paradox can be explained by the nanoscale structure of the surface of the treated sample, which was observed by using FE-SEM. Huijgen et al. (2012) reported that the reduced xylan content of the residue and the changed nature of the lignin enhance enzymatic digestibility in spite of the higher lignin content. Our FE-SEM results showed the changed nature of the lignin and the exposed cellulose nano-fibers by removing xylan, mannan, and partial lignin. The results indicate that one of the most important requirements for enzymatic hydrolysis of treated samples is a surface nanoscale structure to be attacked by cellulase molecules, the sizes of which are approximately 3–5 nm. The glucose yield of EtOH50/W50/HCl0.4 samples greatly increased with the use of ACC1500 rather than acremonium cellulase. The cellulose nanofibers were exposed on the surface of EtOH50/W50/HCl0.4 samples. These results indicated that ACC1500 derived from T. reesei is more suitable than acremonium cellulase for the hydrolysis of high CrI-cellulose. The reason why ACC1500 is suitable for enzymatic hydrolysis of treated JC compared with acremonium cellulase is that ACC1500 derived from T. reesei has a higher cellobiohdrolase activity, which can catalyze the hydrolysis of crystalline cellulose. In summary, ethanol-based organosolv treatment with trace hydrochloric acid promotes the partial exposure of cellulose nanofibers and increases the breathing space for cellulases molecules

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resulting in increased enzymatic digestibility, by partial delignification and the complete removal of hemicelluloses. The high enzymatic digestibility was obtained using ethanol-based organosolv treatment with trace hydrochloric acid but without ball milling. 3.6. Simultaneous saccharification and fermentation of treated samples using thermotolerant yeast In preliminary experiments using commercial cellulose powder, it was found that the ethanol productivity of K. marxianus NBRC1777 strain greatly decreases in the SSF of cellulose using cellulases at temperatures greater than 45 °C for 72 h although this strain can achieve high ethanol productivity from glucose at 45 °C for 24 h (data not shown). Glucose, which was generated by hydrolysis of cellulose, was not used by K. marxianus NBRC1777 and accumulated in the culture of SSF using cellulose at temperature greater than 45 °C and for periods longer than 48 h. Therefore, SSF was performed at 43 °C in this experiment. The production of ethanol from pretreated samples by SSF using this strain is shown in Fig. 4. Raw JC was used as a negative control. The highest ethanol concentration (22.1 g/L) was achieved using the EtOH50/W50/ HCl0.4-treated sample at 72 h. The rates of ethanol production using both samples subjected to ball milling in combination with organosolv treatment (EtOH75/W25/AA1_BM10 and EG75/W25/ AA1_BM10) were higher than that obtained using the EtOH50/ W50/HCl0.4-treated sample, however, the rates slowed down rapidly, and their ethanol concentration soon plateaued. On the other hand, the rates of ethanol production in organosolv treated samples with trace hydrochloric acid rose gently and progressively. These trends in these results shown in Fig. 4 are similar to those seen in the results of enzymatic hydrolyses (Fig. 2 b). However, the ethanol concentration obtained by SSF (Fig. 4) using ACC1500 was higher than the theoretical ethanol concentration calculated from the glucose concentration obtained by enzymatic hydrolysis (Fig. 2a) using acremonium cellulase. These results could be caused by the use of ACC1500 instead of acremonium cellulase, and SSF. Hydrolysates of cellulose including glucose and cellobiose are inhibitors for cellulases. In SSF, the efficiencies of cellulases increase because yeasts immediately consume monosaccharides and disaccharides, and convert them to ethanol. The ethanol concentration of the MeOH50/W50/HCl0.4 sample caught up with that of the EtOH75/W25/AA1_BM10 sample although the glucose yield obtained by the hydrolysis of the MeOH50/W50/HCl0.4 sample using ACC1500 was slightly lower than that of the EtOH75/W25/

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AA1_BM10 sample. In particular, the effect of SSF using MeOH/ W50/HCl0.4 could be high based on the results of Fig. 3. To shorten the time of enzymatic hydrolysis and SSF, the samples subjected to organosolv treatment with trace HCl require increased surface area, which is easily attacked by cellulases. The combination of organosolv treatment with trace HCl and wet disk milling (Hideno et al., 2009), a treatment that consumes low energy and is highly efficient, would contribute to shorten the time of enzymatic hydrolysis and SSF. Investigations of these techniques are required for the next step. In general, organosolv treatment has some advantages as follows: (1) easy recovery of organic solvents by distillation, (2) high recovery yields of fermentable sugars from treated residues, (3) the ability to obtain sulfur-free lignin, with high purity and low molecular weight (Zhao et al., 2009). The lignin can be used as fuel to provide power to the pretreatment process. High-quality lignin can be converted to phenolic powder resins, polyurethane, polyisocyanurate foams, and epoxy resins for polymer materials (Zhang, 2008). In this study, ethanol was produced with high efficiency as the common product by SSF. The ethanol used in treatment and washing can be recovered and re-used, while the produced ethanol can be collected as stock for other purposes such as fuels. Moreover, the insoluble lignin recovered in this study (data not shown), holds promise as a raw material of other chemicals. However, the recovery of ethanol and lignin might appear to have a high operating cost. These problems are common in alcohol organosolv treatment (Pan et al., 2005). Further studies must focus on increasing the efficiency of this process. 4. Conclusions To obtain a high sugar yield from JC, which is a highly recalcitrant material, via a low-energy consuming process, the effects of ethanol-based organosolv treatment containing trace hydrochloric acid were investigated. Ethanol-based organosolv treatment using trace hydrochloric acid greatly improved the enzymatic digestibility by removing hemicelluloses and lignin, and the generation of cellulose nanofibers on the JC. Moreover, the high ethanol production was achieved through an SSF process from the EtOH50/W50/ HCl0.4-treated sample. These results indicate that ethanol-based organosolv treatment with trace hydrochloric acid is an efficient pretreatment for the enzymatic hydrolysis of soft wood such as JC. Acknowledgements

Ethanol conc. (g/ L )

25 This work was supported by a Grant-in-Aid for Scientific Research for the promotion and establishment of a recycling society from the Ministry of the Environment, Japan, the Shorai Foundation for Science and Technology, and by JSPS KAKENHI Grant Number 22780292. The authors thank Dr. Seiya Watanabe (Ehime University), Meiji Seika Co., Genencor Co., Novozyme Co., Shin Nihon Chemical Co., and Hankyu Bioindustry Co. for supplying enzymes.

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Time (h) Fig. 4. Time course of ethanol concentration (g/L) in simultaneous saccharification and fermentation using thermotolerant yeast. White circles, black triangles, white diamonds, white boxes, gray boxes, and black diamonds represent the enzymatic hydrolysis of raw JC, EtOH50/W50/SA0.05, MeOH50/W50/HCl0.4, EtOH75/W25/ AA1_B10, EG75/W25/AA1_B10 and EtOH50/W50/HCl0.4, respectively.

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