Bioresource Technology 102 (2011) 2916–2924 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Pretreatment efficiency and structural characterization of rice straw by an integrated process of dilute-acid and steam explosion for bioethanol production Wen-Hua Chen ⇑, Ben-Li Pen, Ching-Tsung Yu, Wen-Song Hwang Cellulosic Ethanol Program, Institute of Nuclear Energy Research, Taoyuan, Taiwan, ROC a r t i c l e i n f o Article history: Received 17 June 2010 Received in revised form 9 November 2010 Accepted 11 November 2010 Available online 4 December 2010 Keywords: Rice straw Dilute-acid pretreatment Acid-catalyzed steam explosion Integrated pretreatment process Enzymatic hydrolysis a b s t r a c t The combined pretreatment of rice straw using dilute-acid and steam explosion followed by enzymatic hydrolysis was investigated and compared with acid-catalyzed steam explosion pretreatment. In addition to measuring the chemical composition, including glucan, xylan and lignin content, changes in rice straw features after pretreatment were investigated in terms of the straw’s physical properties. These properties included crystallinity, surface area, mean particle size and scanning electron microscopy imagery. The effect of acid concentration on the acid-catalyzed steam explosion was studied in a range between 1% and 15% acid at 180 °C for 2 min. We also investigated the influence of the residence time of the steam explosion in the combined pretreatment and the optimum conditions for the dilute-acid hydrolysis step in order to develop an integrated process for the dilute-acid and steam explosion. The optimum operational conditions for the first dilute-acid hydrolysis step were determined to be 165 °C for 2 min with 2% H2SO4 and for the second steam explosion step was to be carried out at 180 °C for 20 min; this gave the most favorable combination in terms of an integrated process. We found that rice straw pretreated by the dilute-acid/steam explosions had a higher xylose yield, a lower level of inhibitor in the hydrolysate and a greater degree of enzymatic hydrolysis; this resulted in a 1.5-fold increase in the overall sugar yield when compared to the acid-catalyzed steam explosion. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Lignocellulosic materials such as agricultural, hardwood and softwood residues are potential sources of sugars for ethanol production. Currently, a large number of studies regarding the utilization of lignocellulosic biomass as a feedstock for producing bioethanol are being carried out. A number of processes based on the application of enzymes for ethanol production from lignocellulosic biomass have been proposed (Himmel et al., 2007). In all cases, pretreatment of the biomass is required to hydrolyze the hemicellulose, and make the cellulose more accessible to the enzymes. An efficient pretreatment system is crucial to the enzymatic hydrolysis and thus the fermentation process, which are the essential steps in the cellulose conversion process to produce bioethanol (Mosier et al., 2005). Dilute-acid hydrolysis, steam explosion, liquid hot water extraction, alkaline hydrolysis, ammonia treatment, and biological ⇑ Corresponding author. Address: Cellulosic Ethanol Program, Institute of Nuclear Energy Research, Atomic Energy Committee, Executive Yuan, No. 1000, Wenhua Rd, Jiaan Village, Longtan Township, Taoyuan County 32546, Taiwan, ROC. Tel.: +886 3 4711400x5115; fax: +886 3 4711410. E-mail address: wenhua@iner.gov.tw (W.-H. Chen). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.052 processes have been used for pretreatment. Among these various types of pretreatment, dilute-acid hydrolysis and the steam explosion technique are commonly used; these are more effective than others because they have already been successfully developed for pretreatment, are known to improve sugar recovery (particularly xylose in the aqueous phase) and enhance opening up of the pretreated fibers to enzymatic digestion (Wyman et al., 2005; Yang and Wyman, 2008; Alvira et al., 2010). Rice straw is the most abundant agricultural waste around the world, and the amount available in Taiwan has been estimated to be 700–800 million tons every year. With over 90% of the worldwide production, most rice straw is produced in Asia. Therefore, rice straw is considered to be an attractive alternative for fuel ethanol production in Asia (Binod et al., 2010). The Institute of Nuclear Energy Research (INER), a governmental organization in Taiwan, has been developing lignocellulosic fuel ethanol technology using rice straw in order to build a test cellulosic ethanol plant. At present, although many researchers have studied steam explosion (Moniruzzaman, 1996a,b; Nakamura et al., 2001; Jin and Chen, 2006, 2007) and dilute-acid pretreatment (Roberto et al., 2003; Karimi et al., 2006; Guo et al., 2009; Hsu et al., 2010) using rice straw, only a few investigations of the pretreatment effects on overall total sugar recovery when dilute acid and steam explosion W.-H. Chen et al. / Bioresource Technology 102 (2011) 2916–2924 the glucose yield from the subsequent enzymatic hydrolysis. In addition, the structural properties of the pretreated rice straw solid residues after the different pretreatment processes were also investigated by measuring surface area, mean particle size and crystallinity as well as SEM microscopy imagery. The aim was to estimate the pretreatment efficiencies of the different processes and to clarify the effects of acid pretreatment and explosion on the enzymatic hydrolysis. pretreatment of rice straw are combined have been described (Vlasenko et al., 1997). In general in this context, overall sugar yield has been reported to be the most import parameter when evaluating the production cost of bioethanol. Therefore, studying how the pretreatment process affects overall sugar yield in combination with the enzymatic hydrolysis process is very important. It has been shown that highly severe conditions are required during steam pretreatment to improve the enzymatic digestibility of cellulose, but these conditions will also cause greater degradation of xylose. In other words, obtaining both hemicellulose (xylose) recovery and high cellulose utility at the same time is difficult (Thomsen et al., 2006, 2008). Therefore, a two step dilute-acid pretreatment has been proposed. The first step is performed under low severity to give a high recovery of hemicellulose-derived fermentable sugars in the liquid; the second step is carried out under greater severity in order to make the cellulose more accessible to enzymatic attack (Nguyen et al., 2000; Boussaid et al., 2000; Kim et al., 2001; Söderström et al., 2003; Thomsen et al., 2006; Sørensen et al., 2008; Monavari et al., 2009). Based on the above viewpoint, we propose a two-step pretreatment process (an integrated process of dilute-acid and steam explosion) to improve the overall saccharification efficiency with rice straw. The first step is a dilute-acid hydrolysis to maximize the xylose conversion and the second step is steam explosion to destroy the rice straw in order to enhance enzymatic hydrolysis. The flow chart for the acid-catalyzed steam explosion and the integrated process of dilute-acid and steam explosion is shown in Fig. 1. The goal of this study was to define the optimal conditions of the different pretreatment processes of rice straw, including hydrothermal, steam explosion, acid-catalyzed steam explosion, dilute-acid pretreatment and the integrated process. These were evaluated by estimating the xylose yield after pretreatment and (a) 2. Methods 2.1. Biomass materials Air-dried rice straw with a moisture content of 10% was reduced to about 2 cm in length by chopping and used as the raw material. The rice straw was mainly obtained from a single private farm in Taiwan. The obtained composition of the raw rice straw was glucan 31.1 ± 0.8%, xylan 18.7 ± 0.2%, arabinan 3.6 ± 0.1%, extractive 9.0 ± 1.2%, ash 14.5 ± 0.1% and lignin 13.3 ± 0.6% on a dry weight basis. 2.2. Pretreatment The dilute-acid and steam explosion pretreatment of the rice straw was carried out in a steam explosion apparatus. The steam explosion apparatus consisted of a steam generator, a reactor and a receiver. The capacity of the reactor was 6 L, while the maximum working pressure and temperature was 10 kg/cm2 and 180 °C, respectively. Approximately 100 g of chopped rice straw with or without soaking in an appropriate amount of sulfuric acid solution at 15% (w/w) solid loading for 4 h was introduced into the reactor, Rice straw Presoaking (impregnation) Acid-catalyzed steam explosion Dilute H2SO4 Solid Solid/Liquid Separation Enzymatic hydrolysis Liquid Saturation steam Xylose-rich hydrolysate Xylose yield (P) Glucose yield (P) Xylose yield (E) Glucose yield (E) Total saccharification yield (b) Rice straw Presoaking (impregnation) Dilute-acid pretreatment (Step 1) 2917 Dilute H2SO4 Saturation steam Solid/Liquid Solid Separation Steam explosion (Step 2) Solid/Liquid Separation Liquid Solid Saturation steam Xylose-rich hydrolysate Enzymatic hydrolysis Xylose yield (P) Xylose yield (E) Glucose yield (P) Glucose yield (E) Total saccharification yield Fig. 1. The flow charts of (a) the acid-catalyzed steam explosion and (b) the proposed integrated process of dilute-acid and steam explosion in this study. 2918 W.-H. Chen et al. / Bioresource Technology 102 (2011) 2916–2924 which was then steam heated. The reactor heat-up time varied from 10 to 30 s, depending on the pretreatment temperature. After the desired steaming time (2–20 min), a 200 ball valve at the bottom of the reactor was used to rapidly reduce the vessel to atmospheric pressure. This resulted in the solid and liquid (slurry) products being explosively released into the flash receiver with cooling. In the case of the dilute-acid pretreatment experiment, no such explosive release occurred and the pretreated mixture was transferred from the reactor after the selected retention time and collected. The resulting mixtures were filtered and the total volume of liquid in the filtrate and the weight of solid residue were recorded. In the study, the rice straw was pretreated by an acid-catalyzed steam explosion using different sulfuric acid concentrations in a range between 1% and 15% (w/w) at 180 °C (10 kg/cm2) for 2 min. With regard to the integrated process of dilute-acid and steam explosion pretreatment. As shown in Fig. 1(b), the first step is dilute-acid hydrolysis to maximize the xylose conversion and the second step is steam explosion. The xylose-rich hydrolysate after dilute-acid pretreatment was obtained by filter press separation and the cellulose-rich cake was sequentially pretreated by the steam explosion process. The dilute acid (the first step) pretreatment were performed at 125–165 °C for 2–5 min using 1–8% (w/w) acid concentration. The liquid fraction was the xylose-rich hydrolysate and the solid fraction was sequentially treated by the second steam explosion at 180 °C (10 kg/cm2) for 20 min. 2.3. Enzymatic hydrolysis The wet pretreated residue (about 1 g dry weight) was transferred to a 250 ml shake flask and then the pH was adjusted to 4.8 by adding 50 mM acetate buffer at 2% (w/v) solid loading. The flask was put in a shaking water bath (FIRSTEK; Model B603D) at 50 °C and 100 rpm for 30 min. An appropriate amount of cellulase with a filter paper activity of 60 FPU/ml (Genencor, Spezyme CP) was added at this point. The digestion of the cellulose was conducted at a cellulase activity level of 20 FPU/g cellulose. The enzymatic digestion was carried out at 50 °C and 100 rpm for 72 h. All the experiments were performed in triplicate, with the average value being reported. Win2 V. 2.1). A scanning electron microscope (SEM) (Hitachi S-4800 SEM instrument operated at 10–15 kV accelerated voltage) was used to observe surface morphology and size of the rice straw before and after pretreatment (Yu et al., 2009). The structure of the pretreated residues was characterized by powder X-ray diffraction (XRD) (Bruker D8 Advance). The particle size distribution of pretreated residues was measured by a Beckmann Coulter LS 230 Laser Diffraction Particle Size Analyzer. Detail description of the related experiments can be found in our previous report (Hsu et al. 2010). 2.5. The definition of conversion and saccharification yield The conversions of pretreatment and enzymatic hydrolysis are calculated by the following equations: Xylose conversion (P) (%) = [(xylose (g) released in pretreatment)/(total xylose (g) in the feedstock)] 100 Glucose conversion (P) (%) = [(glucose (g) released in pretreatment)/(total glucose (g) in the feedstock)] 100 Xylan conversion (E) (%) = [(xylose (g) released in enzymatic hydrolysis)/(total xylan (g) in the feedstock)] 100 Cellulose conversion (E) (%) = [(glucose (g) released in enzymatic hydrolysis)/(total cellulose (g) in the feedstock) ] 100 The definition of total saccharification yield (overall sugar yield) is the summation of xylose and glucose monomers saccharification yields. The xylose/glucose saccharification yields are calculated by the following equations: Xylose saccharification yield (%) = [(xylose (g) released in pretreatment (P) and enzymatic hydrolysis (E))/(total xylose and glucose (g) in the feedstock)] 100 Glucose saccharification yield (%) = [(glucose (g) released in pretreatment (P) and enzymatic hydrolysis (E))/(total xylose and glucose (g) in the feedstock)] 100 Total saccharification yield (%) = [(xylose and glucose (g) released in pretreatment (P) and enzymatic hydrolysis (E))/ (total xylose and glucose (g) in the feedstock)] 100 3. Results and discussion 2.4. Analysis methods The identified components are extractive, ash, cellulose, hemicellulose and lignin of the untreated and treated rice straw were determined according to Laboratory Analytical Procedures from the National Renewable Energy Laboratory (NREL) (Sluiter et al., 2005). The carbohydrate contents were determined by measuring the hemicellulose (xylan, arabinan) and cellulose (glucan) of derived sugars. The composition of the hydrolysates after pretreatment and enzymatic hydrolysis was found to include cellobiose, glucose, xylose, arabinose, acetic acid and furfural, which were filtered through a 0.45 lm-pore-size filter and measured by high performance liquid chromatography (HPLC) using a packed column (Agilent 1200 series HPLC equipped with Coregrl-87H3 Column Transgenomic Technologies at 65 °C with 4 mM H2SO4 as the eluent at a flow rate of 1.0 mL/min). BET surface area information of the pretreated residues was measured by N2 adsorption/desorption measurements (Quantachrome instrument; model: NOVA 4200e) done at 196 °C (77 K). Prior to measurement, all biomass materials were dried at 40 °C for 48 h and then a sample (0.5–1 g) was put into the sample tube of the Quantachrome instrument and degassed using a vacuum for 16 h. The BET surface area and pore volume were obtained from the N2 adsorption/desorption curves using NOVA software (NOVA 3.1. Effect of the method used for residues recovery on enzymatic digestibility Two different processes for explosion pretreatment can be used; one is to explosively relieve the pressure, such as an ammonia fiber explosion (AFEX) (Teymouri et al., 2005), and the other is steam explosion, which results in pretreated products draining away (Moniruzzaman, 1996a, 1996b; Vlasenko et al., 1997; Cara et al., 2006; Ewanick et al., 2007; Ruiza et al., 2008). To explore the effect of the explosion processes on structure and enzymatic digestibility of pretreated residues, samples pretreated by the different steam explosion processes, namely explosively relieving the pressure (denoted as HR-P) and steam explosion accompanied by drainage of the pretreated products (denoted as HR-SE) were prepared at 180 °C for 20 min and compared to the direct cooling process under the same steam pretreatment condition (hydrothermal process; denoted as HR). In this context, the crystallinity and BET surface area of the lignocellulosic biomass are thought to play important roles in susceptibility to enzyme digestion; therefore, we measured the crystallinity and BET surface area of the pretreated residue in order to use these parameters to reflect pretreatment efficiency. Table 1 summarizes the total solid recovery, sugar composition, physical properties, enzymatic hydrolysis conversion and overall 2919 W.-H. Chen et al. / Bioresource Technology 102 (2011) 2916–2924 Table 1 The total solid recovery, sugar composition, physical properties, enzymatic hydrolysis conversions and overall sugar yield of untreated and treated rice straw. Sample a Untreated HR HR-P HR-SE Total solid recovery (%) – 80.6 77.8 85.4 Sugar composition in residuesb Physical properties c 2 Enzymatic hydrolysis conversion (%)d Glucose (%) Xylose (%) CrI (%) BET (m /g) Cellulose Xylan 34.5 ± 0.8 49.3 ± 0.7 48.7 ± 0.4 46.9 ± 0.9 21.3 ± 0.2 16.6 ± 0.4 18.3 ± 0.3 18.7 ± 0.5 51.3 54.1 55.8 58.4 1.32 4.32 3.77 5.51 5.2 ± 0.5 34.6 ± 3.5 38.7 ± 0.5 54.8 ± 0.9 – 29.7 ± 3.3 32.0 ± 1.4 33.0 ± 0.8 Overall sugar yield (%)e – 32.5 ± 2.5 34.2 ± 4.8 49.2 ± 1.2 a HR represents hydrothermal at 180 °C for 20 min; HR-P represents explosively relieving the pressure after hydrothermal process; HR-SE represents steam explosion accompanied by drainage of the pretreated products after hydrothermal. b Each value is the average of three replicates and calculated on the basis of dry weight. c CrI (%) = (I002 – Iam)/I002 100. Where I002 is intensity of the 002 peak at 2h = 22.4° and Iam is intensity of the background scatter at 2h = 18.7° (Kim and Holtzapple, 2006). d Cellulose to glucose and xylan to xylose conversion after 72 h of enzymatic hydrolysis. Each value is the average of three replicates. e Overall sugar yield is based on the recovered monosugers (glucose and xylose) obtained after 72 h of enzymatic hydrolysis and in the liquid fraction after pretreatment per dry weight of the raw rice straw. (Total sugar content in the raw rice straw is 55.8 g/100 g.). sugar yield of the untreated and treated rice straw. The xylose contents of the three treated samples are similar and were reduced from an untreated value of 21.3% to 16.6% and the glucose contents were increased from 34.5% when untreated up to 49.3%. Furthermore, the RCI (relative crystalline index), specific BET surface area and enzymatic hydrolysis conversion are all found to have the following order: HR-SE > HR-P > HR >> untreated rice straw. Although an enzymatic hydrolysis enhancement of rice straw pretreated by both steam explosion processes was observed, it is clear that HRSE is better at conversion than HR-P. This suggests that the shear stress through thermal explosion decomposition and the flow of treated slurry create tensile forces that break the rice straws into more fragmental small pieces of tissue; this results in further substantial breakdown of the lignocellulosic structure. Therefore, the BET surface area of the rice straw and the accessibility of the cellulose components to degradation by enzymes are greatly increased from 1.32% to 5.51% and 5.2% to 54.8%, respectively. Based on the results above, it can be seen that the method used for product recovery obviously affects the surface area of the rice straw and the enzymatic hydrolysis efficiency. Thus, the steam explosion accompanied by drainage of the pretreated products process was selected for the pretreatment efficiency studies below using rice straw. 3.2. Acid-catalyzed steam explosion pretreatment In general, the hemicellulose fraction of the lignocellulose complex is mainly solubilized, while cellulose and lignin remain in the fiber fraction during the acid-catalyzed steam explosion pretreatment. Xylan is the major component present in the hemicellulose structure of rice straw, so the presence of xylose in the filtrate can be used as an indicator of the solubilization level of hemicellulose, which is easily utilized by the fermentation process. In this work, the rice straw was pretreated by an acid-catalyzed steam explosion using different sulfuric acid concentrations in a range between 1% and 15% at 180 °C (10 kg/cm2) for 2 min. The flow chart for the acid-catalyzed steam explosion is shown in Fig. 1(a). The sugar compositions, physical properties and enzymatic hydrolysis of the pretreated residues obtained by the acid-catalyzed steam explosion using different acid concentration is presented in Table 2. The xylan, glucan and lignin contents obtained in the pretreated residues were 0.5–2.7%, 45.2–53.4% and 20.8–28.9%, respectively. Normally, xylan content has been found to decrease with increasing acid concentration and eventually plateaus at approximately 3% sulfuric acid; by contrast, glucan content increases, mainly due to the removal of most of the hemicelluloses by acid-catalyzed hydrolysis. It has been shown that there is a relative increase in the amount of lignin present after treatment due to the removal of hemicelluloses. With regard to fermentation inhibitors, the con- centrations of furfural and acetic acid were lower than 1 g/L when the acid concentration was lower than 3%. On the other hand, the concentrations of furfural and acetic acid increased to 1.5–3.5 and 2.9–3.5 g/L, respectively, at acid concentration higher than 3%. The sugar conversions of pretreatment (P) and the subsequent enzymatic digestibility (E) of pretreated residues obtained after 72 h are shown in Fig. 2. From Fig. 2, it can be seen that the xylose conversion in the pretreatment step (P) decreases with acid concentration, while the glucose conversion (P) seems independent of acid concentration. A maximum glucose conversion (E) of 72.5% was attained at a 3% acid concentration for subsequent enzymatic hydrolysis after 72 h. Moreover, when the acid concentration was increased to 8%, a significant increase in cellobiose conversion with a corresponding decrease in the amount of glucose was observed. This phenomenon can be ascribed to the presence of a b-glucosidase inhibitor such as lignin, which is produced by high sulfuric acid concentration pretreatment and results in an altered ratio of glucose and cellobiose. In other words, increasingly higher sulfuric acid concentrations during pretreatment lead to the pretreated biomass having greater hydrophobicity; this results in an increased adsorption of cellulose enzyme onto the lignin droplets by means of hydrogen bonds (Selig et al., 2007; Guo et al., 2008). Merino and Cherry (2007) also reported that unwashed acid-pretreated corn stover was found to have a lower cellulose conversion during enzymatic hydrolysis than washed acid-pretreated corn stover. Therefore, washing pretreated biomass or an inhibitor removal step could be essential to preventing a lower conversion yield from pretreated biomass. In our results, the enzymatic hydrolysis result of washing rice straw residues treated by a high acid content (11%) provides further support for the above argument. No cellobiose was observed and the glucose conversion was increased from 25.0% to 85.2% after hydrolysis of the washed steam-exploded rice straw for 72 h. The results show that the optimum acid concentration is 3% at 180 °C (10 kg/cm2) for 2 min by acid-catalyzed steam explosion in the reaction system, and a high acid concentration is disadvantageous to both the xylose yield after pretreatment and the glucose yield from the subsequent enzymatic hydrolysis. The purpose of pretreatment is not only to remove xylose from hemicellulose but also to increase the rate of enzymatic hydrolysis of cellulose to glucose. Thus, it is necessary to estimate pretreatment efficiency by combining total xylose and glucose conversion yield after both the pretreatment and enzymatic hydrolysis processes; this is because the purpose of pretreatment is to produce sugars for conversion to bioethanol. Our results show that only 49.6% of the total saccharification yield was obtained using the acid-catalyzed steam explosion pretreatment at a combined severity factor (CSF = log Ro pH; where Ro = texp [(Tr 100)/ 14.75]) of 1.6 and enzymatic hydrolysis with 20 FPU/g of cellulose 2920 W.-H. Chen et al. / Bioresource Technology 102 (2011) 2916–2924 Table 2 Effect of acid concentration of the acid-catalyzed steam explosion pretreatment on the compositions, physical properties and enzymatic hydrolysis of the pretreated residues. Samplea Total solid recovery (%) Composition content (%)c Glucan AC-SE-1b AC-SE-2 AC-SE-3 AC-SE-8 AC-SE-11 AC-SE-15 a b c d e 59.8 53.0 44.3 54.7 34.5 38.2 45.2 ± 0.9 46.7 ± 1.1 53.4 ± 1.4 48.0 ± 1.0 46.7 ± 1.2 49.0 ± 0.8 Xylan Physical properties d Lignin 2.7 ± 0.6 1.4 ± 0.2 1.2 ± 0.2 0.9 ± 0.1 0.7 ± 0.1 0.5 ± 0.1 ASL AIL Total 4.1 ± 0.2 3.6 ± 0.3 3.7 ± 0.3 3.5 ± 0.4 3.5 ± 0.2 3.2 ± 0.4 16.7 ± 1.4 20.9 ± 1.7 22.6 ± 1.2 24.4 ± 1.5 25.3 ± 1.2 25.0 ± 2.3 20.8 ± 1.4 24.4 ± 1.6 26.3 ± 1.4 28.1 ± 1.5 28.9 ± 1.5 28.2 ± 2.5 2 Enzymatic hydrolysis conversion (%)e CrI (%) BET (m /g) Cellulose Xylan 55.1 56.9 57.1 58.7 56.9 59.6 5.59 9.73 4.82 5.87 5.93 6.63 58.7 ± 1.0 68.0 ± 0.8 72.5 ± 1.1 57.8 ± 0.9 25.0 ± 1.7 10.8 ± 0.3 6.8 ± 0.8 4.8 ± 0.8 4.7 ± 0.5 1.1 ± 0.2 2.4 ± 0.4 9.2 ± 1.1 All the acid-catalyzed steam explosion experiments were performed at 180 °C for 2 min. AC-SE-1 where 1 represents acid concentration (w/w)%. Each value is the average of three replicates and calculated on the basis of dry weight. CrI (%) = (I002 Iam)/I002 100 where I002 is intensity of the 002 peak at 2h = 22.4° and Iam is intensity of the background scatter at 2h = 18.7° (Kim and Holtzapple, 2006). Cellulose to glucose and xylan to xylose conversion after 72 h of enzymatic hydrolysis. Each value is the average of three replicates. Conversion (g/g xylan or glucan) 0.8 Xylose(P) Glucose(P) Xylose(E) Glucose(E) Cellobiose(E) 0.6 0.4 0.2 0.0 0 4 8 12 16 H2SO4(%) Fig. 2. Effect of sulfuric acid concentration on the obtained sugar conversions of pretreatment (P) and the sequential enzymatic digestibility (E) of pretreated residues using acid-catalyzed steam explosion at 180 °C (10 kg/cm2) for 2 min. loading after 72 h (Chum et al., 1990). Again, this implies that reaching both high hemicellulose (xylose) recovery and high cellulose utility at the same time is impossible (Thomsen et al., 2008). 3.3. Two-step pretreatment process (integrated process of dilute-acid and steam explosion) As mentioned above, high glucose conversion during enzymatic hydrolysis can be obtained under severe pretreatment operational conditions, but these higher severity conditions result in a lower xylose recovery. Thus, we propose a two-step pretreatment to improve the overall saccharification efficiency. The first step is diluteacid hydrolysis to maximize the xylose conversion and the second step is steam explosion to break the protective sheath around the cellulose such that it becomes more vulnerable to enzymatic hydrolysis. A wide range of conditions were used to determine the most favorable combination. 3.3.1. The residence time of the steam explosion process The steam explosion pretreatment step was performed using the rice straw residue obtained from the first dilute-acid pretreatment at 145 °C and 3% H2SO4 for 10 min (CSF = 1.6). The xylose conversion was 71.6% and the cellulose-rich residue contained mainly 48.4% glucan, 3.3% xylan and 23.6% lignin. This was then steam treated in the second step for various residence times (1, 3, 5, 10, 15 and 20 min) at 180 °C (10 kg/cm2) and then the cake underwent a sudden explosion. The change in composition, physical properties and enzymatic hydrolysis of the steam explosion pretreated residues are shown in Table 3. As shown in Table 3, glucan content increased from 34.5% to 48.4% and xylan content obvious decreased from 21.3% to 3.3% after the first dilute-acid pretreatment. Moreover, glucan content increased from 48.4% to 55.3% and xylan content decreased from 3.3% to 1.3% after steam explosion. It can be seen that glucan and xylan content showed in the range of 51.6–55.3% and 1.2–1.9% for various steaming residence times. In comparison, the treated residue using the dilute-acid pretreatment shows apparent change in glucan and xylan content, whereas, only slightly decrease in glucan and xylan content in the steam explosion step after 1 min steaming time. On the other hand, glucan and xylan contents almost unchanged with increasing residence time (3–20 min). Therefore, it is noted that the main component of the contents remained practically unchanged with increasing residence time, while variations in glucan and xylan content for the samples before and after steam treated explosion were observed. The results reveal that the steam cooking time seemed to have no apparent influence on glucan, xylan and lignin contents in the treated samples, whereas the explosion process did result in some alterations in those components. In general, an increase in the cellulose crystallinity index with residence time was observed in the steam explosion treated samples and reached a plateau after 5 min. Furthermore, the mean particle size of the pretreated solid suspensions was reduced to ca. 237.7 lm within 10 min and slightly decreased with steaming time, whereas the BET surface area was observed to apparently increase after 10 min. In terms of enzymatic hydrolysis, it was shown that the maximum glucose conversion (90.6%) and BET surface area (19.5 m2/g) were obtained after 20 min steaming time. Based on the above experimental results, the influence of steam explosion is greater than steam cooking on the glucan and xylan contents remaining in the pretreated residues, while the physical characteristics, such as crystallinity, BET surface area and particle size, as well as enzymatic hydrolysis, depend not only on the steam explosion but also on the residence time in the two-step pretreatment process. In other words, it is suggested that the effect of the steam explosion is higher than that of steam cooking in terms of action on the glucan and xylan contents, while the physical characteristics and enzymatic hydrolysis are influenced by both steam cooking and the explosion process during the two-step pretreatment process. A number of researchers have reported that the best model for xylose formation and degradation is a pair of parallel first-order 2921 W.-H. Chen et al. / Bioresource Technology 102 (2011) 2916–2924 Table 3 Effect of the steaming time of the steam explosion step of the integrated process on the compositions, physical properties and enzymatic hydrolysis of the pretreated residues. Residence time min) Untreated 0a 1 3 5 10 15 20 a b c d Composition content (%)b Physical properties Glucan CrI (%)c BET (m2/g) Mean Particle Size (lm) Cellulose Xylan 51.3 52.2 53.1 56.9 57.6 57.7 56.9 59.0 1.32 4.43 4.53 5.34 5.99 14.0 16.4 19.5 – – 429.8 385.9 301.7 237.7 235.5 233.9 5.2 ± 0.5 66.2 ± 1.1 64.2 ± 0.2 68.7 ± 1.3 75.0 ± 0.8 76.2 ± 0.5 82.3 ± 2.2 90.6 ± 1.8 – 75.1 ± 3.4 66.0 ± 0.8 64.4 ± 0.4 87.0 ± 0.8 92.9 ± 5.1 84.3 ± 3.9 82.8 ± 4.5 34.5 ± 0.8 48.4 ± 1.4 52.9 ± 0.9 52.8 ± 1.5 53.3 ± 0.4 51.6 ± 1.5 52.5 ± 0.3 55.3 ± 0.5 Xylan Lignin (%) 21.3 ± 0.2 3.3 ± 0.3 1.9 ± 0.0 1.8 ± 0.1 1.4 ± 0.0 1.2 ± 0.0 1.4 ± 0.1 1.3 ± 0.0 ASL AIL Total 4.7 ± 0.3 4.0 ± 0.5 4.4 ± 0.6 4.1 ± 0.1 4.2 ± 0.4 4.6 ± 0.5 4.2 ± 0.1 3.9 ± 0.6 9.4 ± 0.5 19.6 ± 1.6 22.6 ± 2.2 19.3 ± 0.7 21.7 ± 1.6 18.2 ± 1.4 22.1 ± 1.9 17.9 ± 0.9 13.3 ± 0.6 23.6 ± 1.9 27.1 ± 2.2 23.4 ± 0.8 25.9 ± 1.4 22.8 ± 1.7 26.3 ± 1.8 21.8 ± 1.5 Enzymatic hydrolysis conversion (%)d 0 represents the dilute-acid pretreatment of the integrated process. Each value is the average of three replicates and calculated on the basis of dry weight. CrI (%) = (I002 – Iam)/I002 100 where I002 is intensity of the 002 peak at 2h = 22.4° and Iam is intensity of the background scatter at 2h = 18.7° (Kim and Holtzapple, 2006). Cellulose to glucose and xylan to xylose conversion after 72 h of enzymatic hydrolysis. Each value is the average of three replicates. Table 4 The compositions, physical properties and enzymatic hydrolysis of the pretreated residues after the integrated pretreatment process. Sample The first step (dilute-acid pretreatment) The second step (steam explosion)b Condition Composition content (%)d T (°C) AP-SE-0.15a AP-SE-0.72 AP-SE-0.97 AP-SE-1.02 AP-SE-1.19 AP-SE-1.38 AP-SE-1.91 a b c d e 165 140 125 145 150 165 145 t (min) 2 5 5 5 5 2 5 Acid (%) 0.9 1.1 5.7 2.7 1.6 2.1 8.0 EH conversion (%)c 37.4 ± 4.0 45.6 ± 1.6 42.8 ± 1.6 48.9 ± 3.7 38.4 ± 3.7 11.5 ± 1.0 0.9 ± 0.1 Glucan 50.2 ± 2.1 54.7 ± 0.6 53.8 ± 0.6 56.1 ± 1.7 48.1 ± 0.3 54.5 ± 1.0 47.2 ± 0.6 Xylan 6.2 ± 0.7 2.1 ± 0.0 1.0 ± 0.2 1.1 ± 0.1 1.2 ± 0.2 4.2 ± 0.2 0.9 ± 0.4 Physical properties e Lignin ASL AIL Total 3.5 ± 0.1 3.2 ± 0.2 2.9 ± 0.1 2.8 ± 0.1 3.0 ± 0.1 3.1 ± 0.2 2.6 ± 0.1 25.5 ± 1.7 21.9 ± 0.9 20.3 ± 1.4 25.3 ± 0.6 22.2 ± 1.5 23.1 ± 0.8 32.0 ± 2.4 29.0 ± 1.8 25.1 ± 1.1 23.2 ± 1.3 28.1 ± 0.6 25.2 ± 1.6 26.2 ± 0.5 34.6 ± 2.6 EH conversion (%)c CrI (%) BET (m2/g) Cellulose Xylan 60.5 69.5 59.4 60.7 61.1 61.0 65.4 11.01 9.68 12.88 14.33 10.56 9.75 10.56 60.7 ± 1.0 81.4 ± 2.4 71.4 ± 0.3 93.1 ± 2.0 77.4 ± 1.6 84.2 ± 2.0 73.5 ± 1.7 57.4 ± 1.7 69.0 ± 4.5 46.2 ± 2.0 72.1 ± 2.4 90.0 ± 5.0 68.8 ± 2.5 35.2 ± 1.1 AP-SE-0.15 where 0.15 represents the combined severity factor (CSF) of the first step (dilute-acid pretreatment) where CSF = log Ro – pH; Ro = texp [(Tr 100)/14.75]). Each sample was steam heated at 180 °C (10 kg/cm2) for 20 min and then explosion. Cellulose to glucose conversion after 72 h of enzymatic hydrolysis. Each value is the average of three replicates. Each value is the average of three replicates and calculated on the basis of dry weight. CrI (%) = (I002 – Iam)/I002 100 where I002 is intensity of the 002 peak at 2h = 22.4° and Iam is intensity of the background scatter at 2h = 18.7° (Kim and Holtzapple, 2006). reactions: one for easy hydrolysis and the other for difficult hydrolysis (Himmel et al., 2007). The easily hydrolysable fraction has been reported to be 60–80% of the hemicelluloses in various lignocellulosic materials. Based on this information, we are able to explain why most xylose (72%), which is easy to hydrolyze, was extracted from the xylan of the hemicellulose after the first dilute-acid treated under the low severity (145 °C, 3% H2SO4, and 10 min). The xylan content then did not change, even with an extended steam cooking time as the residual xylan is part of the difficult hydrolysable fraction. However, the explosion is sufficient to affect the stubborn xylan molecules. The removal of xylan increases the porosity of the rice straw and therefore increases water diffusion inside the channels of the substrate. As a result, the physical characteristics of the treated rice straw were changed due to autohydrolysis and adiabatic explosion of water in the pores of the acid-pretreated residue. 3.3.2. The optimum conditions for the dilute-acid process Using the above experimental results, it was found that the maximum glucose conversion and BET surface area were obtained after 20 min of steaming time at 180 °C. Thus, the second steam explosion was selected to operate at 180 °C (10 kg/cm2) for 20 min. The first step of dilute-acid hydrolysis was then performed at different operational conditions (1–8% acid concentration, 125–165 °C and 2–5 min). The liquid fraction was the xylose-rich hydrolysate and the solid fraction was sequentially treated by the second steam explosion at 180 °C (10 kg/cm2) for 20 min. Table 4 summarized the compositions, physical properties and enzymatic hydrolysis of the pretreated solid residues produced by the integrated pretreatment process under different severity conditions of the dilute-acid pretreatment. The xylose and glucose conversions obtained from pretreatment versus combined severity factor (CSF) of the dilute-acid hydrolysis (the first step) and the acid-catalyzed steam explosion are shown in Fig. 3. The xylose conversion reached a plateau (75%) at CSF = 1.0–2.0 and we expect that the xylose conversion would decrease at even higher severities (Kabel et al., 2007). In the case of the subsequent enzymatic digestibility of pretreated residues after the second steam explosion step, glucose conversions of 73–93% were obtained after 72 h at 20 FPU/g of cellulose without b-glucosidase supplementation. The total saccharification yield made by combining the total xylose and glucose conversion yield for both processes is an index of pretreatment efficiency for ethanol bioconversion. Fig. 4 shows a comparison between acid-catalyzed steam explosion and the integrated pretreatment process, using the total saccharification yield versus the combined severity factor (CSF). Xylose was mainly obtained from the pretreatment process, while glucose was mainly obtained from enzymatic hydrolysis. Both of the pretreatment processes have higher glucose yields for the enzymatic hydrolysis (glucose yield (E)) than that expected for acid-catalyzed steam explosion under the higher severity condition (CSF P 1.7) due to 2922 W.-H. Chen et al. / Bioresource Technology 102 (2011) 2916–2924 1.0 Conversion (g/g xylan or glucan) Xylose Glucose Acid-catalyzed SE Two-step process 0.8 0.6 0.4 0.2 0.0 0.0 0.5 1.0 1.5 2.0 CSF Fig. 3. The obtained xylose and glucose conversions for pretreatment versus combined severity factor (CSF) of the dilute-acid hydrolysis (the first step) and the acid-catalyzed steam explosion. ification yield shows an approximately 1.5-fold increase in that achieved with an experiment performed at CSF = 1.6 over the acid-catalyzed steam explosion pretreatment. The result shows that the optimum operational conditions for the first dilute-acid hydrolysis step were determined to be 165 °C for 2 min with 2% H2SO4 and for the second steam explosion step was to be carried out at 180 °C for 20 min; this gave the most favorable combination in terms of an integrated process. Under the condition, a total 75.9% of xylan and 77.1% of glucan were converted to xylose and glucose, respectively. Moreover, Karimi et al. studied the hydrolysis of rice straw using the two step dilute-acid pretreatment and reported that the ability of the two-stage hydrolysis to depolymerize 78.9% xylan to xylose and 46.6% of glucan to glucose (Karimi et al., 2006). In our study, 73.7% of xylan and 7.7% of glucan were converted to xylose and glucose by the integrated pretreatment process. In comparison, the glucose yield from the pretreatment step obtained in our study is lower than the obtained by Karimi et al. using two-step steam pretreatment. However, the goal of pretreatment is to remain a large part of glucan in the treated residues to proceed the subsequent enzymatic hydrolysis for bioethanol production. Saccharification yield (g/g glucan and xylan in raw material) 3.4. Characterization of pretreated rice straw 1.0 0.8 Acid-catalyzed SE Two-step process Xyloe(P) Glucose(P) Xylose(E) Glucose(E) 0.6 0.4 0.2 0.0 1.3 1.5 1.6 1.7 1.9 2.1 0.2 0.7 0.8 0.9 1.0 1.2 1.4 1.9 CSF Fig. 4. Total saccharification yield versus combined severity factor for the acidcatalyzed steam explosion and the combined severity factor (CSF) of the dilute-acid hydrolysis (the first step) of the integrated pretreatment process. Total saccharification yield (g sugar/100 g xylose and glucose in raw rice straw) expressed the sum of glucose (E) and xylose (E) yields obtained after 72 h of the sequential enzymatic hydrolysis step and xylose (P) and glucose (P) yields obtained in the pretreatment step. In our previous study, we showed that features of vascular bundles, epidermis and silica cells of air-dried untreated rice straw could be observed by SEM microscopy and reported that the exterior area of the epidermis revealed multiple areas of damaged after dilute-acid pretreatment because of the hydrophilic nature of silica (Yu et al., 2009). In this report, the microscopic observations of the change in the different pretreatment processes were also studied using scanning electron microscopy (SEM). The exterior epidermis of the residue fibers after dilute-acid pretreatment (AP) reveals multiple structures. Moreover, the vasculofibrous bundles of HR-SE (the residue fibers after steam explosion) show no obvious peeling phenomenon with the steam explosion, whereas comparatively large amounts of microfibers and destructive perforations of the lumen can be observed in SEM images of the residue treated by acid-catalyzed steam explosion (AC-SE). The rice straw treated by the integrated pretreatment process (AP-SE) also shows significant destruction derived structures. This indicates that the proton ions of H2SO4 should be able to penetrate into the rice straw surface and further catalyze the de-acetylation and hydrolysis; this will break the b(1 ? 4)-glycosidic bonds of the hemicelluloses and promote the defibrillation of rice straw by sequential steam explosion. inhibition at high acid concentrations. This suggests the steam explosion process actually contributes to enzymatic hydrolysis efficiency. On the other hand, the xylose yield (P) is variable in terms of the dilute-acid pretreatment for the integrated pretreatment process or acid-catalyzed steam explosion. This suggests the xylose yield from pretreatment (xylose yield (P)) has more weight in terms of total saccharification yield than the glucose yield from enzymatic hydrolysis (glucose yield (E)). As shown in Fig. 4, 75% of the maximum total saccharification yield was obtained using the integrated pretreatment process of dilute-acid and steam explosion (two-step pretreatment process) at a combined severity factor of 1.4 in the first step (dilute-acid pretreatment) and enzymatic hydrolysis with 20 FPU/g of cellulose loading after 72 h. The maximum total sugar yield obtained in our study is about the same as the obtained from sawdust by Söderström et al. (77%) using two-step steam pretreatment (Söderström et al., 2003). Specifically, the maximum total sacchar- Glucose yield (g/g glucan) 1.0 0.8 0.6 0.4 Untreat Acid-catalyzed SE (AC-SE) Two-step process (AP-SE) Dilute-acid pretreatment (AP) 0.2 0.0 2 4 6 8 10 12 14 16 18 BET(m2/g) Fig. 5. The relationship between the BET surface area of the rice straw and the glucose yield in terms of enzyme digestibility by cellulase. W.-H. Chen et al. / Bioresource Technology 102 (2011) 2916–2924 The SEM microscopic image of the residue after acid-catalyzed steam explosion (AC-SE) shows some particles attached to the external surface of fragments of AC-SE. These particles might be released lignin that have been re-deposited and are called lignin droplets, or other surface degradation products (Selig et al., 2007). No similar particles can be observed on the external surface of fragments from the residue treated by the integrated pretreatment process (AP-SE). This might be explained by these particles being removed by the steam washing during the second steam explosion step. In our results, acid-soluble lignin (ASL) contents were observed decreased from 5.3 ± 0.2% to 3.5 ± 0.1% (AP-SE0.15), from 5.2 ± 0.3% to 3.2 ± 0.2% (AP-SE-0.72) and from 3.6 ± 0.1% to 2.6 ± 0.1% (AP-SE-1.91) after the second steam explosion step. In other words, acid-soluble lignin decreased after the second steam explosion step regardless of the combined severity factor (CSF) of the first dilute-acid pretreatment step. This change of acid-soluble lignin provides further support for the above argument. The relationship between the BET surface area of rice straw and enzyme digestibility by cellulase is shown in Fig. 5. The BET surface areas of the pretreated rice straw using AP (dilute-acid pretreatment), AC-SE (acid-catalyzed steam explosion) and AP-SE (integrated pretreatment process) were found to range from 3.5 to 6, 2.5 to 10 and 10.5 to 16.5 m2/g, respectively. The glucose yield in terms of enzyme digestibility of the AC-SE (57.8–72.5%) shows a higher glucose yield (10–30%) than that of the AP (37.4–48.9%) with a similar BET surface area. On the other hand, AP-SE pretreated rice straw shows a higher glucose yield (71.8–93.1%) and a larger BET surface area (10.5–16.5 m2/g), indicating that most of the hemicellulose in AP-SE has been solubilized and removed after the integrated pretreatment process, which increases the surface area and makes enzymatic hydrolysis easier. Overall, in order to convert hemicellulose and cellulose for use in a cellulosic ethanol production process, the two-step pretreatment process has attractive advantages, but further evaluation is required due to disadvantages in terms of increased machinery cost, such as the reactor and filter that are used in the two-step process. In this study, our experiments were carried out in a 6 L reaction system with a maximum steam pressure of 10 kg/cm2. We expect that a higher saccharification yield might be obtained if the acid-catalyzed steam explosion pretreatment was carried out at a higher steam pressure. Therefore, a batch operation reactor system and a bench-scale continuous operation reactor system equipped with a higher base steam pressure for the acid-catalyzed steam explosion pretreatment and the two-step pretreatment process are under construction and investigation at our institute. 4. Conclusions We have proposed a two-step process to improve the overall saccharification efficiency. The first step is dilute-acid hydrolysis to maximize the xylose conversion and the second step is steam explosion to destroy the rice straw structure to enhance enzymatic hydrolysis. The effects of product recovery, dilute-acid and steam explosion on the structural feature properties of the residue were also explored. The integrated process shows attractive advantages for the utility of holocellulose in the feedstock for cellulosic ethanol production. 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