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Pretreatment of sugarcane bagasse with NH4OH–H2O2 and ionic liquid for efficient hydrolysis and bioethanol production
Bioresource Technology 119 (2012) 199–207
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Pretreatment of sugarcane bagasse with NH4OH–H2O2 and ionic liquid for efficient hydrolysis and bioethanol production Zhisheng Zhu, Mingjun Zhu ⇑, Zhenqiang Wu School of Bioscience and Bioengineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, People’s Republic of China
h i g h l i g h t s " An efficient pretreatment method using NH4OH–H2O2 and ionic liquid (IL) was developed for the recovery of cellulose from sugarcane bagasse (SCB). " [Amim]Cl reduced the crystallinity index of SCB. " The pretreatment did not have a negative effect on subsequent bioethanol fermentation.
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Article history: Received 14 February 2012 Received in revised form 19 May 2012 Accepted 22 May 2012 Available online 30 May 2012 Keywords: Sugarcane bagasse Ionic liquid Pretreatment Enzymatic hydrolysis Bioethanol production
a b s t r a c t An efficient pretreatment method using NH4OH–H2O2 and ionic liquid (IL) was developed for the recovery of cellulose from sugarcane bagasse (SCB). The regenerated SCB from the combined pretreatment exhibited significantly enhanced enzymatic digestibility with an efficiency of 91.4% after 12 h of hydrolysis, which was 64% higher than the efficiency observed for the regenerated SCB after the individual NH4OH–H2O2 pretreatment. 1-Allyl-3-methylimidazolium chloride ([Amim]Cl) dissolved the cellulose from the NH4OH–H2O2–pretreated SCB, and the crystallinity index (CrI) detected by X-ray diffraction (XRD) was reduced by 42%. The recycled and fresh [Amim]Cl demonstrated the same performance on the pretreatment of SCB for the enhancement of enzymatic digestibility. The regenerated SCB was subsequently used in simultaneous saccharification and co-fermentation (SScF) for bioethanol production by cellulase and yeast. The pretreatment did not have a negative effect on bioethanol fermentation, and an ethanol yield of 0.42 g/g was achieved with a corresponding fermentation efficiency of 94.5%. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The rapidly growing demand for energy, a dwindling and unstable supply of petroleum, and the emergence of global warming from the use of fossil fuels have rekindled a strong interest in pursuing alternative and renewable energy sources (Nguyen et al., 2010; Beukes and Pletschke, 2010). Lignocellulosic biomass and crop wastes have been considered as potential sustainable feedstocks for energy production. One of the major lignocellulosic materials considered for bioethanol production in tropical countries is sugarcane bagasse (SCB), which is the fibrous residue remaining after extracting the juice from sugarcane in the sugar production process. More than 70% of SCB consists of hydrolysable carbohydrates that can yield fermentable sugars for the production of value-added bioproducts (Paiva and Frollini, 2001). It is estimated that approximately 100 million dry tons of SCB are produced globally every year. Although most of the SCB is burned
⇑ Corresponding author. Tel.: +86 20 39380623; fax: +86 20 39380601. E-mail address: [email protected] (M. Zhu). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.05.111
to produce steam power, there is still a surplus of this material that can be used for bioethanol production (Pandy et al., 2000). A major issue in the conversion of lignocellulosic materials into biofuel is overcoming biomass recalcitrance through pretreatment while still maintaining green and energy-efficient processing. Several pretreatment methods, including chemical, physical and physico-chemical techniques have been reported, and several detailed review papers have been published (Mosier et al., 2005; Chang and Holtzapple, 2000). In general, regardless of the exact method used, the purpose of pretreatment is to alter or remove the lignin and/or hemicellulose, disrupt the crystallinity of the cellulose, and increase its porosity to make the cellulose more accessible to the enzymes, which significantly improves the hydrolysis of the cellulose (Sun and Cheng, 2002). Recent studies showed that ionic liquids (ILs) could be used to dissolve cellulose from lignocellulosic biomasses such as corn stalks, wheat and wood (Nguyen et al., 2010; Li et al., 2010). ILs, which are known as ‘‘green solvents’’, are organic salts that usually melt below 100 °C. Compared with conventional pretreatment methods, pretreatments using ILs as cellulose solvents have several advantages. First, the application of ILs usually occurs at lower
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temperatures and uses less hazardous process chemicals. Second, ILs can be reused after the pretreatment, and they can easily be applied to enhance the enzymatic digestibility of lignocellulosic biomass and to efficiently recover fermentable sugars (Swatloski et al., 2002). Therefore, as a novel and potential technology, the pretreatment of lignocellulosic biomass with ILs has gained attention and is being widely investigated. This process consists of a direct dissolution of cellulose using ILs as solvents, but the use of a potential IL in lignocellulose pretreatment needs to meet several requirements: no occurrence of cellulose decomposition, easy processing and cellulose regeneration, convenient recovery of the ILs, and no toxic or odour emissions (Hermanutz et al., 2008). In our work, the pretreatment of SCB using a combination of NH4OH–H2O2 and ILs was investigated. NH4OH mainly reacts with lignin but not with cellulose and causes delignification by cleaving lignin–carbohydrate linkages at elevated temperatures (Chang and Holtzapple, 2000), which our previous study also validated (Zhu et al., 2012). H2O2, a strong oxidant and bleaching agent used in the paper industry, can also enhance the enzymatic digestibility of biomass. Radicals, including singlet oxygen, superoxide, and hydroxyl radicals generated from H2O2, could change the hemicellulose structure, remove the lignin, and degrade lignin into CO2, H2O, and carboxylic acids (Kang et al., 2011). Our present work focuses on the delignification effects of NH4OH and the solubilisation of cellulose by ILs to enhance the digestibility of lignocellulosic feedstock for enzymatic hydrolysis. Additionally, the physical characteristics of the regenerated SCB, including the cellulose crystallinity and the plant cell wall morphology, were measured and compared using Xray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Moreover, the regenerated bagasse was subsequently used in simultaneous saccharification and co-fermentation (SScF) to investigate any possible adverse effects of using IL-pretreated bagasse in ethanol fermentation. 2. Methods 2.1. Sample preparation and NH4OH–H2O2 pretreatment The SCB was a gift from the Guangzhou Sugarcane Industry Research Institute (Guangdong Province, China). The SCB was ground and sieved until the SCB particles were able to pass through a 60mesh (0.3 mm) sieve, and only these particles were used for the pretreatment experiments (Zhao et al., 2009). The main chemical composition of raw SCB was measured as follows: 39.5% glucan, 19.8% xylan, 2.0% Arabian, 21.0% Klason lignin, 4.9% acid soluble lignin, 5.7% ash and 2.6% benzene–ethanol extractives. The pretreatment of SCB using an NH4OH–H2O2 solution was carried out according to the methods described in our previous study (Zhu et al., 2012). 2.2. ILs and combined pretreatment The pretreatment of SCB with ILs was investigated using 1-allyl3-methylimidazolium chloride ([Amim]Cl) and 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) purchased from the Lanzhou Institute of Chemical Physics (Lanzhou, China). [Amim]Cl is a highly viscous liquid at room temperature, while ([Bmim]Cl exists in its liquid form at higher temperatures between 80 and 90 °C. The combined pretreatment of SCB with ILs was carried out as follows. First, a 3% (w/w) SCB solution was prepared by adding 0.6 g of NH4OH–H2O2–pretreated SCB to 19.4 g of an individual IL in a 250 mL rockered flask. The flask was subsequently heated using a thermostat magnetic stirrer (78HW-1, Union Instruments, JiangSu, China) to 60, 80, 100, 120 or 140 °C and incubated for 0.5, 1, 3, 6 or 9 h. After the incubation, 50 mL of ethanol, which was
used as an antisolvent, was added to the vigorously stirring SCB solution, and the SCB-cellulose was precipitated and regenerated. The precipitated bulky material was then passed through filter paper (Whatman #2) using a Buchner funnel under a reduced pressure and washed with 10 mL of ethanol. The regenerated SCBcellulose was washed thoroughly with distilled water and then dried in a freeze dryer (FD-1C-50, BOYIKANG Instrument Co., Ltd., Beijing, China) for 48 h prior to the carbohydrate analysis and the subsequent enzymatic hydrolysis and fermentation. 2.3. Recovery of IL The filtered liquid containing the IL and ethanol was collected, and the filtrate containing 100 mL of IL was evaporated by using a rotary evaporator (EYELA NE SERIES, CCA-1 110, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) at 80 °C and 0.08 MPa vacuum for about 2 h to remove the antisolvent before the IL was recovered for subsequent reutilisation. And the IL recovery rate was calculated according to the gram of initial IL at the beginning of the pretreatment and the IL recovered in the end. 2.4. Enzymatic hydrolysis The enzymatic saccharification of the NH4OH–H2O2–pretreated and NH4OH–H2O2 + IL-pretreated SCB samples was carried out in 10-mL vials at 40 °C on a rotary shaker set at 200 rpm. The solutions used for the enzymatic hydrolysis were buffered with 50 mM sodium citrate, pH 4.8, and steam sterilised by an autoclave at 121 °C for 30 min. The total batch volume was 5 mL, containing a cellulase (Celluclast 1.5 L, Novozymes, Denmark) that exhibited cellulase and xylanase activities of 12.54 FPU/mL and 0.97 U/mL, respectively, and loaded with 20 FPU/g of SCB with a substrate concentration of 20 g/L. The reaction was monitored by removing 0.1 mL of the supernatant at specific time intervals, boiling it for 3 min to quench the enzymatic reaction, and centrifuging it at 14,580g for 5 min. The total reducing sugars were measured by the 3,5-dinitrosalicylic acid (DNS) assay against a D-glucose standard (Miller, 1959). Glucose and xylose concentrations were also measured by HPLC as described below. All of the assays were performed in duplicate. The error bars represent the standard deviations of the duplicate measurements. 2.5. SScF SHY07-1 yeast, which is an intergeneric protoplast fusant between Saccharomyces cerevisiae (a gift from the Sanhe ethanol factory, Zanjiang, Guangdong Province, China) and Pichia stipitis (a gift from the Guangzhou Sugarcane Industry Research Institute, Guangzhou, China) that possesses the ability to convert not only glucose but also xylose into ethanol, was used in this study (Zhu et al., 2011a). The preparation of the inoculums of SHY07-1 yeast for SScF was performed according to the methods described in our previous study with slight modifications (Zhu et al., 2012). To prepare the inoculums, 1 mL of 80 °C frozen cells was transferred to 10 mL of YPX medium in sterile test tubes and was incubated at 30 °C and 200 rpm for 12 18 h in an incubator shaker (C24KC refrigerated incubator shaker, Edison, NJ, United States). Ten millilitres of the active cells was then, in turn, aseptically transferred to 100 mL of sterile YPX medium in a 250 mL Erlenmeyer flask. For SScF, the initial pH was adjusted to 5.5 in the beginning. 2.6. Analytical methods An analysis of the cellulose and hemicellulose content in the SCB was conducted according to the methods recommended by
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the National Renewable Energy Laboratory (NREL) (NREL, 2011). The Klason lignin content of the SCB was analysed according to the corresponding Chinese Standards: GB/T 2677.8-1994. The total cellulase activity was quantified in filter paper units (FPU) using a Whatman #1 filter paper strip (1.0 6.0 cm, approximately 50 mg) as the substrate. The xylanase activity was assayed using 1% (w/w) xylan from oat spelts (Sigma–Aldrich, St. Louis, MO, USA) as the substrate. The enzyme activities were expressed in international units (IUs), which was the amount of enzyme required to release 1 lmol per minute of either glucose (cellulase) or xylose (xylanase) under the assay conditions (pH 4.8, 50 °C). The amount of reducing sugars was estimated using the 3,5dinitrosalicylic acid (DNS) method (Miller, 1959). To determine the amounts of ethanol and monomeric sugars, 1mL fermentation samples were acidified with 10% sulphuric acid, centrifuged in 1-mL Eppendorf tubes at 9900g for 10 min in a TGL-16H centrifuge (HEMA Medical Instrument Co., Ltd., Zhuhai, China) and filtered through a 0.22-lm filter. The supernatant (pH 1–3) was analysed for the presence of soluble sugars and ethanol using a Waters 2695 HPLC (Waters Corporation, Milford, MA, United States) equipped with a Waters 2414 refractive index detector (RID) (Waters Corporation, Milford, MA, United States). Glucose, xylose, acetic acid, lactic acid and ethanol were analysed using an Aminex HPX-87H column equipped with a Cation H Cartridge Micro-Guard column (Bio-Rad, Hercules, CA, United States), and the column was operated at 60 °C with 2.5 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min (Lynd et al., 1989).
2.7. Cellulose crystallinity measurement Crystallinity is believed to be an important factor affecting the enzymatic saccharification of cellulose (Dadi et al., 2006; Kumar et al., 2009). X-ray powder diffraction patterns of NH4OH–H2O2– pretreated, NH4OH–H2O2 + IL-pretreated and un-pretreated SCB samples were obtained using a D8 ADVANCE diffractometer (Bruker Corporation, Germany) with a LynxExe Array detector. SCB samples were cast on microscope slides with double-sided tape. Scans were collected at 40 kV and 40 mA with a step size of 0.04° and steps of 0.2 s. The SCB crystallinity, as expressed by the crystallinity index (CrI), was determined from the XRD data and calculated using the following formula (Segal et al., 1959; Thygesen et al., 2005):
CrI ¼
I002 Iam 100 I002
where I002 is the intensity of the crystalline portion of the biomass (cellulose) at approximately 2h = 22.5°, and Iam is the peak of the amorphous portion (cellulose, hemicellulose and lignin) at approximately 2h = 16.6°. In this study, the second highest peak after 2h = 22.5° occurred at 2h = 16.6° and was assumed to correspond to the amorphous region (Kumar et al., 2009).
2.8. FTIR spectroscopy FTIR was conducted using a Bruker Optics VECTOR 33 (Bruker Corporation, Germany). The samples were pressed uniformly against the diamond surface using a spring-loaded anvil. The sample spectra were obtained in duplicates using an average of 256 scans over the range 4000–500 cm1 with a spectral resolution of 2 cm1. Baseline corrections were conducted using the rubber band correction method by following the spectrum’s minima (Singh et al., 2009).
2.9. SEM analysis The morphology of NH4OH–H2O2–pretreated, NH4OH– H2O2 + IL-pretreated and un-pretreated SCB samples was analysed by SEM. SEM pictures of untreated and pretreated bagasse were taken at different magnifications such as 600 and 3000 using a ZEISS EVO 18 electron microscope 51-XMX0003, Special Edition (Carl Zeiss Microscopy, Germany) equipped with an X-Max detector (Oxford Instruments, Oxford, UK) operating at 10 or 50 kV. The samples were first dried in a freeze dryer (FD-1C-50, BOYIKANG Instrument Co., Ltd., Beijing, China) for 24 h and coated with a 20-nm layer of gold by high vacuum metallisation using the SBC12 Sputter Coater System (KYKY Technology Development Ltd., Beijing, China) before being stored in a desiccator until analysis. 2.10. Calculations and statistical methods The enzymatic efficiency, fermentation efficiency, ethanol yield, SCB recovery and cellulose recovery were calculated using the following equations:
ðGlucose concentration ; g=L þ xylose concentration ; g=LÞ 100% Solid loadings glucan% 1:11 þ solid loadings xylan% 1:14; g=L Ethanol formed concentration; g=L 100% Fermentation efficiency ¼ Theoretical ethanol concentration; g=L Ethanol formed concentration ; g=L Ethanol yield ¼ Theoretical reducing sugars concentration; g=L Theoretical reducing sugars concentration ¼ Glucan concentration 1:11 þ Xylan concentration 1:14; g=L Enzymatic efficiency ¼
Regenerated SCB; g 100% Initial SCB; g Regenerated SCB cellulose ; g 100% Cellulose recovery ¼ Initial SCB cellulose ; g
SCB recovery ¼
where ⁄ indicates that the reducing sugar of the enzyme solution was deducted from the concentration of sugar in the hydrolysate; 1.11 is the coefficient of glucose obtained from glucan, 1.14 is the coefficient of xylose obtained from xylan; 0.51 is the coefficient of ethanol obtained from glucose, and 0.46 is the coefficient of ethanol obtained from xylose. 3. Results and discussion 3.1. Selection of the IL for the combined pretreatment of the SCB The NH4OH–H2O2–pretreated SCB samples dissolved in both [Amim]Cl and [Bmim]Cl and the SCB solutions in these ILs were highly viscous at moderate temperatures (70–80 °C). An effective pretreatment for lignocellulosic biomass that is to be used as a feedstock for a biological process must overcome a highly ordered structure and enhance the hydrolysis process to make it viable for producing fermentable sugars (Hendriks and Zeeman, 2009; Wyman et al., 2005). Therefore, the efficiency of the SCB pretreatment using an IL was estimated by the SCB and cellulose recovery from the IL dissolution as well as by the enzymatic efficiency during the hydrolysis. SCB recoveries of approximately 80% were obtained from both regenerated [Amim]Cl–SCB and [Bmim]Cl–SCB. However, the cellulose recovery of 89.4% obtained from regenerated [Amim]Cl– SCB was 10% higher than the cellulose recovery obtained from regenerated [Bmim]Cl–SCB. Both ILs have the same anion ([Cl]) but different cations ([Amim]+ and [Bmim]+). Recent NMR studies on the dissolution mechanism of cellulose in [Cl]-containing ILs indicate that [Cl] acts as a hydrogen bond acceptor, which interacts with the hydroxyl groups of the cellulose (Moulthrop et al., 2005). However, the different cations also affected the dissolution of the cellulose. The [Amim]+ cation has a smaller size than the [Bmim]+ cation because it has a substituent with only three carbon atoms and a double bond, resulting in an increase in the electron deficient state of the cation, which makes it easier for [Amim]+ to
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attack the oxygen atoms of cellulose’s hydroxyl groups. These two factors make [Amim]Cl perform better as a solvent of cellulose (Wu et al., 2004). Moreover, [Amim]Cl has a better lignin solubility (>300 g/kg) than [Bmim]Cl (approximately 100 g/kg) according to others’ research (Lee et al., 2009). Additionally, we evaluated the enzymatic hydrolysis of the SCB pretreated with [Amim]Cl and [Bmim]Cl. As demonstrated in Fig. 1, the content of the total reducing sugars and the enzymatic efficiency increased rapidly with time, indicating that the combined pretreated SCB was hydrolysed rapidly by cellulase compared with the NH4OH–H2O2–pretreated SCB. However, the efficiency of the SCB pretreatment using [Amim]Cl was higher than the pretreatment using [Bmim]Cl because when compared with the regenerated [Bmim]Cl–SCB, the concentration of the total reducing sugars and the enzymatic efficiency of the regenerated [Amim]Cl–SCB was slightly higher. Therefore, [Amim]Cl shows more promise as an IL for use in lignocellulose pretreatment with a higher efficiency of cellulose dissolution. Moreover, [Amim]Cl is non-corrosive, non-toxic, has a pleasant odour and a lower melting point and viscosity than [Bmim]Cl at room temperature. These advantages are very important with respect to technical processes. In our study, [Amim]Cl appeared to be the better selection; therefore, the following investigations were carried out with this IL.
3.2. Influence of the temperature and length of the IL pretreatment The incubation temperature for the combined pretreatment of the SCB was varied from 60 to 140 °C (Table 1). During the pretreatment with [Amim]Cl, the IL suspension turned a darker brown colour as the temperature increased, presumably as a result of lignin extraction. A higher content of glucan and a lower content of lignin were detected as the pretreatment temperatures were increased (Table 1). Approximately 41% of the initial lignin was extracted after 1 h at 140 °C. However, the content of xylan decreased as the temperature increased; a pretreatment for 1 h at 140 °C removed approximately 10% of the initial xylan. These results demonstrated that the SCB underwent delignification with substantially smaller losses of xylan when the combined pretreatment with [Amim]Cl was used. A similar phenomenon has been reported by Lee et al. (2009). As Table 1 shows, the SCB and cellulose recovery both declined with increasing pretreatment temperatures and were reduced by 40.4% and 33.4%, respectively. An explanation for this result could be that the cellulose chains were broken down into soluble oligosaccharides that could not be regenerated into amorphous cellulose; thus, the cellulose recovery was decreased (Nguyen et al., 2010).
Fig. 2A and B shows the time course of the glucose + xylose release and the enzymatic efficiency of the cellulase-catalysed hydrolysis of the combined pretreated SCB. Both the initial rate and enzymatic efficiency were enhanced after 4 h with increasing pretreatment temperatures. Specifically, the initial rate of enzymatic hydrolysis of the SCB was 2.49 and 1.21 g/(L h) for the combined pretreated SCB (1 h at 140 °C) and the NH4OH–H2O2– pretreated SCB, respectively. Compared with the NH4OH–H2O2– pretreated SCB alone, the concentration of the glucose + xylose and the enzymatic efficiency of the combined pretreated SCB were always higher at any sampling time. At the end of the hydrolysis (36 h), the concentration of the glucose + xylose released from the combined pretreated SCB (1 h at 140 °C) was 16.2 g/L (Fig. 2A), which was 66.5% higher than the glucose + xylose released from the SCB treated with NH4OH–H2O2 alone, and the corresponding enzymatic efficiency was 62.3% higher (Fig. 2B). The addition of fresh cellulase did not result in increased enzymatic efficiency, indicating that the termination of the reaction was not due to the loss of cellulase activity. To further understand the influence of incubation time, several samples of [Amim]Cl–pretreated SCB were prepared by changing the incubation time in the IL. Thus, the incubation time for the combined pretreatment of SCB with [Amim]Cl was varied from 0.5 to 9 h at 100 °C (Table 2). The increased pretreatment time led to increased lignin extraction; 21% of the lignin was extracted after 1 h of pretreatment, and approximately 39% of the lignin was extracted after 9 h of pretreatment. We also observed that approximately 7% of the initial xylan had been removed at 100 °C after 9 h of pretreatment. The glucan content in the combined pretreated SCB slightly increased and the content of the lignin rapidly decreased after the pretreatment with [Amim]Cl. Fig. 3A and B depict the time course of the glucose + xylose release and the enzymatic efficiency during the hydrolysis of the pretreated SCB. Similar to our results when we investigated the role of temperature, both the initial rate and the enzymatic efficiency increased with increasing pretreatment times; enzymatic efficiencies of at least 75% were achieved by the time the reactions were terminated. However, the content of glucose + xylose and the enzymatic efficiency of the combined pretreated SCB increased insignificantly as the pretreatment time increased from 1 to 9 h. After 36 h of hydrolysis, the concentration of glucose + xylose and the enzymatic efficiency of the combined pretreated SCB (100 °C for 9 h) were 61.8% (Fig. 3A) and 65.3% (Fig. 3B) higher, respectively, than the concentration of glucose + xylose and the enzymatic efficiency of the NH4OH–H2O2–pretreated SCB. Nguyen et al. achieved 82% of cellulose recovery and 97% of enzymatic glucose conversion when they pretreated rice straw
Fig. 1. Effects of the different ionic liquids pretreatments on the enzymatic hydrolysis of the NH4OH–H2O2–pretreated sugarcane bagasse. The results are the means ± standard deviation for duplicate experiments. The following enzymatic reaction conditions were used: 2% substrate loading; 50 mM sodium citrate buffer, pH 4.8; 20 FPU/g sugarcane bagasse Celluclast 1.5 L cellulase; 40 °C; 200 rpm; and 36 h.
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Z. Zhu et al. / Bioresource Technology 119 (2012) 199–207 Table 1 Effect of the pretreatment temperature on the composition and enzymatic hydrolysis of the sugarcane bagasse. Ionic liquid pretreatmenta
Compositions of the combined pretreated sugarcane bagasse
Enzymatic hydrolysis of sugarcane bagasseb (12 h)
Temperature (°C)
Sugarcane bagasse recovery (%)
Cellulose recovery (%)
Glucan (%)
Xylan (%)
Klason lignin (%)
Released glucose + xylose (g/L)
Enzymatic efficiency (%)
Untreatedc 60 80 100 120 140
100 83.7 82.8 80.6 76.2 59.6
100 87.0 86.5 85.8 84.5 66.6
51.3 53.3 53.5 54.6 56.9 57.3
20.0 19.4 19.3 19.0 18.9 18.0
10.1 9.8 8.0 8.0 6.7 6.0
7.3 10.9 11.3 12.2 13.8 13.9
55.8 74.6 86.7 91.4 92.2 92.6
a
0.6 g of NH4OH–H2O2–pretreated sugarcane bagasse was incubated in 19.4 g of [Amim]Cl for 1 h. Reaction conditions: 2% substrate loading; 5 mL total volume; 50 mM sodium citrate buffer, pH 4.8; 20 FPU/g sugarcane bagasse Celluclast 1.5 L cellulase; 40 °C; 200 rpm; and 36 h. c Untreated by ionic liquids; treated by NH4OH–H2O2 alone. b
(RS) with 10% ammonia and 1-ethyl-3-methylimidazolium acetate at 100 °C for 6 h (Nguyen et al., 2010). Li et al. (2010) also dissolved switchgrass by 1-ethyl-3-methylimidazolium acetate at 160 °C for 3 h and obtained a glucan yield of 96% in 24 h. For the combined NH4OH–H2O2 and [Amim]Cl pretreatment of SCB, 85.8% of cellulose recovery and 91.42% of enzymatic efficiency within 12 h was achieved at 100 °C for 1 h, indicating a significant improvement in the rate and yield of enzymatic hydrolysis of SCB. The low temperature and short pretreatment time coupled with high cellulose conversion rate and yield show the significant advancement of the NH4OH–H2O2 and [Amim]Cl combined pretreatment of SCB. 3.3. SScF of the combined pretreated SCB The regenerated SCB was also employed in SScF for ethanol production by incubating it with cellulase and yeast. The time profiles of the fermentation experiments are presented in Fig. 4A–C. It is evident that the ethanol concentration accumulated rapidly to approximately 13 g/L after 48 h of the SScF using the IL combined pretreated SCB compared with the other samples. After 120 h of fermentation, the acetic acid concentration of the combined pretreated SCB was reduced by 5.26% and 36.8%, compared with the NH4OH–H2O2–pretreated SCB and the un-pretreated SCB, respectively, due to the removal of acetyl groups from the hemicellulose. By the end of the SScF (120 h) with the combined pretreated SCB, the ethanol concentration was as high as 14.1 g/L, which corresponded to an ethanol yield of 0.42 g/g with a fermentation efficiency of 94.5%. Kuo and Lee (2009) obtained 14.5 g/L ethanol and 0.44 g ethanol/g glucose in SSF of Z. molibis as they dissolved SCB by N-methylmorpholine-N-oxide (NMMO) at 100 °C for 1 h. However, xylose was not calculated in the report because it was not consumed in the SSF. In addition, the SCB recovery from the pre-treatment steps is more than 80%, which can be reduced significantly in scale-up. The observed loss after regeneration probably resulted from the loss of extractives of SCB and the loss occurred during the recovery of precipitated SCB. The total cellulose conversion from SCB to ethanol increased from 0.20 g/g raw SCB to 0.35 g/ g combined pre-treated SCB due to the combined pre-treatment. Our results indicated that SCB pretreatment with [Amim]Cl at 100 °C for 1 h had no adverse effects on the production of ethanol through fermentation by yeast. 3.4. Structure of the regenerated SCB To gain insight into the possible mechanism underlying the enhancement of the enzymatic hydrolysis, the structural features of regenerated SCB were examined using XRD and compared to the corresponding untreated SCB samples. Fig. 5A shows that unpretreated and NH4OH–H2O2–pretreated SCB samples displayed
little change in cellulose crystallinity, but the cellulose crystallinity of the IL combined pretreated SCB samples was altered significantly and had a broad diffraction peak. The CrIs for all of the samples were calculated from the XRD data, and the results are summarised in Table 3. As shown in Table 3, the un-pretreated SCB is highly crystalline (38.9), and there is an obvious decrease in the CrI (35.9) for the NH4OH–H2O2–pretreated SCB sample. In the case of the [Amim]Cl combined pretreatment, the peak at 22.5° (due to the 0 0 2 crystalline plane) that appeared in the diffraction pattern was significantly weakened and shifted to 20.2°, indicating that there was minimal structural order in the IL-pretreated SCB cellulose (Kuo and Lee, 2009; Lee et al., 2009); the minimal structural order was likely due to its transformation from celluloses I to II (Sun et al., 2009). The CrI calculated from the IL combined pretreated SCB sample was significantly lower (20.81) than the CrIs of either the un-pretreated or NH4OH–H2O2–pretreated SCB samples. To further validate the above alteration, the structural differences between the regenerated and untreated SCB were also investigated by FTIR spectroscopy measurements (Fig. 5B). The FTIR fingerprints of the regenerated and untreated SCB were almost the same, indicating that the IL dissolution of the cellulose is a derivative dissolution process (Wu et al., 2004). Furthermore, the infrared absorbance ratios A1374/A2917 and A1427/A898 were proposed by Nelson and O’Connor (Nelson and O’Connor, 1964; Oh et al., 2005) as the total crystalline index (TCI) and the lateral order index (LOI) of a cellulosic material, respectively. Higher index values represent materials with higher crystallinity and an ordered structure. As shown in Table 3, the TCI and LOI of the SCB decreased significantly from 1.66 and 2.48 to 1.01 and 2.26, respectively, after the [Amim]Cl combined pretreatment of the SCB at 100 °C for 1 h. Kuo et al. also observed a decrease in TCI and LOI from 1.397 and 1.443 to 1.116 and 1.129, respectively as they dissolved SCB using N-methylmorpholine-N-oxide (NMMO) at 100 °C for 7 h (Kuo and Lee, 2009). Moreover, the decreases in these two indices have also been reported for wheat straw that had been regenerated from IL dissolution (Liu and Chen, 2006). These decreases indicate that the crystalline structure of the lignocellulosic material was transformed into an amorphous form after IL pretreatment. Several FTIR bands were used to monitor the chemical changes of lignin and carbohydrates (Fig. 5B). When compared to the untreated and NH4OH–H2O2 pretreated SCB, the intensity of bands at 1510 cm1 (aromatic skeletal from lignin) and 1329 cm1 (syringyl and guaiacyl condensed lignin) decreased significantly for the combined pretreated SCB (Fig. 5C), indicating the removal of lignin. In addition, a decrease in the peak at 1098 cm1 (referring to the crystalline cellulose) and an increase in the peak at 900 cm1 (referring to amorphous cellulose) were observed for the combined pretreated
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Fig. 2. Influence of the pretreatment temperature on the enzymatic hydrolysis of the sugarcane bagasse samples. The results are the means ± standard deviation for duplicate experiments. The following enzymatic reaction conditions were used: 2% substrate loading; 50 mM sodium citrate, buffer, pH 4.8; 20 FPU/g sugarcane bagasse Celluclast 1.5 L cellulase; 40 °C; 200 rpm; and 36 h.
Table 2 Effect of the pretreatment time on the composition and enzymatic hydrolysis of the sugarcane bagasse. Ionic liquid pretreatmenta
Compositions of the combined pretreated sugarcane bagasse
Enzymatic hydrolysis of sugarcane bagasseb (12 h)
Time (h)
Sugarcane bagasse recovery (%)
Cellulose recovery (%)
Glucan (%)
Xylan (%)
Klason lignin (%)
Released glucose + xylose (g/ L)
Enzymatic efficiency (%)
Untreatedc 0.5 1 3 6 9
100 84.2 81.4 80.7 77.7 61.3
100 86.2 87.4 87.4 86.1 68.2
51.3 52.4 55.1 55.5 56.8 57.1
20.0 19.8 19.7 19.8 19.0 18.7
10.1 9.2 8.0 7.6 7.0 6.2
7.3 11.3 12.7 12.9 13.3 13.5
55.8 81.8 90.7 93.3 93.1 95.0
(82.3)d (89.5) (93.1) (93.7) (94.2)
All the other footnotes are as described in the legend to Table 1. a 0.6 g of NH4OH–H2O2–pretreated sugarcane bagasse was incubated in 19.4 g of [Amim]Cl at 100 °C. d Pretreated with reused [Amim]Cl.
Fig. 3. Influence of the pretreatment time on the enzymatic hydrolysis of the sugarcane bagasse samples. The results are the means ± standard deviation for duplicate experiments. The following enzymatic reaction conditions were used: 2% substrate loading; 50 mM sodium citrate buffer, pH 4.8; 20 FPU/g sugarcane bagasse Celluclast 1.5 L cellulase; 40 °C; 200 rpm; and 36 h.
SCB (Fig. 5C), exhibiting a transformation of the crystalline cellulose to amorphous cellulose after the combined pretreatment of SCB, which was consistent with the XRD pattern, indicating a decrease in cellulose crystallinity. The same results were also observed in the IL pretreated switchgrass by Li et al. (2010). During the regeneration process, the rapid precipitation with ethanol probably prevents the dissolved bagasse from returning to its original crystalline structure. Evidently, the fragmented and porous regenerated bagasse with an amorphous structure provides more surfaces for the enzymes to attack. It is clear that the cellu-
lase was not able to penetrate the intact structure of the untreated bagasse for hydrolysis. Thus, a low hydrolysis rate and yield were obtained. In contrast, the intact structure was disrupted by the IL combined pretreatment, resulting in a porous and amorphous regenerated bagasse with greatly enhanced enzymatic hydrolysis. The results may explain the difference in the amounts of total reducing sugars released for the IL combined pretreated SCB (14.1 g/L for [Amim]Cl and 13.7 g/L for [Bmim]Cl) and the NH4OH–H2O2–pretreated SCB (6.67 g/L) during the first 12 h of the enzymatic hydrolysis (Fig. 1).
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Fig. 4. Substrate and product concentration trends over a time course during simultaneous saccharification and co-fermentation. The results are the means for duplicate experiments. The fermentations were conducted at 30 °C with an initial pH of 5.5 and agitation at 200 rpm under largely anaerobic conditions for 120 h. The initial sugarcane bagasse loading was 4%, and the enzyme loading was 20 FPU/g of sugarcane bagasse Celluclast 1.5 L cellulase. (A) Un-pretreated sugarcane bagasse, (B) NH4OH–H2O2– pretreated sugarcane bagasse and (C) NH4OH–H2O2 + [Amim]Cl–pretreated sugarcane bagasse.
Fig. 5. The X-ray diffraction pattern (A) and Fourier transform infrared spectroscopy fingerprints (B) of the sugarcane bagasse samples.
Table 3 The crystallinity index of X-ray diffraction and the infrared ratios of Fourier transform infrared spectroscopy were measured for the sugarcane bagasse derived from different pretreatments. Samples
Crystallinity index (CrI) X-ray diffraction
Total crystalline index (TCL) A1374/ A2917
Lateral order index (LOI) A1427/ A898
Un-pretreated NH4OH–H2O2–pretreated NH4OH–H2O2 + [Amim]Cl-pretreated (100 °C for 1 h)
38.9 35.9 20.8
1.66 1.52 1.01
2.48 2.34 2.26
In addition, the morphology of the NH4OH–H2O2–pretreated, NH4OH–H2O2 + IL-pretreated and un-pretreated SCB samples was examined by SEM. A well-defined fibre bundle with a smooth sur-
face was observed in the un-pretreated bagasse. After pretreatment with NH4OH–H2O2, the bagasse fibre bundle became much thinner and had a very rough, irregular, and corrugated surface.
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In contrast, the IL combined pretreatment significantly altered the fibrillar structure so that it no longer showed any fibrous structure (Supporting information Fig. S1). The same result was also observed by Li et al. (2010) as they dissolved switchgrass using 1ethyl-3-methylimidazolium acetate at 160 °C for 3 h. Swatloski et al. (2002) observed that the morphology of the material was significantly changed, displaying a rough but conglomerate texture when they dissolved pulp using 1-butyl-3-methylimidazolium chloride and regenerated it with water. Singh et al. (2009) also observed a significant difference in the surface structure between unpretreated and regenerated fibres as they pretreated switchgrass with IL 1-n-ethyl-3-methylimidazolium acetate at 120 °C, indicating that IL pretreatment effectively weakened the van der Waal’s interaction between cell wall polymers, leading to better enzyme accessibility and increased binding sites in regenerated cellulose fibres. Our results are consistent with the observations that faster hydrolysis rates and higher glucan yields are obtained for the IL combined pretreated SCB than for the NH4OH–H2O2–pretreated and un-pretreated SCBs. 3.5. Reuse of the [Amim]Cl for the pretreatment of the SCB A major disadvantage to using ILs as pretreatment solvents for lignocellulosic materials is their relatively high cost. Although many processes have been developed to decrease the production costs of ILs (Waterkamp et al., 2007), typical ILs remain expensive. Therefore, the reuse of ILs is important in the commercial processing of biomass. In this work, [Amim]Cl could easily be recovered by evaporating the filtrate, and the recovery efficiency of [Amim]Cl was approximately 92%. However, the loss can be reduced significantly in scale-up. Tsioptsias et al. (2008) reported a 90% solvent recovery in the production of cellulose foams with the IL 1-allylmethylimidazolium chloride. Duchemin et al. (2009) obtained a 93% IL recovery as they dissolved microfibrillated cellulose using 1-butyl-3-methylimidazolium chloride. Without further purification, the [Amim]Cl solution was reused for the pretreatment of SCB, and no significant difference was observed between the hydrolyses of the bagasse pretreated with the recycled [Amim]Cl (see Table 2) and the bagasse pretreated with the fresh [Amim]Cl. The result indicates that the recycled [Amim]Cl is an effective solvent for lignocellulose pretreatment, which makes the lignocellulose pretreatment process economically feasible for future applications. 4. Conclusion This study presented a promising combined method of SCB pretreatment using ILs and NH4OH–H2O2. Our combined pretreatment resulted in a cellulose recovery of 85.8%, and 91.4% of the enzymatic efficiency was achieved within 12 h, which were significantly higher than the values obtained after the individual NH4OH–H2O2 pretreatment. The analyses by XRD and FTIR demonstrated that the SCB regenerated from the combined pretreatment had a lower crystallinity than the SCB regenerated after the NH4OH–H2O2 pretreatment alone. Both the recycled and fresh [Amim]Cl performed equally well for SCB pretreatment. SScF demonstrated that the pretreatment had no negative effects on the fermentation. Acknowledgements The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 51078147), the Fundamental Research Funds for the Central Universities, SCUT (2012ZM0081), and the Guangdong Provincial Science and Tech-
nology Program 2010A010500005).
(2011B090400033, 2010B031700022, and
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Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy. Carbohydr. Res. 340, 2376–2391. Paiva, J.M.F., Frollini, E., 2001. Sugarcane bagasse reinforced phenolic and lignophenolic composites. J. Appl. Polym. Sci. 83, 880–888. Pandy, A., Soccol, C.R., Nigam, P., Socclo, V.T., 2000. Biotechnological potential of agro-industrial residues. I: Sugarcane bagasse. Bioresour. Technol. 74, 69–80. Segal, L., Creely, J.J., Martin Jr, A.E., Conrad, C.M., 1959. An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text. Res. J. 29, 786–794. Singh, S., Simmons, B.A., Vogel, K.P., 2009. Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass. Biotechnol. Bioeng. 104, 68–75. Sun, Y., Cheng, J.Y., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83, 1–11. Sun, N., Rahman, M., Qin, Y., Maxim, M.L., Rodriguez, H., Rogers, R.D., 2009. Complete dissolution and partial delignification of wood in the ionic liquid 1ethyl-3-methylimidazolium acetate. Green Chem. 11, 646–655.
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Pretreatment of sugarcane bagasse with NH4OH–H2O2 and ionic liquid for efficient hydrolysis and bioethanol production Zhisheng Zhu, Mingjun Zhu ⇑, Zhenqiang Wu School of Bioscience and Bioengineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, People’s Republic of China
h i g h l i g h t s " An efficient pretreatment method using NH4OH–H2O2 and ionic liquid (IL) was developed for the recovery of cellulose from sugarcane bagasse (SCB). " [Amim]Cl reduced the crystallinity index of SCB. " The pretreatment did not have a negative effect on subsequent bioethanol fermentation.
a r t i c l e
i n f o
Article history: Received 14 February 2012 Received in revised form 19 May 2012 Accepted 22 May 2012 Available online 30 May 2012 Keywords: Sugarcane bagasse Ionic liquid Pretreatment Enzymatic hydrolysis Bioethanol production
a b s t r a c t An efficient pretreatment method using NH4OH–H2O2 and ionic liquid (IL) was developed for the recovery of cellulose from sugarcane bagasse (SCB). The regenerated SCB from the combined pretreatment exhibited significantly enhanced enzymatic digestibility with an efficiency of 91.4% after 12 h of hydrolysis, which was 64% higher than the efficiency observed for the regenerated SCB after the individual NH4OH–H2O2 pretreatment. 1-Allyl-3-methylimidazolium chloride ([Amim]Cl) dissolved the cellulose from the NH4OH–H2O2–pretreated SCB, and the crystallinity index (CrI) detected by X-ray diffraction (XRD) was reduced by 42%. The recycled and fresh [Amim]Cl demonstrated the same performance on the pretreatment of SCB for the enhancement of enzymatic digestibility. The regenerated SCB was subsequently used in simultaneous saccharification and co-fermentation (SScF) for bioethanol production by cellulase and yeast. The pretreatment did not have a negative effect on bioethanol fermentation, and an ethanol yield of 0.42 g/g was achieved with a corresponding fermentation efficiency of 94.5%. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The rapidly growing demand for energy, a dwindling and unstable supply of petroleum, and the emergence of global warming from the use of fossil fuels have rekindled a strong interest in pursuing alternative and renewable energy sources (Nguyen et al., 2010; Beukes and Pletschke, 2010). Lignocellulosic biomass and crop wastes have been considered as potential sustainable feedstocks for energy production. One of the major lignocellulosic materials considered for bioethanol production in tropical countries is sugarcane bagasse (SCB), which is the fibrous residue remaining after extracting the juice from sugarcane in the sugar production process. More than 70% of SCB consists of hydrolysable carbohydrates that can yield fermentable sugars for the production of value-added bioproducts (Paiva and Frollini, 2001). It is estimated that approximately 100 million dry tons of SCB are produced globally every year. Although most of the SCB is burned
⇑ Corresponding author. Tel.: +86 20 39380623; fax: +86 20 39380601. E-mail address: [email protected] (M. Zhu). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.05.111
to produce steam power, there is still a surplus of this material that can be used for bioethanol production (Pandy et al., 2000). A major issue in the conversion of lignocellulosic materials into biofuel is overcoming biomass recalcitrance through pretreatment while still maintaining green and energy-efficient processing. Several pretreatment methods, including chemical, physical and physico-chemical techniques have been reported, and several detailed review papers have been published (Mosier et al., 2005; Chang and Holtzapple, 2000). In general, regardless of the exact method used, the purpose of pretreatment is to alter or remove the lignin and/or hemicellulose, disrupt the crystallinity of the cellulose, and increase its porosity to make the cellulose more accessible to the enzymes, which significantly improves the hydrolysis of the cellulose (Sun and Cheng, 2002). Recent studies showed that ionic liquids (ILs) could be used to dissolve cellulose from lignocellulosic biomasses such as corn stalks, wheat and wood (Nguyen et al., 2010; Li et al., 2010). ILs, which are known as ‘‘green solvents’’, are organic salts that usually melt below 100 °C. Compared with conventional pretreatment methods, pretreatments using ILs as cellulose solvents have several advantages. First, the application of ILs usually occurs at lower
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temperatures and uses less hazardous process chemicals. Second, ILs can be reused after the pretreatment, and they can easily be applied to enhance the enzymatic digestibility of lignocellulosic biomass and to efficiently recover fermentable sugars (Swatloski et al., 2002). Therefore, as a novel and potential technology, the pretreatment of lignocellulosic biomass with ILs has gained attention and is being widely investigated. This process consists of a direct dissolution of cellulose using ILs as solvents, but the use of a potential IL in lignocellulose pretreatment needs to meet several requirements: no occurrence of cellulose decomposition, easy processing and cellulose regeneration, convenient recovery of the ILs, and no toxic or odour emissions (Hermanutz et al., 2008). In our work, the pretreatment of SCB using a combination of NH4OH–H2O2 and ILs was investigated. NH4OH mainly reacts with lignin but not with cellulose and causes delignification by cleaving lignin–carbohydrate linkages at elevated temperatures (Chang and Holtzapple, 2000), which our previous study also validated (Zhu et al., 2012). H2O2, a strong oxidant and bleaching agent used in the paper industry, can also enhance the enzymatic digestibility of biomass. Radicals, including singlet oxygen, superoxide, and hydroxyl radicals generated from H2O2, could change the hemicellulose structure, remove the lignin, and degrade lignin into CO2, H2O, and carboxylic acids (Kang et al., 2011). Our present work focuses on the delignification effects of NH4OH and the solubilisation of cellulose by ILs to enhance the digestibility of lignocellulosic feedstock for enzymatic hydrolysis. Additionally, the physical characteristics of the regenerated SCB, including the cellulose crystallinity and the plant cell wall morphology, were measured and compared using Xray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Moreover, the regenerated bagasse was subsequently used in simultaneous saccharification and co-fermentation (SScF) to investigate any possible adverse effects of using IL-pretreated bagasse in ethanol fermentation. 2. Methods 2.1. Sample preparation and NH4OH–H2O2 pretreatment The SCB was a gift from the Guangzhou Sugarcane Industry Research Institute (Guangdong Province, China). The SCB was ground and sieved until the SCB particles were able to pass through a 60mesh (0.3 mm) sieve, and only these particles were used for the pretreatment experiments (Zhao et al., 2009). The main chemical composition of raw SCB was measured as follows: 39.5% glucan, 19.8% xylan, 2.0% Arabian, 21.0% Klason lignin, 4.9% acid soluble lignin, 5.7% ash and 2.6% benzene–ethanol extractives. The pretreatment of SCB using an NH4OH–H2O2 solution was carried out according to the methods described in our previous study (Zhu et al., 2012). 2.2. ILs and combined pretreatment The pretreatment of SCB with ILs was investigated using 1-allyl3-methylimidazolium chloride ([Amim]Cl) and 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) purchased from the Lanzhou Institute of Chemical Physics (Lanzhou, China). [Amim]Cl is a highly viscous liquid at room temperature, while ([Bmim]Cl exists in its liquid form at higher temperatures between 80 and 90 °C. The combined pretreatment of SCB with ILs was carried out as follows. First, a 3% (w/w) SCB solution was prepared by adding 0.6 g of NH4OH–H2O2–pretreated SCB to 19.4 g of an individual IL in a 250 mL rockered flask. The flask was subsequently heated using a thermostat magnetic stirrer (78HW-1, Union Instruments, JiangSu, China) to 60, 80, 100, 120 or 140 °C and incubated for 0.5, 1, 3, 6 or 9 h. After the incubation, 50 mL of ethanol, which was
used as an antisolvent, was added to the vigorously stirring SCB solution, and the SCB-cellulose was precipitated and regenerated. The precipitated bulky material was then passed through filter paper (Whatman #2) using a Buchner funnel under a reduced pressure and washed with 10 mL of ethanol. The regenerated SCBcellulose was washed thoroughly with distilled water and then dried in a freeze dryer (FD-1C-50, BOYIKANG Instrument Co., Ltd., Beijing, China) for 48 h prior to the carbohydrate analysis and the subsequent enzymatic hydrolysis and fermentation. 2.3. Recovery of IL The filtered liquid containing the IL and ethanol was collected, and the filtrate containing 100 mL of IL was evaporated by using a rotary evaporator (EYELA NE SERIES, CCA-1 110, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) at 80 °C and 0.08 MPa vacuum for about 2 h to remove the antisolvent before the IL was recovered for subsequent reutilisation. And the IL recovery rate was calculated according to the gram of initial IL at the beginning of the pretreatment and the IL recovered in the end. 2.4. Enzymatic hydrolysis The enzymatic saccharification of the NH4OH–H2O2–pretreated and NH4OH–H2O2 + IL-pretreated SCB samples was carried out in 10-mL vials at 40 °C on a rotary shaker set at 200 rpm. The solutions used for the enzymatic hydrolysis were buffered with 50 mM sodium citrate, pH 4.8, and steam sterilised by an autoclave at 121 °C for 30 min. The total batch volume was 5 mL, containing a cellulase (Celluclast 1.5 L, Novozymes, Denmark) that exhibited cellulase and xylanase activities of 12.54 FPU/mL and 0.97 U/mL, respectively, and loaded with 20 FPU/g of SCB with a substrate concentration of 20 g/L. The reaction was monitored by removing 0.1 mL of the supernatant at specific time intervals, boiling it for 3 min to quench the enzymatic reaction, and centrifuging it at 14,580g for 5 min. The total reducing sugars were measured by the 3,5-dinitrosalicylic acid (DNS) assay against a D-glucose standard (Miller, 1959). Glucose and xylose concentrations were also measured by HPLC as described below. All of the assays were performed in duplicate. The error bars represent the standard deviations of the duplicate measurements. 2.5. SScF SHY07-1 yeast, which is an intergeneric protoplast fusant between Saccharomyces cerevisiae (a gift from the Sanhe ethanol factory, Zanjiang, Guangdong Province, China) and Pichia stipitis (a gift from the Guangzhou Sugarcane Industry Research Institute, Guangzhou, China) that possesses the ability to convert not only glucose but also xylose into ethanol, was used in this study (Zhu et al., 2011a). The preparation of the inoculums of SHY07-1 yeast for SScF was performed according to the methods described in our previous study with slight modifications (Zhu et al., 2012). To prepare the inoculums, 1 mL of 80 °C frozen cells was transferred to 10 mL of YPX medium in sterile test tubes and was incubated at 30 °C and 200 rpm for 12 18 h in an incubator shaker (C24KC refrigerated incubator shaker, Edison, NJ, United States). Ten millilitres of the active cells was then, in turn, aseptically transferred to 100 mL of sterile YPX medium in a 250 mL Erlenmeyer flask. For SScF, the initial pH was adjusted to 5.5 in the beginning. 2.6. Analytical methods An analysis of the cellulose and hemicellulose content in the SCB was conducted according to the methods recommended by
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the National Renewable Energy Laboratory (NREL) (NREL, 2011). The Klason lignin content of the SCB was analysed according to the corresponding Chinese Standards: GB/T 2677.8-1994. The total cellulase activity was quantified in filter paper units (FPU) using a Whatman #1 filter paper strip (1.0 6.0 cm, approximately 50 mg) as the substrate. The xylanase activity was assayed using 1% (w/w) xylan from oat spelts (Sigma–Aldrich, St. Louis, MO, USA) as the substrate. The enzyme activities were expressed in international units (IUs), which was the amount of enzyme required to release 1 lmol per minute of either glucose (cellulase) or xylose (xylanase) under the assay conditions (pH 4.8, 50 °C). The amount of reducing sugars was estimated using the 3,5dinitrosalicylic acid (DNS) method (Miller, 1959). To determine the amounts of ethanol and monomeric sugars, 1mL fermentation samples were acidified with 10% sulphuric acid, centrifuged in 1-mL Eppendorf tubes at 9900g for 10 min in a TGL-16H centrifuge (HEMA Medical Instrument Co., Ltd., Zhuhai, China) and filtered through a 0.22-lm filter. The supernatant (pH 1–3) was analysed for the presence of soluble sugars and ethanol using a Waters 2695 HPLC (Waters Corporation, Milford, MA, United States) equipped with a Waters 2414 refractive index detector (RID) (Waters Corporation, Milford, MA, United States). Glucose, xylose, acetic acid, lactic acid and ethanol were analysed using an Aminex HPX-87H column equipped with a Cation H Cartridge Micro-Guard column (Bio-Rad, Hercules, CA, United States), and the column was operated at 60 °C with 2.5 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min (Lynd et al., 1989).
2.7. Cellulose crystallinity measurement Crystallinity is believed to be an important factor affecting the enzymatic saccharification of cellulose (Dadi et al., 2006; Kumar et al., 2009). X-ray powder diffraction patterns of NH4OH–H2O2– pretreated, NH4OH–H2O2 + IL-pretreated and un-pretreated SCB samples were obtained using a D8 ADVANCE diffractometer (Bruker Corporation, Germany) with a LynxExe Array detector. SCB samples were cast on microscope slides with double-sided tape. Scans were collected at 40 kV and 40 mA with a step size of 0.04° and steps of 0.2 s. The SCB crystallinity, as expressed by the crystallinity index (CrI), was determined from the XRD data and calculated using the following formula (Segal et al., 1959; Thygesen et al., 2005):
CrI ¼
I002 Iam 100 I002
where I002 is the intensity of the crystalline portion of the biomass (cellulose) at approximately 2h = 22.5°, and Iam is the peak of the amorphous portion (cellulose, hemicellulose and lignin) at approximately 2h = 16.6°. In this study, the second highest peak after 2h = 22.5° occurred at 2h = 16.6° and was assumed to correspond to the amorphous region (Kumar et al., 2009).
2.8. FTIR spectroscopy FTIR was conducted using a Bruker Optics VECTOR 33 (Bruker Corporation, Germany). The samples were pressed uniformly against the diamond surface using a spring-loaded anvil. The sample spectra were obtained in duplicates using an average of 256 scans over the range 4000–500 cm1 with a spectral resolution of 2 cm1. Baseline corrections were conducted using the rubber band correction method by following the spectrum’s minima (Singh et al., 2009).
2.9. SEM analysis The morphology of NH4OH–H2O2–pretreated, NH4OH– H2O2 + IL-pretreated and un-pretreated SCB samples was analysed by SEM. SEM pictures of untreated and pretreated bagasse were taken at different magnifications such as 600 and 3000 using a ZEISS EVO 18 electron microscope 51-XMX0003, Special Edition (Carl Zeiss Microscopy, Germany) equipped with an X-Max detector (Oxford Instruments, Oxford, UK) operating at 10 or 50 kV. The samples were first dried in a freeze dryer (FD-1C-50, BOYIKANG Instrument Co., Ltd., Beijing, China) for 24 h and coated with a 20-nm layer of gold by high vacuum metallisation using the SBC12 Sputter Coater System (KYKY Technology Development Ltd., Beijing, China) before being stored in a desiccator until analysis. 2.10. Calculations and statistical methods The enzymatic efficiency, fermentation efficiency, ethanol yield, SCB recovery and cellulose recovery were calculated using the following equations:
ðGlucose concentration ; g=L þ xylose concentration ; g=LÞ 100% Solid loadings glucan% 1:11 þ solid loadings xylan% 1:14; g=L Ethanol formed concentration; g=L 100% Fermentation efficiency ¼ Theoretical ethanol concentration; g=L Ethanol formed concentration ; g=L Ethanol yield ¼ Theoretical reducing sugars concentration; g=L Theoretical reducing sugars concentration ¼ Glucan concentration 1:11 þ Xylan concentration 1:14; g=L Enzymatic efficiency ¼
Regenerated SCB; g 100% Initial SCB; g Regenerated SCB cellulose ; g 100% Cellulose recovery ¼ Initial SCB cellulose ; g
SCB recovery ¼
where ⁄ indicates that the reducing sugar of the enzyme solution was deducted from the concentration of sugar in the hydrolysate; 1.11 is the coefficient of glucose obtained from glucan, 1.14 is the coefficient of xylose obtained from xylan; 0.51 is the coefficient of ethanol obtained from glucose, and 0.46 is the coefficient of ethanol obtained from xylose. 3. Results and discussion 3.1. Selection of the IL for the combined pretreatment of the SCB The NH4OH–H2O2–pretreated SCB samples dissolved in both [Amim]Cl and [Bmim]Cl and the SCB solutions in these ILs were highly viscous at moderate temperatures (70–80 °C). An effective pretreatment for lignocellulosic biomass that is to be used as a feedstock for a biological process must overcome a highly ordered structure and enhance the hydrolysis process to make it viable for producing fermentable sugars (Hendriks and Zeeman, 2009; Wyman et al., 2005). Therefore, the efficiency of the SCB pretreatment using an IL was estimated by the SCB and cellulose recovery from the IL dissolution as well as by the enzymatic efficiency during the hydrolysis. SCB recoveries of approximately 80% were obtained from both regenerated [Amim]Cl–SCB and [Bmim]Cl–SCB. However, the cellulose recovery of 89.4% obtained from regenerated [Amim]Cl– SCB was 10% higher than the cellulose recovery obtained from regenerated [Bmim]Cl–SCB. Both ILs have the same anion ([Cl]) but different cations ([Amim]+ and [Bmim]+). Recent NMR studies on the dissolution mechanism of cellulose in [Cl]-containing ILs indicate that [Cl] acts as a hydrogen bond acceptor, which interacts with the hydroxyl groups of the cellulose (Moulthrop et al., 2005). However, the different cations also affected the dissolution of the cellulose. The [Amim]+ cation has a smaller size than the [Bmim]+ cation because it has a substituent with only three carbon atoms and a double bond, resulting in an increase in the electron deficient state of the cation, which makes it easier for [Amim]+ to
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attack the oxygen atoms of cellulose’s hydroxyl groups. These two factors make [Amim]Cl perform better as a solvent of cellulose (Wu et al., 2004). Moreover, [Amim]Cl has a better lignin solubility (>300 g/kg) than [Bmim]Cl (approximately 100 g/kg) according to others’ research (Lee et al., 2009). Additionally, we evaluated the enzymatic hydrolysis of the SCB pretreated with [Amim]Cl and [Bmim]Cl. As demonstrated in Fig. 1, the content of the total reducing sugars and the enzymatic efficiency increased rapidly with time, indicating that the combined pretreated SCB was hydrolysed rapidly by cellulase compared with the NH4OH–H2O2–pretreated SCB. However, the efficiency of the SCB pretreatment using [Amim]Cl was higher than the pretreatment using [Bmim]Cl because when compared with the regenerated [Bmim]Cl–SCB, the concentration of the total reducing sugars and the enzymatic efficiency of the regenerated [Amim]Cl–SCB was slightly higher. Therefore, [Amim]Cl shows more promise as an IL for use in lignocellulose pretreatment with a higher efficiency of cellulose dissolution. Moreover, [Amim]Cl is non-corrosive, non-toxic, has a pleasant odour and a lower melting point and viscosity than [Bmim]Cl at room temperature. These advantages are very important with respect to technical processes. In our study, [Amim]Cl appeared to be the better selection; therefore, the following investigations were carried out with this IL.
3.2. Influence of the temperature and length of the IL pretreatment The incubation temperature for the combined pretreatment of the SCB was varied from 60 to 140 °C (Table 1). During the pretreatment with [Amim]Cl, the IL suspension turned a darker brown colour as the temperature increased, presumably as a result of lignin extraction. A higher content of glucan and a lower content of lignin were detected as the pretreatment temperatures were increased (Table 1). Approximately 41% of the initial lignin was extracted after 1 h at 140 °C. However, the content of xylan decreased as the temperature increased; a pretreatment for 1 h at 140 °C removed approximately 10% of the initial xylan. These results demonstrated that the SCB underwent delignification with substantially smaller losses of xylan when the combined pretreatment with [Amim]Cl was used. A similar phenomenon has been reported by Lee et al. (2009). As Table 1 shows, the SCB and cellulose recovery both declined with increasing pretreatment temperatures and were reduced by 40.4% and 33.4%, respectively. An explanation for this result could be that the cellulose chains were broken down into soluble oligosaccharides that could not be regenerated into amorphous cellulose; thus, the cellulose recovery was decreased (Nguyen et al., 2010).
Fig. 2A and B shows the time course of the glucose + xylose release and the enzymatic efficiency of the cellulase-catalysed hydrolysis of the combined pretreated SCB. Both the initial rate and enzymatic efficiency were enhanced after 4 h with increasing pretreatment temperatures. Specifically, the initial rate of enzymatic hydrolysis of the SCB was 2.49 and 1.21 g/(L h) for the combined pretreated SCB (1 h at 140 °C) and the NH4OH–H2O2– pretreated SCB, respectively. Compared with the NH4OH–H2O2– pretreated SCB alone, the concentration of the glucose + xylose and the enzymatic efficiency of the combined pretreated SCB were always higher at any sampling time. At the end of the hydrolysis (36 h), the concentration of the glucose + xylose released from the combined pretreated SCB (1 h at 140 °C) was 16.2 g/L (Fig. 2A), which was 66.5% higher than the glucose + xylose released from the SCB treated with NH4OH–H2O2 alone, and the corresponding enzymatic efficiency was 62.3% higher (Fig. 2B). The addition of fresh cellulase did not result in increased enzymatic efficiency, indicating that the termination of the reaction was not due to the loss of cellulase activity. To further understand the influence of incubation time, several samples of [Amim]Cl–pretreated SCB were prepared by changing the incubation time in the IL. Thus, the incubation time for the combined pretreatment of SCB with [Amim]Cl was varied from 0.5 to 9 h at 100 °C (Table 2). The increased pretreatment time led to increased lignin extraction; 21% of the lignin was extracted after 1 h of pretreatment, and approximately 39% of the lignin was extracted after 9 h of pretreatment. We also observed that approximately 7% of the initial xylan had been removed at 100 °C after 9 h of pretreatment. The glucan content in the combined pretreated SCB slightly increased and the content of the lignin rapidly decreased after the pretreatment with [Amim]Cl. Fig. 3A and B depict the time course of the glucose + xylose release and the enzymatic efficiency during the hydrolysis of the pretreated SCB. Similar to our results when we investigated the role of temperature, both the initial rate and the enzymatic efficiency increased with increasing pretreatment times; enzymatic efficiencies of at least 75% were achieved by the time the reactions were terminated. However, the content of glucose + xylose and the enzymatic efficiency of the combined pretreated SCB increased insignificantly as the pretreatment time increased from 1 to 9 h. After 36 h of hydrolysis, the concentration of glucose + xylose and the enzymatic efficiency of the combined pretreated SCB (100 °C for 9 h) were 61.8% (Fig. 3A) and 65.3% (Fig. 3B) higher, respectively, than the concentration of glucose + xylose and the enzymatic efficiency of the NH4OH–H2O2–pretreated SCB. Nguyen et al. achieved 82% of cellulose recovery and 97% of enzymatic glucose conversion when they pretreated rice straw
Fig. 1. Effects of the different ionic liquids pretreatments on the enzymatic hydrolysis of the NH4OH–H2O2–pretreated sugarcane bagasse. The results are the means ± standard deviation for duplicate experiments. The following enzymatic reaction conditions were used: 2% substrate loading; 50 mM sodium citrate buffer, pH 4.8; 20 FPU/g sugarcane bagasse Celluclast 1.5 L cellulase; 40 °C; 200 rpm; and 36 h.
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Z. Zhu et al. / Bioresource Technology 119 (2012) 199–207 Table 1 Effect of the pretreatment temperature on the composition and enzymatic hydrolysis of the sugarcane bagasse. Ionic liquid pretreatmenta
Compositions of the combined pretreated sugarcane bagasse
Enzymatic hydrolysis of sugarcane bagasseb (12 h)
Temperature (°C)
Sugarcane bagasse recovery (%)
Cellulose recovery (%)
Glucan (%)
Xylan (%)
Klason lignin (%)
Released glucose + xylose (g/L)
Enzymatic efficiency (%)
Untreatedc 60 80 100 120 140
100 83.7 82.8 80.6 76.2 59.6
100 87.0 86.5 85.8 84.5 66.6
51.3 53.3 53.5 54.6 56.9 57.3
20.0 19.4 19.3 19.0 18.9 18.0
10.1 9.8 8.0 8.0 6.7 6.0
7.3 10.9 11.3 12.2 13.8 13.9
55.8 74.6 86.7 91.4 92.2 92.6
a
0.6 g of NH4OH–H2O2–pretreated sugarcane bagasse was incubated in 19.4 g of [Amim]Cl for 1 h. Reaction conditions: 2% substrate loading; 5 mL total volume; 50 mM sodium citrate buffer, pH 4.8; 20 FPU/g sugarcane bagasse Celluclast 1.5 L cellulase; 40 °C; 200 rpm; and 36 h. c Untreated by ionic liquids; treated by NH4OH–H2O2 alone. b
(RS) with 10% ammonia and 1-ethyl-3-methylimidazolium acetate at 100 °C for 6 h (Nguyen et al., 2010). Li et al. (2010) also dissolved switchgrass by 1-ethyl-3-methylimidazolium acetate at 160 °C for 3 h and obtained a glucan yield of 96% in 24 h. For the combined NH4OH–H2O2 and [Amim]Cl pretreatment of SCB, 85.8% of cellulose recovery and 91.42% of enzymatic efficiency within 12 h was achieved at 100 °C for 1 h, indicating a significant improvement in the rate and yield of enzymatic hydrolysis of SCB. The low temperature and short pretreatment time coupled with high cellulose conversion rate and yield show the significant advancement of the NH4OH–H2O2 and [Amim]Cl combined pretreatment of SCB. 3.3. SScF of the combined pretreated SCB The regenerated SCB was also employed in SScF for ethanol production by incubating it with cellulase and yeast. The time profiles of the fermentation experiments are presented in Fig. 4A–C. It is evident that the ethanol concentration accumulated rapidly to approximately 13 g/L after 48 h of the SScF using the IL combined pretreated SCB compared with the other samples. After 120 h of fermentation, the acetic acid concentration of the combined pretreated SCB was reduced by 5.26% and 36.8%, compared with the NH4OH–H2O2–pretreated SCB and the un-pretreated SCB, respectively, due to the removal of acetyl groups from the hemicellulose. By the end of the SScF (120 h) with the combined pretreated SCB, the ethanol concentration was as high as 14.1 g/L, which corresponded to an ethanol yield of 0.42 g/g with a fermentation efficiency of 94.5%. Kuo and Lee (2009) obtained 14.5 g/L ethanol and 0.44 g ethanol/g glucose in SSF of Z. molibis as they dissolved SCB by N-methylmorpholine-N-oxide (NMMO) at 100 °C for 1 h. However, xylose was not calculated in the report because it was not consumed in the SSF. In addition, the SCB recovery from the pre-treatment steps is more than 80%, which can be reduced significantly in scale-up. The observed loss after regeneration probably resulted from the loss of extractives of SCB and the loss occurred during the recovery of precipitated SCB. The total cellulose conversion from SCB to ethanol increased from 0.20 g/g raw SCB to 0.35 g/ g combined pre-treated SCB due to the combined pre-treatment. Our results indicated that SCB pretreatment with [Amim]Cl at 100 °C for 1 h had no adverse effects on the production of ethanol through fermentation by yeast. 3.4. Structure of the regenerated SCB To gain insight into the possible mechanism underlying the enhancement of the enzymatic hydrolysis, the structural features of regenerated SCB were examined using XRD and compared to the corresponding untreated SCB samples. Fig. 5A shows that unpretreated and NH4OH–H2O2–pretreated SCB samples displayed
little change in cellulose crystallinity, but the cellulose crystallinity of the IL combined pretreated SCB samples was altered significantly and had a broad diffraction peak. The CrIs for all of the samples were calculated from the XRD data, and the results are summarised in Table 3. As shown in Table 3, the un-pretreated SCB is highly crystalline (38.9), and there is an obvious decrease in the CrI (35.9) for the NH4OH–H2O2–pretreated SCB sample. In the case of the [Amim]Cl combined pretreatment, the peak at 22.5° (due to the 0 0 2 crystalline plane) that appeared in the diffraction pattern was significantly weakened and shifted to 20.2°, indicating that there was minimal structural order in the IL-pretreated SCB cellulose (Kuo and Lee, 2009; Lee et al., 2009); the minimal structural order was likely due to its transformation from celluloses I to II (Sun et al., 2009). The CrI calculated from the IL combined pretreated SCB sample was significantly lower (20.81) than the CrIs of either the un-pretreated or NH4OH–H2O2–pretreated SCB samples. To further validate the above alteration, the structural differences between the regenerated and untreated SCB were also investigated by FTIR spectroscopy measurements (Fig. 5B). The FTIR fingerprints of the regenerated and untreated SCB were almost the same, indicating that the IL dissolution of the cellulose is a derivative dissolution process (Wu et al., 2004). Furthermore, the infrared absorbance ratios A1374/A2917 and A1427/A898 were proposed by Nelson and O’Connor (Nelson and O’Connor, 1964; Oh et al., 2005) as the total crystalline index (TCI) and the lateral order index (LOI) of a cellulosic material, respectively. Higher index values represent materials with higher crystallinity and an ordered structure. As shown in Table 3, the TCI and LOI of the SCB decreased significantly from 1.66 and 2.48 to 1.01 and 2.26, respectively, after the [Amim]Cl combined pretreatment of the SCB at 100 °C for 1 h. Kuo et al. also observed a decrease in TCI and LOI from 1.397 and 1.443 to 1.116 and 1.129, respectively as they dissolved SCB using N-methylmorpholine-N-oxide (NMMO) at 100 °C for 7 h (Kuo and Lee, 2009). Moreover, the decreases in these two indices have also been reported for wheat straw that had been regenerated from IL dissolution (Liu and Chen, 2006). These decreases indicate that the crystalline structure of the lignocellulosic material was transformed into an amorphous form after IL pretreatment. Several FTIR bands were used to monitor the chemical changes of lignin and carbohydrates (Fig. 5B). When compared to the untreated and NH4OH–H2O2 pretreated SCB, the intensity of bands at 1510 cm1 (aromatic skeletal from lignin) and 1329 cm1 (syringyl and guaiacyl condensed lignin) decreased significantly for the combined pretreated SCB (Fig. 5C), indicating the removal of lignin. In addition, a decrease in the peak at 1098 cm1 (referring to the crystalline cellulose) and an increase in the peak at 900 cm1 (referring to amorphous cellulose) were observed for the combined pretreated
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Fig. 2. Influence of the pretreatment temperature on the enzymatic hydrolysis of the sugarcane bagasse samples. The results are the means ± standard deviation for duplicate experiments. The following enzymatic reaction conditions were used: 2% substrate loading; 50 mM sodium citrate, buffer, pH 4.8; 20 FPU/g sugarcane bagasse Celluclast 1.5 L cellulase; 40 °C; 200 rpm; and 36 h.
Table 2 Effect of the pretreatment time on the composition and enzymatic hydrolysis of the sugarcane bagasse. Ionic liquid pretreatmenta
Compositions of the combined pretreated sugarcane bagasse
Enzymatic hydrolysis of sugarcane bagasseb (12 h)
Time (h)
Sugarcane bagasse recovery (%)
Cellulose recovery (%)
Glucan (%)
Xylan (%)
Klason lignin (%)
Released glucose + xylose (g/ L)
Enzymatic efficiency (%)
Untreatedc 0.5 1 3 6 9
100 84.2 81.4 80.7 77.7 61.3
100 86.2 87.4 87.4 86.1 68.2
51.3 52.4 55.1 55.5 56.8 57.1
20.0 19.8 19.7 19.8 19.0 18.7
10.1 9.2 8.0 7.6 7.0 6.2
7.3 11.3 12.7 12.9 13.3 13.5
55.8 81.8 90.7 93.3 93.1 95.0
(82.3)d (89.5) (93.1) (93.7) (94.2)
All the other footnotes are as described in the legend to Table 1. a 0.6 g of NH4OH–H2O2–pretreated sugarcane bagasse was incubated in 19.4 g of [Amim]Cl at 100 °C. d Pretreated with reused [Amim]Cl.
Fig. 3. Influence of the pretreatment time on the enzymatic hydrolysis of the sugarcane bagasse samples. The results are the means ± standard deviation for duplicate experiments. The following enzymatic reaction conditions were used: 2% substrate loading; 50 mM sodium citrate buffer, pH 4.8; 20 FPU/g sugarcane bagasse Celluclast 1.5 L cellulase; 40 °C; 200 rpm; and 36 h.
SCB (Fig. 5C), exhibiting a transformation of the crystalline cellulose to amorphous cellulose after the combined pretreatment of SCB, which was consistent with the XRD pattern, indicating a decrease in cellulose crystallinity. The same results were also observed in the IL pretreated switchgrass by Li et al. (2010). During the regeneration process, the rapid precipitation with ethanol probably prevents the dissolved bagasse from returning to its original crystalline structure. Evidently, the fragmented and porous regenerated bagasse with an amorphous structure provides more surfaces for the enzymes to attack. It is clear that the cellu-
lase was not able to penetrate the intact structure of the untreated bagasse for hydrolysis. Thus, a low hydrolysis rate and yield were obtained. In contrast, the intact structure was disrupted by the IL combined pretreatment, resulting in a porous and amorphous regenerated bagasse with greatly enhanced enzymatic hydrolysis. The results may explain the difference in the amounts of total reducing sugars released for the IL combined pretreated SCB (14.1 g/L for [Amim]Cl and 13.7 g/L for [Bmim]Cl) and the NH4OH–H2O2–pretreated SCB (6.67 g/L) during the first 12 h of the enzymatic hydrolysis (Fig. 1).
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Fig. 4. Substrate and product concentration trends over a time course during simultaneous saccharification and co-fermentation. The results are the means for duplicate experiments. The fermentations were conducted at 30 °C with an initial pH of 5.5 and agitation at 200 rpm under largely anaerobic conditions for 120 h. The initial sugarcane bagasse loading was 4%, and the enzyme loading was 20 FPU/g of sugarcane bagasse Celluclast 1.5 L cellulase. (A) Un-pretreated sugarcane bagasse, (B) NH4OH–H2O2– pretreated sugarcane bagasse and (C) NH4OH–H2O2 + [Amim]Cl–pretreated sugarcane bagasse.
Fig. 5. The X-ray diffraction pattern (A) and Fourier transform infrared spectroscopy fingerprints (B) of the sugarcane bagasse samples.
Table 3 The crystallinity index of X-ray diffraction and the infrared ratios of Fourier transform infrared spectroscopy were measured for the sugarcane bagasse derived from different pretreatments. Samples
Crystallinity index (CrI) X-ray diffraction
Total crystalline index (TCL) A1374/ A2917
Lateral order index (LOI) A1427/ A898
Un-pretreated NH4OH–H2O2–pretreated NH4OH–H2O2 + [Amim]Cl-pretreated (100 °C for 1 h)
38.9 35.9 20.8
1.66 1.52 1.01
2.48 2.34 2.26
In addition, the morphology of the NH4OH–H2O2–pretreated, NH4OH–H2O2 + IL-pretreated and un-pretreated SCB samples was examined by SEM. A well-defined fibre bundle with a smooth sur-
face was observed in the un-pretreated bagasse. After pretreatment with NH4OH–H2O2, the bagasse fibre bundle became much thinner and had a very rough, irregular, and corrugated surface.
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In contrast, the IL combined pretreatment significantly altered the fibrillar structure so that it no longer showed any fibrous structure (Supporting information Fig. S1). The same result was also observed by Li et al. (2010) as they dissolved switchgrass using 1ethyl-3-methylimidazolium acetate at 160 °C for 3 h. Swatloski et al. (2002) observed that the morphology of the material was significantly changed, displaying a rough but conglomerate texture when they dissolved pulp using 1-butyl-3-methylimidazolium chloride and regenerated it with water. Singh et al. (2009) also observed a significant difference in the surface structure between unpretreated and regenerated fibres as they pretreated switchgrass with IL 1-n-ethyl-3-methylimidazolium acetate at 120 °C, indicating that IL pretreatment effectively weakened the van der Waal’s interaction between cell wall polymers, leading to better enzyme accessibility and increased binding sites in regenerated cellulose fibres. Our results are consistent with the observations that faster hydrolysis rates and higher glucan yields are obtained for the IL combined pretreated SCB than for the NH4OH–H2O2–pretreated and un-pretreated SCBs. 3.5. Reuse of the [Amim]Cl for the pretreatment of the SCB A major disadvantage to using ILs as pretreatment solvents for lignocellulosic materials is their relatively high cost. Although many processes have been developed to decrease the production costs of ILs (Waterkamp et al., 2007), typical ILs remain expensive. Therefore, the reuse of ILs is important in the commercial processing of biomass. In this work, [Amim]Cl could easily be recovered by evaporating the filtrate, and the recovery efficiency of [Amim]Cl was approximately 92%. However, the loss can be reduced significantly in scale-up. Tsioptsias et al. (2008) reported a 90% solvent recovery in the production of cellulose foams with the IL 1-allylmethylimidazolium chloride. Duchemin et al. (2009) obtained a 93% IL recovery as they dissolved microfibrillated cellulose using 1-butyl-3-methylimidazolium chloride. Without further purification, the [Amim]Cl solution was reused for the pretreatment of SCB, and no significant difference was observed between the hydrolyses of the bagasse pretreated with the recycled [Amim]Cl (see Table 2) and the bagasse pretreated with the fresh [Amim]Cl. The result indicates that the recycled [Amim]Cl is an effective solvent for lignocellulose pretreatment, which makes the lignocellulose pretreatment process economically feasible for future applications. 4. Conclusion This study presented a promising combined method of SCB pretreatment using ILs and NH4OH–H2O2. Our combined pretreatment resulted in a cellulose recovery of 85.8%, and 91.4% of the enzymatic efficiency was achieved within 12 h, which were significantly higher than the values obtained after the individual NH4OH–H2O2 pretreatment. The analyses by XRD and FTIR demonstrated that the SCB regenerated from the combined pretreatment had a lower crystallinity than the SCB regenerated after the NH4OH–H2O2 pretreatment alone. Both the recycled and fresh [Amim]Cl performed equally well for SCB pretreatment. SScF demonstrated that the pretreatment had no negative effects on the fermentation. Acknowledgements The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 51078147), the Fundamental Research Funds for the Central Universities, SCUT (2012ZM0081), and the Guangdong Provincial Science and Tech-
nology Program 2010A010500005).
(2011B090400033, 2010B031700022, and
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