Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5664-0
BIOENERGY AND BIOFUELS
Investigation of a novel acid-catalyzed ionic liquid pretreatment method to improve biomass enzymatic hydrolysis conversion Qing Qing & Rong Hu & Yucai He & Yue Zhang & Liqun Wang
Received: 28 October 2013 / Revised: 26 February 2014 / Accepted: 4 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Pretreatment of lignocellulosic materials is a prerequisite to facilitate the disruption of the natural recalcitrance of their carbohydrate–lignin shield and to allow enzymes to easily access the crystalline cellulose surfaces. Recently, pretreatment of ionic liquids (ILs) has been widely studied as a promising pretreatment technique; however, it is too expensive to be commercialized. In this study, an efficient acidcatalyzed aqueous IL pretreatment process was developed to optimize the total sugar conversion of pretreated biomass and to reduce IL usage. The experimental results demonstrated that the total sugar conversion was raised to 92.7 % with the synergistic effects of IL (1,3-dimethylimidazolium dimethylphosphate, [MMIM]DMP) and dilute hydrochloric acid (HCl) under pretreatment conditions of 110 °C for 2 h, compared to the conversion of only 27.3 % obtained with untreated corn stover. Moreover, the addition of the inorganic acids, especially HCl, to the IL pretreatment was found to not only significantly destroy the crystalline structure of cellulose in corn stover, promoting the conversion of cellulose and hemicellulose to monomeric sugars, but also provide an opportunity to reduce the usage of expensive IL solvents.
Keywords Ionic liquid . Pretreatment . Acid . Lignocellulose . Enzymatic hydrolysis
Q. Qing : R. Hu : Y. He : Y. Zhang : L. Wang (*) Department of Biochemical Engineering, College of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu 213164, China e-mail: [email protected] L. Wang e-mail: [email protected]
Introduction Lignocellulosic materials are the most abundant resources in nature that could be biologically converted to biofuels and other value-added chemicals (Adsul et al. 2011; Zhong et al. 2009). Plant cell walls are complex structures composed mostly of cellulose (35–50 %), hemicelluloses (20–35 %), and lignin (5–30 %), which form a matrix of a cross-linked three dimensional polysaccharide network. Cellulose is a linear macromolecular compound composed of glucose units that are linked by β-1,4-glycosidic bonds (Ninomiya et al. 2012) and is mainly composed of two regions, the amorphous and crystalline regions. The amorphous structure is relatively loose and easily degraded, whereas the crystalline structure is stable and difficult to degrade. Hemicellulose is a polymer mainly made up of hetero-1,4-β-D-xylan with an amorphous and highly branched structure and is easily digested by enzymes. Lignin is a cross-linked racemic macromolecule that works as a physical barrier, surrounding the cellulose and hemicelluloses to prevent microorganisms and enzymes from accessing the cellulose surface (Alvira et al. 2010). Thus, lignin is widely believed to be an obstacle to the effective conversion of cellulose by the process of enzymatic hydrolysis. Pretreatment process has been considered as a prerequisite for breaking or removing lignin and for increasing the porosity of cellulose in order to make lignocellulosic materials to be more easily hydrolyzed (Chandra et al. 2007; Galbe and Zacchi 2007). Over the years, many pretreatment methods have been intensively studied and used to improve the degradation of lignocellulosic materials (Hendriks and Zeeman 2009; Mosier et al. 2005). These leading pretreatment processes include physical methods, chemical methods, or a combination of both. Physical processes include mechanical grinding, steam explosion, radiation, etc. However, these methods usually require high energy consumption and high
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cost. On the other hand, chemical methods mainly include acid hydrolysis, alkaline hydrolysis, and organic solvent treatmen t us ing m e tha nol , eth ano l, a cet one , an d N methylmorpholine N-oxide (NMMO) (Mosier et al. 2005). The corrosive nature of the chemicals and solvents involved in these methods results in high requirements for equipment (Xu et al. 2010). Therefore, the exorbitant cost of the processing methods mentioned remains a dominant concern preventing the commercialization of cellulosic ethanol (Nguyen et al. 2010). Ionic liquids (ILs), also called “green solvents,” are thermally stable organic salts (Zhang et al. 2012; Roosen et al. 2008). Their low volatility effectively eliminates a major source of environmental release and contamination. IL pretreatment is a newly emerged method that has been proven to reduce the crystallinity of cellulose and remove portions of hemicellulose and lignin. IL pretreatment does not produce degradation products that inhibit enzymes or fermenting microorganisms (Dadi et al. 2007; Lee et al. 2009; da Costa Lopes et al. 2013a). In addition, supplementation of antisolvents such as water, ethanol, or acetone can make cellulose to be easily regenerated from the IL–cellulose solution (Ha et al. 2011), facilitating recovery and recycling of the expensive ILs. In most IL systems, water is the most commonly used antisolvent since its content above 1 % has been shown to dramatically reduce the solubility of lignocellulose (Swatloski et al. 2002). Nevertheless, some ILs can effectively pretreat lignocellulose even in the presence of large amounts of water (Brandt et al. 2011; Fu and Mazza 2011a, b). Although IL pretreatment has gained considerable attention as a novel and promising technology, it is currently too expensive for industrial application, primarily due to the high cost of ILs. Therefore, recycling ILs will help to increase the economical feasibility of IL pretreatment (da Costa Lopes et al. 2013a). In addition, it is essential to concentrate efforts toward the development of new and advanced IL pretreatment systems or modification of the existing methods to achieve higher efficiency as well as cost reduction (Nguyen et al. 2010). In this study, an efficient pretreatment method was developed and systematically studied using an acid-catalyzed ionic liquid solution at a lower pretreatment temperature (110 °C) than previously reported. Hydrochloric acid (HCl) was found to be the most effective catalyst to use with the chosen IL (1,3dimethylimidazolium dimethylphosphate, [MMIM]DMP). This combination was found to deconstruct plant cell wall structure and led to a significant higher cellulose conversion compared to untreated samples. Optimization of the acidcatalyzed IL system, including the type of ILs, the usage of ILs and acid, and the pretreatment conditions, resulted in a noticeable increase of cellulose conversion and an obvious reduction of IL usage. In addition, the comparatively lower pretreatment temperature applied in this study may potentially
help to decrease energy consumption and lower the requirements for equipment for pretreatment processes.
Materials and methods Materials Rice straw and corn stover were obtained from nearby farms in Changzhou (Jiangsu Province, People’s Republic of China). These materials were milled into particle size smaller than 2 mm, washed, dried at 60 °C for 48 h in a vacuum oven, and then stored at room temperature for future use. The chemical compositions of rice straw and corn stover determined following NREL LAP procedure (Sluiter et al. 2008) are summarized in Table 1. Ionic liquid 1-butyl-3-methylimida-zolium chloride ([BMIM]Cl, purity ≥99 %), MMIM]DMP (purity ≥99 %), and 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc, purity ≥99 %) were purchased from Shanghai Cheng Jie Chemical Co. Ltd (Shanghai, People’s Republic of China). Spezyme CP cellulase (105 FPU/mL) and Novozyme 188 (665 CBU/mL) were purchased from Sigma (St. Louis, MO, USA). Acetic acid, propionic acid, citric acid, malic acid, succinic acid, phosphoric acid, hydrochloric acid, and sulfuric acid (purity ≥99.5 %) were purchased from Changzhou Run You Commercial and Trading Co. Ltd (Changzhou, People’s Republic of China). All other chemicals used were from a commercial source and of reagent grade. Ionic liquid pretreatment and regeneration A 0.6-g biomass (rice straw or corn stover) was added to a certain amount of IL and acid mixture in a 100-mL threenecked round bottom flask and mixed well. The flask was sealed by a lid to prevent water loss and immersed into a silicone oil bath to preheat to the desired temperature. The heating element was equipped with a magnetic stirring device to ensure uniform temperature in the system. After pretreatment, 20 mL of deionized water was used as antisolvent to add into the reaction mixture. The solution was vigorously mixed for 20 min and separated by vacuum filtration to collect the solid residue. The solid residue was washed repeatedly with Table 1 Chemical composition of rice straw and corn stover Biomass component
Rice straw (%)
Corn stover (%)
Glucan Xylan Arabinan Lignin Ash
39.53 15.77 3.06 28.33 2.64
40.90 17.46 1.61 29.00 0.80
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deionized water to remove possible IL residue and then collected. All pretreatment conditions mentioned in the “Results” section were repeated for three to five times to prove its repeatability and generate enough samples for enzymatic hydrolysis experiments. Samples prepared by repeated experiments were collected together and mixed well. Solid samples were then dried at 60 °C in a vacuum oven (Model no. DHG9140A, Shanghai Jing Hong Experimental Equipment Co. Ltd, Shanghai, People’s Republic of China) and stored in sealed bags at ambient temperature for future use.
spectrophotometer (Model no. GD6309007, Shanghai Ling Guang Technology Co. Ltd, Shanghai, People’s Republic of China). The concentrations of total reducing sugars were calculated by the DNS assay using D-glucose as a standard according to the absorbance of saccharification liquids (Miller 1959). Yield of reducing sugar was calculated based on following equations: Yield of reducing sugara ð%Þ ¼ Total reducing sugar wieght ðgÞ =Regenerated biomass weight ðgÞ 100%
Enzymatic hydrolysis Enzymatic hydrolysis of the regenerated biomass was carried out following the NREL Laboratory Analytical Procedure (Selig et al. 2008) at a solid loading of 1 % (w/v) in 50 mL Erlenmeyer flasks to which 0.05 M acetate buffer (pH 4.8) was added. To prevent possible microorganism contamination, 80 μL of 10 mg/mL tetracycline antibiotic was added to the hydrolysis broth before adding enzymes. The total volume was 20 mL with enzyme loadings of 60 FPU/g and 120 CBU/ g regenerated solid. Enzymatic hydrolysis was performed at 50 °C in a thermostated shaker (Model no. THZ-072HT, Shanghai Bo Cai Biotechnology Co. Ltd, Shanghai, People’s Republic of China) at a rotating speed of 160 rpm. Substrate blanks without enzyme and enzyme blanks without substrate were tested in parallel with other samples. To follow the reaction course and determine final sugar yields, samples taken periodically at 1, 4, 24, 48, 72, and 96 h of hydrolysis were analyzed by the 3,5-dinitrosalicylic acid (DNS) method or with a Waters HPLC (Waters Corporation, Shanghai Branch, People’s Republic of China). All samples were prepared in triplicates and run under parallel conditions. The error bars shown in all the figures were the standard deviation of triplicate measurements. The standard deviation was calculated based on the following equation: vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u N u X u ¯ 2 X i −X u t i−1 S¼ N −1
where S is the standard deviation of triplicate measurements, N is the number of parallel experiments and equals to 3, and X is the average value of three parallel experiments. Analytical methods Sugar determination by the DNS method Total reducing sugar contents derived from enzymatic hydrolysis of the pretreated straw were measured by a UV-vis
Yield of reducing sugarb ð%Þ ¼ Total reducing sugar wieght ðgÞ =Regenerated biomass weight ðgÞ 100%
a
Based on regenerated biomass weight Based on the total sugar weight in regenerated biomass
b
High-performance liquid chromatography (HPLC) analysis The content of glucose released in enzymatic hydrolysis broth after 96 h was determined by high-performance liquid chromatography (Waters Alliance HPLC system, serial number no. G1215P359A, Waters Corporation, Shanghai Branch, People’s Republic of China) equipped with a HPX-87H column (Bio-Rad, Hercules, CA, USA) and a reflective index detector (Waters 2414, Waters Corporation, Shanghai Branch, People’s Republic of China). The mobile phase was 0.005 M H2SO4 and run at a flow rate of 0.6 mL/min at 60 °C. Collected liquid samples were filtered through 0.22 μm syringe filters by centrifuging at 12,000 rpm and then pipetted into 1.5 mL glass HPLC vials and stored refrigerated at 4 °C until analyzed. Scanning electron microscopy (SEM) analysis A scanning electron microscope (Model no. JSM-6360LA, JEOL, Tokyo, Japan) operated at 15 kV was used to image the samples to record the surface morphological features of corn stover before and after pretreatment. Fourier transform infrared spectroscopy (FTIR) analysis FTIR was performed using a Nicolet PROTÉGÉ 460 FT-IR Spectrometer (Nicolet, Thermo Scientific, Shanghai, People’s Republic of China). FTIR spectra of the samples were recorded between 4,000 and 400 cm−1. Samples were ground and mixed with the spectroscopic grade KBr then pressed in a standard device to produce diameter pellets.
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X-ray diffraction (XRD) analysis Powder X-ray diffractometry (PXRD) was used to examine changes in the crystallinity of corn stover before and after pretreatment. PXRD spectra were recorded by using a D/max 2500 PC diffractometer with Cu Kα radiation (Rigaku Corporation, Tokyo, Japan). It was operated at a voltage of 60 kV and a current of 300 mA. The 2θ range was from 5° to 40° in steps of 0.02°. Crystallinity index (CrI) was calculated by the formula (Li et al. 2010a): CrI ¼ ðI 002 −I am Þ=I 002 100
where I002 is the maximum intensity of the 002 lattice diffraction, near 2θ=22.5°, and Iam is the minimum intensity between the 101 and 002 lattice planes, near 2θ=18.0°.
Results Selection of ionic liquid type A qualified IL that could be used in pretreatment requires properties, such as no toxicity and cellulose degradation as well as easy cellulose dissolution and regeneration (Hermanutz et al. 2008). To fulfill these requirements, this study started with a screening of qualified ILs. Three types of ILs, [BMIM]Cl, [MMIM]DMP, and [EMIM]OAc, were applied in rice straw pretreatment at 130 °C for 30 min. After pretreatment, the solid residue was regenerated and washed by deionized water. Following this, the regenerated solids were hydrolyzed by sufficient enzyme loadings of commercial cellulase and β-glucosidase to determine the performance of different ILs. As shown in Fig. 1, the reducing sugar yields of regenerated rice straw pretreated by all three types of ILs after 96 h of hydrolysis were higher than that of untreated rice straw. The highest reducing sugar concentrations obtained with [BMIM]Cl-, [MMIM]DMP-, and [EMIM]OAcpretreated samples were 4.67, 5.61, and 5.18 mg/mL, respectively. The reducing sugar yield showed substantial improvement compared to that of the untreated sample, especially when a phosphate containing IL was used as the pretreatment solvent. In light of the enzymatic hydrolysis performance of these three ILs as well as their physical and chemical characteristics, [MMIM]DMP was chosen as the favorable IL for biomass pretreatment in this study. Effect of acid type Although IL pretreatment could obviously improve the enzymatic digestibility of biomass, the reducing sugar yields shown in Fig. 1 are still too low to justify its practical value.
Thus, a novel acid-catalyzed aqueous IL pretreatment method was developed to improve the pretreatment performance. Five organic acids (acetic acid, propionic acid, citric acid, malic acid, and succinic acid) and three inorganic acids (phosphoric acid, hydrochloric acid, and sulfuric acid) were applied as catalysts in the [MMIM]DMP pretreatment system to evaluate their synergistic effects with the IL. The total volume of the reaction system was 40 mL with an acid concentration of 8 % (w/w) and an [MMIM]DMP loading of 12 mL. Biomass was pretreated using this system at 110 °C for 2 h. After pretreatment, all samples were hydrolyzed following the enzymatic hydrolysis method described in the “Materials and methods” section. As indicated in Fig. 2a, in the acid-catalyzed aqueous [MMIM]DMP system, pretreatments with inorganic acids were more effective than that with organic acids. Pretreatments catalyzed by organic acids (all five) resulted in a slight increase of the hydrolysis rate and conversion compared to the control sample. By contrast, inorganic acids (all three) showed a greater ability to improve pretreatment efficacy, thus increasing the enzymatic hydrolysis conversion of pretreated samples. The reducing sugar yields were 4.34, 6.63, and 5.55 mg/ mL, respectively, for this group after 96 h of enzymatic hydrolysis. The reducing sugar yield (based on regenerated biomass weight) of the samples pretreated by the HClcatalyzed [MMIM]DMP solution at 96 h was 66.3 % and the highest among all the samples. In addition, the effect of acid type on the amount of glucose produced was also investigated, as shown in Fig. 2b. Similar to the reducing sugar results, the concentration of glucose after 96 h of enzymatic hydrolysis using the [MMIM]DMP–HCl system was the highest at 6.12 mg/mL. Based on these results, HCl was selected as the desired acid catalyst. Effect of volume of ionic liquid To optimize the loading of IL in the aqueous acid-catalyzed IL system, different volumes of [MMIM]DMP (0, 3, 6, 12, 18, and 24 mL) were mixed with HCl as the acid catalyst. This system was pretreated at 110 °C for 2 h in a total volume of 40 mL with an acid concentration of 8 % (w/w). As illustrated in Fig. 3a, the reducing sugar yield for enzymatic hydrolysis of the regenerated biomass increased with the addition of 0– 18 mL [MMIM]DMP in the pretreatment system and then decreased with a loading of higher volumes of [MMIM]DMP. The optimal reducing sugar yield (based on regenerated biomass weight) after 96 h was 76.1 % for the regenerated solids pretreated with 18 mL of [MMIM]DMP. In addition, the corresponding glucose yield was 6.38 mg/mL, as shown in Fig. 3b. These results suggest that a combination of 18 mL of [MMIM]DMP with HCl as the pretreatment system could significantly promote the destruction of the lignocellulose structure, thereby enhancing the cellulose enzymatic conversion of regenerated corn stover.
Appl Microbiol Biotechnol Fig. 1 Enzymatic hydrolysis of regenerated rice straw pretreated with different types of ionic liquids. Conditions: rice straw was pretreated at 130 °C for 30 min and the concentration of enzymatic hydrolysis solid loading was 20 mg/mL
Effect of acid concentration The impact of different HCl acid concentrations in the pretreatment system was also investigated in this study. Corn stover was pretreated with 0 (control sample), 2, 4, 6, 8, and 10 % of HCl (w/w), respectively, and 18 mL of [MMIM]DMP at 110 °C for 2 h, and then the regenerated solids were hydrolyzed using the enzyme loading protocol described above. The concentration of HCl acid had a vital impact on the yield of reducing sugars as reflected in Fig. 4a. Within the range of concentration of diluted acid (acid concentration ≤10 %), the reducing sugar yield (based on regenerated biomass weight) increased with an increase in HCl concentration and was the highest at 82.5 % with 10 % HCl after 96 h of enzymatic hydrolysis. A similar pattern was also observed for glucose produced after 96 h of enzymatic hydrolysis (Fig. 4b).
Effect of pretreatment temperature and time Unlike other IL pretreatment studies, comparatively lower temperatures (90–110 °C) and pretreatment times (1–10 h) were used in the present study. Based on the optimal conditions obtained from previous experiments, a total reaction volume of 40 mL with an HCl concentration of 10 % (w/w) and 18 mL of [MMIM]DMP was used for pretreatment. As indicated in Fig. 5, the reducing sugar concentrations of the regenerated biomass showed an initial increase with pretreatment temperatures of 90, 100, and 110 °C, and then decreased with longer pretreatment time. The highest reducing sugar yield (based on regenerated biomass weight) obtained after 96 h of enzymatic hydrolysis for samples pretreated at temperatures of 90, 100, and 110 °C was 82.6 % at 7 h, 84.9 % at 6 h, and 82.9 % at 2 h, respectively. Considering the sugar
recovery yield, possible energy consumption, and possible cost of the equipment involved in this process, pretreatment using temperature of 110 °C and a time period of 2 h was chosen as the optimal condition.
Characteristics of pretreated corn stover To further understand the mechanism of acid-catalyzed IL pretreatment, SEM analysis was conducted to monitor the changes in the surface and inner structure of corn stover pretreated with [MMIM]DMP–citric acid and [MMIM]DMP–HCl acid as well as untreated corn stover. As shown in the SEM photographs displayed in Fig. 6a, the surface of corn stover pretreated with ILs showed increased breakages and cracks. Compared to the smooth and compact surface of the untreated sample, the [MMIM]DMP–HCl pretreatment rendered the structure of corn stover extremely porous. The results of the FTIR analysis of untreated, aqueous [MMIM]DMP–citric acid-pretreated, and [MMIM]DMP– HCl-pretreated corn stover are shown in Fig. 6b. The peaks at 1,375, 1,162, and 1,055 cm−1 are specifically attributed to C–H bending vibration, C–O–C asymmetric bridge stretching vibration and C–O stretching vibration in cellulose and hemicellulose, respectively (Labbé et al. 2005). These peaks were weaker for acid-catalyzed [MMIM]DMP-pretreated samples compared to the untreated sample. The peak at 1,250 cm−1 is attributed to ether bonds (Liu et al. 2009) and was found to reduce in the spectrum of aqueous [MMIM]DMP–citric acidpretreated corn stover and almost disappeared in the spectrum of aqueous [MMIM]DMP–HCl-pretreated corn stover. A similar tendency was observed for the characteristic peak of ester bond at 1,732 cm−1 (Liu et al. 2009).
Appl Microbiol Biotechnol Fig. 2 a Enzymatic hydrolysis of regenerated rice straw pretreated with different acid types (control sample: pretreated with 40 mL IL only). b Glucose concentrations in enzymatic hydrolysis broth of regenerated rice straw after 96 h. Condition: rice straw was pretreated at 110 °C for 2 h and the concentration of enzymatic hydrolysis solid loading was 10 mg/mL
XRD analysis was conducted to measure the crystallinity of different corn stover samples (pretreated and untreated). Two typical peaks were observed near 2θ=18.0° and 22.5°, which were corresponding to (101) and (002) lattice planes of crystalline cellulose I (Fig. 6c). After acid-catalyzed aqueous [MMIM]DMP pretreatment, these two peaks became weaker than the untreated control sample. Based on the equation provided in the “Materials and methods” section, XRD data were calculated. The CrI of untreated corn stover was found to be 69.7 %, while the CrI of [MMIM]DMP–citric acid-
pretreated and [MMIM]DMP–HCl acid-pretreated corn stover decreased to 56.5 and 61.1 %, respectively (Table 2).
Discussion Pretreatment is the key to effective conversion of lignocelluloses to biofuels (Yang and Wyman 2008). ILs have been employed for lignocellulose pretreatment for several years for the removal of portions of hemicellulose and lignin.
Appl Microbiol Biotechnol Fig. 3 a Enzymatic hydrolysis of regenerated corn stover using the [MMIM]DMP–HCl system with different volumes of ILs. b Glucose concentrations in hydrolysis liquid after 96 h of enzymatic hydrolysis. Pretreatment condition: corn stover was pretreated with 8 % (w/w) of HCl at 110 °C for 2 h. The concentration of enzymatic hydrolysis solid loading was 10 mg/mL
Several types of ILs have been identified as effective solvents for cellulose dissolution, which usually contain anions of chloride, formate, acetate, or alkylphosphonate because of their strong ability to form hydrogen bonds with cellulose (Remsing et al. 2008). It was reported that 1-ethyl-3methylimidazolium acetate ([emim][CH3COO]) can be used to pretreat and separate wheat straw into cellulose, hemicellulose, and lignin (da Costa Lopes et al. 2013b). Pretreatment with cheap and easily available dilute inorganic acids has long since been considered as an inexpensive and effective alternative method (Kim et al. 2005). For example, strong acids such as sulfuric acid (H2SO4) and HCl are widely used for pretreatment process since they are powerful agents for
lignocellulose hydrolysis (Sun and Cheng 2002). In this study, a pretreatment technique combining ILs and dilute acids was employed to increase the enzymatic hydrolysis yield of regenerated biomass. Five factors, namely, the IL type, acid type, IL volume, acid concentration, and pretreatment temperature and time were investigated. Firstly, [MMIM]DMP, a phosphate-based IL, was selected to use as the pretreatment solvent in this study due to its prominent effect on pretreatment of corn stover. It is possible that DMP− confers favorable solubility and biocompatibility on [MMIM][DMP] and also lowers inhibition on enzyme activities (Fig. 1) (Li et al. 2010b). On the other hand, [BMIM]Cl, which is a chloride-based IL, is a corrosive, toxic,
Appl Microbiol Biotechnol Fig. 4 a Enzymatic hydrolysis of regenerated corn stover pretreated with different concentrations of HCl acid. b Glucose concentration in hydrolysis liquid after 96 h of enzymatic hydrolysis. Pretreatment condition: corn stover was pretreated with 18 mL [MMIM]DMP at 110 °C for 2 h. The concentration of enzymatic hydrolysis solid loading was 10 mg/mL
and extremely hygroscopic solid (m.p. ~70 °C) (Li et al. 2009) with inhibitory effects on enzyme activities due to its high anion concentration. As a result, samples pretreated with [BMIM]Cl have to be thoroughly washed before enzymatic hydrolysis to remove possible IL residue. Therefore, considering both the physical properties and lignocellulose dissolving properties of the three ILs used in this study, [MMIM]DMP was chosen as the pretreatment solvent. To further optimize the pretreatment performance, a synergistic acid-catalyzed aqueous [MMIM]DMP pretreatment system was developed. In this system, a combination of [MMIM]DMP and HCl was found to be more efficient, possibly because the chloride ion is more electronegative than other anions (Fig. 2). The chloride ion is known to form
hydrogen bonds with the cellulose hydroxyl hydrogen in biomass, which in turn destroys the intramolecular and intermolecular hydrogen bonds of cellulose (Swatloski et al. 2002). Moreover, the addition of an inorganic acid to ILs could significantly reduce the usage and the cost of IL solvents incurred in the pretreatment process. In addition, the results show that pretreatment with inorganic acids is more effective than that with organic acids, possibly because of the higher amount of hydrogen ions in inorganic acids at the same acid concentration, which could result in a higher proportion of xylan removal and, subsequently, higher enzymatic saccharification conversion (Zhang et al. 2012). Pretreatment efficiency of biomass was also estimated according to the amount of glucose released during enzymatic hydrolysis of the
Appl Microbiol Biotechnol Fig. 5 Effect of pretreatment temperature and time on the acidcatalyzed IL pretreatment. Pretreatment condition: corn stover was pretreated with 18 mL [MMIM]DMP and 10 % (w/w) HCl and regenerated by water. The concentration of enzymatic hydrolysis solid loading was 10 mg/mL
regenerated sample (Zhu et al. 2006). Regenerated biomass pretreated by ILs containing essentially amorphous cellulose was believed to be more accessible to enzymes. In this study, [MMIM]DMP–HCl pretreatment was the most efficient system in terms of reducing sugars and glucose production. The reducing sugar yield (based on regenerated biomass weight) of corn stover pretreated with HCl-catalyzed [MMIM]DMP solutions reached 82.9 % under the optimal pretreatment conditions summarized in this study. This is higher than the previously reported sugar yield of 71.2 % for [AMIM]Clpretreated wheat straw (Chen et al. 2012). The volume of IL is also an important factor affecting the [MMIM]DMP–HCl pretreatment system. Our results indicated that the enzymatic saccharification conversion increased with an increase in the amount of ILs in the reaction system (Fig. 3), which may be due to the fact that with the addition of [MMIM]DMP into the pretreatment system, part of the cellulose hydrogen bonds becomes easily decomposed, resulting in improved hydrolysis rate and overall cellulose conversion. However, beyond a specific threshold of the IL volume, the viscosity of the reaction system became too high, thereby hindering the contact between acid catalysts and cellulose fibers and causing a decline in enzymatic saccharification conversion (Li et al. 2008). In addition, enzymatic hydrolysis conversion was found to improve with an increase in HCl concentration (Fig. 4). The increased hydrogen ion concentration in the solution may provide more hydrogen ions to attack the oxygen atoms of the cellulose glycosidic bond, improving the catalytic effect and enzymatic hydrolysis conversion (Li et al. 2008). Temperature and time are two important factors that directly affect the efficiency of pretreatment systems. Although higher pretreatment temperature and longer reaction time
usually lead to a more severe destruction of lignocellulosic structure, these conditions also result in further degradation of monosaccharide sugars into furfural, HMF, and other undesirable inhibitory products (Zhou et al. 2012). In a previously reported study, the combined use of IL and ammonia pretreatment showed good efficiency, but required a high temperature of 130 °C and a long reaction time of 24 h (Nguyen et al. 2010), whereas the method developed in this study dramatically reduced the pretreatment temperature and time (110 °C, 2 h) with a comparable cellulose conversion. It is evident from the SEM photographs that compared to the compact fibers and smooth surface of the untreated sample, the pretreated fibers were partially disrupted. This was probably due to the partial dissolution of the corn stover cell wall and partial removal of lignin during IL pretreatment. [MMIM]DMP–HCl-pretreated corn stover showed a porous and fragmented structure, and these breakages and fissures may facilitate the access of enzymes to the cellulose surface, in turn improving the efficiency of enzymatic saccharification as revealed in this study. To compare the FTIR data obtained from these three samples, a number of bonds were used to monitor the chemical changes in lignin and carbohydrates. The decline of these peaks illustrated that the chemical bonds of lignocellulose were destroyed after acid-catalyzed aqueous [MMIM]DMP pretreatment. The peak at 1,250 cm−1, assigned to ether bonds (Liu et al. 2009), reduced in the spectrum of aqueous [MMIM]DMP–citric acid-pretreated corn stover and almost disappeared in the spectrum of aqueous [MMIM]DMP–HClpretreated corn stover. This reduction of peak intensity at 1,250 cm −1 confirmed that pretreatment with aqueous [MMIM]DMP/HCl solution was more effective in breaking
Appl Microbiol Biotechnol Fig. 6 a SEM micrographs, b FTIR spectra, and c X-ray diffraction spectra of untreated corn stover (a), corn stover pretreated by aqueous [MMIM]DMP/citric acid (b), and corn stover pretreated with aqueous [MMIM]DMP/HCl (c). Pretreatment conditions: 18 mL of ([MMIM]DMP), 10 % HCl, and 100 °C for 6 h
ether linkages between lignin and carbohydrates than aqueous [MMIM]DMP/citric acid solution. A similar tendency was observed for the ester bond at 1,732 cm−1 (Liu et al. 2009), suggesting that some ester linkages between lignin and carbohydrates are cleaved during pretreatment. Table 2 CrI values determined from XRD spectra of corn stover before and after pretreatment Pretreatment method
CrI (%)
Untreated Pretreated with aqueous [MMIM]DMP/citric acid Pretreated with aqueous [MMIM]DMP/HCl
69.7 56.5 61.1
According to the XRD analysis, the crystallinity index was found to decrease after acid-catalyzed aqueous [MMIM]DMP pretreatment. This may be due to the disruption of the hydrogen bonds in cellulose by ILs (Zhao et al. 2009). The crystallinity index of aqueous [MMIM]DMP–citric acid-pretreated corn stover was lower than that of aqueous [MMIM]DMP– HCl-pretreated corn stover, possibly due to the severe disruption of the amorphous hemicellulose structure by HCl. In light of these data and the SEM and FTIR analyses, it can be concluded that the structure of the acid-catalyzed ILpretreated samples was obviously destroyed, and the crystalline structure of cellulose was partially destroyed during pretreatment, leading to improved enzymatic hydrolysis of the regenerated corn stover.
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In conclusion, an effective pretreatment method using acidcatalyzed aqueous [MMIM]DMP solution was established in this study. Through a series of optimization processes of the reaction conditions, the enzymatic hydrolysis conversion of regenerated biomass was significantly improved with a higher total sugar conversion of 92.7 % obtained for the pretreated corn stover, compared to 27.3 % for the untreated control sample. Moreover, the optimized acid-catalyzed IL system established in this research may potentially facilitate reduced IL usage, pretreatment temperatures, and requirements for equipment. The detailed study of the structural and chemical changes of the regenerated corn stover further demonstrated the efficacy of this novel pretreatment method and revealed the mechanism of the improved enzymatic hydrolysis conversion. Acknowledgments We are grateful to China Petroleum Jilin Chemical Group Company for funding this research work through Contract Number Petrochina 2013-1630. We also acknowledge the Laboratory of Cellulosic Biofuels, Changzhou University for providing the facilities and equipments used in this research.
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Appl Microbiol Biotechnol Remsing RC, Hernandez G, Swatloski RP, Massefski WW, Rogers RD, Moyna G (2008) Solvation of carbohydrates in N, N′-dialkylimidazolium ionic liquids: a multinuclear NMR spectroscopy study. J Phys Chem B 112:11071–11078. doi:10.1021/jp8042895 Roosen C, Müller P, Greiner L (2008) Ionic liquids in biotechnology: applications and perspectives for biotransformations. Appl Microbiol Biotechnol 81(4):607–614. doi:10.1007/s00253-0081730-9 Selig M, Weiss N, Ji Y (2008) Enzymatic saccharification of lignocellulosic biomass. National Renewable Energy Laboratory, Golden, CO, USA Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory, Golden, CO, USA Sun Y, Cheng JY (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 83:1–11. doi:10. 1002/chin.200301272 Swatloski RP, Spear SK, Holbrey JD, Rogers RD (2002) Dissolution of cellulose with ionic liquids. J Am Chem Soc 124:4974–4975. doi: 10.1021/ja025790m Xu J, Thomsen MH, Thomsen AB (2010) Investigation of acetic acidcatalyzed hydrothermal pretreatment on corn stover. Appl Microbiol Biotechnol 86(2):509–516. doi:10.1007/s00253-009-2340-x
Yang B, Wyman CE (2008) Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod Bioref 2:26–40. doi:10.1002/ bbb.49 Zhang Z, O’Hara IM, Doherty WOS (2012) Pretreatment of sugarcane bagasse by acid-catalysed process in aqueous ionic liquid solutions. Bioresour Technol 120:149–156. doi:10.1016/j.biortech.2012.06. 035 Zhao H, Jones CL, Baker GA, Xia S, Olubajo O, Person VN (2009) Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis. J Biotechnol 139(1):47–54. doi:10.1016/j.jbiotec. 2008.08.009 Zhong C, Lau MW, Balan V, Dale BE, Yuan Y-J (2009) Optimization of enzymatic hydrolysis and ethanol fermentation from AFEX-treated rice straw. Appl Microbiol Biotechnol 84:667–676. doi:10.1007/ s00253-009-2001-0 Zhou N, Zhang YM, Gong XW, Wang QH, Ma YH (2012) Ionic liquidsbased hydrolysis of Chlorella biomass for fermentable sugars. Bioresour Technol 118:512–517. doi:10.1016/j.biortech.2012.05. 074 Zhu S, Wu Y, Chen Q, Yu Z, Wang C, Jin S, Ding Y, Wu G (2006) Dissolution of cellulose with ionic liquids and its application: a mini-review. Green Chem 8(4):325–327. doi:10.1039/b601395c
BIOENERGY AND BIOFUELS
Investigation of a novel acid-catalyzed ionic liquid pretreatment method to improve biomass enzymatic hydrolysis conversion Qing Qing & Rong Hu & Yucai He & Yue Zhang & Liqun Wang
Received: 28 October 2013 / Revised: 26 February 2014 / Accepted: 4 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Pretreatment of lignocellulosic materials is a prerequisite to facilitate the disruption of the natural recalcitrance of their carbohydrate–lignin shield and to allow enzymes to easily access the crystalline cellulose surfaces. Recently, pretreatment of ionic liquids (ILs) has been widely studied as a promising pretreatment technique; however, it is too expensive to be commercialized. In this study, an efficient acidcatalyzed aqueous IL pretreatment process was developed to optimize the total sugar conversion of pretreated biomass and to reduce IL usage. The experimental results demonstrated that the total sugar conversion was raised to 92.7 % with the synergistic effects of IL (1,3-dimethylimidazolium dimethylphosphate, [MMIM]DMP) and dilute hydrochloric acid (HCl) under pretreatment conditions of 110 °C for 2 h, compared to the conversion of only 27.3 % obtained with untreated corn stover. Moreover, the addition of the inorganic acids, especially HCl, to the IL pretreatment was found to not only significantly destroy the crystalline structure of cellulose in corn stover, promoting the conversion of cellulose and hemicellulose to monomeric sugars, but also provide an opportunity to reduce the usage of expensive IL solvents.
Keywords Ionic liquid . Pretreatment . Acid . Lignocellulose . Enzymatic hydrolysis
Q. Qing : R. Hu : Y. He : Y. Zhang : L. Wang (*) Department of Biochemical Engineering, College of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu 213164, China e-mail: [email protected] L. Wang e-mail: [email protected]
Introduction Lignocellulosic materials are the most abundant resources in nature that could be biologically converted to biofuels and other value-added chemicals (Adsul et al. 2011; Zhong et al. 2009). Plant cell walls are complex structures composed mostly of cellulose (35–50 %), hemicelluloses (20–35 %), and lignin (5–30 %), which form a matrix of a cross-linked three dimensional polysaccharide network. Cellulose is a linear macromolecular compound composed of glucose units that are linked by β-1,4-glycosidic bonds (Ninomiya et al. 2012) and is mainly composed of two regions, the amorphous and crystalline regions. The amorphous structure is relatively loose and easily degraded, whereas the crystalline structure is stable and difficult to degrade. Hemicellulose is a polymer mainly made up of hetero-1,4-β-D-xylan with an amorphous and highly branched structure and is easily digested by enzymes. Lignin is a cross-linked racemic macromolecule that works as a physical barrier, surrounding the cellulose and hemicelluloses to prevent microorganisms and enzymes from accessing the cellulose surface (Alvira et al. 2010). Thus, lignin is widely believed to be an obstacle to the effective conversion of cellulose by the process of enzymatic hydrolysis. Pretreatment process has been considered as a prerequisite for breaking or removing lignin and for increasing the porosity of cellulose in order to make lignocellulosic materials to be more easily hydrolyzed (Chandra et al. 2007; Galbe and Zacchi 2007). Over the years, many pretreatment methods have been intensively studied and used to improve the degradation of lignocellulosic materials (Hendriks and Zeeman 2009; Mosier et al. 2005). These leading pretreatment processes include physical methods, chemical methods, or a combination of both. Physical processes include mechanical grinding, steam explosion, radiation, etc. However, these methods usually require high energy consumption and high
Appl Microbiol Biotechnol
cost. On the other hand, chemical methods mainly include acid hydrolysis, alkaline hydrolysis, and organic solvent treatmen t us ing m e tha nol , eth ano l, a cet one , an d N methylmorpholine N-oxide (NMMO) (Mosier et al. 2005). The corrosive nature of the chemicals and solvents involved in these methods results in high requirements for equipment (Xu et al. 2010). Therefore, the exorbitant cost of the processing methods mentioned remains a dominant concern preventing the commercialization of cellulosic ethanol (Nguyen et al. 2010). Ionic liquids (ILs), also called “green solvents,” are thermally stable organic salts (Zhang et al. 2012; Roosen et al. 2008). Their low volatility effectively eliminates a major source of environmental release and contamination. IL pretreatment is a newly emerged method that has been proven to reduce the crystallinity of cellulose and remove portions of hemicellulose and lignin. IL pretreatment does not produce degradation products that inhibit enzymes or fermenting microorganisms (Dadi et al. 2007; Lee et al. 2009; da Costa Lopes et al. 2013a). In addition, supplementation of antisolvents such as water, ethanol, or acetone can make cellulose to be easily regenerated from the IL–cellulose solution (Ha et al. 2011), facilitating recovery and recycling of the expensive ILs. In most IL systems, water is the most commonly used antisolvent since its content above 1 % has been shown to dramatically reduce the solubility of lignocellulose (Swatloski et al. 2002). Nevertheless, some ILs can effectively pretreat lignocellulose even in the presence of large amounts of water (Brandt et al. 2011; Fu and Mazza 2011a, b). Although IL pretreatment has gained considerable attention as a novel and promising technology, it is currently too expensive for industrial application, primarily due to the high cost of ILs. Therefore, recycling ILs will help to increase the economical feasibility of IL pretreatment (da Costa Lopes et al. 2013a). In addition, it is essential to concentrate efforts toward the development of new and advanced IL pretreatment systems or modification of the existing methods to achieve higher efficiency as well as cost reduction (Nguyen et al. 2010). In this study, an efficient pretreatment method was developed and systematically studied using an acid-catalyzed ionic liquid solution at a lower pretreatment temperature (110 °C) than previously reported. Hydrochloric acid (HCl) was found to be the most effective catalyst to use with the chosen IL (1,3dimethylimidazolium dimethylphosphate, [MMIM]DMP). This combination was found to deconstruct plant cell wall structure and led to a significant higher cellulose conversion compared to untreated samples. Optimization of the acidcatalyzed IL system, including the type of ILs, the usage of ILs and acid, and the pretreatment conditions, resulted in a noticeable increase of cellulose conversion and an obvious reduction of IL usage. In addition, the comparatively lower pretreatment temperature applied in this study may potentially
help to decrease energy consumption and lower the requirements for equipment for pretreatment processes.
Materials and methods Materials Rice straw and corn stover were obtained from nearby farms in Changzhou (Jiangsu Province, People’s Republic of China). These materials were milled into particle size smaller than 2 mm, washed, dried at 60 °C for 48 h in a vacuum oven, and then stored at room temperature for future use. The chemical compositions of rice straw and corn stover determined following NREL LAP procedure (Sluiter et al. 2008) are summarized in Table 1. Ionic liquid 1-butyl-3-methylimida-zolium chloride ([BMIM]Cl, purity ≥99 %), MMIM]DMP (purity ≥99 %), and 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc, purity ≥99 %) were purchased from Shanghai Cheng Jie Chemical Co. Ltd (Shanghai, People’s Republic of China). Spezyme CP cellulase (105 FPU/mL) and Novozyme 188 (665 CBU/mL) were purchased from Sigma (St. Louis, MO, USA). Acetic acid, propionic acid, citric acid, malic acid, succinic acid, phosphoric acid, hydrochloric acid, and sulfuric acid (purity ≥99.5 %) were purchased from Changzhou Run You Commercial and Trading Co. Ltd (Changzhou, People’s Republic of China). All other chemicals used were from a commercial source and of reagent grade. Ionic liquid pretreatment and regeneration A 0.6-g biomass (rice straw or corn stover) was added to a certain amount of IL and acid mixture in a 100-mL threenecked round bottom flask and mixed well. The flask was sealed by a lid to prevent water loss and immersed into a silicone oil bath to preheat to the desired temperature. The heating element was equipped with a magnetic stirring device to ensure uniform temperature in the system. After pretreatment, 20 mL of deionized water was used as antisolvent to add into the reaction mixture. The solution was vigorously mixed for 20 min and separated by vacuum filtration to collect the solid residue. The solid residue was washed repeatedly with Table 1 Chemical composition of rice straw and corn stover Biomass component
Rice straw (%)
Corn stover (%)
Glucan Xylan Arabinan Lignin Ash
39.53 15.77 3.06 28.33 2.64
40.90 17.46 1.61 29.00 0.80
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deionized water to remove possible IL residue and then collected. All pretreatment conditions mentioned in the “Results” section were repeated for three to five times to prove its repeatability and generate enough samples for enzymatic hydrolysis experiments. Samples prepared by repeated experiments were collected together and mixed well. Solid samples were then dried at 60 °C in a vacuum oven (Model no. DHG9140A, Shanghai Jing Hong Experimental Equipment Co. Ltd, Shanghai, People’s Republic of China) and stored in sealed bags at ambient temperature for future use.
spectrophotometer (Model no. GD6309007, Shanghai Ling Guang Technology Co. Ltd, Shanghai, People’s Republic of China). The concentrations of total reducing sugars were calculated by the DNS assay using D-glucose as a standard according to the absorbance of saccharification liquids (Miller 1959). Yield of reducing sugar was calculated based on following equations: Yield of reducing sugara ð%Þ ¼ Total reducing sugar wieght ðgÞ =Regenerated biomass weight ðgÞ 100%
Enzymatic hydrolysis Enzymatic hydrolysis of the regenerated biomass was carried out following the NREL Laboratory Analytical Procedure (Selig et al. 2008) at a solid loading of 1 % (w/v) in 50 mL Erlenmeyer flasks to which 0.05 M acetate buffer (pH 4.8) was added. To prevent possible microorganism contamination, 80 μL of 10 mg/mL tetracycline antibiotic was added to the hydrolysis broth before adding enzymes. The total volume was 20 mL with enzyme loadings of 60 FPU/g and 120 CBU/ g regenerated solid. Enzymatic hydrolysis was performed at 50 °C in a thermostated shaker (Model no. THZ-072HT, Shanghai Bo Cai Biotechnology Co. Ltd, Shanghai, People’s Republic of China) at a rotating speed of 160 rpm. Substrate blanks without enzyme and enzyme blanks without substrate were tested in parallel with other samples. To follow the reaction course and determine final sugar yields, samples taken periodically at 1, 4, 24, 48, 72, and 96 h of hydrolysis were analyzed by the 3,5-dinitrosalicylic acid (DNS) method or with a Waters HPLC (Waters Corporation, Shanghai Branch, People’s Republic of China). All samples were prepared in triplicates and run under parallel conditions. The error bars shown in all the figures were the standard deviation of triplicate measurements. The standard deviation was calculated based on the following equation: vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u N u X u ¯ 2 X i −X u t i−1 S¼ N −1
where S is the standard deviation of triplicate measurements, N is the number of parallel experiments and equals to 3, and X is the average value of three parallel experiments. Analytical methods Sugar determination by the DNS method Total reducing sugar contents derived from enzymatic hydrolysis of the pretreated straw were measured by a UV-vis
Yield of reducing sugarb ð%Þ ¼ Total reducing sugar wieght ðgÞ =Regenerated biomass weight ðgÞ 100%
a
Based on regenerated biomass weight Based on the total sugar weight in regenerated biomass
b
High-performance liquid chromatography (HPLC) analysis The content of glucose released in enzymatic hydrolysis broth after 96 h was determined by high-performance liquid chromatography (Waters Alliance HPLC system, serial number no. G1215P359A, Waters Corporation, Shanghai Branch, People’s Republic of China) equipped with a HPX-87H column (Bio-Rad, Hercules, CA, USA) and a reflective index detector (Waters 2414, Waters Corporation, Shanghai Branch, People’s Republic of China). The mobile phase was 0.005 M H2SO4 and run at a flow rate of 0.6 mL/min at 60 °C. Collected liquid samples were filtered through 0.22 μm syringe filters by centrifuging at 12,000 rpm and then pipetted into 1.5 mL glass HPLC vials and stored refrigerated at 4 °C until analyzed. Scanning electron microscopy (SEM) analysis A scanning electron microscope (Model no. JSM-6360LA, JEOL, Tokyo, Japan) operated at 15 kV was used to image the samples to record the surface morphological features of corn stover before and after pretreatment. Fourier transform infrared spectroscopy (FTIR) analysis FTIR was performed using a Nicolet PROTÉGÉ 460 FT-IR Spectrometer (Nicolet, Thermo Scientific, Shanghai, People’s Republic of China). FTIR spectra of the samples were recorded between 4,000 and 400 cm−1. Samples were ground and mixed with the spectroscopic grade KBr then pressed in a standard device to produce diameter pellets.
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X-ray diffraction (XRD) analysis Powder X-ray diffractometry (PXRD) was used to examine changes in the crystallinity of corn stover before and after pretreatment. PXRD spectra were recorded by using a D/max 2500 PC diffractometer with Cu Kα radiation (Rigaku Corporation, Tokyo, Japan). It was operated at a voltage of 60 kV and a current of 300 mA. The 2θ range was from 5° to 40° in steps of 0.02°. Crystallinity index (CrI) was calculated by the formula (Li et al. 2010a): CrI ¼ ðI 002 −I am Þ=I 002 100
where I002 is the maximum intensity of the 002 lattice diffraction, near 2θ=22.5°, and Iam is the minimum intensity between the 101 and 002 lattice planes, near 2θ=18.0°.
Results Selection of ionic liquid type A qualified IL that could be used in pretreatment requires properties, such as no toxicity and cellulose degradation as well as easy cellulose dissolution and regeneration (Hermanutz et al. 2008). To fulfill these requirements, this study started with a screening of qualified ILs. Three types of ILs, [BMIM]Cl, [MMIM]DMP, and [EMIM]OAc, were applied in rice straw pretreatment at 130 °C for 30 min. After pretreatment, the solid residue was regenerated and washed by deionized water. Following this, the regenerated solids were hydrolyzed by sufficient enzyme loadings of commercial cellulase and β-glucosidase to determine the performance of different ILs. As shown in Fig. 1, the reducing sugar yields of regenerated rice straw pretreated by all three types of ILs after 96 h of hydrolysis were higher than that of untreated rice straw. The highest reducing sugar concentrations obtained with [BMIM]Cl-, [MMIM]DMP-, and [EMIM]OAcpretreated samples were 4.67, 5.61, and 5.18 mg/mL, respectively. The reducing sugar yield showed substantial improvement compared to that of the untreated sample, especially when a phosphate containing IL was used as the pretreatment solvent. In light of the enzymatic hydrolysis performance of these three ILs as well as their physical and chemical characteristics, [MMIM]DMP was chosen as the favorable IL for biomass pretreatment in this study. Effect of acid type Although IL pretreatment could obviously improve the enzymatic digestibility of biomass, the reducing sugar yields shown in Fig. 1 are still too low to justify its practical value.
Thus, a novel acid-catalyzed aqueous IL pretreatment method was developed to improve the pretreatment performance. Five organic acids (acetic acid, propionic acid, citric acid, malic acid, and succinic acid) and three inorganic acids (phosphoric acid, hydrochloric acid, and sulfuric acid) were applied as catalysts in the [MMIM]DMP pretreatment system to evaluate their synergistic effects with the IL. The total volume of the reaction system was 40 mL with an acid concentration of 8 % (w/w) and an [MMIM]DMP loading of 12 mL. Biomass was pretreated using this system at 110 °C for 2 h. After pretreatment, all samples were hydrolyzed following the enzymatic hydrolysis method described in the “Materials and methods” section. As indicated in Fig. 2a, in the acid-catalyzed aqueous [MMIM]DMP system, pretreatments with inorganic acids were more effective than that with organic acids. Pretreatments catalyzed by organic acids (all five) resulted in a slight increase of the hydrolysis rate and conversion compared to the control sample. By contrast, inorganic acids (all three) showed a greater ability to improve pretreatment efficacy, thus increasing the enzymatic hydrolysis conversion of pretreated samples. The reducing sugar yields were 4.34, 6.63, and 5.55 mg/ mL, respectively, for this group after 96 h of enzymatic hydrolysis. The reducing sugar yield (based on regenerated biomass weight) of the samples pretreated by the HClcatalyzed [MMIM]DMP solution at 96 h was 66.3 % and the highest among all the samples. In addition, the effect of acid type on the amount of glucose produced was also investigated, as shown in Fig. 2b. Similar to the reducing sugar results, the concentration of glucose after 96 h of enzymatic hydrolysis using the [MMIM]DMP–HCl system was the highest at 6.12 mg/mL. Based on these results, HCl was selected as the desired acid catalyst. Effect of volume of ionic liquid To optimize the loading of IL in the aqueous acid-catalyzed IL system, different volumes of [MMIM]DMP (0, 3, 6, 12, 18, and 24 mL) were mixed with HCl as the acid catalyst. This system was pretreated at 110 °C for 2 h in a total volume of 40 mL with an acid concentration of 8 % (w/w). As illustrated in Fig. 3a, the reducing sugar yield for enzymatic hydrolysis of the regenerated biomass increased with the addition of 0– 18 mL [MMIM]DMP in the pretreatment system and then decreased with a loading of higher volumes of [MMIM]DMP. The optimal reducing sugar yield (based on regenerated biomass weight) after 96 h was 76.1 % for the regenerated solids pretreated with 18 mL of [MMIM]DMP. In addition, the corresponding glucose yield was 6.38 mg/mL, as shown in Fig. 3b. These results suggest that a combination of 18 mL of [MMIM]DMP with HCl as the pretreatment system could significantly promote the destruction of the lignocellulose structure, thereby enhancing the cellulose enzymatic conversion of regenerated corn stover.
Appl Microbiol Biotechnol Fig. 1 Enzymatic hydrolysis of regenerated rice straw pretreated with different types of ionic liquids. Conditions: rice straw was pretreated at 130 °C for 30 min and the concentration of enzymatic hydrolysis solid loading was 20 mg/mL
Effect of acid concentration The impact of different HCl acid concentrations in the pretreatment system was also investigated in this study. Corn stover was pretreated with 0 (control sample), 2, 4, 6, 8, and 10 % of HCl (w/w), respectively, and 18 mL of [MMIM]DMP at 110 °C for 2 h, and then the regenerated solids were hydrolyzed using the enzyme loading protocol described above. The concentration of HCl acid had a vital impact on the yield of reducing sugars as reflected in Fig. 4a. Within the range of concentration of diluted acid (acid concentration ≤10 %), the reducing sugar yield (based on regenerated biomass weight) increased with an increase in HCl concentration and was the highest at 82.5 % with 10 % HCl after 96 h of enzymatic hydrolysis. A similar pattern was also observed for glucose produced after 96 h of enzymatic hydrolysis (Fig. 4b).
Effect of pretreatment temperature and time Unlike other IL pretreatment studies, comparatively lower temperatures (90–110 °C) and pretreatment times (1–10 h) were used in the present study. Based on the optimal conditions obtained from previous experiments, a total reaction volume of 40 mL with an HCl concentration of 10 % (w/w) and 18 mL of [MMIM]DMP was used for pretreatment. As indicated in Fig. 5, the reducing sugar concentrations of the regenerated biomass showed an initial increase with pretreatment temperatures of 90, 100, and 110 °C, and then decreased with longer pretreatment time. The highest reducing sugar yield (based on regenerated biomass weight) obtained after 96 h of enzymatic hydrolysis for samples pretreated at temperatures of 90, 100, and 110 °C was 82.6 % at 7 h, 84.9 % at 6 h, and 82.9 % at 2 h, respectively. Considering the sugar
recovery yield, possible energy consumption, and possible cost of the equipment involved in this process, pretreatment using temperature of 110 °C and a time period of 2 h was chosen as the optimal condition.
Characteristics of pretreated corn stover To further understand the mechanism of acid-catalyzed IL pretreatment, SEM analysis was conducted to monitor the changes in the surface and inner structure of corn stover pretreated with [MMIM]DMP–citric acid and [MMIM]DMP–HCl acid as well as untreated corn stover. As shown in the SEM photographs displayed in Fig. 6a, the surface of corn stover pretreated with ILs showed increased breakages and cracks. Compared to the smooth and compact surface of the untreated sample, the [MMIM]DMP–HCl pretreatment rendered the structure of corn stover extremely porous. The results of the FTIR analysis of untreated, aqueous [MMIM]DMP–citric acid-pretreated, and [MMIM]DMP– HCl-pretreated corn stover are shown in Fig. 6b. The peaks at 1,375, 1,162, and 1,055 cm−1 are specifically attributed to C–H bending vibration, C–O–C asymmetric bridge stretching vibration and C–O stretching vibration in cellulose and hemicellulose, respectively (Labbé et al. 2005). These peaks were weaker for acid-catalyzed [MMIM]DMP-pretreated samples compared to the untreated sample. The peak at 1,250 cm−1 is attributed to ether bonds (Liu et al. 2009) and was found to reduce in the spectrum of aqueous [MMIM]DMP–citric acidpretreated corn stover and almost disappeared in the spectrum of aqueous [MMIM]DMP–HCl-pretreated corn stover. A similar tendency was observed for the characteristic peak of ester bond at 1,732 cm−1 (Liu et al. 2009).
Appl Microbiol Biotechnol Fig. 2 a Enzymatic hydrolysis of regenerated rice straw pretreated with different acid types (control sample: pretreated with 40 mL IL only). b Glucose concentrations in enzymatic hydrolysis broth of regenerated rice straw after 96 h. Condition: rice straw was pretreated at 110 °C for 2 h and the concentration of enzymatic hydrolysis solid loading was 10 mg/mL
XRD analysis was conducted to measure the crystallinity of different corn stover samples (pretreated and untreated). Two typical peaks were observed near 2θ=18.0° and 22.5°, which were corresponding to (101) and (002) lattice planes of crystalline cellulose I (Fig. 6c). After acid-catalyzed aqueous [MMIM]DMP pretreatment, these two peaks became weaker than the untreated control sample. Based on the equation provided in the “Materials and methods” section, XRD data were calculated. The CrI of untreated corn stover was found to be 69.7 %, while the CrI of [MMIM]DMP–citric acid-
pretreated and [MMIM]DMP–HCl acid-pretreated corn stover decreased to 56.5 and 61.1 %, respectively (Table 2).
Discussion Pretreatment is the key to effective conversion of lignocelluloses to biofuels (Yang and Wyman 2008). ILs have been employed for lignocellulose pretreatment for several years for the removal of portions of hemicellulose and lignin.
Appl Microbiol Biotechnol Fig. 3 a Enzymatic hydrolysis of regenerated corn stover using the [MMIM]DMP–HCl system with different volumes of ILs. b Glucose concentrations in hydrolysis liquid after 96 h of enzymatic hydrolysis. Pretreatment condition: corn stover was pretreated with 8 % (w/w) of HCl at 110 °C for 2 h. The concentration of enzymatic hydrolysis solid loading was 10 mg/mL
Several types of ILs have been identified as effective solvents for cellulose dissolution, which usually contain anions of chloride, formate, acetate, or alkylphosphonate because of their strong ability to form hydrogen bonds with cellulose (Remsing et al. 2008). It was reported that 1-ethyl-3methylimidazolium acetate ([emim][CH3COO]) can be used to pretreat and separate wheat straw into cellulose, hemicellulose, and lignin (da Costa Lopes et al. 2013b). Pretreatment with cheap and easily available dilute inorganic acids has long since been considered as an inexpensive and effective alternative method (Kim et al. 2005). For example, strong acids such as sulfuric acid (H2SO4) and HCl are widely used for pretreatment process since they are powerful agents for
lignocellulose hydrolysis (Sun and Cheng 2002). In this study, a pretreatment technique combining ILs and dilute acids was employed to increase the enzymatic hydrolysis yield of regenerated biomass. Five factors, namely, the IL type, acid type, IL volume, acid concentration, and pretreatment temperature and time were investigated. Firstly, [MMIM]DMP, a phosphate-based IL, was selected to use as the pretreatment solvent in this study due to its prominent effect on pretreatment of corn stover. It is possible that DMP− confers favorable solubility and biocompatibility on [MMIM][DMP] and also lowers inhibition on enzyme activities (Fig. 1) (Li et al. 2010b). On the other hand, [BMIM]Cl, which is a chloride-based IL, is a corrosive, toxic,
Appl Microbiol Biotechnol Fig. 4 a Enzymatic hydrolysis of regenerated corn stover pretreated with different concentrations of HCl acid. b Glucose concentration in hydrolysis liquid after 96 h of enzymatic hydrolysis. Pretreatment condition: corn stover was pretreated with 18 mL [MMIM]DMP at 110 °C for 2 h. The concentration of enzymatic hydrolysis solid loading was 10 mg/mL
and extremely hygroscopic solid (m.p. ~70 °C) (Li et al. 2009) with inhibitory effects on enzyme activities due to its high anion concentration. As a result, samples pretreated with [BMIM]Cl have to be thoroughly washed before enzymatic hydrolysis to remove possible IL residue. Therefore, considering both the physical properties and lignocellulose dissolving properties of the three ILs used in this study, [MMIM]DMP was chosen as the pretreatment solvent. To further optimize the pretreatment performance, a synergistic acid-catalyzed aqueous [MMIM]DMP pretreatment system was developed. In this system, a combination of [MMIM]DMP and HCl was found to be more efficient, possibly because the chloride ion is more electronegative than other anions (Fig. 2). The chloride ion is known to form
hydrogen bonds with the cellulose hydroxyl hydrogen in biomass, which in turn destroys the intramolecular and intermolecular hydrogen bonds of cellulose (Swatloski et al. 2002). Moreover, the addition of an inorganic acid to ILs could significantly reduce the usage and the cost of IL solvents incurred in the pretreatment process. In addition, the results show that pretreatment with inorganic acids is more effective than that with organic acids, possibly because of the higher amount of hydrogen ions in inorganic acids at the same acid concentration, which could result in a higher proportion of xylan removal and, subsequently, higher enzymatic saccharification conversion (Zhang et al. 2012). Pretreatment efficiency of biomass was also estimated according to the amount of glucose released during enzymatic hydrolysis of the
Appl Microbiol Biotechnol Fig. 5 Effect of pretreatment temperature and time on the acidcatalyzed IL pretreatment. Pretreatment condition: corn stover was pretreated with 18 mL [MMIM]DMP and 10 % (w/w) HCl and regenerated by water. The concentration of enzymatic hydrolysis solid loading was 10 mg/mL
regenerated sample (Zhu et al. 2006). Regenerated biomass pretreated by ILs containing essentially amorphous cellulose was believed to be more accessible to enzymes. In this study, [MMIM]DMP–HCl pretreatment was the most efficient system in terms of reducing sugars and glucose production. The reducing sugar yield (based on regenerated biomass weight) of corn stover pretreated with HCl-catalyzed [MMIM]DMP solutions reached 82.9 % under the optimal pretreatment conditions summarized in this study. This is higher than the previously reported sugar yield of 71.2 % for [AMIM]Clpretreated wheat straw (Chen et al. 2012). The volume of IL is also an important factor affecting the [MMIM]DMP–HCl pretreatment system. Our results indicated that the enzymatic saccharification conversion increased with an increase in the amount of ILs in the reaction system (Fig. 3), which may be due to the fact that with the addition of [MMIM]DMP into the pretreatment system, part of the cellulose hydrogen bonds becomes easily decomposed, resulting in improved hydrolysis rate and overall cellulose conversion. However, beyond a specific threshold of the IL volume, the viscosity of the reaction system became too high, thereby hindering the contact between acid catalysts and cellulose fibers and causing a decline in enzymatic saccharification conversion (Li et al. 2008). In addition, enzymatic hydrolysis conversion was found to improve with an increase in HCl concentration (Fig. 4). The increased hydrogen ion concentration in the solution may provide more hydrogen ions to attack the oxygen atoms of the cellulose glycosidic bond, improving the catalytic effect and enzymatic hydrolysis conversion (Li et al. 2008). Temperature and time are two important factors that directly affect the efficiency of pretreatment systems. Although higher pretreatment temperature and longer reaction time
usually lead to a more severe destruction of lignocellulosic structure, these conditions also result in further degradation of monosaccharide sugars into furfural, HMF, and other undesirable inhibitory products (Zhou et al. 2012). In a previously reported study, the combined use of IL and ammonia pretreatment showed good efficiency, but required a high temperature of 130 °C and a long reaction time of 24 h (Nguyen et al. 2010), whereas the method developed in this study dramatically reduced the pretreatment temperature and time (110 °C, 2 h) with a comparable cellulose conversion. It is evident from the SEM photographs that compared to the compact fibers and smooth surface of the untreated sample, the pretreated fibers were partially disrupted. This was probably due to the partial dissolution of the corn stover cell wall and partial removal of lignin during IL pretreatment. [MMIM]DMP–HCl-pretreated corn stover showed a porous and fragmented structure, and these breakages and fissures may facilitate the access of enzymes to the cellulose surface, in turn improving the efficiency of enzymatic saccharification as revealed in this study. To compare the FTIR data obtained from these three samples, a number of bonds were used to monitor the chemical changes in lignin and carbohydrates. The decline of these peaks illustrated that the chemical bonds of lignocellulose were destroyed after acid-catalyzed aqueous [MMIM]DMP pretreatment. The peak at 1,250 cm−1, assigned to ether bonds (Liu et al. 2009), reduced in the spectrum of aqueous [MMIM]DMP–citric acid-pretreated corn stover and almost disappeared in the spectrum of aqueous [MMIM]DMP–HClpretreated corn stover. This reduction of peak intensity at 1,250 cm −1 confirmed that pretreatment with aqueous [MMIM]DMP/HCl solution was more effective in breaking
Appl Microbiol Biotechnol Fig. 6 a SEM micrographs, b FTIR spectra, and c X-ray diffraction spectra of untreated corn stover (a), corn stover pretreated by aqueous [MMIM]DMP/citric acid (b), and corn stover pretreated with aqueous [MMIM]DMP/HCl (c). Pretreatment conditions: 18 mL of ([MMIM]DMP), 10 % HCl, and 100 °C for 6 h
ether linkages between lignin and carbohydrates than aqueous [MMIM]DMP/citric acid solution. A similar tendency was observed for the ester bond at 1,732 cm−1 (Liu et al. 2009), suggesting that some ester linkages between lignin and carbohydrates are cleaved during pretreatment. Table 2 CrI values determined from XRD spectra of corn stover before and after pretreatment Pretreatment method
CrI (%)
Untreated Pretreated with aqueous [MMIM]DMP/citric acid Pretreated with aqueous [MMIM]DMP/HCl
69.7 56.5 61.1
According to the XRD analysis, the crystallinity index was found to decrease after acid-catalyzed aqueous [MMIM]DMP pretreatment. This may be due to the disruption of the hydrogen bonds in cellulose by ILs (Zhao et al. 2009). The crystallinity index of aqueous [MMIM]DMP–citric acid-pretreated corn stover was lower than that of aqueous [MMIM]DMP– HCl-pretreated corn stover, possibly due to the severe disruption of the amorphous hemicellulose structure by HCl. In light of these data and the SEM and FTIR analyses, it can be concluded that the structure of the acid-catalyzed ILpretreated samples was obviously destroyed, and the crystalline structure of cellulose was partially destroyed during pretreatment, leading to improved enzymatic hydrolysis of the regenerated corn stover.
Appl Microbiol Biotechnol
In conclusion, an effective pretreatment method using acidcatalyzed aqueous [MMIM]DMP solution was established in this study. Through a series of optimization processes of the reaction conditions, the enzymatic hydrolysis conversion of regenerated biomass was significantly improved with a higher total sugar conversion of 92.7 % obtained for the pretreated corn stover, compared to 27.3 % for the untreated control sample. Moreover, the optimized acid-catalyzed IL system established in this research may potentially facilitate reduced IL usage, pretreatment temperatures, and requirements for equipment. The detailed study of the structural and chemical changes of the regenerated corn stover further demonstrated the efficacy of this novel pretreatment method and revealed the mechanism of the improved enzymatic hydrolysis conversion. Acknowledgments We are grateful to China Petroleum Jilin Chemical Group Company for funding this research work through Contract Number Petrochina 2013-1630. We also acknowledge the Laboratory of Cellulosic Biofuels, Changzhou University for providing the facilities and equipments used in this research.
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