Article pubs.acs.org/EF
Comparative Study on Four Chemical Pretreatment Methods for an Efficient Saccharification of Corn Stover Heng Yu,† Min Zhang,† Jia Ouyang,*,†,‡ and Yang Shen† †
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China
‡
ABSTRACT: Corn stover is a potential feedstock for cellulosic ethanol production. In this study, the performances of four chemical pretreatment methods, low-temperature moderate acid (LTMA), high-temperature dilute acid (HTDA), alkali pretreatment (AP), and sulfite pretreatment (SP), were compared in pretreating non-wood bioresource (corn stover) for an efficient saccharification. Under the investigated conditions, LTMA and HTDA could directly convert most xylan into xylose but had lower enzymatic digestibility from forming more fermentation inhibitors. AP pretreatment achieved better delignification, while its effect on removing xylan was very weak. SP pretreatment not only dissolved about 83.75 and 63.14% of xylan and lignin, respectively, but also caused less known inhibitors than acidic pretreatments. More interestingly, a large amount of xylooligosaccharides (about 17.12 g/L) was found in spent liquor from SP pretreatment, which is a potential high-value co-product. After 48 h of enzymatic hydrolysis, the cellulose saccharification yields were 24% for the LTMA substrate, 47% for the HTDA substrate, 60% for the SP substrate, and 65% for the AP substrate, respectively. These results suggested that SP pretreatment was a more suitable pretreatment method for bioethanol production of corn stover.
1. INTRODUCTION For a long time, energy has been considered as a strong driving force for economic development. Until now, as a result of the rapid increase in the world energy demand,1 the development of biofuels has become increasingly important.2,3 The International Energy Agency (IEA) survey shows that a bioenergy source provides about 10.0% of the annual global total primary energy supply (TPES) from biofuels and waste in 2011.4 However, at present, most ethanol is produced from food crops that have been increasingly questioned over concerns, such as competition with food and feed supplies and effects on the environment and climate change. There is a strong need to develop the second bioethanol from the abundant low-cost lignocellulosic biomass for energy security.5 Because of this, many research efforts have been focused on lignocellulose biomass for ethanol production. Currently, processing lignocellulosic biomass to obtain bioenergy is still hampered by economic and technical obstacles.6 Lignocellulose biomass is the most abundant organic renewable resource. It mainly includes agricultural and forestry residues, woods, and grasses.7 Because of the complex cell-wall structure of lignocellulose biomass, liberation of fermentable sugars from recalcitrant biomass is the most difficult and expensive step in the cellulosic ethanol production. Many factors, such as lignin content, crystallinity of cellulose, and particle size, limit the digestibility of the hemicellulose and cellulose present in the lignocellulosic biomass. Therefore, it was thought that pretreatment is necessary to reduce recalcitrance and improve enzymatic hydrolysis and fermentable sugar yield in cellulosic ethanol production. It aims at breaking the recalcitrant cell wall structure and increasing the accessibility of enzyme to lignocellulose. Considering that pretreatment is a upstream process to remove biomass recalcitrance for downstream microbial and enzymatic processing,8 the criterion of an excellent pretreatment © 2014 American Chemical Society
should include reducing wastage of valuable components (cellulose and hemicellulose), enhancing the enzyme hydrolysis performance, producing less fermentation inhibitor, and costing lower expenditure (energy and time) in the pretreatment process.9 Moreover, some minor concerns, such as the recycle of high-value-added side products10 and the choice of the pretreatment catalyst,11 are also worth considering. Through several decades of efforts, available pretreatment technologies have been developed for all kinds of lignocellulose, such as physical pretreatment, physicochemical pretreatment, chemical pretreatment, and biological pretreatment.12 Each pretreatment process has its own merit and defect. Several studies found that agricultural residues were particularly well-suited to dilute acid pretreatment.13 However, this method would lead to various environmental problems and the formation of many inhibiting byproducts.14,15 Alkali treatment could solubilize both lignin and hemicellulose from lignocellulosic biomass. The main drawbacks of this process are the difficult recovery of hemicellulose sugars and that NaOH recovery is very expensive.16 Sulfite pretreatment was recently another attractive pretreatment method. This process is very effective when applied to woody biomass.17 However, its commercial scalability needs to be demonstrated, especially applied to agriculture residues. Corn is one of the important traditional crops, which is widely planted in the northern part of China. Corn stover, as a lignocellulose biomass, is one of the most abundant agricultural wastes in China.18 It has the potential to serve as a low-cost feedstock for biofuel production. The aim of this work is to Special Issue: International Biorefinery Conference Received: January 17, 2014 Revised: March 12, 2014 Published: March 13, 2014 4282
dx.doi.org/10.1021/ef5001612 | Energy Fuels 2014, 28, 4282−4287
Energy & Fuels
Article
All experiments were performed in replicates, and each data point was the average of two replicates. 2.4. Compositional Analysis. All of the samples were washed by 10-fold distilled water (w/w), filtered, and stored in sealed plastic bags at 4 °C for more than 2 days. This process ensured that the distribution of moisture was uniform. The dry matter content were measured by infrared moisture determination balance (FD-720), which is produced by KETT.19 The components of raw material and pretreated solid residue were determined according to the National Renewable Energy Laboratory (NREL, Golden, CO) analytical methods for biomass.20 Duplicate experiments were run for each sample. 2.5. Scanning Electron Microscopy (SEM) Analysis. All samples for SEM were dried at room temperature and then dried in a vacuum refrigerating machine (VirTis) at −55 °C for at least 24 h. The SEM images were performed using a FEI Quanta 200 (FEI, Eindhoven, Netherlands), operated at 10−20 kV. All samples were coated with gold/palladium (Au/Pd) by a SC7640 automatic/manual high-resolution sputter coater (Quorum Technologies, Newhaven, U.K.). 2.6. X-ray Powder Diffraction (XRD) Measurement. XRD measurements were performed with an Ultima IV diffractometer by Rigaku Corporation operating at 40 kV, 30 mA, and λ (Cu Kα) = 0.154 06 nm. The scan speed was 4° (2θ)/min over the range of 5−50° (2θ). Before the XRD analysis, the samples were freeze-dried under vacuum conditions. Then, they were ground into powder and tiled on a glass sample holder (35 × 50 × 5 mm). The average crystallite size (D) of cellulose fibrils was calculated by Scherrer’s equation21
evaluate the effect of four different pretreatment methods, lowtemperature moderate acid (LTMA), high-temperature dilute acid (HTDA), alkali pretreatment (AP), and sulfite pretreatment (SP), on corn stover for sugar and ethanol production. Furthermore, the solid recovery, chemical composition, fermentation inhibitors, and producing soluble sugars were investigated and compared in detail. The suitable pretreatment method of corn stover for ethanol production was obtained.
2. MATERIALS AND METHODS 2.1. Materials. Corn stover, harvested in early winter of 2012, was obtained from Jiangsu province in China. The raw material contained 15.84% extractives (10.20% water-soluble extractives and 5.64% ethanol-soluble extractives) and 1.17% ash. It is air-dried, crushed, and screened. The fraction collected between 20 and 80 mesh was used in the experiments. Commercial enzymes, Celluclast 1.5L (Cat C2730, Lot SLBB4803V) and Novozyme 188 (Cat C6105, Lot 021M1799V), were purchased from Sigma-Aldrich. The activity of cellulase was 121.54 filter paper units (FPU)/mL, and the activity of β-glucosidase was 574.82 cellobiase units (CBU)/mL. All of the chemical reagents used in this study were purchased from Nanjing Chemical Reagent Co., Ltd. Magnesium sulfite hexahydrate was prepared by mixing sodium sulfite anhydrous with magnesium chloride hexahydrate in a mole ratio of 1:1. 2.2. Pretreatments. In all pretreatment processes, 3 g (oven dry) of corn stover was loaded into a 30 mL sealed stainless-steel tank and heated by an oil bath with glycerol. Different chemical solutions were used in the pretreatment process: 1.82% (w/v) H2SO4 for HTDA pretreatment, 2% (w/v) NaOH for AP pretreatment, and 4% (w/v) Mg(HSO3)2 for SP pretreatment. The ratios of solid/liquid were 1:6 for HTDA and SP pretreatments and 1:10 for AP pretreatment, respectively. In HTDA pretreatment, the pretreated temperature was raised to 160 °C and kept for 60 min. The AP pretreatment required the pretreated temperature be 105 °C and was kept for 60 min. The SP pretreatment needed 170 °C and was kept for 20 min. The spent liquor of HTDA, AP, and SP was used for analysis without diluting. Furthermore, in LTMA pretreatment, the corn stover was soaked in 4% (w/v) H2SO4 overnight at room temperature. Afterward, H2SO4 liquid was squeezed out, and the stock content was about 60% H2SO4 liquid and then heated to 105 °C for 600 min. The pretreated solid residue was washed with 10 times as heavy as distilled water, and this liquid was perceived as spent liquor for analysis. LTMA, HTDA, AP, and SP were performed in replicates, and each data point was the average of two replicates. The details of pretreatment conditions for four pretreatments were listed in Table 1.
D=
LTMA HTDA AP SP a
temperature (°C) 105 160 105 170
time (min) 600 60 60 20
S/L ratio a
N/A 1:6 1:10 1:6
pH
catalyst (w/v)
N/A 0.43 13.71 5.07
4% H2SO4 1.82% H2SO4 2% NaOH 4% Mg(HSO3)2
(1)
where K is Scherrer’s constant (0.9), λ is the wavelength (0.154 06 nm), and β is the full width at half maximum (fwhm) value. For the above calculation, The Gaussian curve was fitted to the top of the 002 peak for determining the fwhm and the position using the Origin 8.5 program.22 The crystallinity index (CI) was estimated depending upon the spectra of XRD by the “peak height method”.23 CI calculation formula is as follows CI (%) =
I002 − IAM × 100 I002
(2)
where I002 and IAM are the maximum intensity of the 002 peak (22.0°, 2θ) and the intensity at 18° (2θ). XRDs of the samples were measured in duplicate. 2.7. Sugar and Fermentation Inhibitor Analyses in Liquids. The concentration of glucose, xylose, arabinose, cellobiose, and known inhibitors [formic acid, acetic acid, levulinic acid, furfural, and 5-hydroxymethylfurfural (HMF)] were measured by a high-performance liquid chromatography system (HPLC, Agilent Technology 1200 Series, Waldbronn, Germany) equipped with a Bio-Rad Aminex HPX87H column (300 × 7.8 mm) and a refractive index detector. Analysis was performed with a mobile phase of 5 mM H2SO4 at a flow rate of 0.6 mL/min at 55 °C.24 Xylo- and cello-oligosaccharide analyses were conducted using a Dionex high-performance anion-exchange chromatography (HPAEC) system (ICS-3000) equipped with an integrated amperometric detector and Carbopac PA200 guard and analytical columns at 30 °C. The eluent was provided at a rate of 0.3 mL/min, according to the following gradient: 0−9 min, 100% 0.1 M NaOH; 9−26 min, from 0 to 50% 0.5 M NaOAc; and 26−40 min, 0.5 M NaOH, at a rate of 0.3 mL/min, used as the post-column eluent.25 The cellulose hydrolysis and glucose yields were calculated according to the following equations:
Table 1. Details of Pretreatment Conditions for Four Pretreatments of Corn Stover method
Kλ β cos θ
N/A = not detected.
2.3. Enzymatic Hydrolysis. Enzymatic hydrolysis was performed at a 3% (w/v) cellulose concentration in 20 mL of citrate buffer (50 mM, pH 4.8) on a thermostat shaker set at 50 °C and 150 rpm for 48 h. The total working volume was 20 mL in 50 mL Erlenmeyer flasks. A Cellulast 1.5L loading of 15 FPU/g of cellulose with the addition of Novozyme 188 at a loading of 30 CBU/g of cellulose was used for all hydrolysis experiments. At specific time intervals, aliquots were withdrawn and centrifuged to remove the insoluble materials. The supernatants were subsequently filtered through a 0.22 mm syringe filter (Millipore, Bedford, MA) and used for subsequent analysis.
saccharification yield (%) =
(Cglu + 1.053C biose) Ccel
× 0.9 × 100 (3)
4283
dx.doi.org/10.1021/ef5001612 | Energy Fuels 2014, 28, 4282−4287
Energy & Fuels
Article
Table 2. Mass of Three Key Components and Solid Recovery of Corn Stover by Four Different Pretreatmentsa solid residue (g/100 g of dry biomass) method
glucan
untreated LTMA HTDA AP SP a
34.66 30.24 29.99 29.22 29.88
± ± ± ± ±
0.13 0.09 0.14 0.01 0.09
xylan 19.26 1.97 0.28 10.05 3.13
± ± ± ± ±
0.20 0.01 0.01 0.01 0.02
lignin
solid recovery (g/100 g of dry biomass)
± ± ± ± ±
100 57.79 ± 0.34 56.09 ± 0.20 50.88 ± 0.47 49.09 ± 0.69
26.15 19.76 22.10 3.72 9.64
0.31 0.02 0.04 0.03 0.25
Data are on a dry weight basis.
Table 3. Concentration of Monomeric Sugars and Fermentation Inhibitors in Pretreated Spent Liquors methoda sugars (g/L)
LTMA glucose xylose arabinose formic acid acetic acid levulinic acid furfural HMF
inhibitors (g/L)
2.03 18.39 2.61 0.25 3.42 0.15 0.14 0.07
± ± ± ± ± ± ± ±
0.08 0.36 0.10 0.01 0.14 0.01 0.01 0.01
HTDA
AP
SP
± ± ± ± ± ± ± ±
0.28 ± 0.01 0.43 ± 0.02 0.05 ± 0.01 0.05 ± 0.04 3.86 ± 0.19 N/Ab N/A N/A
0 0.69 ± 0.02 0.07 ± 0.04 0.09 ± 0.01 3.53 ± 0.18 N/A N/A N/A
5.56 23.30 3.88 0.92 4.67 0.63 3.11 0.92
0.17 0.46 0.12 0.03 0.14 0.02 0.09 0.03
a
The pretreated solid residue was washed 5 times water, and the liquid was perceived as pretreated spent liquors for analysis during LTMA processing. bN/A = not detected.
Table 4. Chemical Composition of Corn Stover Residues before/after Pretreatmenta method untreated LTMA HTDA AP SP a
Cglu Ccel
× 0.9 × 100
± ± ± ± ±
0.13 0.14 0.25 0.01 0.17
xylan (%) 19.26 3.41 0.51 19.76 6.38
± ± ± ± ±
0.20 0.02 0.02 0.03 0.03
lignin (%) 26.15 34.20 39.40 7.31 19.65
± ± ± ± ±
0.31 0.02 0.09 0.06 0.50
Data are on a dry weight basis.
3.1. Mass Recovery of Key Components in Solid Residues after Pretreatments. Table 2 summarized a detailed mass of glucan, xylan, lignin, and total solid residue for four different pretreatments. There was an obvious reduction on the recovery of solid residue for all of the pretreatment methods, ranging from 49.09 to 57.79%. When the spruce was treated by dilute acid and sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) pretreatment, the recovery of solid residue was 64.1 and 60.5%, respectively.13 This weight loss was mainly caused by delignification, partial hydrolysis of hemicellulose, and dissolution of non-structural components, extractives, and ashes.26 Further analysis showed that acidic pretreatments (LTMA and HTDA) had the strongest ability to remove the xylan fraction from the solid. However, their effects on the removal of lignin were weak, especially for HTDA. Higher solid recovery could be partly explained by the retention of the original lignin. In comparison to the raw corn stover (19.26 g of xylan), the content of xylan in 100 g of dry corn stover was 1.97 and 0.28 g by LTMA and HTDA. In opposition to acidic pretreatments, about 52.21% of xylan remained in the solid residue after AP pretreatment. Therefore, the maximum total sugar recovery (29.22 g of glucan and 10.05 g of xylan) was obtained from AP pretreatment, which was consistent with previous studies.27 Our result also showed that the main effect of AP pretreatment is delignification. Almost 85.77% of lignin was removed from corn stover. As for SP pretreatment, which has a relatively higher pH than those
Figure 1. HPAEC chromatogram of linear xylo-oligosaccharides. Peak identification: X1 + G1, xylose + glucose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose; and X6, xylohexaose.
glucose yield (%) =
glucan (%) 34.66 52.33 53.47 57.43 60.88
(4)
where Cglu is the concentration of glucose in enzymatic hydrolysate, Cbiose is the concentration of cellobiose in enzymatic hydrolysate, and Ccel is the initial concentration of cellulose. The units of measurement were g/L. The total sugar yield was defined on the basis of potential glucose and xylose in the raw material.
3. RESULTS AND DISCUSSION As an upstream process in cellulosic ethanol production, an ideal pretreatment should improve the enzymatic digestibility of biomass as much as possible. It is important as well to maximally recover all available sugars and limit the formation of inhibitors.13 After pretreatments, corn stover was separated into two fractions: solid residue and pretreatment liquor. Hence, the comparison of four different pretreatment methods were proposed on the basis of the results obtained from solid residue and pretreatment liquor studies. 4284
dx.doi.org/10.1021/ef5001612 | Energy Fuels 2014, 28, 4282−4287
Energy & Fuels
Article
Figure 2. SEM images of untreated corn stover fiber and four pretreated corn stover fibers by different pretreatment processes: (A) raw corn stover fiber, (B) pretreated solid residue by LTMA, (C) pretreated solid residue by HTDA, (D) pretreated solid residue by AP, and (E) pretreated solid residue by SP.
reported studies of sulfite pretreatment (
