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Comparison of alkaline and acid pretreatments for enzymatic hydrolysis of soybean hull and soybean straw to produce fermentable sugars
Industrial Crops & Products 109 (2017) 391–397
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Research paper
Comparison of alkaline and acid pretreatments for enzymatic hydrolysis of soybean hull and soybean straw to produce fermentable sugars Qing Qing, Qi Guo, Linlin Zhou, Xiaohang Gao, Xiaoxue Lu, Yue Zhang
MARK
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Department of Biochemical Engineering, College of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, 213164, Jiangsu, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Soybean hull Soybean straw Dilute acid pretreatment Alkaline pretreatment Enzymatic hydrolysis
The specific characteristics of biomass structure and differences in chemical composition of soybean hull (SBH) and soybean straw (SBS) may result in different behavior of pretreatment and enzymatic hydrolysis. In this work, dilute sulfuric acid (DA) pretreatment and alkaline sodium hydroxide (AL) pretreatment in a range of pretreatment temperatures and durations were investigated to improve the enzymatic digestibility of SBH and SBS. Satisfactory enzymatic digestibility was observed in the hydrolysis of both pretreated soybean fractions, but SBH showed higher digestibility than that of SBS, no matter which pretreatment technology was applied. Furthermore, DA pretreatment was more effective than AL pretreatment in conversion of soybean fractions into fermentable sugars, if both pretreatment and enzymatic hydrolysis stages were considered. The highest total sugar yield of SBH and SBS using DA pretreatment and subsequently hydrolyzed with 30 FPU/g-DM cellulase were 86.9% and 70.3%, respectively.
1. Introduction Soybean is an important source of food in many countries, especially in many Asian countries (Cabrera et al., 2015). The oil and protein constituents of soybeans are regarded as valuable products in soybean processing, but little attention has been paid to the producing residues, such as soybean hulls and straws (Cassales et al., 2011). These residues are rich in cellulose and do not require an extra grinding process prior to pretreatment as some other lignocellulosic material. As a major food and energy crop in China, soybean has an annual production of 13 Tg. Soybean straws and hulls, collected after soybean harvest, are partially used for animal feed and the main part of these residues are burnt directly in the fields, causing serious environmental pollution. Therefore, exploiting the potential utilization of soybean carbohydrates can not only increase the added value of soybean industry, but also benefit to environmental protection (Corredor et al., 2008). Soybean hull and straw are basically lignocellulosic material composed of fermentable hextose and pentose sugars, polymerized as cellulose and hemicellulose, in addition to a small proportion of lignin that is composed of phenolic compounds. Therefore, the efficient hydrolysis of cellulose and hemicelluloses in soybean residues to liberate monomer sugars is of potential interest for conversion into biofuels and chemicals with economic interest. However, the complex network of lignocellulosic biomass presents a major resistance for degradation of
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Corresponding author. E-mail address: [email protected] (Y. Zhang).
http://dx.doi.org/10.1016/j.indcrop.2017.08.051 Received 11 May 2017; Received in revised form 14 August 2017; Accepted 28 August 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
polysaccharide fractions into soluble monomeric sugars, which is commonly referred to as the recalcitrance barrier (Himmel et al., 2007; Behera et al., 2014). Consequently, the saccharification efficiency is extremely low unless a suitable pretreatment conducted before enzymatic hydrolysis to break down the lignin and hemicelluloses network and to disrupt the crystalline cellulose structure. Among existing pretreatment technologies, dilute acid pretreatment (DA) and alkaline pretreatment (AL) are believed as the most mature ones that are ready for commercialized application (Lee et al., 2015; Alvira et al., 2010), which also have shown high effectiveness on several agricultural residues (Dagninoa et al., 2013). In DA pretreatments, substantial amounts of sugars, mainly from hemicellulose and partially from cellulose, could be solubilized into the liquid phase of the hydrolysis slurry (Jung et al., 2013). Dilute sulfuric acid, which has been extensively investigated for pretreatment using experimental and theoretical approaches, can effectively degrade hemicellulose in the cell wall network by the catalytic effect of proton H+ (Chen et al., 2015). It was reported that over 90% of hemicellulose could be successfully removed during DA pretreatment under moderate severities, remaining highly digestible residue that mainly composed of cellulose with small part of lignin (Singh et al., 2015). However, due to the strong protonation effect, the monomer sugars obtained during acid pretreatment are ready to be further decomposed into their degradation forms, such as furfural, 5-hydroxymethyl furfural (5-HMF), levulinic acid, and even humins, leading to strong inhibition to enzymes and
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fermentation microorganisms. On the other hand, AL pretreatment is considered as the most effective one with less sugar degradation, lower energy consumption, more lignin removal, and less furan derivatives (Behera et al., 2014; Whitfield et al., 2012). Compared to DA pretreatment, AL methods exhibit higher capacity in breaking the linkages between lignin and carbohydrates, and disrupting lignin structures, with a minor cellulose and hemicelluloses alteration. Acetyl groups and various uronic acid substitutes, which have lower susceptibility to hydrolytic enzymes, are also eliminated by AL pretreatment (Mosier et al., 2005; Zheng et al., 2009). More importantly, with the salvation reactions, the AL pretreatment could swell the material, thus increase the internal surface area of the recovered solids and the accessibility of the enzymes (Jin et al., 2013). In this study, soybean residues including SBH and SBS, as renewable and low-cost lignocellulosic materials for the production of fermentable sugars were investigated. DA and AL pretreatments were performed separately on different soybean residue fractions. Then the digestibility of different pretreated soybean fractions was evaluated with various enzyme dosages, in addition to determination of the structural characteristics and composition changes of the pretreated solids. The impacts of the intrinsic structure differences between SBS and SBH, and different pretreatment methods on the digestibility of the pretreated solids were therefore compared and elucidated.
Hemicellulose removal (%) hemicellulose inpretreated samples (g) × SR% ⎞ = ⎜⎛1 − ⎟ × 100% hemicellulose inone gram native samples (g) ⎠ ⎝ 2.3. Enzymatic hydrolysis Saccharification of the untreated and pretreated samples were performed following the NREL laboratory analytical procedure (Selig et al., 2008). A solid loading of 2% (w/v) with 0.05 M acetate buffer (pH 4.8) were added in 50 mL Erlenmeyer flasks. 800 μg of 20 mg/mL tetracycline antibiotic in DI water was also supplemented before adding enzymes to prevent possible microorganism contamination during hydrolysis. Enzymes were added once the mixture reached 50 °Cand the slurry was stirred at 160 rpm in a thermostated shaker (Model # THZ072HT, Shanghai, China). Samples were taken after 1, 4, 24, 48, and 72 h of hydrolysis and immediately analyzed with a high performance liquid chromatography (HPLC) to determine the monomer sugar yields. All enzymatic hydrolysis samples were prepared in triplicates and run under parallel conditions. 2.4. Analytical method 2.4.1. Sugar analysis Samples were analyzed for carbohydrates using a Waters Alliance HPLC (Model 2695, Waters Corporation, Milford, MA), equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) run at 65 °C and a refractive index detector (Waters 2414). The sample injection volume was 20 μL and 0.005 M sulfuric acid at a flow rate of 0.6 mL/min was set for the mobile phase. The concentration of various monomeric sugars, furfural, HMF, and levulinic acid were quantified based on the calibration curves constructed by standards. The monomer sugar yields were calculated according to the following equations, and the total sugar yields were the sum of monomer sugars and cellobiose.
2. Materials and methods 2.1. Material SBH and SBS were separated after soybean crops collected from the farmland nearby Changzhou (Jiangsu Province, China). The materials were washed with deionized water and dried at 45 °C until constant weight, then milled into size smaller than 3 mm. The composition of the raw and pretreated substrates were determined based upon National Renewable Energy Laboratory Analytical Procedure (Sluiter et al., 2008). Accellerase 1500 (96 FPU/mL) was generously provided by Genencor (Wuxi, Jiangsu province, China). Novozyme 188 (066K0676603) was purchased from Sigma (St. Louis, MO, USA). Sodium hydroxide (NaOH), sulfuric acid (H2SO4) and other chemical reagents were purchased from Sinopharm Group Chemical Reagent Co. Ltd. (Shanghai, China).
Glucose yield(%) =
Xylose yield(%) =
xylose released 3 × 0.88 (g) × 100% intial xylan content in the substrate (g)
2.4.2. Sample characterization Crystallinity index (CrI) was analyzed by X-ray diffraction (XRD) using a D/max 2500 PC diffractometer with Cu Ka radiation (Rigaku Corporation, Tokyo, Japan) and operated at a voltage of 60 kV and a current of 300 mA. The 2θ range was detected from 5 to 40°in a step of 0.02. CrI was calculated according to the following equation:
2.2. Pretreatment The quantitative dried sample was mixed with 1 w/w% H2SO4 or NaOH solution in a 100 mL high-temperature and high-pressure stainless steel reactor (Zhenjiang Dantu Universal Electrical Equipment, China), equipped with a stirring apparatus. The solid to liquid ratio was set to 1: 20. Then the reactor was sealed and electrically heated to the desired temperature using a porcelain-heating jacket. After the reaction, the reactor was quenched in an iced water bath, then filtrated to separated the liquid from the solid. The collected solid was washed for several times until pH neutral. The pretreatment liquid and solid were collected for different analyses. The solid recovery, delignification, and hemicellulose removal of different pretreatments were calculated based on the following equations:
Solid recovery(%) =
glucose released × 0.9 (g) × 100% intial glucan content in the substrate (g)
CrI (%) =
I002 − Iam × 100% I002
Where I002 is the intensity of the crystalline portion (crystalline cellulose) determined at 2θ = 22.2°and Iam is the peak for the amorphous portion (i.e. amorphous cellulose, hemicellulose and lignin) at 2θ = 16.4°. The surface morphological features of different samples were imaged with a scanning electron microscope (SEM, Model # JSM-6360LA, JEOL, Japan) that was operated at 15 kV. Fourier transform infrared (FTIR) was to verify the change of the chemical structure of SBH and SBS before and after pretreatment. The spectra were analyzed using a FTIR spectrometer (Nicolet, USA). The sample spectra were obtained using 32 scans over the range of 500–4000 cm−1. Samples were ground and mixed with the spectroscopic grade KBr then pressed in a standard device to produce diameter pellets (Qing et al., 2016). The porosity of the samples was determined by N2 adsorption/desorption isotherms at 77 K using a Brenauer-Emmett-Teller (BET) surface area analyzer (ASAP 2010 M). Before the measurement, all samples were dried at
substrate recovered after pretreatment (g) × 100% substrate used for pretreatment (g)
lignin inpretreated samples (g) × SR% ⎞ Delignification(%) = ⎜⎛1 − ⎟ lignin in one gram native samples (g) ⎠ ⎝ × 100% 392
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105 °C for 4 h. The specific surface area (SSA), total pore volume (PV) and average pore size (PS) of porous materials were calculated based on the BET method (Qing et al., 2017).
– – 1.05 ± 0.18 0 1.10 ± 0.21 0
La
Q. Qing et al.
– – 0.30 ± 0.09 0 0.60 ± 0.12 0
3.1. Chemical composition of SBH and SBS before and after pretreatment The chemical compositions of untreated SBH and SBS were determined and the results were shown in Table 1. The compositions of soybean residue fractions, similar to other lignocellulosic materials, are comprised of a complex mixture of cellulose, hemicellulose, and lignin, in addition to a small amount of extractives and ash. Notably, the xylan contents of SBS and SBH were comparable, which were 16.7% and 20.0%, respectively. However, the glucan and lignin contents in SBS were obviously higher than in SBH, which might make SBS more difficulty to be decomposed. The total carbohydrate fractions were about 48.6% and 59.0% of the dry biomass basis for SBH and SBS, respectively, making these agricultural residues attractive feedstock for bioethanol production. In order to compare the impacts of different pretreatment methods on the composition and digestibility of soybean fractions, DA (H2SO4) and AL (NaOH) pretreatments as widely recognized technologies were performed under 120 °C for 1 h with 1% catalysts. The cellulose contents of the pretreated SBH and SBS increased noticeably compared to the untreated fractions due to the destruction of the intact structure and the solubilization of lignin and hemicelluloses. In addition, the AL pretreatment removed a larger portion of lignin, whereas the DA pretreatment mainly hydrolyzed hemicelluloses for both SBH and SBS. After AL pretreatment, the amount of lignin in SBH and SBS decreased 37.9 and 32.6%, respectively, indicating a strong delignification capacity of this method. On the other hand, DA pretreatment removed 73.2 and 62.4% of hemicelluloses from the pretreated solids for SBH and SBS, respectively. It is worth mentioning that the solid recovery ratio of SBS was obviously higher than that of SBH no matter for DA or AL pretreatments, indicating that the structure of SBH was more vulnerable to pretreatment than SBS, thus was severely damaged. In addition, the ingredients of the reaction liquid were also determined and shown in Table 1. As expected, the DA pretreatment liquid contained more xylose monomers and degradation products as a result of the stronger capacity in cleavage of hemicellulose linkages by H+ protons that were supplied by acids. On the contrary, smaller amounts of reducing sugars and almost no degradation products were detected in the AL pretreatment liquor since AL methods remove hemicelluloses mainly as oligosaccharides (Gáspár et al., 2007) that cannot be detected by the HPLC method used in this study. The total sugar release determined during 72 h enzymatic hydrolysis and the enzymatic hydrolysis courses were reflected in Fig. 1. Both DA and AL pretreatments significantly enhanced substrate digestibility which could be due the removal of hemicelluloses and lignin compounds and the generation of porous structure. Coincide with the composition results analyzed above, SBH was easier to be decomposed by enzymes than SBS, thus exhibiting a higher enzymatic digestibility than SBS for both DA and AL pretreatments. And for both SBH and SBS, the total sugar yields of AL pretreatment samples were nearly 18% higher than that of the DA pretreatment samples. The highest enzymatic hydrolysis sugar yields of AL-SBH and AL-SBS reached 76.8% and 50.5% at 72 h, respectively, which were 31.6% and 28.4% higher than the corresponding raw soybean fractions.
± ± ± ±
0.42 032 0.45 0.01
– – 1.08 0.21 0.50 0.21 0.2 0.2 0.5 0.5 0.3 0.3 ± ± ± ± ± ± 13.1 21.6 23.7 17.9 30.4 24.2 0.5 0.4 0.5 0.6 0.7 0.6 ± ± ± ± ± ± 20.0 16.7 13.2 22.9 11.7 16.6 – – 40.5 45.5 53.8 60.3
3.2. DA pretreatment
UN-SBH UN-SBS DA-SBH AL-SBH DA-SBS AL-SBS
± ± ± ±
1.5 1.7 1.0 2.3
28.6 42.3 53.5 52.7 56.4 55.1
± ± ± ± ± ±
0.7 0.9 1.1 0.9 0.8 1.0
Hemi SR, % Sample
Table 1 Compositional analysis of untreated and pretreated SBS and SBH.
Lignin
0.2 0.5 0.3 0.2 0.3 0.5
Ash
± ± ± ± ± ±
0.1 0.1 0.1 0.1 0.1 0.1
– – 73.2 47.9 62.4 40.0
– – 26.7 37.9 24.4 32.6
– – 1.48 1.13 0.35 0.11
± ± ± ±
0.03 0.56 0.09 0.02
– – 5.13 0.61 4.78 0.10
xyl glu Hemi Cell
Lignin
Removal, % Composition, %
Pretreatment liquid, mg/mL
ara
± ± ± ±
0.12 0.08 0.11 0.07
– – 0.08 ± 0.02 0 0.03 ± 0.01 0
FF
HMF
3. Results and discussion
The impacts of pretreatment temperature and time on sugar releasing during DA pretreatment and subsequent enzymatic hydrolysis were presented in Fig. 2. Prolongation of the reaction duration under lower temperature (120 °C) clearly enhanced the monomer sugar 393
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degraded into soluble monomers under 140 °C. When the reaction temperature was raised to 160 °C, more polymer carbohydrates were degraded as the solid recovery ratio further decreased compared to 140 °C. However, the sugar yields in the pretreatment step declined at 160 °C, as the monomer sugars released were further degraded to inhibitory compounds. In the meanwhile, the solid recovery decreased with the increase of the pretreatment severity, and this trend was more apparent for SBS than SBH. Enzymatic hydrolysis was conducted to evaluate the digestibility of different soybean residue solids that had been subjected to DA pretreatment under different conditions. The sugar yields of both SBH and SBS during enzymatic hydrolysis stage were comparable for all evaluated DA pretreatment conditions. However, if the monomer sugars released in the pretreatment stage was included, the total sugar yields of SBH varied between 61.3% (120 °C, 60 min) to 86.9% (140 °C, 40 min). This result indicates that DA pretreatment could effectively recover most of the carbohydrates in SBH as monomers under the optimized conditions, and only small quantity of inhibitory compounds were formed. Nevertheless, the total sugar yields of SBS were much lower than SBH, which were only between 41.7% (160 °C, 80 min) and 70.3% (140 °C, 80 min). It was also noticed that the solid recovery of SBS was much higher than that of SBH, indicating the SBS structure was more rigid and complicated than SBH. Moreover, raise of pretreatment temperature and prolongation of reaction duration to disrupt the structure of SBS might also lead to severer sugar degradation and less total sugar recovery.
Fig. 1. Enzymatic hydrolysis of raw soybean fractions and SBS/SBH pretreated by 1%. DA (H2SO4) or AL (NaOH) at 120 °C for 60 min.
3.3. AL pretreatment AL pretreatment, especially dilute sodium hydroxide, was considered as one of most suitable methods for pretreatment of agricultural residues (Galbe and Zacchi, 2007; Sarkar et al., 2012). The impacts of pretreatment temperature (100–140 °C) and time (60–140 min) on the total sugar releases during both AL pretreatment and enzymatic hydrolysis stages were therefore analyzed for SBS and SBH fractions as shown in Fig. 3. Compared to DA pretreatment, much smaller amounts of reducing sugars were detected in the AL pretreatment liquid, corresponding to the findings presented in Table 1. In the meanwhile, the variation of pretreatment conditions had little impacts on the monomer sugar released to the pretreatment liquid, while its influences on the digestibility of the solid residues were obvious. Different from DA pretreated sample, most of the monomeric carbohydrates were released during the enzymatic hydrolysis stage for AL pretreated soybean fractions. Under the investigated conditions, the highest total sugar yields of two stages were achieved under 100 °C, 120 min for SBH and 140 °C, 120 min for SBS, which were 75.6% and 56.7%, respectively. Similar to DA pretreated samples, SBH exhibited higher digestibility than SBS whatever the pretreatment conditions applied. These results reinforced the finding that SBS was much more difficult to deconstruct, thus a higher pretreatment temperature or severer condition might be needed to disrupt the recalcitrance barrier and make cellulose accessible to enzymes. 3.4. The influence of cellulase loading Different cellulase loadings of 15, 30 and 60 FPU/g DM were compared during enzymatic hydrolysis of untreated and pretreated substrates that were obtained by the optimized pretreatment conditions determined above. Both of the DA and AL pretreated soybean fractions exhibited much higher enzymatic hydrolysis yields than the raw materials, supporting the applied pretreatments successfully modified the structural properties of the substrates and increased the accessibility of cellulase to cellulose surface. An obvious increment of sugar yield was observed for all samples when the enzyme loading was raised from 15 to 30 FPU/g-DM. However, when the enzyme loading further increased to 60 FPU/g-DM, no significant increase in digestibility was detected as
Fig. 2. (a) The impacts of pretreatment temperature and duration on solid recovery and the total sugar yields of DA pretreated soybean fractions. (b) Concentrations of soluble sugars released in enzymatic hydrolysate of SBH pretreated at 120 °C for 80 min. (c) Concentrations of soluble sugars released in enzymatic hydrolysate of SBS pretreated by at 140 °C for 80 min.
released to the pretreatment liquid. But under the reaction temperature of 140 °C, this trend was obscure. About 45–47% of the total carbohydrate fraction in the SBH and 29–34% of that in the SBS could be 394
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Fig. 3. (a) The impacts of pretreatment temperature and duration on solid recovery and the total sugar yields of AL pretreated soybean fractions. (b) Concentrations of soluble sugars released in enzymatic hydrolysate of SBH pretreated at 140 °C for 80 min. (c) Concentrations of soluble sugars released in enzymatic hydrolysate of SBS pretreated at 140 °C for 120 min.
illustrated in Fig. 4a and b. Nevertheless, the initial reaction rates of the higher enzyme loading samples displayed evidently promotes, especially for the pretreated samples as shown in Fig. 4c. The maximum initial reaction rate achieved was 4.91 g/L per hour for AL-SBH with cellulase loading of 60 FPU/g DM, which might benefit from the high lignin and hemicelluloses removal of this sample. The enzymatic digestibility of AL-SBH was 87.6% with 60 FPU/g-DM cellulase loading, which was 17.1% higher than DA-SBH hydrolyzed with the same enzyme loading. Compared to SBH, the enzymatic hydrolysis yields of SBS were much lower, which were 63.1% and 50.7% for AL and DA pretreatment, respectively. These results demonstrated that AL pretreatment was more effective to increase enzymatic digestibility of the pretreated solids, which might due to the higher removal ratio of the lignin that sheathed the cellulose. On the other hand, DA pretreatment mainly removes hemicelluloses during the pretreatment stage, thus the enzymatic performance was inferior to AL pretreatment. However, if the monomer sugar released into pretreatment liquid was included, DA pretreatment showed a higher total sugar yield of these two stages as explained in previous section.
Fig. 4. Enzymatic hydrolysis of 2% soybean fractions with different cellulase loadings (a) SBH; (b) SBS; (c)The 72 h total sugar yields and initial reaction rate of the untreated, DA, and AL pretreated SBH and SBS.
3.5. Characterization of SBH and SBS before and after pretreatment Scanning electron micrographs depicting the morphological features of native soybean fractions, DA and AL pretreated soybean fractions, and residues collected after enzymatic hydrolysis were recorded. Untreated SBH and SBS showed smooth and well-ordered fibrous structure, indicating a rigid and highly packed surface structure that constructed by cellulose, hemicellulose, lignin, and other binding materials. It was noticed that DA pretreatment of SBH and SBS resulted in 395
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acetyl and uronic ester groups in hemicellulose or the phenyl ester linkage between hemicellulose and lignin (Kristensen et al., 2008; Phitsuwan et al., 2015). It was noticed that this band attenuated after DA pretreatment and almost disappeared after AL pretreatment, indicating partial removal of hemicelluloses after DA pretreatment and fully cleavage of the ester band from the hemicelluloses and lignin after AL pretreatment. Lignin-related bands in the spectra were assigned to peaks near 1510 cm−1, 1627 cm −1, 1745 cm−1, and 1745 cm−1. The bands at approximately 1510 cm−1 and 1745 cm−1 reflected the C]C stretching of the lignin aromatic ring. The bands at 1627 cm−1 was attributable to lignin aromatic skeletal vibration and C]O stretching (Kumar et al., 2009). The obscurely decrease of the intensity of these three bands for DA-SBS and DA-SBH suggested a limited lignin disruption by the DA pretreatment. However, the reduction of the intensity of these bands became apparent for AL-SBS and AL-SBH, which agreed with the chemical composition analysis in Table 1. All these information manifested that DA pretreatment can remove most of the hemicellulose in biomass by cleaving acetyl linkages and AL pretreatment is more conducive to remove lignin compounds.
Table 2 Characterization of SBH and SBS before and after pretreatment. Sample
CrI, %
Surface Area, m2/g
Pore Volume, cm3/g
Pore Size, nm
DA-SBH AL-SBH DA-SBS AL-SBS UN-SBH UN-SBS
41.4 44.0 43.1 41.7 32.9 35.1
4.4807 1.0925 3.4085 3.2671 0.5752 0.8088
0.003587 0.000895 0.002134 0.001447 0.000378 0.000582
3.20187 3.27858 2.50422 1.77138 2.62658 2.87703
damage to the cellular structure, as evident by the separation of fibers from the pith and the loosening of the fibrous network. Similar structural changes were found in the AL pretreated samples, which could be resulted from the removal of hemicelluloses and lignin from soybean fractions by AL pretreatment. In addition, the surface of the pretreated SBH showed larger pore size than the SBS, which agreed with the BET analysis shown in Table 2. Compared to the pretreated samples, the surface of hydrolyzed residues was more rough and cracked, appearing more porous and disruptions. It was observed that a considerable amount of cell wall material (cellulose and hemicellulose) was dissolved and swelled after enzymatic hydrolysis. The crystallinity index and the crystalline structure of cellulose have been considered as major factors that dominate the efficiency of lignocelluloses enzymatic hydrolysis. The calculated crystallinity index of the raw materials and the pretreated samples were shown in Table 2. The results revealed that the CrI of the untreated SBH increased from 32.9% to 41.4% for DA pretreated sample and 44.0% for AL pretreated sample. Correspondingly, the CrI of SBS increased from 35.1% to 43.1% and 41.7%, respectively, for DA and AL pretreated samples. All these changes could be attributed to the removal of the amorphous components from the soybean fractions, such as lignin, hemicellulose, and extractives. The CrI of native SBS was higher than SBH, which could be due to the higher cellulose content in SBS. The SSA, PV, and PS values were also presented in Table 2 to illustrate the change of exterior and interior surface structure of SBH and SBS that were subjected to different pretreatment technologies. The SSA and PV for native samples were 0.5752 and 0.8088 m2/g, and 0.000378 and 0.000582 cm3/g, respectively, for SBH and SBS. The SSA and PV of SBS increased after pretreatment, but the pore size shrunk due to the formation of new minor pores after hemicelluloses and lignin removal. For SBH, all the three parameters notably increased after DA pretreatment, but the increments were limited after AL pretreatment, indicating the pores might disappear by collapsing when delignification. This phenomenon, which was usually noticed when the lignin removal exceeded 50%, was also reported in several previous research (Zhu et al., 2008; Lee et al., 2015). These research also mentioned that cellulase can efficiently reach cellulose structure after removing of the lignin barrier in this case, thus resulted in enhanced enzymatic digestibility. FTIR was carried out to evaluate the change of chemical function groups before and after pretreatment. At approximately 3400 cm−1 is associated with the −OH stretching vibration of cellulose. The peak at 2918 cm−1 is due to the asymmetrical stretching of CH2 and CH bonds, and the bond at 900 cm−1 is attributed to β-(1–4)-glycosidic bond (CeOeC). As the absorption peaks in the native SBS and SBH were similar to those of the pretreated substrates, it agreed with the composition analysis that most of the crystalline structure cellulose was preserved after DA or AL pretreatments. In addition, these peaks of SBH were obviously smaller than those of SBS, indicating more crystalline cellulose in SBS than in SBH which tallied with the CrI values shown in Table 2. The comparably more crystalline structure cellulose could be a reason that explains the higher digestibility of all SBH samples than SBS samples. The hemicelluloses and lignin related band at approximately 1245 cm−1 was due to the stretching of CeO bonds, which was observed to be notably decreased after DA and AL pretreatment for both SBH and SBS. Absorption bond at 1733 cm−1 was attributed to the
4. Conclusion SBH and SBS as main fractions of soybean residues were pretreated by DA and AL pretreatments under various conditions to compare their behaviors during pretreatment and subsequent enzymatic hydrolysis. SBS contains larger portions of cellulose and lignin, which was more difficult to be pretreated and hydrolyzed than SBH. The AL-SBH and DA-SBH pretreated under optimized conditions exhibited the highest total sugar yields of 86.9% and 75.6%, respectively. AL pretreatment was more effective in delignification and enhancing enzymatic digestibility of pretreated substrates, whereas DA pretreatment showed a higher capacity in removing hemicelluloses and increasing the total sugar recovery of both two stages. Acknowledgements The authors appreciate the financial supports by the funds from Jiangsu Natural Science Funds through the contract Number of BK20140258, Changzhou Sci & Tech Program through the grant Number of CE20145053, and Natural and Science Research Program of Jiangsu Universities through the contract Number of 15KJB530002. We also acknowledge the Analysis and Testing Center of Changzhou University for providing the facilities for sample characterization used in this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2017.08.051. References Alvira, P., Tomas-Pejo, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101 (13), 4851–4861. Behera, S., Arora, R., Nandhagopal, N., Kumar, S., 2014. Importance of chemical pretreatment for bioconversion of lignocellulosic biomass. Renew. Sust. Energy Rev. 36, 91–106. Cabrera, E., Munoz, M.J., Martin, R., Caro, I., Curbelo, C., Diaz, A.B., 2015. Comparison of industrially viable pretreatments to enhance soybean straw biodegradability. Bioresour. Technol. 194, 1–6. Cassales, A., Souza-Cruz, P.B., Rech, R., Záchia, A., Marco, A., 2011. Optimization of soybean hull acid hydrolysis and its characterization as a potential substrate for bioprocessing. Biomass Bioener. 35 (11), 4675–4683. Chen, L., Zhang, H., Li, J., Lu, M., Guo, X., Han, L., 2015. A novel diffusion-biphasic hydrolysis coupled kinetic model for dilute sulfuric acid pretreatment of corn stover. Bioresour. Technol. 177, 8–16. Corredor, D.Y., Sun, X.S., Salazar, J.M., Hohn, K.L., Wang, D., 2008. Enzymatic hydrolysis of soybean hulls using dilute acid and modified steam-Explosion pretreatments. J. Biobased Mater. Bio. 2 (1), 43–50.
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Research paper
Comparison of alkaline and acid pretreatments for enzymatic hydrolysis of soybean hull and soybean straw to produce fermentable sugars Qing Qing, Qi Guo, Linlin Zhou, Xiaohang Gao, Xiaoxue Lu, Yue Zhang
MARK
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Department of Biochemical Engineering, College of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, 213164, Jiangsu, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Soybean hull Soybean straw Dilute acid pretreatment Alkaline pretreatment Enzymatic hydrolysis
The specific characteristics of biomass structure and differences in chemical composition of soybean hull (SBH) and soybean straw (SBS) may result in different behavior of pretreatment and enzymatic hydrolysis. In this work, dilute sulfuric acid (DA) pretreatment and alkaline sodium hydroxide (AL) pretreatment in a range of pretreatment temperatures and durations were investigated to improve the enzymatic digestibility of SBH and SBS. Satisfactory enzymatic digestibility was observed in the hydrolysis of both pretreated soybean fractions, but SBH showed higher digestibility than that of SBS, no matter which pretreatment technology was applied. Furthermore, DA pretreatment was more effective than AL pretreatment in conversion of soybean fractions into fermentable sugars, if both pretreatment and enzymatic hydrolysis stages were considered. The highest total sugar yield of SBH and SBS using DA pretreatment and subsequently hydrolyzed with 30 FPU/g-DM cellulase were 86.9% and 70.3%, respectively.
1. Introduction Soybean is an important source of food in many countries, especially in many Asian countries (Cabrera et al., 2015). The oil and protein constituents of soybeans are regarded as valuable products in soybean processing, but little attention has been paid to the producing residues, such as soybean hulls and straws (Cassales et al., 2011). These residues are rich in cellulose and do not require an extra grinding process prior to pretreatment as some other lignocellulosic material. As a major food and energy crop in China, soybean has an annual production of 13 Tg. Soybean straws and hulls, collected after soybean harvest, are partially used for animal feed and the main part of these residues are burnt directly in the fields, causing serious environmental pollution. Therefore, exploiting the potential utilization of soybean carbohydrates can not only increase the added value of soybean industry, but also benefit to environmental protection (Corredor et al., 2008). Soybean hull and straw are basically lignocellulosic material composed of fermentable hextose and pentose sugars, polymerized as cellulose and hemicellulose, in addition to a small proportion of lignin that is composed of phenolic compounds. Therefore, the efficient hydrolysis of cellulose and hemicelluloses in soybean residues to liberate monomer sugars is of potential interest for conversion into biofuels and chemicals with economic interest. However, the complex network of lignocellulosic biomass presents a major resistance for degradation of
⁎
Corresponding author. E-mail address: [email protected] (Y. Zhang).
http://dx.doi.org/10.1016/j.indcrop.2017.08.051 Received 11 May 2017; Received in revised form 14 August 2017; Accepted 28 August 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
polysaccharide fractions into soluble monomeric sugars, which is commonly referred to as the recalcitrance barrier (Himmel et al., 2007; Behera et al., 2014). Consequently, the saccharification efficiency is extremely low unless a suitable pretreatment conducted before enzymatic hydrolysis to break down the lignin and hemicelluloses network and to disrupt the crystalline cellulose structure. Among existing pretreatment technologies, dilute acid pretreatment (DA) and alkaline pretreatment (AL) are believed as the most mature ones that are ready for commercialized application (Lee et al., 2015; Alvira et al., 2010), which also have shown high effectiveness on several agricultural residues (Dagninoa et al., 2013). In DA pretreatments, substantial amounts of sugars, mainly from hemicellulose and partially from cellulose, could be solubilized into the liquid phase of the hydrolysis slurry (Jung et al., 2013). Dilute sulfuric acid, which has been extensively investigated for pretreatment using experimental and theoretical approaches, can effectively degrade hemicellulose in the cell wall network by the catalytic effect of proton H+ (Chen et al., 2015). It was reported that over 90% of hemicellulose could be successfully removed during DA pretreatment under moderate severities, remaining highly digestible residue that mainly composed of cellulose with small part of lignin (Singh et al., 2015). However, due to the strong protonation effect, the monomer sugars obtained during acid pretreatment are ready to be further decomposed into their degradation forms, such as furfural, 5-hydroxymethyl furfural (5-HMF), levulinic acid, and even humins, leading to strong inhibition to enzymes and
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fermentation microorganisms. On the other hand, AL pretreatment is considered as the most effective one with less sugar degradation, lower energy consumption, more lignin removal, and less furan derivatives (Behera et al., 2014; Whitfield et al., 2012). Compared to DA pretreatment, AL methods exhibit higher capacity in breaking the linkages between lignin and carbohydrates, and disrupting lignin structures, with a minor cellulose and hemicelluloses alteration. Acetyl groups and various uronic acid substitutes, which have lower susceptibility to hydrolytic enzymes, are also eliminated by AL pretreatment (Mosier et al., 2005; Zheng et al., 2009). More importantly, with the salvation reactions, the AL pretreatment could swell the material, thus increase the internal surface area of the recovered solids and the accessibility of the enzymes (Jin et al., 2013). In this study, soybean residues including SBH and SBS, as renewable and low-cost lignocellulosic materials for the production of fermentable sugars were investigated. DA and AL pretreatments were performed separately on different soybean residue fractions. Then the digestibility of different pretreated soybean fractions was evaluated with various enzyme dosages, in addition to determination of the structural characteristics and composition changes of the pretreated solids. The impacts of the intrinsic structure differences between SBS and SBH, and different pretreatment methods on the digestibility of the pretreated solids were therefore compared and elucidated.
Hemicellulose removal (%) hemicellulose inpretreated samples (g) × SR% ⎞ = ⎜⎛1 − ⎟ × 100% hemicellulose inone gram native samples (g) ⎠ ⎝ 2.3. Enzymatic hydrolysis Saccharification of the untreated and pretreated samples were performed following the NREL laboratory analytical procedure (Selig et al., 2008). A solid loading of 2% (w/v) with 0.05 M acetate buffer (pH 4.8) were added in 50 mL Erlenmeyer flasks. 800 μg of 20 mg/mL tetracycline antibiotic in DI water was also supplemented before adding enzymes to prevent possible microorganism contamination during hydrolysis. Enzymes were added once the mixture reached 50 °Cand the slurry was stirred at 160 rpm in a thermostated shaker (Model # THZ072HT, Shanghai, China). Samples were taken after 1, 4, 24, 48, and 72 h of hydrolysis and immediately analyzed with a high performance liquid chromatography (HPLC) to determine the monomer sugar yields. All enzymatic hydrolysis samples were prepared in triplicates and run under parallel conditions. 2.4. Analytical method 2.4.1. Sugar analysis Samples were analyzed for carbohydrates using a Waters Alliance HPLC (Model 2695, Waters Corporation, Milford, MA), equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) run at 65 °C and a refractive index detector (Waters 2414). The sample injection volume was 20 μL and 0.005 M sulfuric acid at a flow rate of 0.6 mL/min was set for the mobile phase. The concentration of various monomeric sugars, furfural, HMF, and levulinic acid were quantified based on the calibration curves constructed by standards. The monomer sugar yields were calculated according to the following equations, and the total sugar yields were the sum of monomer sugars and cellobiose.
2. Materials and methods 2.1. Material SBH and SBS were separated after soybean crops collected from the farmland nearby Changzhou (Jiangsu Province, China). The materials were washed with deionized water and dried at 45 °C until constant weight, then milled into size smaller than 3 mm. The composition of the raw and pretreated substrates were determined based upon National Renewable Energy Laboratory Analytical Procedure (Sluiter et al., 2008). Accellerase 1500 (96 FPU/mL) was generously provided by Genencor (Wuxi, Jiangsu province, China). Novozyme 188 (066K0676603) was purchased from Sigma (St. Louis, MO, USA). Sodium hydroxide (NaOH), sulfuric acid (H2SO4) and other chemical reagents were purchased from Sinopharm Group Chemical Reagent Co. Ltd. (Shanghai, China).
Glucose yield(%) =
Xylose yield(%) =
xylose released 3 × 0.88 (g) × 100% intial xylan content in the substrate (g)
2.4.2. Sample characterization Crystallinity index (CrI) was analyzed by X-ray diffraction (XRD) using a D/max 2500 PC diffractometer with Cu Ka radiation (Rigaku Corporation, Tokyo, Japan) and operated at a voltage of 60 kV and a current of 300 mA. The 2θ range was detected from 5 to 40°in a step of 0.02. CrI was calculated according to the following equation:
2.2. Pretreatment The quantitative dried sample was mixed with 1 w/w% H2SO4 or NaOH solution in a 100 mL high-temperature and high-pressure stainless steel reactor (Zhenjiang Dantu Universal Electrical Equipment, China), equipped with a stirring apparatus. The solid to liquid ratio was set to 1: 20. Then the reactor was sealed and electrically heated to the desired temperature using a porcelain-heating jacket. After the reaction, the reactor was quenched in an iced water bath, then filtrated to separated the liquid from the solid. The collected solid was washed for several times until pH neutral. The pretreatment liquid and solid were collected for different analyses. The solid recovery, delignification, and hemicellulose removal of different pretreatments were calculated based on the following equations:
Solid recovery(%) =
glucose released × 0.9 (g) × 100% intial glucan content in the substrate (g)
CrI (%) =
I002 − Iam × 100% I002
Where I002 is the intensity of the crystalline portion (crystalline cellulose) determined at 2θ = 22.2°and Iam is the peak for the amorphous portion (i.e. amorphous cellulose, hemicellulose and lignin) at 2θ = 16.4°. The surface morphological features of different samples were imaged with a scanning electron microscope (SEM, Model # JSM-6360LA, JEOL, Japan) that was operated at 15 kV. Fourier transform infrared (FTIR) was to verify the change of the chemical structure of SBH and SBS before and after pretreatment. The spectra were analyzed using a FTIR spectrometer (Nicolet, USA). The sample spectra were obtained using 32 scans over the range of 500–4000 cm−1. Samples were ground and mixed with the spectroscopic grade KBr then pressed in a standard device to produce diameter pellets (Qing et al., 2016). The porosity of the samples was determined by N2 adsorption/desorption isotherms at 77 K using a Brenauer-Emmett-Teller (BET) surface area analyzer (ASAP 2010 M). Before the measurement, all samples were dried at
substrate recovered after pretreatment (g) × 100% substrate used for pretreatment (g)
lignin inpretreated samples (g) × SR% ⎞ Delignification(%) = ⎜⎛1 − ⎟ lignin in one gram native samples (g) ⎠ ⎝ × 100% 392
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105 °C for 4 h. The specific surface area (SSA), total pore volume (PV) and average pore size (PS) of porous materials were calculated based on the BET method (Qing et al., 2017).
– – 1.05 ± 0.18 0 1.10 ± 0.21 0
La
Q. Qing et al.
– – 0.30 ± 0.09 0 0.60 ± 0.12 0
3.1. Chemical composition of SBH and SBS before and after pretreatment The chemical compositions of untreated SBH and SBS were determined and the results were shown in Table 1. The compositions of soybean residue fractions, similar to other lignocellulosic materials, are comprised of a complex mixture of cellulose, hemicellulose, and lignin, in addition to a small amount of extractives and ash. Notably, the xylan contents of SBS and SBH were comparable, which were 16.7% and 20.0%, respectively. However, the glucan and lignin contents in SBS were obviously higher than in SBH, which might make SBS more difficulty to be decomposed. The total carbohydrate fractions were about 48.6% and 59.0% of the dry biomass basis for SBH and SBS, respectively, making these agricultural residues attractive feedstock for bioethanol production. In order to compare the impacts of different pretreatment methods on the composition and digestibility of soybean fractions, DA (H2SO4) and AL (NaOH) pretreatments as widely recognized technologies were performed under 120 °C for 1 h with 1% catalysts. The cellulose contents of the pretreated SBH and SBS increased noticeably compared to the untreated fractions due to the destruction of the intact structure and the solubilization of lignin and hemicelluloses. In addition, the AL pretreatment removed a larger portion of lignin, whereas the DA pretreatment mainly hydrolyzed hemicelluloses for both SBH and SBS. After AL pretreatment, the amount of lignin in SBH and SBS decreased 37.9 and 32.6%, respectively, indicating a strong delignification capacity of this method. On the other hand, DA pretreatment removed 73.2 and 62.4% of hemicelluloses from the pretreated solids for SBH and SBS, respectively. It is worth mentioning that the solid recovery ratio of SBS was obviously higher than that of SBH no matter for DA or AL pretreatments, indicating that the structure of SBH was more vulnerable to pretreatment than SBS, thus was severely damaged. In addition, the ingredients of the reaction liquid were also determined and shown in Table 1. As expected, the DA pretreatment liquid contained more xylose monomers and degradation products as a result of the stronger capacity in cleavage of hemicellulose linkages by H+ protons that were supplied by acids. On the contrary, smaller amounts of reducing sugars and almost no degradation products were detected in the AL pretreatment liquor since AL methods remove hemicelluloses mainly as oligosaccharides (Gáspár et al., 2007) that cannot be detected by the HPLC method used in this study. The total sugar release determined during 72 h enzymatic hydrolysis and the enzymatic hydrolysis courses were reflected in Fig. 1. Both DA and AL pretreatments significantly enhanced substrate digestibility which could be due the removal of hemicelluloses and lignin compounds and the generation of porous structure. Coincide with the composition results analyzed above, SBH was easier to be decomposed by enzymes than SBS, thus exhibiting a higher enzymatic digestibility than SBS for both DA and AL pretreatments. And for both SBH and SBS, the total sugar yields of AL pretreatment samples were nearly 18% higher than that of the DA pretreatment samples. The highest enzymatic hydrolysis sugar yields of AL-SBH and AL-SBS reached 76.8% and 50.5% at 72 h, respectively, which were 31.6% and 28.4% higher than the corresponding raw soybean fractions.
± ± ± ±
0.42 032 0.45 0.01
– – 1.08 0.21 0.50 0.21 0.2 0.2 0.5 0.5 0.3 0.3 ± ± ± ± ± ± 13.1 21.6 23.7 17.9 30.4 24.2 0.5 0.4 0.5 0.6 0.7 0.6 ± ± ± ± ± ± 20.0 16.7 13.2 22.9 11.7 16.6 – – 40.5 45.5 53.8 60.3
3.2. DA pretreatment
UN-SBH UN-SBS DA-SBH AL-SBH DA-SBS AL-SBS
± ± ± ±
1.5 1.7 1.0 2.3
28.6 42.3 53.5 52.7 56.4 55.1
± ± ± ± ± ±
0.7 0.9 1.1 0.9 0.8 1.0
Hemi SR, % Sample
Table 1 Compositional analysis of untreated and pretreated SBS and SBH.
Lignin
0.2 0.5 0.3 0.2 0.3 0.5
Ash
± ± ± ± ± ±
0.1 0.1 0.1 0.1 0.1 0.1
– – 73.2 47.9 62.4 40.0
– – 26.7 37.9 24.4 32.6
– – 1.48 1.13 0.35 0.11
± ± ± ±
0.03 0.56 0.09 0.02
– – 5.13 0.61 4.78 0.10
xyl glu Hemi Cell
Lignin
Removal, % Composition, %
Pretreatment liquid, mg/mL
ara
± ± ± ±
0.12 0.08 0.11 0.07
– – 0.08 ± 0.02 0 0.03 ± 0.01 0
FF
HMF
3. Results and discussion
The impacts of pretreatment temperature and time on sugar releasing during DA pretreatment and subsequent enzymatic hydrolysis were presented in Fig. 2. Prolongation of the reaction duration under lower temperature (120 °C) clearly enhanced the monomer sugar 393
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degraded into soluble monomers under 140 °C. When the reaction temperature was raised to 160 °C, more polymer carbohydrates were degraded as the solid recovery ratio further decreased compared to 140 °C. However, the sugar yields in the pretreatment step declined at 160 °C, as the monomer sugars released were further degraded to inhibitory compounds. In the meanwhile, the solid recovery decreased with the increase of the pretreatment severity, and this trend was more apparent for SBS than SBH. Enzymatic hydrolysis was conducted to evaluate the digestibility of different soybean residue solids that had been subjected to DA pretreatment under different conditions. The sugar yields of both SBH and SBS during enzymatic hydrolysis stage were comparable for all evaluated DA pretreatment conditions. However, if the monomer sugars released in the pretreatment stage was included, the total sugar yields of SBH varied between 61.3% (120 °C, 60 min) to 86.9% (140 °C, 40 min). This result indicates that DA pretreatment could effectively recover most of the carbohydrates in SBH as monomers under the optimized conditions, and only small quantity of inhibitory compounds were formed. Nevertheless, the total sugar yields of SBS were much lower than SBH, which were only between 41.7% (160 °C, 80 min) and 70.3% (140 °C, 80 min). It was also noticed that the solid recovery of SBS was much higher than that of SBH, indicating the SBS structure was more rigid and complicated than SBH. Moreover, raise of pretreatment temperature and prolongation of reaction duration to disrupt the structure of SBS might also lead to severer sugar degradation and less total sugar recovery.
Fig. 1. Enzymatic hydrolysis of raw soybean fractions and SBS/SBH pretreated by 1%. DA (H2SO4) or AL (NaOH) at 120 °C for 60 min.
3.3. AL pretreatment AL pretreatment, especially dilute sodium hydroxide, was considered as one of most suitable methods for pretreatment of agricultural residues (Galbe and Zacchi, 2007; Sarkar et al., 2012). The impacts of pretreatment temperature (100–140 °C) and time (60–140 min) on the total sugar releases during both AL pretreatment and enzymatic hydrolysis stages were therefore analyzed for SBS and SBH fractions as shown in Fig. 3. Compared to DA pretreatment, much smaller amounts of reducing sugars were detected in the AL pretreatment liquid, corresponding to the findings presented in Table 1. In the meanwhile, the variation of pretreatment conditions had little impacts on the monomer sugar released to the pretreatment liquid, while its influences on the digestibility of the solid residues were obvious. Different from DA pretreated sample, most of the monomeric carbohydrates were released during the enzymatic hydrolysis stage for AL pretreated soybean fractions. Under the investigated conditions, the highest total sugar yields of two stages were achieved under 100 °C, 120 min for SBH and 140 °C, 120 min for SBS, which were 75.6% and 56.7%, respectively. Similar to DA pretreated samples, SBH exhibited higher digestibility than SBS whatever the pretreatment conditions applied. These results reinforced the finding that SBS was much more difficult to deconstruct, thus a higher pretreatment temperature or severer condition might be needed to disrupt the recalcitrance barrier and make cellulose accessible to enzymes. 3.4. The influence of cellulase loading Different cellulase loadings of 15, 30 and 60 FPU/g DM were compared during enzymatic hydrolysis of untreated and pretreated substrates that were obtained by the optimized pretreatment conditions determined above. Both of the DA and AL pretreated soybean fractions exhibited much higher enzymatic hydrolysis yields than the raw materials, supporting the applied pretreatments successfully modified the structural properties of the substrates and increased the accessibility of cellulase to cellulose surface. An obvious increment of sugar yield was observed for all samples when the enzyme loading was raised from 15 to 30 FPU/g-DM. However, when the enzyme loading further increased to 60 FPU/g-DM, no significant increase in digestibility was detected as
Fig. 2. (a) The impacts of pretreatment temperature and duration on solid recovery and the total sugar yields of DA pretreated soybean fractions. (b) Concentrations of soluble sugars released in enzymatic hydrolysate of SBH pretreated at 120 °C for 80 min. (c) Concentrations of soluble sugars released in enzymatic hydrolysate of SBS pretreated by at 140 °C for 80 min.
released to the pretreatment liquid. But under the reaction temperature of 140 °C, this trend was obscure. About 45–47% of the total carbohydrate fraction in the SBH and 29–34% of that in the SBS could be 394
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Fig. 3. (a) The impacts of pretreatment temperature and duration on solid recovery and the total sugar yields of AL pretreated soybean fractions. (b) Concentrations of soluble sugars released in enzymatic hydrolysate of SBH pretreated at 140 °C for 80 min. (c) Concentrations of soluble sugars released in enzymatic hydrolysate of SBS pretreated at 140 °C for 120 min.
illustrated in Fig. 4a and b. Nevertheless, the initial reaction rates of the higher enzyme loading samples displayed evidently promotes, especially for the pretreated samples as shown in Fig. 4c. The maximum initial reaction rate achieved was 4.91 g/L per hour for AL-SBH with cellulase loading of 60 FPU/g DM, which might benefit from the high lignin and hemicelluloses removal of this sample. The enzymatic digestibility of AL-SBH was 87.6% with 60 FPU/g-DM cellulase loading, which was 17.1% higher than DA-SBH hydrolyzed with the same enzyme loading. Compared to SBH, the enzymatic hydrolysis yields of SBS were much lower, which were 63.1% and 50.7% for AL and DA pretreatment, respectively. These results demonstrated that AL pretreatment was more effective to increase enzymatic digestibility of the pretreated solids, which might due to the higher removal ratio of the lignin that sheathed the cellulose. On the other hand, DA pretreatment mainly removes hemicelluloses during the pretreatment stage, thus the enzymatic performance was inferior to AL pretreatment. However, if the monomer sugar released into pretreatment liquid was included, DA pretreatment showed a higher total sugar yield of these two stages as explained in previous section.
Fig. 4. Enzymatic hydrolysis of 2% soybean fractions with different cellulase loadings (a) SBH; (b) SBS; (c)The 72 h total sugar yields and initial reaction rate of the untreated, DA, and AL pretreated SBH and SBS.
3.5. Characterization of SBH and SBS before and after pretreatment Scanning electron micrographs depicting the morphological features of native soybean fractions, DA and AL pretreated soybean fractions, and residues collected after enzymatic hydrolysis were recorded. Untreated SBH and SBS showed smooth and well-ordered fibrous structure, indicating a rigid and highly packed surface structure that constructed by cellulose, hemicellulose, lignin, and other binding materials. It was noticed that DA pretreatment of SBH and SBS resulted in 395
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acetyl and uronic ester groups in hemicellulose or the phenyl ester linkage between hemicellulose and lignin (Kristensen et al., 2008; Phitsuwan et al., 2015). It was noticed that this band attenuated after DA pretreatment and almost disappeared after AL pretreatment, indicating partial removal of hemicelluloses after DA pretreatment and fully cleavage of the ester band from the hemicelluloses and lignin after AL pretreatment. Lignin-related bands in the spectra were assigned to peaks near 1510 cm−1, 1627 cm −1, 1745 cm−1, and 1745 cm−1. The bands at approximately 1510 cm−1 and 1745 cm−1 reflected the C]C stretching of the lignin aromatic ring. The bands at 1627 cm−1 was attributable to lignin aromatic skeletal vibration and C]O stretching (Kumar et al., 2009). The obscurely decrease of the intensity of these three bands for DA-SBS and DA-SBH suggested a limited lignin disruption by the DA pretreatment. However, the reduction of the intensity of these bands became apparent for AL-SBS and AL-SBH, which agreed with the chemical composition analysis in Table 1. All these information manifested that DA pretreatment can remove most of the hemicellulose in biomass by cleaving acetyl linkages and AL pretreatment is more conducive to remove lignin compounds.
Table 2 Characterization of SBH and SBS before and after pretreatment. Sample
CrI, %
Surface Area, m2/g
Pore Volume, cm3/g
Pore Size, nm
DA-SBH AL-SBH DA-SBS AL-SBS UN-SBH UN-SBS
41.4 44.0 43.1 41.7 32.9 35.1
4.4807 1.0925 3.4085 3.2671 0.5752 0.8088
0.003587 0.000895 0.002134 0.001447 0.000378 0.000582
3.20187 3.27858 2.50422 1.77138 2.62658 2.87703
damage to the cellular structure, as evident by the separation of fibers from the pith and the loosening of the fibrous network. Similar structural changes were found in the AL pretreated samples, which could be resulted from the removal of hemicelluloses and lignin from soybean fractions by AL pretreatment. In addition, the surface of the pretreated SBH showed larger pore size than the SBS, which agreed with the BET analysis shown in Table 2. Compared to the pretreated samples, the surface of hydrolyzed residues was more rough and cracked, appearing more porous and disruptions. It was observed that a considerable amount of cell wall material (cellulose and hemicellulose) was dissolved and swelled after enzymatic hydrolysis. The crystallinity index and the crystalline structure of cellulose have been considered as major factors that dominate the efficiency of lignocelluloses enzymatic hydrolysis. The calculated crystallinity index of the raw materials and the pretreated samples were shown in Table 2. The results revealed that the CrI of the untreated SBH increased from 32.9% to 41.4% for DA pretreated sample and 44.0% for AL pretreated sample. Correspondingly, the CrI of SBS increased from 35.1% to 43.1% and 41.7%, respectively, for DA and AL pretreated samples. All these changes could be attributed to the removal of the amorphous components from the soybean fractions, such as lignin, hemicellulose, and extractives. The CrI of native SBS was higher than SBH, which could be due to the higher cellulose content in SBS. The SSA, PV, and PS values were also presented in Table 2 to illustrate the change of exterior and interior surface structure of SBH and SBS that were subjected to different pretreatment technologies. The SSA and PV for native samples were 0.5752 and 0.8088 m2/g, and 0.000378 and 0.000582 cm3/g, respectively, for SBH and SBS. The SSA and PV of SBS increased after pretreatment, but the pore size shrunk due to the formation of new minor pores after hemicelluloses and lignin removal. For SBH, all the three parameters notably increased after DA pretreatment, but the increments were limited after AL pretreatment, indicating the pores might disappear by collapsing when delignification. This phenomenon, which was usually noticed when the lignin removal exceeded 50%, was also reported in several previous research (Zhu et al., 2008; Lee et al., 2015). These research also mentioned that cellulase can efficiently reach cellulose structure after removing of the lignin barrier in this case, thus resulted in enhanced enzymatic digestibility. FTIR was carried out to evaluate the change of chemical function groups before and after pretreatment. At approximately 3400 cm−1 is associated with the −OH stretching vibration of cellulose. The peak at 2918 cm−1 is due to the asymmetrical stretching of CH2 and CH bonds, and the bond at 900 cm−1 is attributed to β-(1–4)-glycosidic bond (CeOeC). As the absorption peaks in the native SBS and SBH were similar to those of the pretreated substrates, it agreed with the composition analysis that most of the crystalline structure cellulose was preserved after DA or AL pretreatments. In addition, these peaks of SBH were obviously smaller than those of SBS, indicating more crystalline cellulose in SBS than in SBH which tallied with the CrI values shown in Table 2. The comparably more crystalline structure cellulose could be a reason that explains the higher digestibility of all SBH samples than SBS samples. The hemicelluloses and lignin related band at approximately 1245 cm−1 was due to the stretching of CeO bonds, which was observed to be notably decreased after DA and AL pretreatment for both SBH and SBS. Absorption bond at 1733 cm−1 was attributed to the
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