Bisulfite pretreatment changes the structure and properties of oil palm empty fruit bunch to improve enzymatic hydrolysis and bioethanol production.

Biotechnology Journal

Biotechnol. J. 2015, 10, 915–925

DOI 10.1002/biot.201400733

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Research Article

Bisulfite pretreatment changes the structure and properties of oil palm empty fruit bunch to improve enzymatic hydrolysis and bioethanol production Liping Tan1, Wan Sun1, Xuezhi Li1,*, Jian Zhao1,*, Yinbo Qu1, Yuen May Choo2 and Soh Kheang Loh2 1 State

Key Laboratory of Microbial Technology, Shandong University, Ji nan City, China Palm Oil Board, Kuala Lumpur, Malaysia

2 Malaysian

Bisulfite pretreatment is a proven effective method for improving the enzymatic hydrolysis of empty fruit bunch (EFB) from oil palm for bioethanol production. In this study, we set out to determine the changes that occur in the structure and properties of EFB materials and fractions of hemicellulose and lignin during the bisulfite pretreatment process. The results showed that the crystallinity of cellulose in EFB increased after bisulfite pretreatment, whereas the EFB surface was damaged to various degrees. The orderly structure of EFB, which was maintained by hydrogen bonds, was destroyed by bisulfite pretreatment. Bisulfite pretreatment also hydrolyzed the glycosidic bonds of the xylan backbone of hemicellulose, thereby decreasing the molecular weight and shortening the xylan chains. The lignin fractions obtained from EFB and pretreated EFB were typically G-S lignin and with low content of H units. Meanwhile, de-etherification occurred at the β-O-4 linkage, which was accompanied by polymerization and demethoxylation as a result of bisulfite pretreatment. The adsorption ability of cellulase differed for the various lignin fractions, and the water-soluble lignin fractions had higher adsorption capacity on cellulase than the milled wood lignin. In general, the changes in the structure and properties of EFB provided insight into the benefits of bisulfite pretreatment.

Received Revised Accepted Accepted article online

26 DEC 2014 02 FEB 2015 07 APR 2015 20 APR 2015

Supporting information available online

Keywords: Bisulfite pretreatment · Empty fruit bunch · Hemicellulose · Lignin

1 Introduction Lignocellulose renewable biomass can be directly or indirectly used for the production of biofuels and commodity chemicals [1]. Oil palm empty fruit bunch (EFB) is the

Correspondence: Prof. Jian Zhao and Senior Engineer: Xuezhi Li, State Key Laboratory of Microbial Technology, Shandong University, 250100, Ji-nan City, China E-mail: [email protected], [email protected] Abbreviations: EFB, empty fruit bunch; FPA, filter paper activity; FPU, filter paper units; FT-IR, Fourier-transform infrared spectrometry; GPC, gel-permeation chromatography; HBI, hydrogen bonding intensity; HSQC, heteronuclear single-quantum correlation; MWL, milled wood lignin; NMR, nuclear magnetic resonance; pNPC, p-nitrophenyl-β-D-cellobioside; SEM, scanning electron microscopy; SPORL, sulfite pretreatment to overcome recalcitrance of lignocellulose

© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

main waste product of the palm oil processing industry. The main carbohydrates of EFB, such as glucan and xylan, are present in high amounts [2]. EFB has recently been considered as a potential low-cost material and an alternative renewable bioresource for the production of bioethanol [3, 4]. Our previous work proved that the bisulfite pretreatment of EFB is an effective and practical method to improve enzymatic hydrolysis for ethanol production; the optimum pretreatment conditions of bisulfite pretreatment were likewise identified [5, 6]. After bisulfite pretreatment, higher conversion of cellulose and higher ethanol concentrations for pretreated EFB can be obtained. To date, only a few studies have been devoted to the possible mechanisms of EFB pretreatment by bisulfite pretreatment. Zhu et al. [7, 8] and Wang et al. [9] demonstrated that using SPORL is a robust and efficient method for ethanol production and enzymatic cellulose

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conversion. They reported the effects of SPORL pretreatment on substrate morphology, cell wall structures, and specific surface of softwood [10–12]. SPORL pretreatment enabled fiber separation. Consequently, the pore structure and delamination of fibers in the cell wall were very easily observed after SPORL pretreatment. Therefore, the external and internal surface areas increased after SPORL pretreatment. They also found that the removal of almost all hemicellulose in dilute acid pretreatment material did not result in a high cellulose conversion, thereby suggesting that a small amount of lignin removal is necessary to achieve good cellulose enzyme activity. However, the significant structural changes of hemicellulose and lignin in bisulfite-pretreated EFB have not been previously studied. In this study, we set out to determine the changes that occur in the structure and properties of EFB materials and fractions of hemicellulose and lignin during the bisulfite pretreatment process. The surface properties of unpretreated and bisulfite-pretreated EFB were analyzed by SEM, X-ray diffraction, fiber-specific surface area, and FT-IR. Hemicellulose and lignin fractions were isolated from the bisulfite-pretreated EFB, and their structural characteristics were investigated by spectroscopic techniques, such as FT-IR, GPC, 1H-NMR, 13C-NMR, and 1H-13C-2D HSQC. Lignin physically hinders the accessibility of cellulases to cellulose [13], and the soluble lignin fractions may cause cellulase inhibition [14]. Previous work researched the effect of lignosulfonate on pure cellulose saccharification [15], indicating a varying effect (enhancing or inhibitive) on cellulose hydrolysis by different lignosulfonates. Therefore, MWL was separated from unpretreated and bisulfite-pretreated EFB, respectively, and the differences in the adsorption of cellulase on the MWLs were investigated.

2.2 Pretreatment of EFB Bisulfite pretreatment was conducted as previously described [5]. In this process, the EFB materials reacted with chemicals at 180°C, 30 min. The dosage of chemicals was: 8% NaHSO3, 1% H2SO4. The ratio of pretreatment liquor to solid was 4:1. After pretreatment, the analyzed EFB contained 60.8% glucan, 2.5% xylan, and 20.4% lignin.

2.3 Isolation of hemicelluloses Samples were extracted with water and ethanol before they were subjected to ball-milling for 72 h by using a vibratory milling apparatus (Fritsch, Germany). The sample obtained from ball-milling (10 g) was resuspended in water at 55°C for 6 h, with a solid-to-liquid ratio (g/mL) of 1:20. Centrifugation of the resulting mixture was performed at 8000 rpm for 10 min. Concentration of the residue to 50 mL was performed at reduced pressure. Subsequently, the concentrated residue was poured into three volumes of 95% ethanol. The precipitated hemicellulose was subjected to centrifugation, washing with 70% ethanol, and freeze drying. These hemicellulose fractions were designated as H1 (extracted from EFB) and H3 (extracted from bisulfite-pretreated EFB). The remaining residue was further extracted at 55°C for 6 h in 10% KOH with a solid-to-liquid ratio (g/mL) of 1:20. Centrifugation of the resulting mixture was performed at 8000 rpm for 10 min, and the pH value of the solution was adjusted to about pH 5.5 by using HCl. The remaining procedures in the extraction process was performed as decribed above. These hemicellulose fractions were designated as H2 (from EFB) and H4 (from bisulfitepretreated EFB). Finally, all the hemicellulose fractions were analysed by FT-IR and GPC.

2 Materials and methods 2.4 Isolation of lignin 2.1 Materials The oil palm EFB used in this study originated from Malaysia. EFB milling was performed, and particle sizes ranging from 0.30 to 0.45 mm (diameter) were obtained. The EFB was then kept in bags, sealed, and stored at room temperature. The analyzed unpretreated EFB contained 36.8, 19.3, and 17.9% of glucan, xylan, and lignin, respectively. Cellulase (Sino Enzymes R) that exhibits a filter paper activity of 160 IU/g, as well as a β-glucosidase activity of 40 IU/g was obtained from Baiyin Sainuo Technology, Ltd. (Gansu Province, PR China). Analytical grade sodium bisulfite, sulfuric acid, sodium hydroxide, potassium hydroxide, hydrochloric acid, dioxane, acetic acid, 1,2-dichloroethane, ethanol, and diethyl ether were used.

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Extraction of the MWL of EFB was performed by using the method of Björkman [16]. The ball-milled EFB (20 g) was extracted thrice with dioxane/water (200 mL; 96:4, v/v) for 24 h. The precipitated lignin was then filtered, dissolved in 20 mL 1,2-dichloroethane:ethanol (2:1, v/v) and precipitated into 200 mL diethyl ether. The obtained lignin sample was designated as MWL. The lignin fractions were named L1 (from unpretreated EFB) and L2 (from bisulfitepretreated EFB). Each solution was obtained by centrifugation and concentration (100 mL). The crude MWL was dried and subsequently dissolved in 15 mL acetic acid:water (9:1, v/v) and precipitated into water. The solution was dried and designated as water-soluble MWL. These lignin fractions were designated as L3 (from unpretreated EFB) and L4 (from bisulfite-pretreated EFB). The yield of MWL (L1 and L2) and water-soluble MWL (L3 and L4) was 0.164, 0.271, 3.30, and 6.15%, respectively. Final-





ly, all MWL samples were analyzed by FT-IR, GPC, and NMR.

2.5 Adsorption and inhibition of MWL and water-soluble MWL by the enzyme Cellulase (20 FPU) was supplied to 50 mM sodium citrate buffer (pH 4.8) with 0.02% (w/v) sodium azide for the inhibition of microbial contamination. The enzyme was then mixed to the MWL fractions. The amount used for the different lignin fractions (from L1 to L4) was 1.64, 2.71, 33, and 61.5 mg, respectively. The experiments were performed in 100 mL Erlenmeyer flasks with a total reaction volume of 50 mL (the buffer-enzyme mixture). The flasks were then reacted in a rotary shaker at 150 rpm for 72 h at 45°C. The resulting mixture was centrifuged after the reaction. The supernatant was obtained, and the free enzyme was used to measure the cellulase activity, protein concentration, and SDS-PAGE.

2.6 Analytical methods SEM observations were performed on dry samples using a JEOL JSM-6700F scanning electron microscope (JEOL, Japan). The crystallinity of cellulose used the method of X-ray diffraction. X-ray diffraction data were obtained with a Bruker D8 Advance Diffractometer (Bruker, German). The formula of crystallinity was reported by Kim et al. [17]. A V-Sorb X800 Series analyzer (App, China) was used to determine the surface area. The FT-IR spectra of the EFB samples, as well as the hemicellulose and lignin fractions, were recorded on an FT-IR spectrophotometer (Nicolet, USA) with the use of KBr discs. The spectra were recorded in the absorption mode from 4000 cm–1 to 500 cm–1. The hemicellulose (H1 to H4) molecular weights were obtained by using the GPC system, which comprised a Waters 1525 binary HPLC pump, a Waters 717 plus Auto-sampler, a Waters 2414 refractive index detector, and a Breeze (V3.3) GPC work station (Waters, USA). Water was used to dissolve the samples, and the solutions were subsequently injected in series into a TSK-GELG-5000PW xL column (7.8 mm × 300 mm) and then a TSK-GELG-3000PW xL column (7.8 mm × 300 mm; TOSOH, Japan). The molecular weights of the MWL were determined by a GPC system, which comprised a Waters Model 510 HPLC pump, a Waters Model U6K Auto-sampler, a Waters 410 refractive index detector, and a Waters 848 UV detector (Waters, USA). Tetrahydrofuran was used to dissolve the samples, which were then injected into the Waters HR 5E and Waters HR 1 in a series. At room temperature, solution 1D- and 2D-NMR spectra were recorded on a Bruker AVANCE 400 MHz spectrometer. The lignin samples with concentrations of approximately 20 mg in 1.0 mL dimethyl sulfoxide DMSO-d6 for 1H NMR and 180 mg in 1.0 mL DMSO-d6 for 13C-NMR was placed in glass tubes and run at room tem-


perature. The respective spectral widths were 5000 and 20 000 Hz for the 1H and 13C dimensions (for the HSQC experiments). A total of 1024 complex points were collected for the 1H dimension, along with a 1.5 s recycle delay. The total number of transients was 64. A total of 256 time increments were always recorded in the 13C dimension. The central DMSO peak (dC = 40 ppm; dH = 2.5 ppm) was used as chemical shift reference. Filter paper activity (FPA) of the commercial cellulase concentrate was measured according to Ghose [18] and expressed in filter paper units (FPU). Cellobiohydrolase (CBH) activity for 30 min was measured with 1% w/v p-nitrophenyl-β-D-cellobioside (pNPC) [19]. We estimated the amount of p-nitrophenol released by the enzyme reaction. The protein content was quantified by using Bradford assay [20]. The standard used was bovine serum albumin. All the measurements were performed in triplicate. SDS-PAGE was conducted with a DYY-II electrophoresis apparatus (Beijing, China). The concentration of the separating gel was 10%, whereas that of the stacking gel was 5%. Coomassie brilliant blue was subsequently used for staining. The detection and the quantitative evaluation of the bands were conducted with the Image J software (1.42 q).

3 Results and discussion 3.1 Bisulfite pretreatment changes the structure and properties of EFBs 3.1.1 FT-IR analysis of unpretreated and bisulfite-pretreated EFBs FT-IR was used to analyze the unpretreated and bisulfitepretreated EFBs in the region of 500 to 4000 cm–1. The observed spectra are exhibited in Supporting information, Fig. S1, and the functional groups of the FT-IR signals are listed in Supporting information, Table S1. The absorption of the unpretreated and bisulfite pretreated EFB were structurally similar. The absorption at about 3443 to 3447 cm–1 was due to the O-H stretching vibration in the OH groups. The C-H stretching vibration gave a signal at 2901 to 2919 cm–1. The absorption bands in the 1000 to 1650 cm–1 region may provide information about lignin and hemicellulose. The absorptions at 1646, 1511, 1424, and 1270 cm–1 were associated with lignin. The results showed that lignin underwent minimal change after bisulfite pretreatment, which was in agreement with the results for the various chemical components. The lignin contents of unpretreated and bisulfite-pretreated EFBs were 17.9 and 20.4%, respectively. As seen in Supporting information, Fig. S1, absorption bands characteristically appeared at 1163 cm–1, which was assigned to the contributions of the glycosidic linkage (C–O–C) in celluloses and hemicelluloses. These results indicate that some hemicelluloses were degraded after bisulfite pretreatment.

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Table 1. Yield, 72 h cellulose conversion, glucose yield, crystallinity of cellulose, HBI, and specific surface area of untreated and bisulfite pretreated EFB

Sample

Yield (%)a)

Untreated-EFB Bisulfite-pretreated EFB

100 66.44

72 h Cellulose conversion (%) 21.09 82.32

Glucose yieldb) 0.086 0.314

Crystallinity (%)

HBI c)

62.3 74.4

4.75 3.24

Specific surface area (m2/g) 0.90 1.18

a) Yield means the solid content after bisulfite pretreatment process. b) Glucose yield at enzymatic hydrolysis after 72 h based on the oven-dried weight of EFB c) HBI means hydrogen bonding intensity.

The optimum conditions for bisulfite pretreatment were selected. The resulting solid yield, cellulose conversion at 72 h, glucose yield, crystal index changes of cellulose, hydrogen bonding intensity (HBI), and the specific surface area of bisulfite-pretreated EFB are summarized in Table 1. The glucose yield after the enzyme hydrolysis of bisulfite-pretreated EFB was 0.314 g/g EFB after 72 h, whereas that of untreated EFB was only 0.086 g/g EFB. The calculated HBI was the intensity ratio of 4000–2995/1337 cm–1 [21]. The HBI of bisulfite-pretreated EFB was reduced from 4.75 to 3.24. A possible reason for this phenomenon was the disruption of the orderly structure maintained by hydrogen bonds, thereby promoting easy EFB degradation by cellulase.

3.1.2 Bisulfite pretreatment changes the cellulose crystallinity of EFBs The crystallinity of cellulose in the EFB samples changed from 62.3 to 74.4% after bisulfite pretreatment (Table 1). Bisulfite pretreatment likely destroyed the low crystallinity of hemicellulose and some of the cellulose crystallinity in the amorphous region, thereby leading to the relative increase in the cellulose crystallinity of the bisulfite-pretreated samples. Yu et al. [22] studied the crystallinity of cellulosic feedstock treated with an acid solution. This group also concluded that the crystallinity index of residual microcrystalline cellulose was widely enhanced by the removal of amorphous hemicellulose, whereas the crystal structure remained unchanged. In the hot acid solution reaction, amorphous hemicellulose was hydrolyzed, and only the larger perfect cellulose molecules remained, thereby increasing the crystal size.

3.1.3 Bisulfite pretreatment changes the specific surface area of EFBs For an enzymatic reaction to occur, the enzyme and its substrate need to have direct physical contact. An enzyme-substrate complex is created, and such a complex subsequently breaks down into the reaction products. Therefore, the reaction rate is expected to be a function of the cellulose surface area that is accessible to the enzyme. The fiber surface area can be divided into the external and internal surface areas.

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3.1.3.1 External specific surface of EFBs SEM observation has been widely used for characterizing the external surfaces via imaging. The external surface of the unpretreated EFB was tight and smooth (Fig. 1Ai and ii). After pretreatment with bisulfite, the EFB external surface showed damage and became rough (Fig. 1Aiii and iv). Bisulfite pretreatment separated EFB materials into individual fibers by acidic cutting, fiber separation, and fibrillation, as well as some level of hemicellulose and lignin removal, thereby producing a loosened fiber cell wall with significant delamination [23]. Consequently, the external surface area of the EFB materials increased, which consequently increased cellulose accessibility to enzymes and significantly improved the efficiency of enzymatic hydrolysis.

3.1.3.2 Internal specific surface of EFBs In this study, we used the BET method, which measures the adsorption of nitrogen by the pore surfaces. The specific surface area of the EFB and bisulfite-pretreated EFB are summarized in Table 1. The volume of pores with diameters of 50 nm or higher increased after bisulfite pretreatment (Fig. 1B), which implied the increased internal specific area of EFB. The increased pore volume with bigger pore sizes was also beneficial because it improved the penetration of enzymes into the substrate. Bisulfite pretreatment could clearly and effectively increase the specific surface area of the EFB (from 0.99 to 1.18; Table 1). Zhu et al. [24] and Wang et al. [25] demonstrated that milling and pretreatment of lignocelluloses could increase the specific surface area of the raw material. In the meantime, the enzymatic hydrolysis efficiency can be advanced by the increasing specific surface area. Previous research also showed that hemicellulose removal led to a substantial increase of the pore volume of acid-treated softwoods [26]. The lignin removal increases the population of pores and significantly increases the rate of hydrolysis [27]. Therefore, the increased specific surface area (both external and internal) and the larger pore size are important factors that improve the enzymatic hydrolysis of EFB after bisulfite pretreatment.



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Figure 1. (A) SEM of unpretreated (i, ii) and bisulfite-pretreated (iii, iv) EFBs. SEM observations of unpretreated and bisulfite pretreated EFBs were performed on dry samples using a scanning electron microscope. i, iii: 25×; ii, iv: 100×. (B) Pore size and pore volume distribution curves of unpretreated and bisulfitepretreated EFBs. We used the BET method which measures the adsorption of nitrogen by the pore surfaces. Experiments were performed in duplicate.

3.2 Bisulfite pretreatment changes the structure and properties of hemicellulose and lignin fractions isolated from EFBs 3.2.1 Bisulfite pretreatment changes molecular weight of hemicellulose and lignin fractions in EFBs The number-average (Mn) molecular and weight-average (Mw) molecular weights, and the polydispersity (Mw/Mn) of different hemicellulosic preparations were obtained using GPC and are listed in Table 2. Generally, high molecular weight hemicelluloses could be extracted by alkaline solutions. However, the Mw of alkali-soluble hemicellulose fractions (samples H2 and H4) was lower than those of water-soluble hemicellulose (samples H1 and


H3), thereby indicating that hemicelluloses were probably severely degraded during the extraction with strong alkali. Bisulfite pretreatment caused the molecular weights of all the hemicellulose fractions of pretreated samples to decrease to levels lower than that of the raw material. Therefore, heavy degradation of hemicellulose polymers was observed under the sodium-based liquid. The reduction of hemicellulose chain length partly resulted from the action of heat and pressure produced during bisulfite pretreatment. This effect was probably due to the cleavage of glycosidic esther linkages between sugar units, which degraded hemicelluloses substantially. In addition, the relatively low polydispersity (1.87 to 1.89) implied that all

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Table 2. Molecular weight and polydispersity of the isolated hemicellulose and lignin fractions

Hemicellulose fraction

Mn Mw Mw/Mn

Lignin fraction

H1

H2

H3

H4

L1

L2

5645 10663 1.89

950 2840 2.99

3816 7124 1.87

920 1310 1.44

4400 3569 1.23

5200 4334 1.20

of the water-soluble hemicellulose fractions had narrow distributions in terms of their molecular sizes. However, the polydispersity of alkaline-soluble hemicellulose fractions decreased (2.99 to 1.44) after bisulfite pretreatment. Therefore, the molecular sizes of hemicellulose fractions was decreased in bisulfite pretreated EFB because of the degradation of high molecular weight hemicellulose and the dissolution of low molecular weight hemicellulose. The Mw of MWL slightly increased after bisulfite pretreatment (Table 2), and such increase could be due to the slight condensation of lignin during bisulfite pretreatment. Another reasonable explanation for this result might be the difference in the carbohydrate content. The carbohydrate chains connected to lignin can reportedly increase the hydrodynamic volume of lignin, thereby increasing the molar mass of lignin, as measured by GPC [28]. In addition, the dissolution of low molecular weight lignin components might lead to the increase of molecular weight. Compared with L1, the extracted pre-treated lignin (L2) showed a slight decrease in Mw/Mn value. Therefore, some low molecular weight lignin components may have dissolved during bisulfite pretreatment.

3.2.2 FT-IR analysis of hemicellulose fractions from untreated and bisulfite-pretreated EFBs The effect of bisulfite pretreatment on the structures of the hemicellulose fractions were demonstrated by FT-IR analysis in the region of 500 to 4000 cm–1. The spectra of the hemicelluloses fractions are shown in Supporting information, Fig. S2. The spectral profile and relative intensity of most bands appeared to be similar, thereby indicating the similar structure of the respective hemicellulosic fractions (H1 compared with H3 and H2 compared with H4). The absorption at 3416 cm–1 was due to the stretching of the -OH groups. The C-H stretching vibration obtained a signal at 2916 and 2973 cm–1. The band at 1606 cm–1 was caused by the absorbed water in the hemicellulose fractions, as reported in literature [29]. The prominent band at 1047 cm–1 can be assigned to C-O or C-C stretching (or even to C-OH bending) in xylan. Thus, the dominance of xylan in the hemicelluloses is indicated. The absorption bands in the 800 to 1200 cm–1 region may provide information regarding certain types of polysaccharides. The sharp band at 896 cm–1 corresponded to the β-glycosidic linkage between the sugar units. These results implied that the hemicellulose fractions consisted

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of the main chain of xylan, which contained hydroxyl and methyl groups in untreated and bisulfite-pretreated EFBs. The absorptions at 1462, 1419, 1383, 1319, and 1249 cm–1 are associated with alkyl in hemicelluloses. A comparison of H1 with H3 and H2 with H4 revealed that the intensity of the functional groups increased by varying degrees, thereby verifying that the alkyl groups were exposed by acid degradation after bisulfite pretreatment. The presence of a shoulder peak at 1735 cm–1 in the spectra of H1 and H3 implied that the hemicellulose fraction extracted with water had small quantities of the ester bonds of carboxylic groups from alduronic acids. The intensity of 1735 cm–1 in H3 increased because of the formation of ester bonds during pretreatment. The weak absorbance at 1506 cm–1 was only present in H2, and this band originated from the aromatic skeletal vibrations in associated lignin. Thus, the hemicelluloses extracted from unpretreated EFB were slightly contaminated with minimal amounts of bound lignin, which cleaved the bonds between lignin and hemicellulose during pretreatment [30].

3.2.3 FT-IR analysis of lignin fractions from untreated and bisulfite pretreated EFBs The FT-IR spectra of the L1 and L2 lignin fractions are shown in Supporting information, Fig. S3. The main assignments of the corresponding FT-IR bands are summarized in Supporting information, Table S2. The absorption around 3475 cm–1 corresponded to the O-H stretching vibration in aromatic and aliphatic -OH groups. The signals at 2939 and 2840 cm–1 are assigned to the C-H asymmetric and symmetrical vibrations in methyl and methylene groups, respectively. The intensity of these two bands increased in the spectra of L2 to L1, which likely indicated that methyl and methylene groups were formed. The absorptions at approximately 1713 cm–1 in the two spectra could be assigned to the formate ester. This phenomenon was probably caused by the esterification of the aromatic and alcohol groups of the propane chain (Cα and Cγ) during delignification. The bands at 1593 and 1507 cm–1 corresponded to the aromatic ring vibrations of the phenyl-propane skeleton. The vibrations caused by asymmetric C–H deformations (in CH3 and CH2) at 1462 cm–1 could be observed in the two spectra. These results agree with the results of the chemical composition analysis of unpretreated and bisulfite-pretreated EFBs, where in the relative content of lignin increased from 17.9 to 20.4%. Absorptions of syringyl and condensed guaiacyl were observed at 1329 cm–1, whereas C=O stretching vibrations in the guaiacyl units appeared at 1271 cm–1. The band at 1228 cm–1 could be attributed to the C–C, C–O, and C=O stretching. The intensity of the band at 1124 cm–1 was notably assigned to GS lignin. The band at 1033 cm–1 corresponded to the aromatic C–H in-plain deformation. The intensities of all these bands decreased by varying degrees, probably because of the





Figure 2. (A) 1H-NMR of lignin fractions (L1 and L2). 1H-NMR is used to analyze the hydrogen proton of chemicals. (B) 13C-NMR of lignin fractions (L1 and L2). 13C NMR spectroscopy is a method that has sufficient power to investigate lignin’s structural features. (C) 2D HSQC spectra of lignin fractions (L1) (D) HSQC spectra of lignin fractions (L2).1H and 13C NMR spectra, the signals are overlapped due to the very complex and heterogeneous structure of lignin. Lignin fraction L1 meant milled wood lignin that was isolated from untreated EFB. Lignin fraction L2 meant milled wood lignin that was isolated from bisulfite pretreated EFB.

degradation and dissolution of syringyl, condensed guaiacyl, and guaiacyl units. Based on the lignin classification system of Faix [31], the intensity ratio of 1507/1462 cm–1 (which increased in L2, but not in L1) indicated the decreased number of S units in the L2 fraction. Furthermore, the 1462/1593 cm–1 ratio was directly related to the amount of methoxyl groups; this value was smaller in L2 than in L1. Therefore, methoxyl groups could be partial removed by bisulfite pretreatment.

3.2.4 NMR spectra of lignin fractions from untreated and bisulfite-pretreated EFBs 3.2.4.1

1H

NMR spectra of lignin fractions from untreated and bisulfite-pretreated EFBs

1H

NMR spectra were used to investigate the structural characteristics of MWL (Fig. 2A). Analysis of the 1H NMR signal intensity provided a method that can indirectly monitor the level of substitution of the aromatic ring of lignin. The shifting of signals between 3.1 and 3.8 ppm could be attributed to the protons in methoxyl groups, whereas those near 3.4 ppm were assigned to aliphatic


methoxyl groups. Meanwhile, signals between 3.6 and 3.8 ppm were assigned to aromatic methoxyl groups. According to the FT-IR results, the number of methoxyl groups decreased. The aliphatic methoxyl groups rather than the aromatic methoxyl groups could be degraded by bisulfite pretreatment. Signal shifts at 6.5 to 7.3 ppm were related to the aromatic protons in guaiacyl units (G) and syringyl units (S). The important differences between the two spectra included the following: (i) signals of saturated aliphatic side chain protons between 0.7 and 2.3 ppm showed greater intensity in L2 than in L1, thereby suggesting that the side chains in C-units were saturated in L1; and (ii) signals between 4.3 and 5.1 ppm corresponding to the Hα, Hβ, and Hγ attached to carbons increased in intensity in L2. These results suggested that β-O-4 linkages were produced while extracting MWL.

3.2.4.2

13C

NMR spectra of lignin fractions from untreated and bisulfite-pretreated EFBs

13C

NMR spectroscopy is a method that has sufficient power to investigate lignin’s structural features; this method can provide a more comprehensive view of the

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entire lignin macromolecule. The 13C NMR spectra of the lignin fractions L1 and L2 are shown in (Fig. 2B). The 13C-NMR spectra allowed the qualitative detection of some symmetrical signals, which were considered to be caused by lignin molecules. The peaks between 172 and 162 ppm were attributed to the C=O in carboxyl groups, which may come from aliphatic carboxyl and aliphatic esters. The intensity of these signals increased in the spectra of L1 to L2. The same results were observed during the FT-IR analysis. The spectra of the lignin fractions exhibited wellresolved signals in the aromatic region (160 to 103 ppm) and aliphatic carbon region (100 to 60 ppm). In the aromatic region (160 to 103 ppm), the S units are detected via signals at approximately 153 ppm (typical of C-3/C-5 carbons in etherified groups), 148 ppm (C-3/C-5 carbons in non-etherified), 139 ppm (C-4 carbons in etherified), 135 and 133 ppm (C1 carbons in etherified), and 105 ppm (C-2/C-6). The G units produced signals at 149 ppm (C-3 in non-etherified), 148 ppm (C-4 in etherified), 145 ppm (C-4 in non-etherified), 119 ppm (C-6), 113 ppm (C-5), and 111 ppm (C-2). The H units appeared as weak signals at 122 ppm (C-1). The spectra showed that the intensity of C-3/C-5 carbons in the nonetherified groups (148 ppm), C-2/C-6 in the syringyl units (105 ppm), and C-4 in the etherified and in non-etherified groups (145 and 148 ppm), as well as the C-6 (119 ppm), C-5 (113 ppm), and C-2 (111 ppm) in the guaiacyl units, decreased after pretreatment. Therefore, these groups were likely present in lignin and were degraded by bisulfite pretreatment. In the aliphatic carbon region (100 to 60 ppm), the signals at 86, 85, 84, 73, 71, 63, and 60 ppm were assigned to the Cα, Cβ, and Cγ carbons of the propane side chains when β-O-4 linkages were present. Compared with L1, these signals in the L2 spectrum were weaker. Therefore, de-etherification needed to occur at the β-O-4 linkage because of the bisulfite process. The strong signal at 56.3 ppm was attributed to the-OCH3 groups in the syringyl and guaiacyl groups. In addition, the signals between 18 and 23 ppm represented the γ-methyl groups in the propyl side chains of the lignin preparations.

3.2.4.3 2D HSQC spectra of lignin fractions from untreated and bisulfite-pretreated EFBs The lignin fractions L1 and L2 were also subjected to 2D HSQC NMR analysis to study the changes in structure of the lignin fractions. 2D 1H-13C NMR has been widely used to provide important structural information of lignin in either 1H or 13C NMR spectra. The HSQC spectra of lignin samples (L1 and L2) are shown in Fig. 2C and 2D. HSQC cross-signals of lignin were assigned via comparison with those in previous reports [32], and the main cross-signal assignments are listed in Supporting information, Table S3. The HSQC spectra are generally divided into three regions, as follows: the aliphatic (δC/δH, approximately 10–50/0.5–2.5 ppm), lignin side chain (δC/δH,

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approximately 50–100/2.5–6.0 ppm), and aromatic (δC/δH, approximately 100–140/5.5–8.5 ppm) regions. Signals in the aliphatic region failed to reveal structural information and are usually due to impurities such as fatty acids. Useful information on inter-unit linkages can be obtained from the correlations in the lignin side chain region. Cross-signals of methoxyl groups (δC/δH, 56.3/3.77 ppm) and side chains in the β-O-4’ aryl ether linkages are the most prominent. Other intense correlations are correspondingly detected at 72.4/4.91 [(A, A′, A″)α], 84.1/4.34, and 85.7/4.70 ppm (Aβ), and 60.5/3.27–3.65 (Aγ), 63.8/4.34 [(A′, A’″)γ], 71.2–71.8/ 3.36–4.22 (Bγ), and 87.6/5.49 ppm (Ca). The major differences caused by bisulfite pretreatment were the weakened signal intensities of β-O-4 ether. The main cross-signals were due to the substituted phenyl rings of the various lignin units in the aromatic region of the HSQC spectrum. Signals from syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) units were all qualitatively detected. The S lignin units showed a prominent signal for the C2,6-H2,6 correlation at δC/δH 104.5/6.74 ppm, whereas signals corresponding to C2,6-H2,6 correlations in Cα-oxidized S units (S′) (δC/δH 106.9/7.24 ppm) were present in all of the HSQC spectra of these lignin fractions. The G lignin units showed different correlations at C2–H2 (δC/δH, 111.7/7.03 ppm), C5–H5 (δC/δH, 115.7/6.80 ppm), and C6–H6 (dC/dH, 119.6/6.82 ppm). The intensity of these signals decreased, which explained why the S and G units were partly degraded by bisulfite pretreatment. These results were in agreement with those mentioned above in the FT-IR analysis. Trace amounts of p-hydroxyphenyl (H) units were observed from C2,6–H2,6 correlations at δC/δH 127.8/7.19 ppm, thereby indicating that H units (at minor amounts) were present in the lignin fractions. In addition, the C2,6-H2,6 correlations of p-hydroxybenzoate substructures (PB) were observed as a strong signal at δC/δH 131.9/7.69 ppm. The various structural features of the lignin fractions L1 and L2 were investigated in a quantitative manner. The percentage of the lignin side chains involved in the primary substructures and the S/G ratios were calculated from the corresponding HSQC spectra. The main substructures present in the lignin fractions were the β-O-4′ linkages. However, the S/G ratios increased from 0.67 (L1) to 1.0 (L2), and L2 had fewer G units than L1.

3.3 Cellulase adsorption by MWL and water-soluble MWL The MWL (L1 and L2 fractions) and the water-soluble MWL (L3 and L4 fractions) with low molecular weight were isolated from untreated and bisulfite pretreated EFBs, and used to determine the potential effect of lignin chemical characteristics on the interaction between lignin and cellulases, and this interaction was not assessed in previous literature to the best of our knowledge. The adsorption



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Figure 3. (A) Decreased FPA (i), CBH (ii), and cellulase protein (iii) after 72 h adsorption. Decreased FPA, CBH, and cellulase protein were defined by calculating the changes before and after 72 h adsorption. Experiments were performed in triplicate. The data as means ± SD (n = 3). (B) SDS-PAGE of cellulase proteins in the supernatant after 72 h adsorption. C: control (cellulase alone); C + L1: cellulase and MWL of untreated EFB (L1); C + L2: cellulase and MWL of treated EFB (L2); C + L3: cellulase and W-MWL of untreated EFB (L3); C + L4: cellulase and W-MWL of treated EFB (L4). The residual cellulase proteins in the supernatant were taken on SDS-PAGE after 72 h adsorption. Experiments were performed in duplicate.

capacity of cellulase by the lignin fractions was tested with the commercial cellulase (Sino Enzymes R) at 45°C for 72 h. The amount of non-adsorbed enzymes and the rate of enzyme absorption were measured from the supernatant by protein concentration (using Bradford assay)


and cellulase activity assays, as shown in Fig. 3A. All of the lignin fractions showed considerable ability to adsorb protein, and approximately 26, 31, 30, and 35%, plus the total protein in reaction system, were adsorbed onto lignin fractions of L1, L2, L3, and L4, respectively. The

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enzyme activities decreased after the lignin fractions were added to the cellulase enzyme. For the fractions of L1 and L2, approximately 9 and 22% of the filter paper activities, as well as 15 and 19% of CBH activities, respectively, were lost. For the lignin fractions L3 and L4, the loss of enzyme activities were approximately 15 and 32% for the filter paper activities and more than 18 and 19% for the CBH activity, respectively. To study the adsorption profiles of specific enzyme components in the cellulase mixture, we conducted SDSPAGE (Fig. 3B). This figure clearly shows that some protein bands disappeared. The intensity of some protein bands in Fig. 3B notably decreased when the lignin fractions were added to the cellulase enzyme solution. These main components of the cellulase system will be identified in our future work. The adsorption of cellulase enzyme onto lignin showed that lignin fractions from bisulfite-pretreated EFB (fraction L2 and L4) have stronger adsorption ability compared with that from untreated EFB (fraction L1 and L3). The results from the analysis of lignin structures above showed that the S/G ratios in the lignin fraction L2 were higher than that in the lignin fraction L1. Furthermore, the fraction L2 had fewer G units than L1. In our previous work [33], lignin with high S/G ratios showed weak absorption ability on the protein, which seemed to contradict the results of the present study. However, some studies [34] showed that the alcoholic hydroxyl and carboxylic groups had greater effect when lignin was added to the typical hydrolysis of pure cellulose (Avicel). The results mentioned above showed that MWL from EFB contained several chemical groups, including alcoholic hydroxyl, phenolic hydroxyl, carbanyl, and ester groups. Consequently, we hypothesized that the observed phenomenon was due to the presence of various chemical groups in the isolated lignin, especially the alcoholic hydroxyl and carboxylic groups. We also found in Fig. 3A that the adsorption ability of water-soluble MWL was stronger than that of MWL, both in untreated and pretreated EFBs. This result may be explained by following statements: (i) the water-soluble MWLs had a larger specific surface area compared with MWLs because of their small molecular weight and the weak dissolving ability of water on lignin; and (ii) watersoluble MWLs had more uniform lignin fragment sizes, which showed good correlation with the high absorption ability of the protein [33].

4 Conclusions In this study, changes in the structure and properties of EFB substrate and in the fractions of hemicelluloses and lignin (which were extracted from EFB before and after bisulfite pretreatment) were investigated. MWLs and water-soluble MWLs were fractionated and their adsorption ability on cellulase enzymes and effect on enzyme

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activities were evaluated. The bisulfite pretreatment process led to an increase in crystallinity of EFB cellulose and EFB surface damage. The orderly structure of the EFB cell wall was damaged. The glycosidic linkages between xylan backbones in hemicelluloses were hydrolyzed, and in part, hemicellulose and lignin were dissolved. These results led to greater external- and internal specific surfaces of EFB and helped with the reaction between enzyme and cellulose. Lignin fractions obtained from untreated and bisulfite-pretreated EFBs were typical of G-S lignin, which has a low amount of H units. The differences, in terms of active groups in MWLs, the ratio of S/G, and lignin properties, resulted in varied effects on adsorption of cellulase proteins and enzymatic activity.

This study was supported by grants from the National Basic Research Program of China (Grant No. 2011CB707401), the National Natural Science Foundation of China (Grant Nos. 21276143 and 21376141), and the Malaysian Palm Oil Board. The authors declare no financial or commercial conflicts of interest.

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ISSN 1860-6768 · BJIOAM 10 (6) 821–926 (2015) · Vol. 10 · June 2015

Systems & Synthetic Biology · Nanobiotech · Medicine

6/2015

Bioenergy and biorefinery

Biomass Bioprocess engineering Biocatalysis

Cover illustration


Special Issue: Bioenergy and biorefinery. This special issue, in collaboration with the Asian Federation of Biotechnology, is edited by Yinbo Qu and Wen-Teng Wu. It includes contributions on the improvement of enzymes or organisms and pretreatment methods for increasing the refinery efficiency of plant biomass. Furthermore, articles on the use of microalgae not only for α-glucan and protein production but also for the removal of different substances from flue gas are part of this special issue. Image: © Olaf Wandruschka-Fotolia.com.

Biotechnology Journal – list of articles published in the June 2015 issue. Editorial: Bioenergy and biorefinery – biological solution for sustainable development of human society Yinbo Qu and Wen-Teng Wu

Review Improving polyglucan production in cyanobacteria and microalgae via cultivation design and metabolic engineering

http://dx.doi.org/10.1002/biot.201500291

Shimpei Aikawa, Shih-Hsin Ho, Akihito Nakanishi, Jo-Shu Chang, Tomohisa Hasunuma and Akihiko Kondo

Review CO2, NOx and SOx removal from flue gas via microalgae cultivation: A critical review


Hong-Wei Yen, Shih-Hsin Ho, Chun-Yen Chen and Jo-Shu Chang

Rapid Communication Linker length and flexibility induces new cellobiohydrolase activity of PoCel6A from Penicillium oxalicum


Le Gao, Lushan Wang, Xukai Jiang and Yinbo Qu

Review Miscanthus as cellulosic biomass for bioethanol production Wen-Chien Lee and Wei-Chih Kuan


http://dx.doi.org/10.1002/biot.201400734 Research Article Improving protein production of indigenous microalga Chlorella vulgaris FSP-E by photobioreactor design and cultivation strategies

Review Current progress of targetron technology: Development, improvement and application in metabolic engineering

Chun-Yen Chen, Po-Jen Lee, Chung Hong Tan, Yung-Chung Lo, Chieh-Chen Huang, Pau Loke Show, Chih-Hung Lin and Jo-Shu Chang

Ya-Jun Liu, Jie Zhang, Gu-Zhen Cui, Qiu Cui


http://dx.doi.org/10.1002/biot.201400716 Review Steam explosion and its combinatorial pretreatment refining technology of plant biomass to bio-based products Hong-Zhang Chen and Zhi-Hua Liu


Research Article Bisulfite pretreatment changes the structure and properties of oil palm empty fruit bunch to improve enzymatic hydrolysis and bioethanol production Liping Tan, Wan Sun, Xuezhi Li, Jian Zhao, Yinbo Qu, Yuen May Choo and Soh Kheang Loh




Production of Monomeric Aromatic Compounds from Oil Palm Empty Fruit Bunch Fiber Lignin by Chemical and Enzymatic Methods.

An integrative process of bioconversion of oil palm empty fruit bunch fiber to ethanol with on-site cellulase production.

Hydrothermal pretreatment enhanced enzymatic hydrolysis and glucose production from oil palm biomass.

Hemicellulase production by Aspergillus niger DSM 26641 in hydrothermal palm oil empty fruit bunch hydrolysate and transcriptome analysis.

Production and purification of xylooligosaccharides from oil palm empty fruit bunch fibre by a non-isothermal process.

oil palm empty fruit bunch fiber composites.

Optimization of NaOH-catalyzed steam pretreatment of empty fruit bunch.

Use of Empty Fruit Bunches from the oil palm for bioethanol production: a thorough comparison between dilute acid and dilute alkali pretreatment.

Flexural properties of poly(methyl methacrylate) resin reinforced with oil palm empty fruit bunch fibers: a preliminary finding.

Ethanol and lignin production from Brazilian empty fruit bunch biomass.

In vitro and in silico characterization of metagenomic soil-derived cellulases capable of hydrolyzing oil palm empty fruit bunch.

Biological pretreatment of rubberwood with Ceriporiopsis subvermispora for enzymatic hydrolysis and bioethanol production.

Improved physicochemical pretreatment and enzymatic hydrolysis of rice straw for bioethanol production by yeast fermentation.

Isolation, characterization and application of a cellulose-degrading strain Neurospora crassa S1 from oil palm empty fruit bunch.

Effects of aeration rate on degradation process of oil palm empty fruit bunch with kinetic-dynamic modeling.

Ball milling pretreatment of oil palm biomass for enhancing enzymatic hydrolysis.

Combined pretreatment using alkaline hydrothermal and ball milling to enhance enzymatic hydrolysis of oil palm mesocarp fiber.

Effects of pyrolysis temperature on the physicochemical properties of empty fruit bunch and rice husk biochars.

Chemical characterization and hydrothermal pretreatment of Salicornia bigelovii straw for enhanced enzymatic hydrolysis and bioethanol potential.

Comparison of aqueous ammonia and dilute acid pretreatment of bamboo fractions: Structure properties and enzymatic hydrolysis.

Hydrothermal Pretreatment of Date Palm (Phoenix dactylifera L.) Leaflets and Rachis to Enhance Enzymatic Digestibility and Bioethanol Potential.

Mesophilic co-digestion of palm oil mill effluent and empty fruit bunches.

Pretreatment methods for bioethanol production.

High glucose recovery from direct enzymatic hydrolysis of bisulfite-pretreatment on non-detoxified furfural residues.