International Journal of Biological Macromolecules 69 (2014) 158–164
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Structural characterization of residual hemicelluloses from hydrothermal pretreated Eucalyptus fiber Shao-Ni Sun a , Xue-Fei Cao b , Han-Ying Li a , Feng Xu a , Run-Cang Sun a,b,∗ a b
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
a r t i c l e
i n f o
Article history: Received 19 April 2014 Received in revised form 12 May 2014 Accepted 19 May 2014 Available online 24 May 2014 Keywords: Hydrothermal pretreatment Hemicelluloses Structural characterization
a b s t r a c t In this study, an environmental-friendly hydrothermal pretreatment of Eucalyptus fiber followed with alkali post-treatment was developed to produce bioethanol efficiently. This biorefinery process allowed all major components of biomass being converted into high value-added products. The chemical and structural features of the residual hemicelluloses isolated with alkali from the hydrothermal pretreated Eucalyptus fiber, were comparatively investigated. Sugar and spectral analyses indicated that the hemicelluloses were mainly composed of glucuronoxylans, and especially hemicelluloses prepared at higher temperature (180 ◦ C) contained higher contents of glucomannans and ␣-glucan. Hydrothermal pretreatment resulted in a significant hydrolysis of the glycosidic linkages in xylan backbone, and thus the molecular weight of the hemicelluloses was significantly reduced from 56,520 to 7780 g/mol with the increase of temperature. This suggested that a combination of hydrothermal pretreatment at low temperatures (100–140 ◦ C) and alkali post-treatment was an effective technique for isolating of hemicelluloses from Eucalyptus fiber. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A biorefinery is a facility that integrates biomass separation to produce fuels, power, and chemicals from each of the primary components (cellulose, hemicelluloses, and lignin) of lignocellulosic biomass [1,2]. However, biorefinery focusing mainly on ethanol or related biofuel production has limited opportunities for profitability. Recently, the utilizations of hemicelluloses and lignin attract more and more attention. Unlike cellulose, hemicelluloses are amorphous, heterogeneous, and branched polysaccharides, which are surrounded by a complex phenyl propanoic polymer called lignin which provides a protective sheath to the heimicelluloses–cellulose framework [3]. Generally, hemicelluloses can be divided into glucomannans, galactomannans, xylans (arabinoxylans and 4-O-methyl-glucuronoxylans), -d-glucancallose (3-linked), -d-glucans (3- and 4-linked), and xyloglucans (4-linked -d-glucans with attached side chains) [4]. All of the five general groups occur in many structural variations differing in side chain type, distribution, location and/or types and
∗ Corresponding authors at: Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China. Tel.: +86 10 62336903; fax: +86 10 62336903. E-mail address: [email protected] (R.-C. Sun). http://dx.doi.org/10.1016/j.ijbiomac.2014.05.037 0141-8130/© 2014 Elsevier B.V. All rights reserved.
distributions of glycoside linkages in the main macromolecular chain [5]. Hemicelluloses have numerous applications such as biomaterials (film, hydrogel, fiber, biocomposites), special chemicals (drug carrier, food additive, plant growth regulator), and pharmaceuticals (wound management aids), etc. [5–7]. In addition, some of them serve as ethanol, acetone, butanol, or xylitol in monomeric form [8,9]. In order to eventually become an economical process, the full and high value-added utilizations of abundant hemicelluloses and lignin provide a rational comprehensive way of lignocellulosic biomass, getting out of the cost restriction of single-bioethanol production. To date, pretreatment is required as the first and essential step for the biological conversion of lignocellulosic biomass to biofuels. The purpose of pretreatment is usually to reduce biomass recalcitrance by altering cell wall structure so that the polysaccharide fractions (mainly cellulose) locked in the intricacy of plant cell walls can become more accessible and amenable to enzymatic hydrolysis [10,11]. Hydrothermal pretreatment (autohydrolysis) has been considered to be an eco-friendly green processing technology, since the medium only contains biomass and water, avoiding many problems, such as corrosion, acid recycling and the formation of neutralization sludges [12,13]. Water autohydrolysis has the advantage to enable a high recovery of hemicelluloses as soluble polysaccharides, while both cellulose and lignin can be recovered in solid phase with minor losses [14]. The hemicelluloses obtained
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
159
Fig. 1. Scheme for fractionating residual hemicelluloses from hydrothermal pretreatment Eucalyptus fiber.
can be converted into hemicellulosic sugars at good yields with low by-products, resulting in the solution of mono- and oligosaccharides that can be used for a variety of practical purposes [15]. The solid residues obtained from the hydrothermal pretreatment can be used for the production of biofuel. As well known, effective fractionations of hemicelluloses and lignin from the residues using a post-treatment process will be undoubtedly beneficial for the economy of biomass utilization, which is in accordance with the “biorefinery” concept. Based on this rethinking process, the residual hemicelluloses and lignin were successively fractionated with alkali from the hydrothermal pretreated fibers. The isolated hemicelluloses and lignin are under investigation for converting into high valuable materials such as hydrogels and polyurethane in our laboratory. Furthermore, the removal of hemicelluloses and lignin could dramatically improve enzyme digestibility by enhancing the accessibility of cellulose to enzyme [16]. In the present study, hemicelluloses were fractionated from hydrothermal pretreated fibers (100–220 ◦ C). The structural and physicochemical features of the residual hemicelluloses were examined in detail by means of sugar analysis, molecular weights, FT-IR, and NMR spectroscopies. 2. Materials and methods 2.1. Material Thermo-mechanical fiber obtained at 120 ◦ C for 1–2 min from Eucalyptus urophylla, as raw material (Eucalyptus fiber), was kindly supplied by the State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, China. The sample was ovendried at 50 ◦ C for 24 h and dewaxed with methylbenzene/ethanol (2:1, v/v) in a Soxhlet apparatus for 3 h, and the sample was dried in an oven at 60 ◦ C for 16 h. The composition of the dewaxed fiber was determined to be 45.0% cellulose, 21.7% hemicelluloses, 25.8% Klason lignin, and 1.3% acid soluble lignin by National Renewable Energy Laboratory’s (NREL) standard analytical procedure [17]. All chemicals purchased were of analytical or reagent grade and used without further purification. Fig. 1. shows the scheme for fractionating residual hemicelluloses from hydrothermal pretreated Eucalyptus fibers. 2.2. Hydrothermal pretreatment process The hydrothermal process was carried out as follow. A total of 15.0 g Eucalyptus fiber was dispersed in 450 mL distilled water and transferred to a 1 L Parr stirred pressure reactor (Parr Instrument Company, Moline, Illinois). Then the reactor was heated to
100 ◦ C (60 min), 120 ◦ C (60 min), 140 ◦ C (60 min), 160 ◦ C (60 min), 180 ◦ C (15, 30, 45, and 60 min), 200 ◦ C (30 min), and 220 ◦ C (30 min), respectively, at a heating rate of about 4 ◦ C/min. Once the desired operation reached, the reactor was cooled by flowing water through an internal stainless steel loop. At the end of incubation, the mixtures were cooled to room temperature. The liquid stream was separated by filtration with a nylon cloth from the solid residues and then the residues were washed with 450 mL distilled water, dried in an oven at 60 ◦ C for 12 h and labeled as AM100-60 , AM120-60 , AM140-60 , AM160-60 , AM180-15 , AM180-30 , AM180-45 , AM180-60 , AM200-30 , and AM220-30 , respectively, according to the pretreatment temperature and time. The chemical composition of the raw material and hydrothermal pretreated fibers was also determined by National Renewable Energy Laboratory’s (NREL) standard analytical procedure [17]. 2.3. Fractionation of residual hemicelluloses from hydrothermal pretreated fibers The hydrothermal pretreated fibers (5.0 g) was post-treated with 2% NaOH at 90 ◦ C for 2.5 h with a solid-to-liquid ratio of 1:30 (g/mL), and then filtered. Afterwards, the supernatant was adjusted to pH = 5.5–6.0 with 6 M HCl, concentrated to about 60 mL on a rotary evaporator under reduced pressure, and then mixed with three volumes of 95% ethanol, precipitated for 1 h, and finally filtered through filter paper to isolate hemicelluloses. The residual hemicelluloses obtained were freeze-dried and labeled as AH100-60 , AH120-60 , AH140-60 , AH160-60 , AH180-15 , AH180-30 , AH180-45 , AH180-60 , AH200-30 , and AH220-30 , respectively, according to the pretreatment temperature and time. However, no hemicellulosic polymer could be obtained when the hydrothermal pretreatment temperature was higher than 180 ◦ C. For comparison, the control hemicellulosic fraction AH prepared without hydrothermal pretreatment was also fractionated by treating raw material (Eucalyptus fiber 5.0 g) with 2% NaOH using a solid-to-liquid ratio of 1:30 (g/mL) at 90 ◦ C for 2.5 h. All experiments were performed at least in duplicate. 2.4. Chemical characterization The contents of acid insoluble lignin in the residual hemicelluloses were measured according to the Klason method [18]. The composition of neutral sugars and uronic acids of the hemicelluloses were determined by high performance anion exchange chromatography (HPAEC) as previously reported by Sun et al. [19]. The molecular weights and molecular weight distributions of all hemicelluloses were examined by gel permeation chromatograph according to a previous method [19].
160
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
Table 1 The percentage of xylo-oligosaccharides in initial xylan (%). HPTea (◦ C)
100
120
140
160
180
180
180
180
HPTib (min) XOSc (%)
60 2.59
60 3.96
60 11.25
60 37.51
15 38.90
30 43.55
45 34.21
60 28.63
a b c
HPTe, hydrothermal pretreatment temperature. HPTi, hydrothermal pretreatment time. XOS, xylo-oligosaccharide.
2.5. Spectroscopic characterization The FT-IR spectra of the residual hemicelluloses were recorded on a Bruker spectrophotometer in the range of 400–4000 cm−1 with a resolution of 4 cm−1 . A KBr disc containing 1% finely ground sample was used for measurement. The soluble-state 13 C- and heteronuclear single quantum coherence (HSQC) NMR spectra were recorded on a Bruker AV III 400 MHz spectrometer operating in the FT mode at 100.6 MHz. The 13 C- and HSQC NMR experiments were conducted with 60 and 20 mg lignins dissolved in 1 mL D2 O, respectively. The parameters for data acquisition were set according to a previous literature [19].
3. Result and discussion 3.1. Production of xylo-oligosaccharides and chemical composition of hydrothermal pretreated fibers Table 1 shows that the yield of xylo-oligosaccharides increased with the increasing of temperature (100–160 ◦ C) for 60 min and then decreased at 180 ◦ C. The maximum yield of xylooligosaccharide of 43.55% of the initial xylan was achieved at 180 ◦ C for 30 min. A further increase in pretreatment time resulted in a decrease of the xylo-oligosaccharide yield (28.63% of the initial xylan at 180 ◦ C for 60 min). The reason for this was probable that large amounts of xylan and xylo-oligosaccharides were mainly degraded into xylose and sugar degradation products for a longer pretreatment period under this condition. As expected, after the hydrothermal pretreatment, the pretreated fibers consisted mainly of cellulose and lignin together with small amounts of residual hemicelluloses. As can be seen, an increasing the pretreatment temperature from 100 to 180 ◦ C led to a significant decrease of hemicelluloses from 19.51% to 1.69% in the hydrothermal pretreated samples, which was due to the fact that the chemical basis of the autohydrolysis process is the hydrolysis reaction of hemicelluloses in aqueous medium. Meanwhile, the
contents of cellulose and lignin in the pretreated fibers increased from 45.01% to 59.69% and 25.84% to 54.35% with the increasing temperature (100–180 ◦ C), which was mainly due to the removal of hemicelluloses. 3.2. Yields and sugar components of residual hemicelluloses The yield of the hemicelluloses isolated and the content of lignin in the residual hemicelluloses are also given in Table 2. As can be seen, the yield of the hemicelluloses first increased from 7.11% to 11.26% with the increment of the pretreatment temperature from 100 to 140 ◦ C and then decreased (3.28–5.58%) with the temperature at 160–180 ◦ C. Furthermore, it was found that the residual hemicelluloses at low pretreatment temperatures (100–140 ◦ C) or without hydrothermal pretreatment contained relatively low amounts of lignin (5.22–8.53%). However, when the pretreatment temperature increased to 160–180 ◦ C, the content of lignin in the residual hemicelluloses increased to 53.58–74.84%, which was probably due to the degraded lignin associated with hemicelluloses. The result indicated that a combination of hydrothermal pretreatment at low temperatures (100–140 ◦ C) and alkali post-treatment was an effective treatment for the fractionation of hemicelluloses from Eucalyptus fiber. As we known, hemicelluloses are not chemically homogeneous and different hydrolytic technologies and various pretreatment methods are available for both fractionation and solubilization of hemicelluloses from biomass. In this study, to analyze the difference among these hemicelluloses, the released monomeric sugars and uronic acids from hemicelluloses by acid hydrolysis were monitored with HPAEC (Table 3). Sugar composition analysis indicated that the residual hemicelluloses (AH100-60 –AH160-60 ) prepared at temperature lower than 180 ◦ C are rich in xylose (76.73–82.35%), glucuronic acid (GluA, 9.27–10.76%), and galactose (5.04–6.49%). Glucose (0.91–2.32%), arabinose (1.05–2.02%), and rhamnose (0.45–0.82%) were present in small amounts. The results suggested that the residual hemicelluloses prepared from Eucalyptus fiber with hydrothermal pretreatment at low
Table 2 The chemical composition (w/w, %) of the raw fiber and the hydrothermal pretreated fibers, the yields of residual hemicelluloes isolated with 2% NaOH at 90 ◦ C for 2.5 h from the hydrothermal pretreated fibers, and the content of acid insoluble lignin in the isolated residual hemicelluloes. Chemical compositiona (w/w, %)
RM AM100-60 AM120-60 AM140-60 AM160-60 AM180-15 AM180-30 AM180-45 AM180-60 a
Hemicelluloses
AILb
Cellulose
21.66 19.51 17.34 17.17 7.56 4.81 3.65 3.60 1.69
25.79 25.84 25.91 26.17 28.93 29.82 31.79 32.22 33.46
45.01 43.91 43.67 45.88 53.74 55.74 56.04 57.08 59.69
Hemicellulosic fractions
Yieldc
AILCd
AH AH100-60 AH120-60 AH140-60 AH160-60 AH180-15 AH180-30 AH180-45 AH180-60
8.69 7.11 7.26 11.26 5.58 4.00 3.20 4.99 3.28
5.22 6.75 7.33 8.53 53.58 61.22 68.98 69.71 74.84
Absolute weight percentage based on the raw fiber and the hydrothermal pretreated fibers. AIL represents acid insoluble lignin. c Yields of the AH and residual hemicelluloses (AH100-60 –AH180-60 ) represent the percentage of the hemicelluloses in the raw material and the hydrothermal pretreated fibers (AM100-60 –AM180-60 ), respectively. d AILC represents acid insoluble lignin content in the residual hemicelluloses. b
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
161
Table 3 Contents of neutral sugars and uronic acids in the residual hemicelluloses obtained by alkali extraction of the hydrothermal pretreated fibers. Neutral sugars and uronic acidsa
AH AH100-60 AH120-60 AH140-60 AH160-60 AH180-15 AH180-30 AH180-45 AH180-60 a b c d
Rha
Ara
Gal
Glu
Xyl
Man
GluA
GalA
0.95 0.82 0.79 0.54 0.45 0.52 ND ND ND
3.48 2.02 1.66 1.19 1.05 2.09 3.24 5.42 5.38
7.01 6.49 6.33 5.34 5.04 5.02 4.43 3.85 3.74
5.20 2.32 2.16 1.62 0.91 12.47 14.57 18.94 20.36
72.11 76.73 78.03 80.82 82.35 72.09 67.79 66.01 62.79
1.50 0.86 0.71 0.49 0.93 7.81 9.97 5.78 7.73
9.48 10.76 10.32 10.00 9.27 ND ND ND ND
0.28 NDd ND ND ND ND ND ND ND
Xyl/Arab
Xyl/UAc
7.6 7.1 7.6 8.1 8.9 34.5 20.9 12.2 11.7
20.7 38.0 47.0 67.9 78.4 ND ND ND ND
Rha, rhamnose; Ara, arabinose; Gal, galactose; Glu, glucose; Xyl, xylose; Man, mannose; GluA, glucuronic acid; GalA, galacturonic acid. Xylose to arabinose ratio. Xylose to uronic acids ratio. ND, not detected.
temperatures were mainly composed of glucuronoxylans-type polysaccharides (GX), which was the typical hemicellulosic component in hardwoods. Generally, GX consists of a linear backbone of -d-xylopyranosyl units (Xylp) linked by -(1,4) glycosidic bonds. Some xylose units are acetylated at C2 and C3 and one in 10 molecules has an uronic acid group (4-O-methylglucuronic acid) attached by ␣-(1,2) linkages [20]. However, under the condition of alkali treatment, the cleavage of O-acetyl groups occurs and all acetyl groups are split off [21]. Therefore, in GX of Eucalyptus fiber, GluA was originated from 4-O-methyl-d-glucoronic acid as side chain, which can be confirmed by the following NMR analysis. A similar sugar composition was observed in the control hemicellulosic fraction AH and the residual hemicelluloses, except galacturonic acid (GalA, 0.28%) that was only detected with a minor amount in AH. Surprisingly, when the temperature reached 180 ◦ C, the residual hemicelluloses AH180-15 –AH180-60 presented low xylose (62.79–72.09%), galactose (3.74–5.02%), and rhamnose (0–0.52%) contents, but high glucose (12.47–20.36%) and mannose (5.78–9.97%) contents. These data suggested that the residual hemicelluloses prepared at a high temperature contained high glucomannan contents together with a small amount of ␣-glucan. Also notable was that the amount of GluA in AH100-60 –AH160-60 reached around 10%, while GluA was not detected in AH180-15 –AH180-60 . In addition, it can be seen from Table 3 that as the reaction temperature increased from 100 to 160 ◦ C, xylose content increased from 76.73 to 82.35%, while the contents of arabinose, galactose, and glucose decreased from 2.02, 6.49 and 2.32 to 1.05%, 5.04%, and 0.91%, respectively. For AH180-15 –AH180-60 , with pretreating time increasing from 15 to 60 min, the contents of galactose and xylose decreased, but the content of glucose increased. Generally, the ratios of xylose to uronic acid (Xyl/UA) and xylose to arabinose (Xyl/Ara) are indicative of the degree of linearity or branching of hemicelluloses. Obviously, the Xyl/UA and Xyl/Ara ratios increased from 7.1 (AH100-60 ) and 38.0 (AH100-60 ) to 8.9 (AH160-60 ) and 78.4 (AH160-60 ), respectively, indicating that a higher pretreating temperature resulted in the isolation of hemicelluloses with less branches. In light of this, hemicelluloses rich in backbone structure could be obtained at high temperature. On the contrary, at low temperature, hemicelluloses with some side chains and more complex structure were obtained. This can be explained by the fact that the presence of large unsubstituted regions in xylan backbone can lead to strong hydrogen bonds, causing interchain aggregation and more difficult isolation [22]. Consequently, a high isolating temperature by alkali post-treatment was required. However, with the pretreating temperature increasing to 180 ◦ C, hemicelluloses with less branches were obtained, as shown by low Xyl/Ara ratios (11.7–34.5).
3.3. Molecular weight distribution In order to investigate the degradation during hydrothermal pretreatment, all the isolated hemicelluloses were further analyzed by the determination of their weight-average (Mw ), numberaverage (Mn ) molecular weights and polydispersity (Mw /Mn ), and the results are summarized in Table 4. The residual hemicelluloses AH100-60 had a high Mw (56,520 g/mol) and Mn (26,330 g/mol), which was comparable to AH (Mw = 44,040 and Mn = 20,330 g/mol). It was probably ascribed to the effect of hydrothermal swelling of the cell walls, resulting in the release of hemicelluloses with high molecular weights in the following alkaline extraction. However, the Mw (41,870–7780 g/mol) and Mn (20,500–6650 g/mol) of AH120-60 –AH160-60 and AH180-15 –AH180-60 were lower than those of AH. This was probably due to the cleavage of glycosidic ether linkages between sugar units, which led to the difference in degree of degradation of hemicelluloses. Based on this observation and Xyl/Ara ratios, the residual hemicelluloses AH100-60 –AH160-60 with a high Xyl/Ara ratio had a low Mw , indicating that an increase of the hydrothermal pretreatment temperature resulted in a significant degradation of the hemicellulosic polymers, which was in agreement with the previous study by Dervilly et al. [23]. In contrast, the hemicellulosic fractions AH180-15 –AH180-60 obtained at a higher temperature of 180 ◦ C with an increment of the pretreating time from 15 to 60 min showed a decreasing trend of Xyl/Ara ratios and molecular weights, suggesting that an extension of the pretreating time also led to a depolymerision of the hemicelluloses at 180 ◦ C, which was in accordance with the results found by Peng and Roels et al. [24,25]. Polydispersity is an important parameter of macromolecules relative to their applications in chemical industry. The polydispersities of the obtained hemicellulosic fractions ranged from 1.11 to 2.17. It has been reported that polysaccharides with Mw /Mn value below 3 were molecularly uniform polymers and have potential Table 4 Weight-average (Mw ) and number-average (Mn ) molecular weights and polydispersity (Mw /Mn ) of the residual hemicelluloses isolated by 2% NaOH at 90 ◦ C for 2.5 h from the raw fiber and the hydrothermal pretreated fibers. Hemicellulosic fractions
Mw
Mn
Mw /Mn
AH AH100-60 AH120-60 AH140-60 AH160-60 AH180-15 AH180-30 AH180-45 AH180-60
44,040 56,520 41,870 39,570 16,990 13,530 13,470 8910 7780
20,330 26,330 20,500 24,990 13,170 10,790 10,750 7390 6650
2.17 2.15 2.04 1.58 1.29 1.25 1.25 1.21 1.17
162
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
Fig. 3. 13 C-NMR spectra of the residual hemicelluloses AH, AH100-60 , AH160-60 , and AH180-15 .
Fig. 2. FT-IR spectra of the residual hemicelluloses AH, AH100-60 , AH120-60 , AH140-60 , AH160-60 , and AH180-15 .
commercial utilization [26]. It was noticeable that the Mw /Mn value of hemicelluloses decreased with the increased pretreatment temperature. 3.4. FT-IR spectra Fig. 2 shows that the FT-IR spectra of fractions AH, AH100-60 , AH120-60 , AH140-60 , AH160-60 , and AH180-15 were rather similar. Generally, absorptions in the region of 1200–800 cm−1 give the information about the main polysaccharides. Most of the absorption peaks can be assigned according to literatures [19,27,28]. The absorptions at 3443 and 2937 cm−1 are attributed to the OH and C H stretching vibrations, respectively. As expected, the disappearance of the band at about 1730 cm−1 (the carbonyl stretching region of hemicelluloses) revealed that the hydrothermal pretreatment followed with alkaline extraction cleaved the ester bands of hemicelluloses, such as acetyl and uronic ester groups. The CH2 stretching vibration exhibits a signal at 1462 cm−1 and the band at 1414 cm−1 represents symmetric stretching vibration of glucuronic acid group. Moreover, the bands at 1387 and 1248 cm−1 are originated from OH and C O bending vibration in hemicelluloses. The occurrence of a peak at 1119 cm−1 is due to C OH skeletal vibration, whereas the absorption band at 1045 cm−1 is related to C O, C C stretching or C OH bending in hemicelluloses, indicating the typical xylan in the hemicelluloses. In addition, the peak at 989 cm−1 is indicative of arabinofuranosyl, which has been reported to be attached to the position 3 of the xylopyranosyl constituents. In anomeric region (950–700 cm−1 ), an important band at 897 cm−1 is originated from C1 group frequency or ring frequency, which is characteristic of -glycosidic linkages between the sugar units. It
should be noted that in the isolated hemicelluloses, the intensity of the band at 1045 cm−1 decreased but that of the band at 1516 cm−1 (the phenolic ring absorbance of lignin) [29] increased with the raise of the hydrothermal pretreatment temperature, which was probably due to the increasing percentage of lignin in the residual hemicelluloses (Table 2). 3.5. NMR spectra analysis To further characterize the structural features of hemicelluloses, the four fractions AH, AH100-60 , AH160-60 , and AH180-15 were comparatively investigated by 13 C NMR spectra (Fig. 3). Clearly, the five strong signals of (1 → 4) linked -d-Xylp residues were detected at 102.0 (C-1), 76.0 (C-4), 74.9 (C-3), 73.3 (C-2), and 63.4 (C-5) ppm [30]. Weak signals at 177.1, 97.5, 82.7, 72.2, and 59.6 ppm, corresponding to COOH, C-1, C-4, C-5, and OCH3 of the 4O-methyl-d-glucoronic acids in hemicelluloses, respectively, were also observed [31]. Meanwhile, the signals at 55.7 and 181.5 ppm are ascribed to OCH3 in the guaiacyl and syringyl units and carbonyl group, respectively. Obviously, the intensities of -d-Xylp and 4-O-methyl-d-glucoronic acids decreased while the intensities of lignin increased with the increment of hydrothermal pretreatment temperature, corresponding to the content of lignin in the residual hemicelluloses. More specific information about the structural variation among hemicellulosic fractions AH, AH100-60 , AH160-60 , and AH180-15 were further investigated by 2D HSQC NMR spectra (Fig. 4). The marked 1 H 13 C cross-peaks further confirmed the structural elements of (1 → 4)-linked -d-Xylp, 4-O-methyl-d-glucoronic acids, and (1 → 4)--d-Xylp-2-O-(4-OMe-d-GlcpA) units. Specially, in the HSQC spectrum of AH, five dominant cross peaks at 102.1/4.34, 73.0/3.19, 74.5/3.41, 76.0/3.67, and 63.1/3.28 and 3.98 ppm, are assigned to C1–H1, C2–H2, C3–H3, C4–H4, and C5–H5 of (1 → 4)linked -d-Xylp units, respectively. In addition, the signals at 97.4/5.19, 71.5/3.46, 72.2/3.60, 82.4/3.11, and 72.1/4.22 ppm are, respectively assigned to C1–H1, C2–H2, C3–H3, C4–H4, and C5–H5 of 4-O-methyl-d-glucoronic acids units. The signals of
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
163
Fig. 4. HSQC spectra of the residual hemicelluloses AH, AH100-60 , AH160-60 , and AH180-15 . X: (1 → 4)--D-Xylp; UA: 4-O-Methyl-␣-D-GlcpA; XU: (1 → 4)--D-Xylp-2-O-(4OMe-D-GlcpA).
(1 → 4)--d-Xylp-2-O-(4-OMe-d-GlcpA) units with less intensity were detected at 100.8/4.54 (C1–H1) and 76.4/3.33 ppm (C2–H2). Furthermore, it was noticeable that the signals of 4-O-methyl-dglucoronic acids were not detected in the spectrum of AH180-15 , in accordance with the results of sugar analysis. These data concluded that the hemicelluloses isolated with 2% NaOH from the hydrothermal pretreated fibers were assumed to be mainly composed of (1 → 4)-linked -d-Xylp attached with various monosaccharides and small amounts of glucomannans and ␣-d-glucan. 4. Conclusions In summary, the hydrothermal pretreatment significantly hydrolyzed the glycosidic linkages in xylan backbone and thus reduced the molecular weight of the residual hemicelluloses extracted with alkali from hydrothermal pretreated fibers. The residual hemicelluloses isolated at low pretreatment temperatures (100–140 ◦ C) contained low amounts of lignin (6.75–8.53%), but high amounts of lignin (53.58–74.84%) was observed in the hemicelluloses obtained at high pretreatment temperatures (160–180 ◦ C). The residual hemicelluloses isolated from the hydrothermal pretreated fibers at 100–160 ◦ C were rich in xylose and glucuronic acid, while a higher temperature (180 ◦ C) resulted in hemicelluloses being rich in xylose, glucose, and mannose. Moreover, the Xyl/UA and Xyl/Ara ratios increased from 7.1 and 38.0 (AH100-60 pretreated at 100 ◦ C for 60 min) to 8.9 and 78.4 (AH160-60 pretreated at 160 ◦ C for 60 min), respectively, indicating that a
higher temperature resulted in the isolation of hemicelluloses with less branches. In conclusion, the hydrothermal pretreatment at low temperatures followed by alkali treatment is an effective method for the fractionation of hemicelluloses from Eucalyptus fiber.
Acknowledgments The authors are extremely grateful to financial support from the Major State Basic Research Projects of China (973-2010CB732204) and State Forestry Administration (201204803).
References [1] S. Fernando, S. Adhikari, C. Chandrapal, N. Murali, Biorefineries: current status, challenges, and future direction, Energy Fuels 20 (2006) 1727–1737. [2] P. Kaparaju, M. Serrano, A.B. Thomsen, P. Kongjan, I. Angelidaki, Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept, Bioresour. Technol. 100 (2009) 2562–2568. [3] A.S. Mamman, J.M. Lee, Y.C. Kim, I.T. Hwang, N.J. Park, Y.K. Hwang, J.S. Chang, J.S. Hwang, Furfural: hemicellulose/xylose derived biochemical, Biofuels, Bioprod. Biorefin. 2 (2008) 438–454. [4] E. Sjöström, in: E. Sjöström (Ed.), Wood Chemistry: Fundamentals and Applications, Academic Press, New York, NY, 1981. [5] A. Ebringerova, Z. Hromadkova, T. Heinze, in: T. Heinze (Ed.), Polysaccharides I, Springer-Verlag, Berlin, 2005, pp. 1–67. [6] N.M. Hansen, D. Plackett, Sustainable films and coatings from hemicelluloses: a review, Biomacromolecules 9 (2008) 1493–1505. [7] X. Li, X.J. Pan, Hydrogels based on hemicellulose and lignin from lignocellulose biorefinery: a mini-review, J. Biobased Mater. Bioenergy 4 (2010) 289–297.
164
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
[8] S. Mateo, J.G. Puentes, S. Sánchez, A.J. Moya, Oligosaccharides and monomeric carbohydrates production from olive tree pruning biomass, Carbohydr. Polym. 93 (2012) 416–423. [9] B.C. Saha, Hemicellulose bioconversion, J. Ind. Microbiol. Biotechnol. 30 (2003) 279–291. [10] N. Mosier, C. Wyman, B. Dale, R. Elander, Y.Y. Lee, M. Holtzapple, M. Ladisch, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresour. Technol. 96 (2005) 673–686. [11] B. Yang, C.E. Wyman, Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels, Bioprod. Biorefin. 2 (2008) 26–40. [12] T. Ghose, Measurement of cellulase activities, Pure Appl. Chem. 59 (1987) 257–268. [13] P. Gullón, A. Romaní, C. Vila, G. Garrote, J.C. Parajó, Potential of hydrothermal treatments in lignocellulose biorefineries, Biofuels, Bioprod. Biorefin. 6 (2012) 219–232. [14] F. Carvalheiro, T. Silva-Fernandes, L.C. Duarte, F.M. Gírio, Wheat straw autohydrolysis: process optimization and products characterization, Appl. Biochem. Biotechnol. 153 (2009) 84–93. [15] M.J. Taherzadeh, K. Karimi, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review, Int. J. Mol. Sci. 9 (2008) 1621–1651. [16] D. Haverty, K. Dussan, A.V. Piterina, J. Leahy, M. Hayes, Autothermal, singlestage, performic acid pretreatment of Miscanthus × giganteus for the rapid fractionation of its biomass components into a lignin/hemicellulose-rich liquor and a cellulase-digestible pulp, Bioresour. Technol. 109 (2012) 173–177. [17] A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, D. Crocker, Laboratory Analytical Procedure (LAP): determination of structural carbohydrates and lignin in biomass, in: Technical Report NREL/TP-510-42618, National Renewable Energy Laboratory, Golden, CO, 2008. [18] C.W. Dence, in: S.Y. Lin, C.W. Dence (Eds.), Methods in Lignin Chemistry, Springer-Verlag, Berlin, 1992, pp. 33–61. [19] S.L. Sun, J.L. Wen, M.G. Ma, R.C. Sun, Successive alkali extraction and structural characterization of hemicelluloses from sweet sorghum stem, Carbohydr. Polym. 92 (2012) 2224–2231. [20] F. Gírio, C. Fonseca, F. Carvalheiro, L. Duarte, S. Marques, R. Bogel-Łukasik, Hemicelluloses for fuel ethanol: a review, Bioresour. Technol. 101 (2010) 4775–4800.
[21] F. Peng, P. Peng, F. Xu, R.C. Sun, Fractional purification and bioconversion of hemicelluloses, Biotechnol. Adv. 30 (2012) 879–903. [22] A. Höije, M. Gröndahl, K. Tømmeraas, P. Gatenholm, Isolation and characterization of physicochemical and material properties of arabinoxylans from barley husks, Carbohydr. Polym. 61 (2005) 266–275. [23] G. Dervilly, L. Saulnier, P. Roger, J.F. Thibault, Isolation of homogeneous fractions from wheat water-soluble arabinoxylans. Influence of the structure on their macromolecular characteristics, J. Agric. Food Chem. 48 (2000) 270–278. [24] F. Peng, J. Bian, J.L. Ren, P. Peng, F. Xu, R.C. Sun, Fractionation and characterization of alkali-extracted hemicelluloses from peashrub, Biomass Bioenergy 39 (2012) 20–30. [25] S.P. Roels, M. Collado, A.M. Loosveld, P.J. Grobet, J.A. Delcour, Variation in the degree of D-xylose substitution in water-extractable European durum wheat (Triticum durum Desf.) semolina arabinoxylans, J. Agric. Food Chem. 47 (1999) 1813–1816. [26] W.G. Glasser, W.E. Kaar, R.K. Jain, J.E. Sealey, Isolation options for non-cellulosic heteropolysaccharides (HetPS), Cellulose 7 (2000) 299–317. [27] D.K. Buslov, F.N. Kaputski, N.I. Sushko, V.I. Torgashev, L.V. Solov’eva, V.M. Tsarenkov, O.V. Zubets, L.V. Larchenko, Infrared spectroscopic analysis of the structure of xylans, J. Appl. Spectrosc. 76 (2009) 801–805. [28] M. Kaˇcuráková, P.S. Belton, R.H. Wilson, J. Hirsch, A. Ebringerová, Hydration properties of xylan-type structures: an FTIR study of xylooligosaccharides, J. Sci. Food Agric. 77 (1998) 38–44. [29] S.N. Sun, M.F. Li, T.Q. Yuan, F. Xu, R.C. Sun, Effect of ionic liquid/organic solvent pretreatment on the enzymatic hydrolysis of corncob for bioethanol production. Part 1: Structural characterization of the lignins, Ind. Crops Prod. 43 (2013) 570–577. [30] A. Teleman, J. Lundqvist, F. Tjerneld, H. Stålbrand, O. Dahlman, Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1 H and 13 C NMR spectroscopy, Carbohydr. Res. 329 (2000) 807–815. [31] A. Bendahou, A. Dufresne, H. Kaddami, Y. Habibi, Isolation and structural characterization of hemicelluloses from palm of Phoenix dactylifera L., Carbohydr. Polym. 68 (2007) 601–608.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Structural characterization of residual hemicelluloses from hydrothermal pretreated Eucalyptus fiber Shao-Ni Sun a , Xue-Fei Cao b , Han-Ying Li a , Feng Xu a , Run-Cang Sun a,b,∗ a b
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
a r t i c l e
i n f o
Article history: Received 19 April 2014 Received in revised form 12 May 2014 Accepted 19 May 2014 Available online 24 May 2014 Keywords: Hydrothermal pretreatment Hemicelluloses Structural characterization
a b s t r a c t In this study, an environmental-friendly hydrothermal pretreatment of Eucalyptus fiber followed with alkali post-treatment was developed to produce bioethanol efficiently. This biorefinery process allowed all major components of biomass being converted into high value-added products. The chemical and structural features of the residual hemicelluloses isolated with alkali from the hydrothermal pretreated Eucalyptus fiber, were comparatively investigated. Sugar and spectral analyses indicated that the hemicelluloses were mainly composed of glucuronoxylans, and especially hemicelluloses prepared at higher temperature (180 ◦ C) contained higher contents of glucomannans and ␣-glucan. Hydrothermal pretreatment resulted in a significant hydrolysis of the glycosidic linkages in xylan backbone, and thus the molecular weight of the hemicelluloses was significantly reduced from 56,520 to 7780 g/mol with the increase of temperature. This suggested that a combination of hydrothermal pretreatment at low temperatures (100–140 ◦ C) and alkali post-treatment was an effective technique for isolating of hemicelluloses from Eucalyptus fiber. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A biorefinery is a facility that integrates biomass separation to produce fuels, power, and chemicals from each of the primary components (cellulose, hemicelluloses, and lignin) of lignocellulosic biomass [1,2]. However, biorefinery focusing mainly on ethanol or related biofuel production has limited opportunities for profitability. Recently, the utilizations of hemicelluloses and lignin attract more and more attention. Unlike cellulose, hemicelluloses are amorphous, heterogeneous, and branched polysaccharides, which are surrounded by a complex phenyl propanoic polymer called lignin which provides a protective sheath to the heimicelluloses–cellulose framework [3]. Generally, hemicelluloses can be divided into glucomannans, galactomannans, xylans (arabinoxylans and 4-O-methyl-glucuronoxylans), -d-glucancallose (3-linked), -d-glucans (3- and 4-linked), and xyloglucans (4-linked -d-glucans with attached side chains) [4]. All of the five general groups occur in many structural variations differing in side chain type, distribution, location and/or types and
∗ Corresponding authors at: Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China. Tel.: +86 10 62336903; fax: +86 10 62336903. E-mail address: [email protected] (R.-C. Sun). http://dx.doi.org/10.1016/j.ijbiomac.2014.05.037 0141-8130/© 2014 Elsevier B.V. All rights reserved.
distributions of glycoside linkages in the main macromolecular chain [5]. Hemicelluloses have numerous applications such as biomaterials (film, hydrogel, fiber, biocomposites), special chemicals (drug carrier, food additive, plant growth regulator), and pharmaceuticals (wound management aids), etc. [5–7]. In addition, some of them serve as ethanol, acetone, butanol, or xylitol in monomeric form [8,9]. In order to eventually become an economical process, the full and high value-added utilizations of abundant hemicelluloses and lignin provide a rational comprehensive way of lignocellulosic biomass, getting out of the cost restriction of single-bioethanol production. To date, pretreatment is required as the first and essential step for the biological conversion of lignocellulosic biomass to biofuels. The purpose of pretreatment is usually to reduce biomass recalcitrance by altering cell wall structure so that the polysaccharide fractions (mainly cellulose) locked in the intricacy of plant cell walls can become more accessible and amenable to enzymatic hydrolysis [10,11]. Hydrothermal pretreatment (autohydrolysis) has been considered to be an eco-friendly green processing technology, since the medium only contains biomass and water, avoiding many problems, such as corrosion, acid recycling and the formation of neutralization sludges [12,13]. Water autohydrolysis has the advantage to enable a high recovery of hemicelluloses as soluble polysaccharides, while both cellulose and lignin can be recovered in solid phase with minor losses [14]. The hemicelluloses obtained
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
159
Fig. 1. Scheme for fractionating residual hemicelluloses from hydrothermal pretreatment Eucalyptus fiber.
can be converted into hemicellulosic sugars at good yields with low by-products, resulting in the solution of mono- and oligosaccharides that can be used for a variety of practical purposes [15]. The solid residues obtained from the hydrothermal pretreatment can be used for the production of biofuel. As well known, effective fractionations of hemicelluloses and lignin from the residues using a post-treatment process will be undoubtedly beneficial for the economy of biomass utilization, which is in accordance with the “biorefinery” concept. Based on this rethinking process, the residual hemicelluloses and lignin were successively fractionated with alkali from the hydrothermal pretreated fibers. The isolated hemicelluloses and lignin are under investigation for converting into high valuable materials such as hydrogels and polyurethane in our laboratory. Furthermore, the removal of hemicelluloses and lignin could dramatically improve enzyme digestibility by enhancing the accessibility of cellulose to enzyme [16]. In the present study, hemicelluloses were fractionated from hydrothermal pretreated fibers (100–220 ◦ C). The structural and physicochemical features of the residual hemicelluloses were examined in detail by means of sugar analysis, molecular weights, FT-IR, and NMR spectroscopies. 2. Materials and methods 2.1. Material Thermo-mechanical fiber obtained at 120 ◦ C for 1–2 min from Eucalyptus urophylla, as raw material (Eucalyptus fiber), was kindly supplied by the State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, China. The sample was ovendried at 50 ◦ C for 24 h and dewaxed with methylbenzene/ethanol (2:1, v/v) in a Soxhlet apparatus for 3 h, and the sample was dried in an oven at 60 ◦ C for 16 h. The composition of the dewaxed fiber was determined to be 45.0% cellulose, 21.7% hemicelluloses, 25.8% Klason lignin, and 1.3% acid soluble lignin by National Renewable Energy Laboratory’s (NREL) standard analytical procedure [17]. All chemicals purchased were of analytical or reagent grade and used without further purification. Fig. 1. shows the scheme for fractionating residual hemicelluloses from hydrothermal pretreated Eucalyptus fibers. 2.2. Hydrothermal pretreatment process The hydrothermal process was carried out as follow. A total of 15.0 g Eucalyptus fiber was dispersed in 450 mL distilled water and transferred to a 1 L Parr stirred pressure reactor (Parr Instrument Company, Moline, Illinois). Then the reactor was heated to
100 ◦ C (60 min), 120 ◦ C (60 min), 140 ◦ C (60 min), 160 ◦ C (60 min), 180 ◦ C (15, 30, 45, and 60 min), 200 ◦ C (30 min), and 220 ◦ C (30 min), respectively, at a heating rate of about 4 ◦ C/min. Once the desired operation reached, the reactor was cooled by flowing water through an internal stainless steel loop. At the end of incubation, the mixtures were cooled to room temperature. The liquid stream was separated by filtration with a nylon cloth from the solid residues and then the residues were washed with 450 mL distilled water, dried in an oven at 60 ◦ C for 12 h and labeled as AM100-60 , AM120-60 , AM140-60 , AM160-60 , AM180-15 , AM180-30 , AM180-45 , AM180-60 , AM200-30 , and AM220-30 , respectively, according to the pretreatment temperature and time. The chemical composition of the raw material and hydrothermal pretreated fibers was also determined by National Renewable Energy Laboratory’s (NREL) standard analytical procedure [17]. 2.3. Fractionation of residual hemicelluloses from hydrothermal pretreated fibers The hydrothermal pretreated fibers (5.0 g) was post-treated with 2% NaOH at 90 ◦ C for 2.5 h with a solid-to-liquid ratio of 1:30 (g/mL), and then filtered. Afterwards, the supernatant was adjusted to pH = 5.5–6.0 with 6 M HCl, concentrated to about 60 mL on a rotary evaporator under reduced pressure, and then mixed with three volumes of 95% ethanol, precipitated for 1 h, and finally filtered through filter paper to isolate hemicelluloses. The residual hemicelluloses obtained were freeze-dried and labeled as AH100-60 , AH120-60 , AH140-60 , AH160-60 , AH180-15 , AH180-30 , AH180-45 , AH180-60 , AH200-30 , and AH220-30 , respectively, according to the pretreatment temperature and time. However, no hemicellulosic polymer could be obtained when the hydrothermal pretreatment temperature was higher than 180 ◦ C. For comparison, the control hemicellulosic fraction AH prepared without hydrothermal pretreatment was also fractionated by treating raw material (Eucalyptus fiber 5.0 g) with 2% NaOH using a solid-to-liquid ratio of 1:30 (g/mL) at 90 ◦ C for 2.5 h. All experiments were performed at least in duplicate. 2.4. Chemical characterization The contents of acid insoluble lignin in the residual hemicelluloses were measured according to the Klason method [18]. The composition of neutral sugars and uronic acids of the hemicelluloses were determined by high performance anion exchange chromatography (HPAEC) as previously reported by Sun et al. [19]. The molecular weights and molecular weight distributions of all hemicelluloses were examined by gel permeation chromatograph according to a previous method [19].
160
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
Table 1 The percentage of xylo-oligosaccharides in initial xylan (%). HPTea (◦ C)
100
120
140
160
180
180
180
180
HPTib (min) XOSc (%)
60 2.59
60 3.96
60 11.25
60 37.51
15 38.90
30 43.55
45 34.21
60 28.63
a b c
HPTe, hydrothermal pretreatment temperature. HPTi, hydrothermal pretreatment time. XOS, xylo-oligosaccharide.
2.5. Spectroscopic characterization The FT-IR spectra of the residual hemicelluloses were recorded on a Bruker spectrophotometer in the range of 400–4000 cm−1 with a resolution of 4 cm−1 . A KBr disc containing 1% finely ground sample was used for measurement. The soluble-state 13 C- and heteronuclear single quantum coherence (HSQC) NMR spectra were recorded on a Bruker AV III 400 MHz spectrometer operating in the FT mode at 100.6 MHz. The 13 C- and HSQC NMR experiments were conducted with 60 and 20 mg lignins dissolved in 1 mL D2 O, respectively. The parameters for data acquisition were set according to a previous literature [19].
3. Result and discussion 3.1. Production of xylo-oligosaccharides and chemical composition of hydrothermal pretreated fibers Table 1 shows that the yield of xylo-oligosaccharides increased with the increasing of temperature (100–160 ◦ C) for 60 min and then decreased at 180 ◦ C. The maximum yield of xylooligosaccharide of 43.55% of the initial xylan was achieved at 180 ◦ C for 30 min. A further increase in pretreatment time resulted in a decrease of the xylo-oligosaccharide yield (28.63% of the initial xylan at 180 ◦ C for 60 min). The reason for this was probable that large amounts of xylan and xylo-oligosaccharides were mainly degraded into xylose and sugar degradation products for a longer pretreatment period under this condition. As expected, after the hydrothermal pretreatment, the pretreated fibers consisted mainly of cellulose and lignin together with small amounts of residual hemicelluloses. As can be seen, an increasing the pretreatment temperature from 100 to 180 ◦ C led to a significant decrease of hemicelluloses from 19.51% to 1.69% in the hydrothermal pretreated samples, which was due to the fact that the chemical basis of the autohydrolysis process is the hydrolysis reaction of hemicelluloses in aqueous medium. Meanwhile, the
contents of cellulose and lignin in the pretreated fibers increased from 45.01% to 59.69% and 25.84% to 54.35% with the increasing temperature (100–180 ◦ C), which was mainly due to the removal of hemicelluloses. 3.2. Yields and sugar components of residual hemicelluloses The yield of the hemicelluloses isolated and the content of lignin in the residual hemicelluloses are also given in Table 2. As can be seen, the yield of the hemicelluloses first increased from 7.11% to 11.26% with the increment of the pretreatment temperature from 100 to 140 ◦ C and then decreased (3.28–5.58%) with the temperature at 160–180 ◦ C. Furthermore, it was found that the residual hemicelluloses at low pretreatment temperatures (100–140 ◦ C) or without hydrothermal pretreatment contained relatively low amounts of lignin (5.22–8.53%). However, when the pretreatment temperature increased to 160–180 ◦ C, the content of lignin in the residual hemicelluloses increased to 53.58–74.84%, which was probably due to the degraded lignin associated with hemicelluloses. The result indicated that a combination of hydrothermal pretreatment at low temperatures (100–140 ◦ C) and alkali post-treatment was an effective treatment for the fractionation of hemicelluloses from Eucalyptus fiber. As we known, hemicelluloses are not chemically homogeneous and different hydrolytic technologies and various pretreatment methods are available for both fractionation and solubilization of hemicelluloses from biomass. In this study, to analyze the difference among these hemicelluloses, the released monomeric sugars and uronic acids from hemicelluloses by acid hydrolysis were monitored with HPAEC (Table 3). Sugar composition analysis indicated that the residual hemicelluloses (AH100-60 –AH160-60 ) prepared at temperature lower than 180 ◦ C are rich in xylose (76.73–82.35%), glucuronic acid (GluA, 9.27–10.76%), and galactose (5.04–6.49%). Glucose (0.91–2.32%), arabinose (1.05–2.02%), and rhamnose (0.45–0.82%) were present in small amounts. The results suggested that the residual hemicelluloses prepared from Eucalyptus fiber with hydrothermal pretreatment at low
Table 2 The chemical composition (w/w, %) of the raw fiber and the hydrothermal pretreated fibers, the yields of residual hemicelluloes isolated with 2% NaOH at 90 ◦ C for 2.5 h from the hydrothermal pretreated fibers, and the content of acid insoluble lignin in the isolated residual hemicelluloes. Chemical compositiona (w/w, %)
RM AM100-60 AM120-60 AM140-60 AM160-60 AM180-15 AM180-30 AM180-45 AM180-60 a
Hemicelluloses
AILb
Cellulose
21.66 19.51 17.34 17.17 7.56 4.81 3.65 3.60 1.69
25.79 25.84 25.91 26.17 28.93 29.82 31.79 32.22 33.46
45.01 43.91 43.67 45.88 53.74 55.74 56.04 57.08 59.69
Hemicellulosic fractions
Yieldc
AILCd
AH AH100-60 AH120-60 AH140-60 AH160-60 AH180-15 AH180-30 AH180-45 AH180-60
8.69 7.11 7.26 11.26 5.58 4.00 3.20 4.99 3.28
5.22 6.75 7.33 8.53 53.58 61.22 68.98 69.71 74.84
Absolute weight percentage based on the raw fiber and the hydrothermal pretreated fibers. AIL represents acid insoluble lignin. c Yields of the AH and residual hemicelluloses (AH100-60 –AH180-60 ) represent the percentage of the hemicelluloses in the raw material and the hydrothermal pretreated fibers (AM100-60 –AM180-60 ), respectively. d AILC represents acid insoluble lignin content in the residual hemicelluloses. b
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
161
Table 3 Contents of neutral sugars and uronic acids in the residual hemicelluloses obtained by alkali extraction of the hydrothermal pretreated fibers. Neutral sugars and uronic acidsa
AH AH100-60 AH120-60 AH140-60 AH160-60 AH180-15 AH180-30 AH180-45 AH180-60 a b c d
Rha
Ara
Gal
Glu
Xyl
Man
GluA
GalA
0.95 0.82 0.79 0.54 0.45 0.52 ND ND ND
3.48 2.02 1.66 1.19 1.05 2.09 3.24 5.42 5.38
7.01 6.49 6.33 5.34 5.04 5.02 4.43 3.85 3.74
5.20 2.32 2.16 1.62 0.91 12.47 14.57 18.94 20.36
72.11 76.73 78.03 80.82 82.35 72.09 67.79 66.01 62.79
1.50 0.86 0.71 0.49 0.93 7.81 9.97 5.78 7.73
9.48 10.76 10.32 10.00 9.27 ND ND ND ND
0.28 NDd ND ND ND ND ND ND ND
Xyl/Arab
Xyl/UAc
7.6 7.1 7.6 8.1 8.9 34.5 20.9 12.2 11.7
20.7 38.0 47.0 67.9 78.4 ND ND ND ND
Rha, rhamnose; Ara, arabinose; Gal, galactose; Glu, glucose; Xyl, xylose; Man, mannose; GluA, glucuronic acid; GalA, galacturonic acid. Xylose to arabinose ratio. Xylose to uronic acids ratio. ND, not detected.
temperatures were mainly composed of glucuronoxylans-type polysaccharides (GX), which was the typical hemicellulosic component in hardwoods. Generally, GX consists of a linear backbone of -d-xylopyranosyl units (Xylp) linked by -(1,4) glycosidic bonds. Some xylose units are acetylated at C2 and C3 and one in 10 molecules has an uronic acid group (4-O-methylglucuronic acid) attached by ␣-(1,2) linkages [20]. However, under the condition of alkali treatment, the cleavage of O-acetyl groups occurs and all acetyl groups are split off [21]. Therefore, in GX of Eucalyptus fiber, GluA was originated from 4-O-methyl-d-glucoronic acid as side chain, which can be confirmed by the following NMR analysis. A similar sugar composition was observed in the control hemicellulosic fraction AH and the residual hemicelluloses, except galacturonic acid (GalA, 0.28%) that was only detected with a minor amount in AH. Surprisingly, when the temperature reached 180 ◦ C, the residual hemicelluloses AH180-15 –AH180-60 presented low xylose (62.79–72.09%), galactose (3.74–5.02%), and rhamnose (0–0.52%) contents, but high glucose (12.47–20.36%) and mannose (5.78–9.97%) contents. These data suggested that the residual hemicelluloses prepared at a high temperature contained high glucomannan contents together with a small amount of ␣-glucan. Also notable was that the amount of GluA in AH100-60 –AH160-60 reached around 10%, while GluA was not detected in AH180-15 –AH180-60 . In addition, it can be seen from Table 3 that as the reaction temperature increased from 100 to 160 ◦ C, xylose content increased from 76.73 to 82.35%, while the contents of arabinose, galactose, and glucose decreased from 2.02, 6.49 and 2.32 to 1.05%, 5.04%, and 0.91%, respectively. For AH180-15 –AH180-60 , with pretreating time increasing from 15 to 60 min, the contents of galactose and xylose decreased, but the content of glucose increased. Generally, the ratios of xylose to uronic acid (Xyl/UA) and xylose to arabinose (Xyl/Ara) are indicative of the degree of linearity or branching of hemicelluloses. Obviously, the Xyl/UA and Xyl/Ara ratios increased from 7.1 (AH100-60 ) and 38.0 (AH100-60 ) to 8.9 (AH160-60 ) and 78.4 (AH160-60 ), respectively, indicating that a higher pretreating temperature resulted in the isolation of hemicelluloses with less branches. In light of this, hemicelluloses rich in backbone structure could be obtained at high temperature. On the contrary, at low temperature, hemicelluloses with some side chains and more complex structure were obtained. This can be explained by the fact that the presence of large unsubstituted regions in xylan backbone can lead to strong hydrogen bonds, causing interchain aggregation and more difficult isolation [22]. Consequently, a high isolating temperature by alkali post-treatment was required. However, with the pretreating temperature increasing to 180 ◦ C, hemicelluloses with less branches were obtained, as shown by low Xyl/Ara ratios (11.7–34.5).
3.3. Molecular weight distribution In order to investigate the degradation during hydrothermal pretreatment, all the isolated hemicelluloses were further analyzed by the determination of their weight-average (Mw ), numberaverage (Mn ) molecular weights and polydispersity (Mw /Mn ), and the results are summarized in Table 4. The residual hemicelluloses AH100-60 had a high Mw (56,520 g/mol) and Mn (26,330 g/mol), which was comparable to AH (Mw = 44,040 and Mn = 20,330 g/mol). It was probably ascribed to the effect of hydrothermal swelling of the cell walls, resulting in the release of hemicelluloses with high molecular weights in the following alkaline extraction. However, the Mw (41,870–7780 g/mol) and Mn (20,500–6650 g/mol) of AH120-60 –AH160-60 and AH180-15 –AH180-60 were lower than those of AH. This was probably due to the cleavage of glycosidic ether linkages between sugar units, which led to the difference in degree of degradation of hemicelluloses. Based on this observation and Xyl/Ara ratios, the residual hemicelluloses AH100-60 –AH160-60 with a high Xyl/Ara ratio had a low Mw , indicating that an increase of the hydrothermal pretreatment temperature resulted in a significant degradation of the hemicellulosic polymers, which was in agreement with the previous study by Dervilly et al. [23]. In contrast, the hemicellulosic fractions AH180-15 –AH180-60 obtained at a higher temperature of 180 ◦ C with an increment of the pretreating time from 15 to 60 min showed a decreasing trend of Xyl/Ara ratios and molecular weights, suggesting that an extension of the pretreating time also led to a depolymerision of the hemicelluloses at 180 ◦ C, which was in accordance with the results found by Peng and Roels et al. [24,25]. Polydispersity is an important parameter of macromolecules relative to their applications in chemical industry. The polydispersities of the obtained hemicellulosic fractions ranged from 1.11 to 2.17. It has been reported that polysaccharides with Mw /Mn value below 3 were molecularly uniform polymers and have potential Table 4 Weight-average (Mw ) and number-average (Mn ) molecular weights and polydispersity (Mw /Mn ) of the residual hemicelluloses isolated by 2% NaOH at 90 ◦ C for 2.5 h from the raw fiber and the hydrothermal pretreated fibers. Hemicellulosic fractions
Mw
Mn
Mw /Mn
AH AH100-60 AH120-60 AH140-60 AH160-60 AH180-15 AH180-30 AH180-45 AH180-60
44,040 56,520 41,870 39,570 16,990 13,530 13,470 8910 7780
20,330 26,330 20,500 24,990 13,170 10,790 10,750 7390 6650
2.17 2.15 2.04 1.58 1.29 1.25 1.25 1.21 1.17
162
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
Fig. 3. 13 C-NMR spectra of the residual hemicelluloses AH, AH100-60 , AH160-60 , and AH180-15 .
Fig. 2. FT-IR spectra of the residual hemicelluloses AH, AH100-60 , AH120-60 , AH140-60 , AH160-60 , and AH180-15 .
commercial utilization [26]. It was noticeable that the Mw /Mn value of hemicelluloses decreased with the increased pretreatment temperature. 3.4. FT-IR spectra Fig. 2 shows that the FT-IR spectra of fractions AH, AH100-60 , AH120-60 , AH140-60 , AH160-60 , and AH180-15 were rather similar. Generally, absorptions in the region of 1200–800 cm−1 give the information about the main polysaccharides. Most of the absorption peaks can be assigned according to literatures [19,27,28]. The absorptions at 3443 and 2937 cm−1 are attributed to the OH and C H stretching vibrations, respectively. As expected, the disappearance of the band at about 1730 cm−1 (the carbonyl stretching region of hemicelluloses) revealed that the hydrothermal pretreatment followed with alkaline extraction cleaved the ester bands of hemicelluloses, such as acetyl and uronic ester groups. The CH2 stretching vibration exhibits a signal at 1462 cm−1 and the band at 1414 cm−1 represents symmetric stretching vibration of glucuronic acid group. Moreover, the bands at 1387 and 1248 cm−1 are originated from OH and C O bending vibration in hemicelluloses. The occurrence of a peak at 1119 cm−1 is due to C OH skeletal vibration, whereas the absorption band at 1045 cm−1 is related to C O, C C stretching or C OH bending in hemicelluloses, indicating the typical xylan in the hemicelluloses. In addition, the peak at 989 cm−1 is indicative of arabinofuranosyl, which has been reported to be attached to the position 3 of the xylopyranosyl constituents. In anomeric region (950–700 cm−1 ), an important band at 897 cm−1 is originated from C1 group frequency or ring frequency, which is characteristic of -glycosidic linkages between the sugar units. It
should be noted that in the isolated hemicelluloses, the intensity of the band at 1045 cm−1 decreased but that of the band at 1516 cm−1 (the phenolic ring absorbance of lignin) [29] increased with the raise of the hydrothermal pretreatment temperature, which was probably due to the increasing percentage of lignin in the residual hemicelluloses (Table 2). 3.5. NMR spectra analysis To further characterize the structural features of hemicelluloses, the four fractions AH, AH100-60 , AH160-60 , and AH180-15 were comparatively investigated by 13 C NMR spectra (Fig. 3). Clearly, the five strong signals of (1 → 4) linked -d-Xylp residues were detected at 102.0 (C-1), 76.0 (C-4), 74.9 (C-3), 73.3 (C-2), and 63.4 (C-5) ppm [30]. Weak signals at 177.1, 97.5, 82.7, 72.2, and 59.6 ppm, corresponding to COOH, C-1, C-4, C-5, and OCH3 of the 4O-methyl-d-glucoronic acids in hemicelluloses, respectively, were also observed [31]. Meanwhile, the signals at 55.7 and 181.5 ppm are ascribed to OCH3 in the guaiacyl and syringyl units and carbonyl group, respectively. Obviously, the intensities of -d-Xylp and 4-O-methyl-d-glucoronic acids decreased while the intensities of lignin increased with the increment of hydrothermal pretreatment temperature, corresponding to the content of lignin in the residual hemicelluloses. More specific information about the structural variation among hemicellulosic fractions AH, AH100-60 , AH160-60 , and AH180-15 were further investigated by 2D HSQC NMR spectra (Fig. 4). The marked 1 H 13 C cross-peaks further confirmed the structural elements of (1 → 4)-linked -d-Xylp, 4-O-methyl-d-glucoronic acids, and (1 → 4)--d-Xylp-2-O-(4-OMe-d-GlcpA) units. Specially, in the HSQC spectrum of AH, five dominant cross peaks at 102.1/4.34, 73.0/3.19, 74.5/3.41, 76.0/3.67, and 63.1/3.28 and 3.98 ppm, are assigned to C1–H1, C2–H2, C3–H3, C4–H4, and C5–H5 of (1 → 4)linked -d-Xylp units, respectively. In addition, the signals at 97.4/5.19, 71.5/3.46, 72.2/3.60, 82.4/3.11, and 72.1/4.22 ppm are, respectively assigned to C1–H1, C2–H2, C3–H3, C4–H4, and C5–H5 of 4-O-methyl-d-glucoronic acids units. The signals of
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
163
Fig. 4. HSQC spectra of the residual hemicelluloses AH, AH100-60 , AH160-60 , and AH180-15 . X: (1 → 4)--D-Xylp; UA: 4-O-Methyl-␣-D-GlcpA; XU: (1 → 4)--D-Xylp-2-O-(4OMe-D-GlcpA).
(1 → 4)--d-Xylp-2-O-(4-OMe-d-GlcpA) units with less intensity were detected at 100.8/4.54 (C1–H1) and 76.4/3.33 ppm (C2–H2). Furthermore, it was noticeable that the signals of 4-O-methyl-dglucoronic acids were not detected in the spectrum of AH180-15 , in accordance with the results of sugar analysis. These data concluded that the hemicelluloses isolated with 2% NaOH from the hydrothermal pretreated fibers were assumed to be mainly composed of (1 → 4)-linked -d-Xylp attached with various monosaccharides and small amounts of glucomannans and ␣-d-glucan. 4. Conclusions In summary, the hydrothermal pretreatment significantly hydrolyzed the glycosidic linkages in xylan backbone and thus reduced the molecular weight of the residual hemicelluloses extracted with alkali from hydrothermal pretreated fibers. The residual hemicelluloses isolated at low pretreatment temperatures (100–140 ◦ C) contained low amounts of lignin (6.75–8.53%), but high amounts of lignin (53.58–74.84%) was observed in the hemicelluloses obtained at high pretreatment temperatures (160–180 ◦ C). The residual hemicelluloses isolated from the hydrothermal pretreated fibers at 100–160 ◦ C were rich in xylose and glucuronic acid, while a higher temperature (180 ◦ C) resulted in hemicelluloses being rich in xylose, glucose, and mannose. Moreover, the Xyl/UA and Xyl/Ara ratios increased from 7.1 and 38.0 (AH100-60 pretreated at 100 ◦ C for 60 min) to 8.9 and 78.4 (AH160-60 pretreated at 160 ◦ C for 60 min), respectively, indicating that a
higher temperature resulted in the isolation of hemicelluloses with less branches. In conclusion, the hydrothermal pretreatment at low temperatures followed by alkali treatment is an effective method for the fractionation of hemicelluloses from Eucalyptus fiber.
Acknowledgments The authors are extremely grateful to financial support from the Major State Basic Research Projects of China (973-2010CB732204) and State Forestry Administration (201204803).
References [1] S. Fernando, S. Adhikari, C. Chandrapal, N. Murali, Biorefineries: current status, challenges, and future direction, Energy Fuels 20 (2006) 1727–1737. [2] P. Kaparaju, M. Serrano, A.B. Thomsen, P. Kongjan, I. Angelidaki, Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept, Bioresour. Technol. 100 (2009) 2562–2568. [3] A.S. Mamman, J.M. Lee, Y.C. Kim, I.T. Hwang, N.J. Park, Y.K. Hwang, J.S. Chang, J.S. Hwang, Furfural: hemicellulose/xylose derived biochemical, Biofuels, Bioprod. Biorefin. 2 (2008) 438–454. [4] E. Sjöström, in: E. Sjöström (Ed.), Wood Chemistry: Fundamentals and Applications, Academic Press, New York, NY, 1981. [5] A. Ebringerova, Z. Hromadkova, T. Heinze, in: T. Heinze (Ed.), Polysaccharides I, Springer-Verlag, Berlin, 2005, pp. 1–67. [6] N.M. Hansen, D. Plackett, Sustainable films and coatings from hemicelluloses: a review, Biomacromolecules 9 (2008) 1493–1505. [7] X. Li, X.J. Pan, Hydrogels based on hemicellulose and lignin from lignocellulose biorefinery: a mini-review, J. Biobased Mater. Bioenergy 4 (2010) 289–297.
164
S.-N. Sun et al. / International Journal of Biological Macromolecules 69 (2014) 158–164
[8] S. Mateo, J.G. Puentes, S. Sánchez, A.J. Moya, Oligosaccharides and monomeric carbohydrates production from olive tree pruning biomass, Carbohydr. Polym. 93 (2012) 416–423. [9] B.C. Saha, Hemicellulose bioconversion, J. Ind. Microbiol. Biotechnol. 30 (2003) 279–291. [10] N. Mosier, C. Wyman, B. Dale, R. Elander, Y.Y. Lee, M. Holtzapple, M. Ladisch, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresour. Technol. 96 (2005) 673–686. [11] B. Yang, C.E. Wyman, Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels, Bioprod. Biorefin. 2 (2008) 26–40. [12] T. Ghose, Measurement of cellulase activities, Pure Appl. Chem. 59 (1987) 257–268. [13] P. Gullón, A. Romaní, C. Vila, G. Garrote, J.C. Parajó, Potential of hydrothermal treatments in lignocellulose biorefineries, Biofuels, Bioprod. Biorefin. 6 (2012) 219–232. [14] F. Carvalheiro, T. Silva-Fernandes, L.C. Duarte, F.M. Gírio, Wheat straw autohydrolysis: process optimization and products characterization, Appl. Biochem. Biotechnol. 153 (2009) 84–93. [15] M.J. Taherzadeh, K. Karimi, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review, Int. J. Mol. Sci. 9 (2008) 1621–1651. [16] D. Haverty, K. Dussan, A.V. Piterina, J. Leahy, M. Hayes, Autothermal, singlestage, performic acid pretreatment of Miscanthus × giganteus for the rapid fractionation of its biomass components into a lignin/hemicellulose-rich liquor and a cellulase-digestible pulp, Bioresour. Technol. 109 (2012) 173–177. [17] A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, D. Crocker, Laboratory Analytical Procedure (LAP): determination of structural carbohydrates and lignin in biomass, in: Technical Report NREL/TP-510-42618, National Renewable Energy Laboratory, Golden, CO, 2008. [18] C.W. Dence, in: S.Y. Lin, C.W. Dence (Eds.), Methods in Lignin Chemistry, Springer-Verlag, Berlin, 1992, pp. 33–61. [19] S.L. Sun, J.L. Wen, M.G. Ma, R.C. Sun, Successive alkali extraction and structural characterization of hemicelluloses from sweet sorghum stem, Carbohydr. Polym. 92 (2012) 2224–2231. [20] F. Gírio, C. Fonseca, F. Carvalheiro, L. Duarte, S. Marques, R. Bogel-Łukasik, Hemicelluloses for fuel ethanol: a review, Bioresour. Technol. 101 (2010) 4775–4800.
[21] F. Peng, P. Peng, F. Xu, R.C. Sun, Fractional purification and bioconversion of hemicelluloses, Biotechnol. Adv. 30 (2012) 879–903. [22] A. Höije, M. Gröndahl, K. Tømmeraas, P. Gatenholm, Isolation and characterization of physicochemical and material properties of arabinoxylans from barley husks, Carbohydr. Polym. 61 (2005) 266–275. [23] G. Dervilly, L. Saulnier, P. Roger, J.F. Thibault, Isolation of homogeneous fractions from wheat water-soluble arabinoxylans. Influence of the structure on their macromolecular characteristics, J. Agric. Food Chem. 48 (2000) 270–278. [24] F. Peng, J. Bian, J.L. Ren, P. Peng, F. Xu, R.C. Sun, Fractionation and characterization of alkali-extracted hemicelluloses from peashrub, Biomass Bioenergy 39 (2012) 20–30. [25] S.P. Roels, M. Collado, A.M. Loosveld, P.J. Grobet, J.A. Delcour, Variation in the degree of D-xylose substitution in water-extractable European durum wheat (Triticum durum Desf.) semolina arabinoxylans, J. Agric. Food Chem. 47 (1999) 1813–1816. [26] W.G. Glasser, W.E. Kaar, R.K. Jain, J.E. Sealey, Isolation options for non-cellulosic heteropolysaccharides (HetPS), Cellulose 7 (2000) 299–317. [27] D.K. Buslov, F.N. Kaputski, N.I. Sushko, V.I. Torgashev, L.V. Solov’eva, V.M. Tsarenkov, O.V. Zubets, L.V. Larchenko, Infrared spectroscopic analysis of the structure of xylans, J. Appl. Spectrosc. 76 (2009) 801–805. [28] M. Kaˇcuráková, P.S. Belton, R.H. Wilson, J. Hirsch, A. Ebringerová, Hydration properties of xylan-type structures: an FTIR study of xylooligosaccharides, J. Sci. Food Agric. 77 (1998) 38–44. [29] S.N. Sun, M.F. Li, T.Q. Yuan, F. Xu, R.C. Sun, Effect of ionic liquid/organic solvent pretreatment on the enzymatic hydrolysis of corncob for bioethanol production. Part 1: Structural characterization of the lignins, Ind. Crops Prod. 43 (2013) 570–577. [30] A. Teleman, J. Lundqvist, F. Tjerneld, H. Stålbrand, O. Dahlman, Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1 H and 13 C NMR spectroscopy, Carbohydr. Res. 329 (2000) 807–815. [31] A. Bendahou, A. Dufresne, H. Kaddami, Y. Habibi, Isolation and structural characterization of hemicelluloses from palm of Phoenix dactylifera L., Carbohydr. Polym. 68 (2007) 601–608.
