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Microwave-enhanced extraction of lignin from birch in formic acid: Structural characterization and antioxidant activity study
Process Biochemistry 47 (2012) 1799–1806
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Microwave-enhanced extraction of lignin from birch in formic acid: Structural characterization and antioxidant activity study Shuai Zhou a , Lu Liu a , Bo Wang a,∗ , Feng Xu a , Runcang Sun a,b a b
Institute of Biomass Chemistry and Technology, College of Materials Science and Technology, Beijing Forestry University, Beijing 100083, PR China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e
i n f o
Article history: Received 27 March 2012 Received in revised form 6 June 2012 Accepted 10 June 2012 Available online 18 June 2012 Keywords: Birch lignin Microwave-enhanced extraction FTIR HSQC Antioxidant activity
a b s t r a c t A rapid and mild extraction protocol for the preparation of lignin was achieved by microwave-assisted heating in formic acid at 101 ◦ C under atmospheric pressure. In this case, birch lignin was extracted with microwave heating process (ML) in formic acid and characterized by elemental analysis, FTIR, GPC, 1 H NMR and 13 C–1 H HSQC. In addition, the antioxidant activity of the samples was investigated. For comparative study, milled wood lignin (MWL) and lignin extracted with oil bath heating process (OL) were prepared. The results showed that the lignin yield under microwave heating was much higher than that under oil bath heating. A maximal delignification degree (89.77%) was achieved when microwave heating time was 30 min. When double time (60 min) was used under oil bath heating, the delignification degree was 66.11%. The structural characterization showed that the lignin structure of ML did not change dramatically, which is a mixture of GS-type with -O-4 ether bond as the major inter-unit linkage. As for antioxidant activity against DPPH, the radical scavenging index (RSI) of ML was 1.20, which was higher than that of MWL (0.53), suggesting that ML exhibited much higher antioxidant activity than MWL. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction Lignocelluloses, as renewable sources for the production of various chemicals, materials, and fuels, have attracted increasing attention due to the emerging depletion of fossil fuels together with the high pressure of carbon dioxide discharge. Lignocelluloses are principally composed of cellulose, hemicelluloses, and lignin. Cellulose and most of hemicelluloses are structural carbohydrates, forming the bulk of the plant cells’ supporting skeleton. Lignin fills the space between cellulose and hemicelluloses, and cross-links with hemicellulosic polysaccharides [1,2] mainly through covalent bonds (ether and ester) [3,4] and phenyl glycosidic linkages [5]. Lignin gives stiffness to the cell wall, enabling it to serve as a mechanical support to build up the stem and branches. Due to the aromatic features, lignin makes the cell wall hydrophobic, which is a prerequisite for the development of cells for efficient water- and nutrition-transport. In addition, lignin functions as protection against microbial degradation. Owing to its special functional groups, such as phenolic hydroxyl, alcoholic hydroxyl, and carboxyl groups, lignin can be used for the production of materials with unique properties, such as dispersants, absorbents, resin, surfactants, and so forth [6,7]. In addition, lignin also exhibits
∗ Corresponding author. Tel.: +86 10 62336592; fax: +86 10 62336972. E-mail addresses: [email protected], [email protected] (B. Wang).
pharmacological properties. Due to its antioxidant activity [8], for instance, lignin is used as inhibitor or neutralizer in oxidation processes to stabilize reactions induced by oxygen radicals and their derived species [9]. It has been reported that lignin obtained from waste of pulp and paper production (in kraft and prehydrolysis processes) can inhibit mutagenicity and SOS response and it shows protective effect on DNA, which suggests that it is a promising antimutagenic and anticarcinogenic agent [10]. Besides, the potential cytotoxic effect on cells of lignin indicates that it is suitable for the cosmetic production without any harmful effects on human cells [11,12]. The commercially available lignin is mainly obtained as byproduct from chemical pulp mill. In these processes, lignin is liberated from lignocelluloses at elevated temperatures (over 160 ◦ C) under high pressures in alkaline or acidic solution for several hours. The extracted lignin is highly degraded, condensed or modified, thus it has limited usages. To explore more efficient methods to separate components from lignocelluloses, numerous processes, such as organic solvent fractionation [13], steam explosion [14], alkaline hydrogen peroxide extraction [15], microwave extraction [16], etc., have been developed and applied in recent decades. Among them, microwave extraction has attracted increasing attention due to its easy operation as well as specific effect [17]. Microwave-assisted technology is a promising process, which utilizes thermal and non-thermal effects generated by microwaves in aqueous conditions. The vibration of polar molecules and the
1359-5113/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.06.006
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movement of ions lead to the generation of heat and extensive collisions, thus microwave heating can accelerate mass transfer and facilitate the dissolution of products. Generally, microwave is mainly used to extract some bio-active components with low molecular weights such as phenolic compounds [18], notoginseng saponins [19] and polysaccharides [20]. In most cases, water is selected as a medium for the treatment of lignocelluloses at elevated temperatures. The extraction process is conducted in acidic solution in fact, since organic acids are produced under high temperatures. In addition, the components in lignocelluloses can be extracted in acidic, alkaline or organic solvents according to the target products. With respect to the extraction of lignin applying microwave heating, most studies have been conducted by using aqueous alkali [21] and ethanol [22]. However, high temperature and pressure is needed to achieve an ideal delignification in these media. This is because the fundamental cleavages of the linkages between lignin units occur at elevated temperature in aqueous alkali or ethanol. Formic acid, an easily recycled organic acid, is a good solution to extract lignin. It has been applied to pulping of many wood and annual plants in the early stage [23,24] and fractionation of lignocelluloses in a biorefinery manner recently [25,26]. Since formic acid is a polar solvent, it is helpful that it can be used as a medium for microwave irradiation. Therefore, in the present study, birch, a typical hardwood species, was subjected to microwave irradiation in aqueous formic acid, aiming at extracting lignin in a highly efficient way. The microwave-assisted extraction of lignin in formic acid provides a novel process which has the advantages of higher extraction yield, lower extraction temperature, and easier recycling of solvent, as compared to the conventional way of microwave-assisted extraction aforementioned. The structure of the extracted lignin was characterized with a set of wet chemistry (elemental and sugar analysis) and spectroscopy (Fourier transform infrared spectroscopy (FTIR), 1 H nuclear magnetic resonance (1 H NMR) and heteronuclear singular quantum correlation (HSQC)) methods. In addition, in order to find the structural changes of lignin, the extracted lignins under different conditions were compared to that from the oil bath heating process, as well as the native lignin. Furthermore, the antioxidant activity of the lignins was also evaluated since it is closely correlated to the structure. 2. Materials and methods 2.1. Materials Birch (Betula alnoides) was harvested from Yunnan province, southwest of China. The outer and inner layers of bark were peeled off and the stem was chopped into small pieces, and then ground to obtain meal. The wood meal sized in 40–60 mesh was collected. The meal was extracted with toluene/ethanol (2:1, v/v) in a Soxhlet apparatus, air-dried and stored in a hermetic polypropylene container before use. The lignin content in the dewaxed material was determined as 26.36%.
For comparative study, lignin was extracted from birch under the same extracting conditions (88% aqueous formic acid, 101 ◦ C) with oil bath heating. The oil bath was equipped with a magnetic stirring system and water-cooled condenser. The temperature of the oil bath was controlled by an adjustable thermometer inserted into the extraction system. The aqueous formic acid (20 mL) was introduced in a 100 mL three-neck flask and heated to 101 ◦ C. Then the dewaxed birch (2 g) was immersed into a three-neck flask and kept for a given time (10, 20, 30, and 60 min, respectively). After cooking, the mixtures were treated similarly to the procedure of microwave heating extraction. In addition, as milled wood lignin (MWL) is considered close to native lignin of lignocelluloses, it was prepared according to a previous report [27] for comparison. The lignin fraction extracted by microwave heating was defined as ML and that by oil bath heating was marked as OL. The weight of the lignin fraction was determined gravimetrically. Extraction yield was measured gravimetrically after the solid product was drying to constant weight. Lignin yield was calculated according to the formula: lignin yield (%) = (1 − sugar content) × extraction yield/lignin content in raw material × 100. Lignin content in residue was determined according to acetyl bromide method [28] on UV 2300 (Shanghai Tianmei Science and Technology Corporation, China) using 1 cm cells. Delignification degree was calculated according to the formula: delignification degree (%) = (1 − weight of lignin in residue/weight of lignin in raw material) × 100. 2.3. Component analysis and structural characterization Fourier transform infrared (FTIR) spectra were recorded on a spectrophotometer (Nicolet iN10, USA) with a MCT detector in the region of 4000–650 cm−1 at a resolution of 4 cm−1 . Elemental analysis was performed to determine the carbon, hydrogen, and nitrogen content in the lignin samples using a Vario EL III Elemental analyzer instrument (Elementar, Germany). Oxygen content was deduced from the difference with respect to the total samples. The methoxyl content in lignin was determined as described by Mousavioun and Doherty [29]. The molecular weight distribution of lignin was measured by gel permeation chromatography (GPC, Agilent 1200, USA) using a PL-gel 10 mm Mixed-B 7.5 mm ID column, calibrated with monodisperse polystyrene. The samples were acetylated with acetic anhydride before determination. 2 mg acetylated sample was dissolved in 2 mL tetrahydrofuran (THF), and 20 L lignin solutions were injected. The column was operated at ambient temperature and THF was used as eluent with a flow rate of 1 mL/min. The measurements were conducted with three parallels and the relative standard deviation was below 5%. Sugar analysis (neutral sugars and uronic acids) was conducted by using high performance anion exchange chromatography (HPAEC). The neutral sugars and uronic acids in the samples were released by hydrolysis with 72% H2 SO4 for 2 h at 25 ◦ C followed by a high temperature hydrolysis at 105 ◦ C for 4 h after dilution to 6.1% H2 SO4 . After hydrolysis, the samples were filtered, diluted 30-fold and injected into a HPAEC system (Dionex ISC 3000, USA) with an amperometric detector, a CarbopacTMPA-20 column (4 mm × 250 mm, Dionex), and a guard PA-20 column (3 mm × 30 mm, Dionex). Neutral sugars and uronic acids were separated in isocratic 5 mM NaOH (carbonate free and purged with nitrogen) for 20 min, followed by a 0.75 mM NaAc gradient in 5 mM NaOH for 15 min with a flow rate of 0.4 mL/min. Calibration was performed with standard solutions of sugars. Results of the yield and sugar composition of the samples are presented as mean values of three parallels, and the relative standard deviation was below 0.5%. NMR spectra of the samples were acquired on a Bruker AVIII 400 MHz spectrometer at 25 ◦ C. The 1 H NMR spectra were obtained at 100 MHz with 15 mg acetylated sample in 1 mL CDCl3 . Heteronuclear single quantum correlation (HSQC) spectra were acquired by HSQC GE experiment mode with 20 mg lignin sample dissolved in 1 mL d-DMSO. HSQC spectral widths for 1 H and 13 C dimensions were 2200 Hz and 15,400 Hz, respectively. The number of collected complex points for 1 H dimension was 1024 with a recycle delay of 1.5 s. The number of scan was 128, and 256 time increments were recorded in the 13 C-dimension. The 1 JCH was set to 146 Hz. Prior to Fourier transform the data matrixes were zero filled up to 1024 points in the 13 C-dimension. 2.4. Antioxidant activity
2.2. Extraction of lignin from birch Microwave assisted extraction of lignin from birch was conducted in 88% formic acid solution at boiling point (101 ◦ C) under atmospheric pressure. The microwave oven used was a focused single-mode microwave synthesis system (2.45 GHz, Discover, CEM, USA), which was equipped with a magnetic stirring system and a water-cooled condenser. The temperature was measured using thermocouple-type thermometer and controlled by automatically adjusting of microwave power. When the formic acid solution has been heated to 101 ◦ C, the dewaxed material (2 g) was immersed into the preheated 88% aqueous formic acid with a solid to liquor ratio of 1:10 (g/mL). Then the mixtures were treated in the microwave oven under 700 W at 101 ◦ C for various times (5, 10, and 30 min). After cooking, the mixtures were decanted through a tared glass filter crucible (G2). In a run, the solid residue (cellulosic pulp) was washed thoroughly with 88% formic acid followed by washing with water. The filtrate was collected and concentrated with a rotatory evaporator under reduced pressure to about 8 mL. Then the concentrated liquor was added into 60 mL water to precipitate the dissolved lignin. Subsequently, the precipitated lignin was recovered by centrifugation, washed with acidified water (pH = 2), and then dried.
Antioxidant activity was determined according to the radical scavenging activity method using 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH), which was developed by Brand-Williams et al. [30] and modified by Dizhbite et al. [8]. A lignin sample was dissolved in 0.1 mL dioxane–water (9/1, v/v), and then the solution was added to 3.9 mL DPPH solution (25 mg/L in ethanol) as the free radical source. The blank sample consisted of 0.1 mL methanol and 3.9 mL DPPH solution. After a 30 min incubation period at room temperature in the dark, the decrease of the solution absorbance, due to proton donating activity, was immediately measured at 517 nm using a UV 2300 spectrometer (Shanghai Tianmei Science and Technology Corporation, China). The tests were carried out in duplicate. The original data are shown in supplementary data (Table S1). The radical scavenging activity was calculated as follows: I (%) = (A0 − A1 )/A0 ; where A0 is the absorbance of the blank, and A1 is the absorbance in the presence of the test compound at different concentrations. The IC50 (concentration providing 50% inhibition) was calculated graphically using a calibration curve in the linear range by plotting the extract concentration vs. the corresponding scavenging effect. Radical scavenging index (RSI) was defined as the inverse of IC50 .
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Table 1 Extraction yield, lignin yield and total sugar content of lignin fractions extracted with formic acid and delignification degree under microwave heating and oil bath heating. Sample No.a
Heating type
Microwave intensity (W)
Heating time (min)
Extraction yield (%)
Total sugar (%)
FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 FL9 FL10 FL11 MWL
Microwave Microwave Microwave Microwave Microwave Microwave Microwave Oil bath Oil bath Oil bath Oil bath –
400 500 600 700 800 700 700 – – – – –
30 30 30 30 30 5 10 10 20 30 60 –
19.66 21.85 23.38 26.49 26.47 8.10 15.08 6.96 9.80 16.30 18.77 –
11.97 12.02 11.68 11.70 12.32 17.12 15.84 16.88 18.16 11.74 8.33 5.46
3. Results and discussion 3.1. Yield and chemical composition of the extracted lignin samples Extraction yield, lignin yield and total sugar content of lignin fractions extracted with formic acid and delignification degree under microwave heating and oil bath heating are shown in Table 1. As can be seen from the table, as for microwave heating extraction, extraction yield, lignin yield and delignification degree increased along with the increase of the microwave intensity. This was probably caused by the specific and non-thermal microwave effects, which was still controversial [31,32]. It can be confirmed that mixtures are able to absorb microwaves in a controlled and fast manner when high power is applied [33]. And the delignification degrees under 700 W and 800 W were comparable. Therefore, the microwave intensity was set as 700 W for the following research. Besides, lignin yield and delignification degree in microwave heating extraction process are much higher than that in oil bath heating extraction process under the same extraction time. Sugar content in both ML and OL is higher than that in MWL. With the time going on, the sugar content decreased, and the lignin yield and delignification degree increased. When the extraction time was 10 min under microwave heating, the delignification degree reached 55.09%, indicating that half of the lignin in the raw material has been liberated. When the extraction time was prolonged to 30 min, 89.77% of lignin in the raw material has been removed. To achieve comparable delignification degree, the extraction duration using oil bath heating needs to be doubled. This illustrates that microwave heating was more effective to disrupt recalcitrant structures in lignocellulosic biomass to liberate lignin in cell wall than conventional heating [34]. This is probably because that water, cellulose, hemicelluloses, lignin and other low molecular compounds (i.e., the organic acid) belong to dielectrics [35]. Hence, the formic acid solution can be heated by microwaves in a dielectric way owing to its non-thermal or thermal effects [36,37]. Microwaves induce heat at the molecular level by direct conversion of the electromagnetic energy into heat [38] and by means of irradiation and heat
Lignin yield (%) 65.66 72.92 78.34 88.74 88.04 25.46 54.33 21.94 30.42 54.58 65.27 –
Delignification degree (%) 66.84 73.92 79.66 89.77 89.02 26.16 55.09 22.12 31.10 55.33 66.11
flows from inside to outside [39], while conventional heating, such as oil bath heating, by the modes of conduction and convection. For conventional heating, energy is converted to heat primarily, and then transferred along temperature gradients from the surface to the core of the material. This means that microwaves are able to penetrate into the solution immediately. Therefore it needs less energy input than oil bath heating and it can save time. To study the effect of microwave heating on the structural changes of lignin, two typical lignin fractions, ML30 (microwave heating for 30 min) and OL60 (oil bath heating for 60 min), were chosen for further investigation as compared to MWL (milled wood lignin). The elemental composition and methoxyl content of the lignin samples are presented in Table 2 along with the calculated C9 formulae. These C9 formulae were corrected as carbohydrates [40]. The elemental analysis results demonstrate that ML30 has higher carbon and lower oxygen contents than OL60 , which were comparable with that in MWL. In addition, nitrogen was not detected in all samples, implying that the extracted lignins were not contaminated by protein. 3.2. FTIR spectroscopy Fig. 1 shows the FTIR spectra of ML30 , OL60 , and MWL, and Fig. S1 shows the FTIR spectra of the solid residue under microwave heating for 30 min (MR30 ), solid residue under oil bath heating for 60 min (OR60 ) and the raw material. The FTIR spectra of the lignin samples show weak changes in the peak intensities, confirming that the “core” of the lignin structure did not change dramatically during the fractionation processes. The three lignin samples exhibit typical SG-type lignin absorption bands (Fig. 1), which is in accordance with the HSQC spectra afterwards. The bands at 1592, 1502, and 1420 cm−1 are corresponding to aromatic ring vibrations of phenyl-propane (C9 ) skeleton and the C–H deformation combined with aromatic ring vibration at 1456 cm−1 are observed in all spectra, although the intensity of the bands differs. This suggested that the aromaticity of the different samples remained the same and lignin did not change dramatically by the extraction procedure [41]. For ML30 and OL60 , the signal around 3455 cm−1 is assigned
Table 2 Elemental analysis, methoxyl content, and C9 formula of formic acid lignin fractions as compared to MWL. Samplea
MWL ML30 OL60 a b
Element and methoxyl content (%) C
H
O
OCH3
58.01 57.13 56.73
6.35 5.67 5.81
35.64 37.20 37.46
20.47 21.60 21.60
C9 formulab
C9 weight
C9 H9.11 O3.17 (OCH3 )1.49 C9 H7.16 O3.14 (OCH3 )1.71 C9 H7.77 O3.33 (OCH3 )1.67
214.10 218.48 220.89
MWL represents milled wood lignin; ML30 and OL60 correspond to lignin sample extracted under microwave heating for 30 min and oil bath heating for 60 min, respectively. Corrected as carbohydrates.
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Fig. 1. FTIR spectra of birch formic acid lignins (ML30 and OL60 ) as compared to MWL.
to the stretch of OH groups and the intensities change slightly, which means that oxidative cleavage in lignin macromolecules was negligible. The signals at 2940 and 2842 cm−1 are attributed to CH stretch in CH2 and CH3 groups. Except for these typical lignin structure bands, other signals are associated with syringyl (S) and guaiacyl (G). This is confirmed by the absorption at 830 cm−1 (C–H out-of-plane in position 2 and 6 from S unit). In addition, bands for aromatic ring breathing vibration, aromatic in-plane bending, and out-of-plane C–H bending were observed at 1324, 1216, and 1114 cm−1 for S unit, and at 1033 cm−1 and 925 cm−1 for G unit, respectively. Both syringyl and guaiacyl type lignins presented in the lignin molecules of ML30 and OL60 and indicated no drastic changes of lignin molecules occurred during the formic acid fractionation process. Additionally, ML30 and OL60 displayed important formate ester absorption at 1715 cm−1 . This band shows the esterification of the alcohol and phenol of the propane chain (C␣ and C␥ ) occurred during the extraction process, which is in accordance with the studies performed on lignin models in formic acid by Ede et al. [42] and the investigation by Shukry et al. [43]. In addition, similar band was observed during peroxyformic acid pulping of non-woody lignocelluloses [44]. The increase in the intensity of signal at 1735 cm−1 in Fig. S1 shows that esterification also occurred in the solid residue too. In addition, the intensities of signals at 1592 and 1503 cm−1 in ML30 and OL60 decreased and were largely weaker than those in the raw material. This demonstrates that delignification occurred during the extraction process. The negligible intensity of signal at 1503 cm−1 in MR30 further supported that most of the lignin in the raw material was liberated.
3.3. Molecular weight distribution Lignin samples were only partially soluble in tetrahydrofuran, a common solvent used for GPC to evaluate the molecular weight
distribution. Therefore, the lignin samples were acetylated with anhydride/pyridine to enhance their solubility before analysis. The GPC chromatograms of acetylated ML30 , OL60 and MWL are shown in Fig. S2. Chromatogram of MWL presented a bimodal curve in the high Mw part, with a main peak at 8720 g/mol and a small shoulder peak at 21,530 g/mol. The peaks in both ML30 and OL60 shifted to low Mw region. From MWL to ML30 and to OL60 , the peak in the low Mw part decreased from 8720 to 6470 and to 7730 g/mol, and the peak in the high Mw part decreased from 21,530 to 19,630 and to 21,360 g/mol. The weight-average (Mw) and number-average (Mn) molecular weights and polydispersities (Mw/Mn) of MWL, ML30 , and OL60 are calculated from their chromatograms and listed in Table 3. Mw decreased from 10,860 g/mol in MWL to 7290 g/mol in ML30 , which was probably due to the partial cleavage of -O-4 linkages between the lignin units, as revealed by subsequent H NMR spectra. On the other hand, the Mw of OL60 is slightly higher than that of MWL, which was probably attributed to the existence of a high amount of carbohydrates [45]. In addition, the ratio of Mw and Mn is a measure of the homogeneity of the fragment. As compared to MWL, the extracted lignins showed a similar Mw/Mn value indicating the same homogeneity of these samples.
Table 3 Weight-average (Mw) and number-average (Mn) molecular weights and polydispersities (Mw/Mn) of formic acid lignins as compared to MWL. Samplea
MWL
ML30
OL60
Mw (g/mol) Mn (g/mol) Mw/Mn
10,860 5860 1.85
7290 3830 1.90
11,450 5000 2.29
a
Corresponding to the samples in Table 2.
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Table 4 Assignments of signals and protons per C9 structural unit in 1 H NMR spectra of acetylated lignin samples.a Range number
1 2 3 4 5 6 7 8 9 10 11 12 a
Range (ppm)
11.5–8.0 8.0–6.8 6.8–6.1 6.1–5.8 5.8–5.2 5.2–4.9 4.9–4.3 4.3–4.0 4.0–2.5 2.5–2.2 2.2–1.6 1.6–1.3
Main assignments
Carboxylic acid and aldehyde protons Aromatic protons in guaiacyl units Aromatic protons in syringyl units H␣ of -O-4 structures H␣ of -5 structures H of xylan residue H␣ and H of -O-4 structures H␣ of - structures and H of xylan residue H of methoxyl groups H of aromatic acetates H of aliphatic acetates Hydrocarbon protons
MWL
ML30
OL60
0.03 0.96 1.21 0.40 0.30 0.23 1.41 0.71 4.47 0.97 4.57 0.23
0.75 1.21 1.50 0.31 0.52 0.38 1.60 0.76 5.13 1.45 3.09 0.34
0.98 1.17 1.47 0.31 0.51 0.39 1.71 0.74 5.01 1.07 3.66 0.39
Corresponding to the lignin samples in Table 2.
3.4.
1H
3.5. HSQC spectra
NMR spectra
Analysis of the 1 H NMR signal intensity provides an indirect method of monitoring the level of substitution of the aromatic ring of lignin. The 1 H NMR spectra of acetylated ML30 , OL60 and MWL are shown in Fig. S3 and the signals assigned according to Islam et al. [46] are illustrated in Table 4. The functional groups, as estimated from the acetate signals, are listed for each lignin sample in Table 5. The shifts of signals between 6.1 and 6.8 ppm are attributed to aromatic protons in syringyl units (S) and shifts of signals 6.8–7.2 ppm are related to the aromatic protons in guaiacyl units (G). Integral signals of S units were more prominent than those of G units. Therefore, the S units were the main structural components in birch lignin. Both ML30 and OL60 afforded more numbers of phenolic hydroxyl groups and less alcoholic hydroxyl groups. It has been reported that organosolv processes lead to a reduction in the number of aliphatic hydroxyl groups and an increase in phenolic hydroxyl groups [47]. The total hydroxyl groups in ML30 and OL60 (1.51 and 1.59, respectively) were lower than that in MWL (1.84). The increase of the phenolic protons in ML30 and OL60 was due to the enhancement of aryl ether cleavage; while the loss of the alcoholic hydroxyl protons may be partly because that the acidity of the medium promoted intermolecular condensation reactions with the loss of formaldehyde [48]. As for aromatic protons, an average of 2.71 (ML30 ) and 2.64 (OL60 ) aromatic protons/C9 carbon are observed, as compared with 2.17 (MWL). The number of aromatic protons decreased, which indicated that condensation reactions occurred at C6 , corresponding to the literature report [49]. Obviously, the numbers of protons in (-O-4 ) ether were less than those in MWL. In addition, the numbers of protons of -5 linkages in ML30 and OL60 (0.52 and 0.51/C9 , respectively) were slightly higher than that in MWL (0.30/C9 ), which showed the stability of this type of linkages in formic acid medium.
Table 5 Analysis of functional groups of lignin samples (abundance based on C9 ).a Functional group
MWL
ML30
OL60
OCH3 Total OH Phenolic OH Alcoholic OH Total aromatic protons Aromatic protons of G unit Aromatic protons of S unit Carboxyl
4.47 1.84 0.32 1.52 2.17 0.96 1.21 0.03
5.13 1.51 0.48 1.03 2.71 1.21 1.50 0.75
5.01 1.59 0.37 1.22 2.64 1.17 1.47 0.98
a
Protons per C9 unit
Corresponding to the lignin samples in Table 2.
To investigate the inter-unit linkages of lignin in more detail, MWL, ML30 , and OL60 were characterized by HSQC. The structural features, such as the main inter-unit linkages of ether and C–C bonds (Fig. 2), are revealed from the spectra (Fig. 3) and their proportions were estimated according to the integration of the signals (Table 6) [50]. As MWL is considered as the closest to native lignin, its spectra and quantitative data can present structural information of lignin in the raw material. Generally, the HSQC spectra are divided into three regions: aliphatic (approximately ıC /ıH 0–50/0–2.5 ppm), lignin side chain (approximately ıC /ıH 50–100/2.5–6.5 ppm), and aromatic (approximately ıC /ıH 100–160/5.5–9 ppm) regions [47]. Signals in the aliphatic region (non-oxygenated), which do not reveal structural information, are usually attributed to impurities, such as those from extractives like fatty acids [51]. The correlations in the lignin side chain region yield useful information on inter-unit linkages. The -O-4 linkage (A) is the predominant one, as the corresponding correlations are intense and detected at ıC –ıH 71.5/4.77 ppm (A␣-G ), 72.3/4.90 ppm (A␣-S ), 84.0/4.32 ppm (A-G ), 86.4/4.15 ppm (A-S ), and 54.8/3.49 ppm (A␥ ). Strong signals for resinol substructures (- , B) were observed in the spectra, with their C␣ –H␣ , C –H and the double C␥ –H␥ correlations at ıC –ıH 85.3/4.70, 54.0/3.10, and 71.4/3.85 and 4.22 ppm, respectively. Phenylcoumaran substructures (-5 ,
Table 6 Assignments of from birch.
13
C–1 H correlation signals in the 2D HSQC spectrum of the MWL
Labels
ıC /ıH (ppm)
Assignment
C
52.6/3.86
B A␥ –OMe C␥
54.0/3.10 54.8/3.49 56.1/4.62 60.4/3.27
A␣-G B␥ A␣-S A-G B␣ A-S C␣
71.5/4.77 71.4/3.85 and 4.22 72.3/4.90 84.0/4.32 85.3/4.70 86.4/4.15 87.3/5.48
S2,6 S2,6
104.3/6.73 106.6/7.30
G2 G5 G6
111.3/7.03 115.2/6.74 and 6.98 119.3/6.80
C –H in phenylcoumaran substructures (C) C –H in resinol substructures (B) C␥ –H␥ in -O-4 substructures (A) C–H in methoxyls C␥ –H␥ in phenylcoumaran substructures (C) C␣ –H␣ in -O-4 linked to a G unit (A) C␥ –H␥ in resinol substructures (B) C␣ –H␣ in -O-4 linked to a S unit (A) C –H in -O-4 linked to a G unit (A) C␣ –H␣ in resinol substructures (B) C –H in -O-4 linked to a S unit (A) C␣ –H␣ in phenylcoumaran substructures (C) C2,6 –H2,6 in etherified syringyl units (S) C2,6 –H2,6 in oxidized (C␣ O) phenolic syringyl units (S) C2 –H2 in guaiacyl units (G) C5 –H5 in guaiacyl units (G) C6 –H6 in guaiacyl units (G)
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Fig. 2. Main substructures of birch lignins involving different side-chain linkages and aromatic units identified by HSQC. (A) -O-4 linkages; (A ) acylated -O-4 linkages (R, HCOO or H); (B) resinol structures formed by - /␣-O-␥ /␥-O-␣ linkages; (C) phenylcoumaran structures formed by -5 /␣-O-4 linkages; (G) guaiacyl unit; (S) syringyl unit; (S ) oxidized syringyl unit with a carbonyl group at C␣ (phenolic).
Fig. 3. HSQC spectra of birch formic acid lignins (ML30 and OL60 ) as compared to MWL.
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100
Table 7 Relative abundance of main inter-unit linkages (as percentage of side-chains involved) and S/G ratios of formic acid lignins as compared to MWL.a Abundance (%)
a
80
MWL
ML30
OL60
71.4 20.0 8.5 1.2
82.9 16.0 1.1 2.9
85.9 13.6 0.5 3.2
Corresponding to the lignin samples in Table 2.
C) were also found, although in lower amounts, with the signals for their C␣ –H␣ and C –H correlations being observed at ıC –ıH 87.3/5.48 and 52.6/3.86 ppm, respectively, and that of C␥ –H␥ correlation overlapping with other signals around ıC –ıH 60.4/3.27 ppm. Table 7 shows the relative abundance of the inter-unit linkages involved in the main substructures in the extracted birch lignins and a NMR estimation of the molar S/G ratios of birch lignin samples. In all cases, the -O-4 one (A) was the main substructure that accounts for 71.4% in MWL, 82.9% in ML30 and 85.9% in OL60 of all side-chains. The second most abundant linkage in the birch lignin is corresponding to the resinaol substructure (B), which involves 20.0%, 16.0% and 13.6% in MWL, ML30 and OL60 , respectively. While the -5 phenylcoumaran structure (C) was observed in MWL with a percentage of 8.5%, and with 1.1% and 0.5% in ML30 and OL60 , respectively. The main cross-signals in the aromatic region of the HSQC spectra corresponded to the aromatic rings of lignin units. Signals of S and G units can be clearly observed from the aromatic region in all spectra. The S units showed a remarkable signal for the C2,6 –H2,6 (S2,6 ) at ıC –ıH 104.3/6.73 ppm. In addition, signal at ıC –ıH 106.6/7.30 ppm is corresponding to C2,6 and C O in S units (S2,6 ). The signals at ıC –ıH 111.3/7.03 ppm (C2 –H2 ), 119.3/6.80 ppm (C6 –H6 ), 115.2/6.74 ppm (C5 –H5 ) and 115.2/6.98 ppm (C5 –H5 ) exhibited the presence of G units. The double signals of C5 –H5 showed some heterogeneity among the G units, which especially affects the C5 –H5 correlation, probably due to different substituents at C4 , such as phenolic or etherified in different substructures [52]. The signals at ıC –ıH 74.6/6.09 ppm in ML30 and ıC –ıH 74.1/6.07 ppm in OL60 suggested that esterification occurred during the fractionation process under the acidic conditions. Signals of H units are not detected in any of the HSQC spectra and that means birch lignin was a typical SG-type lignin, in agreement with FTIR spectra. ML30 and OL60 had fewer G units than MWL due to the slight lignin condensation during the fractionation processes. 3.6. Antioxidant activity analysis The curves of the antioxidant activity of ML30 , OL60 and MWL at different concentrations are shown in Fig. 4. As seen, the inhibition of DPPH increased with the increase of lignin sample concentrations. Besides, ML30 had significantly higher antioxidant activity than OL60 and MWL. The RSI values of ML30 , OL60 and MWL are 1.20, 0.87 and 0.53, respectively. Higher antioxidant activity results in higher RSI value. It has been reported that some functional groups, including non-etherified OH phenolic groups, ortho-methoxy groups, hydroxyl groups and the double bond between the outermost carbon atoms in the side chain, are favorable to increase the scavenger activity of lignin. Among these, phenolic hydroxyls play the key role in the radical scavenging activity [8,53]. Research into lignin model compounds shows that more free phenolic hydroxyl groups can induce higher antioxidant activity, and, conversely, aliphatic hydroxyl group content has a negative effect on the antioxidant activity of lignins [11]. This is the reason why the radical scavenging activity of MWL was lower than that
Inhibition (%)
-O-4 aryl ether (A, A ) - resinols (B) -5 phenylcoumaran (C) S/G ratio
1805
60
40
MWL ML30
20
OL60 0
0
2
4
6
8
10
12
Concnetration (mg/mL) Fig. 4. Scavenging activity of birch formic acid lignins (ML30 and OL60 ) as compared to MWL.
of both ML30 and OL60 . Additionally, another reason was partly attributed to the existence of high amount of co-existing carbohydrates since hydroxyl groups in hemicelluloses may connect with phenolic groups in lignin via hydrogen bonding [54]. Pan et al. have reported that high molecular weight (Mn and Mw) increased heterogeneity and decreased radical scavenging activity [55]. However, in the present study, OL60 shows higher molecular weight than MWL, and it shows higher antioxidant activity too. This was probably because that hydroxyl groups had more important effect on radical scavenging activity than molecular weight.
4. Conclusions Microwave heating has been successfully used in the extraction of lignin from birch in aqueous formic acid solution. The extracted lignin exhibits typical SG-type structure. A much higher delignification degree has been achieved under microwave heating as compared to oil bath heating, suggesting that microwave heating is an efficient way. The extracted lignin sample has a slightly lower molecular weight than MWL mainly due to the partial cleavage of -O-4 linkages between the lignin units. FTIR analysis indicated there were no significant changes of the “core” of the lignin structure, except for the esterification, which is normal and inevitable in organic acid extraction process. NMR spectra show that the -O-4 linkage was the most predominant inter-unit linkage of birch lignin. As compared to MWL, lignin sample extracted with microwave heating had stronger antioxidant activity, mainly due to the reduction of aliphatic hydroxyls together with the significant increase of free phenolic hydroxyls.
Acknowledgements The authors are grateful for financial supports from the National Natural Science Foundation of China (31170556), Fundamental Research Funds for the Central Universities (YX2011-36 and TD2011-11), Research Fund for the Doctoral Program of Higher Education of China (20100014120007), China Postdoctoral Science Foundation (20110490303), Beijing Forestry University Young Scientist Fund (BLX2009003), Major State Basic Research Development Program of China (973 Program, No. 2010CB732204), and China Ministry of Education (No. 111 project).
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Microwave-enhanced extraction of lignin from birch in formic acid: Structural characterization and antioxidant activity study Shuai Zhou a , Lu Liu a , Bo Wang a,∗ , Feng Xu a , Runcang Sun a,b a b
Institute of Biomass Chemistry and Technology, College of Materials Science and Technology, Beijing Forestry University, Beijing 100083, PR China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e
i n f o
Article history: Received 27 March 2012 Received in revised form 6 June 2012 Accepted 10 June 2012 Available online 18 June 2012 Keywords: Birch lignin Microwave-enhanced extraction FTIR HSQC Antioxidant activity
a b s t r a c t A rapid and mild extraction protocol for the preparation of lignin was achieved by microwave-assisted heating in formic acid at 101 ◦ C under atmospheric pressure. In this case, birch lignin was extracted with microwave heating process (ML) in formic acid and characterized by elemental analysis, FTIR, GPC, 1 H NMR and 13 C–1 H HSQC. In addition, the antioxidant activity of the samples was investigated. For comparative study, milled wood lignin (MWL) and lignin extracted with oil bath heating process (OL) were prepared. The results showed that the lignin yield under microwave heating was much higher than that under oil bath heating. A maximal delignification degree (89.77%) was achieved when microwave heating time was 30 min. When double time (60 min) was used under oil bath heating, the delignification degree was 66.11%. The structural characterization showed that the lignin structure of ML did not change dramatically, which is a mixture of GS-type with -O-4 ether bond as the major inter-unit linkage. As for antioxidant activity against DPPH, the radical scavenging index (RSI) of ML was 1.20, which was higher than that of MWL (0.53), suggesting that ML exhibited much higher antioxidant activity than MWL. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction Lignocelluloses, as renewable sources for the production of various chemicals, materials, and fuels, have attracted increasing attention due to the emerging depletion of fossil fuels together with the high pressure of carbon dioxide discharge. Lignocelluloses are principally composed of cellulose, hemicelluloses, and lignin. Cellulose and most of hemicelluloses are structural carbohydrates, forming the bulk of the plant cells’ supporting skeleton. Lignin fills the space between cellulose and hemicelluloses, and cross-links with hemicellulosic polysaccharides [1,2] mainly through covalent bonds (ether and ester) [3,4] and phenyl glycosidic linkages [5]. Lignin gives stiffness to the cell wall, enabling it to serve as a mechanical support to build up the stem and branches. Due to the aromatic features, lignin makes the cell wall hydrophobic, which is a prerequisite for the development of cells for efficient water- and nutrition-transport. In addition, lignin functions as protection against microbial degradation. Owing to its special functional groups, such as phenolic hydroxyl, alcoholic hydroxyl, and carboxyl groups, lignin can be used for the production of materials with unique properties, such as dispersants, absorbents, resin, surfactants, and so forth [6,7]. In addition, lignin also exhibits
∗ Corresponding author. Tel.: +86 10 62336592; fax: +86 10 62336972. E-mail addresses: [email protected], [email protected] (B. Wang).
pharmacological properties. Due to its antioxidant activity [8], for instance, lignin is used as inhibitor or neutralizer in oxidation processes to stabilize reactions induced by oxygen radicals and their derived species [9]. It has been reported that lignin obtained from waste of pulp and paper production (in kraft and prehydrolysis processes) can inhibit mutagenicity and SOS response and it shows protective effect on DNA, which suggests that it is a promising antimutagenic and anticarcinogenic agent [10]. Besides, the potential cytotoxic effect on cells of lignin indicates that it is suitable for the cosmetic production without any harmful effects on human cells [11,12]. The commercially available lignin is mainly obtained as byproduct from chemical pulp mill. In these processes, lignin is liberated from lignocelluloses at elevated temperatures (over 160 ◦ C) under high pressures in alkaline or acidic solution for several hours. The extracted lignin is highly degraded, condensed or modified, thus it has limited usages. To explore more efficient methods to separate components from lignocelluloses, numerous processes, such as organic solvent fractionation [13], steam explosion [14], alkaline hydrogen peroxide extraction [15], microwave extraction [16], etc., have been developed and applied in recent decades. Among them, microwave extraction has attracted increasing attention due to its easy operation as well as specific effect [17]. Microwave-assisted technology is a promising process, which utilizes thermal and non-thermal effects generated by microwaves in aqueous conditions. The vibration of polar molecules and the
1359-5113/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.06.006
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movement of ions lead to the generation of heat and extensive collisions, thus microwave heating can accelerate mass transfer and facilitate the dissolution of products. Generally, microwave is mainly used to extract some bio-active components with low molecular weights such as phenolic compounds [18], notoginseng saponins [19] and polysaccharides [20]. In most cases, water is selected as a medium for the treatment of lignocelluloses at elevated temperatures. The extraction process is conducted in acidic solution in fact, since organic acids are produced under high temperatures. In addition, the components in lignocelluloses can be extracted in acidic, alkaline or organic solvents according to the target products. With respect to the extraction of lignin applying microwave heating, most studies have been conducted by using aqueous alkali [21] and ethanol [22]. However, high temperature and pressure is needed to achieve an ideal delignification in these media. This is because the fundamental cleavages of the linkages between lignin units occur at elevated temperature in aqueous alkali or ethanol. Formic acid, an easily recycled organic acid, is a good solution to extract lignin. It has been applied to pulping of many wood and annual plants in the early stage [23,24] and fractionation of lignocelluloses in a biorefinery manner recently [25,26]. Since formic acid is a polar solvent, it is helpful that it can be used as a medium for microwave irradiation. Therefore, in the present study, birch, a typical hardwood species, was subjected to microwave irradiation in aqueous formic acid, aiming at extracting lignin in a highly efficient way. The microwave-assisted extraction of lignin in formic acid provides a novel process which has the advantages of higher extraction yield, lower extraction temperature, and easier recycling of solvent, as compared to the conventional way of microwave-assisted extraction aforementioned. The structure of the extracted lignin was characterized with a set of wet chemistry (elemental and sugar analysis) and spectroscopy (Fourier transform infrared spectroscopy (FTIR), 1 H nuclear magnetic resonance (1 H NMR) and heteronuclear singular quantum correlation (HSQC)) methods. In addition, in order to find the structural changes of lignin, the extracted lignins under different conditions were compared to that from the oil bath heating process, as well as the native lignin. Furthermore, the antioxidant activity of the lignins was also evaluated since it is closely correlated to the structure. 2. Materials and methods 2.1. Materials Birch (Betula alnoides) was harvested from Yunnan province, southwest of China. The outer and inner layers of bark were peeled off and the stem was chopped into small pieces, and then ground to obtain meal. The wood meal sized in 40–60 mesh was collected. The meal was extracted with toluene/ethanol (2:1, v/v) in a Soxhlet apparatus, air-dried and stored in a hermetic polypropylene container before use. The lignin content in the dewaxed material was determined as 26.36%.
For comparative study, lignin was extracted from birch under the same extracting conditions (88% aqueous formic acid, 101 ◦ C) with oil bath heating. The oil bath was equipped with a magnetic stirring system and water-cooled condenser. The temperature of the oil bath was controlled by an adjustable thermometer inserted into the extraction system. The aqueous formic acid (20 mL) was introduced in a 100 mL three-neck flask and heated to 101 ◦ C. Then the dewaxed birch (2 g) was immersed into a three-neck flask and kept for a given time (10, 20, 30, and 60 min, respectively). After cooking, the mixtures were treated similarly to the procedure of microwave heating extraction. In addition, as milled wood lignin (MWL) is considered close to native lignin of lignocelluloses, it was prepared according to a previous report [27] for comparison. The lignin fraction extracted by microwave heating was defined as ML and that by oil bath heating was marked as OL. The weight of the lignin fraction was determined gravimetrically. Extraction yield was measured gravimetrically after the solid product was drying to constant weight. Lignin yield was calculated according to the formula: lignin yield (%) = (1 − sugar content) × extraction yield/lignin content in raw material × 100. Lignin content in residue was determined according to acetyl bromide method [28] on UV 2300 (Shanghai Tianmei Science and Technology Corporation, China) using 1 cm cells. Delignification degree was calculated according to the formula: delignification degree (%) = (1 − weight of lignin in residue/weight of lignin in raw material) × 100. 2.3. Component analysis and structural characterization Fourier transform infrared (FTIR) spectra were recorded on a spectrophotometer (Nicolet iN10, USA) with a MCT detector in the region of 4000–650 cm−1 at a resolution of 4 cm−1 . Elemental analysis was performed to determine the carbon, hydrogen, and nitrogen content in the lignin samples using a Vario EL III Elemental analyzer instrument (Elementar, Germany). Oxygen content was deduced from the difference with respect to the total samples. The methoxyl content in lignin was determined as described by Mousavioun and Doherty [29]. The molecular weight distribution of lignin was measured by gel permeation chromatography (GPC, Agilent 1200, USA) using a PL-gel 10 mm Mixed-B 7.5 mm ID column, calibrated with monodisperse polystyrene. The samples were acetylated with acetic anhydride before determination. 2 mg acetylated sample was dissolved in 2 mL tetrahydrofuran (THF), and 20 L lignin solutions were injected. The column was operated at ambient temperature and THF was used as eluent with a flow rate of 1 mL/min. The measurements were conducted with three parallels and the relative standard deviation was below 5%. Sugar analysis (neutral sugars and uronic acids) was conducted by using high performance anion exchange chromatography (HPAEC). The neutral sugars and uronic acids in the samples were released by hydrolysis with 72% H2 SO4 for 2 h at 25 ◦ C followed by a high temperature hydrolysis at 105 ◦ C for 4 h after dilution to 6.1% H2 SO4 . After hydrolysis, the samples were filtered, diluted 30-fold and injected into a HPAEC system (Dionex ISC 3000, USA) with an amperometric detector, a CarbopacTMPA-20 column (4 mm × 250 mm, Dionex), and a guard PA-20 column (3 mm × 30 mm, Dionex). Neutral sugars and uronic acids were separated in isocratic 5 mM NaOH (carbonate free and purged with nitrogen) for 20 min, followed by a 0.75 mM NaAc gradient in 5 mM NaOH for 15 min with a flow rate of 0.4 mL/min. Calibration was performed with standard solutions of sugars. Results of the yield and sugar composition of the samples are presented as mean values of three parallels, and the relative standard deviation was below 0.5%. NMR spectra of the samples were acquired on a Bruker AVIII 400 MHz spectrometer at 25 ◦ C. The 1 H NMR spectra were obtained at 100 MHz with 15 mg acetylated sample in 1 mL CDCl3 . Heteronuclear single quantum correlation (HSQC) spectra were acquired by HSQC GE experiment mode with 20 mg lignin sample dissolved in 1 mL d-DMSO. HSQC spectral widths for 1 H and 13 C dimensions were 2200 Hz and 15,400 Hz, respectively. The number of collected complex points for 1 H dimension was 1024 with a recycle delay of 1.5 s. The number of scan was 128, and 256 time increments were recorded in the 13 C-dimension. The 1 JCH was set to 146 Hz. Prior to Fourier transform the data matrixes were zero filled up to 1024 points in the 13 C-dimension. 2.4. Antioxidant activity
2.2. Extraction of lignin from birch Microwave assisted extraction of lignin from birch was conducted in 88% formic acid solution at boiling point (101 ◦ C) under atmospheric pressure. The microwave oven used was a focused single-mode microwave synthesis system (2.45 GHz, Discover, CEM, USA), which was equipped with a magnetic stirring system and a water-cooled condenser. The temperature was measured using thermocouple-type thermometer and controlled by automatically adjusting of microwave power. When the formic acid solution has been heated to 101 ◦ C, the dewaxed material (2 g) was immersed into the preheated 88% aqueous formic acid with a solid to liquor ratio of 1:10 (g/mL). Then the mixtures were treated in the microwave oven under 700 W at 101 ◦ C for various times (5, 10, and 30 min). After cooking, the mixtures were decanted through a tared glass filter crucible (G2). In a run, the solid residue (cellulosic pulp) was washed thoroughly with 88% formic acid followed by washing with water. The filtrate was collected and concentrated with a rotatory evaporator under reduced pressure to about 8 mL. Then the concentrated liquor was added into 60 mL water to precipitate the dissolved lignin. Subsequently, the precipitated lignin was recovered by centrifugation, washed with acidified water (pH = 2), and then dried.
Antioxidant activity was determined according to the radical scavenging activity method using 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH), which was developed by Brand-Williams et al. [30] and modified by Dizhbite et al. [8]. A lignin sample was dissolved in 0.1 mL dioxane–water (9/1, v/v), and then the solution was added to 3.9 mL DPPH solution (25 mg/L in ethanol) as the free radical source. The blank sample consisted of 0.1 mL methanol and 3.9 mL DPPH solution. After a 30 min incubation period at room temperature in the dark, the decrease of the solution absorbance, due to proton donating activity, was immediately measured at 517 nm using a UV 2300 spectrometer (Shanghai Tianmei Science and Technology Corporation, China). The tests were carried out in duplicate. The original data are shown in supplementary data (Table S1). The radical scavenging activity was calculated as follows: I (%) = (A0 − A1 )/A0 ; where A0 is the absorbance of the blank, and A1 is the absorbance in the presence of the test compound at different concentrations. The IC50 (concentration providing 50% inhibition) was calculated graphically using a calibration curve in the linear range by plotting the extract concentration vs. the corresponding scavenging effect. Radical scavenging index (RSI) was defined as the inverse of IC50 .
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Table 1 Extraction yield, lignin yield and total sugar content of lignin fractions extracted with formic acid and delignification degree under microwave heating and oil bath heating. Sample No.a
Heating type
Microwave intensity (W)
Heating time (min)
Extraction yield (%)
Total sugar (%)
FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8 FL9 FL10 FL11 MWL
Microwave Microwave Microwave Microwave Microwave Microwave Microwave Oil bath Oil bath Oil bath Oil bath –
400 500 600 700 800 700 700 – – – – –
30 30 30 30 30 5 10 10 20 30 60 –
19.66 21.85 23.38 26.49 26.47 8.10 15.08 6.96 9.80 16.30 18.77 –
11.97 12.02 11.68 11.70 12.32 17.12 15.84 16.88 18.16 11.74 8.33 5.46
3. Results and discussion 3.1. Yield and chemical composition of the extracted lignin samples Extraction yield, lignin yield and total sugar content of lignin fractions extracted with formic acid and delignification degree under microwave heating and oil bath heating are shown in Table 1. As can be seen from the table, as for microwave heating extraction, extraction yield, lignin yield and delignification degree increased along with the increase of the microwave intensity. This was probably caused by the specific and non-thermal microwave effects, which was still controversial [31,32]. It can be confirmed that mixtures are able to absorb microwaves in a controlled and fast manner when high power is applied [33]. And the delignification degrees under 700 W and 800 W were comparable. Therefore, the microwave intensity was set as 700 W for the following research. Besides, lignin yield and delignification degree in microwave heating extraction process are much higher than that in oil bath heating extraction process under the same extraction time. Sugar content in both ML and OL is higher than that in MWL. With the time going on, the sugar content decreased, and the lignin yield and delignification degree increased. When the extraction time was 10 min under microwave heating, the delignification degree reached 55.09%, indicating that half of the lignin in the raw material has been liberated. When the extraction time was prolonged to 30 min, 89.77% of lignin in the raw material has been removed. To achieve comparable delignification degree, the extraction duration using oil bath heating needs to be doubled. This illustrates that microwave heating was more effective to disrupt recalcitrant structures in lignocellulosic biomass to liberate lignin in cell wall than conventional heating [34]. This is probably because that water, cellulose, hemicelluloses, lignin and other low molecular compounds (i.e., the organic acid) belong to dielectrics [35]. Hence, the formic acid solution can be heated by microwaves in a dielectric way owing to its non-thermal or thermal effects [36,37]. Microwaves induce heat at the molecular level by direct conversion of the electromagnetic energy into heat [38] and by means of irradiation and heat
Lignin yield (%) 65.66 72.92 78.34 88.74 88.04 25.46 54.33 21.94 30.42 54.58 65.27 –
Delignification degree (%) 66.84 73.92 79.66 89.77 89.02 26.16 55.09 22.12 31.10 55.33 66.11
flows from inside to outside [39], while conventional heating, such as oil bath heating, by the modes of conduction and convection. For conventional heating, energy is converted to heat primarily, and then transferred along temperature gradients from the surface to the core of the material. This means that microwaves are able to penetrate into the solution immediately. Therefore it needs less energy input than oil bath heating and it can save time. To study the effect of microwave heating on the structural changes of lignin, two typical lignin fractions, ML30 (microwave heating for 30 min) and OL60 (oil bath heating for 60 min), were chosen for further investigation as compared to MWL (milled wood lignin). The elemental composition and methoxyl content of the lignin samples are presented in Table 2 along with the calculated C9 formulae. These C9 formulae were corrected as carbohydrates [40]. The elemental analysis results demonstrate that ML30 has higher carbon and lower oxygen contents than OL60 , which were comparable with that in MWL. In addition, nitrogen was not detected in all samples, implying that the extracted lignins were not contaminated by protein. 3.2. FTIR spectroscopy Fig. 1 shows the FTIR spectra of ML30 , OL60 , and MWL, and Fig. S1 shows the FTIR spectra of the solid residue under microwave heating for 30 min (MR30 ), solid residue under oil bath heating for 60 min (OR60 ) and the raw material. The FTIR spectra of the lignin samples show weak changes in the peak intensities, confirming that the “core” of the lignin structure did not change dramatically during the fractionation processes. The three lignin samples exhibit typical SG-type lignin absorption bands (Fig. 1), which is in accordance with the HSQC spectra afterwards. The bands at 1592, 1502, and 1420 cm−1 are corresponding to aromatic ring vibrations of phenyl-propane (C9 ) skeleton and the C–H deformation combined with aromatic ring vibration at 1456 cm−1 are observed in all spectra, although the intensity of the bands differs. This suggested that the aromaticity of the different samples remained the same and lignin did not change dramatically by the extraction procedure [41]. For ML30 and OL60 , the signal around 3455 cm−1 is assigned
Table 2 Elemental analysis, methoxyl content, and C9 formula of formic acid lignin fractions as compared to MWL. Samplea
MWL ML30 OL60 a b
Element and methoxyl content (%) C
H
O
OCH3
58.01 57.13 56.73
6.35 5.67 5.81
35.64 37.20 37.46
20.47 21.60 21.60
C9 formulab
C9 weight
C9 H9.11 O3.17 (OCH3 )1.49 C9 H7.16 O3.14 (OCH3 )1.71 C9 H7.77 O3.33 (OCH3 )1.67
214.10 218.48 220.89
MWL represents milled wood lignin; ML30 and OL60 correspond to lignin sample extracted under microwave heating for 30 min and oil bath heating for 60 min, respectively. Corrected as carbohydrates.
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Fig. 1. FTIR spectra of birch formic acid lignins (ML30 and OL60 ) as compared to MWL.
to the stretch of OH groups and the intensities change slightly, which means that oxidative cleavage in lignin macromolecules was negligible. The signals at 2940 and 2842 cm−1 are attributed to CH stretch in CH2 and CH3 groups. Except for these typical lignin structure bands, other signals are associated with syringyl (S) and guaiacyl (G). This is confirmed by the absorption at 830 cm−1 (C–H out-of-plane in position 2 and 6 from S unit). In addition, bands for aromatic ring breathing vibration, aromatic in-plane bending, and out-of-plane C–H bending were observed at 1324, 1216, and 1114 cm−1 for S unit, and at 1033 cm−1 and 925 cm−1 for G unit, respectively. Both syringyl and guaiacyl type lignins presented in the lignin molecules of ML30 and OL60 and indicated no drastic changes of lignin molecules occurred during the formic acid fractionation process. Additionally, ML30 and OL60 displayed important formate ester absorption at 1715 cm−1 . This band shows the esterification of the alcohol and phenol of the propane chain (C␣ and C␥ ) occurred during the extraction process, which is in accordance with the studies performed on lignin models in formic acid by Ede et al. [42] and the investigation by Shukry et al. [43]. In addition, similar band was observed during peroxyformic acid pulping of non-woody lignocelluloses [44]. The increase in the intensity of signal at 1735 cm−1 in Fig. S1 shows that esterification also occurred in the solid residue too. In addition, the intensities of signals at 1592 and 1503 cm−1 in ML30 and OL60 decreased and were largely weaker than those in the raw material. This demonstrates that delignification occurred during the extraction process. The negligible intensity of signal at 1503 cm−1 in MR30 further supported that most of the lignin in the raw material was liberated.
3.3. Molecular weight distribution Lignin samples were only partially soluble in tetrahydrofuran, a common solvent used for GPC to evaluate the molecular weight
distribution. Therefore, the lignin samples were acetylated with anhydride/pyridine to enhance their solubility before analysis. The GPC chromatograms of acetylated ML30 , OL60 and MWL are shown in Fig. S2. Chromatogram of MWL presented a bimodal curve in the high Mw part, with a main peak at 8720 g/mol and a small shoulder peak at 21,530 g/mol. The peaks in both ML30 and OL60 shifted to low Mw region. From MWL to ML30 and to OL60 , the peak in the low Mw part decreased from 8720 to 6470 and to 7730 g/mol, and the peak in the high Mw part decreased from 21,530 to 19,630 and to 21,360 g/mol. The weight-average (Mw) and number-average (Mn) molecular weights and polydispersities (Mw/Mn) of MWL, ML30 , and OL60 are calculated from their chromatograms and listed in Table 3. Mw decreased from 10,860 g/mol in MWL to 7290 g/mol in ML30 , which was probably due to the partial cleavage of -O-4 linkages between the lignin units, as revealed by subsequent H NMR spectra. On the other hand, the Mw of OL60 is slightly higher than that of MWL, which was probably attributed to the existence of a high amount of carbohydrates [45]. In addition, the ratio of Mw and Mn is a measure of the homogeneity of the fragment. As compared to MWL, the extracted lignins showed a similar Mw/Mn value indicating the same homogeneity of these samples.
Table 3 Weight-average (Mw) and number-average (Mn) molecular weights and polydispersities (Mw/Mn) of formic acid lignins as compared to MWL. Samplea
MWL
ML30
OL60
Mw (g/mol) Mn (g/mol) Mw/Mn
10,860 5860 1.85
7290 3830 1.90
11,450 5000 2.29
a
Corresponding to the samples in Table 2.
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Table 4 Assignments of signals and protons per C9 structural unit in 1 H NMR spectra of acetylated lignin samples.a Range number
1 2 3 4 5 6 7 8 9 10 11 12 a
Range (ppm)
11.5–8.0 8.0–6.8 6.8–6.1 6.1–5.8 5.8–5.2 5.2–4.9 4.9–4.3 4.3–4.0 4.0–2.5 2.5–2.2 2.2–1.6 1.6–1.3
Main assignments
Carboxylic acid and aldehyde protons Aromatic protons in guaiacyl units Aromatic protons in syringyl units H␣ of -O-4 structures H␣ of -5 structures H of xylan residue H␣ and H of -O-4 structures H␣ of - structures and H of xylan residue H of methoxyl groups H of aromatic acetates H of aliphatic acetates Hydrocarbon protons
MWL
ML30
OL60
0.03 0.96 1.21 0.40 0.30 0.23 1.41 0.71 4.47 0.97 4.57 0.23
0.75 1.21 1.50 0.31 0.52 0.38 1.60 0.76 5.13 1.45 3.09 0.34
0.98 1.17 1.47 0.31 0.51 0.39 1.71 0.74 5.01 1.07 3.66 0.39
Corresponding to the lignin samples in Table 2.
3.4.
1H
3.5. HSQC spectra
NMR spectra
Analysis of the 1 H NMR signal intensity provides an indirect method of monitoring the level of substitution of the aromatic ring of lignin. The 1 H NMR spectra of acetylated ML30 , OL60 and MWL are shown in Fig. S3 and the signals assigned according to Islam et al. [46] are illustrated in Table 4. The functional groups, as estimated from the acetate signals, are listed for each lignin sample in Table 5. The shifts of signals between 6.1 and 6.8 ppm are attributed to aromatic protons in syringyl units (S) and shifts of signals 6.8–7.2 ppm are related to the aromatic protons in guaiacyl units (G). Integral signals of S units were more prominent than those of G units. Therefore, the S units were the main structural components in birch lignin. Both ML30 and OL60 afforded more numbers of phenolic hydroxyl groups and less alcoholic hydroxyl groups. It has been reported that organosolv processes lead to a reduction in the number of aliphatic hydroxyl groups and an increase in phenolic hydroxyl groups [47]. The total hydroxyl groups in ML30 and OL60 (1.51 and 1.59, respectively) were lower than that in MWL (1.84). The increase of the phenolic protons in ML30 and OL60 was due to the enhancement of aryl ether cleavage; while the loss of the alcoholic hydroxyl protons may be partly because that the acidity of the medium promoted intermolecular condensation reactions with the loss of formaldehyde [48]. As for aromatic protons, an average of 2.71 (ML30 ) and 2.64 (OL60 ) aromatic protons/C9 carbon are observed, as compared with 2.17 (MWL). The number of aromatic protons decreased, which indicated that condensation reactions occurred at C6 , corresponding to the literature report [49]. Obviously, the numbers of protons in (-O-4 ) ether were less than those in MWL. In addition, the numbers of protons of -5 linkages in ML30 and OL60 (0.52 and 0.51/C9 , respectively) were slightly higher than that in MWL (0.30/C9 ), which showed the stability of this type of linkages in formic acid medium.
Table 5 Analysis of functional groups of lignin samples (abundance based on C9 ).a Functional group
MWL
ML30
OL60
OCH3 Total OH Phenolic OH Alcoholic OH Total aromatic protons Aromatic protons of G unit Aromatic protons of S unit Carboxyl
4.47 1.84 0.32 1.52 2.17 0.96 1.21 0.03
5.13 1.51 0.48 1.03 2.71 1.21 1.50 0.75
5.01 1.59 0.37 1.22 2.64 1.17 1.47 0.98
a
Protons per C9 unit
Corresponding to the lignin samples in Table 2.
To investigate the inter-unit linkages of lignin in more detail, MWL, ML30 , and OL60 were characterized by HSQC. The structural features, such as the main inter-unit linkages of ether and C–C bonds (Fig. 2), are revealed from the spectra (Fig. 3) and their proportions were estimated according to the integration of the signals (Table 6) [50]. As MWL is considered as the closest to native lignin, its spectra and quantitative data can present structural information of lignin in the raw material. Generally, the HSQC spectra are divided into three regions: aliphatic (approximately ıC /ıH 0–50/0–2.5 ppm), lignin side chain (approximately ıC /ıH 50–100/2.5–6.5 ppm), and aromatic (approximately ıC /ıH 100–160/5.5–9 ppm) regions [47]. Signals in the aliphatic region (non-oxygenated), which do not reveal structural information, are usually attributed to impurities, such as those from extractives like fatty acids [51]. The correlations in the lignin side chain region yield useful information on inter-unit linkages. The -O-4 linkage (A) is the predominant one, as the corresponding correlations are intense and detected at ıC –ıH 71.5/4.77 ppm (A␣-G ), 72.3/4.90 ppm (A␣-S ), 84.0/4.32 ppm (A-G ), 86.4/4.15 ppm (A-S ), and 54.8/3.49 ppm (A␥ ). Strong signals for resinol substructures (- , B) were observed in the spectra, with their C␣ –H␣ , C –H and the double C␥ –H␥ correlations at ıC –ıH 85.3/4.70, 54.0/3.10, and 71.4/3.85 and 4.22 ppm, respectively. Phenylcoumaran substructures (-5 ,
Table 6 Assignments of from birch.
13
C–1 H correlation signals in the 2D HSQC spectrum of the MWL
Labels
ıC /ıH (ppm)
Assignment
C
52.6/3.86
B A␥ –OMe C␥
54.0/3.10 54.8/3.49 56.1/4.62 60.4/3.27
A␣-G B␥ A␣-S A-G B␣ A-S C␣
71.5/4.77 71.4/3.85 and 4.22 72.3/4.90 84.0/4.32 85.3/4.70 86.4/4.15 87.3/5.48
S2,6 S2,6
104.3/6.73 106.6/7.30
G2 G5 G6
111.3/7.03 115.2/6.74 and 6.98 119.3/6.80
C –H in phenylcoumaran substructures (C) C –H in resinol substructures (B) C␥ –H␥ in -O-4 substructures (A) C–H in methoxyls C␥ –H␥ in phenylcoumaran substructures (C) C␣ –H␣ in -O-4 linked to a G unit (A) C␥ –H␥ in resinol substructures (B) C␣ –H␣ in -O-4 linked to a S unit (A) C –H in -O-4 linked to a G unit (A) C␣ –H␣ in resinol substructures (B) C –H in -O-4 linked to a S unit (A) C␣ –H␣ in phenylcoumaran substructures (C) C2,6 –H2,6 in etherified syringyl units (S) C2,6 –H2,6 in oxidized (C␣ O) phenolic syringyl units (S) C2 –H2 in guaiacyl units (G) C5 –H5 in guaiacyl units (G) C6 –H6 in guaiacyl units (G)
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Fig. 2. Main substructures of birch lignins involving different side-chain linkages and aromatic units identified by HSQC. (A) -O-4 linkages; (A ) acylated -O-4 linkages (R, HCOO or H); (B) resinol structures formed by - /␣-O-␥ /␥-O-␣ linkages; (C) phenylcoumaran structures formed by -5 /␣-O-4 linkages; (G) guaiacyl unit; (S) syringyl unit; (S ) oxidized syringyl unit with a carbonyl group at C␣ (phenolic).
Fig. 3. HSQC spectra of birch formic acid lignins (ML30 and OL60 ) as compared to MWL.
S. Zhou et al. / Process Biochemistry 47 (2012) 1799–1806
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Table 7 Relative abundance of main inter-unit linkages (as percentage of side-chains involved) and S/G ratios of formic acid lignins as compared to MWL.a Abundance (%)
a
80
MWL
ML30
OL60
71.4 20.0 8.5 1.2
82.9 16.0 1.1 2.9
85.9 13.6 0.5 3.2
Corresponding to the lignin samples in Table 2.
C) were also found, although in lower amounts, with the signals for their C␣ –H␣ and C –H correlations being observed at ıC –ıH 87.3/5.48 and 52.6/3.86 ppm, respectively, and that of C␥ –H␥ correlation overlapping with other signals around ıC –ıH 60.4/3.27 ppm. Table 7 shows the relative abundance of the inter-unit linkages involved in the main substructures in the extracted birch lignins and a NMR estimation of the molar S/G ratios of birch lignin samples. In all cases, the -O-4 one (A) was the main substructure that accounts for 71.4% in MWL, 82.9% in ML30 and 85.9% in OL60 of all side-chains. The second most abundant linkage in the birch lignin is corresponding to the resinaol substructure (B), which involves 20.0%, 16.0% and 13.6% in MWL, ML30 and OL60 , respectively. While the -5 phenylcoumaran structure (C) was observed in MWL with a percentage of 8.5%, and with 1.1% and 0.5% in ML30 and OL60 , respectively. The main cross-signals in the aromatic region of the HSQC spectra corresponded to the aromatic rings of lignin units. Signals of S and G units can be clearly observed from the aromatic region in all spectra. The S units showed a remarkable signal for the C2,6 –H2,6 (S2,6 ) at ıC –ıH 104.3/6.73 ppm. In addition, signal at ıC –ıH 106.6/7.30 ppm is corresponding to C2,6 and C O in S units (S2,6 ). The signals at ıC –ıH 111.3/7.03 ppm (C2 –H2 ), 119.3/6.80 ppm (C6 –H6 ), 115.2/6.74 ppm (C5 –H5 ) and 115.2/6.98 ppm (C5 –H5 ) exhibited the presence of G units. The double signals of C5 –H5 showed some heterogeneity among the G units, which especially affects the C5 –H5 correlation, probably due to different substituents at C4 , such as phenolic or etherified in different substructures [52]. The signals at ıC –ıH 74.6/6.09 ppm in ML30 and ıC –ıH 74.1/6.07 ppm in OL60 suggested that esterification occurred during the fractionation process under the acidic conditions. Signals of H units are not detected in any of the HSQC spectra and that means birch lignin was a typical SG-type lignin, in agreement with FTIR spectra. ML30 and OL60 had fewer G units than MWL due to the slight lignin condensation during the fractionation processes. 3.6. Antioxidant activity analysis The curves of the antioxidant activity of ML30 , OL60 and MWL at different concentrations are shown in Fig. 4. As seen, the inhibition of DPPH increased with the increase of lignin sample concentrations. Besides, ML30 had significantly higher antioxidant activity than OL60 and MWL. The RSI values of ML30 , OL60 and MWL are 1.20, 0.87 and 0.53, respectively. Higher antioxidant activity results in higher RSI value. It has been reported that some functional groups, including non-etherified OH phenolic groups, ortho-methoxy groups, hydroxyl groups and the double bond between the outermost carbon atoms in the side chain, are favorable to increase the scavenger activity of lignin. Among these, phenolic hydroxyls play the key role in the radical scavenging activity [8,53]. Research into lignin model compounds shows that more free phenolic hydroxyl groups can induce higher antioxidant activity, and, conversely, aliphatic hydroxyl group content has a negative effect on the antioxidant activity of lignins [11]. This is the reason why the radical scavenging activity of MWL was lower than that
Inhibition (%)
-O-4 aryl ether (A, A ) - resinols (B) -5 phenylcoumaran (C) S/G ratio
1805
60
40
MWL ML30
20
OL60 0
0
2
4
6
8
10
12
Concnetration (mg/mL) Fig. 4. Scavenging activity of birch formic acid lignins (ML30 and OL60 ) as compared to MWL.
of both ML30 and OL60 . Additionally, another reason was partly attributed to the existence of high amount of co-existing carbohydrates since hydroxyl groups in hemicelluloses may connect with phenolic groups in lignin via hydrogen bonding [54]. Pan et al. have reported that high molecular weight (Mn and Mw) increased heterogeneity and decreased radical scavenging activity [55]. However, in the present study, OL60 shows higher molecular weight than MWL, and it shows higher antioxidant activity too. This was probably because that hydroxyl groups had more important effect on radical scavenging activity than molecular weight.
4. Conclusions Microwave heating has been successfully used in the extraction of lignin from birch in aqueous formic acid solution. The extracted lignin exhibits typical SG-type structure. A much higher delignification degree has been achieved under microwave heating as compared to oil bath heating, suggesting that microwave heating is an efficient way. The extracted lignin sample has a slightly lower molecular weight than MWL mainly due to the partial cleavage of -O-4 linkages between the lignin units. FTIR analysis indicated there were no significant changes of the “core” of the lignin structure, except for the esterification, which is normal and inevitable in organic acid extraction process. NMR spectra show that the -O-4 linkage was the most predominant inter-unit linkage of birch lignin. As compared to MWL, lignin sample extracted with microwave heating had stronger antioxidant activity, mainly due to the reduction of aliphatic hydroxyls together with the significant increase of free phenolic hydroxyls.
Acknowledgements The authors are grateful for financial supports from the National Natural Science Foundation of China (31170556), Fundamental Research Funds for the Central Universities (YX2011-36 and TD2011-11), Research Fund for the Doctoral Program of Higher Education of China (20100014120007), China Postdoctoral Science Foundation (20110490303), Beijing Forestry University Young Scientist Fund (BLX2009003), Major State Basic Research Development Program of China (973 Program, No. 2010CB732204), and China Ministry of Education (No. 111 project).
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