Characterization of Lignins Isolated from Alkali Treated Prehydrolysate of Corn Stover

BIOTECHNOLOGY AND BIOENGINEERING Chinese Journal of Chemical Engineering, 21(4) 427—433 (2013) DOI: 10.1016/S1004-9541(13)60468-1

Characterization of Lignins Isolated from Alkali Treated Prehydrolysate of Corn Stover* LEI Mingliu (雷鸣柳)1,2, ZHANG Hongman (张红漫)1,2,**, ZHENG Hongbo (郑洪波)1, LI Yuanyuan (李媛媛)1,2, HUANG He (黄和)2 and XU Rong (徐蓉)1,2 1 2

College of Science, Nanjing University of Technology, Nanjing 210009, China China State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China

Abstract Lignins were isolated and purified from alkali treated prehydrolysate of corn stover. The paper presents the structural features of lignins in a series purification processes. Fourier transform infrared spectroscopy, ultraviolet-vis spectroscopy and proton nuclear magnetic resonance spectroscopy were used to analyze the chemical structure. Thermogravimetric analysis was applied to follow the thermal degradation, and wet chemical method was used to determine the sugar content. The results showed that the crude lignin from the prehydrolysate of corn stover was a heterogeneous material of syringyl, guaiacyl and p-hydroxyphenyl units, containing associated polysaccharides, lipids, and melted salts. Some of the crude lignin was chemically linked to hemicelluloses (mainly xylan). The lipids in crude lignin were probably composed of saturated and/or unsaturated long carbon chains, fatty acids, triterpenols, waxes, and derivatives of aromatic. The sugar content of purified lignin was less than 2.11%, mainly composed of guaiacyl units. DTGmax of purified lignin was 359 °C. The majority of the hydroxyl groups were phenolic hydroxyl groups. The main type of linkages in purified lignin was β-O-4. Other types of linkages included β-5, β-β and α-O-4. Keywords lignin structure, cellulose ethanol, alkaline pretreatment, corn stover, purification

1

INTRODUCTION

With the depletion of fossil fuels, the fraction of energy and chemicals supplied by renewable resources such as biomass is expected to increase in the future [1]. The development of biorefineries, producing fuels and commodity chemicals from lignocellulosic biomass, is viewed as a potential alternative to our current reliance on fossil fuels [2, 3]. The removal of lignin is needed in the cellulosic ethanol production, because the lignin not only prevents enzymes to access cellulose and hemicellulose, but also adsorbs some cellulose enzyme, reducing the catalytic efficiency of cellulase [4-7]. The removed lignin is usually burned to generate energy. However, the production of cellulosic ethanol is not economically competitive without an effective utilization of the lignin, which, full of aromatic rings, is a potential new source of aromatic chemicals and other products. The trend is to chemically modify or fractionate residual lignins, or propose new uses [8]. Some of the applications being developed are lignin-based carbonfibers [9], biopolymers [10], surfactants [11], high value-added phenolic aldehydes chemicals [12], and so on. Lignins used for industrial applications must have acceptable purity and desired chemical and physical properties. Currently, there are some problems such as low purity, heterogeneity, odor and color of lignin-based products, and lacking of reliable analytical methods

[13]. Various purification procedures [14-17] have been attempted to reduce polysaccharide content in lignin preparations. The two-step precipitation method [18] is a typical way to obtain the lignin fractions that are relatively free of polysaccharides. However, it is necessary to investigate the purification in more detail. Further purification of lignin is closely related to its thermal stability and the amount of chemical functional groups [19, 20]. In this work, for all samples, the portion of lignin is quantified and characterized by a set of analytical methods, including thermogravimetric analysis (TGA), ultraviolet-vis (UV) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance spectroscopy (1H NMR), and wet chemical method. We investigate the chemical changes in a series purification processes and characterize the purified lignin structure, in order to evaluate its potential for producing value-added products.

2 2.1

EXPERIMENTAL Material

The corn stover used in this study was collected from Nanjing, China. This material was milled until the sample passed through a 2 mm sieve, and then it was screened to obtain a 180-830 μm fraction, and stored in a polyethylene container kept at −4 °C before use. The composition of the corn stover, as determined

Received 2011-12-26, accepted 2012-08-06. * Supported by the National Natural Science Foundation of China (20876078, 21176124), the National High Technology Research and Development Program of China (2011AA02A207), the National Basic Research Program of China (2009CB724700), the Key Program of the National Natural Science Foundation of China (20936002), and the Independent Innovation Project of Jiangsu Province (CX(11)2051). ** To whom correspondence should be addressed. E-mail: [email protected]

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by the method of NREL LAP (laboratory analytical procedures of National Renewable Energy Laboratory) [21], was 35.23% glucan, 17.33% xylan, 4.16% arabinan, 3.26% galactan, 18.90% lignin, 6.90% ash, 5.44% protein, and 8.78% others by dry mass. All experiments were performed in duplicate under the same conditions and their average values were reported. The method for alkaline pretreatment of corn stover was chosen according to the optimized protocol [22]. Chips of corn stover were subjected to an alkaline extraction by 2% (by mass) NaOH at 85 °C for 75 min at a ratio of solid to liquid of 1︰8. This was followed by filtration. The chemical components of the alkali treated prehydrolysate were total base number 29.3 g·L−1, effective alkali 2.2 g·L−1, solid content 78.5 g·L−1, organic matter 57.7 g·L−1, inorganic matter 20.8 g·L−1, density 1.35 g·cm−3, and chemical oxygen demand (COD) 72000 mg·L−1. 2.2

Isolation and purification of lignin

As shown in Fig. 1, the alkali treated prehydrolysate was acidified carefully with 20% (by mass) sulfuric acid to pH 2 with rapid stirring. Crude lignin (L1) was collected by filtering the acidified liquor through a Whatman No 4 (pore size 20-25 µm) filter paper and then air-dried. The dried lignin was exhaustively extracted with hexane in a Soxhlet apparatus to obtain extractive-free lignin. For purification of the crude lignin, 1 g of extractive-free lignin was dissolved in 10 ml of dioxane-water (volume ratio 96︰4). The lignin solution was refluxed at 85 °C under nitrogen for 2 h. And the insoluble residue (L2) was removed by centrifugation. L2 was washed with fresh dioxane until the filtrate was clear. The filtrated solutions were rotary-evaporated at 45 °C to about 1/5 of the initial volume. The evaporated supernatant was added dropwise into 250 ml of Et2O under vigorous stirring. The dioxane lignin (L3) was collected, washed successively

Figure 1

with small portions of Et2O and water, and then vacuum-dried to obtain the purified lignin (L4). 2.3

Analytical procedure

The lignin content, carbohydrate analysis, and extractive content (by acetone) were determined according to the NREL LAP [21]. Elemental analysis (C, H and N) was performed on Perkin-Elmer 1400C. The methoxyl contents of the lignin samples were determined according to the Zeisel method. Thermal analysis of the lignin was performed using TGA on a simultaneous thermal analyzer (DMA242C-DSC204STA449, NETZSCH, Germany). After drying in an oven at 105 °C for 24 h, the samples were heated from 30 °C to 900 °C at a heating rate of 10 °C·min−1, using a constant nitrogen flow as inert atmosphere in the experiments. UV spectra were obtained using a Perkin Elmer Lambda 25 spectrophotometer. Lignin sample (5 mg) was dissolved in dioxane-water (volume ratio 95︰5) (10 ml). 1 ml aliquot was diluted to 4 ml with dioxanewater (volume ratio 50︰50), and the absorbance between 260 and 400 nm was measured. Fourier transform infrared (FT-IR) spectroscopy was performed on a Nicolet Avatar360 FT-IR spectrophotometer using a KBr disk containing 1% finely ground sample. The 1H NMR spectrum of the acetylated lignin in the solution was recorded on a Bruker MSI-250 spectrometer using 25 mg of lignin in 1.0 ml of DMSO-d6. 3 3.1

RESULTS AND DISCUSSION Chemical composition of isolated lignins

To verify the purity of the isolated lignins, the preliminary search for non-ligneous components of the lignins was carried out by analysis of the bound polysaccharides composition. Table 1 gives the composition

Scheme for isolation and purification of lignin from alkali treated prehydrolysate of corn stover

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of polysaccharides of lignins from the sequential purification steps. L1 (crude lignin) contains about 16% polysaccharides, which are degraded from polysaccharides in alkaline pretreatment. Lignin can easily dissolve in dioxane [23], while polysaccharides cannot. Thus, polysaccharides should be separated from lignin by dioxane dissolution. A relatively high quantity of lignin (31%) in L2 (dioxane insoluble residue) indicates that some polysaccharides (mainly xylose) link to L1 chemically. Obviously, the mild alkaline treatment does not completely cleave the α-ether linkages between lignin and polysaccharides. It is widely accepted that the complex structure of the lignin polysaccharides (especially hemicellulose) network is the major reason for the difficult isolation of pure lignin. Milled wood lignin, after a series of purification steps, still contains 5%-10% polysaccharides [24-26]. Interestingly, the sugar content of L4 (purified lignin) is less than 2.11%. Crude lignin (L1), purified by three steps, leads to a collection of purified lignin (L4), which is 63.4% of the initial Klason lignin. The elementary composition contents determined for the lignins are presented in Table 1. Elemental analysis results demonstrate that L4 has the highest carbon and lowest sulfur contents compared to the other lignins. The nitrogen content reflects the contamination by the protein residues, and implies the strong chemical bond between proteins and lignins, which is the same result found previously [14, 27]. The sulfur content of lignin is due to the acid precipitation Table 1

by sulphuric acid. 3.2

Thermal stability

TGA curves reveal the mass loss of substances in relation to the temperature of thermal degradation, while the first derivative of the curve (DTG) shows the corresponding rate of mass loss. The peak of this curve (DTGmax) may be expressed as a single thermal decomposition temperature and can be used to compare thermal stability characteristics of different materials. Fig. 2 shows that the TG curves of the four lignins exhibit similar three-stage degradation process. In the first stage (30-200 °C), the mass loss is mainly attributed to the loss of moisture, bound water and some low molecular mass fractions (such as volatile acid). Pyrolysis of lignin mainly happens in the second stage (200-500 °C). Pyrolytic degradation in this region involves fragmentation of inter-unit linkages, releasing monomeric phenols into the vapor phase, and the decomposition of some aromatic rings [28, 29]. In the third stage (500-900 °C), the rate of mass loss is lower. Possibly further decomposition and recondensation of aromatic rings happen in this stage [30]. The DTG curves of the four lignins are different from each other, due to their different chemical compositions. It has been shown that hemicelluloses degrade at a much faster rate than lignins between 200 and 300 °C, while cellulose degrades faster than lignins

Polysaccharides content, klason lignin composition and elemental analysis of lignin samples

Lignin

Glucose/%

Xylose/%

Arabinose/%

L1

4.42

9.71

2.3

L2

8.06

25.87

4.60

L3

1.99

1.32

0.44

L4

0.71

1.06

0.34

Klason lignin/%

Elemental composition/% C

H

N

S

O

55.5

47.41

5.40

2.07

3.295

41.82

30.97

44.89

6.11

3.59

2.49

42.90

82.91

57.69

6.03

1.81

2.103

32.37

89.78

58.98

5.99

1.72

2.025

33.30

Note: L1—crude lignin; L2—dioxane insoluble residue; L3—dioxane lignin; L4—purified lignin.

Figure 2 TGA and DTG curves of lignin samples (S+L: 50% sodium sulfate plus 50% purified lignin)

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between 315 °C and 400 °C [31]. L2 contains the largest amount of hemicelluloses. The decomposition temperature of hemicelluloses is lower than those of the others. Thus, L2 should degrade at a lower temperature, which is in accordance with the DTGmax results in Table 2. Table 2

Mass loss parameters of lignin samples

Lignin

Temperature of 50% mass loss/°C

Residue at 900 °C /%

DTGmax /°C

L1

414

31

304, 770

L2

377

34

290

L3

430

35

343

L4

416

34

359

Different from other three lignins, the TG curves of L1 show distinct downtrend between 750 °C and 800 °C. This may be due to the existence of metal salt Na2SO4, which is originated from the neutralizing step by adding 20% (by mass) H2SO4 to acidify the prehydrolysate. In order to verify it, TGA and DTG were performed on the mixture of 50% (by mass) sodium sulfate and 50% (by mass) purified lignin (S + L). Fig. 2 shows that S + L has a DTGmax at 770 °C. Similar phenomenon was also observed by other researchers. Wu et al. [32] pointed out that molten salt reaction occurs in the high temperature region, followed by the reaction between the carbonized lignin and the melted salt. 3.3

Chemical characterization of lignin

3.3.1 UV Figure 3 shows that UV spectra of L3 and L4 are similar, typical UV spectra of lignins with a maximum at 280 nm, originated from non-conjugated phenolic groups in the lignins [33], such as sinapyl alcohol, coniferyl alcohol and a smaller amount of p-coumaryl alcohol. The absorption around 310 nm indicates the presence of structures containing unsaturated moieties conjugated with aromatic moieties, which has been previously reported as indicator of the presence of hydroxycinnamic acid type structures [34], and will be verified by FT-IR spectra and 1H NMR spectrum.

Figure 3 UV spectra of lignin samples

UV spectrum is able to semi-quantify the purity

of lignin. Obviously, L4 gives higher absorption than L3, indicating that some water soluble substances were washed away by water. There is almost no absorption for L1 and L2, due to the nonlignin material such as associated hemicelluloses. 3.3.2 FT-IR To further investigate the chemical structures of the lignins, FT-IR spectra are shown in Fig. 4. The corresponding bands and assignments are in accordance with the published data [35-39]. The FT-IR spectrum of L4 clearly shows the typical signal pattern of a lignin. The broad band at 3430 cm−1 is attributed to hydroxyl groups. The absorption at 1652 cm−1 is due to the carboxylic groups or acetate. Bands at 2934, 2850 and 1462 cm−1 are from C H stretching, asymmetric vibrations of CH3 and CH2, respectively. Aromatic skeletal vibrations give three strong peaks at 1587, 1507, and 1421 cm−1. Syringyl (1329 cm−1) and guaiacyl (1263 cm−1) ring breathing, syringyl (1126 cm−1) and guaiacyl (1033 cm−1) aromatic C H in-plane deformation, syringyl C H in-plane deformation (1227 cm−1), C H out of plane bending (836 cm−1), and p-hydroxyphenyl (1166 cm−1) C O stretching demonstrate that L4 is a typical annual (GSH) lignin. In short, the FT-IR spectra of L4 show that lignin from prehydrolysate of corn stover is of GSH type with a small content of hydroxycinnamic acids [40]. Besides, it contains substantial hydroxyl and carbonyl groups, and a large amount of unsaturated bonds. L3 and L4 have rather similar FT-IR spectra, but a careful examination reveals a significant difference in the range of 3000-2800 cm−1, corresponding to C H stretching in aliphatic moieties. In terms of UV spectral results, L4 has higher purity than L3. The bands of L1 at 1651, 1513, 1457, 1422, 1332, 1127, 835 cm−1 and those of L2 at 1657, 1518, 1418 cm−1 are the lignin absorption peaks. The wave number characteristic for typical xylan is 1043 cm−1, which is assigned to C O and C C stretching and glycosidic linkage (C O C) contributions. L1 has the band intensities at 1380 cm−1 (C H deformation vibration) and 1240 cm−1 (C H, C O or O H bending vibration), and L2 has the band intensities at 1382 cm−1 (C H deformation vibration), 1248 cm−1 (acetyl) and 899 cm−1 (β-glycosidic linkages), which are the further evidence for polysaccharides moieties in L1 and L2, as shown in Table 2. Marques et al. [40] indicated that the existence of lipids might cause interference in the lignin utilization. Thus, FT-IR analysis of lipids was also carried out on hexane extractive of L1 in this study. In Fig. 4 (d), there are two sharp peaks at 2924 cm−1 and 2849 cm−1. In addition, the intensity of 2924 cm−1 is much stronger than that of 2894 cm−1. Thus, there should be much more methyl than methylene. This indicates that long carbon chains may exist. Signal at 1712 cm−1 is due to C O stretching. 1162, 1095 and 1049 cm−1 can be assigned to C O stretching of tertiary alcohol, primary alcohol and secondary alcohol, respectively. The aromatic skeletal vibrations at 1647, 1514 and

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Figure 4 FT-IR spectra of L1, L2, L3, L4 and E (E—hexane extractive of L1)

Figure 5

1

H NMR spectra of purified lignin (L4)

1462 cm−1 are observed. In conclusion, the lipids in crude lignin are probably composed of saturated and/or unsaturated long carbon chains, fatty acids, triterpenols, waxes, and derivatives of aromatic. 3.3.3 1H NMR The 1H NMR spectrum of acetylated lignin (Fig. 5)

allows the estimation of relative abundance of several substructures. Signal assignments are made on the basis of literature data [24, 38, 41]. Calculations for all structural elements are made using the resonance of methoxyl protons (3.6-4.0) as an internal standard. The results are shown in Table 3. The shoulder at 5.0 arises from the protons of xylan residual, in

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Chin. J. Chem. Eng., Vol. 21, No. 4, April 2013 Table 3

Signal assignment for 1H NMR spectroscopy of acetylated lignin (L4)

Structural element

Number

aromatic proton in p-hydroxyphenyl units

7.43-8

0.11

aromatic proton in guaiacyl units

6.70-7.43

0.48

aromatic proton in syringyl units

6.28-6.7

0.17

Hα of β-O-4 and β-1 structure

5.75-6.15

0.09

Hα of β-5 and Hα of α-O-4

5.2-5.75

0.07

Hα of β-β

4.45-5.2

0.20

Hγ of β-O-4

3.95-4.45

0.33

H of methoxyl groups

3.5-3.95

1.00

H of aromatic acetates

2.15-2.4

0.29

H of aliphatic acetates

1.6-2.15

0.88

0.6-1.6

0.65

CH2

and

CH3

accordance with the results from sugar analysis. An intensive signal at 2.4 is indicative of protons in DMSO. The integrals of signals in the range of 7.43-8.00, 6.70-7.43 and 6.28-6.70 are attributed to aromatic protons of p-hydroxyphenyl, guaiacyl and syringyl units, respectively. The marked signals in this range illustrate the presence of these three units in the lignins, which is also reflected in the FT-IR spectra. The calculated ratio of S︰G︰H is 14︰63︰23. As long as the ratio of the syringyl units of this lignin is lower than that of technical lignin, steric hindrance by the methoxyl groups should be smaller. In the aliphatic regions, signals of typical lignin units such as β-O-4 (5.75-6.15, 3.95-4.45), β-5 (5.2-5.75), β-β (4.45-5.2) and α-O-4 (5.2-5.75) moieties can be easily seen. Clearly, β-ether (β-O-4) units are the major inter-unit structure. Strong signals of pinoresinol (β-β) units are also remarkably presented. Thus the presence of hydroxycinnamic acid type structures in lignins is supported by the FT-IR spectra (Fig. 4) and 1H NMR spectra (Fig. 5). In FT-IR spectra, characteristic bands at 1705 and 1630 cm−1 are assigned to C O stretching of carboxylic acids and C C moieties conjugated with aromatic rings, respectively [41]. In Fig. 5, the broad signal around 7.3-7.6 spectra can be assigned to the aromatic protons in positions 2 and 6, within the structures containing a C O group [42]. 4

Spectral area

than 2.11%, mainly composed of guaiacyl units, and β-O-4 ether bonds as a predominant linkage together with small amounts of β-β, β-5 and α-O-4 linkages. Its DTGmax is 359 °C. Structural and thermal analyses suggest that purified lignin from prehydrolysate of corn stover may be a better biopolymer for utilization, as it presents higher amount of activated free ring positions, higher purity and appropriate thermal decomposition temperature. REFERENCES 1 2

3

4

5 6

CONCLUSIONS 7

The crude lignin from prehydrolysate of corn stover is a heterogeneous material of syringyl, guaiacyl and p-hydroxyphenyl units, containing associated polysaccharides, lipids and melted salts. Some of the lignin is chemically linked to hemicelluloses (mainly xylan). The lipids in crude lignin are probably composed of saturated and/or unsaturated long carbon chains, fatty acids, triterpenols, waxes, and derivatives of aromatic. The purified lignin contains sugar of less

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