Chemical reactivity of alkali lignin modified with laccase

b i o m a s s a n d b i o e n e r g y 5 5 ( 2 0 1 3 ) 1 9 8 e2 0 4

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Chemical reactivity of alkali lignin modified with laccase Yong Sun a,c, Xueqing Qiu b,*, Yunquan Liu a a

School of Energy Research, Xiamen University, Xiamen 361005, PR China State Key Laboratory of Pulp and Paper Engineering, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510641, PR China c Key Laboratory of Energy Thermal Conversion and Control of Ministry of Education, South East University, Nanjing 210018, PR China b

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abstract

Article history:

The modification of alkali lignin with laccase was investigated. The structural change of lignin

Received 12 May 2012

was analyzed. The sulfonation reactivity was measured by the content of sulfonic group. The

Received in revised form

results showed the sulfonation reactivity increased to some extent under the condition of

31 January 2013

atmosphere pressure, but decreased under the condition of 0.3 MPa oxygen pressure. The

Accepted 3 February 2013

analysis of Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR)

Available online 7 March 2013

and gel permeation chromatography (GPC) showed the cleavage of various ether linkages and demethylation took place in the structure of lignin to certain extent during modification with

Keywords:

laccase, which contributed to the improvement of sulfonation reactivity. Under the condition

Alkali lignin

of 0.3 MPa oxygen pressure, the ratio of s/g (guaiacyl/syringyl) increased after modification,

Laccase

which reduced the sulfonation reactivity of lignin. Simultaneously partial polymerization

Modification

reaction, such as 4-O-50 , b-5, 5-5 and other reaction in the aromatic ring decreased the activity

Sulfonation reactivity

sites of C2, C5 and C6. Abundant polymerization reaction of a-O increased steric hindrance of C2

FTIR

and C6 in aromatic ring, resulting in low sulfonation reactivity of lignin. ª 2013 Elsevier Ltd. All rights reserved.

13

C NMR

1.

Introduction

Lignin is a natural polymer found in biomass and one of the most abundant biomacromolecules, second only to cellulose in natural abundance [1]. Its structure is composed of three different types of phenolic precursor units (e.g. p-coumaryl-, coniferyl- and sinapyl-alcohols), which linked by ether and carbonecarbon bonds formed an irregular network biopolymer [2,3]. At present, lignin mostly comes from pulp and paper industry, where it usually serves as a fuel or for the recovery of inorganic cooking chemicals [4]. Only a limited amount is isolated from the spent pulping liquors and used in various specialty products such as biomaterials, fuels, biocides and biostabilisers, animal feed, health products and

crops cultivations [5]. However, in many pulp mills, which do not install the recovery system, the black liquor is simply treated or discarded. In this case, the valuable chemical properties and functionality of lignin are not utilized. Thereby, the utilization of this renewable natural product, lignin, could have economic and environmental benefits [4]. Alkali lignin is one kind of technical lignin, coming from pulp and paper making industry. Its insolubility is the disadvantage limiting its applications. Thereby, alkali lignin must be modified before application. However the high-valueadded application of lignin depended entirely on the structures of monolignols, the functional group in the aromatic ring and various linkages in lignin. The main chemical functional groups in lignin are the hydroxyl(phenolic or alcoholic),

* Corresponding author. Tel./fax: þ86 020 87114722. E-mail addresses: [email protected] (Y. Sun), [email protected] (X. Qiu). 0961-9534/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biombioe.2013.02.006

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methoxyl, carbonyl and carboxylic groups. The abundant interunit linkages in all lignin are the b-aryl ether (b-O-4), 4-O5(diaryl ether), 5-50 (biphenyl), b-5 (phenylcoumaran), b-b, and b-1 structures [6]. The proportion of these groups and linkages depend on the genetic origin and the isolation processes employed [7]. During the violent isolation of alkali lignin, its various linkages may be broken or polymerized, which usually results in the decrease of chemical reactivity and hindering the further chemical modification [8]. Due to these reasons, the lignin must be activated before modification. The current activation methods of alkali lignin could be either chemical or biological. The chemical methods including demethylation [9], hydroxymethylation [10,11], oxidation [12] and so on, have already used in the industry [11], but the biological one is still at the research stage. Compared to the chemical ones, the biological method has the advantage of mild reaction conditions and less chemical reagents, which could be the promising direction in future. In this study, the wheat straw alkali lignin (WAL) was treated with laccase, then the treated wheat straw alkali lignin (TWAL) was modified by sulfonation reaction in order to increase its solubility and surface activity. The chemical reactivity of WAL was evaluated by the content of sulfonic group. Its structural change was also analyzed before and after treatment with laccase to understand the mechanisms of modification.

2.

Material and method

2.1.

Modification of lignin with laccase

Crude laccase was supplied by Shanghai Denykem Co., Ltd, which was kept at the low temperature of 4  C. The alkali lignin was supplied by Quanlin Paper Co., Ltd. in Shandong province, China, which was separated from wheat straw pulping black liquor using acid precipitation. The wheat straw was collected around Quanlin Paper Mill in Shandong region. The lignin purification process was as follows. Firstly, the lignin was dissolved in alkali solution, and then the impurity was separated from lignin solution by centrifugation. At last, the lignin solution was adjusted to pH 5.0 with hydrochloric acid for further experiment. The experiment was conducted in a 500 mL reaction vessel, which was agitated at 2.5 Hz under the temperature of 60  C. After the treatment, the lignin was washed and freezingly desiccated. 0503, 0506, 05012 and 0524 represent the lignin samples treated for 3 h, 6 h, 12 h and 24 h under atmosphere pressure respectively; whilst O-0503, O-0506 and O-0509 represent those treated for 3 h, 6 h and 9 h under 0.3 MPa oxygen pressure respectively. The dosage of crude laccase for all samples is a mass fraction of 5% on the lignin. About 25 mg of dry lignin was put in a 1:1 mixture of acetic anhydride/pyridine (2.00 mL) and stirred at room temperature for 24 h. Ethanol (25.0 mL) was added to the reaction mixture, leaving it for 30 min, and then removed by a rotary evaporator. The addition and removal of ethanol was repeated until all traces of acetic acid were removed from the sample. The residue was dissolved in chloroform (2.0 mL) and precipitated with diethyl ether (100.0 mL). The precipitate was centrifuged, washed with diethyl ether (3), and dried under vacuum prior to the NMR analysis.

2.2.

Sulfonation of lignin

10 g of lignin was dissolved in 100 mL of NaOH (concentration of 25 kg m3), and pH is 10.7. 0.5 g formaldehyde was added to the solution and reacted at 70  C for 1.0 h. Then the temperature was raised to 90  C; Added 2.5 g Na2SO3 to the solution and let them react for 2.5 h.

2.3.

Sulfonic group content measurements

The potentiometric titration method was used to measure the sulfonic group content of the samples with an automatic potentiometric titrator (809 Titrando, Metrohm Corp.). Before titration, the lignosulfonate sample was ion-exchanged through anion exchange resin and cation exchange resin to remove the low molecule organic acid, inorganic salt and other impurities. The Sulfonic group content was calculated as follows: Sulfonic group content ¼

  Mole of sulfonic group 1 mo lg Mass of dry lignin

2.4. Fourier transform infrared spectroscopy (FTIR) analysis FTIR spectra were recorded between 4000 and 400 cm1, using a Nexus spectrometer (Thermo Nicolet, Madison state, USA). Discs were prepared by firstly mixing 2 mg of dried sample with 200 mg of KBr (for spectroscopy) in an agate mortar. The resulted mixture was successively pressed at 10 MPa for 3 min. The spectra were normalized by Omnic software.

2.5.

Liquid-state NMR analysis

The NMR spectra were recorded on a Bruker DRX-400 spectrometer using DMSO-d6 as the solvent. Chemical shifts were referenced to TMS (0.0 ppm). The 13C NMR spectra were recorded at 100.59 MHz using 5 mm-diameter tubes with the following parameters: 90 pulse angle; 12 s relaxation delay and 18,000 scans. The acetylated lignin was resolved in DMSO-d6.

2.6.

Molecular weight distribution analysis

The molecular weight distribution of lignin was determined by using aqueous gel permeation chromatography (GPC) which consisted of Waters 1515 Isocratic HPLP pump, Waters 2487 UV Absorbance Detector (Waters Corp., USA) and Ultrahydragel 120 and Ultrahydragel 250 columns. The mobile phase was 0.10 mol L1 NaNO3 solution with pH 10.7 and ran at a flow rate of 0.50 mL min1. The polystyrene sulfonate was used as the standard substance. Samples were filtrated by a 0.22 mm filter and analyzed in duplicate.

3.

Results and discussion

3.1.

Sulfonation reactivity of lignin

The scheme of sulfomethylation was shown as Fig. 1. The sulfonation reactivity of lignin was described by the content of sulfonic group. Table 1 showed the reactivity of modified

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Fig. 1 e The scheme of sulfomethylation in lignin.

lignin was improved after treatment under atmosphere pressure, thus reduced quickly under 0.3Mpa oxygen pressure.

3.2.

Fig. 2 e Gel permeation chromatography of lignin.

Molecular weight distribution

The modification of lignin structure with laccase was investigated by evaluating the changes in molecular weight distribution. Fig. 2 recorded the chromatograms obtained by GPC for lignin treated with laccase. The weight-average (Mw), number-average (Mn) molecular weights and the polydispersity (Mw M1 n ) were given in Table 2. For control sample, the molecular weight distributed between 100 and 55000, among which the fraction with molecular weight more than 1000 occupied a mass fraction of 74.75%, and that less than 1000 occupied a mass fraction of 25.25%, about a mass fraction of 65.46% had the molecular weight within 1000e30000. These results proved the structural heterogeneity of the lignin macromolecule and the polydispersity of molecular mass distribution. After being treated with laccase, molecular weight gradually increased with the treatment time. These results generally demonstrated a partial polymerization of lignin during treatment with laccase.

3.3.

FT-IR spectroscopy analysis

The FT-IR spectra of lignin samples were shown in Fig. 3. A wide absorption band focused at 3420 cm1 was assigned to stretching vibration of aromatic and aliphatic OH groups while bands at 2935 cm1, 2847 cm1 were related to the CeH stretching vibration of CH2, CH3 and CH3O groups [4,13]. All lignin samples showed bands at 1607 cm1, 1515 cm1 and 1425 cm1 corresponding to aromatic ring stretching vibrations [14]. Bands between 1778 cm1 and 1679 cm1 was attributed to the non-conjugated carbonyl groups, the wide

Table 1 e The content of sulfonic group in lignin. No. 1 2 3 4 5 6 7 8

Item Control 0503 0506 0512 0524 O-0503 O-0506 O-0509

Sulfonic group content (mmol g1) 2.09 1.92 2.06 2.58 2.68 1.42 1.09 1.24

absorption band at 1650 cm1 band was related to the presence of conjugated carbonyl groups in its structure [4,13,15e17]. The bands at 1460 cm1 was assigned to asymmetric vibration of CeH asymmetric deformations in CH3 and CH2 [13,17], bands at 1365 cm1 was assigned to in-plane deformation vibration of phenolic hydroxyl and CH3 [13], bands at 1328 cm1 and 1270 cm1 were assigned to syringyl ring breathing with CeO stretching vibration and guaiacyl ring breathing respectively [4,6,13,18,19], bands at 1215 cm 1 was assigned to CeO stretching vibration of phenolic hydroxyl and phenolic ether [13,16,18], bands at 1117 cm1 was assigned to Aromatic CeH in-plane deformation [13,17,18,20], bands at 1034 cm1 was assigned to Aromatic CeH in-plane deformation, CeO deformation in primary alcohols and CeH stretching [6,13,16e18], bands at 855 and 810 cm1 were assigned to C-H out-of-plane deformation vibration of guaiacyl [13,15,16,18], bands at 825 cm1 was assigned to C-H out-ofplane deformation vibration of syringyl [3,21]. The Fig. 3 showed the peaks around 2847 cm1 became less apparent under atmosphere pressure and 0.3 MPa oxygen pressure, there may be some methoxyls were removed from the aromatic ring during the treatment with laccase. However, the peak around 1365 cm1 in the sample 0524 is more obvious than that in the other two samples, this result was likely to be caused by the increase of phenolic hydroxyl. When the ether bonds, such as b-O-4, broke during the treatment could lead to the more phenolic hydroxyl in lignin. Meanwhile, the signal

Table 2 e Number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity (Mw ML1 n ) of lignin. Time Day1

Mn

Mw

Mw M1 n

Control 0503 0506 0512 0524 O-0503 O-0506 O-0509

860 877 895 916 987 993 1065 1249

3687 4001 4140 4231 4645 4405 4898 5964

4.29 4.57 4.63 4.62 4.71 4.44 4.60 4.78

b i o m a s s a n d b i o e n e r g y 5 5 ( 2 0 1 3 ) 1 9 8 e2 0 4

201

Fig. 3 e FTIR spectra of lignin.

intensity around 1328 cm1 increased slightly in samples 0524 and O-0509, but the peak around 1270 cm1 became less obvious in sample 0524, maybe some guaiacyl moieties converted to syringyl moieties during the treatment process. The signal intensity between 11631 and 1679 cm1 increased obviously which indicated the more conjugated carbonyl groups formed.

3.4. 13

13

C NMR analysis

C NMR spectroscopy was a reliable method to investigate the structure of the carbon skeleton in lignin. Though signals of some lignin structures were overlapped in the 13C NMR spectra each other, the mostly bond-linkages of lignin had been confirmed. The specific lignin moieties and bondlinkages were detected in various source of lignin Refs. [15,22e27]. The structural changes during treatments with laccase were also investigated by 13C NMR spectroscopy in this experiment. The 13C NMR spectra of acetylated lignin were shown in Fig. 4. The integral of the 162e102 ppm region was set as the reference, assuming that it includes six aromatic carbons and 0.12 vinylic carbons. It follows that the integral value divided by 6.12 was equivalent to one aromatic ring (Ar) [22]. Table 3 listed the various spectral regions identified in the quantitative 13C spectra with results reported as the number of moieties per aromatic ring. Analysis of the carbonyl region (210e190 ppm) of the 13C NMR spectra revealed the differences among three lignin preparations. The total amount of conjugated carbonyl moieties (integral at 200-190 ppm) was 0.091 Ar1, the integration of the resonance of nonconjugated C]O groups (210-200 ppm) gave the value of 0.121 Ar1 for control sample (Fig. 3). However, the total amount of conjugated carbonyl moieties (integral at 200-190 ppm) increased to 0.122 Ar1 and 0.137 Ar1 for 0524 and O-0509 samples respectively. The amount of noconjugated carbonyl noietyes also increased after

modification with laccase. The conjugated carbonyl included a-ketone moieties, a-carbonyl moieties, vanillin, coniferaldehyde and so on. The increased amounts of conjugated carbonyl mostly resulted from these moieties, which indicated Ca in moieties of lignin was easy to be oxidized during modification with laccase. Although the content of these moieties in the lignin was relatively low, the increased amount of benzaldehyde structures such as vanillin further suggested that modification process leads to not only oxidation but also degradation of side-chain to some extent. The amount of hydroxyl groups was estimated in the acetylated control sample from the region of 173e166 ppm. The total amount of hydroxyl groups was about 0.783 Ar1 in control sample, but increased to 0.592 Ar1 and 0.929 Ar1 in 0524 and O-0509 samples respectively, which indicated the ether bonds and ester bonds partially broke. The amounts of primary OH were 0.136 Ar1 in control, but increased to 0.189 Ar1 and 0.203 Ar1 in 0524 and O-0509 samples respectively. The amount of secondary OH only marginally increased. The amount of phenolic OH was estimated by integrating the region of 168.7e166 ppm. However the minor amount of Ar-COOR moieties also resonated in the same region of the spectrum [22]. Though the amount of phenolic OH was overestimated, the increase of content indicated modification of lignin with laccase resulted in the cleavage of bond b-O-4. The increase of primary OH indicated the side chain of lignin broke during the process of modification. Integration of the control sample methoxyl peak at 58e53.5 ppm yielded a methoxyl group content of 1.642 Ar1. However, NMR analysis of 0524 and O-0529 samples spectra determined the methoxyl content to be 1.330 Ar1 and 1.444 Ar1 respectively. These results indicated modification with laccase was able to decrease the content of methyl group in the structure of lignin. The characteristic tertiary carbon resonances from s units at 102e108.5 ppm (C2, 6), g units at 108.5e123.5 ppm (C2, 5, 6)

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Fig. 4 e 13C NMR spectra of acetylated technical lignin.

were used to assess the s:g ratios of lignin. The C3, 5 signals of h units arose at about 123 ppm, which overlapped with signals of C5, 6 in g units. The Cb signals of h units arose at about 118 ppm. There were still the problems that the unequal proportion noncondensed moieties in g and s units. To

Table 3 e Signal assignment in 13C NMR spectra of technical lignin and results from quantification of functional groups. NO.

Peaks

Assignment

Amount (Ar1) Control 0524 O-0509

1 2 3 4 5 6 7

8 9 10 11 12 13

210e200

Non-conjugated C]O 200e190 Conjugated C]O 173e171 primary aliphatic OH 171e168.8 secondary aliphatic OH 168.8e166 phenolic OH, conjugated COOR 114.5e108.5 C2 in g units 81e78.2 Ca, Cb in Alk-OeAr and a-O-Alk 58e53.5 OMe, C1 and Cb in spirodienone 108.5e102 s 123.5e108.5 g 125e102 “CAr-H” 142e125 “CAr-C” 162e142 “CAr-O”

0.121

0.130

0.150

0.091 0.136

0.122 0.189

0.137 0.203

0.267

0.299

0.268

0.380

0.404

0.459

0.593 0

0.543 0.094

0.541 0.307

1.642

1.330

1.444

0.760 1.734 2.668 2.086 1.371

0.671 1.558 2.392 2.173 1.550

0.812 1.539 2.5s58 2.091 1.472

eliminate this shortcoming, Capanema [23] suggested estimating the amount of g units from the integral of g-2 at 114.5e108.5 ppm. The amount of g-2 condensed moieties was negligible, and the substitution at g-5 and g-6 did not affect the chemical shift of g-2 significantly. The percentage of s-condensed carbons was also rather low as compared to the total amounts of s-2, 6 carbons, and it was in the range of the accuracy of the NMR experiment. The ratios of s:g was 0.642, 0.618 and 0.750 for control, 0524 and O-0509 samples respectively. Analysis of 13C NMR showed the amounts of CAr-H decreased during modification with laccase, which indicated the aromatic rings can polymerize each other with linkgages such as 4-O-50 , b-5 or 5-5. The amount of CAr-O and CAr-C increased slightly attested this result. In the 13C-NMR spectrum of control, the peak between 82 and 78.2 was negligible. After the alkali lignin was treated with laccase, the obvious peak arose between 82 and 78.2 ppm in the 13C NMR spectra of 0524 and O-0509 samples, especial in O-0509 sample, which contribute to the chemical shifts of Ca such as a-O-4 with b-O-4 or a-O-alkyl(H) and Cb with a-ketone

Table 4 e The charge density of lignin model compounds on the C2, C5 and C6. Compounds

C2 C5 C6

Charge density LC

LD

0.159439 0.130515 0.115399

0.158504 0.129620 0.121352

b i o m a s s a n d b i o e n e r g y 5 5 ( 2 0 1 3 ) 1 9 8 e2 0 4

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Fig. 5 e The charge density of lignin model compounds. The potential energy of molecular conformation optimized to minimum by molecular mechanics method.

moieties or a-O-4, Cg ¼ O [27e30]. This indicated partial moieties polymerized each other with a-O linkgages. The 13C-NMR analysis attested the results of FTIR analysis, such as the demethylation function, bond cleavage of b-O-4, increase in the content of hydroxyl group (including aliphatic hydroxyl and phenolic hydroxyl), conjugated and nonconjugated carbonyl groups.

3.5.

Molecular simulation analysis

The hydroxylmethylation was electrophilic addition reaction. The methoxyl, etherified and nonetherified hydroxyl groups were electron-donating groups. The hydroxylmethylation was easy to take place on the carbon atom rich in electron cloud. Table 4 showed structural analysis of two lignin model compounds. For compound LC, because of the grant electron effect of methoxyl group, the electron density on C2 of the aromatic ring was higher than that on C5 and C6. For compound LD, the electron density on C6 was higher than that on LC, which indicated the grant electron effect of hydroxyl was more than methoxyl. The demethylation function usually transformed methyl group to hydroxyl group [31e33]. From Fig. 5, the steric hindrance effect of methoxyl or hydroxyl group on C3 and side chains on Ca to C2 was more prominent than that to C5 and C6. Thereby the hydroxylmethylation can first take place on C5, then on C6 or C2. The analysis of GPC, FTIR and NMR showed that more content of guaiacyl and hydroxy, less content of methoxyl, syringyl can enhance the sulfonation reactivity of lignin. The steric hindrance was also the importance factor affecting the chemical reactivity of lignin. During the treatment with laccase, various bonds cleavage, such as b-O-4, b-5 and b-1, can also increase the content of hydroxyl group (including aliphatic hydroxyl and phenolic hydroxyl). The demethylation function usually transformed methyl group to hydroxyl group. These may be beneficial to the increase of sulfonation reactivity. Under atmosphere pressure, the contents of methoxyl decreased obviously after treatment with laccase, which increased the contents of phenolic hydroxyl and tertiary carbon atom. Simultaneously, the polymerization of Ca-O was not obvious, the effect of steric hinderanc was small. Thereby, the sulfonation reaction reactivity of lignin was enhanced. But under 0.3Mpa oxygen pressure, the polymerization of Ca-O was

very obvious, which increased the effect of steric hindrance. The sulfonation reaction reactivity of lignin dropped quickly.

4.

Conclusions

The modification of alkali lignin with laccase was investigated under different conditions. The results showed the reasctivity of modified lignin is improved after treatment under atmosphere pressure, but reduced quickly under 0.3Mpa oxygen pressure. The analysis of GPC, FTIR and NMR showed that more content of guaiacyl and hydroxy, the less content of methoxyl, syringyl can enhance the sulfonation reactivity of lignin. The steric hindrance also was the important factor to affect the chemical reactivity of lignin. During modification with laccase, the various bonds cleavage, such as b-O-4, b-1, b-5 and 5-5, can also increase the content of hydroxyl group (including aliphatic hydroxyl and phenolic hydroxyl). The demethylation function usually transforms methyl group to hydroxyl group. These contributed to the improvement of the sulfonation reactivity. Under atmosphere pressure, the contents of methoxyl decreased after treatment with laccase, which increased the contents of phenolic hydroxyl or tertiary carbon atom. Simultaneously, the polymerization of Ca-O was not obvious, the steric hindrance on C2 and C6 in the structure of aromatic ring was small. These contributed to the increase of sulfonation reactivity of lignin. But under 0.3 MPa oxygen pressure, the partial polymerization reaction and other reaction on the aromatic ring decreased the activity sites of C2, C5 and C6. Abundant polymerization reaction of a-O also increased the steric hindrance on C2 and C6 in the structure of aromatic ring, which resulted in low sulfonation reaction reactivity of lignin.

Acknowledgments The authors are grateful to the financial support from National Key Basic Research Program (2010CB732201) from the Ministry of Science and Technology of China, Natural Science Foundation of China (21106121), Basic Foundation for Scientific Research of Universities (2010121077) from the Ministry of Education of China, Provincial R&D Program (1270-K42004) from Economic and Trade Committee of Fujian Province of

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China, Key Laboratory of Energy Thermal Conversion and Control of Ministry of Education.

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