Effect of mechanical activation on structure changes and reactivity in further chemical modification of lignin

Accepted Manuscript Title: Effect of mechanical activation on structure changes and reactivity in further chemical modification of lignin Author: Xiaohong Zhao Yanjuan Zhang Huayu Hu Zuqiang Huang Mei Yang Dong Chen Kai Huang Aimin Huang Xingzhen Qin Zhenfei Feng PII: DOI: Reference:

S0141-8130(16)30620-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.06.074 BIOMAC 6247

To appear in:

International Journal of Biological Macromolecules

Received date: Accepted date:

21-6-2016 23-6-2016

Please cite this article as: Xiaohong Zhao, Yanjuan Zhang, Huayu Hu, Zuqiang Huang, Mei Yang, Dong Chen, Kai Huang, Aimin Huang, Xingzhen Qin, Zhenfei Feng, Effect of mechanical activation on structure changes and reactivity in further chemical modification of lignin, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.06.074 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of mechanical activation on structure changes and reactivity in further chemical modification of lignin

Xiaohong Zhao a,c, Yanjuan Zhang a,b,*, Huayu Hu a, Zuqiang Huang a,b,*, Mei Yang a,b, Dong Chen b, Kai Huang b, Aimin Huang a, Xingzhen Qin a, Zhenfei Feng a

a

School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004,

China b

State Key Laboratory of Non-Food Biomass and Enzyme Technology, Guangxi Academy of

Sciences, Nanning 530007, China c

College of Chemistry and Biology Engineering, Hezhou University, Hezhou 542899, China

*

Corresponding authors. Telephone: +86 771 3233718. Fax: +86 771 3233728.

E-mail address: [email protected] (Y. Zhang); [email protected] (Z. Huang).

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Highlights 

MA significantly changed the molecular and morphological structure of lignin.



The reactivity of lignin was significantly improved after mechanical activation (MA).



The activation mechanism of MA treatment of lignin was investigated.



MA is a simple, efficient and green method for enhancing the reactivity of lignin.

ABSTRACT: Lignin was treated by mechanical activation (MA) in a customized stirring ball mill, and the structure and reactivity in further esterification were studied. The chemical structure and morphology of MA-treated lignin and the esterified products were analyzed by chemical analysis combined with UV/Vis spectrometer, FTIR ,NMR, SEM and particle size analyzer. The results showed that MA contributed to the increase of aliphatic hydroxyl, phenolic hydroxyl, carbonyl and carboxyl groups but the decrease of methoxyl groups. Moreover, MA led to the decrease of particle size and the increase of specific surface area and roughness of surface in lignin. The reactivity of lignin was enhanced significantly for the increase of hydroxyl content and the improvement of mass transfer in chemical reaction caused by the changes of molecular structure and morphological structure. The process of MA is green and simple, and is an effective method for enhancing the reactivity of lignin.

Keywords: Lignin; Mechanical activation; Reactivity

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1. Introduction Lignin is one of the principal components of lignocellulosic biomass. It is the second abundant polymer in land plants and the only renewable aromatic compounds in nature [1]. Lignin is a three dimensional amorphous polymer, composed of three phenyl-propanoid units: p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S), linked through a variety of ether (C-O) and condensed (C-C) linkages. There are also a variety of functional groups in lignin. The proportion of these linkages and groups in lignin changes with the source (softwood, hardwood, or annual plants), growing environment, harvesting time, drying and isolation pattern, and so on [1,2]. The industrial lignin is usually obtained as a byproduct of pulp in paper-making and biofuels industries. There are annually about 60 million metric tons lignin being generated worldwide [3]. The relevant department of U.S. and Europe set goals of mass biofuels production [4,5]. Large quantities of lignin will be continuously produced as the implementation of these policies. If the lignin can be properly used, the economy and feasibility of papermaking and biofuels industries will be further improved. Although lignin is abundant in source and has many characteristics such as diverse rich functional groups, renewable and biodegradable, non-toxic, strong antioxidant properties and weather resistance, thermoplastic, glassy state transformation, and so on [6-8], but only approximate 2% of the lignin is used commercially today due to the weaknesses such as the brittle nature of lignin and its incompatibility with other polymer systems. The remainder still mainly been burned as energy source or discard as waste, leading to the waste of resources and growing environmental problems. Therefore, chemical modification of lignin, which

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could improve its performances and expand its applications, would have important economic and social significance. Lignin was widely used as raw material to prepare lignin-based functional materials, such as composites, phenol-formaldehyde, epoxy resins, biomedical materials, smart materials, carbon fibers, and so on, through modification or graft polymerization [9]. While the isolation of lignin from lignocellulosic biomass usually adopts physical and/or chemical and biochemical treatment [1]. During the violent isolation process, its various linkages may be broken or formed by repolymerization for the influence of strong acid, alkali, or enzyme, which usually results in the decrease of chemical reactivity and the hindering of further chemical modification [10]. Therefore, lignin had better be activated before modification. For example, lewis acid treatment of lignin was adopted to improve the reactivity of lignin with diisocyanate monomers in order to get better lignin polyurethane properties [11]. Phenolation of lignin was also adopted to improve its reactivity before synthesizing lignin base epoxy resin [12]. The current activation methods of lignin mainly include chemical, biological, and physical methods [13-15]. Physical methods are operated through various physical actions to change the basic phenyl propane structure of lignin (C6−C3), such as heat treatment [16], microwave [17], ultrasonic [18], etc. For example, the aryl methyl ether bonds can be destroyed and the unsaturated oxygen bonds in side chain can be reduced by the physical actions, contributing to the generation of new reaction centers and the improvement of the reactivity of lignin [17]. Mechanical activation (MA) refers to the use of mechanical actions to change the structures and physicochemical properties of the solids. The chemical reactivity of the solids

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can be improved for a part of the mechanical energy changes into the internal energy of material under the mechanical actions during the MA process [19]. MA is widely used in the mineral processing, preparation of nanomaterials, pharmaceutical, etc. [20], for its application is not limited by reagent and equipment, and the equipment is also simple and easy for mass production. In our previous works [19,21,22], MA was used for the pretreatment of starches, sugarcane bagasse, and cassava stillage residue, and the results showed that MA could improve their accessibility and reactivity because the strong mechanical actions resulted in the destruction of stable aggregate structure, the decrease of particle size and crystallinity, and the weakening of inter- and intra-molecular hydrogen bonding, which also enhanced the reaction efficiency for the good dispersion and contact between the reactants. Moreover, our group has developed the MA-assisted solid phase reaction for efficient chemical modification of starch and cellulosic materials [23,24]. Similar to starch and cellulose, lignin also has amounts of hydroxyl groups, and it is promising that MA may affect its structure and improve its reactivity. Compared with common activation methods, MA can be considered as a green, economical and simple process for the activation of lignin without the use of any solvent as active medium, and it is also easy for mass production. Therefore, it has great significance to introduce MA technology into the chemical modification of lignin. But it is difficult to evaluate the interrelation between structure changes and reactivity of lignin during the process of simultaneous MA and chemical modification. In this work, lignin was first treated by MA to investigate the structure changes during MA, and then the esterification was chosen to study the effect of MA on the reactivity of hydroxyl groups in lignin with acetic anhydride as esterifying agent in both liquid phase and solid phase. The

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reasons of increased reactivity of MA-treated lignin were investigated and discussed by comparative analyses of the original lignin, MA-treated lignin and their esterified products, and the activation mechanism also could be proposed. 2. Materials and methods 2.1. Materials In this study, enzymatic hydrolysis lignin, kindly supplied by Ji'nan Yang Hai Chemical Co., Ltd., China, was a softwood lignin got from the pine which grew in the north of China. It contained 95.92% of lignin (10.60% of acid soluble and 85.32% of klason lignin), 1.05% of ash, and 3.02% of polysaccharide, with pH of 7. All chemical reagents were of analytical grade and were obtained commercially without further purification. 2.2. MA treatment of lignin The MA treatment of lignin was performed in a customized stirring ball mill driven by a commercially available drill press (maximum drilling diameter = 16 mm) equipped with a speed-tuned motor (Fig. 1). A fixed amount of milling balls (300 mL, 5 mm diameter, ratio of grinding media to material was 6 mL/g) was first added into a jacketed stainless steel chamber (1200 mL), and then 50.0 g of lignin was added into the chamber and was subjected to milling at the speed of 300 rpm at a constant temperature of 50 °C by circulating the thermostatic water in the jacket of chamber. When the mixture was milled for desired time (0.25, 0.50, 1.00, and 1.50 h), the balls were removed from the resulting sample by a sieve. The sample was sealed for chemical modification and characterization. 2.3. Esterification of lignin The esterification of lignin was carried out in two ways: liquid phase synthesis (LPS) and

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solid phase synthesis (TPS). LPS: It was performed according to the method reported in references [25,26] with minor modification: lignin sample (10 g) was added in a 250 mL Erlenmeyer flask using 4-dimethyl amino pyridine (DMAP, 0.5 g) as catalyst and 20 mL of 1,4-dioxane as solvent. Acetic anhydride was added to the flask as esterifying agent with a weight ratio of 2:1 (acetic anhydride:lignin). The flask was sealed and shaken for about 2 min in order to accelerate the dispersion of lignin and catalyst in acetic anhydride, and then was put in a constant temperature water bath to react at 50 °C for 8 h. The deionized water was added to quench the reaction. Then, the mixture was filtered and washed repeatedly with deionized water to remove solvent, catalyst and residual acid until the filtrate was neutral. The filter cake was oven dried at 50 °C for 24 h, and then the esterified lignin was obtained. SPS: The mixture of lignin, acetic anhydride and DMAP as the same amount in LPS was placed in a 100 mL Erlenmeyer flask. The flask was sealed and put in an oven to react at 50 °C for 8 h after being shaken for 2 min. Then, the mixture was taken out and washed with deionized water. The products were filtered, washed and dried as the same processes in LPS. 2.4. Functional groups analysis of MA-treated lignin Total hydroxyl content was analyzed by acetylation and titration [27]; phenolic hydroxyl and carboxyl contents were analyzed by non-aqueous potentiometry method [28]; aliphatic hydroxyl content was the difference of total hydroxyl and phenol hydroxyl; carbonyl content was analyzed by wet chemical methods [28]. All tests were performed in triplicate, and the results represented the mean values of three independent experiments. 2.5. Characterization

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The chemical structure and morphology of MA-treated lignin and the esterified products were analyzed by UV/Vis spectrometer, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), and particle size analyzer. The operating conditions of these analyses were described in supplementary material. 3. Results and discussion 3.1. Effect of MA on the chemical structure of lignin There are a large number of functional groups in the lignin molecules, such as phenolic hydroxyl, aliphatic hydroxyl, carbonyl, methoxyl, carboxyl, conjugated double bond, and so on. The relative amount of these groups will directly affect the reactivity of lignin. In order to study the effect of MA on the molecular structure of lignin, the functional groups of original lignin and MA-treated lignin were analyzed by chemical analysis combined with UV/Vis spectrometer, FTIR and NMR, and the results are shown in Table 1 and Figs. 2−5. 3.1.1. Chemical analysis It is an important aspect that functionalization of hydroxyl groups or introducing new groups at the ortho- or para- positions of phenolic groups with making use of the activated hydroxyl for modification of lignin [1]. So the content of hydroxyl is an important factor for impacting the reactivity of lignin, and increasing its relative content is the main aim for the activation of lignin. As shown in Table1, the total hydroxyl and aliphatic hydroxyl contents increased obviously after MA, and the 0.50 h MA-treated lignin had the maximum values of 7.004 and 5.497 mmo1/g, which increased by 39.74% and 50.60% compared with those of original lignin, respectively. At the same time, the phenolic hydroxyl content also increased,

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and the 1.0 h MA-treated lignin had the maximum value of 1.523 mmo1/g, which increased by 11.90%. In addition, the carbonyl and carboxyl contents also slightly increased, but the content of methoxyls decreased. 3.1.2. UV/Vis spectroscopy analysis At present, UV/Vis spectroscopy is used in the study of lignin for qualitative and quantitative analyses. The lignin model compound is also widely used to study the structure and chemical behavior of lignin due to its definite structure and properties, and the results can reflect the real lignin preferably. In this study, the ionization difference spectrum was used to analyze the type and content of the phenolic hydroxyl based on relate theory of lignin model compound. According to the research of Lin [29], there is a linear relationship between the difference molar absorptivity and the frequency of ultraviolet light at the maximum absorption for different types of lignin (Figs. S1 and S2). The maximum absorption of lignin in this study was about 350 nm (Fig. 2a, b and c), which was corresponding to phenolic hydroxyl of the type 3. Since the absorbance obeys the Beer-Lambert law, the content of phenolic hydroxyl could be calculated by the following formula (1) [26]. q 



max



 100 %

(1)

where Δαmax (l g−1 cm−1) is the difference in absorptivity between ionized and non-ionized compounds, and Δε (l mol−1 cm−1) is the difference of molar absorptivity. Δε is the same for all the lignin is the same type, q  Δαmax. So Δαmax can be directly used to show the phenol hydroxyl content. As show in Fig. 2a and c, the difference absorptivity of lignin enhanced after MA treatment. The Δαmax of MA-treated lignin with the MA time of 0, 9

0.25, 0.50, 1.00, and 1.50 h at 350 nm was measured to be 29.9, 31.3, 33.1, 33.5, and 33.0, respectively. So the phenolic hydroxyl content was improved by MA. The increment was obvious in the MA time of 0−0.50 h but there was nearly no change for the MA time of more than 0.50 h. 3.1.3 FTIR analysis As show in Fig. 3, the peaks at 1600, 1510, 1460 and 1423 cm−1 corresponding to aromatic skeleton vibrations, the strong broad band centered at about 3400 cm−1 corresponding to O−H stretching vibration, and the peaks at 2939/2848 cm−1 corresponding to methyl/methylene groups are distinguishable for all the samples [30,31]. So MA did not change the main structure of lignin. The intensity of the bands at 1514 and 1600 cm−1 can be used to differentiate softwood and hardwood lignin [32], and the intensity of 1514 cm−1 was stronger than that of 1600 cm−1, which indicates that the lignin used in this study was indeed softwood lignin. For the MA-treated lignin, the absorption peak of O−H became narrow and shifted from 3390 cm−1 to a higher wavenumber of 3407 cm−1, which is similar to the phenomenon of MA-treated cellulose [22]. The intense impact of ball milling could break the inter- and intra-molecular hydrogen bonds in main chains of lignin and increased the content of free hydroxyl groups, promoting the reactions between lignin and reagents. But the effect of MA on lignin was more complex due to the presence of other active groups. To facilitate the comparison of spectra, the absorption peak attributing to aromatic skeleton vibration (A1600 cm−1) were chosen to normalize each spectrum. Although in the process of MA, the aromatic skeleton may be destroyed by the breakage of C−C or the formation of quinone, it would not

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lead to large errors due to the absorption peak of C=C and its conjugate state is at 1620−1680 and 1600 cm−1, respectively, which is close to the aromatic skeleton vibration. The effect of MA on the functional groups of lignin is shown in three aspects: (1) C=O: the peaks at 1693 cm−1 corresponding to carbonyl groups became stronger after MA, which may be the result of oxidation of alcohols or phenols and generation of aldehyde, ketone and carboxyl. (2) O−H: the content of hydroxyl groups increased since the enhanced intensities of the peaks at 1365, 1222, 1130 and 1030 cm−1, which are attributed to the characteristic absorption peaks of plane vibration of phenolic O−H and the −CH3, phenolic hydroxyl and phenol ether, tertiary alcohol, and primary alcohols, respectively; (3) −OCH3: the content of methoxyl decreased for the weakening of the peaks at 2848 cm−1 (−CH3 and methoxyl) and 1330 cm−1 (C−O and C−C of syringyl ring), which may be the results of demethylation and demethoxylation, while the bands at 1260 and 1160 cm−1 ( C−H plane vibration of G unit) became stronger. The decrease of methoxyl would reduce steric hindrance and improve the reactivity of lignin. Similar to the pretreatments of laccase and ultrasonication [15,18], MA could increase the content of hydroxyls in lignin but decrease the content of methoxyls, and the breakage of C−C and C−O in β-O-4, β-1, β-5 and 5-5' or demethylation may also happen during the process of MA. 3.1.4 NMR analysis It can be observed that no new peaks appear in the 1H and 13C NMR spectra of MA-treated lignin (Figs.4 and 5), indicating that no new groups generated during the process of MA, but the amount of each group had some changes. The addition of trioxane which was used as internal standards in order to study the change of the content of functional groups had nearly no influence on the signal of functional groups. For the 1H NMR spectrum of

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MA-treated lignin, the integrated area of trioxane (5.4-4.9 ppm) was chosen as reference, the integrated area of ArH (8−6.2 ppm), methoxyl groups (3.5−4.1 ppm), alkyl group (0−2.5 ppm), aliphatic hydroxyl groups (3−3.5 ppm), phenolic hydroxyl groups (9.9−10.1 ppm) and COOH (12−12.6 ppm) [25,26,30,31] were calculated (Table 2). For the 13C NMR spectrum of 0.50 h MA-treated lignin, the integrated area of trioxane (98-90 ppm) was chosen as reference, the integration area of methoxyl (50−60 ppm) decreased from 3.08 to 2.69, while the integration of carbonyl (150−170 ppm) increased from 1.02 to 1.33. Comprehensive analysis of these two NMR spectra also shows that the contents of hydroxyl and carbonyl increased after MA, particularly the aliphatic hydroxyl, but the content of methoxyl decreased. The passivation and steric effect of carbonyl and carboxyl groups may decrease the reactivity of hydroxyls in lignin, but the increment of them was less than that of hydroxyl, so the MA-treated lignin may still have better reactivity. 3.1.5 Discussion about the possible reaction during MA process The content of hydroxyl groups may change with the reaction such as redox [33], demethylation [34], and depolymerization/repolymerization of lignin by ether (C−O) and condensed (C−C) linkages. For example, the phenolic hydroxyl can increase with the depolymerization but decrease with the repolymerization of lignin by methyl-aryl ether and β-O-4 bond. Furthermore, the main reaction depends on the temperature and heating mode [35,36]. Then, what happened to the lignin during the process of MA? If the increase of hydroxyl content was due to the reduction of carbonyl groups, it should lead to the decrease of carbonyl content. However, as shown in above, the carbonyl and carboxyl contents also increased, and the variation trend was consistent with that of the total hydroxyl. So it can be

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deduced that the increase of hydroxyl content did not depend on redox reaction but demethylation and depolymerization/repolymerization of lignin. The studies on the depolymerization of lignin mainly focus on pyrolysis, autohydrolysis, steam explosion, etc. [37,38], and the cleavage of ether (C−O) and condensed (C−C) linkages are closely related to the temperature in these processes. Free radicals reaction easily happens at high temperature, and repolymerization reaction is found to take place simultaneously with the depolymerization reaction during these processes. The similar reaction could also be caused in the process of MA. The researches of mechanochemistry have reported that there is a huge impact force at the point of collision in a part of the system, which may result in instantaneous high-temperature and the destruction of the structure of materials, forming the ions or free radicals [39,40] in the process of mechanical actions. In addition, the researches of the free radical reaction of lignin and its model compounds showed that the redox reaction of the side chains could occur at the same time [38]. Therefore, the instantaneous high-temperature may cause a series of free radical reaction in the process of MA. First of all, a large number of free radicals, such as phenoxyl radical, hydrogen radical, alkyl radical, and so on, were generated from the homolytic cleavage of ether (β-O-4, α-O-4, 4-O-5) and C−C bonds. Then the aliphatic and phenolic hydroxyls were formed by the rearrangement and repolymerization. The processes of demethylation and depolymerization/repolymerization are illustrated in Fig. 6. Similar to photooxidation reaction of lignin [41], the phenolic hydroxyls may also be converted to quinone and the C=O in side chain was converted to C−OH (Fig. 7).

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Too short MA time could not induce the reaction sufficiently, while too long MA time may lead to the consumption of hydroxyls by the repolymerization of lignin with methyl-aryl ether and β-O-4 bonds. In addition, since the surface of lignin was directly exposed to air, similar with the MA of graphite, additional aliphatic hydroxyl, phenolic hydroxyl and carbonyl may also be generated on the surface [42]. So there was an optimum MA time to get the desired hydroxyl content. The phenolic hydroxyls were prone to convert to quinone, so the phenolic hydroxyl content was less than aliphatic hydroxyl content. The oxygen may cause the partial oxidation of aldehyde to carboxylic groups, leading to a slight increase of carboxyl content after MA treatment. 3.2. Effect of MA on the morphological structure of lignin The particle size of material may affect its reactivity due to the influence on the mass transfer and heat transfer, especially for mass transfer [43]. The particle size of material can change not only the reaction rate constant, reaction order, and activation energy, but also the mechanism of reaction and product for the influence on dissolving, dispersion, contact and surface hydroxyl of reactant [44]. Herein, the morphological structure was analyzed by SEM and particle size analyzer, and the results are shown in Fig. 8 and Table 3. The original lignin was irregular with large particle size, and the surface structure was compact and smooth. But the smooth surface was destroyed by the mechanical actions during the process of MA .The particle size of MA-treated lignin was greatly reduced, especially for the MA time of 0.25 and 0.50 h. Compared with the original lignin, the d(0.5) and d(0.9) of 0.50 h MA-treated lignin reduced from 49.151 and 110.640 µm to 14.525 and 52.379 µm, respectively, and the specific surface area increased from 0.222 to 0.864 m2/g. As the MA

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time of more than 0.50 h, the particle size, roughness and specific surface area of MA-treated lignin had nearly no change due to the balance between fragmentation and agglomeration. The smaller particle size of material indicates it possesses larger surface area, contributing to the lower mass transfer resistance, more surface hydroxyl and higher reactivity. The study on the effect of MA on the reactivity of fly ash showed that the decrease in particle size drastically enhanced the reactivity of fly ash [45]. 3.3 Effect of MA on the esterification of lignin In order to evaluate the effect of MA on the reactivity of lignin, esterification was carried out with acetic anhydride as esterifying agent. The MA-treated lignin samples with different time were esterified by two traditional methods (LPS and SPS). The esterification was proved by structure analysis of products with UV/Vis spectrometer, FTIR and NMR, and reactivity was evaluated by degree of esterification (DE) of lignin.The results are shown in Figs. 2−5 and Table 4. 3.3.1. The structure of esterified lignin For the UV/Vis analysis of the acetylated lignin, the absorbance of differential spectrum (Fig.

2b) obviously decreased because majority of the phenolic hydroxyls had participated in

the esterification. Both the aliphatic hydroxyl and phenolic hydroxyl in lignin could participate in esterification, and the former would result in a red shift for the introduction of carbonyls in the side chain, while the latter would result in a blue shift for the consumption of auxochrome. The neutral spectrum (Fig. 2d and e) was used to analyze the effect of esterification on the structure of lignin and the results show that the maximum absorption had

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a red shift from 300 to 315 nm, indicating that the amount of alcohol esters was more than that of phenolic esters due to more aliphatic hydroxyl in lignin. For the FTIR analysis, the appearance of new peaks at 1740, 1762 and 1200 cm−1 corresponding to aliphatic ester link, phenolic ester link and C−O−C of aromatic acetyl groups respectively [25,26,30,31], can be clearly seen in Fig. 3c and d. The peak at 1365 cm−1 (plane vibration of phenolic O−H and −CH3) transferred to 1370 cm−1, and the intensity also enhanced, resulting from the introduction of acetyl groups. The peak at 1643 cm−1 (conjugated C=O and enol) could be seen in all the samples, but it was more obvious in the esterified lignin, which may be that esterification promoted the shift of 1693 cm−1 in original lignin. For the NMR analysis, the appearance of new peaks at 2.3 and 2.15 ppm corresponding to −CH3 of aliphatic ester and phenolic ester in 1H NMR spectra (Fig. 4d and e), and 170 and 20 ppm corresponding to C=O and acetyl −CH3 in 13C NMR spectra (Fig. 5d and e) can be clearly observed. Comprehensive analyses of these three characterizations implied that the lignin was successfully esterified both in LPS and SPS. 3.3.2. Reactivity of MA-treated lignin DE of the products was analyzed and calculated according to the method of Nevárez, et al. [26]. It was analyzed by UV/Vis spectrometer based on the change of phenol hydroxyl because esterification of either the phenol hydroxyl or aliphatic hydroxyl will lead to the decrease of phenol hydroxyl content. The reason why the esterification of aliphatic hydroxyl will still lead to the decrease in concentrate of phenol hydroxyl was explained in

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supplementary material as explanatory notes. DEs could be calculated by comparing Δαmax before and after the esterification, and the results are shown in Table 4. The DEs of original lignin were only 42.3% and 27.5% in the LPS and SPS, respectively. Because solvent guarantee homogeneous of reactants and stable heat exchange, LPS gave higher DEs than SPS. The esterification of MA-treated lignin had higher DEs both in LPS and SPS. The enhancement of DEs for 0.25 and 0.50 h MA-treated lignin were obvious, and the DEs of the 0.50 h MA-treated lignin reached the maximum values of 80.4% and 63.9% in LPS and SPS, respectively. However, when the MA time was more than 0.5 h, the DEs of MA-treated lignin did not increase but showed a slight decrease. As expected, more activated hydroxyl group, smaller particle size and larger specific surface in lignin certainly lead to better reactivity of lignin. As the MA time increasing from 0 to 0.50 h, the reactions of generating hydroxyl and fragmentation of particle in lignin took advantage, so the DE increased with the MA time, and the DE of 0.50 h MA-treated lignin reached the maximum for the maximum hydroxyl content, minimum particle size and maximum specific surface area of lignin (Tables 1−3). When the MA time was more than 0.50 h, the generation and consumption of hydroxyls, fragmentation and agglomeration of particle reached dynamic equilibrium, contributing to that the hydroxyl content, specific surface and DE of MA-treated lignin no longer increased with the MA time. Furthermore, long MA time also consumed too much energy. Therefore, the optimum MA time was determined to be 0.50 h. 4. Conclusions

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Mechanical actions results in the changes of molecular structure and morphological structure of lignin. The change of molecular structure mainly showed that the contents of aliphatic hydroxyl, phenolic hydroxyl, carbonyl and carboxyl groups increased while the content of methoxyl groups decreased after MA treatment. The change of morphological structure indicated that MA led to the decrease of particle size and the increase of specific surface area and roughness of surface in lignin. The esterification of lignin in both liquid phase and solid phase was effectively enhanced by MA, ascribing to that mechanical actions improved the reactivity of lignin for the increase of hydroxyl content and enhancing the mass transfer in chemical reaction. MA, a green and simple process, can be considered as an effective method for improving the reactivity of lignin. It should be significant and feasible to combine MA and reaction in the same equipment for solid phase modification of lignin. Acknowledgements This research was supported by Natural Science Foundation of China (No. 51163002 and 51463003), Guangxi Natural Science Foundation of China (No. 2013GXNSFDA019004 and 2013GXNSFAA019025), Guangxi Scientific Research and Technological Development Project of China (No. 1598020-1), Guangxi Distinguished Experts Special Foundation of China, and the Scientific Research Foundation of Guangxi University, China (Grant No. XTZ140787).

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Figure Captions Fig. 1. Equipment drawing of stirring ball mill.

25

Fig. 2. UV spectra of lignin and its esters: (a) ionization difference spectra of original lignin, (b) ionization difference spectra of acetylated lignin, (c) ionization difference spectra of 0.50 h MA treated lignin, (d) neutral spectrum of original lignin, and (e) neutral spectrum of acetylated lignin.

26

Fig. 3. FTIR spectra of lignin and its ester: (a) original lignin, (b) 0.50 h MA-treated lignin, (c) acetylated (b) in solid phase, (d) acetylated (b) in liquid phase.

27

Fig. 4. 1H NMR spectra of (a) original lignin with trioxane as internal standards, (b) original lignin, (c) 0.50 h MA-treated lignin, (d) acetylated (c) in solid phase, and (e) acetylated (c) in liquid phase.

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Fig. 5. 13C NMR spectra of (a) original lignin with trioxane as internal standards, (b) original lignin, (c) 0.50 h MA-treated lignin, (d) acetylated (c) in solid phase, and (e) acetylated (c) in liquid phase.

29

Fig. 6. The formation of hydroxyl groups: (A) demethylation, (B) homolytic cleavage of β-O-4.

30

Fig. 7. The formation of quinone and hydroxyl groups.

31

Fig. 8. The SEM analysis of original lignin (a1, a2) and MA-treated lignin with different time at 1,000× and 3,000× magnifications: (b1, b2) 0.25 h, (c1, c2) 0.50 h, (d1, d2) 1.00 h, and (e1, e2) 1.50 h a1

b1

c1

d1

e1

a2

b2

c2

d2

e2

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Table 1. Functional groups analysis of original and MA-treated lignin Total hydroxyl Carbonyl MA time (mmol/g) (mmol/g) (h)

Carboxyl (mmol/g)

Phenolic hydroxyl (mmol/g)

Aliphatic hydroxyl (mmol/g)

0

5.012±0.021

0.556±0.004

0.020±0.000

1.361±0.011

3.651

0.25

5.503±0.028

0.567±0.005

0.030±0.001

1.426±0.007

4.077

0.50

7.004±0.032

0.575±0.003

0.091±0.002

1.507±0.008

5.497

1.00

5.477±0.022

0.568±0.005

0.163±0.003

1.523±0.012

3.954

1.50

5.308±0.027

0.566±0.006

0.182±0.003

1.489±0.005

3.819

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Table 2. Integration of the functional groups of original and MA-treated lignin by 1H NMR MA time (h)

CH3O

AlOH

COOH

ArOH

ArH

C-H

CH2 of trioxane

0

3.02

1.66

0

0.04

1.80

0.79

1.00

0.25

2.92

2.36

0

0.05

1.75

0.7

1.00

0.50

2.46

4.70

0.08

0.09

1.78

1.09

1.00

1.00

2.49

3.01

0.12

0.09

1.76

0.92

1.00

1.50

2.36

2.68

0.12

0.09

1.75

0.95

1.00

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Table 3. Particle size and specific surface area of original and MA-treated lignin MA time (h)

Specific surface (m2/g)

d(0.5) (µm)

d(0.9) (µm)

0

49.151

110.640

0.222

0.25

32.403

81.438

0.530

0.50

14.525

52.379

0.864

1.00

14.188

47.460

0.896

1.50

14.839

47.212

0.867

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Table 4. The degree of esterification (DE) of original and MA-treated lignin MA time

Lignin

Liquid phase method

(h)

Δαmax

Δαmax

DE (%)

Δαmax

DE (%)

0

29.9

17.3

42.3

21.7

27.5

0.25

31.3

11.8

62.2

13.9

55.5

0.50

33.1

6.5

80.4

12.0

63.9

1.00

33.5

6.7

80.1

12.3

63.4

1.50

33.0

6.8

79.4

12.3

62.8

36

Solid phase method