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Abstract： Recalcitrance of lignocellulosic biomass is closely related to the presence of lignin in secondary cell walls， which has a negative effect on enzyme digestibility， biomass-to-biofuels conversion， and chemical pulping. The lignification process and structural heterogeneity of the cell wall for various parts of moso bamboo were investigated. There were slight differences among three different column parts of moso bamboo in terms of chemical compositions， including cellulose， hemicelluloses， and lignin. However， the detailed analysis of the fractionated lignin indicated that the acid-soluble lignin was first biosynthesized， and the largest molecular weight value was detected from the bottom part of the moso bamboo， as well as the highest syringyl-to-guaiacyl ratio. Although the main b-O-4 aryl ethers and resinol structures were clearly present in all lignin samples examined by NMR analysis， the relatively small lignin biomacromolecule in the top part of the moso bamboo lead to poor thermal stability. For the bioconversion process， no significant difference was found among all the moso bamboo samples， and the relatively higher hydrolysis efficiency was largely dependent on the low crystallinity of cellulose rather than the degree of lignin biosynthesis.
Keywords： lignification process; enzymatic efficiency; moso bamboo; biorefinery
1 Introduction
Social dependence on renewable resources is often viewed as an important contributor to the sustainable development of industrial society and effective management of greenhouse gas emission[1]. As the use of petroleum-based resources is being reduced， lignocellulosic biomass has been regarded as one of the potentially sustainable resources for the production of energy and chemicals due to its availability in large quantities and its sustainability[2]. One of the primary challenges for efficient utilization of lignocellulosic biomass is clarifying its complicated components and structures in the cell wall as a guide to develop pretreatment and bioconversion strategies. Moreover， the influence of the different parts on the following treatment should also be considered， and the mechanism underlying the degradation barrier is worth investigating[3].
Bamboo is an abundant natural resource that has been used traditionally as a structural material and cellulosic material for pulping and papermaking. According to available data， 1000 species of bamboo are distributed worldwide， in which approximately 500 species across 6 million hectares can be found in China[4]. Moso bamboo is the most widely cultivated bamboo， occupying approximately 65% of the total area of bamboo forest[5]. Recently， bamboo has been considered as a potential feedstock for biofuel and biochemical production because of its fast growth， short renovation， and easy propagation. The variation in the main composition during the lignification process is critical for optimizing the parameters of pretreatment， enzymatic hydrolysis， and yeast fermentation. For bamboo， the lignin content of 16%～34% is similar to hardwood， except for some specific species[5]. For example， Neosinocalamus has about 28.2% lignin， while the lignin component in Pseudostachyum is lower （22.1%）[6-7]. Besides， the biosynthesis of lignin in different species affects the lignification with significant differences. Exponential increment of lignin with age was observed in one to eight years Neosinocalamus[8-9]， and the content of lignin in Bambusa was stable （24.3%） after one year of growth. The growth location， altitude， and climatic condition also influence the level of lignification in bamboo significantly. Studies on the structural characteristics of lignin are usually conducted to understand the biosynthesis mechanism and with the aim of determining the category of bamboo. Based on the concept of biorefinery， effective removal and then modification of lignin for improving the bioconversion efficiency or the overall utilization of bamboo has attracted increasing attention[10-11]. The purpose of this study is to examine the structural changes of lignin polymer during the lignification process of moso bamboo and the bioconversion efficiency of moso bamboo substrate. Swollen residual enzyme lignin （SREL） was chosen to represent the original structure of lignin. Several spectroscopic and chromatographic nondestructive techniques， including Fourier transform infrared （FT-IR）， gel permeation chromatography （GPC）， heteronuclear single-quantum coherence Nuclear Magnetic Resonance （HSQC NMR） spectroscopy， and thermogravimeric analysis （TGA）， were used to determine the variation of the lignin macromolecule in the lignification process. Enzymatic hydrolysis for C6 and C5 sugar recovery was further performed to test its influence on the bioconversion efficiency. 2 Experimental
2.1 Raw material
Moso bamboo columns were harvested at the end of May 2015 from Anhui Province， China. The columns were subsequently subdivided into 13 sections， each 1 m in length， and three samples （positioned 1， 6， and 12 m above the ground） were selected and labeled as BB （bottom）， BM （middle）， and BT （top）. These moso bamboo samples were ground and sieved to obtain particles with sizes between 40 and 60 mesh. Each resulting sample was subsequently treated with toluene/ethanol （2：1， V/V） in a Soxhlet extractor to remove most of the extractives. The dewaxed sample was then ball-milled with a planetary ball mill （Fritsch， Germany） for 5 h using alternating sequences of 10 min of ball-milling followed by 10 min breaks to prevent overheat. Commercial cellulase （145 FPU/g） was supplied by Novozyme （China） Investment Co. Ltd. （Beijing）. All other chemicals are of analytical grade unless otherwise mentioned.
2.2 Preparation of swollen residual enzyme lignin （SREL）
The preparation of SREL was mainly based on the previous literature[12]. Briefly， the ball-milled moso bamboo powder was swollen by being fully distributed into 4% （w/V） sodium hydroxide aqueous solution for 24 h at room temperature. The mixture was directly pH-adjusted to 4.8 using acetic acid， and then subjected to enzymatic hydrolysis by loading cellulase （100 FPU/g substrate）. After 48 h of incubation in a rotary shaker at 150 r/min， the hydrolyzed carbohydrates were removed by centrifugation， and the solid residue was finally obtained after the complete elimination of the residual enzyme and sugars by washing with acidic boiling water （pH value=2.0） and freeze-drying. According to the naming convention， the lignin obtained was labeled as LB， LM， and LT.
2.3 Physicochemical property of the SREL fractionated lignin
2.3.1 Molecular weight distribution
Three SREL samples were first dissolved in 2 mL tetrahydrofuran （THF）， and 20 mL solutions were injected after being filtered with 0.22 mm filter. The weight-average （Mw） and number-average （Mn） molecular weights were determined by GPC on a PL-gel 10 mm Mixed-B 7.5 mm column， calibrated with monodisperse polystyrene （Polymer Laboratories Ltd.， UK） of known molecular weight according to previous literature[13].
2.3.2 FT-IR analysis
The FT-IR spectra of SREL samples were recorded from a Thermo Scientific Nicolet iN10 FT-IR Microscope （ThermoNicolet Corporation， USA） in the absorption mode， ranging from 400 to 4000 cm-1. The fingerprint region was baseline corrected between 800 and 2000 cm-1， and the background spectrum of pure potassium bromide was subtracted. 2.3.3 Thermal behavior
Thermal stability was examined with a simultaneous thermal analyzer （DTG-60， Shimadzu， Japan） under nitrogen atmosphere. The SREL samples were heated from 40℃ to 600℃ at a heating rate of 10℃/min.
2.3.4 NMR spectra of SERL samples
The 1H-13C Two-Dimensional Correlation HSQC NMR spectra of the lignin samples were recorded on a 400-MHz NMR spectrometer （Avance III， Bruker， Germany） with a z-gradient triple-resonance probe at room temperature. Typically， 50 mg lignin sample was dissolved in 0.5 mL dimethyl sulfoxide （DMSO-d6）， the number of transients was 64， and 256 time increments were always recorded in the 13C dimension. The experiment was conducted for 22 h. Prior to Fourier transformation， the data matrixes were zero-filled up to 1024 points in the 13C dimension， and the data processing was performed using standard Bruker’s Topspin NMR software.
2.3.5 Characterization and bioconversion of ball-milled moso bamboo
The crystallinity indices of three ball-milled moso bamboo samples were determined with a powder X-ray diffractometer （D8 ADVANCE， Bruker， Germany） with Ni-filtered Cu Ka radiation （l=1.54?）. The operating voltage and current were kept constant at 40.0 kV and 40.0 mA， respectively， and the diffraction angle （2q） was scanned from 5° to 40°. The enzymatic bioconversion was performed in 25 mL Erlenmeyer-flask， containing 1 g ball-milled moso bamboo samples， enzyme （10 FPU cellulase/g substrate）， and 10 mL acetate buffer （50 mmol/L， pH value=4.8）. The mixture was incubated at 50℃ and stirred in a rotary shaker at 150 r/min. The supernatant samples of 0.1 mL were taken periodically， and the released monosaccharides were analyzed by a high-performance anion exchange chromatography system （ICS-3000， Thermo Scientific， USA） equipped with pulse amperometric detector and PA-20 column （4×250 mm）. All enzymatic hydrolysis experiments were performed in duplicate， and the data were presented as the average value.
3 Results and discussion
3.1 Characteristic analysis of lignin fraction
Lignin is a complex phenolic polymer with the main functions of providing stability to the vascular part of the plant and forming a barrier against microbial infections. Natural lignin polymer generally comprises p-hydroxyphenyl （H）， guaiacyl （G）， and syringyl （S） units， which are produced by three primary monolignols， p-coumaryl， coniferyl， and sinapyl alcohols， respectively. Other phenolic acids （e.g.， ferulic and p-coumaric acids） are also widely distributed within the cell wall， forming covalent linkage with lignin and polysaccharide components known as lignin-carbohydrate complex （LCC）. Most enzymes involved in the biosynthesis of lignin monomers have been well documented; however， the biosynthetic pathway has been revised by the recent discovery of caffeoyl shikimate esterase[14]. The component analysis indicates that the content of total lignin （AIL and ASL） increased gradually from 29.3% to 34.7% with lignification process （Table 1）. It is interesting to note that the acid-soluble lignin was first biosynthesized， as evidenced by the obvious increment of acid-soluble lignin from the top to the bottom part in both the moso bamboo samples and the SREL samples. Correspondingly， the molecular weight of the SERL lignin increased from 6680 to 8310; the polydispersity （Mw/Mn） also increased. As can be seen from the distribution curves （Fig.1）， two peaks， assigned to the high and low molecular weight parts， respectively， are clearly right-shifted from LT to LB. The relative content of total lignin further increased by the lignification process， mainly focusing on the acid-insoluble lignin. This observation indicated that the biosynthesis between monolignols formed the complex subunits， which are difficult to be acidic hydrolyzed. However， the molecular weight and polydispersity were slightly decreased from LB to LT， suggesting that the lignification process was concentrated on the formation of more condensed/stable linkages， rather than the generation of larger biomacromolecules. In terms of the carbohydrate components， glucose and xylose are the main monosaccharides from cellulose and hemicelluloses， respectively， and a little amount of arabinose and galactose were also detected. In the SREL samples， nearly half of the fractioned solid were carbohydrates， mainly due to the partial hydrolysis of polysaccharides and the coprecipitation of oligosaccharides in the hydrolysate.
Two spectral technologies were employed to characterize the main linkages and functional groups in lignin. FT-IR technology has been widely used to quickly determine the structural property of lignin （Fig.2）. The typical FT-IR spectra of lignin are clearly shown： the absorptions around 1597， 1506， and 1425 cm-1 indicate the aromatic skeleton vibrations; the band at 1460 cm-1 is assigned to the C—H asymmetric deformations; and the peak at 1665 cm-1 is attributed to the C=O stretching in conjugated p-substituted aryl ketones. It is worthy to note that the band at 1114 cm-1 represents the aromatic C—H in-plane deformation in syringyl type， and the intensity of this adsorption in LB is much higher than in the other two samples， corresponding to its highest S/G ratio （Table 1） from NMR analysis below. Due to the extensive presence of carbohydrate in lignin samples， the typical peaks at 1052 and 1038 cm-1 are assigned to xylose and glucose， respectively， and are clearly observed in all samples[15].
To further investigate the aromatic units and the different inter-unit linkages present in the lignin macromolecule， all SREL samples were analyzed using the 2D HSQC NMR technique， and the signal assignments were primarily in accordance with previous literatures[16-17]. The main structural characteristics of lignin， including basic monolignols （S， G， and H units） and various substructures linked by ether， ester， and carbon-carbon bonds （b-O-4’， b-b’， b-5’， etc.） can be observed in Fig.3. In the side-chain region of the spectrum， the prominent substructure， b-O-4’ aryl ethers （substructure A）， is reflected from the Ca—Ha correlation （dC/dH， 72/（4.7～4.9） ppm）， Cb—Hb correlation （dC/dH， 84/4.3 ppm for the G unit and 86/4.1 ppm for the S unit， respectively）， and Cg—Hg correlation （dC/dH， 60.1/（3.4～3.7） ppm）. Resinol （b-b’， substructure B） appeared in all spectra in noticeable amounts as indicated by their Ca—Ha （dC/dH， 84.8/4.66）， Cb—Hb （dC/dH， 53.5/3.07）， and the double Cg—Hg correlations （dC/dH， 71.2/（3.82 and 4.18））， respectively. Accompanied by the lignification process， these two substructures were simultaneously synthesized and the main difference in those fractionated lignin samples was the S/G ratio. By integrating the area of C2，6/H2，6 in the S and G units， the S/G ratios were calculated to be 1.6， 2.1， and 3.3 for LT， LM， and LB， respectively. The thermal stability of lignin samples was also studied by TGA （Fig.4）. Among the lignocellulosic materials， lignin is the most thermo-stable component mainly due to the inherent structure of aromatic rings with various branches， resulting in the wide temperature range of thermal degradation. According to the previous report， the thermal degradation of lignin can occur through the following steps： （1） cleavage of a- and b-aryl-alkyl-ether linkages occurs between 150℃ and 300℃; （2） aliphatic side chains start splitting off from the aromatic ring at around 300℃; （3） the carbon-carbon linkage between lignin structural units is cleaved at 370～400℃; Decomposition or condensation of aromatic rings is believed to occur at 400～600℃[18-19]. As illustrated in Fig.4， the onset of thermal degradation occurs at around 250℃， and the temperature at the maximum weight loss rate is about 320℃ for all SREL samples. However， the non-volatile residues of LM and LB were clearly higher than LT. This indicates that higher thermal stability corresponds with higher molecular weights， along with the lignification process.
3.2 Lignification effect on bioconversion
The crystalline structure of cellulose plays a major role in recalcitrating the bioconversion process. The ball-milling process can partially destroy the intermolecular hydrogen bond between cellulose chains and then decrease the crystallinity. Therefore， it is reasonable to observe that the typical X-ray diffraction pattern of cellulose I disappeared and a relatively amorphous cellulose is presented from Fig.5（a）. Correspondingly， the CrI values of all the samples were small， ranging from 10.9% to 14.1%. However， it could be predicted that the difference in crystallinity， together with the bioconversion efficiency， were mainly ascribed to the degree of lignification of the moso bamboo， since the same ball-milling process was performed on all moso bamboo samples. As shown in Fig.5（b）， almost complete bioconversion was achieved for all samples， mainly due to the ball-milling treatment， and the slight difference was also noticed in detail. The chart shows that both initial hydrolysis rate and final bioconversion of the sample BM was the highest among all the moso bamboo samples. Since the physicochemical property of LM showed no remarkable difference in all SREL samples concerning molecular weight， S/G ratio， and lignin substructure， the improved bioconversion of BM was probably due to the property of cellulose， especially its lowest crystallinity among all the moso bamboo samples. 4 Conclusion
Bamboo is considered a grass biomass material that has great potential as a future bioresource for biorefining. Three different parts （bottom， middle， top） of moso bamboo were comparatively examined in this study to determine their characteristic patterns in terms of chemical composition， lignin structure， and bioconversion efficiency. Although the difference between the three different parts in bioconversion was not obvious， the results indicated that the relatively higher hydrolysis efficiency of bamboo samples in the middle was mainly dependent on the low crystallinity of cellulose rather than the degree of lignin biosynthesis.
Acknowledgments
The authors gratefully acknowledge the financial support from the Natural Science Foundation of China （31770622） and the Innovation Program of College of Materials Science and Technology.
Reference
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Keywords： lignification process; enzymatic efficiency; moso bamboo; biorefinery
1 Introduction
Social dependence on renewable resources is often viewed as an important contributor to the sustainable development of industrial society and effective management of greenhouse gas emission[1]. As the use of petroleum-based resources is being reduced， lignocellulosic biomass has been regarded as one of the potentially sustainable resources for the production of energy and chemicals due to its availability in large quantities and its sustainability[2]. One of the primary challenges for efficient utilization of lignocellulosic biomass is clarifying its complicated components and structures in the cell wall as a guide to develop pretreatment and bioconversion strategies. Moreover， the influence of the different parts on the following treatment should also be considered， and the mechanism underlying the degradation barrier is worth investigating[3].
Bamboo is an abundant natural resource that has been used traditionally as a structural material and cellulosic material for pulping and papermaking. According to available data， 1000 species of bamboo are distributed worldwide， in which approximately 500 species across 6 million hectares can be found in China[4]. Moso bamboo is the most widely cultivated bamboo， occupying approximately 65% of the total area of bamboo forest[5]. Recently， bamboo has been considered as a potential feedstock for biofuel and biochemical production because of its fast growth， short renovation， and easy propagation. The variation in the main composition during the lignification process is critical for optimizing the parameters of pretreatment， enzymatic hydrolysis， and yeast fermentation. For bamboo， the lignin content of 16%～34% is similar to hardwood， except for some specific species[5]. For example， Neosinocalamus has about 28.2% lignin， while the lignin component in Pseudostachyum is lower （22.1%）[6-7]. Besides， the biosynthesis of lignin in different species affects the lignification with significant differences. Exponential increment of lignin with age was observed in one to eight years Neosinocalamus[8-9]， and the content of lignin in Bambusa was stable （24.3%） after one year of growth. The growth location， altitude， and climatic condition also influence the level of lignification in bamboo significantly. Studies on the structural characteristics of lignin are usually conducted to understand the biosynthesis mechanism and with the aim of determining the category of bamboo. Based on the concept of biorefinery， effective removal and then modification of lignin for improving the bioconversion efficiency or the overall utilization of bamboo has attracted increasing attention[10-11]. The purpose of this study is to examine the structural changes of lignin polymer during the lignification process of moso bamboo and the bioconversion efficiency of moso bamboo substrate. Swollen residual enzyme lignin （SREL） was chosen to represent the original structure of lignin. Several spectroscopic and chromatographic nondestructive techniques， including Fourier transform infrared （FT-IR）， gel permeation chromatography （GPC）， heteronuclear single-quantum coherence Nuclear Magnetic Resonance （HSQC NMR） spectroscopy， and thermogravimeric analysis （TGA）， were used to determine the variation of the lignin macromolecule in the lignification process. Enzymatic hydrolysis for C6 and C5 sugar recovery was further performed to test its influence on the bioconversion efficiency. 2 Experimental
2.1 Raw material
Moso bamboo columns were harvested at the end of May 2015 from Anhui Province， China. The columns were subsequently subdivided into 13 sections， each 1 m in length， and three samples （positioned 1， 6， and 12 m above the ground） were selected and labeled as BB （bottom）， BM （middle）， and BT （top）. These moso bamboo samples were ground and sieved to obtain particles with sizes between 40 and 60 mesh. Each resulting sample was subsequently treated with toluene/ethanol （2：1， V/V） in a Soxhlet extractor to remove most of the extractives. The dewaxed sample was then ball-milled with a planetary ball mill （Fritsch， Germany） for 5 h using alternating sequences of 10 min of ball-milling followed by 10 min breaks to prevent overheat. Commercial cellulase （145 FPU/g） was supplied by Novozyme （China） Investment Co. Ltd. （Beijing）. All other chemicals are of analytical grade unless otherwise mentioned.
2.2 Preparation of swollen residual enzyme lignin （SREL）
The preparation of SREL was mainly based on the previous literature[12]. Briefly， the ball-milled moso bamboo powder was swollen by being fully distributed into 4% （w/V） sodium hydroxide aqueous solution for 24 h at room temperature. The mixture was directly pH-adjusted to 4.8 using acetic acid， and then subjected to enzymatic hydrolysis by loading cellulase （100 FPU/g substrate）. After 48 h of incubation in a rotary shaker at 150 r/min， the hydrolyzed carbohydrates were removed by centrifugation， and the solid residue was finally obtained after the complete elimination of the residual enzyme and sugars by washing with acidic boiling water （pH value=2.0） and freeze-drying. According to the naming convention， the lignin obtained was labeled as LB， LM， and LT.
2.3 Physicochemical property of the SREL fractionated lignin
2.3.1 Molecular weight distribution
Three SREL samples were first dissolved in 2 mL tetrahydrofuran （THF）， and 20 mL solutions were injected after being filtered with 0.22 mm filter. The weight-average （Mw） and number-average （Mn） molecular weights were determined by GPC on a PL-gel 10 mm Mixed-B 7.5 mm column， calibrated with monodisperse polystyrene （Polymer Laboratories Ltd.， UK） of known molecular weight according to previous literature[13].
2.3.2 FT-IR analysis
The FT-IR spectra of SREL samples were recorded from a Thermo Scientific Nicolet iN10 FT-IR Microscope （ThermoNicolet Corporation， USA） in the absorption mode， ranging from 400 to 4000 cm-1. The fingerprint region was baseline corrected between 800 and 2000 cm-1， and the background spectrum of pure potassium bromide was subtracted. 2.3.3 Thermal behavior
Thermal stability was examined with a simultaneous thermal analyzer （DTG-60， Shimadzu， Japan） under nitrogen atmosphere. The SREL samples were heated from 40℃ to 600℃ at a heating rate of 10℃/min.
2.3.4 NMR spectra of SERL samples
The 1H-13C Two-Dimensional Correlation HSQC NMR spectra of the lignin samples were recorded on a 400-MHz NMR spectrometer （Avance III， Bruker， Germany） with a z-gradient triple-resonance probe at room temperature. Typically， 50 mg lignin sample was dissolved in 0.5 mL dimethyl sulfoxide （DMSO-d6）， the number of transients was 64， and 256 time increments were always recorded in the 13C dimension. The experiment was conducted for 22 h. Prior to Fourier transformation， the data matrixes were zero-filled up to 1024 points in the 13C dimension， and the data processing was performed using standard Bruker’s Topspin NMR software.
2.3.5 Characterization and bioconversion of ball-milled moso bamboo
The crystallinity indices of three ball-milled moso bamboo samples were determined with a powder X-ray diffractometer （D8 ADVANCE， Bruker， Germany） with Ni-filtered Cu Ka radiation （l=1.54?）. The operating voltage and current were kept constant at 40.0 kV and 40.0 mA， respectively， and the diffraction angle （2q） was scanned from 5° to 40°. The enzymatic bioconversion was performed in 25 mL Erlenmeyer-flask， containing 1 g ball-milled moso bamboo samples， enzyme （10 FPU cellulase/g substrate）， and 10 mL acetate buffer （50 mmol/L， pH value=4.8）. The mixture was incubated at 50℃ and stirred in a rotary shaker at 150 r/min. The supernatant samples of 0.1 mL were taken periodically， and the released monosaccharides were analyzed by a high-performance anion exchange chromatography system （ICS-3000， Thermo Scientific， USA） equipped with pulse amperometric detector and PA-20 column （4×250 mm）. All enzymatic hydrolysis experiments were performed in duplicate， and the data were presented as the average value.
3 Results and discussion
3.1 Characteristic analysis of lignin fraction
Lignin is a complex phenolic polymer with the main functions of providing stability to the vascular part of the plant and forming a barrier against microbial infections. Natural lignin polymer generally comprises p-hydroxyphenyl （H）， guaiacyl （G）， and syringyl （S） units， which are produced by three primary monolignols， p-coumaryl， coniferyl， and sinapyl alcohols， respectively. Other phenolic acids （e.g.， ferulic and p-coumaric acids） are also widely distributed within the cell wall， forming covalent linkage with lignin and polysaccharide components known as lignin-carbohydrate complex （LCC）. Most enzymes involved in the biosynthesis of lignin monomers have been well documented; however， the biosynthetic pathway has been revised by the recent discovery of caffeoyl shikimate esterase[14]. The component analysis indicates that the content of total lignin （AIL and ASL） increased gradually from 29.3% to 34.7% with lignification process （Table 1）. It is interesting to note that the acid-soluble lignin was first biosynthesized， as evidenced by the obvious increment of acid-soluble lignin from the top to the bottom part in both the moso bamboo samples and the SREL samples. Correspondingly， the molecular weight of the SERL lignin increased from 6680 to 8310; the polydispersity （Mw/Mn） also increased. As can be seen from the distribution curves （Fig.1）， two peaks， assigned to the high and low molecular weight parts， respectively， are clearly right-shifted from LT to LB. The relative content of total lignin further increased by the lignification process， mainly focusing on the acid-insoluble lignin. This observation indicated that the biosynthesis between monolignols formed the complex subunits， which are difficult to be acidic hydrolyzed. However， the molecular weight and polydispersity were slightly decreased from LB to LT， suggesting that the lignification process was concentrated on the formation of more condensed/stable linkages， rather than the generation of larger biomacromolecules. In terms of the carbohydrate components， glucose and xylose are the main monosaccharides from cellulose and hemicelluloses， respectively， and a little amount of arabinose and galactose were also detected. In the SREL samples， nearly half of the fractioned solid were carbohydrates， mainly due to the partial hydrolysis of polysaccharides and the coprecipitation of oligosaccharides in the hydrolysate.
Two spectral technologies were employed to characterize the main linkages and functional groups in lignin. FT-IR technology has been widely used to quickly determine the structural property of lignin （Fig.2）. The typical FT-IR spectra of lignin are clearly shown： the absorptions around 1597， 1506， and 1425 cm-1 indicate the aromatic skeleton vibrations; the band at 1460 cm-1 is assigned to the C—H asymmetric deformations; and the peak at 1665 cm-1 is attributed to the C=O stretching in conjugated p-substituted aryl ketones. It is worthy to note that the band at 1114 cm-1 represents the aromatic C—H in-plane deformation in syringyl type， and the intensity of this adsorption in LB is much higher than in the other two samples， corresponding to its highest S/G ratio （Table 1） from NMR analysis below. Due to the extensive presence of carbohydrate in lignin samples， the typical peaks at 1052 and 1038 cm-1 are assigned to xylose and glucose， respectively， and are clearly observed in all samples[15].
To further investigate the aromatic units and the different inter-unit linkages present in the lignin macromolecule， all SREL samples were analyzed using the 2D HSQC NMR technique， and the signal assignments were primarily in accordance with previous literatures[16-17]. The main structural characteristics of lignin， including basic monolignols （S， G， and H units） and various substructures linked by ether， ester， and carbon-carbon bonds （b-O-4’， b-b’， b-5’， etc.） can be observed in Fig.3. In the side-chain region of the spectrum， the prominent substructure， b-O-4’ aryl ethers （substructure A）， is reflected from the Ca—Ha correlation （dC/dH， 72/（4.7～4.9） ppm）， Cb—Hb correlation （dC/dH， 84/4.3 ppm for the G unit and 86/4.1 ppm for the S unit， respectively）， and Cg—Hg correlation （dC/dH， 60.1/（3.4～3.7） ppm）. Resinol （b-b’， substructure B） appeared in all spectra in noticeable amounts as indicated by their Ca—Ha （dC/dH， 84.8/4.66）， Cb—Hb （dC/dH， 53.5/3.07）， and the double Cg—Hg correlations （dC/dH， 71.2/（3.82 and 4.18））， respectively. Accompanied by the lignification process， these two substructures were simultaneously synthesized and the main difference in those fractionated lignin samples was the S/G ratio. By integrating the area of C2，6/H2，6 in the S and G units， the S/G ratios were calculated to be 1.6， 2.1， and 3.3 for LT， LM， and LB， respectively. The thermal stability of lignin samples was also studied by TGA （Fig.4）. Among the lignocellulosic materials， lignin is the most thermo-stable component mainly due to the inherent structure of aromatic rings with various branches， resulting in the wide temperature range of thermal degradation. According to the previous report， the thermal degradation of lignin can occur through the following steps： （1） cleavage of a- and b-aryl-alkyl-ether linkages occurs between 150℃ and 300℃; （2） aliphatic side chains start splitting off from the aromatic ring at around 300℃; （3） the carbon-carbon linkage between lignin structural units is cleaved at 370～400℃; Decomposition or condensation of aromatic rings is believed to occur at 400～600℃[18-19]. As illustrated in Fig.4， the onset of thermal degradation occurs at around 250℃， and the temperature at the maximum weight loss rate is about 320℃ for all SREL samples. However， the non-volatile residues of LM and LB were clearly higher than LT. This indicates that higher thermal stability corresponds with higher molecular weights， along with the lignification process.
3.2 Lignification effect on bioconversion
The crystalline structure of cellulose plays a major role in recalcitrating the bioconversion process. The ball-milling process can partially destroy the intermolecular hydrogen bond between cellulose chains and then decrease the crystallinity. Therefore， it is reasonable to observe that the typical X-ray diffraction pattern of cellulose I disappeared and a relatively amorphous cellulose is presented from Fig.5（a）. Correspondingly， the CrI values of all the samples were small， ranging from 10.9% to 14.1%. However， it could be predicted that the difference in crystallinity， together with the bioconversion efficiency， were mainly ascribed to the degree of lignification of the moso bamboo， since the same ball-milling process was performed on all moso bamboo samples. As shown in Fig.5（b）， almost complete bioconversion was achieved for all samples， mainly due to the ball-milling treatment， and the slight difference was also noticed in detail. The chart shows that both initial hydrolysis rate and final bioconversion of the sample BM was the highest among all the moso bamboo samples. Since the physicochemical property of LM showed no remarkable difference in all SREL samples concerning molecular weight， S/G ratio， and lignin substructure， the improved bioconversion of BM was probably due to the property of cellulose， especially its lowest crystallinity among all the moso bamboo samples. 4 Conclusion
Bamboo is considered a grass biomass material that has great potential as a future bioresource for biorefining. Three different parts （bottom， middle， top） of moso bamboo were comparatively examined in this study to determine their characteristic patterns in terms of chemical composition， lignin structure， and bioconversion efficiency. Although the difference between the three different parts in bioconversion was not obvious， the results indicated that the relatively higher hydrolysis efficiency of bamboo samples in the middle was mainly dependent on the low crystallinity of cellulose rather than the degree of lignin biosynthesis.
Acknowledgments
The authors gratefully acknowledge the financial support from the Natural Science Foundation of China （31770622） and the Innovation Program of College of Materials Science and Technology.
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