Structural and Thermal Characterization of Milled Wood Lignin from Bamboo (Phyllostachys pubescens) Grown in Korea

The structural and thermal characterization of milled wood lignin (MWL) prepared from bamboo (Phyllostachys pubescens) grown in Korea was investigated, and the results were compared with bamboo MWLs from other studies. The C9 formula of the bamboo MWL was C9H7.76O3.23N0.02 (OCH3)1.41. The Mw and Mn of MWL were 13,000 and 4400 Da, respectively, which resulted in a polydispersity index (PDI) of 3.0. The PDI of the prepared MWL was higher than other bamboo MWLs (1.3–2.2), suggesting a broader molecular weight distribution. The structural features of MWL were elucidated using FT-IR spectroscopy and NMR techniques (1H, 13C, HSQC, 31P NMR), which indicate that MWL is of the HGS-type lignin. The major lignin linkages (β-O-4, β-β, β-5) were not different from other bamboo MWLs. The syringyl/guaiacyl ratio, determined from 1H NMR, was calculated as 0.89. 31P NMR revealed variations in hydroxyl content, with a higher aliphatic hydroxyl content in MWL compared to other bamboo MWLs. Thermal properties were investigated through TGA, DSC, and pyrolysis-GC/MS spectrometry (Py-GC/MS). The DTGmax of MWL under inert conditions was 287 °C, and the Tg of MWL was 159 °C. Py-GC/MS at 675 °C revealed a syringyl, guaiacyl, p-hydroxyphenyl composition of 17:37:47.


Introduction
According to the Food and Agriculture Organization (FAO), the annual rate of deforestation was estimated at 10 million ha between 2015 and 2020.Over the past three decades, an estimated 420 million ha of forest have been lost since 1990 [1].This rampant forest destruction contributes to approximately 20% of global carbon emissions [2].Moreover, the excessive use of wood has faced strong criticism due to the continuous depletion of forest resources worldwide [3].As the world's wood resources decline, there is a growing need for lignocellulosic biomass to replace them.Among these lignocellulosic biomass, bamboo is an emerging biomass that can replace wood because of its short production cycle and high amount of biomass per unit area.In addition, the remarkable regeneration capability and minimal maintenance requirements make bamboo an environmentally friendly alternative [3][4][5].The Food and Agriculture Organization (FAO) reports that bamboo is widely grown in tropical and subtropical climatic zones, including in East, Southeast, and South Asia [6].In historical China, bamboo found diverse applications, such as arrowheads, baskets, writing scrolls, pens, paper, boats, shoes, and construction materials [7].In modern-day China, bamboo utilization spans various applications, including bamboo shoots, ceiling/flooring interiors, scaffolding, timber, furniture, and crafts [3,8].According to China's 9th National Forest Inventory, China's bamboo forest area gradually increased from 2014 to 2018, with a total bamboo forest area of 6.73 million ha [6].In this light, China's research on bamboo remained active over these years.However, in Korea, bamboo consumption is declining, leading to the desolation of domestic bamboo forests [9]; hence, there has not been much research on bamboo in Korea.
Bamboo can be processed into a variety of products, including particleboards, plywood, laminated bamboo, bamboo composites, and bamboo fiber [10][11][12].Truly, the utilization of bamboo is limited due to its hollow structure, and certain bamboo stems may not be affected by certain types of chemicals due to their waxy skin [13].These impermeable properties may limit chemical treatment to improve mechanical properties for potential applications [3].In addition to these problems, numerous obstacles still impede the efficient utilization of bamboo resources.Therefore, overcoming these hurdles requires further research efforts.
On the other hand, bamboo can be easily delignified compared to other woody biomass [14,15].In other words, it offers the advantage of producing pulps, as well as lignin, more readily compared to other conventional methods.Additionally, bamboo lignin contains a substantial amount of p-hydroxyphenyl units, a feature nearly absent in softwood and hardwood lignins [16].The high reactivity of this unit, with vacant positions at 3 and 5, is expected to significantly contribute to future lignin utilization.
In this study, the structural and thermal characteristics of lignin were investigated as a fundamental study for the future utilization of bamboo grown in Korea.In addition, the results obtained were compared with previously reported bamboo lignin.formula weight of MWL and other bamboo MWLs are listed.The formula weight of MWL was approximately 214 Da, higher than that of the other two bamboo MWLs.[20] MWL: P. pubescens, A: P. acuta, X: unknown bamboo species.Methoxyl content was calculated by the integrations of the aromatic and methoxyl signals in 1 H NMR spectrum [18].[20] MWL: P. pubescens, A: P. acuta, X: unknown bamboo species.

Molecular Weight (MW) Distribution, Average MW, and Polydispersity
The weight-average (Mw) and number-average (Mn) MW, along with the polydispersity index (PDI), of acetylated MWL (Ac-MWL), were determined using gel permeation chromatography (GPC).The MW distribution of Ac-MWL is illustrated in Figure 1, and the corresponding Mw, Mn, and PDI values are presented in Table 4.For comparison, data on previously reported bamboo MWLs are provided in Table 4.The Mw and Mn of Ac-MWL were approximately 13,000 Da and 4400 Da, respectively.The Mw of Ac-MWL prepared in this study was higher than that of all other bamboo MWLs.In addition, the PDI of Ac-MWL was also higher than that of all other bamboo MWLs, indicating a broader distribution of MW.The Mw, Mn, and PDI differed even from the same bamboo species.This result is likely attributed to the different climatic zones wherein the bamboo was grown, since the Korean-grown bamboo is from temperate regions and the Chinese-grown bamboo is from subtropical regions.The Korean-grown bamboo was believed to have thicker and more rigid culms to withstand colder temperatures and harsher climatic conditions.This robustness was thought to contribute to the greater strength and density of Korean-grown bamboo, potentially resulting in a higher Mw.

FT-IR Spectroscopy
FT-IR spectroscopy was conducted for the determination of functional groups in MWL.In Figure 2, the FT-IR spectrum of MWL is presented.The bands have been assigned according to the work of Faix [24], and the assignments are detailed in Table 5.The observed spectral features of MWL aligned with those of HGS-type lignins found in other bamboo MWLs [19,21,24].Notably, the band at 834 cm −1 , corresponding to C-H outof-plane vibrations in H units, along with a characteristic shoulder at 1160 cm −1 -typical for HGS type-was observed.In addition, the C=O stretching related to unconjugated ketone, carbonyl, and ester groups was assigned at 1718 cm −1 , aromatic skeleton vibrations at 1594, 1503, and 1419 cm −1 , syringyl (S)-related bands at 1130 and 1123 cm −1 , and guaiacyl (G)-related bands at 1330, 1266, 1222, and 1033 cm −1 .

FT-IR Spectroscopy
FT-IR spectroscopy was conducted for the determination of functional groups in MWL.In Figure 2, the FT-IR spectrum of MWL is presented.The bands have been assigned according to the work of Faix [24], and the assignments are detailed in Table 5.The observed spectral features of MWL aligned with those of HGS-type lignins found in other bamboo MWLs [19,21,24].Notably, the band at 834 cm −1 , corresponding to C-H out-of-plane vibrations in H units, along with a characteristic shoulder at 1160 cm −1 -typical for HGS type-was observed.In addition, the C=O stretching related to unconjugated ketone, carbonyl, and ester groups was assigned at 1718 cm −1 , aromatic skeleton vibrations at 1594, 1503, and 1419 cm −1 , syringyl (S)-related bands at 1130 and 1123 cm −1 , and guaiacyl (G)-related bands at 1330, 1266, 1222, and 1033 cm −1 .

1 H NMR Analysis
The 1 H NMR spectrum of Ac-MWL is presented in Figure 3.The aromatic region (7.20-6.25 ppm) revealed the presence of G and S phenylpropane (C 9 ) units.The aliphatic content was higher than the aromatic content.

1 H NMR Analysis
The 1 H NMR spectrum of Ac-MWL is presented in Figure 3.The aromatic region (7.20-6.25 ppm) revealed the presence of G and S phenylpropane (C9) units.The aliphatic content was higher than the aromatic content.Signal assignments in the 1 H NMR spectrum of Ac-MWL are listed in Table 6, based on literature data [25,26].To estimate the distribution of protons per C9 structural unit in Ac-MWL, integration ratios and their C9 molecular formulas were used [27].The methoxyl content in the C9 molecular formula for MWL was 1.41, multiplied by 3 to yield 4.23-the total number of protons in the methoxyl groups.Integration values for other structural components were normalized to the methoxyl protons in a single C9 unit.However, certain quantitative conclusions cannot be drawn due to overlapping signals, carbohydrate inclusions, and uncertainties in range assignments.

Ppm
Main Assignments Ac-MWL 7.20-6.80* Aromatic proton in G units 1.05 6.80-6.25 Aromatic proton in S units 0.93 6.25-5.75 Hα of β-O-4 and β-1 structures 0.47 Signal assignments in the 1 H NMR spectrum of Ac-MWL are listed in Table 6, based on literature data [25,26].To estimate the distribution of protons per C 9 structural unit in Ac-MWL, integration ratios and their C 9 molecular formulas were used [27].The methoxyl content in the C 9 molecular formula for MWL was 1.41, multiplied by 3 to yield 4.23-the total number of protons in the methoxyl groups.Integration values for other structural components were normalized to the methoxyl protons in a single C 9 unit.However, certain quantitative conclusions cannot be drawn due to overlapping signals, carbohydrate inclusions, and uncertainties in range assignments.The arylglycerol β-O-4 aryl ether linkage (6.25-5.75ppm, 4.90-4.30ppm) is the main intermonomeric linkage found in native lignin [25].The H α and H β contents from Acβ-O-4 structures were highest among the linkages.The aromatic protons per C 9 unit for Ac-MWL were determined to be 0.93 for S units and 1.05 for G units.The S/G molar ratio of MWL based on 1 H NMR was determined to be 0.89.

13 C NMR Analysis
The structural features and linkages within MWL were further elucidated through 13 C NMR analysis.In Figure 4, the 13 C NMR spectrum of MWL is presented, and Table 7 provides the chemical shifts along with their assignments based on previously reported works [16,21,28,29].The NMR spectrum was divided into four regions: C=O, aromatic, side chain, and aliphatic.

13 C NMR Analysis
The structural features and linkages within MWL were further elucidated through 13 C NMR analysis.In Figure 4, the 13 C NMR spectrum of MWL is presented, and Table 7 provides the chemical shifts along with their assignments based on previously reported works [16,21,28,29].The NMR spectrum was divided into four regions: C=O, aromatic, side chain, and aliphatic.The signals at positions 1 and 2 correspond to carbonyl groups in MWL.Signals at positions 25, 26, 31, 32, and 33 were attributed to residual carbohydrates, which likely originated from impurities such as traces of hemicelluloses associated with some MWL substructures.The intense signal at position 40 is indicative of OCH 3 in both S and G units.
The etherified S/nonetherified S unit ratio was estimated based on the peak height ratio at 152.2/147.1 ppm, while the etherified G/nonetherified G ratio was derived from resonance ratios at 149.4/145.5 ppm [34].The values of etherified S/nonetherified S (4.4) and etherified G/nonetherified G (1.5) suggested a greater involvement of S units in ether linkages with other lignin units compared to G structures.This finding agrees with previously reported results [21,34].
Since MWL is a macromolecule, some overlapping signals were observed in the 13 C NMR spectrum.Therefore, 2D HSQC NMR analysis was performed to enhance spectral resolutions in intercoupling bonds and linkages within lignin substructures.

2D HSQC NMR Analysis
Figure 5 depicts the side chain (120-70/5.0-3.0 ppm) and aromatic (160-100/8.0-6.0 ppm) regions of MWL in the HSQC spectrum.The aliphatic region was excluded from the discussion, as notable information was not provided.Cross-signals and their assignments, derived from previously reported works by Wen et al. [21,35,36], are shown in Table 8.The substructures present in MWL, along with their corresponding notations, are presented in Figure 6.
Since MWL is a macromolecule, some overlapping signals were observed in the 13 C NMR spectrum.Therefore, 2D HSQC NMR analysis was performed to enhance spectral resolutions in intercoupling bonds and linkages within lignin substructures.

2D HSQC NMR Analysis
Figure 5 depicts the side chain (120-70/5.0-3.0 ppm) and aromatic (160-100/8.0-6.0 ppm) regions of MWL in the HSQC spectrum.The aliphatic region was excluded from the discussion, as notable information was not provided.Cross-signals and their assignments, derived from previously reported works by Wen et al. [21,35,36], are shown in Table 8.The substructures present in MWL, along with their corresponding notations, are presented in Figure 6.    ), as reported by Kim and Ralph [37] and Wen et al. [35,36].
In the aromatic region (Figure 5b), signals from S, G, and H moieties were highly visible.S moieties (S 2,6 , S ′ 2,6 ′ , and S ′′ 2,6 ) were in the range of 104.8-107.2/7.30-6.69ppm.G moieties were situated at 111.9/6.97,115.4/6.68, and 129.8/6.80 ppm for C 2 -H 2 , C 5 -H 5 , and C 6 -H 6 , respectively.H moieties were observed at 113.8/6.67 ppm for C 3,5 -H 3,5 and 128.3/7.17ppm for C 2,6 -H 2,6 .Additionally, three cross signals were assigned to C The hydroxyl and carboxyl content of MWL were determined using the 31 P NMR method based on Argyropoulos et al. [38], which allows the quantification of different types of hydroxyl groups, including aliphatic and phenolic hydroxyl groups, as well as G, S, H, and C 5 condensed phenolic hydroxyl groups.In Figure 7a, the full 31 P NMR spectrum of phosphitylated MWL is presented, and Figure 7b shows the enlarged hydroxyl group region of interest (150-134 ppm).A sharp peak at 174 ppm was attributed to the excess amount of unreacted TMDP, indicating the complete derivatization of all hydroxyl groups in MWL.The hydroxyl and carboxyl content of MWL were determined using the 31 P NMR method based on Argyropoulos et al. [38], which allows the quantification of different types of hydroxyl groups, including aliphatic and phenolic hydroxyl groups, as well as G, S, H, and C5 condensed phenolic hydroxyl groups.In Figure 7a, the full 31 P NMR spectrum of phosphitylated MWL is presented, and Figure 7b shows the enlarged hydroxyl group region of interest (150-134 ppm).A sharp peak at 174 ppm was attributed to the excess amount of unreacted TMDP, indicating the complete derivatization of all hydroxyl groups in MWL.The aliphatic and phenolic (C5-substituted + S, G, and H) hydroxyl, and carboxyl contents of MWL, calculated from the 31 P NMR result, are listed in Table 9.The hydroxyl content was compared to other bamboo MWLs based on published data [23,39].For MWL, the total hydroxyl content was found to be 8.19 mmol/g MWL.The hydroxyl group of the H units was similar in all bamboo MWLs.The aliphatic and total hydroxyl contents in MWL were notably higher, while the C5-substituted+S content was lower compared to MWL-Y and MWL-N.The carboxyl content of MWL was determined to be 0.23 mmol/g MWL, close to MWL-N and slightly lower than MWL-Y.The aliphatic and phenolic (C 5 -substituted + S, G, and H) hydroxyl, and carboxyl contents of MWL, calculated from the 31 P NMR result, are listed in Table 9.The hydroxyl content was compared to other bamboo MWLs based on published data [23,39].For MWL, the total hydroxyl content was found to be 8.19 mmol/g MWL.The hydroxyl group of the H units was similar in all bamboo MWLs.The aliphatic and total hydroxyl contents in MWL were notably higher, while the C 5 -substituted+S content was lower compared to MWL-Y and MWL-N.The carboxyl content of MWL was determined to be 0.23 mmol/g MWL, close to MWL-N and slightly lower than MWL-Y.TGA is widely employed for examining the thermal behavior and thermal and thermooxidative stability of lignin.Figure 8a,b show the thermogravimetric (TG) and derivative TG (DTG) curves of MWL, respectively, under oxidative and inert conditions.In the TG and DTG curves, two crucial temperatures are observed.The onset temperature marks the point at which decomposition begins, indicating the initiation of gas release during thermal oxidation or pyrolysis experiments.The other temperature, DTG max , represents the point at which maximum thermal degradation occurs and is considered a parameter for determining the thermal stability of lignins [40].

TGA
TGA is widely employed for examining the thermal behavior and thermal and thermo-oxidative stability of lignin.Figure 8a,b show the thermogravimetric (TG) and derivative TG (DTG) curves of MWL, respectively, under oxidative and inert conditions.In the TG and DTG curves, two crucial temperatures are observed.The onset temperature marks the point at which decomposition begins, indicating the initiation of gas release during thermal oxidation or pyrolysis experiments.The other temperature, DTGmax, represents the point at which maximum thermal degradation occurs and is considered a parameter for determining the thermal stability of lignins [40].The decomposition process of the lignin sample can be divided into several stages.In the initial stage before 120 °C, the weight loss is attributed to the evaporation of moisture remaining in the lignin samples [41] and low MW volatiles.Under oxidative conditions at 120 °C, a weight loss of 1.9% in MWL was noted (Table 10).The onset temperature was 234 °C and DTGmax was 544 °C.The weight losses at 400 °C and 500 °C were approximately 43% and 65%, respectively.There were no weight changes beyond 590 °C, and the ash content obtained at 800 °C was 0.3%.
Under inert conditions at 120 °C, a weight loss of 1.8% in MWL was observed (Table 10).The onset temperature of MWL was found to be 235 °C, nearly the same as that of oxidative condition (234 °C).In the 200-400 °C region of the DTG curve, two major bands were observed at 287 °C and 370 °C (Figure 8b).The weight loss in this region is attributed to the cleavage of interunit linkages in lignin, releasing monomeric phenols into the vapor phase [42].Specifically, the peak at 287 °C signifies the degradation of aliphatic side chains, particularly the scission of β-O-4 ether linkages, while the peak at 370 °C indicates the degradation of methoxyl groups [43], based on information regarding gases released during pyrolysis [44].Between 400 and 600 °C, the weight loss is primarily attributed to the decomposition or condensation of the aromatic ring [42,45].The weight losses at 400 °C and 500 °C were approximately 11% and 36%, respectively.MWL underwent The decomposition process of the lignin sample can be divided into several stages.In the initial stage before 120 • C, the weight loss is attributed to the evaporation of moisture remaining in the lignin samples [41] and low MW volatiles.Under oxidative conditions at 120 • C, a weight loss of 1.9% in MWL was noted (Table 10).The onset temperature was 234 • C and DTG max was 544 • C. The weight losses at 400 • C and 500 • C were approximately 43% and 65%, respectively.There were no weight changes beyond 590 • C, and the ash content obtained at 800 • C was 0.3%.MWL: P. pubescens, N: Neosinocalamus affinis, Y: unknown bamboo species.
Under inert conditions at 120 • C, a weight loss of 1.8% in MWL was observed (Table 10).The onset temperature of MWL was found to be 235 • C, nearly the same as that of oxidative condition (234 • C).In the 200-400 • C region of the DTG curve, two major bands were observed at 287 • C and 370 • C (Figure 8b).The weight loss in this region is attributed to the cleavage of interunit linkages in lignin, releasing monomeric phenols into the vapor phase [42].Specifically, the peak at 287 • C signifies the degradation of aliphatic side chains, particularly the scission of β-O-4 ether linkages, while the peak at 370 • C indicates the degradation of methoxyl groups [43], based on information regarding gases released during pyrolysis [44].Between 400 and 600 • C, the weight loss is primarily attributed to the decomposition or condensation of the aromatic ring [42,45].The weight losses at 400 • C and 500 • C were approximately 11% and 36%, respectively.MWL underwent continuous carbonization at temperatures ranging from 600 to 800 • C, with a residual content of 28.7% at 800 • C.
In Table 10, the DTG max and residual content of other bamboo MWLs are presented in comparison to MWL.The DTG max of MWL was lower (287 • C) than that of other bamboo MWLs (360 and 367 • C).This difference may be attributed to higher S unit content.The residue content of MWL was higher than MWL-Y and lower than MWL-N.
Furthermore, under inert conditions, the DTG max (287 • C) was lower than that obtained under oxidative conditions (544 • C).This can be attributed to the difficulty involved in degrading oxidized condensed aromatic moieties [43].

DSC
The thermal behavior of MWL, expressed as heat flow with respect to temperature, was determined over the range of 20-240 • C. In Figure 9, the 1st heating, cooling, and 2nd heating cycles of MWL are illustrated.During the 1st heating cycle, the thermal history of MWL, i.e., moisture absorbed during storage conditions, residual solvents, and drying methods, was removed.Upon reaching 240 • C, a temperature exceeding its melting transition, MWL was allowed to cool at 20 • C.An exothermic peak observed during the cooling cycle indicated the solidification of the lignin melt.The 2nd heating cycle reveals the true thermal behavior of the sample and is known to provide a reliable estimate of the glass transition temperature (T g ).T g is an important transition temperature, at which amorphous polymers shift from a glassy to a rubbery state.In the 2nd heating cycle, the T g of MWL was determined to be 159 Furthermore, under inert conditions, the DTGmax (287 °C) was lower than that obtained under oxidative conditions (544 °C).This can be attributed to the difficulty involved in degrading oxidized condensed aromatic moieties [43].

DSC
The thermal behavior of MWL, expressed as heat flow with respect to temperature, was determined over the range of 20-240 °C.In Figure 9, the 1st heating, cooling, and 2nd heating cycles of MWL are illustrated.During the 1st heating cycle, the thermal history of MWL, i.e., moisture absorbed during storage conditions, residual solvents, and drying methods, was removed.Upon reaching 240 °C, a temperature exceeding its melting transition, MWL was allowed to cool at 20 °C.An exothermic peak observed during the cooling cycle indicated the solidification of the lignin melt.The 2nd heating cycle reveals the true thermal behavior of the sample and is known to provide a reliable estimate of the glass transition temperature (Tg).Tg is an important transition temperature, at which amorphous polymers shift from a glassy to a rubbery state.In the 2nd heating cycle, the Tg of MWL was determined to be 159 °C.[46,47], along with bamboo Py-GC/MS data [20,22,48].The pyrogram of MWL is shown in Figure 10, and the resulting pyrolysis products with their relative compositions are listed in Table 11.Sixteen monolignol compounds were identified during pyrolysis at 675 • C, encompassing typical H-, G-, and S-related pyrolysis products.The pyrolysis products of MWL were categorized into three groups: H lignin derivatives (peaks 1, 2, 4, 6), G lignin derivatives (peaks 3,5,8,10,11,12,13), and S lignin derivatives (peaks 7, 9, 14, 15, 16).The major pyrolysis products released were 4-vinylphenol (6), 4-vinylguaiacol (8), guaiacol (3), syringol (9), and 4-methylphenol (2).These five major pyrolysis products constituted 68% of the total relative composition of MWL.Among them, 4-vinylphenol was the most abundant, accounting for approximately 30% of the total relative composition, consistent with previously reported findings from Saiz-Jimenez and De Leeuw [48] and Li et al. [20].The S:G:H composition of MWL was determined to be 16:37:47.The calculated S/G ratio for MWL was 0.43, aligning with a previously published result (0.4) from Bai et al. [22].The S/G ratio derived from Py-GC/MS differed from the S/G ratios obtained through 1 H NMR (0.89).However, both methods concurred that the S content was lower than the G content.

Materials
The bamboo powder, prepared from a 2-3-year-old bamboo (P.pubescens) culm, was supplied by Songjuk Industry, located in Hamyang, Gyeongnam, Republic of Korea.The bamboo powder was air dried at room temperature for a week, 40 mesh passed powders were used.

Preparation of MWL
The thoroughly dried, extractive-free bamboo powder was used for the preparation of MWL.Six grams of bamboo powder were placed in a 500-mL stainless-steel jar and filled with toluene.The jar, containing the sample, was then mounted on a vibratory ball mill and treated for 100 h.After milling, the MWL was isolated and purified according to the Björkman method [57].

Elemental Analysis
The MWL was vacuum dried under P 2 O 5 at ambient temperature for 24 h prior to elemental analysis.C, H, O, N, and S analyses were performed using an Elemental Analyzer (IT/Flash 2000, Thermo Fisher Scientific, Waltham, MA, USA) at the Center for University-wide Research Facility, Jeonbuk National University (CURF, JBNU).

Acetylation of MWL
For the acetylation, 50 mg of MWL was dissolved in 1 mL of pyridine and 1 mL of acetic anhydride.The reactions of quenching, filtering, washing, and drying were carried out in the same manner as described by Mun et al. [58].The acetylated MWL was designated as Ac-MWL.

Determination of MW
The average MW of MWL was determined by gel permeation chromatography (GPC).One milligram of Ac-MWL was dissolved in 1 mL of THF in a 10-mL conical beaker.The beaker was sonicated for 5 s and then filtered through a 0.45 µm PTFE syringe filter (Chemco Scientific, Seoul, Republic of Korea).The filtrate was transferred into a 2-mL vial and diluted 2 times with THF.The GPC (Waters, Milford, MA, USA) was conducted at CURF under the conditions shown in Table 12.

FT-IR Spectroscopy
FT-IR analysis was conducted utilizing a diamond attenuated total reflectance (ATR) accessory on an FT-IR spectrophotometer (Frontier, Perkin Elmer, Shelton, CT, USA), equipped with a deuterated triglycine sulfate (DTGS) detector.The spectrum was acquired in the wavelength range of 4000-500 cm -1 with a resolution of 4 cm -1 .The analysis was performed at the CURF, JBNU.

1 H NMR Analysis
Ten milligrams of the Ac-MWL sample was dissolved in 0.4 mL of CDCl 3 in a 10-mL conical beaker.The beaker was sonicated for 1-2 min to dissolve the sample.The mixture was filtered through a fine glass wool suspended inside a Pasteur pipette, which was directly connected to a clean NMR tube.The conical beaker was rinsed with additional 0.3 mL of CDCl 3 and the contents were transferred as described in a previous filtration method.The measurement was conducted using the NMR spectrometer (500 MHz FT-NMR, JNM-ECZ500R, JEOL, Tokyo, Japan) at the CURF, JBNU.
3.9. 13C and 2D HSQC NMR Analysis A 120 mg MWL was placed into a 5-mL vial and vacuum dried under P 2 O 5 at ambient temperature for 24 h before sample preparation.The moisture-free MWL was dissolved at 0.75 mL DMSO-d 6 at 50 • C. The filtration was carried out in the same manner as for 1 H NMR samples mentioned above.The 13 C and HSQC NMR analyses were conducted using an NMR spectrometer (600 MHz, JEOL, Tokyo, Japan) at the CURF, JBNU.

31 P NMR Analysis
The hydroxyl and carboxyl group content of MWL was determined through 31 P NMR analysis following the procedure outlined by Argyropoulos et al. [38].The sample was prepared using pyridine/CDCl 3 (1.6:1v/v) solvent with an internal standard NHND, a relaxation agent (chromium (III) acetylacetonate), and a phosphitylating agent (TMDP).Throughout the process, maintaining a moisture-free condition was crucial.The 31 P NMR analysis was conducted using an NMR spectrometer (600 MHz, JEOL, Tokyo, Japan) at the CURF, JBNU.The spectrum was obtained using an inverse-gated decoupling pulse sequence, a 10 s relaxation delay, and 64 scans.

TGA
A 4-8 mg MWL was placed in a standard aluminum pan and secured in a thermogravimetric analyzer (Q600 SDT, TA Instruments).The sample was heated from 20 to 800 • C at 10 • C/min under nitrogen and oxidative conditions.TGA was performed at the CURF, JBNU.

DSC
A 2-6 mg MWL was loaded in a standard aluminum pan, and the heat flow was measured by a differential scanning calorimeter (DSC Q20, TA Instruments, New Castle, DE, USA).The sample was heated from 40 to 240 • C at 10 • C/min under a nitrogen

Figure 2 .
Figure 2. FT-IR (ATR) spectrum of MWL.Table 5. Assignment of FT-IR spectrum of MWL.Band (cm −1 ) Assignments 3441 O-H stretching 2843-2937 C-H stretching in methyl, methylene groups 1718 C=O stretching in unconjugated ketone, carbonyl, and ester groups 1664 C=O stretching in conjugated p-substituted aryl ketone 1594 Aromatic skeleton vibration plus C=O stretching; S > G: Gcondensed > Getherified 1503 Aromatic skeleton vibration (G > S) 1462 C-H deformations (asymm in -CH3 and -CH2-) 1419 Aromatic skeleton vibration combined with C-H in plane deformations 1365 Aliphatic C-H stretching in CH3 and phenolic OH 1330 Condensed S and G ring (G ring bound via position 5) 1266 G ring plus C=O stretching (G-methoxyl C-O) 1222 C-O + C-O + C=O stretching (Gcondensed > Getherified) 1160 Typical for HGS lignins; C=O in ester groups (conj.)1123 Aromatic C-H in-plane deformation (S) 1089 C-O deformation in sec-alcohols and aliphatic ethers 1033 Aromatic C-H in-plane deformation (G > S) + C-O deformation in primary alcohols + C-H stretching (unconjugated) 921 C-H out of plane (aromatic ring) 834 C-H out of plane in positions (2 and 6 of S + in all positions of H units)

Figure 8 .
Figure 8.(a) TG and (b) DTG curves of MWL under oxidative and inert conditions.

Figure 8 .
Figure 8.(a) TG and (b) DTG curves of MWL under oxidative and inert conditions.

Table 2 .
Elemental analyses and methoxyl contents of MWLs.

Table 3 .
C 9 formula and formula weight of MWLs.

Table 4 .
Average MW and PDI of Ac-MWLs.

Table 4 .
Average MW and PDI of Ac-MWLs.

Table 5 .
Assignment of FT-IR spectrum of MWL.

Table 6 .
1H NMR assignments and distribution of protons per C9 structural unit of Ac-MWL.

Table 6 .
1H NMR assignments and distribution of protons per C 9 structural unit of Ac-MWL.From reference, it was 7.25-6.80,but CDCl 3 solvent peak was detected at 7.24, thus, the chemical shift was adjusted. *

Table 8 .
Assignments of13C/ 1 H correlation signals in the HMQC spectra of MWL.
The HSQC NMR results confirmed MWL as an HGS-type lignin, consistent with the FT-IR and13C NMR results.Signals related to spirodienone, ferulate, and cinnamaldehyde endgroups were observed in bamboo MWL from P. pubescens grown in China [21], but not in bamboo MWL grown in Korea.

Table 9 .
Hydroxyl and carboxyl contents of MWLs.

Table 11 .
Pyrolysis products and relative composition at 675 °C.

Table 11 .
Pyrolysis products and relative composition at 675 • C. PCA: p-coumaric acid, and FA: ferulate; a RRT: relative retention time, guaiacol as the reference; b m/z values in bold: base peak; only m/z values > 30% of the base peak are included.