- Email: [email protected]
Sequential utilization of bamboo biomass through reductive catalytic fractionation of lignin
Accepted Manuscript Sequential utilization of bamboo biomass through reductive catalytic fractionation of lignin Kaili Zhang, Helong Li, Ling-Ping Xiao, Bo Wang, Run-Cang Sun, Guoyong Song PII: DOI: Article Number: Reference:
S0960-8524(19)30557-7 https://doi.org/10.1016/j.biortech.2019.121335 121335 BITE 121335
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 February 2019 8 April 2019 10 April 2019
Please cite this article as: Zhang, K., Li, H., Xiao, L-P., Wang, B., Sun, R-C., Song, G., Sequential utilization of bamboo biomass through reductive catalytic fractionation of lignin, Bioresource Technology (2019), doi: https:// doi.org/10.1016/j.biortech.2019.121335
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.
Sequential utilization of bamboo biomass through reductive catalytic fractionation of lignin
Kaili Zhang,a Helong Li,a Ling-Ping Xiaob,*, Bo Wanga, Run-Cang Sunb, Guoyong Songa,*
a
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University,
Beijing 100083, China. b
Center for Lignocellulose Science and Engineering, Liaoning Key Laboratory of
Pulp and Papermaking Engineering, School of Light Industry and Chemical Engineering, Dalian Polytechnic University, Dalian 116034, China.
*
Corresponding authors:
E-mail addresses: [email protected] (L.-P. Xiao) [email protected] (G. Song)
1
ABSTRACT Reductive catalytic fractionation (RCF) has emerged as a new biorefinery paradigm for the fractionation and sequential utilization of entire components of biomass. Herein, we investigated the RCF of bamboo, a highly abundant herbaceous feedstock, in the presence of Pd/C catalyst. The lignin fraction in bamboo was preferentially depolymerized into well-defined low-molecular-weight phenols, with leaving carbohydrates pulp as a solid residue. In the soluble fraction, four major phenolic compounds, e.g., methyl coumarate/ferulate derived from hydroxycinnamic units and propanol guaiacol/syringol derived from β-O-4 units, were generated up to 41.7 wt% yield based on original lignin. In the insoluble fraction, the carbohydrates of bamboo were recovered with high retentions of cellulose (68%) and hemicellulose (49%), which upon treatment with enzyme gave glucose (90%) and xylose (85%). Overall, the three major components of bamboo could efficient to be fractionated and converted into useful platform chemicals on the basis of this study.
Keywords: Bamboo; Reductive catalytic fractionation; Lignin; Biorefinery; Hydrogenolysis
2
1. Introduction Lignocellulosic biomass, which is composed by cellulose, hemicellulose and lignin, represents the most abundant renewable carbon source with significant potential for the production of sustainable chemicals and fuels (Besson et al., 2014; Somerville et al., 2010; Tuck et al., 2012). Lignin is a complex aromatic biopolymer, accounting for 20-30% of lignocellulosic biomass by weight (Boerjan et al., 2003). Depolymerization of lignin into well-defined mono-aromatic chemicals amenable to downstream processes is regarded as an important starting point for lignin valorization (Cao et al., 2018; Galkin & Samec, 2016; Li et al., 2015; Rinaldi et al., 2016; Sun et al., 2018; Wang et al., 2019). However, current lignocellulosic biorefineries have focused on the utilization and valorization of carbohydrate fractions, wherein lignin was considered as an inferior component (Kamm, 2007; Wyman, 2013). As a consequent, the objective of current biomass pretreatment (e.g., soda and kraft pulping) is to get rid of lignin to facilitate carbohydrate valorization. Under such severe conditions, the reactive C–O bonds in native lignin undergo cleavage to generate some reactive intermediates, which may irreversibly form more refractory C–C bonds via recondensation reactions (Constant et al., 2016; Renders et al., 2017; Schutyser et al., 2018). The biorefinery lignins features lower reactivity than the native biopolymer, and they most often can be used as a cheap energy source for burning. Recent studies have demonstrated that direct reductive catalytic fractionation (RCF), that is lignin first strategy, can transfer the lignin in its native form in 3
lignocellulose into monomeric phenols in high yield, together with well-preservation of carbohydrate components (Renders et al., 2017). The heterogeneous transition metal catalysts were usually employed, which can accelerate the cleavage of β-O-4 units and avoid the recondensation/repolymerization processes through reductive pathways (Galkin & Samec, 2014; Huang et al., 2017; Klein et al., 2015; Kumaniaev et al., 2017; Parsell et al., 2015; Renders et al., 2018; Song et al., 2013; Sun et al., 2019; Van den Bosch et al., 2017; Van den Bosch et al., 2015a; Van den Bosch et al., 2015b; Yan et al., 2008; Zhai et al., 2017). Generally, hardwoods and genetically engineered plants with high syringyl-to-guaiacyl (S/G) monolignol ratios are the preferred feedstock for the RCF processes, from which monomeric phenols having an end-chain could be obtained in close-to-theoretical maximum yields (Van den Bosch et al., 2015b). Commercial transition metal catalysts (e.g. Rh/C (Yan et al., 2008) Ru/C (Van den Bosch et al., 2015b), Ni/C (Song et al., 2013)) or bimetallic catalysts (e.g. Zn/Pd/C (Klein et al., 2016; Parsell et al., 2015) gave 4-propyl-subtituted guaiacol and syringol as major products. In the case of Pd/C catalyst, the end-chain of phenols can be modulated by choice of hydrogen donor. For examples, saturated 4propanol-substituted phenols were generated under H2 (Van den Bosch et al., 2015a), while unsaturated 4-propenyl-substituted phenols were produced in EtOH/H2O (Galkin & Samec, 2014). Recent result has described that Ni@ZIF-8 can act as a chemodivergent catalyst for hydrogenolysis of eucalyptus biomass, selectively producing 4-propanol- or 4-propyl-substituted phenols under different reaction conditions (Liu et al., 2019). The heterogeneous Mo catalysts, which feature a lower 4
activity to hydrogenation, afforded allyl ether substituted phenols in a selective manner (Sun et al., 2019; Xiao et al., 2017b). In the case of softwoods with the high ratio of guaiacol units and low content of cleavable linkages, RCF became more challenging, which resulted in the drops in both monomers yield and delignification. Herbaceous plants are important fast-growing crops. The lignin in herbaceous plants features higher proportion of hydroxycinnamic acid, i.e., p-coumaric acid (pCA) and ferulic acid (FA) moieties, in addition to β-O-4 structures (Buranov & Mazza, 2008; Helm & Ralph, 1992; Kim et al., 2017; Peng et al., 2017). Sels (Van den Bosch et al., 2015b) and Abu-Omar (Luo et al., 2016) independent reported the RCF of miscanthus grass by using Ru/C and Ni/C catalysts, respectively, which afforded 4-propyl-, 4-propanol and 4-propionate-substituted phenols from lignin component. Beckham and Román-Leshkov described the RCF process of corn stover with Ni/C and H3PO4 (Anderson et al., 2016). We recently reported that ZnMoO4/MCM-41-catalyzed RCF of corncob, which gave p-hydroxycinnamic esters in a selective version (Wang et al., 2018). Bamboo is a perennial herbaceous plant, which features advantages of fast growth, rapid renovation and readily propagation, has been recognized as a promising lignocellulosic resource in the global bioeconomy (Hong et al., 2011; Li et al., 2012; Zhang et al., 2017). To the best of our knowledge, the reductive catalytic fractionation of bamboo biomass has not been realized. Herein, we demonstrate the first example of direct RCF of bamboo by using Pd/C as a catalyst, which resulted in monomeric phenols in high yield from lignin depolymerization, together with the retention of carbohydrate pulp as a solid residue 5
amenable to valorization. A systematic study on the effects of solvent, temperature and reaction time, are also presented in view of lignin-derived monomers yields and the carbohydrate retentions. This study provides an economically pathway for fractionation of bamboo biomass, and in in extension, sequential utilization of all three components of bamboo biomass.
2. Material and methods 2.1. Material Bamboo (Dendrocalamus brandisii, three year old) was harvested from Yunnan Province, China (Bai et al., 2013). It was crushed, screened into powders in size of 40-60 meshes, extracted with toluene/ethanol and dried under vacuum at 50 oC. Pd/C catalyst (where Pd content is 5 wt%) was purchased from Energy Chemical. Methanol (MeOH), ethanol (EtOH), isopropanol (iPrOH) and tetrahydrofuran (THF) were purchased from Fisher Chemical and used as received without further purification. The commercial enzyme (Cellic@CTec2, 100 FPU/mL) was kindly provided from Novozymes (Beijing, China). Authentic samples for identification of lignin- and carbohydrate- derived monomers were acquired from commercially or synthesized independently.
2.2. General catalytic reaction Extracted bamboo sawdust (1.0 g), Pd/C (150 mg, 15 wt% based on bamboo sawdust) and solvent (40 mL) were charged into a stainless steel batch reactor (100 6
mL, Parr instruments Co.), which was flushed with N2 and pressurized with H2 at room temperature. The reactor was heated at 220 oC, 240 oC, 260 oC, 280 oC and 320 o
C for 4 h or at 260 oC for 2, 4, 8, 12, 16 h. After reaction, the reactor was cooled to
room temperature with water-flow. The reaction mixture was filtered through a nylon 66 membrane filter (0.2 μm), and the insoluble fraction was washed with methanol, thus forming soluble fraction containing phenolic compounds and insoluble fractions containing carbohydrate pulp and catalyst.
2.3. Lignin characterization For the soluble fraction, the methanol was removed under vacuum and then extracted with dichloromethane and water. Subsequent removal of dichloromethane of the organic phase gave a brown “lignin oil”. The lignin oil and a standard (tetradecane) were dissolved in dichloromethane in a 10 mL volumetric flask, which was then analyzed by gas chromatography coupled with mass spectrometer (GC/MS) and gas chromatography (GC) to identify and quantify the monomeric phenols. Gel permeation chromatography (GPC) analysis was performed on Shimadzu LC-20A equipped with a PL-gel 10 μm Mixed-B 7.5 mm I.D. column (mixed) and UV detection detector (254 nm) at 50 °C, using THF as the solvent (1 mL/min). Twodimensional heteronuclear single quantum coherence (2D HSQC) NMR analyses were acquired on a Bruker Avance 400 MHz spectrometer. Generally, DMSO-d6 was used as a NMR solvent and the central solvent peak at δC/δH = 39.5/2.49 ppm was used as an internal reference. 7
Lignin oil (%) =
m(extracted soluble fraction ) × 100% m(bamboo saudust)
Phenolic monomers (%) =
Solid residue (%) =
m(total monomers) × 100% m(total lignin)
m(insoluble fraction) − m(catalyst) × 100% m(bamboo saudust)
The water of aqueous phase from the extraction was removed under vacuum, which was then mixed with anhydrous pyridine (0.5 mL) and N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA) (0.5 mL). After heated at 80 oC for 30 min, the reaction mixture was submitted to GC/MS analysis.
2.4. Compositional analysis of carbohydrate pulp For the insoluble fraction, the Pd/C catalyst could be separated and recycled in 90% yield from carbohydrate pulp through a mesh screening (300 mesh). The spent catalyst was subjected to simple washing and was used directly in the following cycle at 260 oC, H2 (4 MPa) for 4 h. After first run, the ICP-MS (inductively coupled plasma mass spectrometry) of soluble fraction was performed on Agilent 7800 ICP-MS. The carbohydrate pulp was then subjected to composition analysis using National Renewable Energy Laboratory (NREL) standard acid hydrolysis procedure (Sluiter et al., 2008). The carbohydrate pulp (300 mg) was treated in 72 wt % sulfuric acid (3 mL) for 1 h at 30 °C. Deionized water (84.0 mL) was added to dilute sulfuric acid (ca. 3%), which was then heated at 120 °C for 1 h. The mixture was filtered through a mixed cellulose ester (MCE) membrane filter (0.2 µm). The determination and 8
quantification of monomeric sugars in the aqueous soluble fraction was performed on high performance liquid chromatography (HPLC, Shimadzu, LC-20A) by comparison with authentic samples. The concentration of acid soluble lignin (ASL) was determined by UV spectra by measuring the absorbance of the soluble fraction at 205 nm. The degrees of delignification were assessed based on the weight of the residual lignin in carbohydrate pulp and the original Klason lignin weight. Delignification (%) =
m(original Klason lignin) − m(residual lignin ) × 100% m(original Klason lignin )
2.5. Enzymatic hydrolysis of carbohydrate pulp In a typical experiment, 100 mg of dry material was suspended to a 25 mL flask with 5 mL of 0.05 M sodium acetate buffer (pH = 4.8), to which enzyme Cellic@CTec2 (Novozymes, 100 FPU/mL) was added in the form of 35 FPU/g substrate. The hydrolysis reaction was carried out at 50 oC on a rotary shaker at 200 rpm. In the enzymatic hydrolysis process, an aliquot (0.2 mL) was picked, deactivated enzymes, centrifuged and filtered, which was then analyzed on a HPLC system (Shimadzu, LC20A) with a Bio-Rad Aminex HPX-87H column by using 0.05 mM H2SO4 solution as mobile phase.
3. Results and discussion 3.1. Monomeric phenols from RCF of bamboo sawdust The lignin, cellulose and hemicellulose contents of bamboo used in this study was 9
determined as 24.5%, 47.7% and 21.9% by weight, respectively, being consistent with previous report (Huang et al., 2018). Pre-extracted bamboo sawdust was treated with Pd/C (15 wt% based on bamboo sawdust) at 260 oC and 4 MPa H2 for 4 h, which resulted in a soluble fraction containing phenolic compounds and insoluble carbohydrate pulp. The soluble fraction underwent extraction with CH2Cl2-water to afford the organic phase and aqueous phase. The lignin oil, which was obtained from organic phase, corresponds to the total lignin products being the sum of phenolic mono-, di- and oligomers (Sun et al., 2019). The aqueous phase contained some sugars and alcohols from degradation of (hemi)cellulose. The insoluble fraction, contained most (hemi)cellulose components, some lignin component and catalyst; the degrees of delignification and the retentions of (hemi)cellulose were determined by biomass compositional analysis (Fig. 1). The lignin monomers distribution determined on GC and GC/MS by comparison with authentic samples, and the average molecular weight of lignin oil measured on gel permeation chromatography, were summarized in Table 1. In the case of MeOH used as solvent, 82.9 wt% of original lignin has been solubilized into MeOH (Table 1, entry 1). The analysis of lignin oil by GPC exhibited an obvious decrease in average molecular weight (422 g/mol) compared to the isolated milled wood lignin (MWL) (13000 g/mol) from bamboo (Li et al., 2012). This RCF reaction of bamboo sawdust produced monomeric phenols in 32.2 wt% combined yield based on Klason lignin, in which compounds 1-4 were identified as major products by GC/MS. As a grass species, bamboo lignin features coumaric and ferulic acids, which are bonded with α-OH or γ-OH of β-O-4 10
moieties through ester or ether linkages (Helm & Ralph, 1992). Correspondingly, methyl 3-(4-hydroxyphenyl)propionate 1 (8.2 wt%) and methyl 3-(4-hydroxy-3methoxyphenyl)propanoate 2 (2.0 wt%) were produced through the cleavage of ether or ester bonds and subsequent esterification with MeOH and reduction of C=C bonds. 4-Ethylphenol (0.1 wt%) and 4-ethyl guaiacol (0.5 wt%), which are generated from decarboxylation reaction of coumaric and ferulic acids and subsequent hydrogenation, were both found in this lignin oil (Wang et al., 2018). The phenolic compounds derived from β-O-4 units, such as 4-propanol guaiacol 3 (5.5 wt%) and 4-propanol syringol 4 (11.1wt%), were also generated through C–O bonds cleavage and C=C hydrogenation. The observation of small amount of 4-propyl guaiacol/syringol is in line with the Pd/C-catalyzed RCF of woody biomass (Van den Bosch et al., 2015a). The cleavage of β-O-4 structure is a redox transformation, where MeOH may act as a hydrogen donor to release formaldehyde (HCHO), and Pd/C-catalyzed hydrogenation of HCHO can regenerate MeOH (Li & Song, 2019). Theoretically, the production of either compounds 1 and 2 from hydroxycinnamic species or compounds 3 and 4 from β-O-4 structures requires two equivalent of H2 for hydrogenolysis of C-O bond and hydrogenation of C=C bond. To analyze of the aqueous phase from extraction of soluble fraction, a derivatization process through trimethylsilylation with MSTFA was performed before GC/MS analysis. 1,2,5-Pentanetriol, xylofuranose and 2ketoglutaric acid derived from hemicellulose, as well as methylated glucose and hexane-1,2,5,6-tetraol derived from cellulose, were detected. The direct catalytic treatment of bamboo sawdust also afforded an insoluble fraction containing most 11
unreacted carbohydrate component, where the retentions of cellulose (C6) and hemicellulose (C5) were measured as 73.4% and 57.4% by NREL standard acid hydrolysis procedure, respectively, being lower than that from RCF treatment of woody sawdust (Schutyser et al., 2015; Van den Bosch et al., 2015a). A control experiment in the absence of Pd/C was performed under similar conditions; the drops in both delignification degree (55.3%) and phenolic monomers yield (13.2 wt%) was observed (Table 1, entry 2). Further analysis indicated that methyl coumarate (3.9 wt%) and methyl ferulate (1.3 wt%) were generated, with no observation of hydrogenated products. The monomers from cleavage β-O-4 units such as 4-propenyl substituted phenols were also generated in control reaction with a low yield (0.8 wt%). Thereby, the role of Pd/C in RCF bamboo was established as to accelerate the cleavage of C–O linkage bonds and to avoid recondensation through hydrogenation at end-chain. A biomass compositional analysis implied 64.8% and 80.6% retentions of hemicellulose and cellulose in insoluble fraction, being slight higher than those from catalytic experiment; this indicated that Pd/C has a less effect on depolymerization of carbohydrate components. 3.2. Solvent effect To combine efficient lignin hydrogenolysis together with high retention of carbohydrate, several solvents were screened in the reductive catalytic fractionation delignification of bamboo sawdust in presence of Pd/C under 260 oC and H2 atmosphere (Table 1, entries 3-6). Compared to methanol (where ET(30) = 55.4 kcal 12
S0960-8524(19)30557-7 https://doi.org/10.1016/j.biortech.2019.121335 121335 BITE 121335
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 February 2019 8 April 2019 10 April 2019
Please cite this article as: Zhang, K., Li, H., Xiao, L-P., Wang, B., Sun, R-C., Song, G., Sequential utilization of bamboo biomass through reductive catalytic fractionation of lignin, Bioresource Technology (2019), doi: https:// doi.org/10.1016/j.biortech.2019.121335
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.
Sequential utilization of bamboo biomass through reductive catalytic fractionation of lignin
Kaili Zhang,a Helong Li,a Ling-Ping Xiaob,*, Bo Wanga, Run-Cang Sunb, Guoyong Songa,*
a
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University,
Beijing 100083, China. b
Center for Lignocellulose Science and Engineering, Liaoning Key Laboratory of
Pulp and Papermaking Engineering, School of Light Industry and Chemical Engineering, Dalian Polytechnic University, Dalian 116034, China.
*
Corresponding authors:
E-mail addresses: [email protected] (L.-P. Xiao) [email protected] (G. Song)
1
ABSTRACT Reductive catalytic fractionation (RCF) has emerged as a new biorefinery paradigm for the fractionation and sequential utilization of entire components of biomass. Herein, we investigated the RCF of bamboo, a highly abundant herbaceous feedstock, in the presence of Pd/C catalyst. The lignin fraction in bamboo was preferentially depolymerized into well-defined low-molecular-weight phenols, with leaving carbohydrates pulp as a solid residue. In the soluble fraction, four major phenolic compounds, e.g., methyl coumarate/ferulate derived from hydroxycinnamic units and propanol guaiacol/syringol derived from β-O-4 units, were generated up to 41.7 wt% yield based on original lignin. In the insoluble fraction, the carbohydrates of bamboo were recovered with high retentions of cellulose (68%) and hemicellulose (49%), which upon treatment with enzyme gave glucose (90%) and xylose (85%). Overall, the three major components of bamboo could efficient to be fractionated and converted into useful platform chemicals on the basis of this study.
Keywords: Bamboo; Reductive catalytic fractionation; Lignin; Biorefinery; Hydrogenolysis
2
1. Introduction Lignocellulosic biomass, which is composed by cellulose, hemicellulose and lignin, represents the most abundant renewable carbon source with significant potential for the production of sustainable chemicals and fuels (Besson et al., 2014; Somerville et al., 2010; Tuck et al., 2012). Lignin is a complex aromatic biopolymer, accounting for 20-30% of lignocellulosic biomass by weight (Boerjan et al., 2003). Depolymerization of lignin into well-defined mono-aromatic chemicals amenable to downstream processes is regarded as an important starting point for lignin valorization (Cao et al., 2018; Galkin & Samec, 2016; Li et al., 2015; Rinaldi et al., 2016; Sun et al., 2018; Wang et al., 2019). However, current lignocellulosic biorefineries have focused on the utilization and valorization of carbohydrate fractions, wherein lignin was considered as an inferior component (Kamm, 2007; Wyman, 2013). As a consequent, the objective of current biomass pretreatment (e.g., soda and kraft pulping) is to get rid of lignin to facilitate carbohydrate valorization. Under such severe conditions, the reactive C–O bonds in native lignin undergo cleavage to generate some reactive intermediates, which may irreversibly form more refractory C–C bonds via recondensation reactions (Constant et al., 2016; Renders et al., 2017; Schutyser et al., 2018). The biorefinery lignins features lower reactivity than the native biopolymer, and they most often can be used as a cheap energy source for burning. Recent studies have demonstrated that direct reductive catalytic fractionation (RCF), that is lignin first strategy, can transfer the lignin in its native form in 3
lignocellulose into monomeric phenols in high yield, together with well-preservation of carbohydrate components (Renders et al., 2017). The heterogeneous transition metal catalysts were usually employed, which can accelerate the cleavage of β-O-4 units and avoid the recondensation/repolymerization processes through reductive pathways (Galkin & Samec, 2014; Huang et al., 2017; Klein et al., 2015; Kumaniaev et al., 2017; Parsell et al., 2015; Renders et al., 2018; Song et al., 2013; Sun et al., 2019; Van den Bosch et al., 2017; Van den Bosch et al., 2015a; Van den Bosch et al., 2015b; Yan et al., 2008; Zhai et al., 2017). Generally, hardwoods and genetically engineered plants with high syringyl-to-guaiacyl (S/G) monolignol ratios are the preferred feedstock for the RCF processes, from which monomeric phenols having an end-chain could be obtained in close-to-theoretical maximum yields (Van den Bosch et al., 2015b). Commercial transition metal catalysts (e.g. Rh/C (Yan et al., 2008) Ru/C (Van den Bosch et al., 2015b), Ni/C (Song et al., 2013)) or bimetallic catalysts (e.g. Zn/Pd/C (Klein et al., 2016; Parsell et al., 2015) gave 4-propyl-subtituted guaiacol and syringol as major products. In the case of Pd/C catalyst, the end-chain of phenols can be modulated by choice of hydrogen donor. For examples, saturated 4propanol-substituted phenols were generated under H2 (Van den Bosch et al., 2015a), while unsaturated 4-propenyl-substituted phenols were produced in EtOH/H2O (Galkin & Samec, 2014). Recent result has described that Ni@ZIF-8 can act as a chemodivergent catalyst for hydrogenolysis of eucalyptus biomass, selectively producing 4-propanol- or 4-propyl-substituted phenols under different reaction conditions (Liu et al., 2019). The heterogeneous Mo catalysts, which feature a lower 4
activity to hydrogenation, afforded allyl ether substituted phenols in a selective manner (Sun et al., 2019; Xiao et al., 2017b). In the case of softwoods with the high ratio of guaiacol units and low content of cleavable linkages, RCF became more challenging, which resulted in the drops in both monomers yield and delignification. Herbaceous plants are important fast-growing crops. The lignin in herbaceous plants features higher proportion of hydroxycinnamic acid, i.e., p-coumaric acid (pCA) and ferulic acid (FA) moieties, in addition to β-O-4 structures (Buranov & Mazza, 2008; Helm & Ralph, 1992; Kim et al., 2017; Peng et al., 2017). Sels (Van den Bosch et al., 2015b) and Abu-Omar (Luo et al., 2016) independent reported the RCF of miscanthus grass by using Ru/C and Ni/C catalysts, respectively, which afforded 4-propyl-, 4-propanol and 4-propionate-substituted phenols from lignin component. Beckham and Román-Leshkov described the RCF process of corn stover with Ni/C and H3PO4 (Anderson et al., 2016). We recently reported that ZnMoO4/MCM-41-catalyzed RCF of corncob, which gave p-hydroxycinnamic esters in a selective version (Wang et al., 2018). Bamboo is a perennial herbaceous plant, which features advantages of fast growth, rapid renovation and readily propagation, has been recognized as a promising lignocellulosic resource in the global bioeconomy (Hong et al., 2011; Li et al., 2012; Zhang et al., 2017). To the best of our knowledge, the reductive catalytic fractionation of bamboo biomass has not been realized. Herein, we demonstrate the first example of direct RCF of bamboo by using Pd/C as a catalyst, which resulted in monomeric phenols in high yield from lignin depolymerization, together with the retention of carbohydrate pulp as a solid residue 5
amenable to valorization. A systematic study on the effects of solvent, temperature and reaction time, are also presented in view of lignin-derived monomers yields and the carbohydrate retentions. This study provides an economically pathway for fractionation of bamboo biomass, and in in extension, sequential utilization of all three components of bamboo biomass.
2. Material and methods 2.1. Material Bamboo (Dendrocalamus brandisii, three year old) was harvested from Yunnan Province, China (Bai et al., 2013). It was crushed, screened into powders in size of 40-60 meshes, extracted with toluene/ethanol and dried under vacuum at 50 oC. Pd/C catalyst (where Pd content is 5 wt%) was purchased from Energy Chemical. Methanol (MeOH), ethanol (EtOH), isopropanol (iPrOH) and tetrahydrofuran (THF) were purchased from Fisher Chemical and used as received without further purification. The commercial enzyme (Cellic@CTec2, 100 FPU/mL) was kindly provided from Novozymes (Beijing, China). Authentic samples for identification of lignin- and carbohydrate- derived monomers were acquired from commercially or synthesized independently.
2.2. General catalytic reaction Extracted bamboo sawdust (1.0 g), Pd/C (150 mg, 15 wt% based on bamboo sawdust) and solvent (40 mL) were charged into a stainless steel batch reactor (100 6
mL, Parr instruments Co.), which was flushed with N2 and pressurized with H2 at room temperature. The reactor was heated at 220 oC, 240 oC, 260 oC, 280 oC and 320 o
C for 4 h or at 260 oC for 2, 4, 8, 12, 16 h. After reaction, the reactor was cooled to
room temperature with water-flow. The reaction mixture was filtered through a nylon 66 membrane filter (0.2 μm), and the insoluble fraction was washed with methanol, thus forming soluble fraction containing phenolic compounds and insoluble fractions containing carbohydrate pulp and catalyst.
2.3. Lignin characterization For the soluble fraction, the methanol was removed under vacuum and then extracted with dichloromethane and water. Subsequent removal of dichloromethane of the organic phase gave a brown “lignin oil”. The lignin oil and a standard (tetradecane) were dissolved in dichloromethane in a 10 mL volumetric flask, which was then analyzed by gas chromatography coupled with mass spectrometer (GC/MS) and gas chromatography (GC) to identify and quantify the monomeric phenols. Gel permeation chromatography (GPC) analysis was performed on Shimadzu LC-20A equipped with a PL-gel 10 μm Mixed-B 7.5 mm I.D. column (mixed) and UV detection detector (254 nm) at 50 °C, using THF as the solvent (1 mL/min). Twodimensional heteronuclear single quantum coherence (2D HSQC) NMR analyses were acquired on a Bruker Avance 400 MHz spectrometer. Generally, DMSO-d6 was used as a NMR solvent and the central solvent peak at δC/δH = 39.5/2.49 ppm was used as an internal reference. 7
Lignin oil (%) =
m(extracted soluble fraction ) × 100% m(bamboo saudust)
Phenolic monomers (%) =
Solid residue (%) =
m(total monomers) × 100% m(total lignin)
m(insoluble fraction) − m(catalyst) × 100% m(bamboo saudust)
The water of aqueous phase from the extraction was removed under vacuum, which was then mixed with anhydrous pyridine (0.5 mL) and N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA) (0.5 mL). After heated at 80 oC for 30 min, the reaction mixture was submitted to GC/MS analysis.
2.4. Compositional analysis of carbohydrate pulp For the insoluble fraction, the Pd/C catalyst could be separated and recycled in 90% yield from carbohydrate pulp through a mesh screening (300 mesh). The spent catalyst was subjected to simple washing and was used directly in the following cycle at 260 oC, H2 (4 MPa) for 4 h. After first run, the ICP-MS (inductively coupled plasma mass spectrometry) of soluble fraction was performed on Agilent 7800 ICP-MS. The carbohydrate pulp was then subjected to composition analysis using National Renewable Energy Laboratory (NREL) standard acid hydrolysis procedure (Sluiter et al., 2008). The carbohydrate pulp (300 mg) was treated in 72 wt % sulfuric acid (3 mL) for 1 h at 30 °C. Deionized water (84.0 mL) was added to dilute sulfuric acid (ca. 3%), which was then heated at 120 °C for 1 h. The mixture was filtered through a mixed cellulose ester (MCE) membrane filter (0.2 µm). The determination and 8
quantification of monomeric sugars in the aqueous soluble fraction was performed on high performance liquid chromatography (HPLC, Shimadzu, LC-20A) by comparison with authentic samples. The concentration of acid soluble lignin (ASL) was determined by UV spectra by measuring the absorbance of the soluble fraction at 205 nm. The degrees of delignification were assessed based on the weight of the residual lignin in carbohydrate pulp and the original Klason lignin weight. Delignification (%) =
m(original Klason lignin) − m(residual lignin ) × 100% m(original Klason lignin )
2.5. Enzymatic hydrolysis of carbohydrate pulp In a typical experiment, 100 mg of dry material was suspended to a 25 mL flask with 5 mL of 0.05 M sodium acetate buffer (pH = 4.8), to which enzyme Cellic@CTec2 (Novozymes, 100 FPU/mL) was added in the form of 35 FPU/g substrate. The hydrolysis reaction was carried out at 50 oC on a rotary shaker at 200 rpm. In the enzymatic hydrolysis process, an aliquot (0.2 mL) was picked, deactivated enzymes, centrifuged and filtered, which was then analyzed on a HPLC system (Shimadzu, LC20A) with a Bio-Rad Aminex HPX-87H column by using 0.05 mM H2SO4 solution as mobile phase.
3. Results and discussion 3.1. Monomeric phenols from RCF of bamboo sawdust The lignin, cellulose and hemicellulose contents of bamboo used in this study was 9
determined as 24.5%, 47.7% and 21.9% by weight, respectively, being consistent with previous report (Huang et al., 2018). Pre-extracted bamboo sawdust was treated with Pd/C (15 wt% based on bamboo sawdust) at 260 oC and 4 MPa H2 for 4 h, which resulted in a soluble fraction containing phenolic compounds and insoluble carbohydrate pulp. The soluble fraction underwent extraction with CH2Cl2-water to afford the organic phase and aqueous phase. The lignin oil, which was obtained from organic phase, corresponds to the total lignin products being the sum of phenolic mono-, di- and oligomers (Sun et al., 2019). The aqueous phase contained some sugars and alcohols from degradation of (hemi)cellulose. The insoluble fraction, contained most (hemi)cellulose components, some lignin component and catalyst; the degrees of delignification and the retentions of (hemi)cellulose were determined by biomass compositional analysis (Fig. 1). The lignin monomers distribution determined on GC and GC/MS by comparison with authentic samples, and the average molecular weight of lignin oil measured on gel permeation chromatography, were summarized in Table 1. In the case of MeOH used as solvent, 82.9 wt% of original lignin has been solubilized into MeOH (Table 1, entry 1). The analysis of lignin oil by GPC exhibited an obvious decrease in average molecular weight (422 g/mol) compared to the isolated milled wood lignin (MWL) (13000 g/mol) from bamboo (Li et al., 2012). This RCF reaction of bamboo sawdust produced monomeric phenols in 32.2 wt% combined yield based on Klason lignin, in which compounds 1-4 were identified as major products by GC/MS. As a grass species, bamboo lignin features coumaric and ferulic acids, which are bonded with α-OH or γ-OH of β-O-4 10
moieties through ester or ether linkages (Helm & Ralph, 1992). Correspondingly, methyl 3-(4-hydroxyphenyl)propionate 1 (8.2 wt%) and methyl 3-(4-hydroxy-3methoxyphenyl)propanoate 2 (2.0 wt%) were produced through the cleavage of ether or ester bonds and subsequent esterification with MeOH and reduction of C=C bonds. 4-Ethylphenol (0.1 wt%) and 4-ethyl guaiacol (0.5 wt%), which are generated from decarboxylation reaction of coumaric and ferulic acids and subsequent hydrogenation, were both found in this lignin oil (Wang et al., 2018). The phenolic compounds derived from β-O-4 units, such as 4-propanol guaiacol 3 (5.5 wt%) and 4-propanol syringol 4 (11.1wt%), were also generated through C–O bonds cleavage and C=C hydrogenation. The observation of small amount of 4-propyl guaiacol/syringol is in line with the Pd/C-catalyzed RCF of woody biomass (Van den Bosch et al., 2015a). The cleavage of β-O-4 structure is a redox transformation, where MeOH may act as a hydrogen donor to release formaldehyde (HCHO), and Pd/C-catalyzed hydrogenation of HCHO can regenerate MeOH (Li & Song, 2019). Theoretically, the production of either compounds 1 and 2 from hydroxycinnamic species or compounds 3 and 4 from β-O-4 structures requires two equivalent of H2 for hydrogenolysis of C-O bond and hydrogenation of C=C bond. To analyze of the aqueous phase from extraction of soluble fraction, a derivatization process through trimethylsilylation with MSTFA was performed before GC/MS analysis. 1,2,5-Pentanetriol, xylofuranose and 2ketoglutaric acid derived from hemicellulose, as well as methylated glucose and hexane-1,2,5,6-tetraol derived from cellulose, were detected. The direct catalytic treatment of bamboo sawdust also afforded an insoluble fraction containing most 11
unreacted carbohydrate component, where the retentions of cellulose (C6) and hemicellulose (C5) were measured as 73.4% and 57.4% by NREL standard acid hydrolysis procedure, respectively, being lower than that from RCF treatment of woody sawdust (Schutyser et al., 2015; Van den Bosch et al., 2015a). A control experiment in the absence of Pd/C was performed under similar conditions; the drops in both delignification degree (55.3%) and phenolic monomers yield (13.2 wt%) was observed (Table 1, entry 2). Further analysis indicated that methyl coumarate (3.9 wt%) and methyl ferulate (1.3 wt%) were generated, with no observation of hydrogenated products. The monomers from cleavage β-O-4 units such as 4-propenyl substituted phenols were also generated in control reaction with a low yield (0.8 wt%). Thereby, the role of Pd/C in RCF bamboo was established as to accelerate the cleavage of C–O linkage bonds and to avoid recondensation through hydrogenation at end-chain. A biomass compositional analysis implied 64.8% and 80.6% retentions of hemicellulose and cellulose in insoluble fraction, being slight higher than those from catalytic experiment; this indicated that Pd/C has a less effect on depolymerization of carbohydrate components.