The effect of lignin degradation products on the generation of pseudo-lignin during dilute acid pretreatment

Industrial Crops & Products 146 (2020) 112205

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The effect of lignin degradation products on the generation of pseudo-lignin during dilute acid pretreatment

T

Juan Hea,1, Caoxing Huanga,1, Chenhuan Laia, Chen Huanga, Mi Lib,c, Yunqiao Puc, Arthur J. Ragauskasb,c,d,**, Qiang Yonga,* a

Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing, 210037, China Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, TN, 37996, USA c Joint Institute for Biological Sciences, Biosciences Division Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA d Department of Forestry, Wildlife and Fisheries, Center for Renewable Carbon, The University of Tennessee Institution of Agriculture, Knoxville, TN 37996, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Acid-pretreatment Lignin Phenolic compound Pseudo-lignin

Pseudo-lignin is an insoluble material that tends to deposit on acidic pretreated fiber surface and negatively impacts the biological conversion of biomass to valued added products. Employing a laboratory generated acidic hydrolysate solution from dilute acid pretreatment of bamboo residues, we sought to seek an improved understanding of pseudo-lignin formation from fragmented carbohydrates. In addition, we also performed experiments to investigate how lignin model compounds interact with carbohydrates en route to pseudo-lignin’s formation. The yields of pseudo-lignin generated from xylose (CXPL) were higher than those generated from glucose (CGPL). Gas chromatography-Mass spectrometry and Foloin-Ciocalteau analysis suggested that there were more aromatic compounds in hydrolyzate during CGPL’s formation. The presence of lignin phenolic model compounds had both a positive and negative effect upon quantitative yields of pseudo-lignin. Gel permeation chromatography and NMR analysis (31P and 2D-HSQC NMR) of the pseudo-lignin samples generated in the presence and absence of 4-hydroxybenzoic acid (4-HA), employed as a lignin model compound, revealed drastic differences in molecular weight, changes in hydroxyl group content, and hydrocarbon bonds, suggesting significant differences in the pseudo-lignin reaction pathways when certain lignin model compounds are present. Our findings demonstrate the effects of both hemicellulose carbohydrate makeup and lignin model compound impact on the formation of pseudo-lignin and its chemical properties, suggesting a potential way to minimize pseudo-lignin formation.

1. Introduction Lignocellulosic materials containing large amounts of polysaccharides (∼70 wt%), are a sustainable and abundant resource for the production of carbohydrate-derived bio-products like ethanol, butanol, succinic acid and many other bio-derived products (Xu et al., 2018; Ragauskas et al., 2014; Kumar et al., 2016; Lou et al., 2019). The process by which these products are produced is commonly referred to as biorefining, a sustainable analog to the petroleum refinery. Biological conversion, involving enzymatic depolymerization and microbial fermentation, is now well known to be an energetically favorable biorefinery process for converting plant polysaccharides into valuable end-products (Ragauskas et al., 2014).

The efficient liberation of monosaccharides from plant polysaccharides is often the most challenging step rather than the subsequent biological conversion process. The complex characteristic of lignocellulosic form the basis for biomass recalcitrance toward cellulases and this has been a subject of many studies (Meng et al., 2016; Qi et al., 2019). An efficient pretreatment process is typically required to render lignocellulosic materials into processable polysaccharides. It is generally agreed upon that a key goal of pretreatment for generating useful polysaccharides is to minimize the recalcitrant nature of biomass and this can be accomplished owing to various factors including: (1) increasing enzyme accessibility to cellulose and hemicellulose in the plant cell wall that is typically covered by lignin, and (2) reducing the nonspecifically binding between lignin associated compounds and

⁎

Corresponding author at: Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China. Corresponding author at: Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, TN 37996, USA. E-mail addresses: [email protected] (A.J. Ragauskas), [email protected] (Q. Yong). 1 Contributed equally to this work, regarding as the first author. ⁎⁎

https://doi.org/10.1016/j.indcrop.2020.112205 Received 17 October 2019; Received in revised form 31 January 2020; Accepted 3 February 2020 0926-6690/ © 2020 Published by Elsevier B.V.

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what components of biomass lead to pseudo-lignin formation and additional insights into the structural information of resultant pseudolignin from acid pretreatment, thus aiding in the development of strategies to overcome its barriers to the biorefinery process.

cellulase (Shinde et al., 2018; Yao et al., 2018). Of the many pretreatment technologies, two of the most thoroughly investigated processes are dilute acid and hydrothermal pretreatment because these two processes have been deemed economically feasible at larger scale when compared with other pretreatments, primarily based on reagent and energy costs (Chen et al., 2018; dos Santos Rocha et al., 2017; Redding et al., 2011; Huang et al., 2019a). During both of these pretreatments, the hydronium ion plays a role in hydrolyzing both polysaccharides and lignin, resulting in a net disruption of the tightly woven lignocellulosic matrix. Physicochemical changes of biomass, such as depolymerization, morphological changes, as well as many additional structural changes take place to varying degrees depending on pretreatment severity (Donohoe et al., 2011; Kumar et al., 2009). As a result, the pretreatment of biomass generates a liquid and solid phase that contains lignin fragments and processable saccharides (i.e., poly-, oligo-, and mono-) amongst other decomposition products (Huang et al., 2019b). Along with the cellulose-enriched solid phase generated from pretreatment, several fermentable monosaccharides and their degradation compounds, including furfural (FF), 5-hydroxymethylfurfural (5-HMF), levulinic acid and formic acid are found in the liquid phase. Some of these degradation products can undergo condensation reactions and aromatization reactions that lead to the generation of pseudo-lignin that is problematic for the enzymatic depolymerization of cellulose (Shinde et al., 2018; He et al., 2018). Pseudo-lignin, like lignin, is commonly observed as insoluble droplets deposited on cellular surface of biomass that has been subjected to acidic biorefinery pretreatments (Shinde et al., 2018). Despite originating from carbohydrates, pseudolignin is a polyaromatic phenolic material and contributes to the Klason lignin content of pretreated biomass. Considering the complex conditions of biomass pretreatment, researchers have begun to examine the use of model systems to investigate the formation of pseudo-lignin from polysaccharides and characterize its structure. Sannigrahi et al. pretreated poplar holocellulose with 0.10 or 0.20 M sulfuric acid solution at 160−180 °C to prepare pseudo-lignin. They confirmed that pseudo-lignin is a polyphenolic material with carbonyl, carboxylic, methoxyl, aromatic, and aliphatic structures (Sannigrahi et al., 2011). Ma et al. pretreated holocellulose bamboo chips with deionized water at 170 °C at four time intervals from 30 to 240 min. X-ray photoelectron spectroscopy (XPS) of the pretreated material revealed that the generation of pseudo-lignin could cause a drastic increase in the C1 category (CeC/CeH), and a decrease in C2 (CeO), C3(C]O/OeCeC), which suggests that there were cyclic structures and hydroxyl groups in pseudo-lignin (Ma et al., 2015). Our previous work focusing on glucose and xylose as model compounds to investigate the reaction pathways involved in pseudolignin formation which provided pseudo-lignin samples with differences in the quantities of aliphatic hydroxyls groups and carboxylic acids (He et al., 2018). Lam et al. suggested a kinetic model of pseudolignin’s formation and indicated that the proposed pseudo-lignin formation pathway is a combination of reaction kinetics models of xylan depolymerization, lignin solubilization and lignin condensation in acid pretreatment, which suggest that lignin degradation products are probably participating in the formation of pseudo-lignin (Lam et al., 2009). To better understand the influence of lignin upon pseudo-lignin formation, we performed dilute acid pretreatments with model solutions including mixtures of monosaccharides and lignin model compounds (vanillin, vanillic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, syringaldehyde, syringic acid, ferulic acid and paracomaric acid). Various forms of chromatography were employed to detect the variety of reaction products that remain soluble prior to pseudo-lignin formation. The surface morphology of the pseudo-lignin formed from model compounds was characterized by SEM. In addition, GPC, FTIR, 31P NMR and HSQC NMR were used to characterize molecular mass and chemical structures of the formed pseudo-lignin. The results of this study provided a more fundamental understanding of

2. Materials and methods 2.1. Materials The biomass used in this study, bamboo residues (Phyllostachys heterocycle), were obtained from the He Qi Chang Bamboo Processing Factory (Fujian, China). The air-dried samples were first milled to 20–80 mesh particle size. Chemical characterization of the bamboo residues was performed using the NREL method (Sluiter et al., 2008), and results were as follows (wt% on a dry basis): 39.5 % glucan, 20.8 % xylan, 30.7 % total lignin. p-Coumaric acid, syringaldehyde, syringic acid, vanillin, vanillic acid, ferulic acid, 4-hydroxybenzoic acid and 4hydroxybenzaldehyde were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents used were purchased from Ronghua Chemicals in Nanjing, China and used as received. 2.2. Dilute sulfuric acid pretreatment Bamboo residues were pretreated with dilute sulfuric acid pretreatment, which was carried out in 30 ml autoclave reaction vessels, and heated in an oil bath at 180 °C for 1 h. The sulfuric acid charge was 2 % (w/v) with a solid to liquid ratio of 1:10 (w/w). When the pretreatment was finished, the mixture was centrifuged at 10,000 rpm for 5 min, the supernatant liquid was separated for lignin degradation products analysis. D-glucose and D-xylose were utilized as carbohydrate sources to generate blank-pseudo-lignin under the same reaction conditions as bamboo residues. The reaction mixture was then centrifuged at 10,000 rpm for 5 min, the supernatant liquid separated for further analysis and solid pseudo-lignin was washed with DI water until the effluent was pH neutral and the solid residues were defined as Blank-CGPL and BlankCXPL, respectively. Additional experiments were performed containing a model system of 10.00 g/L monosaccharides (D-glucose or D-xylose) and 50 mg/L of lignin model compound, including either p-coumaric acid, syringaldehyde, syringic acid, vanillin, vanillic acid, ferulic acid, 4-hydroxybenzoic acid or 4-hydroxybenzaldehyde. All lignin model compounds were added independently and reaction conditions remained constant as described above. After pretreatment, all mixtures were centrifuged at 10,000 rpm for 5 min, the solid pseudo-lignin and the supernatant liquid were separated for further analysis. 2.3. Analysis of hydrolyzate 2.3.1. Total phenolics The total amount of polyphenols in the hydrolyzate was determined according to the Folin-Ciocalteau method using gallic acid as a calibration standard (Singleton and Rossi, 1965). Specifically, a 1.00 mL liquid sample or gallic acid standard solution was mixed with 1.00 mL Folin-Ciocalteau reagent and 5.00 mL deionized water. Next, 3.00 ml Na2CO3 solution (6 wt% concentration) was added to terminate the reaction. The terminated mixture was finally incubated at 30 °C for 2 h, and then the concentration of phenolics was determined with a UV–vis spectrophotometer at 745 nm. 2.3.2. High-pressure liquid chromatography analysis The dehydration compounds in the hydrolyzates from the pretreatments, specifically formic acid, acetic acid, levulinic acid, hydroxymethylfurfural (HMF) and furfural were quantified using a highperformance liquid chromatography system equipped with an Aminex HPX-87H column (300 × 7.8 mm) and a refractive index (RI) detector. 2

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A 5 mM H2SO4 solution was used as the mobile phase, flowing at a rate of 0.6 mL/min. This analysis was conducted in duplicate. Phenolic compounds (lignin degradation products from DAP pretreated bamboo residues) were measured using a RP-HPLC system. The system included a Zorbax Eclipse XDB-C18 separation column (4.6 × 250 mm, 5 μm) and a UV detector with an Agilent 1100 system. Elution conditions for the detection of phenolic compounds in hydrolyzate contained 1.5 % acetic acid as mobile phase at 30 °C with a flow rate of 0.8 mL/min. The detection wavelengths were set to 254 and 280 nm. Quantification was accomplished using external calibration with pure standards. The linear range of detection was from 9.6 mg/L to 960 mg/ L (correlation coefficient, R2 = 0.999), and the detection limit was at least 0.10 mg/L.

2.4.3. Gel permeation chromatography (GPC) for molecular weight determination Weight-average (Mw) and number-average (Mn) of pseudo-lignin samples were measured using a GPC following a procedure from literature (Hao et al., 2018). Prior to analysis, all pseudo-lignin samples (CGPL, CXPL, HA-CGPL and HA-CXPL) were extracted with 1, 4-dioxane then concentrated, freeze dried, and defined as GPL, XPL, HAGPL and HA-XPL, respectively. GPC analyses were performed on an Agilent 1200 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, US) equipped with a UV detector (270 nm) and Waters Styragel columns (HR1, HR2, and HR6; Waters corporation, Milford, MA, US). THF was used as the mobile phases at a flow rate of 1.0 mL/min. Polystyrene narrow standards were used as calibration standards. 2.4.4. Solid-state NMR analysis The original and extracted pseudo-lignin samples were packed into 4 mm ZrO rotors and analyzed with a Brucker Avance-400 MHz spectrometer operating at 75.48 MHz for 13C. CP/MAS experiments with a spinning speed of 8 kHz. The parameter are as followings: 90° proton pulse, 1.5 ms contact pulse, 3072 scans, and 4 s recycle delay (Hao et al., 2018).

2.3.3. Gas chromatography-mass spectrometry (GC–MS) analysis Certain pretreatment separated liquids from Blank-CGPL, BlankCXPL, HA-CGPL (pseudo-lignin generated from glucose with 4-hydrobenzoic acid) and HA-CXPL (pseudo-lignin generated from xylose with 4-hydrobenzoic acid) were further characterized by GC–MS to characterize the low molecular weight compounds formed. An aliquot of the select hydrolyzates were subjected to liquid-liquid extraction using ethyl acetate (3/1, v/v) to isolate the pseudo-lignin precursors. The ethyl acetate extracts were combined and dried with MgSO4. The supernatant (500 μL) was silylated in a mixture of N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA, 400 μL) and pyridine (100 μL) at 70 °C for 30 min (Cao et al., 2020). The prepared liquid samples were analyzed using a GC–MS with splitless injection on a Thermo Finnigan Trace DSQ instrument. An Agilent 0.25 mm × 30 m DB-5 fused silica capillary column with a 0.25 μm film thickness coated stationary phase was used for the chromatographic separation. The temperature of the injector was set at 280 °C. The column temperature program was first 150 °C and held at this value for 15 min, after which it was heated at the rate of 3 °C min−1 until reaching 210 °C. Once reaching this temperature, further heating at a rate of 10 °C min−1 took place until reaching 280 °C (which was then held 10 min) (Kang et al., 2012). Identification of the eluted components was achieved by comparison of their mass fragments with the National Institute of Standards and Technology (NIST) mass spectral library.

2.4.5. 31P and 2D NMR spectroscopic analysis 31 P nuclear magnetic resonance (NMR) analysis of 1, 4-dioxaneextracted pseudo-lignin samples were acquired using a Bruker Avance/ DMX 400-MHz spectrometer. Spectral processing was then performed using Bruker Topspin 3.5 (Mac) software. Chromium acetylacetonate (relaxation regent) and endo-N-hydroxy-5-norbornene-2, 3-dicarboximide (internal standard) were added in the stock solution of pyridine/ CDCl3 (v/v = 1.6/1). All samples (40 mg) were dissolved in the solution mixture, and then derivatized with 2-choloro-4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaphospholane (TMDP). The 31P NMR spectra experiments were carried out with a BBO probe using an inverse-gated decoupling pulse sequence (Waltz-16), 90° pulse, 25-spulse delay with 64 scans (Meng et al., 2019; Li et al., 2017). For HSQC NMR analysis, 50 mg of extracted pseudo-lignin samples (GPL, HA-GPL, XPL, HA-XPL) were dissolved in 0.5 mL dimethyl sulfoxide (DMSO)-d6. The acquisition parameters are as followings: 2048 data points in F2 (1H) dimension with 256.1 ms acquisition time, and 256 increments (acquisition time 6.1 ms) with a 1.5-s delay, a 1JC–H of 145 HZ, and 32 scans. 3. Results and discussion

2.4. Analysis of generated pseudo-lignin

3.1. Analysis of hydrolyzate from acid pretreatment of bamboo residues

2.4.1. Scanning electron microscopy (SEM) analysis Images of pseudo-lignins were acquired using an FEI Quanta 400 (HITACHI, Japan), operated at 15 kV. All samples subjected to SEM were first freeze-dried and coated with gold/palladium (Au/Pd) using a SC7640 automatic/manual high-resolution sputter coater (Quorum Technologies, Newhaven, U.K.).

During acid catalyzed pretreatment of biomass, one well documented reaction that takes place is the depolymerization of lignin by acid catalyzed fragmentation of the β-O-4 ether linkages, releasing lignin fragments with free phenolic functional groups (Narron et al., 2016; Pielhop et al., 2017; Pu et al., 2013). We employed a highpressure liquid chromatography (RP-HPLC system), a rapid qualitative and quantitative analysis for lignin degradation products, to detect the low molecular phenolic compounds (lignin fragments) degraded from lignin (Pecina et al., 1986). With an internal standard, the hydrolyzate from acid pretreatment of bamboo residues was analyzed (Table 1) for vanillin, syringaldehyde, 4-hydroxybenzoic acid, ferulic acid, syringic acid, and 4-hydroxybenzaldehyde. The total amount of aromatics generated from lignin was 82.9 mg/L as can be seen in Table 1. Cao et al.

2.4.2. FTIR-ATR spectroscopic analysis The functional groups of pseudo-lignins were characterized using a Spectrum One FT-IR system (Themor, USA, Thermo Nicolet 360) with a universal attenuated total reflection (ATR) accessory. All samples were first dried at 60 °C and flaked with KBr. The wavelength region analyzed was 4000 to 400 cm−1 at 2 cm−1 resolution.

Table 1 Concentration of lignin degradation products in the hydrolyzates of bamboo residue pretreatmenta (mg/L).

Bamboo residues a

Syringaldehyde

Syringic acid

Vanillin

Vanillic acid

4-hydroxybenzaldehyde

4-hydroxybenzoic acid

Ferulic acid

p-coumaric acid

285.4

2.0

31.5

0.0

2.0

11.5

7.4

0.0

Bamboo residues was pretreated under acid condition, and the hydrolyzate were analyzed by HPLC. 3

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Table 2 Yield of pseudo-lignin with different additives. Additivea

Yield of CGPb (%)

Percentage of enhancementc (%)

Yield of CXPLb (%)

Percentage of enhancementc (%)

Blank Syringaldehyde Syringic acid Vanillin Vanillic acid 4-Hydroxybenzaldehyde 4-Hydroxybenzoic acid Ferulic acid para-coumaric acid Alld

21.6 ± 0.8 20.1 ± 0.4 20.0 ± 1.1 21.1 ± 0.4 20.0 ± 0.2 21.6 ± 0.6 22.3 ± 0.1 21.0 ± 0.4 22.4 ± 1.0 23.1 ± 0.9

– −6.9 −7.4 −2.3 −7.4 0.0 3.2 −2.8 3.7 6.5

32.2 ± 0.3 34.8 ± 0.2 33.7 ± 0.9 32.6 ± 0.7 34.0 ± 0.5 31.9 ± 0.3 36.0 ± 0.9 33.2 ± 0.9 32.2 ± 0.5 34.1 ± 0.2

– 8.1 4.7 1.2 5.6 −0.9 11.8 3.1 0.0 5.9

a

The concentration of phenolic model compound is 50 mg/L. Pseudo-lignin, which was gained from sulfuric acid pretreated glucose and xylose, defined as CGPL and CXPL.Calculated as following: recovered dried solids / initial dried solids*100%. c Calculated as following: (yield of additive pseudo lignin－yield of blank pseudo lignin)/yield of blank pseudo-lignin*100%. d Including syringaldehyde, syringic acid, vanillin, vanillic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, ferulic acid, and para-coumaric acid which were added into the solution. b

3.3. Hydrolyzates from acid pretreatment of monosaccharides

suggested that during dilute acid pretreatment, acid catalyzed condensation would occur between aromatic units which gave rise to new CeC linkage between lignin units and generated a more heterogeneous structure (Cao et al., 2020). Since pseudo-lignin contains lignin-like aromatic structures, it was hypothesized that acid-catalyzed condensation reactions are probably occurring with low molecular weight lignin compounds and pseudo-lignin, which are described below (He et al., 2018; Pielhop et al., 2017; Jiang et al., 2016).

Exploring the mechanism of pseudo-lignin formation during acid pretreatment is a pressing to facilitate novel methodologies to hinder its formation. Fig. S1 shows the concentration of intermediate products in the hydrolyzates from acid pretreatment of monosaccharides with additives of lignin model compounds. Compared with xylose, glucose tends to lead to greater amounts of levulinic acid and formic acid, but less amount of furfural and acetic acid. The results are in good agreement with the study by Rasmussen et al. that depending on pretreatment conditions, glucose can be converted into 5-(hydroxymethyl)-2furaldehyde (HMF) or levulinic acid and formic acid while xylose resulting in the formation of furan-2-carbaldehyde (furfural) or various C1 and C-4 compounds (Rasmussen et al., 2014). Fig. S1A showed that there was no HMF detected in the hydrolyzate from acid pretreatment of glucose in this study. HMF is an intermediate in the generation of levulinic acid and 2, 5-dioxo-6-hydroxy-hexanal, which could undergo aldol condensation or addition with HMF to form pseudo-lignin (Patil and Lund, 2011; Wan et al;, 2019). It appeared that the phenolic compounds additives play a promoting role in generating levulinic acid and formic acid from acid pretreatment of glucose during the formation of CGPL. However, for xylose, only 4-hydrobenzaldehyde and 4-hydrobenzoic acid could increase the amount of furfural and formic acid in the hydrolysate during the formation of CXPL (Fig. S1B). Moreover, Fig. S1A demonstrated that during dilute acid pretreatment of glucose, the lignin model compound had little effect on the residual glucose amount. But for xylose, as showed in Figure S1B, most of lignin model compounds (i.e., syringaldehyde, syringic acid, vanillic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and ferulic acid) played a negative role in the consuming of xylose. Based on these results, it could be suggested that during dilute sulfuric acid pretreatment, glucose and xylose would generate different intermediate products and lignin phenolic model compounds have different effects on the formation of pseudo-lignin based on different reaction pathways (Rasmussen et al., 2014; Patil and Lund, 2011; Forssk et al., 1976; Popoff and Theander, 1970; Bhat and Vaidyanathan, 1976). The soluble phenolic compounds present in the hydrolyzates from acid pretreatment of monosaccharides (i.e., glucose and xylose) were measured using the Folin-Ciocalteau assay (Iacopini et al., 2008). Fig. 1 summarizes the total phenolic content in the hydrolyzates with or without lignin phenolic compounds additive. The results showed that the total phenolic content of Blank-CGPL’ hydrolyzate (4.51 g/L) was found to be higher than that of Blank-CXPL (3.79 g/L). In addition, various phenolic compounds were pretreated separately with dilute sulfuric acid under the same condition as a control set in comparison with phenolic compounds pretreated with mono-sugars. For all of the

3.2. Yield of pseudo-lignin from acid pretreatment of monosaccharides We used a model system consisting of monosaccharides and lignin model compounds to investigate the effects of lignin compounds on the formation of pseudo-lignin. Acid pretreatments of D-glucose and D-xylose were carried out in the presence and absence of phenolic lignin model compounds. The phenolic model compounds used in this study are summarized in Table 2. These compounds were selected for three reasons: (1) most of them were found in substantial amounts in the hydrolyzate from the bamboo pretreatment, even some cannot be detected here, it was reported to be lignin degradation products; (2) it was reported that methoxyl groups would increase the steric hindrance effect of aromatic ring, these model compounds all contain a phenyl group and differed in the side chains or the number of methoxyl groups (3) the presence of acrylic acid structure in the side chains of p-coumaric acid and ferulic acid may affect the reaction between aromatic rings. (Li et al., 2015; Jiang et al., 2016; Schorr et al., 2014; Okamoto et al., 1996). The yields of generated pseudo-lignin in the present study were performed in triplicate and the results are shown in Table 2. For the control glucose sample, the pseudo-lignin yield with 21.6 % ± 0.8 was achieved under the used reaction condition. The addition of each lignin model compound had little pronounced effect on the pseudolignin yield. In fact, pseudo-lignin yield generated in the presence of vanillic acid decreased by ∼7.4 % in one case and increased by 3.7 % when para-coumaric acid was added. These results suggest that these lignin model compounds, may not play a significant positive role in the formation of pseudo-lignin derived from glucose (CGPL) when added separately. Moreover, Table 2 also showed that the existence of methoxyl groups in lignin model compounds played a negative role on the generation of CGPL. For xylose, however, it was found that the additives exhibited a significant positive effect on the generation of CXPL (pseudo-lignin generated from xylose). As shown in Table 2, the additives impacted the yields of CXPL ranging from -0.9%–11.8% relative to the xylose control. The different phenomena between CGPL and CXPL indicate that lignin model compounds may have different impacts on the generation of pseudo-lignin from various sources. 4

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Table 4 Molecular weights of pseudo-lignin extracted from pretreated monosaccharides with or without additives. Sample

Mw (g/mol)

Mn (g/mol)

GPL XPL HA-GPL HA-XPL

2680 2710 2950 7070

480 390 490 490

which would go through the condensation reaction with 4-HA (Patil and Lund, 2011). It has been reported that D-xylose could yield aromatic compounds such as 3, 8-dihydroxy-2-methylchromone, 6, 7, 8, 8a-tetrahydro-3, 8adihydroxy-2-methyl-8-oxobenzopyrone and 2, 3-dihydroxyacetophenone under acidic condition (pH 3.5, 96 °C, 48 h) (Popoff and Theander, 1970). As shown in Table 3, there were no monomer or low molecular weight aromatic compounds detected in the hydrolyzate of Blank-CXPL through GC–MS analysis. However, there were aromatics detected in hydrolyzate of HA-CXPL, most of them were the derivatives of 4-HA, such as 4-hydroxybenzaldehyde and 3-hydroxymandelic acid (Bhat and Vaidyanathan, 1976). It has been reported that furfural was the key intermediate product during the formation of xylose based pseudo-lignin (Hu, 2014; Cheng et al., 2018). Several possible pathways have been reported by which furfural (derived from xylose) could undergo self-condensation or react with isomerized xylose and other intermediate products. 4-HA probably participated in the further polymerization with pseudo-lignin (Cheng et al., 2018).

Fig. 1. Concentration of total phenolic compound in different hydrolyzates.

pretreated lignin compounds, the yields reported were similar with the contents of phenolics averaging around 2.70 g/L. With the addition of 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, ferulic acid or p-coumaric acid, the amount of phenolic compounds in CGPL’ hydrolyzate increased up to 4.90 g/L. However, the amount of phenolics in CXPL’s hydrolyzate slightly decreased with addition of vanillin, vanillin acid, 4-hydroxybenzoic acid, ferulic acid and p-coumaric acid. Generally, under acid-catalyzed hydration reactions, glucose generated more soluble phenolic compounds than xylose, and the phenolic model compound additives resulted in augment effects on soluble phenolic compounds during the formation of CGPL than CXPL. In Tables 2, 4-HA showed obvious enhancement in the yield of pseudo-lignin both generated from glucose (CGPL) and xylose (CXPL). To further understand the generation mechanism of pseudo-lignin with lignin model compounds, GC–MS was used to analyze the aromatic products found in hydrolyzates with or without the 4-hydroxybenzoic acid (4-HA) additive. The total identified aromatic compounds in the hydrolyzates of Blank-CGPL, HA-CGPL, and HA-CXPL are summarized in Table 3. It is interesting that both hydroquinone and pyrogallol were detected in Blank-CGPL. Forsskhl et al. has also reported that phenolic compounds like 1, 2-benzenediol, 1, 2, 3-benzenetriol and 3-methyl-1, 2-benzenediol were formed from hexuronic acid in aqueous solutions of pH 4.5 at 96 or 160 °C (Forssk et al., 1976). From this finding, it can be proposed that during the formation of CGPL, the phenolic compounds could be an intermediate for various polycondensation reactions. With the addition of 4-HA, the hydrolyzate of HA-CGPL yielded a new phenolic compound-homosalate. Patil and Lund also indicated that 2, 5dioxo-6-hydroxy (derived from HMF) could tautomerize to an enol,

3.4. Analysis of pseudo-lignins Apart from the in-depth analysis of soluble pseudo-lignin precursors developed across a variety of model hydrolyzates of CGPL and CXPL, we investigated the physicochemical properties of different solid pseudo-lignin samples. Fig. S2 shows the FT-IR spectra of pseudo-lignin formed from a blank sugar solution (CGPL or CXPL) and from a sugar solution with 4-HA (the most influential simple phenolic as discussed in previous sections) (i.e., HA-CGPL or HA-CXPL). From the spectral data it can be observed that different pseudo-lignin samples contain hydroxyl, carbonyl, and aromatic chemical structures. The signal at 2910 cm−1 was assigned to C–H bonds in CHO groups. The strong bands at 1701 cm−1, 1597 cm−1 and, 1597 cm−1 were attributed to C]O stretching in unconjugated ketones and, aromatic ring in stretching and aromatic C]C stretching (in the ring), respectively. The adsorption bands in the region of 1320-1000 cm−1 can be assigned to CeO stretching in alcohols, ethers, or carboxylic acids (Shinde et al., 2018; He et al., 2018; Sun et al., 2019). In addition, four pseudo-lignin samples were analyzed by CP/MAS

Table 3 GC–MS analysis of aromatics composition in hydrolyzates. Sample Number 1 2 3 4 5 6 7 8

Compounds 4-Hydroxybenzaldehyde 3-Phenylpropanol Hydroquinone 3-Hydroxybenzoic acid Pyrogallol 4-Hydroxybenzoic acid 3-Hydroxymandelic acid Homosalate

RTa(min) 11.36 11.53 12.13 14.46 14.66 15.18 16.47 17.78

CGPLb

HA-CGPLb

+

+

CXPLc

HA-CXPLc + + +

+ + + +

a

Retention time. Hydrolyzates were separated with pseudo-lignin generated from dilute acid pretreated glucose without addition of 4-hydroxybenzoic acid (CGPL) or with addition of 4-hydroxybenzoic acid (HA-CGPL). c Hydrolyzates were separated with pseudo-lignin generated from dilute acid pretreated xylose without addition of 4-hydroxybenzoic acid (CXPL) or with addition of 4-hydroxybenzoic acid (HA-CXPL). b

5

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Fig. 2. SEM images of CGPL (pseudo-lignin generated from dilute acid pretreated glucose), CXPL (pseudo-lignin generated from dilute acid pretreated xylose), additive in CGPL and CXPL. 13

after extracting. The spectrum analysis suggested that the performed extractions did not change/fractionate the chemical structure of pseudo-lignin. The weight-average (Mw) and number average (Mn) molecular weight distributions of pseudo-lignin are listed in Table 4. The estimated Mw of GPL and XPL is 2680 and 2710 g/mol, respectively. When the phenolic compound 4-HA was presented during the formation of pseudo-lignin, the Mw was found to be significantly increased. The Mw of XPL increased from 2710 g/mol to 7070 g/mol (HA-XPL), and for GPL, the value of Mw increased from 2680 g/mol to 2950 g/mol. The Mn of GPL, HA-GPL, XPL and HA-XPL showed the same tendency. These results suggested that the addition of 4-HA modified the Mw or Mn of the recovered pseudo-lignin, and the amplification of Mw or Mn were much lower in GPL than XPL, which indicated that 4-HA may broaden the molecular weight distribution of pseudo-lignin generated from xylose pretreated by acid. Therefore, it is hypothesized that 4-HA could be condensed with compounds generated from acid pretreated sugars or lignin-like aromatic structure of pseudo-lignin, which may be the mechanism for the increased molecular weight of pseudo-lignin. To further characterize the functional groups of the pseudo-lignins, 31P NMR spectrum was used to analyze GPL, HA-GPL, XPL, and HA-XPL (Pu et al., 2014; Meng et al., 2019). Table 5 represented that amongst the various hydroxyl groups, the phenolic hydroxyl signal is typically the dominant in pseudo-lignin. With the addition of 4-HA to the glucose acid pretreatment, the content of aliphatic OH, condensed phenolic OH, noncondensed phenolic OH, and carboxylic acid in pseudo-lignin (GPL) increased from 0.11, 0.63, 0.74 and 0.53 to 0.44, 1.30, 1.57 and 0.94 mmol/g, respectively (Table 5). These results suggest that the presence of 4-HA

C NMR. Fig. S3 clearly illustrates that four pseudo-lignin samples exhibited function groups including carbonyl (220−187 ppm), aromatic (162−95 ppm), methylene carbon CH2 (32 ppm), and methyl carbon CH3 (23 ppm) (Ben and Ragauskas, 2012). Both FT-IR and CP/ MAS 13C NMR indicated that pseudo-lignin with the addition of 4-HA has similar spectra assignments with the ones from control sugars both glucose and xylose. These spectral findings showed that the addition of 4-HA (intended to represent soluble lignin monomers) did not significantly alter the functional groups of the different pseudo-lignins produced with model solutions. SEM images of the pseudo-lignin produced from 4-HA mixed with glucose (CGPL) or xylose (CXPL) are shown in Fig. 2. Based on our previous work, pseudo-lignin is a kind of aggregate droplet with the spherical droplets (He et al., 2018). Fig. 2 shows that phenolic compound (4-HA) seemingly played an import role in the formation or assemble platform of spherical droplets when it is added during the formation of pseudo-lignin. Intuitively, phenolic compound (4-HA) could participated in the formation of pseudo-lignin droplets and affect its aggregate structures. To further explore the nature of the formed pseudo-lignin under the studied reaction condition, their molecular properties were examined (i.e., CGPL, HA-CGPL, CXPL, and HA-CXPL). Hence, the solid residues from acid pretreatment were extracted with 1, 4-dioxane then concentrated, freeze dried, and defined as GPL, XPL, HA-GPL and HA-XPL, respectively. The CP/MAS 13C NMR of extracted pseudo-lignin was showed in Fig. S4. Compared with Fig. S3, extracted pseudo-lignin consisted of carbonyl, aromatic, methylene carbon CH2, and methyl carbon CH3 functional groups (Ben and Ragauskas, 2012), besides, carboxyl groups (173 ppm) of pseudo-lignins were assigned obvious 6

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4. Conclusions

Table 5 Functional group contents of pseudo-lignin extracted from pretreated monosaccharides with or without additives by quantitative 31P NMR. Sample

GPL XPL HA-GPL HA-XPL

Two source's pseudo-lignin (i.e., CGPL and CXPL) were prepared by dilute sulfuric acid pretreatment, and eight representative phenolic compounds were added to mimic the effect of depolymerized lignin in the tested model solution systems. The yields of pseudo-lignin increased from −7.4% to −11.8% for the different model solutions when a variety of similar phenolic compounds were also present during pretreatment. GCeMS characterization revealed that compounds' composition in hydrolyzates changed by phenolic compounds. SEM images indicated that phenolic compounds seemingly play a role in assembling platform of spherical droplets of pseudo-lignin. The molecular weight of two pseudo-lignins (GPL and XPL) have been increased from 2680 and 2710 g/mol to 2950 and 7070 g/mol with phenolic compound adding (HA-GPL and HA-XPL). 31P NMR analysis represented that phenolic compound increased the various hydroxyl groups in GPL, but play a slight role in hydroxyl groups of XPL. And the 2D-HSQC NMR analysis showed that phenolic compound changed the hydrocarbon bonds both in GPL and XPL. Analysis of hydrolyzates and pseudo-lignin provide a hypothesis that soluble lignin model compound could combine with lignin-like aromatic structure to participate in pseudo-lignin’s formation during acid pretreatment. These observed changes are crucial to understanding the fundamental chemistry associated with the pseudolignin formation during pretreatment for the future utilization of biomass.

OH content (mmol/g) Aliphatic OH

Condensed phenolic OH

Noncondensed phenolic OH

Total OH

COOH

0.11 0.74 0.44 0.64

0.63 0.96 1.30 0.96

0.74 1.07 1.57 1.01

1.37 2.03 2.87 1.97

0.53 1.52 0.94 1.36

affected the generation pathway of pseudo-lignin which changed the original structure or participated in the aromatization of glucose during the formation of pseudo-lignin (Bhat and Vaidyanathan, 1976; Cheng et al., 2018). According to Table 5, there were slight differences between the functional groups of XPL and HA-XPL, which indicated that 4-HA played a minor role in the functional groups of pseudo-lignin derived from xylose. Fig. S5-S6 presented the 2D HSQC spectra and assignments of C–H bonds in pseudo-lignin (GPL, XPL, HA-GPL, and HA-XPL). The detailed structural information showed in Fig. S5 are based on published literature (Ben and Ragauskas, 2012, 2011; Hao et al., 2018). The spectrum indicated that carbonyl group (compound G in Fig. S7), furans (compounds F, E1 and E2 in Fig. S7) and side-chain of phenols are the major structural features in pseudo-lignin. Compared with GPL, XPL has weaker C–H signal value of G in δ 90-10/6-0.8 ppm region; however, in aromatic region, there are stronger hydrocarbon signals in E2, E1, A3, A2, and A1. In addition, from Fig. S5–S6, it showed that 4-hydroxynbenzoic acid do affect the structure of pseudolignin. In δ 90-10/6-0.8 ppm region, HA-GPL showed more undefined bonds than GPL. The HA-XPL sample had a stronger C–H signal value in G and weaker in F than XPL. In aromatic region, the C–H bonds in B3 disappeared in HA-GPL, and for HA-XPL the C–H bonds in B2, B1 disappeared but the signal of A2 increased. The spectra assignments indicated that under sulfuric acid pretreatment, glucose and xylose could form pseudo-lignin with different structures, and the phenolic compounds like 4-HA would participate in the generating pathways then change pseudo-lignin’s structure. Above all, the different reaction pathway of CGPL and CXPL with 4HA addition are proposed in Figs. S8 and S9, respectively. As mentioned before that 2, 5-dioxo-6-hydroxyhexanal (DHH) which has alpha hydrogens at carbons 3, 4, would be formed from 5-HMF (Patil and Lund, 2011). Fig. S8 suggests that during acid pretreatment, DHH would undergo aldol condensation with R1 (5-HMF, DHH, and 5-methyl furfural) at carbons 3, 4, 1, or addition/condensation with R1/R2 (aromatic rings, including 4-HA and other generated aromatic rings) at carbons 1, 6 (Patil and Lund, 2011; Wan et al., 2019). Besides, 4-HA could undergo esterification with an alcoholic hydroxyl in the pathway. According to the results from GPC and 31P NMR analysis, we speculated that 4-HA replaced R1 or generated aromatic rings and participated in the generation of CGPL. Just as shown in the 2D-NMR, it can be found that there is a little difference between GPL and HA-GPL in aromatic regions. For the formation of CXPL, furfural could undergo aldol condensation with xylose or condensation with R1 or R2 as suggested in Fig. S9 (Wan et al., 2019). Based on GPC and 31P NMR data, it is suggested that 4-HA may participate in condensation reactions mostly by esterification and increased the dispersion of XPL, then the exposed Ph−OH would be consumed in further reactions. During acid pretreatment, glucose and xylose inevitably undergo different dehydration polymerization to form the pseudo-lignin, in which 4-HA played a differential but promotive role in the reaction pathway Therefore, it is better to modify the severity of acid pretreatment to avoid the deep degradation of lignin or carbohydrate, which would be an efficiency way to reduce the formation of pseudo-lignin and increase the utilization of biomass.

Credit authorship contribution statement JH designed the project, performed experiments, analyzed data, and prepared the manuscript. CXH helped to analyze the data and revised the manuscript. CHL helped to design the project. CH helped to revise the manuscript. ML, YQP helped to carry out NMR analysis and revise the manuscript. AJR helped to analyze data and revise the manuscript. QY supervised the project and revised the manuscript. All authors read and approve the final manuscript. Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments The research was supported by the National Natural Science Foundation of China (31800501), and Natural Science Foundation of Jiangsu Province for youth (BK20180772). The authors thank the National First-class Disciplines (PNFD), the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD), and the Doctorate Fellowship Foundation of Nanjing Forestry University for supporting the work. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2020.112205. References Ben, H.X., Ragauskas, A.J., 2011. Heteronuclear single-quantum correlation–nuclear magnetic resonance (hsqc–nmr) fingerprint analysis of pyrolysis oils. Energ. Fuel. 25, 5791–5801. Ben, H.X., Ragauskas, A.J., 2012. Torrefaction of loblolly pine. Green Chem. 14, 72–76.

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