Alkaline Pretreatment Severity Leads to Different Lignin Applications

Research Article pubs.acs.org/journal/ascecg

Alkaline Pretreatment Severity Leads to Different Lignin Applications in Sugar Cane Biorefineries Fabrícia Farias de Menezes,*,†,∥ Jorge Rencoret,‡,∥ Simone Coelho Nakanishi,†,§ Viviane Marcos Nascimento,† Vinicius Fernandes Nunes Silva,† Ana Gutiérrez,‡ José C. del Río,‡ and George Jackson de Moraes Rocha† †

Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Caixa Postal 6192, Rua Giuseppe Máximo Scolfaro, 10.000, CEP 13083-970 Campinas, São Paulo, Brazil ‡ Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Avenida Reina Mercedes, 10, 41012-Seville, Spain § Departamento de Biotecnologia, Escola de Engenharia de Lorena, US. Estrada Municipal do Campinho, s/n - Pte. Nova, Lorena SP, CEP 12602-810 Lorena, São Paulo, Brazil ABSTRACT: Lignin, a multifunctional major biomass component, has a prominent potential as feedstock to be converted into high value-added products. Lignin is available in high amounts as side streams during cellulosic ethanol production, and within the biorefinery context, it is important to assess its structural characteristics in order to explore its potential to replace some petroleum-based reactants. In this study, some important features were evaluated for different lignins such as lignin purity and the amounts of syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) units. Four alkaline lignins, generated from a pilot-scale pretreatment of sugar cane bagasse (NaOH 1.5%, 30 min), were evaluated according to the severity of the alkaline pretreatment (130 or 170 °C, with or without the addition of anthraquinone). The different pretreatments produced lignins with different chemical characteristics that can be used for different purposes in sugar cane biorefineries. As the severity of alkaline pretreatment increased, the recovered lignins presented higher amounts of H- and lower amounts of S-lignin units. In particular, the lignin obtained at 170 °C with the addition of anthraquinone presented the highest content of H- and the lowest content of S-lignin units, which would present higher reactivity toward formaldehyde in phenolic resins. KEYWORDS: Sugar cane industry, Pretreatment, Alkaline-lignins, Chemical features, Biorefinery

■

INTRODUCTION The large amounts of sugar cane bagasse generated for global sugar-alcohol industry (∼540 million metric tons per year basis) make this industrial wastes attractive lignocellulosic feedstocks to produce cellulosic ethanol.1 This ethanol production mainly consists of three steps: (i) a pretreatment to remove and/or modify the lignin polymer; (ii) saccharification of cellulose; and (iii) fermentation of released sugars.2 The pretreatment is the most expensive step in the conversion of cellulosic biomass to fermentable sugars.2 The pretreatment step aims to remove lignin and disrupt the crystalline phase of cellulose to maximize the sugar production during the enzymatic hydrolysis step.3 The pretreatment efficiency (mass yield and solubilization rates) can be affected by various factors, including residence time, solids loading, temperature, and catalyst addition.4 One of the most promising pretreatments for sugar cane bagasse is the alkaline delignification because it is efficient, relatively inexpensive, and energy-efficient.5 This method leads to a high lignin removal and decreases cellulose crystallinity and © 2017 American Chemical Society

the polymerization degree of the carbohydrates cellulose and hemicelluloses.6 However, the alkaline pretreatment also promotes carbohydrates losses. A catalyst that could prevent such loss is anthraquinone, AQ.7 A lignin-rich liquid stream is obtained after the alkaline pretreatment of sugar cane bagasse in a cellulosic ethanol plant. Extending the concept of biorefinery in cellulosic ethanol production is a key strategy to achieve economic and environmental sustainability. Thus, instead of burning the lignin for power cogeneration, a higher-value utilization of the lignin is desirable. Lignin has a high potential for the production of aromatic specialties and fine chemicals due to its high-functionality and aromatic nature.4 Lignin is a complex aromatic macromolecule mainly composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, differing in the degree of methoxylation of the Received: January 23, 2017 Revised: May 19, 2017 Published: May 22, 2017 5702

DOI: 10.1021/acssuschemeng.7b00265 ACS Sustainable Chem. Eng. 2017, 5, 5702−5712

Research Article

ACS Sustainable Chemistry & Engineering

numbered according to experiment number at pilot scale: L1 (130 °C without AQ); L2 (130 °C with AQ); L3 (170 °C without AQ); and L4 (170 °C with AQ). Chemical Composition and Solubilization Rates. The chemical composition of the lignin samples (L1−L4) obtained from NaOH pretreatment at pilot scale was determined in triplicate, as performed by Rocha and coauthors.9 The acid insoluble and soluble lignin, cellulose, hemicelluloses, and ash contents were determined in the four lignin samples. Solubilization rates of the main components were calculated as shown by Nascimento and coauthors.7 Elemental Analysis and C9-Formulas. PerkinElmer2400 Series II CHNS/O Elemental Analyzer was used to determine carbon, hydrogen, nitrogen, and sulfur contents in lignin samples. The oxygen content was estimated by difference. Duplicates were performed. The C9 formula for lignins were estimated based on elemental analysis for the determination of carbon, hydrogen, and oxygen contents and PyGC/MS for estimation of the methoxyl group. Determination of Higher Heating Values (HHVs). The HHVs of the lignins were determined in triplicate according to the ABNT NBR 8633/84 standard,10 using approximately 1.0 g of the lignins in a IKA C-200 oxygen bomb calorimeter. Pyrolysis-Gas Chromatography−Mass Spectrometry (PyGC/MS). Around 0.1 mg of the lignin samples was pyrolyzed (at 500 °C) in an EGA/PY-3030D microfurnace system (Frontier Lab Ltd.) coupled to an Agilent 7820A gas chromatograph (GC) connected to an Agilent 5975 mass selective detector (MSD). The GC oven was equipped with a DB-1701 fused-silica capillary column (60 m × 0.25 mm i.d., 0.25 μm film thickness), and the temperature was programmed from 45 °C (4 min) to 280 °C (10 min) at a rate of 4 °C min−1. The carrier gas (helium) was 1 mL min−1. The compounds were identified by comparison of their mass spectra with those published in commercial libraries (Wiley and NIST 14) and with those reported in the literature,11,12 and when possible, by comparison of their retention times and mass spectra with those of authentic standards. The peak areas were expressed as percentages. The data for two technical replicates were averaged. 2D-Nuclear Magnetic Resonance Spectroscopy (2D NMR). 2D-NMR spectra were acquired on a Bruker Avance III 500 MHz instrument equipped with a cryogenically cooled 5 mm TCI gradient probe using the experimental conditions previously described.8 Briefly, around 40 mg of lignin samples (L1−L4) was dissolved in DMSO-d6, and HSQC (heteronuclear single quantum coherence) experiments were performed using the standard Bruker’s “hsqcetgpsisp2.2” pulse program. The signals were assigned by comparing with the literature.8,13,14 A semiquantitative analysis was performed using Bruker’s Topspin 3.5 processing software. The relative abundances of the different interunit linkages were estimated from the volume integrals of their Cα−Hα correlation signals, although the Cγ−Hγ correlations were used for the cinnamyl alcohol end-groups. The signals for the C2−H2 correlations from H, G, and S lignin units, as well as from PCA and FA were used to estimate their relative abundances. The content of the different interunit linkages (as well as the content of the PCA and FA) were referred as per 100 aromatic lignin units (H + G + S = 100). Molecular Weight by Size Exclusion Chromatography (SEC)AKTA System. Molecular weight of the lignins L1−L4 were estimated using size exclusion chromatography (SEC) analysis using a handmade Superdex 30 Prep grade GETM column (65 cm × 1.6 cm) attached to an AKTA automated system equipped with a UV detector (280 nm) and 0.1 M NaOH as eluent. The flow was 0.5 mL min−1 at room temperature and 1.0 mL of sample injection volume. Tannic acid, hydroquinone, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine4′,4″-disulfonic acid sodium salt from Sigma-Aldrich, and a lignin with known molecular weight were used as internal standards. The samples were not previously acetylated since they are fully soluble in 0.1 M NaOH solution. The average molecular weights were determined comparing the lignin curves with the standards. The peaks of the chromatograms generated in these analyses were integrated using Origin 9.0 software (peak analyzer function). A normalization was made in order to provide a better comparison between the results.

aromatic ring. Grass lignins also contain p-coumaric acids (PCA) and ferulic acids (FA).8 The lignin units are cross-linked to each other by different chemical bonds (β−O−4′, β−5′, β−β′, 5−5′, and 5−O−4′).4,8 In order to provide a suitable use of lignin as feedstock for the production of value-added products, it is imperative to assess its structural characteristics. These structural features are influenced by several factors, including the biomass source, the extraction method, and even the severity of the extraction process.4 Alkaline pretreatment of sugar cane bagasse at different conditions could produce different lignins with different compositions that can be used for different purposes. Thus, it is possible to produce tailormade lignins with desired properties by modifying the pretreatment conditions. Bearing this in mind, in this article we evaluated the effect of temperature and the addition of AQ on the chemical features of sugar cane bagasse lignins. The major structural features of the lignins assessed in this study included the carbohydrates content (lignin purity), the relative amounts of S, G, and H aromatic units, the S/G ratios, the abundance of the different interunit linkages, and the content of cinnamic acids (PCA and FA). The chemical composition, elemental analysis, and higher heating value (HHV) of these lignins were determined. In addition, the detailed structural characterization of the alkaline lignins was carried out by analytical pyrolysis coupled to gas chromatography and mass spectroscopy (Py-GC/MS), and two-dimensional nuclear magnetic resonance (2D NMR).

■

MATERIALS AND METHODS

Lignins from sugar cane bagasse were analyzed in order to evaluate their structural changes according to the difference in the severity of the pretreatment process. Lignins were recovered by acidification of black liquors from sugar cane bagasse pretreatments with sodium hydroxide at different severity conditions. The increase in the severity level of pretreatment occurs by both adding anthraquinone (C14H8O2, AQ) and increasing the temperature of the process. The particular experimental conditions of the alkaline pretreatment processes that lead to the different lignins and the analytical techniques applied are detailed below. Alkaline Pretreatments of Sugar Cane Bagasse and Obtaining Lignins. The raw sugar cane bagasse was supplied by Pedra Agroindustrial S/A mill (Serrana, São Paulo, Brazil). This bagasse was submitted to a pretreatment process without any previous milling process, as received from the sugar and ethanol mill. About 95% of the sugar cane bagasse had an average particle diameter between 0.2 mm and 11.0 mm. The other 5% had an average particle diameter less than 0.15 mm. The raw sugar cane bagasse was composed of 42% of cellulose, 26% of hemicelluloses, 22% of lignin, 4% of ashes, and 5% of extractives on dry matter. Alkaline pretreatment experiments at pilot scale were performed in duplicate under four different conditions, previously optimized by Nascimento and coauthors:7 (1) 130 °C, without AQ; (2) 130 °C, with AQ (0.15% w/w); (3) 170 °C without AQ; and (4) 170 °C with AQ (0.15% w/w). Residence time (30 min), loading NaOH (1.5% w/ v), and solid/liquid ratio (1:15 w/v) were the same for all experiments. Twelve kilograms (wt % on dry basis) of sugar cane bagasse was used for each reaction held on a Hastelloy C-276 steel reactor (350 L; Pope Scientific Inc.).7 A Nutshe filter (100 L, Hastelloy C-276, Pope Scientific Inc.) was used to separate from the black liquor (lignin-rich soluble fraction). Further details are described by Nascimento and coauthors.7 Lignins were precipitated from the black liquors by acidification. This step was performed in a 500 L stirred tank, adding 98% sulfuric acid to black liquor until pH 2 (≈1.5 L). Then, a filtration step was carried out to separate the precipitated lignin that was washed to reach pH 6. The washed lignin was dried in an oven at 50 °C until 10% moisture and stored for further analyses. The lignin samples were 5703


Research Article


■

RESULTS AND DISCUSSION The conditions (temperature and AQ addition) and the severity of the different alkaline pretreatments used in this study are shown in Table 1. Lignins were isolated from the

since it forms a redox system with carbohydrates and lignin, promoting the cleavage of β−O−4 linkages in lignin.17,18 Elemental Analysis. Table 2 depicts the elemental composition (C, H, and O), methoxyl content, and C9-

Table 1. Effect of the AQ Addition and the Temperature in Alkaline Pretreatments of Sugarcane Bagasse on Solubilization Rates and Chemical Composition of the Lignins (L1−L4)

Table 2. Elemental Composition (C/H/O) and C9Formulas of the Alkaline Lignins (L1−L4) Isolated from Sugarcane Bagasse

L1 temperature (°C) catalysta severity factorb

130

L2

L3

130 170 AQ 2.52 2.59 3.74 Solubilization Rate in Black Liquor (%) lignin 79.6 73.3 83.7 hemicelluloses 56.1 41.1 57.9 cellulose 28.2 9.2 22.8 Chemical Compositional (% w/w, Dry Basis) cellulose 2.9 (2.1) 0.7 (0.1) 1.5 (0.2) hemicelluloses 13.6 (1.6) 10.1 (2.5) 16.6 (0.2) total lignin 81.2 (3.3) 88.1 (2.8) 81.0 (0.1) ashes 2.0 (0.4) 1.2 (0.3) 1.0 (0.1) higher heating 22.8 23.3 24.2 value (MJ/kg)

L4 170 AQ 3.72 87.6 53.8 16.3

lignins

%C

%H

%O

OCH3

C9-formulas

L1 L2 L3 L4

55.7 57.2 59.4 63.2

6.9 6.5 7.0 6.7

36.6 35.3 33.0 29.3

1.38 1.35 1.07 1.01

C9.0H11.0O3.7(OCH3)1.4 C9.0H10.0O3.5(OCH3)1.4 C9.0H10.8O3.1(OCH3)1.1 C9.0H9.5O2.5(OCH3)1.0

formulas for each lignin. These data show the effects of anthraquinone and temperature on the elemental composition and methoxyl content of the lignins obtained from the different alkaline pretreatments. A steady enrichment of carbon and a depletion of oxygen were observed in the lignins obtained at higher temperatures and with AQ addition (from lignin L1 to L4). The addition of AQ hardly affected the methoxyl content. Thus, the methoxyl content of lignins can be directly related to the temperature (severity factor) of the pretreatment (Table 1). The C9formula for each lignin was estimated from elemental analysis and Py-GC/MS results (methoxyl content). Lignins obtained at 170 °C (L3 and L4) showed C9-formulas with lower oxygen content and higher carbon content. Py-GC/MS. Py-GC/MS gave valuable information regarding the composition of the alkaline lignins. The pyrograms of the alkaline lignins L1−L4 obtained in this study are shown in Figure 1. A peak corresponding to AQ could also be detected in the pyrograms of L2 (130 °C, AQ) and L4 (170 °C, AQ), which is consistent with the use of AQ during the alkaline pretreatment of these samples. The lignin-derived phenolic compounds released upon pyrolysis and their relative molar abundances are listed in Table 3. Pyrolysis released phenolic compounds resulting from the H, G, and S-lignin units, as well as from the p-hydroxycinnamic acids: PCA and FA. The main lignin-derived compounds were phenol (1), guaiacol (2), 4methylphenol (4), 4-methylguaiacol (5), 4-ethylguaiacol (8), 4vinylguaiacol (11), 4-vinylphenol (12), syringol (15), transisoeugenol (19), 4-methylsyringol (20), vanillin (21), 4ethylsyringol (22), 4-vinylsyringol (24), trans-4-propenylsyringol (29), and acetosyringone (32), among others. However, the high contents of 4-vinylphenol (12) and 4-vinylguaiacol (11) released from these lignins upon pyrolysis is due, to a large extent, to the occurrence of PCA and FA, which decarboxylate during pyrolysis, as already observed in the pyrolysis of lignins from grasses, including sugar cane bagasse.8,19,20 Different compositional parameters can be obtained from the Py-GC/MS of the alkaline lignins L1−L4, such as H/G/S compositions, S/G ratios, and the ratio of lignin compounds with shorter side chains (0−2 carbon atoms) with respect to those with intact side chains (3 carbon atoms), as reflected in Table 3. The relative abundances of the H, G, and S-lignin, as well as the S/G ratios, had to be estimated without using 4vinylphenol (12) and 4-vinylguaiacol (11), which in the case of grasses are also produced from PCA and FA upon decarboxylation (and without the respective 4-vinylsyringol, 24). The pyrolysis data indicate that the lignins are comparatively depleted in S-lignin units and enriched in H-

0.6 (0.1) 12.9 (5.2) 85.8 (5.0) 0.7 (0.3) 25.3

a

Anthraquinone (AQ) concentration of 0.15% (w/w). bData are from Nascimento et al.7

different treatments used, which were labeled as follows: L1, 130 °C without AQ; L2, 130 °C with AQ; L3, 170 °C without AQ; and L4, 170 °C with AQ. The isolated lignins were subsequently analyzed by different analytical techniques to assess their purity and their structural composition. The effect of the temperature and the addition of anthraquinone on the structural features of lignins derived from alkaline pretreatments were thoroughly studied. Chemical Composition. Solubilization rates of the components (lignin, cellulose, and hemicelluloses) in liquor stemmed by alkaline pretreatments are reported in Table 1, which depicts the effects of the different conditions of the alkaline pretreatments on the purity of the obtained lignins. Solubilization rates of lignin and hemicelluloses increased with the temperature increase from 130 to 170 °C. The effect of AQ addition and temperature on the purity of the obtained lignins after alkaline pretreatments can be observed. Lignins with higher amounts of carbohydrates and lower ash contents were obtained (L3 and L4) at higher temperature (170 °C). The AQ addition preserved the carbohydrates during the pulping process generating a purer lignin (>84%). This preservation of cellulose can be due to the formation of aldonic acid in the terminal groups of polysaccharide which drives the stabilization of alkaline degradation.15 Nascimento et al.7 demonstrated that the alkaline pretreatment at 130 °C with AQ addition provided the highest conversion of carbohydrates into sugars by an enzymatic hydrolysis process from the pilot plant of cellulosic ethanol. The estimation of the severity factors of the pretreatment processes takes into account the time and the temperature.16 According to the severity factors shown in Table 1, temperature significantly influenced them, being about 40−50% higher at 170 °C. The addition of AQ is not included in this estimate; however, AQ can be considered as an intensifier of severity 5704


Research Article


Figure 1. Py-GC/MS chromatograms of the alkaline lignins isolated from sugar cane bagasse. (a) L1 (130 °C without AQ), (b) L2 (130 °C with AQ), (c) L3 (170 °C without AQ), and (d) L4 (170 °C with AQ).

noticed in these lignins, as indicated by the low abundances of α-oxidized lignin compounds (i.e., peaks 21, vanillin; 23, acetoguaiacone; 30, syringaldehyde; 31, syringic acid methyl ester; and 32, acetosyringone) in the pyrograms (Figure 1). 2D-HSQC-NMR. 2D-NMR provided further information about the structure of the lignins L1−L4. The side chain (δC/ δH 50−90/2.8−6.0) and the aromatic (δC/δH 100−150/5.5− 8.0) regions of the HSQC spectra of the alkaline lignins are shown in Figures 2 and 3, respectively, together with the main substructures found. The main lignin signals present in the spectra are listed in Table 4. A semiquantitative analysis of the different lignin interunit linkages, the H, G, and S lignin units, and the p-hydroxycinnamic acids (PCA and FA), present in the lignins was performed by integration of the volume contours of the different signals and was referred to as per 100 lignin aromatic units (Table 5). Signals from xylan polysaccharides (X2, X3, X4, and X5) were also observed in the aliphatic region of the spectra (Figure 2). The intensity of these signals is slightly higher in the lignins obtained from alkaline pretreatments without AQ addition (L1 and L3). This fact supports the chemical composition data

lignin units with increasing severity of pretreatments, as a result of the preferential removal and degradation of S-lignin during alkaline delignification.21 It is noted that the G-lignin units virtually remained constant in all experiments. Hence, the S/G ratio of lignins L1−L4 steadily decreases from 1.4 to 1.0 as the delignification conditions become more severe. In the Kraft cellulose process, it is known that softwoods (lignin rich in Gunits) require more severe cooking conditions (higher residence times, temperatures, and alkali charges) than hardwoods (lignin consisting of G- and S-units). The ratio of lignin markers with shorter side chains with respect to those with intact side chains (Ph−C0−2/Ph−C3 ratio) are also shown in Table 3. The increase in the Ph−C0−2/Ph−C3 ratio also indicates the extent of the side chain degradation of the lignins with the extent of delignification conditions, which is evident in lignins L1−L4. Comparing L1 and L2 (NaOH 1.5%, 130 °C, 30 min, with and without AQ) on the one hand, and L3 and L4 (NaOH 1.5%, 170 °C, 30 min, with and without AQ) on the other hand, it is evident that the addition of AQ in alkaline cooking decreases the S/G ratio and increases the Ph− C0−2/Ph−C3 ratio, indicating an improvement of the delignification reaction. Finally, no major oxidations were 5705


Research Article


Table 3. Identities and Relative Abundances of the Lignin-Derived Phenolic Compounds Released after Py-GC/MS of the Alkaline Lignins (L1−L4) Isolated from Sugarcane Bagassea label

compounds

origin

L1 (130 °C)

L2 (130 °C/AQ)

L3 (170 °C)

L4 (170 °C/AQ)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

phenol guaiacol 2-methylphenol 4-methylphenol 4-methylguaiacol C2-phenol 4-ethyl-phenol 4-ethylguaiacol C3-phenol C3-phenol 4-vinylguaiacol 4-vinylphenol eugenol 4-propylguaiacol syringol p-coumaric acid 4-trans-propenylphenol cis-isoeugenol trans-isoeugenol 4-methylsyringol vanillin 4-ethylsyringol acetoguaiacone 4-vinylsyringol guaiacylacetone 4-allylsyringol ferulic acid cis-4-propenylsyringol trans-4-propenylsyringol syringaldehyde syringic acid methyl ester acetosyringone syringylacetone propiosyringone ferulic acid methyl ester H/G/Sb S/G ratiob Ph-C0-2/Ph-C3c

H G H H H H H G H H G/FA H/PCA G G S PCA H G G S G S G S G S FA S S S S S S S FA

2.3 5.5 0.7 2.4 5.3 0.6 1.4 3.1 0.2 0.2 13.7 21.2 0.5 0.3 7.3 0.1 0.0 0.7 2.7 6.1 2.3 2.6 0.6 4.8 0.8 1.8 0.3 1.3 5.1 1.8 0.3 2.4 0.9 0.4 0.5 13:36:51 1.4 5.6

2.9 7.3 0.9 2.1 5.3 0.7 1.8 3.3 0.2 0.2 12.8 21.8 0.5 0.3 9.5 0.1 0.2 0.7 2.6 5.3 1.3 2.8 0.4 3.9 0.6 1.5 0.3 1.1 4.5 1.0 0.6 2.1 0.7 0.3 0.3 15:37:49 1.3 6.4

5.4 6.0 1.1 4.0 5.8 0.8 6.9 5.0 0.5 0.4 8.9 18.4 0.4 0.3 6.6 0.5 1.1 0.4 1.7 5.5 2.0 3.2 0.8 3.0 0.6 1.0 0.4 0.7 2.0 1.9 0.7 2.3 1.1 0.3 0.2 30:33:37 1.1 8.5

5.8 6.9 1.3 4.6 5.7 0.9 7.6 5.1 0.7 0.5 6.3 21.8 0.4 0.4 8.0 0.6 1.2 0.6 1.7 5.2 0.9 3.0 0.6 2.5 0.3 0.9 0.5 0.7 1.7 0.8 0.3 1.7 0.5 0.2 0.2 33:33:34 1.0 9.1

a H, p-hydroxyphenyl units; G, guaiacyl units; S, syringyl units; PCA, p-coumarates; FA, ferulates. bEstimated without using 4-vinylphenol (12) and 4-vinylguaiacol (11), also arising from p-coumaric and ferulic acids upon decarboxylation, and the respective 4-vinylsyringol (24). cRatio of lignin phenolic markers bearing side chains of 0−2 carbon atoms with respect to lignin phenolic markers bearing side chains of 3 carbon atoms.

tetrahydrofuran structures, which are originally γ-p-coumaroylated in sugar cane bagasse,8 are completely hydrolyzed, but the core tetrahydrofuran backbone still remains. A reduction in the abundance of interunit linkages can be observed from lignins L1 to L4, as the severity of the delignification increases (Table 5). Interestingly, lignins L1 (130 °C) and L2 (130 °C, AQ) still present a high content of β−O−4′ alkyl-aryl ether linkages (43 and 35 linkages per 100 aromatic units), which are drastically reduced (about 81% and 89% lower, respectively) in L3 (170 °C) and L4 (170 °C, AQ) due to the significant increase of the delignification severity at higher temperatures. The addition of AQ significantly enhances the delignification reactions, and thus, the abundances of β− O−4′ ether linkages (A) in the lignins with AQ (35 and 4 linkages per 100 aromatic units at 130 and 170 °C) was significantly lower than those in the corresponding lignins

where L1 and L3 contained about 16−18% of carbohydrates, while L2 and L4 contained about 11−13%. The main lignin interunit linkages observed (Figure 2) in the lignin samples were β−O−4′ alkyl-aryl ethers (A), β−5′ phenylcoumarans (B), β−β′ resinols (C), α,β-diaryl ethers (E), and β−1′ spirodienones (F), together with cinnamyl alcohol end-groups (I). Signals for oxidized lignin side chains were absent in the 2D-NMR spectra in these lignins indicating the absence of oxidative reactions, as already observed by Py-GC/ MS. Signals for a β−β′-linked tetrahydrofuran structure (C′) were also observed in the spectra of these lignins, with the characteristic Cα−Hα and Cβ−Hβ correlations at δC/δH 81.7/ 4.89 (C′α) and 53.3/2.12 (C′β). This structure results from the β−β′ -homocoupling of two γ-p-coumaroylated sinapyl alcohol monomers and has already been reported in the lignin of sugar cane bagasse.8 In the alkaline lignins L1−L4, however, the 5706


Research Article


Figure 2. Side chain (δC/δH 50−90/1.8−6.0) region in the 2D HSQC NMR spectra of the alkaline lignins isolated from sugar cane bagasse. (a) L1 (130 °C without AQ), (b) L2 (130 °C with AQ), (c) L3 (170 °C without AQ), and (d) L4 (170 °C with AQ). β-O-4′ Alkyl-aryl ethers (A); phenylcoumarans (B); resinols (C); β−β′ tetrahydrofuran (C′); α-O-4′ diaryl ethers (E); spirodienones (F); and cinnamyl alcohol end-groups (I).

Finally, it is important to note the occurrence of phydroxycinnamic acids (PCA and FA) in these lignins. Previous work8 has indicated the important amounts of PCA and FA in the lignin from sugar cane bagasse, accounting for 68% and 26%, respectively (with respect to all lignin units). The HSQC data indicate that PCA, which in sugar cane bagasse occurs acylating the γ−OH of the lignin side chains,8 has been completely hydrolyzed and is present in free form in the alkaline lignins (as indicated by the characteristic signals from the Cβ-Hβ correlations at δC/δH 115.1/6.29, PCAβ). The same happens with FA, which is also present in free form as indicated by the Cβ-Hβ correlations at δC/δH 116.5/6.37 (FAβ). Our data indicate that the relative amounts of PCA and FA in these lignins were reduced by around 75% of the original amounts present in the native lignin. Molecular Weight by the SEC-AKTA System. Molecular weight values of lignins largely depend on the analytical

without AQ (43 and 8 linkages per 100 aromatic units at 130 and 170 °C). The main cross-signals observed in the aromatic regions of the spectra (Figure 3) are from the different lignin and phydroxycinnamic acid units. Signals from H, G, and S-lignin units were present in the spectra of the alkaline lignins. S-units decreased about 36%, and H-units increased about 76−81% in lignins when the temperature increased from 130 to 170 °C (Table 5). In general terms, as the severity of the alkaline pretreatments increases, lignins are depleted in S-units and enriched in H, while G-units remain practically intact, as already shown by Py-GC/MS. This is particularly evident in the series of lignins from L1 to L4, where the S/G ratio gradually decreases from 2.6 to 1.4. The amounts of H-lignin units were much lower in the native lignin from sugar cane bagasse (3% of all lignin units)8 when compared to that of lignin from pretreatment at 170 °C. 5707


Research Article


Figure 3. Aromatic/unsaturated (δC/δH 100−150/5.5−8.0) region in the 2D HSQC NMR spectra of the alkaline lignins isolated from sugar cane bagasse. (a) L1 (130 °C without AQ), (b) L2 (130 °C with AQ), (c) L3 (170 °C without AQ), and (d) L4 (170 °C with AQ). p-Hydroxyphenyl units (H); guaiacyl units (G); syringyl units (S); oxidized syringyl units (S′); p-coumaric acid (PCA); and ferulic acid (FA).

conditions, such as, the column systems, nature of solvents used, acetylation of the lignin, type of calibration standard, and others.23 Figure 4 depicts the chromatograms of tannic acid, hydroquinone, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″disulfonic acid sodium salt from Sigma-Aldrich, and a lignin with known molecular weight which were used as internal standards to estimate the average molecular weight of the lignins (L1−L4). Figure 5 depicts the SEC chromatograms of the lignins L1−L4, while Table 5 shows the values of the peaks from the SEC chromatogram generated by Origin software. A major peak (43−65 mL) was observed in all chromatogram profiles of the lignins. In L1 and L2 chromatograms, this peak corresponds to about 80% of the total area, while in L3 and L4 chromatograms, it corresponds to 68% and 75% (Table 6), respectively. In Figure 5b, it can be observed that the first peak region is equivalent to a molecular weight range of 4.4− 1.7 kDa, as per standard chromatograms (Figure 4). Doherty and Mousavioun22 have reported the molecular weight of alkaline bagasse lignin (170 °C, 1.5 h) of 2410 Da.

The second larger peak (70−80 mL) that appears in all lignin profiles corresponds to a molecular weight range of

Alkaline Pretreatment Severity Leads to Different Lignin Applications

Recommend Documents