Alkaline Pretreatment Severity Leads to Different Lignin Applications

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Alkaline pretreatment severity leads to different lignin applications in sugarcane biorefineries Fabrícia Farias de Menezes, Jorge Rencoret, Simone Coelho Nakanishi, Viviane Marcos Nascimento, Vinicius F. N. Silva, Ana Gutierrez, Jose Carlos Del Rio, and George Jackson Moraes Rocha ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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ACS Sustainable Chemistry & Engineering

Alkaline pretreatment severity leads to different lignin applications in sugarcane biorefineries

Fabrícia Farias de Menezes1§*, Jorge Rencoret2§, Simone Coelho Nakanishi1,3, Viviane Marcos Nascimento1, Vinicius Fernandes Nunes Silva1, Ana Gutiérrez2, José C. del Río2, George Jackson de Moraes Rocha1 *e-mail: [email protected]

[1] 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, Brasil.

[2] Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Avenida Reina Mercedes, 10, 41012-Seville, Spain.

[3] 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, Brasil. §

These authors contributed equally

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Abstract Lignin, a multi-functional major biomass component, has a prominent potential as feedstock for being 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 pilotscale pretreatment of sugarcane bagasse (NaOH 1.5%, 30 min), were evaluated according to the severity of the alkaline pretreatment (130 °C 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 sugarcane 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 towards formaldehyde in phenolic resins.

Keywords: Sugarcane industry, pretreatment, alkaline-lignins, chemical features, biorefinery.

INTRODUCTION

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The large amounts of sugarcane 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 sugarcane 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 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 sugarcane 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 production of aromatic specialties and fine chemicals due to its high-functionality and aromatic nature.4 3



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Lignin is a complex aromatic macromolecule mainly composed by p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, differing in the degree of methoxylation of the 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–4ʹ, β–5ʹ, β–βʹ, 5–5ʹ and 5–O–4ʹ).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 sugarcane bagasse at different conditions could produce different lignins with different compositions that can be used for different purposes. Thus, it is possible to produce tailor-made lignins with desired properties by modifying the pretreatment conditions. Bearing this in mind, in this paper we evaluated the effect of temperature and the addition of AQ on the chemical features of sugarcane 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 inter-unit 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 4


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Lignins from sugarcane 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 sugarcane 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 leads to the different lignins and the analytical techniques applied are detailed bellow.

Alkaline pretreatments of sugarcane bagasse and the obtainment of lignins The raw sugarcane bagasse was supplied by Pedra Agroindustrial S/A mill (Serrana, São Paulo, Brazil). This bagasse was submitted to pretreatment process without any previous milling process, as received from the sugar and ethanol mill. About 95% of the sugarcane 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 sugarcane bagasse was composed by 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 co-authors7: (1) 130 °C, without AQ; (2) 130 °C, with AQ (0.15% w/w); (3) 170 °C without AQ; (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.

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12 kg (wt% on dry basis) sugarcane 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 (ligninrich soluble fraction). Further details was described by Nascimento and co-authors7. Lignins were precipitated from the black liquors by acidification. This step was performed in a 500 L stirred tank, adding 98% sulphuric 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 oven at 50 °C until 10% moisture and stored for further analyzes. The lignin samples were 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 Rocha and co-authors.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 co-authors.7

Elemental analysis and C9-formulae PerkinElmer®2400 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 6


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estimated based on elemental analysis for determination of carbon, hydrogen and oxygen contents and Py-GC/MS for estimation of methoxyl group.

Determination of Higher Heating Values (HHVs) The HHVs of the lignins were determined in triplicate according to the ABNT NBR 8633/84 standard10, using approximately 1.0 g of the lignins in a IKA C-200 oxygen bomb calorimeter.

Pyrolysis-Gas Chromatography-Mass Spectrometry (Py-GC/MS) Around of 0.1 mg the lignin samples were pyrolyzed (at 500 °C) in an EGA/PY3030D micro-furnace system (Frontier Lab Ltd.) coupled to an Agilent 7820A gas chromatograph (CG) connected to an Agilent 5975 mass selective detector (MSD). The GC oven was equipped with a DB-1701 fused-silica capillary column (60 m x 0.25 mm i.d., 0.25 µm film thickness) and the temperature was programmed from 45 ºC (4 min) to 280 ºC (10 min) at a rate of 4 ºC 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 literature11,12, 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)

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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) were 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 literature8,13,14. A semiquantitative analysis was performed using Bruker’s Topspin 3.5 processing software. The relative abundances of the different inter-unit 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 inter-unit 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 x 1.6 cm) attached to an AKTA® automated system equipped with UV detector (280 nm) and NaOH 0.1 M 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-(2Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,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 8


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average molecular weights were determinate comparing the lignins 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.

RESULTS AND DISCUSSION The conditions (temperature, AQ addition) and the severity of the different alkaline pretreatments used in this study are shown in Table 1. Lignins were isolated from the different treatments used, which were labeled as follows: L1, 130 ºC without AQ; L2, 130 ºC with AQ; L3, 170 ºC without AQ; L4, 170 ºC 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

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amounts of carbohydrates and lower ash contents were obtained (L3 and L4) at higher temperature (170 ºC). The AQ addition preserved the carbohydrates during pulping process generating a purer lignin (>84%). This preservation of cellulose can be due to formation of aldonic acid in the terminal groups of polysaccharide which drives to a stabilization of alkaline degradation.15 Nascimento et al. (2016)7 demonstrated that the alkaline pretreatment at 130 °C with AQ addition provided the highest conversion of carbohydrates into sugars by 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 since it forms 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 compositional (C, H, O), methoxyl content and C9formulae 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 10


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can be directly related to the temperature (severity factor) of the pretreatment (Table 1). C9-formula to each lignin was estimated from elemental analysis and Py-GC/MS results (methoxyl content). Lignins obtained at 170 °C (L3 and L4) showed C9-formulae 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 phydroxycinnamic acids: PCA and FA. The main lignin-derived compounds were phenol (1), guaiacol (2), 4-methylphenol (4), 4-methylguaiacol (5), 4-ethylguaiacol (8), 4vinylguaiacol (11), 4-vinylphenol (12), syringol (15), trans-isoeugenol (19), 4methylsyringol (20), vanillin (21), 4-ethylsyringol (22), 4-vinylsyringol (24), trans-4propenylsyringol (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 sugarcane bagasse.8,19,20

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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 4-vinylphenol (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 indicates that the lignins are comparatively depleted in S-lignin units and enriched in Hlignin units with increasing the severity of the pretreatment, as a result of the preferential removal and degradation of S-lignin during alkaline delignification21. It is noted that the Glignin 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 G-units) 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 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 the lignins L1-L4. Comparing L1 and L2 (NaOH 1.5%, 130 °C, 30 min, with and without AQ) on 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 the 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 noticed in these lignins, as 12


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indicated by the low abundances of α-oxidized lignin compounds (i.e. peaks 21: vanillin; 23: acetoguaiacone; 30: syringaldehyde; 31: syringic acid methyl ester; 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 Figure 2 and Figure 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 inter-unit 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, 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 where the L1 and L3 contained about 16-18% of carbohydrates, while L2 and L4 contained about 11-13%. The main lignin inter-unit linkages observed (Figure 2) in the lignin samples were β–O–4ʹ alkyl-aryl ethers (A), β–5ʹ phenylcoumarans (B), β–βʹ resinols (C), α,β-diaryl ethers (E) and β–1ʹ 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–βʹ13



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linked tetrahydrofuran structure (Cʹ) 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 (Cʹα) and 53.3/2.12 (Cʹβ). This structure results from the β–βʹ -homo-coupling of two γ-p-coumaroylated sinapyl alcohol monomers and has already been reported in the lignin of sugarcane bagasse.8 In the alkaline lignins L1-L4, however, the tetrahydrofuran structures, which are originally γ-p-coumaroylated in sugarcane bagasse8, are completely hydrolysed, but the core tetrahydrofuran backbone still remains. A reduction in the abundance of inter-unit 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–4ʹ 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 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–4ʹ 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 in the corresponding lignins 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 p-hydroxycinnamic acid units. Signals from H, G and Slignin 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, whilst G-units remain practically intact, as 14


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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 sugarcane bagasse (3% of all lignin units)8 when compared to lignin from pretreatment at 170 °C. Finally, it is important to note the occurrence of p-hydroxycinnamic acids (PCA and FA) in these lignins. Previous work8 has indicated the important amounts of PCA and FA in the lignin from sugarcane bagasse, accounting for 68% and 26%, respectively (with respect to all lignin units). The HSQC data indicates that PCA, which in sugarcane bagasse occurs acylating the γ-OH of the lignin side-chains8, 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 SEC - AKTA system Molecular weight values of lignins largely depend on the analytical conditions, such as, the column systems, nature of solvents used, acetylation of the lignin, type of calibration standard, and others (Constant et al., 2016). 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

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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 5 b, it can be observed that the first peak region is equivalent to 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) being of 2410 Da. The second larger peak (70-80 mL) that appears in all lignin profiles correspond to molecular weight range of ˂1700-500 Da. In L1 and L2 chromatograms, the peak at 73 mL corresponds to about 20% of the total area, while in L3 and L4 chromatograms, it corresponds to 18% and 15%, respectively. Thus, the lignins obtained at 130 °C (L1 and L2) had a larger area of fractions with higher molecular weight than the lignins obtained at 170 °C (L3 and L4). With respect to the AQ addition, it has influenced on the L4, its fraction area with higher molecular weight (4.4 - 1.7 kDa) was 7% higher than in lignin without AQ (L3). Other difference between the lignins chromatograms is a presence of two minor peaks with lower molecular weight (500 - ˂200 Da) for L3 and L4. The areas sum of these peaks correspond to 14% (L3) and 10% (L4). The AQ addition resulted in a lignin with fractions of higher molecular weight. Thus, the L3 that was obtained at higher temperature and without AQ addition presented larger fractions with lower molecular weight.

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According to literature4,23,24, the higher severity of the obtainment lignin process, the higher recondensation of the low molecular weight, resulting in streams more recalcitrant and with higher molecular weight fractions. However, our results indicate that at higher temperature, lignin fractions with lower molecular weight were obtained. This could be related to the pretreatment duration (30 min) that may have not been enough to occur the recondensation process.

Different applications of the alkaline lignins in biorefineries Throughout this study, it was observed that the alkaline pretreatments of sugarcane bagasse at different conditions caused changes in the chemical structures of lignins. Furthermore, in this section, suitable applications of lignins in value-added products will be suggested according to their chemical features with regard to the need to innovate and extend the portfolio of ethanol biorefinery. As can be seen in Table 1, the severity of alkaline pretreatment increased the HHV of lignins. The lignin L4 (170 °C, AQ) showed the highest HHV, being 11% higher than from lignin obtained with lowest severity factor (L1). Considering the traditional scenario of ethanol industry, lignin L4 could be applied for thermochemical conversion purposes where the bagasse or lignin is preferentially burned. Lignins L1 and L2 could be used to obtain aromatic monomers due to the high amount of β–O–4ʹ linkages.25 A high fraction of these linkages in lignin is important for achieving higher amounts of aromatic monomers.4 The yield of monomers from the lignin depolymerization reaction is often related with its abundance of β-ether units, since this linkage is easier to break, requiring milder conditions.4 17



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Based on lignin purity, lignins L2 and L4 could be used for product synthesis in which the presence of carbohydrates would be less desired. Sugars could be unstable under basic conditions, generating furans or humins, which may subsequently lead to undesirable reactions.4 On the other hand, lignins L1 and L3 could be used, for example, to produce lignin-phenol matrix composites reinforced with cellulose microfibers. Lignins with high amounts of H units could be engaged for synthesis of products that need ortho positions of phenyl rings unblocked by methoxyl groups. The lignin with higher content of H-units and lower content of S-units (L4) would be more reactive in the synthesis of phenolic resins, which have lower content of methoxyl groups, leading to a higher reactivity of the phenol toward the formaldehyde.26 On the other hand, the S-units have no vacant the 5-position, therefore, lignin L1 that have the highest content of S-units (69%), would not be suitable for the production of phenolic resin, but could be indicated, for example, for methanol production from their methoxy groups.27 Molecular weight distribution of lignin is one of the principle properties to evaluate reactivity and physicochemical characteristics for potential applications. With respect to molecular weight, all lignins showed a largest fraction ranging from 4.4 to 1.7 kDa. The smaller weight fractions of the L3 and L4 could be applied, for example, to produce low molecular weight aromatic compounds. However, if these lignins were latter submitted to thermal process, it should take into account that the low molecular weight species have tendency to undergo recondensation, resulting in streams more recalcitrant, difficult to upgrade.4,24 Bearing all this in mind, it is clear that changes in the severity of the alkaline pretreatments could lead to the production of different lignins with different compositions 18


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that can be used for different applications. It is then possible to produce tailor-made lignins by carefully controlling the pretreatment conditions. In the cellulosic ethanol industry, it is an interesting alternative to target not only cellulosic pulp, but also the lignin stream in order to make the overall process even more profitable and more environmentally friendly.

CONCLUSIONS This study has demonstrated how the temperature and the addition of AQ in alkaline pretreatments greatly influence the structural features of the lignins isolated from sugarcane bagasse. In alkaline pretreatment experiments with anthraquinone addition (L2 and L4), carbohydrates were preserved in pretreated bagasse, thus, purer lignins were obtained. The higher the temperature, the lower the oxygen and methoxyl content in lignins. G-units remained practically intact in all lignins, whilst the H-units were enriched and S-units were depleted as the severity of the alkaline delignification increased. The amounts of β–O–4ʹ linkages was much more influenced by temperature than by the addition of AQ. Lignins obtained from the pretreatment at higher temperatures (170 °C) were depleted in β–O–4ʹ linkages (4-8 linkages per 100 aromatic units). In L1 and L2 chromatograms, 80% of lignin fractions ranged from 4.4 to 1.7 kDa. In L3 and L4 profiles, it corresponds to 68% and 75%, respectively. Alkaline pretreatments at higher temperature (170 °C) generated lignins (L3 and L4) with fractions with lower molecular weight (500 - ˂200 Da). In this study, it was possible to observe that the choice of the pretreatment conditions for sugarcane bagasse can be also based on what features of lignin are desired for its application in valueadded products. Most industries that use lignocellulosic biomass as feedstock have sought to maximize the potential of the cellulose stream, while lignin has only been considered as 19



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a by-product or a waste. However, finding conditions that are suitable to obtain both topquality cellulose and lignin is essential to increase the economic and environmental sustainability, expanding the portfolio in sugarcane biorefineries.

ACKNOWLEDGMENTS This study has been partially funded by the Spanish projects AGL2014-53730-R and CTQ2014-60764-JIN (co-financed by FEDER funds) and the CSIC project 2014-40E-097. We thank Dr. Manuel Angulo (from the NMR facilities of the General Research Services of the University of Seville – SGI-CITIUS) for performing the NMR analyses that were acquired on a Bruker Avance III 500 MHz instrument. We also thank the São Paulo Research Foundation (FAPESP) for partially financial support (project 2013/11961-1) and the Amazonas Research Foundation (FAPEAM) for providing a doctoral scholarship (project 253/2014).

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REFERENCES (1)

Bezerra, L. T.; Ragauskas, A. J. A review of sugarcane bagasse for secondgeneration bioethanol and biopower production. Biofuels, Bioprod. biorefining 2016, 10, 634–647.

(2)

Singh, R.; Shukla, A.; Tiwari, S.; Srivastava, M. A review on delignification of lignocellulosic biomass for enhancement of ethanol production potential. Renew. Sustain. Energy Rev. 2014, 32, 713–728.

(3)

Rocha, G. J. de M.; Nascimento, V. M.; Silva, V. F. N. da; Chandel, A. K. Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw for Second-Generation Ethanol Production. In Biofuels in Brazil; Silva, S. S. da, Chandel, A. K., Eds.; Springer International Publishing Switzerland, 2014; pp 151–172.

(4)

Rinaldi, R.; Jastrzebski, R.; Clough, M. T.; Ralph, J.; Kennema, M.; Bruijnincx, P. C. A.; Weckhuysen, B. M. Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis. Angew. Chemie - Int. Ed. 2016, 55 (29), 8164–8215.

(5)

Kim, J. S.; Lee, Y. Y.; Kim, T. H. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresour. Technol. 2016, 199, 42–48.

(6)

McIntosh, S.; Vancov, T. Optimisation of dilute alkaline pretreatment for enzymatic saccharification of wheat straw. Biomass Bioenerg. 2011, 35 (7), 3094–3103.

(7)

Nascimento, V. M.; Nakanishi, S. C.; Rocha, G. J. M.; Rabelo, S. C.; Pimenta, M. T. B.; Rossell, C. E. V. Effect of Anthraquinone on Alkaline Pretreatment and Enzymatic Kinetics of Sugarcane Bagasse Saccharification: Laboratory and Pilot Scale Approach. ACS Sustain. Chem. Eng. 2016, 4 (7), 3609–3617. 21



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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(8)

Page 22 of 39

del Río, J. C.; Lino, A. G.; Colodette, J. L.; Lima, C. F.; Gutiérrez, A.; Martínez, Á. T.; Lu, F.; Ralph, J.; Rencoret, J. Differences in the chemical structure of the lignins from sugarcane bagasse and straw. Biomass Bioenerg. 2015, 81, 322–338.

(9)

Rocha, G. J. D. M.; Nascimento, V. M.; Gonçalves, A. R.; Silva, V. F. N.; Martín, C. Influence of mixed sugarcane bagasse samples evaluated by elemental and physical– chemical composition. Ind. Crops Prod. 2015, 64, 52–58.

(10)

ABNT. NBR 8633: Charcoal - Determination of calorific value - Test method. In Brazilian National Standards Organization; 1984; p 13.

(11)

Ralph, J.; Hatfield, R. D. Pyrolysis-GC-MS characterization of forage materials. J. Agric. Food Chem. 1991, 39 (8), 1426–1437.

(12)

Faix, O.; Meier, D.; Fortmann, I. Thermal degradation products of wood. Holz als Roh- und Werkst. 1990, 48 (7), 281–285.

(13)

Rencoret, J.; Marques, G.; Gutiérrez, A.; Nieto, L.; Santos, J. I.; Martínez, J. J.-B. Á. T.; Río, J. C. del. HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state 10th EWLP. Holzforschung 2009, 63 (6), 691–698.

(14)

Ralph, S. A.; Ralph, J.; Landucci, L. L. NMR database of lignin and cell wall model compounds. https://www.glbrc.org/databases_and_software/nmrdatabase/. Accessed on July 2015

(15)

Wang, Y.; Shoaib, A.; Lindström, M. E.; Henriksson, G. Stabilization of Polysaccharides During Alkaline Pre-Treatment of Wood Combined with EnzymeSupported Extractions in a Biorefinery. J. Wood Chem. Technol. 2015, 35 (2), 91– 101. 22


Page 23 of 39

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(16)

Overend, R. P.; Chornet, E.; Gascoigne, J. A. Fractionation of lignocellulosics by steam-aqueous pretreatments. R. Soc. Publ. 1987, 321, 523–536.

(17)

Kubes, G. J.; Fleming, B. I.; MacLeod, J. M.; Bolker, H. I. Alkaline pulping with additives. A review. Wood Sci. Technol. 1980, 14 (3), 207–228.

(18)

Kaplunova, T. S.; Saipov, Z. K.; Abduazimov, K. A. Hydrogenolysis of rice husk protolignin. II. Chem. Nat. Compd. 1987, 23 (5), 613–616.

(19)

del Río, J. C.; Prinsen, P.; Rencoret, J.; Nieto, L.; Jiménez-Barbero, J.; Ralph, J.; Martínez, Á. T.; Gutiérrez, A. Structural characterization of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems. J. Agric. Food Chem. 2012, 60 (14), 3619–3634.

(20)

del Río, J. C.; Rencoret, J.; Prinsen, P.; Martínez, Á. T.; Ralph, J.; Gutiérrez, A. Structural characterization of wheat straw lignin as revealed by analytical pyrolysis, 2D-NMR, and reductive cleavage methods. J. Agric. Food Chem. 2012, 60 (23), 5922–5935.

(21)

Prinsen, P.; Rencoret, J.; Gutiérrez, A.; Liitiä, T.; Tamminen, T.; Colodette, J. L.; Berbis, M. Á.; Jiménez-Barbero, J.; Martínez, Á. T.; Del Río, J. C. Modification of the lignin structure during alkaline delignification of eucalyptus wood by kraft, sodaAQ, and soda-O2 cooking. Ind. Eng. Chem. Res. 2013, 52 (45), 15702–15712.

(22)

Doherty, P.; Mousavioun, W. O. S. Chemical and Thermal Properties of Bagasse Soda Lignin. Ind. Crops Prod. 2010, 31, 52–58.

(23)

Constant, S.; Wienk, H. L. J.; Frissen, A. E.; Peinder, P. de; Boelens, R.; van Es, D. S.; Grisel, R. J. H.; Weckhuysen, B. M.; Huijgen, W. J. J.; Gosselink, R. J. A.; et al. New insights into the structure and composition of technical lignins: a comparative 23



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characterisation study. Green Chem. 2016, 18 (9), 2651–2665. (24)

Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; Davis, M. F.; Davison, B. H.; Dixon, R. A.; Gilna, P.; Keller, M.; et al. Lignin Valorization: Improving Lignin Processing in the Biorefinery. Science. 2014, 344 (6185) 1246843.

(25)

Xu, C.; Arneil, R.; Arancon, D.; Labidi, J.; Luque, R. Lignin depolymerisation strategies: towards valuable chemicals and fuels. Chem. Soc. Rev 2014, 43, 7485– 7500.

(26)

Menezes, F. F. de; Silva, R. H. F. da; Rocha, G. J. de M.; Maciel Filho, R. Physicochemical characterization of residue from the enzymatic hydrolysis of sugarcane bagasse in a cellulosic ethanol process at pilot scale. Ind. Crops Prod. 2016, 94, 463–470.

(27)

Ibrahim, V.; Mamo, G. Enzymatic monitoring of lignin and lignin derivatives biooxidation. J. Microbiol. Methods 2016, 120, 53–55.

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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). L1 Temperature (°C) 130 Catalysta b Severity factor 2.52 Solubilization rate in black liquor (%) Lignin 79.6 Hemicelluloses 56.1 Cellulose 28.2 Chemical compositional (% w/w, dry basis) Cellulose 2.9 (2.1) Hemicelluloses 13.6 (1.6) Total lignin 81.2 (3.3) Ashes 2.0 (0.4) 22.8 Higher Heating Value (MJ/Kg) a b

L2

L3

L4

130 AQ 2.59

170 3.74

170 AQ 3.72

73.3 41.1 9.2

83.7 57.9 22.8

87.6 53.8 16.3

0.7 (0.1) 10.1 (2.5) 88.1 (2.8) 1.2 (0.3) 23.3

1.5 (0.2) 16.6 (0.2) 81.0 (0.1) 1.0 (0.1) 24.2

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

Anthraquinone (AQ) concentration of 0.15% (w/w). Data from Nascimento et al. (2016).

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Table 2. Elemental composition (C/H/O) and C9-formulae of the alkaline lignins (L1-L4) isolated from sugarcane bagasse. Lignins L1 L2 L3 L4

%C 55.7 57.2 59.4 63.2

%H 6.9 6.5 7.0 6.7

%O 36.6 35.3 33.0 29.3

OCH3 1.38 1.35 1.07 1.01

C9-formulae 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

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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 bagasse. Label Compounds 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

Origin

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:Sa S/G ratioa Ph-C0-2/Ph-C3b

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

L1 (130 ºC) 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

L2 (130 ºC/AQ) 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

L3 (170 ºC) 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

L4 (170 ºC/AQ) 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

13:36:51 1.4 5.6

15:37:49 1.3 6.4

30:33:37 1.1 8.5

33:33:34 1.0 9.1

H: p-hydroxyphenyl units; G: guaiacyl units; S: syringyl units; PCA: p-coumarates; FA: ferulates. a Estimated 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). b Ratio of lignin phenolic markers bearing side-chains of 0–2 carbon atoms respect to lignin phenolic markers bearing side-chains of 3 carbon atoms. 27



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Table 4. Assignments of the lignin 13C–1H correlation peaks in the 2D HSQC spectra of the alkaline lignins from sugarcane bagasse. Label Bβ Cʹβ Cβ -OCH3 Aγ Iγ Bγ Cγ Aα Eα Fα Cʹα Aβ(G)

δC/δH (ppm) 53.1/3.43 53.3/2.12 53.5/3.05 55.6/3.73 59.4 /3.40 and 3.72 61.3/4.08 62.6/3.67 71.0/3.81 and 4.17 71.8/4.85 78.6/5.57 81.2/5.01 81.7/4.89 83.4/4.27

Cα Aβ(S)

84.8/4.65 85.9/4.10

Bα S2,6 G2 FA2 G5/G6

86.8/5.43 103.8/6.69 110.9/6.99 111.0/7.32 114.9/6.72 and 6.94 118.7/6.77 115.1/6.29 116.5/6.37 115.5/6.77 122.3/7.08 127.5/7.16 129.8/7.50 143.9/7.48

PCAβ FAβ PCA3,5 FA6 H2,6 PCA2,6 PCAα/FAα

Polysaccharide cross-peak signals X5 63.2/3.26 and 3.95 X2 72.9/3.14 X3 74.1/3.32 X4 75.6/3.63

Assignment Cβ–Hβ in β-5ʹ phenylcoumaran substructures (B) Cβ–Hβ in β–βʹ tetrahydrofuran substructures (Cʹ) Cβ–Hβ in β–βʹ resinol substructures (C) C–H in methoxyls Cγ–Hγ in β–O–4ʹ substructures (A) Cγ–Hγ in cinnamyl alcohol end-groups (I) Cγ–Hγ in β-5ʹ phenylcoumaran substructures (B) Cγ–Hγ in β–βʹ resinol substructures (C) Cα–Hα in β–O–4ʹ substructures (A) Cα–Hα in α–O–4ʹ substructures (E) Cα–Hα in spirodienone substructures (F) Cα–Hα in β–βʹ tetrahydrofuran structures (Cʹ) Cβ–Hβ in β–O–4ʹ substructures (A) linked to a G unit Cα–Hα in β–βʹ resinol substructures (C) Cβ–Hβ in β–O–4ʹ substructures linked (A) to a S unit Cα–Hα in β-5ʹ phenylcoumaran substructures (B) C2–H2 and C6–H6 in etherified syringyl units (S) C2–H2 in guaiacyl units (G) C2–H2 in ferulic acid (FA) C5–H5 and C6–H6 in guaiacyl units (G) Cβ–Hβ in free p-coumaric acid (PCA) Cβ–Hβ in free ferulic acid (FA) C3–H3 and C5–H5 in p-coumaric acid (PCA) C6–H6 in ferulic acid (FA) C2,6–H2,6 in p-hydroxyphenyl units (H) C2–H2 and C6–H6 in p-coumaric acid (PCA) Cα–Hα in p-coumaric (PCA) and ferulic (FA) acids

C5–H5 in β-D-xylopyranoside C2–H2 in β-D-xylopyranoside C3–H3 in β-D-xylopyranoside C4–H4 in β-D-xylopyranoside

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Table 5. Structural characteristics from integration of 13C-1H correlation peaks in the HSQC contour map of the alkaline lignins (L1-L4) from sugarcane bagasse. L1 (130 °C)

L2 (130 °C/AQ)

L3 (170 °C)

L4 (170 °C/AQ)

Lignin inter-unit linkagesa β-O-4´ aryl ethers (A) 43 35 8 4 Phenylcoumarans (B) 4 3 1 1 4 3 1 1 Resinols (C) 6 5 3 2 Tetrahydrofurans (C´) α,β-diaryl ethers (E) 5 4 1 1 Spirodienones (F) 1 1 0 0 2 2 1 1 Cinnamyl alcohol end-groups (I) b Lignin aromatic units H (%) 5 7 27 29 G (%) 26 29 29 30 S (%) 69 64 44 41 S/G ratio 2.6 2.2 1.5 1.4 p-Hydroxycinnamatesc p-Coumarates (%) 16 16 3 1 Ferulates (%) 11 10 5 3 p-Coumarates/ferulates ratio 1.5 1.6 0.6 0.5 a Expressed as per 100 aromatic units. b Molar percentages (H+G+S=100). c p-Coumarate and ferulate molar contents expressed as percentages of lignin content (H+G+S=100).

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Table 6. Data values of the peaks from chromatogram lignins. Lignins L1 130 °C L2 130 °C/AQ L3 170 °C

L4 170 °C/AQ

Range peak (mL) 42-65 70-82 43-64 70-80 43-63 70-78 83-88 88-96 44-63 70-77 82-88 88-94

Central point (mL) 49 73 50 73 52 73 86 91 52 73 85 91

Area peak (%) 79 21 80 20 68 18 5 9 75 15 5 5

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FIGURE CAPTIONS Figure 1. Py-GC/MS chromatograms of the alkaline lignins isolated from sugarcane 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). 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 sugarcane 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); cinnamyl alcohol end-groups (I). 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 sugarcane 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); ferulic acid (FA). Figure 4. Overlapped standard chromatograms. (a) tannic acid, 3-(2-Pyridyl)-5,6-diphenyl1,2,4-triazine-4′,4′′-disulfonic acid sodium salt from Sigma Aldrich® and a lignin with known molecular weight; (b) hydroquinone and ferulic acid. Figure 5. Lignin chromatograms (L1-L4) from gel permeation analyses in AKTA system. (a) chromatograms arranged in stack mode (40-160 mL). (b) Overlapped chromatograms of the L1-L4 (40-100 mL).

31



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TOC/Abstract Graphic. Scheme of the obtainment process of the alkaline lignins isolated from sugarcane bagasse and the major structural features of the lignins assessed in this study.

32


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(a)

Relative response

15

5

2

8 4

1

20

25

910

30

25 26

21

13

29

24 22

19

7 6

3

20

14 18

23

35

40

28

31

20

15

4

22 19

3

20

25

17 14 910 13 18

6

30

35

21

32

24

25 23

40

26

29 30

28

45

20

13 18 10 14 9

6

25

30

21

35

28

40

31 33 AQ 3435

45

50

55

(d)

11+12

2

5

8

15

20

4

1

3

55

23

30

Retention time (min)

22 24

19

50

32

25 26

7

31 33 3435


7

100%

Relative response

1

2

55

(c)

7 8

4

29

22 24

19

3

50

11+12

5

8

33 3435

45

20

5

1


100%

15

2

32 30

(b)

11+12

100%

Relative response

11+12

100%

Relative response

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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20

17

6

25

9 14 18 10

30

35

32

29 25 26 21

23

40

28

30

45

AQ 31 33 3435


50

Figure 1


55


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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

C'β δ C

(b)

Bβ

Aγ

Iγ X5

60

Cα 5

4

(c)

Bβ

δH

Cβ

C'β δ C

Iγ X5

γ

δH

γ′

Cγ

X3 X4

Eβ

C'

OMe

Cα 4

X5

3

δH

70

OMe

O

X2 α

HO

O

β γ

1′

γ′

α′ β′

OMe O

Aβ(S) 4

F 3

OH

γ

80

Cα 5

4′

E

X4

C'α

Bα

O

O

Aβ(G)

Aβ(S)

β

α

60

X3

Aβ(G)

O

γ

OMe

Cγ

80

HO

OMe

Aγ

Eβ

C'α

OMe

C

C'β δ C

Aα

X2

O

α

O

4′

X5 70

O

α′

β

HO

OMe

β′

γ

β′

O

OMe

OH

γ′

O α′

α

Bβ C β

X5

B

O

β

Iγ

OMe

OMe

3

(d)

60

Aα

5

A

80

4

OMe

O α

O

-OMe Aγ

HO

O

5

-OMe

Bα

X2

Aβ(S)

Bα 3

70

Aβ(G)

Cα

Aβ(S)

Bα

Fα

β

OMe

X3 X4

C'α

5′

γ

4′

O

O

Eβ

Aβ(G)

β

α

X5

Cγ

80

γ

OMe

Aα

70

X3 X4

Eβ

Bγ

HO

60

Iγ X5

X2

Cγ

HO

Aγ

X5

Aα

Fα

C'β δ C

-OMe

-OMe

C'α

Cβ

Page 34 of 39

β

α

OH OMe O

I

δH

Figure 2


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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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)


S'2,6

FA2

G2 G5/6 FA6

PCA2,6

G6

S2,6

δC

H3,5

110

PCAβ/FAβ PCAβ(f ) PCA3,5

H2,6

7.5

(c)

7.0

140

6.5

6.0

FA2

G2 G5/6 FA6

G6

PCA2,6

PCAβ/FAβ PCAβ(f ) PCA3,5

7.0

6.5

6.0

δH

PCAβ(f ) PCA3,5

H2,6

7.0

6.5

6.0

OMe O

H

120

G6 PCA2,6

OMe

O

MeO

OMe O

S

S′

120

O

γ

α

β

130 OH

PCAα(f )

6.0

α

O

O

PCAβ(f ) PCA3,5

140

6.5

R

OH

110

H2,6

7.0

G

MeO

δC

G2 H3,5

FA2

OH

α

O

δH

S2,6

OH

120

α

S'2,6

7.5

α

140

PCAα(f )

(d)

110

130

G5/6

140

PCAα(f )

7.5

110

130

H2,6

PCAβ/FAβ

G6

PCA2,6

7.5

δC

H3,5

H3,5

G2

FA6

δH

S2,6

S'2,6

FA2

120

δC

S2,6

S'2,6

G5/6

130

FAα/PCAα

(b)

PCA

O

O

γ β

α

O

OMe

FA

δH

Figure 3



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Overlapped standard chromatograms. (a) tannic acid, 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. 279x215mm (300 x 300 DPI)


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Overlapped standard chromatograms. (b) hydroquinone and ferulic acid. 279x215mm (300 x 300 DPI)



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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Lignin chromatograms (L1-L4) from gel permeation analyses in AKTA system. (a) chromatograms arranged in stack mode (40-160 mL). 228x254mm (300 x 300 DPI)


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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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Lignin chromatograms (L1-L4) from gel permeation analyses in AKTA system. (b) Overlapped chromatograms of the L1-L4 (40-100 mL). 304x254mm (300 x 300 DPI)


Alkaline Pretreatment Severity Leads to Different Lignin Applications

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