Structural characteristics of lignin in pruning residues of olive tree (Olea europaea L.)

Samples

The OTPs (Olea europaea L., Picual variety), composed of thin branches (<5 cm diameter), were collected after fruit-harvesting from a local orchard in Jaén, Southern Spain. The samples were manually debarked, chipped, homogenized, air-dried and grounded in a IKA MF10 knife mill (IKA, Staufen, Germany) to pass 1 mm screen. The air-dried samples were then successively Soxhlet extracted with acetone (8 h), methanol (8 h) and water (3 h). Finally, the extractive free samples were finely ball-milled in a Restch PM-100 planetary mill (Retsch, Haan, Germany) equipped with a 500 ml agate jar and agate balls (20×20 mm), at 400 rpm, during 10 h (alternating 10 min of pause every 20 min of milling). The Klason lignin content was estimated as the residue after sulfuric acid hydrolysis of the pre-extracted material, corrected for ash and protein content, according to the TAPPI method T222 om-88 (Tappi, 2004). The acid-soluble lignin was determined at 205 nm based on the extinction coefficient of 110 l cm⁻¹ g⁻¹. The average yields of three replicates are presented.

Milled wood lignin (MWL) according to Björkman (1956)

Around 50 g of ball-milled material were extracted with 1 l of dioxane-water (96:4, v/v) under continuous stirring in the dark for 24 h. The solution was centrifuged and the lignin containing supernatant was collected. The extraction process was repeated twice with fresh dioxane-water solution each time and the supernatants were combined and subsequently evaporated at 40°C at reduced pressure until dryness. The crude MWL was then purified according to the solubilization/precipitation procedure described elsewhere (del Río et al. 2012). The MWL yield was around 20% of the total lignin content.

2D-NMR analyses

One hundred milligram of finely ball-milled OTP sample (the whole cell wall, OTP_total) were swelled in 0.75 ml of DMSO-d₆ and the resulting gel was submitted to spectroscopy according to the literature (Kim et al. 2008; Rencoret et al. 2009a). In the case of MWL, 50 mg of sample was completely dissolved in 0.75 ml of DMSO-d₆, forming a clear solution. 2D-NMR-HSQC experiments were recorded at 300K on an AVANCE III 500 MHz instrument (Bruker, Karlsruhe, Germany) fitted with a 5 mm TCI gradient cryoprobe at the NMR facilities of the General Research Services of the University of Seville. The HSQC spectra were acquired using the standard adiabatic pulse program of Bruker “hsqcetgpsisp2.2”. The spectra were obtained from 10 to 0 ppm in ¹H dimension, with acquisition times of 145 ms (MWL) and 100 ms (OTP_total) and a recycle delay (d1) of 1 s. For the ¹³C dimension, the spectral width was from 165 to 0 ppm, being collected 256 increments of 32 scans for total experiment times of 2 h 40 min and 2 h 34 min for MWL and gel sample, respectively. The ¹J_CH used was 145 Hz. Processing is based on the typical matched Gaussian apodization in ¹H (LB=−0.1 and GB=0.001) and a squared cosine bell in ¹³C (LB=0.3 and GB=0.1). The signal of residual DMSO served as an internal reference (δ_C/δ_H 39.5/2.49). Lignin correlation signals in the HSQC spectrum of the OTP-MWL were assigned according to the literature (Rencoret et al. 2008, 2018; Ralph et al. 2009; Lourenço et al. 2015). A semi-quantitative analysis of lignin units and linkages, based on measuring the contour volume integrals of correlation signals, was performed according to Rencoret et al. (2013). Concerning the aromatic signal evaluation of S_2,6, H_2,6, the integrals were halved to get the equivalent for the G₂ signal. The different inter-unit linkages were quantitated via the volume integrals of the A_α, B_α, C_α, D_α and F_α correlation signals, corresponding to chemically analogous C_α/H_α with similar ¹J_CH coupling values. The relative abundance of cinnamyl alcohol end-groups (I) were estimated by integration of the signal I_γ, whereas the abundance of cinnamaldehyde end-groups (J) was determined by integrating the signal J_β and comparing it with I_β.

Thioacidolysis and subsequence Raney-nickel desulphuration of MWL

The method was described by Rolando et al. (1992). The reagent was immediately prepared before use by pouring 0.25 ml of BF₃ etherate and 1 ml of ethanetiol into a 10 ml flask containing some dioxane (1–2 ml), and the final volume was adjusted to 10 ml with dioxane resulting in a 0.2 M BF₃ etherate in dioxane/ethanethiol (8.75:l, v:v) solution. 5 mg of MWL were treated with 5 ml of thioacidolysis reagent in a 10 ml screw-cap reaction vial under N₂ at 100°C during 4 h with occasional shaking. The reaction products together with a 15 ml of water were transferred into a separatory funnel with CH₂Cl₂ via rinsing the vial (3×5 ml) and the internal standard was added (0.2 ml, 1 mg ml⁻¹ octadecane in dioxane). The aqueous phase was adjusted to pH 3–4 by the addition of aqueous 0.4 M NaHCO₃ and the mixture was vigorously mixed. The aqueous phase was extracted twice with CH₂Cl₂. The combined CH₂Cl₂ fractions were dried over anhydrous Na₂SO₄, and then evaporated to dryness in a rotary evaporator at 40°C. The residue was completely redissolved in 1 ml of CH₂Cl₂ and 20 μl was trimethylsilylated with 50 μl with N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and 10 μl of pyridine. The GC analysis of the thioacidolysis monomers was performed with a gas chromatography-mass spectrometry (GCMS)-QP2010 Ultra instrument (Shimadzu, Japan) via a capillary column (DB-5HT, 30 m×0.25 mm I.D. 0.10 μm film thickness). Temperature program: 50°C (1.5 min)→90°C (2.0 min) at 30°C min⁻¹, → 250°C (8°C min⁻¹), 15 min holding time. The injector and transfer line temperatures were 250°C and 300°C, respectively. He was the carrier gas (1 ml min⁻¹). For dimer analysis, 0.9 ml of the CH₂Cl₂ solution containing the thioacidolysis products was subjected to a Raney-nickel desulfuration according to Lapierre et al. (1991). The dimeric compounds were trimethylsilylated with BSTFA and pyridine and then analyzed by GCMS on a Saturn 4000 (Varian, Walnut Creek, CA, USA) equipment. A short capillary column DB-5HT (12 m) was applied. The temperature program: 50°C→90°C (2 min) at 30°C min⁻¹, 90°C→250°C (8°C min⁻¹), holding time 2 min. The GCMS transfer line and injector temperatures were 300°C and 250°C, respectively, and He served as carrier gas (2 ml min⁻¹). Identification of dimers was done according to the literature (Lapierre et al. 1991; Rencoret et al. 2008; del Río et al. 2009; Kishimoto et al. 2010; Yue et al. 2017).

2D-NMR of OTP and MWL

The OTP_total was analyzed in situ by 2D-HSQC in the gel state according to Kim et al. (2008) and Rencoret et al. (2009a), and the spectrum was compared with that of the MWL-OTP. The aliphatic-oxygenated (δ_C/δ_H 50–90/2.5–6.0) and the aromatic/unsaturated (δ_C/δ_H 100–155/5.8–7.8) regions of the HSQC spectra and that of MWL are presented in Figure 1. The HSQC spectrum of OTP_total (Figure 1a,b) reveal signals from carbohydrates and lignin. The former are dominated by hemicelluloses as cellulose signals cannot be detected in the gel state due to its high crystallinity (Kim et al. 2008). The main carbohydrate signals correspond to xylans (β-d-xylopyranoside X₂, X₃, X₄, X₅), including O-acetylated xylans (2- and 3-O-acetyl-β-d-xylopyranoside, X′₂ and X′₃), and 4-O-methyl-α-d-glucuronic acid (U₄). The signals in the MWL spectrum belong almost exclusively to lignin (Figure 1c,d). Although some modifications in lignin structure and its S/G ratio have been reported as a result of the isolation procedure (Balakshin et al. 2008; Capanema et al. 2015), comparison of the isolated MWL and the OTP_total spectra indicates that the former can be considered as representative of the native lignin in the OTP residue. The lignin correlation signals assigned in the HSQC spectra are listed in Table 1, and the lignin substructures identified are depicted in Figure 2.

Figure 1:

Side-chain (δ_C/δ_H 50–90/2.5–6.0, top) and aromatic/unsaturated (δ_C/δ_H 100–155/5.8–7.8, bottom) regions of the 2D HSQC NMR spectra of OTP_total (a and b) and OTP-MWL (c and d). The assignments of the lignin signals are listed in Table 1 and the lignin structures identified are depicted in Figure 2.

Figure 2:

Main lignin structures and units identified in the 2D HSQC NMR spectra of OTP_total and OTP-MWL: β-O-4′ alkyl-aryl ethers (A); β-5′ phenylcoumarans (B); β-β′ resinols (C); 5-5′ dibenzodioxocins (D); β-1′ spirodienones (F); cinnamyl alcohol end-groups (I); cinnamaldehyde end-groups (J); p-hydroxyphenyl units (H); guaiacyl units (G); syringyl units (S); Cα-oxidized syringyl units (S′).

The aliphatic-oxygenated region of the spectra gives information about the different inter-unit linkages of lignin. Here, the most intense correlation signals correspond to β-O-4′ alkyl aryl ethers (structure A) and methoxy groups of the benzene ring in G and S type lignin units. β-O-4′ linkages were found involving both G- and S- lignin units as two different C_β/H_β correlation signals are seen, depending on whether the 4′-O- is forming part of a guaiacyl (A_β(G)) or a syringyl (A_β(S)) lignin unit. Signals from carbon-carbon linkages, such as β-5′ phenylcoumarans (structure B) and β-β′ resinols (structure C), were also readily observed in the HSQC spectra of the OTP_total and the MWL. Interestingly, two clearly differentiated signals for the C_α/H_α correlations of β-5′ phenylcoumarans, at δ_C/δ_H 86.8/5.44 (B_α(G)) and at δ_C/δ_H 87.7/5.59 (B_α(S)), are detected only in the MWL spectrum. Although double phenylcoumaran C_α/H_α signals that could belong to phenolic and non-phenolic β-5′ structures can be observed in softwood lignin, in the OTP lignins they largely correspond to phenylcoumaran substructures involving G- or S-lignin units, as already shown for other hardwood lignins (Rencoret et al. 2018). Several signals are better resolved in the HSQC spectrum of MWL, such as 5-5′ dibenzodioxocins (structure D), and β-1′ spirodienones (structure F). The presence of dibenzodioxocin units is remarkable, which are important branching points in softwoods lignins (Karhunen et al. 1995), and which are seldom observed in hardwood lignins (Ämmälahti et al. 1998; Kukkola et al. 2004; Rencoret et al. 2009b). Dibenzodioxocin structures involve condensed 5-5 biphenyl C-C bonds, which are especially resistant to most chemical pretreatments, thus the reactivity of OTP lignin is probably lower than in other hardwoods.

In the aromatic/unsaturated region of the spectra, the typical correlation signals of S-, G-, and H-lignin units are seen, together with other signals from lignin end-groups, including cinnamyl alcohols (I) and cinnamaldehydes (J), and spirodienone substructures (F). According to the NMR data, OTP lignin is mainly composed of G- and S-lignin units in almost equal amounts (S/G ratio of 0.9–1.0), and with minor amounts of H-units (only 1–2% of the total lignin units).

The essential data estimated from the HSQC spectra of OTP_total and MWL are presented in Table 2, which include the quantification of inter-unit linkages and cinnamyl end-groups (as per 100 aromatic units, and as percentages of total linkages), the relative abundances of the H-, G-, and S-lignin units, and the S/G ratios. In general terms, the HSQC data indicate again that the MWL represents well the total lignin in the cell wall. It is also obvious that the MWL spectra have a higher resolution, an enhanced signal-to-noise ratio (i.e. minor lignin signals are detectable), and show correlation signals that appear overlapped with those from carbohydrates in the spectrum of OTP_total. In OTP lignin, the β-O-4′ alkyl-aryl ether linkages (A) are predominant accounting for 75% of the total detected inter-unit linkages, followed by β-5′ phenylcoumarans (12%), and β-β′ resinols (8%), together with lower proportions of β-1′ spirodienones (3%), and 5-5′ dibenzodioxocins (2%). Cinnamyl end-groups, including cinnamyl alcohols and cinnamaldehydes, accounted for 2 and 3% with respect to the total side-chains, respectively. The relatively high abundance of condensed (C-C) linkages, phenylcoumarans, dibenzodioxocins and G-lignin units (51%) are indicative that OTP lignin is probably less reactive than other hardwoods with higher S/G ratios and less C-C linkages, as, for example, in eucalyptus wood lignins (Rencoret et al. 2007, 2008; Prinsen et al. 2012). Lignocellulosic feedstocks with lower S/G lignin ratios are more difficult to delignify by alkaline pulping and therefore require higher amounts of alkali (Chang and Sarkanen 1973; Tsutsumi et al. 1995; del Río et al. 2005).

The 2D-NMR data demonstrate again the structural differences between the native lignin and the technical lignins of OTP by Santos et al. (2017). The lignins obtained from bioethanol production process (after steam explosion, followed by simultaneous saccharification and fermentation of the released sugars), contain less β-O-4′ alkyl-aryl ethers (49% of all detected linkages) and increased amounts of phenylcoumarans (15%) and, particularly, resinols (34%), because of the cleavage of β-O-4′ linkages during steam explosion (Santos et al. 2017). The lignins obtained after alkaline pulping of OTP suffered more severe degradation with a drastic decrease of all types of lignin linkages, particularly of β-O-4′ linkages, being resinols the only ones detected in alkaline lignins, which also have more phenolic content (Santos et al. 2017). The technical lignins recovered after bioethanol or alkaline pulping were highly enriched in syringyl units (S/G ratios around 4.0–5.7) compared with the native lignin (S/G around 1), most probably due to the enrichment of resinol substructures.

Analysis of lignin monomers and condensed linkages

2D-HSQC provided semiquantitative information regarding the lignin composition and the main inter-unit linkages. The data with this regard obtained by thioacidolysis are quantitative, where the yields of monomeric products are indicative for the selective cleavage of β-O-4′ linkages. The analysis of dimeric products gives information on the ‘condensed’ (C-C) and diaryl ether linkages including 5-5′, 4-O-5′, β-1′, β-5′, and β-β′ linkages. The chromatogram in Figure 3 shows the monomeric thioacidolysis degradation products as TMS ether derivatives. Thioacidolysis released similar amounts of G and S type units (710 and 830 μmoles g⁻¹ lignin, respectively), with small amounts of H monomers (17 μmoles g⁻¹ lignin), as already observed by 2D-NMR. As expected, the S/G ratio of 1.2 obtained upon thioacidolysis was slightly higher than that estimated by 2D-NMR, as thioacidolysis only releases lignin monomers involved in β-O-4′ bonds, which are more abundant among the syringyl units. Therefore, it should be indicated that the thioacidolysis data underestimates the amount of the condensed units.

Figure 3:

Total ion chromatogram of the monomeric thioacidolysis degradation products (as TMS ethers) released from OTP-MWL.

The chromatogram of the thioacidolysis dimeric products (as TMS ethers) is shown in Figure 4, together with their structures, and their mass spectral data, and their relative abundances are summarized in Table 3. The main dimeric compounds released are of the type 5-5′ (peaks 1–3), 4-O-5′ (peaks 4 and 6), β-1′ (peaks 5, 9, 11, 14 and 24), β-5′ (peaks 7, 8, 10, 15, 16, 21 and 22), and β-β′ (peaks 12, 13, 17–19 and 25). The 5-5′ dimers arise in part from dibenzodioxocin structures after the breakdown of their α-O-4′ and β-O-4′′ ethers, and also from others 5-5′ etherified and non-etherified units (Capanema et al. 2004). The β-1′ dimers are produced from spirodienones after the cleavage of the α-O-α′ ether bond whereas the β-β′ tetralin dimers arise from the cleavage of α-O-γ ether bonds in resinols and subsequent recyclization through the formation of an additional bond between Cα and C₆ (Lapierre et al. 1995). Two types of β-5′ dimers were detected: (1) those simply derived from an unopened phenylcoumaran, such as dimer 22 that was recently established as authentic β-5′ dimer by synthesized standards (Yue et al. 2017), and (2) those β-5′ dimers arising from the breakdown of α-O-4′ ether in phenylcoumaran substructures (dimers 7, 8, 10, 15, 16 and 21).

Figure 4:

Total ion chromatogram of the dimeric products (as TMS ethers) obtained by thioacidolysis degradation followed by Raney-nickel desulphuration of OTP-MWL.

The mass spectral data are detailed in Table 3.

The relative proportions of the different dimers released upon thioacidolysis-desulfuration are listed in Table 4. The quantity of the most prominent dimers (based on all dimers) are β-β′ tetralin (≈37%) and β-5′ compounds (≈25%), followed by β-1′ (≈17%), 5-5′ (≈13.3%) and 4-O-5′ (≈7.8%) structures. 2D-NMR analysis, however, showed a higher abundance of β-5′ than β-β′ structures in OTP lignin. One possible explanation is that β-5′ linkages occur among G units (65% of all β-5′ dimers involve two G-units), that can also be connected to other lignin units, which are not degraded by thioacidolysis resulting in underrepresented β-5′ substructures among thioacidolysis degradation products. On the contrary, the vast majority of the β-β′ linkages in OTP lignin are of the S-S type (85% of all β-β′ linkages) (Table 4). This observation is also true for lignins in other plants (Rencoret et al. 2008; del Río et al. 2009). Such S-S dimers can only be integrated into the lignin macromolecule via their 4-OH groups, which leads to formation of β-O-4′ alkyl-aryl ethers, which are cleaved by thioacidolysis. β-β′ resinols are mainly syringaresinol and this could explain the high S/G ratios of OTP technical lignins obtained by steam explosion and alkaline pulping (Santos et al. 2017), because these pretreatments mostly cleave β-O-4′ linkages and produce a lignin highly enriched in β-β′ resinols.