Chemical, Thermal and Antioxidant Properties of Lignins Solubilized during Soda/AQ Pulping of Orange and Olive Tree Pruning Residues

molecules Article Chemical, Thermal and Antioxidant Properties of Lignins Solubilized during Soda/AQ Pulping of Orange and Olive Tree Pruning Residues María E. Eugenio 1, Raquel Martín-Sampedro 1, JoséI. Santos 2, Bernd Wicklein 3and David Ibarra 1,*   Citation: Eugenio, M.E.; Martín-Sampedro, R.; Santos, J.I.; Wicklein, B.; Ibarra, D. Chemical, Thermal and Antioxidant Properties of Lignins Solubilized during Soda/AQ Pulping of Orange and Olive Tree Pruning Residues. Molecules 2021,26, 3819. https://doi.org/10.3390/ molecules26133819 Academic Editors: Margit Schulze and Birgit Kamm Received: 31 May 2021 Accepted: 18 June 2021 Published: 23 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Forest Research Center (INIA, CSIC), Ctra. de la Coruña Km 7.5, 28040 Madrid, Spain; [email protected] (M.E.E.); [email protected] (R.M.-S.) 2NMR of Facility of Research (SGIker), University of the Basque Country (UPV/EHU), Avenida Tolosa 72, 20018 Donostia-San Sebastián, Spain; [email protected] 3Materials Science Institute of Madrid (ICMM), Consejo Superior de Investigaciones Científicas (CSIC), Sor Juana Inés de la Cruz 3, 28049 Madrid, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-913473948 Abstract: Some agroforestry residues such as orange and olive tree pruning have been extensively evaluated for their valorization due to its high carbohydrates content. However, lignin-enriched residues generated during carbohydrates valorization are normally incinerated to produce energy. In order to find alternative high added-value applications for these lignins, a depth characterization of them is required. In this study, lignins isolated from the black liquors produced during soda/anthraquinone (soda/AQ) pulping of orange and olive tree pruning residues were analyzed by analytical standard methods and Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (solid state 13 C NMR and 2D NMR) and size exclusion chromatography (SEC). Thermal analysis (thermogravimetric analysis (TGA), differential scanning calorimetry (DSC)) and antioxidant capacity (Trolox equivalent antioxidant capacity) were also evaluated. Both lignins showed a high OH phenolic content as consequence of a wide breakdown of β -aryl ether linkages. This extensive degradation yielded lignins with low molecular weights and polydispersity values. Moreover, both lignins exhibited an enrichment of syringyl units together with different native as well as soda/AQ lignin derived units. Based on these chemical properties, orange and olive lignins showed relatively high thermal stability and good antioxidant activities. These results make them potential additives to enhance the thermo-oxidation stability of synthetic polymers. Keywords: lignin purity and composition; lignin structural characterization; lignin thermal properties; lignin antioxidant properties; olive tree pruning; orange tree pruning; soda/AQ pulping 1. Introduction Agroforestry residues are one of the major resources of unexploited potential lignocellulosic feedstocks. Most of these residues are underutilized or burned in situ, generating serious environmental pollutions. Thus, the circular bioeconomy, as a way to reach a sustainable development, should be a chance to manage these lignocellulosic residues towards the production of energy and high added-value products. In addition, the success of this target should have a significant impact on the mitigation of both petroleum consumption and greenhouse gas (GHG) emissions. An interesting and abundant agroforestry residue is orange tree pruning, the result of trimming away unneeded branches of orange trees. Orange tree cultivation is significant in Mediterranean countries, including Spain, where 2.82 × 10 6 tons of oranges are produced annually [ 1 ]. This agricultural activity generates a quantity of orange tree pruning residue around 2.25 × 10 6 tons each year [ 1 ]. In the same way, Spain is also the main olive oil producer worldwide, with about 2.3 M ha of olive trees cultivated that generates Molecules 2021,26, 3819. https://doi.org/10.3390/molecules26133819 https://www.mdpi.com/journal/molecules Molecules 2021,26, 3819 2 of 21 1.3 tons ha−1year−1 (3.0 tons ha −1 biennial pruning) of olive tree pruning residue [ 2 ]. These tree pruning residues, which include a main woody fraction and a remaining fraction consisting of leaves and thin branches, have been extensively evaluated for their valorization due to its high carbohydrates (cellulose and hemicellulose) content. Then, cellulose has been used for production of bioethanol [ 3 ], cellulosic pulp and advanced materials such as nanocellulose, among others [ 1 , 4 – 6 ]. Hemicelluloses have been exploited for production of xylitol and xylooligosacharides [ 7 , 8 ]. However, lignin-enriched residues generated during these processes, solubilized in black liquors from pulp and paper industry or as nonfermentable residues resulting from bioethanol production, are currently underutilized, being normally incinerated to generate energy i.e., heat and electricity, which supplies part of the demands of the pulp and paper and bioethanol industries. Lignin is a complex aromatic macromolecule composed by p-hydroxyphenyl (H) (from p-coumaryl alcohol), guaiacyl (G) (from coniferyl alcohol), and syringyl (S) (from sinapyl alcohol) phenylpropane units [ 9 ]. These units are linked through a variety of inter-units linkages, including aryl ether and carbon–carbon (C–C) bonds [ 9 ]. Among them, β -aryl ether linkage ( β -O-4’) represents the most abundant bond, followed by resinol ( β - β ’) and phenylcoumaran ( β -5’) among others. Depending on the lignocellulosic feedstock, extraction process and conditions, lignin displays a range of properties, i.e., aromatic structure, chemical functionalities, hydrophobicity, thermal stability and treatability, antioxidant, etc., which makes it interesting as a feedstock to produce chemicals and materials [ 10 ]. Then, lignin has been assessed for polymer and material applications such as carbon materials, resins, hydrogels, and polymer modifiers and/or as a source to produce bulk and fine chemicals such as benzene, toluene and xylene (BTX), phenols and vanillin [ 10 , 11 ]. Therefore, in addition to helping to the competitive and sustainable production of energy and high added-value products from carbohydrates, the valorization of lignin-enriched residues generated during carbohydrates transformation will also contribute for the implementation of the circular bioeconomy, which aims to maximize the usage and value of all raw materials, products, and residues. Kraft pulping with NaOH and Na 2 S is the alkaline process most widely employed for delignification of woods, i.e., hardwoods and softwoods, in the pulp and paper production [ 12 ]. Yearly, 120,000 tons of kraft lignin is generated [ 13 ]. On the other hand, soda/anthraquinone (soda/AQ) pulping, with AQ as a pulping additive to limit the carbohydrate degradation [ 12 ], is generally used for agriculture residues [ 14 ]. In this sense, soda/AQ pulping has been extensively used to produce paper pulp from orange and olive tree pruning residues [ 1 , 6 ]. Although smaller volumes of soda lignin are produced annually (5000 tons) compared to kraft lignin [ 13 ], soda lignin is sulfur-free, which makes it more attractive for the production of bio-based products. Nevertheless, prior to its valorization, a thorough knowledge of the purity, the type and proportion of the different inter-unit linkages (either native or derived extraction process linkages), the content of syringyl and guaiacyl units, the molecular weight, the hydroxyl proportion, and the thermal behavior is required to select the best way for lignin valorization. In this sense, multitude of studies have analyzed different residual lignins to elucidate its features depending on the biomass origin, i.e., hardwood, softwood, and non-woody materials [ 15 – 17 ], and the extraction technology, i.e., kraft pulping, organosolv, acid hydrolysis and steam explosion [ 18 – 21 ]. Lignins solubilized during soda/AQ pulping have also been characterized [22–24]. In this study, lignins solubilized during soda/AQ pulping of orange and olive tree pruning residues were recovered and their chemical composition and structural features were analyzed by analytical standard methods and Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (solid state 13 C NMR and 2D NMR) and size exclusion chromatography (SEC). Thermal analysis (thermogravimetric analysis (TGA), differential scanning calorimetry (DSC)) and antioxidant abilities (Trolox equivalent antioxidant capacity) were also evaluated. According to all this information, possible valorization ways are discussed for these lignins. Molecules 2021,26, 3819 3 of 21 2. Results and Discussion 2.1. Chemical Composition of Lignins Soda/AQ pulping was carried out using orange and olive tree pruning residues as raw materials. The resulting soda/AQ pulps were filtered, and the black liquors containing solubilized lignins recovered. Then, acidification at pH 2.5 of soda/AQ black liquors generated orange and olive soda/AQ lignins (denoted as soda/AQ-orange and soda/AQ-olive lignins, respectively). Orange lignin sample showed a lignin content of 75.1% (64.5 ±1.6% of acid insoluble lignin and 10.6 ± 0.6% of acid soluble lignin), whereas olive lignin sample exhibited a lignin content of 69.9% (61.3 ± 0.22% of acid insoluble lignin and 8.6 ± 0.0% of acid soluble lignin). Some carbohydrates impurities were also determined in both lignins, being much higher in soda/AQ-olive lignin (11.0 ± 0.0% glucan, 4.7 ±0.0% xylan and 1.6 ± 0.0% arabinan) compared to soda/AQ-orange lignin ( 2.7 ±0.1% glucan, 3.1 ± 0.1% xylan and 0.6 ± 0.0% arabinan). Domínguez-Robles et al. [ 22 ] also described the presence of carbohydrates in soda lignins from agricultural residues such as wheat straw and barley straw as well as soda/AQ lignins from fast growing plants such as Leucaena leucocephala and Chamaecytisus proliferus. Part of the carbohydrates in the lignocellulosic materials is dissolved during alkaline processes, starting immediately when the lignocellulosic fibres come in contact with the alkaline pulping liquor and proceeding rapidly even at temperatures around 100 ◦ C, especially the xylans. At temperatures about 170 ◦ C, or higher, a random alkaline hydrolysis of glucosidic bonds may also take place [ 12 ]. These dissolved carbohydrates can partially precipitate during lignin precipitation due to their lower solubility under acidic conditions, explaining the carbohydrates content determined in both lignin samples [ 18 ]. Nevertheless, they can also be attributed to lignin-carbohydrate complexes [25]. 2.2. FTIR Spectra Analysis of Lignins The FTIR spectra of orange and olive soda/AQ lignins are showed in Figure 1, being the visible bands identified according to previous studies [ 21 – 23 , 26 – 28 ]. Both spectra displayed characteristic lignin patterns, with a broad band at 3400–3300 cm −1 attributed to the O–H stretching vibration in aromatic and aliphatic lignin structures. The bands at 2930 cm−1 and 2850 cm −1 are associated to the symmetrical and asymmetrical C–H stretching in the methyl and methylene groups, respectively, together with the band at 1455 cm−1 associated to the C–H asymmetric vibrations and deformation (asymmetric in methyl and methylene). The absorption intensity at 1700 cm −1 , corresponding to the carbonyl in the unconjugated ketones and ester groups stretching from lignin oxidation [ 26 ], was also clearly visible in both lignin samples. Nevertheless, carbonyl groups in hemicelluloses that are remaining in both lignin samples as impurities (Section 2.1) could also be contributing to this absorption [22]. Both lignin spectra displayed bands at 1595 cm −1 , 1507 cm −1 , and a shoulder at 1417 cm−1 corresponding to aromatic skeleton lignin vibrations. Other bands were associated to syringyl (S) and guaiacyl (G) units, including bands at 1315 cm −1 (aromatic ring breathing, S and G condensed units), 1264 cm −1 (G ring breathing with C=O stretching), 1208 cm −1 (G ring breathing with C–C, C–O, and C=O stretching), 1110 cm −1 (C–H bond deformation in S units), 1025 cm −1 (C–H bond deformation in G units) and 819 cm −1 (C–H out of plane deformation of S units). These lignin bands are typical of lignins from hardwoods such as eucalypt, poplar, black locust and elm materials [19,20,27,29]. In addition to carbonyl groups from hemicelluloses absorbing at 1700 cm −1 , other bands in both lignin spectra can also reflect the carbohydrate impurities determined by chemical composition analysis (Section 2.1). Thus, cellulose and hemicellulose bands at 1110 cm −1 (C–OH skeletal vibration) and 1025 cm −1 (C–O stretching vibration) were observed. Finally, the signal at 617 cm −1 is attributed to C–S bending generated from the use of H2SO4during the precipitation of lignins from soda/AQ black liquors [22]. Molecules 2021,26, 3819 4 of 21 Molecules 2021, 26, x FOR PEER REVIEW 4 of 23 observed. Finally, the signal at 617 cm−1 is attributed to C–S bending generated from the use of H2SO4 during the precipitation of lignins from soda/AQ black liquors [22]. Figure 1. FTIR spectra, 4000–600 cm−1 region, of soda/AQ-orange lignin (discontinuous line) and soda/AQ-olive lignin (continuous line). 2.3. Solid State 13C NMR Spectra Analysis of Lignins The 13C NMR spectra of orange and olive soda/AQ lignins are displayed in Figure 2, being the signals identified based on those described in bibliography [22,26,29,30]. In accordance with FTIR patterns (Section 2.2), both 13C NMR spectra exhibited a signal at δC 175 ppm, especially in soda/AQ-olive lignin, assigned to carbonyl groups and aliphatic COOR from lignin oxidation during alkaline pulping [31]. Nevertheless, hemicelluloses impurities can also contribute to this signal [32]. Moreover, the aromatic regions (around δC 152–95 ppm) of both spectra were dominated by signals corresponding to phenolic units. Then, the signals at δc 147 ppm, associated to C3 and C5 in S units and C3 and C5 in G units, and at δc 133 ppm, endorsed to C1 and C4 in S units and C1 in G units, showed a great intensity. Contrary, only a small shoulder at δC 152 ppm from non-phenolic units was visible in both spectra. This high content of phenolic units observed by 13C NMR in both lignins (slightly higher for soda/AQ-orange lignin (317.6 ± 9.2 g GAE mg−1 lignin) than soda/AQ-olive lignin (294.3 ± 1.6 g GAE mg−1 lignin) after reaction with Folin–Ciocalteau reagent) indicates an abundant degradation of them produced during soda/AQ pulping. As it is well known, the β-O-4’ substructures is the dominant linkage in native lignin [9], representing around 50−60% of all linkages. Alkaline pulping processes, either kraft or soda pulping, generate phenolic units by cleaving β-O-4’ ether bonds, which help to solubilize lignin [23,24,28]. Other signals in the aromatic region of both spectra were also visible at δC 128 ppm, C2 and C6 in p-hydroxyphenyl (H) units; δC 120 ppm, C6 in G units; δC 115 ppm, C5 in G 1100 16002100 2600 31003600 cm-1 600 3330 2930 2850 1700 1595 1507 1452 1417 1315 1264 1208 1110 1025 818 3400 617 Figure 1. FTIR spectra, 4000–600 cm −1 region, of soda/AQ-orange lignin (discontinuous line) and soda/AQ-olive lignin (continuous line). 2.3. Solid State 13C NMR Spectra Analysis of Lignins The 13 C NMR spectra of orange and olive soda/AQ lignins are displayed in Figure 2 , being the signals identified based on those described in bibliography [ 22 , 26 , 29 , 30 ]. In accordance with FTIR patterns (Section 2.2), both 13 C NMR spectra exhibited a signal at δC 175 ppm, especially in soda/AQ-olive lignin, assigned to carbonyl groups and aliphatic COOR from lignin oxidation during alkaline pulping [ 31 ]. Nevertheless, hemicelluloses impurities can also contribute to this signal [ 32 ]. Moreover, the aromatic regions (around δC 152–95 ppm) of both spectra were dominated by signals corresponding to phenolic units. Then, the signals at δ c 147 ppm, associated to C 3 and C 5 in S units and C 3 and C 5 in G units, and at δ c 133 ppm, endorsed to C 1 and C 4 in S units and C 1 in G units, showed a great intensity. Contrary, only a small shoulder at δC 152 ppm from non-phenolic units was visible in both spectra. This high content of phenolic units observed by 13 C NMR in both lignins (slightly higher for soda/AQ-orange lignin (317.6 ± 9.2 g GAE mg −1 lignin) than soda/AQ-olive lignin (294.3 ± 1.6 g GAE mg −1 lignin) after reaction with Folin–Ciocalteau reagent) indicates an abundant degradation of them produced during soda/AQ pulping. As it is well known, the β -O-4’ substructures is the dominant linkage in native lignin [ 9 ], representing around 50 − 60% of all linkages. Alkaline pulping processes, either kraft or soda pulping, generate phenolic units by cleaving β -O-4’ ether bonds, which help to solubilize lignin [23,24,28]. Other signals in the aromatic region of both spectra were also visible at δC 128 ppm, C 2 and C 6 in p-hydroxyphenyl (H) units; δC 120 ppm, C 6 in G units; δC 115 ppm, C 5 in G units; and δC 102 ppm, C 2 and C 6 in S units. Nevertheless this signal is overlapped by C 1 in hemicellulose, or shifted to δC 105 ppm of C 1 in cellulose [ 32 , 33 ], according to the carbohydrates contamination determined by chemical composition analysis (Section 2.1). Regarding to oxygenated aliphatic region (around δC 95–50 ppm), lignin signals from native β -O-4’ substructure (A), including signals at δC 81 ppm for C β in β -O-4’, δC73 ppm for C α in β -O-4’ and δC 62 ppm for C γ in β -O-4’ were detected in both spectra. A signal at δC71 ppm for Cγin native β-β’ resinol substructure (B) was also visible, together with Molecules 2021,26, 3819 5 of 21 a signal at δC 56 ppm corresponding to methoxyl groups (–OCH 3 ). Nevertheless, the existence of carbohydrates signals in this region complicates the interpretation of lignin substructures [ 32 , 33 ]. Then, in agreement with the carbohydrates impurities determined in both soda/AQ lignin samples (Section 2.1), cellulose signals at δC 81 ppm of C 4 (amorphous), δC 73 ppm of C 2 , C 3 , C 5 and δC 62 ppm of C 6 (amorphous), and hemicelluloses signals at δC 73 ppm of C 2 , C 3 , C 5 and δC 62 ppm of C 6 can interfere with the signals identified for lignin substructures. 2D NMR analysis was applied for soda/AQ lignins analysis (Section 2.4) to resolve the lignin and carbohydrates signals overlapping. Finally, the non-oxygenated alyphatic region (around δC 50–0 ppm) showed a broad signal at δC 30 ppm, generally attributed to alkyl carbons such as the γ -methyl, as well as the α - and β - methylene groups in n-propyl side chains of lignins, the acetyl of hemicelluloses and saturated aliphatic moieties associated with lipid extractives [32]. Molecules 2021, 26, x FOR PEER REVIEW 5 of 23 units; and δC 102 ppm, C2 and C6 in S units. Nevertheless this signal is overlapped by C1 in hemicellulose, or shifted to δC 105 ppm of C1 in cellulose [32,33], according to the carbohydrates contamination determined by chemical composition analysis (Section 2.1). Regarding to oxygenated aliphatic region (around δC 95–50 ppm), lignin signals from native β-O-4’ substructure (A), including signals at δC 81 ppm for Cβ in β-O-4’, δC 73 ppm for Cα in β-O-4’ and δC 62 ppm for Cγ in β-O-4’ were detected in both spectra. A signal at δC 71 ppm for Cγ in native β-β’ resinol substructure (B) was also visible, together with a signal at δC 56 ppm corresponding to methoxyl groups (–OCH3). Nevertheless, the existence of carbohydrates signals in this region complicates the interpretation of lignin substructures [32,33]. Then, in agreement with the carbohydrates impurities determined in both soda/AQ lignin samples (Section 2.1), cellulose signals at δC 81 ppm of C4 (amorphous), δC 73 ppm of C2, C3, C5 and δC 62 ppm of C6 (amorphous), and hemicelluloses signals at δC 73 ppm of C2, C3, C5 and δC 62 ppm of C6 can interfere with the signals identified for lignin substructures. 2D NMR analysis was applied for soda/AQ lignins analysis (Section 2.4) to resolve the lignin and carbohydrates signals overlapping. Finally, the non-oxygenated alyphatic region (around δC 50–0 ppm) showed a broad signal at δC 30 ppm, generally attributed to alkyl carbons such as the γ-methyl, as well as the αand βmethylene groups in n-propyl side chains of lignins, the acetyl of hemicelluloses and saturated aliphatic moieties associated with lipid extractives [32]. Figure 2. 13C NMR spectra, δC 200.0–0.0 ppm, of soda/AQ-orange lignin (discontinuous line) and soda/AQ-olive lignin (continuous line). 2.4. 2D NMR Spectra Analysis of Lignins The 13C–1H two dimensional nuclear magnetic resonance (2D NMR) spectra of orange and olive soda/AQ lignins are showed in Figures 3 and 4, respectively, including the whole spectra (δC/δH 0.0–150.0/0.0–9.0) and the spectra corresponding to the oxygenated aliphatic (δC/δH 45.0–95.0/2.5–6.0 ppm) and the aromatic (δC/δH 90.0–150.0/5.0–9.0 ppm) regions. The main 13C–1H lignin correlation signals identified in HSQC spectra are listed in Table 1, assigned according to those reported by different studies [19,20,22–24,29,34– 150 100 50 ppm 175 175 152 147 152 133 128 133 128 115 115 102 105 81 73 81 71 62 62 55 30 120 Figure 2. 13 C NMR spectra, δC 200.0–0.0 ppm, of soda/AQ-orange lignin (discontinuous line) and soda/AQ-olive lignin (continuous line). 2.4. 2D NMR Spectra Analysis of Lignins The 13 C– 1 H two dimensional nuclear magnetic resonance (2D NMR) spectra of orange and olive soda/AQ lignins are showed in Figures 3and 4, respectively, including the whole spectra ( δC / δH 0.0–150.0/0.0–9.0) and the spectra corresponding to the oxygenated aliphatic ( δC / δH 45.0–95.0/2.5–6.0 ppm) and the aromatic ( δC / δH 90.0–150.0/5.0–9.0 ppm) regions. The main 13 C– 1 H lignin correlation signals identified in HSQC spectra are listed in Table 1, assigned according to those reported by different studies [ 19 , 20 , 22 – 24 , 29 , 34 – 37 ]. The lignin substructures and carbohydrates identified are represented in Figures 5and 6. The non-oxygenated aliphatic region (around δC / δH 0.0–50.0/0.0–5.0 ppm) exhibited a variety of saturated aliphatic moieties with quite high intensities, especially in soda/AQ-olive lignin spectrum (Figure 4a). Some of these signals could be associated to extractives [ 38 ], whereas others could be assigned to groups neighbouring alkene and oxygen-containing groups such as ethers, carbonyl and alcohol, which could originate from lignin degradation [39]. The oxygenated aliphatic region of both spectra displayed the information about the different inter-unit linkages of lignin samples, including those from native and soda/AQ lignin derived linkages (Figures 3b and 4b). The predominant signals corresponding to native lignin linkages observed in both spectra were assigned to β - β ’ resinol substructures, including correlations of C α –H α (B α ), C β –H β (B β ) and the double C γ –H γ (B γ ). The resinol Molecules 2021,26, 3819 6 of 21 substructures with C–C bonds are usually stable to alkaline pulping processes [24,40] . Other signals from native lignin linkages were also visible, although in a lesser extent probably due to its degradation during alkaline pulping. Then, C β –H β correlation (C β ) from β -5’ phenylcoumaran substructures was observed in soda/AQ-orange lignin spectrum (Figure 3b), whereas C γ –H γ correlation (B γ ) was found in both orange and olive soda/AQ lignins spectra (Figures 3b and 4b, respectively). Signals for spirodienones were clearly observed in soda/AQ-orange lignin (Figure 3b), containing C α –H α (E α ) and C α’ –H α’ (E α’ ) correlations, and in a lesser extent in soda/AQ-olive lignin (Figure 4b), whereas C γ –H γ correlation signal for cinnamyl alcohol end-groups (I γ ) was detected in both lignin samples. Regarding β -O-4’ substructures, a weak intensity signal of C α –H α for β -O-4’ substructures (A α ) was noticed in both spectra, involving S units in soda/AQ-orange and soda/AQ-olive lignins (Figures 3b and 4b, respectively) and also G units in soda/AQ-olive lignin (Figure 4b). C γ –H γ (A γ ) correlations from β -O-4’ substructures were also observed in both lignin samples, which in part are overlapped with other signals. This scarce presence of signals attributed to β -O-4’ substructures is explained by the preferential β -O-4’ linkage breakdown under alkaline conditions [ 23 , 24 , 28 ], which supports the high phenolic content previously described by 13C NMR for both lignins (Section 2.3). Signals from soda/AQ derived lignin linkages were also observed in the oxygenated aliphatic region of both spectra. Aryl-glycerol substructure (AG), with correlations of C α –H α (AG α ), C β –H β (AG β ) and C γ –H γ (AG γ ), could be tentatively identified in both spectra (Figures 3b and 4b), especially in soda/AQ-orange lignin. This substructure is produced from the non-phenolic β -aryl ether linkage under alkaline pulping processes, especially in soda pulping rather than kraft pulping [ 24 ]. C α –H α correlation signal of lignin terminal structures with a carboxyl group in C β (Ar–CHOH–COOH; F α ), which overlaps with C α –H α correlation signal of aryl-glycerol substructure, could also be assigned in both spectra. This kind of lignin terminal structures has recently been described during alkaline processes such as kraft pulping of poplar, elm and spruce [ 19 , 24 , 29 ]. Finally, signals from epiresinol (B’), a diastereomer resulting from the conversion of native resinol ( β - β ’) substructure during kraft process [ 35 ], were also found in both spectra (Figures 3b and 4b). C α –H α (B’ α ), C β –H β (B’ β ) and C γ –H γ (B’ γ ) correlation signals were detected in the case of soda/AQ-orange lignin (Figure 3b), whereas C γ –H γ (B’ γ ) in the case of soda/AQ-olive lignin (Figure 4b). Carbohydrates signals were also observed in the oxygenated aliphatic region of orange and olive soda/AQ-lignin spectra (Figures 3b and 4b, respectively). These signals comprised mainly correlations of xylan chain for C 2 –H 2 (X 2 ), C 3 –H 3 (X 3 ), C 4 –H 4 (X 4 ), and C 5 –H 5 (X 5 ). Moreover, both spectra showed the C-1 cross peak for (1-4) β -D-Xylp of xylan (Figures 3a and 4a). In the aromatic region of both spectra (Figures 3c and 4c), the characteristic correlation signals of S, G, and H lignin units were seen, in the same way that other hardwoods such as eucalypt, poplar, elm and black locust [ 19 , 20 , 29 , 40 ]. The S lignin units presented correlation signals of C 2,6 –H 2,6 (S 2,6 ). C 2,6 –H 2,6 in oxidized S units (S’ 2,6 ) with a ketone group (acetosyringone) or aldehyde end-group (syringaldehyde) in C α was also observed. The G lignin units showed correlation signals for C 2 –H 2 (G 2 ), C 5 –H 5 (G 5 ), and C 6 –H 6 (G 6 ). Signals from oxidized G units were also visible, including correlations in soda/AQorange lignin attributed to C 2 –H 2 (G’ 2 ) and C 6 –H 6 (G’ 6 ) in C α oxidized G units bearing an aldehyde-end group (vanillin), C 2 –H 2 (G” 2 ) and C 6 –H 6 (G” 6 ) in C α oxidized G units bearing a ketone group (acetovanillone) and C 2 –H 2 (G”’ 2 ) and C 6 –H 6 (G”’ 6 ) in C α oxidized G units bearing a carboxylic group (vanillic acid) (Figure 3c). Some of these oxidized G signals were also observed in soda/AQ-olive lignin spectrum (Figure 4c), confirming the lignin oxidation observed by FTIR (Section 2.2) and 13 C NMR (Section 3.3) produced during soda/AQ pulping process. In this sense, Prinsen et al. [ 23 ] reported a higher lignin oxidation, with an increment between 200 − 400% of carboxylic groups, during soda/AQ pulping of eucalypt. Lastly, the H lignin units showed correlation signals of C 2,6 –H 2,6 (H 2,6 ) and C3,5–H3,5 (H3,5). Molecules 2021,26, 3819 7 of 21 Molecules 2021, 26, x FOR PEER REVIEW 8 of 23 Figure 3. HSQC 2D-NMR spectra of soda/AQ-orange lignin. (a) whole spectrum, δC/δH 0.0– 150.0/0.0–9.0; (b) aliphatic oxygenated region, δC/δH 45.0–95.0/2.5–6.0 ppm; (c) aromatic region, δC/δH 90.0–150.0/5.0–9.0 ppm. 50.0 60.0 70.0 80.0 90.0 ppm B γ A α (S) / F α AG α E α /B' α B α E α´ B' α X 4 X 3 X 2 /AG β X 5 X 5 A γ and others B β I γ B' β B' γ 3.03.54.0 4.5 5.05.5 ppm B' γ V trans α V cis α S 2/6 S ' 2/6 SB1 α H 2/6 SB5 α SB5 β G' 2 G'' 2 G'' 6 G' 6 G 2 G 5 + H 3/5 G 6 100.0 110.0 120.0 130.0 6.0 ppm 140.0 7.0 8.0 ppm MeO B' γ FA 2 FA α FA β C γ 50.0 100.0 1.02.0 3.0 4.0 5.0 6.0 ppm ppm 7.0 8.0 G''' 6 G''' 2 β-D-X C1-H1 DMSO / AG γ a b c Figure 3. HSQC 2D-NMR spectra of soda/AQ-orange lignin. ( a ) whole spectrum, δC / δH 0.0–150.0/0.0–9.0 ; ( b ) aliphatic oxygenated region, δC / δH 45.0–95.0/2.5–6.0 ppm; ( c ) aromatic region, δC/δH90.0–150.0/5.0–9.0 ppm. Molecules 2021,26, 3819 8 of 21 Molecules 2021, 26, x FOR PEER REVIEW 9 of 23 Figure 4. HSQC 2D-NMR spectra of soda/AQ-olive lignin. (a) whole spectrum, δC/δH 0.0–150.0/0.0– 9.0; (b) aliphatic oxygenated region, δC/δH 45.0–95.0/2.5–6.0 ppm; (c) aromatic region, δC/δH 90.0– 150.0/5.0–9.0 ppm. 50.0 60.0 70.0 80.0 90.0 ppm Bγ Aα (S) Bα X4 X3 X2 /AGβ Bβ Iγ B'γ B'γ Aγ and others Aα (G) Cβ MeO B'γ Cγ S2/6 S ' 2/6 SB1α H2/6 SB5α G''6 G'2 G2 G5+ H3/5 G6 100.0 110.0 120.0 130.0 6.0 ppm 140.0 7.0 8.0 ppm 3.03.54.0 4.5 5.05.5 ppm 50.0 100.0 1.02.0 3.0 4.0 5.0 6.0 ppm ppm 7.0 8.0 X5 Jβ J2,6(S) G'''6 β-D-XC1-H1 DMSO G'6 / Fα AGα Eα´ a b c Figure 4. HSQC 2D-NMR spectra of soda/AQ-olive lignin. ( a ) whole spectrum, δC / δH 0.0–150.0/0.0–9.0 ; ( b ) aliphatic oxygenated region, δC / δH 45.0–95.0/2.5–6.0 ppm; ( c ) aromatic region, δC/δH90.0–150.0/5.0–9.0 ppm. Molecules 2021,26, 3819 9 of 21 Other native lignin units could also be identified in the aromatic region of both lignin spectra. Then, low intensity signals corresponding to ferulates, including correlations for C 2 –H 2 (FA 2 ), C α –H α (FA α ) and C β –H β (FA β ), were observed in soda/AQ-orange lignin spectrum (Figure 3c). Ferulates can be found in the structure of non-woody plants lignins such as wheat straw and elephant grass [ 41 , 42 ], acylating cell wall carbohydrates and contributing to lignin-carbohydrates cross-coupling reactions, becoming integrally bound into the lignin molecule [ 43 ]. Nevertheless, ferulates have been also reported in Quercus suber L. [ 44 ], and more recently in Ulmus minor Mill. [ 29 ]. On the other hand, soda/AQ-olive lignin spectrum showed weak correlation signals corresponding to C β –H β (Jβ) and C2,6–H2,6 (J2,6) of cinnamaldehyde end-groups (Figure 4c). Molecules 2021, 26, x FOR PEER REVIEW 10 of 23 Figure 5. Main lignin and carbohydrate substructures identified in aliphatic oxygenated region of orange and olive soda/AQ lignins: (A), β-O-4’ alkyl-aryl ether; (AG), aryl-glycerol; (B), resinols; (B’), epiresinol; (C), phenylcoumarans; (E), spirodienones; (F), Ar–CHOH–COOH; (I), cinnamyl alcohol end-groups; (X), xylopyranose (R, OH). A HO O OCH3 H3COHO OCH3 O H3CO αβ γ 1 2 3 4 5 6 1' 6' 5' 4' 3' 2' B O OCH3 OCH3 O O O OCH3 H3CO α β γ 1 2 3 4 5 6 β' γ' α' 1' 2' 3' 4' 5' 6' O HO OCH3 O OCH3 H3CO C α β γ 1 2 3 4 5 6 1' 2' 3' 4 ' 5' 6' O 2 6 53 4 O 1 O OCH3 α β γ O H OAr α ´β´ γ´ OH OCH3 H3CO H3C O E O β 1 2 3 4 5 6 OCH3 α H3CO OH AG OH HO γ F O OH HO O OCH3 H3CO αβ 1 2 3 4 5 6 H3CO O OCH3 OH αβ γ 1 2 3 4 5 6 I 1 2 3 4 5 OH O R O OH X O OCH3 OCH3 O O O OCH3 H3CO B' α β γ 1 2 3 4 5 6 β' γ' 1' 2' 3' 4' 5' 6' α' Figure 5. Main lignin and carbohydrate substructures identified in aliphatic oxygenated region of orange and olive soda/AQ lignins: ( A ), β -O-4’ alkyl-aryl ether; ( AG ), aryl-glycerol; ( B ), resinols; (B’), epiresinol; ( C ), phenylcoumarans; ( E ), spirodienones; ( F ), Ar–CHOH–COOH; ( I ), cinnamyl alcohol end-groups; (X), xylopyranose (R, OH). Signals from soda/AQ derived lignin linkages were also observed in the aromatic region of both lignin spectra. Correlation signals attributed to C α –H α in β 1 stilbene (SB1 α ), and C α –H α and C β –H β in β 5 stilbene (SB5 α and SB5 β , respectively) were detected in soda/AQ-orange lignin (Figure 3c), some of them also observed in soda/AQ-olive lignin Molecules 2021,26, 3819 16 of 21 comparable with thus showed by soda/AQ lignin in our work (35–47%, for 12.5mg L −1 of lignin). Finally, García et al. [ 57 ] reported antioxidant capacities between 16.1% and 46.8% (percentage with respect to the inhibition observed for the DPPH with Trolox after 60 min of reaction) for lignin with molar weight (Mw) between 1.1 KDa and 5.6 KDa, extracted by different methods from Miscanthus sinensis. They observed a clear relation between antioxidant capacity and the molar weight and polydispersity, apart from phenolic content. Moreover, among these lignins, they study a soda lignin with similar molar weight than those presented in our work (Mw 5.7 KDa) which showed an antioxidant capacity of 16.1%, similar to those observed for soda/AQ-orange and -olive lignins: 15.3% and 20.5% (percentage with respect to the inhibition observed for the ABTS •+ with Trolox after 6 min of reaction). Table 4. Trolox equivalent antioxidant capacity of soda/AQ lignins. Soda/AQ-Orange Soda/AQ-Olive mg TE g−1lignin 149.7 ±1.2 598.2 ±4.7 mM TE g−1lignin 202.4 ±5.1 808.9 ±20.3 Soda/AQ-orange, solubilized orange lignin recovered from soda/AQ black liquor; soda/AQ-olive, solubilized olive lignin recovered from soda/AQ black liquor. This antioxidant property could be exploited in mixtures with other synthetic polymers (e.g., polyethylene, polypropylene, poly (vinyl alcohol)) with the objective to reduce their oxidative degradation [ 58 – 60 ], particularly those governed by radical mechanisms. Then, commercial antioxidants such as hindered phenols and amines traditionally applied to polymers could be partially substituted by these natural soda/AQ lignins. Furthermore, lignin could be used also in pharmaceutical, cosmetic, food and packaging industries as a substitute of commonly used cytotoxic synthetic antioxidant like butylated hidroxytoluene (BHT) or butylated hydroxyanisole (BHA) since it has shown higher antioxidant power according to different authors [51,55,56,61,62]. Together with the antioxidant activity, the relatively high thermal stability described for both soda/AQ lignins (Section 2.6) make them potential additives to improve the thermo-oxidation stability of synthetic polymers [ 63 ]. This could particularity improve the flame resistance performance of synthetic polymers [ 64 ], wherein these soda/AQ lignins might become potential substitutes of organic flame retardants like halogenated bisphenol A or polybrominated diphenyl ethers [ 65 , 66 ]. Nevertheless, at this stage only conjectures can be made about the applicability of these orange and olive soda/AQ lignins as environmentally benign, flame retardant antioxidants, while these properties need to be verified experimentally in the mentioned polymer mixtures. 3. Materials and Methods 3.1. Raw Materials and Chemicals Orange tree (Citrus sinensis) and olive tree (Olea europea) pruning residues were kindly provided by Universidad de Córdoba and Universidad de Jaén, respectively. The samples were chipped, homogenized, and stored until their use. On average, the orange tree pruning residue presented the following composition: 3.6%, extractives; 3.4%, ash; 19.9%, lignin; and 73.2% holocellulose [ 1 ]; whereas the composition for olive tree pruning residue was: 8.0% extractives; 1.4%, ash; 20.7% lignin; 89.2% and holocellulose [6]. All chemicals were reagent-grade and were purchased from Merck (Barcelona, Spain), Panreac (Barcelona, Spain) or Sigma-Aldrich (Madrid, Spain). 3.2. Pulps and Lignins Production Pulp from orange tree pruning residue was obtained according to Fillat et al. [ 1 ]. The material was cooked with soda/anthraquinone (soda/AQ) under the following conditions: 185 ◦ C, 60 min, 20% (w/w) NaOH, 1% (w/w) AQ (both oven-dried material) and 8:1 liquid/solid ratio. Regarding olive tree pruning residue the cooking conditions employed Molecules 2021,26, 3819 17 of 21 for pulp production were: 175 ◦ C, 120 min, 15% (w/w) NaOH, 1% (w/w) AQ (both ovendried material) and 8:1 liquid/solid ratio [ 6 ]. Resulting soda/AQ pulps were filtered, collecting the black liquors for lignin precipitation. Orange soda/AQ pulp showed a kappa number, viscosity, and brightness values of 22.0, 430 mL g −1 and 33.5% ISO, respectively, whereas olive soda/AQ pulp showed a kappa number and viscosity values of 38.7 and 794 mL g−1and a brightness of 18.4% ISO. Orange and olive soda/AQ lignins (referenced as soda/AQ-orange and soda/AQolive, respectively) were extracted from recovered black liquors by acid precipitation. Then, the pH of the black liquors was lowered to 2.5 by slow addition of sulphuric acid (98% w/w) and left under stirring for 30 min. The precipitated lignins were filtered and washed twice with acidified water (pH 2.5) and dried at room temperature. 3.3. Lignins Characterization Chemical composition of soda/AQ lignins was examined according to the Laboratory Analytical Procedures for biomass analysis from the National Renewable Energies Laboratory [ 67 ], employing the protocol NREL/TP-510-42618. After the acid hydrolysis of lignin samples, the acid insoluble solid residue (klason lignin) was recovered, whereas the liquid fraction was examined for carbohydrates content by high-performance liquid chromatography (1260 HPLC, Agilent, Waldbronn, Germany, equipped with a G1362A refractive index (RI) detector and an Agilent Hi-PlexPb column) [ 30 ]. Mean values and standard deviations were calculated from the triplicates. The total phenols content of soda/AQ lignins was evaluated according to a slightly modified version of the Folin-Ciocalteau protocol [ 30 ]. Then, the absorbance of a mixture with Folin-Ciocalteau solution, Na 2 CO 3 and lignin samples (previously dissolved in dimethylsulfoxide) was measured at 760 nm in a UV-Vis spectrophotometer (Lambda 365, PerkinElmer, Boston, MA, USA). The total phenols content was calculated from a calibration curve prepared from a standard solution of gallic acid (1–20 mg L −1 ) and expressed as g gallic acid equivalent (GAE) mg−1of lignin (on a dry basis). Mean values and standard deviations were calculated from the triplicates. FTIR spectra of soda/AQ lignins were acquired by a JASCO FT/IR 460 Plus spectrometer (Jasco, Japan), with an accessory single reflection diamond, working at a resolution of 1 cm−1, 100 scans, and a spectral range of 4000–600 cm–1 [30]. Solid-state 13 C nuclear magnetic resonance ( 13 C NMR) analyses of soda/AQ lignins were carried out in a Bruker Avance III 400MHz (Bruker, Billerica, MA, USA) at 100.64 MHz with the cross polarization/magic angle spinning (CP/MAS) technique at the conditions described by Jiménez-López et al. [30]. 13 C– 1 H two dimensional nuclear magnetic resonance (2D NMR) analyses of soda/AQ lignins were recorded in a Bruker AVANCE 500 MHz (Bruker, Billerica, MA, USA) with a zgradient double resonance probe. Soda/AQ lignins were dissolved in deuterated dimethylsulfoxide (DMSO-d 6 ) and HSQC (heteronuclear single quantum correlation) experiments were recorded at the conditions reported by Eugenio et al. [ 29 ] and Martín-Sampedro et al. [19]. Residual DMSO (at δC/δH39.6/2.5 ppm) was used as an internal reference. Size exclusion chromatography (SEC) of soda/AQ lignins was conducted on a HPLC (1260 HPLC, Agilent, Waldbronn, Germany, equipped with a G1362A refractive index (RI) detector and two columns PLgel 10 µ m MIXED B 300 × 7.5 mm). N,N-dimethylformamide (DMF) was pumped as mobile phase at the conditions described by Jiménez-López et al. [ 30 ]. Columns were calibrated with polystyrene standards (peak of average molecular weights of 570, 8900, 62,500, 554,000, Sigma-Aldrich, San Luis, MO, USA). 3.4. Thermal Lignins Characterization Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of soda/AQ lignins were carried out in air and nitrogen (SDT Q600, TA Instruments, New Castle, DE, USA), using a heating rate of 10 ◦ C min −1 [ 20 ]. In addition, differential scanning calorimetry (DSC) was carried out under nitrogen (Q2000 calorimeter, TA Instruments), using a Molecules 2021,26, 3819 18 of 21 heating rate of 20 ◦C min−1[19] . In order to remove any previous thermal history from soda/AQ lignins, the samples were dried (60 ◦ C for 48 h) and a heating and cooling cycle from 20 to 160 ◦C in the DSC instrument was performed [68]. 3.5. Antioxidant Activity of Lignins The antioxidant activity of soda/AQ lignins was performed using Trolox equivalent antioxidant capacity methods according to Re et al. [ 69 ], with slight modifications [ 52 ]. ABTS +• was produced by the reaction between a stock solution of 2,2 0 -azinobis(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) and potassium persulfate. Prior to use, the ABTS +• stock solution was diluted with phosphate buffer saline to get an absorbance of 0.7 ± 0.02 at 734 nm. Then, 1 mL of the ABTS +• stock solution was mixed with 10 µ L sample (1 mg mL −1 ) or control (buffer). The absorbance of the reaction mixture was measured at 734 nm during 6 min in a UV–Vis spectrophotometer Lambda 365 (PerkinElmer, Boston, MA, USA). Trolox was employed as a standard and results were showed in mg of Trolox equivalent (TE) g −1 lignin. Mean values and standard deviations were calculated from the triplicates. 4. Conclusions In order to increase the competitive and sustainable production of energy and high added-value products from carbohydrates contained in lignocellulosic materials, including agroforestry residues, the valorization of lignin-enriched residues generated during these transformation processes is crucial. In addition, the use of these lignin-enriched residues will also contribute for the establishment of the circular bioeconomy, which seeks to maximize the usage and value of all raw materials, products and residues. Nevertheless, depth knowledge of lignin is necessary to define its valorization route. In this study, residual lignins solubilized during soda/AQ pulping of orange and olive tree pruning residues, two of the most abundant agroforestry residues generated in Spain, were isolated and chemical, thermal and antioxidant properties analyzed. Both lignins showed extensive β -O-4’ linkages degradation, as revealed 13 C– 1 H two dimensional nuclear magnetic resonance analyses. Consequently, a high OH phenolic content, inferred by solid state 13 C NMR, and low molecular weight and polydispersity values, showed by size exclusion chromatography, were observed for both lignins. In addition, lignins displayed a high proportion of syringyl units, containing different native as well as soda/AQ lignin derived units. Based on these chemical properties, orange and olive lignins exhibited relatively high thermal stability and good antioxidant properties. This antioxidant capacity could be used in blends with other synthetic polymers in order to reduce their oxidative degradation. Moreover, the relatively high thermal stability described for both lignins makes them potential additives to improve the thermo-oxidation stability of synthetic polymers. Author Contributions: Conceptualization, M.E.E. and D.I.; methodology, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; software, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; validation, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; formal analysis, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; investigation, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; resources, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; data curation, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; writing—original draft preparation, D.I.; writing—review and editing, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; visualization, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; supervision, M.E.E., and D.I.; project administration, M.E.E., B.W. and D.I.; funding acquisition, M.E.E., B.W. and D.I. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Comunidad de Madrid and MCIU/AEI/FEDER, EU via Projects SUSTEC-CM S2018/EMT-4348 and RTI2018-096080-B-C22, respectively. B.W. acknowledges financial support from MINECO (Spain) and FEDER (EU) (project MAT2015-71117-R) and from MICINN (Spain) for a JIN contract (PID2019-107022RJ-I00). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Molecules 2021,26, 3819 19 of 21 Acknowledgments: Universidad de Jaén and Universidad de Córdoba are acknowledging for olive and orange tree pruning residues, respectively. The contribution of COST Action LignoCOST (CA17128), supported by COST (European Cooperation in Science and Technology), in promoting interaction, exchange of knowledge and collaborations in the field of lignin valorization is gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Not applicable. References 1. Fillat, Ú.; Martín-Sampedro, R.; González, Z.; Ferrer, A.; Ibarra, D.; Eugenio, M.E. 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