Lignin composition in wood of gymnosperms, angiosperms, in biomass from monocotyledons and barks.
The objective of this chapter is to provide a concise overview of lignin composition and structure in different species and materials (wood, barks and nonwood plants). It includes a brief review on the lignin precursors and their polymerization as well as of the analytical tools used for lignin characterization from wet chemical to spectroscopic methods. Wood of gymnosperms is characterized by high lignin content (25–35%) and a HG-type of lignin with more guaiacyl (G) units and a small portion of p-hydroxyphenyl (H) units. Wood of angiosperms has a lignin content of 15–28%, with a GS-lignin having different proportions of syringyl (S) units. Nonwoody monocotyledon species have different lignin content (9–20%) and a HGS type of lignin, characterized by a high proportion of H units. Bark lignin content ranges from 13 to 43% and is of HGS-type with species-specific composition and different in the bark components, phloem and cork. Lignin composition and macromolecular structure are key issues to understand the properties of lignocellulosic materials and to design a lignin-based pathway within biomass biorefineries. The available information on lignin composition is still limited to a few species and plant components. This is certainly an area where more research is needed.
- analytical tools
- lignin composition
- S/G ratio
Lignin has been the subject of continuous and intensive research over the last century. The Web of Science shows that since 1908 more than 27,000 publications were published with the topic “lignin,” including articles, reviews, book chapters, notes and proceedings, under different subject areas, e.g., plant science, biotechnology, applied microbiology, chemistry, wood, pulp & paper, materials, energy and fuels.
Lignin is the second most abundant biopolymer in nature and accounts for almost 30% of the plants . Its deposition in the cell wall is of great importance for plant development: (i) it provides rigidity and strength to the cell wall, giving mechanical support for the plant organs; (ii) it presents hydrophobicity favoring the transport of water and solutes in the vascular system and (iii) it protects the cell against pathogens [1, 2, 3, 4, 5]. Lignin is linked to the other structural components of the cell wall—cellulose and hemicelluloses—by covalent linkages, forming lignin-carbohydrate complex (LCC) .
The first lignin studies were mainly driven by its importance for the pulp & paper industry, where the objective is to remove lignin from the wood cells to obtain a fibrous product rich in cellulose. Thus, the studies on lignin, whether related to content, composition or structure, were focused on pulpwoods [7, 8, 9, 10, 11]. Lignin was also studied in herbaceous plants [12, 13, 14, 15, 16] partly triggered by digestability and dietary conversion issues in animal feed . More recently, other wood species as well as various lignocellulosic residues and wastes attracted attention within the biorefinery concept providing opportunities for production of green chemicals, bioproducts and energy, calling for the need to include lignin valorization.
The lignin content shows a large variability between species: in general, in monocotyledons, it ranges between 5 and 12%, in softwoods between 25 and 35% and in hardwoods between 15 and 30%. The structural arrangement of lignin also differs between these three groups. This chapter makes a review on the compositional variability of lignin in various species and biomass components after an introductory compilation of the macromolecular assembly and the analytical tools used in lignin research.
2. The molecular construction of lignin
2.1. Precursors and monomers
Lignin is a heterogeneous aromatic polymer mainly constituted by three precursors:
The deposition of lignin and cellulose in the cell wall proceeds in three phases and starts after the deposition of pectins and the formation of the secondary wall S1 layer has begun [3, 20]. The first phase starts by the lignification at cell corners and middle lamella; the second phase corresponds mainly to the deposition in the S2 layer of cellulose in microfibrils and of xylan and mannan, with lignin being slowly added; in the third phase, lignin deposition proceeds extensively across the cell wall after the deposition of cellulose in the microfibrils of the S3 layer.
The composition of lignin changes during cell development. According to Terashima and Fukushima , in conifers, the middle lamella and cell corners are first enriched in
Lignin heterogeneity may be related to enzyme diversity and specificity regarding substrates, thereby affecting the metabolite flux into diverse branches of the biosynthetic pathway . A clear example is the difference in lignin type between softwoods and hardwood: softwood lignin is mainly constituted by G units and minor amounts of H units, whereas hardwood lignin has G and S units.
2.2. Polymerization and molecular assembly
The theory underlying lignification was presented by Freudenberg and Neish  based on chemical processes involving the oxidative coupling of phenols and addition of the available phenolic substrates to the polymer [23, 24]. The oxidation produces a phenolic radical with unpaired electron density delocalized at the C1, C3, C5 and O-4 positions of the aromatic ring and at the propanolic Cβ, forming resonance structures.
The lignin polymerization starts with the coupling of two monomeric radicals and continues by coupling of monomer radicals with phenoxy radicals formed on the growing polymer . This concept explains some features of the lignin composition and structure, e.g., the evidences that other monomers such as coniferyl and sinapyl acetates and coumarates are also incorporated in the polymer [17, 18, 19]. Although a lignin polymerization model based on a protein-controlled radical coupling was proposed [26, 27, 28, 29], the idea was not fully accepted by the scientific community given its flaws . It was proved that lignification is malleable to plant needs and the polymer can be manipulated by changing the lignin-biosynthetic pathway genes [17, 31], and plants may incorporate other monomers into the lignin [32, 33].
The dehydrogenation of the lignin monomers is made by peroxidases (or peroxidase-H2O2 system) that are capable of removing a proton from the phenolic hydroxyl forming the resonance-stabilized free radicals, using the H2O2 produced by the peroxidase enzyme as an electron-acceptor substrate . Laccase is a phenoloxidase also related to lignin biosynthesis .
After formation of the phenoxy radicals, the reaction is no longer controlled by enzymes but is a random radical polymerization process at the reactive sites . The most reactive positions are the phenoxy oxygen and the Cβ that readily couple into aryl-ether linkages; the β-O-4′ linkage is predominant in lignin, e.g., almost 50% of all intermonomeric linkages in softwoods and 60% in hardwoods . Overall, the coupling of the lignin monomers may be by ether bonds (β-O-4′, 4-O-5′, 1-O-4′) and by carbon-carbon bonds (5–5′, β-5′, β-β′, β-1′) is often called condensed bonds . Some of these linkages are shown in Figure 2.
2.3. Analytical tools and lignin compositional indicators
Lignin quantification is usually made through wet chemistry by acid hydrolysis with sulfuric acid, using standard methods, e.g., TAPPI T222 om-11 and UM 205 om-83, respectively, for Klason lignin (obtained as a solid residue) and acid-soluble lignin (measured at 205 mm in the solution) that together make up the total lignin in the sample . The procedure was optimized for wood and may lead to overestimation if the raw material is rich in ash and proteins or contains carbohydrate degradation products such as furfural and hydroxymethyl furfural. In spite of these shortcomings, most of the available data on lignin content of lignocellulosic materials refer to Klason lignin determinations and therefore establish a comparative reference. More recently, lignin content has been calculated from analytical pyrolysis or estimated using FTIR and NIR spectra modeling, as described subsequently.
As regards the study of lignin composition, the ideal would be to have an isolated pure lignin, e.g., recovered after removal of extractives, cellulose and hemicelluloses, without chemical modification of the original lignin. This proved unfeasible although some procedures approach the requirements . Lignin is frequently isolated by the classical Bjӧrkman method  and called milled wood lignin (MWL) if wood is the starting material. The lignin is obtained by milling the extracted sample in a planetary ball mill, followed by extraction with dioxane; after evaporation, the lignin is dissolved in acetic acid, precipitated into water, dried and dissolved in dichloroethane:ethanol solution and precipitated into ethyl ether . MWL is still contaminated with carbohydrates  and represents only a part of the total cell wall lignin  whose structural features are correlated with the yield and, to a less degree, milling time . Other procedures involving enzymes, e.g., cellulolytic enzymes, to remove the carbohydrates are used to increase the lignin yield .
Much of the present understanding of the composition and structure of lignin is based upon interpretation and extrapolation of data obtained from chemical degradative methods that include thioacidolysis, nitrobenzene oxidation, derivatization followed by reductive cleavage and analytical pyrolysis. Nondestructive methods, such as Fourier transform infrared (FTIR), near-infrared spectroscopy (NIRS) and nuclear magnetic resonance (NMR), are also used for lignin characterization.
Thioacidolysis involves the solvolysis of the extractive-free material in dioxane/ethanediol (9/1) containing boron trifluoride etherate [43, 44]. The reaction depolymerizes part of the lignin and the released monomers can be analyzed, e.g., by GC-MS allowing to estimate the amount and composition of uncondensed alkyl ether structures . Most studies were focused on wood but thioacidolysis was also applied to other materials, e.g., cork . Calculation of the S/G ratio is usually made.
2.3.2. Nitrobenzene oxidation (NO)
Alkaline nitrobenzene oxidation leads to formation of aromatic aldehydes (
2.3.3. Derivatization followed by reductive cleavage (DFRC)
DFRC provides information on the occurrence of acylated γ-OH units [50, 51, 52, 53]. The derivatization is made with acetyl bromide in acetic acid (called DFRC) or propionyl bromide in propionic acid (DFRC modified) at 50°C. The products are dissolved in dioxane/propionic acid/water (5:4:1, v/v/v), with the addition of zinc for the reductive cleavage, and the final derivatization step made with acetic anhydride or propionic anhydride depending of the method chosen (DFRC or DFRC modified) . The lignin degradation compounds are collected after evaporation and analyzed by GC/MS.
2.3.4. Analytical pyrolysis
Pyrolysis transforms a nonvolatile compound into a volatile degradation mixture by heat in the absence of oxygen and by the breaking of chemical bonds using thermal energy [54, 55]. The ground biomass sample (particle sizes from mm to μm) is heated at temperatures from 400 to 1000°C, in the absence of oxygen, for 300 s to less than 0.5 s, producing charcoal (solid), bio-oil (liquid) and fuel gas products [56, 57]. Analytical pyrolysis was perfected for the pyrolysis of small samples for analytical purposes: a mixture of volatile compounds derived from the three macromolecular constituents of biomass (cellulose, hemicelluloses and lignin) is obtained and separated through a capillary column, identified by mass spectrometry (MS) and quantified by a flame ionization detector (FID). The MS identification of pyrolysis products was mostly made by Faix et al. [58, 59, 60, 61] and by Ralph and Hatfield . Nowadays, quantification is also made by Py-GC/MS . Pyrolysis is particularly interesting to characterize the lignin monomeric composition into the phenolic S, G and H precursor monomers. The ratio of monomers is given in the form of H:G:S or, more frequently, as the S/G ratio.
2.3.5. Fourier transform infrared (FTIR)
FTIR spectroscopy is a rapid technique for lignin characterization that permits determination of the lignin monomeric composition, of methoxy and carbonyl groups, and the calculation of the ratio of phenolic to aliphatic groups . It is based on the interaction of infrared radiation (4000–500 nm wavelength) with the sample and the fact that each molecule absorbs energy characteristic from its specific intramolecular bonds. Some of the bands related to lignin are found at 1600 and 1500 cm−1 due to the aromatic skeleton vibration of the benzene ring, at 1300 and 1200 cm−1 related to syringyl and guaiacyl lignin units and the bands at 1716 and 1711 cm−1 are attributed to phenol esterification and the alcohol of the propanoic chain (Cα and Cγ) [65, 66]. The spectral acquisition data can be made in transmission mode (TR), attenuated total reflectance (ATR), diffuse reflectance (DR) and photoacoustic (PA). Most lignin studies have used FTIR-TR but FTIR-DR is suitable to study the wood surface oxidation and weathering . Watkins et al.  used FTIR-ATR to characterize organosolv lignins. FTIR spectral data were also used to model lignin content in wood of, e.g.,
2.3.6. Near-infrared spectroscopy (NIRS)
NIRS is a fast nondestructive technique that requires minimal or no sample preparation. The NIR region spans the wavelength range 780–2500 nm (12,821–4000 cm−1), in which absorption bands correspond mainly to overtones and combinations of fundamental molecular vibrations, especially stretching and bending [70, 71]. The NIR spectrum is a superposition of scatter and light absorbance signals  and consequently contains information specific to the molecular vibrational aspects and their physical environments. NIRS methods require multivariate calibration algorithms (PCA, SIMCA, PCR or PLSR) usually referred to as chemometric methods to model spectral response to chemical or physical properties of a calibration sample set . Spectra pre-processing is used to eliminate or minimize variability not related to the investigated property  and include normalization, derivatives (usually first or second), multiplicative scatter correction (MSC), standard normal variate (SNV), de-trending (DT) or a combination thereof . NIRS has been successfully applied to evaluate different features of lignocellulosic materials [73, 74]. For instance, it was used to estimate Klason lignin content in
2.3.7. Two-dimensional nuclear magnetic resonance (2D NMR)
Nuclear magnetic resonance spectrometry (NMR) is based on the measurement of absorption of electromagnetic radiation in the radiofrequency region of 4–900 MHz . The NMR parameters, chemical shifts (δ), coupling constants (J), relaxation times and signal intensities, are related to the electronic structure and chemical environments of nuclei involved in the resonance phenomenon . NMR spectroscopy can be proton (1H) NMR or carbon (13C), and a two-dimensional (2D) 13C-1H correlation spectrum has also been used. The acquisition can be made by: i) homonuclear correlation spectroscopy: COZY, TOCSY meaning that the protons correlate with the other protons; and ii) heteronuclear correlation (13C-1H): HMQC or HSQC where each carbon correlates with its attached proton; HMQC-TOCSY or HSQC-TOCSY where a carbon correlates with the proton attached to him and to other protons in the same coupling network . The 2D HSQC NMR spectra are analyzed in the aromatic and the aliphatic regions. The aromatic region contains the correlations of C2-H2 at δC/δH 100–150/6.0–9.0 and includes the hydroxycinnamates and cinnamyl alcohol end-groups . The H:G:S relation is quantified by volume integration of the contours. Also, the presence of acylated structures in lignin is identified in this region (especially in Cγ). The aliphatic-oxygenated region (around δC/δH 50–90/2.5–6.0) provides information on the inter-unit bonds in lignin. Signals from the β-O-4′ alkyl-aryl ethers, phenylcoumarans, resinols and dibenzodioxocins are a few examples of signals that are visualized in this region. The presence of polysaccharides signals is also detected here . Overall, NMR is a powerful tool for structural investigation, since it allows to accurately assess the chemical structures, functionalities and nature of chemical bonds in the lignin macromolecule [78, 79], including an accurate measurement of the S/G ratio.
3. Lignin composition in biomass
Lignin differs naturally in content and composition between biomass materials at various levels, e.g., between species, within species and between components (such as wood and bark), and is influenced by plant growth stage and environmental stress [2, 15, 62, 65, 80].
Most studies on lignin composition were focused on the economic important pulpwoods, such as the pine and spruce softwoods, and the eucalyptus and birch hardwoods. The S/G ratio has been the mostly evaluated lignin composition parameter due to its importance in the pulping reactions; it is considered a pulpwood quality trait and a selection parameter in breeding programmes. Lignin composition has also been investigated in herbaceous plants in relation with their use in animal nutrition. More recently, and with the growing interest of biomass as a feedstock for biorefineries, lignin has increasingly been investigated in different species and components, namely in barks.
Table 1 makes a synthesis of the available information on lignin composition in wood of gymnosperms and angiosperms, in biomass from monocotyledons and barks. Figure 3 compares the monomeric composition of lignin (S/G) in wood and bark for the species for which this determination was made.
|wood||25-29%||-||-||69%, 18%, 10%||no||[37, 68, 80, 93, 84]|
|wood||23-30%||-||0.041||-||-||[91, 92, 93, 94]|
|wood||19-25%||-||0.048*||-||-||[96, 97, 98]|
|wood||15-28%||-||1.5-2.9||76%, 2%, 17%||no||[105, 106, 107, 108, 109, 110, 111, 112, 113]|
|younger trees||-||1:4:6||1.3||68%, 5%, 18%||-|||
|older trees||-||1:10:39||3.8||69%, 1%, 19%||-|||
|bark (cork)||14%||1:43:6||0.1||[176, 177]|
|wood||24%||1:44:55||1.2||77%, 9%, 8%||-|||
|phloem||38%||1:58:41||0.7||71%, 13%, 7%||-|||
|bark (cork)||27%||1:43:7||0.1||68%, 20%, 4%||48%, at G-units||[125, 170, 173]|
|stalks||16-19%||0:58:42||0.7||70%, 14%, 7%||12%, acetates (S-units)||[131, 134, 135, 136]|
|stalks||20%||1:61:38||0.62||79%, 8%, 10%||43%||[139, 142]|
|internode||16-22%||1:2:0.5||1.2||49%, -, -||-||[140, 143]|
|stalks||13%||1:13:11||0.7||93%, -, -||46% acetate or ||[144, 145, 146]|
|stalks||5-16%||1:15:5||75%, 11%, -||10%, acetates in G-units (12%), in S (1%)||[14, 15, 149, 154]|
|rachis||11%||1:0.7:1||1.4||0.32/C6, -, -||-||[159, 160]|
|leaf sheaths||13%||1:2:0.5||0.25||-||-||[159, 160]|
|floral stalks||11%||1:1.6:1||0.63||0.12/C6, -, -||-||[159, 160]|
|leaf blades||24%||1:9.3:6.3||0.60||-||-||[159, 160]|
3.1. Wood of gymnosperms
Softwoods are gymnosperms (mostly conifers), generally needle-leaved evergreen trees, e.g., pines (
Maritime pine (
Loblolly pine (
Lignin content of
Douglas-fir wood is used for timber and pulping, mainly in North America and also in Europe. The lignin content can range from 19.7 to 32.8% [100, 101]. The MWL was characterized as a HG-type, presenting an total amount of β-O-4′ aryl ether bonds of approximately 1700 μmol/g and a total amount of phenolic hydroxyl groups of 1500 μmol/g, with 40% of condensed structures, and the average molecular weight was 7400 g/mol .
3.2. Wood of angiosperms
Hardwoods belong to angiosperms, typically broadleaf deciduous trees. Lignin in hardwoods is constituted mainly by guaiacyl and syringyl units (GS-lignin), with a methoxyl content of 21%, and the β-O-4′ as the most common linkage, with a proportion of 71% or higher of the intermonomeric linkages [81, 102, 103].
Lignin content differs between eucalyptus species: Neiva et al.  reported values of 21.6% (
The European beech (
Acacia species are fast-growing trees used for timber and pulp production. Total lignin in
3.3. Biomass of monocotyledons
Monocotyledons are angiosperm flowering plants with seeds typically containing only one cotyledon that include the families
Wheat is extensively cultivated for seed production, leaving the straw as a widely available residue with great potential for bioenergy, including bioethanol [15, 149]. The lignin content in wheat straw ranges from 5 to 16% [15, 149]. It is a HGS-type of lignin with a proportion of 1:11:5  and 1:10:9 , corresponding to S/G ratios of 0.45 and 0.9, respectively. Milled straw lignin presents
Barks are complex and heterogeneous components of plants that include phloem and periderm and eventually rhytidome (periderms interspersed by phloem), as schematically represented in Sen et al. . Phloem is produced by the cambium and the periderm by the phellogen . Barks are a largely available residue from the timber and pulp industries mostly used for energy but increasingly considered as potential feedstocks for biorefineries given their chemical and structural diversity [163, 164]. Cork is one component of bark periderms that may attain considerable proportions in some species . The cork from
Lourenço et al.  also studied the lignin content and composition in the bark phloem after isolation by Bjӧrkman method. The lignin content was 38.4% (% o.d material), with a H:G:S ratio of 1:58:41 and a S/G ratio of 0.7 (from NMR). The lignin was characterized mainly by β-O-4′ alkyl-aryl ethers (71%), with low amounts of condensed linkages, and was scarcely acetylated, mainly over S units.
Lignin from eucalypt bark was isolated by mild acidolysis and characterized by nitrobenzene oxidation (NO) and NMR (13C and 31P NMR) . Bark lignin was of HGS-type, with a H:G:S relation of 1:6:18 (13C NMR). The S/G ratio values differed between techniques, with the higher values attained by the nitrobenzene oxidation since it only quantifies the noncondensed structures: 1.5 (NMR), 1.5 (31P NM), 3.17 (13C NMR) and 5.9 (NO).
3.5. Variability of lignin composition at tissue and cell levels
Few studies have compared lignin composition in different biomass components of the same plant (Figure 3). For
Variation of lignin composition also occurs at cellular level. Lignin formation and composition are cell specific, e.g., lignin differs between tracheary elements, vessels and sclerenchyma cells, and presents a distinctive feature at subcellular localization [2, 180]. During the early phases of xylem lignification, the H units are incorporated in the cell and G units are present in the middle lamella and cell corners, whereas in the next phase, the lignification of the cell primary wall and outer layers of secondary wall is mainly by G units [181, 182, 183]. In Arabidopsis, xylem vessels have a predominance of H units and cell corners and middle lamella have a G-lignin, while the fibers are rich in S units . In white birch, the vessels secondary wall has a G:S relation of 88:12, the fibers 12:88 and the ray parenchyma 49:51 . In
4. Concluding remarks
This chapter provides an overview on lignin content and composition, showing its complexity as a polymer and variability between and within plant species. The lignin polymer is built with three monomers (
Lignin diversity is well demonstrated when analyzing the available data on the lignin monomeric composition of various species and biomass components. The usual classification of lignin in three types—HG-lignin, GS-lignin and HGS-lignin—broadly assigned to softwoods, hardwoods and monocots, respectively, is a crude generalization and do not encompass the diversity found within each group. It is true that the knowledge on lignin composition and structure is still restricted to a limited number of species and plant components. This is clearly an area in which more research is needed. The various analytical tools that have been developed, including wet chemistry, spectroscopic, magnetic and pyrolytic methodologies allow a better insight into lignin structure and the possibility of making a much more extensive coverage of biomass materials.
Lignin plays an important role in plant cell walls providing support and protection, and it is the second most abundant polymer in nature after cellulose. Increased knowledge on lignin will therefore contribute to our understanding of plant physiology and adaptation, as well as support a lignin platform within future biorefineries providing combined valorization routes for chemicals, materials and energy.
The support for this work was provided by Fundação para a Ciência e a Tecnologia (FCT) through funding of the Forest Research Center (UID/AGR/00239/2013). Ana Lourenço acknowledges support from FCT through a postdoctoral grant (SFRH/BPD/95385/2013). A word of appreciation to Vanda Oliveira, Duarte M. Neiva and Jorge Gominho for their help.