Lignin, a major component of lignocellulosic biomass, is crucial to plant growth and development but is a major impediment to efficient biomass utilization in various processes. Valorizing lignin is increasingly realized as being essential. However, rapid condensation of lignin during acidic extraction leads to the formation of recalcitrant condensed units that, along with similar units and structural heterogeneity in native lignin, drastically limits product yield and selectivity. Catechyl lignin (C-lignin), which is essentially a benzodioxane homopolymer without condensed units, might represent an ideal lignin for valorization, as it circumvents these issues. We discovered that C-lignin is highly acid-resistant. Hydrogenolysis of C-lignin resulted in the cleavage of all benzodioxane structures to produce catechyl-type monomers in near-quantitative yield with a selectivity of 90% to a single monomer.

Bioengineered biomass could be used to achieve higher hydrogenolysis yields and simpler product mixtures. For example, the recent use of formaldehyde protection during lignin extraction from a high- S poplar lignin ( 7 , 16 ) that has up to 98% syringyl S units and ~90% β–O–4 linkages [from nuclear magnetic resonance (NMR) estimates] prevented condensation reactions and allowed an unprecedentedly high monomer yield (78%) under hydrogenolytic conditions ( 7 ). However, even in this high- S lignin, some 10% of the linkages are C–C bonds that do not cleave. The use of formaldehyde to protect the lignin from condensation reactions also resulted in some formaldehyde addition to the ring, complicating the hydrogenolysis products with methyl-substituted aromatics. Without formaldehyde, the lignin extracted under acidic conditions had significant condensation, thwarting the production of monomers and resulting in a hydrogenolysis monomer yield of only 26% ( 7 ). Although new methods for displacing formaldehyde for the protection from acid-catalyzed condensation reactions, retaining much of the yield (70%) and producing a simpler monomer mix, have recently been revealed ( 17 ), extra protection chemicals remain necessary during the lignin extraction.

The use of extracted lignins rather than whole biomass has the advantage that the material can be fully dissolved in organic solvents, facilitating catalyst recovery and continuous processing. However, acidic industrial lignin fractionation is known to cause some β-ether cleavage and condensation between units via the electrophilic substitution of acid-generated benzylic carbocation intermediates on the electron-rich aromatic rings ( 7 , 11 ), limiting depolymerization yields ( Scheme 1A ) ( 12 – 14 ). There are some elegant solutions focusing on suppressing the condensation reaction, either using a capping agent ( 7 , 15 ) or using two-step strategies ( 4 , 5 , 11 ). However, extra chemicals or catalysts are needed to achieve this goal.

Although mild depolymerization methods, such as oxidative ( 4 , 5 ) and hydrogenolytic ( 6 – 8 ) procedures, have produced encouraging results in laboratory-scale experiments, their applicability in industrial processes has been limited. Direct hydrogenolysis, that is, the hydrogenation of unprocessed solid biomass by a heterogeneous metal catalyst, remains one of the most promising methods for cleaving lignin’s ether bonds and producing aromatic monomers in high yields ( 8 – 10 ). However, hydrogenolysis still suffers from product complexity issues. In most wild-type biomass, the lignin polymer is composed of three phenylpropanoid subunits—p-hydroxyphenyl ( H ), guaiacyl ( G ), and syringyl ( S )—derived by combinatorial radical coupling from the three main monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). Although H units are typically at low-levels, this results in at least three different types of monomers ( H , G , and S ), each with a selection of side chains, as the primary hydrogenolysis products, which makes monomer separation and utilization difficult. Lignin’s principal alkyl-aryl-ether units with their β–O–4 interunit bonds (45 to 85%) can be selectively cleaved, but other linkages including β–5 (1 to 12%), β–β (5 to 12%), 5–5 (1 to 9%), 4–O–5 (~2%), and β–1 (1 to 2%), which are also present in lignins, remain largely uncleaved ( 8 ); carbon-carbon (C–C) and diaryl ether (4–O–5) units typically result from dimeric or higher oligomeric products.

Lignin is a polymeric material composed of phenylpropanoid subunits and is one of the largest sources of naturally produced aromatics on the planet. Because of its aromatic nature, lignin has a higher energy density than polysaccharide polymers, as well as a higher potential commercial value ( 1 ). However, because of lignin’s complexity, its efficient utilization, either as a polymer or from its derivable small-molecule products, is currently problematic ( 1 – 3 ).

RESULTS

An “ideal lignin” archetype On the basis of the plethora of information stemming from the lignin biosynthetic research community over the last decade, and with the revelations regarding lignins’ structural malleability from studies on lignin pathway mutants and transgenics as well as on various “natural” plants discovered to have unusual lignins, researchers have been able to contemplate designing lignins for improved utilization (18). It is now a realistic juncture to posit the characteristics for an ideal lignin archetype for biomass processing. For the depolymerization of the polymer to monomers, lignin should have at least the following three characteristics. First, if acidic pretreatment is used, then it should be stable under acidic conditions to prevent condensation and the generation of undesired new C–C bonds. Second, it should contain only ether (C–O) interunit linkages in its backbone so that it can be fully depolymerized. Finally, it should be generated in planta from a single phenylpropanoid monomer to allow the production of the simplest array of compounds. We have reported the discovery of an unusual catechyl lignin (C-lignin) present in the seed coats of vanilla (Vanilla planifolia) (19) and various members of the Cactaceae of the genus Melocactus (20). In this special case, the lack of O-methyltransferase (OMT) activity for conversion from catechyl C to guaiacyl G and, subsequently, on to syringyl S, aromatic-level precursors, results in 100% C units in the cell wall (CW). This C-lignin was, somewhat surprisingly, found to be essentially a homopolymer synthesized almost purely by β–O–4 coupling of caffeyl alcohol with the growing polymer chain, producing benzodioxanes as the dominant unit in the polymer (Fig. 1A). If it has particular stability toward biomass pretreatment conditions, then this C-lignin might therefore represent an example of such an ideal lignin that can, in principle, be depolymerized to a single product by hydrogenolysis. Furthermore, this substrate has the potential to produce valuable catechol monomers, whereas the large majority of monomers produced from lignin have been S or G derivatives (1, 2). Expanding the arsenal of lignin-derived platform molecules could play an important role in the successful use of this fraction within future biorefineries. Here, we describe the ideal nature of this lignin via a revised compositional characterization of the vanilla seed coat fiber, new features of the C-lignin’s reactivity and stability, and our successful attempts at converting it to monomers in near-quantitative yields. Fig. 1 NMR spectra. Partial 2D HSQC NMR spectra of (A) EL, (B) KL, and (C) LBL from vanilla (V. planifolia) seed coat. There are no obvious lignin structural changes after the acidic lignin extraction processes. Cellulose was labeled following the conventional monosaccharide nomenclature; NR is the nonreducing end of the cellulose. Protein residuals were labeled by the aromatic amino acid. Tyr, l-tyrosine; Phe, l-phenylalanine; ppm, parts per million.

Acid stability of C-lignin Because of the lack of an accessible and eliminable benzylic hydroxyl group in C-lignin units (Scheme 1B), condensation reactions due to the formation of benzyl cations might be mitigated under acidic conditions. We therefore examined the acid stability of the polymer to determine whether acidolytic methods could be used to purify the lignin. Comparison of the two-dimensional (2D) heteronuclear single-quantum coherence (HSQC) NMR spectra from the enzyme lignin (EL) (derived by removing polysaccharides via crude cellulases treatment) (21) and Klason lignin (KL) showed no significant differences in the lignin structure (Fig. 1, A and B) (22). The C-lignin survives even the harshest of acidic pretreatment methods—the KL isolation procedure includes a 1-hour treatment in 72% (w/w) sulfuric acid, followed by dilution to 4% (w/w) sulfuric acid and autoclaving at 121°C for 1 hour—while retaining its original lignin structure. An efficient acidic lithium bromide (LiBr) pretreatment method was also used to purify the lignin. This treatment method is known for its quick and near-quantitative removal of the polysaccharides to give an LiBr lignin (LBL) (Fig. 1C) (23). The molecular weight of the LBL was shown to be similar to that of the EL (Fig. 2). The C-lignin polymer appeared to survive this pretreatment based on the retention of its key lignin structural features in its NMR spectra and little change in its molecular weight distribution. On the basis of these results, we can conclude that, unlike normal S-G lignins, polysaccharides can be removed via acid pretreatment from C-lignin without its suffering from unwanted condensation reactions. After removing the polysaccharides, the resulting lignins (EL, KL, and LBL) were completely soluble in various organic solvents [for example, acetone, dioxane, or tetrahydrofuran (THF)] mixed with water to match lignin solubility parameters (24). Efficient lignin solubilization should greatly facilitate continuous processing in an industrial setting. Fig. 2 Molecular weight profiles. Molecular weight profiles of EL (cyan) and LBL (magenta) from V. planifolia seed coat measured by gel-permeation chromatography (GPC). The x axis indicates the apparent molecular weight of individual lignin polymers and is shown as a log scale. The y axis shows the response of a UV-light detector (at 280 nm) normalized to the most abundant signal in each chromatogram. The most abundant signal in the each of the two samples corresponds to a molecular weight of ~13,000 Da (determined via polystyrene standards); comparison shows that there was no obvious lignin polymer degradation during the acid pretreatment. PDI is the polydispersity index. a.u., arbitrary units. MW, molecular weight.

Response of C-lignin to traditional degradative methods To investigate the potential for C-lignin depolymerization, we applied two traditional lignin degradative analytical methods, alkaline nitrobenzene oxidation (NBO) and thioacidolysis, to a C-lignin model compound, the caffeyl alcohol dimer D1 (C-dimer), and to the vanilla bean seed coat CW (Fig. 3 and fig. S3). Although relatively low yields of the corresponding monomeric products (30 to 60%) were obtained from the dimeric compound, the use of the CW gave monomeric products in extremely low yields (<1%). As discussed widely in the past, both thioacidolysis and alkaline oxidation need the involvement of a free benzylic hydroxyl group on the lignin side chain (25, 26). It was therefore concluded that, because of the stability of the 1,4-benzodioxane structure, especially under the tested acidic and alkaline oxidative conditions, traditional lignin chemical degradation methods are ineffective for the depolymerization of C-lignin. A computational approach to evaluate the bond dissociation energy (BDE) of C-lignin using density functional theory models suggested that depolymerization of C-lignin is theoretically possible (27). Although the benzodioxane β–O–4 bond calculates to have a slightly higher BDE value than a conventional β–O–4 bond, it is still much lower than the BDEs of lignin’s C–C bonds (28).