Biofuels and green chemicals from plant lignocellulose offer the promise of decreased petroleum consumption with the caveat that technological and economic barriers remain that impede large-scale industrial implementation. The polysaccharide portion of plant cell walls (cellulose and hemicellulose) is ideally suited to conversion via
biochemical transformations due to the central placement of carbohydrates in cellular metabolism. A diverse set of enzymes and metabolic pathways currently exist that are capable of converting carbohydrates to a wide range of useful metabolites and recent progress in metabolic and protein engineering is expanding this range. The challenge to realizing the potential of plant cell wall polysaccharides is primarily due to the set of plant properties collectively known as “biomass recalcitrance” [1
] that limit the availability of polysaccharides for biological conversion by enzymatic or catabolic routes. This recalcitrance is primarily derived from the heterogeneous supramolecular organization of the plant cell wall matrix or higher order structures in the plants and necessitates a chemical or physical pretreatment step prior to biological conversion [2
]. These higher order structures include considerations such as overall plant anatomy, cell wall thickness, covalent and non-covalent interactions between macromolecules (cellulose, hemicellulose, and lignin) as well as distribution of these macromolecules within the cell wall matrix.
Polysaccharides within secondary cell walls are embedded within a matrix of lignin that limits their accessibility. Lignin’s physiological role in the plant cell wall and the reason for its contribution to recalcitrance is to protect vulnerable carbohydrates from attack by pathogens, provide structural stability to the cell wall, and present a hydrophobic barrier to water penetration through cell types that serve the purpose of fluid transport. While lignin’s role in cell wall recalcitrance is universally accepted, the precise set of factors that contribute to this recalcitrance are not universally acknowledged. Factors specific to lignin’s role in recalcitrance have been proposed to include the total lignin abundance [3
], lignin location within the cell wall [6
], and the properties of lignin such as hydrophobicity [7
], as well as indirect impacts such as lignin’s ability to bind enzymes [8
Lignin is a polymer composed primarily from three “canonical” p
-hydroxycinnamyl alcohols (monolignols) including coniferyl, sinapyl, and to a lesser extent p-
coumaryl alcohols which form the guaiacyl (G), sinapyl (S), and p
-hydroxyphenyl (H) units in lignin [9
]. Other “non-canonical” aromatic monomers taken from other points of the monolignol biosynthesis grid are known to be incorporated into the lignin polymeric framework as well [10
]. The linkages between monolignols can be through ethers such as the β-O-4 (β-aryl ether) and 5-O-4 (biphenyl ether) linkages, C-C (“condensed”) bonds such as 5–5 (biphenyl) linkages, or a combination of C-C and ether linkages as in β–5
α-O-4 (phenylcoumaran) and β–β
α-O-γ (resinol) among others [12
], as well as ester linkages involving, for example, p
]. The total content and relative abundance of monolignols, their linkages, and degree of crosslinking with polysaccharides varies by plant taxa, plant developmental stage, and plant tissue [14
]. Other features of lignin that differ between plant source include potentially the degree of polymerization (although this is not clear) and the number of free phenolic groups. Free phenolics provide initiation sites for alkaline and oxidative delignification and contribute to lignin’s alkali solubility [17
] due to deprotonation of phenolic hydroxyls at a lower pH than aliphatic hydroxyls.
A significant number of promising bioenergy feedstocks are commelinid monocots, specifically the Poaceae
or grasses, and include agricultural wastes [19
] such as corn stover, wheat straw, rice straw, and sugar cane bagasse and dedicated perennial energy crops such as switchgrass and Miscanthus
spp. among others. Lignin composition and cell wall structural organization in grasses is significantly different from herbaceous and woody dicots (forbs and hardwoods, respectively) or gymnosperm lignins. One distinguishing feature of the monocot lignins is the considerable incorporation of the p
-hydroxycinnamic acids including ferulic and p
-coumaric acids [13
]. Ferulate monomers and dimers are known to be ester-linked to glucuronoarabinoxylan [22
] and are proposed to be involved in ether and C-C linkages to the lignin polymer that act as crosslinks between hemicellulose and lignin polymer chains [11
]. Monomers of p-
coumaric acid are proposed to be esterified to the lignin polymer at the γ-carbon of the side chain region of β-O-4 linked syringyl moieties [24
] and to a lesser degree esterified to glucuronoarabinoxylan. Grass lignins are significantly more condensed (i.e.
contain more C-C linkages between monolignols) and have higher phenolic hydroxyl contents than the lignins of dicots [21
] and an important implication of this is that more than 50% of grass lignins can be solubilized by treatment with alkali [26
] due to the destruction of alkali-labile ester linkages along with the high free phenolic content improving lignin solubility in alkali [17
Plants have typically neither been under selective evolutionary constraint nor bred to yield phenotypes that would yield high polysaccharide conversion for a bioenergy process, although the identification and propagation of forage crops with the phenotype for high digestibility in ruminants [27
] represents an important starting point. Examples include forage improvement studies on corn stover [28
] and the identification of the brown midrib mutations in grasses including maize, millet, and sorghum [29
] which have been known as having the phenotype for improved ruminant digestibility in corn for more than 50
]. The brown midrib lines of maize are known to contain less lignin as well as altered monolignol ratios, and altered inter-monolignol linkages which the present work aims to exploit in comparing differences in the lignin contents and structures.
Alkaline hydrogen peroxide (AHP) pretreatment has been studied as a chemical pretreatment [33
] and as a delignifying post-treatment [37
] and is based on treatment of biomass with hydrogen peroxide at alkaline pH (optimally at pH 11.5) at ambient or near-ambient temperatures and pressures. Due to the distinctive properties of their lignins and structural organization of their cell walls as described above, alkaline pretreatments are particularly effective for grasses, and it is known that AHP is less effective on forbs [36
] and woody dicots (unpublished observations). We hypothesize that the cellulose enzymatic digestibility improvement resulting from AHP pretreatment may be attributable to the destruction of ferulate crosslinks as well as mild oxidation and solubilization of lignin. These outcomes have the net effect of improving the overall hydrophilicity of the cell wall matrix which can allow for water and hydrolytic enzyme penetration.
In this work, AHP pretreatment is used to generate a set of biomass samples exhibiting a diverse range of lignin contents and abundance of the p-hydroxycinnamates with the goal of better characterizing the relationship between a delignifying pretreatment, the composition and properties of the cell wall, and the digestibility by cellulolytic enzymes in grasses displaying a diverse natural range of lignin phenotypes. These lignin phenotypes include differences in total lignin content, differences in the relative abundance of monolignols incorporated into the cell wall with the consequence of altered linkages between monolignols, and differences in the ferulate content resulting in differences in the extent of cross-linking between cell wall polymers. Specifically, these biomass types consist of a switchgrass cultivar, a commercial hybrid corn stover, and two inbred brown midrib lines of maize (bm1 and bm3). The relationship between S/G ratios as determined by Py-GC/MS, thioacidolysis-GC/MS, and HSQC NMR are compared in parallel for the first time for grasses, while HSQC NMR is applied to characterize structural changes associated with the whole cell wall, with the overall intention of identifying and linking properties associated with grass lignins to improved cellulolytic enzyme digestibility.