Lignin is a polymeric material composed of phenylpropanoid units derived primar­ily from three cinnamyl alcohols (monolignols): p-coumaryl, coniferyl, and sinapyl alcohols. Polymer formation is thought to occur via oxidative (radical-mediated) coupling between monolignols and the growing oligomer/polymer [52, 53] and is commonly believed to occur in a near-random fashion [54], although some recent studies suggest an ordered and protein-regulated lignin synthesis [55]. In any case, the resulting polymer is complex, heterogeneous, and recalcitrant to biological degradation. Although lignin loss is minimal during thermal-acidic/neutral pre­treatments, it can undergo structural and chemical changes [56] that significantly influence downstream enzymatic conversion.

Although enzymes thoroughly penetrate cell walls after high severity pre­treatments [49], incomplete cellulose conversion by cellulases suggests additional barriers exist at the ultrastructural level. One potential barrier is occlusion of the cel­lulose microfibrils by residual lignin or hemicellulose that would sterically prevent

cellulases from binding to cellulose [42]. Other indirect mechanisms that impede complete cellulose hydrolysis are also possible such as non-productive binding of cellulases to lignin [34-36], however reports that contradict this theory also exist [57].

Enzymatic hydrolysis of biomass pretreated under alkaline conditions, which hydrolyzes less xylan than acidic pretreatments, supports the steric hindrance concept. Elevated cellulolytic activity is observed on alkaline pretreated biomass when cellulases are supplemented with xylanases and other hemicellulose degrading enzymes, likely a function of removing additional barriers to cellulose accessibility [58, 59]. A study in pretreatment variability by Selig and co-workers suggested that cellulose digestibility is improved directly by xylan removal, but only indirectly by lignin removal [47]. Removal of lignin by pretreatment appeared to increase enzymatic removal of xylan, which in turn increased cellulose digestibility. Lignin removal alone had little impact on cellulose digestion. Lignin modifying enzymes, however, have been shown to synergistically work with cellulases during digestion of steam-pretreated biomass, improving sugar yields through at least partial removal of the lignin barrier [60]. In spite of a general consensus in the scientific community about the significance of the lignin barrier to cellulose digestibility, only limited attention has been given to the fate of lignin during widely used high tempera­ture dilute acid, hot water, and steam pretreatments which only partially remove lignin [1,8].

A recent study investigated the fate of lignin during high temperature acid and neutral pretreatments using electron microscopy and spectroscopy techniques [40]. This study revealed that lignin could be mobilized within the cell wall matrix at temperatures as low at 120°C during both neutral and low pH pretreatments, and appears to be, at least in part, dependent on pretreatment severity. On a relatively macro scale, part of the mobilized lignin deposits back on to biomass surfaces as spherical bodies, suggesting that lignin undergoes the following sequence of events during these pretreatments — phase-transition or melting, mobilization into bulk solution, coalescence, and deposition onto solid surfaces. Scanning- and transmis­sion electron microscopy (SEM and TEM) of pretreated cell walls shows that the lignin droplets (stained with KMnO4) take a wide range of sizes (<50 nm to 2 ^m) and shapes (Fig. 4a, b and Fig. 5), though the “free” shapes are uniformly spheri­cal. Other shapes observed appear to be dictated by the physical constraints of the structures surrounding them. In addition to redeposition, there also appears to be a reorganization of lignin structure within the cell walls. A fraction of the lignin remains within the walls during pretreatment. This fraction apparently melts, but is unable to escape into the bulk liquid phase before coalescing back into droplets, as evidenced by the KMnO4 stained lignin droplets that appear between layers in the cell wall (Fig. 4b-d).

Aside from the obvious implications of lignin mobility, coalescence, and rede­position observed during high temperature pretreatments, chemical modification of the lignin should also be considered. These may range from covalent bond break­age and formation to changes in inter — and intramolecular interactions. Although FTIR and NMR studies did not distinctly show chemical changes in the mobilized

Fig. 1.4 TEM micrograph of lignin droplets re-deposited on cellulose surfaces after being transported from the cell wall matrix during high temperature pretreatments (a). Electron tomograph images of coalesced lignin within cell walls. The boxed region in b has been segmented to show the 3D volume of coalesced lignin (c). Large lignin globules can form in openings like pits (arrow b, d). Scale bars = 200 nm a; 500 nm b, c; 200 nm d

lignin in this study, it is possible that chemical alteration could be part of the lignin removal and transport process because lignin can partially dissolve and react in acid solutions under appropriate conditions [56]. It is further possible that part of this mobilized lignin could contain lignin-carbohydrate complexes that might sequester cellulases as observed in some studies [34, 36].

Another recent study [42] showed that purified lignin preparations as well as native lignin from corn stover could be redeposited onto clean cellulose surfaces such as filter paper. More severe pretreatments (higher temperature or acid concen­trations) resulted in finer redeposited droplets. Under these conditions, digestibility of filter paper was lower by up to 15% in comparison with treatments that did not contain lignin. Since these digestions were performed at very high enzyme load­ings to circumvent issues related to non-productive binding to lignin, it appears that physical blockage of the cellulose surface by lignin resulted in lower digestibility. Although redeposited lignin inhibited digestion of pure cellulose substrates in the study by Selig and coworkers [42], it is also probable that the mass transport of lignin could enhance enzymatic cellulose degradation in biomass. For example, we could visualize that as a result of lignin mass transport, the lignin sheath coating cellulose surfaces gets concentrated into droplets rendering a greater cellulose sur­face area available for enzymatic attack. Removal of lignin could also improve cell wall porosity allowing enzymes better access for penetration. Much work needs to be done to completely understand the nature and implications of lignin transport.