Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment

Abstract

Plant cell walls are composed primarily of cellulose, hemicelluloses, lignins, and pectins. Of these components, lignins exhibit unique chemistry and physiological functions. Although lignins can be used as a product feedstock or as a fuel, lignins are also generally seen as a barrier to efficient enzymatic breakdown of biomass to sugars. Indeed, many pretreatment strategies focus on removing a significant fraction of lignin from biomass to better enable saccharification. In order to better understand the fate of biomass lignins that remain with the solids following dilute acid pretreatment, we undertook a structural investigation to track lignins on and in biomass cell walls. SEM and TEM imaging revealed a range of droplet morphologies that appear on and within cell walls of pretreated biomass; as well as the specific ultrastructural regions that accumulate the droplets. These droplets were shown to contain lignin by FTIR, NMR, antibody labeling, and cytochemical staining. We provide evidence supporting the idea that thermochemical pretreatments reaching temperatures above the range for lignin phase transition cause lignins to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls. This decompartmentalization and relocalization of lignins is likely to be at least as important as lignin removal in the quest to improve the digestibility of biomass for sugars and fuels production.

Side by Side Comparison of Chemical Compounds Generated by Aqueous Pretreatments of Maize Stover, Miscanthus and Sugarcane Bagasse

“…Even at 180°C, acidic pretreament was ineffective at reducing the lignin fraction of the feedstock residue, compared to alkaline. This is in accordance with the observation that acid pretreatment at temperatures above the glass transition temperature of lignin (~130°C) is able to melt and redistribute lignin in the cell wall, but not to remove lignin from the biomass [28]. Table 3 Effects of Pretreatments on the Release of Phenolics and Fatty Acids…”

“…However, it cannot be excluded that the dimers in 20°C samples originate Fig. 5 Compounds structurally characterised in alkaline and acid pretreatment liquors (22)(23)(24)(25)(26)(27)(28)(29). See Supplemental Fig.…”

Effects of thermo-hygro-mechanical (THM) treatment on the viscoelasticity of in-situ lignin

“…The fact that CS treatment leads to a decrease in T g accompanied by an increasing ΔH a may be interpreted in terms of competitive depolymerization and re-condensation reactions. One should however be aware of that some parts of the lignin could have been rearranged and become inactive due to redeposition on cell walls at these high temperatures (Selig et al 2007;Donohoe et al 2008;Navi and Pizzi 2015), thus affecting the viscoelastic properties of the remaining lignin.…”

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

“…Consistent with these studies, we find homologs for almost every gene in the phenylpropanoid synthesis pathway present within at least one of our lignin abundance-or saccharification yieldrelated QTL regions (Supplemental Table S6). However, the effect of our pretreatment removes lignin recalcitrance by melting and partially removing it from the cell wall (Donohoe et al, 2008). Even though the B73 inbred line is a derived stiff-stalk line (Liu et al, 2003) and has slightly more stem lignin than does Mo17 (Penning et al, 2014), QTL were established with both Mo17 and B73 as the dominant parent (Supplemental Table S6).…”

“…In switchgrass (Panicum virgatum), acid pretreatment increased saccharification yield in transgenic lines with reduced cinnamyl alcohol dehydrogenase (Fu et al, 2011b). Thermochemical pretreatments of biomass such as steam or ammonia explosion that reach temperatures above the range for lignin phase transition cause lignin to coalesce into molten bodies that redistribute within the biomass (Donohoe et al, 2008). We treated postharvest stem (stover) cell walls isolated from members of maize recombinant inbred lines (RILs) with steam explosion (Selig et al, 2011) and subsequently determined that quantitative trait loci (QTL) for lignin abundance and those for saccharification yield of Glc or Xyl do not overlap under these conditions.…”