High throughput determination of glucan and xylan fractions in lignocelluloses

Abstract

The analysis of structural glucan and xylan in lignocellulose was scaled down from original two-stage sulfuric acid hydrolysis methods (Moore WE and Johnson DB 1967 Procedures for the chemical analysis of wood and wood products. U.S. Forest Products Laboratory, U.S. Department of Agriculture., Madison, WI) and integrated into a recently-developed, high throughput pretreatment and enzymatic saccharification system. Novel 96 9 1.8 ml-well Hastelloy reactor plates (128 9 86 9 51 mm) based on previously described 96-well pretreatment reactor plates were paired with custom aluminum filler plates (128 9 86 9 18 mm) for use in Symyx Powdernium solids dispensing systems. The incorporation of glucose oxidase and xylose dehydrogenase linked assays to speed post-hydrolysis sugar analysis dramatically reduced the time for analysis of large lignocellulosic sample sets. The current system permits the determination of the glucan and xylan content of 96 replicates (per reactor plate) in under 6 h and parallel plate processing increases the analysis throughput substantially.

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

“…We identified two QTL for m/z 120 (4-vinylphenol) abundance. Two years of the equivalent cell wall materials used in PyMBMS were available for the studies of saccharification yield by a semiautomated, high-throughput saccharification screen (Selig et al, 2009(Selig et al, , 2011. Values for Glc and Xyl varied 1.5-to 2.2-fold over the population in each year.…”

“…Glc and Xyl were assayed enzymatically with Glc oxidase and Xyl dehydrogenase as described (Selig et al, 2011) and quantified by comparison with standards treated similarly. All assays were validated statistically.…”

“…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. Pyrolysis molecular-beam mass spectrometry (PyMBMS) gives the relative abundance of mass-to-charge ratio (m/z) fragments diagnostic for monomeric units of phenylpropanoid, hydroxycinnamic acid, and cell wall polysaccharides (Evans and Milne, 1987;Boon, 1989;Tuskan et al, 1999;Yin et al, 2010).…”

“…We validated diagnostic fragments for guaiacyl (G) and S lignin as reliable measures of abundance in maize cell walls (Penning et al, 2014). In parallel, we used a high-throughput protocol to quantify saccharification yields of Glc and Xyl using a cocktail of polysaccharide-hydrolyzing enzymes (Selig et al, 2009(Selig et al, , 2011.…”

The Increasing Importance and Capabilities of Biomass Characterization

“…12,13 These allow for analysis of smaller samples and complement prior work with near infrared (NIR) and pyrolysis molecular beam mass spectrometry. [14][15][16][17] Lastly, screens of biomass-degrading enzymes have also been improved to facilitate screening for known or new degradative activities.…”