Cell Wall Analysis
In use from 2008-09-01
Samples of vegetative biomass from all treatments are sent to the Cell Wall Facility each year to determine their digestibility and composition. The composition analysis provides concentrations of monosaccharides, including rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, and crystalline cellulose. It also provides lignin content and lignin monomer composition. The digestibility analysis provides data on the % conversion to glucose and xylose. These data can be ultimately used to determine ethanol yield. However, the Cell Wall Facility requests that GLBRC scientists discuss these calculations with them before publishing any determinations of ethanol yield.
After recording fresh weight and dry weight, grind plant samples on a mill with a 1mm screen. Obtain pre-coded sample tubes from the Cell Wall Facility, and fill each tube with 0.5 ml of sample. Enter the codes on the Enabling Technologies website http://glbrc.bch.msu.edu/cwsc/ (you will first need to obtain access to the site) and deliver the samples.
Cell Wall Composition (Enabling Technologies 5.6.2)
Using the dried biomass, a 60 mg aliquot is ball milled with the iWall grinding and feeding robot. The milled material is then used to prepare the alcohol insoluble residue (AIR) which involves aqueous and solvent washes to extract the soluble components, such as soluble sugars, proteins, lipids, pigments, DNA, and RNA. The AIR is then incubated with amylase and pullulanase and subsequently washed with water to remove the starch content to yield isolated lignocellulosic material for cell wall analysis (Foster et al. 2010a).
The isolated lignocellulosic cell wall material is dried and weighed into three, 2mg technical replicates to assay the matrix polysaccharide composition and crystalline cellulose content. The polysaccharide composition is analyzed via GC-MS (Agilent 7890A GC / 58975C MS) after a 2M trifluoroacetic acid (TFA) hydrolysis and subsequent alditol acetate derivatization of the neutral monosaccharides present the hydrolysate. The crystalline cellulose is isolated and purified from the insoluble residue remaining from the TFA hydrolysis and then hydrolyzed in 72% sulfuric acid and assayed using the colorimetric anthrone assay to determine the crystalline cellulose content of the lignocellulosic material (Foster et al. 2010b).
Digestibility (Enabling Technologies 5.6.3)
The samples are pretreated in two ways: with dilute sodium hydroxide (6.25mM) at 90°C and with dilute sulfuric acid (2% w/v) at 120°C. Next, enzyme digestion occurs, at a target of one third or less of the total glucan. Keeping the percent of total glucose released below 50% provides higher resolution for identifying modified biomass with increased as well as decreased digestibility. The process is completed with a separation step. The soluble digestion fraction is removed from the residual biomass by centrifugation. Aliquots are removed from the digested solutions, and enzyme-based assays are performed for glucose and xylose (Santoro et al. 2010).
Cell Wall Composition, Cliff Foster, firstname.lastname@example.org
Cell Wall Digestibility, Nick Santoro, email@example.com
Foster, C. E., Martin, T. M., Pauly, M. 2010a. Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin. http://www.jove.com/details.php?id=1745 doi: 10.3791/1745. J Vis Exp. 37.
Foster, C. E., Martin, T. M., Pauly, M., 2010b. Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates. http://www.jove.com/details.php?id=1837 doi: 10.3791/1837. J Vis Exp. 37.
Santoro, N., Cantu, S., Tornqvist, C.E., Falbel, T., Bolivar, J., Patterson, S., Pauly, M., Walton, J. 2010. A High-Throughput Platform for Screening Milligram Quantities of Plant Biomass for Lignocellulose Digestibility. Bioenrg.Res. 3:93–102.