Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.
1. Cell wall isolation
2. Matrix Polysaccharide composition
This method is essentially a modification of the method published by Albersheim 1.
3. Crystalline Cellulose Content
This method is essentially described by Updegraf8. There are a number of starting materials for this procedure: Isolated cell wall material (see 1) or wall material that has already been treated with 2M TFA (see 2.8) either the remaining pellet immediately after the acid treatment (see 2.8) or a TFA pellet, that has been washed with 2-propanol and dried.
4. Representative Results
An example of a wall analysis is presented in Figure 2. In this case poplar stem (wood) was analyzed by the various procedures outlined in the protocol section. The matrix polysaccharide composition is highlighted by an example chromatogram identifying the typical sugar present in plant cell walls, fucose, rhamnose, xylose, arabinose, galactose, mannose and glucose (and the internal standard inositol). The main hemicellulosic component of poplar is xylan as demonstrated by the high xylose content. However, the abundance of these sugars will vary depending on the feedstock used4. The glucose in this analysis is derived from the hemicellulose xyloglucan and amorphous cellulose. Due to the analysis the data can be presented as mol% or ug/ mg wall material (or dry weight). The content of crystalline cellulose is self-explanatory, one can expect values of between 20-50% of the wall dry weight. Based on the results presented here and in Part I3 the lignocellulosic composition of poplar wood is 21% lignin, 30% hemicelluloses, and 41% crystalline cellulose. The remainder would be ash.
Figure 1: Overview of lignocellulosic analysis. Cell walls (lignocellulosics) are isolated from crude dried plant material. The wall material is then weighted into aliquots and subdivided for the various assays. Matrix polysaccharide composition is established after treating the wall material with a weak acid (2M TFA), derivatizing the resulting solubilized monosaccharides to their alditol acetates, and analysis by GC-MS. The residue of the weak acid treatment is washed with the so-called Updegraff reagent leaving only insoluble crystlline cellulose behind. The cellulose is solubilized by sulfuric acid and quantified by a colorimetric assay determining the glucose content. In parallel, the content and composition of lignin can be determined as described in Part I3.
Figure 2: Comprehensive lignocellulosic analysis of poplar wood. Wood chips from poplar (Populus tremoloides) were subjected to the described protocol.
Upper left: Matrix polysaccharide composition; Fuc fucose; Rha rhamnose; Ara arabinose; Xyl xylose; Man mannose; Gal galactose; Glc glucose; inositol internal standard.
The described methods enable a rapid quantitative assessment of the composition of lignocellulosic plant biomass. The method allows the determination of the composition of such materials including the sugar composition of the matrix polysaccharides namely the hemicelluloses, the crystalline cellulose content. The throughput of the various analytical methods per person varies. Using the protocols described here, 20 samples can be processed for matrix polysaccharide compositions and 30 for crystalline cellulose content. Due to the quantitative nature of the data optimal feedstock crops, variety or genotypes can be assessed in terms of their suitability for biofuel production.
We are grateful to Matthew Robert Weatherhead for excellent technical service and John Ralph, University of Wisconsin for valuable advice, discussions, and the poplar wood sample. This work was funded by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (award no. DE-FG02-91ER20021) and by the US Department of Energy (DOE) Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494).
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Trifluoroacetic acid | Sigma-Aldrich | T6508 | ||
myo-Inositol | Sigma-Aldrich | I5125 | ||
Sodium Borohydride | Sigma-Aldrich | 213462 | ||
Pyridine | J.T. Baker | 3348-01 | ||
Acetic Anhydride | Sigma-Aldrich | 320102 | ||
Spectromax Plus 384 | Molecular Devices | Plus384 | ||
GC-MS | Agilent | 7890A GC/5975C MSD | ||
5.5mm Stainless Steel Balls | Salem Ball Company | (N/A) | ||
96 well plate heat spreader | Biocision | Coolsink 96F | ||
Retsch Mill | Qiagen | TissueLyser II | ||
Heating block | Techne | Dri-block DB-3D | ||
Sample concentrator | Techne | FSC400D |