Use of the Simons' Staining Technique to Assess Cellulose Accessibility in Pretreated Substrates

The specificity of the Simons' staining technique for determining the specific surface area (SSA) of cellulose—the amount of cellulose in a pretreated substrate that is accessible to cellulases—was assessed over a range of cellulosic substrates. Both the Direct Blue (DB) dye and the high molecular weight (HMW) fraction (>100 kda) of the Direct Orange (DO) dye were shown to bind with high specificity to the cellulose rather than hemicellulose and/or lignin. The use of the DO dye by itself could also indicate differences in the accessibility/SSA of various types of cellulose resulting in an R2 value of 0.87 when plotted against enzymatic hydrolysis yields. The previously reported fluctuations in the ratio of DO:DB when the dyes were adsorbed to similar cellulosic substrates were shown to be primarily due to differences in the method used for the isolation of the high molecular weight fraction of the DO dye. A procedure for the enrichment of the >100 kDa HMW fraction of the DO using a minimum of two rounds of ultrafiltration is described. The ability of the modified assays to assess the specific surface area of pretreated and enzymatically hydrolyzed substrates showed good reproducibility.