Simons’ stain

The accessibility of unpretreated switchgrass was estimated by a down-scaled Simons’ stain procedure with DB 1 and DO 15 dyes, as described previously [22, 34]. In detail, extractive-free switchgrass (10.00 mg oven-dry basis) was weighed into seven micro-centrifuge tubes (2 mL) individually. Phosphate-buffered saline solution (pH 6.0, 0.30 M PO₄^3-, 1.40 mM NaCl) (0.10 mL) was added to each tube. A set of dye solutions containing 0.20, 0.40, 0.60, 0.80, 1.20, 1.60, and 2.00 mg/mL of both DO and DB was made and 0.50 mL of the solution was added to each tube. DI water (0.40 mL) was added to each tube to make up the final volume to 1.00 mL to give concentrations of DO and DB of 0.10, 0.20, 0.30, 0.40, 0.60, 0.80, and 1.00 mg/mL. The centrifuge tubes were incubated at 70 °C for 6 h with shaking at 200 rpm to allow dye adsorption to reach equilibrium. The absorbance of the supernatant solution after centrifugation was obtained on a Lambda 35 UV–Vis spectrophotometer at 410 and 599 nm, which represent the wavelengths of maximum absorbance for DO and DB, respectively. The amount of each dye adsorbed by the biomass sample was determined using the difference between the concentration of the initial added dye and the concentration of the dye in the supernatant. The maximum amount of DO or DB adsorbed to the substrate was calculated from the slope of a plot of C/A versus C according to the Langmuir adsorption equation shown below:

where C (mg/mL) is the concentration of free dye in supernatant, A (mg/g biomass) is the amount of dye adsorbed by the substrate, K (mL/mg) is the Langmuir adsorption constant, and A _m (mg/g biomass) is the maximum amount of dye adsorbed.