The dispute against direct dyes as molecular probes for evaluating enzyme accessible cellulose in lignocellulosic biomass

This study outlines the general experimental guidelines for using direct dyes as molecular probes and interpretation of the results thereof to visualize surface architecture of cellulosic materials. The modified Simons’ staining technique aims at quantifying enzyme accessibility to cellulose and suggests a correlation between the orange to blue dye adsorption ratio and enzymatic hydrolysis yields for lignocellulosic biomass¹. Results from our application of this technique to poplar pretreated at aqueous conditions in batch and flowthrough reactors indicated that the orange to blue dye ratio may not be accurate as an indicator of enzyme accessibility to cellulose. The retention of orange dye above the membrane for the shorter periods of ultrafiltration currently employed in the modified Simons’ staining technique may be due to formation of aggregates that result from local high concentrations seen near the membrane surface, similar to that reported for proteins such as BSA². This outcome may give the illusion of the orange dye being of similar size to the Stokes-Einstein value for T. ressei cellulase. Furthermore, multiple layers of dye may form on the cellulose surface in certain electrical environments as adsorption curves were found to deviate from Langmuir behavior at high concentrations. Aggregation and dye adsorption of any of the dyes were also found to be highly salt dependent, with the absence of salt resulting in no adsorption onto biomass. The interaction of cellulosic direct dyes with hemicellulose and lignin in pretreated lignocellulosic biomass was also studied for the first time.