10-22: Simple, rapid, and in situ quantifications of productive and nonproductive cellulase adsorption during enzymatic hydrolysis of lignocelluloses using UV-vis spectrophotometry

      Enzyme adsorption kinetics in the initial period of lignocellulose hydrolysis is very important to achieve high saccharification efficiency.  Batch sampling and offline measurement methods used in the literature are tedious and incapable of resolving adsorption kinetics.  Spectral absorption by lignin prevented the application of spectroscopic methods for in situ quantification of cellulase. We will present two simple, rapid, and robust methods for in situ and temporally resolved measurements of cellulase adsorption onto lignocelluloses by UV-vis spectrophotometry.  We will demonstrate a dual-wavelength and a spectral derivative method to successfully correct spectral interferences by leached lignin and solid particles present in the enzyme-lignocellulose suspensions on cellulase adsorption spectra. By subtracting the measured cellulase in the hydrolysate from the initial cellulase applied, cellulose adsorption onto lignocellulose was obtained.  We then applied the method to determine cellulase absorption kinetics of lignocellulosic substrates pretreated by different methods (dilute acid, SPORL, and alkaline).  We will reveal the dynamic cellulase adsorption characteristics onto these substrates with the applications of a surfactant (Tween 80) and metal salts (MgSO4, CaSO4).  Both types of compounds have shown to be effective to prevent non-productive absorption of cellulase by lignin.  We used the present method to facilitate in situ cellulose accessibility measurements during hydrolysis using a technique by Hong et al. (Langmuir 2007). We will report rapid, in situ determination of total, nonproductive, and productive adsorptions of cellulase using the same set of substrates.  The results will provide new insights to understand the effectiveness of different pretreatment methods for removal biomass recalcitrance.