Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.

The crystallinity index of cellulose is an important parameter to establish because of the effect this property has on the utilization of cellulose as a material and as a feedstock for biofuels production. However, it has been found that the crystallinity index varies significantly depending on the choice of instrument and data analysis technique applied to the measurement. We introduce in this study a simple and straightforward method to evaluate the crystallinity index of cellulose. This novel method was developed using solid state 13 C NMR and subtraction of the spectrum of a standard amorphous cellulose. The crystallinity indexes of twelve different celluloses were measured and the values from this method were compared with the values obtained by other existing methods, including methods based on X-ray diffraction. An interesting observation was that the hydration of the celluloses increased their crystallinity indexes by about 5%, suggesting that addition of water increased cellulose order for all the cellulose samples studied.

Enzyme accessibility has been proposed as a limiting factor in the enzymatic conversion of the cellulose in biomass to glucose. Prior work has shown a strong correlation between porosity, measured as the change in the volume of pores accessible to a cellulase-sized molecule, and the initial digestibility of biomass pretreated by various methods. The goal of this work was to determine if porosity was one of the factors governing the overall enzymatic digestibility of the cellulose in dilute acid pretreated biomass. The porosity of wet pretreated corn stover was determined using the methods of solute exclusion and 1H nuclear magnetic resonance (NMR) thermoporometry. The solute exclusion method identified differences in the accessible pore volume of the pretreated samples compared to untreated corn stover; however, only very small differences in porosity were observed among samples pretreated with a range of severities, giving ethanol yields from 70 to 96%. No correlation was found between the volume accessible to an enzyme-sized molecule (diameter estimated to be 51 A) and the digestibility of the cellulose in dilute acid pretreated corn stover. 1H NMR thermoporometry was used to measure the amount of water in pores ranging from 20 to 200 A. As was the case for the solute exclusion method, a difference was observed in the pore volume of untreated and acid pretreated corn stover, but no significant differences in pore volume were measured for the different pretreated samples.

It has previously been shown that the improved digestibility of dilute acid pretreated corn stover is at least partially due to the removal of xylan and the consequent increase in accessibility of the cellulose to cellobiohydrolase enzymes. We now report on the impact that lignin removal has on the accessibility and digestibility of dilute acid pretreated corn stover. Samples of corn stover were subjected to dilute sulfuric acid pretreatment with and without simultaneous (partial) lignin removal. In addition, some samples were completely delignified after the pretreatment step using acidified sodium chlorite. The accessibility and digestibility of the samples were tested using a fluorescence-labeled cellobiohydrolase (Trichoderma reesei Cel7A) purified from a commercial cellulase preparation. Partial delignification of corn stover during dilute acid pretreatment was shown to improve cellulose digestibility by T. reesei Cel7A; however, decreasing the lignin content below 5% (g g -1 ) by treatment with acidified sodium chlorite resulted in a dramatic reduction in cellulose digestibility. Importantly, this effect was found to be enhanced in samples with lower xylan contents suggesting that the near complete removal of xylan and lignin may cause aggregation of the cellulose microfibrils resulting in decreased cellulase accessibility.