Do Enzymatic Hydrolyzability and Simons' Stain Reflect the Changes in the Accessibility of Lignocellulosic Substrates to Cellulase Enzymes?

Abstract

In an attempt to elucidate the impact of substrate accessibility to cellulases on the susceptibility of lignocellulosic substrates to enzymatic hydrolysis, a hydrogen peroxide treated, Douglas fir kraft pulp was dried using several methods with varying levels of intensity. Oven-drying at 50 and 100 degrees C, air-drying, and freeze-drying methods were employed to remove the interfibrillar water from the pulp samples. Subsequently, the never-dried and variably dried pulps were hydrolyzed using a commercial cellulase preparation supplemented with additional beta-glucosidase. Drying reduced the susceptibility of the substrates to enzymatic hydrolysis, which can be attributed to the hornifying effect that drying has on fibers. This effect was more pronounced for the fibers that were oven-dried at 100 degrees C (23% reduction) and 50 degrees C (15% reduction), and there was a good correlation between the Simons's stain results and the enzymatic digestibility of the dried pulps. These observations indicated that drying significantly reduced the population of larger pores and that the partial closure of larger pores created a large number of smaller pores that were not accessible to the displacement dye molecules (orange dye). The inaccessibility of the cellulose to the enzymes, due to the collapse or closure of the large pores, appears to be the primary reason for the lower susceptibility of the dried pulps to enzymatic hydrolysis.

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

“…In particular, the COSLIF-pretreated corn stover has nearly twofold higher hemicellulose composition (6.2%) than has DA-pretreated corn stover (3.4%) but the former has much higher digestibility and faster hydrolysis rates. A common belief is that lignin removal promotes faster and more efficient enzymatic cellulose hydrolysis Chang and Holtzapple, 2000;Esteghlalian et al, 2001;Lee et al, 1994;Mooney et al, 1998;Ohgren et al, 2007;Zhu et al, 2008), but the data presented here suggest that increasing CAC is more important for achieving fast hydrolysis rates and high glucan digestibility because NCAC of the Table II. Total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) values of corn stover samples before pretreatment and after COSLIF and DA pretreatment.…”

“…The total substrate accessibility has been measured previously by using cellulase-size molecule exclusion (Burns et al, 1989;Esteghlalian et al, 2001;Grethlein, 1985;Thompson et al, 1992), low-temperature cellulase adsorption (Gerber et al, 1997;Kumar and Wyman, 2008;Lee et al, 1994;Lu et al, 2002;Mooney et al, 1998) or labeled cellulase (Jeoh et al, 2007;Palonen et al, 2004). However, in pretreated lignocellulose materials it remains relatively challenging to quantitatively differentiate accessibilities for cellulose and non-cellulose fractions.…”

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

“…5). This concept has been discussed in the literature as relating changes in the porosity of the biomass sample to digestibility (Esteghlalian et al, 2001;Grethlein, 1985). NMR measurements conducted on these samples showed that dilute acid pretreatment increased the porosity in corn stover (Ishizawa et al, 2007), suggesting that the observed increase in T. reesei Cel7A access to the cellulose in PCS is due to improved physical access within the biomass structure.…”

“…The opposite of ''opening up'' the structure of biomass to increase cellulase access is to collapse the structure to limit access, which is thought to occur when a biomass sample is dried (Esteghlalian et al, 2001;Grous et al, 1986). The water content in biomass affects the degree of swelling (Browning, 1975), the crystallinity (Fan et al, 1981) and the digestibility (Focher et al, 1981) of cellulose microfibrils.…”

“…The water content in biomass affects the degree of swelling (Browning, 1975), the crystallinity (Fan et al, 1981) and the digestibility (Focher et al, 1981) of cellulose microfibrils. Air-drying of biomass samples is thought to irreversibly collapse the capillary structure (Esteghlalian et al, 2001;Weise, 1998), significantly decreasing internal surface area (Stone and Scallan, 1968;Browning, 1975). Consequently, biomass sample drying is generally understood to negatively affect the enzymatic digestibility of the cellulose fraction in biomass by limiting enzyme access (Esteghlalian et al, 2001;Fan et al, 1980a;Grous et al, 1986).…”