Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

Abstract

A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates) were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase) that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion) of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber) required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine). Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic cleavage of the cellulose chains per se, but rather the limited accessibility of the enzymes to the cellulose chains due to the physical structure of the cellulosic substrate.

Figure 1

Typical time course of (A) the enzymatic hydrolysis of cellulose; (B) cellulose hydrolysis with increasing protein loadings.

Figure 2

Response surface fitted to the experimental data corresponding to the hydrolysis of a broad range of pretreated substrates. Hydrolysis times and solids loadings were kept constant at 72 hours and 2% (w/v), respectively.

Figure 3

Effect of hydrolysis time and solids loading on the minimum protein requirement for efficient hydrolysis of a variety of lignocellulosic substrates. Cellulase and β-glucosidase were kept constant according to the protein level shown in Table 6.

Figure 4

Relationship between maximum protein adsorption capacity of a range of pretreated lignocellulosic biomass and the optimized minimum protein loading for efficient hydrolysis.

Figure 5

Relationship between minimum protein loading for efficient hydrolysis and external surface area determined as average initial fiber length.

Figure 6

Relationship between glucan content and distribution of large and small pores (combination interior/exterior surface area) determined by the Simons' staining technique.

Figure 7

Relationship between distribution of large and small pores (combination interior/exterior surface area) and minimum protein loading for efficient hydrolysis.