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.
Highlights
Bioethanol derived from the bioconversion of lignocellulosic feedstocks continues to attract global interest as a potentially environmentally compatible alternative to current petroleum-based transportation fuels
A statistical experimental design was used to define the minimum amounts of protein required for efficient hydrolysis of the pretreated substrates, in order to establish a reliable assessment of the factors governing the minimum protein loading required for efficient hydrolysis of each of the pretreated substrates
Previous work has suggested that the limited available surface area of cellulose is a key factor that necessitates the need for relatively high enzyme dosages to attain effective cellulose hydrolysis
Summary
Bioethanol derived from the bioconversion of lignocellulosic feedstocks continues to attract global interest as a potentially environmentally compatible alternative to current petroleum-based transportation fuels. One of the major limitations of this process is the consistently high cost of the enzymes involved in the conversion of the cellulose component into fermentable sugars [1]. This is primarily due to the comparatively. In a typical batch enzyme-based process, cellulose conversion-time experiments are characterized by a three-phase curve (Figure 1A). This usually starts with the rapid adsorption of the cellulases onto the readily accessible cellulose, followed by an initial, fast rate of hydrolysis. In some cases, depending on the nature of the substrate and the pretreatment method used, even at very high protein loadings of the commercially available cellulase mixtures (Figure 1B, curve D) and extensive hydrolysis times, complete cellulose hydrolysis cannot be achieved (Figure 1B) [3,4]
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