High cellulase loading is still a major impediment in the production of fermentative sugars from high-solids enzymatic hydrolysis of lignocellulosic substrates in the enzyme-based “biorefinery” industry. This study attempted a high-solids (20%) enzymatic hydrolysis of lignocellulosic substrate at a very low cellulase loading with mixed use of additives and accessory enzymes by fed-batch mode. To avoid the high initial biomass viscosity, the high-solids enzymatic hydrolysis of lignocellulosic substrates was initiated with a solids content of 8%. Thereafter, 4% of the additional substrates were consecutively fed into the hydrolysis system after 6, 12, and 18 h to reach a final solids content of 20%. Some additive mixtures (40 mg/g substrateTween 80 + 10 mg/g substrate tea saponin +20 mg/g substrate BSA) were observed to enable this fed-batch hydrolysis to increase 30% of the glucose yield after the 48 h. The combination of these additives and accessory enzymes (2.4 mg/g substrate xylanase and 1 mg/g substrate AA9) in the high-solids hydrolysis system further boosted the sugar release. This allowed us to achieve an industrially relevant sugar yield (83% cellulose and 90% xylan hydrolysis) and fermentable sugar titer (∼160 g/L) after 72 h, with a low cellulase enzyme loading (3 FPU/g substrate). Our results indicate that the fed-batch substrate addition process is a favorable model for high-solids enzymatic hydrolysis of lignocellulosic substrates. Moreover, the synergism between the additives and accessory enzymes can greatly boost the high-solids enzymatic hydrolysis of lignocellulosic substrates.

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Conventional atmospheric glycerol organosolv pretreatment is energy-intensive with the requirement of long time and/or high temperature. Herein, acid-catalyzed atmospheric glycerol organosolv (ac-AGO) pretreatment was developed under a mild condition to modify the sugarcane bagasse structure for improving enzymatic hydrolyzability. Using single factor and central composite design experiments, ac-AGO pretreatment was optimized at 200 °C for 15 min with 0.06% H2SO4 addition, wherein the hemicellulose and lignin removal rates were 82 and 52%, respectively, with extremely high cellulose retention of 98%. The ac-AGO-pretreated substrate exhibited good enzymatic hydrolyzability at a modest cellulase loading, affording a 70% glucose yield after 72 h. Multiple analysis tools were used to correlate the hydrolyzability of the substrate with its structural features. The results indicated that the mild ac-AGO pretreatment can modify the lignocellulosic biomass structure to achieve good hydrolyzability, mainly resulting in significant hemicellulose removal.

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