Inducing the synthesis of active cellulase is critical for biorefineries. The effective inducer was prepared from stevioside, which contains sophorose.

Jerusalem artichoke (Helianthus tuberosus L.) is emerging as one of the energy plants considered for biofuel production. Alkali and alkali-involved pretreatment methods have been widely used for the bioconversion of cellulosic materials due to their high sugar yield and low inhibitor release. However, the recovery and treatment of wastewater (black liquor) have been poorly studied. Here, we present a novel two-stage pretreatment process design for recycling black liquor. Jerusalem artichoke stalk (JAS) was first treated with 2% (w/v) NaOH, after which lignin was recovered by H2SO4 at pH 2.0 from the black liquor. The recycled solutions were subsequently used to treat the NaOH-pretreated JAS for the second time to dissolve hemicellulose. CO-pretreated JAS, hydrolysates, and acid-insoluble lignin were obtained after the above-mentioned two-stage pretreatment. A reducing sugar yield of 809.98 mg/g Co-pretreated JAS was achieved after 48 h at 5% substrate concentration using a cellulase dosage of 25 FPU/g substrate. In addition, hydrolysates containing xylose and acid-insoluble lignin were obtained as byproducts. The pretreatment strategy described here using alkali and acid combined with wastewater recycling provides an alternative approach for cellulosic biorefinery.

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Knockout of the transcriptional repressor Trctf1 is known to enhance the yield of cellulose-induced cellulase synthesis in Trichoderma reesei. However, different inducers possess distinct induction mechanisms, and the effect of Trctf1 on cellulase synthesis with soluble inducers remains unknown. To evaluate the effect of the Trctf1 gene on cellulase synthesis and develop a high-yielding cellulase strain, we established a CRISPR–Cas9 genome editing system in T. reesei Rut C30 using codon-optimized Cas9 protein and in vitro transcribed RNA. This study demonstrated that T. reesei ΔTrctf1 with the Trctf1 gene knocked out showed no statistically significant differences in cellulase, cellobiohydrolase, endoglucanase, and β−glucosidase production when induced with MGD (the mixture of glucose and sophorose). However, when induced with lactose, the activities of these enzymes increased by 20.2%, 12.4%, and 12.9%, respectively, with no statistically significant differences in β−glucosidase activity. The hydrolysis efficiency on corn stover of cellulases produced by T. reesei ΔTrctf1 under different inducers was not significantly different from that of wild-type cellulases, indicating that Trctf1 gene deletion has little effect on the cellulase cocktail. These findings contribute to a better understanding of the molecular mechanisms underlying the regulation of T. reesei cellulase synthesis by different soluble inducers, as well as the construction of high-yield cellulase gene−engineered strains.