Boosting softwood hemicellulose hydrolysis: Enzymes from a new fungi Penicillium rotoruae remarkably improve CTec-2 hydrolysis efficiency and reduce sugar production costs

Economic production of fermentable sugars from lignocellulosic biomass is critical for the biorefinery applications in the bioeconomy industry. This study demonstrates effective enzymatic hydrolysis of recalcitrant softwood using newly identified fungus Penicillium rotoruae. Initially, nineteen fungal isolates were screened on softwood galactoglucomannan (GGM), with nine showing strong responses in the liquid culture. Trichoderma viride, Penicillium rotoruae, and Amorphotheca resinae showed highest β-mannanase, β-mannosidase, and α-galactosidase activities. P. rotoruae demonstrated superior main chain cleaving enzyme activities, while A. resinae excelled in the side chain cleaving activity. The crude enzyme of P. rotoruae was evaluated on two Pinus radiata substrates. Using soluble GGM, P. rotoruae released 34.3 % monomeric sugars (32.1 g/L reducing sugars), outperforming commercial CTec-2 (22.9 % and 23.2 g/L respectively). Co-application of CTec-2 with P. rotoruae enzymes increased monomeric sugar yield to 56.3 %, with galactose, mannose, and glucose increasing 20-, 3.6-, and 2.2-fold respectively. Using insoluble pulp, co-application yielded 88 % of monomeric sugars (20.2 g/L reducing sugars) representing an increase of 20 % soluble sugars relative to CTec-2 used alone. Techno-economic analysis indicated an increase in annual EBITDA, a positive ROCE and sugar cost savings of NZD 125/t demonstrating significant economic potential for softwood biorefineries.