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Title: The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei

Source: Biotechnology for Biofuels. 14(1). 4406. 30 p.

Author(s)Novy, Vera ; Nielsen, Fredrik ; Cullen, Daniel ; Sabat, Grzegorz ; Houtman, Carl J.; Hunt, Christopher G.

Publication Year: 2021  View PDF »

Category: Journal Articles
Associated Research Project(s):   FPL-4707-1C   FPL-4709-1C   FPL-4712-4C

Abstract: Background: On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharifcation by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With diferent pretreatments of similar starting materials, we presented the fungus with systematically modifed substrates. This allowed the elucidation of substrateinduced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates. Results: Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifcally, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharifcation efciencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efciencies reached those of a state-of-the-art commercial enzyme preparation. Conclusion: Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specifc substrate requirements.

Keywords: Softwood substrates; enzyme production; Trichoderma reesei; Secretome; substrate sensing; enzymatic hydrolysis

Publication Review Process: Formally Refereed

File size: 3,072 kb(s)

Date posted: 06/26/2021

This publication is also viewable on Treesearch:  view
RITS Product ID: 10178
Current FPL Scientists associated with this product (listed alphabetically)
Cullen, Daniel
Research Microbiologist
Houtman, Carl J.
Chemical Engineer
Hunt, Christopher G.
Research Chemist

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