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

Abstract

Background On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification 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 different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced 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. Specifically, 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 fiber 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 saccharification efficiencies 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, efficiencies 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 specific substrate requirements.

Parascedosporium putredinis NO1 tailors its secretome for different lignocellulosic substrates

“…This is especially interesting considering the somewhat similar proportions of cellulose, hemicellulose, and lignin for the empty fruit bunch from oil palm ( 29 ), rice straw ( 30 ), sugar cane bagasse ( 31 ), and wheat straw ( 32 ). Differences in fungal secretomes have been demonstrated previously when grown on compositionally distinct carbon sources ( 33 – 35 ). However, the variation on broadly similar lignocellulosic substrates demonstrated here is important as it may suggest a tailoring of the lignocellulose-degrading enzymes produced by P. putredinis NO1 which could be utilized for substrate-specific cocktail development.…”

“…The similarity in the proportion of GH class CAZymes in the supernatant fraction even for compositionally distinct substrates such as wheat bran and kraft lignin could be the result of the transcriptional activation of GH class CAZymes like cellulases and xylanases. Mono- and disaccharides, which could be present in all substrates investigated here, have been demonstrated to act as inducers of the expression of large numbers of hydrolytic CAZymes in other ascomycete fungi previously ( 35 , 51 ). This may be occurring for all substrates despite the target polysaccharides of induced enzymes not necessarily being present in similar amounts across all substrates investigated.…”

Investigating the role of AA9 LPMOs in enzymatic hydrolysis of differentially steam-pretreated spruce

“…The enzymes were loaded based on activity measurements. The cellulolytic activity in Celluclast and Cellic CTec2 was determined using the filter paper unit (FPU) assay [ 50 ] with some adjustments [ 51 ]. The β-glucosidase activity of Novozym 188 was determined as described previously [ 52 ] with some adjustments [ 13 ].…”

Culture of Trichoderma sp. with biochar to produce high-activity cellulase in a laboratory

“…Fujii et al (2009) estimated the hydrolysis ability of the produced enzyme (Acremonium cellulolyticus) against woody biomass, Eucalyptus and Douglus fir, and conversion rates for both were above 69%, which was higher than the commercial enzyme (Accellase 1000), which was below 60%. Novy et al (2021) customized the enzyme mixture of Celluclast 1.5 L and cellulase from T. reesei and treated it with pretreated lodgepole pine, which represented approximately the same conversion rate as Cellic CTec 3 (80%). Aksenov et al (2020) observed that the conversion rate of bleached hardwood and softwood pulp with cellulase from Penicillium verruculosum and the reduced sugar yield were approximately 60%.…”