Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

Enzymatic hydrolysis of cellulose provides a renewable source of monosaccharides for production of variety of biochemicals and biopolymers. Unfortunately, the enzymatic hydrolysis of cellulose is often incomplete, and the reasons are not fully understood. We have monitored enzymatic hydrolysis in terms of molecular density, ordering and autofluorescence of cellulose structures in real time using simultaneous CARS, SHG and MPEF microscopy with the aim of contributing to the understanding and optimization of the enzymatic hydrolysis of cellulose. Three cellulose-rich substrates with different supramolecular structures, pulp fibre, acid-treated pulp fibre and Avicel, were studied at microscopic level. The microscopy studies revealed that before enzymatic hydrolysis Avicel had the greatest carbon-hydrogen density, while pulp fibre and acid-treated fibre had similar density. Monitoring of the substrates during enzymatic hydrolysis revealed the double exponential SHG decay for pulp fibre and acid-treated fibre indicating two phases of the process. Acid-treated fibre was hydrolysed most rapidly and the hydrolysis of pulp fibre was spatially non-uniform leading to fractioning of the particles, while the hydrolysis of Avicel was more than an order of magnitude slower than that of both fibres

Impact of the supramolecular structure of cellulose on the efficiency of enzymatic hydrolysis

Background: The efficiency of enzymatic hydrolysis is reduced by the structural properties of cellulose. Although efforts have been made to explain the mechanism of enzymatic hydrolysis of cellulose by considering the interaction of cellulolytic enzymes with cellulose or the changes in the structure of cellulose during enzymatic hydrolysis, the process of cellulose hydrolysis is not yet fully understood. We have analysed the characteristics of the complex supramolecular structure of cellulose on the nanometre scale in terms of the spatial distribution of fibrils and fibril aggregates, the accessible surface area and the crystallinity during enzymatic hydrolysis. Influence of the porosity of the substrates and the hydrolysability was also investigated. All cellulosic substrates used in this study contained more than 96% cellulose. Results: Conversion yields of six cellulosic substrates were as follows, in descending order: nano-crystalline cellulose produced from never-dried soda pulp (NCC-OPHS-ND)∈>∈never-dried soda pulp (OPHS-ND)∈>∈dried soda pulp (OPHS-D)∈>∈Avicel∈>∈cotton treated with sodium hydroxide (cotton∈+∈NaOH)∈>∈cotton. Conclusions: No significant correlations were observed between the yield of conversion and supramolecular characteristics, such as specific surface area (SSA) and lateral fibril dimensions (LFD). A strong correlation was found between the average pore size of the starting material and the enzymatic conversion yield. The degree of crystallinity was maintained during enzymatic hydrolysis of the cellulosic substrates, contradicting previous explanations of the increasing crystallinity of cellulose during enzymatic hydrolysis. Both acid and enzymatic hydrolysis can increase the LFD, but no plausible mechanisms could be identified. The sample with the highest initial degree of crystallinity, NCC-OPHS-ND, exhibited the highest conversion yield, but this was not accompanied by any change in LFD, indicating that the hydrolysis mechanism is not based on lateral erosio

Effect of highly branched hyphal morphology on the enhanced production of cellulase in Trichoderma reesei DES-15

The morphology of Trichoderma reesei is a vitally important factor for cellulase productivity. This study investigated the effect of hyphal morphology on cellulase production in the hyper-cellulolytic mutant, T. reesei DES-15. With a distinct morphology, T. reesei DES-15 was obtained through Diethyl sulfite (DES) mutagenesis. The hyphal morphology of DES-15 batch-cultured in a 5-L fermentor was significantly shorter and more branched than the parental strain RUT C30. The cellulase production of DES-15 during batch fermentation was 66 % greater than that of RUT C30 when cultured the same conditions. DES-15 secreted nearly 50 % more protein than RUT C30. The gene expression level of a set of genes (cla4, spa2, ras2, ras1, rhoA, cdc42, and racA) known to be involved in hyphae growth and hyphal branching was measured by quantitative real-time PCR. The transcriptional analysis of these genes demonstrated that a decrease in gene expressions might contribute to the increased hyphal branching seen in DES-15. These results indicated that the highly branching hyphae in DES-15 resulted in increased cellulase production, suggesting that DES-15 may be a good candidate for use in the large-scale production of cellulase. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13205-016-0516-5) contains supplementary material, which is available to authorized users