Defence of doctoral thesis: Salla Koskela – Lytic polysaccharide monooxygenases for green production of cellulose nanomaterials

Cellulose is the main structural polymer in wood, and its potential in the form of nanomaterial building blocks, nanocelluloses, has now been recognized. Nanocelluloses, including cellulose nanofibers (CNFs) and cellulose nanocrystals (CNCs), have become increasingly important in development of modern sustainable materials. Nanocelluloses are typically produced from wood pulp fibers by chemical pre-treatments that deposit charged functional groups onto cellulose microfibril surfaces, thereby promoting disintegration of the fiber cell wall during mechanical fibrillation. Due to environmental risks related to the use of harsh chemical treatments, it is crucial to develop greener, nature-inspired alternatives. As renowned decomposers of wood, fungi secrete cellulose-active enzymes that work in aqueous reaction conditions. Of these, lytic polysaccharide monooxygenases (LPMOs) have piqued a special interest in green production of nanocellulose owing to their ability to introduce charged carboxyl groups onto cellulose surfaces. However, little is known about the properties of LPMO-oxidized nanocelluloses, their mechanical performance in bulk materials, and the mechanism how LPMOs facilitate fibrillation of the wood fiber cells.

This PhD thesis aimed to dissect the potential of C1-oxidizing LPMOs in the production of nanocelluloses and to clarify the mechanism of LPMO oxidation that facilitates the disintegration of wood cell wall. LPMOs with and without attached carbohydrate-binding modules (CBMs) were recombinantly produced in Pichia pastoris and studied for the production of CNFs and CNCs, which were further processed into bulk materials. The morphology and properties of the nanocelluloses, and the optical and mechanical properties of the bulk materials were characterized. In addition, delignified wood with a preserved cellular structure was used as a model substrate for LPMO oxidation, and the LPMO-induced changes in the wood cell wall structure were investigated using advanced scattering techniques.

The results on CNF production showed that LPMO-oxidized wood pulp fibers can be transformed into discrete and colloidal CNFs by mild mechanical disintegration, analogous to chemical pre-treatments such as 2,2,6,6-tetramethylpyperidine-1-oxy radical (TEMPO)-mediated oxidation. Importantly, these CNFs were well individualized with an average width of 4 nm, resembling that of cellulose microfibrils in wood. Such CNFs were obtained from softwood holocellulose- and kraft pulp fibers with a hemicellulose content of 16–19%, but not from dissolving pulp with a lower hemicellulose content of 4%. Nanopapers prepared from the LPMO-oxidized CNFs were transparent and they demonstrated tensile strengths of ca. 260 MPa and Young’s moduli of ca. 17 GPa. The water suspensions of LPMO-oxidized CNFs also exhibited acid-triggered gelation behavior due to the enzymatically introduced carboxyl groups.

LPMO oxidation was also found applicable in the preparation of CNCs from microcrystalline cellulose. The LPMO-oxidized CNCs had a needle-like morphology and they formed stable colloidal suspensions in water that demonstrated flow-induced birefringence. Solution cast films showed that the CNCs bearing C1 carboxyl groups possessed the pivotal ability to undergo self-assembly into an anisotropic phase. As some LPMOs are appended to a non-catalytic CBM, the effect of this module on nanocellulose production was also determined. CBM was found to increase the release of carboxyl groups from cellulose microfibril surfaces in the form of soluble cello-oligosaccharides. By contrast, a non-modular LPMO introduced more carboxyl groups to the cellulose surfaces, up to 0.53 mmol g^-1 on CNFs, and 0.70 mmol g^-1 on CNCs. Indeed, a non-modular LPMO was found advantageous in production of both CNFs and CNCs.

Despite the important role of LPMOs for natural and biotechnological degradation of wood biomass, the LPMO-induced changes in the wood cell wall structure have remained unknown. In this work, these changes were characterized for the first time. It was shown that a C1-oxiding LPMO can modulate cellulose microfibrils and disrupt the wood cell wall ultrastructure by modifying cellulose surface chemistry. After the LPMO oxidation, the average distance between cellulose microfibril centers increased from 4.1 nm to 10.7 nm, signifying the separation of microfibrils in a microfibril bundle. This result revealed a previously unidentified role for C1-oxidizing LPMOs in degradation of cellulose at the nanoscale. Remarkably, LPMO-treated wood veneers could be further compressed into anisotropic, transparent films with an ultrahigh tensile strength of 824 MPa.

In summary, this PhD thesis clarified the potential of C1-oxidizing LPMOs in green production of nanocelluloses and showed that LPMO oxidation is a suitable method to obtain high-performing isotropic and anisotropic bulk materials from wood. On the basis of the obtained findings, a new model was also proposed which elucidates the mechanism of cellulose degradation at the nanoscale. This study broadened the understanding of LPMOs including their biological- and biotechnological significance and provided new insights into the use of LPMOs for the preparation of cellulose-based nanomaterials.

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