ForestValue – FunEnzFibres (From fundamentals to valorization: Enzymatic oxidation of cellulosic fibres and underlying mechanisms)

Cellulose from wood can be used to replace many products that today are made from fossil raw materials. The breakdown of cellulose into sugar, which can then be used to produce, for example, second-generation biofuels, is well known. Other applications of cellulose make greater use of the wonderful fiber that cellulose is, and are aimed at producing new, environmentally friendly materials. In this project, we have developed enzyme-based processes to produce improved and novel cellulose-based materials. The goal was to develop better varieties of the processes and products that already exist, but also to develop new products. Enzyme technology is well suited for this because it is a green technology that does not involve toxic chemicals or extreme reaction conditions. The project has been based on the use of so-called "Lytic Polysaccharide Monoxogygenases" (LPMOs). LPMOs were discovered at NMBU in 2010 and are currently used worldwide for the breakdown of cellulose. However, it is clear that much more can be done with LPMOs, for example, by using these enzymes to only break down “irregularities” in otherwise homogeneous fibers. Furthermore, LPMOs lead to partial, and nicely controllable oxidation of cellulose fibres. Using LPMOs for fiber treatment may improve fiber functionality and may also allow the development , of ne cellulose-based new materials. In order to develop new processes for the production of new types of cellulose, one needs advanced and complicated analytical methods. The Norwegian partner, NMBU, has contributed to the development of a set of new methods by partner BOKU (Austria) for analyzing how cellulose fibers are affected by various enzyme treatments. These methods allow us to determine the extent of fiber modification, which, when using LPMOs, means the same as the extent of fiber oxidation. These methods allow for simultaneous determination of average fiber length and fiber length distribution (polydispersity). As a highlight, these novel methods include an analytical tool to determine the “three dimensional” distribution of oxidized sites through the cellulose fiber. In other words, we can now see whether oxidation primarily happens on the fiber surface or also deeper in the fiber core. The fiber properties discussed above are all important for fiber functionality. Such (industrially relevant) functionality has been studied through fiber dissolution studies, assessment of water binding capacity, and through microscopy by partner VTT (Finland). The development of these analytical methods allowed detailed investigations of how different types of LPMOs affect different preparations of cellulose fibers. Thus, we were able to unravel the connection between the choice of the LPMO and the properties of the resulting LPMO-treated cellulose fibers. Next to providing information to end users regarding the optimal LPMO for their purpose (= specified fiber properties), this work also revealed a remarkable functional variation among cellulose-active LPMOs found in nature. Cellulose-active LPMOs are abundant in nature, and it seems that this related to the fact that cellulose occurs in multiple forms that each require their specialized LPMOs. Some LPMOs contain additional substrate-binding domains, and we showed that the presence of such domains has considerable impact on how the LPMO modifies the cellulose fibre. Next to studying the impact of a wide variety of LPMOs, we have also studied how the production of oxidized fibers can be optimized by manipulation of process conditions, focusing on parameters such as the supply of reductants and/or hydrogen peroxide that are needed to drive the LPMO reaction. Several reactions have been scaled up, to generate sufficient amounts of oxidized fibers for the functional studies at VTT. The results of this project have been or will be reported in approximately 10 peer-reviewed scientific publications and one PhD thesis. The FunEnzFibres project has given NMBU (and Norway) a unique opportunity to connect to world-leading research groups in cellulose fiber engineering and analysis. These groups have learned from NMBU’s enzyme competence, whereas NMBU has acquired new knowledge and methods, that have improved NMBU’s enzyme research and, in particular, NMBU’s ability to make industrially relevant contributions to the biorefining of forest biomass in Norway. The project has led to new scientific connections within Europe that will benefit future research project applications, future projects, future competence development, and future industrialization of biotechnological processes.

The FunEnzFibres project has given NMBU (and Norway) a unique opportunity to connect to world-leading research groups in cellulose fiber engineering and analysis. These groups have learned from NMBU’s unique enzyme competence, whereas NMBU has been able to acquire new knowledge and methods, that have improved NMBU’s enzyme research and, in particular, NMBU’s ability to make industrially relevant contributions to the biorefining of forest biomass in Norway. During the project, concrete application testing of novel technologies for cellulose fiber engineering has been conducted with Norwegian industry. Next to yielding a clear increase in industrially relevant cellulose competence and multiple high-level scientific publications, the project has yielded: - New scientific connections in Europe that will benefit future research project applications, future projects, future competence development, and future industrialization of biotechnological processes. - New methods for analysis of cellulose fibers, in particular of enzymatically refined fibers, in particular fibers enzymatically refined with LPMOs. These methods include methods for specific labeling of oxidized cellulose fibers and methods for measuring the distribution of enzymatically modified sites in the cellulose fibers. - New bioprocesses for converting forest biomass to nanocelluloses and regenerated cellulose products, with controlled water-binding and charge properties, and unprecedented insight in underlying properties such as fiber length and depth, and the distribution of oxidized sites. Of note, primarily thanks to partner VTT, the work has been done, in part, at mini-pilot scale, bringing the project’s output closer to industrial reality. The developed methods and processes will be published late 2023, early 2024, and we expect them to be adopted in both academia and industry.

Wood cellulose is a future material for replacement of many fossil-based products. Modification of the wood pulp is needed for preparation of value-added products. Enzymes are specific, non-toxic biodegradable tools for the modification of the pulps in mild reaction conditions. Recently discovered lytic polysaccharide monooxygenases (LPMOs) oxidize cellulose in the crystalline parts, having thus capability to modify the most recalcitrant celluloses. This project will explore the potential of LPMOs in oxidative modification of cellulosic fibres. The research aims at developing sustainable refining and dissolving processes. The consortium brings together top-class expertise in enzymatic modification of pulp and fibres, LPMO enzymology, cellulose analytics and applications. Six industrial partner are committed to follow and advise the project.