Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely Enzymatic Pretreatments Claire Holland, † Alixander Perzon, † Pierre R. C. Cassland, ‡ John P. Jensen, § Birger Langebeck, § Ole Bandsholm Sørensen, ∥ Eric Whale, ⊥ David Hepworth, ⊥ Robyn Plaice-Inglis, ⊥ Øjvind Moestrup, # Peter Ulvskov, † and Bodil Jørgensen* ,† † Department of Plant and Environmental Sciences, Section for Glycobiology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark ‡ Novozymes, Krogshoejvej 36, 2880 Bagsvaerd, Denmark § Nordzucker, Technology and Innovation, Falckvænget 1, 4900 Nakskov, Denmark ∥ KMC, Herningvej 60, 7330 Brande, Denmark ⊥ Cellucomp Ltd., Unit 3, West Dock, Harbour Place, Burntisland, Fife KY3 9DW, United Kingdom # Department of Biology, University of Copenhagen, 2100 København Ø, Denmark * S Supporting Information ABSTRACT: Cellulose fibers can be freed from the cell-wall skeleton via high-shear homogenization, to produce cellulose nanofibers (CNF) that can be used, for example, as the reinforcing phase in composite materials. Nanofiber production from agro-industrial byproducts normally involves harsh chemical-pretreatments and high temperatures to remove noncellulosic polysaccharides (20−70% of dry weight). However, this is expensive for large-scale processing and environmentally damaging. An enzyme-only pretreatment to obtain CNF from agro-industrial byproducts (potato and sugar beet) was developed with targeted commercial enzyme mixtures. It is hypothesized that cellulose can be isolated from the biomass, using enzymes only, due to the low lignin content, facilitating greater liberation of CNF via high-shear homogenization. Comprehensive Microarray Polymer Profiling (CoMPP) measured remaining extractable polysaccharides, showing that the enzyme-pretreatment was more successful at removing noncellulosic polysaccharides than alkaline- or acid-hydrolysis alone. While effective alone, the effect of the enzyme-pretreatment was bolstered via combination with a mild high-pH pretreatment. Dynamic rheology was used to estimate the proportion of CNF in resultant suspensions. Enzyme-pretreated suspensions showed 4-fold and 10-fold increases in the storage modulus for potato and sugar beet, respectively, compared to untreated samples. A greener yet facile method for producing CNF from vegetable waste is presented here. ■ INTRODUCTION Cellulose, the predominant plant cell wall polysaccharide and most abundant natural biopolymer, is a long, linear homo- polysaccharide comprised of thousands of unbranched β-(1→ 4)-D-glucopyranose residues. Hydrogen-bond formation be- tween adjacent glucose residues on several neighboring cellulose chains produces densely packed and highly rigid microfibrils (approximately 3 nm wide and several micrometers long). 1,2 Cellulose microfibrils are separate entities that bundle within the cell wall to form larger fibers that associate to form the main load bearing scaffold of the plant cell wall, providing resistance to the internal osmotic pressure of the cell as a result of its high tensile strength. 3,4 This microfibril arrangement is combined with hemicelluloses, pectin, lignin, and proteoglycan, with propor- tions of each dependent on the plant species and type of cell wall. Received: September 26, 2018 Revised: November 25, 2018 Published: December 10, 2018 Article pubs.acs.org/Biomac Cite This: Biomacromolecules 2019, 20, 443-453 © 2018 American Chemical Society 443 DOI: 10.1021/acs.biomac.8b01435 Biomacromolecules 2019, 20, 443−453 Downloaded via COPENHAGEN UNIV LIBRARY on February 20, 2019 at 11:27:55 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.