Sustainable production of cellulose nanofiber gels and paper from sugar beet waste using enzymatic pre-treatment

Journal Pre-proof Sustainable production of cellulose nanofiber gels and paper from sugar beet waste using enzymatic pe-treatment Alixander Perzon, Bodil Jørgensen, Peter Ulvskov

PII:

S0144-8617(19)31249-4

DOI:

https://doi.org/10.1016/j.carbpol.2019.115581

Reference:

CARP 115581

To appear in:

Carbohydrate Polymers

Received Date:

10 September 2019

Revised Date:

1 November 2019

Accepted Date:

6 November 2019

Please cite this article as: Perzon A, Jørgensen B, Ulvskov P, Sustainable production of cellulose nanofiber gels and paper from sugar beet waste using enzymatic pe-treatment, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115581

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Sustainable production of cellulose nanofiber gels and paper from sugar beet waste using enzymatic pre-treatment Alixander Perzona, Bodil Jørgensena, Peter Ulvskova * a

Department of Plant and Environmental Sciences, Section for Glycobiology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark

Contacts: Alixander Perzon ([email protected], +45 3533 6206), Bodil Jørgensen ([email protected], +45 3533 3466), Peter Ulvskov ([email protected], +45 3533 2580, *corresponding)

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Key Words: Cellulose nanofibers (CNF), nanopapers, gels, sugar beet, agro-industrial waste, primary cell wall,

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enzymatic degradation

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Graphical abstract

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Highlights

Sugar beet nanofibers were produced with lower water consumption using enzymatic pre-treatments



More nanofibers were obtained by pre-incubating pulp at pH 9 prior to enzymatic treatment



Trace polysaccharides remaining on nanofibers had a strong impact on their rheological behavior

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

Abstract: Removal of non-cellulosic polymers from vegetable pulp to obtain cellulose nanofibers (CNF) is

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normally achieved by chemical pre-treatments which requires several washing steps. In the present study, it is

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demonstrated how incubation of sugar beet pulp at pH 9, followed by treatment with polysaccharide-degrading enzymes and subsequent bleaching can be done in a one-pot procedure to make CNF. The new method consumes

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67% less water and removes non-cellulosic polysaccharides with similar efficiency as a chemical method. In addition, CNF produced by the new method contained slightly more pectin and formed gels with 2.7 times higher

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storage modulus. Nanopapers cast from chemically- and enzymatically produced CNF showed similar mechanical properties. However, without the pH 9 incubation step, the enzymes accessibility to cell-wall polymers was limited

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resulting in lower gel and paper strengths. In conclusion, the new method offers a sustainable route for producing

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high quality CNF from sugar beet waste.

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1

Introduction

Sugar beet pulp (SBP) is a major waste product of sugar industry in Europe and North America. Due to the sucrose content of sugar beet being no more than 10-20% per dry weight (Artschwager, 1930), a substantial amount of pulp remains after processing. Some of the waste pulp is sold as animal fodder, but a large part of it is discarded. Considering that several million tons of SBP waste is generated yearly (Stenmarck et al., 2016), it constitutes a potential source of renewable biopolymers. This is also the case for industries that produces starch, juice, pectin, etc. The leftover SBP consists of roughly 20-30% cellulose, 30% pectin, and 30% hemicellulose dry weight (Michel, Thibault, Barry, & de Baynast, 1988; Thibault, Renard, & Guillon, 1994). The cellulose fraction is

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particularly interesting as it can be used to construct materials such as gels, paper, or as reinforcing agent in

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composites. Therefore, deriving cellulose from waste products serves an opportunity to achieve a more sustainable bioeconomy, especially in the future when demands for natural biopolymers might increase due to necessity of

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replacing petroleum derived plastics.

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Over the last decades, new concept designs of cellulose-materials have emerged. Instead of using traditional plant fibers (cotton, hemp, paper), cellulose elements of nanometer scale (nanocellulose) are derived and used to

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assemble materials with novel properties as presented in the seminal papers by Herrick et al (Herrick, Casebier, Hamilton, & Sandberg, 1983) and Turbak et al (Turbak, Snyder, & Sandberg, 1983). Two extensive reviews on recent developments within nanocellulose production and applications have been provided by Klemm et al

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(Klemm et al., 2018; Klemm et al., 2011). Nanocellulose includes various subcategories of materials, these comprise cellulose nanofibers (CNF), cellulose nanocrystals (CNC), and bacterial nanocellulose (BNC). Cellulose nanofibers are derived by high-shear homogenization of plant fibers using a microfluidizer, sonication, or steamexplosion. These fibers are of high aspect ratio (length to width ratio), typically 5-60 nm in width and 0.1-2 µm in

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length, which makes them useful in composite materials (Saïd Azizi Samir, Alloin, Paillet, & Dufresne, 2004). In contrast, cellulose nanocrystals (CNC) consist of smaller fragments (5-70 nm in width, 100-250 nm in length) obtained by degrading the amorphous regions of cellulose, usually resulting in colloidal suspensions. Another type of cellulose is bacterial nanocellulose (BNC) which is typically produced by Acetobacter xylinum. Bacterial nanocellulose have a different structure from plant-derived cellulose and the fibers are 20-100 nm in width and several micrometers in length.

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In this study, the focus is on production of CNF from sugar beet pulp for fabrication of gels and nanopapers. Compared to traditional paper made of larger fibers, nanopapers possess properties such as higher strength, transparency, and reduced gas permeability (Aulin, Gällstedt, & Lindström, 2010; Henriksson, Berglund, Isaksson, Lindstrom, & Nishino, 2008; Nogi, Iwamoto, Nakagaito, & Yano, 2009). Moreover, CNF have found applications within packaging (films, coating, paper, reinforcement), filtration of water, preventing attrition of paint, stabilization of concrete, and flexible electronics (Cowie, Bilek, Wegner, & Shatkin, 2014; Shatkin, Wegner, Bilek, & Cowie, 2014).

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A major concern regarding production of nanocellulose are the economically costly pre-treatments required to loosen cell walls in plant fibers. This involves the use of solvents, strong base or acid, water, and energy. When

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considering a life-cycle analysis of nanocellulose, sustainability and environmental impact of these materials have been questioned (Nascimento et al., 2018). Within plant cell walls, cellulose exists as a mesh of microfibrils, each

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fibril being 3 nm wide and several micrometers long (McNamara, Morgan, & Zimmer, 2015; Somerville et al., 2004). A single cellulose microfibril is made of hundreds to thousands of β-(1→4)-D-glucopyranose residues that

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hydrogen bond laterally making this structure remarkably robust (Moon, Martini, Nairn, Simonsen, &

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Youngblood, 2011). How cellulose interacts with other biopolymers in the wall is not known in detail. Apart from cellulose, primary cell walls contain mostly pectin, hemicelluloses, and proteoglycans which makes them less recalcitrant than secondary cell walls that contain lignin (up to 25%) and hemicelluloses (Bidlack & Dashek,

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2016). Producing CNF from secondary cell walls (e.g. wood) requires delignification prior to homogenization. This is achieved by kraft-pulping (NaOH and sodium sulfide), high temperatures, and several washing steps at industrial

scale.

An

additional

pre-treatment

with

TEMPO-oxidation

(oxidation

by

2,2,6,6

tetramethylpiperidinyloxyl) has shown to be effective, as it introduces negative charges onto the cellulose which

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facilitates separation of larger fibers into CNF during homogenization, and also prevents subsequent fiberflocculation (Saito, Kimura, Nishiyama, & Isogai, 2007). Primary cell walls require milder pre-treatments to produce CNF during homogenization, however there is a substantial amount of pectin and hemicelluloses (2070% of the dry weight) that needs to be removed (Rani & Kawatra, 1994).

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The most common pre-treatment for production of CNF from sugar beet, potato, and carrot (predominately primary walls) is incubation of the pulp in 0.5 M sodium hydroxide at 80 °C, followed by bleaching with 1% sodium chlorite at 70 °C (Dinand, Chanzy, & Vignon, 1999; Dufresne, Dupeyre, & Vignon, 2000; Siqueira, Oksman, Tadokoro, & Mathew, 2016). Sodium hydroxide treatment serves to riddance the pulp of non-cellulosic polymers, which drastically facilitates cell wall disassembly into CNF during subsequent homogenization. Sodium chlorite oxidizes lignin (<1%) and tannins in the pulp and thus bleaches it. While showing effective result, this pretreatment demands several washing steps to remove the chemicals and solubilized compounds. A more precise measurement of how much water is consumed has not been carried out. Washing concentrated pulp on an industrial

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scale is non-trivial, and it is a bottleneck in making production of CNF more cost-effective and environmentally

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friendly.

In the present study, it is hypothesized that CNF can be derived effectively, and with reduced water consumption,

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using enzymatic pre-treatments that hydrolyze non-cellulosic polysaccharides in sugar beet cell walls. Enzymatic pre-treatments (with and without pH 9 pre-incubation) developed in one of our previous studies (Holland et al.,

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2018) are compared to the standard chemical treatment for producing CNF from sugar beet pulp. In addition, in

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each pre-treatment procedure a bleaching step (NaClO2) was implemented due to previous findings that suberized fragments (tissue such as peels) are not broken down by the enzymes. The water consumption throughout the pretreatments is measured, and the properties of resulting CNF are analyzed in terms of morphology, polysaccharide

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composition, rheology, and mechanical properties of cast nanopapers. Moreover, a relationship between polysaccharides remaining attached to cellulose and physical properties of CNF is discussed. The aim is to reduce the liquid waste to achieve a more cost-effective and environmentally friendly process for producing CNF from

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sugar beet waste.

2.1

Material and Methods Pre-homogenization of sugar beet

Fresh sugar beet ground into particles of 1-2 cm in size (dry weight of 26% w/w) was obtained from Nordic Sugar post sugar extraction and stored at -20 °C.

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An amount of 20 g (dry weight) of sugar beet was dispersed in 2.5 L of distilled water, and subsequently homogenized in a Silverson L5A homogenizer at 6000 rpm for 30 minutes using a slotted disintegrating head. After homogenization, 15 g (dry weight) of the sugar beet pulp was retained on a 38 µm sieve for subsequent pretreatment.

2.2

Chemical pre-treatment of sugar beet pulp

An amount of 15 g (dry weight) of pre-homogenized sugar beet pulp (section 2.1) was soaked in 500 mL of 0.8 M NaOH which resulted in a final concentration of 0.5 M NaOH. The sample was stirred thoroughly at 80 °C for 2

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hours. For stirring, an over-heard stirrer (IKA RW 16 basic) was used in all instances. After the given time, the remaining pulp was collected on a sieve (38 µm). Sodium hydroxide (NaOH) was washed out by resuspending the

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pulp in 5 L of water and collecting it on the sieve again; this procedure was repeated until pH 6.5 was measured in the pulp (see section 2.6). The pulp was then submerged in 500 mL of 1.45% (v/v) NaClO2 containing 50 mM

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sodium acetate (pH 5.0), resulting in a final NaClO2 concentration of 1% (v/v). The sample was mixed thoroughly

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at 70 °C for 2 hours. After that, the pulp was collected on a sieve (38 µm). Chlorite (NaClO2) was washed out by resuspending the pulp in 5 L of water and collecting it on the sieve again; this procedure was repeated until no

°C until further use.

Enzymatic pre-treatment of sugar beet pulp

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2.3

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oxidation activity could be detected in the pulp (see section 2.7). The pre-treated and washed pulp was stored at 4

An amount of 15 g (dry weight) of pre-homogenized sugar beet pulp (section 2.1) was soaked in 500 mL of distilled water. Following enzymes were subsequently added, Viscozyme L (10 μL/g pulp), Pectinex Ultra Clear (10 μL/g pulp), Pulpzyme HC (10 μL/g pulp), and Aquazym 240 L (10 μL/g pulp). Description of each enzyme is shown in

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Table 1. The enzymes were incubated with the pulp at 40 °C for 2 hours under stirring. After the given time, the enzymes were inactivated and the pulp was bleached by addition of NaClO2 to a final concentration of 1% (v/v), the temperature was raised to 70 °C, and the sample was stirred for 2 hours. After the reaction, the remaining pulp was collected on a sieve (38 µm) and resuspended in 5 L of water. This was repeated five times (25 L of water) until no oxidation activity was detected (see section 2.7). The pre-treated and washed pulp was stored at 4 °C until further use.

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Table 1 Declared main enzyme-activities in Novozymes products which were used in the pre-treatments of sugar beet pulp. FBG = Fungal Beta-Glucanase Units, AXU = Endo Xylanase Units, ECU = endo cellulase units, PGNU = Polygalacturonase units, KNU = Kilo Novo Units alpha-amylase units, all units as specified by Novozymes.

Product

Main enzyme activity

Declared activity

Viscozyme L

Beta-glucanase (endo-1,3(4)-β-glucanase)

100 FBG/g

Pectinex Ultra Clear Polygalacturonase (endo-1,4-α-galacturonidase) 7900 PGNU/ml Endo-xylanase (endo-1,4-β-xylanase)

1000 AXU/g

FiberCare R

Cellulase (endo-1,4-β-glucanase)

4500 ECU/g

Aquazym 240 L

Alpha-amylase (endo-1,4-α-glucanase)

240 KNU/g

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2.4

Pulpzyme HC

pH 9 and enzymatic pre-treatment of sugar beet pulp

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An amount of 15 g (dry weight) of pre-homogenized sugar beet pulp (section 2.1) was soaked in 500 mL of distilled

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water and heated to 80 °C under stirring. The pH was kept at 9 by drop-wise addition of 4 M NaOH over 2 hours, 8 mL was added in total. After that, 36 mL of 3.3 M Acetic acid was added to bring the pH to 5.0. The sample was

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cooled down to 40 °C and enzymes were added in the same quantities as described in section 2.3 and the sample was stirred for 2 hours. After the given time, the enzymes were inactivated, and the pulp was bleached by addition

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of NaClO2 to a final concentration of 1% (v/v), the temperature was raised to 70 °C, and the sample was stirred for 2 hours. After the reaction, remaining pulp was collected on a sieve (38 µm) and resuspended in 5 L of water.

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This procedure was repeated five times (25 L of water) until no oxidation activity was detected (see section 2.7). The pre-treated and washed pulp was stored at 4 °C until further use.

2.5

Production of cellulose nanofibers

The pre-treated pulps (sections 2.2-2.4) were diluted to 1% (w/w) with distilled water. A volume of 200 mL of

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each sample was subsequently circulated in a high-shear homogenizer (Microfluidizer materials processor M110P with two orifices of 200 and 400 μm). The pressure in the microfluidizer was set to 500 bars and each sample was circulated for 18 minutes, corresponding to 11 passes through the system, to produce the nanofibers. The samples were stored at 4 °C for further characterization.

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2.6

Measurement of pH in pulp

The water required for washing out NaOH in the chemical pre-treatment of sugar beet pulp (section 2.2) was monitored by measuring the pH of the pulp. After each wash with 5 L of water, 5 g (wet weight) of the pulp was centrifuged, and the pH of resulting supernatant was measured. Washing was done until pH of the supernatant measured 6.5, which was the pH of water by itself.

2.7

Measurement of oxidation activity in pulp

The water required for washing out NaClO2 in the chemical pre-treatment of sugar beet pulp (section 2.2) was

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monitored by measuring the oxidation of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) added to the pulp suspension. Oxidation of ABTS results in absorption of light at 420 nm. After each wash (5 L of water),

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5 g (wet weight) of pulp was centrifuged, and 10 µL of the resulting supernatant was added to 240 µl of 0.5 mM

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ABTS. After 15 minutes, the absorption of the supernatant was measured using a SpectraMax 190 spectrophotometer (Molecular Devices) set at 420 nm. The absorption value obtained at this wavelength was used

Preparation of nanopapers

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2.8

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to quantify oxidizing compounds remaining in the pulp after washing.

The 1% (w/w) cellulose nanofiber suspensions were diluted to 0.5% (w/w) with distilled water to a volume of 30 mL and vortexed at maximum speed for 2 minutes. The suspensions were then de-gassed under vacuum and mixing

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for 5 minutes to prevent formation of air-bubbles in subsequent drying process. The degassed nanofiber suspensions were carefully poured onto separate petri-dishes and placed in an oven at 50 °C for 48 hours to produce nanopapers.

Scanning electron microscopy

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2.9

Scanning Electron Microscopy (SEM) images were acquired with a Quanta 3D FEG (FEI company, Netherlands). A volume of 10 µL cellulose nanofibers (0.01% w/w in water) was air dried on a metal plate at 40 °C and subsequently coated with a gold layer of 2 nm. In case of nanopapers, a square (1x1 cm) of the material was cut out, attached to the metal plate, and subsequently coated with a gold layer (2 nm).

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2.10 Sugar composition of nanofibers A volume of 150 µL trifluoroacetic acid (TFA) was added to 850 µL of 1% (w/w) cellulose nanofibers (CNF) in water and the samples were heated to 110 °C for 1 hour to degrade the non-cellulosic polysaccharides. The samples were centrifuged at 30 000 X g for 10 minutes and the supernatants containing soluble sugars were collected, and the pellet with cellulose was discarded. The solvent was evaporated from the samples under vacuum, and the dried samples were re-dispersed in 300 µl of water. This procedure was repeated 3 times in total to lower pH of the samples which were saved for sugar analysis. The samples (diluted 1:100) were run on a Dionex-HPAEC chromatographic system (Dionex, Germany) for quantitative determination of sugars. Following sugars from

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Sigma Aldrich were used as standards at a concentration of 10 µg/mL in water: D-glucose, D-mannose, D-

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galactose, D-xylose, L-arabinose, L-rhamnose, L-fucose, D-galacturonic acid, and D-glucuronic acid.

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2.11 Fourier transform infrared

The FTIR spectra were recorded on a MB100 spectrometer (ABB Bomem Inc.) by averaging 64 scans from 4000

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to 400 cm−1 at 4 cm−1 resolution. All samples were freeze dried before their FTIR spectra were obtained. Each FTIR spectra was normalized at 1056 cm−1 (C–O stretching vibration of glucose ring) and the baseline was

2010).

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2.12 Dynamic rheology

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corrected using adaptive iteratively reweighted penalized least squares in MATLAB (Zhang, Chen, & Liang,

Dynamic rheology measurements were carried on a Discovery HR-3 Rheometer (TA Instruments). The geometry used was a 40 mm cone (1° angle) with the gap set to 22 μm. Before each measurement, the sample was allowed to rest for 5 minutes at 25 °C. The linear viscoelastic region was determined by strain sweeps for all samples.

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Oscillation sweeps were measured between strains of 0.01−100% at a frequency of 1 Hz. The chosen strain for the frequency sweep measurements was 0.1% for all samples. The frequency sweeps were carried out in the range of 0.1−100 Hz. The viscosity measurements were carried out using the same geometry (40 mm cone, 1°angle) under rotational movement measuring the viscosity at shear rates between 0.1-100 1/s.

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2.13 Mechanical testing Tensile tests of nanopapers were performed with a TA-XT plus texture analyser (TTC company) with grip accessory and a 50 kg load cell. The nanopapers were cut into 5 x 40 mm strips of 10-30 µm thickness. The tests were carried out at a crosshead speed of 4 mm/min. The relative humidity was kept at 50% at a temperature of 22 °C. The measured parameters (Young’s modulus, tensile strength, toughness, and strain-to-failure) were calculated from the obtained stress-strain curves. The results for each material are based on 5-7 replicas.

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Results and Discussion Water consumption

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Three different pre-treatments were applied to sugar beet pulp to remove non-cellulosic polysaccharides and facilitate degradation of cell walls into cellulose nanofibers (CNF) (Figure 1). The amount of water required in the

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standard chemical pre-treatment was at least 30 L for neutralizing the pH after treatment with 0.5 M sodium hydroxide, and 15 L to wash out oxidizing agent (chlorite, NaClO2) (Figure 2). Neutralization of pH is necessary

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to enable buffering of the oxidant solution to pH 5, at which chlorite works effectively for oxidizing non-cellulosic polymers and preserving cellulose (Kantouch, Hebeish, & El-Rafie, 1970). By replacing 0.5 M sodium hydroxide

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with commercial enzymes (pectinases, hemicellulases, amylase, endo-glucanase), the first washing step can be removed completely (Figure 1 b-c), thus saving 30 L of water or avoiding use of strong acid for neutralization.

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This is made possible due to the optimal pH for the enzymes being within the same range as for chlorite, so the two treatments can be carried out sequentially in one pot. Furthermore, a step comprising pre-incubation of the pulp at pH 9 was added to open the cell wall structure and increase accessibility for the enzymes (Figure 1c). This was done because, as can be seen in the dry weight losses throughout the pre-treatment steps (Table 2), the enzymes

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by themselves did not reduce the dry weight content to the same extent as the chemical treatment. Considering that sugar beet pulp consists of 20-30% cellulose per dry weight, as a rough estimate it is expected that 20% of the dry weight should remain after a successful pre-treatment. By incubating the pulp at pH 9 prior to the enzyme treatment, the dry weight was further reduced to 18%. This compares well with the standard chemical treatment.

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a)

30 L water

15 L water Cellulose fibers

Sugar beet pulp (15 g dry weight)

Microfluidizer

1) 0.5 M NaOH, 80 °C

2) 1% NaClO 2, pH 5.0, 70 °C

b)

Cellulose nanofibers (cCNF)

15 L water Cellulose fibers

Sugar beet pulp (15 g dry weight)

Microfluidizer

1) Enzymes, pH 5.0, 40°C 2) 1% NaClO 2, pH 5.0, 70 °C

c)

Cellulose nanofibers (eCNF)

Sugar beet pulp (15 g dry weight)

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15 L water Cellulose fibers

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Cellulose nanofibers (pHeCNF)

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Microfluidizer

1) pH 9, 80 °C 2) Enzymes, pH 5.0, 40 °C 3) 1% NaClO2, pH 5.0, 70 °C

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Figure 1 Different pre-treatment methods for producing cellulose nanofibers from sugar beet pulp using a) chemical pre-treatment, b) enzymatic pre-treatment, and c) pH 9 combined with enzymatic treatment. After each pre-treatment, the fibers were bleached with NaClO2 and circulated in a microfluidizer to produce CNF. The enzymes used were commercial hemicellulases, pectinases, amylase, and endoglucanase.

a) Pulp washed after treatment with 0.5 M NaOH

pH

10

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8

Oxidation activity (a.u.)

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b) Pulp washed after treatment with 1 % NaClO2 0.5

0.4

0.3

0.2

0.1

6

0

5

10

0.0

15

20

25

30

0

35

5

10

15

20

25

Water consumption (L)

Water consumption (L)

Figure 2 a) The pH of pulp after 0.5 M sodium hydroxide treatment and washing with varying amount of water, and b) the oxidative activity in pulp after treatment with 1 % NaClO2 and washing with varying amount of water. The oxidizing potential in the pulp was measured by oxidation of ABTS, causing it to absorb light at a wavelength of 420 nm.

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Table 2 Dry weight losses throughout the various pre-treatments for preparing cellulose nanofibers.

No treatment

100.0

a) Chemical

23

b) Enzymes

65

c) pH 9 followed by enzymes

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Dry weight (%) remaining after treatment

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3.2

Treatment

Analysis of nanofibers

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The degree of fibrillation in the three materials was assessed using scanning electron microscopy (SEM). Images

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of cellulose nanofibers (CNF) produced are shown in Figure 3. The thickest fibers measured are 20-40 nm in width and the thinnest are 5 nm. This is close to the resolution of the microscope so the occurrence of thinner fibers

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cannot be excluded. The fiber lengths appear to be 1 µm or longer in all samples. The thickest nanofibers are found in CNF prepared using the chemical pre-treatment (cCNF), followed by CNF produced using the pH-enzymatic pre-treatment (pHeCNF), and CNF produced using the enzymatic pre-treament (eCNF) consisted mainly of thin

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nanofibers as well as plant debris. The results indicate that different types of cell walls within the raw material are affected by the treatments. For example, enzymes may primarily break down primary cell walls, while 0.5 M

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sodium hydroxide also breaks down secondary cell walls, which produces a fraction of coarser nanofibers.

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Figure 3 SEM images of cellulose nanofibers produced using a) chemical pre-treatment (cCNF), b) enzymatic pre-treatment (eCNF), and c) pH 9 combined with enzymatic pre-treatment (pHeCNF). The arrows highlight one thin and one thick nanofiber in each sample. The magnification of nanofibers is 100 000× and the scale bar represents 1 µm.

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To assess how much non-cellulosic polysaccharides remained in the CNF samples, their sugar compositions were analyzed. Each sample was treated with trifluoroacetic acid to degrade the non-cellulosic fraction as described in section 2.10. The results in Figure 4 show that around 15% (w/w) of cCNF consists of non-cellulosic sugars, which is within the range of what a previous study has reported for sugar beet nanofibers (Dinand et al., 1999), while corresponding values for eCNF and pHeCNF are 41 and 16.7% (w/w). In these calculations, glucose was not accounted for as it seems to mainly originate from breakdown of cellulose to varying degree depending on the amount of non-cellulosic polysaccharides remaining in a sample. It is noteworthy that even though the amount of non-cellulosic sugars is similar between cCNF and pHeCNF, the sugar composition differs. Most apparent for

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pHeCNF is the lower amount of xylose, higher amount of arabinose, and slightly higher amount of galacturonic

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and glucuronic acids compared to cCNF. These differences may originate from the treatments varying effectivity in removing polysaccharides from cellulose. The eCNF sample contains more of every type of non-cellulosic sugar

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compared to the other samples. Fourier Transform Infrared (FTIR) was used to indicate compositional differences in the CNF preparations (Figure 5). The FTIR spectrum of eCNF is distinguished from the other samples by larger

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peaks at 1735 cm-1, 1615 cm-1, and 1245 cm-1 which are generally associated with pectin and hemicelluloses (Abidi, Cabrales, & Haigler, 2014). The FTIR spectra of cCNF and pHeCNF have subtle differences at 1735 cm(C=O stretch, associated with carboxyl groups) and 1245 cm-1 which confirm that slightly more pectin and

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hemicelluloses remained on pHeCNF. Apart from these differences, typical absorption bands associated with

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cellulose such as 1056 cm−1 for C–O stretch in the glucose ring, and 897 cm−1 for the glycosidic linkage (Abidi

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et al., 2014) do not differ between the samples.

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Ara

14

Percent of dry weight (%)

Fuc Ara Rha Gal Glu Xyl Man GalA GlcA

12 10

Rha GalA

8

Glu

Xyl Xyl

6 Xyl

Ara

4 2 0

Glu

Gal

Man

Man

Ara Rha Gal

Rha Man

GalA GlcA

Glu Fuc

Fuc

GalA

GlcA

Gal

GlcA

Fuc

eCNF

pHeCNF

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cCNF

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Figure 4 Sugar composition of cellulose nanofibers produced using chemical pre-treatment (cCNF), enzymatic pre-treatment (eCNF), and pH 9 combined with enzymatic pre-treatment (pHeCNF). The samples were treated with trifluoroacetic acid and the soluble fractions were analyzed with a Dionex-HPAEC chromatographic system. Error bars indicate standard error.

1056 cm-1

1245 cm-1 1735 cm-1

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Normalized Absorbance (a.u.)

897 cm-1

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1615 cm-1

1700

1600

1500

1400

1300

1200

Pulp cCNF eCNF pHeCNF

1100

1000

900

800

Wavenumber (cm-1)

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Figure 5 FTIR spectra for sugar beet pulp and cellulose nanofibers produced using chemical pre-treatment (cCNF), enzymatic pretreatment (eCNF), and pH 9 combined with enzymatic pre-treatment (pHeCNF).

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3.3

Rheology of nanofibers

The non-cellulosic polysaccharides that remain bound to primary-wall derived CNF, even after extensive pretreatments, have been discussed in previous studies (Dinand et al., 1999; Siqueira et al., 2016). It has been indicated that negatively charged pectic molecules (containing carboxyl groups) that remain bound to cellulose improve the ability of CNF to disperse in water (Dinand et al., 1999; Hietala, Sain, & Oksman, 2017). In Figure 6 a-b, it is shown that the rheological properties of the CNF gels (1% w/w in water) differ drastically. The storage modulus (G′), which is a measure of the deformation-energy stored by a sample, is 2.7 times higher for pHeCNF than for cCNF, while G′ for eCNF is 17 times lower. The higher G′ observed in pHeCNF means that the nanofibers form

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a more elastic and stronger gel. The loss modulus (G′′), which is a measurement of the dissipated energy of a

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sample, is lower than G′ in all samples which means that the CNF-preparations exhibit typical gel-behavior (Pääkkö et al., 2007). It appears that the rheological differences are due to slightly more pectin remaining on the

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pHeCNF compared to cCNF, while the larger amount of pectin in eCNF remain within cell wall structures (Figure 5). A similar difference between the samples can be seen in viscosity measurements over increasing shear rate

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(Figure 6c). Shear thinning is present in all samples, but most pronounced in pHeCNF followed by cCNF, and lastly eCNF. The stronger gel formation of pHeCNF, which can be distinguished by its thicker consistency, may

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find applications within rheological modification of various formulations. It seems that, by preserving the “natural polysaccharide coating” of CNF they are functionalized with minimal effort, without the need of using harmful

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chemistry to change the surface properties of cellulose (Dufresne, 2013). Considering the variety of polysaccharides that remain on CNF (Figure 4), it poses an interesting question whether the natural polysaccharide

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coating can be further fine-tuned using specialized enzymes.

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a)

cCNF eCNF pHeCNF

10000

Loss modulus, G'' (Pa)

10000

Storage modulus, G' (Pa)

b)

cCNF eCNF pHeCNF

1000

100

1000

100

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10 10

1

0.1

100

10

1

0.1

1400

cCNF eCNF pHeCNF

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1200 1000 800

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Viscosity (Pa.s)

100

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c)

10

Frequency (Hz)

Frequency (Hz)

600 400

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200 0

0.1

1

10

100

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Shear rate (1/s)

Figure 6 a) storage modulus (G′) as a function of frequency, b) loss modulus (G″) as a function of frequency, and c) viscosity as a function of shear rate. The geometry used for the measurements was a 40 mm cone (1°). Temperature was set to 25 °C. The different samples are cellulose nanofibers (1% w/w in water) produced using chemical pre-treatment (cCNF), enzymatic pre-treatment (eCNF), and pH 9 combined with enzymatic pre-treatment (pHeCNF).

Nanopaper properties

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3.4

To investigate how the different types of CNF performed in materials, nanopapers were cast from each sample and the mechanical properties were tested. As seen in Figure 7, the eCNF-nanopaper has a smaller radius than nanopapers prepared from the other CNF, despite being cast under the same conditions (section 2.8). The eCNFnanopaper is also thicker than the other samples, measuring 20-30 µm compared to 10-20 µm for cCNF and pHeCNF. The SEM images of the nanopapers show that the nanofibers are assembled in tight networks (Figure 7).

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It is noteworthy that the eCNF nanopaper has regions where nanofibers appear aligned, which might explain the shrinkage of this sample during drying. An interesting question arises weather the nanofibers are aligned due to physical interactions between different types of polysaccharides in this sample. Moreover, Figure 7 shows that the nanopapers are translucent, which is characteristic for materials consisting of densely packed nanoscale cellulose fibers (Nogi et al., 2009). Varying tints of yellow between the samples are also observed. The stronger tint in the

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eCNF nanopaper may arise due to remaining phenolic compounds or polysaccharides.

Figure 7 Pictures and SEM images of nanopapers made of cellulose nanofibers produced using chemical pre-treatment (cCNF), enzymatic pre-treatment (eCNF), and pH 9 combined with enzymatic pre-treatment (pHeCNF). The arrows highlight 1) aligned nanofibers and 2) randomly oriented nanofibers in the eCNF sample. The magnification of nanofibers is 100 000× and the scale bar represents 1 µm.

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The mechanical properties of the nanopapers are listed in Table 3, and the stress-strain curves are shown in appendix (Figure A.1). The Young’s moduli and tensile strengths of the nanopapers, especially cCNF and pHeCNF, are higher compared to corresponding values found in the literature. In previous studies, papers prepared from sugar beet nanofibers (obtained using chemical pre-treatments) measured a Young’s modulus of 1.8-9.3 GPa and tensile strength of 104 MPa (Dufresne, Cavaillé, & Vignon, 1997; Leitner, Hinterstoisser, Wastyn, Keckes, & Gindl, 2007).

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In comparison, cCNF- and pHeCNF-nanopapers prepared in the present study measured Young’s moduli around 15 GPa and tensile strengths around 250 MPa. These values are more consistent with mechanical properties of nanopapers cast from carrot nanofibers (Siqueira et al., 2016). One important similarity between the present study and Siqueira et al is that the raw plant material was never dried. This was not the case in the previous studies on sugar beet nanofibers where the raw materials were pressed and dried (Dufresne et al., 1997; Leitner et al., 2007). Drying plant material makes it more recalcitrant due to hornification between cellulose fibers. The hornification process involve irreversible hydrogen bonding that are difficult to break without hydrolyzing cellulose (Diniz, Gil, & Castro, 2004; Luo & Zhu, 2011). Additionally, the values obtained in the present study are close to nanopapers

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prepared from de-lignified wood pulp (Young’s modulus of 10.4-13.7 GPa, and tensile strength of 129-214 MPa),

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which likewise does not go through drying and re-suspension prior to mechanical homogenization (Henriksson et al., 2008). From these observations it seems that a never-dried raw material is critical for obtaining nanopapers of

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higher stiffness and strength.

Comparing the mechanical properties of nanopapers in Table 3, it is evident that the eCNF-nanopaper had

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significantly lower Young’s modulus and tensile strength than the other two samples. However, the lower

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toughness and strain-to-failure of the eCNF-nanopaper compared to the other samples were not significant (Table A.1). The lower mechanical properties of the eCNF-nanopaper are likely due to some intact cell wall structures remaining in the sample. There were no significant differences between cCNF- and pHeCNF-nanopapers in regard

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to any mechanical property (Table A.1). Nevertheless, it is speculated that the slightly over-all lower values of pHeCNF-nanopaper compared to cCNF-nanopaper is due to higher trace amount of non-cellulosic polysaccharides remaining on pHeCNF. A layer of non-cellulosic polysaccharides may prevent cellulose-cellulose interaction and form brittle interfaces between the nanofibers. However, in fabrication of composites, the layer of polysaccharides

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may conversely become useful due to better dispersion of CNF within a matrix, which results in stronger materials.

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Table 3 Mechanical properties of nanopapers made by cellulose nanofibers produced using chemical pre-treatment (cCNF), enzymatic pre-treatment (eCNF), and pH 9 combined with enzymatic pre-treatment (pHeCNF). The ± values are the sample standard deviations.

Sample number

Young’s modulus (GPa)

Tensile strength (MPa)

Toughness (MJ/m3)

Strain to failure (%)

7

16.2 ± 1.3 256.6 ± 50.1

3.7 ± 2.6

3.2 ± 1.3

eCNF

5

8.2 ± 0.8

123.8 ± 41.7

1.5 ± 1.3

2.3 ± 0.9

pHeCNF

7

15.3 ± 1.0 236.5 ± 67.8

3.1 ± 2.1

3.1 ± 1.0

3.5

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cCNF

Towards sustainable production of nanofibers

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The lower amount of liquid waste produced by the enzymatic pre-treatments compared to the chemical treatment demonstrate how production of CNF from vegetable waste can be made more sustainable. No attempts were made

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to recycle water in this study as would be done in an industrial setting. However, the relative water saving would

processes are scaled up.

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probably be similar and the simplicity of a one-pot process without intermediate filtrations are desirable as the

Sugar beet has been shown to be a particularly recalcitrant material compared to other agro-industrial waste

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products such as potato and carrot (Holland et al., 2018; Varanasi, Henzel, Sharman, Batchelor, & Garnier, 2018). Thus, it remains to see how modified enzyme protocols may work on other crops. It is possible that the water consumption could be completely omitted if the bleaching (NaClO2) step could be removed. However, for sugar beet we have found that oxidative chemistry is still necessary for obtaining similar quality of CNF as with the

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chemical pre-treatment. This is potentially due to a fraction of more recalcitrant tissue present within sugar beet pulp.

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4

Conclusion

Enzymatic pre-treatments comprising pectinases, hemicellulases, amylase, and endo-glucanase can be used to produce cellulose nanofibers from sugar beet waste, resulting in 67% lower water consumption and less toxic effluent compared to a chemical treatment. By additionally preincubating the pulp at pH 9, the enzymes were able to remove non-cellulosic polysaccharides to similar degree as the chemical treatment. The pH-enzymatically produced cellulose nanofibers, pHeCNF, and chemically produced cellulose nanofibers, cCNF, resulted in nanopapers of comparable mechanical properties. The nanopapers reached tensile strengths around 250 MP and Young’s moduli around 15 GPa, which is on par with wood-based nanopapers. However, pHeCNF contained

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slightly more pectin bound to cellulose and produced 2.7 times stronger gels in water. Thus, the new pre-treatment

be used to obtain nanofibers with tailored functional properties.

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Acknowledgement

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method opens for a more sustainable way of preparing CNF from sugar beet and demonstrates how enzymes may

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We would like to thank the Innovation Foundation Denmark for funding (grant number 5152-00001B), Birger Langebeck and John P. Jensen of Nordic Sugar useful discussion and for providing sugar beet pulp, and

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Novozymes for donating enzymes. We also acknowledge the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen for help obtaining SEM images.

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Appendix A. Supplementary data Table A.1 P-values for differences in mechanical properties between nanopapers prepared from cellulose nanofibers produced using chemical pre-treatment (cCNF), enzymatic pre-treatment (eCNF), and pH 9 combined with enzymatic pre-treatment (pHeCNF). The Pvalues were calculated using two sample welsh T-test, 95 % confidence interval, H0: µ = 0. Significance is highlighted by an asterisk (*) where P < 0.05.

P-values Factors cCNF and eCNF cCNF and pHeCNF pHeCNF and eCNF < 0.001*

0.15

< 0.001*

Tensile strength

< 0.001*

0.54

0.0053*

Toughness

0.07849

0.62

Strain-to-failure

0.154

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Youngs’s modulus

0.13

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Figure A.1 Stress-strain curves for nanopapers prepared from cellulose nanofibers which were produced using chemical pre-treatment (cCNF), enzymatic pre-treatment (eCNF), and pH 9 combined with enzymatic pre-treatment (pHeCNF). Each curve represents one measurement. Testing was done at 50% relative humidity and 22 °C.

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