Totally-Chlorine-Free Bleaching Alternatives to Produce Sugar Beet Pulp Cellulose Nanofibers

Totally-Chlorine-Free Bleaching Alternatives to Produce Sugar Beet Pulp Cellulose Nanofibers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Totally-Chlorine-Free Bleaching Alternatives to Produce Sugar Beet Pulp Cellulose Nanofibers Christian Donohoe, Ellen Engquist, Nicolas Carstens, Thomas Kinsella, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7917067/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract 1.1 Background A simple, one-pot process can be applied to sugar beet pulp (SBP), a low-lignin biomass, to produce cellulose nanofibers (CNF). However, this process uses sodium chlorite for bleaching. This work explores both the replacement of sodium chlorite (SC) with the environmentally friendly peracetic acid (PAA), both exogenous and in-situ generated, and how the choice of peroxyl or free-radical bleaching mechanism affects this reaction. 1.2 Results PAA bleached CNFs (PAA-CNF) were very close to SC bleached CNF (SC-CNF) in terms of colour and whilst the peroxyl mechanism whitened the pulp, free-radical bleaching darkened the pulp. PAA and Fenton-oxidised CNFs showed higher levels of lignin content than SC oxidised CNF but were more viscous. The CNFs all showed stability incipiently, with zeta potentials from − 17 to -23 mV. PAA-CNF formed thicker films with a lower tensile strength and higher Youngs modulus than SC-CNF films due to their higher lignin content. Moreover, the in-situ generation of PAA CNFs were similar quality to CNF bleached by exogenous PAA. 1.3 Conclusions In this study, we demonstrate three successful alternatives for replacing sodium chlorite in our CNF production method. After PAA oxidation the lignin remains, yet the resultant CNF suspensions are more viscous and equal in whiteness to SC-CNF. Overall this work shows how we can innovate an entirely chlorine-free, enzyme-mediated production processing that represent an advance for the safety and reduction of toxicity of the process, and further the development of upcycling of secondary biomass into sustainable alternatives for biomaterials. Sugar beet Oxidation Nanocellulose Peracetic acid Glucose oxidase Green Chemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 2 Background Sugar beet pulp (SBP), a by-product of sugar production, is an abundant cellulose-rich, low-lignin biomass that has potential for value-added products in biorefineries. SBP consists, on a dry weight basis, of approximately 60 to 80% of polysaccharides, where 40% is cellulose, 30% hemicellulose, and 30% pectin. The two other major components are protein and lignin, which constitutes 9 and 4-6%, respectively. (1,2). Of this low-lignin biomass, the main phenolic components are gallic acid, epicatechin, and quercetin-3-O-rutinoside (3). In addition, ferulic acid decorates the arabinan and galactan side chains of pectin (4). Cellulose, the main polymer in SBP, can be made into cellulose nanofibers (CNF) that can be used as thickeners, emulsion stabilisers, fillers in papers and biocomposites, among other applications (5). There are several methods to produce CNFs from SBP, with the differences being in pretreatments and methodology (6–8). An enzymatic approach published by Perzon et al. (2020) starts by swelling the SBP in sodium hydroxide, followed by digestion with mainly pectinases and hemicellulases, to be oxidised by sodium chlorite (SC), to lastly, being microfluidised. The oxidation, also known as bleaching, should remove lignin and molecules that are coloured or give rise to discoloration (9), and also helps with fibrillation (10). However, the use of chlorite is problematic due to production of chlorine gas which is why totally-free chlorine (TCF) methods, among other reasons, have been developed. Among several different TCF methods, three common ones are the use of peracetic acid (PAA) (8), hydrogen peroxide (HP) (2), and hydroxyl radicals (11). Peracetic acid in solution creates a mixture of peracetic acid, acetic acid and hydrogen peroxide (12). Hydrogen peroxide oxidises ester bonds, introducing hydroxyl groups that can further be oxidised into carboxyl group like aldehydes and ketones (13,14). Hydrogen peroxide, in the presence of a catalyst like Fe 2+ or Cu + , breaks down into hydroxyl radicals. This is also known as the Fenton reaction. Hydroxyl radicals can oxidise glycosidic bonds, both in pectin and hemicellulose (15,16) and can create peroxyl radicals in the presents of polysaccharides that cleave low-molecular polymers (17). Notably, the energy needed to cleave peracetic acid is lower than hydrogen peroxide, which is why hydroxyl radicals are easier created from peracetic acid than hydrogen peroxide (18). Nonetheless, hydroxyl radicals have a brief half-life and can only travel roughly the equivalent of two glucose residues in a cellulose chain (19). In comparison to peracetic acid and hydroxyl radical bleaching, sodium chlorite targets the phenolic groups, or the methyl and methylene groups in allylic position. Chlorite also oxidises the reducing ends of polysaccharides. (20) It is also possible to generate the oxidising agent peracetic acid in situ . This is done by the usage of hydrogen peroxide and a hydrogen peroxide activator. The activators react with the hydrogen peroxide to produce peracids, which is favourable due to the peracids higher redox potential and stronger oxidising properties. Several activators have been researched and used, and two of them are tetraacetylethylenediamine (TAED) and triacetin. TAED is widely used in industrial processes but is more complex to synthesise and has lower solubility than triacetin. (21,22) Sodium percarbonate (SPC) can be used instead of pure hydrogen peroxide to generate peracid in situ with TAED or triacetate. In water, SPC slowly decompose into hydrogen peroxide. This creates a steady process of peracids instead of the short-lived reactive species hydrogen peroxide produces. Additionally, TAED reacts better in slight alkaline solutions, which SPC can offer. (21) Peracetic acid is also possible to create enzymatically in situ . While keeping the hydrogen peroxide activator in the solution, hydrogen peroxide is replaced with glucose oxidase, EC 1.1.3.4. Glucose oxidase catalyses the conversion of glucose to gluconic acid, whilst reducing molecular oxygen to hydrogen peroxide (23). Therefore, in the presents of SBP where glucose is available, glucose oxidase should generate small amount of hydrogen peroxide. This concept has been shown to work in previous studies of bleaching of cotton (24,25). In this work to improve our previous CNF production method from SBP (7,26), it is hypothesised that sodium chlorite can be replaced with an environmentally friendly oxygen-based bleaching step, with little to no comparative loss in whitening or physical properties. To investigate this, peracetic acid and hydrogen peroxide, as a peroxyl and free-radical oxidants respectively, were compared to sodium chlorite oxidation for the production of CNF suspensions and their subsequent casted films. The bleaching of CNF was equal for peracetic acid and sodium chlorite; however, hydrogen peroxide darkened the suspension. This did not seem to affect the viscosity, where the three suspensions were similar. However, it could be seen that the elongation before the breakpoint of the film, where peracetic acid bleached CNF was used, decreased. Lastly, based on the above-mentioned findings, in situ generation of peracetic acid, both chemically and enzymatically, was tested and showed to bleach the CNF to the same whiteness as exogenously supplied peracetic acid. 3 Methods and Materials 3.1 Sugar Beet Pulp Fresh post-sugar extraction sugar beet, in pieces of 1–2 cm, was obtained from Nordic Sugar and stored at − 20°C. 3.2 Materials All chemicals, both powders and liquids, and glucose oxidase (from Aspergillus niger ) were purchased from Sigma-Aldrich. The enzymes used in section 3.3.1 were kindly provided by Novozymes (Denmark). The antibodies used in 3.4.4 were either bought or given. The LM and JIM antibodies including MAC207 were donated from Plant Probes, Paul Knox lab, the INRA- RUI and INRA-RUII antibodies donated from INRA (France), and the CCRS, BS-400-4 and CBM3a antibodies were bought from Carbosource, University of Georgia (USA), Biosupplies (Australia) and NZYtech (Portugal), respectively. 3.3 Cellulose Nanofiber Production Method 3.3.1 Enzyme-treated Sugar Beet Pulp The production of enzyme-treated SBP is adapted from Perzon et al . protocol ( 26 ). 20 grams (dry weight) sugar beet pulp cossettes were dispersed in 2.5 L of distilled water and subsequently homogenised (Silverson L5M homogenizer fitted with the General Purpose Disintegrating Head) for 30 minutes at 6000 RPM. The pulp was filtered on Miracloth and added to a beaker together with water to a total volume of 500 ml. The pH was raised to pH 9 by the addition of 8 ml 4 M sodium hydroxide. The suspension was heated at 80°C for two hours with mechanical stirring. Afterwards, the reaction was cooled to 40°C. When the temperature was cooled down, 1.5 ml of 12.1 M glacial acetic acid was added under mixing to reach pH 5. Enzymes, according to Table 1 , were added and the reaction was incubated at 40°C for 2 hours with mechanical stirring. If no bleaching method was applied, the enzymes were inactivated by heating at 80°C for 15 minutes. Table 1 Identity, activity and volume of enzymes applied to sugar beet pulp for nanocellulose production. Adapted from ( 26 ). 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 Volume of enzyme solution / gram dry mass SBP (µL / g) Viscozyme L Beta-glucanase (endo-1,3( 4 )-β-glucanase) 100 FBG/g 10 Pectinex Ultra Clear Polygalacturonase (endo-1,4-α-galacturonidase) 7900 PGNU/ml 10 Pulpzyme HC Endo-xylanase (endo-1,4-β-xylanase) 1000 AXU/g 10 FiberCare R Cellulase (endo-1,4-β-glucanase) 4500 ECU/g 5 Aquazym 240 L Alpha-amylase (endo-1,4-α-glucanase) 240 KNU/g 10 3.3.2 Bleaching with sodium chlorite, peracetic acid or hydrogen peroxide The enzyme-treated SBP suspension was heated to 70°C. To the suspension the oxidant was added, up to a concentration of 100 mM, and the reaction was incubated for 2 hours with mechanical stirring. The different oxidants used were 2.8 M sodium chlorite, 5.1 M peracetic acid, or 8.8 M hydrogen peroxide with 1 mM iron chloride. Afterwards, pulp was collected on Miracloth and washed thoroughly with deionised water. Normally, the amount of deionised water was three times the volume of the reaction. 3.3.3 Bleaching via peracetic acid activators combined with a source of hydrogen peroxide For the sodium percarbonate reaction, sodium percarbonate was added to final concentration of 100 mM in 500 ml of 4% (w/v) SBP suspension in water. Glacial acetic acid was added to obtain pH 10. For the glucose oxidase reaction, 5 mg of the enzyme (Glucose Oxidase from Aspergillus niger , Sigma Aldrich, 100,000-250,000 units/g solid) was added to 500 ml of 4% (w/v) SBP suspension in 0.1 M sodium phosphate, pH 7. Either tetraacetylethylenediamine (TAED) or triacetin was added to a concentration of 50 mM or 33 mM, respectively, to equate 100 mM peracetic acid. The sodium percarbonate mixture was incubated at 70°C for 2 hours while the glucose oxidase reaction was incubated at 35°C for 12 h, both with mechanical stirring. After the incubation, the pulp was collected on Miracloth and washed thoroughly with deionised water, approximately three times the mixture volume. 3.3.4 Microfluidisation The enzyme-treated and bleached pulps were resuspended to 1% (w/v) in distilled water with 0.05% (w/v) chlorobutanol as a bacteriostatic agent. The suspensions were microfluidised (Microfluidizer materials processor M110-P) at 600 bars, with a set-up of two chambers of 400 µm followed by 200 µm. The suspensions were passed six times though the system to obtain cellulose nanofibers (CNF). The dry content of the pulp was analysed with a moisture analyser (Kern DLB moisture analyser). 3.3.5 Film Preparation 25 ml of 0.5% (w/v) CNF suspension was spread in polystyrene petri dish (Ø 85 mm) and slowly evaporated at 35°C for 72 hours until completely dry. To ensure uniform moisture content and avoid film shrinkage, all films were kept in airtight sealed desiccator containing saturated sodium bromide, maintaining a relative humidity of 60% at 20°C prior to analysis. 3.4 Analysis Methods 3.4.1 Whiteness Index The whitening of the oxidised CNF was analysed with ImageJ FIJI software (Fig. 1 ). For each sample, a photo was taken and processed into an 8-bit grey-scale image. The software measures absolute brightness from a scale of 0 to 255, where zero refers to black and 255 refers to white. Several points in each photo were analysed, and the average of the absolute brightness of each sample was converted into an index scale from 0–100% as a measure of whiteness. 3.4.2 Rheological Measurements The rheology of the CNF suspensions was measured with a rheometer (TA Discovery HR-3) with a 40 mm cone plate (0.9767° angle) with a 22 µm gap. 670 µl of 0.1 to 0.4% (w/w) CNF suspensions was added to plate. The oscillation sweep was measure from 0.1% to 100% at 1 Hz, the shear sweep was measured from 0.11 to 1000 Hz, and the frequency sweep was measured at 0.1% strain from 0.01 to 100 Hz. All measurements were performed at 25°C. 3.4.3 Lignin Content The lignin content of the samples was determined with the acetyl bromide assay, adapted from Gui et al ( 27 ). 1 mg of dried biomass was placed in a 2 ml screwcap Sarstedt tube. 100 µl of 25% (v/v) acetyl bromide, diluted in glacial acetic acid, was added and the samples incubated for 3 hours at 50°C and 600 rpm. Afterwards, the samples were cooled on ice to room temperature. 400 µl of 2 M sodium hydroxide was added followed by 70 µl of 0.5 M hydroxylamine hydrochloride. The samples were vortexed, followed by addition of 1.43 ml of glacial acetic acid and vortexed again. The samples were centrifuged at 13,000 rpm for 3 minutes and 200 µl of the supernatant of samples was transferred to an UV-transparent 96-well plate. The absorbance was read at 280 nm in a spectrophotometer (Spectromax 190, Molecular Devices, USA) and the concentrations were calculated from a calibration curve of alkali lignin. 3.4.4 Array-based Microfibril Surface Assessment The surface compositions of the CNF suspensions were analysed with the array-based microfibril surface assessment (AMSA) procedure, as previously performed in Perzon et al. (2020). The samples were spotted with a robot (synQUAD Pixsys, Digilab Inc.) in arrays. The samples were done in four replicates, in eight dilutions. The arrays were blocked in 5% fat-free milk protein in phosphate-buffered saline (PBS), followed by being probed with primary monoclonal antibodies (mAb) according to Table 2 in 5% fat-free milk protein in phosphate-buffered saline. The arrays were washed in PBS, followed by being probed with secondary mAbs. The arrays were washed in PBS, then water, before being developed in 0.02% 5-bromo-4-chloro-3-indolylphosphate (BCIP) and 0.03% nitro-blue tetrazolium chloride (NBT) in 100 mM tris pH 9.5 with 100 mM sodium chloride and 5 mM magnesium chloride. After development, the arrays were scanned at 1200 dpi, loaded into ArrayProAnalyzer version 6.3.1 software for detection and relative quantification of the composition of the samples. Table 2 List of the used primary monoclonal antibodies and their target epitopes, together with their references. Primary antibodies were used at concentrations ranging from 1–50 mM) † XGL nomenclature of describing xyloglucan substructures as developed by Fry et al. (1993) Antibody Epitope Reference LM18 Homogalacturonanon ( 28 ) LM19 Homogalacturonanon ( 28 , 29 ) LM5 β-1,4-galactan ( 30 ) LM6 α-1,5-arabinan ( 31 ) INRA-RU1 Rhamnogalacturonan-I ( 32 ) INRA-RU2 Rhamnogalacturonan-I ( 32 ) BS-400-4 (1→4)-β-mannan/galacto-(1→4)-β-mannan ( 33 ) LM21 Heteromannan ( 34 ) LM22 Glucomannan ( 34 ) CCRC-M167 Galactomannan ( 35 ) CCRC-M170 Galactomannan: Acetylated ( 35 , 36 ) CCRC-M175 ( 1 , 4 )-β-D-xylosyl ( 35 ) CCRC-M145 Xylan: 4-O-MeGlcA ( 37 ) CCRC-M147 β-1,4-linked xylosyl ( 35 , 37 , 38 ) LM11 ( 1 , 4 )-β-D-xylosyl ( 37 ) LM28 Glucuronosyl xylan ( 35 , 37 , 38 ) LM14 Arabinogalactan ( 39 ) JIM8 Arabinogalactan ( 40 ) JIM13 Arabinogalactan ( 41 ) MAC207 Arabinogalactan w. GlcA ( 42 ) LM15 XXXG (xyloglucan) † ( 37 , 43 ) LM25 XLLG, XXLG, XXXG (xyloglucan) † ( 37 , 44 ) CBM3a Cellulose ( 45 ) LM12 Ferulates ( 44 ) 3.4.5 Electronic Stability of Suspension The zeta potential of the CNF suspensions was measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) equipped with a DTS1070 unit cell and analysed with Malvern ZetaSizer software. The suspensions were prepared at 0.1% (w/v) in deionised water. The measurements were taken at 25°C with an angle of 17°. Each sample was recorded as three technical replicates. 3.4.6 Scanning Electron Microscopy The CNF films were visualised with field-emission scanning electron microscopy (FE-SEM) by a Quanta 3D FEG (FEI company, Netherlands). For imaging of the surface, a 1x1 cm 2 square of the film was cut out, placed on a metal stud, and coated with 2 nm layer of gold. To image the cross-section, a strip of the CNF film was frozen in liquid nitrogen for 30 seconds and snapped in half. The broken edge was attached to the metal plate facing upwards and coated with 2 nm layer of gold. 3.4.7 Mechanical Testing The Young’s modulus and tensile strength of the films were tested with a TA-XT Texture Analyser (TTC company) with a grip accessory and 50 kg load cell. Films were cut into 5x0.4 cm 2 strips, placed in the instrument and the analysis was performed at a cross-head speed of 4 mm/min. Relative humidity was kept at 60% at a temperature of 22°C. Young’s modulus, the tensile strength, and the strain were calculated from the obtained stress-strain curves. The results were derived from 6–10 replicates per film. 4 Results and Discussion 4.1 Sugar beet pulp nanofibers The method of Cellulose nanofiber production from SBP as published by Perzon et al. (2020) which this paper further develops, follows two key steps: matrix polysaccharide removal (chemically or enzymatically) followed by an oxidation step. The enzyme mediated removal of matrix polysaccharides was accomplished with a mild alkaline swelling step followed by digestion with enzymes active on pectic and hemicellulosic polysaccharides. This process has been reported before and the material thoroughly characterised. Removal of matrix polysaccharides is intentionally not exhaustive as a residual coating of the nanofibers is required for colloidal stability ( 46 ). Based on quantification of trifluoroacetic acid soluble monosaccharides, the material is ~ 85% cellulose ( 26 ). The polymers that could be extracted from the CNF were found to comprise most notably of extensin, xyloglucan, mannan and rhamnogalacturonan-I ( 7 ). Direct detection of glycans on the CNF corroborated these observations and demonstrated that different populations of mannans exist, one of which is very strongly adhering to the nanofiber surface ( 47 ). This product constitutes starting material for investigations of oxidation methods alternative to chlorite. The oxidising effects of peracetic acid, hydrogen peroxide and hydroxyl radicals on sugar beet pulp CNF were evaluated by the whitening of the pulp, lignin removal, polysaccharide composition, rheology, and suspension stability. The oxidising agents were compared to sodium chlorite to evaluate the potential in chlorine-free bleaching. 4.1.1 Whitening of the pulp The starting substrate, the enzyme treated sugar beet pulp (SBPe), was visibly brown with a measured whiteness index (WI) of 59% (Table 3 ). Sodium chlorite oxidised the SBPe to a pale white colour with a WI of 68%. Peracetic acid was more effective than hydrogen peroxide in whitening the material with a WI of 67% and 63%, respectively. However, none of the oxidising agents could exceed the sodium chlorite bleached pulp. Table 3 The whiteness index of the solids of the sugar beet pulp suspensions, calculated from triplicates. SBPe = Enzyme-treated sugar beet pulp, SC = sodium chlorite bleached pulp, PAA = peracetic acid bleached pulp, and HP = hydrogen peroxide bleached pulp. Sample Whiteness index (%) Uncatalysed Catalysed with FeCl 2 SBPe 58.5 ± 1.4 53.6 ± 2.5 SC 67.6 ± 1.5 69.9 ± 3.5 PAA 67.4 ± 5.1 46.0 ± 4.6 HP 62.9 ± 5.6 38.7 ± 2.8 To evaluate the free radical bleaching, the same oxidising agents were tried with the catalytic presence of iron ions (Fe 2+ ), to initiate the Fenton reaction. In general, the bleaching effect seems to be reversed, with a darkening of the pulp. The SBPe with the catalyst had a WI of 54%, while the sodium chlorite bleached suspension had a slight increase, compared to without the catalyst, of the WI to 70%. The chlorite-free oxidising agents together with the catalyst darkened the pulp considerably to a WI of 46% and 39%, respectively. While there is no strict correlation between whitening and lignin removal, oxidation is believed to remove both lignin and chromophoric groups, resulting in a whitening of materials. SBP is primarily formed from the roots of Beta vulgaris , the colour of which is formed from the type and ratio of betalins ( 48 ). It has been shown that peracetic acid is more selective towards lignin than sodium chlorite across a wide range of substrate ( 46 , 49 , 50 ), but this seems not to influence the whitening of SBP in this study. Furthermore, free radicals are known to react non-selectively due to their high-energy state. It is possible the free radicals through the Fenton reaction does not degrade colour-giving compounds but oxidises other compounds that leads to darkening of the pulp. From this initial assessment, the uncatalyzed PAA and SC together with the catalysed HP oxidised pulps were chosen to be analysed further, since these represented the whitest and darkest pulps respectively. 4.1.2 Lignin Content To investigate the hypothesis of bleaching correlating with lignin removal, we performed an acetyl bromide assay to measure the total lignin content of these oxidised samples (Table 4 ). Table 4 The lignin content of sugar beet pulp suspensions and availability of ferulates of the SBP CNF suspensions. The lignin content of each sample measured by the acetyl-bromide assay. The content is the average ± standard deviation of three replicates. SBP = sugar beet pulp, SBPe = Enzyme-treated sugar beet pulp CNF, SC-CNF = sodium chlorite bleached CNF, PAA-SBPe = peracetic acid bleached CNF, and HP.F = Fenton catalysed hydrogen peroxide bleached CNF. Sample Lignin content (% of dry mass) SBP 6.2 ± 1.0 SBPe-CNF 4.4 ± 1.1 SC-CNF 3.9 ± 1.5 PAA-CNF 8.6 ± 1.2 HP.F-CNF 9.9 ± 1.1 The raw biomass material, SBP, already has a low total lignin content (Table 4 ) of 6.2% to begin with. After enzymatic treatment (SBPe), the lignin content decreases to 4.4%. It is possible that the lignin co-solubilises with the digested hemicellulose or pectin, but it is more likely the decrease should be attributed to the loss of xylan. It has been shown before that starch and xylan can increase the measured lignin content ( 51 ). The oxidations applied did not seem to cause delignification; SC-CNF was not significantly different from SBPe-CNF, and the other oxidants PAA and HP.F actually resulted in CNFs with higher measured levels of lignin after oxidation. This could be due to the oxidation and solubilisation of the other cell-wall components, seen previously with hydrogen peroxide oxidation ( 2 ). 4.1.3 Surface availability of ferulates LM12, an antibody that binds to ferulic residues ( 44 ), was used to estimate the phenolic availability of the surfaces of the oxidised SBP CNFs. Typically SBP would contain high amounts of ferulic acid in its pectin fraction ( 52 – 54 ). Due to the pre-enzymatic digestion step before any oxidation, the vast majority of surface-available pectin and hemicellulose were removed (Additional file 1). This explains why the availability of ferulates decrease between the starting material SBP to the SBPe (Fig. 2 ). After oxidation of SBPe, there is a significant increase in LM12 binding and hence surface-available ferulates. Hydrogen peroxide bleached CNF (HP.F-CNF) had the highest surface availability of ferulates, up to almost twice of the availability of SBP, and in turn a near twenty-time fold increase in comparison to SBPe CNF. Sodium chlorite and peracetic acid bleached CNFs did not have the same high availability as HP-F-CNF, but they did increase the availability of ferulates by five-fold and almost three-fold compared to SBPe CNF. Whether this increase, or lack thereof, in LM12 binding to the oxidised samples compared to SBPe-CNF accurately portrays the oxidising effect of the oxidants is unclear. Whilst an increase in LM12 binding levels indicates that ferulates have become exposed due to oxidation, it is also true that a smaller increase does not preclude that that the oxidant was less effective. It could also mean that ferulates are being exposed and then solubilised by the oxidation. Ferulic acid has been previously shown to be more resistant to HP than PAA ( 55 ) which suggests that the small increase of LM12 binding to PAA-CNF means that the ferulic acid has been solubilised. 4.1.4 Rheology of Suspensions The physical properties of the oxidised materials were evaluated by their rheological properties in response to a frequency sweep and shear sweep. An ideal oxidant replacement for SC would create a CNF suspension of equal or higher viscosity. The lack of crossover in the storage modulus (G’) and loss modulus (G’’) of the frequency sweep (Fig. 3 A and 3 B) in any of the samples demonstrated that they were all cross-linked gels ( 56 ), which was expected. This behaviour has been previously observed in both unbleached CNF suspensions ( 57 , 58 ) and the previous reported SC-CNF ( 26 ). Only the PAA-CNF shows a higher recorded G’, indicating that it forms a stronger gel. SC-CNF in general is the most similar to the unbleached CNF in both G’ and G’’. Finally, Fenton catalysed HP.F-CNF has the weakest G’ and G’’ out of the set, which is ten times lower than PAA-CNF, implying a weakly formed gel. These observations mostly related to what can be observed in the shear sweep tests of the samples (Fig. 3 C), even if complex and shear viscosities are not typically directly comparable at high shear rates ( 59 ). The viscosity of PAA-CNF was 4500 cP, which was twice as large as SBPe-CNF and SC-CNF of 2000 cP. The exception here was HP.F-CNF with a viscosity of 2500 cP which also was more viscous than SBPe-CNF at low shear rates. 4.1.5 Suspension stability The electrostatic stabilities of the CNF suspensions were evaluated by their zeta potential (ZP). This property represents the ionic interaction of the particles of the suspension as millivolt (mV). As the voltage increases, the repulsive forces between the particles increases which indicates that the suspension will be more resistant to flocculation or aggregation. A zeta potential between 0 mV and − 10 mV would mean the suspension is inherently unstable. Going further, a suspension ZP between − 10 mV and − 30 mV mildly stable, and suspensions with a ZP below − 30 mV would be functionally stable. ( 60 ) Table 5 The zeta potentials of the enzyme-treated sugar beet pulp and the oxidised SBP CNF suspensions. The potential is the average ± standard deviation of triplicates. SBPe = Enzyme-treated sugar beet pulp, SC-CNF = sodium chlorite bleached CNF, PAA-SBPe = peracetic acid bleached CNF, and HP.F = Fenton catalysed hydrogen peroxide bleached CNF. Samples Zeta Potential (mV) SBPe-CNF -11.1 ± 1.6 SC-CNF -20.3 ± 1.4 PAA-CNF -17.4 ± 2.5 HP.F-CNF -23.7 ± 1.9 The SBPe-CNF was already partially stable at -11.1 mV (Table 5 ). However, as the number indicates, the suspension is only initially stable and will settle over a few minutes. The measured ZP of all bleached CNF suspensions have a ZP of roughly 20 mV, with hydrogen peroxide and peracetic with the highest and lowest ZP, respectively. The suspensions are only partially stable, which leads to aggregation after hours of resting or under cold conditions. Whilst it was thought that a more negative ZP would correlate with a higher viscosity, the inverse was observed with these samples; the least electronegative oxidised sample PAA was the most viscous. In this case it could be argued that the differences in ZP between the samples are too minor to be determinant. Previous work studying CNCs showed that samples between − 10mV and − 20mV being broadly similar in terms of shear viscosity ( 61 ), and this might be the same for CNFs. 4.2 Morphology and Mechanical Testing of CNF Films To understand what implication the different CNF suspensions could have on being further processed, the different CNF were casted into films. The morphology and rheology of the films were analysed with SEM and mechanical testing (Fig. 4 and Table 6 ), respectively. Hydrogen peroxide-bleached CNF was excluded due to its’ low whitening effect. Films from SBPe-CNF were also produced, but they were too brittle for mechanical testing and hence were excluded from these analyses. Pinkl et al. ( 62 ) has reported similar problems with SBP CNF films, where the low content of cellulose was assumed to be the reason. From the SEM images, it can be seen that the surface of the sodium chlorite film is smooth while the peracetic acid film has more visible, exposed microfibrils (Fig. 4 ). Previous work, on the SC bleached CNF films, showed rough surface covered by visible nanofibrils ( 26 ). The cross sections (XC) of the films also show that the peracetic acid film was thicker in comparison to the sodium chlorite film. This could be reasoned by the idea that the fibrils are rougher and hence less consistently layered. Supported by Galland et al. (2015) reasoning, this would indicate the peracetic acid film has a weaker structural reinforcement since the jagged layering would be more susceptible for stress transfer. This is also what the mechanical testing of the films further shows, where the peracetic acid film failed at a lower stress than the sodium chlorite film (Table 6 ). The peracetic acid film showed lower tensile strength than the sodium chlorite film, possibly due to the higher lignin content in the peracetic acid films. Table 6 Measured thickness from three cross-section SEM images of each oxidised CNF film as shown in Fig. 4 , as well as subsequent calculated density and the stress/strain of each film at failure. Oxidative treatment Film Thickness (µm) Density (g/cm 3 ) Stress (MPa) Strain (%) Young’s modulus (GPa) Sodium chlorite (SC) 13.0 ± 0.4 1.69 ± 0.05 32.6 ± 1 5.0 ± 1 6.5 ± 1 Peracetic acid (PAA) 18.5 ± 0.3 1.19 ± 0.02 24.8 ± 3 3.4 ± 1 7.4 ± 4 Typically density correlates with the Young’s modulus of a material ( 63 ) but in our study, the peracetic acid bleached CNF film is still more elastic despite its reduced packing. It has previously been discussed that lignin lowers hydrogen bond formation between the fibers which leads to lower mechanical properties ( 64 , 65 ). The peracetic acid film is stiffer but slightly weaker and less elastic than the sodium chlorite film which aligns with the rheological results (Table 6 ). Both, however, are similar to previously produced CNF films ( 46 , 66 ). 4.3 In situ Generation of Peracetic Acid The bleaching with chemicals of SBP are not only evaluated based on their effect on final properties, but also how it can be integrated at large-scale production of SBP CNF. The chemical oxidants can be limited due to their explosive risk and health hazards ( 67 – 69 ), which in situ generation of peracetic acid could circumvent and possible provide both a safer and in-line oxidation. For the in situ generation of peracetic acid, one of the hydrogen peroxide activators TAED or triacetin was combined with hydrogen peroxide from either sodium percarbonate (SPC) or the enzyme glucose oxidase. The WI of CNFs treated by SPC with TAED and triacetin was 37% and 36% (Table 7 ), respectively. The WI of the CNFs treated by glucose oxidase with TAED and triacetin was 34% and 27%, respectively. This shows SPC showed to be a more effective bleaching agent than glucose oxidase, although this could be due to the alkaline swelling effects of the dissolved compound. Table 7 The whiteness index of SBP CNF suspensions, bleached with in situ generation of peracetic acid with either Tetraacetylethylenediamine (TAED) or triacetin (TriA) together with sodium percarbonate (SPC) or glucose oxidase (GOx). Oxidative treatment Whiteness Index (%) SPC 32.9 ± 1.0 SPC-TAED 37.1 ± 1.0 SPC-TriA 36.2 ± 1.1 GOx 21.1 ± 1.3 GOx-TAED 34.3 ± 1.4 GOx-TriA 26.8 ± 1.5 The viscosities of the in situ peracetic acid bleached CNFs, in comparison to peracetic acid CNF, are decreased. The glucose oxidase samples, regardless of using TAED or triacetin, have similar loss of viscosity in comparison to bleaching with peracetic acid exogenously (Fig. 5 A). For the SPC samples, even though the two bleach activators have very similar WIs, the triacetin resulted in a much larger loss of viscosity than TAED (Fig. 5 B). Finally, the amount of available ferulates on the surface were analysed via LM12 antibody binding levels. Considering that SBPe has very low availability of ferulates on its’ surface (Fig. 2 ), when oxidised by SPC or glucose oxidised alone ferulates were exposed (Fig. 5 C). When TAED is present though, the surface availability of ferulates is similar to PAA-SBPe. The shows that peracetic acid, whether in situ or exogenously sourced, is solubilising the available ferulates whereas hydrogen peroxide exposes but does not solubilise them. Overall, these results shows that SPC with either TAED or triacetin could be a possible replacement of PAA, and thereby, provide an in-line, safer oxidation of SBP. However, the reaction is limited to a concentration of 10 mM by the strong alkalinity of SPC, that would require neutralisation if higher amounts were used. Similar systems involving sodium perborate and TAED ( 70 ) have been implemented previously for cotton bleaching with similar effects on WI. Enzymatic in situ generation of PAA shows the choice of hydrogen peroxide activator, or the set-up, matters. Glucose oxidase together with TAED show similar results in whitening, viscosity, and elongation before breakpoint of both PAA and SPC with TAED or triacetin. Glucose oxidase with TAED has been shown to bleach cotton linter ( 24 ), and together with these results, it demonstrates the potential of enzymatic bleaching with TAED. However, pairing glucose oxidase with triacetin did not show any significant enhancement of bleaching. This is likely due to the pH that was chosen to be a compromise between the optimal pH for the enzyme and triacetin but resulted in suboptimal reaction for both. Here, SPC could create a reaction with pH 10 to 11 that is the optimal range for PAA generation with both hydrogen peroxide activators ( 22 ). Furthermore, the differing pH of the methods seems to have affected the physical properties of the CNFs. SPC oxidation resulted in higher viscosity along with higher amounts of exposed surface pectin. It is likely the pectin would increase the viscosity, as it has been shown to do in plant cell wall suspension ( 26 , 71 ). 5 Conclusions In this work, peracetic acid has been shown to be a feasible replacement for sodium chlorite as a bleaching agent in the production of cellulose nanofibers from sugar beet pulp. The resultant CNF has the same whiteness index and increased viscosity. In contrast, Fenton-catalysed hydrogen peroxide creates CNF that are darker in colour but still with a slight increase in viscosity compared to sodium chlorite. The lignin content of the enzymatically digested SBP was not affected by any of the oxidants applied, and that the small differences in the relative lignin content shows be attributed to the removal of polysaccharides rather than lignin. However increased amounts of ferulates were detectable on the surface of the oxidised CNFs, especially after free-radical oxidation. Nevertheless, our data shows that peracetic acid can replace sodium chlorite when focusing on whitening. Finally, it has been shown that peracetic acid can be directly generated in-situ and used immediately for bleaching SBP in this process, thus making PAA a safer, in-line oxidant. Chemically, peracetic acid can be released from either TAED or triacetin together with sodium percarbonate. Enzymatically, peracetic acid can be created from glucose oxidase with TAED which makes this process entirely enzyme mediated. Triacetin with sodium percarbonate allows for a simplification of the method where the swelling and bleaching steps occur in one. Overall, this work demonstrates the successful replacement of chlorine-bleaching with the novel application of two possible in-situ PAA bleaching methods to the secondary biomass sugar beet pulp. 6 Abbrevations SBP Sugar Beet Pulp TAED Tetraacetylethylenediamine CNF Cellulose nano-fibers SC Sodium chlorite HP Hydrogen peroxide PAA Peracetic acid SPC Sodium percarbonate %w/w Weight/ weight %v/v Volume / volume AMSA Array-based microfibril surface PBS Phosphate-buffered saline BCIP 5-bromo-4-chloro-3-indolylphosphate NBT Nitro-blue tetrazolium chloride SEM Scanning electron microscopy FEG Field emission gun WI% Whiteness index SBPe SBP, enzymatically digested SC-SBPe Sodium chlorite - oxidised SBPe PAA-SBPe Peracetic acid - oxidised SBPe HP.F-SBPe Hydrogen peroxide - Fenton - oxidised SBPe G' Storage modulus G'' Loss modulus ZP Zeta potential TriA Triacetin GOx Glucose oxidase Declarations Competing interests The authors declare no conflicts of interest. Funding This work was supported by a grant from the Novo Nordisk Foundation: NNF22OC0072911. Author Contribution Christian Donohoe; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Ellen Engquist; Conceptualization, Methodology, Writing – review & editing. Nicolas Carstens; Data curation, Formal analysis, Investigation, Validation, Visualisation . Thomas Kinsella; Data curation, Formal analysis, Investigation, Validation, Visualisation. Bodil Jørgensen; Conceptualisation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing. Marwa Faisal; Formal analysis, Methodology, Resources, Writing – review & editing. Peter Ulvskov; Conceptualisation, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing. Declaration of competing interest Data Availability The data supporting this article has been included in this text and as part of the Supplementary Information. References Dinand E, Chanzy H, Vignon MR. Parenchymal cell cellulose from sugar beet pulp: preparation and properties. Cellulose. 1996;3(1):183–8. Whale E, Bulling AEK, Fry SC. Biochemical characterisation of cellulose and cell-wall-matrix polysaccharides in variously oxidised sugar-beet pulp preparations differing in viscosity. International Journal of Biological Macromolecules. 2023;253:127356. Arjeh E, Khodaei SM, Barzegar M, Pirsa S, Sani IK, Rahati S, et al. Phenolic compounds of sugar beet (Beta vulgaris L.): Separation method, chemical characterization, and biological properties. Food Science & Nutrition. 2022 Sept 13;10(12):4238. Ralet MC, André-Leroux G, Quéméner B, Thibault JF. Sugar beet ( Beta vulgaris ) pectins are covalently cross-linked through diferulic bridges in the cell wall. Phytochemistry. 2005;66(24):2800–14. Lasseuguette E, Roux D, Nishiyama Y. Rheological properties of microfibrillar suspension of TEMPO-oxidized pulp. Cellulose. 2008 June 1;15(3):425–33. Hassan ML, Berglund L, Abou Elseoud WS, Hassan EA, Oksman K. Effect of pectin extraction method on properties of cellulose nanofibers isolated from sugar beet pulp. Cellulose. 2021;28(17):10905–20. Holland C, Perzon A, Cassland PRC, Jensen JP, Langebeck B, Sørensen OB, et al. Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely Enzymatic Pretreatments. Biomacromolecules. 2019;20(1):443–53. Yang W, Feng Y, He H, Yang Z. Environmentally-Friendly Extraction of Cellulose Nanofibers from Steam-Explosion Pretreated Sugar Beet Pulp. Materials. 2018 July;11(7):1160. Sarkanen KV. The chemistry of delignification in pulp bleaching. Pure and Applied Chemistry. 1962;5(1–2):219–32. Galland S, Berthold F, Prakobna K, Berglund LA. Holocellulose Nanofibers of High Molar Mass and Small Diameter for High-Strength Nanopaper. Biomacromolecules. 2015;16(8):2427–35. Liu K, Yan K, Sun G. Mechanism of H2O2/bleach activators and related factors. Cellulose. 2019;26(4):2743–57. Hickman WS. Peracetic acid and its use in fibre bleaching. Review of Progress in Coloration and Related Topics. 2002;32(1):13–27. Afsahi G, Rojalin T, Vuorinen T. Chemical characteristics and stability of eucalyptus kraft pulps bleached with tertiary amine catalyzed hypochlorous acid. Cellulose. 2019;26(3):2047–54. Li K, Tang B, Zhang W, Shi Z, Tu X, Li K, et al. Formation Mechanism of Bleaching Damage for a Biopolymer: Differences between Sodium Hypochlorite and Hydrogen Peroxide Bleaching Methods for Shellac. ACS Omega. 2020 Sept 8;5(35):22551–9. Fry SC, Dumville JC, Miller JG. Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell walls of ripening pear fruit. Biochem J. 2001;357(Pt 3):729–37. Miller JG, Fry SC. Characteristics of xyloglucan after attack by hydroxyl radicals. Carbohydr Res. 2001 June 15;332(4):389–403. Zimin YuS, Borisova NS, Kutlugildina GG, Mudarisova RKh, Borisov IM, Mustafin AG. Oxidation and destruction of arabinogalactan and pectins under the action of hydrogen peroxide and ozone-oxygen mixture. Reac Kinet Mech Cat. 2017;120(2):673–90. Carlos TD, Bezerra LB, Vieira MM, Sarmento RA, Pereira DH, Cavallini GS. Fenton-type process using peracetic acid: Efficiency, reaction elucidations and ecotoxicity. Journal of Hazardous Materials. 2021;403:123949. Airianah OB, Vreeburg RAM, Fry SC. Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method. Annals of Botany. 2016;117(3):441–55. Mishra SP, Manent AS, Chabot B, Daneault C. The Use of Sodium Chlorite in Post-Oxidation of TEMPO-Oxidized Pulp: Effect on Pulp Characteristics and Nanocellulose Yield. Journal of Wood Chemistry and Technology. 2012;32(2):137–48. Li Q, Lu R, Liang Y, Gao K, Jiang H. Energy-Saving One-Step Pre-Treatment Using an Activated Sodium Percarbonate System and Its Bleaching Mechanism for Cotton Fabric. Materials. 2022;15(17):5849. Zhou Y, Zheng G, Zhang J, Wang Q, Zhou M, Yu Y, et al. An eco-friendly approach to low-temperature and near-neutral bleaching of cotton knitted fabrics using glycerol triacetate as an activator. Cellulose. 2021;28(12):8129–38. Müller D. Studies on a new enzyme glycose-odydase. I. Biochem Z. 1928;199:136–70. Tavčer PF. Low-temperature bleaching of cotton induced by glucose oxidase enzymes and hydrogen peroxide activators. Biocatalysis and Biotransformation. 2012;30(1):20–6. Tzanov T, Costa SA, Gübitz GM, Cavaco-Paulo A. Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching. J Biotechnol. 2002;93(1):87–94. Perzon A, Jørgensen B, Ulvskov P. Sustainable production of cellulose nanofiber gels and paper from sugar beet waste using enzymatic pre-treatment. Carbohydrate Polymers. 2020;230. Gui J. Acetyl Bromide Method for Total Lignin Content Determination in Plant Biomass. In: Jiang L, Shen J, Gao C, Wang X, editors. Plant Protein Secretion: Methods and Protocols [Internet]. New York, NY: Springer US; 2024 [cited 2025 Feb 26]. p. 95–100. Available from: https://doi.org/10.1007/978-1-0716-4059-3_8 Verhertbruggen Y, Marcus SE, Haeger A, Ordaz-Ortiz JJ, Knox JP. An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohydr Res. 2009 Sept 28;344(14):1858–62. Kilcoyne M, Gerlach JQ. Glycan Microarrays: Methods and Protocols. 1st ed. 2022. New York, NY: Springer US; 2022. xi + 253. (Methods in Molecular Biology, 2460). Jones L, Seymour GB, Knox JP. Localization of Pectic Galactan in Tomato Cell Walls Using a Monoclonal Antibody Specific to (1[->]4)-[beta]-D-Galactan. Plant Physiol. 1997;113(4):1405–12. Willats WGT, Marcus SE, Knox JP. Generation of a monoclonal antibody specific to (1→5)-α-l-arabinan. Carbohydrate Research. 1998;308(1):149–52. Ralet MC, Tranquet O, Poulain D, Moïse A, Guillon F. Monoclonal antibodies to rhamnogalacturonan I backbone. Planta. 2010;231(6):1373–83. Pettolino FA, Hoogenraad NJ, Ferguson C, Bacic A, Johnson E, Stone BA. A (1–>4)-beta-mannan-specific monoclonal antibody and its use in the immunocytochemical location of galactomannans. Planta. 2001;214(2):235–42. Marcus SE, Blake AW, Benians TAS, Lee KJD, Poyser C, Donaldson L, et al. Restricted access of proteins to mannan polysaccharides in intact plant cell walls. Plant J. 2010;64(2):191–203. Pattathil S, Avci U, Baldwin D, Swennes AG, McGill JA, Popper Z, et al. A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies. Plant Physiol. 2010 June;153(2):514–25. Zhang X, Rogowski A, Zhao L, Hahn MG, Avci U, Knox JP, et al. Understanding How the Complex Molecular Architecture of Mannan-degrading Hydrolases Contributes to Plant Cell Wall Degradation*. Journal of Biological Chemistry. 2014;289(4):2002–12. Ruprecht C, Bartetzko MP, Senf D, Dallabernadina P, Boos I, Andersen MCF, et al. A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies. Plant Physiol. 2017;175(3):1094–104. Peralta AG, Venkatachalam S, Stone SC, Pattathil S. Xylan epitope profiling: an enhanced approach to study organ development-dependent changes in xylan structure, biosynthesis, and deposition in plant cell walls. Biotechnology for Biofuels. 2017;10(1):245. Moller I, Marcus SE, Haeger A, Verhertbruggen Y, Verhoef R, Schols H, et al. High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles. Glycoconj J. 2008;25(1):37–48. Pennell RI, Janniche L, Kjellbom P, Scofield GN, Peart JM, Roberts K. Developmental Regulation of a Plasma Membrane Arabinogalactan Protein Epitope in Oilseed Rape Flowers. Plant Cell. 1991;3(12):1317–26. Yates EA, Valdor JF, Haslam SM, Morris HR, Dell A, Mackie W, et al. Characterization of carbohydrate structural features recognized by anti-arabinogalactan-protein monoclonal antibodies. Glycobiology. 1996;6(2):131–9. Pennell RI, Knox JP, Scofield GN, Selvendran RR, Roberts K. A family of abundant plasma membrane-associated glycoproteins related to the arabinogalactan proteins is unique to flowering plants. J Cell Biol. 1989;108(5):1967–77. Marcus SE, Verhertbruggen Y, Hervé C, Ordaz-Ortiz JJ, Farkas V, Pedersen HL, et al. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls. BMC Plant Biology. 2008;8(1):60. Pedersen HL, Fangel JU, McCleary B, Ruzanski C, Rydahl MG, Ralet MC, et al. Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research. J Biol Chem. 2012;287(47):39429–38. Blake AW, McCartney L, Flint JE, Bolam DN, Boraston AB, Gilbert HJ, et al. Understanding the Biological Rationale for the Diversity of Cellulose-directed Carbohydrate-binding Modules in Prokaryotic Enzymes *. Journal of Biological Chemistry. 2006 Sept 29;281(39):29321–9. Yang X, Reid MS, Olsén P, Berglund LA. Eco-Friendly Cellulose Nanofibrils Designed by Nature: Effects from Preserving Native State. ACS Nano. 2020;14(1):724–35. Perzon A, Kračun SK, Jørgensen B, Ulvskov P. Array-based microfibril surface assessment (AMSA): a method for probing surface-exposed polysaccharides on cellulose nanofibres. Cellulose. 2020;27(15):8635–51. Dong J, Jiang W, Gao P, Yang T, Zhang W, Huangfu M, et al. Comparison of betalain compounds in two Beta vulgaris var. cicla and BvCYP76AD27 function identification in betalain biosynthesis. Plant Physiology and Biochemistry. 2023 June 1;199:107711. Kumar R, Hu F, Hubbell CA, Ragauskas AJ, Wyman CE. Comparison of laboratory delignification methods, their selectivity, and impacts on physiochemical characteristics of cellulosic biomass. Bioresour Technol. 2013;130:372–81. Yang X, Berthold F, Berglund LA. Preserving Cellulose Structure: Delignified Wood Fibers for Paper Structures of High Strength and Transparency. Biomacromolecules. 2018 July 9;19(7):3020–9. Hatfield RD, Grabber J, Ralph J, Brei K. Using the Acetyl Bromide Assay To Determine Lignin Concentrations in Herbaceous Plants: Some Cautionary Notes. J Agric Food Chem. 1999;47(2):628–32. Colquhoun IJ, Ralet MC, Thibault JF, Faulds CB, Williamson G. Structure identification of feruloylated oligosaccharides from sugar-beet pulp by NMR spectroscopy. Carbohydrate Research. 1994;263(2):243–56. Mathew S, Abraham TE. Ferulic Acid: An Antioxidant Found Naturally in Plant Cell Walls and Feruloyl Esterases Involved in its Release and Their Applications. Critical Reviews in Biotechnology. 2004;24(2–3):59–83. Saeidy S, Omidi P, Nasirpour A, Keramat J. Physicochemical and functional properties of cross linked and high pressure homogenized sugar beet pectin: A comparative study. Food Hydrocolloids. 2023;134:108041. Pan GX, Spencer L, Leary GJ. Reactivity of Ferulic Acid and Its Derivatives toward Hydrogen Peroxide and Peracetic Acid. J Agric Food Chem. 1999;47(8):3325–31. Stojkov G, Niyazov Z, Picchioni F, Bose RK. Relationship between Structure and Rheology of Hydrogels for Various Applications. Gels. 2021;7(4):255. Li MC, Wu Q, Song K, Lee S, Qing Y, Wu Y. Cellulose Nanoparticles: Structure–Morphology–Rheology Relationships. ACS Sustainable Chem Eng. 2015;3(5):821–32. Xu J, Wang P, Yuan B, Zhang H. Rheology of cellulose nanocrystal and nanofibril suspensions. Carbohydrate Polymers. 2024;324:121527. Li S, Zhao G, Chen H. The Relationship between Steady Shear Viscosity and Complex Viscosity. Journal of Dispersion Science and Technology. 2005 July 1;26(4):415–9. Lunardi CN, Gomes AJ, Rocha FS, De Tommaso J, Patience GS. Experimental methods in chemical engineering: Zeta potential. The Canadian Journal of Chemical Engineering. 2021;99(3):627–39. Boluk Y, Lahiji R, Zhao L, McDermott MT. Suspension viscosities and shape parameter of cellulose nanocrystals (CNC). Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2011;377(1):297–303. Pinkl S, Veigel S, Colson J, Gindl-Altmutter W. Nanopaper Properties and Adhesive Performance of Microfibrillated Cellulose from Different (Ligno-)Cellulosic Raw Materials. Polymers. 2017;9(8):326. Wei L, Bian H, Agarwal UP, Sabo RC, Matuana LM, Stark NM. Correlation between morphology and performance of cellulose nanofibril-based films. Current Research in Green and Sustainable Chemistry. 2023;6:100363. Almeida R, Ramos A, Håkonsen V, Maloney T, Gamelas J. Functionalized cellulose nanofiber films as potential substitutes for Japanese paper. Carbohydrate Polymer Technologies and Applications. 2024;8:100573. Trovagunta R, Zou T, Österberg M, Kelley SS, Lavoine N. Design strategies, properties and applications of cellulose nanomaterials-enhanced products with residual, technical or nanoscale lignin—A review. Carbohydrate Polymers. 2021;254:117480. Xu J, Sagnelli D, Faisal M, Perzon A, Taresco V, Mais M, et al. Amylose/cellulose nanofiber composites for all-natural, fully biodegradable and flexible bioplastics. Carbohydr Polym. 2021;253:117277. Churchill DG. Chemical Structure and Accidental Explosion Risk in the Research Laboratory. J Chem Educ. 2006;83(12):1798. Joint FAO/WHO Expert Committee on Food Additives. Meeting. 73th, World Health Organization. Safety evaluation of certain food additives and contaminants: prepared by the Seventy-third meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). 2011 [cited 2025 June 26]; Available from: https://iris.who.int/handle/10665/44521 Pechacek N, Osorio M, Caudill J, Peterson B. Evaluation of the toxicity data for peracetic acid in deriving occupational exposure limits: A minireview. Toxicology Letters. 2015;233(1):45–57. Shafie AE, Fouda MMG, Hashem M. One-step process for bio-scouring and peracetic acid bleaching of cotton fabric. Carbohydrate Polymers. 2009 Sept 5;78(2):302–8. Dinand E, Chanzy H, Vignon RM. Suspensions of cellulose microfibrils from sugar beet pulp. Food Hydrocolloids. 1999;13(3):275–83. Additional Declarations No competing interests reported. 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09:56:52","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166739,"visible":true,"origin":"","legend":"","description":"","filename":"f6aa855a7ae242839e05fb1592c4a2df1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/b2ee36637db03e058bff5217.xml"},{"id":96939397,"identity":"a9d732e5-b31d-40e1-ab86-ba6ee642c20f","added_by":"auto","created_at":"2025-11-27 17:30:09","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":177704,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/0d60ccbe1cb1b3ac32946e71.html"},{"id":96939375,"identity":"e5a80cff-5655-4e63-9149-8f9093312c0b","added_by":"auto","created_at":"2025-11-27 17:30:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102773,"visible":true,"origin":"","legend":"\u003cp\u003eVisualisation of how the whiteness index was calculated for each oxidised pulp sample. The raw image was put into ImageJ FIJI software where the image was converted into 8-bit grey scale. The absolute brightness, also known as the pixel index value, was measured in triplicates and the average number was calculated. The average number was then converted into 0 to 100 % whiteness, i.e. the average number was divided by 255.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/10cfda1acb1f07b084e7171f.png"},{"id":97248886,"identity":"e1cd7fd1-b4c7-4a20-85b3-1a5346c557b3","added_by":"auto","created_at":"2025-12-02 13:07:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":171542,"visible":true,"origin":"","legend":"\u003cp\u003eThe surface availability of ferulates measured by LM12 antibody binding in AMSA. The error bars represent the standard deviation of three replicates. SBP = sugar beet pulp, SBPe = Enzyme-treated sugar beet pulp CNF, SC-CNF = sodium chlorite bleached CNF, PAA-SBPe = peracetic acid bleached CNF, and HP.F = Fenton catalysed hydrogen peroxide bleached CNF.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/c58fb997b9173984bf07407e.png"},{"id":96939378,"identity":"8d5cb787-d11b-4e56-8148-bfb8655fa24b","added_by":"auto","created_at":"2025-11-27 17:30:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":198176,"visible":true,"origin":"","legend":"\u003cp\u003eThe rheology of the differently oxidised CNF suspensions. (A) The storage modulus (G’) of the different suspensions. (B) The loss modulus modulus (G’’) of the suspensions. (C) The viscosity of the suspensions. SBPe-CNF = Enzyme-treated sugar beet pulp CNF, SC-SBPe = sodium chlorite bleached SBPe, PAA-SBPe = peracetic acid bleached SBPe, and HP.F = Fenton catalysed hydrogen peroxide bleached SBPe.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/a4a47bcafdc4c8cfc707463a.png"},{"id":96939376,"identity":"55c20ec5-f065-4ca4-8bff-7d6670b8b4ed","added_by":"auto","created_at":"2025-11-27 17:30:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":536215,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM imagery of sodium chlorite and peracetic acid CNF films. (a) The topology and cross section (XC) of the films. SC = sodium chlorite bleached CNF films, and PAA = peracetic acid bleached CNF films.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/78923f4dea782258a6d5f142.png"},{"id":96939382,"identity":"0036fb82-10c2-42f9-9123-ed5ba49c8c21","added_by":"auto","created_at":"2025-11-27 17:30:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":214733,"visible":true,"origin":"","legend":"\u003cp\u003eViscosity and available surface ferulates of in situ generation of peracetic CNF samples. (A) The viscosity of glucose oxidase by itself, or together with TAED or triacetin. (B) The viscosity of SPC by itself, or together with TAED or triacetin. (C) The available surface detection of ferulates of the samples, measured by the antibody LM12 detection. GOx = glucose oxidase, TAED = Tetraacetylethylenediamine, TriA = triacetin, and SPC = sodium percarbonate.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/5820a0684772431c0603f7e6.png"},{"id":97664726,"identity":"033a59a9-755c-417b-876b-5cd5f0d815bc","added_by":"auto","created_at":"2025-12-08 09:13:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2402092,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/4a52180c-69d8-4591-9782-39d8f41cc3ca.pdf"},{"id":96939384,"identity":"4c4202cb-ffcd-4ea5-b5f3-c459aa6da26a","added_by":"auto","created_at":"2025-11-27 17:30:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":366733,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/b9c91992a457b4f8d6d28b64.docx"},{"id":97135834,"identity":"044ca688-c6cb-4539-bc7d-9178ddb6a228","added_by":"auto","created_at":"2025-12-01 09:53:57","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":156528,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7917067/v1/d601d069a20652e2c2822702.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Totally-Chlorine-Free Bleaching Alternatives to Produce Sugar Beet Pulp Cellulose Nanofibers","fulltext":[{"header":"2\tBackground","content":"\u003cp\u003eSugar beet pulp (SBP), a by-product of sugar production, is an abundant cellulose-rich, low-lignin biomass that has potential for value-added products in biorefineries. SBP consists, on a dry weight basis, of approximately 60 to 80% of polysaccharides, where 40% is cellulose, 30% hemicellulose, and 30% pectin. The two other major components are protein and lignin, which constitutes 9 and 4-6%, respectively.\u0026nbsp;(1,2). Of this low-lignin biomass, the main phenolic components are gallic acid, epicatechin, and quercetin-3-O-rutinoside\u0026nbsp;(3). In addition, ferulic acid decorates the arabinan and galactan side chains of pectin\u0026nbsp;(4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCellulose, the main polymer in SBP, can be made into cellulose nanofibers (CNF) that can be used as thickeners, emulsion stabilisers, fillers in papers and biocomposites, among other applications\u0026nbsp;(5). There are several methods to produce CNFs from SBP, with the differences being in pretreatments and methodology\u0026nbsp;(6–8). An enzymatic approach published by Perzon \u003cem\u003eet al.\u003c/em\u003e (2020) starts by swelling the SBP in sodium hydroxide, followed by digestion with mainly pectinases and hemicellulases, to be oxidised by sodium chlorite (SC), to lastly, being microfluidised. The oxidation, also known as bleaching, should remove lignin and molecules that are coloured or give rise to discoloration\u0026nbsp;(9), and also helps with fibrillation\u0026nbsp;(10). However, the use of chlorite is problematic due to production of chlorine gas which is why totally-free chlorine (TCF) methods, among other reasons, have been developed.\u003c/p\u003e\n\u003cp\u003eAmong several different TCF methods, three common ones are the use of peracetic acid (PAA)\u0026nbsp;(8), hydrogen peroxide (HP)\u0026nbsp;(2), and hydroxyl radicals\u0026nbsp;(11). Peracetic acid in solution creates a mixture of peracetic acid, acetic acid and hydrogen peroxide\u0026nbsp;(12). Hydrogen peroxide oxidises ester bonds, introducing hydroxyl groups that can further be oxidised into carboxyl group like aldehydes and ketones\u0026nbsp;(13,14). Hydrogen peroxide, in the presence of a catalyst like Fe\u003csup\u003e2+\u003c/sup\u003e or Cu\u003csup\u003e+\u003c/sup\u003e, breaks down into hydroxyl radicals. This is also known as the Fenton reaction. Hydroxyl radicals can oxidise glycosidic bonds, both in pectin and hemicellulose\u0026nbsp;(15,16)\u0026nbsp;and can create peroxyl radicals in the presents of polysaccharides that cleave low-molecular polymers\u0026nbsp;(17). Notably, the energy needed to cleave peracetic acid is lower than hydrogen peroxide, which is why hydroxyl radicals are easier created from peracetic acid than hydrogen peroxide\u0026nbsp;(18). Nonetheless, hydroxyl radicals have a brief half-life and can only travel roughly the equivalent of two glucose residues in a cellulose chain\u0026nbsp;(19).\u0026nbsp;In comparison to peracetic acid and hydroxyl radical bleaching, sodium chlorite targets the phenolic groups, or the methyl and methylene groups in allylic position. Chlorite also oxidises the reducing ends of polysaccharides.\u0026nbsp;(20)\u003c/p\u003e\n\u003cp\u003eIt is also possible to generate the oxidising agent peracetic acid \u003cem\u003ein situ\u003c/em\u003e. This is done by the usage of hydrogen peroxide and a hydrogen peroxide activator. The activators react with the hydrogen peroxide to produce peracids, which is favourable due to the peracids higher redox potential and stronger oxidising properties. Several activators have been researched and used, and two of them are tetraacetylethylenediamine (TAED) and triacetin. TAED is widely used in industrial processes but is more complex to synthesise and has lower solubility than triacetin.\u0026nbsp;(21,22)\u0026nbsp;Sodium percarbonate (SPC) can be used instead of pure hydrogen peroxide to generate peracid \u003cem\u003ein situ\u0026nbsp;\u003c/em\u003ewith TAED or triacetate. In water, SPC slowly decompose into hydrogen peroxide. This creates a steady process of peracids instead of the short-lived reactive species hydrogen peroxide produces. Additionally, TAED reacts better in slight alkaline solutions, which SPC can offer.\u0026nbsp;(21)\u0026nbsp;Peracetic acid is also possible to create enzymatically \u003cem\u003ein situ\u003c/em\u003e. While keeping the hydrogen peroxide activator in the solution, hydrogen peroxide is replaced with glucose oxidase, EC 1.1.3.4. Glucose oxidase catalyses the conversion of glucose to gluconic acid, whilst reducing molecular oxygen to hydrogen peroxide\u0026nbsp;(23). Therefore, in the presents of SBP where glucose is available, glucose oxidase should generate small amount of hydrogen peroxide. This concept has been shown to work in previous studies of bleaching of cotton\u0026nbsp;(24,25).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this work to improve our previous CNF production method from SBP\u0026nbsp;(7,26), \u0026nbsp;it is hypothesised that sodium chlorite can be replaced with an environmentally friendly oxygen-based bleaching step, with little to no comparative loss in whitening or physical properties. To investigate this, peracetic acid and hydrogen peroxide, as a peroxyl and free-radical oxidants respectively, were compared to sodium chlorite oxidation for the production of CNF suspensions and their subsequent casted films. The bleaching of CNF was equal for peracetic acid and sodium chlorite; however, hydrogen peroxide darkened the suspension. This did not seem to affect the viscosity, where the three suspensions were similar. However, it could be seen that the elongation before the breakpoint of the film, where peracetic acid bleached CNF was used, decreased. Lastly, based on the above-mentioned findings, \u003cem\u003ein situ\u003c/em\u003e generation of peracetic acid, both chemically and enzymatically, was tested and showed to bleach the CNF to the same whiteness as exogenously supplied peracetic acid.\u0026nbsp;\u003c/p\u003e"},{"header":"3 Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Sugar Beet Pulp\u003c/h2\u003e\u003cp\u003eFresh post-sugar extraction sugar beet, in pieces of 1\u0026ndash;2 cm, was obtained from Nordic Sugar and stored at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Materials\u003c/h2\u003e\u003cp\u003eAll chemicals, both powders and liquids, and glucose oxidase (from \u003cem\u003eAspergillus niger\u003c/em\u003e) were purchased from Sigma-Aldrich. The enzymes used in section 3.3.1 were kindly provided by Novozymes (Denmark). The antibodies used in 3.4.4 were either bought or given. The LM and JIM antibodies including MAC207 were donated from Plant Probes, Paul Knox lab, the INRA- RUI and INRA-RUII antibodies donated from INRA (France), and the CCRS, BS-400-4 and CBM3a antibodies were bought from Carbosource, University of Georgia (USA), Biosupplies (Australia) and NZYtech (Portugal), respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Cellulose Nanofiber Production Method\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1 Enzyme-treated Sugar Beet Pulp\u003c/h2\u003e\u003cp\u003eThe production of enzyme-treated SBP is adapted from Perzon \u003cem\u003eet al\u003c/em\u003e. protocol (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). 20 grams (dry weight) sugar beet pulp cossettes were dispersed in 2.5 L of distilled water and subsequently homogenised (Silverson L5M homogenizer fitted with the General Purpose Disintegrating Head) for 30 minutes at 6000 RPM. The pulp was filtered on Miracloth and added to a beaker together with water to a total volume of 500 ml. The pH was raised to pH 9 by the addition of 8 ml 4 M sodium hydroxide. The suspension was heated at 80\u0026deg;C for two hours with mechanical stirring. Afterwards, the reaction was cooled to 40\u0026deg;C. When the temperature was cooled down, 1.5 ml of 12.1 M glacial acetic acid was added under mixing to reach pH 5. Enzymes, according to Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, were added and the reaction was incubated at 40\u0026deg;C for 2 hours with mechanical stirring. If no bleaching method was applied, the enzymes were inactivated by heating at 80\u0026deg;C for 15 minutes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIdentity, activity and volume of enzymes applied to sugar beet pulp for nanocellulose production.\u003c/p\u003e \u003cdiv class=\"Credit\"\u003e\u003cp\u003eAdapted from (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). FBG\u0026thinsp;=\u0026thinsp;Fungal Beta-Glucanase Units, AXU\u0026thinsp;=\u0026thinsp;Endo Xylanase Units, ECU\u0026thinsp;=\u0026thinsp;endo cellulase units, PGNU\u0026thinsp;=\u0026thinsp;Polygalacturonase units, KNU\u0026thinsp;=\u0026thinsp;Kilo Novo Units alpha-amylase units, all units as specified by Novozymes.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProduct\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMain enzyme activity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDeclared activity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVolume of enzyme solution / gram dry mass SBP (\u0026micro;L / g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eViscozyme L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBeta-glucanase (endo-1,3(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)-β-glucanase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100 FBG/g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePectinex Ultra Clear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePolygalacturonase (endo-1,4-α-galacturonidase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7900 PGNU/ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePulpzyme HC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEndo-xylanase (endo-1,4-β-xylanase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1000 AXU/g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFiberCare R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCellulase (endo-1,4-β-glucanase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4500 ECU/g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAquazym 240 L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlpha-amylase (endo-1,4-α-glucanase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e240 KNU/g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2 Bleaching with sodium chlorite, peracetic acid or hydrogen peroxide\u003c/h2\u003e\u003cp\u003eThe enzyme-treated SBP suspension was heated to 70\u0026deg;C. To the suspension the oxidant was added, up to a concentration of 100 mM, and the reaction was incubated for 2 hours with mechanical stirring. The different oxidants used were 2.8 M sodium chlorite, 5.1 M peracetic acid, or 8.8 M hydrogen peroxide with 1 mM iron chloride. Afterwards, pulp was collected on Miracloth and washed thoroughly with deionised water. Normally, the amount of deionised water was three times the volume of the reaction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e3.3.3 Bleaching via peracetic acid activators combined with a source of hydrogen peroxide\u003c/h2\u003e\u003cp\u003eFor the sodium percarbonate reaction, sodium percarbonate was added to final concentration of 100 mM in 500 ml of 4% (w/v) SBP suspension in water. Glacial acetic acid was added to obtain pH 10. For the glucose oxidase reaction, 5 mg of the enzyme (Glucose Oxidase from \u003cem\u003eAspergillus niger\u003c/em\u003e, Sigma Aldrich, 100,000-250,000 units/g solid) was added to 500 ml of 4% (w/v) SBP suspension in 0.1 M sodium phosphate, pH 7. Either tetraacetylethylenediamine (TAED) or triacetin was added to a concentration of 50 mM or 33 mM, respectively, to equate 100 mM peracetic acid. The sodium percarbonate mixture was incubated at 70\u0026deg;C for 2 hours while the glucose oxidase reaction was incubated at 35\u0026deg;C for 12 h, both with mechanical stirring. After the incubation, the pulp was collected on Miracloth and washed thoroughly with deionised water, approximately three times the mixture volume.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e3.3.4 Microfluidisation\u003c/h2\u003e\u003cp\u003eThe enzyme-treated and bleached pulps were resuspended to 1% (w/v) in distilled water with 0.05% (w/v) chlorobutanol as a bacteriostatic agent. The suspensions were microfluidised (Microfluidizer materials processor M110-P) at 600 bars, with a set-up of two chambers of 400 \u0026micro;m followed by 200 \u0026micro;m. The suspensions were passed six times though the system to obtain cellulose nanofibers (CNF). The dry content of the pulp was analysed with a moisture analyser (Kern DLB moisture analyser).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e3.3.5 Film Preparation\u003c/h2\u003e\u003cp\u003e25 ml of 0.5% (w/v) CNF suspension was spread in polystyrene petri dish (\u0026Oslash; 85 mm) and slowly evaporated at 35\u0026deg;C for 72 hours until completely dry. To ensure uniform moisture content and avoid film shrinkage, all films were kept in airtight sealed desiccator containing saturated sodium bromide, maintaining a relative humidity of 60% at 20\u0026deg;C prior to analysis.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Analysis Methods\u003c/h2\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1 Whiteness Index\u003c/h2\u003e\u003cp\u003eThe whitening of the oxidised CNF was analysed with ImageJ FIJI software (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For each sample, a photo was taken and processed into an 8-bit grey-scale image. The software measures absolute brightness from a scale of 0 to 255, where zero refers to black and 255 refers to white. Several points in each photo were analysed, and the average of the absolute brightness of each sample was converted into an index scale from 0\u0026ndash;100% as a measure of whiteness.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2 Rheological Measurements\u003c/h2\u003e\u003cp\u003eThe rheology of the CNF suspensions was measured with a rheometer (TA Discovery HR-3) with a 40 mm cone plate (0.9767\u0026deg; angle) with a 22 \u0026micro;m gap. 670 \u0026micro;l of 0.1 to 0.4% (w/w) CNF suspensions was added to plate. The oscillation sweep was measure from 0.1% to 100% at 1 Hz, the shear sweep was measured from 0.11 to 1000 Hz, and the frequency sweep was measured at 0.1% strain from 0.01 to 100 Hz. All measurements were performed at 25\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.4.3 Lignin Content\u003c/h2\u003e\u003cp\u003eThe lignin content of the samples was determined with the acetyl bromide assay, adapted from Gui et al (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). 1 mg of dried biomass was placed in a 2 ml screwcap Sarstedt tube. 100 \u0026micro;l of 25% (v/v) acetyl bromide, diluted in glacial acetic acid, was added and the samples incubated for 3 hours at 50\u0026deg;C and 600 rpm. Afterwards, the samples were cooled on ice to room temperature. 400 \u0026micro;l of 2 M sodium hydroxide was added followed by 70 \u0026micro;l of 0.5 M hydroxylamine hydrochloride. The samples were vortexed, followed by addition of 1.43 ml of glacial acetic acid and vortexed again. The samples were centrifuged at 13,000 rpm for 3 minutes and 200 \u0026micro;l of the supernatant of samples was transferred to an UV-transparent 96-well plate. The absorbance was read at 280 nm in a spectrophotometer (Spectromax 190, Molecular Devices, USA) and the concentrations were calculated from a calibration curve of alkali lignin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.4.4 Array-based Microfibril Surface Assessment\u003c/h2\u003e\u003cp\u003eThe surface compositions of the CNF suspensions were analysed with the array-based microfibril surface assessment (AMSA) procedure, as previously performed in Perzon et al. (2020). The samples were spotted with a robot (synQUAD Pixsys, Digilab Inc.) in arrays. The samples were done in four replicates, in eight dilutions. The arrays were blocked in 5% fat-free milk protein in phosphate-buffered saline (PBS), followed by being probed with primary monoclonal antibodies (mAb) according to Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e in 5% fat-free milk protein in phosphate-buffered saline. The arrays were washed in PBS, followed by being probed with secondary mAbs. The arrays were washed in PBS, then water, before being developed in 0.02% 5-bromo-4-chloro-3-indolylphosphate (BCIP) and 0.03% nitro-blue tetrazolium chloride (NBT) in 100 mM tris pH 9.5 with 100 mM sodium chloride and 5 mM magnesium chloride. After development, the arrays were scanned at 1200 dpi, loaded into ArrayProAnalyzer version 6.3.1 software for detection and relative quantification of the composition of the samples.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of the used primary monoclonal antibodies and their target epitopes, together with their references. Primary antibodies were used at concentrations ranging from 1\u0026ndash;50 mM) \u003csup\u003e\u0026dagger;\u003c/sup\u003e XGL nomenclature of describing xyloglucan substructures as developed by Fry et al. (1993)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntibody\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEpitope\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHomogalacturonanon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHomogalacturonanon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eβ-1,4-galactan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eα-1,5-arabinan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eINRA-RU1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRhamnogalacturonan-I\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eINRA-RU2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRhamnogalacturonan-I\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBS-400-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(1\u0026rarr;4)-β-mannan/galacto-(1\u0026rarr;4)-β-mannan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHeteromannan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGlucomannan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCCRC-M167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGalactomannan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCCRC-M170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGalactomannan: Acetylated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCCRC-M175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)-β-D-xylosyl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCCRC-M145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXylan: 4-O-MeGlcA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCCRC-M147\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eβ-1,4-linked xylosyl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)-β-D-xylosyl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGlucuronosyl\u0026nbsp;xylan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArabinogalactan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJIM8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArabinogalactan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJIM13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArabinogalactan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMAC207\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArabinogalactan\u0026nbsp;w.\u0026nbsp;GlcA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXXXG\u0026nbsp;(xyloglucan) \u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXLLG,\u0026nbsp;XXLG,\u0026nbsp;XXXG (xyloglucan) \u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCBM3a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCellulose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLM12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFerulates\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.4.5 Electronic Stability of Suspension\u003c/h2\u003e\u003cp\u003eThe zeta potential of the CNF suspensions was measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) equipped with a DTS1070 unit cell and analysed with Malvern ZetaSizer software. The suspensions were prepared at 0.1% (w/v) in deionised water. The measurements were taken at 25\u0026deg;C with an angle of 17\u0026deg;. Each sample was recorded as three technical replicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.4.6 Scanning Electron Microscopy\u003c/h2\u003e\u003cp\u003eThe CNF films were visualised with field-emission scanning electron microscopy (FE-SEM) by a Quanta 3D FEG (FEI company, Netherlands). For imaging of the surface, a 1x1 cm\u003csup\u003e2\u003c/sup\u003e square of the film was cut out, placed on a metal stud, and coated with 2 nm layer of gold. To image the cross-section, a strip of the CNF film was frozen in liquid nitrogen for 30 seconds and snapped in half. The broken edge was attached to the metal plate facing upwards and coated with 2 nm layer of gold.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.4.7 Mechanical Testing\u003c/h2\u003e\u003cp\u003eThe Young\u0026rsquo;s modulus and tensile strength of the films were tested with a TA-XT Texture Analyser (TTC company) with a grip accessory and 50 kg load cell. Films were cut into 5x0.4 cm\u003csup\u003e2\u003c/sup\u003e strips, placed in the instrument and the analysis was performed at a cross-head speed of 4 mm/min. Relative humidity was kept at 60% at a temperature of 22\u0026deg;C. Young\u0026rsquo;s modulus, the tensile strength, and the strain were calculated from the obtained stress-strain curves. The results were derived from 6\u0026ndash;10 replicates per film.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4 Results and Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Sugar beet pulp nanofibers\u003c/h2\u003e\u003cp\u003eThe method of Cellulose nanofiber production from SBP as published by Perzon et al. (2020) which this paper further develops, follows two key steps: matrix polysaccharide removal (chemically or enzymatically) followed by an oxidation step. The enzyme mediated removal of matrix polysaccharides was accomplished with a mild alkaline swelling step followed by digestion with enzymes active on pectic and hemicellulosic polysaccharides. This process has been reported before and the material thoroughly characterised. Removal of matrix polysaccharides is intentionally not exhaustive as a residual coating of the nanofibers is required for colloidal stability (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Based on quantification of trifluoroacetic acid soluble monosaccharides, the material is ~\u0026thinsp;85% cellulose (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The polymers that could be extracted from the CNF were found to comprise most notably of extensin, xyloglucan, mannan and rhamnogalacturonan-I (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Direct detection of glycans on the CNF corroborated these observations and demonstrated that different populations of mannans exist, one of which is very strongly adhering to the nanofiber surface (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). This product constitutes starting material for investigations of oxidation methods alternative to chlorite. The oxidising effects of peracetic acid, hydrogen peroxide and hydroxyl radicals on sugar beet pulp CNF were evaluated by the whitening of the pulp, lignin removal, polysaccharide composition, rheology, and suspension stability. The oxidising agents were compared to sodium chlorite to evaluate the potential in chlorine-free bleaching.\u003c/p\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e4.1.1 Whitening of the pulp\u003c/h2\u003e\u003cp\u003eThe starting substrate, the enzyme treated sugar beet pulp (SBPe), was visibly brown with a measured whiteness index (WI) of 59% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Sodium chlorite oxidised the SBPe to a pale white colour with a WI of 68%. Peracetic acid was more effective than hydrogen peroxide in whitening the material with a WI of 67% and 63%, respectively. However, none of the oxidising agents could exceed the sodium chlorite bleached pulp.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe whiteness index of the solids of the sugar beet pulp suspensions, calculated from triplicates. SBPe\u0026thinsp;=\u0026thinsp;Enzyme-treated sugar beet pulp, SC\u0026thinsp;=\u0026thinsp;sodium chlorite bleached pulp, PAA\u0026thinsp;=\u0026thinsp;peracetic acid bleached pulp, and HP\u0026thinsp;=\u0026thinsp;hydrogen peroxide bleached pulp.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eWhiteness index (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eUncatalysed\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eCatalysed with FeCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSBPe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e53.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e67.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e69.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e67.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e46.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e62.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the free radical bleaching, the same oxidising agents were tried with the catalytic presence of iron ions (Fe\u003csup\u003e2+\u003c/sup\u003e), to initiate the Fenton reaction. In general, the bleaching effect seems to be reversed, with a darkening of the pulp. The SBPe with the catalyst had a WI of 54%, while the sodium chlorite bleached suspension had a slight increase, compared to without the catalyst, of the WI to 70%. The chlorite-free oxidising agents together with the catalyst darkened the pulp considerably to a WI of 46% and 39%, respectively.\u003c/p\u003e\u003cp\u003eWhile there is no strict correlation between whitening and lignin removal, oxidation is believed to remove both lignin and chromophoric groups, resulting in a whitening of materials. SBP is primarily formed from the roots of \u003cem\u003eBeta vulgaris\u003c/em\u003e, the colour of which is formed from the type and ratio of betalins (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). It has been shown that peracetic acid is more selective towards lignin than sodium chlorite across a wide range of substrate (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), but this seems not to influence the whitening of SBP in this study. Furthermore, free radicals are known to react non-selectively due to their high-energy state. It is possible the free radicals through the Fenton reaction does not degrade colour-giving compounds but oxidises other compounds that leads to darkening of the pulp.\u003c/p\u003e\u003cp\u003eFrom this initial assessment, the uncatalyzed PAA and SC together with the catalysed HP oxidised pulps were chosen to be analysed further, since these represented the whitest and darkest pulps respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e4.1.2 Lignin Content\u003c/h2\u003e\u003cp\u003eTo investigate the hypothesis of bleaching correlating with lignin removal, we performed an acetyl bromide assay to measure the total lignin content of these oxidised samples (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe lignin content of sugar beet pulp suspensions and availability of ferulates of the SBP CNF suspensions. The lignin content of each sample measured by the acetyl-bromide assay. The content is the average\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three replicates. SBP\u0026thinsp;=\u0026thinsp;sugar beet pulp, SBPe\u0026thinsp;=\u0026thinsp;Enzyme-treated sugar beet pulp CNF, SC-CNF\u0026thinsp;=\u0026thinsp;sodium chlorite bleached CNF, PAA-SBPe\u0026thinsp;=\u0026thinsp;peracetic acid bleached CNF, and HP.F\u0026thinsp;=\u0026thinsp;Fenton catalysed hydrogen peroxide bleached CNF.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLignin content (% of dry mass)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSBP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSBPe-CNF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSC-CNF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePAA-CNF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHP.F-CNF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe raw biomass material, SBP, already has a low total lignin content (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) of 6.2% to begin with. After enzymatic treatment (SBPe), the lignin content decreases to 4.4%. It is possible that the lignin co-solubilises with the digested hemicellulose or pectin, but it is more likely the decrease should be attributed to the loss of xylan. It has been shown before that starch and xylan can increase the measured lignin content (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). The oxidations applied did not seem to cause delignification; SC-CNF was not significantly different from SBPe-CNF, and the other oxidants PAA and HP.F actually resulted in CNFs with higher measured levels of lignin after oxidation. This could be due to the oxidation and solubilisation of the other cell-wall components, seen previously with hydrogen peroxide oxidation (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e4.1.3 Surface availability of ferulates\u003c/h2\u003e\u003cp\u003eLM12, an antibody that binds to ferulic residues (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), was used to estimate the phenolic availability of the surfaces of the oxidised SBP CNFs. Typically SBP would contain high amounts of ferulic acid in its pectin fraction (\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Due to the pre-enzymatic digestion step before any oxidation, the vast majority of surface-available pectin and hemicellulose were removed (Additional file 1). This explains why the availability of ferulates decrease between the starting material SBP to the SBPe (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After oxidation of SBPe, there is a significant increase in LM12 binding and hence surface-available ferulates. Hydrogen peroxide bleached CNF (HP.F-CNF) had the highest surface availability of ferulates, up to almost twice of the availability of SBP, and in turn a near twenty-time fold increase in comparison to SBPe CNF. Sodium chlorite and peracetic acid bleached CNFs did not have the same high availability as HP-F-CNF, but they did increase the availability of ferulates by five-fold and almost three-fold compared to SBPe CNF.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhether this increase, or lack thereof, in LM12 binding to the oxidised samples compared to SBPe-CNF accurately portrays the oxidising effect of the oxidants is unclear. Whilst an increase in LM12 binding levels indicates that ferulates have become exposed due to oxidation, it is also true that a smaller increase does not preclude that that the oxidant was less effective. It could also mean that ferulates are being exposed and then solubilised by the oxidation. Ferulic acid has been previously shown to be more resistant to HP than PAA (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) which suggests that the small increase of LM12 binding to PAA-CNF means that the ferulic acid has been solubilised.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e4.1.4 Rheology of Suspensions\u003c/h2\u003e\u003cp\u003eThe physical properties of the oxidised materials were evaluated by their rheological properties in response to a frequency sweep and shear sweep. An ideal oxidant replacement for SC would create a CNF suspension of equal or higher viscosity.\u003c/p\u003e\u003cp\u003eThe lack of crossover in the storage modulus (G\u0026rsquo;) and loss modulus (G\u0026rsquo;\u0026rsquo;) of the frequency sweep (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) in any of the samples demonstrated that they were all cross-linked gels (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e), which was expected. This behaviour has been previously observed in both unbleached CNF suspensions (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e) and the previous reported SC-CNF (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Only the PAA-CNF shows a higher recorded G\u0026rsquo;, indicating that it forms a stronger gel. SC-CNF in general is the most similar to the unbleached CNF in both G\u0026rsquo; and G\u0026rsquo;\u0026rsquo;. Finally, Fenton catalysed HP.F-CNF has the weakest G\u0026rsquo; and G\u0026rsquo;\u0026rsquo; out of the set, which is ten times lower than PAA-CNF, implying a weakly formed gel.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese observations mostly related to what can be observed in the shear sweep tests of the samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), even if complex and shear viscosities are not typically directly comparable at high shear rates (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). The viscosity of PAA-CNF was 4500 cP, which was twice as large as SBPe-CNF and SC-CNF of 2000 cP. The exception here was HP.F-CNF with a viscosity of 2500 cP which also was more viscous than SBPe-CNF at low shear rates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e4.1.5 Suspension stability\u003c/h2\u003e\u003cp\u003eThe electrostatic stabilities of the CNF suspensions were evaluated by their zeta potential (ZP). This property represents the ionic interaction of the particles of the suspension as millivolt (mV). As the voltage increases, the repulsive forces between the particles increases which indicates that the suspension will be more resistant to flocculation or aggregation. A zeta potential between 0 mV and \u0026minus;\u0026thinsp;10 mV would mean the suspension is inherently unstable. Going further, a suspension ZP between \u0026minus;\u0026thinsp;10 mV and \u0026minus;\u0026thinsp;30 mV mildly stable, and suspensions with a ZP below \u0026minus;\u0026thinsp;30 mV would be functionally stable. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe zeta potentials of the enzyme-treated sugar beet pulp and the oxidised SBP CNF suspensions. The potential is the average\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of triplicates. SBPe\u0026thinsp;=\u0026thinsp;Enzyme-treated sugar beet pulp, SC-CNF\u0026thinsp;=\u0026thinsp;sodium chlorite bleached CNF, PAA-SBPe\u0026thinsp;=\u0026thinsp;peracetic acid bleached CNF, and HP.F\u0026thinsp;=\u0026thinsp;Fenton catalysed hydrogen peroxide bleached CNF.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSamples\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZeta Potential (mV)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSBPe-CNF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSC-CNF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePAA-CNF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHP.F-CNF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe SBPe-CNF was already partially stable at -11.1 mV (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, as the number indicates, the suspension is only initially stable and will settle over a few minutes. The measured ZP of all bleached CNF suspensions have a ZP of roughly 20 mV, with hydrogen peroxide and peracetic with the highest and lowest ZP, respectively. The suspensions are only partially stable, which leads to aggregation after hours of resting or under cold conditions.\u003c/p\u003e\u003cp\u003eWhilst it was thought that a more negative ZP would correlate with a higher viscosity, the inverse was observed with these samples; the least electronegative oxidised sample PAA was the most viscous. In this case it could be argued that the differences in ZP between the samples are too minor to be determinant. Previous work studying CNCs showed that samples between \u0026minus;\u0026thinsp;10mV and \u0026minus;\u0026thinsp;20mV being broadly similar in terms of shear viscosity (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e), and this might be the same for CNFs.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Morphology and Mechanical Testing of CNF Films\u003c/h2\u003e\u003cp\u003eTo understand what implication the different CNF suspensions could have on being further processed, the different CNF were casted into films. The morphology and rheology of the films were analysed with SEM and mechanical testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table \u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), respectively. Hydrogen peroxide-bleached CNF was excluded due to its\u0026rsquo; low whitening effect. Films from SBPe-CNF were also produced, but they were too brittle for mechanical testing and hence were excluded from these analyses. Pinkl \u003cem\u003eet al.\u003c/em\u003e (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) has reported similar problems with SBP CNF films, where the low content of cellulose was assumed to be the reason.\u003c/p\u003e\u003cp\u003eFrom the SEM images, it can be seen that the surface of the sodium chlorite film is smooth while the peracetic acid film has more visible, exposed microfibrils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Previous work, on the SC bleached CNF films, showed rough surface covered by visible nanofibrils (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The cross sections (XC) of the films also show that the peracetic acid film was thicker in comparison to the sodium chlorite film. This could be reasoned by the idea that the fibrils are rougher and hence less consistently layered. Supported by Galland \u003cem\u003eet al.\u003c/em\u003e (2015) reasoning, this would indicate the peracetic acid film has a weaker structural reinforcement since the jagged layering would be more susceptible for stress transfer. This is also what the mechanical testing of the films further shows, where the peracetic acid film failed at a lower stress than the sodium chlorite film (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The peracetic acid film showed lower tensile strength than the sodium chlorite film, possibly due to the higher lignin content in the peracetic acid films.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMeasured thickness from three cross-section SEM images of each oxidised CNF film as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, as well as subsequent calculated density and the stress/strain of each film at failure.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOxidative treatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFilm Thickness (\u0026micro;m)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDensity (g/cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStress (MPa)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStrain (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eYoung\u0026rsquo;s modulus (GPa)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium chlorite (SC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e32.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeracetic acid (PAA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTypically density correlates with the Young\u0026rsquo;s modulus of a material (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e) but in our study, the peracetic acid bleached CNF film is still more elastic despite its reduced packing. It has previously been discussed that lignin lowers hydrogen bond formation between the fibers which leads to lower mechanical properties (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). The peracetic acid film is stiffer but slightly weaker and less elastic than the sodium chlorite film which aligns with the rheological results (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Both, however, are similar to previously produced CNF films (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e4.3 \u003cem\u003eIn situ\u003c/em\u003e Generation of Peracetic Acid\u003c/h2\u003e\u003cp\u003eThe bleaching with chemicals of SBP are not only evaluated based on their effect on final properties, but also how it can be integrated at large-scale production of SBP CNF. The chemical oxidants can be limited due to their explosive risk and health hazards (\u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e), which \u003cem\u003ein situ\u003c/em\u003e generation of peracetic acid could circumvent and possible provide both a safer and in-line oxidation.\u003c/p\u003e\u003cp\u003eFor the \u003cem\u003ein situ\u003c/em\u003e generation of peracetic acid, one of the hydrogen peroxide activators TAED or triacetin was combined with hydrogen peroxide from either sodium percarbonate (SPC) or the enzyme glucose oxidase. The WI of CNFs treated by SPC with TAED and triacetin was 37% and 36% (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), respectively. The WI of the CNFs treated by glucose oxidase with TAED and triacetin was 34% and 27%, respectively. This shows SPC showed to be a more effective bleaching agent than glucose oxidase, although this could be due to the alkaline swelling effects of the dissolved compound.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe whiteness index of SBP CNF suspensions, bleached with in situ generation of peracetic acid with either Tetraacetylethylenediamine (TAED) or triacetin (TriA) together with sodium percarbonate (SPC) or glucose oxidase (GOx).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOxidative treatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhiteness Index (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSPC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e32.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSPC-TAED\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e37.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSPC-TriA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGOx\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGOx-TAED\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e34.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGOx-TriA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe viscosities of the \u003cem\u003ein situ\u003c/em\u003e peracetic acid bleached CNFs, in comparison to peracetic acid CNF, are decreased. The glucose oxidase samples, regardless of using TAED or triacetin, have similar loss of viscosity in comparison to bleaching with peracetic acid exogenously (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). For the SPC samples, even though the two bleach activators have very similar WIs, the triacetin resulted in a much larger loss of viscosity than TAED (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Finally, the amount of available ferulates on the surface were analysed via LM12 antibody binding levels. Considering that SBPe has very low availability of ferulates on its\u0026rsquo; surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), when oxidised by SPC or glucose oxidised alone ferulates were exposed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). When TAED is present though, the surface availability of ferulates is similar to PAA-SBPe. The shows that peracetic acid, whether \u003cem\u003ein situ\u003c/em\u003e or exogenously sourced, is solubilising the available ferulates whereas hydrogen peroxide exposes but does not solubilise them.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOverall, these results shows that SPC with either TAED or triacetin could be a possible replacement of PAA, and thereby, provide an in-line, safer oxidation of SBP. However, the reaction is limited to a concentration of 10 mM by the strong alkalinity of SPC, that would require neutralisation if higher amounts were used. Similar systems involving sodium perborate and TAED (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e) have been implemented previously for cotton bleaching with similar effects on WI. Enzymatic \u003cem\u003ein situ\u003c/em\u003e generation of PAA shows the choice of hydrogen peroxide activator, or the set-up, matters. Glucose oxidase together with TAED show similar results in whitening, viscosity, and elongation before breakpoint of both PAA and SPC with TAED or triacetin. Glucose oxidase with TAED has been shown to bleach cotton linter (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), and together with these results, it demonstrates the potential of enzymatic bleaching with TAED. However, pairing glucose oxidase with triacetin did not show any significant enhancement of bleaching. This is likely due to the pH that was chosen to be a compromise between the optimal pH for the enzyme and triacetin but resulted in suboptimal reaction for both. Here, SPC could create a reaction with pH 10 to 11 that is the optimal range for PAA generation with both hydrogen peroxide activators (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Furthermore, the differing pH of the methods seems to have affected the physical properties of the CNFs. SPC oxidation resulted in higher viscosity along with higher amounts of exposed surface pectin. It is likely the pectin would increase the viscosity, as it has been shown to do in plant cell wall suspension (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003cp\u003eIn this work, peracetic acid has been shown to be a feasible replacement for sodium chlorite as a bleaching agent in the production of cellulose nanofibers from sugar beet pulp. The resultant CNF has the same whiteness index and increased viscosity. In contrast, Fenton-catalysed hydrogen peroxide creates CNF that are darker in colour but still with a slight increase in viscosity compared to sodium chlorite. The lignin content of the enzymatically digested SBP was not affected by any of the oxidants applied, and that the small differences in the relative lignin content shows be attributed to the removal of polysaccharides rather than lignin. However increased amounts of ferulates were detectable on the surface of the oxidised CNFs, especially after free-radical oxidation. Nevertheless, our data shows that peracetic acid can replace sodium chlorite when focusing on whitening.\u003c/p\u003e\u003cp\u003eFinally, it has been shown that peracetic acid can be directly generated \u003cem\u003ein-situ\u003c/em\u003e and used immediately for bleaching SBP in this process, thus making PAA a safer, in-line oxidant. Chemically, peracetic acid can be released from either TAED or triacetin together with sodium percarbonate. Enzymatically, peracetic acid can be created from glucose oxidase with TAED which makes this process entirely enzyme mediated. Triacetin with sodium percarbonate allows for a simplification of the method where the swelling and bleaching steps occur in one. Overall, this work demonstrates the successful replacement of chlorine-bleaching with the novel application of two possible \u003cem\u003ein-situ\u003c/em\u003e PAA bleaching methods to the secondary biomass sugar beet pulp.\u003c/p\u003e\u003c/div\u003e"},{"header":"6 Abbrevations","content":"\u003cp\u003eSBP Sugar Beet Pulp\u003c/p\u003e\u003cp\u003eTAED Tetraacetylethylenediamine\u003c/p\u003e\u003cp\u003eCNF Cellulose nano-fibers\u003c/p\u003e\u003cp\u003eSC Sodium chlorite\u003c/p\u003e\u003cp\u003eHP Hydrogen peroxide\u003c/p\u003e\u003cp\u003ePAA Peracetic acid\u003c/p\u003e\u003cp\u003eSPC Sodium percarbonate\u003c/p\u003e\u003cp\u003e%w/w Weight/ weight\u003c/p\u003e\u003cp\u003e%v/v Volume / volume\u003c/p\u003e\u003cp\u003eAMSA Array-based microfibril surface\u003c/p\u003e\u003cp\u003ePBS Phosphate-buffered saline\u003c/p\u003e\u003cp\u003eBCIP 5-bromo-4-chloro-3-indolylphosphate\u003c/p\u003e\u003cp\u003eNBT Nitro-blue tetrazolium chloride\u003c/p\u003e\u003cp\u003eSEM Scanning electron microscopy\u003c/p\u003e\u003cp\u003eFEG Field emission gun\u003c/p\u003e\u003cp\u003eWI% Whiteness index\u003c/p\u003e\u003cp\u003eSBPe SBP, enzymatically digested\u003c/p\u003e\u003cp\u003eSC-SBPe Sodium chlorite - oxidised SBPe\u003c/p\u003e\u003cp\u003ePAA-SBPe Peracetic acid - oxidised SBPe\u003c/p\u003e\u003cp\u003eHP.F-SBPe Hydrogen peroxide - Fenton - oxidised SBPe\u003c/p\u003e\u003cp\u003eG' Storage modulus\u003c/p\u003e\u003cp\u003eG'' Loss modulus\u003c/p\u003e\u003cp\u003eZP Zeta potential\u003c/p\u003e\u003cp\u003eTriA Triacetin\u003c/p\u003e\u003cp\u003eGOx Glucose oxidase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by a grant from the Novo Nordisk Foundation: NNF22OC0072911.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eChristian Donohoe; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Ellen Engquist; Conceptualization, Methodology, Writing \u0026ndash; review \u0026amp; editing. Nicolas Carstens; Data curation, Formal analysis, Investigation, Validation, Visualisation . Thomas Kinsella; Data curation, Formal analysis, Investigation, Validation, Visualisation. Bodil J\u0026oslash;rgensen; Conceptualisation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing \u0026ndash; review \u0026amp; editing. Marwa Faisal; Formal analysis, Methodology, Resources, Writing \u0026ndash; review \u0026amp; editing. Peter Ulvskov; Conceptualisation, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing \u0026ndash; review \u0026amp; editing. Declaration of competing interest\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting this article has been included in this text and as part of the Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDinand E, Chanzy H, Vignon MR. Parenchymal cell cellulose from sugar beet pulp: preparation and properties. Cellulose. 1996;3(1):183\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWhale E, Bulling AEK, Fry SC. Biochemical characterisation of cellulose and cell-wall-matrix polysaccharides in variously oxidised sugar-beet pulp preparations differing in viscosity. International Journal of Biological Macromolecules. 2023;253:127356.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArjeh E, Khodaei SM, Barzegar M, Pirsa S, Sani IK, Rahati S, et al. Phenolic compounds of sugar beet (Beta vulgaris L.): Separation method, chemical characterization, and biological properties. Food Science \u0026amp; Nutrition. 2022 Sept 13;10(12):4238.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRalet MC, Andr\u0026eacute;-Leroux G, Qu\u0026eacute;m\u0026eacute;ner B, Thibault JF. Sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e) pectins are covalently cross-linked through diferulic bridges in the cell wall. Phytochemistry. 2005;66(24):2800\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLasseuguette E, Roux D, Nishiyama Y. Rheological properties of microfibrillar suspension of TEMPO-oxidized pulp. Cellulose. 2008 June 1;15(3):425\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHassan ML, Berglund L, Abou Elseoud WS, Hassan EA, Oksman K. Effect of pectin extraction method on properties of cellulose nanofibers isolated from sugar beet pulp. Cellulose. 2021;28(17):10905\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHolland C, Perzon A, Cassland PRC, Jensen JP, Langebeck B, S\u0026oslash;rensen OB, et al. Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely Enzymatic Pretreatments. Biomacromolecules. 2019;20(1):443\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang W, Feng Y, He H, Yang Z. Environmentally-Friendly Extraction of Cellulose Nanofibers from Steam-Explosion Pretreated Sugar Beet Pulp. Materials. 2018 July;11(7):1160.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSarkanen KV. The chemistry of delignification in pulp bleaching. Pure and Applied Chemistry. 1962;5(1\u0026ndash;2):219\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalland S, Berthold F, Prakobna K, Berglund LA. Holocellulose Nanofibers of High Molar Mass and Small Diameter for High-Strength Nanopaper. Biomacromolecules. 2015;16(8):2427\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu K, Yan K, Sun G. Mechanism of H2O2/bleach activators and related factors. Cellulose. 2019;26(4):2743\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHickman WS. Peracetic acid and its use in fibre bleaching. Review of Progress in Coloration and Related Topics. 2002;32(1):13\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAfsahi G, Rojalin T, Vuorinen T. Chemical characteristics and stability of eucalyptus kraft pulps bleached with tertiary amine catalyzed hypochlorous acid. Cellulose. 2019;26(3):2047\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi K, Tang B, Zhang W, Shi Z, Tu X, Li K, et al. Formation Mechanism of Bleaching Damage for a Biopolymer: Differences between Sodium Hypochlorite and Hydrogen Peroxide Bleaching Methods for Shellac. ACS Omega. 2020 Sept 8;5(35):22551\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFry SC, Dumville JC, Miller JG. Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell walls of ripening pear fruit. Biochem J. 2001;357(Pt 3):729\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiller JG, Fry SC. Characteristics of xyloglucan after attack by hydroxyl radicals. Carbohydr Res. 2001 June 15;332(4):389\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZimin YuS, Borisova NS, Kutlugildina GG, Mudarisova RKh, Borisov IM, Mustafin AG. Oxidation and destruction of arabinogalactan and pectins under the action of hydrogen peroxide and ozone-oxygen mixture. Reac Kinet Mech Cat. 2017;120(2):673\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarlos TD, Bezerra LB, Vieira MM, Sarmento RA, Pereira DH, Cavallini GS. Fenton-type process using peracetic acid: Efficiency, reaction elucidations and ecotoxicity. Journal of Hazardous Materials. 2021;403:123949.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAirianah OB, Vreeburg RAM, Fry SC. Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method. Annals of Botany. 2016;117(3):441\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMishra SP, Manent AS, Chabot B, Daneault C. The Use of Sodium Chlorite in Post-Oxidation of TEMPO-Oxidized Pulp: Effect on Pulp Characteristics and Nanocellulose Yield. Journal of Wood Chemistry and Technology. 2012;32(2):137\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Q, Lu R, Liang Y, Gao K, Jiang H. Energy-Saving One-Step Pre-Treatment Using an Activated Sodium Percarbonate System and Its Bleaching Mechanism for Cotton Fabric. Materials. 2022;15(17):5849.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou Y, Zheng G, Zhang J, Wang Q, Zhou M, Yu Y, et al. An eco-friendly approach to low-temperature and near-neutral bleaching of cotton knitted fabrics using glycerol triacetate as an activator. Cellulose. 2021;28(12):8129\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eM\u0026uuml;ller D. Studies on a new enzyme glycose-odydase. I. Biochem Z. 1928;199:136\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTavčer PF. Low-temperature bleaching of cotton induced by glucose oxidase enzymes and hydrogen peroxide activators. Biocatalysis and Biotransformation. 2012;30(1):20\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTzanov T, Costa SA, G\u0026uuml;bitz GM, Cavaco-Paulo A. Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching. J Biotechnol. 2002;93(1):87\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerzon A, J\u0026oslash;rgensen B, Ulvskov P. Sustainable production of cellulose nanofiber gels and paper from sugar beet waste using enzymatic pre-treatment. Carbohydrate Polymers. 2020;230.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGui J. Acetyl Bromide Method for Total Lignin Content Determination in Plant Biomass. In: Jiang L, Shen J, Gao C, Wang X, editors. Plant Protein Secretion: Methods and Protocols [Internet]. New York, NY: Springer US; 2024 [cited 2025 Feb 26]. p. 95\u0026ndash;100. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-0716-4059-3_8\u003c/span\u003e\u003cspan address=\"10.1007/978-1-0716-4059-3_8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVerhertbruggen Y, Marcus SE, Haeger A, Ordaz-Ortiz JJ, Knox JP. An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohydr Res. 2009 Sept 28;344(14):1858\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKilcoyne M, Gerlach JQ. Glycan Microarrays: Methods and Protocols. 1st ed. 2022. New York, NY: Springer US; 2022. xi\u0026thinsp;+\u0026thinsp;253. (Methods in Molecular Biology, 2460).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJones L, Seymour GB, Knox JP. Localization of Pectic Galactan in Tomato Cell Walls Using a Monoclonal Antibody Specific to (1[-\u0026gt;]4)-[beta]-D-Galactan. Plant Physiol. 1997;113(4):1405\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWillats WGT, Marcus SE, Knox JP. Generation of a monoclonal antibody specific to (1\u0026rarr;5)-α-l-arabinan. Carbohydrate Research. 1998;308(1):149\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRalet MC, Tranquet O, Poulain D, Mo\u0026iuml;se A, Guillon F. Monoclonal antibodies to rhamnogalacturonan I backbone. Planta. 2010;231(6):1373\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePettolino FA, Hoogenraad NJ, Ferguson C, Bacic A, Johnson E, Stone BA. A (1\u0026ndash;\u0026gt;4)-beta-mannan-specific monoclonal antibody and its use in the immunocytochemical location of galactomannans. Planta. 2001;214(2):235\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarcus SE, Blake AW, Benians TAS, Lee KJD, Poyser C, Donaldson L, et al. Restricted access of proteins to mannan polysaccharides in intact plant cell walls. Plant J. 2010;64(2):191\u0026ndash;203.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePattathil S, Avci U, Baldwin D, Swennes AG, McGill JA, Popper Z, et al. A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies. Plant Physiol. 2010 June;153(2):514\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang X, Rogowski A, Zhao L, Hahn MG, Avci U, Knox JP, et al. Understanding How the Complex Molecular Architecture of Mannan-degrading Hydrolases Contributes to Plant Cell Wall Degradation*. Journal of Biological Chemistry. 2014;289(4):2002\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRuprecht C, Bartetzko MP, Senf D, Dallabernadina P, Boos I, Andersen MCF, et al. A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies. Plant Physiol. 2017;175(3):1094\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeralta AG, Venkatachalam S, Stone SC, Pattathil S. Xylan epitope profiling: an enhanced approach to study organ development-dependent changes in xylan structure, biosynthesis, and deposition in plant cell walls. Biotechnology for Biofuels. 2017;10(1):245.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoller I, Marcus SE, Haeger A, Verhertbruggen Y, Verhoef R, Schols H, et al. High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles. Glycoconj J. 2008;25(1):37\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePennell RI, Janniche L, Kjellbom P, Scofield GN, Peart JM, Roberts K. Developmental Regulation of a Plasma Membrane Arabinogalactan Protein Epitope in Oilseed Rape Flowers. Plant Cell. 1991;3(12):1317\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYates EA, Valdor JF, Haslam SM, Morris HR, Dell A, Mackie W, et al. Characterization of carbohydrate structural features recognized by anti-arabinogalactan-protein monoclonal antibodies. Glycobiology. 1996;6(2):131\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePennell RI, Knox JP, Scofield GN, Selvendran RR, Roberts K. A family of abundant plasma membrane-associated glycoproteins related to the arabinogalactan proteins is unique to flowering plants. J Cell Biol. 1989;108(5):1967\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarcus SE, Verhertbruggen Y, Herv\u0026eacute; C, Ordaz-Ortiz JJ, Farkas V, Pedersen HL, et al. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls. BMC Plant Biology. 2008;8(1):60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePedersen HL, Fangel JU, McCleary B, Ruzanski C, Rydahl MG, Ralet MC, et al. Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research. J Biol Chem. 2012;287(47):39429\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlake AW, McCartney L, Flint JE, Bolam DN, Boraston AB, Gilbert HJ, et al. Understanding the Biological Rationale for the Diversity of Cellulose-directed Carbohydrate-binding Modules in Prokaryotic Enzymes *. Journal of Biological Chemistry. 2006 Sept 29;281(39):29321\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang X, Reid MS, Ols\u0026eacute;n P, Berglund LA. Eco-Friendly Cellulose Nanofibrils Designed by Nature: Effects from Preserving Native State. ACS Nano. 2020;14(1):724\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerzon A, Kračun SK, J\u0026oslash;rgensen B, Ulvskov P. Array-based microfibril surface assessment (AMSA): a method for probing surface-exposed polysaccharides on cellulose nanofibres. Cellulose. 2020;27(15):8635\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDong J, Jiang W, Gao P, Yang T, Zhang W, Huangfu M, et al. Comparison of betalain compounds in two \u003cem\u003eBeta vulgaris\u003c/em\u003e var. \u003cem\u003ecicla\u003c/em\u003e and \u003cem\u003eBvCYP76AD27\u003c/em\u003e function identification in betalain biosynthesis. Plant Physiology and Biochemistry. 2023 June 1;199:107711.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar R, Hu F, Hubbell CA, Ragauskas AJ, Wyman CE. Comparison of laboratory delignification methods, their selectivity, and impacts on physiochemical characteristics of cellulosic biomass. Bioresour Technol. 2013;130:372\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang X, Berthold F, Berglund LA. Preserving Cellulose Structure: Delignified Wood Fibers for Paper Structures of High Strength and Transparency. Biomacromolecules. 2018 July 9;19(7):3020\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHatfield RD, Grabber J, Ralph J, Brei K. Using the Acetyl Bromide Assay To Determine Lignin Concentrations in Herbaceous Plants: Some Cautionary Notes. J Agric Food Chem. 1999;47(2):628\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eColquhoun IJ, Ralet MC, Thibault JF, Faulds CB, Williamson G. Structure identification of feruloylated oligosaccharides from sugar-beet pulp by NMR spectroscopy. Carbohydrate Research. 1994;263(2):243\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMathew S, Abraham TE. Ferulic Acid: An Antioxidant Found Naturally in Plant Cell Walls and Feruloyl Esterases Involved in its Release and Their Applications. Critical Reviews in Biotechnology. 2004;24(2\u0026ndash;3):59\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaeidy S, Omidi P, Nasirpour A, Keramat J. Physicochemical and functional properties of cross linked and high pressure homogenized sugar beet pectin: A comparative study. Food Hydrocolloids. 2023;134:108041.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePan GX, Spencer L, Leary GJ. Reactivity of Ferulic Acid and Its Derivatives toward Hydrogen Peroxide and Peracetic Acid. J Agric Food Chem. 1999;47(8):3325\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStojkov G, Niyazov Z, Picchioni F, Bose RK. Relationship between Structure and Rheology of Hydrogels for Various Applications. Gels. 2021;7(4):255.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi MC, Wu Q, Song K, Lee S, Qing Y, Wu Y. Cellulose Nanoparticles: Structure\u0026ndash;Morphology\u0026ndash;Rheology Relationships. ACS Sustainable Chem Eng. 2015;3(5):821\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu J, Wang P, Yuan B, Zhang H. Rheology of cellulose nanocrystal and nanofibril suspensions. Carbohydrate Polymers. 2024;324:121527.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi S, Zhao G, Chen H. The Relationship between Steady Shear Viscosity and Complex Viscosity. Journal of Dispersion Science and Technology. 2005 July 1;26(4):415\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLunardi CN, Gomes AJ, Rocha FS, De Tommaso J, Patience GS. Experimental methods in chemical engineering: Zeta potential. The Canadian Journal of Chemical Engineering. 2021;99(3):627\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoluk Y, Lahiji R, Zhao L, McDermott MT. Suspension viscosities and shape parameter of cellulose nanocrystals (CNC). Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2011;377(1):297\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePinkl S, Veigel S, Colson J, Gindl-Altmutter W. Nanopaper Properties and Adhesive Performance of Microfibrillated Cellulose from Different (Ligno-)Cellulosic Raw Materials. Polymers. 2017;9(8):326.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei L, Bian H, Agarwal UP, Sabo RC, Matuana LM, Stark NM. Correlation between morphology and performance of cellulose nanofibril-based films. Current Research in Green and Sustainable Chemistry. 2023;6:100363.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlmeida R, Ramos A, H\u0026aring;konsen V, Maloney T, Gamelas J. Functionalized cellulose nanofiber films as potential substitutes for Japanese paper. Carbohydrate Polymer Technologies and Applications. 2024;8:100573.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrovagunta R, Zou T, \u0026Ouml;sterberg M, Kelley SS, Lavoine N. Design strategies, properties and applications of cellulose nanomaterials-enhanced products with residual, technical or nanoscale lignin\u0026mdash;A review. Carbohydrate Polymers. 2021;254:117480.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu J, Sagnelli D, Faisal M, Perzon A, Taresco V, Mais M, et al. Amylose/cellulose nanofiber composites for all-natural, fully biodegradable and flexible bioplastics. Carbohydr Polym. 2021;253:117277.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChurchill DG. Chemical Structure and Accidental Explosion Risk in the Research Laboratory. J Chem Educ. 2006;83(12):1798.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoint FAO/WHO Expert Committee on Food Additives. Meeting. 73th, World Health Organization. Safety evaluation of certain food additives and contaminants: prepared by the Seventy-third meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). 2011 [cited 2025 June 26]; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://iris.who.int/handle/10665/44521\u003c/span\u003e\u003cspan address=\"https://iris.who.int/handle/10665/44521\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePechacek N, Osorio M, Caudill J, Peterson B. Evaluation of the toxicity data for peracetic acid in deriving occupational exposure limits: A minireview. Toxicology Letters. 2015;233(1):45\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShafie AE, Fouda MMG, Hashem M. One-step process for bio-scouring and peracetic acid bleaching of cotton fabric. Carbohydrate Polymers. 2009 Sept 5;78(2):302\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDinand E, Chanzy H, Vignon RM. Suspensions of cellulose microfibrils from sugar beet pulp. Food Hydrocolloids. 1999;13(3):275\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sugar beet, Oxidation, Nanocellulose, Peracetic acid, Glucose oxidase, Green Chemistry","lastPublishedDoi":"10.21203/rs.3.rs-7917067/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7917067/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003e1.1 Background\u003c/h2\u003e\u003cp\u003eA simple, one-pot process can be applied to sugar beet pulp (SBP), a low-lignin biomass, to produce cellulose nanofibers (CNF). However, this process uses sodium chlorite for bleaching. This work explores both the replacement of sodium chlorite (SC) with the environmentally friendly peracetic acid (PAA), both exogenous and \u003cem\u003ein-situ\u003c/em\u003e generated, and how the choice of peroxyl or free-radical bleaching mechanism affects this reaction.\u003c/p\u003e\u003ch2\u003e1.2 Results\u003c/h2\u003e\u003cp\u003ePAA bleached CNFs (PAA-CNF) were very close to SC bleached CNF (SC-CNF) in terms of colour and whilst the peroxyl mechanism whitened the pulp, free-radical bleaching darkened the pulp. PAA and Fenton-oxidised CNFs showed higher levels of lignin content than SC oxidised CNF but were more viscous. The CNFs all showed stability incipiently, with zeta potentials from \u0026minus;\u0026thinsp;17 to -23 mV. PAA-CNF formed thicker films with a lower tensile strength and higher Youngs modulus than SC-CNF films due to their higher lignin content. Moreover, the \u003cem\u003ein-situ\u003c/em\u003e generation of PAA CNFs were similar quality to CNF bleached by exogenous PAA.\u003c/p\u003e\u003ch2\u003e1.3 Conclusions\u003c/h2\u003e\u003cp\u003eIn this study, we demonstrate three successful alternatives for replacing sodium chlorite in our CNF production method. After PAA oxidation the lignin remains, yet the resultant CNF suspensions are more viscous and equal in whiteness to SC-CNF. Overall this work shows how we can innovate an entirely chlorine-free, enzyme-mediated production processing that represent an advance for the safety and reduction of toxicity of the process, and further the development of upcycling of secondary biomass into sustainable alternatives for biomaterials.\u003c/p\u003e","manuscriptTitle":"Totally-Chlorine-Free Bleaching Alternatives to Produce Sugar Beet Pulp Cellulose Nanofibers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-27 17:30:04","doi":"10.21203/rs.3.rs-7917067/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-26T08:42:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T21:28:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34094138627707956071189290943382434357","date":"2025-12-08T13:17:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-18T11:04:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-24T03:28:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-24T03:28:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2025-10-21T13:23:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cb4f7523-39d1-4e9a-aa37-4cf4a9eabd4b","owner":[],"postedDate":"November 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-03T10:53:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-27 17:30:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7917067","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7917067","identity":"rs-7917067","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}