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# Chemical degradations of kraft pulp carbohydrates molar mass distributions and their consequences on fibres strength

Etienne Montet, Dominique Lachenal, Christine Chirat

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Etienne Montet, Dominique Lachenal, Christine Chirat. Chemical degradations of kraft pulp carbohydrates molar mass distributions and their consequences on fibres strength. 2024. hal-04890217

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# Chemical degradations of kraft pulp carbohydrates molar mass distributions and their consequences on fibres strength

Etienne Montet<sup>a,b,c*</sup>, Dominique Lachenal<sup>a</sup>, and Christine Chirat<sup>a</sup>

<sup>a</sup> Univ. Grenoble Alpes, CNRS, Grenoble INP (Institute of Engineering Univ. Grenoble Alpes), LGP2, 38000 Grenoble, France

<sup>b</sup> The French Agency for Ecological Transition (ADEME), 20, avenue du Grésillé, BP 90406, 49004 Angers Cedex 01, France

<sup>c</sup> Institut Polytechnique UniLaSalle, Université d'Artois, ULR 7519, 19 Rue Pierre Waguet, BP 30313, 60026 Beauvais, France

* Corresponding author's e-mail address: etienne.montet@unilasalle.fr

## Abstract

Industrial oxygen-delignified and fully-bleached hardwood kraft pulps were treated with chemicals to provoke carbohydrates depolymerisation: ozone, hypochlorous acid, and cellulase. The degrees of polymerisation (DP) of cellulose obtained by pulp viscosity measurement in Cuen and by size-exclusion chromatography after direct dissolution in DMAc/LiCl indicated the extent of the chemical degradation inflicted to the fibres. Molar mass distributions (MMD) described the carbohydrates depolymerisation patterns. Fibre strength was assessed by measuring the zero-span tensile index at never-dried state. Fibre strength deterioration seemed to be mainly driven by the topochemistry of the cellulose degradation (homogeneous or localised), rather than by its intensity usually measured as an average DP loss. In fact, depolymerisation by cellulase was found critically detrimental to fibres strength whereas ozone and hypochlorous acid induced little harm to the fibres despite a significant cellulose depolymerisation. According to these results, in line with several past studies, fibre strength measurements should be performed systematically as the sole pulp viscosity is an inadequate indicator. Alternatively, albeit being insufficient strength predictors on their own MMD can give valuable insight on the topochemistry of the cellulose degradation, a key aspect when monitoring fibre strength preservation during pulping and bleaching.

## Keywords

kraft pulp; ozone bleaching; fibre strength; degree of polymerisation; molar mass distributions; SEC
# 1. Introduction

Bleached kraft pulp is produced for various end-uses such as bleached packaging, wrappings, printing papers, smooth papers, or tissues. A common requirement for virgin kraft fibres is their high strength. The pulping, bleaching, and paper-making steps can alter the kraft fibres strength by mechanically and chemically introducing local defects (Mohlin & Alfredson 1990, Hartler 1995, Mohlin et al. 1996, Palme et al. 2016). Bleaching is the chemical process of removing residual lignin from unbleached pulp by the successive use of strong oxidants and alkaline extraction stages. Chlorine dioxide is today the most widely used bleaching agent and commonly employed in Elemental Chlorine Free (ECF) bleaching sequences. Its main drawback resides in the generation of bioaccumulable chlorinated organics in the bleach plant effluent. Totally Chlorine Free (TCF) bleaching sequences may perform better environmentally but have suffered from technical limitations such as insufficient brightness, poor brightness stability, or high cost (Rennel 1995). Among TCF chemicals, ozone is a candidate for totally substituting chlorine dioxide but provokes an inevitable depolymerisation of the pulp carbohydrates (cellulose and hemicelluloses).

Today, the cellulose degree of polymerisation (DP) is generally considered the main parameter for kraft fibre strength prediction. Although various DP/strength correlations have been established in the past, there is no such thing as a unique linear trend encompassing the large diversity of chemicals affecting cellulose integrity. In fact, some chemical treatments would target weak spots in the fibres (ie. kinks or curls). Derivatisation reactions (e.g. carbanilation, etherification) performed without proper swelling or dissolution of the fibres induces incomplete and localised reactions due to inaccessible cellulose domains (Terbojevich et al. 1995, Mischnick et al. 2021). Other examples include both acid and enzymatic (cellulase) hydrolyses of cellulosic fibres (Gurnagul et al. 1992). On the contrary, homogeneous degradations like those triggered by alkaline hydrolysis would not substantially decrease fibre strength despite a critical DP loss (Sjöström & Brolin 1996, Iribarne & Schroeder 1997, Oglesby et al. 2016). Therefore, knowing the homogeneity of cellulose degradation both at the macro and ultrastructure levels would be key to predict fibre strength loss, and not solely the average carbohydrates DP commonly measured as the pulp viscosity dissolved in Cuen (DPv) (Figure 1). Discriminating homogeneous and heterogeneous/localised degradation patterns is not an easy task. Heterogeneous degradations encompass localised degradations affecting specific parts of the fibres - usually local defects - and degradations selective towards specific cell wall layers or cellulose chains lengths. Cellulase enzymes and acid hydrolysis fall in the first category (Engström et al. 2006) whereas treatments such as sonication would preferentially affect long cellulose chains (Stefanovic et al. 2014). A sure fact is that the DPv gives no information on the topochemistry of cellulose degradation occurring within the fibres. Alternatively, Size Exclusion Chromatography (SEC) seems more relevant when dealing with pulp chemical degradations (Godsay 1984, Berggren et al. 2001). For hardwood pulps, the now widely-used
method for SEC is the direct dissolution of fibres in a DMAc/LiCl (8%) mixture (McCormick et al. 1985, Henniges et al. 2014). In this work, the direct dissolution method has been favoured over the carbanilation for reasons mentioned above.

<figure><img src="image_3.png" /><figcaption>**Figure 1** – Representation of a wood fibre with close-up on its cell walls organisation (cross-section). Carbohydrates chemical degradation can occur at two distinct levels. Degradations occurring at the fibres weak spots and fibre ends (in red) and degradations affecting the whole of the fibre which can be discriminated at the ultrastructure level (in blue). Crossed green dots represent degradations mostly confined to external layers (P, S1 and S3) and purple dots illustrate more homogeneous degradations affecting the wide S2 layer as well.</figcaption></figure>

The present study aims at getting more insight on the unclear ties between cellulose DP and fibres strength. The assumption that ECF pulps perform better than TCF pulps have led many to believe that ozone TCF cannot compete with ECF bleaching (Pedrola et al. 2004). In fact, the absence of scientific consensus on ozone-bleached fibres mechanical quality hitherto represents a significant obstacle to the industrial implementation of water-saving and less polluting bleaching processes such as TCF bleaching.
# 2. Materials and Methods

## 2.1. Materials

The hardwood oxygen-delignified kraft "Ko" and issuing DEpDD fully-bleached kraft "Fb" pulps were provided by Fibre Excellence Saint-Gaudens kraft mill in France (Table 1). Major wood species in the two hardwoods mix pulps were poplar, beech, oak, and chestnut (80% of the total).

<table><caption>Table 1 - Main characteristics of the two starting pulps used in this work: kraft oxygen-delignified "KO" and fully-bleached "FB". Kappa number, hexA content, pulp brightness and pulp viscosity were determined according to ISO 302, Tappi T 282 pm-07, ISO 3688 and ISO 5351-1 standards, respectively.</caption><thead><tr><td>Pulp</td><td>Kappa number</td><td>hexA (µmol/g)</td><td>Brightness (%ISO)</td><td>DPv</td></tr></thead><tbody><tr><td>Ko</td><td>10</td><td>66.9</td><td>49</td><td>1580</td></tr><tr><td>Fb</td><td>-</td><td>-</td><td>89.8</td><td>1370</td></tr></tbody></table>

Sodium borohydride was bought from Fisher Scientific, hydrogen peroxide and Novozymes Celluclast® (Trichoderma Reesei source) from Millipore Sigma, and Endo-1,4-β-D-glucanase (Aspergillus Niger source) from Megazyme (endoglucanase). MgSO₄ in its heptahydrated form and the other chemicals were purchased from Roth (Germany). All the chemicals were used without further purification. Ozone was generated in the laboratory with an Ozonia generator.

## 2.2. Pulp preparation and chemical oxidations

Upon reception, the industrial pulps were washed with tap water in large sieves prior to storage in PET bags for freezing. Prior to and after any chemical reaction the pulp was washed with deionised (DEI) water until the washing filtrate conductivity was below 5 µS.cm⁻¹. All the chemical charges presented in this article are expressed as % wt./wt. of pulp (oven dry basis). The chemical charge of each treatment is indicated within open brackets in the sample's name, e.g. Fb-Z(1)Eo indicates a 1% O₃ charge on o.d. pulp. For the cellulase treatments, namely Cₐₐₐ and Cₐₐₐ, the number between brackets indicate the enzymatic charge (U/g, units per o.d. gram of pulp). The conditions of the various stages performed in this study were as follow:

**A, E, P, H:** reactions were performed in PE bags. DEI water and chemicals were added to the pulp and the PE bag kneaded by hand for 2 minutes before place in a heated water bath. At half the duration of the reaction, samples were taken off the bath and briefly kneaded again, then replaced in the bath. Other conditions are reported in Table 2.

**Eo:** oxygen-reinforced alkaline extraction stages were performed in a rotating multi-shell reactor heated with an oil bath. The required amount of NaOH (40 g/L solution) and
water was added to the pulp to reach pH 11.5 and 10% PC. 2 bars of oxygen were introduced in the shell(s) before starting the reaction.

**C<sub>cell</sub> and C<sub>endo</sub>:** the Celluclast® treatment C<sub>cell</sub> took place in 4 x 250 mL double-walled glass reactors set up in series, each of them positioned on magnetic stirrers. The reactors were connected to a closed water loop system with set temperature 2 g of o.d. pulp were introduced in a 100 mL glass bottle with thread cap. 50 mL of pH 5.3 citrate-phosphate buffer as well as DEI water to reach 5% PC were added. Temperature was set so the temperature of the pulp suspension within the threaded bottles reached 40 °C. After 15 min the enzymes were introduced and the stirring set on 200 rpm for a 24 h reaction time. This system allowed for 4 simultaneous enzymatic hydrolyses with constant stirring and temperature. After 24 h, the glass bottle was taken off the reactor and enzymes deactivated (10 min boiling water treatment followed by 1 h in iced bath). For the C<sub>endo</sub> treatment the same procedure was employed but using an acetate buffer (pH 4.6) and a reaction temperature of 55 °C. Numbers in open brackets indicate the number of charged enzymatic units per gram of o.d. pulp.

**Z:** high consistency ozonation consisted of pulp pH adjustment to 2.5 with 4N H<sub>2</sub>SO<sub>4</sub> solution at 1-2 % Pulp Consistency (PC), followed by the pulp filtration on no. 2 crucible and centrifugation of the pulp to obtain 38-42 % PC. The pulp was fluffed and put in a spherical glass reactor for ozonation. The ozone generator settings were: input oxygen pressure = 2 bars; current = 0.7 A; ozone concentration ≈ 65 mg/L. The real ozone concentration in the gas flow was titrated from a potassium iodide trap solution by adding sodium thiosulfate. Ozone consumption was calculated the same way by titrating the excess ozone in the reactor's exhaust gas. At the end of the experiment, if the Z stage was followed by a E, Eo, or P stage, the pulp was directly stabilised to the desired (alkaline) pH value and PC by adding DEI water and a 40 g/L NaOH stock solution.

<table><caption>Table 2 - Experimental conditions of the treatments applied to pulps</caption><thead><tr><th></th><th>Chemicals (% on o.d. pulp)</th><th>PC (%)</th><th>pH</th><th>T (°C)</th><th>t (h)</th></tr></thead><tbody><tr><td>A</td><td>H<sub>2</sub>SO<sub>4</sub></td><td>10</td><td>2.5</td><td>95</td><td>2</td></tr><tr><td>Z</td><td>H<sub>2</sub>SO<sub>4</sub>, O<sub>3</sub></td><td>38-42</td><td>2.5</td><td>20</td><td>0-0.3</td></tr><tr><td>E</td><td>1% NaOH</td><td>10</td><td>11.5</td><td>70</td><td>1</td></tr><tr><td>Eo</td><td>1.5-2% NaOH; 2 bar O<sub>2</sub></td><td>10</td><td>11.5</td><td>70</td><td>1</td></tr><tr><td>P</td><td>0.5 % H<sub>2</sub>O<sub>2</sub>; 0.8 % NaOH; 0.3% MgSO<sub>4</sub></td><td>10</td><td>12</td><td>70</td><td>2</td></tr><tr><td>P<sub>100c</sub></td><td>1% H<sub>2</sub>O<sub>2</sub>; 0.8% NaOH</td><td>10</td><td>12</td><td>100</td><td>2</td></tr><tr><td>H</td><td>0.6-5% NaClO; acetate buffer</td><td>10</td><td>4.9</td><td>70</td><td>1</td></tr><tr><td>C<sub>cell</sub></td><td>0-100 U/g Celluclast®; citrate-phosphate buffer</td><td>5</td><td>5.3</td><td>40</td><td>24</td></tr><tr><td>C<sub>endo</sub></td><td>0-80 U/g endo-1,4-N-β-glucanase; acetate buffer</td><td>5</td><td>4.6</td><td>55</td><td>24</td></tr></tbody></table>

TCF fully-bleached pulp was produced in the lab by bleaching Ko pulp with TCF sequence A-Z(0.2)Eo-Z(0.2)Eo-Z(0.1)P.
## 2.3. Cellulose DP determination and MMD deconvolution

Prior to any DP measurement, the unbleached and semi-bleached samples were treated according to the Holocellulose procedure: 2 g of o.d. pulp were bleached with 20 mL of a sodium chlorite $NaClO_2$ solution (34 g/L), 20 mL of acetate buffer (pH 4.9) and 160 mL added DEI water within threaded cap bottle. The bottle was plunged in a water bath at 70 °C for 2h after vigorous shaking at t=0 and t=1h. Pulp was then washed thoroughly before storage or use. All the pulp samples containing “oxycellulose” ie. cellulose with substantial amounts of carbonyl groups introduced in the ZEo, H, $Z_{alk}E$, and $P_{100C}$ stages were first treated with sodium borohydride $NaBH_4$. Sodium carbonate was added to reach pH 9, 1% $NaBH_4$ was introduced for 2 hours reacting at room temperature. The reduction was performed on 2 g of o.d. pulp in 250 mL Erlenmeyer with deionised water (PC = 2%) and under vigorous magnetic stirring. DPv was calculated using the formula established by Sihtola et al. (1963) after measuring pulp viscosity according to ISO 5351-1: DPv = $0.75 \times (954 \times \log(v) - 325)^{1.105}$ where v is the viscosity of the pulp sample dissolved in cupriethylenediamine, expressed in mPa.s.

Other DP values were calculated from the $M_w$ mass molar mass average obtained with Size Exclusion Chromatography (SEC) as: $DPw=(M_w-18.02)/(180.16-18.02)$. $M_n$, $M_w$, $M_z$ show, from left to right, an increased sensitivity towards high molar masses (Figure 2) and can be used to calculate molecular dispersity $D_M$. $D_M$ is generally defined as $D_M = M_w/M_n$ but can also be expressed as $M_z/M_w$ ratio when looking at the high-$M_r$ fraction (Vizárová et al. 2012). In this work, as the focus was set on the cellulose fraction and especially the long cellulose chains, dispersity was calculated from the deconvoluted cellulose molar masses as $D_M(C) = M_z(C)/M_w(C)$.

The analytical method was reproduced from Dupont (2003) and based on a three solvent exchange process: water/methanol/DMAc. The chromatographic system was from Malvern company, using the data collection software OmniSEC. Dual angle laser light scattering detectors Viscotek 270 (RALS/LALS) were coupled with a Viscotek VE 3580 Refractive Index (RI) detector. Two PL gel Mixed-A LS (20 µm porosity, 300x7.5mm) columns preceded by a guard column (10 µm porosity, 50x7.5mm) from Agilent were used as stationary phase. SEC chromatograms were smoothed (Savitsky-Golay) with SciDAVis.
<figure><img src="image_4.png" /><figcaption>Figure 2 - MMD of Fb pulp. The left and right peaks represent the hemicelluloses (HC) and cellulose (C) fractions of the pulp, respectively, which have been deconvoluted by pseudo-Voigt functions. Number-average Mn, weight-average Mw, and zeta-average Mz are represented on the logarithmic scale, as well as their mathematical formula.</figcaption></figure>

DPw(C) calculated on the cellulose fraction were obtained after peaks deconvolution (Figure 2) performed on SciDAVis software using a double or triple Gaussian model (for bimodal cellulose fraction). For highly degraded samples we used the pseudo-Voigt function pV(x) that embodies both Gaussian and Lorentzian profiles relevant in SEC chromatograms (Hopgood et al. 2019, Weißpflog et al. 2021):

$$ \frac{dWF}{dLog(M)} = pV(x) = Am_n \left( k_n \frac{\sqrt{\ln(2)}}{w_n \sqrt{\pi}} + \frac{1-k_n}{\pi w_n} \right)^{-1} \left( k_n \frac{\sqrt{\ln(2)}}{w_n \sqrt{\pi}} \exp \left( -4 \ln(2) \times \left( \frac{x-xc_n}{w_n} \right)^2 \right) + \frac{1-k_n}{\pi w_n \left( 1+4 \left( \frac{x-xc_n}{w_n} \right)^2 \right)} \right) + Am_{n+1} \dots $$

Where x is Log(M), k_n the degree of Gaussian or Lorentzian profile of peak n (0<k_n<1), Am_n the amplitude of peak n, w_n the full width at half maximum of the peak n, xc_n the Log(M) value of the centre of peak n.

## 2.4. Other characterisations

The single fibre tensile strength was measured in zero-span experiments on never-dried dewatered pulp pads (unrefined pulp). These experiments have been performed by the Pulp and Paper Research & Technical Centre (CTP, 38044 Grenoble, France) on never-dried pulp as it avoids hornification mechanism. Up to 80% cellulose in bleached pulp, the influence of hemicelluloses on the zero-span tensile index is deemed negligible (Page et al. 1985, Molin & Teder 2002). Furthermore, zero-span tensile measurements at the wet state would best represent chemical-induced local defects (Oksanen et al. 1997, Bennington et al. 1999).
The analysis of fibres morphological characteristics like fibre length, width, coarseness, curl, kink, flexibility, fines etc. analysis of fibres was performed on a MorFi® analyser (TECHPAP, Grenoble, France). 40 mg (air-dry basis) of the pulp sample is dispersed in 1 L tap water within a 1.5 L Erlenmeyer until suspension is clear (no visible flocculation). The sample is then poured in a 1 L plastic beaker of the MorFi device. Measurements were carried out in triplicates. More information about the use of MorFi is described by Passas et al. (2001).

**3. Experimental evidence for the insufficiencies of the DPv**

Although a robust and widespread method, the DPv represents only an average value of the
cellulose degree of polymerisation. On the other hand, SEC allows for the description of the
MMD alongside the determination of the molecular weight averages M<sub>r</sub> = M<sub>n</sub>, M<sub>w</sub>, M<sub>z</sub>, and
therefore represents a more fitting analysis method than the sole DPv to understand
carbohydrates degradation patterns in cellulosic pulps (Emsley et al. 2000, Sjöholm et al.
2000). The first intent of this investigation was to compare the MMD of kraft pulps chemically
degraded down to the same DPv to see if different MMD evolution patterns would arise, on
both unbleached Ko and fully-bleached Fb pulps.

**3.1. MMD of bleached pulp samples degraded down to the same DPv**

Various treatments were performed to lower Fb pulp DPv to approximately 1150. These
treatments include endoglucanase (C<sub>endo</sub>) and Celluclast (C<sub>cell</sub>) cellulase hydrolyses,
hypochlorous acid HOCl oxidation (H), ozone followed by alkaline extraction (ZEo) or
hydrogen peroxide (ZP), and hydrogen peroxide stage at 100°C (P<sub>100C</sub>). Details of the
experimental conditions are given in the Materials & Methods section. The resulting
degraded Fb pulp samples showed identical MMD and minor differences in DP values (Table
3). The only exception was pulp mix Fb<sup>40%</sup>+Fb-Z(0.5)Eo<sup>60%</sup>, represented in Figure 3 along Fb-
H(0.6) pulp. This sample was a blend made with 40% of starting pulp Fb and 60% of ozonated
Fb-Z(0.5)Eo pulp. Compared to other samples, this pulp had a higher cellulose fraction
dispersity D<sub>M</sub>(C) (Table 3). This is a direct example of the superior level of details granted by
SEC over pulp viscosity measurements.
<figure><img src="image_5.png" /><figcaption>Figure 3 - mass distributions of hypochlorous acid- and blended ozonated/starting pulps of equal DPv (~1150). Fb is the starting fully-bleached kraft pulp.</figcaption></figure>

<table><caption>Table 3 - DP values and dispersity of the cellulose fraction of bleached pulp FB degraded at ~1150 and ~850 DPv values by various treatments.</caption><thead><tr><td>Pulp sample</td><td>DPv</td><td>DPw(C)</td><td>Δ<sub>M</sub>(C)</td></tr></thead><tbody><tr><td>Fb</td><td>1370</td><td>4670</td><td>2.1</td></tr><tr><td>Fb-P<sub>100C</sub></td><td>1140</td><td>3850</td><td>2.0</td></tr><tr><td>Fb-H(0.6)</td><td>1150</td><td>3880</td><td>2.3</td></tr><tr><td>Fb-Z(0.17)Eo</td><td>1170</td><td>4290</td><td>2.0</td></tr><tr><td>Fb<sup>40%</sup>+Fb-Z(0.5)Eo<sup>60%</sup></td><td>1130</td><td>3550</td><td>2.7</td></tr><tr><td>Fb-C<sub>endo</sub>(20)</td><td>1100</td><td>3970</td><td>1.8</td></tr><tr><td>Fb-H(1.5)</td><td>830</td><td>2190</td><td>1.7</td></tr><tr><td>Fb-Z(0.35)P</td><td>860</td><td>3230</td><td>2.8</td></tr><tr><td>Fb<sup>30%</sup>+Fb-Z(1)Eo<sup>70%</sup></td><td>890</td><td>2960</td><td>2.9</td></tr><tr><td>Fb-C<sub>endo</sub>(80)</td><td>880</td><td>3630</td><td>1.8</td></tr><tr><td>Fb-C<sub>cell</sub>(10)</td><td>870</td><td>2520</td><td>1.8</td></tr></tbody></table>

At a lower DPv of ~850, differences between MMD appeared (Figure 4). Treatment H seemed to degrade the fibres more homogeneously than the ZP treatment leaving more long cellulose chains untouched and depolymerising hemicelluloses significantly (Figure 4a). These visual interpretations were confirmed by the Δ<sub>M</sub>(C) values reported in Table 3. The two cellulase treatments C<sub>endo</sub> and C<sub>cell</sub> changed the starting pulp MMD differently (Figure 4b). Endoglucanase (C<sub>endo</sub> treatment) was probably constrained to some parts of the fibres as some long cellulose chains remained intact, as observed on the high-<i>M</i><sub>r</sub> end of the MMD. This observation is in accordance with past studies on endoglucanase stating that their action would be mostly limited to fibres surface (Hildén et al. 2005). On the other hand, the Celluclast® cocktail could penetrate the fibres down to their core (Cheng et al. 2011), ultimately breaking them as well as forming flocculated debris (see MorFi analyses in Table 4 and SEM micrographs in Supplementary file) (Jackson et al. 1993). Part of the low-<i>M</i><sub>r</sub>
material generated during the hydrolyses such as short polysaccharides, oligosaccharides and monomeric sugars was probably lost during the pulp washing. The MMD thus represent the insoluble fibre fraction of these enzymatic hydrolyses. Discrepancies between DPv and DPw(C) values (Table 3) are briefly discussed in the Supplementary file.

<figure><img src="image_6.png" /><figcaption>Figure 4 - MMD of Fb pulp degraded samples at DPv ~850 with Z(0.35)P and H(1.5) (a) and enzyme-treated with either pure endoglucanase (80 U/g) or cellulase cocktail Celluclast® (10 U/g) (b).</figcaption></figure>

It is interesting to note that a purely homogeneous degradation of paper cellulose, ie. randomly cleaving carbohydrates chains, would provoke a decrease of the dispersity down to D<sub>M</sub> = 2, whereas values below 2 would point out preferential scissions at the middle of the chain (Guita et al. 1990). Here, the two cellulase hydrolysed samples, and Fb-H(1.5) have cellulose deconvoluted dispersity values D<sub>M</sub>(C) < 2 although it can be assumed that the enzymatic hydrolysis targeted specific spots of the fibres with a preference for short chains (Bragatto et al. 2012). Here, it is probably the loss of very short cellulose chains with pulp washing that yield more monodisperse cellulose MMD instead of an expected increase of
dispersity. Hence, at first glance, data derived from MMD do not seem enough *per se* for characterising the homogeneity degree of cellulose depolymerisation occurring within fibres. However, using MMD for monitoring the effect of each depolymerising chemical can help one to make assumptions on the topochemical aspects of the degradation.

## 4. Evolution of pulps molar mass distributions with depolymerisation treatments

A better understanding of how each cellulose-degrading chemical affects the fibres can be obtained by making series of pulps degradations and analyse the resulting MMD. Knowledge of the chemical degradation mechanisms coupled with interpretation of the MMD and some optical tools (e.g. SEM micrographs available in Supplementary file, MorFi fibre analysis results in Table 4) can help scientists to make reasonable conclusions regarding the topochemistry at play.

<table><caption>Table 4 - Fibres characteristics from various pulp samples collected on the MorFi analyser</caption><thead><tr><th>Sample</th><th>DPv</th><th>fibre length (µm)</th><th>Broken fibres content (%)</th><th>kinked fibres (%)</th><th>curl (%)</th><th>Macro-fibrillation index (%)</th></tr></thead><tbody><tr><td><strong>Fb</strong></td><td><strong>1370</strong></td><td><strong>820</strong></td><td><strong>28</strong></td><td><strong>75</strong></td><td><strong>19</strong></td><td><strong>1.3</strong></td></tr><tr><td>Fb-Ccell(10)</td><td>870</td><td>630</td><td>35</td><td>69</td><td>16</td><td>1.4</td></tr><tr><td>Fb-Ccell(50)</td><td>-</td><td>350</td><td>49</td><td>50</td><td>15</td><td>3.2</td></tr><tr><td>Fb-Cendo(30)</td><td>990</td><td>830</td><td>27</td><td>70</td><td>15</td><td>1.4</td></tr><tr><td>Fb-H(4)</td><td>250</td><td>830</td><td>28</td><td>74</td><td>18</td><td>1.4</td></tr><tr><td>Fb-Z(0.35)Eo</td><td>1060</td><td>800</td><td>26</td><td>73</td><td>19</td><td>1.3</td></tr><tr><td>Fb-Z(1)Eo</td><td>740</td><td>830</td><td>26</td><td>70</td><td>16</td><td>1.2</td></tr><tr><td><strong>Ko</strong></td><td><strong>1580</strong></td><td><strong>770</strong></td><td><strong>29</strong></td><td><strong>66</strong></td><td><strong>16</strong></td><td><strong>1.7</strong></td></tr><tr><td>Ko-Ccell(35)</td><td>-</td><td>380</td><td>48</td><td>54</td><td>16</td><td>3.1</td></tr><tr><td>Ko-Z(0.5)Eo</td><td>960</td><td>830</td><td>27</td><td>71</td><td>16</td><td>1.1</td></tr><tr><td>Ko-Z(4)Eo</td><td>330</td><td>690</td><td>31</td><td>67</td><td>16</td><td>1.9</td></tr><tr><td>Ko-Z<sub>alk</sub>(1.3)E</td><td>800</td><td>800</td><td>27</td><td>72</td><td>17</td><td>1.3</td></tr><tr><td>TCF</td><td>1060</td><td>830</td><td>25</td><td>58</td><td>12</td><td>1.1</td></tr></tbody></table>

### 4.1. Chemical degradation of fully-bleached kraft pulp

The fully-bleached pulp Fb was degraded with ZEo, H, and enzymatic $C_{endo}$ treatments using increasing chemical charges. When introduced on fully-bleached pulps, ozone is assumed to directly depolymerise carbohydrates and form some carbonyl groups leading to a further depolymerisation in the subsequent alkaline extraction stage via a β-elimination mechanism. In the acidic conditions of Z (pH 2.5) it is suspected that ozone mostly attacks accessible cellulose, probably the more amorphous regions as well as fibrils surface
(Johansson & Lind 2005). For low ozone doses - up to 0.5% on o.d. pulp - ZEo did not cut in the long cellulose chains and so the cellulose fraction lost its true gaussian profile (Figure 5a). For an ozone dose as high as 1%, the long chains were also affected. This was confirmed by using a tri-pVoigt deconvolution model which exposed the presence of two cellulose fractions C<sub>High-Mr</sub> and C<sub>Low-Mr</sub> in those pulps (Figure 5b). This suggests that some cellulose chains are more difficult for ozone to access than others and that others are just purely unavailable. The intact cellulose fraction may represent more ordered regions with higher average crystallinity. Another interesting point is that the ozone-cleaved cellulose chains represented by the C<sub>Low-Mr</sub> peak seem to be preserved from further depolymerisation by ozone (Figure 5b), even for higher ozone doses (unshown results). This result points out the difficulty for ozone to degrade cellulose crystallites.

<figure><img src="image_7.png" /><figcaption>Figure 5 – MMD of ozone-treated Fb pulp in Fb-ZEo experiments. (a) Set of Fb-ZEo pulps with increasing ozone doses: 0%, 0.17%, 0.5%, and 1% on o.d. pulp. (b) Deconvolution of the cellulose fraction of Fb-Z(0.5)Eo and Fb-Z(1)Eo show the existence of two distinct peaks of different molecular weight: C<sub>High-Mr</sub> in green and C<sub>Low-Mr</sub> in red.</figcaption></figure>

Contrarily to ozone oxidation performed at very acidic pH thus strongly limiting
ozone decomposition into radicals, hypochlorous acid affects cellulose via both ionic and
radical pathways (Zhou et al. 2008). We assume that it is the effect of radicals that allowed
the H treatment to provoke such a contraction of the high-$M_r$ end of the MMD on Figure 6. The attack of crystalline cellulose domains would give access to hitherto untouched amorphous cellulose regions, further increasing the extent of the oxidation. Noticeably, the H treatment generated a lot of low $M_r$ material compared to ZEo. This is probably due to the absence of an alkaline stage that might wash off part of the cleaved polysaccharides.

<figure><img src="image_8.png" /><figcaption>Figure 6 - MMD of hypochlorous acid-treated Fb pulp with increasing HOCl doses: 0%, 0.6%, 4%, 5%. The presence of high-Mr cellulose chains can be observed, although for the most degraded samples this cellulose fraction seems to be gradually attacked.</figcaption></figure>

Results of the enzymatic treatment $C_{endo}$ shown on Figure 7 showed the limited cellulose availability for endoglucanase. Increasing the enzymatic dose seemed to mostly affect the low-$M_r$ cellulose chains, illustrating the steric hindrance of cellulase (Bragatto et al. 2012).

<figure><img src="image_9.png" /><figcaption>Figure 7 - MMD of Fb pulp hydrolysed by endoglucanase (20 to 80 U/g of o.d. pulp).</figcaption></figure>

## 4.2. Chemical degradation of O-delignified kraft pulp
The O-delignified kraft pulp Ko contained approximately 1.5% lignin in weight, according to its Kappa number (Table 1). This small amount of lignin embodies a major difference in the reactivity of the chemicals previously applied on the fully-bleached pulp Fb.

For instance, in the case of ozone the presence of lignin implies an inevitable depolymerisation of polysaccharides throughout the *in-situ* generation of free radicals, rather than by a direct attack of ozone (Kang et al. 2001). The MMD of the Ko-ZEo series in Figure 8a show the apparition of a shoulder in the high-*M*<sub>r</sub> end with increasing ozone charge. This is foreshadowing the presence of a *C*<sub>High-Mr</sub> peak. But unlike that of the Fb-ZEo pulps, the peak is now contracting towards lower *M*<sub>r</sub> values with increasing ozone dose. The similarity with the effect described for the Fb-H series (Figure 6) may confirm that the contraction of the *C*<sub>High-Mr</sub> peak is a phenomenon attributed to the cleavage of cellulose crystallites. Indeed, this contraction of the high-Mr end was also observed for Fb-Ccell pulps (Figure 4b), with Celluclast® having the ability of attacking crystalline regions (Suurnäkki et al. 2000). We assume that this high *M*<sub>r</sub> cellulose is mostly present in the fibre outer parts (P+S1, S3, outer S2) as ozone bleaching follows a shrinking-core reaction model (Bennington et al. 1999, tran 2006), which was further exemplified by the macrofibrillation index of Ko increasing with the ozone charge (Table 4). Alternatively, “Z<sub>alk</sub>”, an ozone treatment in alkaline conditions was performed (pH 12.5), followed by alkaline extraction E and illustrated in Figure 8b. At this pH, the oxidation mechanisms taking place are almost exclusively due to free radicals issued from the rapid ozone decomposition. During the Z<sub>alk</sub>(1.3)E treatment the very reactive radicals unselectively affected polysaccharides in their immediate vicinity. The resulting MMD was somehow similar to that of Ko-Z(2)Eo sample but seemed to hold longer cellulose chains. In fact, the Z<sub>alk</sub>E treatment produced a pulp with the highest Φ<sub>M</sub>(C) value by far in this work (3.4 vs. maximum 2.8 for other samples, see Supplementary file for more details). This result is rather surprising since one could expect a more homogeneous degradation such as HOCl oxidation, due to the high fibre swelling at alkaline pH and the presence of radicals that should provoke random chain scissions. A possible explanation is that lignin being a good radical scavenger the *C*<sub>High-Mr</sub> peak in Ko-Z<sub>alk</sub>(1.3)E would, in this case, represent protected cellulose chains which are adjacent to residual lignin in Ko rather than cellulose located in highly organised domains.
<figure><img src="image_10.png" /><figcaption>Figure 8 – MMD of ozone-treated Ko pulp in KO-ZEo experiments. (a) Set of Ko-ZEo pulps with increasing ozone doses: 0%, 0.15%, 0.5%, 2% and 4% on o.d. pulp. (b) Comparison of similar MMD: Ko-Z<sub>alk</sub>(1.3)E (DPv 800) and Ko-Z(2)Eo (DPv 520).</figcaption></figure>

Interestingly, the H treatment degraded less the low-$M_r$ polysaccharides on the O-delignified Ko pulp than on fully-bleached Fb pulp (cf. Figure 9), a possible consequence of the oxidation quenching effect of lignin.
<figure><img src="image_11.png" /><figcaption>Figure 9 - MMD of hypochlorous acid-treated KO pulp in KO-H experiments with increasing HOCl doses: 0%, 2% and 4%.</figcaption></figure>

**5. Consequences on fibres and paper strength**

For this attempt at unveiling the link between cellulose DP and fibre strength, the wet zero-span tensile index was chosen as a representation of single fibre strength and performed on never-dry pulp samples. All the zero-span results have been plotted against either DPv or deconvoluted DPw(C) for the cellulose fraction (Figure 10). As expected, the two cellulase-treated pulps Fb-Ccell and Fb-Cendo fibre strength was significantly reduced despite a mild DPv loss, due to the localised nature of the degradation at the macro level. Overall, trends encompassing most data points were observable.

The degraded Fb pulps set showed a curvilinear relationship between zero-span tensile index and DPv while the Ko pulps set trend looked linear (Figures 10a and 10b, respectively). Both Fb and Ko degraded samples followed a linear trend with deconvoluted DPw(C), down to a critical value of ~1300. This is equivalent to DPv 590, hence lower than the 885 value recently reported by Brogdon and Lucia (2023). Past this threshold, only the two most degraded samples that had been oxidised by hypochlorous acid showed very low zero-span tensile values: Fb-H(4) and Fb-H(5) (details given in Supplementary file).

The most intriguing result of this study was the low strength of Ko-ZalkE: its zero-span tensile index value was remarkably below those reported for other degraded Ko samples (Figures 10b and 10c). Looking back at its MMD presenting a large fraction of relatively intact cellulose (Figure 8c), the low fibre strength can be regarded as an anomaly as it is contradicting a previous theory: the presence of a high-Mr cellulose fraction under the bimodal MMD of cellulose in pulp would be associated with retained mechanical properties despite an average DP loss (Berggren et al. 2003). It is plausible that it is actually the location of this high-Mr cellulose fraction within the fibre that matters. As detailed before, the cellulose least depolymerised by ZalkE treatment might be close to lignin fragments in Ko, ie. distributed along the cell wall layers. S2, the layer responsible for the high tensile strength
of fibres with its low microfibrillar angle, is the thickest layer in wood fibres (Wathén 2006). For this reason we assume that contrarily to Ko-ZEo pulps being more damaged on the fibre surface, a substantial part of the S2 cellulose of Ko-Z<sub>alk</sub>(1.3)E has been depolymerised by radicals so it could explain the bad mechanical performance of this sample.

<figure><img src="image_12.png" /><figcaption>Figure 10 – Zero-span tensile values for a wide diversity of never-dried degraded pulp samples: bleached pulp Fb (a) and O-delignified pulp Ko (b) samples plotted against the pulp DPv, or the deconvoluted SEC-issued cellulose fraction DPw(C) (c). The TCF pulp was bleached according to A-ZEo-ZEo-ZP sequence presented in the Materials and Methods section.</figcaption></figure>
Looking back to the first figure presented in this article, Ko-ZEo degradation type would be embodied by the crossed green dots concentrated in the outer parts of the fibre with limited consequence on fibre strength (Figure 1). Conversely, purple dots more evenly distributed in the cell wall layers would illustrate the Ko-Z<sub>alk</sub>E degradation and is associated with a decrease of the zero-span tensile index due to the major role of S2 layer on tensile strength. These interpretations cannot be fully demonstrated with SEC and would require complementary analyses. Nano-indentation measurements on fibres cross-sections could be relevant in that case (Adusumalli et al. 2014).

**6. Conclusions**

It has been shown through a handful of studies in the last decades that the homogeneous or localised nature of the cellulose depolymerisation taking place within fibres when subject to various treatments (pulping, bleaching, aging...) is the driving factor regarding fibre strength deterioration, rather than the cellulose average degree of polymerisation (DP). Here, a fully-bleached hardwood kraft pulp has been depolymerised with various chemicals to two DP levels: from initial DP 1370 down to 1150 and 850. Differences in MMD shapes only appear at the lower DP level, while giving no clear indication on the topochemistry of the degradation due to opposite mechanisms affecting cellulose peak dispersity.

However, looking at how the MMD evolve with an increased chemical charge for each treatment (ozone, hypochlorous acid, cellulase) applied on fully-bleached and O-delignified pulps can be relevantly correlated to existing knowledge of these chemicals to make reasonable assumptions on the types of degradation they inflict to fibres.

Linking these visual degradation patterns to fibre strength evaluated by zero-span tensile measurements further corroborate some conclusions from the past. Ozone and hypochlorous acid treatments which affect almost indifferently the whole of the fibre have less impact on fibre strength than localised attacks like those of cellulase enzymes, even though the DP can drop significantly. The superior level of details given by SEC over simple pulp viscosity measurements proves that both ozone and hypochlorous acid leave a bimodal cellulose fraction, with some cellulose being relatively intact. This fraction had hitherto been thought responsible for maintaining fibre strength despite the DP loss. Our new results include an unconventional ozone treatment performed in alkaline conditions and resulting a substantial fibre strength loss. Consequently, we believe that it is the degree of degradation of the fibre core (S2 layer) that would rule kraft fibre strength deterioration through various chemical treatments. This could explain why conventional ozone bleaching, by depolymerising cellulose mostly located in the fibres surface, does not cause substantial strength loss.

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