Jonathan Leboucher

Philippe Bazin

Didier Goux

Hussein El Siblani

Arnaud Travert

Antoine Barbulée

Joel Bréard

Benoît Duchemin

Benoit Duchemin
email: benoit.duchemin@univ-lehavre.fr

High-yield cellulose hydrolysis by HCl vapor: cocrystallization, deuterium accessibility and hightemperature thermal stability

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Cellulose nanocrystals (CNC) are needle-like organic crystals that can be extracted from the lignocellulosic biomass. CNC may be useful in multiple applications. For instance, they can reinforce polymer matrices and improve their stiffness, damping, oxygen barrier properties or provide shape-memory effects. [START_REF] Eichhorn | Review: current international research into cellulose nanofibres and nanocomposites[END_REF][START_REF] Klemm | Nanocelluloses: A New Family of Nature-Based Materials[END_REF][START_REF] Dufresne | Nanocellulose: From Nature to High Performance Tailored Materials[END_REF][START_REF] Lourdin | Concentration driven cocrystallisation and percolation in all-cellulose nanocomposites[END_REF] Multiple applications in the field of functional materials exist: CNC can for instance be used as photonic materials, biocatalysts, conductive materials, biosensors or crack-healing drilling fluids. [START_REF] Lam | Applications of functionalized and nanoparticlemodified nanocrystalline cellulose[END_REF][START_REF] Lin | Preparation, properties and applications of polysaccharide nanocrystals in advanced functional nanomaterials: a review[END_REF] The mass-production of CNC is limited by the yield of current production methods. The most common preparation method of CNC is a sulfuric acid hydrolysis. Initially, this process was proposed by Rånby 70 years ago [START_REF] Rånby | Aqueous Colloidal Solutions of Cellulose Micelles[END_REF] and has since been improved to enhance the final yield of CNC. For instance, yields as high as 66-69% for wood-based CNC were expected, based on the results of a central composite design study. [START_REF] Dong | Analysis of the sulfuric acid hydrolysis of wood pulp for cellulose nanocrystal production: A central composite design study[END_REF]) A predicted maximum yield of 54% was announced for CNC made from microcrystalline cellulose using surface response methodology. [START_REF] Bondeson | Optimization of the isolation of nanocrystals from microcrystalline cellulose by acid hydrolysis[END_REF] It has also been demonstrated that other acids could successfully hydrolyze cellulose. For instance, hydrochloric acid, formic acid, phosphoric acid, bromhydric acid or phosphotungstic acid can be used. [START_REF] Nevell | The hydrolysis of cotton cellulose by hydrochloric acid in benzene[END_REF]Habibi et al. 2010;[START_REF] Yu | Facile extraction of thermally stable cellulose nanocrystals with a high yield of 93% through hydrochloric acid hydrolysis under hydrothermal conditions[END_REF][START_REF] Espinosa | Isolation of Thermally Stable Cellulose Nanocrystals by Phosphoric Acid Hydrolysis[END_REF][START_REF] Liu | A novel approach for the preparation of nanocrystalline cellulose by using phosphotungstic acid[END_REF][START_REF] Li | Cellulose nanocrystals prepared via formic acid hydrolysis followed by TEMPO-mediated oxidation[END_REF][START_REF] Du | Preparation and characterization of thermally stable cellulose nanocrystals via a sustainable approach of FeCl3-catalyzed formic acid hydrolysis[END_REF] In particular, the yield of CNC produced using FeCl3-catalyzed formic acid can reach values of 75% and the yield of CNC using an hydrothermal hydrolysis pathway in HCl can reach 93%. However, a recent breakthrough regarding the yield of the acid hydrolysis was recently reported. [START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF][START_REF] Lorenz | Cellulose nanocrystals by acid vapour: towards more effortless isolation of cellulose nanocrystals[END_REF][START_REF] Pääkkönen | From vapour to gas: optimising cellulose degradation with gaseous HCl[END_REF]) Instead of using conventional "wet-chemistry" reaction pathways, this method exposes cotton cellulose to a vapor of HCl. An impressive yield of 97-99% could be achieved and the neutralization of the acid was tremendously simplified. In comparison with the classical sulfuric acid method, HCl hydrolysis produces CNC that have a lower amount of surface charges, which leads to an unstable colloidal dispersion but to a better thermal stability of the dry particles [START_REF] Araki | Flow properties of microcrystalline cellulose suspension prepared by acid treatment of native cellulose[END_REF][START_REF] Klemm | Nanocelluloses: A New Family of Nature-Based Materials[END_REF][START_REF] Dufresne | Nanocellulose: From Nature to High Performance Tailored Materials[END_REF][START_REF] Fang | Noncovalent Dispersion and Functionalization of Cellulose Nanocrystals with Proteins and Polysaccharides[END_REF][START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF][START_REF] Lorenz | Cellulose nanocrystals by acid vapour: towards more effortless isolation of cellulose nanocrystals[END_REF]. The HCl vapor process also uses less water and practically solves the issues surrounding the recycling of the acid at the end of the process. [START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF][START_REF] Lorenz | Cellulose nanocrystals by acid vapour: towards more effortless isolation of cellulose nanocrystals[END_REF][START_REF] Niinivaara | The Effect of Polymorphism on the Kinetics of Adsorption and Degradation: A Case of Hydrogen Chloride Vapor on Cellulose[END_REF] The hydrolysis of cellulose with HCl needs to be well-understood in order to control some important parameters such as the dimensions of the CNC or their redispersibility in various solvents. [START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF][START_REF] Lorenz | Cellulose nanocrystals by acid vapour: towards more effortless isolation of cellulose nanocrystals[END_REF]) So far, it has been demonstrated that HCl hydrolysis in the vapor phase induces the adsorption of a monolayer of HCl molecules on the surface of the solid cellulosic substrate, at a concentration above the water solubility limit. [START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF][START_REF] Niinivaara | The Effect of Polymorphism on the Kinetics of Adsorption and Degradation: A Case of Hydrogen Chloride Vapor on Cellulose[END_REF][START_REF] Pääkkönen | From vapour to gas: optimising cellulose degradation with gaseous HCl[END_REF]) However, the presence of residual water is paramount to dissociate the HCl molecules and proceed with the hydrolysis. [START_REF] Pääkkönen | From vapour to gas: optimising cellulose degradation with gaseous HCl[END_REF]) Hydrolysis consists in a scission of the cellulose chains and it yields a marginal quantity of glucose. Since there is no mass transfer during the exposure to the acidic vapor, the glucose is rinsed away when the hydrolysis is quenched by immersing the substrate in water. [START_REF] Niinivaara | The Effect of Polymorphism on the Kinetics of Adsorption and Degradation: A Case of Hydrogen Chloride Vapor on Cellulose[END_REF] It is also well established that the degree of polymerization (DP) decreases non-linearly with the duration of the hydrolysis. The depolymerization kinetics is well-described by a two-stage model (first order kinetic). [START_REF] Calvini | On the kinetics of cellulose degradation: looking beyond the pseudo zero order rate equation[END_REF][START_REF] Pääkkönen | From vapour to gas: optimising cellulose degradation with gaseous HCl[END_REF] In the early stages of the hydrolysis, the DP undergoes a fast decrease that is mostly assigned to the hydrolysis of the 1,4 glycosidic bonds of the readily accessible regions. [START_REF] Battista | Level-Off Degree of Polymerization[END_REF][START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF][START_REF] Niinivaara | The Effect of Polymorphism on the Kinetics of Adsorption and Degradation: A Case of Hydrogen Chloride Vapor on Cellulose[END_REF]) After long hydrolytic processes, the amount of chain scission reach a plateau value that is characterized by a "level-off DP" (LODP). [START_REF] Battista | Level-Off Degree of Polymerization[END_REF][START_REF] Calvini | The influence of levelling-off degree of polymerisation on the kinetics of cellulose degradation[END_REF][START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF] An increased exposure to HCl leads to a slow DP reduction consistent with an endwise depolymerization of the low DP paracrystallites or segments. [START_REF] Pääkkönen | From vapour to gas: optimising cellulose degradation with gaseous HCl[END_REF]) Kinetic studies can be completed by microscopy studies. It was demonstrated by AFM that HCl hydrolysis performed on carboxylated cellulose nanofibrils occurs preferentially at kinks. [START_REF] Nyström | Nanocellulose Fragmentation Mechanisms and Inversion of Chirality from the Single Particle to the Cholesteric Phase[END_REF] Kinks in cellulose nanofibers are best described as crystalline defects, rather than amorphous regions. [START_REF] Nishiyama | Periodic Disorder along Ramie Cellulose Microfibrils[END_REF][START_REF] Usov | Understanding nanocellulose chirality and structure-properties relationship at the single fibril level[END_REF] Therefore, previous results agree on the deterministic nature of HCl hydrolysis: this type of hydrolysis will preferentially target the water-accessible regions and the kinks that are located along the nanofibril length. On the other hand, it is still unclear whether a possible recrystallization of cellulose when exposed to an HCl vapor could play a role. This work hypothesis was put forward to explain the high yields of the HCl vapor method, the slight crystallinity increase and the moderate crystallite thickening observed by solid-state NMR and X-ray diffraction. [START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF] It is indeed possible to envisage some level of recrystallization once the fibrillar structure of the cellulosic substrate is hydrolyzed: chains that were previously structurally constrained become free to reorder once split into smaller fragments. Nevertheless, the moderate crystallinity increase of the product after it was fragmented to CNC and the exceptionally high yield of the method challenge our conception of CNC: should CNC be considered as single nanocrystals, or as rigid, hydrolytically stable fragments bearing some structural disorder?

In the present work, cellulose nanocrystals were produced with a high yield from chlorinefree bleached flax shives, using the HCl vapor method proposed by [START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF] In the past, CNC have been produced from unbleached flax shives -vascular bundles (phloem and xylem) akin to normal wood tissues -using the ammonium persulfate method. [START_REF] Leung | Characteristics and Properties of Carboxylated Cellulose Nanocrystals Prepared from a Novel One-Step Procedure[END_REF]) . The hydrolysis time was varied and the resulting nanoparticles were characterized in terms of width, length and thickness using TEM and AFM. WAXD was used to determine the size and shape of the nanocrystals under the assumption of a bimodal size distribution and taking into account possible Iα/Iβ stacking faults. Eventually, an isotopic exchange monitored by an ATR-FTIR analysis from room temperature to 260°C was performed in order to quantify the water accessibility and the structural changes undergone by the cellulose during this HCl hydrolysis. To the best of our knowledge, the combination of isotopic labelling and high-temperature FTIR analysis has never been performed on such materials. The production of uncharged cellulose nanoparticles with a high yield gives us an opportunity to gain further understanding as to the novel production method using gaseous HCl as well as a better insight into the nanocellulose structure itself.

Experimental section

Materials

Flax shives were provided by SAS Teillage Vandecandelaere (Depestele company), located in Bourguébus (Normandy, France). Flax shives were ground and sieved with a 315 µm mesh. The flax shives were bleached and the procedure as well as the analytical results (POM, SEM, WAXD, ATR-FTIR and 13 C CP/MAS ss-NMR) are given in supplementary information. Deuterium oxide (D2O, 99.8% atom D, Roth), toluene (≥ 99.5%, AnalaR normapur, VWR), absolute ethanol (≥ 99.5%, empura, Merck), sodium hydroxide (NaOH, ≥ 99%, Carl Roth), hydrogen peroxide (35 %, stabilized aqueous solution, Alfa Aesar), hydrochloric acid (32% for analysis, emsure, Merck) and deionized water (18.2 MΩ*cm) were used as received. Filtrations were made on filter paper (Whatman n°1) unless otherwise stated. Glass fiber filters (90 mm diameter, MN85/70, Macherey-Nagel) and nylon filters (0.45 µm pore size, 90 mm diameter, Whatman) were also used for filtration purposes.

Methods

Acid hydrolysis

Bleached flax shives were suspended in deionized water (2.1 wt. %) and mixed with an Ultraturrax (IKA) for 3 minutes at 7,000 rpm and 2 minutes at 11,000 rpm. The suspensions were filtered on a nylon filter in order to produce a film with an even thickness for subsequent hydrolysis. After filtration, the films were dried at ambient conditions under a fume hood. The films were then separated from the nylon filter. The bottom of a vacuum desiccator was filled with hydrochloric acid (80 ml in a desiccator 100 mm in diameter). In order to reach an equilibrium (PHCl = 2.9 kPa), the valve of the desiccator was left open for 2 days. It was then shut one day before the start of the hydrolysis experiment. The cellulose films (~1.0 g; water content: 30 ± 6 wt. %) were introduced in the desiccator (Table S2). Hydrolysis was performed for 30 min, 1 h, 2 h, 4 h and 8 h. A chemical quench was performed in deionized water (250 mL) in order to stop the hydrolysis. The hydrolyzed material was then filtered and suspended again in 250 mL of deionized water. The suspension was dispersed with high intensity ultrasounds (Bioblock Scientific, model vibracell 75115, 500 W, 20 kHz) set at 40% output power with 100 min of treatment time (3 s on, 2 s off). The average energy transmitted to the solution was 171 ± 5 kJ. A sample of reference (control sample) was also produced using the same protocol with the exception of the hydrolysis.

Characterization X-ray diffraction Measurements

Powder diffractograms were acquired in reflection mode with a θ-θ Bragg-Brentano configuration using an Xpert-Pro Powder (PANalytical B.V., Almelo, Nederland) with a cobalt radiation source (λCoKα = 1.790307 Å) and a PIXcel 1D detector. The operating voltage and current were maintained at 40 kV and 40 mA, respectively. Measurements were made between 10 and 80° with 0.0131° steps and for a total duration of 8 h. The lateral divergence of the incident beam path was controlled with 0.04° Soller slits and a 10 mm mask. The divergence in the 2.θ plane was controlled with a ½° fixed slit.

Anisotropic size broadening

The results were analyzed using a Rietveld method that was adapted from a recently published work. (Duchemin 2017b) This method describes the cellulose Iβ crystallites as anisotropic crystallites with a deterministic shape and size in the direct space. The method was slightly modified in the present work to provide a more accurate three-dimensional description of the crystallites. The anisotropic size distribution is described in the direct space by a parametric superellipsoid hull obeying the following equation set:

𝑥 = 𝑟 𝑥 . 𝑠𝑖𝑔𝑛(𝑐𝑜𝑠𝜔. 𝑠𝑖𝑛). |𝑠𝑖𝑛| 𝑚 . |𝑐𝑜𝑠𝜔| 𝑛 (1) 𝑦 = 𝑟 𝑦 . 𝑠𝑖𝑔𝑛(𝑠𝑖𝑛𝜔. 𝑠𝑖𝑛). |𝑠𝑖𝑛| 𝑚 . |𝑠𝑖𝑛𝜔| 𝑛 (2) 𝑧 = 𝑟 𝑧 . 𝑠𝑖𝑔𝑛(𝑐𝑜𝑠). |𝑐𝑜𝑠| 𝑚 (3)
With 𝜔  [- ;  [ and  [- ;  [. The x axis (resp. z) is oriented along the crystallographic a (resp. c) axis. Additionally, m (resp. n) is the "squareness" parameter in the z (resp. x and y) axis. Values close to 0 describe a square shape, a value of 1 describes a perfect ellipsoid, a value of 2 describes a "diamond shape" (rhombus) and values > 2 create a recess between the corners of this rhombus; all intermediary values are adequate. (Duchemin 2017b) For each diffracting crystallographic plane (hkl), the diameter of the crystallites in the direction orthogonal to the plane is:

𝑑 ℎ𝑘𝑙 = 2√𝑥² + 𝑦² + 𝑧²
(4) The link between the FWHM of the Bragg band and dhkl is given by Scherrer equation with a value K = 0.9. (Guinier 1963;Duchemin 2017a) Therefore, a homothetic relationship is found between the 3D shape of the particles and the FWHM spectrum of the diffractogram. For each (hkl) pane, there exists a vector n hkl normal to this plane that obeys the equation:

𝒏 ℎ𝑘𝑙 = ( 𝑛 𝑥 ℎ𝑘𝑙 𝑛 𝑦 ℎ𝑘𝑙 𝑛 𝑧 ℎ𝑘𝑙 ) = 𝛼. ( 𝑥 𝑦 𝑧 ) (5)
The vector n hkl is a function of the lattice parameters of the Iβ phase and it is expressed in the direct space ℝ 3 . The equation set is solved algebraically to determine α, ω and χ for each plane, as a function of the five superellipsoid parameters. Therefore, solving the size and shape problem of an experimental diffractogram requires determining the values of rx, ry, rz, m and n. This can easily be performed using the least-square method to minimize the difference between the experimental diffractograms and their models. The parametric shape representation was plotted using the matplotlib and numpy Python libraries.

Implementation and triphasic model

The signal was defined as the sum of three components: a background, small diffusing domains of cellulose IVI with no more than 2-3 chains in thickness, and larger cellulose Iβ crystallites, usually in the 25-60 Å diameter range. (Duchemin 2017b) The background comprised a Gaussian halo centered at 2.θ = 0° and a zero-order background; this form essentially captures the main background contributions: incoherent (Compton) scattering, coherent scattering and air scattering. [START_REF] Ramírez | Microcrystalline cellulose (MCC) analysis and quantitative phase analysis of ciprofloxacin/MCC mixtures by Rietveld XRD refinement with physically based background[END_REF] Cellulose IVI is selected as a disordered form of cellulose Iβ, as discussed in previous works. [START_REF] Marrinan | Infrared spectra of the crystalline modifications of cellulose[END_REF][START_REF] Wada | Polymorphism of Cellulose I Family: Reinvestigation of Cellulose IVI[END_REF][START_REF] Newman | Simulation of X-ray diffractograms relevant to the purported polymorphs cellulose IVI and IVII[END_REF]Duchemin 2017b) The preferred orientation of cellulose Iβ was modelled in a simple manner using the March-Dollase approach along the c axis. (Duchemin 2017b) Furthermore, the amount of Iα/Iβ stacking faults was estimated using the structure factor list published by Driemeier and Francisco in their seminal work using the Diffax software. [START_REF] Driemeier | X-ray diffraction from faulted cellulose I constructed with mixed Iα-Iβ stacking[END_REF] These structure factors were integrated in our analysis. The problem was solved on a desktop workstation using the Microsoft ® Excel Solver Add-in and the least-square method. The following parameters were refined for cellulose Iβ: lattice dimensions, monoclinic angle, March-Dollase strength, relative phase intensity, rx, ry, rz, m, n and the amount of stacking faults. The parameters relative to cellulose IVI crystallites that were refined were their relative amount and width of crystallites. As to the background, the height and width of the central Gaussian function and the zero-order function were refined. The refinements were run until a variation of the correlation coefficient < 1 ‱ between the experimental data and the simulated ones was obtained.

ζ potential The ζ potential was measured with a Zeta Sizer Nano-Z, with a Dip Cell (Malvern Instrument, Malvern UK). Measurements were repeated 5 times and the solid particle concentration amounted to 0.1 g.L -1 . The results were analyzed with the proprietary software (version 7.11, Malvern, UK) and Schmoluchowski equation.

Atomic force microscopy

The cellulose nanocrystals were imaged using an atomic force microscope (Innova, Brücker). Suspensions were diluted to 0.06 g.L -1 , a drop was cast on a freshly cleaved mica and the drop was allowed to dry. The scans were performed using the tapping mode in air. Ultrasharp tips (Tespa V2, Brüker, 320 kHz, 42 N/m, 7 nm tip radius) were used. The height of the CNC was measured using the software Nanoscope analysis (Brücker).

Transmission electron microscopy

The width and length of the CNC were measured by transmission electron microscopy using a Jeol JEM-1011 TEM equipped with a tungsten filament at 80.0 kV acceleration voltage.

Copper grids covered by a formvar film (0.5% in dichloromethane) were used. The grids were also placed in a glow discharge (Elmo, Cordouan Technologies) before suspension casting.

Each suspension (0.06 g.L -1 ) was dispersed with high intensity ultrasounds (Vibracell 75115) for 5 minutes (3s pulse on, 2s pulse off, 40% intensity, average energy: 13 367 J). One drop of 3 µl was then deposited on the grid. After 10 min, excess suspension was eliminated, 3 µl of uranyl acetate (1.5%) were put on the grid for 1 min in a black box. Excess was eliminated and the grids were dried under the fume hood. Two grids were made for each sample.

High temperature ATR-FTIR All the samples were studied with an FTIR spectrometer (Nicolet iS50 FT-IR, Thermo scientific) equipped with a temperature-controlled ATR cell (diamond ATR "golden gate high temperature", SPECAC). The measurements were performed by applying a 2°C/min temperature ramp from room temperature to 260°C and one spectrum was recorded every 40s. Each spectrum resulted from the accumulation of 64 scans at a 4 cm -1 resolution. The acquisition was done using the OMNIC (v.9.6) software. One spectrum was always acquired after a return to room temperature. The background was also measured at all temperatures on an empty cell in order to correct the temperature-induced background shifts. The plots of the raw data plots were performed using the Python libraries matplotlib and numpy. The data treatment was performed using the spectroscopy plugin implemented in Orange. [START_REF] Demšar | Orange: data mining toolbox in Python[END_REF] The measurements were performed on the non-hydrolyzed sample and on the hydrolyzed samples. They were performed on (i) control samples (ii) deuterated samples and (iii) deuterated samples that were further back-exchanged with ultrapure ordinary water. In order to perform the hydrogen-deuterium exchange, 0.1 g of never dried material (control sample and 8 h hydrolysis) was immersed in 5 mL of D2O in a 25 ml sealed glass bottle and the bottle was placed in an ultrasonic bath (VWR, 160 W) for 1 h. The suspension was then centrifuged and the supernatant was discarded. A fresh amount (5 mL) of D2O was then added to the solid and the mixture was left in the ultrasonic bath for 4 h. This duration was found to be sufficient to reach a complete exchange of the accessible groups. (Mann and Marrinan 1956a, b, c) The solid substrate was again isolated by centrifugation. It was then dried overnight under a constant flux of dry N2 (ultra-pure spectroscopy grade, purity > 99.999%) supplied by a Parker domnick hunter G1010E generator. Half of the dried deuterated mass was then swiftly placed in a chamber with a 75 % RH obtained by saturating D2O with NaCl for one week. The chamber was sealed and reopened only for FTIR measurement purposes. The other half of the sample was reprotonated in order to assess the amount of deuterium trapped by drying hysteresis. To do so, the deuteration procedure was simply repeated by replacing D2O with ultrapure H2O. The sample was also stored in a sealed container with a known relative humidity (75% H2O). Two "never deuterated" control samples were also produced by applying the same complete protocol with ultrapure H2O. A ratio of exchanged groups was calculated using the formula:

𝑟 𝑎𝑐𝑐𝑒𝑠𝑠 = 1.34 * 𝐴 𝜈 𝑂𝐷 1.34 * 𝐴 𝜈 𝑂𝐷 +𝐴 𝜈 𝑂𝐻 (6)
Where 𝐴 𝜈 𝑂𝐷 is the area of the spectrogram taken between 2800 and 2360 cm -1 and 𝐴 𝜈 𝑂𝐻 is the area of the spectrum taken between 3680 and 3050 cm -1 . The areas were measured after a linear baseline correction. 2-D correlation spectroscopy was not performed because of large band shifts. The formula is sometimes found without the 1.34 correction factor. [START_REF] Horikawa | Varietal difference in cellulose microfibril dimensions observed by infrared spectroscopy[END_REF][START_REF] Thybring | Hydroxyl accessibility in wood cell walls as affected by drying and re-wetting procedures[END_REF] This 1.34 factor stems from the νOH to νOD wavenumber shift after deuteration. [START_REF] Hofstetter | Moisture uptake in native cellulose -the roles of different hydrogen bonds: a dynamic FT-IR study using Deuterium exchange[END_REF][START_REF] Driemeier | Hydrated fractions of cellulosics probed by infrared spectroscopy coupled with dynamics of deuterium exchange[END_REF] Indeed, since the band intensity is proportional to this wavenumber, a true measure of raccess requires this intensity correction to be performed. (Mann and Marrinan 1956b;[START_REF] Janoschek | Calculated and observed isotope effects with easily polarizable hydrogen and deuterium bonds[END_REF] Results and discussions

Acid hydrolysis Yield, microscopy and ζ potential

The production of cellulose nanocrystals from bleached flax shives was performed with gaseous HCl for various durations, from 30 min to 8 h. The yields were characterized by a gravimetric method and were found to lie in the 89-92 wt.% range for all hydrolysis times (Table 1). A 2 wt.% weight loss was measured for the non-hydrolyzed sample which was attributed to the other processing steps (filtration, ultrasounds, etc). The nanocrystals were then observed using TEM and this technique was useful to assess their lengths and widths (Figure 1, left column). Generally speaking, the nanoparticles appeared as rods with "kinks" along their lengths. These kinks were observed in other works. [START_REF] Nishiyama | Periodic Disorder along Ramie Cellulose Microfibrils[END_REF][START_REF] Usov | Understanding nanocellulose chirality and structure-properties relationship at the single fibril level[END_REF][START_REF] Nyström | Nanocellulose Fragmentation Mechanisms and Inversion of Chirality from the Single Particle to the Cholesteric Phase[END_REF] Nanoparticles with widths in the 10-22 nm range and with average lengths ranging from 160 to 800 nm were observed (Figure 2). These values decreased with the hydrolysis time but they were found to be relatively stable after 2 h of exposure to HCl (Figure 2). The dispersion of the data also decreased with time, probably due to the fractionation of the most refractory particles after longer exposure times to the acid vapor.

The nanoparticles often appeared as aggregates of smaller nanocrystallites packed side-byside. These nanocrystals were about 2-4 nm in width. Small disc-like fragments in the 10-35 nm diameter range were also sometimes observed (Figure 1). Due to their shape, these fragments can be attributed to remnants of lignin. [START_REF] Beisl | Lignin from micro-to nanosize: production methods[END_REF] Atomic force microscopy can provide complementary information by giving an assessment of the height of the nanofragments as deposited on the mica substrates (Figure 1, right column).

The values before hydrolysis are not reported because of the bias induced by the remnants of large (micron-sized) unaffected shives coexisting with scarce submicron microfibrils produced by the ultrasound treatment. The height of the nanoparticles evolved from ~30 nm after 30 min of hydrolysis to ~12 nm after 8 h. As observed using TEM, the average dimension was relatively stable after a 2 h hydrolysis (Figure 2), but the statistical dispersion was lower at 4 and 8 h. A 24 h hydrolysis was also trialed and no statistically significant evolutions of the dimensions were observed relative to the 8 h hydrolysis (results not shown). Both sets of results (TEM and AFM) underline the strong efficiency of the HCl vapor method when one wants to produce cellulose nanoparticles in a simple manner. [START_REF] Leung | Characteristics and Properties of Carboxylated Cellulose Nanocrystals Prepared from a Novel One-Step Procedure[END_REF] reported the production of carboxylated cellulose nanocrystals from flax shives using ammonium persulfate (APS) in a one-pot method at 60°C for 16 h, without preliminary bleaching. [START_REF] Leung | Characteristics and Properties of Carboxylated Cellulose Nanocrystals Prepared from a Novel One-Step Procedure[END_REF] The nanocrystals produced in their work were thinner (5.1 ± 0.1 nm) and longer (296 ± 16 nm) at the expense of a lower yield: 22% when compared to 49% in this work (taking the bleaching step in consideration).

Crystallinity

Cellulose is often assessed in the light of X-ray diffraction. Yet, the analysis of the diffractograms is often simplified in the sense that the analyses are not based on a thorough physical model that would describe the state of the cellulosic matter. A first common method is the conventional peak fitting technique. This technique focuses on a small number of arbitrarily prominent peaks. Therefore, it largely ignores a large number of low to medium intensity diffraction rays. These rays are then mistakenly accounted for in the so-called amorphous background. [START_REF] Thygesen | On the determination of crystallinity and cellulose content in plant fibres[END_REF][START_REF] French | Cellulose polymorphy, crystallite size, and the Segal Crystallinity Index[END_REF][START_REF] Ahvenainen | Comparison of sample crystallinity determination methods by X-ray diffraction for challenging cellulose I materials[END_REF]Duchemin 2017b) Furthermore, the shape of the crystallites cannot be obtained using such a routine. The second method relies on the simulation of whole powder diffractograms using the Rietveld method.(De Figueiredo and Ferreira 2014; Ramírez and Bucio 2018) The Rietveld method is powerful, but it doesn't always implement an anisotropic line broadening routine that describes the size and shapes of non-spherical nanocrystals that display ample peak broadening and overlap. When it does, the implementation of the anisotropic size broadening is not necessarily well-adapted to the needle-like and convex shape of cellulose nanocrystallites. These issues are discussed in detail elsewhere. (Duchemin 2017b) In the present work, a parametric model is proposed in which the size and shape (assuming no microstrain contribution) of the native Iβ cellulose crystallites are modelled together and fitted to the experimental diffractograms. Five main parameters can be adjusted to provide simulated diffractograms of Iβ crystallites: three radii and two shape parameters, as described in the experimental section. Examples of simulated diffractograms and the corresponding crystallite shapes are illustrated in Figure 3. As expected, size and aspect ratio have a strong impact on the diffractograms (Figure 3a andb). It is also of specific interest to notice how the ( 110) and (11 ̅ 0) peaks are affected by the lateral shape (parameter n). Depending on n, the peaks can overlap or appear as two well separated Bragg rays (Figure 3c). The longitudinal shape parameter m influences the diffractogram in a subtler way by affecting the (102) ray and other reflections such as the (h k 3) reflections located above 2.θ = 30° (Figure 3d). These reflections are usually not analyzed when performing the analysis on powder patterns by curve fitting, demonstrating the potential use of this model if one wants to get the three-dimensional line-broadening information instead of the sole "cross-sectional" information contained in the three main diffraction bands (110), (11 ̅ 0) and (200). It is also possible to plot a discrete three-dimensional hull where each point is oriented perpendicular to a diffraction plane and at a distance from the origin equal to half the diameter of the crystallite in this direction (Figure S5). The model was then fitted to the experimental diffractograms of CNC produced in this work (Figure 4). The results show that the nanocrystals belong to the cellulose I family and remain fairly unaffected throughout the hydrolysis process in terms of shape (Table 2). The lateral shape of the crystallites was found to be that of a diamond (1.84 < n < 2.25), in accordance with the most common model currently employed for cellulose nanocrystals. [START_REF] Viëtor | A priori crystal structure prediction of native celluloses[END_REF][START_REF] Nishiyama | Diffraction from nonperiodic models of cellulose crystals[END_REF]) Interestingly, the longitudinal shape was also that of an elongated and recessed bipyramid (2.85 < m < 3.59). This shape suggests either a straight dimensional cutoff (i.e. a finite length) or, alternatively, lattice periodicity discontinuities introduced by the presence of placement defects, vacancies, twinning, bending or twisting. [START_REF] Hosemann | The Alpha-Star-Constant, Equilibrium State and Bearing Net Planes in Polymers, Bio-Polymers and Catalysts[END_REF][START_REF] Nishiyama | Diffraction from nonperiodic models of cellulose crystals[END_REF][START_REF] Newman | Wide-Angle X-Ray Scattering and Solid-State Nuclear Magnetic Resonance Data Combined to Test Models for Cellulose Microfibrils in Mung Bean Cell Walls[END_REF][START_REF] Lindner | Determination of cellulose crystallinity from powder diffraction diagrams[END_REF] The helical twist hypothesis induces geometric frustrations along the microfibril axis and excess displacements of the outer chains with respect to the chains located at (or near) the helix axis. [START_REF] Bruss | Non-Euclidean geometry of twisted filament bundle packing[END_REF][START_REF] Panaitescu | Measuring geometric frustration in twisted inextensible filament bundles[END_REF] The lattice displacement induced by the twist grows larger from the center to the periphery, which is consistent with the size-broadening distribution measured here: all the (hkl) planes with l ≠ 0 would be very sensitive to such a displacement. However, the cross-sectional dimensions of the crystallites as measured by XRD would be rather invariant and insensitive to the twist since the same amount of scatterers is found throughout the cross-section by translation along the microfibril axis: this applies to the standard measurements performed on the (hk0) bands. If this the case, the current work would constitute the first "in-situ" evidence of crystallographic twist. In addition, the strains induced by the twist would also create very periodic crystallographic/topological defects (i.e. dislocations) under the continuum theory. [START_REF] Azadi | Defects in crystalline packings of twisted filament bundles. II. Dislocations and grain boundaries[END_REF][START_REF] Panaitescu | Measuring geometric frustration in twisted inextensible filament bundles[END_REF]) For a given microfibril size (i.e. 18 chains, 36 chains, etc), these periodic defects could explain the LODP and the presence of Iα/Iβ stacking faults. These defects acting as hydrolysis loci would also explain why the longitudinal shape measured here is insensitive to the hydrolysis. [START_REF] Nyström | Nanocellulose Fragmentation Mechanisms and Inversion of Chirality from the Single Particle to the Cholesteric Phase[END_REF] The diameter of the crystallites in the direction orthogonal to the most intense (200) plane is ~2.rx ± 7% and the diameter in the direction orthogonal to the (010) plane is 2.ry. Hydrochloric acid hydrolysis induced a manifest increase of diameter (+22%) in the directions orthogonal to the (010) plane; this unidirectional thickening is attributed to a co-crystallization along the hydrophilic {110}, {11 ̅ 0} and possibly {010} planes (Table 2). It can be explained by a progressive adsorption of cellulose chains (co-crystallization) on the sides of the crystallites by creation of hydrogen bonds. (Newman 2004;[START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF]) These adsorbed chains are probably chain segments that were granted extra mobility by the chain scissions occurring during the hydrolysis. 13 C CP/MAS NMR showed that the samples that were not hydrolyzed displayed a C4 ratio consistent with 59% of the cellulose chains in the amorphous or exposed regions whereas the sample hydrolyzed for 8 h had a ratio of 55% (Figure S3). Both XRD and NMR results are therefore in good agreement with the slight co-crystallization observed in the initial report by Kontturi et al. [START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF] As the crystallite length is 2rz, the longitudinal dimension of the nanocrystals was estimated to be in the 16.4 to 25.8 nm range and there was no statistical effect of the hydrolysis on this length.

The maximum lateral dimensions of the crystallites are 2-3 times less than that of the nanoparticles as discussed using TEM or AFM. Therefore, the nanoparticles produced by HCl hydrolysis are lateral aggregates of nanocrystals. Their crystallographic length is 6-40 times smaller than that measured by microscopy; at that stage possible explanations include longrange lattice displacements produced by bending and twisting, as detailed above.

There is still more information that can be extracted from the diffractograms. The stacking fault probability q could be estimated using the method proposed by Driemeier et al. [START_REF] Driemeier | X-ray diffraction from faulted cellulose I constructed with mixed Iα-Iβ stacking[END_REF] In this work, q amounted to no more than 20% (Table 2).

The Iα/Iβ ratio was also estimated using the C-1 signal of the 13 CP/MAS NMR experiment. It was found to be no more than 10% for the control sample and the sample hydrolyzed for 8 h, confirming (i) a good agreement between XRD and NMR and (ii) the large prevalence of the Iβ allomorph in such vascular tissues. The crystallinity was also of interest. The crystallinity as assessed by XRD in this work was found to be invariant as variations in cellulose I, cellulose IV and background signal displayed no clear trend (Table 2). For the sake of discussion, the popular crystallinity index devised by Segal (1959) in his seminal work is also reported (Table 2). (Segal et al. 1959) Its value slightly increased after hydrolysis. As demonstrated in other works, this increase can here be attributed to a crystallite thickening without necessarily involving a change in the amorphous/crystalline ratio stricto sensu. (Duchemin et al. 2007;[START_REF] French | Cellulose polymorphy, crystallite size, and the Segal Crystallinity Index[END_REF] The paracrystalline cellulose IVI modelled in this work was isotropic with a size of 7.4-8 Å, meaning domains no more than 2-3 glucose monomers packed side-by-side. These domains produce a Bragg scatter with a well-defined interplanar distance. They are thus relatively ordered, supporting the hypothesis of a biphasic model comprising comparatively large "bi-pyramidal coherent" Iβ crystallites and small IVI stacks. It seems to indicate that elementary fibrils in the native state are in fact aggregates of crystallites and tightly packed small paracrystalline domains acting as anchor knots (interconnections) between the crystallites. The presence of amorphous material stricto sensu in the form of individual and randomly oriented chains was not accounted for in the present model. However, the data could be described with a good level of accuracy, which means that only minor amounts of such amorphous material, if any, could be present.

Other nanocrystals produced by sulfuric acid or ammonium persulfate hydrolysis run in our laboratory comprise almost no cellulose IVI domains (data not shown here). This point is of particular interest since the HCl vapor method produces nanoparticles with no surface charges and with a minimum amount of mass loss. In this work, the control material as well as the hydrolyzed material (8 h) were studied. Both materials were either used (i) "as is", in their protonated form, (ii) labelled by deuterium exchange and (iii) back-exhanged and thoroughly dried in order to assess the amount of drying hysteresis. The ATR-FTIR experiment was conducted from 20°C to 260°C (Figure 4), and the samples were then cooled down to look at possible thermal hysteretic effects and phase changes.

Background Replacing H2O with D2O in a cellulose suspension at room temperature leads to a limited isotopic exchange. In contrast, the use of high temperatures or swelling agents can lead to a complete replacement of hydrogen by deuterium. [START_REF] Sepall | Hydrogen exchange between cellulose and water: II. Interconversion of accessible and inaccessible regions[END_REF][START_REF] Nishiyama | Intracrystalline deuteration of native cellulose[END_REF]) Such harsh conditions will not be discussed in the present work. It is particularly informative to monitor the isotopic exchange by FTIR spectroscopy because the exchanged -OD groups will have an elongational vibration νOD near 2500 cm -1 instead of 3600-3200 cm -1 for νOH. Therefore, the -OD signal can easily be distinguished from the -OH one. The -OH signal is a superimposition of several bands. In general, it is agreed that the small band at 3650-3600 cm -1 is attributed to unbound water molecules. [START_REF] Fengel | Influence of water on the OH valency range in deconvoluted FTIR spectra of cellulose[END_REF][START_REF] Kondo | A fourier transform infra-red spectroscopic analysis of the character of hydrogen bonds in amorphous cellulose[END_REF][START_REF] Olsson | The association of water to cellulose and hemicellulose in paper examined by FTIR spectroscopy[END_REF] Bands at 3580 and 3555 cm -1 were assigned to loosely bond water or perhaps to non-hydrogen bonded O(2)H and O(6)H groups in amorphous cellulose. [START_REF] Kondo | A fourier transform infra-red spectroscopic analysis of the character of hydrogen bonds in amorphous cellulose[END_REF][START_REF] Michell | The Absence of Free Hydroxyl Groups in Cellulose[END_REF][START_REF] Olsson | The association of water to cellulose and hemicellulose in paper examined by FTIR spectroscopy[END_REF][START_REF] Hofstetter | Moisture uptake in native cellulose -the roles of different hydrogen bonds: a dynamic FT-IR study using Deuterium exchange[END_REF]Watanabe et al. 2006a) Regarding cellulose Iβ, there is a general agreement on the following band assignment: an intramolecular O( 2 6) band at 3230 cm -1 . [START_REF] Marechal | The hydrogen bond network in Iβ cellulose as observed by infrared spectrometry[END_REF][START_REF] Hofstetter | Moisture uptake in native cellulose -the roles of different hydrogen bonds: a dynamic FT-IR study using Deuterium exchange[END_REF]Watanabe et al. 2006a;[START_REF] Hayakawa | Evaluation of hydrogen bond networks in cellulose Iβ and II crystals using density functional theory and Car-Parrinello molecular dynamics[END_REF] Recently, it was demonstrated by CPMD calculation that the vibrations at 3340 and 3270 cm -1 were resolved and could be assigned without ambiguity. [START_REF] Hayakawa | Evaluation of hydrogen bond networks in cellulose Iβ and II crystals using density functional theory and Car-Parrinello molecular dynamics[END_REF]) However, the complexity of the band perpendicular to the chain direction and with peaks at 3410, 3345 and 3305 cm -1 was explained by variations in elongational frequencies between the -OH groups found in the origin and center chains. In contrast, the -OD signal obtained on surface deuterated samples only shows one broad νOD band near 2500 cm -1 . The presence of a single broad band that is not further resolved by polarization, suggests the presence of unstructured -OD groups. [START_REF] Priest | Determination of the accessibility of cellulose films by infra-red spectroscopy[END_REF][START_REF] Hofstetter | Moisture uptake in native cellulose -the roles of different hydrogen bonds: a dynamic FT-IR study using Deuterium exchange[END_REF][START_REF] Driemeier | Hydrated fractions of cellulosics probed by infrared spectroscopy coupled with dynamics of deuterium exchange[END_REF] This result is confirmed by inelastic neutron scattering. [START_REF] Müller | All Disordered Regions of Native Cellulose Show Common Low-Frequency Dynamics[END_REF] Both cellulose I and cellulose IVI contain similar well-identified νOH bands in the 3800-3000 cm -1 area of the spectrum, reinforcing the assumption that cellulose IVI is just a form of small and imperfect Iβ crystallites, as already inferred in the XRD analysis conducted in this work. [START_REF] Marrinan | Infrared spectra of the crystalline modifications of cellulose[END_REF][START_REF] O'connor | Applications of Infrared Absorption Spectroscopy to Investigations of Cotton and Modified Cottons[END_REF] A "soft" room temperature H2O-D2O isotopic exchange was used in numerous works to evaluate the water accessibility of different cellulose substrates. [START_REF] Priest | Determination of the accessibility of cellulose films by infra-red spectroscopy[END_REF][START_REF] Rousselle | Accessibility of Cotton Cellulose by Deuterium Exchange[END_REF][START_REF] Reishofer | Deuterium and Cellulose: A Comprehensive Review[END_REF][START_REF] Lindh | Surface accessibility of cellulose fibrils studied by hydrogendeuterium exchange with water[END_REF] Only accessible hydroxyl groups are then prone to deuterium exchange, meaning that the hydroxyl groups located on the cellulose chains localized at the interior of the crystallites are essentially left unaffected. [START_REF] Priest | Determination of the accessibility of cellulose films by infra-red spectroscopy[END_REF][START_REF] Marechal | The hydrogen bond network in Iβ cellulose as observed by infrared spectrometry[END_REF][START_REF] Reishofer | Deuterium and Cellulose: A Comprehensive Review[END_REF][START_REF] Lindh | Surface accessibility of cellulose fibrils studied by hydrogendeuterium exchange with water[END_REF] Consequently, the corresponding OD groups labelled by the exchange establish no hydrogen bonds with their surroundings. [START_REF] Marechal | The hydrogen bond network in Iβ cellulose as observed by infrared spectrometry[END_REF] Sometimes, the exchange or back-exchange procedure involves a certain amount of drying, which can lead to deuterium trapping due to hornification (co-crystallization). (Mann and Marrinan 1956a;[START_REF] Priest | Determination of the accessibility of cellulose films by infra-red spectroscopy[END_REF][START_REF] Wadehra | Irreversible exchange of hydrogen in the drying of cellulose at high temperature[END_REF][START_REF] Rousselle | Accessibility of Cotton Cellulose by Deuterium Exchange[END_REF][START_REF] Suchy | Impact of Drying on Wood Ultrastructure Observed by Deuterium Exchange and Photoacoustic FT-IR Spectroscopy[END_REF][START_REF] Lemke | New insights into nano-crystalline cellulose structure and morphology based on solid-state NMR[END_REF]Agarwal et al. 2016;[START_REF] Lindh | Surface accessibility of cellulose fibrils studied by hydrogendeuterium exchange with water[END_REF] These possible artefacts were anticipated as early as 1962 by Rousselle and Nelson who devised a method to assess the accessibility of cellulose by an isotopic back-exchange procedure in the wet state, without any drying. [START_REF] Rousselle | Accessibility of Cotton Cellulose by Deuterium Exchange[END_REF] The effect of temperature on the molecular structure of cellulose has also been studied by FTIR spectroscopy. [START_REF] Marechal | The hydrogen bond network in Iβ cellulose as observed by infrared spectrometry[END_REF][START_REF] Kokot | Two-dimensional correlation spectroscopy and principal component analysis studies of temperature-dependent IR spectra of cotton-cellulose[END_REF][START_REF] Wada | Lateral thermal expansion of cellulose Iβ and IIII polymorphs[END_REF]Watanabe et al. 2006c, b) In general, the band positions are affected much more by temperatures above 100°C than they are by humidity variations. (Watanabe et al. 2006a) These studies were mainly focused on the hydroxyl group regions and demonstrated a good stability of the intramolecular O(2)H•••O(6) bond. In contrast, a progressive change of the O(3)H•••O(5) above 130 °C with a marked transition near 184°C-220°C was observed and assigned to a glass transition. [START_REF] Kokot | Two-dimensional correlation spectroscopy and principal component analysis studies of temperature-dependent IR spectra of cotton-cellulose[END_REF]Watanabe et al. 2006c) This transition is linked to the so-called "high-temperature form" of cellulose Iβ which has been evidenced by non-linearities in the lateral unit cell dimensions above 180°C of both Cladophora nanocrystals and tension wood. [START_REF] Hori | The Thermal Expansion of Wood Cellulose Crystals[END_REF][START_REF] Wada | X-ray diffraction study on the thermal expansion behavior of cellulose Iβ and its high-temperature phase[END_REF] The formation of carbonyl groups above 220°C has also been observed and attributed to dehydration reactions, i.e. thermal degradation of the glucosidic backbone. [START_REF] Pastorova | Cellulose char structure: a combined analytical Py-GC-MS, FTIR, and NMR study[END_REF][START_REF] Nam | Method for identifying the triple transition (glass transition-dehydration-crystallization) of amorphous cellulose in cotton[END_REF] The monitoring of this band, however, has never been performed in-situ.

Figure 5. Absorbance FTIR spectra of the control sample (top row) and 8 h hydrolyzed sample (bottom row) as obtained after the deuterium-free control process (first column), after isotopic labelling with D2O (second column) and after an isotopic labelling, drying, and hydrogen re-exchange (third column). The measurements were performed between room temperature and 260°C.

Effect of heating on hydration

The FTIR spectra show drastic changes in the νOH region with ample band shifts as seen directly (Figure 5), by peak maximum measurement (Figure S7) or by second derivative analysis (Figure S8). In contrast to the νOH results, the νOD region is less affected and the broad νOD band is only moderately shifted in frequency (Figure S7). The most prominent νOD shift occurs below ca. 100°C, which is concomitant with the drying (Figures 5 andS7). When compared to the νOH intensity, the νOD intensity is largely reduced by the temperature sweep.

It is of particular interest to calculate the ratio raccess of deuterium exchanged hydroxyl groups and bound water over the total amount of non-exchanged hydroxyl groups (Figure 6a). This was done with equation ( 6). In the present experimental setup, raccess could decrease due to combined dehydration and back-exchange. The dehydration occurred due to heating. The back-exchange was triggered by the moisture of the air surrounding the sample that enables deuterium-hydrogen exchanges. However, comparisons between the samples are still of interest. In particular, the initial values were not affected by possible back-exchange because the measurements were performed immediately after opening the air-tight container with the labelled samples. This raccess calculation shows that the non-hydrolyzed sample initially contained about 47% of labelled deuteroxyl groups on cellulose and D2O against 38% for the sample that was hydrolyzed. This initial ratio ranking is counterintuitive because the hydrolyzed sample is expected to have a higher surface to volume ratio and similar crystallinity (XRD results) when compared to the non-hydrolyzed material. Therefore, this difference at low-temperature doesn't originate from a larger amount of hygroscopic amorphous regions in the hydrolyzed material. Bound water might then be an explanation. A higher water content is indeed confirmed by the intensity of the band at 1640 cm -1 (Figures 7 andS13). The samples were expected to contain only bound water since they were dried with dry N2 before being equilibrated for a week with H2O or D2O under the water saturation point for cellulose. Bound water could be trapped in pores or defects. These pores or defects are probably opened or suppressed during the production of nanoparticles, explaining the initial differences in D2O content and also the absence of trapped D2O in the nanoparticles after the H2O back-exchange procedure (Figure 6b). [START_REF] Strømme | Fractal Dimension of Cellulose Powders Analyzed by Multilayer BET Adsorption of Water and Nitrogen[END_REF] When the samples were heated, both raccess ratios decreased with temperature (Figure 6). The decrease was initially much faster for the non-hydrolyzed sample. The two curves crossed each other near 100°C and then stabilized with a fairly linear behavior all the way from 100°C to 260°C, confirming moisture evaporation in a process also involving polymer dehydration and dynamic reprotonation. On this high temperature range, raccess was always higher for the nanoparticles, as expected given their higher surface to volume ratio. Water evaporation was confirmed by plotting the intensity of the band at 1640 cm -1 against temperature (Figure 7). This plot shows a signal decreasing in intensity from 20°C to ~100° for the non-hydrolyzed sample and to ~120°C for the hydrolyzed sample. As opposed to the raccess data, the intensity of the band remains lower for the hydrolyzed sample up to the onset of cellulose dehydration near 180°C, in accordance with a lower water content. Therefore, the deuterium exchange procedure should be used with care when assessing cellulose crystallinity or "accessibility" since the intrinsic hygroscopic moisture content of the substrate can introduce an important bias in the measurement. This moisture content can be largely affected by the transient porosity and it doesn't necessarily depend on the crystallinity. This bias is minimized above ~100°C and high temperature FTIR brings a critical information to deuterium labelling experiments. Figure 7. Intensity of the molecular water maxima near 1640 cm -1 as a function of the temperature for the sample that was not hydrolyzed () and the sample that was hydrolyzed for 8 h (). The signals were normalized with respect to intensity of the -CH band near 2900 cm -1 . The intensity increase observed after 180°C for the hydrolyzed sample is attributed to the onset of cellulose dehydration (see Figures 7 andS12).

Effect of heating on specific bonds

It is explanatory to look at the νOH region in more detail. The plot of the second derivative shows a linear shift of the three main peaks (Figure S8). The maxima of the second derivative plots were sharp for the intermolecular O( 6 6) intensities decrease. The hydrolysis has not marked effect on these bonds in this temperature range. This is a strong indication of a redistribution of the hydrogen bond network upon dehydration, with cellulose-water bonds supposedly replaced by cellulosecellulose bonds. The acid hydrolysis has however a marked effect above ca. 230°C: the intensities of all the bonds undergo a sharp decrease between 230°C and 260°C on the hydrolyzed material only (Figure 5 andS9). This intensity drop is concomitant with a disruption of the whole hydrogen bond network. The explanation cannot come from a crystallinity difference, as evidenced by X-ray diffraction. Therefore, this transition can be attributed to cellulose thermal degradation or thermal motion. Thermal degradation is known to be triggered by the cellulose reducing ends and the higher number of reducing ends after hydrolysis can explain the difference between the two materials. [START_REF] Matsuoka | Thermal glycosylation and degradation reactions occurring at the reducing ends of cellulose during low-temperature pyrolysis[END_REF][START_REF] Agustin | The thermal stability of nanocellulose and its acetates with different degree of polymerization[END_REF] During this degradation, intra-ring condensation and rearrangements lead to the formation of furans and phenols in place of anhydroglucose units in the cellulose chain. [START_REF] Pastorova | Cellulose char structure: a combined analytical Py-GC-MS, FTIR, and NMR study[END_REF][START_REF] Nam | Method for identifying the triple transition (glass transition-dehydration-crystallization) of amorphous cellulose in cotton[END_REF] This mutation forms carbonyl groups. In this work, the hydrolyzed material was found much more sensitive than the non-hydrolyzed material to this temperature degradation: in particular, a strong carbonyl C=O band near 1702 cm -1 was formed between 180 and 200°C and its intensity increased abruptly up to 260°C during the heating cycle (Figures 5, 8a and S12), confirming chemical degradation. The evolution of the band at 1590 cm -1 (aromatic skeletal vibration of lignin) could be attributed to lignin polycondensation in the course of the thermal cycle. These thermo-chemical transformations also correspond to the two α2,2 and α2,1 viscoelastic relaxations of cellulose I and the creation of high-temperature cellulose Iβ. [START_REF] Kokot | Two-dimensional correlation spectroscopy and principal component analysis studies of temperature-dependent IR spectra of cotton-cellulose[END_REF][START_REF] Hori | The Thermal Expansion of Wood Cellulose Crystals[END_REF]Watanabe et al. 2006c;[START_REF] Wada | X-ray diffraction study on the thermal expansion behavior of cellulose Iβ and its high-temperature phase[END_REF][START_REF] Duchemin | High-Temperature Viscoelastic Relaxation in All-Cellulose Composites[END_REF]) Therefore, the thermally induced reorganization of the hydrogen bond network can also be considered as a thermal process during which chain segments gain extra mobility. Shorter segments produced by hydrolysis ease thermal mobility.

Hysteretic effects

The spectra were compared at room temperature before and after completion of the heating cycle as performed during the ATR-FTIR scan. The hydrolysis had nearly no effect on the positions of the hydrogen bonds (Figure 6c). However, the heating cycle markedly displaced the intermolecular O(6)H•••O'(3) which can be attributed to both an evaporation of bound water and chain degradation. This evaporation also induced a distortion of the hydrogen bond network resulting in hornification or co-crystallisation. Interestingly, the two intramolecular bonds were poorly affected by this process, which is an indication that the distortion is structural (long-range aggregation). This result also means that the high temperature collapse of the hydrogen bond network undergone in the hydrolyzed sample (Figure S9) is reversible to a large extent.

The positions of the maxima of the -CH band near 2900 cm -1 were significantly affected by the heat cycle and shifted by ~5 cm -1 after the samples were hydrolyzed (Figure 6d). This displacement is concomitant with a thermal degradation favored by reducing chain ends, as detailed above. The intense carbonyl band appearing during heating in the hydrolyzed material remains stable after cooling (Figure 8b), as observed in other works. [START_REF] Pastorova | Cellulose char structure: a combined analytical Py-GC-MS, FTIR, and NMR study[END_REF][START_REF] Nam | Method for identifying the triple transition (glass transition-dehydration-crystallization) of amorphous cellulose in cotton[END_REF] Furthermore, the dismantlement of the cellulose system into smaller nanofragments granted extra longitudinal and lateral mobility to the cellulose chains for reorganization during heating and dehydration, as seen above. [START_REF] Matsuoka | What is active cellulose in pyrolysis? An approach based on reactivity of cellulose reducing end[END_REF][START_REF] Nam | Method for identifying the triple transition (glass transition-dehydration-crystallization) of amorphous cellulose in cotton[END_REF] This could explain the shift of the -CH band, a band that is otherwise known to be sensitive to the crystallinity of the material. [START_REF] Marrinan | Infrared spectra of the crystalline modifications of cellulose[END_REF][START_REF] Schwanninger | Effects of short-time vibratory ball milling on the shape of FT-IR spectra of wood and cellulose[END_REF][START_REF] Ciolacu | Amorphous cellulose-structure and characterization[END_REF] Regarding the C-O intrachain bonds as measured using the positions of the C-O stretching region, small hysteretic variations can be observed (Figure 6e and S11) but the cellulosic backbone remained essentially unaffected. [START_REF] Marechal | The hydrogen bond network in Iβ cellulose as observed by infrared spectrometry[END_REF]Watanabe et al. 2006b) For all the samples, the least affected bond was C3-O3 (Δ0h and Δ8h = 0 cm -1 ) and the most affected bonds were C2-O2 (Δ0h = -2 cm -1 and Δ8h = -1 cm -1 ) and especially C6-O6 (Δ0h = -2 cm -1 and Δ8h = -3 cm -1 ). The glycosidic C1-O-C4 bond was affected in an intermediary way (Δ0h and Δ8h = -1 cm -1 ). The C3-O3 relative "inertness" is certainly due its in-plane conformation and to its orientation running parallel to the C1-O-C4 bond, along the chain axis. The singularity of the C6-O6 band is most certainly linked to its central role in the hydrogen bond network. This singular band shift can be explained by the higher sensitivity of the hydrogen bond network to temperature-induced deformations when the polymer chain fringe structure is dismantled and when the long-range polymer constrains are alleviated, i.e.

when the longitudinal chain connectivity is lost due to chain scission.

Conclusions

Hydrochloric acid hydrolysis in the vapor phase offers a unique opportunity to open up the cellulose structure without affecting the crystallinity and with almost no monomer production when compared with the popular sulfuric acid hydrolysis. [START_REF] Kontturi | Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals[END_REF] In the present work, a cellulose source from vascular flax bundles was bleached and submitted to such an acid hydrolysis. A three-dimensional X-ray diffraction Rietveld analysis showed that the crystallinity changes were not significant whereas the lateral size variations were substantial, in the order of 22% on the hydrophilic surfaces. The crystallographic model measures a certain paracrystalline content consisting of stacks containing 2 or 3 monomers arranged in a periodic manner. In that sense, the hypothesis of cellulose microfibrils being fractal aggregates of 3 × 2 chains, in accordance with the current knowledge on hexameric terminal complexes, is plausible. [START_REF] Usov | Understanding nanocellulose chirality and structure-properties relationship at the single fibril level[END_REF] This paracrystalline content is not affected in size or relative content by the hydrolysis. This result suggests that the HCl hydrolysis does not proceed by full hydrolysis of the least ordered regions, unlike H2SO4 hydrolysis, which explains the high yield of the method. This result is compatible a progression of the HCl hydrolysis through accessible regions or kinks. [START_REF] Nyström | Nanocellulose Fragmentation Mechanisms and Inversion of Chirality from the Single Particle to the Cholesteric Phase[END_REF] Isotopic labelling with D2O and thermal ATR-FTIR results demonstrated that temperatures above 100°C can be used to reveal the higher amount of labelled sites in the hydrolyzed material when the material is conditioned at 75 % RH. This result also means that hygroscopic moisture can induce an important bias in the quantitative assessment of accessibility (or "crystallinity") by deuterium labelling. In this work, two materials with very close crystallinities but with different morphologies (fibres and nanoparticles) had very different accessibilities, meaning that deuterium labelling cannot measure crystallinity. Thermal ATR-FTIR also evidenced small hysteretic effects of the hydrogen bond network and cellulose backbone (C-O stretching region) to temperature variations after hydrolysis. The effect is more prevalent for all interatomic linkages relative to the hydroxymethyl group (C6). The irreversible formation of a carboxylic band at 1702 cm -1 is characteristic of the cellulose degradation by dehydration reactions.

Finally, the nanoparticles produced in this work were stable aggregates regarding the effect of wetting and drying cycles (deuterium trapping was cancelled by the hydrolysis). However, their polymer chains were more susceptible to thermal motion and degradation above ca. 200°C was evidenced by the rise of the carbonyl group signal. These two effects should be attributed to the topology of the cellulosic substrate (chain ends, chain entanglement, adjacency, chain continuity between highly ordered regions) rather than to their crystallinity, as measured by XRD. source (λCoKα = 1.790307 Å) and a PIXcel 1D detector. The operating voltage and current were maintained at 40 kV and 40 mA, respectively. Measurements were made between 10 and 90° with 0.0525° steps and for a total duration of 3.5 h. The lateral divergence of the incident beam path was controlled with 0.04° Soller slits and a 15 mm mask and its divergence in the 2.θ plane was controlled with a ½° programmable divergence slit and a 1° fixed anti-scatter slit. The crystallite lateral thickness in the direction perpendicular to the (200) plane was estimated using Scherrer equation (K=0.9) and the crystallinity index (CrI) was calculated following the work of Segal (1959). (Segal et al. 1959;Guinier 1963;Duchemin et al. 2007) 13 C CP/MAS NMR spectroscopy Solid-state 13 C CP/MAS NMR experiments were performed on a Bruker AV III 400 spectrometer with a 13 C resonance frequency of 100.66 MHz, equipped with a Bruker 4 mm double resonance probe. The spectra were obtained at spinning rate of 12 KHz and 12,288 transients were added with a recycling delay of 1.5 s. A contact time of 2,000 ms was employed. The 13 C chemical shifts were referenced to TMS.

b. Results

Microscopy and yield

Flax shives are part of the flax stem and they are produced by scutching the flax stems during the production of textile fibers. They mainly consist of xylem residues. Polarized optical microscopy of flax stem cross-sections evidences the presence of crystalline cellulose in the xylem because of its birefringence (Figure S5). Therefore, this cheap material was deemed as a possible source for the production of CNC. The starting material was ground to a fine powder in order to render the two main subsequent treatments (bleaching and hydrolysis) as homogeneous as possible. SEM (Figure S5b) shows elongated fragments 25-80 µm in length and 8-12 µm in diameter. The flax shives that were received underwent a chlorine-free bleaching treatment in three steps. The yields of the Soxhlet extraction, NaOH treatment and bleaching step were respectively 91 wt.%, 55 wt.% and 54 wt.% (with respect to the initial weight). At the end of the final step, the total yield was slightly more to that usually found in the literature: it is admitted that flax shives contain 34-53 wt.% cellulose, 23-31 wt.% lignin and 13-26 wt.% hemicellulose as well as moderate amounts of ash (1-2 wt.%) and wax (3-4 wt.%). [START_REF] Sain | Flax shives refining, chemical modification and hydrophobisation for paper production[END_REF][START_REF] Kim | Extraction and Separation of Carbohydrates and Phenolic Compounds in Flax Shives with pH-Controlled Pressurized Low Polarity Water[END_REF][START_REF] Del Río | Structural Characterization of Guaiacyl-rich Lignins in Flax (Linum usitatissimum) Fibers and Shives[END_REF] ATR-FTIR ATR-FTIR of the bleached material confirm the presence of cellulose with typical cellulose I bands (Figure S6). The distinctive carbonyl band of hemicellulose at 1732 cm -1 was removed. This band can sometimes be found in glucomannan, xyloglucan or glucuronic acid-substituted xylan. The disappearance of this band coincides with the NaOH treatment step which resulted in a ~36 % weight loss. Since the hemicellulose content is expected to lie in the 13-26 wt.% range, one assumes that most hemicelluloses were removed as well as some other components. The final yield found here (54 wt.%) is in the upper range with respect to the cellulose content expected in flax shives. Possible remnants of cell wall compounds could explain this discrepancy. In particular, a portion of recalcitrant lignin might have resisted the different treatments: ATR-FTIR (Figure S6) shows remaining lignin bands at 1660 cm -1 (C=O), 1590 and 1508 cm -1 (aromatic skeletal vibration), 1460 cm -1 (C-H deformation) and 1265 cm -1 (ring stretching). [START_REF] Marchessault | Application of infra-red spectroscopy to cellulose and wood polysaccharides[END_REF][START_REF] Pandey | A study of chemical structure of soft and hardwood and wood polymers by FTIR spectroscopy[END_REF][START_REF] Duarte | Eucalyptus globulus Kraft Pulp Residual Lignins. Part 1. Effects of Extraction Methods upon Lignin Structure[END_REF][START_REF] Derkacheva | Investigation of Lignins by FTIR Spectroscopy[END_REF][START_REF] Duchemin | Treatment of Harakeke fiber for biocomposites[END_REF]Huang et al. 2019) The bands found at 2920 and 2850 cm -1 (C-H stretching of aromatic groups) were removed after the Soxhlet extraction. These bands are sometimes attributed to lignin. Given the strong presence of many lignin bands throughout the process and the removal of these two C-H stretching bands in the toluene:ethanol mixture, they were rather assigned to soluble wax compounds.

Figure S2. Absorbance FTIR spectra of (top to bottom): the as-received ground flax shives, the shives that were solvent extracted using a Soxhlet apparatus and a toluene:ethanol (2:1) reflux, the shives treated with NaOH and the shives bleached with H2O2 in an alkaline environment.

13 C CP/MAS NMR spectroscopy 13 C CP/MAS NMR spectroscopy is complementary to FTIR spectroscopy and X-ray diffraction with respect to the information provided on cellulose (allomorphic nature, crystallinity, crystallite size) and on the nature of non-cellulosic polysaccharides or phenolic compounds. In this work, two bands were removed by the "bleaching" treatment (Figure S7). These bands are visible on the signal obtained on the as-received flax shives. The first band is a peak near 172 ppm and it is most likely attributed to galacturonan or other various acetyl or carboxyl moieties. [START_REF] Newman | Solid-State 13C Nuclear Magnetic Resonance Characterization of Cellulose in the Cell Walls of Arabidopsis thaliana Leaves[END_REF][START_REF] Jarvis | Macromolecular biophysics of the plant cell wall: Concepts and methodology[END_REF][START_REF] Maunu | NMR studies of wood and wood product[END_REF][START_REF] Martins | A solid state 13C high resolution NMR study of raw and chemically treated sisal fibers[END_REF][START_REF] Duchemin | Ultrastructure of cellulose crystallites in flax textile fibers[END_REF]) the second peak was found at 20.7 ppm and it is assigned to the acetyl group in hemicelluloses [START_REF] Newman | Determination of the degree of cellulose crystallinity in wood by carbon-13 nuclear magnetic resonance spectroscopy[END_REF][START_REF] Girault | Galactans and cellulose in flax fibers: putative contributions to the tensile strength[END_REF][START_REF] Jarvis | Macromolecular biophysics of the plant cell wall: Concepts and methodology[END_REF][START_REF] Maunu | NMR studies of wood and wood product[END_REF][START_REF] Martins | A solid state 13C high resolution NMR study of raw and chemically treated sisal fibers[END_REF]) Therefore, and in accordance with FTIR results, it is reasonable to infer that hemicelluloses and pectins were removed during the pretreatment step. Other bands remained throughout the process. In particular, several small bands in the 120-160 ppm region with peaks at 151.2, 147.1 and 132.9 ppm remained. These bands are attributed to the C=C bonds in guaiacyl and syringil units. [START_REF] Josefsson | Changes in cellulose supramolecular structure and molecular weight distribution during steam explosion of aspen wood[END_REF][START_REF] Maunu | NMR studies of wood and wood product[END_REF][START_REF] Newman | Leaf-fibre lignins of Phormium varieties compared by solid-state 13C NMR spectroscopy[END_REF] The lignin peak at 55.5 ppm also remained. [START_REF] Josefsson | Changes in cellulose supramolecular structure and molecular weight distribution during steam explosion of aspen wood[END_REF][START_REF] Maunu | NMR studies of wood and wood product[END_REF][START_REF] Newman | Leaf-fibre lignins of Phormium varieties compared by solid-state 13C NMR spectroscopy[END_REF]) Therefore, the pre-treatment used wasn't able to remove the lignin recalcitrant portion. This result is also in line with FTIR results. Regarding cellulose, all the characteristic bands were present: C-1 near 105 ppm (Iβ :104.1 and 105.3 ppm; Iα: 104.7 ppm), C-4 "interior" near 88.5 ppm, C-4 "exterior" with a band at 83.6-83.8 ppm, C-2,3,5 with intense bands near 74.6 and 72.0 ppm and C-6 at 62.4 and 64.6 ppm. [START_REF] Newman | Solid-State 13C Nuclear Magnetic Resonance Characterization of Cellulose in the Cell Walls of Arabidopsis thaliana Leaves[END_REF][START_REF] Larsson | A CP/MAS13C NMR investigation of molecular ordering in celluloses[END_REF][START_REF] Newman | Estimation of the relative proportions of cellulose Iα and Iβ in wood by carbon-13 NMR spectroscopy[END_REF][START_REF] Maunu | NMR studies of wood and wood product[END_REF] Figure S3. Nuclear magnetic resonance spectra of "raw" ground flax shives (top), the reference non-hydrolyzed sample (middle) and the sample that was hydrolyzed for 8 h (bottom). The C-1 to C-6 carbons are cellulose bands. The "h" letter indicates typical hemicellulose shoulders. All other bands are explicitly attributed.

X-ray diffraction

XRD unambiguously confirms the presence of crystalline cellulose with typical cellulose I diffractograms (Figure S8) and the relatively broad diffractograms indicate the presence of small cellulose crystallites in the flax shives, relatively to that found in cotton, flax fibers or tunicate. The dimensions of the crystallites in the direction orthogonal to the (200) plane were estimated using Scherrer equation. They were found to be ~27 Å initially and ~36 Å after the alkaline treatments (Table S2). It is well-known that NaOH can dissolve very small crystallites and provide extra-mobility to 102), ( 200) and ( 004) planes are materialized by red dots on both figures.

3. Hydrolysis: complementary information 13 C CP/MAS NMR The amount of cellulose chains located in the amorphous regions or at the surface of the crystallites was obtained by measuring the area between the baseline and the spectral line between 79 and 86 ppm. The amount of interior chains was estimated as the corresponding area between 86 and 94 ppm. The Iα:Iβ ratio was estimated using the three C-1 bands of cellulose in the 100-108 ppm range. [START_REF] Duchemin | Ultrastructure of cellulose crystallites in flax textile fibers[END_REF] The calculation was based on the relative areas of the phases after a fitting three Gaussian function corresponding to the Iβ phase (104.1 and 105.3 ppm) and Iα phase (104.7 ppm). Figure S13. FTIR absorbance spectra of the never deuterated samples in the initial conditions. The molecular water region is plotted after normalization relative to the -CH band at 2900 cm -1 (intensity method).

Figure 1 .

 1 Figure 1. Transmission electron microscopy of CNC deposited on formvar films (left column, scale bars are 50 nm) and atomic force microscopy of CNC deposited on mica (right column, images are 2 µm x 2 µm). .Nanocrystals produced by sulfuric acid or APS bear repulsive surface charges due to the presence of grafted ionic groups. In general, the ζ-potential of such particles is much more negative than -20 mV and typically around -50 to -70 mV. In this work, the ζ-potential (> -30 mV) resulting from the process is not sufficient to generate the required amount of electrostatic repulsion that would prevent the nanoparticles from aggregating (Table1).

Figure 3 .

 3 Figure 3. Simulated diffractograms of (a) isotropic Iβ crystallites with m = n = 1 and rx = ry = rz, (b) elongated Iβ crystallites with m = n = 1 and rx = ry = 2 nm, (c) laterally deformed Iβ crystallites with rx = ry = rz = 2 nm and m = 1 and (d) longitudinally deformed Iβ crystallites with rx = ry = 2 nm, rz = 5 nm and n = 1.

Figure 4 .

 4 Figure 4. X-ray diffractograms (black lines) and simulated diffractograms (grey circles) after various hydrolysis times. The simulation of the control samples (0 h) is shown at the bottom with the background signal (light grey), the cellulose IVI signal (grey) and the cellulose Iβ signal (dark grey). An insert (top right) shows the crystallographic shape of the nanocrystals obtained after an 8 h long hydrolysis.

  )H•••O(6) band at 3460-3436 cm -1 , an intermolecular O(6)H•••O'(3) at 3412 cm -1 , an intramolecular O(3)H•••O(5) band at 3340-3360 cm -1 , an intramolecular O(2)H•••O(6) band at 3270 cm -1 and an intermolecular O(3)H•••O'(

Figure 6 .

 6 Figure 6. (a) Relative areas of the νOD region expressed as raccess for the non-hydrolyzed sample () and the sample that was hydrolyzed for 8 h (). Comparisons of various parameters at room temperature before (dark grey) and after (light grey) the completion of the heating cycle for the normally protonated samples (H), the deuterated samples (D) and the samples that were deuterated and then reprotonated (DH). These parameters are (b) the relative deuterated fraction, (c) the position of the main inter-and intramolecular bands in the νOH region, (d) the maximum of the band in νCH region and (e) the positions of the three main C-O bonds found in cellulose chains.

  )H•••O'(3) band at ~3410 cm -1 , the intramolecular O(3)H•••O(5) band at ~3340 cm -1 and the other intramolecular O(2)H•••O(6) band at ~3280 cm - 1 (band frequencies at room temperature). The positions of the maxima were used to assess the amplitudes of the three bands at all temperatures (Figure S9). It can be seen that the intensities of the intermolecular O(6)H•••O'(3) become more intense during heating between 100°C and ca. 200°C, whereas both the intramolecular O(3)H•••O(5) and O(2)H•••O(

Figure 8 .

 8 Figure 8. Evolution of the carbonyl signal on the absorbance spectra during (a) heating from 20°C (blue) to 260°C (yellow) and (b) cooling from 260°C (blue) to 30°C (yellow) of the nondeuterated sample hydrolyzed for 8 h.

Figure S1 .

 S1 Figure S1. Polarized optical microscopy (a) of a flax stem displaying the epidermis (e), the fibers (f) and the xylem (x); SEM micrographs (b) of the ground flax shives used in this work (scale bar: 100 µm).

  Figure S5. Plot of a "standard" calculated diffractogram (left) and corresponding superellispoid hull (right). The hull is a discrete representation of the points projected on the superellipsoid shape for each (hkl) plane. The most intense Bragg bands corresponding to the (110), (11 ̅ 0), (102), (200) and (004) planes are materialized by red dots on both figures.

Figure S6 .

 S6 Figure S6. Difference between the area-normalized diffractograms obtained on the hydrolyzed sample and on the non-hydrolyzed reference sample.

Figure S7 .

 S7 Figure S7. Peak positions in the elongational -OH and -OD zones for the reference sample (continuous line) and 8h hydrolyzed sample (dotted line) that were deuterated (black) or reprotonated (grey).

Figure S10 .

 S10 Figure S10. Temperature dependence of the main C-O bands during the heating cycle of the normally protonated non-hydrolyzed sample (○) and the hydrolyzed sample (•).

Figure S11 .Figure S12 .

 S11S12 Figure S11. Band shifts of the main C-O bonds after completion of the heating and coolingcycle with respect to the initial position (adapted from Figure8d).

Table 1 .

 1 Summary of the dimensions (arithmetic mean 𝑥̅ ± mean absolute deviation) measured by TEM, AFM, ζ potential and yield of the hydrolysis as a function of the HCl exposure duration.

	Hydrolysis	Length (nm) Width (nm)	Aspect	Height	ζ potential	Yield
	time			ratio	(nm)	(mV)	(%)
	Control	2368 ± 3342	96 ± 128	22 ± 12	/	-29.1	98
	30 min	803 ± 734	22 ± 13	31 ± 15	30 ± 14	-24.4	89
	1 h	475 ± 380	21 ± 15	25 ± 14	26 ± 13	-29.6	90
	2 h	247 ± 158	12 ± 5	23 ± 14	13 ± 7	-19.2	92
	4 h	196 ± 131	13 ± 5	16 ± 8	15 ± 12	-9.8	90
	8 h	144 ± 80	10 ± 4	16 ± 9	12 ± 9	-9.4	90

Table 2

 2

	. Results of the simulation of X-ray diffractograms using a Rietveld analysis routine with a superellipsoid model to describe the three-dimensional shape of the cellulose nanocrystals.
	Hydrolysis time	Control	30	1 h	2 h	4 h	8 h
	Segal's CrI (%)(Segal et al. 1959)	53%	min 55%	56%	57%	57%	56%
	Lattice parameter a (Å)	7.96	7.96	7.97	7.94	7.94	7.98
	Lattice parameter b (Å)	8.17	8.18	8.18	8.15	8.17	8.22
	Lattice parameter c (Å)	10.36	10.36 10.36 10.34 10.36 10.37
	γ (°)	95.53	95.75 95.73 95.31 95.52 95.82
	March-Dollase strength	1.40	1.52	1.54	1.38	1.54	1.65
	rx (Å)	18	18	18	19	19	19
	ry (Å)	27	28	29	27	30	33
	rz (Å)	113	114	102	82	129	129
	Superellispoid longitudinal squareness m	3.44	3.59	3.36	2.85	3.46	3.33
	Superellispoid radial squareness n	2.02	1.84	1.87	2.25	2.26	2.01
	Cellulose IVI size (Å)	7.4	7.8	7.8	7.3	7.6	8.0
	Background (%)	42%	42%	42%	38%	41%	44%
	Iβ (%)	22%	23%	23%	25%	23%	21%
	IVI (%)	36%	35%	35%	37%	36%	35%
	Stacking fault probability q (%)	10	10	10	20	10	10
	Correlation coefficient	0.99896	0.9991	0.9991	0.9990	0.9989	0.999
			5	3	5	9	01
	R-pattern	1.97%	1.76% 1.83% 1.86% 1.90% 2.05%
	Temperature-dependent ATR-FTIR

Table S2 .

 S2 RH measurement of the films prior to acid hydrolysis

	Time (h)		0	0,5	1	2	4	8
	Water	content	24,0
	(w/w)		%	42,7%	26,8%	29,2%	25,2%	34,1%
	Standard deviation 1,9% 3,4%	5,1%	1,9%	4,5%	3,7%

Acknowledgements:

The authors thank the National Technical and Research Association (ANRT) and the Public Investment Bank (BPIfrance) for their financial support, Nicolas Hucher (Normandie Université, URCOM EA 3221 FR CNRS 3038, 76600 Le Havre, France) for his help with the zetasizer and FTIR measurements (bleaching process), Bernard Cathala (BIA INRA Nantes, France) for his input on this work and Maëlle Coquemont (Normandie Université, CMABio 3 , Caen, France) for her expertise in microtoming. We also thank Yoshiharu Nishiyama (CERMAV, Grenoble) for the useful discussions on thermal degradation.

Supporting information:

The analysis of the bleaching steps, the 13 C CP/MAS NMR spectra, additional ATR-FTIR spectroscopy data treatment are provided as supplementary information, after the references.

REFERENCES:

Agarwal UP, Ralph SA, Reiner RS, Baez C (2016) Probing crystallinity of never-dried wood cellulose with Raman spectroscopy. Cellulose

Supplementary information

1. Preparation of the flax shives a. Materials and methods

Cellulose isolation

Prior to hydrolysis, the shive meal was pre-treated in order to separate the cellulose from the other non-cellulosic components following a chlorine-free procedure described elsewhere. [START_REF] Jiang | Cellulose nanocrystal isolation from tomato peels and assembled nanofibers[END_REF] The isolation procedure started with a dewaxing step in a Soxhlet apparatus with toluene-ethanol (2:1, v/v) for 20 h. The resulting product was filtered and dried at 70 °C for 24h and 103°C for Dewaxed shives (FSd) were obtained. FSd were then treated with NaOH (5 wt.%) for 24 h at room temperature and 5 h at 90°C. Shives were then sequentially filtered and washed 3 times in order to obtain clear filtrates with a neutral pH. After neutralization, the flax shives were dried at 70°C for 24 h and at 103°C for 2 h. The shives obtained are called FSNaOH. Bleaching was then performed for 6h at 45 °C with hydrogen peroxide (4%) at a pH of 11.5 adjusted with NaOH. The treated material was filtered, neutralized and dried using the same protocol as that described in the previous step. The shives obtained at the end of this step are called bleached flax shives (FSb), or cellulose in the rest of this work.

Yield

Aliquots (30 ml) were sampled from the CNC suspensions (~3 g.L -1 ) and left to dry under a fume hood. The effectiveness of the drying was controlled by measuring the dried CNC powders three times at half-day intervals. The weight of the dry residue was then used to estimate the yield of the hydrolysis expressed as the dry mass of CNC relative to the initial mass of bleached flax shives before hydrolysis.

Microscopy of the flax shives and stems

Flax stems were embedded in paraffin and microtomed sections (~2-3 µm in thickness) were observed in transmission mode using cross-polarizers with an Olympus BX51 optical microscope. The ground flax shives were observed with a field-emission scanning electron microscope (FE-SEM) Hitachi S3000N, in back-scattered electrons mode (BSE) under partial vacuum (5 Pa) with a 15 kV accelerating voltage. The samples were carbon-coated using an automated carbon coated JEC-530 (JEOL Tokyo, Japan) with a preset cycle (6s, 40 V, 2 cycles).

Infrared spectroscopy

FTIR spectra of all the samples were obtained using an attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR, Frontier, Perkin Elmer). Spectra were collected at ambient temperature from an accumulation of 32 scans at 2 cm -1 resolution over the 4000-650 cm -1 region. For each sample, 3 spectra were collected, baseline corrected, normalized and averaged (using Spekwin32) in order to obtain a final spectrum for each treatment step.

X-ray diffraction

The diffractograms of the samples before and after bleaching were measured. Powder diffractograms were acquired in reflection mode with a θ-θ Bragg-Brentano configuration using an Xpert-Pro Powder (PANalytical B.V., Almelo, Nederland) with a cobalt radiation cellulose oligomers, which translates into an average thickening of the cellulose crystallites left behind such treatments; the removal of non-crystalline components also induces an increased crystallinity (Table S1). (Newman 2004;[START_REF] Daicho | The Crystallinity of Nanocellulose: Dispersion-Induced Disordering of the Grain Boundary in Biologically Structured Cellulose[END_REF]) Additionally, one notices the presence of quartz in the sample due to the ( 101) and (101 ̅ ) diffraction peaks at 31° (Figures S8 and4). Due to the industrial nature of the flax shive extraction process, it is indeed plausible to find small amounts of quartz in the resulting product. An XRD Rietveld analysis using the Maud software was performed (data not shown here) and the amount of quartz in the bleached material was found to be ~0.4 wt.%.

Figure S4. Powder diffractograms of (top to bottom): the as-received ground flax shives, the shives that were solvent extracted using a soxhlet apparatus and a toluene:ethanol (2:1) reflux, the shives treated with NaOH and the shives bleached with H2O2 in an alkaline environment.

Table S1. XRD results of the bleaching process