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Humidity and multiscale structure govern mechanical properties and deformation modes in films of native cellulose nanofibrils Alejandro J. Benitez, Jose Torres-Rendon, Mikko Poutanen, and Andreas Walther Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 18 Nov 2013 Downloaded from http://pubs.acs.org on November 19, 2013

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Humidity and multiscale structure govern mechanical properties and deformation modes in films of native cellulose nanofibrils

Alejandro J. Benítez, Jose Torres-Rendon, Mikko Poutanen†, Andreas Walther*

DWI at RWTH Aachen University – Institute for Interactive Materials Research, Forckenbeckstr. 50, D-52056 Aachen, Germany; †

present address: Molecular Materials, Department of Applied Physcis, Aalti Univeristy, Helsinki,

FI-00076 Finland Email: [email protected]

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KEYWORDS. Nanofibrils, Nanocellulose, Mechanical Properties, Humidity, Deformation modes, Colloidal Properties. ABSTRACT. Nanopapers formed by stiff and strong native cellulose nanofibrils are emerging as mechanically robust and sustainable materials to replace high-performance plastics or as flexible, transparent and “green” substrates for organic electronics. The mechanical properties endowed by nanofibrils crucially depend on mastering structure formation processes and on understanding interfibrillar interactions as well as deformation mechanism in bulk. Herein, we show how different dispersion states of cellulose nanofibrils, i.e. unlike tendencies to interfibrillar aggregation, and different relative humidities influence the mechanical properties of nanopapers. The materials undergo a humidity-induced transition from a predominantly linear elastic behavior in dry state to films displaying plastic deformation due to disengagement of the hydrogen-bonded network and lower nanofibrillar friction at high humidity. A concurrent loss of stiffness and tensile strength of one order of magnitude is observed, while maximum elongation stays near constant. Scanning electron microscopy imaging in plastic failure demonstrates pull-out of individual nanofibrils, bundles of nanofibrils, as well as of larger mesoscopic layers, stemming from structures organized on different length scales. Moreover, multiple yielding phenomena and substantially increased elongation in strongly disengaged networks, swollen in water, show that strain at break in such nanofibril-based materials is coupled to relaxation of structural entities, such as cooperative entanglements and aggregates, which depend on the pathway of material preparation. The results demonstrate the importance of controlling the state of dispersion and aggregation of nanofibrils by mediating their interactions, and highlight the complexity associated with understanding hierarchically structured nanofibrillar networks under deformation. TABLE OF CONTENTS ENTRY

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INTRODUCTION Stiff and strong nanofibrils emerge as viable building blocks for materials with extraordinary mechanical properties and advanced functionalities. These include self-assembled fibrillizing peptides1, 2 and proteins (e.g. amyloids, collagen)3-7, chitin8, 9 or aramide10, 11 nanofibrils obtained in specific solvents, or native chitin and cellulose nanofibrils isolated from sustainable resources.12-15 Most of these materials maintain the nanofibrillar state when preparing macroscale bulk materials via filtration (paper making), film casting or spinning. During late stages of solvent removal, the concentration of nanofibrils drastically rise, which can lead to interfibrillar agglomeration and further floc formation, hence defining the resulting structure on larger length scales. Such mesoscopic agglomerates are generally very difficult to identify, due to lack of suitable experimental techniques, but are of decisive importance as they may act as material flaws and can crucially influence material failure under loading. Moreover, the large amount of hydrogen bonds found among many of these nanofibrils defines material cohesion, interfibrillar sliding and dissipative frictional movement to allow for toughness. Hydrogen bonds are prone to changes in humidity, yet very little attention has so far been drawn on this aspect. From natural high-performance composites, it is known that they are only tough in hydrated form, when hydrogen bonds are substantially weakened to allow yielding and energy dissipation.16-18 This implies that a rational design approach to drastically improved properties in man-made nanofibrillar materials requires understanding the effect of moisture. Herein, we will focus on how different dispersion states of nanofibrils, i.e. unlike tendencies to interfibrillar aggregation during material preparation, and different relative humidities influence the mechanical properties of films formed by native cellulose nanofibrils. Native nanocellulose is one of the most promising nanoparticle building blocks for future sustainable high performance and functional materials.13, 14 They can be divided in short and highly crystalline cellulose nanocrystals and long and entangled nanofibrillated cellulose (NFC). Their high mechanical properties originate from the exceptional stiffness of the underlying cellulose 1 crystal of up to 140 GPa at a low density of ca. 1.58 g/cm3.13, 19 NFC allows the formation of stiff, strong and tough self-standing nanopapers with high gas barrier, transparency and low thermal expansion.20-26 Such materials have proven to be viable substrates for organic light emitting diodes, organic field effect transistors and other “green” electronic devices.27-29 We and others reported on the spinning of macrofibers and their use for controlled release, magnetic actuation or the preparation of bio-based conducting fibers.30-32 Additionally, NFC can be used as an intrinsically ductile, nanofibrillar matrix material for stiff, strong and tough clay/NFC and graphene/NFC nanocomposites with advanced fire shielding and increased gas barrier.33-36 Furthermore, the addition of small amounts of polysaccharides or tailored block copolymers could be used to provide efficient lubrication between the nanofibrils and allow frictional sliding to increase the strain at break and thus toughness.20, 37, 38 Scientifically the latter is an extremely relevant parameter, as nature has solved the problem of yielding of highly reinforced fibrillar composites at high stress levels by providing a soft and ACS Paragon Plus Environment

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deformable interstitial phase.16-18 In wood, this phase contains hemicellulose and lignin as major components between the cellulose microfibrils, and is thus rich in non-covalent hydrogen bonds. When hydrated, shear deformation occurs more easily due to increased dynamics in the soft phase and hence, toughness correlates with water content. One of the main bottlenecks in the use and understanding of properties endowed by NFC is its high polarity and ability to undergo strong hydrogen bonding; both factors are inherently coupled to the moisture content. Hence, the effect of humidity on the mechanical properties is of profound relevance to be able to better compare mechanical performance and to understand fundamental aspects such as nanofibrillar sliding and inelastic deformations in such nanofibrillar networks. Surprisingly, to the best of our knowledge, there is no comprehensive experimental study addressing the changes occurring in films based on nanofibrillated cellulose (NFC), or as a matter of fact for other nanofibril materials (e.g. polyaramide, peptide), upon exposure to different humidities. The importance of such an understanding is yet underscored by comparing to other waterborne high-performance materials. For instance, we recently showed in a related waterborne bioinspired material, how humidity changes the mechanical fracture behavior of nacre-inspired, highly reinforced lamellar polymer/clay composites from a linear elastic and more brittle behavior at low relative humidity to a ductile and tough material at high humidity.39 Furthermore, Weder’s group reported on tunicate nanofibril/polymer composites that undergo changes from the percolated network structure to isolated reinforced state when placed into water.40-42 Herein we will demonstrate a substantial influence of humidity on the mechanical properties of NFC nanopapers by comparing data obtained from mechanical tensile tests in a range of different humidities (0 – 100 %RH, fully immersed in water). We will further show that, when swollen in water at different pH, the mechanical cohesion of the network depends on the amount of charges present due to the weak polyelectrolyte character of the carboxylic acid groups found on the surface of the nanofibrils. We will relate the observed changes and deformation modes to altered interfibrillar interactions in terms of hydrogen bonding and polyelectrolyte repulsion, as well as to the presence of different mesostructures formed in the films as a results of unlike stability of nanofibrillar dispersions prior to film preparation. Scheme 1 summarizes preparation, multiscale structure and the effect of tension on the nanofibrillar network.

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Scheme 1. Simplified scheme describing the formation process, hierarchical structure and effect of mechanical tension on nanopapers made from native cellulose nanofibrils. The material preparation starts with a filtration of a dispersion of native cellulose nanofibrils (a) to give a film which contains structural units on the mesoscale induced by aggregation in the late stages of filtration (b, c see Figure 4 for electron microscopy). Yielding of nanoscale network linkages (d, red) and interfibrillar sliding allow inelastic deformation once the network is set under tension (σ). (e) Fringe-fibrillar model used to describe amorphous and highly crystalline domains inside cellulose nanofibrils. The degree of polymerization of the underlying cellulose chains regulates the amount of tie chains and bridging of single chains along various crystalline segments. (f) Chemical groups, responsible for interfibrillar hydrogen bonds, found at the surface of the crystalline nanofibrils. TEMPO mediated NaOCl oxidation partly converts glucopyranose units at their C6 position into carboxylic acids. Either carboxylic acids or their sodium salts are found depending on the degree of neutralization.

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MATERIALS AND METHODS Preparation of Nanocellulose A 1.6 wt% suspension of TEMPO/NaOCl-oxidized43 bleached softwood sulfite pulp (oxidation in alkaline conditions) was provided by PTS Hagenau and homogenized in a microfluidizer (Microfluidicscorp MRT CR5) applying three shear cycles (500, 1400 and 1400 bar). From the aforementioned suspension, the following 0.1 wt % NFC dispersions were prepared: (i) adjusted to pH = 9 with NaOH and subsequent dialysis against pH 9, (ii) dialyzed extensively against Milliq water pH = 7 with pure water, (iii) dialyzed against Milliq and adjusted to pH = 3 with HCl. The dispersions were centrifuged (25 min, 8000 rpm) to remove visible impurities. Preparation of Nanopapers The 0.1 wt % dispersions were filtered through OmniporeTM Membrane filters with a pore size of 1.0 µm. Afterwards, a transparent hydrogel is obtained, which was dried in an oven at 60 °C for 5 days under pressure (approx. 2.5 kPa) to yield films with thicknesses of 40-50 µm. The densities were measured by weighing a piece of nanopapers of known dimensions at 40 %RH. Adsorbed water is included in the density value. Analysis Mechanical Properties. Mechanical tests were carried out on a DEBEN minitester equipped with a 200 N load cell at room temperature. The samples were conditioned at different relative humidities (RH) in the range of 0 - 100 % and swollen in water for at least 2 days. The specimen sizes used were typically in the range of 1 cm x 1.25 mm x 40-50 µm. At least 5 specimens were tested for each condition. A nominal strain rate of 0.1 mm/min was used. The slope of the linear region of the stressstrain curves was used to determine the Young’s modulus (E). The yield points were determined by the intersection of the two lines of the elastic region with the strain hardening region. Water Sorption. Water sorption isotherms were performed with a HIDEN ISOCHEMA IGAsorp device at 25 °C and 35 °C in a range of 5-95 % of relative humidity. Samples were exhaustively dried (60 °C) before being placed into the machine. The dried samples weighed approx. 20 mg were quickly placed into the machine and each isotherm was performed for a period of 60 min, starting from the lowest RH, giving enough time for a complete sorption and equilibration. Fourier Transform Infrared (FTIR) spectra were recorded using Thermo Nicolet Nexus 470 spectrometer with a smart split ATR single reflection Si crystal over a frequency range from 1800 to 1500 cm-1 with a resolution of 4 cm-1. Field-Emission Scanning Electron Microscopy (FE-SEM). Films surfaces and fracture surfaces were observed by a Hitachi S4800 FE-SEM. The samples were sputter coated with a thin gold/palladium layer. X-ray diffraction (XRD). Diffractograms were recorded from 10 to 30 ͦ of diffraction angle 2Θ in Bragg Brentano wide-angle x-ray scattering mode with Χ-φ-Z stage 240 mm (reflection) configuration using ACS Paragon Plus Environment

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EPPYREAN diffractometer system. Data was recorded using Cu Kα (0.1541 nm) x-ray incident beam from an Empyrean Cu LFF HR x-ray tube at 40 kV voltage and 40 mA current and a PIXcel3D detector. Atomic Force Microscopy (AFM). Atomic force micrographs of NFC-TEMPO nanofibrils were taken using a NanoScope V (Digital Instruments Veeco Instruments Santa Barbara, CA) AFM operating in tapping mode. The sample was obtained by dip-coating from a diluted suspension in water (0.005 %) onto mica. Polarized optical microscopy (POM) was performed on a ZEISS Axioplan 2 microscope using a 20 x lens at room temperature (23 ͦC). Samples were conditioned at the respective relative humidities and in water for 2 days.

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RESULTS The starting point for our investigations is NFC, prepared by TEMPO-mediated NaOCl oxidation of wood pulp and subsequent homogenization in a microfluidizer.43 The oxidation selectively converts parts of the C6 hydroxyl groups of the glucopyranose units into carboxylic acids and adds 0.54 mmol/g charged acid groups to the surface of the nanofibrils. A clear dispersion of the nanofibrils (c = 1.6 wt%) is obtained after homogenization and AFM shows long and entangled nanofibrils (Figure 1) with diameters in the range of 2 - 5 nm.

Figure 1. Microscopic characterization of individual cellulose nanofibrils and nanopapers surfaces. (a) AFM height image of NFC after deposition from a dilute aqueous dispersion onto freshly cleaved mica (z-scale = 13 nm). The inset shows a transparent NFC gel at a concentration of 1.6 wt%. (b) Top surface of a nanopaper prepared by filtration of NFC, depicting the entangled fibrillar network.

We conditioned these nanofibrils to different pH values and hence colloidal stability due to electrostatic repulsion imparted by surface carboxyl groups. Extensive dialysis of 0.1 wt% dispersions at acid (pH = 3), neutral (pH = 7) and basic (pH = 9) conditions yields purified TEMPO-NFC fibrils with the native COOH function (termed NFC-COOH) for the lowest pH, whereas the neutral conditions and adjustment with NaOH to pH = 9 leads to the formation of the salt form, COONa (termed NFC-Na+). FTIR confirms these chemical modifications with absorption bands (C=O stretching) for COONa at ca. 1600 cm-1 and COOH groups at 1720 - 1740 cm-1 (see Figure S1, Supporting Information).44 The COONa band is dominant for NFC fibrils conditioned to pH = 9 and pH = 7, with a slightly higher fraction of COOH groups for the TEMPO-NFC at pH = 7, as expected. Once the pH is reduced, the relative intensity of both bands inverts and the COOH functionality is dominant We chose these three different pHvalues to understand the influence of colloidal stability of dispersion, and also chemical interactions. It is well known that TEMPO-NFC, like other electrostatically stabilized nanoparticles, shows a pHdependent behavior with a tendency for aggregation at low pH due to insufficient repulsion, while basic pH provides the most efficient repulsion.15, 45 Neutral conditions at pH 7 are used as an intermediate value. Indeed we find aggregation at pH 3, which can also not be disrupted at prolonged sonication. We will later exploit this agglomeration to prepare films containing purposefully induced ACS Paragon Plus Environment

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aggregates, yet initially we will focus on films prepared from pH = 7 and pH = 9 to even reveal subtle differences in the mechanical performance. Nanofibrils at pH 7 are less efficiently stabilized as compared to pH = 9 and more prone to aggregation during filtration. Subsequently, we prepared nanopapers with NFC-Na+ form (pH = 7 and pH = 9) by simple vacuum filtration. During this process the concentration at the filter surface increases strongly. This leads to concentration-induced aggregation and floc formation of nanofibrils prior ultimate film formation and hence to deposition of those agglomerates in the final material. These phenomena are known for a long time for cellulose microfibers and paper-making.46, 47 However, the details of such aggregation are experimentally extremely difficult to understand for native cellulose nanofibrils, due to lack of proper experimental techniques. Yet it becomes clear that the films contain additional structural length scales on the mesoscale (see Scheme 1 and Figure 4 for electron microscopy). These define the material in addition to the size and structure of the nanofibrils, being composed of the highly crystalline and amorphous domains along the fibrillar axis, and their interfibrillar bonding. Such a hierarchical structure adds considerable complexity to understanding the mechanical deformation behavior. It is important to emphasize that phenomena found in NFC-based nanopapers require a fundamental and quantitative understanding as they are different to established concepts of classical papers based on sub-millimeter scale pulp fibers. This is due to the fact that dimensions and hence dynamics, associated energy scales, tendency and mechanism of aggregation, as well as structures and properties differ considerably. NFC nanopapers are largely defined by the interfaces due to the nanoscale building blocks. This motivates in particular to understand the effect of pH and interfibrillar sliding and humidity. In addition, structure formation during filtration is different because of unlike colloidal stability and interactions. While highly charged TEMPO-NFC can be described adequately by the DLVO theory45, pulp fibers contain additional components of steric stabilization due to dangling chains or fibrils.48, 49 In consequence, concentration-induced aggregation mechanisms and formed structures are different. The inherent flexibility (lower persistence length) of nanoscale fibers also leads to tighter packing, less porous films and transparency. Not to the least, this is the reason why NFC films outperform classical paper by one order of magnitude in the mechanical properties, or even more in gas barrier. These differences in interface and structure are important to understand in order to advance the properties of NFC materials and cannot be quantitatively derived or anticipated by looking at results from microscopic pulp fibers. Nanopapers contain a small fraction of porosity (≲20 %), which mostly depends on the diameter of the nanofibrils.37, 50, 51 Larger diameter nanofibrils typically pack less tightly and lead to larger pores and often higher fraction of porosity.51 Our nanopapers are based on very thin nanofibrils and hence we find high densities at 1.46 ± 0.04 g/cm3, close to the values reported for NFC or crystalline cellulose (1.46 - 1.58 g/cm3).21, 37 This indicates very little porosity. We give an estimation of the porosity as a function of the assumed density of NFC nanofibrils in the supporting information (See Figure S2). Attempts to measure the surface area and pore-size distribution using N2 and even Kr sorption ACS Paragon Plus Environment

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isotherms failed, hence confirming insufficient available surface and low porosity. Although only slightly porous, the pores are important for two aspects: First, the nanoscopic size of the pores leads to strong capillary forces to hold moisture inside the material. Second, the porous space allows room for unconstrained deformation under tension. For mechanical testing, we conditioned the samples for several days at the respective relative humidities and subjected them to tensile testing. The tensile testing curves are depicted in Figure 2 and all relevant data are summarized in Table 1.

Figure 2. Mechanical properties of nanopapers formed by NFC-Na+ as a function of relative humidity. (a) Stress-strain curves, (b) elastic modulus (Young’s modulus, E) and yield stress (σy), (c) tensile strength (σb) and strain at break (εb). All data are obtained considering the true cross section that enlarges with increasing humidity (see also Figure 3)

The strong effect of humidity on the mechanical performance is evident in all samples. The tensile testing curves show softening, as seen by decreased Young’s modulus and tensile strength, as well as an increased strain at break for higher humidities. One of the most pronounced transition occurs when passing from 0 %RH to 25 - 40 %RH, thus when introducing the first moisture at all. In complete absence of moisture, the materials demonstrate an almost purely linear elastic behavior and yielding hardly takes place. Already in presence of small amounts of moisture (25 %RH) a distinct yielding can

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be observed. NFC-Na+ (pH = 7) requires slightly higher relative humidity to undergo larger inelastic deformation compared to the dried state. We will comment on these differences further below. A clear decline of the Young’s modulus and tensile strength of one order of magnitude can be observed. This demonstrates limitations for water-borne NFC-based films as future structural materials or as substrate for organic electronics at high humidity, and has previously hardly been addressed. A humidity-induced decline of the Young’s modulus was reported for carboxymethylated microfibrillated cellulose24 using dynamic mechanical analysis. DMA is however restricted to small scale elastic deformations and incapable of capturing inelastic deformation phenomena and fracture. A quantification of the yield points and strain at break reveals that yielding is promoted by higher humidity as seen by the drop of the yield stress and longer strain at break. Interestingly, the strain at break changes only slightly (4.5 – 6 %) after passing the level of 23 or 40 % RH for NFC-Na+ (pH = 9) or NFC-Na+ (pH = 7), respectively. Another distinct transition occurs for completely hydrated samples submerged in water. Here significantly lower Young’s moduli, yield points and most importantly much higher elongations are found.

Table 1. Overview of mechanical properties of NFC-Na+ nanopapers prepared at two different pH values as a function of relative humidity. Relative

NFC-Na+ (pH = 9)

NFC-Na+ (pH = 7)

Young’s

Yield

Tensile

Strain at

Young’s

Yield

Tensile

Strain at

modulus

stress

strength

break

modulus

stress

strength

break

E (GPa)

σy (MPa)

σb (MPa)

ɛb (%)

E (GPa)

σy (MPa)

σb (MPa)

ɛb (%)

0

20 ± 1

231 ± 20

360 ± 25

4±1

17 ± 2

203 ± 11

326 ± 25

4.2 ± 0.7

23

17 ± 2

163 ± 8

264 ± 19

3.6 ± 0.6

14 ± 1

116 ± 4

203 ± 8

4.0 ± 0.2

42

15 ± 2

162 ± 18

271 ± 27

5.8 ± 0.7

11 ± 1

114 ± 7

203 ± 16

5.8 ± 0.6

60

13 ± 2

137 ± 14

232 ± 17

5.6 ± 0.4

11 ± 1

98 ± 6

178 ± 14

5.7 ± 0.8

80

12 ± 1

103 ± 10

199 ± 22

5±1

9.0 ± 1

66 ± 6

164 ± 12

5.9 ± 0.8

95

4±1

51 ± 8

123 ± 15

5.6 ± 0.7

5.0 ± 1

39 ± 5

89 ± 9

5.0 ± 0.6

100

1.5 ± 0.2

14 ± 2

34 ± 6

5±1

2.0 ± 0.4

12 ± 6

38 ± 9

5±1

soaked in water

0.7 ± 0.1

1.5 ± 0.2

5±1

17 ± 3

0.5 ± 0.2

1.5 ± 0.6

2.5 ± 0.5

12 ± 3

Humidity RH (%)

DISCUSSIONS The deformation mechanisms in NFC nanopapers are complex as we deal with a material that is structured on several length scales (Scheme 1). Considering the individual nanofibrils, it is important to realize that humidity is only able to effectively plasticize the amorphous domains, into which it can easily diffuse, while it leaves the crystalline parts unaffected. This is also confirmed by x-ray diffraction (XRD) comparing dry state, humid state (near 100 %RH) and soaked in water. The diffractograms show persistent diffraction peaks for the crystalline lattice (Figure S3), but slightly more pronounced amorphous background signals upon water uptake, which is due to an increased volume fraction of ACS Paragon Plus Environment

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amorphous material. Furthermore, water molecules adsorb on the nanofibril surface and act as competitive agent for interfibrillar hydrogen bonding. Let us first inspect the elastic regions. The value of the Young’s modulus is associated with four main deformation modes: Elongation, bending, bonding energy stored in the contact points and shear. Earlier finite element simulations on the elastic properties of nanocellulose papers discuss elongation of fibrils and bending as main components contributing to elastic deformation in networks of long nanofibrils with tight bonding at the contact points.52 The elastic behavior does not depend significantly on the bonding stiffness, as long as the fibrils are of sufficient length. Obviously increasing the humidity plasticizes the amorphous regions of the nanofibrils and allows easier elastic elongation and possibly bending, thus reducing the Young’s modulus. In addition, one has to take into account that the material swells with increasing humidity, which thereby decreases the overall density and density of junction points, and contributes to an overall decreased elastic modulus. The degree of moisture uptake is quite considerable and shows a non-linear dependence with respect to relative humidity, as seen in the water sorption isotherms displayed in Figure 3. The water content reaches up to 20 – 30 wt% at above 95% relative humidity, rather independently of the chemical modification of the NFC. A similar uptake was found for carboxymethylated microfibrillated cellulose (MFC) and a lower uptake was found for more hydrophobic melamine formaldehyde/MFC composites.24, 53 Higher temperatures lead to slightly enhanced water uptake, which is most visible at highest humidity.

Figure 3. Water uptake as a function of relative humidity. Water sorption isotherms of NFC-Na+ nanopapers (pH = 7 and pH = 9) describing the mass uptake of water as a function of relative humidity at two different temperatures and for both chemical modifications.

The importance of reduced density of junction points on the elastic modulus can also be seen in experimental work targeting nanopapers of different porosity, which can for instance be obtained in mixed solvents or via freeze drying.51, 54 Increasing porosity leads to lower stiffness, yet higher elongation. However, even at similar total porosity it was found that structures of lower surface area, thus containing more interfibrillar network junctions, have higher stiffness and strength.51 ACS Paragon Plus Environment

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The transition from linear to plastic behavior is associated with a yield point, followed by a fairly significant strain-hardening region. The strain at yielding shows only a minor decrease, yet the yield stress falls constantly while increasing the relative humidity. Aside of plastifying the amorphous regions of the NFC, the increased humidity has a significant effect on the strength of interfibrillar connections. Hence it is reasonable to associate the yield point with interfibrillar debonding and subsequent fibrillar sliding, aided by residual porosity in the material. The strong hydrogen bonding at 0 %RH almost completely prevents inelastic deformation. Added water molecules compete with the interfibrillar hydrogen bonding, and as adsorbed moisture increase, the amount of direct interfibrillar bonds decreases. This weakens the interfibrillar binding strength and allows easier debonding and promotes interfibrillar sliding. It is interesting to realize that the slope in the strain hardening regime after the yield point continuously decreases with increasing humidity, thus reflecting reduced interfibrillar friction and eased alignment of plasticized chains of the amorphous regions within the fibrils (see Figure S4 and fringe fibrillar structure in Scheme 1e). To gain a deeper understanding of the differences in the mechanical behavior and failure modes, we conducted careful scanning electron microscopy (SEM) imaging of the cross sections fractured at different relative humidity. An instructive comparison of fracture surfaces obtained in the dehydrated (0 %RH) and in the highly hydrated state (100 %RH) is presented in Figure 4. The fracture surface of the dried film shows moderate roughness reminiscent of typical brittle fracture. Yet the interface is not completely flat and micrometer sized structural features such as protrusion and holes can be identified (white arrows), likely stemming from the most loosely bound interfaces of agglomerated flocs. Individual fibrils cannot be identified on the nanoscale (inset, Figure 4a), clearly confirming nanofibril fracture without any pull-out as main fracture mechanism. The situation is drastically different in the hydrated state. Most striking is the appearance of the layered structure originating from pull-out of mesoscopic layers (50 – 100 nm thick layers), as well as the observation of bundles of nanofibrils and individual nanofibrils being pulled-out from these layers at considerable length scales up to few hundred nanometers. The layer formation stems from concentration-induced aggregation and floc formation during late stage of the filtration, as also suggested for polymer/NFC composites.20, 37

Pull-out on several length scales clearly originates from weakened interfibrillar interactions by

increased humidity and contributes to achieving toughness and inelastic deformation. On a larger length scale (several µm) an even larger scale roughness can be observed as some layers are clearly pulled out together (white arrows), which is reminiscent of the structural feature size found in the fracture surface of the dry specimen (see also Supplementary Information, Figure S6, S7). Additionally, the red arrows highlight nanofibrils, which are stretched up to several hundred nanometers inbetween various layers, forming mechanical interlayer connections. The amount of nanofibrils connecting these layers influences the probability and force for layer pull-out, and most probably also influence the strain at break. Samples fractured at intermediate humidity fall in-between the two shown cases, displaying torn fibrils and mesoscopic layers (Figure S7c). In summary the observed ACS Paragon Plus Environment

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deformation modes clearly indicate relevant processes happening on several length scales, from pullout of individual nanofibrils and bundles to mesoscale cooperative movement of nanofibrils as layers and associated layer pull-out, as well as fragmentation along micron-sized grains. These observations underscore the subtleness of the hierarchical deformation mechanisms encountered in nanofibrillar materials.

Figure 4. Different deformation mechanism in dry and moist conditions. Cross sections of films fractured at 0 %RH (a) and at 100 %RH (b). A clear transition of the fracture mechanism from brittle fracture with roughness in the micrometer regime (a) to pull-out phenomena on multiple length scales (mesoscale layers, bundles and individual nanofibrils) is visible (b). The red arrows highlight nanofibrils bridging from various layers into each other. The white arrows point to areas of larger scale roughness and pull-out of coupled layers (see also Figure S5, S6 and Figure S7c for fracture surfaces at 60 %RH).

Next, let us inspect the strain-to-failure. Surprisingly, the strain-to-failure in the range of 25 – 95 %RH is almost constant (4.5-6 %). From earlier studies on NFC nanopapers it is known that the underlying degree of polymerization (DP) of the cellulose chains is a strain-limiting factor at ACS Paragon Plus Environment

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intermediate humidity (50 – 60 %, εb ~ DP).21, 55, 56 The dependence on DP can be understood considering that entangled nanofibrils may only flow to a certain extent and subsequent excessive force concentrates within the nanofibrils. Thereupon pull-out of cellulose polymer chains from crystalline areas occurs and because shorter chains are packed into less crystals along the fibrillar axis, less tie chains result and reduced total strain-to-failure is observed (see Scheme 1e for model). This mechanism implies that nanofibril fracture is the dominant failure mechanism, simply because fracture-free and complete nanofibril pull-out should be rather independent of the DP as stress does not concentrate to a critical limit. The possible transition from nanofibril fracture to pull-out is mainly a function of the aspect ratio and interfibrillar bonding. Herein we deal with nanofibrils of exactly the same underlying DP and length, but change the strength of fibrillar contacts and the degree of plasticization of the amorphous cellulose segments. Despite allowed inelastic deformation and frictional sliding of the cellulose nanofibrils at intermediate to high humidity, a persistent structural characteristic still obviously limits interfibrillar reorientation beyond a certain point and the nanopapers fail at the same elongation. Since we observe similar strain-to-failures, it becomes evident that nanofibrils still fracture, as a complete transition to fracturefree nanofibril pull-out, due to weakened interfibrillar interactions at very high humidities, would lead to substantial improvements in strain-to-failure. The most pronounced change in strain-to-failure occurs upon passing from high humidity to the swollen state in water. NFC nanopapers, once dried, are stable in water and form strong hydrogels. This is different to freely dissolving, molecular carboxymethylcellulose (a close polymeric analogue) and hence points to some very persistent hydrogen-bonded junctions and strong colloidal entanglements. Substantially larger strain-to-failures are found for fully hydrated nanopapers in water (2-3 times higher) as compared to 95 – 100 %RH, with the value of the NFC-Na+ (pH = 9) being slightly higher than NFC-Na+ (pH = 7) (Figure 2). As a distinct advantage, the conditioning in water also allows to study the effect of pH, which can be used to mediate the interfibrillar interactions by providing additional electrostatic repulsion in water. High pH leads to increased electrostatic repulsion due to deprotonation of the present surface carboxyl groups. Note that different structures of the nanopapers can be excluded as these experiments are conducted with exactly the same material. Herein, we chose NFC-Na+ (pH = 9) as it gives an overall higher strain. Figure 5 clearly shows that increased repulsion at higher pH values leads to a consistent reduction of the elastic modulus, the yield stress and the stress level of fibrillar sliding, thus in agreement with the proposed weakening of interfibrillar interactions. All samples rupture at similar strain values, which again points to the fact that a structural feature limits the strain and that the interfibrillar interactions are not strain-limiting.

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Figure 5. Effect of interfibrillar repulsion in wet NFC films by adjusting the pH. Representative stress-strain curves of wet NFC-Na+ (pH = 9) nanopapers rehydrated in water at different pH-values as indicated. The cross sections were measured for each film. The inset depicts the evolution of the Young’s modulus as a function of the pH-value. The black and cyan arrows indicate primary yielding and secondary and tertiary yielding regions, respectively.

The most striking and unexpected observation is the identification of regions of increasing strain hardening (upturn in stress-strain curves, highlighted by green arrows) followed by another yielding. Two of these transitions can be found independently of the pH conditions, suggesting them to be independent of nanoscale chemical interactions. In fact, additional strain hardening and yielding must be related to a second kind of activation and motion of a structurally organized length scale that requires higher activation energy. Strikingly, the first additional yield phenomenon (6 - 8 % strain) occurs in the same strain region of the breaking point of the samples at lower humidity (ca. 6 % strain). High humidity alone is insufficient but full hydration in water is required. The strongly reduced hydrogen bonding and the ionization and repulsion of the nanofibrils induced by exposure to water are decisive to allow for a break-up of these points. We suggest that this yield point is associated with the release of nanofibrils or movement of strong cooperative entanglements. The additional yield point at ca. 14 % strain and the same strain at break independent of the adjusted interfibrillar repulsion (different pH) are indications for the breakup and resistance of additional mesostructures with even lower dynamics, such as mesoscopic layers or larger grains, respectively. Two further points underscore the assumption that mesoscale structures are decisive in defining the mechanical properties. First, we purposely induced interfibrillar aggregation into the NFC dispersion at low pH (pH = 3), whereupon the freely flowing 0.1 wt% dispersion turned into a light gel, and prepared films by filtration. Already macroscopically these films are different as they are fairly more opaque compared to fully transparent NFC-Na+ films discussed above (Figure 6b). Polarized optical microscopy also reveals the presence of grain boundaries (Figure S8). The tensile tests at 60 %RH reveal a significantly different behavior, that is a much lower Young’s modulus and to some surprise a larger strain-to-failure (Figure 6a,c). The density of the nanopapers prepared at pH = 3 is slightly lower (ca. 1.33 ± 0.04 g/cm3) compared to the other films, thus explaining lower Young’s ACS Paragon Plus Environment

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modulus and also slightly higher strain. Fujisawa et al. suggest that an individualization of these NFCCOOH by sonication favors denser films and increased mechanical properties.44 Herein we found that aggregation could not be broken efficiently and hence the resulting temporal changes in colloid stability of poorly stabilized NFC prevent reproducible materials preparation. The opaqueness is related to scattering at internal interfaces due to higher porosity (Figure S2) and also to the presence of larger grain boundaries. SEM images indeed depict larger flocs and aggregates in addition to a layered structure, whereas the cross sections for nanopapers prepared after extensive dialysis against water or basic conditions (NFC-Na+) are without discernible differences (Figure S7). Secondly, if we compare the stress-strain curve of all three materials, distinctly different values for the yield stress can be observed. Hence, considering that NFC-Na+ nanopapers prepared from pH = 7 and pH = 9 contain almost the same chemical structure, the different values indicate that even the first yield point, which is associated with interfibrillar debonding and requires the lowest stress, is cooperatively linked to local structural organization (see Figure 2 and Table 1). The detailed origin for this behavior must be sought in the better stability of the NFC at high pH, leading to retarded aggregation and probably subtle differences in local structural arrangement. These differences however remain elusive with the present level of experimental detail and call for further experiments e.g. using synchrotron small angle x-ray scattering to characterize the aggregation behavior in the concentrated regime and study structure formation in-situ during filtration with high spatial and temporal resolution.57

Figure 6. Effect of dispersion conditions on the mechanical properties of NFC nanopapers. (a) Tensile tests obtained for nanopapers at 60 %RH based on (i) NFC-Na+ conditioned to pH = 9 (subsequently dialyzed overnight to remove excess of base), (ii) NFC-Na+ conditioned to pH = 7 and (dialyzed overnight in pure water) and NFC-COOH conditioning to pH = 3 with HCl (also dialyzed overnight) with HCl (iii). (b) A digital photograph showing the comparison of clarity for transparent films of NFC-Na+ prepared after extensive dialysis against water (right) and after adjusting the pH to 3 with HCl (left). The films are ca. 2.5 x 2 cm and are placed in a distance of 5 cm in front of the printed text. Note that both samples show a similar transparency when placed directly in front of the text. (c) Comparison of the Young’s modulus of the NFC films as a function of the pH value. See Figure S7 for SEMs of cross sections.

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For structural materials, it is desirable to have a high Young’s modulus and a large elastic region (high yield point), simply because this allows to fully elastically recover larger stresses imparted on a material. Herein, we find that such a behavior is preferred for nanofibrillar dispersion prepared from less aggregated, more individualized fibrils. Even though this might be qualitatively anticipated, the quantitative differences in stiffness and tensile strength are remarkable with an almost doubling of the respective material characteristics when properly handled. In summary, this demonstrates the paramount importance of understanding and controlling the dispersion state of nanofibrils during the material preparation.58 These results also call for developing experimental techniques capable of capturing the complex aggregation phenomena of strongly interacting (cellulose) nanofibrils during late stages of solvent removal.

CONCLUSION We showed for the first time in a quantitative experimental approach how humidity influences the mechanical properties of nanopapers formed by native cellulose nanofibrils. The NFC films undergo a transition from a predominantly linear elastic fracture behavior in dry state to materials capable of plastic deformation. Two distinct moisture-induced transitions in the mechanical behavior are observed. The first from completely dry state to 25 – 40 %RH enables inelastic deformation. It is caused by the competition of water molecules with the strong interfibrillar hydrogen bonds, leading to disengagement of the network and lower nanofibrillar friction to allow for flow. The second occurs upon immersion in water, which drastically enhances interfibrillar disengagement – also controlled by pH and electrostatic repulsion – to overcome strain-limiting cooperative entanglements and mesoscale structures of nanofibrils and substantially extend the maximum strain by 200 - 300 %. From a materials perspective, the loss in mechanical properties from the dry state to the humidified state (95 – 100 %RH) is considerable – almost one order of magnitude – and a consistent decrease of the Young’s modulus, yield points and tensile strength are observed. This demonstrates limitations for water-borne NFC-based films as future structural materials, as substrate for organic electronics, or for separation membranes at high humidity, and calls for developing efficient ways to stabilize them against water-induced decay of mechanical properties. On a fundamental level, it becomes clear that controlling the colloidal state of the dispersion and the tendency of interfibrillar aggregation is decisive to govern the multiscale structure formation of nanofibril-based films. Drastically poorer mechanical properties are found for nanofibrils with higher tendency of aggregation, or even purposefully aggregated ones. Careful SEM imaging demonstrates pull-out and deformation modes on various length scales. Additionally, the multiple yielding phenomena found only in significantly disengaged networks in pure water allow concluding that ultimate strain is also coupled to relaxation of structural entities, such as cooperative entanglements and aggregates.

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Looking out to the future, this study motivates to find efficient ways to control interfibrillar interactions, and look in closer detail how small amounts of water-soluble polymers affect nanofibrillar dispersions to achieve better mechanical properties. From a materials perspective it becomes important to find ways towards water-resistant nanocellulose materials to better suit application needs. Moreover, the subject of “flowing” nanofibrils provokes questions how theories of entangled polymer melts and reptation can be applied to nanofibrillar materials and how other types of polar nanofibrils undergo humidity-induced changes in mechanical deformation.

SUPPORTING INFORMATION Supporting FT-IR, XRD, SEM and POM images and additional information of the strain hardening can be found in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding author

Email: [email protected] Notes The authors declare no conflicting interests.

ACKNOWLEGDMENTS We thank Baolei Zhu, Manuel Noack and Baochun Wang for supporting AFM measurements and Paramita Das for XRD measurements. We further acknowledge financial support from the BMBF in the framework of the AQUAMAT research group, the “ERANET Woodwisdom Program” financed by the BMELV and the “Fonds der Chemischen Industrie”. We thank Lars Berglund and Olli Ikkala for discussions. Andreas Walther gratefully acknowledges continuous support by Martin Möller.

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