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Unraveling the rapid assembly process of stiff cellulosic fibers from mistletoe berries Nils Horbelt, Michaela Eder, Luca Bertinetti, Peter Fratzl, and Matthew J. Harrington Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00648 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Unraveling the rapid assembly process of stiff cellulosic fibers from mistletoe berries Nils Horbelt,† Michaela Eder,† Luca Bertinetti,† Peter Fratzl,† Matthew J. Harrington*,‡

†

Dept. of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam 14424,

Germany

‡

Dept. of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec

H3A 0B8, Canada KEYWORDS. bio-inspired materials, nanocellulose, green material processing, selfassembly

ABSTRACT The mucilaginous viscin tissue within mistletoe berries possesses an extraordinary ability to be rapidly processed under ambient conditions into stiff cellulosic fibers (>14 GPa) through simple mechanical drawing. This rapid and extreme transformation process is hydration-dependent and involves an astonishing >200-fold increase in length, providing a relevant role model for efforts to produce advanced composites from cellulose-based structures such as cellulose nanocrystals (CNCs) or 1 ACS Paragon Plus Environment

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cellulose nanofibrils (CNF). Using a combination of in situ polarized light microscopy (PLM), synchrotron X-ray scattering and humidity-controlled mechanical analysis, we examine here the dynamic transition of viscin cell bundle (VCB) from hydrogel-like tissue to high-performance fiber. Our findings indicate a massive phase transition in which cellulose microfibrils (CMFs)1 containing high aspect ratio crystalline domains undergo dramatic reorganization, facilitated by a water-responsive non-cellulosic matrix. Transition from an aligned, yet flowing state to a stiff fiber is likely triggered by rapid water loss below 45% relative humidity (RH). These findings not only help understanding the adaptive success of mistletoe, but may also be relevant for the development of new facile processing methods for next-generation cellulosic composites.

Introduction A future in which humans are less reliant on petroleum-based polymers requires development of sustainable material paradigms. Many biopolymeric materials have emerged as important role models for inspiring how humans might produce polymeric materials with improved properties under greener processing conditions. Indeed, recent investigations of the fabrication of spider silk,2 mussel byssus,3 and the slime fibers from velvet worms4 and hagfish5 offer possibly transformative insights in this direction. In the case of silk, bio-inspired concepts have even been applied for the commercial production

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of artificial silks with near native properties.6 Along these lines, we examine here the remarkable fiber formation behavior exhibited by mistletoe berries7 as an intriguing system for understanding the processing of cellulose-based materials under ambient conditions. This is especially relevant in light of recent efforts to process nanocellulose isolated from biological sources (e.g. plant tissues, algae, bacteria, tunicates) into highperformance composite materials8 9 10 11 12 The European mistletoe Viscum album is a shrubby hemiparasitic plant species that predominantly grows on hardwoods.13 14 Its fruits are shiny white berries with seeds inside (Figure 1C). The seeds of V. album are typically roundish or heart shaped,15 flattened on two sides and surrounded by a thick spherical and sticky layer named viscin, a mucilaginous glue. Due to its adhesive nature, viscin was traditionally used to produce birdlime (Latin: viscum) – a natural glue made from mistletoe berries, which was spread on branches to capture birds since ancient times.13

16

When the berries ripen in winter,

they serve as a food source for birds, which act as the primary vector for spreading the seeds of V. album. This primarily occurs in one of two ways: 1) Birds peck into the berries and the sticky seed adheres to bird’s beak, after which it dislodges the seed onto nearby branches, forming small sticky threads that help to secure the seed onto the branch where it can germinate.13

14 16 17

2) Birds rapidly swallow a couple of whole berries, which pass

through the digestive system within minutes and are excreted onto lower branches onto which the viscin adhesive glues the seed.13 16 17 This results in formation of long cellulosic


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adhesive fibers, often connecting numerous seeds hanging loose like a string of pearls,16 which by shaking in the wind can come into contact with a nearby branch where they adhere and germinate.

D C E

ex

hc

VCB

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A

vc

500µm

P

s A

20µm p

B

fruit skin

P

10mm

F

ft

fiber

p

transition zone

viscin cell bundle

10mm seed

50µm 50µm

A Figure 1. Morphology of mistletoe berry and viscin bundles. A) Photograph showing the typical formation of adhesive viscin fibers connecting the seed with the fruit skin B) White berries of V. album with small floral traces (ft) on the top collected in groups of 3 including the peduncle (p). C) Schematic section of a mistletoe berry showing the inner organization with the green seed (s) in the center, surrounded by radially oriented hairy cells (hc). Viscin 4 ACS Paragon Plus Environment

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cell bundles (VCB) are connected to the bottom of the seed arcing towards the top of the berry covered by vacuolated cells (vc) towards the exocarp (ex). D) Polarized light microscopy (PLM) image of manually isolated and hydrated VCB viewed under crossed polarizers. E) PLM image of a single viscin cell showing the perpendicular arrangement of cellulosic filaments. F) PLM image of a typical mistletoe viscin fiber. Early microscopic investigations15 17 and later ultrastructural analysis of berries from V. album7 and a distantly related species Phtirusa pyrifolia18 reported that formation of mistletoe fibers emerges from specific bundles of elongated cells (Figure 1D) known as viscin cells that are embedded within the mucilaginous tissue surrounding the seed. In both species, these cells possess an irregular cross-sectional shape with massive cell walls and small central lumen, in which cellulose fibrils are arranged in an unusual manner, perpendicular to the cell long axis by an angle close to 90° (Figure 1E). Viscin cells were reported to be easily stretched leading to the formation of numerous micron-sized elongated filaments with highly aligned cellulose fibrils.7 18 However, the dynamic nature of this processability and the relationships between force, hydration, cellulose alignment and the resulting mechanical properties of the mistletoe fibers have not been fully elucidated. Clearly, a deeper understanding of this process would be highly relevant to current efforts in nanocellulose based materials fabrication, which currently involve diverse and intricate processing methods based on, for example, application of electrical



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and magnetic fields, mechanically deforming cellulose reinforced gels and alignment of cellulose by flow in a microfluidic device.19 20 21 22 In order to address this critical question in biological materials self-assembly, we performed a multiscale mechanical and structural characterization of the fiber formation process utilizing polarized light microscopy (PLM) and relative humidity (RH) controlled tensile testing, as well as both wide-angle and small-angle X-ray scattering (WAXS/SAXS). Our results indicate that viscin bundle cells contain pre-organized microfibrils with high aspect ratio nanocrystalline domains, that undergo a rapid and collective transition in both orientation and higher order structural arrangement during mechanical drawing, during which the nascent fibers possess a high degree of cellulose alignment, yet flow like a polymer melt. Through this simple mechanical processing, the viscin bundle transitions from a viscous hydrogel of several millimeters in length to a stiff (>14 GPa) fiber of more than one meter, containing highly aligned CMFs. The key to this remarkable process is a water-responsive matrix component between CMFs that behaves as a sacrificial, shearthinning polymer network above 45% relative humidity (RH), allowing CMFs to align and glide past one another and below 45% RH, as an extremely effective cement, locking CMFs in place to produce a stiff, yet flexible polymer fiber. This behavior is a highly adaptive response that has aided the proliferation of this hemiparasitic plant species, and which may also have potential to inspire novel and sustainable composite fabrication methods. Experimental Section


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Material. V. album ssp. album plants were collected from apple trees in Golm, Germany in winter. Berries were either used immediately for experiments or were flash frozen by collecting berries with peduncle intact in 50 ml falcon tubes sealed with parafilm and submerged into liquid nitrogen for 5 minutes. Falcon tubes were stored at -20 °C and single berries were thawed for later use. The freeze-thaw process did not result in noticeable differences in fiber forming ability. Picking a single berry usually damages the skin near the peduncle creating a small opening. Gentle compression between fingertips squeezes the seed through the opening. Using razor blades to cut the skin, seeds were removed from the berry using fine tweezers, maintaining the integrity of the viscin layer and bundles. Light Microscopy (LM) and Polarized Light Microscopy (PLM). For investigation of fiber formation, VCBs were cut near the seed interface and the fiber was cut a few centimeters from the tip of the VCB. The obtained samples were carefully transferred onto a glass slide with the help of fine tweezers. Exploiting the natural stickiness, the sample was glued on a glass slide. The samples were investigated with a digital microscope (Keyence VHXS550E) equipped with a universal objective (VH-Z100UR) operated in transmission mode for conventional LM or under crossed polarizers with optional use of a quarter-wave plate for PLM. To reduce the influence of the sample thickness on the interference colors observed in PLM, dried samples were trimmed parallel to the surface of the glass slide with a rotational microtome (Leica RM2245) to obtain a homogeneous sample thickness.



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Environmental Scanning Electron Microscopy. Fibers were investigated in an SEM in low vacuum mode (FEI Quanta FEG 600). Images were obtained at an acceleration voltage of 4kV using a secondary electron detector. Mechanical Testing. For mechanical testing, fibers were drawn manually by pulling the seed out of the berry with fine tweezers. The resulting twin fibers were glued to a laboratory stand using their natural stickiness and dried under the seed’s own weight at ambient conditions overnight (Figure S1A). Small homogenous segments from individual fibers were glued onto a foliar frame using cyanoacrylate superglue (Loctite 454) and cured overnight. The initial free fiber length was determined by taking micrographs with a digital microscope (Keyence VHX-S550E) and by analyzing with Fiji 1.51n resulting in a fiber length of ~5.4 mm. Mechanical experiments were conducted using a custom-built tensile tester operated with custom-built software (1D force table by K. Bienert), which allowed the control of the RH and the T inside the testing chamber. With the help of a custom-built humidity generator (Dr. Wernecke Feuchtemesstechnik Humigen, Potsdam, Germany) RH and T were regulated during the experiment and the tensile tester T was kept constant at ~20 °C via a thermostat (Huber Ministat 125cc). Samples were loaded in foliar frames and fixed between two clamps on the tensile tester. Frames were cut on both sides flanking the fiber so only the fiber was connecting both ends of the tensile apparatus. Force was measured in a sealed chamber with a load cell (Honeywell 31E) with a maximum capacity of 0.5 N at a testing speed of 5 µm s-1, while deformation was


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recorded using a digital camera (Basler acA1920-40um) mounted on a stereo microscope (Olympus SZX7). All samples were stored in a sealed box with silica gel before testing. Each fiber was equilibrated for at least 3 h at 0% RH and 20 °C before it was successively tested at the following RH levels: 0%, 20%, 30%, 45%, 60%, 75% and 90% with an equilibration time of 30 min after each successive change of the RH. Each test consisted of 3 cycles with a motor travel distance of 25 µm (0% to 45% RH) or 75 µm (60% to 90% RH). Tensile stiffness was determined as the mean value of the slope of the initial linear region of the stress-strain curves of 3 cycles. Stress was calculated on the basis of the fiber diameter at each RH level assuming a cylindrical cross section as confirmed with environmental scanning electron microscopy (Figure S1). Diameter was determined at each RH level by calculating the mean of 10 measurement points per sample with a digital microscope (see LM section). 5 fibers from 5 different berries were tested successfully. PLM with In Situ Fiber Drawing. Dissected seeds were spiked on a sample holder connected to a high precision load cell (Honeywell 31E) with a maximum capacity of 0.5 N to measure the drawing forces. The initial short fiber was attached to a spinning wheel connected to a LEGO® Technic step motor with a tunable rotation speed ranging from ~30 µm s-1 to ~6 mm s-1 placed under a digital microscope (Keyence VHX-S550E equipped with a VH-Z100UR universal objective) with crossed polarizers in transmission mode. The drawing direction was set to an angle of 45° relative to the polarizers via a rotational stage and videos of the transition zone were recorded during the drawing



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process (Video S1). Videos were recorded with a frame size of 1600 x 1200 pixels and a frame rate of 15 Hz. Additionally, a commercial table-top testing machine (Zwick zwickiLine Z2.5; 10 N load cell) was utilized, which allowed higher drawing speeds up to 1.66 cm s-1 with a maximum linear travel distance of ~65 cm. Seeds were clamped on one side of the device and the remaining part of the berry was clamped on the other side without cutting the fiber or the tissue (Video S2). Thermogravimetric Analysis. The moisture content (MC) as a function of relative humidity was measured with a thermogravimetric analysis system (Setaram, Sensys Evo TG-DSC),

connected

to

a

custom-built

humidity

generator

(Dr.

Wernecke

Feuchtemesstechnik Humigen, Potsdam, Germany). The water sorption–desorption isotherms of ~17 mg of dried fibers were measured at 29 °C by applying a step-by-step humidity program with 10% RH steps and equilibration time of 4 h. The MC was calculated using Equation (1): 𝑀𝐶 =

𝑚𝑤𝑒𝑡 ― 𝑚𝑑𝑟𝑦 𝑚𝑑𝑟𝑦

∙ 100%

Small Angle X-ray Scattering and Wide Angle X-ray Scattering. The synchrotron scattering experiments were conducted at the mySpot beamline23 at the BESSY II synchrotron radiation facility (Helmholtz-Zentrum Berlin, Adlershof). Fibers were prepared as described for mechanical testing and mounted vertically and perpendicular to the incident X-ray beam in a custom-built tensile tester replacing glass windows with Kapton 10 ACS Paragon Plus Environment

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foils. The fiber was pre-stretched at low forces at ambient conditions to ensure a straight vertical alignment. During the entire measurement the force was kept constant. A humidity generator (Setaram, Wetsys) was connected to the tensile testing chamber applying a step-by-step humidity program with 10% RH steps with an equilibration time of 1.5 h. Continuous line scan across the fiber were performed to measure changes during hydration/dehydration with 5 adjacent measuring points with a beam size of 50 µm and a measurement time of 120 s were performed. To avoid beam damage each successive line scan was shifted vertically by 100 µm along the fiber. The wavelength of the incident beam was set to 0.082656 nm using a MoBC multilayer monochromator. SAXS patterns were collected with a 2D CCD detector (Rayonix MAR Mosaic225, USA) with total area of 3072 x 3072 pixels and a pixel size of 73.2 µm x 73.2 µm at a sample-to-detector distance of ~85 cm. WAXS patterns were collected at a sample-to-detector distance of ~30 cm. Samples from the transition zone between VCB and fiber were mounted vertically and perpendicular to the incident X-ray beam. Mesh scans were performed with a 2 x 2 detector binning and a measurement time of 45 s per point. Two mesh scans with a grid of 25 x 9 and 10 x 5 scan points with a beam diameter and step size of 50 µm resulted in two adjacent scanned total areas of 1.25 mm x 0.45 mm for the VCB and 0.5 mm x 0.25 mm for the fiber, respectively. Dimensions of crystalline cellulose domains were determined using a Si 111 DC monochromator to reduce signal broadening due to the



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experimental setup of the beam line. Three spots were measured on each of two fibers harvested in two different seasons in winter 2016 and 2017. The measurement time was 120 s. The beam diameter was 50 µm. The 2D scattering patterns were further processed and analyzed with dpdak v.1.3, an open source XRD analysis tool.24 Radial intensity profiles in this work are presented as a function of the scattering vector q according to Equation (2): sin 𝜃 𝑞 = 4𝜋 𝜆 Q-values from reciprocal space were transformed into real space using Equation (3): 𝑑=

2𝜋 𝑞

Radial profiles were obtained from small sections with an azimuthal width of 10° along the meridian and the equator and from a region showing no defined reflections used as an estimate for the contribution of amorphous scattering. Dimensions of crystalline domains were calculated using the Scherrer equation (Equation (4)) in which Lhkl is the mean crystal size normal to the indexed reflection plane; K is a dimensionless shape factor close to unity (0.9); λ is the wavelength of the incident X-ray beam (0.082656 nm); Bhkl is the analyzed FWHM of the indexed reflection after subtracting the instrumental line broadening and ϴ is the Bragg angle. 𝐿ℎ𝑘𝑙 =

𝐾𝜆 𝐵ℎ𝑘𝑙cos 𝜃

Results and discussion 12 ACS Paragon Plus Environment

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From Ultra-Soft Cells to Stiff Fibers. Translucent white berries from V. album were collected in early winter from apple trees and were carefully dissected to remove the seed with the nearly transparent gelatinous viscin layer fully intact. Along the flat surfaces of the seeds, two mirrored whitish strands (~1 mm wide by several mm long) are connected to the bottom of the seed, which we henceforth refer to as viscin cell bundles (VCBs). The VCBs arc towards the top of the berry where they are connected to an inner layer of the fruit skin, as illustrated in the schematic in Figure 1C. VCBs were previously described as clusters of densely packed elongated viscin cells, which provide the source of fiber formation and thus, the focus of the current investigation.7 18 Applying a small tensile load to the VCBs with tweezers resulted in the formation of two individual viscin fibers (Figure 1B) emerging from the ends of the VCBs (Figure 1A). Initially these fibers were only a few mm to cm long, extremely sticky and highly extensible. Upon further manual drawing, they readily extend into thin fibers (diameter 20 - 100 µm) with a final length of more than 1 m each – a more than 200-fold increase in length. If the two individual fibers come into contact during the drawing process, they fuse into a single fiber. While the gelatinous layer surrounding the seed remains rather sticky for several hours, the fibers lose their viscous, adhesive character after only a few minutes of drying under ambient conditions and become quite stiff, but remain flexible in bending and twisting. In order to characterize this rapid and dramatic change in material properties, micro-tensile tests were performed using small segments of fibers. When tested under



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dry ambient conditions (23 °C; 20% RH), fibers exhibited a linear elastic behavior with no observed yield point, followed by brittle failure at ~2% maximum strain. Notably, fibers possessed an average tensile stiffness of more than 10 GPa, which is comparable to that of dragline spider silk25 or wood cell wall.26

Figure 2. Humidity-dependent mechanics and swelling of viscin fibers. A) Exemplar stress strain curves for two viscin fibers measured at 0% RH (green) and 90% RH (black). Underlying red triangles represent the linear region used to determine the tensile stiffness. B) The mean tensile stiffness of 5 successfully tested viscin fibers as a function of the RH. C) Sorption isotherm at 29 °C from thermogravimetric analysis (TGA) presented 14 ACS Paragon Plus Environment

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as the moisture content as a function of the RH for the sorption (black triangle) and desorption (red triangle) cycle. D) The volume swelling of tested fibers from Figure 2B as a function of the RH. An alternative presentation of the tensile stiffness as a function of the moisture content, approximated from TGA analysis, can be found in Figure S2. To further investigate the contribution of hydration to the formation and mechanical performance of viscin fibers, micro-tensile tests were performed under controlled RH and T conditions. Fiber material stiffness is highly dependent on humidity as illustrated by the extremely different stress-strain curves observed going from 0% to 90% RH, which resulted in a 35-fold drop in stiffness from ~14 GPa down to ~0.4 GPa (Figure 2A-B). Examining the stiffness of fibers equilibrated over a range of different RH conditions reveals that stiffness plummets between 30% and 60% RH, clearly indicating that dehydration plays a key role in mediating the transition from viscous hydrogel to stiff fiber. A subsequent macroscopic analysis of the fiber swelling as a function of the RH showed almost no change of the volume from 0% RH up until 45% RH, followed by a sudden increase from 45% to 90% RH to a maximum volume swelling of ~23% (Figure 2D), exhibiting a change in the mean fiber diameter from 36 µm ± 4 µm at 0% RH to 40 µm ± 4 µm at 90% RH (Fig. S1). A water sorption isotherm (Figure 2C) obtained from thermogravimetric analysis (TGA) shows a similar behavior in which there is almost no water uptake from 0% to 30% RH, followed by a sudden increase in the moisture content (MC) up to ~34% MC at 95% RH, which is a highly unusual behavior compared with that 15 ACS Paragon Plus Environment


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of other natural cellulosic fiber materials.27 The desorption isotherm indicates that this process is fully reversible with almost no hysteresis observed. Microscopic Investigation of Fiber Formation. To better understand this dramatic transition, we performed a multiscale structural analysis on the VCB with a specific focus on the transition zone where the VCB transforms into a thin fiber. After carefully dissecting VCBs from the seed, they were first examined using conventional light microscopy. The tissue was almost transparent, showing little contrast and revealing few details of its structure. On the other hand, polarized light microscopy (PLM) provided improved contrast due to the natural birefringence of the VCB arising from the anisotropic arrangement of cellulose. As previously reported,7

18

PLM reveals that the VCB tissue

consists of aligned and densely packed viscin cells with an elongated shape and a typical diameter between ~20 - 40 µm. The individual cells possess massive cell walls with irregular cross sections and a small lumen. Towards the edges of the VCB, the cells showed a more relaxed order with some viscin cells sticking out sideways, revealing their slender tipped ends. Also in agreement with earlier studies,7 18 each cell showed the presence of submicron sized filaments oriented perpendicular to the long axis of the cell (Figure 1E). In the hydrated state, the bundle as well as the individual cells were highly flexible while upon drying they lose their flexibility and finally become brittle. While the bulk of the VCB is relatively uniform, an abrupt structural change was observed near the tip where fiber formation is initiated – numerous long filaments of ~2-3


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µm in diameter were observed to extend from the VCB. These filaments were highly aligned along the fiber direction and glued together by a non-birefringent matrix material to form the macroscopic viscin fiber. The specific orientation of the cellulose within the VCB, transition zone and emergent fibers was investigated using a PLM equipped with a quarter-wave plate, which exhibits a change in the polarization color depending on molecular orientation. As shown in Figure 3A, within the transition zone, the cellulosic filaments abruptly change from a perpendicular arrangement in the VCB to a highly aligned parallel orientation in the resulting fiber with respect to the long axis, as revealed by the sharp transition of the polarization color from blue to yellow. In situ Microscopic Investigation of the Dynamic Fiber Formation Process. To gain further insights into the dynamic nature of the dramatic structural changes occurring at the transition zone, a custom-built fiber drawing device was developed to follow the transformation in situ during PLM measurements. With the seed fixed and a short initial fiber from the VCB attached to a spinning wheel from the drawing device, the VCB was initially stretched at low drawing speed of ~30 µm s-1 until a steady state was achieved, after which continuous drawing resulted in fiber formation almost exclusively at the tip of the VCB, while the bulk VCB remains mostly unchanged. Although the bulk VCB shows only diffuse birefringence throughout the whole drawing process (Video S1), the drawn fiber exhibited much stronger birefringence shortly after emerging from the bulk VCB. The extremely high degree of cellulose alignment in the fibers was further emphasized by



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the nearly total extinction of the birefringence upon aligning the fiber axis parallel to the polarizer (Video S1), a characteristic reminiscent of hemp bast fibers in which cellulose orientation is nearly parallel to the cell long axis.28 Increasing the drawing speed stepwise up to a maximum of ~0.6 cm s-1 did not affect the fiber formation process. Higher automated drawing speeds (up to 1.66 cm s-1) achieved using a commercial Zwick tensile tester (Video S2) also revealed a similar fiber formation mechanism localized at the VCB transition zone and showed that the fiber formation process can initiate from both ends of the VCB (Figure 1A). At maximum drawing speed, several samples broke before the maximum travel distance of ~65 cm was reached, which limited the maximum fiber length achieved by automated drawing. Remarkably, however, manually drawing fibers with tweezers (drawing speeds in the range of m s-1), frequently resulted in the formation of fibers from a single berry longer than 2 m. With both tensile test devices, throughout a range of pulling speeds, we measured constant drawing forces of ~20 mN during the simultaneous drawing process of two fibers emerging from a single berry. WAXS and SAXS Characterization of CMFs in Viscin Cell Bundles and Fibers. Dynamic PLM studies revealed that nanoscale structural reorganization within the VCB localized at the transition zone plays a critical role during fiber formation. In order to investigate this hierarchical length scale and the potential role of cellulose orientation, we employed synchrotron WAXS, which is a well-established technique for investigating


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structural organization of cellulosic materials. Manually drawn and dried fibers were mounted in an X-ray beam (50 µm spot size) and scattering patterns were collected in a mesh over the transition zone going from bulk VCB to fiber. An image of the sample grid can be seen in Figure 3B where each pixel equals one 2D scattering pattern of the scanned volume. Three selected scattering patterns (Figure 3C to 3E) were chosen from the bulk VCB, the transition zone and the fiber in order to illustrate the large nano-structural changes occurring during transformation process. Azimuthal intensity profiles obtained from the most intense cellulose reflection (200) – notation for cellulose 1β29 – were used for a qualitative analysis of the cellulose orientation. Within the VCB, we deduce a preferred, but loosely organized cellulose orientation perpendicular to the drawing direction (i.e. perpendicular to the long axis of the VCB), based on the azimuthal profile (Figure 3E), which shows two corresponding maxima with broad distributions centered around 100° and 280°. The shift of ~10° from expected peak centers at 90° and 270° arises from a slightly tilted arrangement of the individual cells along the length axis of the VCB. In stark contrast, within the drawn fiber, cellulose is highly aligned along the fiber axis based on the two narrow peaks in the azimuthal intensity plot at 0° and 180° (Figure 3C). The transition zone shows evidence for cellulose oriented both perpendicular and parallel to the fiber drawing axis (Figure 3D) based on two pairs of maxima observed at 0°/180° and at 100°/280°. This is consistent with the observation using PLM that the transition



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zone is the site where the cellulose within the viscin cells flows and reorients to form the emerging fiber.

Figure 3. WAXS and PLM investigation of cellulose orientation across the VCB transition zone. A) PLM image of a VCB with an emerging fiber viewed under crossed polarizers and a quarter-wave plate. Changing polarization colors from blue (VCB) to yellow (fiber) indicate the sudden change of the cellulose orientation. B) Image of a mesh scan over the VCB transition zone where each ‘pixel’ represents a scattering pattern of the scanned


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volume with a beam diameter of 50 µm. C-E) Selected scattering patterns from the fiber (C), the transition zone (D) and the bulk VCB (E) and the corresponding azimuthal intensity profiles of the (200) cellulose reflection. In addition to the broad intense (200) reflection in the radial scattering profile, viscin fiber scattering patterns (Figure 4A) showed the presence of an extremely sharp (004) cellulose reflection, which originates from the regular stacking of crystal unit cells along the crystal length axis.29 Radial intensity profiles of small azimuthal sections along the meridian (004) and the equator (200) are shown in Figure 4B where a strong amorphous background arising from the matrix has been subtracted. Typical peak broadening due to a small crystallite width is evident in the equatorial profile based on the overlapping (110)/(110) reflections and broad (200) reflection.30 In contrast, the crystalline domains appear to be quite long based on the extremely sharp (004) reflection in the meridional profile. The (200) and (004) peak widths were further analyzed by Lorentzian peak fitting allowing extraction of the full width at half maximum (FWHM) which was used to calculate the dimensions of the crystalline domains with the help of the Scherrer equation (Equation 4). Apparent mean width and length of L200 ~2.5 nm and L004 ~67 nm, respectively, were calculated leading to a quite high aspect ratio of ~27. While the obtained width is comparable to findings from other primary cell wall systems,31

32

the apparent length is

extraordinarily high compared to literature values based on WAXS analysis reported from spruce wood,33 34 sugarcane,35 and bamboo.36 Moreover, considering that sources besides 21 ACS Paragon Plus Environment


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instrumental line broadening were not accounted for (e.g. disorder from micro-stresses,37 twisting,38 structural inhomogeneities39 or contaminations from neighboring reflections), the reported values are likely an underestimate of the true dimensions and reported values should be interpreted as apparent minimum values.

Figure 4. SAXS/WAXS investigation of the dimensions of crystalline cellulose domains and higher order structural organization. A) Viscin fiber WAXS pattern. B) Radial intensity profiles as a function of the scattering vector q of sectors along the equatorial reflections (blue) and the meridional reflections (orange), where background scattering due to air and amorphous scattering was subtracted. Highlighted broad (200) and sharp (004) 22 ACS Paragon Plus Environment

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reflections indicate a strong anisotropy of the crystalline domains. C) (200) azimuthal intensity profile revealing the nearly perfect cellulose alignment along the viscin fiber. D) Zoomed in detail of (A) with strong lateral SAXS reflections indicating a highly regular structural organization of the CMFs with a center-to-distance of ~5.7 nm at ambient conditions. E) Radial intensity profiles at low q along the SAXS reflections at the following RH-levels: a) ~95%, b) ~85%, c) ~75%, d) ~65%, e) ~55%, f) ~43%. A reversible peak shift towards lower q can be observed during dehydration accompanied by a dramatic decrease in scattering intensity. CMF center-to-center (ctc) distances ranging from ~7.8 nm in the wet state and ~5.5 nm in the dry state were calculated based on peak maxima. F) Schematic of the changing CMF spacings during dehydration with dimensions of the crystalline domains calculated from (200) and (004) peak width analysis using the Scherrer equation. Apart from the WAXS reflections, fiber scattering patterns exhibited intense lateral SAXS peaks centered at q ~ 1.1 nm-1 (Figure 4D), interpreted as a highly regular packing of the CMFs with a mean center-to-center distance of ~5.7 nm when measured under ambient conditions (~50% RH). Experiments under changing RH conditions showed that the mean center-to-center distance changed reversibly from ~5.5 nm at ~30% RH to ~7.8 nm at ~95% RH as deduced from shifts of the SAXS peak (Figure 4E). Due to very low scattering intensities it was impossible to analyze the peak position below ~30% RH. However, with rising RH the scattering intensity dramatically increased as illustrated by logarithmic 23 ACS Paragon Plus Environment


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scaling. With regard to the small crystallite width of ~2.5 nm the calculated mean centerto-center distances indicate an interfibrillar spacing ranging from ~3 nm to ~5.3 nm. These values are considerably higher than what was measured with SAXS or small angle neutron scattering for other primary39 40 or secondary cell wall systems.38 41 42 Even in the dried state, these data suggest that the cellulose microfibrils always maintain a large and well-defined minimum distance and that there is little or no direct fibril-fibril contact. Interestingly, the macroscopic swelling predicted for a homogenous sample by this change in crystal-to-crystal distance is larger than that observed in our swelling studies (Fig. 2C-D). This suggests a heterogenous structure and the presence of levels of structural hierarchy that we do not yet understand – both of which are consistent with the fact that the VCB originates from plant cell wall material. Processability of Mistletoe Viscin Fibers. The findings of the current study reveal an extreme hydration dependent mechanical transformation, which under wet conditions facilitates simple low-force mechanical drawing of fibers of up to several meters in length from a starting tissue just a few millimeters long. In the dry state (< 45% RH), on the other hand, the drawn fiber becomes remarkably stiff and strong, yet remains flexible. Remarkably, this hydration-dependent 35-fold change in stiffness, is fully reversible as humidity conditions are modulated. Concomitant with this moisture-dependent mechanical transformation is a drastic structural reorganization of the cellulose orientation and higher order packing as the material flows like a fluid polymer melt with


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liquid crystal-like properties and exhibits an incredible draw ratio over a wide range of drawing speeds. In this light, it is clear that mistletoe viscin exhibits a highly appealing ease of processability that is not currently found in the fabrication of typical polymeric materials, nor in current methods for producing cellulosic composites.19-22,

43

Indeed, viscin fiber

formation process functions under ambient conditions with water as an already integrated solvent. It does not require external heat since it naturally functions within a T range starting from below 0 °C outside in winter up until ~40 °C when passing a bird’s digestive system before excretion, where it even tolerates short-term biochemical attack. Furthermore, it does not require a coagulation bath – rather, just a few minutes of air drying under ambient conditions to solidify. Thus, from the perspective of green and sustainable polymer fiber fabrication, mistletoe viscin presents an appealing role model for bio-inspired materials fabrication, and is especially relevant to current efforts aimed at developing nanocellulose-based composites as a veritable alternative to petroleumbased plastics. The Critical Role of the Non-Cellulosic Matrix in Viscin Fiber Processing. Considering that microfluidic-based processing of CNCs can yield fibers with stiffness of over 80 GPa (following chemical cross-linking),43 it is not at all surprising that the aligned cellulose in mistletoe viscin leads to a stiffness of more than 14 GPa. However, what is surprising and quite remarkable is the ease of processing by which this is achieved. Most



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notable is the critical role of the water absorbent matrix in guiding this process. Based on the sharp SAXS reflections (Figure 4D-E), we posit a minimalistic view of mistletoe VCB and fibers consisting of a dispersion of CMFs with a highly-defined interfibrillar spacing, which is completely controlled by a water-responsive matrix material. Indeed, the matrix appears to mediate the fully reversible interfibrillar swelling and deswelling during hydration cycles (Figure 5) correlated to the large water-dependent changes in fiber volume (Figure 2D). The matrix additionally appears to prevent the CMFs from aggregating or fusing since even the lowest calculated mean center-to-center distance in the dry state suggests a minimum interfibrillar spacing on the order of ~3 nm. Furthermore, the exceptionally high length of the crystalline domains obtained from WAXS measurements hints that the matrix compounds bind to the CMFs such that their axial coherence is not disturbed. The resulting high aspect ratio of the crystalline domains is certainly beneficial for CMF reorientation along the drawing direction during the fiber formation process as illustrated in Figure 5. Likely, the matrix acts like a viscous stimuliresponsive gel in the wet state, whereas in the dry state it behaves like a strong cement, binding the CMFs together. This is somewhat reminiscent of a glass transition, only that it does not depend on temperature, but rather on the moisture content. Based on the fact that the CMFs originate from cell wall material, it seems plausible that during this radical transformation process, there are important contributions to fiber formation occurring at different hierarchical levels than those illustrated in Figure 5 (e.g. uncoiling of elongated


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CMFs). This is hinted at in a previous study7; however, we were not able to observe these dynamic structural changes in the present study. Clues to the molecular origin of this moisture-responsive behavior of the tissue can be extracted from previous compositional analysis of VCB tissue from different V. album variants,44 indicating that mistletoe viscin contains not only cellulose (~45 dry wt%), but also hemicelluloses (~53 dry wt%) and charged groups (e.g. ~2 dry wt% uronic acid). The moisture-mediated reversibility of fiber drawing indicates non-covalent stabilization of the matrix. Thus, it seems plausible based on these compositional studies that the hemicelluloses and ionic groups may serve as a water-responsive matrix between CMFs as indicated by SAXS. Indeed, earlier studies appear to indicate that at least some hemicelluloses are covalently attached to the CMF surfaces,44 which may explain why the interfibrillar distances extracted from SAXS are extremely well defined. We hypothesize that electrostatic interactions mediated by charged groups in the matrix provide weak sacrificial cross-link in the presence of water (as has been observed in bone,45 wood46 and insect silks47), but upon drying, water loss results in a stronger interaction between electrostatic bonds which stabilizes the matrix. While this model of water-dependent fiber processing is intriguing based on the current data, further work is required to determine its validity.



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Figure 5. Schematic minimalistic model of mistletoe viscin fiber formation. Based on current evidence, we posit an assembly model in which mistletoe viscin fiber assembly proceeds via hydration-dependent reorganization of crystalline domains of high aspect ratio from a perpendicular orientation relative to the fiber axis in the VCB to a highly aligned axial configuration in the fiber mediated through a structurally aligned, yet fluidlike flowing phase in the transition zone. The VCB provides an astonishing degree of hidden length in which the initial tissue undergoes an extreme >200-fold length increase and ~35-fold stiffness increase as it transforms into a fiber. SAXS data indicate this process depends on a decrease in the interfibrillar spacing during drying, proposed to arise from 28 ACS Paragon Plus Environment

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deswelling of the matrix material as it changes from a deformable hydrogel to a strong cement. Sacrificial Bonds and Hidden Length as a Means for Fast Fiber Formation. It is worth considering, from a basic geometrical perspective, how the millimeter-scale VCB tissue is able to physically transform into a fiber of several meters. For reasons of simplicity, we consider an idealized cylindrical shape of a single viscin cell cross section with a typical diameter of ~25 µm in which the initial cross section area is reduced via drawing by a factor of 100 to a cylindrical filament with a diameter ~2.5 µm. If one further assumes volume conservation, a 1 mm long viscin cell would yield a 100 mm long filament. Considering the whole viscin bundle (diameter x length: ~500 µm x ~10 mm, see Figure 5), one would predict a theoretical final length of 1 m with a corresponding fiber diameter of ~50 µm, which is in good agreement with observations of typical dimensions of mechanically drawn mistletoe fibers. Taking into consideration that often diameters of 200-fold draw ratio and 35-fold increase in stiffness by simple mechanical drawing and drying at ambient conditions. The ability to mimic this process


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in industrial level manufacturing of cellulose-based composite materials would represent a paradigm shift in efforts to mass produce sustainable material alternatives to petroleumderived plastics. However, before this can happen, the intricate biochemical and biological details of the viscin fiber formation process must be further elucidated – in particular, the nature of the interfibrillar matrix material and the mechanism of moisture-dependent deformation must be determined. Along these lines, comparative studies of other mistletoe species that form adhesive fibers16

18

are likely to reveal further important

insights into the underlying mechanisms of fiber formation, enabling the eventual transfer of these design principles towards green manufacture of materials using bio-renewable starting materials. ASSOCIATED CONTENT Supporting Information The following files are available free of charge. Fiber sample preparation and determination of fiber dimensions. (Figure S1) (PDF) The tensile stiffness of mistletoe fibers as a function of the moisture content. (Figure S2) (PDF) Linear radial intensity profiles along the equatorial SAXS reflections of viscin fibers at varying relative humidity (RH) levels. (Figure S3) (PDF) Visualization of the mistletoe fiber formation process. PLM video of the automated mistletoe fiber drawing process with a custom-built LEGO® Technic drawing device. 31 ACS Paragon Plus Environment


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Drawing speed: ~ 250µm s-1. Playback speed: 4x. Total field of view: 4.64 mm x 3.38 mm. (Video S1) (AVI) Macroscopic mistletoe fiber drawing. Automated mistletoe fiber drawing using a zwickiLine Z2.5 universal testing machine. Drawing speed: 1.66 cm s-1. Playback speed: 8x. (Video S2) (AVI) AUTHOR INFORMATION Corresponding Author *Prof. M. J. Harrington, E-mail: [email protected] *Prof. P. Fratzl, E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by the Max Planck Society. MJH acknowledges support from the Natural Sciences and Engineering Research Council of Canada (NSERC Discovery Grant RGPIN-2018-05243). ACKNOWLEDGMENT


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We thank Stefan Siegel and Chenghao Li at the mySpot beamline (BESSY, AdlershofBerlin, Germany) for scientific and technical support. We also thank Klaus Bienert for technical support of the humidity controlled mechanical testing. ABBREVIATIONS CNC, cellulose nanocrystals; CNF, cellulose nanofibers; CMF, cellulose microfibril; PLM, polarized light microscopy; VCB, viscin cell bundle; RH, relative humidity; WAXS, wide angle X-ray scattering; SAXS, small angle X-ray scattering. REFERENCES 1.

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