Strong and tough chitin film from Î±-chitin nanofibers prepared by high

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Strong and tough chitin film from #-chitin nanofibers prepared by high pressure homogenization and chitosan addition NGESA EZEKIEL MUSHI, Takashi Nishino, Lars A. Berglund, and Qi Zhou ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05452 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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ACS Sustainable Chemistry & Engineering

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Strong and tough chitin film from -chitin

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nanofibers prepared by high pressure

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homogenization and chitosan addition

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Ngesa Ezekiel Mushi,a,b Takashi Nishino,c Lars A Berglund,a Qi Zhoua,d,*

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a

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Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of

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Technology, SE-100 44 Stockholm, Sweden

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b

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Technology, University of Dar es Salaam, P.O. Box 35131, Dar es Salaam, Tanzania

Wallenberg Wood Science Center, Department of Fiber and Polymer Technology, School of

Department of Mechanical and Industrial Engineering, College of Engineering and

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c

Department of Chemical Science and Engineering, Kobe University, Kobe 657-8501, Japan

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d

Division of Glycoscience, Department of Chemistry, School of Engineering Sciences in

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Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova

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University Centre, SE-106 91 Stockholm, Sweden

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KEYWORDS. chitin nanofibers, chitin film, chitosan, mechanical, nanostructure.

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ACS Paragon Plus Environment

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ABSTRACT.

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Chitin nanofibers is an interesting biological nanomaterial for advanced applications, for

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example, in medicine, electronics, packaging and water purification. The challenge is to

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separate chitin nanofibers from protein in the exoskeleton structure of arthropods and avoid

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nanofibril aggregation to realize the mechanical potential of chitin. In this work, we

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developed a new method for the preparation of chitin nanofibers from lobster shell

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exoskeleton using 10 wt.% chitosan as a sacrificial polymer. The addition of chitosan in the

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raw chitin colloidal suspension during high pressure homogenization process at pH 3

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significantly reduced the agglomeration of chitin nanofibers as revealed by dynamic light

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scattering and transmission electron microscopy. Chitin film prepared from the chitin

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nanofiber suspension by vacuum filtration exhibited a true nanofibrils network structure

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without fibril aggregations as characterized by scanning electron microscopy. The presence

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of chitosan not only improves the colloidal stability of chitin nanofibers suspension but also

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facilitates the formation of chitin nanofiber network structure in the film as indicated by wide

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angle X-ray diffraction analysis. The chitin nanofiber film with 4 ± 1 wt.% residual chitosan

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showed high tensile strength (187.2  5.6 MPa) and high work of fracture (12.1  0.4

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MJ/m3), much higher than those chitin and chitosan films reported previously in literature.

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Introduction

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Chitin is an important building block in load-bearing exoskeleton structures in arthropods and

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the second most abundant natural biopolymer with vast mechanical potential for structural

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applications in nanotechnology. Chitin nanofibers have been prepared from -chitin

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resources such as the exoskeletons of crustaceans (e.g. crabs, lobsters, and prawns) and

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fungal cell walls (e.g. mushroom) by chemical pretreatment combined with mechanical

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homogenization.1–4 The preparation of chitin nanofibers of high aspect ratio (length to width

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ratio) from the less abundant -chitin resources such as squid pen and tubeworm has been

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achieved by simple mechanical treatment under acid conditions, which is easier compared to

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-chitin.5–6 This is possibly because there is no inter-sheet hydrogen bonds in -chitin and

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polar solvents such as water and alcohol are able to penetrate into -chitin.7 In addition, -

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chitin forms strong complexes with proteins through histidyl and aspartyl residues in nature,8

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which makes it more difficult to extract pristine -chitin nanofibers. Moreover, the nature of

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chitin agglomeration in water suspensions after extraction is a major obstacle for enhancing

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colloidal stability of -chitin nanofibers. Agglomeration may compromise mechanical

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performance of -chitin nanofiber network structures.

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In our previous work, crude chitin powder from crab shells was treated with 2 M HCl for 2

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days at room temperature, then boiled in 8 wt% NaOH for 2 days, and finally refluxed in

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ethanol for 6 h to remove minerals, protein, and pigments, respectively. -Chitin nanofibers

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with 7.0 wt% residual proteins were prepared by mechanical homogenization.9 Films

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prepared from such chitin-protein composite fibers showed a tensile strength of 77  5.6

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MPa. In addition, we have developed a mild process by performing all the chemical

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pretreatments at room temperature.10 -Chitin nanofibers with a very small diameter (3.6 –

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3.9 nm) and lower residual proteins content (4.7 wt%) were successfully prepared. Hydrogel


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prepared from such nanofibers showed a storage modulus of 13 kPa and a compression

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modulus of 309 kPa at 2 wt.% chitin content, the highest reported for chitin hydrogels.11

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Films prepared from such nanofibers demonstrated much higher transparency and enhanced

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mechanical properties (tensile strength of 153  10.6 MPa). However, aggregates of chitin

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nanofibers were still observed in the film. The mechanical potential of chitin was still not

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fully realized. The axial elastic modulus of -chitin crystals is estimated at 41 GPa as

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measured by an X-ray diffractometer equipped with a stretching device and a load cell.12 The

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strength of single -chitin nanofibrils is 1.6 GPa as estimated via sonication-induced

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fragmentation.6 A key challenge is to improve the individualization of chitin nanofibers and

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reduce the presence of chitin/protein aggregates, and at the same time preserve the native

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structure of chitin.

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Chitosan is a deacetylated chitin derivative and a cationic polyelectrolyte with good colloidal

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stability in water. The cationic polyelectrolyte properties, the biocompatibility, the linear

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chain structure and solubility in aqueous system have made chitosan a very attractive

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polymer. Fan et al.13 and Ifuku et al.14 performed surface deacetylation on chitin nanofibers

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and produced chitin films by solution casting, reporting a tensile strength of 140 ± 48 MPa

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and 156.5 ± 10.0 MPa, respectively. Previously, we discovered that nanocomposites of chitin

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nanofibers and chitosan have a unique combination of modulus, strength and strain-to-failure

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owing to reorientation, slippage and straightening of chitin nanofibers during deformation of

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the ductile chitosan matrix.15 Nanocomposites with a chitosan content of 30 wt.% showed

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considerable strength (140 MPa) and strain-to-failure (11%) due to good dispersion of chitin

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nanofibers in the chitosan matrix. In the present work, chitosan was utilized as a sacrificial

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polymer for improving the individualization of chitin nanofibers during mechanical

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disintegration by increasing electrostatic repulsive forces between chitin nanofibers. The

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advantage of this method is that it eliminates the need of direct chemical surface


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deacetylation of chitin nanofibers and avoids the associated hydrolysis of chitin. The

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structure and colloidal stability of the chitin nanofibers were characterized by electron

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microscopy and dynamic light scattering. Chitin films was prepared by vacuum filtration of

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the water suspension of chitin nanofibers. The structure and mechanical properties of the

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chitin films were studied and compared with chitin nanofibers prepared without the addition

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of chitosan.

90 91

Experimental section

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Materials. Chitin was extracted from lobster shell (Homarus Americanus of Northwest

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Atlantic, Canada) by removing minerals, pigments and proteins according to our previous

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work.10, 15 After the treatments of 2 M HCl and ethanol, the exoskeleton of lobster was treated

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with 20 wt% NaOH for 2 weeks at room temperature. The degree of acetylation (DA) of the

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extracted chitin was ca. 87% as measured by solid state

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(NMR) spectroscopy. Chitosan powder from shrimp (high viscous, Sigma, Germany) with a

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degree of acetylation of less than 15% was purchased from Sigma-Aldrich.

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Preparation of chitin nanofibers. Chitosan was first dissolved in 4% acetic acid. The

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extracted whitish chitin slurry was also suspended in 4% acetic acid with the addition of the

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chitosan solution. The total solid content and the solid content of chitosan were 1 wt.% and

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0.1 wt.% in the suspension, respectively. Thus, the initial content of chitosan was 10 wt.% in

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the chitin/chitosan mixture. The suspension was mixed in a kitchen blender (Vita-Prep® 3,

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Vita-Mix Corporation, USA) for 5 min and then passed through a Microfluidizer (M-110EH,

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Microfluidics Ind., Newton, MA, USA) to obtain a chitin nanofiber hydrocolloidal

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suspension. The suspension was first passed five times through the 400 and 200 μm

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microfluidic chambers at a pressure of 900 bar and then five times through the 200 and 100

13C


Nuclear Magnetic Resonance

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μm chambers at a pressure of 1600 bar. This sample was coded as ChNF-c. The control

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sample was prepared in the same manner without the addition of the chitosan solution and

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coded as ChNF.

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Preparation of chitin nanofiber film. The chitin nanofiber suspension was diluted to 0.1

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wt.% using an Ultra Turrax Mixer (IKA, D125 Basic, USA) at a speed of 1200 rpm for 10

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min to allow uniform dispersion and complete disentanglement of the nanofiber aggregates.

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The suspension was then vacuum filtrated using a 0.65 μm pore size filter membrane (DVPP,

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Millipore, USA) to form a wet cake with water content of ca. 90 %. The wet cake was dried

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in the drying stage of a Rapid Köthen sheet former (Germany) at 70 mbar and 93 °C for 10

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min to obtain a chitin nanofiber film. The film was further dried to constant weight at 105 oC.

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The weight of the residue chitosan in the film was measured from the weight of the chitin

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nanofiber/chitosan mixture before filtration to the weight of the film after filtration and

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drying. The initial solid weight of the sample was based on the solid content of the colloidal

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suspension (0.1 wt.%) and total weight of the suspension before filtration. The weight loss of

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chitin nanofiber after filtration has not been observed for neat ChNF. Chitosan is completely

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filtered through the filter membrane when neat chitosan solution was applied. Five films were

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prepared for ChNC-c to measure the residual content of chitosan.

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Characterization. Transmission electron microscopy (TEM) of chitin nanofibers was

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performed using the Hitachi Model HT7700 transmission electron microscope operated in

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high-contrast mode at 100 kV. Chitin nanofiber sample was deposited on a carbon coated

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copper grid (Ultra-thin Carbon Type-A, Ted pella) and stained with 1% uranyl acetate.

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The charge and size of the chitin nanofiber hydrocolloids were studied by dynamic light

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scattering (DLS) using Zetasizer Nano (Model ZEN3600, Malvern Instruments Ltd, UK).

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The light source was operated at a wavelength of 633 nm. Chitin nanofiber suspension was


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diluted to a concentration of 0.1 mg/mL at pH 3 and scanned three times in a Poly (methyl

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methacrylate) cuvette at 21 °C. The size of chitin nanofiber aggregates was determined based

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on Smoluchowski's approximations.

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The topographical and cross-sectional morphology of the chitin nanofiber films was

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characterized by using field emission scanning electron microscopy (FE-SEM, Hitachi S-

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4800, Japan). The samples were conditioned in a desiccator overnight and then sputtered with

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a thin layer of gold/palladium using Agar HR sputter coater (Cressington scientific

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instruments ltd, UK). FE-SEM images were captured from secondary electrons at an

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acceleration voltage of 1.0 kV.

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To measure the bulk density of the chitin nanofiber films, rectangular samples with edge size

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ranging between 1 and 2 cm were accurately weighed. Bulk volume was measured using

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Mercury Intrusion Porosimeter (Micrometrics, USA). The samples were placed in a

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penetrometer chamber. The air was evacuated and filled with mercury at atmospheric

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pressure. The bulk volume was obtained from the difference between volume of the

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penetrometer with and without the sample. Porosity was calculated from measured bulk

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density of the sample using the following equation,

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𝑃𝑜𝑟𝑜𝑠𝑖𝑡𝑦 = 1 ― 𝜌𝑐ℎ

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where b corresponds to the bulk density of the film and ch corresponds to the density for

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chitin, which is assumed to be 1.425 g/cm3.16

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Tensile test of the chitin nanofiber films was performed using a universal tensile testing

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machine (Model 5944, Instron, UK) equipped with a 500 N load cell at a strain rate of 4

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mm/min. The specimens with a width of 5 mm and a length of 40 mm were preconditioned at

𝜌𝑏

(1)


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50% relative humidity and 23 °C overnight. Mechanical properties such as modulus, tensile

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strength, and strain to failure were obtained based on conventional analysis of tensile stress-

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strain curves. Toughness is defined as work to fracture and is calculated as the area under the

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stress−strain curve.17

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To study the orientation of the chitin nanofibers in the film before and after tensile stretching,

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wide angle X-ray diffraction (WAXD) measurement was performed. The samples were

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irradiated by Cu K radiation in the direction both perpendicular (in-the-plane) and parallel

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(cross-section) to the film surface and the diffraction photographs were recorded. From

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azimuthal intensity distribution graphs for the 110 equatorial reflection, the degree of

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orientation () was calculated according to the following equation,

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𝛱=

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where FWHM is the full width at half-maximum.

180 ― 𝐹𝑊𝐻𝑀 180

(2)

166 167

Results and discussion

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Structure of chitin nanofibers

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The morphology of the chitin nanofibers (ChNFs) prepared using microfluidizer was directly

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characterized by TEM analysis with negative staining using uranyl acetate. As shown Figure

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1a, ChNFs with a width of ca. 4–6 nm can be individualized by mechanical homogenization

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without the addition of chitosan. However, large aggregates of ChNFs or chitin/protein

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bundles with a width of ca. 20–100 nm are also found in the sample. This is probably because

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individualized ChNF tends to form aggregates together with the residual protein. It could also

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be due to incomplete fibrillation of chitin-protein fibril bundles during the mechanical


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treatment. In our previous work,10 such aggregates were further disintegrated by

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ultrasonication in very dilute solution (0.005 wt.%) before structural analysis. A fiber width

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of ca. 3.6 nm was measured by AFM. However, nanofiber aggregates formed again after the

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suspension was stored overnight. Interestingly, with the addition of chitosan to the extracted

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lobster chitin, ChNFs with a uniform width of ca. 4–6 nm was completely individualized after

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homogenization, see Figure 1b. This suggests that the addition of chitosan during

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homogenization facilitates the fibrillation of the extracted lobster chitin fibrils, and prevent

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the aggregation of individualized ChNFs. Such colloidal nanofiber suspension is stable upon

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storage for a few weeks.

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Figure 1. Typical transmission electron microscopy (TEM) images of chitin nanofibers from

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lobster shell (a) disintegrated without the addition of chitosan and (b) disintegrated in the

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presence of chitosan in suspension.

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Figure 2. Estimated size distributions of chitin nanofibers in water suspensions for neat chitin

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(ChNF) and chitin with the addition of 10 wt.% chitosan (ChNF-c) in suspension during

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mechanical homogenization, as obtained from dynamic light scattering (DLS) measurements.

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Figure 2 presents the size (hydrodynamic radius) distribution of the ChNFs based on DLS

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data, for neat ChNF and the ChNF-c which was prepared with the addition of chitosan. Two

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peaks (a bimodal distribution) can be observed for neat ChNF, one with the particle size in

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the range of 350–1300 nm and the other with particle size between 60–150 nm. For the

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ChNF-c prepared with the addition of chitosan, there is only one peak (a unimodal

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distribution) with lower particle size in the range of 400–700 nm and higher peak intensity

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compared to the neat ChNF. This indicates that the size distribution of ChNF-c particles is

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more uniform as compared to the neat ChNF particles. With the addition of chitosan during

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mechanical homogenization, the colloidal chitin system appears more stable. Similar DLS


10

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results were observed with core-shell cellulose nanofibrils/xyloglucan (CNF/XG) system, in

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which adsorbed XG provides steric stabilization of CNF nanofibrils.18 Quartz Crystal

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Microbalance (QCM) analysis results from our previous work indicated that there is no

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evidence of chitosan adsorption to chitin nanofiber at pH 3, due to strong electrostatic

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repulsive forces between protonated amine groups from both chitin nanofibers and chitosan.15

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Thus, chitosan is possibly strongly associated between ChNF nanofibirls, contributing to

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electrostatic stabilization of the whole suspension. In our previous study, the colloidal

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stability of ChNFs was significantly improved by adding 30 wt.% chitosan into the aqueous

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suspension of ChNFs and the aggregation of ChNFs was completely reduced as revealed by

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DLS analysis.15 However, such stabilization effect was not achieved for chitosan addition

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lower than 30 wt.% to the aqueous suspension of ChNFs. Thus, it is essential to add chitosan

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during the homogenization process of lobster chitin to prepare colloidal stable ChNFs.

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Structure and mechanical properties of chitin films

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Nanostructured chitin films were prepared from water suspension of chitin nanofibers by an

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Ultra Turrax mixing step followed by vacuum filtration and drying with a Rapid Köthen sheet

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former.17,19 In Figure 3a, aggregates and bundles of chitin nanofibers with a width of 20–100

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nm are observed in the surface SEM micrograph of a of a neat ChNF film, similar to the

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result by TEM analysis (Figure 1a). Chitin films prepared from ChNF-c shows much more

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homogeneous nanofibril network structure with much less aggregates of ChNFs, as shown in

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Figure 3b. Free chitosan was vacuum filtered during the film preparation process. 4 ± 1 wt.%

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chitosan remained in the chitin film after filtration. It is evident from both SEM (Figure 3b)

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and TEM (Figure 1b) analysis that the nanofiber length is several micrometers and nanofiber

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ends are not apparent. The predominant chitin orientation in both chitin films appears to be


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random-in-the-plane. The layered structure in the cross section of fractured surfaces of both

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chitin films is apparent, similar to cellulose nanopaper structures.17 The layered structure of

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neat ChNF (Figure 3c) is rather distinct as compared to ChNF-c sample. This is possibly due

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to facilitated floc formation of larger ChNF fibril aggregates in the high concentration region

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just above the filter membrane. Individualized ChNFs associated with chitosan show

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interfibril repulsion, are better dispersed and able to form a nanofibril network linking the

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adjacent layers (Figure 3d). Such structure is further supported by WAXD analysis discussed

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latter.

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237 238

Figure 3. Scanning electron microscopy (SEM) micrographs of the chitin nanofibrillar

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network on the surfaces of (a) neat ChNF and (b) ChNF-c films, and layered structure on the

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cross section of fracture surfaces of (c) neat ChNF and (d) ChNF-c films.


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Figure 4. Typical tensile stress–strain curves of ChNF and ChNF-c chitin films under

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uniaxial tensile deformation.

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Table 1. Properties of the chitin films. (The values in parentheses are the sample standard

245

deviations.) Elastic

Yield

Tensile

Strain-

Work to

modulus

strength

strength

to-failure

fracture

(GPa)

(MPa)

(MPa)

(%)

(MJ/m3) 7.3 (1.2)

Porosity Density (%)

(g/cm3)

ChNF

16

1.19

6.5 (0.5)

57.4 (2.2)

153.0 (10.6)

7.6 (0.6)

ChNF-c

18

1.17

6.0 (0.6)

54.1 (1.2)

187.2 (5.6)

10.1(0.2) 12.1 (0.4)

246 247

Figure 4 presents the stress–strain curves of chitin film samples of neat ChNF and the ChNF

248

with added chitosan during mechanical homogenization (ChNF-c). Mechanical properties of

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the chitin films are summarized in Table 1 together with porosity and density data. The

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ChNF-c film has slightly higher porosity. The ChNF sample shows higher variations in

251

mechanical properties due to the presence of aggregates in the film. The stress–strain

252

behavior of both samples follows the same curve with an elastic deformation region at lower

253

strain (below 2%) followed by a linear and strong strain-hardening (plastic deformation)


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region. The modulus and yield strength of the nanostructured chitin ChNF-c film decreased

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from 6.5 ± 0.5 to 6.0 ± 0.6 GPa and from 57.4 ± 2.2 to 54.1 ± 1.2 MPa, respectively, due to

256

the presence of a soft matrix from 4 ± 1 wt.% residual chitosan. However, the ChNF-c film

257

has higher strain-to-failure and ultimate tensile strength of 10.1 ± 0.2% and 187.2 ± 5.6 MPa,

258

respectively. This tensile strength value is higher than the neat ChNF sample (153.0 ± 10.6

259

MPa) and higher than for chitin films from chitin-protein composite fibers (77  5.6 MPa) in

260

our previous work.9 This value is also higher than the chitin films from surface deacetylated

261

chitin nanofibers from crab shell reported by Ifuku et al.14 (156.5 MPa) and Fan et al.13 (140

262

MPa), as well as the film from TEMPO-oxidized α-chitin nanowhiskers (110 MPa).13 The

263

work to fracture of the ChNF-c film is 12.1 ± 0.4 MJ/m3, 66% higher than that for the neat

264

ChNF film. Although the nanocomposite film of ChNF with 30 wt.% content of chitosan can

265

achieve a work to fracture of 12 MJ/m3, its tensile strength can only reach 141 MPa.15

266

In the strain-hardening region, the highly individualized nanoscale chitin nanofibers can slide

267

and reorient much easier in the presence of 4 ± 1 wt.% residual chitosan compared to the

268

larger aggregates and bundles of nanofibers in the neat chitin film, particularly for out-of-

269

plane nanofibers between the layers parallel to the surface plan. This and fewer aggregate

270

defects result in much higher strain-to-failure and ultimate tensile strength for the ChNF-c

271

film. The orientation of the chitin nanofibers in the film before and after the tensile test was

272

characterized by WAXD. The X-ray diffractograms perpendicular and parallel to the film

273

surface are shown in Figure 5. Before tensile stretching, the orientation of chitin in the plane

274

of the films is completely random. The plan parallel to the film surface is strongly ordered.

275

The degree of in-plane orientation () for the ChNF-c film is 74.2%, lower than that for neat

276

ChNF film (76.4%), indicating more out-of-plane nanofibers in the layered structure.

277

Interestingly this correlates with the lower modulus of ChNF-c compared with ChNF films,

278

see Table 1. These results confirm the observations by SEM (Figure 3). After tensile


14

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279

stretching, the orientation in the surface plane of the films is still completely random,

280

although there may be local orientation effects close to failure sites. However, the  value for

281

the ChNF-c film parallel to the film surface is increased by 4.8% after tensile testing, and this

282

is substantially higher than the 2.5% increase for the neat ChNF film. It means that the out-

283

of-plane nanofibers become more strongly oriented in the ChNF-c film during the tensile test.

284

285 286

Figure 5. X-ray diffractograms perpendicular (through) and parallel (edge) to the membrane

287

surfaces for the ChNF and ChNF-c samples before and after uniaxial tensile stretching. The

288

degree of orientation () was calculated according to equation (2) from azimuthal intensity

289

distribution graphs for the 110 equatorial reflection (Figure S1, Supporting Information).

290 291

Conclusions

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The main problem of -chitin nanofibers extracted from crustaceans such as lobster is the

293

poor colloidal stability due to aggregate formation in the suspension. These aggregates form


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294

defects in the nanofiber network and lead to strong variations in mechanical properties. We

295

have demonstrated that addition of chitosan during homogenization of the chitin slurry

296

facilitates complete individualization of chitin nanofibers. Chitosan associates with chitin

297

nanofibers and increases the electrostatic repulsion forces and stability of the colloidal

298

suspension. Chitosan is used as a sacrificial polymer and is partially removed by vacuum

299

filtration during film forming process. The chitosan addition leads to increased tensile

300

strength from 156 MPa to 187 MPa and increased work to fracture (area under stress-strain

301

curve) from 7.3 to 12.1 MJ/m3 for the nanostructured chitin ChNF-c film. The improved

302

mechanical properties are attributed to the true nanoscale network structure from better

303

individualization of chitin nanofibers stabilized by the residual chitosan. The new method of

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using sacrificial chitosan for preparation of colloidal stable chitin nanofibers from -chitin

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resources is simple and more environmentally friendly than chemical methods for colloidal

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stabilization such as surface deacetylation and TEMPO-mediated oxidation.

307 308

ASSOCIATED CONTENT

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Supporting Information.

310

The following file is available free of charge.

311

Azimuthal intensity distribution graphs for the 110 reflection for the stretched and

312

unstretched ChNF and ChNF-c films (PDF)

313

AUTHOR INFORMATION

314

Corresponding Author

315

* To whom correspondence should be addressed, Tel: +46 8 790 96 25. E-mail: [email protected]

316

ACKNOWLEDGMENT


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317

The Wallenberg Wood Science Center (WWSC) is acknowledged for financial support for

318

this work. LB acknowledges funding from the Knut and Alice Wallenberg foundation

319

through a Wallenberg Scholar grant.

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REFERENCES

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BRIEFS. Strong and tough chitin film from highly individualized -chitin nanofibers that are

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prepared by using chitosan as a sacrificial polymer during homogenization.

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SYNOPSIS.

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Figure 1. Typical transmission electron microscopy (TEM) images of chitin nanofibers from lobster shell (a) disintegrated without the addition of chitosan and (b) disintegrated in the presence of chitosan in suspension. 70x142mm (300 x 300 DPI)


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Figure 2. Estimated size distributions of chitin nanofibers in water suspensions for neat chitin (ChNF) and chitin with the addition of 10 wt.% chitosan (ChNF-c) in suspension during mechanical homogenization, as obtained from dynamic light scattering (DLS) measurements. 81x60mm (300 x 300 DPI)



Figure 3. Scanning electron microscopy (SEM) micrographs of the chitin nanofibrillar network on the surfaces of (a) neat ChNF and (b) ChNF-c films, and layered structure on the cross section of fracture surfaces of (c) neat ChNF and (d) ChNF-c films. 142x106mm (300 x 300 DPI)


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Figure 4. Typical tensile stress–strain curves of ChNF and ChNF-c chitin films under uniaxial tensile deformation. 79x61mm (300 x 300 DPI)



Figure 5. X-ray diffractograms perpendicular (through) and parallel (edge) to the membrane surfaces for the ChNF and ChNF-c samples before and after uniaxial tensile stretching. The degree of orientation (Π) was calculated according to equation (2) from azimuthal intensity distribution graphs for the 110 equatorial reflection (Figure S1, Supporting Information). 165x82mm (300 x 300 DPI)


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Strong and tough chitin film from Î±-chitin nanofibers prepared by high

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