Native cellulose nanosheets from Japanese Cedar via mild delignification and mild homogenization

Native cellulose nanosheets from Japanese Cedar via mild delignification and mild homogenization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Native cellulose nanosheets from Japanese Cedar via mild delignification and mild homogenization Makiko Imai (Koyama), Shogo Suzuki, Yuki Nakamura, Katsuhiro Isozaki, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9423095/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Since the discovery of graphene, two-dimensional nanosheets have attracted attention for their unique shapes and properties. The simplest production method is the top-down approach, in which layered materials are mechanically cleaved or exfoliated. However, preparing biomaterial nanosheets by the top-down method is challenging, and to date, only a few reports have been published. In this study, we developed native cellulose nanosheets with a thickness of several tens of nanometers, which maintained the native uniaxial orientation of cellulose fibers from wood cell walls through a combination of mild delignification and mild homogenization. The wood cell wall is a complex structure composed of three main polymers, cellulose, hemicellulose, and lignin. The elementary unit of wood cellulose is a fiber 3–4 nm in width, which exists as bundles within the cell wall. Although cellulose has been disintegrated into nanofibers in various ways, cellulose nanosheets have not previously been isolated from wood cell walls. Nanocellulose is a developing material for various applications, taking advantage of its lightweight nature, high strength, excellent thermal dimensional stability, and superior gas barrier properties. A wide variety of performances can be achieved by controlling its morphology. The resulting nanosheets preserved the native orientation of the cellulose microfibrils in the cell walls, providing evidence in support of the presence of a lamellar cellulose structure within the cell wall. Biomaterials Native cellulose nanosheet one-step reaction cell wall Japanese cedar lamella delignification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Two-dimensional nanosheets are materials of several to tens of nanometers thick and several µm meters square. There are numerous reports of nanosheets of organic (Rodríguez-San-Miguel et al. 2020 ) and inorganic compounds of platinum (Takimoto et al. 2023 ), titanium (Sasaki et al. 1996 ), and graphene (Novoselov et al. 2004 ; Li et al. 2008), attracting attention in the fields of electronics, optoelectronics, catalysts, energy storage, energy generation, sensors, separation, and biomedicines 6 . For biomass materials, various artificial nanosheets produced by stacking biomass-based nanofibers have been reported (You et al. 2017 ; Wilson et al. 2018 ; Kuga and Wu 2019 ). However, only a few studies have described nanosheets derived directly from biomaterials. To the best of our knowledge, native nanosheets obtained by the top-down method have been reported from silkworm cocoons (Cheng et al. 2022 ), agave leaves (Chávez-Guerrero et al. 2017 ; Silva-Mendoza et al. 2019 ), Banana stems (Chávez-Guerrero et al. 2019; Flores-Jerónimo et al. 2021 ). Cellulose is the most abundant natural polymer. Further, given the versatility of its potential applications, it has been essential in human societies throughout history. Indeed, to date, purified cellulose has been used in various ways, including as a raw material in the paper and textile industries. In particular, wood cell walls contain large amounts of cellulose. However, high-temperature processes requiring large amounts of increasingly expensive energy to remove lignin and hemicellulose are needed to obtain pure cellulose. Therefore, new processes with lower energy consumption have become a research priority (Francis et al. 2004 ). Recently, research on methods for lignin removal with low ecological impact has made substantial progress. Thus, for example, woody biomass was treated with peracetic acid containing sulfuric acid (H 2 SO 4 ) as a catalyst at 90 ˚C (Kundu et al. 2021 ); similarly, in another study, it was pretreated with hydrogen peroxide (H 2 O 2 ) and acetic acid at 80 ˚C for saccharification (Lee et al. 2021 ). The Japanese cedar ( Cryptomeria japonica ) is endemic to Japan and occupies the largest plantation area among all tree species in the country. Although Japanese cedar trees planted decades ago have reached maturity for logging, they cannot be readily used as biomass materials, largely due to the difficulties associated with delignification. As guaiacyl lignin units, the main components of softwood, are difficult to decompose, harsh reaction conditions are required to remove softwood lignin (Shimizu et al. 2012 ). According to our method, Japanese cedar powder was treated with diluted H 2 O 2 at 60 ˚C under ambient pressure, using a versatile organic acid as the catalyst. This one-step reaction leads to delignification, and the resulting cellulosic residue resembles what is known as a “pulp” that retains the shape of the tracheids (Nakamura et al. 2024 ) Recently, the development of novel applications for nanocellulose, among which nanofibers are a typical example, has been actively pursued. Thus, for example, pulp is now generally fibrillated to cellulose nanofibers several to hundreds of nanometers wide using a blender (Uetani and Yano 2011 ), a high-pressure homogenizer (Iwamoto et al. 2005 ), a grinder (Iwamoto et al. 2005 ; Abe and Yano 2012 ), or some counter-collision method (Tsuboi et al. 2014 ). This has allowed the fabrication of highly transparent sheets from wood cellulose nanofibers (Nogi et al. 2009 ) which, owing to their light weight, high strength, high thermal dimensional-stability, and good gas-barrier properties, are a highly promising material under development for various applications. In this study, sheet-structured cellulose was obtained from cellulose isolated via a mild process using a common homogenizer. To date, thin plates several tens of nanometers thick from agave leaf parenchyma (Chávez-Guerrero et al. 2017 , 2018 ) and banana pseudo-stem parenchyma (Flores-Jerónimo et al. 2021 ) have been reported as native thin-cellulose structures. However, these are parts of the parenchyma with thin cell walls composed of randomly arranged cellulose fibrils. On the other hand, nanometer-thick sheets have not been generated from highly lignified, thick cell walls such as tracheids and fiber cells characteristic of wood. Instead, the shapes of the sheets obtained were quite different from those of thin plates, nanofibers, nanocrystals, and microfibers reported to date. Herein, we report detailed observations and measurements of this new structure. Methods Cellulose separation Air-dried Japanese cedar ( Cryptomeria japonica ) wood was milled using a Wiley mill equipped with a 1 mm sieve. The resulting wood powder (10 g) was placed into a 0.2 L three-neck round-bottom flask; then, a mixture of 10 mL of 30 wt% H 2 O 2 and 0.86 g of dodecylbenzenesulfonic acid (DBSA) was added. After adding of 50 mL of toluene, the reaction mixture was heated at 60 ˚C on an aluminum block for 24 h with stirring at 100 rpm with a blade. The obtained solid was separated by vacuum filtration using filter paper and washed with ultrapure water until a neutral pH was attained. Measurement of acid-insoluble lignin extracted from cedar powder and the separated cellulosic materials Cedar powder or the cellulosic material (20 mg) obtained was heated in 0.3 mL of 72 wt% H 2 SO 4 at 30 ˚C for 60 min, with occasional shaking. The reaction solutions were diluted with 8.4 mL of ultra-pure water to 4 wt% H 2 SO 4 , and heated in an autoclave at 120 ˚C for 60 min (Effland 1977 ). The residues (acid-insoluble lignin) were washed with ultrapure water until a neutral pH was attained, and then weighed. Finally, the corresponding ratios relative to the initial solids were calculated. Fourier-transform infrared (FT-IR) spectroscopy The FT-IR spectra of the freeze-dried cellulosic materials and wood powder were recorded using an FT-IR spectrometer (FT/IR-4700, JASCO, Tokyo, Japan) in the attenuated total reflection (ATR) mode in the 4000 − 500 cm − 1 range at a resolution of 4 cm − 1 , with 64 scans. Homogenization An ultrahigh-speed homogenizer (Physcotron, Microtec Co., Ltd., Funabashi, Japan) equipped with an inner and an outer blade (with the inner blade rotating at an ultrahigh speed) was used to homogenize the material, which subsequently entered the space between the rotating inner and outer blades, where it was ground to a powder. In addition, high-frequency vibrations were generated between the two blades, further contributing to the refinement of the pulverized material. This homogenizer has been widely employed for the disintegration of cellulose (Nishiyama et al. 1997 ; Kose and Kondo 2013 ). Then, 0.3 wt% aqueous suspension slurries were homogenized at 13,000 rpm for 60 min, with occasional cooling in ice water. A small portion was removed from the suspension after 5 and 10 min for microscopic analysis. X-ray diffractometry Freeze-dried cellulosic materials were analyzed via X-ray diffraction (XRD) using an XRD-7000 diffractometer (Shimadzu, Kyoto, Japan) in the reflection mode. Diffraction patterns were collected stepwise using Cu Kα radiation (λ=1.5406 Å) in the range of 5°–40°, at a step of 0.02° and a scan speed of 1° min − 1 . The Segal crystallinity index was calculated using the following equation (Segal et al. 1959 ): $$\:Crystallinity\:index\:\left(\%\right)=100{\times\:(I}_{200}-{I}_{a})/{I}_{200}$$ \(\:{I}_{200}\) Intensity of the (200) peak for cellulose I β \(\:{I}_{a}\) Intensity of amorphous Scanning electron microscopy (SEM) We used a nanopercolator (JEOL Ltd., Tokyo, Japan), which is a disposable SEM sample stage equipped with a polycarbonate membrane filter (pore size:0.6 µm). By performing filtration with a syringe, the solution can be removed, and the sample can be deposited onto the membrane. The 0.06 wt% aqueous suspension of cellulosic materials (50 µL) was dropped on a nanopercolator. Excess water was removed using a 10-mL syringe. The adaptor for SEM, to which the nanopercolator was attached, was placed in liquid nitrogen. The sample was freeze-dried on the nanopercolator and coated with Au by using a DII-29010SCTR Smart Coater (JEOL). Images were obtained using a scanning electron microscope SEM (JCM-7000 NeoScope™, JEOL) equipped with a tungsten filament operating at 15 kV. White light-interference Laser Microscopy The homogenized cellulose slurry was diluted with ultrapure water to 0.01 wt%. Then, it was dropped onto a freshly cleaved mica surface and air-dried. Surface morphology was observed using a white light-interference laser microscope (VK-X3000, Keyence, Osaka, Japan). Atomic force microscopy (AFM) The homogenized slurry was diluted with ultrapure water to 0.01 wt%, dropped onto a freshly cleaved mica surface, air-dried, and subjected to atomic force microscopy (AFM; Dimension Icon SPM; Bruker, Yokohama, Japan and SPM-Nanoa; Shimadzu, Kyoto, Japan) equipped with a microcantilever. Images were recorded for a 5 × 5 µm and 30 × 30 µm wide square in the tapping mode. Height analysis was performed using the NanoScope9.4R1 software and SPM-Nanoasoftware. Production of the nanofilms The cellulose slurry homogenized using a Physcotron at 13,000 rpm for 60 min was vacuum-filtered through a membrane filter (pore size: 0.45 µm) and pressed under 5 MPa at room temperature for 4 min. The obtained films were dried at 105 ˚C for 10 min. Results Separation and certification of cellulose Cedar powder was heated in H 2 O 2 containing DBSA to form a white solid (Fig. 1 ). The yield of the solid was 57% of the oven-dried cedar wood powder. In turn, acid-insoluble lignin of the cedar material was 33.5% and that of the obtained cellulosic material was 4.1%, corresponding to 2.3% of the cedar powder, all on a weight basis. Lignin decreased by 93% compared to the content measured before the reaction. The ATR-IR spectrum of the obtained cellulosic material confirmed that the absorbance of the lignin aromatic skeletal vibration at 1509 cm − 1 (Huang et al. 2012 ), which was observed in the spectrum of the cedar powder, completely disappeared (Fig. 2 ). Thus, despite the recalcitrance of softwood lignin, our mild oxidative reaction facilitated the delignification of cedar powder and yielded a cellulose-based material, which constitutes a significant achievement. We call this material the Molecular Cellulose Assembly (MCA). Our XRD analysis confirmed that the MCA before and after homogenization (60 min) consisted of cellulose I (Fig. 3 ). The crystallinities before and after homogenization were 80% and 77%, respectively, and there was no detectable change. Microscopic observation of MCA We used SEM to observe MCA before and after homogenization of the cellulosic material obtained as described above. Tracheids are high aspect ratio cells that function in water transport and provide mechanical support to plants. Further, they constitute 95% of softwood total mass such as Japanese cedar (Perré and Turner 2001 ). Although cellulose fibrils were exposed on the surface of tracheids before homogenization of the material, the latter maintained their shape (Fig. 4 a), and after 5 or 10 min of homogenization, thin sheets were exfoliated from the outer surface of the tracheids (Fig. 4 b, 4 c). After 60 min of homogenization, sheet structures occupied most, whereas shape-maintaining tracheids occupied a minimum portion of the homogenized MCA. Cell walls appeared thinner (Fig. 4 d). The scarce shape-maintaining tracheids in the slurry were removed by mild centrifugation at 600 × g. Measurement of cellulose sheet thickness by AFM and white light-interference laser microscopy The fine structure of the cellulose sheet visualized by SEM was analyzed by AFM, and cellulose microfibrils were clearly visualized in the sheet. Numerous sheets were also observed by AFM, with the side length of some sheets measured at over 10 µm. Cellulose fibers were highly oriented in one direction within the sheet. Their thickness was less than 90 nm and most measured approximately 30 nm (Fig. 6 ). As shown in Fig. 6 a, the remaining relatively thick fragments of cell walls, which had not yet been exfoliated into nanosheets, were also observed. Additionally, thin fibers unraveled from the edge of the sheet with a height of 3–4 nm. The width of cellulose microfibrils from wood is approximately 3 nm (Jakob et al. 1995 ), therefore, the height observed in this study corresponds to the width of the microfibrils in wood cell walls, and the thickness of the sheet corresponds to that of 10 microfibrils. To our knowledge, this is the first report of native nanosheets of uniaxially oriented cellulose. Cellulose exists as aggregates (10–30 nm), and several aggregates form lamellae in tangential direction within the secondary walls of Norwegian spruce (Fahlén and Salmén 2002 , 2003 ). The sheets are assumed to be composed of several stacked lamellae exfoliated from the cell wall. The lignin and hemicellulose matrices filled the interlamellar regions between the cellulose lamellae, and the cellulose sheets were easily exfoliated upon removal of the matrix, such that nanosheets were observed using white light-interference laser microscopy. Circular structures, presumably corresponding to the borders of bordered pits, were identified on the sheets (Fig. 7 ), and their height was approximately 230 nm. The average height of the area enclosed within the square is 21 nm (Fig. 7 ). We observed that thin sheets exfoliated from the outer surface of the tracheids and that the cellulose microfibrils were oriented in one direction within a sheet, and pit borders were also observed. From the above observations, we inferred that the sheets were not formed by the reassembly of fibrillated microfibrils but were native cellulose sheets that maintained the native orientation of the microfibrils. We named this sheet, native cellulose nanosheet (NCNS). Transparency of cellulose films Cellulose films were produced from NCNS and commercial bleached softwood pulp and homogenized under the same conditions. The NCNS film was more transparent than the pulp film (Fig. 8 ). This finding means that the MCA was homogenized to yield nanometer-sized NCNS. Discussion In this study, DBSA was used as the catalyst for the delignification reaction. DBSA is an amphiphilic substance with a benzene ring, a long-chain alkyl, and a sulfo group. Furthermore, DBSA is soluble in aqueous H 2 O 2 solution and is accessible to hydrophobic lignin. Therefore, it is possible to selectively degrade lignin rather than cellulose and hemicellulose. The cedar cell walls consist of a primary wall and a three-layered secondary wall (S1, S2, and S3) with different thicknesses and microfibril angles. In particular, S2 is the thickest, and its microfibrils are oriented at an angle nearly parallel to the long axis of the tracheid (Booker and Sell 1998 ). The NCNS maintained this fiber orientation. No significant decrease in the crystallinity was observed, indicating that exfoliating occurred without damaging the crystals. To date, thin plates several tens of nanometers thick from agave leaf parenchyma (Chávez-Guerrero et al. 2017 , 2018 ) and banana pseudostem parenchyma (Flores-Jerónimo et al. 2021 ) have been reported as native thin-cellulose structures. However, these are parts of the parenchyma with thin cell walls composed of randomly arranged cellulose fibrils. Conversely, nanometer-thick sheets have not been isolated from highly lignified, thick cell walls, such as wood tracheids and fiber cells. As for NCNSs, these were predominantly approximately 30–50 nm thick, which is equivalent to 10–17 cellulose microfibrils with a width of 3–4 nm (Fig. 6 ). Secondary walls reportedly have a lamellar structure in which cellulose and lignin are alternately arranged parallel to the lumen surface (Ruel et al. 1978 , 2006 ; Tanahashi 1990 ). In this study, it was presumed that spaces were formed between the cellulose lamellae by selective delignification under mild conditions and that cellulose lamellae became easier to exfoliate. On the other hand, we speculated that hydrogen bonds binding cellulose molecules within the sheet to each other, or cellulose to hemicellulose, were not broken, and that new hydrogen bonds were formed between lamellae. As a result, sheet structure was preserved, as shown in Fig. 9 . This sheet formation process also provided strong evidence of the lamellar structure of wood cell walls. According to our SEM observations, the one-sided lengths of some NCNSs exceeded 50 µm, which corresponds to the perimeter of the tracheid. Therefore, it was assumed that the cellulose in lamellae encircled the tracheid cell walls. When kraft pulp or dissolving pulp is treated by a disc mill, high-pressure homogenizer, or refiner, it is fibrillated into nanofibers without forming a sheet structure (Nakagaito and Yano 2004 ; Stelte and Sanadi 2009 ; Kumagai et al. 2019 ). The mild process used in this study caused cellulose to be degraded into nanosheets and further homogenization caused it to be further degraded into nanofibers. Additionally, we found that the shape of nanocellulose can be adjusted to a sheet- or fiber-like shape by adjusting the disintegrating method and processing time, which together will help produce cellulose structures suitable for future applications. Moreover, the method described can be used for biomass sources other than the Japanese cedar biomass used herein. One of the distinctive features of NCNS is that its cellulose microfibrils are uniaxially oriented. Artificially oriented sheets, films, and papers have been reported to exhibit various properties. In particular, the ball milling of cellulose powder with silicon oil (Zhao et al. 2016 ) or without additives (Zhang et al. 2019 ) gave uniaxially-oriented cellulose nanosheets via the rearrangement of cellulose microfibrils. Cellulose nanofiber films of aligned cellulose have reportedly better piezoelectric properties (Zhai et al. 2020 ). Furthermore, nanopaper with oriented TEMPO cellulose nanofibers shows better mechanical properties (Sehaqui et al. 2012 ). Oriented nanocellulose materials will become increasingly popular as functional materials. As we aim for a sustainable, decarbonized society, cellulose has great potential for the development of unprecedented new materials in addition to its traditional uses. Conclusions Cellulose was effectively obtained from Japanese cedar powder by selective delignification under mild reaction conditions. The NCNSs exfoliated sequentially from the outermost layer of the cell wall by homogenization. The NCNSs with uniaxially oriented cellulose microfibrils were obtained for the first time. We believe these materials have great potential for development of a range of applications. The successful formation of NCNS strongly indicates the possibility of controlling the size and shape of nano cellulose. Furthermore, from the viewpoint of cell wall structure, this proves that cellulose has a lamellar structure within the wood cell walls. Declarations Competing interest The authors have no relevant financial or non-financial interests to disclose. However, Makiko Imai (Koyama), Shogo Suzuki, Yuki Nakamura, Katsuhiro Isozaki, Hiroyuki Matsumura, Masaharu Nakamura are inventors on an international patent application related to the Native Cellulose Nanosheets (WO2023162263A1). Makiko Imai (Koyama), Yuki Nakamura, Katsuhiro Isozaki, Hiroyuki Matsumura, Masaharu Nakamura hold a patent for the Methods for producing lignin and polysaccharides (JPWO2021125362A1). Funding This work was supported by JSPS KAKENHI, Grant Number JP23K1399, and by the Analysis and Development System for Advanced Materials (ADAM) of the Research Institute for Sustainable Humanosphere (RISH) at Kyoto University. Author contributions Makiko Imai (Koyama), Katsuhiro Isozaki, Hiroyuki Matsumura, and Masaharu Nakamura contributed to the conception and design of the study. Shogo Suzuki, Yuki Nakamura, and Hiroyuki Matsumura prepared the materials. Makiko Imai (Koyama), Maki Kishimoto, Yoshiki Ueyama, and Hiroyuki Matsumura performed data collection, analysis, and microscopic observations. The first draft of the manuscript was written by Makiko Imai (Koyama), and all authors commented on previous versions of the manuscript. All the authors have read and approved the final version of the manuscript. Acknowledgements We gratefully acknowledge the staff members of the Laboratory of Material Biology at the Research Institute RISH for their generous support in providing homogenization. References Abe K, Yano H (2012) Cellulose nanofiber-based hydrogels with high mechanical strength. Cellulose 19:1907–1912. https://doi.org/10.1007/s10570-012-9784-3 Booker RE, Sell J (1998) The nanostructure of the cell wall of softwoods and its functions in a living tree. Holz als Roh-und Werkst 56:1–8. https://doi.org/10.1007/s001070050255 Chávez-Guerrero L, Sepúlveda-Guzmán S, Rodríguez-Liñan C et al (2017) Isolation and characterization of cellulose nanoplatelets from the parenchyma cells of Agave salmiana. Cellulose 24:3741–3752. https://doi.org/10.1007/s10570-017-1376-9 Chávez-Guerrero L, Sepúlveda-Guzmán S, Silva-Mendoza J, Aguilar-Flores C, Pérez-Camacho O (2018) Eco-friendly isolation of cellulose nanoplatelets through oxidation under mild conditions. Carbohydr Polym 181:642–649. https://doi.org/10.1016/j.carbpol.2017.11.100 Cheng B, Lei Z, Wu P (2022) Bio-derived crystalline silk nanosheets for versatile macroscopic assemblies. Nano Res 15:5538–5544. https://doi.org/10.1007/s12274-022-4124-x Effland MJ (1977) Modified procedure to determine acid-insoluble lignin in wood and pulp. Tappi; (United States) 60:143–144 Fahlén J, Salmén L (2002) On the lamellar structure of the tracheid cell wall. Plant Biol 4:339–345. https://doi.org/10.1055/s-2002-32341 Fahlén J, Salmén L (2003) Cross-sectional structure of the secondary wall of wood fibers as affected by processing. J Mater Sci 38:119–126. https://doi.org/10.1023/A:1021174118468 Flores-Jerónimo G, Silva-Mendoza J, Morales-San Claudio PC et al (2021) Chemical and mechanical properties of films made of cellulose nanoplatelets and cellulose fibers obtained from banana pseudostem. Waste Biomass Valor 12:5715–5723. https://doi.org/10.1007/s12649-021-01377-2 Francis DW, Towers MT, Browne TC (2004) Energy cost reduction in the pulp and paper industry—An energy benchmarking perspective Huang Y, Wang L, Chao Y et al (2012) Analysis of lignin aromatic structure in wood based on the IR spectrum. J Wood Chem Technol 32:294–303. https://doi.org/10.1080/02773813.2012.666316 Iwamoto S, Nakagaito AN, Yano H, Nogi M (2005) Optically transparent composites reinforced with plant fiber-based nanofibers. Appl Phys A 81:1109–1112. https://doi.org/10.1007/s00339-005-3316-z Jakob HF, Fengel D, Tschegg SE, Fratzl P (1995) The elementary cellulose fibril in Picea abies: Comparison of transmission electron microscopy, small-angle X-ray scattering, and wide-angle X-ray scattering results. Macromolecules 28:8782–8787. https://doi.org/10.1021/ma00130a010 Kose R, Kondo T (2013) Size effects of cellulose nanofibers for enhancing the crystallization of poly(lactic acid). J Appl Polym Sci 128:1200–1205. https://doi.org/10.1002/app.38308 Kuga S, Wu M (2019) Mechanochemistry of cellulose. Cellulose 26:215–225. https://doi.org/10.1007/s10570-018-2197-1 Kumagai A, Endo T, Adachi M (2019) Evaluation of cellulose Nanofibers by Using Sedimentation Method. J Tappi J 73:461–469. https://doi.org/10.2524/jtappij.1901 Kundu C, Samudrala SP, Kibria MA, Bhattacharya S (2021) One-step peracetic acid pretreatment of hardwood and softwood biomass for platform chemicals production. Sci Rep 11:11183. https://doi.org/10.1038/s41598-021-90667-9 Lee D-S, Lee Y-G, Cho EJ, Song Y, Bae H-J (2021) Hydrolysis pattern analysis of xylem tissues of woody plants pretreated with hydrogen peroxide and acetic acid: Rapid saccharification of softwood for economical bioconversion. Biotechnol Biofuels 14:37. https://doi.org/10.1186/s13068-021-01889-y Nakagaito AN, Yano H (2004) The effect of morphological changes from pulp fiber towards nano-scale fibrillated cellulose on the mechanical properties of high-strength plant fiber based composites. Appl Phys A 78:547–552. https://doi.org/10.1007/s00339-003-2453-5 Nakamura M, Isozaki K, Lu S, Jeon J, Wang J, Kitayama K, Ogata Y, Matsumura H, Imai M, Wu B (2024) the Methods for producing lignin and polysaccharides. (Japan) Patent No. JPWO2021125362A1 Nishiyama Y, Kuga S, Wada M, Okano T (1997) Cellulose Microcrystal Film of High Uniaxial Orientation. Macromolecules 30:6395–6397. https://doi.org/10.1021/ma970503 Nogi M, Iwamoto S, Nakagaito AN, Yano H (2009) Optically transparent nanofiber paper. Adv Mater 21:1595–1598. https://doi.org/10.1002/adma.200803174 Novoselov KS, Geim AK, Morozov SV et al (2004) Electric Field Effect in Atomically Thin Carbon Films. Science 306:666–669. https://doi.org/10.1126/science.1102896 Perré P, Turner I (2001) Determination of the material property variations across the growth ring of softwood for use in a heterogeneous drying model Part 1. Capillary pressure,tracheid model and absolute permeability. Holzforschung 55:318–323. https://doi.org/10.1515/HF.2001.052 Ruel K, Barnoud F, Goring DAI (1978) Lamellation in the S2 layer of softwood tracheids as demonstrated by scanning transmission electron microscopy. Wood Sci Technol 12:287–291. https://doi.org/10.1007/BF00351930 Ruel K, Chevalier-Billosta V, Guillemin F, Berrio-sierra J, Joseleau JP (2006) The wood cell wall at the ultrastructural scale—Formation and topochemical organization. Maderas Cienc Tecnol 8:107–116. https://doi.org/10.4067/S0718-221X2006000200004 Rodríguez-San-Miguel D, Montoro C, Zamora F (2020) Covalent organic framework nanosheets: preparation, properties and applications. Chem Soc Rev 49:2291–2302. https://doi.org/10.1039/C9CS00890J Sasaki T, Watanabe M, Hashizume H et al (1996) Macromolecule-like Aspects for a Colloidal Suspension of an Exfoliated Titanate. Pairwise Association of Nanosheets and Dynamic Reassembling Process Initiated from It. J Am Chem Soc 118:8329–8335 Segal L, Creely JJ, Martin AE, Conrad CM (1959) An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer. Text Res J 29:786–794. https://doi.org/10.1177/004051755902901003 Sehaqui H, Ezekiel Mushi N, Morimune S et al (2012) Cellulose nanofiber orientation in nanopaper and nanocomposites by cold drawing. ACS Appl Mater Interfaces 4:1043–1049. https://doi.org/10.1021/am2016766 Shimizu S, Yokoyama T, Akiyama T, Matsumoto Y (2012) Reactivity of lignin with different composition of aromatic syringyl/guaiacyl structures and erythro/threo side chain structures in β-O-4 type during alkaline delignification: As a basis for the different degradability of hardwood and softwood lignin. J Agric Food Chem 60:6471–6476. https://doi.org/10.1021/jf301329v Silva-Mendoza J, Cantú-Cárdenas ME, Vazquez-Rodriguez S et al (2019) Biopolymeric films obtained from the parenchyma cells of Agave salmiana leaves. Cellulose 26:1869–1879. https://doi.org/10.1007/s10570-018-2173- Stelte W, Sanadi AR (2009) Preparation and characterization of cellulose nanofibers from two commercial hardwood and softwood pulps. Ind Eng Chem Res 48:11211–11219. https://doi.org/10.1021/ie9011672 Takimoto D, Toma S, Suda Y et al (2023) Platinum nanosheets synthesized via topotactic reduction of single-layer platinum oxide nanosheets for electrocatalysis. Nat Commun 14:19. https://doi.org/10.1038/s41467-022-35616-4 Tanahashi M (1990) Characterization and Degradation Mechanisms of Wood Components by Steam Explosion and Utilization of Exploded Wood. Wood research: Bull Wood Res Inst Kyoto Univ 77:49–117 Tsuboi K, Yokota S, Kondo T (2014) Difference between bamboo- and wood-derived cellulose nanofibers prepared by the aqueous counter collision method. Nord Pulp Pap Res J 29:69–76. https://doi.org/10.3183/npprj-2014-29-01-p069-076 Uetani K, Yano H (2011) Nanofibrillation of wood pulp using a high-speed blender. Biomacromolecules 12:348–353. https://doi.org/10.1021/bm101103p Wilson BP, Yliniemi K, Gestranius M et al (2018) Structural distinction due to deposition method in ultrathin films of cellulose nanofibres. Cellulose 25:1715–1724. https://doi.org/10.1007/s10570-018-1665-y You J, Li M, Ding B et al (2017) Crab Chitin-Based 2D Soft Nanomaterials for Fully Biobased Electric Devices. Adv Mater 29:1606895. https://doi.org/10.1002/adma.201606895 Zhai L, Kim HC, Kim JW, Kim J (2020) Alignment effect on the piezoelectric properties of ultrathin cellulose nanofiber films. ACS Appl Bio Mater 3:4329–4334. https://doi.org/10.1021/acsabm.0c00364 Zhang Y, Kuga S, Wu M, Huang Y (2019) Cellulose nanosheets formed by mild additive-free ball milling. Cellulose 26:3143–3153. https://doi.org/10.1007/s10570-019-02282-7 Zhao M, Kuga S, Jiang S, Wu M, Huang Y (2016) Cellulose nanosheets induced by mechanical impacts under hydrophobic environment. Cellulose 23:2809–2818. https://doi.org/10.1007/s10570-016-1033-8 Additional Declarations The authors declare potential competing interests as follows: The authors have no relevant financial or non-financial interests to disclose. However, Makiko Imai (Koyama), Shogo Suzuki, Yuki Nakamura, Katsuhiro Isozaki, Hiroyuki Matsumura, Masaharu Nakamura are inventors on an international patent application related to the Native Cellulose Nanosheets (WO2023162263A1). Makiko Imai (Koyama), Yuki Nakamura, Katsuhiro Isozaki, Hiroyuki Matsumura, Masaharu Nakamura hold a patent for the Methods for producing lignin and polysaccharides (JPWO2021125362A1). Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9423095","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":623415858,"identity":"dfe7d781-cc24-4a30-9ccd-74b1f2809c44","order_by":0,"name":"Makiko Imai (Koyama)","email":"","orcid":"https://orcid.org/0009-0003-5671-8994","institution":"Institute for chemical research, Kyoto university","correspondingAuthor":false,"prefix":"","firstName":"Makiko","middleName":"Imai","lastName":"(Koyama)","suffix":""},{"id":623415859,"identity":"ff8fcb1f-b024-45ca-bf67-280176b5594f","order_by":1,"name":"Shogo Suzuki","email":"","orcid":"","institution":"Institute for chemical research, Kyoto university","correspondingAuthor":false,"prefix":"","firstName":"Shogo","middleName":"","lastName":"Suzuki","suffix":""},{"id":623415860,"identity":"1ed629e0-591a-4d03-92fb-9bdec54d0bc5","order_by":2,"name":"Yuki Nakamura","email":"","orcid":"","institution":"Institute for chemical research, Kyoto university","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Nakamura","suffix":""},{"id":623415861,"identity":"1b0ee7b0-f2f5-4af6-85b9-c3d56db7dce5","order_by":3,"name":"Katsuhiro Isozaki","email":"","orcid":"https://orcid.org/0000-0002-0990-1708","institution":"Institute for chemical research, Kyoto university","correspondingAuthor":false,"prefix":"","firstName":"Katsuhiro","middleName":"","lastName":"Isozaki","suffix":""},{"id":623415862,"identity":"c08bfb07-bd69-44f5-9877-eec02405ddd9","order_by":4,"name":"Maki Kishimoto","email":"","orcid":"","institution":"Daicel corporation","correspondingAuthor":false,"prefix":"","firstName":"Maki","middleName":"","lastName":"Kishimoto","suffix":""},{"id":623415863,"identity":"1decd1aa-3ecc-4744-82b2-3d2e8238f030","order_by":5,"name":"Yoshiki Ueyama","email":"","orcid":"","institution":"Daicel corporation","correspondingAuthor":false,"prefix":"","firstName":"Yoshiki","middleName":"","lastName":"Ueyama","suffix":""},{"id":623415864,"identity":"9230d587-b6e8-4d61-b29a-0e18f3590911","order_by":6,"name":"Hiroyuki Matsumura","email":"","orcid":"","institution":"Daicel corporation","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Matsumura","suffix":""},{"id":623415865,"identity":"592fcd0e-6222-4e11-86f4-191f9eb3cc09","order_by":7,"name":"Masaharu Nakamura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYJACZhDBz8DAeIDhAJIwYwMBLZJABWAtPERrMTiArgUX0G0//vBzQc29xM3nDz84wHDmsJw9ewObBEONHQPzbOzWmJ3JMZaecaw4cduNNKBFNw4b8/AcAGo5lszAOOcAdi0HchikedgSgFp4gM76cDixRyL/mwQD2wEGxhkJ2LWcf/74N8+/hMTN/WdgWhKAtvzDo+VGgpk0b1tC4gaGHAaQwyBaGNvwaXljZs3bl2A8A+SXhDPpxjxnDjBbJPYl8+D0y/n0x7d5viXI9vcffvjgwzFrOfb2BsYbH77ZyRniCDFUkMDQDGMw8BjOIEIHENQhmPISxGkZBaNgFIyCYQ8A9dtjq3nvzekAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1419-2117","institution":"Institute for chemical research, Kyoto university","correspondingAuthor":true,"prefix":"","firstName":"Masaharu","middleName":"","lastName":"Nakamura","suffix":""}],"badges":[],"createdAt":"2026-04-15 07:14:30","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9423095/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9423095/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107410407,"identity":"7286fc5a-1b96-4950-b51b-cbe8e12a2d60","added_by":"auto","created_at":"2026-04-21 08:59:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":180353,"visible":true,"origin":"","legend":"\u003cp\u003eMild cedar powder-delignification reaction and homogenization of the cellulosic material obtained\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/33e2bebe6a0bff8acc3d6160.png"},{"id":107410413,"identity":"7bd1c717-0dfe-4883-8ec6-39379fc01b30","added_by":"auto","created_at":"2026-04-21 08:59:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46150,"visible":true,"origin":"","legend":"\u003cp\u003eATR-IR spectra. a) Cedar powder; b) MCA, the arrow indicates absorbance at 1509 cm\u003csup\u003e−1\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/9ba1cda6e6ed22aa6fae62b7.png"},{"id":107410444,"identity":"023ee53c-7623-4da0-90b9-668877d668dc","added_by":"auto","created_at":"2026-04-21 08:59:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56458,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction profiles of MCA. a) Before homogenization; b) after homogenization\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/07340a1d446fd9aa444bb64f.png"},{"id":107410409,"identity":"61f6b6b6-c0c0-47f4-a60c-d8f774d9e402","added_by":"auto","created_at":"2026-04-21 08:59:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":706169,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopic images: a) before homogenization, b) after 5 min of homogenization, c) after 10 min of homogenization, and d) after 60 min of homogenization. The arrow indicates sheets exfoliating from the tracheids, and arrowheads show thin cell walls\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/b6a9d8219314cb525050cd92.png"},{"id":107410410,"identity":"e609e27f-d234-4644-a6f0-72813c606183","added_by":"auto","created_at":"2026-04-21 08:59:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":151460,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopic image of native cellulose nanosheets with residual tracheids removed by centrifugation\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/d5ffac9f78c98bfaf1cf0c80.png"},{"id":107410408,"identity":"b324e2a2-30b1-4d51-a17e-af4c2b1e19d4","added_by":"auto","created_at":"2026-04-21 08:59:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":446881,"visible":true,"origin":"","legend":"\u003cp\u003eAFM image of the native cellulose nanosheets and height profiles. a) Image in a large area. The double-headed arrow indicates the arranging directions of cellulose fibers. The arrow indicates a remaining thick fragment of cell wall. b) Image in a smaller area. The double-headed arrow indicates the arranging directions of cellulose fibers. The arrowhead indicates an unraveling cellulose fiber from the edge.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/44bd7e33bb612833b9da0f58.png"},{"id":107487968,"identity":"3fadd54b-070e-4ccc-912d-94a4680bba1e","added_by":"auto","created_at":"2026-04-22 02:43:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":616533,"visible":true,"origin":"","legend":"\u003cp\u003eWhite light-interference laser microscopy\u003cstrong\u003e \u003c/strong\u003eimage of the native cellulose nanosheets. The arrow indicates a pit border. The height of the area enclosed in the square is 21 nm\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/7c9d16b447a02cde3c24563a.png"},{"id":107410418,"identity":"14285bb7-9379-483b-aa3d-71edb63e43e4","added_by":"auto","created_at":"2026-04-21 08:59:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":196848,"visible":true,"origin":"","legend":"\u003cp\u003eCellulose films. left) films from native cellulose nanosheets of MCA, right) films from commercial bleached softwood pulp\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/cbb895fed6134f749a1a90c2.png"},{"id":107410406,"identity":"4364240d-ae14-475a-8661-cd2e59bfc072","added_by":"auto","created_at":"2026-04-21 08:59:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":466511,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of exfoliating of native cellulose nanosheets\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/74315b4ba7902f5a183a5f1c.png"},{"id":107489377,"identity":"4ec51a37-9498-4148-8995-9b49dc602496","added_by":"auto","created_at":"2026-04-22 02:47:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3061262,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9423095/v1/40be29e1-0c47-487a-a8c3-f9e9de1b61f2.pdf"}],"financialInterests":"The authors declare potential competing interests as follows: The authors have no relevant financial or non-financial interests to disclose. However, Makiko Imai (Koyama), Shogo Suzuki, Yuki Nakamura, Katsuhiro Isozaki, Hiroyuki Matsumura, Masaharu Nakamura are inventors on an international patent application related to the Native Cellulose Nanosheets (WO2023162263A1). Makiko Imai (Koyama), Yuki Nakamura, Katsuhiro Isozaki, Hiroyuki Matsumura, Masaharu Nakamura hold a patent for the Methods for producing lignin and polysaccharides (JPWO2021125362A1).","formattedTitle":"\u003cp\u003e\u003cstrong\u003eNative cellulose nanosheets from Japanese Cedar via mild delignification and mild homogenization\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTwo-dimensional nanosheets are materials of several to tens of nanometers thick and several \u0026micro;m meters square. There are numerous reports of nanosheets of organic (Rodr\u0026iacute;guez-San-Miguel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and inorganic compounds of platinum (Takimoto et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), titanium (Sasaki et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), and graphene (Novoselov et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Li et al. 2008), attracting attention in the fields of electronics, optoelectronics, catalysts, energy storage, energy generation, sensors, separation, and biomedicines\u003csup\u003e6\u003c/sup\u003e. For biomass materials, various artificial nanosheets produced by stacking biomass-based nanofibers have been reported (You et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wilson et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kuga and Wu \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, only a few studies have described nanosheets derived directly from biomaterials. To the best of our knowledge, native nanosheets obtained by the top-down method have been reported from silkworm cocoons (Cheng et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), agave leaves (Ch\u0026aacute;vez-Guerrero et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Silva-Mendoza et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), Banana stems (Ch\u0026aacute;vez-Guerrero et al. 2019; Flores-Jer\u0026oacute;nimo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCellulose is the most abundant natural polymer. Further, given the versatility of its potential applications, it has been essential in human societies throughout history. Indeed, to date, purified cellulose has been used in various ways, including as a raw material in the paper and textile industries. In particular, wood cell walls contain large amounts of cellulose. However, high-temperature processes requiring large amounts of increasingly expensive energy to remove lignin and hemicellulose are needed to obtain pure cellulose. Therefore, new processes with lower energy consumption have become a research priority (Francis et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Recently, research on methods for lignin removal with low ecological impact has made substantial progress. Thus, for example, woody biomass was treated with peracetic acid containing sulfuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) as a catalyst at 90 ˚C (Kundu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); similarly, in another study, it was pretreated with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and acetic acid at 80 ˚C for saccharification (Lee et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Japanese cedar (\u003cem\u003eCryptomeria japonica\u003c/em\u003e) is endemic to Japan and occupies the largest plantation area among all tree species in the country. Although Japanese cedar trees planted decades ago have reached maturity for logging, they cannot be readily used as biomass materials, largely due to the difficulties associated with delignification. As guaiacyl lignin units, the main components of softwood, are difficult to decompose, harsh reaction conditions are required to remove softwood lignin (Shimizu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). According to our method, Japanese cedar powder was treated with diluted H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at 60 ˚C under ambient pressure, using a versatile organic acid as the catalyst. This one-step reaction leads to delignification, and the resulting cellulosic residue resembles what is known as a \u0026ldquo;pulp\u0026rdquo; that retains the shape of the tracheids (Nakamura et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eRecently, the development of novel applications for nanocellulose, among which nanofibers are a typical example, has been actively pursued. Thus, for example, pulp is now generally fibrillated to cellulose nanofibers several to hundreds of nanometers wide using a blender (Uetani and Yano \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), a high-pressure homogenizer (Iwamoto et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), a grinder (Iwamoto et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Abe and Yano \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), or some counter-collision method (Tsuboi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This has allowed the fabrication of highly transparent sheets from wood cellulose nanofibers (Nogi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) which, owing to their light weight, high strength, high thermal dimensional-stability, and good gas-barrier properties, are a highly promising material under development for various applications.\u003c/p\u003e \u003cp\u003eIn this study, sheet-structured cellulose was obtained from cellulose isolated via a mild process using a common homogenizer. To date, thin plates several tens of nanometers thick from agave leaf parenchyma (Ch\u0026aacute;vez-Guerrero et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and banana pseudo-stem parenchyma (Flores-Jer\u0026oacute;nimo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have been reported as native thin-cellulose structures. However, these are parts of the parenchyma with thin cell walls composed of randomly arranged cellulose fibrils. On the other hand, nanometer-thick sheets have not been generated from highly lignified, thick cell walls such as tracheids and fiber cells characteristic of wood. Instead, the shapes of the sheets obtained were quite different from those of thin plates, nanofibers, nanocrystals, and microfibers reported to date. Herein, we report detailed observations and measurements of this new structure.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCellulose separation\u003c/h2\u003e \u003cp\u003eAir-dried Japanese cedar (\u003cem\u003eCryptomeria japonica\u003c/em\u003e) wood was milled using a Wiley mill equipped with a 1 mm sieve. The resulting wood powder (10 g) was placed into a 0.2 L three-neck round-bottom flask; then, a mixture of 10 mL of 30 wt% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 0.86 g of dodecylbenzenesulfonic acid (DBSA) was added. After adding of 50 mL of toluene, the reaction mixture was heated at 60 ˚C on an aluminum block for 24 h with stirring at 100 rpm with a blade. The obtained solid was separated by vacuum filtration using filter paper and washed with ultrapure water until a neutral pH was attained.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of acid-insoluble lignin extracted from cedar powder and the separated cellulosic materials\u003c/h3\u003e\n\u003cp\u003eCedar powder or the cellulosic material (20 mg) obtained was heated in 0.3 mL of 72 wt% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e at 30 ˚C for 60 min, with occasional shaking. The reaction solutions were diluted with 8.4 mL of ultra-pure water to 4 wt% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and heated in an autoclave at 120 ˚C for 60 min (Effland \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). The residues (acid-insoluble lignin) were washed with ultrapure water until a neutral pH was attained, and then weighed. Finally, the corresponding ratios relative to the initial solids were calculated.\u003c/p\u003e\n\u003ch3\u003eFourier-transform infrared (FT-IR) spectroscopy\u003c/h3\u003e\n\u003cp\u003eThe FT-IR spectra of the freeze-dried cellulosic materials and wood powder were recorded using an FT-IR spectrometer (FT/IR-4700, JASCO, Tokyo, Japan) in the attenuated total reflection (ATR) mode in the 4000\u0026thinsp;\u0026minus;\u0026thinsp;500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with 64 scans.\u003c/p\u003e\n\u003ch3\u003eHomogenization\u003c/h3\u003e\n\u003cp\u003eAn ultrahigh-speed homogenizer (Physcotron, Microtec Co., Ltd., Funabashi, Japan) equipped with an inner and an outer blade (with the inner blade rotating at an ultrahigh speed) was used to homogenize the material, which subsequently entered the space between the rotating inner and outer blades, where it was ground to a powder. In addition, high-frequency vibrations were generated between the two blades, further contributing to the refinement of the pulverized material. This homogenizer has been widely employed for the disintegration of cellulose (Nishiyama et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Kose and Kondo \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Then, 0.3 wt% aqueous suspension slurries were homogenized at 13,000 rpm for 60 min, with occasional cooling in ice water. A small portion was removed from the suspension after 5 and 10 min for microscopic analysis.\u003c/p\u003e\n\u003ch3\u003eX-ray diffractometry\u003c/h3\u003e\n\u003cp\u003eFreeze-dried cellulosic materials were analyzed via X-ray diffraction (XRD) using an XRD-7000 diffractometer (Shimadzu, Kyoto, Japan) in the reflection mode. Diffraction patterns were collected stepwise using Cu Kα radiation (λ=1.5406 \u0026Aring;) in the range of 5\u0026deg;\u0026ndash;40\u0026deg;, at a step of 0.02\u0026deg; and a scan speed of 1\u0026deg; min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The Segal crystallinity index was calculated using the following equation (Segal et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1959\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Crystallinity\\:index\\:\\left(\\%\\right)=100{\\times\\:(I}_{200}-{I}_{a})/{I}_{200}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{I}_{200}\\)\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e \u003cp\u003eIntensity of the (200) peak for cellulose I\u003csub\u003eβ\u003c/sub\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{I}_{a}\\)\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e \u003cp\u003eIntensity of amorphous\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eWe used a nanopercolator (JEOL Ltd., Tokyo, Japan), which is a disposable SEM sample stage equipped with a polycarbonate membrane filter (pore size:0.6 \u0026micro;m). By performing filtration with a syringe, the solution can be removed, and the sample can be deposited onto the membrane. The 0.06 wt% aqueous suspension of cellulosic materials (50 \u0026micro;L) was dropped on a nanopercolator. Excess water was removed using a 10-mL syringe. The adaptor for SEM, to which the nanopercolator was attached, was placed in liquid nitrogen. The sample was freeze-dried on the nanopercolator and coated with Au by using a DII-29010SCTR Smart Coater (JEOL). Images were obtained using a scanning electron microscope SEM (JCM-7000 NeoScope\u0026trade;, JEOL) equipped with a tungsten filament operating at 15 kV.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhite light-interference Laser Microscopy\u003c/h3\u003e\n\u003cp\u003eThe homogenized cellulose slurry was diluted with ultrapure water to 0.01 wt%. Then, it was dropped onto a freshly cleaved mica surface and air-dried. Surface morphology was observed using a white light-interference laser microscope (VK-X3000, Keyence, Osaka, Japan).\u003c/p\u003e\n\u003ch3\u003eAtomic force microscopy (AFM)\u003c/h3\u003e\n\u003cp\u003eThe homogenized slurry was diluted with ultrapure water to 0.01 wt%, dropped onto a freshly cleaved mica surface, air-dried, and subjected to atomic force microscopy (AFM; Dimension Icon SPM; Bruker, Yokohama, Japan and SPM-Nanoa; Shimadzu, Kyoto, Japan) equipped with a microcantilever. Images were recorded for a 5 \u0026times; 5 \u0026micro;m and 30 \u0026times; 30 \u0026micro;m wide square in the tapping mode. Height analysis was performed using the NanoScope9.4R1 software and SPM-Nanoasoftware.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProduction of the nanofilms\u003c/h2\u003e \u003cp\u003eThe cellulose slurry homogenized using a Physcotron at 13,000 rpm for 60 min was vacuum-filtered through a membrane filter (pore size: 0.45 \u0026micro;m) and pressed under 5 MPa at room temperature for 4 min. The obtained films were dried at 105 ˚C for 10 min.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSeparation and certification of cellulose\u003c/h2\u003e \u003cp\u003eCedar powder was heated in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e containing DBSA to form a white solid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The yield of the solid was 57% of the oven-dried cedar wood powder. In turn, acid-insoluble lignin of the cedar material was 33.5% and that of the obtained cellulosic material was 4.1%, corresponding to 2.3% of the cedar powder, all on a weight basis. Lignin decreased by 93% compared to the content measured before the reaction. The ATR-IR spectrum of the obtained cellulosic material confirmed that the absorbance of the lignin aromatic skeletal vibration at 1509 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which was observed in the spectrum of the cedar powder, completely disappeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, despite the recalcitrance of softwood lignin, our mild oxidative reaction facilitated the delignification of cedar powder and yielded a cellulose-based material, which constitutes a significant achievement. We call this material the Molecular Cellulose Assembly (MCA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur XRD analysis confirmed that the MCA before and after homogenization (60 min) consisted of cellulose I (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The crystallinities before and after homogenization were 80% and 77%, respectively, and there was no detectable change.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopic observation of MCA\u003c/h2\u003e \u003cp\u003eWe used SEM to observe MCA before and after homogenization of the cellulosic material obtained as described above. Tracheids are high aspect ratio cells that function in water transport and provide mechanical support to plants. Further, they constitute 95% of softwood total mass such as Japanese cedar (Perr\u0026eacute; and Turner \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Although cellulose fibrils were exposed on the surface of tracheids before homogenization of the material, the latter maintained their shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and after 5 or 10 min of homogenization, thin sheets were exfoliated from the outer surface of the tracheids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). After 60 min of homogenization, sheet structures occupied most, whereas shape-maintaining tracheids occupied a minimum portion of the homogenized MCA. Cell walls appeared thinner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The scarce shape-maintaining tracheids in the slurry were removed by mild centrifugation at 600 \u0026times; g.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of cellulose sheet thickness by AFM and white light-interference laser microscopy\u003c/h2\u003e \u003cp\u003eThe fine structure of the cellulose sheet visualized by SEM was analyzed by AFM, and cellulose microfibrils were clearly visualized in the sheet. Numerous sheets were also observed by AFM, with the side length of some sheets measured at over 10 \u0026micro;m. Cellulose fibers were highly oriented in one direction within the sheet. Their thickness was less than 90 nm and most measured approximately 30 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, the remaining relatively thick fragments of cell walls, which had not yet been exfoliated into nanosheets, were also observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, thin fibers unraveled from the edge of the sheet with a height of 3\u0026ndash;4 nm. The width of cellulose microfibrils from wood is approximately 3 nm (Jakob et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), therefore, the height observed in this study corresponds to the width of the microfibrils in wood cell walls, and the thickness of the sheet corresponds to that of 10 microfibrils. To our knowledge, this is the first report of native nanosheets of uniaxially oriented cellulose. Cellulose exists as aggregates (10\u0026ndash;30 nm), and several aggregates form lamellae in tangential direction within the secondary walls of Norwegian spruce (Fahl\u0026eacute;n and Salm\u0026eacute;n \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The sheets are assumed to be composed of several stacked lamellae exfoliated from the cell wall. The lignin and hemicellulose matrices filled the interlamellar regions between the cellulose lamellae, and the cellulose sheets were easily exfoliated upon removal of the matrix, such that nanosheets were observed using white light-interference laser microscopy.\u003c/p\u003e \u003cp\u003eCircular structures, presumably corresponding to the borders of bordered pits, were identified on the sheets (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), and their height was approximately 230 nm. The average height of the area enclosed within the square is 21 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe observed that thin sheets exfoliated from the outer surface of the tracheids and that the cellulose microfibrils were oriented in one direction within a sheet, and pit borders were also observed. From the above observations, we inferred that the sheets were not formed by the reassembly of fibrillated microfibrils but were native cellulose sheets that maintained the native orientation of the microfibrils. We named this sheet, native cellulose nanosheet (NCNS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTransparency of cellulose films\u003c/h2\u003e \u003cp\u003eCellulose films were produced from NCNS and commercial bleached softwood pulp and homogenized under the same conditions. The NCNS film was more transparent than the pulp film (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This finding means that the MCA was homogenized to yield nanometer-sized NCNS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, DBSA was used as the catalyst for the delignification reaction. DBSA is an amphiphilic substance with a benzene ring, a long-chain alkyl, and a sulfo group. Furthermore, DBSA is soluble in aqueous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution and is accessible to hydrophobic lignin. Therefore, it is possible to selectively degrade lignin rather than cellulose and hemicellulose. The cedar cell walls consist of a primary wall and a three-layered secondary wall (S1, S2, and S3) with different thicknesses and microfibril angles. In particular, S2 is the thickest, and its microfibrils are oriented at an angle nearly parallel to the long axis of the tracheid (Booker and Sell \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The NCNS maintained this fiber orientation. No significant decrease in the crystallinity was observed, indicating that exfoliating occurred without damaging the crystals.\u003c/p\u003e \u003cp\u003eTo date, thin plates several tens of nanometers thick from agave leaf parenchyma (Ch\u0026aacute;vez-Guerrero et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and banana pseudostem parenchyma (Flores-Jer\u0026oacute;nimo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have been reported as native thin-cellulose structures. However, these are parts of the parenchyma with thin cell walls composed of randomly arranged cellulose fibrils. Conversely, nanometer-thick sheets have not been isolated from highly lignified, thick cell walls, such as wood tracheids and fiber cells.\u003c/p\u003e \u003cp\u003eAs for NCNSs, these were predominantly approximately 30\u0026ndash;50 nm thick, which is equivalent to 10\u0026ndash;17 cellulose microfibrils with a width of 3\u0026ndash;4 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Secondary walls reportedly have a lamellar structure in which cellulose and lignin are alternately arranged parallel to the lumen surface (Ruel et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1978\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tanahashi \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). In this study, it was presumed that spaces were formed between the cellulose lamellae by selective delignification under mild conditions and that cellulose lamellae became easier to exfoliate. On the other hand, we speculated that hydrogen bonds binding cellulose molecules within the sheet to each other, or cellulose to hemicellulose, were not broken, and that new hydrogen bonds were formed between lamellae. As a result, sheet structure was preserved, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis sheet formation process also provided strong evidence of the lamellar structure of wood cell walls. According to our SEM observations, the one-sided lengths of some NCNSs exceeded 50 \u0026micro;m, which corresponds to the perimeter of the tracheid. Therefore, it was assumed that the cellulose in lamellae encircled the tracheid cell walls.\u003c/p\u003e \u003cp\u003eWhen kraft pulp or dissolving pulp is treated by a disc mill, high-pressure homogenizer, or refiner, it is fibrillated into nanofibers without forming a sheet structure (Nakagaito and Yano \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Stelte and Sanadi \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kumagai et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The mild process used in this study caused cellulose to be degraded into nanosheets and further homogenization caused it to be further degraded into nanofibers. Additionally, we found that the shape of nanocellulose can be adjusted to a sheet- or fiber-like shape by adjusting the disintegrating method and processing time, which together will help produce cellulose structures suitable for future applications. Moreover, the method described can be used for biomass sources other than the Japanese cedar biomass used herein.\u003c/p\u003e \u003cp\u003eOne of the distinctive features of NCNS is that its cellulose microfibrils are uniaxially oriented. Artificially oriented sheets, films, and papers have been reported to exhibit various properties. In particular, the ball milling of cellulose powder with silicon oil (Zhao et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) or without additives (Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) gave uniaxially-oriented cellulose nanosheets via the rearrangement of cellulose microfibrils. Cellulose nanofiber films of aligned cellulose have reportedly better piezoelectric properties (Zhai et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, nanopaper with oriented TEMPO cellulose nanofibers shows better mechanical properties (Sehaqui et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Oriented nanocellulose materials will become increasingly popular as functional materials. As we aim for a sustainable, decarbonized society, cellulose has great potential for the development of unprecedented new materials in addition to its traditional uses.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCellulose was effectively obtained from Japanese cedar powder by selective delignification under mild reaction conditions. The NCNSs exfoliated sequentially from the outermost layer of the cell wall by homogenization. The NCNSs with uniaxially oriented cellulose microfibrils were obtained for the first time. We believe these materials have great potential for development of a range of applications. The successful formation of NCNS strongly indicates the possibility of controlling the size and shape of nano cellulose. Furthermore, from the viewpoint of cell wall structure, this proves that cellulose has a lamellar structure within the wood cell walls.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interest\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose. However, Makiko Imai (Koyama), Shogo Suzuki, Yuki Nakamura, Katsuhiro Isozaki, Hiroyuki Matsumura, Masaharu Nakamura are inventors on an international patent application related to the Native Cellulose Nanosheets (WO2023162263A1). Makiko Imai (Koyama), Yuki Nakamura, Katsuhiro Isozaki, Hiroyuki Matsumura, Masaharu Nakamura hold a patent for the Methods for producing lignin and polysaccharides (JPWO2021125362A1).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by JSPS KAKENHI, Grant Number JP23K1399, and by the Analysis and Development System for Advanced Materials (ADAM) of the Research Institute for Sustainable Humanosphere (RISH) at Kyoto University.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eMakiko Imai (Koyama), Katsuhiro Isozaki, Hiroyuki Matsumura, and Masaharu Nakamura contributed to the conception and design of the study. Shogo Suzuki, Yuki Nakamura, and Hiroyuki Matsumura prepared the materials. Makiko Imai (Koyama), Maki Kishimoto, Yoshiki Ueyama, and Hiroyuki Matsumura performed data collection, analysis, and microscopic observations. The first draft of the manuscript was written by Makiko Imai (Koyama), and all authors commented on previous versions of the manuscript. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe gratefully acknowledge the staff members of the Laboratory of Material Biology at the Research Institute RISH for their generous support in providing homogenization.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbe K, Yano H (2012) Cellulose nanofiber-based hydrogels with high mechanical strength. Cellulose 19:1907\u0026ndash;1912. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10570-012-9784-3\u003c/span\u003e\u003cspan address=\"10.1007/s10570-012-9784-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBooker RE, Sell J (1998) The nanostructure of the cell wall of softwoods and its functions in a living tree. Holz als Roh-und Werkst 56:1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s001070050255\u003c/span\u003e\u003cspan address=\"10.1007/s001070050255\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCh\u0026aacute;vez-Guerrero L, Sep\u0026uacute;lveda-Guzm\u0026aacute;n S, Rodr\u0026iacute;guez-Li\u0026ntilde;an C et al (2017) Isolation and characterization of cellulose nanoplatelets from the parenchyma cells of Agave salmiana. Cellulose 24:3741\u0026ndash;3752. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10570-017-1376-9\u003c/span\u003e\u003cspan address=\"10.1007/s10570-017-1376-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCh\u0026aacute;vez-Guerrero L, Sep\u0026uacute;lveda-Guzm\u0026aacute;n S, Silva-Mendoza J, Aguilar-Flores C, P\u0026eacute;rez-Camacho O (2018) Eco-friendly isolation of cellulose nanoplatelets through oxidation under mild conditions. Carbohydr Polym 181:642\u0026ndash;649. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carbpol.2017.11.100\u003c/span\u003e\u003cspan address=\"10.1016/j.carbpol.2017.11.100\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng B, Lei Z, Wu P (2022) Bio-derived crystalline silk nanosheets for versatile macroscopic assemblies. Nano Res 15:5538\u0026ndash;5544. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12274-022-4124-x\u003c/span\u003e\u003cspan address=\"10.1007/s12274-022-4124-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEffland MJ (1977) Modified procedure to determine acid-insoluble lignin in wood and pulp. Tappi; (United States) 60:143\u0026ndash;144\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFahl\u0026eacute;n J, Salm\u0026eacute;n L (2002) On the lamellar structure of the tracheid cell wall. Plant Biol 4:339\u0026ndash;345. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1055/s-2002-32341\u003c/span\u003e\u003cspan address=\"10.1055/s-2002-32341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFahl\u0026eacute;n J, Salm\u0026eacute;n L (2003) Cross-sectional structure of the secondary wall of wood fibers as affected by processing. J Mater Sci 38:119\u0026ndash;126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1021174118468\u003c/span\u003e\u003cspan address=\"10.1023/A:1021174118468\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlores-Jer\u0026oacute;nimo G, Silva-Mendoza J, Morales-San Claudio PC et al (2021) Chemical and mechanical properties of films made of cellulose nanoplatelets and cellulose fibers obtained from banana pseudostem. Waste Biomass Valor 12:5715\u0026ndash;5723. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12649-021-01377-2\u003c/span\u003e\u003cspan address=\"10.1007/s12649-021-01377-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrancis DW, Towers MT, Browne TC (2004) Energy cost reduction in the pulp and paper industry\u0026mdash;An energy benchmarking perspective\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Y, Wang L, Chao Y et al (2012) Analysis of lignin aromatic structure in wood based on the IR spectrum. J Wood Chem Technol 32:294\u0026ndash;303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/02773813.2012.666316\u003c/span\u003e\u003cspan address=\"10.1080/02773813.2012.666316\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwamoto S, Nakagaito AN, Yano H, Nogi M (2005) Optically transparent composites reinforced with plant fiber-based nanofibers. Appl Phys A 81:1109\u0026ndash;1112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00339-005-3316-z\u003c/span\u003e\u003cspan address=\"10.1007/s00339-005-3316-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJakob HF, Fengel D, Tschegg SE, Fratzl P (1995) The elementary cellulose fibril in Picea abies: Comparison of transmission electron microscopy, small-angle X-ray scattering, and wide-angle X-ray scattering results. Macromolecules 28:8782\u0026ndash;8787. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ma00130a010\u003c/span\u003e\u003cspan address=\"10.1021/ma00130a010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKose R, Kondo T (2013) Size effects of cellulose nanofibers for enhancing the crystallization of poly(lactic acid). J Appl Polym Sci 128:1200\u0026ndash;1205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/app.38308\u003c/span\u003e\u003cspan address=\"10.1002/app.38308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuga S, Wu M (2019) Mechanochemistry of cellulose. Cellulose 26:215\u0026ndash;225. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10570-018-2197-1\u003c/span\u003e\u003cspan address=\"10.1007/s10570-018-2197-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumagai A, Endo T, Adachi M (2019) Evaluation of cellulose Nanofibers by Using Sedimentation Method. J Tappi J 73:461\u0026ndash;469. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2524/jtappij.1901\u003c/span\u003e\u003cspan address=\"10.2524/jtappij.1901\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKundu C, Samudrala SP, Kibria MA, Bhattacharya S (2021) One-step peracetic acid pretreatment of hardwood and softwood biomass for platform chemicals production. Sci Rep 11:11183. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-021-90667-9\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-90667-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee D-S, Lee Y-G, Cho EJ, Song Y, Bae H-J (2021) Hydrolysis pattern analysis of xylem tissues of woody plants pretreated with hydrogen peroxide and acetic acid: Rapid saccharification of softwood for economical bioconversion. Biotechnol Biofuels 14:37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13068-021-01889-y\u003c/span\u003e\u003cspan address=\"10.1186/s13068-021-01889-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakagaito AN, Yano H (2004) The effect of morphological changes from pulp fiber towards nano-scale fibrillated cellulose on the mechanical properties of high-strength plant fiber based composites. Appl Phys A 78:547\u0026ndash;552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00339-003-2453-5\u003c/span\u003e\u003cspan address=\"10.1007/s00339-003-2453-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura M, Isozaki K, Lu S, Jeon J, Wang J, Kitayama K, Ogata Y, Matsumura H, Imai M, Wu B (2024) the Methods for producing lignin and polysaccharides. (Japan) Patent No. JPWO2021125362A1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishiyama Y, Kuga S, Wada M, Okano T (1997) Cellulose Microcrystal Film of High Uniaxial Orientation. Macromolecules 30:6395\u0026ndash;6397. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ma970503\u003c/span\u003e\u003cspan address=\"10.1021/ma970503\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNogi M, Iwamoto S, Nakagaito AN, Yano H (2009) Optically transparent nanofiber paper. Adv Mater 21:1595\u0026ndash;1598. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.200803174\u003c/span\u003e\u003cspan address=\"10.1002/adma.200803174\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovoselov KS, Geim AK, Morozov SV et al (2004) Electric Field Effect in Atomically Thin Carbon Films. Science 306:666\u0026ndash;669. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1102896\u003c/span\u003e\u003cspan address=\"10.1126/science.1102896\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerr\u0026eacute; P, Turner I (2001) Determination of the material property variations across the growth ring of softwood for use in a heterogeneous drying model Part 1. Capillary pressure,tracheid model and absolute permeability. Holzforschung 55:318\u0026ndash;323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1515/HF.2001.052\u003c/span\u003e\u003cspan address=\"10.1515/HF.2001.052\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuel K, Barnoud F, Goring DAI (1978) Lamellation in the S2 layer of softwood tracheids as demonstrated by scanning transmission electron microscopy. Wood Sci Technol 12:287\u0026ndash;291. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00351930\u003c/span\u003e\u003cspan address=\"10.1007/BF00351930\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuel K, Chevalier-Billosta V, Guillemin F, Berrio-sierra J, Joseleau JP (2006) The wood cell wall at the ultrastructural scale\u0026mdash;Formation and topochemical organization. Maderas Cienc Tecnol 8:107\u0026ndash;116. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4067/S0718-221X2006000200004\u003c/span\u003e\u003cspan address=\"10.4067/S0718-221X2006000200004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-San-Miguel D, Montoro C, Zamora F (2020) Covalent organic framework nanosheets: preparation, properties and applications. Chem Soc Rev 49:2291\u0026ndash;2302. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C9CS00890J\u003c/span\u003e\u003cspan address=\"10.1039/C9CS00890J\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSasaki T, Watanabe M, Hashizume H et al (1996) Macromolecule-like Aspects for a Colloidal Suspension of an Exfoliated Titanate. Pairwise Association of Nanosheets and Dynamic Reassembling Process Initiated from It. J Am Chem Soc 118:8329\u0026ndash;8335\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSegal L, Creely JJ, Martin AE, Conrad CM (1959) An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer. Text Res J 29:786\u0026ndash;794. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/004051755902901003\u003c/span\u003e\u003cspan address=\"10.1177/004051755902901003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSehaqui H, Ezekiel Mushi N, Morimune S et al (2012) Cellulose nanofiber orientation in nanopaper and nanocomposites by cold drawing. ACS Appl Mater Interfaces 4:1043\u0026ndash;1049. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/am2016766\u003c/span\u003e\u003cspan address=\"10.1021/am2016766\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimizu S, Yokoyama T, Akiyama T, Matsumoto Y (2012) Reactivity of lignin with different composition of aromatic syringyl/guaiacyl structures and erythro/threo side chain structures in β-O-4 type during alkaline delignification: As a basis for the different degradability of hardwood and softwood lignin. J Agric Food Chem 60:6471\u0026ndash;6476. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf301329v\u003c/span\u003e\u003cspan address=\"10.1021/jf301329v\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva-Mendoza J, Cant\u0026uacute;-C\u0026aacute;rdenas ME, Vazquez-Rodriguez S et al (2019) Biopolymeric films obtained from the parenchyma cells of Agave salmiana leaves. Cellulose 26:1869\u0026ndash;1879. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10570-018-2173-\u003c/span\u003e\u003cspan address=\"10.1007/s10570-018-2173-\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStelte W, Sanadi AR (2009) Preparation and characterization of cellulose nanofibers from two commercial hardwood and softwood pulps. Ind Eng Chem Res 48:11211\u0026ndash;11219. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ie9011672\u003c/span\u003e\u003cspan address=\"10.1021/ie9011672\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakimoto D, Toma S, Suda Y et al (2023) Platinum nanosheets synthesized via topotactic reduction of single-layer platinum oxide nanosheets for electrocatalysis. Nat Commun 14:19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-022-35616-4\u003c/span\u003e\u003cspan address=\"10.1038/s41467-022-35616-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanahashi M (1990) \u0026lt;review article=\"\"\u0026gt;Characterization and Degradation Mechanisms of Wood Components by Steam Explosion and Utilization of Exploded Wood\u0026lt;/review\u0026gt;. Wood research: Bull Wood Res Inst Kyoto Univ 77:49\u0026ndash;117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsuboi K, Yokota S, Kondo T (2014) Difference between bamboo- and wood-derived cellulose nanofibers prepared by the aqueous counter collision method. Nord Pulp Pap Res J 29:69\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3183/npprj-2014-29-01-p069-076\u003c/span\u003e\u003cspan address=\"10.3183/npprj-2014-29-01-p069-076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUetani K, Yano H (2011) Nanofibrillation of wood pulp using a high-speed blender. Biomacromolecules 12:348\u0026ndash;353. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/bm101103p\u003c/span\u003e\u003cspan address=\"10.1021/bm101103p\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson BP, Yliniemi K, Gestranius M et al (2018) Structural distinction due to deposition method in ultrathin films of cellulose nanofibres. Cellulose 25:1715\u0026ndash;1724. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10570-018-1665-y\u003c/span\u003e\u003cspan address=\"10.1007/s10570-018-1665-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYou J, Li M, Ding B et al (2017) Crab Chitin-Based 2D Soft Nanomaterials for Fully Biobased Electric Devices. Adv Mater 29:1606895. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.201606895\u003c/span\u003e\u003cspan address=\"10.1002/adma.201606895\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhai L, Kim HC, Kim JW, Kim J (2020) Alignment effect on the piezoelectric properties of ultrathin cellulose nanofiber films. ACS Appl Bio Mater 3:4329\u0026ndash;4334. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsabm.0c00364\u003c/span\u003e\u003cspan address=\"10.1021/acsabm.0c00364\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Kuga S, Wu M, Huang Y (2019) Cellulose nanosheets formed by mild additive-free ball milling. Cellulose 26:3143\u0026ndash;3153. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10570-019-02282-7\u003c/span\u003e\u003cspan address=\"10.1007/s10570-019-02282-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao M, Kuga S, Jiang S, Wu M, Huang Y (2016) Cellulose nanosheets induced by mechanical impacts under hydrophobic environment. Cellulose 23:2809\u0026ndash;2818. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10570-016-1033-8\u003c/span\u003e\u003cspan address=\"10.1007/s10570-016-1033-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Kyoto University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Native cellulose nanosheet, one-step reaction, cell wall, Japanese cedar, lamella, delignification","lastPublishedDoi":"10.21203/rs.3.rs-9423095/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9423095/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSince the discovery of graphene, two-dimensional nanosheets have attracted attention for their unique shapes and properties. The simplest production method is the top-down approach, in which layered materials are mechanically cleaved or exfoliated. However, preparing biomaterial nanosheets by the top-down method is challenging, and to date, only a few reports have been published. In this study, we developed native cellulose nanosheets with a thickness of several tens of nanometers, which maintained the native uniaxial orientation of cellulose fibers from wood cell walls through a combination of mild delignification and mild homogenization. The wood cell wall is a complex structure composed of three main polymers, cellulose, hemicellulose, and lignin. The elementary unit of wood cellulose is a fiber 3\u0026ndash;4 nm in width, which exists as bundles within the cell wall. Although cellulose has been disintegrated into nanofibers in various ways, cellulose nanosheets have not previously been isolated from wood cell walls. Nanocellulose is a developing material for various applications, taking advantage of its lightweight nature, high strength, excellent thermal dimensional stability, and superior gas barrier properties. A wide variety of performances can be achieved by controlling its morphology. The resulting nanosheets preserved the native orientation of the cellulose microfibrils in the cell walls, providing evidence in support of the presence of a lamellar cellulose structure within the cell wall.\u003c/p\u003e","manuscriptTitle":"Native cellulose nanosheets from Japanese Cedar via mild delignification and mild homogenization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 08:59:04","doi":"10.21203/rs.3.rs-9423095/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"690d23fd-331f-4ec7-b893-c827dfbd7eee","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":66586861,"name":"Biomaterials"}],"tags":[],"updatedAt":"2026-04-21T08:59:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 08:59:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9423095","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9423095","identity":"rs-9423095","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}