Review: Catalytic oxidation of cellulose with nitroxyl radicals under aqueous conditions

2,2,6,6-Tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation is a unique reaction to native and regenerated celluloses, and has advantages in terms of position-selective reaction at room temperature under aqueous conditions. When the TEMPO/NaBr/NaClO oxidation is applied to native celluloses in water at pH 10 under suitable conditions, the C6-primary hydroxy groups present on crystalline cellulose microfibril surfaces are mostly converted to sodium C6-carboxylate groups. Anionic sodium glucuronosyl units are densely, regularly, and position-selectively formed on crystalline cellulose microfibril surfaces, while maintaining the original cellulose morphology, cellulose I crystal structure, crystallinity, and crystal width. When TEMPO-oxidized celluloses (TOCs) prepared from, for example, wood cellulose have sodium


Introduction
Cellulose is a linear homopolysaccharide consisting of ␤-(1→4)linked glucosyl units, each of which has one primary hydroxy group (C6-OH) and two secondary hydroxy groups (C2-OH and C3-OH). These hydroxy groups promote a hydrophilic nature of cellulosic materials. In addition, abundant C H groups to the axial direction of glucosyl units in cellulose play a significant role in hydrophobic interactions between cellulose molecules and also between cellulose and other hydrophobic compounds in water ( Fig. 1) [1,2]. Native celluloses form tightly packed crystal structures of cellulose I with various crystallinities and crystal sizes, in which numerous intra-and inter-molecular hydrogen bonds and hydrophobic interactions are regularly present [3][4][5]. The native cellulose I is further classified to two crystal structures of celluloses I ␣ and I ␤ , and most of terrestrial plant celluloses have cellulose I ␤ [5,6].
Degrees of polymerization (DPs), or corresponding molar masses, of native celluloses vary from DP ≈800-≈5000, depending on cellulose resources used and isolation/purification conditions. Purity or ␣-cellulose content of native celluloses is also a significant factor, because non-cellulosic polysaccharides or hemicelluloses present in cellulosic materials can influence chemical reactions and properties of end products. Although the definition of the chemical structure of cellulose is simple, as described above, the properties of commercially available and laboratory-prepared cellulose samples vary in terms of the crystal structure, crystallinity, crystal size, DP/DP distribution, purity, depending on the original plant resource, which should all be taken into account.
Cotton lint and cotton linters celluloses, ramie and sisal fibers, and bleached wood chemical pulps are commercially available as cellulose samples with the native cellulose I structure and DPs >800 [7]. Microcrystalline cellulose powders are industrially produced from the above wood celluloses or cotton linters by dilute acid hydrolysis, and have relatively high cellulose purities and high cellulose I crystallinities, but low DPs of 200-300 [7]. Regenerated cellulose fibers such as viscose rayon, lyocell, and cuprammonium rayon have cellulose II crystal structures with various crystallinities and DPs of 350-800 [7][8][9]. These regenerated celluloses are prepared from cellulose/NaOH/CS 2 /water, cellulose/N-methylmorpholine-N-oxide/water, and cellulose/Cu(NH 3 ) 4 (OH) 2 /water solutions, respectively, by wet or wet/dry spinning [8,9]. Bacterial cellulose prepared by incubating Acetobacter xylinum, and algal and tunicate celluloses can be used in laboratory experiments after isolation and purification, and each of them has a unique crystal structure, crystallinity, crystal size of cellulose I, and high DP > 4000 [10].
Chemical reactions of cellulose have been intensively and extensively studied to add new properties or functions to native celluloses, and some of them have already been used at the industrial level to produce cellulose derivatives, and their films and fibers [4,5,[11][12][13][14]. Cellulose triacetate, cellulose diacetate, and cellulose nitrate are typical cellulose esters used as functional films or cellulose-based manmade fibers. Carboxymethylcellulose, methylcellulose, and hydroxyethyl-and hydroxypropyl-celluloses are water-soluble cellulose ethers used as stable thickeners.
The typical cellulose reactions such as esterification and etherification are condensations mostly in the presence of organic solvents, which generally require high temperatures for long reaction times. The presence of water in the starting cellulosic materials or in reaction media causes, therefore, preferential side reactions formed between water and an etherifying or esterifying reagent rather than reactions between cellulose hydroxy groups and reagents [4,5]. The reaction efficiencies between cellulose and these reagents are generally low, and simultaneously large amounts of reaction effluents containing byproducts, and other residual chemicals and solvents that require treatment are formed. These cellulose reactions are not environmentally friendly under their present conditions. Although a huge number of publications detailing chemical reactions of cellulose or preparation methods of cellulose derivatives have been reported, only limited cellulose derivatives are industrially produced at present. Some breakthrough processes, such as catalytic reactions at ambient temperatures under aqueous conditions like enzymatic reactions in living bodies, should be developed for further utilizations of chemically modified celluloses. The N-oxyl radical-mediated oxidation using, for example, 2,2,6,6tetramethylpiperidine-1-oxyl (TEMPO) is one of the promising candidates in new cellulose reactions fitting to the above purpose.
Cellulose is advantageous as the most abundantly present and renewable biomass on the earth. Plants adsorb CO 2 and simultaneously emit O 2 (or proceed the reduction of CO 2 , producing carbon-containing plant components and O 2 ) by photosynthesis during growing stage. The effective and continuous cycling of plantation, logging, utilization of plant biomass as materials, and their recycling can, therefore, reduce CO 2 in the atmosphere and subsequently retard global warming. The enhancement of qualitative and quantitative utilization of plant cellulose is the key issue for establishment of a sustainable society, partly in place of the present fossil resource-based society. Hydrophilic and hydrophobic planes of cellulose molecules, marked with the primary C6-OH (with tg-conformation corresponding to cellulose I crystal structure), and secondary C2-OH and C3-OH groups in glucosyl unit.

Chemistry of TEMPO-mediated oxidation
de Nooy et al. first reported that catalytic reactions using TEMPO radical can position-selectively oxidize C6-primary hydroxy groups of water-soluble starch, pullulan, and dextran to sodium C6carboxylate groups in water at pH 10 and ambient temperatures in the presence of NaBr and NaClO, which are used as a co-catalyst and a primary oxidant, respectively [32]. TEMPO is a water-soluble, stable, and commercially available N-oxyl radical with the Ames Test negative data (Ames Test: a fast and sensitive assay of the ability of a chemical compound or mixture to induce mutations in DNA) [33]. The TEMPO-mediated oxidation has opened a new research field of polysaccharide chemistry, because this reaction is different from conventional esterification, etherification, and oxidation of cellulose in terms of reaction efficiency and position selectivity under aqueous and moderate reaction conditions.
The TEMPO/NaBr/NaClO oxidation of saccharides and other compounds has been extensively studied for selective conversion of primary hydroxy groups to carboxy groups [34][35][36]. In this system, NaClO used as a primary oxidant first oxidizes TEMPO radical to the N-oxoammonium-type structure or TEMPO + , which then oxidize primary hydroxy groups to carboxy groups through C6-aldehydes. The reduced TEMPO or N-hydroxy-TEMPO is then oxidized by NaBrO, which is formed from NaBr by oxidation with NaClO. Thus, TEMPO and NaBr behave as catalysts, and only inexpensive NaClO is consumed during the oxidation. Because acidic carboxy groups are formed by the oxidation, a small amount of 0.5 M NaOH solution is consumed to maintain the aqueous reaction medium at pH 10 ( Fig. 3). A part of aldehydes are directly oxidized to carboxy groups with NaClO and/or NaBrO [32,37].
More detailed reaction mechanism for the selective oxidation of primary hydroxy groups is illustrated in Fig. 4 [38]. The cationic TEMPO + ion reacts with dissociated primary hydroxy groups to form a covalent bond as an intermediate structure. Because of steric hindrance, TEMPO + ions form the covalent bond with the dissociated primary hydroxy groups much more preferentially than secondary hydroxy groups. Moreover, the dissociation of primary hydroxy groups is enhanced under alkaline conditions so that the formation of covalent bonds proceeds more rapidly under alkaline conditions than neutral or acidic conditions. The cleavage of the covalent bonds forms aldehyde groups and N-hydroxy-TEMPO molecules. The hydration of aldehydes and successive dissociation of the hydroxy groups under alkaline conditions causes the next covalent bond formation with TEMPO + , resulting in the formation of carboxy groups and N-hydroxy-TEMPO molecules. The optimum pH in water at ≈10 to form carboxy groups from primary hydroxy groups in the TEMPO/NaBr/NaClO oxidation is probably caused by a balance between the following two factors. The degrees of dissociation of primary hydroxy groups are enhanced under alkaline conditions, whereas the frequency to form the covalent bonds between dissociated hydroxy groups and TEMPO + rather than those between OH − ions and TEMPO + molecules is preferential under weakly alkaline conditions.

Structures and properties of TEMPO-oxidized celluloses
When the TEMPO/NaBr/NaClO oxidation is applied to cellulose suspended in water at pH 10, it is possible to selectively convert C6-OH groups to sodium C6-carboxylate groups through C6-aldehydes (Fig. 3). If almost all C6-OH groups of water-insoluble cellulose are oxidized to sodium C6-carboxylate groups by oxidation, new water-soluble ␤-(1→4)-linked sodium polyglucuronates (or cellouronic acid Na salt) with homogeneous chemical structures should be obtained.
However, chemical structures and properties of TEMPOoxidized products vary, depending on cellulose samples used as starting materials and the oxidation conditions. When regenerated celluloses, mercerized celluloses (prepared from native celluloses by soaking in NaOH solutions at >15% concentrations), and ballmilled/disordered cellulose powders are used as starting materials under suitable conditions, almost all C6-OH groups are oxidized to sodium C6-carboxylate groups to form water-soluble cellouronic acids Na salt (Fig. 5) [39][40][41]. However, the presence of small and unknown signals in 13 C NMR spectrum of sodium cellouronate indicates some side reactions other than the oxidation of C6-OH groups.
Moreover, significant depolymerization is inevitable during the oxidation. The obtained cellouronic acids have DPs of ≈60, although the original celluloses used as starting materials have DPs >350 [41]. ␤-Alkoxy elimination occurring at C6-aldehyde groups formed as intermediate structures in water under alkaline conditions and/or some radical species formed in situ as byproducts during oxidation probably cause depolymerization [39,[41][42][43][44].
Water-soluble sodium polyuronates with homogeneous chemical structures consisting of the same uronosyl repeating units are prepared from water-insoluble polysaccharides such as curdlan, paramylon, ␣-chitin, and starch, in a similar manner to that for sodium cellouronates [45][46][47][48][49][50][51]. Amine groups are unstable to the TEMPO-mediated oxidation; therefore, chitosan and proteins are degraded to water-soluble and low-molar-mass compounds, and position-selective oxidation of primary hydroxy groups in these polymers cannot be achieved [39]. When TEMPO-mediated oxidation is applied to native celluloses, consisting of crystalline cellulose microfibrils as the smallest elements next to cellulose chains, dif- Fig. 4. TEMPO-mediated oxidation mechanism of primary hydroxyls to carboxyl groups via aldehydes [36,37]. The covalent bond formation between a dissociated primary hydroxyl group and cationized TEMPO + is the initial key reaction.

Fig. 5.
Preparation of sodium ␤-(1→4)-poly(glucoronate) (or cellouronic acid Na salt) from regenerated cellulose by TEMPO-mediated oxidation, and 13 C NMR spectra of cellulose with DP 7 in DMSO-d6 and cellouronic acid Na salt in D2O [38,39]. The cellulose C6-OH signal at ≈62 ppm completely disappears, and the C6-carboxylate signal appears for the oxidized product. ferent oxidized products are obtained. Details are discussed in the following chapter.

Structures and properties of TEMPO-oxidized native celluloses
When a commercial softwood bleached kraft pulp (papermaking grade), containing ≈90% cellulose and ≈10% hemicelluloses, is oxidized by the TEMPO/NaBr/NaClO system in water at pH 10, TEMPO-oxidized celluloses (TOCs) are obtained as water-insoluble products in the yields of ≈90%. The fibrous morphology, crystal structure of cellulose I, crystallinity, and crystal size of the original wood cellulose are maintained during the oxidation. Neutral sugar composition analysis have shown that mannose and xylose originating from hemicelluloses present in the wood cellulose are mostly removed from TOCs during the oxidation and washing processes. Carboxylate content of TOCs increases from 0.01 mmol/g up to ≈1.7 mmol/g with increasing the amount of NaClO added as the primary oxidant; a significant number of sodium C6-carboxylate groups are formed in TOCs ( Fig. 6) [52][53][54][55][56]. Small amounts of aldehyde groups are present as intermediate structures in TOCs (Fig. 3).
When TOCs are dissolved in 0.5 M copper ethylenediamine [Cu(EDA) 2 (OH) 2 ] (EDA: ethylenediamine) solution to determine their viscosity average DPs, TOCs have DPs lower than 300, which corresponds to the levelling-off DPs observed for plant celluloses by dilute acid hydrolysis [57][58][59]. However, after the TOCs are postoxidized with NaClO 2 in water at pH 4-5, the TOCs have higher DPs >500. This is because small amounts of C6-aldehydes are formed and remain in the disordered regions periodically present along the cellulose microfibril direction in TOCs. These C6-aldehydes cause depolymerization of TOCs through ␤-alkoxy elimination in alkaline copper ethylenediamine solution during the DP measurement [60]. However, as shown in Fig. 6, even after post oxidation with NaClO 2 , significant depolymerization is unavoidable during the TEMPO/NaBr/NaClO oxidation in water at pH 10 [60].
When TOCs are prepared from softwood and hardwood bleached kraft pulps, TOCs have the maximum sodium carboxylate contents of ≈1.7 mmol/g (Fig. 6). The maximum sodium carboxylate contents of TOCs depend on the original native celluloses used [38,61]. Fig. 7 shows the relationship between the maximum sodium C6-carboxylate content in TOCs and crystal width of cellulose I for various native celluloses used as starting materials [61]. Moreover, glucose/glucuronic acid alternating co-polysaccharides can be prepared from TOCs by surface peeling with aqueous NaOH (Fig. 8) [62].
These results show that almost all C6-OH groups present on crystalline cellulose microfibril surfaces in native celluloses are converted to sodium C6-carboxylate groups by TEMPO/NaBr/NaClO oxidation under suitable conditions. The high crystallinities and original crystalline microfibril structures of native celluloses cause a highly position-selective oxidation of C6-OH groups to sodium C6-carboxylate groups without oxidation of cellulose molecules inside the crystalline cellulose microfibrils ( Fig. 9) [38,[61][62][63]. Consequently, sodium C6-carboxylate groups are densely, regularly, and position-selectively formed on native cellulose microfibril surfaces by TEMPO/NaBr/NaClO oxidation, and abundantly present in TOCs. Other hypotheses concerning TEMPO-oxidized cellulose microfibril structures have been also proposed based on smallangle X-ray diffraction data [64][65][66].

Kinetics of TEMPO-mediated oxidation of native cellulose
The conversion mechanism of the C6-OH groups exposed on crystalline cellulose microfibril surfaces of native celluloses to sodium C6-carboxylate groups by TEMPO/NaBr/NaClO oxidation in water at pH 10 are shown in Figs. 3 and 4, and the corresponding equation is shown in Fig. 10 [48]. The kinetics of oxidation of regenerated cellulose and lignin-containing mechanical pulp have been studied from carboxylate contents of the oxidized celluloses [75,76]. In the case of TEMPO/NaBr/NaClO oxidation of native celluloses, the kinetics can be studied indirectly by monitoring changes in NaClO concentration in the reaction media [70,77] or those of 0.5 M NaOH consumption to oxidation time [78]. The NaClO concentration in the reaction media can be determined by iodometric titration/UV-vis spectroscopy [77].
The effect of NaBr addition in TEMPO/NaClO oxidation of wood cellulose (1 g) in water at pH 10 on the formation rates of sodium  13 C NMR spectrum of the obtained product (measured in D2O) is also shown (B) [62].  . Seven reaction formulae participating in catalytic oxidation of primary hydroxyl groups of polysaccharides to sodium C6-carboxylate groups by TEMPO/NaBr/NaClO system in water at pH 10 [48]. TEMPO and NaBr behave as catalysts; these two compounds are therefore not included in the equation below dotted line. Copyright 2003. Reproduced with permission from Elsevier Ltd. Fig. 11. Relationships between the oxidation time and the amount of 0.5 M NaOH consumed in TEMPO/NaBr/NaClO and TEMPO/NaClO oxidations of wood cellulose (1 g) in water at pH 10 [78]. The presence of NaBr as a co-catalyst remarkably improves the oxidation rate.
C6-carboxylate groups in oxidized celluloses was investigated by monitoring the amount of 0.5 M NaOH consumed (Fig. 11) [78]. According to the equation in Fig. 10, one mole of NaOH is consumed to form one mole of C6-carboxylate groups from one mole of C6 − OH groups. NaBrO is formed from NaClO and NaBr, and has much higher efficiency to oxidize TEMPO and N-hydroxy-TEMPO to TEMPO + than NaClO [32,35,39,79].
As shown in Fig. 11, the TEMPO/NaBr/NaClO-oxidized cellulose had a carboxylate content of 1.44 mmol/g after the 0.5 M NaOH consumption of 3.05 mL, which corresponds to 1.52 mmol NaOH. Therefore, ≈95% ( = 1.44/1.52 × 100) of the added NaOH was consumed for the formation of sodium C6-carboxylate groups; the patterns of 0.5 M NaOH consumption can be used for calculation of oxidation kinetics. In the case of TEMPO/NaClO system, 1.09 mmol NaOH (or 2.17 mL of 0.5 M NaOH) was consumed to form sodium C6-carboxylate groups of 0.67 mmol/g; ≈38% ( = 100 − 100 × 0.67/1.09) of the added NaOH was consumed in side reactions.

TEMPO-oxidized cellulose nanofibrils (TOCNs)
When TOCs prepared from wood celluloses, cotton, cotton linters, algal, and tunicate celluloses have sufficient amounts of sodium C6-carboxylate groups, highly viscous transparent gels are obtained by gentle mechanical disintegration of TOC/water slurries [38,60,[80][81][82][83][84]. An aqueous slurry of 0.1% (w/v) oxidized cellulose with a sodium carboxylate content of 1.5 mmol/g, prepared from hardwood cellulose by the TEMPO/NaBr/NaClO system in water at pH 10, has been stirred with a magnetic stirring bar at 500 rpm and room temperature (Fig. 12A). The originally fibrous wood TOC subsides at the bottom in the mixture before stirring. The TOC gradually swells in water by stirring, and a transparent dispersion with a 97% cellulose content in the supernatant fraction is obtained after stirring for 10 days [81]. The weight ratio of cellulose present in the supernatant fraction in Fig. 12A has been determined by centrifugation at 12 000g for 30 min to remove fibers or agglomerates present in the mixture.
The sodium carboxylate content of TOCs influences the transparency of the dispersions after mechanical disintegration in water. Fig. 12B shows photographs of a 0.1% (w/v) slurry of the original hardwood cellulose and 0.1% dispersions of hardwood TOCs with various carboxylate contents, after magnetic stirring in water for 10 days. The TOC with a carboxylate content of 1.0 mmol/g is homogeneously dispersed in water, but its dispersion is not transparent. On the other hand, a highly transparent gel can be obtained from the TOC with a carboxylate content of 1.5 mmol/g by disintegration in water under the same conditions.
When an ultrasonic homogenizer is used at the laboratory scale (in place of magnetic stirring in Fig. 12) for mechanical disintegration of the TOC with a carboxylate content of 1.5 mmol/g, transparent highly viscous gels are obtained within 8 min [60]. Disintegration of the TOC/water slurry using a laboratory-scale high-pressure homogenizer (such as Star Burst Labo HJP-25005, SUGINO, Japan) efficiently gives transparent gels within 4 cycles.
Transmission electron microscopy (TEM) images of the transparent wood TOC dispersions shown in Fig. 12, after dilution with water and air-drying on TEM grids, show that the dispersions consist of mostly individual cellulose fibrils with uniform lateral size of ≈3 nm and lengths >1 m (the left low TEM image in Fig. 13) [81]. Vascular or terrestrial plant celluloses have crystalline microfibrils approximately 3 × 3 nm 2 cross-section in cell walls during biosynthesis [83,84]. Each cellulose microfibril consists of 30-40 fully extended cellulose chains. The TEM observation shows that the fibrous wood TOCs with 20-40 m widths and 1-3 mm lengths are converted to individualized TOCNs with ≈3-nm widths and lengths >500 nm by gentle mechanical disintegration in water, when the sodium carboxylate contents of wood TOCs are ≈1 mmol/g or higher (Fig. 13).
As shown in Fig. 9, the sodium C6-carboxylate groups are position-selectively, densely, and regularly formed on crystalline cellulose microfibril surfaces in wood cellulose by TEMPO/NaBr/NaClO oxidation in water at pH 10. These surface anionic groups on cellulose microfibrils cause the complete individualization of cellulose microfibrils formed through osmotic effects and electrostatic repulsions efficiently working among surface anionic TOCN elements in water. High degrees of nanofibrillation for aqueous TOCN dispersions are confirmed from their high light transmittances without any light scattering (Fig. 14A) and birefringence behaviour by observation between cross-polarizers ( Fig. 14B) [38,80,81,85].
Various plant holocelluloses are prepared from Japanese cedar (softwood), eucalyptus and birch (hardwood), ginkgo (gymnosperm), and rice straw and kenaf (grass) by repeated delignification with NaClO 2 in water at pH 4-5 and ≈70 • C. These holocelluloses are oxidized to TOCs with carboxylate contents of 1.3-1.7 mmol/g by TEMPO/NaBr/NaClO oxidation in water at pH 10. These TOCs are then converted to TOCNs with homogeneous ≈3nm widths and high aspect (length/width) ratios >150 by gentle mechanical disintegration in water ( Fig. 15 and Table 1) [86].
TOCs prepared from softwood bleached kraft pulp (papermaking grade) gives the highest efficiency to prepare TOCNs, when TOCs are prepared by the TEMPO/NaBr/NaClO oxidation and subsequent mechanical disintegration in water under the same conditions. Aqueous 0.1% (w/v) TOCN dispersions prepared from softwood bleached kraft pulp have the highest transmittance (>90%) to visible light with wavelengths ranging 400-800 nm and the highest nanofibrillation yield (>90%), which is determined from dry weight of unfibrillated or partly fibrillated fraction present in the TOCN/water dispersion separated by centrifugation. Glucomannan molecules, i.e., predominant hemicelluloses in softwood cellulose, have C6 − OH groups; therefore, 10-15% hemicelluloses in softwood cellulose are preferentially degraded to water-soluble and low-molar-mass compounds during TEMPO-mediated oxidation, resulting in the formation of almost pure water-insoluble TOCs, consisting of only glucosyl and glucuronosyl units. Because hardwood celluloses and hardwood holocelluloses contain hydrophobic xylan as a predominant hemicellulose, a part of xylan remains in TOCs, resulting lower light transmittances of TOCN/water dispersions and lower nanofibrillation yields [88,100].

TEMPO-oxidized cellulose nanocrystals (TO-CNCs)
When wood TOCs with carboxylate contents of 1.2-1.7 mmol/g are sonicated in water for an extended time such as totally 60-120 min with sufficient intervals to maintain the dispersion temperatures at 20-25 • C, needle-like TO-CNCs are obtained in the yields of ≈94% based on the starting wood cellulose weight (Fig. 16). These TO-CNCs have average lengths of 150-200 nm, homogeneous widths of ≈3 nm, and the same sodium carboxylate Fig. 13. Preparation of TEMPO-oxidized cellulose nanofibrils (TOCNs) dispersed in water from fibrous TEMPO-oxidized wood celluloses by mechanical disintegration [38,81].

Table 1
Average widths (nm) of TEMPO-oxidized cellulose nanofibrils prepared from various plant holocelluloses, determined from the AFM height images in Fig. 15  contents of 1.2-1.7 mmol/g on the surfaces as those of the original TOCs [101]. Conventional CNCs are prepared from wood, cotton, tunicate, and algal celluloses with ≈64% H 2 SO 4 at 70 • C, followed by washing with water using centrifugation and dialysis [102]. In the case of conventional wood CNCs with sulfate ester contents of ≈0.3 mmol/g, the yields decrease to ≈40% by acid hydrolysis. These CNCs have spindle-like morphologies with widths of 10-20 nm [103]. Wood TO-CNCs are different from the conventional wood CNCs in terms of yields, sizes, surface charges, and morphologies. Both TOCNs and TO-CNCs are, therefore, prepared from TOCs by controlling sonication time in water.

Characterization of TEMPO-oxidized celluloses (TOCs) and TEMPO-oxidized cellulose nanofibrils (TOCNs)
Structural characterizations of TOCs and TOCNs are significant for their application as new plant-based renewable nanofibers to high-tech material fields. Establishment of suitable analytical protocols are, therefore, required both in academia and industry. Carboxy and aldehyde contents of TOCs and TOCNs are determined by conductivity titration before and after post-oxidation with NaClO 2 [53,54,60,104].
The C6-carboxy groups in TOCs and TOCNs can be positionselectively and completely converted to methyl ester groups with trimethylsilyldiazomethane. These carboxy groups-methylated TOCs and TOCNs as well as the original celluloses and holocelluloses are completely soluble in 8% (w/w) lithium chloride/N,Ndimethylacetamide (LiCl/DMAc) [105][106][107]. The DPs and DP distributions of TOCs and TOCNs can be then determined using a size-exclusion chromatography system attached with a multiangle laser-light scattering (SEC-MALLS) detector. The carboxy groups-methylated TOC and TOCN solutions as well as cellulose and hemicellulose-containing holocellulose solutions are diluted to 1% (w/v) LiCl/DMAc and subjected to the SEC-MALLS analysis. The original softwood cellulose used as the starting material for preparation of TOCs has one major peak and a small shoulder peak in its molar-mass distribution, corresponding to cellulose-rich and hemicellulose-rich fractions, respectively. The peak top of TOCs shifts to the low-molar-mass direction, as the amount of NaClO added is increased (Fig. 17A) [108]. The shoulder peak originating from hemicelluloses becomes small and disappears as the amount of NaClO added is increased; hemicelluloses are preferentially degraded to water-soluble compounds during TEMPO/NaBr/NaClO oxidation and removed from the water-insoluble TOCs. This is because plant hemicelluloses such as glucomannan and xylan have low DPs and disordered structures, different from cellulose, and are susceptible to degradation during oxidation.
When the fibrous TOC with a carboxylate content of 1.7 mmol/g (prepared from softwood cellulose) is sonicated in water, the average molar mass decreased with increasing sonication time in water (Fig. 17B) [108]. Significant depolymerization is, therefore, inevitable during the TEMPO/NaBr/NaClO oxidation and subsequent sonication in water.
Small amounts of C6-aldehyde groups and C2/C3 ketones are formed as side reactions in TOCs during TEMPO/NaBr/NaClO oxidation, and are present in TOCs and TOCNs. After post-reduction of TOCs with NaBH 4 in water at pH 8-10, intrinsic viscosities [Á] of TOCs obtained using 0.5 M copper ethylenediamine solution as the solvent are convertible to viscosity-average molar-masses (M v ) according to the Mark-Houwink-Sakurada equation of [Á] (mL/g) = 0.094 × M v 0.67 , which is obtained from SEC-MALLS data for carboxy groups-methylated TOCs and TOCNs [108].
Crystallinities and crystal sizes of TOCs and TOCNs are determined from their X-ray diffraction patterns and solid-state 13 C NMR spectra. TEM and AFM are used to determine average widths of TOCNs. The length and length distribution of TOCNs are measured from TEM images, although only a limited number of TOCN elements (<300) is measurable in this case. Dilute TOCN/water dispersions consist of completely individualized TOCN elements without agglomeration. Therefore, rheological properties of TOCN/water dispersions have been studied to characterize TOCNs [8,[109][110][111]. Average lengths of TOCNs prepared from wood, cotton linters, and algal celluloses under various conditions can be determined from shear viscosity behaviour of TOCN/water dispersions [110,111]. When intrinsic viscosities [Á] are obtained for various TOCNs and acid-hydrolyzed TOCNs, their average lengths are calculated using the following empirical formula, 1.6 × [Á] = 0.15 × p 1.9 , where p is the aspect ratio of TOCNs and acid-  hydrolyzed TOCNs (Fig. 18) [111]. The p values are obtained as average lengths of hundreds of millions of TOCN elements present in dispersions; therefore, the length information obtained by the viscosity method is more reliable. However, no suitable method has been developed to date for determination of length distributions. The viscosity method cannot be applied to nanocelluloses with heterogeneous 3-100 nm widths that form network structures without dispersion at the individual nano-element level in water.
Average tensile strengths of TOCNs have been determined for aqueous TOCN dispersions by sonication or cavitation treatment and subsequent AFM analysis to measure limiting fracture lengths (or levelling-off and minimum lengths after sonication) and widths of TOCNs [112]. The validity of this cavitation method has been shown for carbon nanotubes, protein fibrils, and metal nanowires [113,114]. Wood and tunicate TOCNs have average tensile strengths of 1-6 GPa, when determined by the cavitation/AFM method [112]. These values are comparable with those of multi-walled carbon nanotubes and polyamide polymers such as Kevlar ® , and much higher than that of steel although the density of TOCNs (≈1.6 g/cm 3 ) is ≈1/5 that of steel. Tensile elastic moduli of TOCNs have been measured only for those prepared from tunicate cellulose with large microfibril widths from stress-strain curves obtained using a cantilever in AFM, and are ≈140 GPa [115]. TOCNs are, therefore, new plant-based renewable nanofibers with high mechanical strengths. Agglomeration behaviour of aqueous TOCN dispersions in the presence of salts or cationic polymers, nanofibrillation of TOCs at high solid concentrations in water, swelling behaviour of TOCN network structures in the presence of salts, simulations of TOCN structures in water, rheological behaviour of aqueous TOCN dispersions, and others have been studied to characterize TOCNs in water [116][117][118][119][120][121][122].

Effect of TEMPO-analogous compounds on oxidation efficiency of native cellulose
The effect of TEMPO derivatives and analogous compounds on oxidation efficiency of C6-OH groups of wood cellulose has been studied using the NaBr/NaClO system in water at pH 10 under the same conditions as those of TEMPO/NaBr/NaClO oxidation [123]. Among six-and five-membered N-oxyl radicals tested, 4-acetamido-TEMPO (4-AcNH-TEMPO) and 4-methoxy-TEMPO give TOCs with sufficiently high sodium C6-carboxylate contents (>1.1 mmol/g) after reaction for 4 h, and afford TOCN/water dispersions with high nanofibrillation yields when disintegrated in water under the same conditions. These two TEMPO derivatives can, therefore, be used as catalysts in place of TEMPO in oxidation of native celluloses with NaBr and NaClO in water at pH 10. The relationship between the sodium carboxylate content of TOCs prepared with various N-oxyl radicals and nanofibrillation yield is shown in Fig. 19 [123]. In contrast, 4-hydroxy-TEMPO and 4-oxo-TEMPO give the lowest efficiency in oxidation; the sodium C6-carboxylate contents of TOCs prepared with these TEMPO derivatives are lower than 0.3 mmol/g even after oxidation under the same conditions. The redox potentials of the TEMPO-analogous compounds in Fig. 19 have no correlation to the corresponding nanofibrillation yields   18. Relationship between aspect ratio of various TEMPO-oxidized cellulose nanofibrils (TOCNs) and acid-hydrolyzed TOCNs measured from transmission electron microscopy (TEM) images and their intrinsic viscosities of aqueous dispersions [110]. These TOCNs and acid-hydrolyzed TOCNs are prepared from wood, cotton linters, and algal celluloses.  19. Relationship between carboxylate content and nanofibrillation yield of TEMPO-oxidized wood celluloses prepared using TEMPO and its analogs by TEMPO/NaBr/NaClO oxidation in water at pH 10 [123]. Copyright 2010. Reproduced with permission from Elsevier Ltd. of oxidized celluloses. Degradation behaviour or stability of the TEMPO-analogous compounds in water containing NaBr and NaClO primarily influences the resulting nanofibrillation yield of oxidized celluloses.
When wood cellulose is oxidized with the same amount of 2azaadamantane N-oxyl (AZADO) or 1-methyl-AZADO as that of TEMPO in NaBr/NaClO system in water at pH 10, the oxidation time required for preparing oxidized celluloses with sodium C6carboxylate contents of 1.2-1.3 mmol/g can be reduced from 80 to 10-15 min. The oxidation efficiency is, therefore, significantly improved using AZADO or 1-methyl-AZADO in place of TEMPO [124,125]. Moreover, the molar amounts of AZADO or 1-methyl-AZADO added for preparation of oxidized celluloses with sodium C6-carboxylate contents of 1.2-1.3 mmol/g can be reduced to 1/32 or 1/16 that of TEMPO, respectively, when the oxidation times are extended to 90 min as in the case of TEMPO/NaBr/NaClO oxidation. Almost no C6-aldehyde groups are formed in the oxidized celluloses.
The DP v values of the AZADO-and 1-methyl-AZADO-oxidized celluloses, irrespective of their sodium C6-carboxylate contents, decrease from 1090 to ≈200, which is determined using 0.5 M copper ethylenediamine solution as the solvent. However, when the AZADO-or 1-methyl-ASADO-oxidized celluloses are post-reduced with NaBH 4 , their DP v values increase to 600-800. These results indicate that C2/C3 ketones are formed to some extent as side reactions in the oxidized celluloses.
The original crystallinity and crystal width of wood cellulose are unchanged after oxidation. The sodium C6-carboxylate groups and C2/C3 ketones formed as primary and side reactions, respectively, are present on crystalline cellulose microfibril surfaces. Highly transparent AZADO-oxidized cellulose nanofibril/water dispersions consisting of individually dispersed nanofibrils with a nanofibrillation yield of 89% are obtained from the AZADO-oxidized cellulose with a sodium carboxylate content of 1.2 mmol/g by mechanical disintegration in water. The AZADO and 1-methyl-AZADO oxidations of cellulose microfibrils with NaBr/NaClO in water at pH 10 are schematically illustrated in Fig. 20 [125].
When a regenerated cellulose fiber (Bemliese ® ) is oxidized by AZADO/NaBr/NaClO in water at pH 10 under suitable conditions, water-soluble sodium 2,3,6-tricarboxylate cellulose (NaTCC) or sodium mesotartarate/monohydrated glyoxilate alternating copolyacetal is obtained in a yield of 82% by one-step oxidation (Fig. 21) [126]. Almost all C6-OH groups are converted to sodium C6-carboxylate groups and also almost all the C C bonds of C2/C3 are cleaved to form sodium C2-and C3-carboxylate groups by AZADO/NaBr/NaClO oxidation. The weight-and number-average molar-masses of the obtained NaTCC are 10,700 and 7000, respectively; the original DP v (680) of regenerated cellulose decreases to weight-and number-average DPs (DP w and DP n , respectively) of 52 and 34, respectively, for the NaTCC [126]. When AZADOmediated oxidation is applied to wood cellulose, water-soluble oxidized products are obtained. However, they have more heterogeneous chemical structures indicating that complete oxidation of the C2, C3, and C6 hydroxy groups to carboxy groups is difficult to achieve for wood cellulose [126].

TEMPO/NaClO/NaClO 2 oxidation of cellulose and other polysaccharides
The TEMPO/NaBr/NaClO oxidation in water at room temperature and pH 10 under suitable conditions efficiently converts native celluloses to TOCs with high sodium carboxylate contents within 90 min. However, significant depolymerization is inevitable   22. Oxidation mechanism of C6-OH groups of cellulose by TEMPO/NaClO/NaClO2 in buffer at pH 4.8 or 6.8 [38,127,128]. No C6-aldehydes are present in oxidized celluloses because oxidation of C6-aldehydes to C6-carboxys occurs with NaClO2 present as the primary oxidant. on cellulose and oxidized cellulose molecules in TOCs during the oxidation [60,108]. One of the alternative oxidation systems is TEMPO/NaClO/NaClO 2 in water at high temperatures under weakly acidic or neutral conditions for long times >1 d (Fig. 22). Here, TEMPO and NaClO behave as catalysts and NaClO 2 behaves as a primary oxidant to be consumed [127][128][129][130]. In this oxidation system, no C6-aldehyde groups are formed in TOCs, and depolymerization of oxidized celluloses, caused by ␤-alkoxy elimination preferentially occurring under alkaline conditions, is avoidable.
When wood cellulose is oxidized by TEMPO/NaClO/NaClO 2 or 4-AcNH-TEMPO/NaClO/NaClO 2 system in a buffer at pH 4.8 or 6.8 and 40 • C for 2 d under suitable conditions, the sodium C6carboxylate content in the TOCs increases up to ≈1.3 mmol/g. The DP v values of the obtained TOCs slightly decrease from 1270 for wood cellulose to ≈1100 during the oxidation (Fig. 23) [130]. Depolymerization is, therefore, mostly avoidable for TOCs prepared by TEMPO/NaClO/NaClO 2 oxidation in comparison with TEMPO/NaBr/NaClO oxidation in water at pH 10. The 4-AcNH-TEMPO is better than TEMPO in terms of high amounts of sodium C6-carboxylate groups introduced and high DP v values of the TOCs.
The original crystallinity and crystal width of cellulose I for wood cellulose are maintained during the oxidation, and transparent highly viscous gels are obtained by mechanical disintegration of the wood cellulose-derived TOCs, with sodium C6-carboxylate contents >0.8 mmol/g, in water [128,130]. Softwood cellulose gives aqueous TOCN gels with higher light transmittances than those from hardwood cellulose when mechanically disintegrated in water under the same conditions. The gels consist of mostly individualized TOCN elements with homogeneous ≈3-nm widths similar to those of TOCNs prepared by the TEMPO/NaBr/NaClO system in water at pH 10. However, the obtained TOCNs have higher average lengths >1 m (Fig. 24) than those of TOCNs prepared by TEMPO/NaBr/NaClO oxidation [128,130].
The TEMPO/NaClO/NaClO 2 oxidation of water-insoluble curdlan with suitable amounts of NaClO 2 in a buffer at pH 4.8 or 6.8 gives the corresponding water-soluble sodium polyuronates with high DPs consisting of glucuronosyl repeating units are obtained in high yields [131,132]. However, when commercial regenerated cellulose fibers, beads for chromatographic use, and mercerized celluloses prepared from native cellulose by swelling with 20% aqueous NaOH under various conditions are used as starting materials for TEMPO/NaClO/NaClO oxidation in water at pH 4.8 and 60 • C, weight recovery ratios as water-insoluble fractions and their sodium C6-carboxylate contents are in the range 67-80% and 1.8-2.2 mmol/g, respectively [133][134][135][136]. The sodium C6carboxylate content of water-soluble pure sodium cellouronates consisting of glucuronosyl repeating units is 5.05 mmol/g; therefore, the sodium carboxylate contents of water-insoluble fractions of oxidized regenerated cellulose fibers and beads, and mercerized celluloses are lower than that of sodium cellouronates. The water-insoluble fractions of the oxidized products are converted to almost transparent dispersions by mechanical disintegration in water, consisting of mostly individualized TEMPO-oxidized CNCs ( Fig. 25) [135]. These results indicate that oxidation of C6-OH groups selectively occurs on cellulose II crystallite surfaces. The TEMPO/NaClO/NaClO 2 or 4-AcNH-TEMPO/NaClO/NaClO 2 oxidation has been applied to bacterial cellulose, alkali-treated wood cellulose, and cotton fabrics, and the oxidized products have been characterized [137][138][139].

TEMPO electro-mediated oxidation of cellulose
TEMPO electro-mediated oxidation is another alternative to TEMPO/NaBr/NaClO oxidation for cellulose in water under neutral conditions, in which no chlorine-containing reagents are needed for oxidation (Fig. 26) [140][141][142][143][144][145][146][147]. When the electro-mediated oxidation is applied to wood cellulose with TEMPO or 4-AcNH-TEMPO in a buffer at pH 6.8 and room temperatures for 2 d, the solid recovery ratio of the water-insoluble oxidized cellulose is more than 90%, and the sodium C6-carboxylate content increases to ≈0.9 mmol/g. A significant amount of C6-aldehyde groups (≈0.4 mmol/g) is formed simultaneously in the oxidized cellulose. The DP v value of wood cellulose decreases from 1270 to 810 during the oxidation (Fig. 27) [147]. The original cellulose I crystallinity and crystal width of wood cellulose are maintained after the oxidation, showing that all the sodium C6-carboxylate and C6-aldehyde groups are positionselectively formed on crystalline cellulose microfibril surfaces. The oxidized celluloses with carboxylate contents >0.9 mmol/g are convertible to TOCNs dispersed at the individual nanofibril level by disintegration in water, and nanofibrillation yields are more than 80%.
Sodium C6-carboxylate groups are predominantly formed in the oxidized celluloses prepared by the TEMPO electro-mediated oxidation without using NaClO. This result shows that the oxidized TEMPO or TEMPO + molecules can oxidize not only C6-OH groups to C6-aldehydes but also C6-aldehyde groups, formed as interme-   diates, to C6-carboxy groups during TEMPO/NaBr/NaClO oxidation. Not only NaClO and/or NaBrO but also TEMPO + molecules can, therefore, oxidize C6-aldehyde groups to C6-carboxylate groups, as shown in Fig. 3 in the 2nd chapter.
When water-insoluble curdlan, amylodextrin, and regenerated cellulose fiber are used in electro-mediated oxidation with 4-AcNH-TEMPO in a buffer at pH 6.8, more than 90% of the C6-OH groups of curdlan and amylodextrin are oxidized to sodium C6carboxylate groups to form corresponding water-soluble sodium polyuronates [145]. Molar masses of the oxidized products prepared are much higher than those prepared from the same polysaccharides by the TEMPO/NaBr/NaClO system in water at pH 10. When a regenerate cellulose fiber (viscose rayon) is treated by TEMPO electro-mediated oxidation in a buffer at pH 6.8 and room temperature for 45 h, sodium C6-carboxylate and C6-aldehyde groups of 1.1 and 0.6 mmol/g, respectively, are formed in the oxidized cellulose fiber. The fine surface morphology and cellulose II crystal structure of the original viscose rayon are maintained, and nearly no weight losses during the oxidation are observed [145]. The TEMPO electro-mediated oxidation is, therefore, a useful chemical modification for regenerated cellulose fibers. Algal celluloses and cotton fabrics have been applied to TEMPO electro-mediated oxidation, and the oxidized products have been characterized [148][149][150].

TEMPO/laccase/O 2 oxidation
The TEMPO/laccase/O 2 system in a buffer at pH 4.5 is advantageous, because no chorine-containing oxidant is used [151]. The optimum conditions for wood cellulose to prepare TOCs with high sodium C6-carboxylate contents have been studied. However, laccase molecules are degraded by oxidized TEMPO or TEMPO + molecules formed by laccase-mediated oxidation with O 2 , which competes with the oxidation of C6-OH groups in wood cellulose. Consequently, large amounts of laccase and TEMPO, and long reaction times are needed to introduce sodium C6-carboxylate groups of ≈0.6 mmol/g to wood cellulose. The TEMPO/laccase/O 2 system undergoes a one-way reaction from TEMPO to reduced TEMPO through TEMPO + without reconversion to TEMPO [152]. When the TEMPO/laccase/O 2 oxidation is repeated to oxidize and then isolated/purified TOC, the amount of sodium C6-carboxylate groups increases to ≈1.1 mmol/g, which is sufficient for conversion of the TOC to TOCNs by sonication in water. However, the higher the carboxylate content of the TOCs, the lower their DP v values. Because the TEMPO/laccase/O 2 oxidation in water under neutral conditions efficiently introduces C6-aldehyde groups together with C6-carboxylate groups to cellulose, this oxidation has been used to improve wet strengths of cellulose sheets by formation of interfiber hemiacetal linkages [153][154][155][156].

N-Hydroxyphthalimide/co-catalyst/NaClO oxidation
Coseri et al. have reported other position-selective and nitroxyl radical-mediated oxidations of regenerated cellulose fiber (viscose rayon) with catalytic amounts of N-hydroxyphthalimide, a co-catalyst, and NaClO in water or a water/acetonitrile mixture at pH 10 or 10.5 and room temperature [157][158][159][160]. The oxidation proceeds via a phthalimide N-oxyl radical intermediate (Fig. 28). The following various co-catalysts have been studied in the Nhydroxyphthalimide/NaClO oxidation in terms of the amounts of sodium C6-carboxylate groups introduced to oxidized fibers, crystallinities, and DPs: anthraquinone, Pb(OAc) 4 , CuCl 2 , UV irradiation, Ce(NH 4 ) 2 (NO 3 ) 6 , and NaBr. Sodium C6-carboxylate groups up to ≈0.3 mmol/g are introduced into the regenerated cellulose fibers, which maintain the original cellulose II crystallinities. However, significant depolymerization is unavoidable during the oxidation; the original DP 235 of the viscose rayon decreases to 40-90 after oxidation.

TEMPO/Na 2 SO 4 /NaClO oxidation
A cotton fabric is oxidized using TEMPO/NaBr/NaClO and TEMPO/Na 2 SO 4 /NaClO systems in water at pH 10 under various conditions [78]. Sodium C6-carboxylate contents increase from 0.06 to 0.30-0.44 mmol/g, DP v values decrease from 2200 to 1000-1220 [161]. The degrees of whiteness reduction of TEMPOoxidized cotton fabrics after heating are clearly decreased by post-NaBH 4 treatment (but not by post-NaClO 2 oxidation); small amounts of C2/C3 ketone groups are likely to be present in the oxidized cotton fabrics, resulting in the whiteness reduction after heating. The TEMPO/Na 2 SO 4 /NaClO-oxidized cotton fabrics with carboxylate contents of ≈0.3 mmol/g have higher DP v values and lower degrees of whiteness reduction after heating than those with similar carboxylate contents prepared by the TEMPO/NaBr/NaClO oxidation [161]. The former oxidation system has, therefore, some advantages in terms of suitable functionalization of cotton fabrics as clothing materials without using expensive NaBr. Scanning electron microscopy combined with energy-dispersive X-ray analysis of the oxidized cotton fibers shows that the TEMPO/Na 2 SO 4 /NaClO oxidation proceeds homogeneously to the fiber cross section [161]. The TEMPO-oxidized cotton fabrics with sodium C6-carboxylate contents of 0.31-0.32 mmol/g have sufficient burst strength and stiffness values for clothing materials, and deodorizing efficiencies to ammonia and acetic acid gases even after 100 repetitions of laundry treatment. The TEMPO-oxidized cotton fabrics have, therefore, potential application as new functional clothing materials. TEMPOoxidation behaviour can monitored in terms of time-dependent changes in TEMPO radical concentration of reaction solutions using electron spin resonance (ESR; Fig. 29) [162,163].

Sonication-assisted TEMPO-mediated oxidation
Sonication-assisted TEMPO-mediated oxidation has been applied to cotton linters, hardwood, bagasse, and palm empty fruit celluloses to efficiently introduce C6-carboxy groups, in which TEMPO or 4-AcNH-TEMPO is used [164][165][166][167][168][169]. The sonication of celluloses during TEMPO-mediated oxidation enhances nanofibrillation of oxidized celluloses and also formation of CNCs in some cases. TOCNs and TEMPO-oxidized CNCs are, therefore, formed in part and dispersed at the individual nano-element level together with water-insoluble oxidized products in the oxidation mixtures. Isolation and purification processes of these nano-dispersed TOCNs and CNCs from oxidation mixtures are required, separated from TEMPO and other various water-soluble reagents present in the mixtures.

Other TEMPO-mediated oxidations
Sodium carboxylate groups of 0.9 mmol/g are introduced to xylan-removed hardwood cellulose by the TEMPO/NaClO oxidation in water at pH 9, and the oxidized celluloses are convertible to TOCNs by mechanical disintegration in water [170]. TEMPO/ClO 2 oxidation under neutral conditions [171], 4-AcNH-TEMPO/peracid system [172], and TEMPO/NaBr/NaIO 4 system under acidic conditions [173] have been investigated in terms of oxidation efficiency, molar mass, carboxy group content, etc. These alternative TEMPOmediated oxidations of cellulose have some advantageous points. However, the TEMPO/NaBr/NaClO system in water at pH 10 and TEMPO/NaClO/NaClO 2 system in water under acidic-neutral conditions are advantageous to introduce C6-carboxy groups sufficient in amounts for conversion to TOCNs.

Side reactions and nanodispersibility of oven-dried TOCs in water
Never-dried and wet TOCs, that are prepared from wood cellulose by the TEMPO/NaBr/NaClO system in water at pH 10 and have sodium C6-carboxylate contents of 1.0-1.7 mmol/g, can be converted to TOCNs with homogeneous widths of ≈3 nm in high nanofibrillation yields by gentle mechanical disintegration in water. However, oven-dried TOCs have low nanodispersibilities even after harsh disintegration in water. When TOCs are reduced with NaBH 4 under suitable conditions, almost all C6-aldehydes and C2/C3 ketones present in low amounts are converted to hydroxy groups. The NaBH 4 -reduced TOCs, even after oven-drying at 105 • C for 3 h, have sufficiently high nanodispersibilities in water in a similar manner to that of never-dried TOCs [174]. Interfibrillar hemiacetal linkages are formed during heating between hydroxy groups and low amounts of either C6-aldehydes or C2/C3 ketones present on crystalline microfibril surfaces of TOCs, resulting in the low nanodispersibilities in water.
The heat-induced discoloration of TOCs is also avoided using NaBH 4 -reduced TOCs. The C6-aldehydes and C2/C3 ketones present in oven-dried TOCs can be semi-quantitatively measured from the specific UV absorbance spectra at 260 and 290 nm, respectively, for the corresponding TOCN films heated at 105 • C for 3 h [174]. The UV method can, therefore, be used to determine whether TOC samples undergo NaClO 2 post-oxidation or NaBH 4 post-reduction. Complete position-selectivity cannot, therefore, be achieved for oxidation of C6 − OH groups of native celluloses in the TEMPO/NaBr/NaClO oxidation (Fig. 30) [174].
The continuous reactions of TEMPO/NaBr/NaClO oxidation of wood cellulose and subsequent NaBH 4 -reduction in a one-pot system in water at pH 10 (without intermediate isolation or purification process) converts the C6-aldehyde and C2/C3 ketone groups to hydroxy groups. The TEMPO-oxidized and NaBH 4 -reduced TOCs can significantly improve nanodispersibilities in water, even after oven drying, and prevent heat-induced discoloration. The postoxidation of TOCs with NaClO 2 can improve the nanodispersibilities to some extent, although the C2/C3 ketones still remain in the NaClO 2 -treated TOCs [174].

Differences between TOCN and other nanocelluloses
Various nanocelluloses including cellulose nanofibrils and nanocrystals are prepared from wood cellulose fibers with or without pretreatment followed by mechanical disintegration in water [38,82,102,192,193]. Representative morphologies of TOCNs, conventional CNCs, cellulose nanofibrils prepared only by mechanical disintegration in water, and microfibrillated celluloses (MFCs) are shown in Fig. 34. Wood CNCs have spindle-like morphologies with heterogeneous widths of 3-15 nm [103]. Cellulose nanofibrils prepared only by mechanical disintegration have heterogeneous widths of 10-50 nm with some branches, and intricate network structures [194]. MFCs contain not only nanofibrils but also micronsized fibers.
Characteristic points of wood TOCNs different from other wood nanocelluloses are as follows. TOCNs are completely individualized nanofibers with homogeneous widths of ≈3 nm and high aspect ratios. TOCNs can be prepared from TOCs with low energy consumption for disintegration in water. TOCNs have carboxylate groups densely, regularly, and position-selectively on their surfaces, and diverse cationic counterions with a variety of functions can be introduced to TOCN surfaces through ion-exchange. TOCNs are convertible to TO-CNCs with the same homogeneous widths and carboxylate contents as those of TOCNs through extended sonication in water.

Preparations and properties of TOCN bulk materials
Numerous fundamental and application studies on films, fibers, hydrogels, aerogels, foams, and nano-sized network structures prepared from TOCNs have been reported for preparing new biobased and functional TOCN bulk materials (Fig. 35) . Because TOCNs have sodium C6-carboxylate groups densely on crystalline cellulose microfibril surfaces, the TOCN elements form self-assemble and nematic-ordered structures in water and some organic solvents through electric repulsion working between TOCN elements [197]. The characteristic self-assembling structures of TOCNs lead to the formation of various TOCN bulk materials with different properties and functions. When TOCN/water dispersions are cast and dried on a plate, transparent and flexible TOCN films with high mechanical strength, high oxygen-barrier, and low coefficient thermal expansion properties are obtained [195]. These properties are accompanied by nano-layered and highly dense film structures formed from the nematic-ordered TOCN/water dispersions during drying [197,216].
When sodium C6-carboxylate groups of TOCNs are protonated with dilute HCl in TOCN/water dispersions followed by thoroughly washing with water, self-standing TOCN-COOH hydrogels with high stiffness are obtained even at low TOCN  [81], conventional wood cellulose nanocrystal (B) [103], mechanically fibrillated wood cellulose nanofibril (C) [194], optical microscopy image of microfibrillated wood cellulose (D), and morphology models of A-C. A, B, and C: Copyrights 2007, 1998, and 2003, respectively. Reproduction with permissions with the American Chemical Society, Springer Nature, and Springer Nature, respectively.  .
concentrations of ≈0.1% [197]. These TOCN-COOH hydrogels are convertible to transparent aerogels with high strength, bendable, and high thermal insulation properties by solvent exchange and subsequent critical point drying [197,199,208]. When TOCN-COONa/water/tert-butanol dispersions are freeze-dried, spider web-like network structures with nano-sized pores can be obtained, which are applicable to high performance air filters [201].

TOCN-containing composite materials
Light-weight and highly strong TOCN-containing composite materials are one of the most promising applications for TOCNs. This is because tensile strengths and Young's moduli of single TOCN elements are ≈3 GPa and ≈140 GPa, respectively, [112,115] regardless of low densities of ≈1.6 g/cm 3 . TOCNs are, however, highly hydrophilic, owing to the abundant sodium C6-carboxylate groups present on the TOCN surfaces, it is generally difficult to prepare TOCN/polymer composite films, in which hydrophilic TOCN elements are individually dispersed without agglomeration in hydrophobic polymer matrices.
The preparation and characterization of TOCN/polymer or inorganic filler/TOCN composite films have been investigated using poly(vinyl alcohol) [217], poly(acrylamide) [218,219], carbon nanotubes [220], and nanoclays [221,222]. The TOCN-COONa/water dispersions and water-soluble polymers dissolved in water or H-NBR latex dispersed in water were mixed at various weight ratios followed by casting and drying to prepare TOCN/polymer composite films. Single-wall carbon nanotube and nanoclays dispersed in water were mixed with TOCN/water dispersions to prepare composite films at various filler/TOCN weight ratios. Some of them have unique mechanical, transparent, excellent oxygen barrier, thermal, and electric properties, owing to sufficiently nanodispersed TOCN elements or filler nanoparticles without agglomeration in polymer or TOCN matrices, which were supported from their TEM images [220][221][222].
Some organic solvents were used to prepare TOCN-containing hydrophobic polymer composite films, such as poly(styrene) [226], poly(lactic acid) (PLA) [186,227], and cellulose triacetate (CTA) [228]. The counterions of TOCN-COONa have to be converted to TOCN-COOH nanodispersible in DMF [178,226] or hydrophobic TOCN-PEG nanodispersible in DMAc, toluene, chloroform, and tetrahydrofuran [186,227,228]. Because poly(styrene) and PLA are soluble in DMF and chloroform, respectively, the TOCN-COOH/poly(styrene) and TOCN-PEG/PLA composite films were prepared using DMF and chloroform as the media, respectively, by mixing the two components at various weight ratios and successive casting/drying [186,227,228]. The TOCN-PEG/CTA composite films were prepared by mixing the TOCN-PEG/chloroform dispersion and CTA/dichloromethane-methanol (9:1 by vol.) solution at various ratios and subsequent casting/drying [228]. These composite films exhibited significant improvements in mechanical and thermal properties at low TOCN contents. These preparation methods of TOCN/polymer composite films using organic solvents were performed as model experiments to prepare ideal composite films consisting of individually nanodispersed TOCN elements in polymer matrices (Fig. 36).
However, it is difficult to prepare TOCN/polymer composite films consisting of individually nanodispersed TOCN elements through general melt-molding or melt-extrusion processes without using any organic solvent. The TOCN elements initially nanodispersed in water turn to large agglomerates through waterremoval process during thermal molding, resulting in almost no improvement in mechanical properties for the TOCN/polymer composites. Another shortcoming for TOCNs is a decrease in thermal degradation temperature from ≈300 • C for the original cellulose to ≈200 • C, which initially occurs through decarboxylation at the abundant carboxylate groups present in TOCNs.

Conclusions and future prospects
N-Oxyl radical-mediated oxidation is a unique reaction and has some advantages in terms of efficiency to introduce anionic sodium C6-carboxylate groups to various cellulosic materials under aqueous conditions. The TEMPO/NaBr/NaClO oxidation proceeds without using any organic solvents, which is different from the conventional cellulose esterifications and etherifications. When native celluloses with cellulose I crystal structures are used as starting materials and TEMPO-mediated oxidation is applied under suitable conditions, the C6-OH groups present on crystalline cellulose microfibril surfaces in native celluloses are position-selectively oxidized to sodium C6-carboxylate groups in TOCs. New cellulosebased nanofibers, TOCNs, with homogeneous widths of ≈3 nm and high aspect ratios can be prepared from wood TOCs with sufficient amounts of sodium C6-carboxylate groups by gentle mechanical disintegration in water.
There are some shortcomings in TEMPO-mediated oxidation of cellulose, which have to be solved for further applications of TOCs and TOCNs in industry. Depolymerization should be controlled to prepare TOCs and TOCNs with high DPs and TOCNs with high aspect ratios. If possible, the use of chlorine-containing oxidants such as NaClO and NaClO 2 should be avoided because of environmental aspects. The O 2 , H 2 O 2 , O 3 , or other chlorine-free compounds are preferable as the primary oxidant, if sufficient amounts of sodium C6-carboxylate groups can efficiently be introduced to oxidized celluloses. Safety issues of TEMPO, TOCs, and TOCNs have to be clarified for their industrial production and application. The treatment or recycling of TEMPO or its related compounds present in effluents formed during industrial production of TOCs and TOCNs should be taken into account for establishing environmentally friendly processes of cellulose modification [229,230].
TOCNs and related materials have been commercialized as an ink dispersant of ballpoint pens developed by Daiichi Kogyo Seiyaku and Mitsubishi Pencil since 2013. Nippon Paper Industries and Nippon Paper Crecia have commercialized TOCN-containing disposable diapers for adults with super deodorizing functions that alleviate the burden for care assistants, also since 2013. The world's largest plant to produce TOCNs at a capacity of 500 t/y was installed in the Ishinomaki mill of Nippon Paper Industries, Miyagi, Japan, and TOCN production has started in 2017. Three additional TOCN production plants were installed in Japan (Daiichi-Kogo Seiyaku Co.), Finland (Betulium Oy), and United States (Forest Products Laboratory, Forest Service, United States Department of Agriculture) [231].
TOCs, TOCNs, and their related bulk and composite materials have been developed since 2006, and are, therefore, still new biobased materials. More fundamental information and data based on experimental and theoretical studies have to be accumulated for further industrial applications of these new materials. The polymer, metal, glass, paper, and other organic and inorganic materials widely used in the present high-tech and commodity fields have long histories of more than several decades. Numerous science and technologies have been accumulated for these materials to be produced and used under the conditions with high reproducibility, runnability, and reliability.