O2 System

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Cellulose Nanofibers Prepared using the TEMPO/Laccase/O2 System Jie Jiang, Wenbo Ye, Liang Liu, Zhiguo Wang, Yimin Fan, Tsuguyuki Saito, and Akira Isogai Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01682 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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Cellulose Nanofibers Prepared using the TEMPO/Laccase/O2 System Jie Jiang,† Wenbo Ye,† Liang Liu,† Zhiguo Wang,† Yimin Fan,†* Tsuguyuki Saito,‡ and Akira Isogai‡ †

Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources,

Jiangsu Key Lab of Biomass-based Green Fuel & Chemicals, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China ‡

Department of Biomaterials Science, Graduate School of Agricultural and Life Sciences, The

University of Tokyo, Tokyo 113-8657, Japan

ACS Paragon Plus Environment

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ABSTRACT: The 2,2,6,6-tetramethylpiperidiine-1-oxyl (TEMPO)/laccase/O2 system was used to prepare cellulose nanofibers from wood cellulose without requiring any chlorine-containing oxidant. Laccase was degraded by oxidized TEMPO (TEMPO+) formed by laccase-mediated oxidation with O2, which competed with the oxidation of wood cellulose. Thus, large amounts of laccase and TEMPO and a long reaction time were needed to introduce ~0.6 mmol g−1 of C6carboxylate groups onto wood cellulose. The TEMPO/laccase/O2 system underwent one-way reaction from TEMPO to reduced TEMPO through TEMPO+. When the oxidation was applied again to the oxidized wood cellulose following isolation and purification, the C6-carboxylate groups increased to ~1.1 mmol g−1, which was sufficient to convert the sample to cellulose nanofibers by sonication in water. However, the higher the carboxylate content of the oxidized celluloses, the lower their degree of polymerization.

KEYWORDS: Cellulose, laccase, TEMPO, oxidation, nanofiber


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INTRODUCTION Cellulose is a primary component in plant cell walls, and is therefore among the most abundant and renewable biopolymers on earth. Cellulose molecules in plant cell walls exist as fine fibrils ~3 nm wide with high aspect ratios. Each cellulose microfibril consists of 30–40 cellulose molecules organized as fully extended chains. Because versatile and energy efficient procedures to prepare cellulose nanofibers (CNFs) from plant cellulose microfibrils have been reported this decade, much attention has been paid to the modification, compositing, and application of CNFs.1–5 In particular, some pretreatments of wood cellulose fibers or paper/dissolving pulps are effective at lowering the energy consumed to prepare CNFs with widths of 0.8 V or large molecular weights such as lignin, which cannot directly enter the active site of the enzyme, can also be oxidized using a mediator together with a laccase. Mediators usually have a low molecular weight and act as an electron shuttle. After mediator is oxidized by a laccase, it diffuses away from the active site of the enzyme, and in turn oxidizes the target substrate.17–19 Efficient mediators reported so far are 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole, N-hydroxyphthalimide, violuric acid, N-hydroxyacetanilide, and TEMPO.20–23 The TEMPO/laccase/O2 oxidation system was first applied to low-molecular-weight alcohols and sugars to prepare the corresponding oxidized compounds containing carboxyls, ketones, and aldehydes,24–27 and recently to cellulose.28–30 Aracri et al.28–30 studied the TEMPO/laccase/O2 oxidation of sisal cellulose fibers in water to prepare C6-aldehyde- and C6-carboxylatecontaining pulps, which improved the wet and dry strengths of paper sheets prepared from them. However, the C6-carboxylate contents of the oxidized sisal celluloses were lower than 0.312 mmol g−1, which is insufficient to convert to individual T-CNFs even by harsh disintegration treatment in water.29 In this paper, we determine the optimum oxidation conditions to prepare oxidized celluloses with high C6-carboxylate contents from wood cellulose (hardwood bleached kraft pulp, HBKP) using the TEMPO/laccase/O2 system at room temperature in water under neutral conditions.


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Although the maximum C6-carboxylate content of the oxidized wood cellulose prepared under suitable conditions is ~0.6 mmol g−1, the C6-carboxylate content increases to ~1.1 mmol g−1 when the oxidation is repeated under the same conditions after washing, isolation, and purification. The twice-oxidized wood cellulose is then converted to individual T-CNFs by sonication in water.

MATERIALS AND METHODS Materials. All chemicals including TEMPO and ABTS were purchased from Sigma Aldrich. Laccase from Trametes versicolor was a lyophilized powder with an enzyme activity of 12.9 U mg−1 (data from the supplier). Copper ethylenediamine (cuen) solution (1 M) was provided by China National Pulp and Paper Research Institute. Commercial HBKP (bleached acacia kraft pulp produced by Asia Symbol Pulp and Paper Co., Ltd, Shandong, China) with an α-cellulose content of ~80% was used as the wood cellulose sample. Enzyme Assay. Laccase activity in solutions was monitored using an ultraviolet–visible (UVvis) spectrophotometer (UV-1800, Shimadzu, Japan) at 420 nm after oxidation with ABTS. Sodium acetate buffer (100 µL, pH 4.5) containing laccase was centrifuged to remove HBKP if necessary, and added to a cuvette containing ABTS solution (3 mL, 0.5 mM). One activity unit (U) was defined as the amount of laccase to transform 1 µmol of ABTS to its cation radical per minute at 25 °C.31 Preparation of Oxidized Celluloses. HBKP (1 g) was suspended in a beaker containing 0.1 M sodium acetate buffer (100 mL, pH 4.5) in which designated amounts of TEMPO and laccase were dissolved. The mixture was magnetically stirred at 500 rpm and room temperature in the beaker, which was covered with a plastic bag. After oxidation time for 0, 24, and 48 h, the bag


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was filled with pure O2 gas from an O2 cylinder. After oxidation, the mixture was poured into ice water for 30 min, and then washed with distilled water by repeated centrifugation (more than 6 times) at 8228×g for 6 min to obtain the TEMPO/laccase/O2-oxidized cellulose (TLO-cellulose) as a water-insoluble fraction. Conventional TEMPO-oxidized cellulose was prepared from the same HBKP using the TEMPO/NaBr/NaClO system (10 mmol NaClO to 1 g HBKP) in water at pH 10 according to a reported method9 for use as a reference. Mechanical Disintegration. A 0.1% (w/v) suspension of TLO-cellulose was sonicated using an ultrasonic homogenizer (VCX500, Sonics & Materials, Inc., USA) at 500 W and 20 kHz for 20 min with start/stop intervals to avoid temperature increases. The diameter of the homogenizer tip was 1.2 cm. After centrifugation at 12857×g for 6 min to remove an unfibrillated fraction, a TLO-cellulose nanofiber (TLO-CNF)/water dispersion was obtained as a supernatant. Analyses. The carboxylate and aldehyde contents of the oxidized celluloses were determined according to a reported method.31 The TLO-oxidized celluloses were freeze-dried and then pressed to form disk pellets (~0.1 g each) before recording their X-ray diffraction (XRD) patterns from 2θ 5° to 30° with the reflection method using an Ultima IV diffractometer at 40 kV and 30 mA. The crystallinity index (C.I.) and crystal width of the (2 0 0) plane of cellulose I were determined according to a reported method.32 The freeze-dried samples (0.1 g each) were postoxidized with NaClO2 in water at pH 4.5 for 2 days. The resulting TLO/NaClO2-celluloses were dissolved in 0.5 M cuen (30 mL). The intrinsic viscosities of the solutions were measured using an Ubbelohde viscometer at 20 °C. The viscosity-average degrees of polymerization (DPv) of the TLO/NaClO2-celluloses were calculated from the intrinsic viscosities using the Mark–Houwink– Sakurada equation.33 The light transmittance spectra of 0.1% (w/v) CNF dispersions were recorded in the range of 400–800 nm using the UV-vis spectrophotometer. The TLO-CNF


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dispersion was diluted to ~0.001% (w/v) with water, added dropwise onto a mica substrate, and dried at room temperature. Surface images were obtained by atomic force microscopy (AFM; (Dimension Edge, Bruker, Germany) in tapping mode with a standard silicon cantilever in air.

RESULTS AND DISCUSSION Time- and TEMPO-Dependent Laccase Activity. The laccase activity in 0.1 M acetate buffer with or without HBKP was monitored as the buffer solution was stirred (Figure 1A). The laccase activity gradually decreased to ~65% of the original value as the stirring increased to 40 h, irrespective of the presence of HBKP. This result indicates that the shear force of the stirred buffer solution may have caused partial degradation of laccase molecules, and that the presence of HBKP hardly influenced laccase activity. When TEMPO was present in the buffer solution containing laccase, its activity clearly decreased with increasing stirring time. The laccase activity decreased to ~50% of the original value after stirring the mixture containing 5 mM TEMPO for 10 min. When 30 or 50 mM TEMPO was present in the buffer solution, the laccase activity rapidly decreased to almost zero after stirring the buffer solution for 15 min. In the TEMPO/laccase/O2 oxidation system, laccase oxidizes both TEMPO and N-hydroxyl-TEMPO (reduced TEMPO) to the N-oxoammonium compound (TEMPO+), which can in turn oxidize primary hydroxyl groups to aldehyde and carboxyl groups.28–30 However, under the experimental conditions shown in Figure 1B, TEMPO+ molecules accumulate in the buffer solution, because the oxidation of TEMPO proceeds in the presence of laccase and O2. The primary amine groups of chitosan react with TEMPO+ to form low-molecular-mass compounds.34 Therefore, some of the laccase molecules containing primary amine groups are probably degraded in the mixtures, and lose their activity. Thus, in


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TEMPO/laccase/O2 oxidation, the reactions between the formation of C6-aldehyde/C6carboxylate groups and degradation of laccase molecules compete with each other because TEMPO+ coexist in the system, which should be taken into account. 100

Laccase activity (%)

A 80

60

40

20 without HBKP with HBKP

0 0

10

20

30

40

Time (h) 100

B Laccase avtivity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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TEMPO concentration (mM)

80

5 30 50

60

40

20

0 0

5

10

15

20

25

Time (h)

Figure 1. Time-dependent laccase activity (A) in acetate buffer at pH 4.5 with or without HBKP, and (B) in the presence of TEMPO (5–30 mM) and laccase (2 U mL−1).

Figure 2A illustrates the time-dependent laccase activity in mixtures containing HBKP during TEMPO/laccase/O2 oxidation at a constant laccase concentration of 3 U mL−1 with various TEMPO concentrations from 5 to 70 mM. The decrease of laccase activity shown in Figure 2A are similar to those in Figure 1B, indicating that the presence of HBKP did not markedly influence the instability of laccase in the oxidation system.


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100

Laccase activity (%)

A 80

60

40

20

TEMPO concetration (mM) 5 30 50 70

0 0

2

4

6

8

10

12

Time (h) 100

B Laccase activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-1

Laccase concentration (U mL )

80

3.1 4.0 4.8 7.2 8.2

60

40

20

0 0

2

4

6

8

10

12

Time (h)

Figure 2. Time-dependent laccase activity in buffer solution at pH 4.5 containing HBKP during TEMPO/laccase/O2 oxidation (A) at a constant laccase concentration (3 U mL−1) with various TEMPO concentrations and (B) at a constant TEMPO concentration (50 mM) with various laccase concentrations in buffer solution.

Figure 2B presents the changes in laccase activity during oxidation of HBKP by the TEMPO/laccase/O2 system at a constant TEMPO concentration of 50 mM with various laccase concentrations from 3.1 to 8.2 U mL−1. The time-dependent inactivation or degradation of laccase could not be avoided, even when the concentration of laccase was increased, indicating that TEMPO+ molecules formed in the mixture by the TEMPO/laccase/O2 system degraded


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laccase. 100

A 80

0.6

60 0.4

Carboxylate content Yield

40

0.2

Yield (%)

Carboxylate content (mmol/g)

0.8

20

0.0

0 0

20

40

60

Concentration of TEMPO (mM) 100

B 80

0.6

60 0.4 40 0.2

Yield (%)


0.8

20


0.0

0 0

2

4

6

8 -1

Laccase concentration (U mL ) 0.8

100

C 80

0.6

60 0.4 40 0.2

Yield (%)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20


0.0

0 0

20

40

60

80

100

120

140

Time (h)

Figure 3. Carboxylate contents and yields of TLO-celluloses at (A) laccase concentration of 3 U mL−1 with various TEMPO concentrations for 24 h, (B) a TEMPO concentration of 50 mM with various laccase concentrations for 48 h, and (C) laccase and TEMPO concentrations of 5 U mL−1 and 50 mM, respectively, for 0–140 h.


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Oxidation

Conditions

of

HBKP

in

the

TEMPO/Laccase/O2

System.

The

TEMPO/laccase/O2 oxidation conditions were further studied in terms of the carboxylate content and yield (or the weight recovery ratio as a water-insoluble fraction) of the TLO-celluloses (Figure 3). When HBKP was oxidized by the TEMPO/laccase/O2 system in acetate buffer at pH 4.5 and room temperature for 24 h at a constant laccase concentration of 3 U mL−1 with various TEMPO concentrations from 5 to 70 mM, the carboxylate content increased substantially from 0.07 to 0.41 mmol g−1 (Figure 3A). The yield of TLO-cellulose slightly increased from 75% to 80% with rising TEMPO concentration. Thus, TEMPO concentration of 70 mM is the best in terms of efficient carboxylate group formation in TLO-celluloses. The yield of TLO-celluloses of ~80% indicates that almost all hemicelluloses present in the original HBKP were degraded and removed as water-soluble fractions during oxidation, washing, purification, and isolation processes. When the concentration of TEMPO and reaction time were fixed to 50 mM and 48 h, respectively, and the laccase concentration was varied from 4.0 to 8.2 U mL−1, the carboxylate contents of the TLO-celluloses were almost constant at ~0.55 mmol g−1 (Figure 3B). The yield increased slightly from 75% to 82%, indicating that the optimum laccase concentration to provide TLO-celluloses with both high carboxylate content and yield ranged from 4 to 5 U mL−1. In the next experiment, the TEMPO and laccase concentrations in the mixture containing 1 g HBKP were fixed to 50 mM and 5 U mL−1, respectively, and the reaction time was extended to 140 h (Figure 3C). The yield of TLO-celluloses was almost constant at ~80% when the reaction time exceeded 40 h. Conversely, the carboxylate content increased up to 0.62 mmol g−1 after stirring the mixture for 140 h. Thus, a longer reaction time seems to be preferable to a shorter one to prepare TLO-celluloses with higher carboxylate contents.


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Some discrepancy was observed between the results for the carboxylate contents of TLOcelluloses shown in Figure 3 and those of laccase activities presented in Figures 1 and 2. Although the laccase activity in the mixture containing 50 mM TEMPO was almost zero after reaction for 20 min, the carboxylate group content increased to 0.62 mmol g−1 as the oxidation time was extended to 140 h.

Table 1. Yield, carboxylate and aldehyde contents, and degree of polymerization (DPv) of the original HBKP, TLO-celluloses, and TEMPO/NaBr/NaClO-cellulose.

No.

Sample

Oxidation conditions

Yield (%)

C: Carboxylate content (mmol/g)

A: Aldehyde content (mmol/g)

C+A (mmol/g)

DPv

–

91

0.098

0.016

0.114

810

1

Original HBKP

2

TO-cellulose

50 mM TEMPO, 96 h

86

0.189

0.028

0.217

760

3

LO-cellulose

5 U mL–1 laccase, 96 h

84

0.118

0.032

0.150

780

4

TLO-cellulose-4

50 mM TEMPO, 5 U mL–1 laccase, 96 h

89

0.596

0.241

0.837

430

5

TLO-cellulose-5

50 mM recycled TEMPO*, 5 U mL–1 laccase, 96 h

83

0.229

0.093

0.332

550

6

TLO-cellulose-6

50 mM TEMPO, two additions of 2.5 U mL–1 laccase at 0 and 48 h, totally 96 h

89

0.767

0.195

0.962

220

7

TLO-cellulose-7

Twice oxidations under conditions for sample #4

86

1.063

0.056

1.119

180

8

TEMPO/NaBr/ NaClO-cellulose

1.5 h

85

1.104

0.087

1.191

190

*

The filtrate of the reaction mixture used to prepare sample #4 was used with fresh laccase and HBKP.

Based on the results in Figure 3, the optimum TEMPO/laccase/O2 oxidation conditions were selected as follows: 1 g HBKP in 100 mL buffer solution at pH 4.5, 50 mM TEMPO, 5 U mL−1 laccase, and a reaction time of 96 h. Table 1 summarizes the yield, carboxylate and aldehyde


12

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contents, and DPv values of the oxidized celluloses prepared under various conditions. Using a TEMPO/O2 or laccase/O2 system did not markedly increase the carboxylate content of the oxidized celluloses (samples #2 and #3 in Table 1), showing that the three-component system with TEMPO, laccase, and O2 is required to form C6-carboxylate groups. Compared with the TEMPO/NaBr/NaClO-oxidized cellulose (sample #8) used as a reference, the oxidation efficiency of the TEMPO/laccase/O2 system to introduce carboxylate groups is much lower in terms of the amounts of reagents added and reaction time, although no chlorine-containing oxidant is required in the latter case. The aldehyde content of sample #4 was higher than that of sample #8, which is also characteristic of the TEMPO/laccase/O2 system. When 50 mM TEMPO and 3 U mL−1 laccase were added twice to the same reaction batch during the oxidation for 96 h, the carboxylate content increased somewhat from 0.596 to 0.767 mmol g−1. Conversely, when the isolated and purified TLO-cellulose (sample #4) was oxidized again under the same conditions, the resulting TLO-cellulose (sample #7) had the highest carboxylate content of the samples of 1.063 mmol g−1. This value was almost the same as that of the reference sample #8, which was prepared using the TEMPO/NaBr/NaClO system in water at pH 10. According to the literature,17–30 when catalytic amounts of TEMPO and laccase are used to oxidize cellulose, the resulting oxidized celluloses contain very small amounts of carboxylate groups and large amounts of aldehyde groups. However, as shown in Figure 3, when large amounts of TEMPO and laccase and longer reaction times were used, the oxidized celluloses contain considerable amounts of carboxylate groups. The carboxylate content was higher than the aldehyde content for each TLO-cellulose in Table 1. The relationship between the carboxylate and aldehyde contents and DPv of the oxidized celluloses are plotted in Figure 4. The higher the content of oxidized groups, the lower the DPv


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value of the oxidized cellulose. If the TEMPO/laccase/O2 system promotes the oxidation of C6primary hydroxyl groups to C6-aldehydes and C6-carboxylate groups alone during the reaction, β-elimination is only one plausible mechanism for depolymerization of the oxidized celluloses. However, because the reaction was performed in water at pH 4.5, β-elimination is likely to be restricted under such acidic conditions.13 Thus, some oxidant species formed from TEMPO and/or laccase as by-products or degradation products during the reaction may have participated in the depolymerization, as in the case of TEMPO/NaBr/NaClO oxidation in water at pH 10.33,35 1000 #1

800 #3

#2

600 DPv

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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#5

#4

400 #8

#6

200

#7

0 0.0

0.5

1.0

1.5

Content of [carboxylate + aldehyde] groups (mmol/g)

Figure 4. Relationship between carboxylate and aldehyde contents and DPv of the oxidized celluloses. The sample numbers correspond to those in Table 1.

The XRD patterns of the original HBKP (sample #1), TLO-cellulose (sample #4), and TEMPO/NaBr/NaClO-cellulose (sample #8) are provided in Figure 5. Both the C.I. and crystal width of cellulose I in HBKP were almost unchanged after TEMPO/laccase/O2 oxidation, suggesting that the carboxylate groups formed in sample #4 were present on the crystalline cellulose microfibril surfaces. That is, only the exposed C6-primary hydroxyl groups on the crystalline cellulose microfibril surfaces are oxidized, as in other examples of TEMPO-mediated oxidation of native celluloses.36


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C.I. Crystal width HBKP 0.48 4.5 nm TLO-cellulose 0.46 4.6 nm TNN-cellulose 0.47 4.0 nm

10

15

20

25

30

Diffraction angle 2θ ( ° )

Figure 5. X-ray diffraction patterns of HBKP (sample #1), TLO-cellulose (sample #4), and TEMPO/NaBr/NaClO-cellulose (sample #8). The number corresponds to that in Table 1.

Oxidation Mechanism of HBKP by the TEMPO/Laccase/O2 System. Figure 6A shows the UV-vis spectra of the reaction mixtures during oxidation after removal of fibrous HBKP. The absorbance at 245 nm is caused by TEMPO, and that at 300 nm originates from TEMPO+.37 The changes in the relative absorbance of TEMPO and TEMPO+ are depicted in Figure 6B. The decrease of absorbance of TEMPO and increase of absorbance of TEMPO+ in Figure 6B were much faster than the decrease of laccase activity shown in Figure 2. This reveals that the formation of TEMPO+ from TEMPO, which is mediated by O2 and laccase, proceeds much faster than the degradation of laccase by TEMPO+. When the conventional TEMPO/laccase/O2 oxidation mechanism proposed in the literature is used, the results in Figures 2 and 3 are inconsistent with each other; although the original laccase activity (5 U mL−1) almost disappeared in the mixture containing 50 mM TEMPO and 1 g HBKP after 20 h of oxidation, the content of carboxylate groups still increased up to the reaction time of 96 h.


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2.0

A 0h 0.33 h 1h 4h

Absorbance

1.6

1.2

0.8

0.4

0.0 240

260

280

300

320

340

Wavelength (nm) 100

B

TEMPO +

Relative amount (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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TEMPO (or N-oxoammonium compound)

80

60

40

20

0 0

1

2

3

4

Reaction time (h)

Figure 6. (A) Changes in the UV-vis absorbance of TEMPO during the TEMPO/laccase/O2 oxidation of HBKP, measured after the removal of fibrous HBKP from the mixture. (B) The relative amounts of TEMPO and TEMPO+ in the mixtures.

According to the literature, the TEMPO/laccase/O2 system with catalytic amounts of TEMPO and laccase in buffer solution generates the reactive TEMPO+, which oxidizes primary hydroxyl groups to aldehyde groups. The TEMPO+ is in turn reduced to N-hydroxyl-TEMPO by the oxidation.38 However, because TEMPO+ attacks both the primary hydroxyl groups of polysaccharides and laccase molecules in competing reactions under the conditions used in this study, a redox cycle between TEMPO+ and N-hydroxyl-TEMPO molecules does not form in our experiments. That is, only one-way conversion of TEMPO to TEMPO+ occurs with O2 in the


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buffer solution containing laccase, and the N-hydroxyl-TEMPO molecules formed from TEMPO+ are not re-oxidized to TEMPO+. Instead, N-hydroxyl-TEMPO accumulates in the reaction medium because there is almost no active laccase in the system (Figure 7). This is the reason why such large amounts of TEMPO and laccase are required to oxidize the C6-primary hydroxyl groups of HBKP in the TEMPO/laccase/O2 system, although no chlorine-containing primary oxidant is required. Because the carboxylate content of the TLO-celluloses increased after 20 h of oxidation, the oxidation of C6-primary hydroxyl groups of HBKP gradually proceeds in the buffer solution at pH 4.5 and room temperature, which is similar to the TEMPO/NaClO/NaClO2 oxidation of HBKP in buffer solution at pH 4.8 and room temperature.13 Oxidized laccase

TEMPO

Cellulose microfibril HOH2C HOH2C CH2OH

HOH2C

CH2OH

HOH2C

O2

Oxidized TEMPO+

Laccase

CH2OH

HOH2C

CH2OH

(N-oxoammonium)

CH2OH

OOC HOH2C

Degraded laccase

Reduced TEMPO (N-hydroxyl-TEMPO)

COO

OHC

CHO

OOC

COO

OOC

COO COO

TEMPO/laccase/O2-oxidized cellulose microfibril

Figure 7. Possible oxidation mechanism of C6-primary hydroxyl groups of HBKP by the TEMPO/laccase/O2 system in buffer solution at pH 4.5.

Nanofibrillation of Fibrous TLO-Celluloses by Sonication in Water. The never-dried fibrous TLO-celluloses were suspended in water, then the TLO-cellulose/water slurries were sonicated under the same conditions. The light transmittance spectra of the 0.1% TLO-


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cellulose/water dispersions are displayed in Figure 8, together with a photograph of the dispersion of sample #4 after centrifugation of the unfibrillated fraction observed between crosspolarizers as an inset. The higher the carboxylate content of the TLO-cellulose, the higher the transparency of the TLO-CNF/water dispersion. When the dispersion of sample #4 was centrifuged to remove the unfibrillated fraction, an almost transparent dispersion similar to that of sample #7 in Figure 8 was obtained. This sample showed clear birefringence when observed between cross-polarizers.8–10,39

Figure 8. Light transmission spectra of 0.1% dispersions of TLO-celluloses after sonication under the same conditions. Inset is a photograph of the dispersion of sample #4 after centrifugation, observed between cross-polarizers.

TLO-cellulose sample #4 was sonicated, and the fibrillated dispersion was centrifuged to remove the unfibrillated fraction. The TLO-CNFs present in the supernatant were obtained with a yield of 65%. This supernatant was further diluted with water to ~0.001% (w/v), and then added dropwise onto a mica plate. AFM images of TLO-CNF sample #4 are depicted in Figure 9. Although some nanofibers formed bundles and network structures without being completely isolated (probably because of their lower carboxylate content, the morphologies are similar to


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those prepared from TEMPO/NaBr/NaClO-oxidized celluloses with carboxylate contents of 1 µm and 4–8 nm, respectively, as determined from the AFM images. Thus, CNFs can be prepared from HBKP by TEMPO/laccase/O2 oxidation and successive sonication under suitable conditions without using any chlorine-containing oxidant.

Figure 9. AFM images of TLO-CNFs prepared from TLO-cellulose sample #4 by sonication and centrifugation.

CONCLUSIONS The TEMPO/laccase/O2 system in buffer solution at pH 4.5 was used to oxidize fibrous HBKP. The laccase molecules were unstable in the presence of TEMPO+. Thus, the reaction of TEMPO+ with the C6-primary hydroxyl groups of HBKP, which is mediated by laccase and O2, and degradation of laccase by TEMPO+ competed with each other in the oxidation media. Almost all laccase molecules degraded after reaction for 15 h. As a result, large amounts of TEMPO and laccase are required to oxidize HBKP to introduce ~0.6 mmol g−1 carboxylate groups. Probably, one-way formation of TEMPO+ from TEMPO occurs in the system; TEMPO+ is not reformed from reduced TEMPO or N-hydroxyl-TEMPO. When 1 g HBKP was subjected to TEMPO/laccase/O2 oxidation with 50 mM TEMPO and 5 U mL−1 twice, including washing and purification, the carboxylate content of the resulting TLO-cellulose reached 1.063 mmol g−1.


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However, the higher the carboxylate content of the TLO-cellulose, the lower its DPv; remarkable depolymerization of the TLO-celluloses occurred during the oxidation. The TLO-cellulose a carboxylate content of 1.063 mmol g−1 was mostly nanodispersed in water by sonication to form a transparent gel consisting of individual TLO-CNFs.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Phone: +86 2585 42 7587. Funding Sources This research was supported by the National Forestry Public Welfare Industry Research Project (No. 201304609), the National Natural Science Foundation of China (No. 31100426), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20133204110008), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Table of Contents

Cellulose Nanofibers Prepared using the TEMPO/Laccase/O2 system

Jie Jiang, Wenbo Ye, Liang Liu, Zhiguo Wang, Yimin Fan, Tsuguyuki Saito, and Akira Isogai


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Figure 1. Time-dependent laccase activity (A) in acetate buffer at pH 4.5 with or without HBKP, and (B) in the presence of TEMPO (5–30 mM) and laccase (2 U mL−1). Figure 1 227x343mm (150 x 150 DPI)


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Figure 2. Time-dependent laccase activity in buffer solution at pH 4.5 containing HBKP during TEMPO/laccase/O2 oxidation (A) at a constant laccase concentration (3 U mL−1) with various TEMPO concentrations and (B) at a constant TEMPO concentration (50 mM) with various laccase concentrations in buffer solution. Figure 2 725x1109mm (96 x 96 DPI)


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Figure 3. Carboxylate contents and yields of TLO-celluloses at (A) laccase concentration of 3 U mL−1 with various TEMPO concentrations for 24 h, (B) a TEMPO concentration of 50 mM with various laccase concentrations for 48 h, and (C) laccase and TEMPO concentrations of 5 U mL−1 and 50 mM, respectively, for 0–140 h. Figure 3 237x479mm (150 x 150 DPI)


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Figure 4. Relationship between carboxylate and aldehyde contents and DPv of the oxidized celluloses. The sample numbers correspond to those in Table 1. Figure 4 365x259mm (150 x 150 DPI)


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Figure 5. X-ray diffraction patterns of HBKP (sample #1), TLO-cellulose (sample #4), and TEMPO/NaBr/NaClO-cellulose (sample #8). The number corresponds to that in Table 1. Figure 5 691x614mm (96 x 96 DPI)


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Figure 6. (A) Changes in the UV-vis absorbance of TEMPO during the TEMPO/laccase/O2 oxidation of HBKP, measured after the removal of fibrous HBKP from the mixture. (B) The relative amounts of TEMPO and TEMPO+ in the mixtures. Figure 6 258x391mm (150 x 150 DPI)


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Figure 7. Possible oxidation mechanism of C6-primary hydroxyl groups of HBKP by the TEMPO/laccase/O2 system in buffer solution at pH 4.5. Figure 7 325x177mm (150 x 150 DPI)


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Figure 8. Light transmission spectra of 0.1% dispersions of TLO-celluloses after sonication under the same conditions. Inset is a photograph of the dispersion of sample #4 after centrifugation, observed between cross-polarizers. Figure 8 759x579mm (96 x 96 DPI)


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Figure 9. AFM images of TLO-CNFs prepared from TLO-cellulose sample #4 by sonication and centrifugation. Figure 9 336x159mm (143 x 143 DPI)


O2 System

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