Simple fabrication of cellulose nanofibers via electrospinning of dissolving pulp and tunicate

Issei Otsuka, Christian Nana Njinang, Redouane Borsali

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Issei Otsuka, Christian Nana Njinang, Redouane Borsali. Simple fabrication of cellulose nanofibers via electrospinning of dissolving pulp and tunicate. Cellulose, 2017, 24 (8), pp.3281-3288. 10.1007/s10570-017-1360-4 . hal-02114427

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(Original article published in Cellulose 24, 3281–3288 (2017) by Springer Nature, DOI:
10.1007/s10570-017-1360-4)

Simple Fabrication of Cellulose Nanofibers via Electrospinning of Dissolving Pulp and
Tunicate

Issei Otsuka*, Christian Nana Njinang, Redouane Borsali*

Univ. Grenoble Alpes, CERMAV, F-38000 Grenoble, France

CNRS, CERMAV, F-38000 Grenoble, France

*Corresponding authors: Centre de Recherche sur les Macromolécules Végétales (CERMAV, UPR-CNRS 5301), BP53, 38041 Grenoble Cedex 9, France.

E-mail: issei.otsuka@cermav.cnrs.fr (I.O) and borsali@cermav.cnrs.fr (R.B.)

Tel: +33 (0)4 76 03 76 82 (I.O.) and +33 (0)4 76 03 76 27 (R.B.)

**ABSTRACT:** This work demonstrates a simple fabrication of cellulose nanofibers by direct electrospinning of dissolved cellulose solutions. The hard- and softwood pulps and the outer mantles of tunicate were dissolved in a mixture of trifluoroacetic acid and dichloroethane by stirring and ultrasonication to give highly viscoelastic clear solutions. These solutions were electrospun to form continuous nanofibers made of unsubstituted cellulose, which were confirmed by scanning electron microscopy (SEM) and IR spectroscopy. Statistical analysis of the SEM images of the nanofibers suggested that there are positive correlations between diameters of the nanofibers and concentration of the cellulose solution. The mean diameters of the nanofibers obtained from softwood pulp (DP of cellulose ≈ 1,200) solutions were larger than those from hardwood pulp (DP of cellulose ≈ 500) at the same concentrations. This
indicates that the DP of cellulose is one of the important parameters to control the diameters of
the electrospun cellulose nanofibers.

**KEYWORDS:** electrospinning, nanofiber, cellulose, dissolution, wood pulp, tunicate

**Graphical Abstract:**

<figure><img src="image_2.png" /><figcaption>**Electrospun Nanofibers**
(plant-derived cellulose)</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>plant</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>animal</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Electrospun Nanofibers**
(animal-derived cellulose)</figcaption></figure>
INTRODUCTION

Cellulose, the most abundant organic polymer on earth, has been attracted huge range of interest of scientists. Recently, there has been a great focus on nano-structured celluloses, so- called “nanocellulose”, such as microfibrillated cellulose, nanocrystalline cellulose, and bacterial nanocellulose as new bio-based macromolecular bricks not only for a replacement of petroleum-based materials but also for various potential applications in interdisciplinary fields of research and industry (Abitbol et al. 2016; Klemm et al. 2011). Nanocellulose combines general nature of cellulose and specific feature of nano scale materials originated basically with their large surface area to volume ratio due to the nano-scale diameter of the fibers. Such a unique property of nanocellulose has attracted academic and industrial interests, and thus numerous studies have been made to elucidate how nanocellulose can be isolated from plant- and non-plant-based sources. Today, several industrial companies produce nanocellulose at capacities beyond pilot plant scale. However, the production cost of nanocellulose is still high enough to deter industry for practical applications. Therefore, there is an urgent need to develop facile and cost-effective method of nanocellulose fabrication.

Electrospinning is a simple and versatile method that uses strong electric fields to draw polymer solutions or melts for the production of continuous fibers with diameters ranging from tens to hundreds nanometers. Nanofibers and nonwovens produced by electrospinning of natural polymers including proteins, nucleic acids, and polysaccharides have attracted ever increased attention for their diverse potential in technical and biomedical applications such as functional textiles, filtration membranes, tissue engineering scaffolds, wound healing, drug delivery, etc. (Khadka and Haynie 2012; Li et al. 2005; Schiffman and Schauer 2008; Sill and von Recum 2008). Among them, the electrospinning of polysaccharides, especially cellulose and its derivatives, have been widely studied from the beginning of the electrospinning technology (Ohkawa 2015). Since cellulose is insoluble in most organic solvents, most of the
studies focused on cellulose derivatives, e.g. cellulose acetate, to electrospin using volatile
solvents, followed by deacetylation to regenerate cellulose nanofibers (Liu and Hsieh 2002).
On the other hand, only few attempts have been made for direct electrospinning of dissolving
cellulose (Freire et al. 2011; Kulpinski 2005; Ohkawa 2008; Quan et al. 2010). In particular,
electrospinning of cellulose derived from animals, e.g. tunicate cellulose, has never reported
probably due to its low solubility arisen from higher molecular weight and crystallinity
comparing to general plant-derived cellulose (Eichhorn et al. 2009).

Dissolving native cellulose has been one of the most fundamental challenges for scientists,
and thus various kinds of solvents for cellulose has been reported so far (Cai and Zhang 2005;
Chanzy et al. 1980; Heinze et al. 2000; McCormick et al. 1985; Zhang et al. 2002).
Trifluoroacetic acid (TFA) is one good example of nonaqueous solvents for cellulose. Unlike
the other nonaqueous solvents such as DMSO, DMF, and ionic liquids, TFA has high volatility,
which is a great advantage for electrospinning. According to the report by Patel and Gilbert
(Patel and Gilbert 1981), the solubility of cellulose increases by mixing TFA with 1,2-
Dichloroethane (DCE) in the ratio of TFA:DCE = 7:3 (v/v) by forming cholesteric liquid-
crystalline phase. Hence, the plant- and the animal-derived cellulose were dissolved in the
mixture of TFA and DCE for electrospinning in this study. Herein, we demonstrate (1)
electrospinning of dissolved hard- and softwood pulps and outer mantles of tunicate in the
mixture of TFA and DCE (TFA:DCE = 7:3 (v/v)) and (2) characterization of obtained fibers by
means of Fourier transform infrared (FTIR) and scanning electron microscopy (SEM).

## EXPERIMENTAL

**Materials.** Hardwood pulp (content: cellulose 95% and hemicellulose 5%, degree of polymerization (DP) of cellulose calculated from the intrinsic viscosity: *ca.* 500) and softwood pulp (content: cellulose 96% and hemicellulose 4%, DP of cellulose calculated from the
intrinsic viscosity: ca. 1200) were gifts from Södra Co., Växjö, Sweden and TEMBEC Co.,
Montreal, Canada respectively and used as received. Outer mantles of tunicate, *Halocynthia
roretzi*, cultured in Onagawa, Miyagi, Japan, were gift from Okasei Co. Ltd., Japan.
Trifluoroacetic acid (TFA, purity: 99%) was purchased from Sigma-Aldrich Chemicals Co., St.
Louis, U.S.A. and used as received. 1,2-Dichloroethane (DCE, purity: 99.99%) was purchased
form Carlo Erba Reagents Co., Milano, Italy and used as received. Disposable needles used for
the electrospinning with their inner diameters 0.84 mm and 0.51 mm were purchased from
Nordson Co., Westlake, U.S.A. and used as received.

**Preparation of Spinning Solution.** The hardwood and softwood pulps were dissolved in a mixture of TFA and DCE with various concentrations. A typical procedure was as follows: the hardwood pulp (0.108 g) was added to a 5 mL of the mixture of TFA/DCE (7/3, v/v) in a round flask, then the mixture was stirred for 3 days at room temperature to obtain a clear viscous solution (1.5 w%) ready to be electrospun.

Tunicate-derived cellulose was purified from the outer mantles of tunicate by alternating treatment with 1% sodium chlorite solution in acetate buffer (pH 5.4) and 5% NaOH aqueous solution. The sample was further cut into small pieces of about 5 mm and fibrillated by double cylinder type homogenizer in water. The suspension was filtered and the filtrate was washed with distilled water several times, and then put into acetone and stirred for 2 days. The suspension was filtered and the filtrate was dried in vacuum for 1 night just before dissolution. There were two different methods for the dissolution of tunicate-derived cellulose. The first method was as follows: the purified tunicate-derived cellulose (0.022 g) was added to a 5 mL of the mixture of TFA/DCE (7/3, v/v) in a round flask, then the mixture was stirred for 40 days at room temperature to obtain a clear viscous solution (0.3 wt%) ready to be electrospun. The second method was as follows: the purified tunicate-derived cellulose (0.072 g) was added to 1.5 mL of DCE in a round flask, and then the mixture was exposed to ultrasonic wave for 12 h
at room temperature. TFA (3.5 mL) was added to the mixture and stirred for 18 days, while exposing to ultrasonic wave from time to time, to obtain clear viscous solution (1.0 wt%) ready to be electrospun.

**Electrospinning.** The electrospinning was performed using Esprayer ES-2000S2A equipment (Fuence Co., Ltd., Tokyo, Japan) at room temperature (c.a. 25 °C) and humidity (c.a. 30%). A metallic electrode plate wrapped with aluminum foil were placed 7-14 cm (depending on the sample) away from the needle. High voltage (20-30 kV) was applied between the needle and the electrode plate while the cellulose solution flows with the flow late of (20-40 µL/min) from the needle to collect the electrospun fibers on the aluminum foil. The applied voltage and the flow late were precisely controlled to keep stable cone-like shaped structure of the cellulose solution on the tip of the needle during electrospinning process.

**Characterization of the Electrospun Fibers.** Fourier transform Infrared (FTIR) spectra of the nanofibers were obtained with an attenuated total reflection (ATR) accessory coupled to a Spectrum Two FTIR spectrometer (PerkinElmer Inc., Waltham, U.S.A.). Morphology of the nanofibers was characterized by scanning electron microscopy (SEM) using a QUANTA FEG 250 scanning electron microscope (FEI Co., Hillsboro, U.S.A.) operating at an accelerating voltage of 2.5 kV. The electrospun fibers on the aluminum foil were coated with a 4 nm thick layer of lead/gold (Pb/Au) prior to SEM observation. The diameters of the fibers and their distributions were statistically analyzed for at least 50 fibers from the SEM image by using ImageJ open-sourced software.

## RESULTS AND DISCUSSION

**Dissolutions of Wood Pulps and Tunicate.** The hard- and softwood pulps and the tunicate were dissolved in the mixture of TFA and DCE. TFA is one of the highly volatile solvents of cellulose, which is an advantage for electrospinning comparing to other solvents such as
DMSO, DMF, ionic liquids, *N*-methylmorpholine-*N*-oxide, etc. In fact, cellulose is dissolved in TFA by forming trifluoroacetyl esters selectively at the C6 hydroxyl groups (Hasegawa et al. 1992). By mixing TFA with chlorinated alkanes such as DCE, the solubility of cellulose increases (Patel and Gilbert 1981). Here, it should be noted that the trifluoroacetyl groups in cellulose are not stable in air and they can be gradually hydrolyzed with moisture, and TFA thus formed is removed by natural evaporation (Hasegawa et al. 1992) once the cellulose solution is electrospun and forms nanofibers. Accordingly, the electrospinning using TFA/DCE as a mixed solvent can be carried out with dry process while previously reported electrospinning systems using ionic liquids (Freire et al. 2011; Quan et al. 2010) or *N*-methylmorpholine-*N*-oxide (Kulpinski 2005) needed wet process where the electrospun fibers were collected on the electrode in the water coagulation bath.

Both the hard- and softwood pulps were completely dissolved by stirring in the mixed solvent of TFA and DCE (TFA/DCE = 7/3 (v/v)) at room temperature for 3 days (when the concentration was in range from 1.2 to 2.0 wt%) to give viscoelastic clear solutions. The dissolution of purified tunicate takes longer time due to higher molecular weight and crystallinity of tunicate-derived cellulose than wood pulp-derived cellulose (Ono et al. 2016; Tamai et al. 2004). The 0.3 wt% tunicate solution was obtained after stirring at room temperature for 40 days in the mixed solvent of TFA and DCE. The 1.0 wt% tunicate solution was obtained by combination of ultrasonication and stirring in the mixed solvent at room temperature for 18 days.

**Electrospinning.** The electrospinning of obtained wood pulp- and tunicate-derived cellulose solutions were carried out at ambient temperature and humidity. The distance between the electrodes (7-14 cm), the applied voltage (20-30 kV), and the flow rate of the solution (10-40 µL/min) were adjusted for each experiment to form cone-like shaped structure of the solution on the tip of the needle, which indicates stable electrospinning process. Firstly, we investigated
the electrospinning of the hardwood pulp (DP of cellulose ≈ 500) solutions of various concentrations. The 1.2 wt% solution was not electrospinnable due to its very low viscosity and gave only electrosprayed beads. On the other hand, the 1.3 wt% solution was stably electrospun and gave fibers including some beads as shown in the SEM image of the corrected sample (Figure 1a). By increasing the concentration to 1.5 wt%, the amount of beads decreased (Figure 1b) and well-defined fibers were observed in the corrected samples from 1.6, 1.75, and 1.9 wt% solutions as shown in Figure 1c, 1d, and 1e, respectively. However, the 2.0 wt% solution was not electrospinnable due to its very high viscosity and surface tension.

The electrospinning of the softwood pulp (DP of cellulose ≈ 1200) solution was then investigated. Similar to the hardwood pulp, the softwood pulp solutions of the concentrations below 1.3 wt% were not electrospinnable due to their low viscosity. On the other hand, the solutions of 1.3-1.6 wt% gave well-defined fibers (Figure 1f-1i). The 0.3 wt% tunicate solution was not electrospinnable due to its low viscosity, while electrospinning of the 1.0 wt% tunicate solution gave well-defined fibers as shown in Figure 1j.
<figure><img src="image_6.png" /><figcaption>**Figure 1.** SEM images of the electrospun nanofibers obtained from hardwood pulp (a-e), softwood pulp (f-i), and tunicate (j) solutions with various concentrations.</figcaption></figure>
**Characterization of the Electrospun Fibers.** The chemical characterization of the electrospun fibers was carried out by ATR-FTIR. As shown in Figure 2, the IR spectra of the electrospun fibers obtained from the hardwood pulp, the softwood pulp, and the tunicate solutions showed typical absorption bands of cellulose, e.g. O-H stretching vibration at 3354 cm⁻¹, C-H stretching vibration at 2889 cm⁻¹, absorbed water at 1643 cm⁻¹, ring breathing at 1155 cm⁻¹, and C-O stretching vibration at 1016 cm⁻¹. In contrast, no absorption bands due to C=O stretching vibrations of trifluoroacetyl ester groups around 1790 cm⁻¹ were observed in the IR spectra of all the fibers. This indicates that the trifluoroacetyl esters formed at the C6 hydroxyl groups of cellulose in TFA/DCE solution had been hydrolyzed with moisture in the air during formations of the fibers and deposits on the electrode plate wrapped with aluminum foil. Therefore, these obtained electrospun fibers are made of unsubstituted cellulose derived from hard- and softwood pulps and tunicates.
<figure><img src="image_7.png" /><figcaption>**Figure 2.** ATR-FTIR spectra of the electrospun nanofibers obtained from hardwood pulp (top), softwood pulp (middle), and tunicate (bottom) solutions.</figcaption></figure>

The diameters of the electrospun fibers were estimated from the SEM images and summarized as histograms shown in Figure 3. The diameters of the fibers ranged from 16 to 270 nm depending on the source of cellulose and the concentration. The mean diameters and the diameter ranges of the fibers were also determined and listed in Table 1. The mean fiber diameters of the fibers obtained from both hard- and softwood pulp solutions are increased according to the concentrations of the electrospinning solutions as shown in Figure 4. This result supports general trend between concentration and diameter observed in electrospinning of any polymer solutions. The mean diameters of the fibers made from softwood pulp (DP of cellulose ≈ 1200) are larger than those made from hardwood pulp (DP of cellulose ≈ 500) when
the concentrations of the both spinning solutions are the same as shown in Figure 4. This result
suggests that the DP of cellulose is one of the important parameters for the diameter of
electrospun cellulose nanofibers. Further investigation to control the diameter of cellulose
nanofibers is in progress and will be discussed in a forthcoming paper.
<figure><img src="image_8.png" /><figcaption>**Figure 3.** Histograms of the diameter distribution of the electrospun nanofibers obtained from hardwood pulp (a-e), softwood pulp (f-i), and tunicate (j) with various concentrations.</figcaption></figure>
<table><caption>**Table 1.** The diameter ranges and the mean diameters of the nanofibers obtained from different cellulose solutions</caption><thead><tr><th>sample</th><th>concentration (%)</th><th>diameter range (nm)</th><th>mean diameter (nm)</th></tr></thead><tbody><tr><td rowspan="5">hardwood pulp (DP 500)</td><td>1.3</td><td>18 - 80</td><td>40</td></tr><tr><td>1.5</td><td>16 - 97</td><td>46</td></tr><tr><td>1.6</td><td>20 - 187</td><td>60</td></tr><tr><td>1.75</td><td>18 - 186</td><td>68</td></tr><tr><td>1.9</td><td>40 - 203</td><td>96</td></tr><tr><td rowspan="4">softwood pulp (DP 1200)</td><td>1.3</td><td>39 - 155</td><td>78</td></tr><tr><td>1.4</td><td>17 - 167</td><td>80</td></tr><tr><td>1.5</td><td>24 - 202</td><td>83</td></tr><tr><td>1.6</td><td>22 - 270</td><td>84</td></tr><tr><td>tunicate</td><td>1.0</td><td>20 - 177</td><td>61</td></tr></tbody></table>
<figure><img src="image_9.png" /><figcaption>**Figure 4.** Variation of the mean diameters of the nanofibers obtained from hardwood pulp (green circle) and softwood pulp (blue square) as function of their concentrations.</figcaption></figure>

CONCLUSION

In this study, we described the fabrication of cellulose nanofibers by electrospinning of dissolved hard- and softwood pulps and tunicate in the mixture of TFA and DCE (TFA/DCE = 7/3 (v/v)). The high solubility of cellulose in the mixture of TFA and DCE, which are both highly volatile solvents, was the key of the successful direct electrospinning of both plant- and animal-derived cellulose. The obtained nanofibers were made of unsubstituted cellulose and the mean diameters ranged from 40 to 100 nm depending on the source and the DP of the cellulose as well as the concentration of the electrospun solution. To the best of our knowledge, this is the first report on the direct electrospinning of dissolved plant- and animal-derived cellulose in the mixture of TFA and DCE. Notably, the nanofibers of tunicate-derived cellulose, which is an abundantly available biomass from the sea, were successfully obtained by electrospinning for the first time. These simply fabricated cellulose nanofibers will broaden the versatility of nanocellulose for various applications in interdisciplinary fields such as nano-biotechnology.
ACKNOWLEDGMENT

This work was financially supported by the Institut Carnot PolyNat (ANR-11-CANR-0007-
01), ANR "sweetmemory" project (ANR-14-CE08-0021), and the CNRS. The authors thank
Dr. B. Carré, Dr. B. Arnoul-Jarriault, and Dr. D. Lachenal for providing the soft- and hardwood
pulp samples. The authors also thank Dr. Y. Nishiyama for providing the tunicate samples and
Dr. C. Lancelon-Pin for SEM observation.

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