Production of Nanocellulose from Pineapple Leaf Fibers via High-Shear Homogenization and Ultrasonication

: In this study, the isolation and characterization of nanocellulose from pineapple leaf ﬁbers (PLF) were carried out. Chemical pretreatment included pulping, bleaching, and acid hydrolysis to remove lignin, hemicellulose, and extractive substances were conducted. This was followed by high-shear homogenization and ultrasonication to produce nanocellulose. Morphological changes to the PLF due to treatment were investigated using scanning electron microscopy (SEM). This showed that the PLF had a diameter of 1–10 µ m after high-shear homogenizing. Transmission electron microscopy (TEM) indicated that the nanoﬁbers after ultrasonication for 60 min showed 40–70 nm diameters. Particle size analysis (PSA) indicates that the ﬁbers had an average diameter of 68 nm. Crystallinity index was determined by X-ray diffraction (XRD) and had the highest value after acid hydrolysis at 83% but after 60 min ultrasonication, this decreased to 62%. Meanwhile, Fourier transform infrared (FTIR) spectroscopy showed there was no chemical structure change after acid hydrolysis. The most signiﬁcant ﬁnding from thermal gravimetric analysis (TGA) is that the higher degradation temperature of nanoﬁbers indicates superior thermal stability over untreated ﬁber. These results indicate that PLF waste could become a viable source of commercially valuable nanocellulose. high-shear homogenization, and ultrasonication.


Introduction
Over the last few decades, research into the development of biodegradable materials from renewable sources has led to an increasing interest in biocomposites manufactured from various natural fibers. These natural fibers are abundant and renewable, and their utilization can minimize negative environmental effects and are able to generate profits [1][2][3].
Natural fibers consist of lignin, cellulose, and hemicelluloses. Lignin and hemicelluloses are amorphous in structure while cellulose is semicrystalline and thus, differs in its physical and mechanical properties [4]. Cellulose is used to manufacture textiles and paper. It is a long linear glucose polymer linked by β-1,4 glycosidic bonds [5]. Cellulose has relatively high strength, high stiffness, low density and good thermal stability [2]. Therefore, it is a promising candidate for reinforcing biocomposites.
To obtain pure cellulose, the raw material is treated with alkali and bleached [3,6,7]. This chemical treatment breaks intermolecular and intramolecular hydrogen bonding between the hydroxyl groups of cellulose and hemicellulose and can increase the hydrophilicity of fibers [2,3]. However, alkali treatment and bleaching do not significantly increase the crystallinity of cellulose fiber. Some studies suggest that subsequent acid hydrolysis can increase crystallinity and reduce the diameter of fibers [8,9].
Pineapple cultivation occurs throughout the South East Asian region. The Philippines, Thailand, and Indonesia are three of the largest producers of this crop globally. In 2014, in Indonesia alone, 1.8 million tons of fruit were harvested [21]. Old leaves must also be removed regularly. The weight of these leaves is about 7% that of the fruit. Hence, in Indonesia, 126,000 tons of fiber-rich pineapple leaves are removed from the plants each year. PLF from these leaves are used for mulch but much of this resource is yet to be used optimally. One possible way to increase the profitability of pineapple growing would be to use these discarded leaves commercially to produce nanofiber for biocomposite reinforcement.
PLF has a unique combination of properties that make it an ideal source for nanofiber production such as has high strength, stiffness and low density [1,3]. Its cellulose content is higher than that of OPEFB [22], water hyacinth and mengkuang (screw pine) leaves [23]. Cherian et al. reported that PLF has 81.27% cellulose, 12.31% hemicellulose, 3.46% lignin and 10.52% moisture content [9]. However, these values will depend on the variety and environmental factors where the plant is grown.
It has been shown that high-shear homogenization is a more environmentally friendly method for production of nanocellulose than chemical treatment and results in higher crystallinity, thermal stability, and transparency. Moreover, nanocellulose production via ultrasonication is a very effective way to depolymerize cellulose [8]. The method used in this study is new and uses both high-shear homogenization and ultrasonication. This has several advantages compared to preparation methods of PLF used in previous studies as it requires only two repeats of the bleaching and acid hydrolysis processes compared to the six-step bleaching and eight-step hydrolysis Cherian et al. used for PLF fiber [9]. This, in turn, minimized the use of expensive and potentially environmentally damaging chemicals. Furthermore, the crystallinity index of the PLF in this study is higher and the size of the nanofibers is comparable.
The characteristics of PLF before and after treatments were observed by using Fourier Transform Infrared (FTIR), X-ray Diffraction (XRD), Thermal Gravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), Particle Size Analysis (PSA) and Transmission Electron Microscopy (TEM).

Preparation of Cellulose Fibers from Pineapple Leaf Fibers
The cellulose was isolated based on previously published techniques with certain modifications [14].
Pulping procedure. Two-hundred grams of dried PLF was cut into 15 mm lengths then cooked in a digester with 25% NaOH solution for 60 min at 150 • C and 9 bar. The ratio of distilled water to fibers used was 10:1. The fiber was washed with distilled water until pH 7 and dried in a drying oven at 60 • C.
Bleaching process. The fiber was broken up in a blender for 10 min and mixed with NaClO 2 : CH 3 COOH (4:1) at 60 • C and 600 rpm for 2 h. Then, it was soaked again in 5% NaOH solution at 50 • C for 4 h. The suspension of bleached fiber was washed to pH 7 before acid hydrolysis.
Acid hydrolysis. The suspension was hydrolyzed with 3.5M HCl at 50 • C for 12 h then was washed until pH 7 followed by hydrolyzation, a second time with 7.5M HCl at 50 • C overnight. It was washed until a neutral pH was achieved.

Preparation of Nanocellulose from Pineapple Leaf Fibers
High-shear homogenization. High-shear homogenization was performed to reduce the diameter size of the PLF in suspension. A WiseTis Homogenizer HG-15D DAIHAN Scientific Co., Ltd. from Gangwon-do, Korea was used. The diameter of the stator and rotor were 180 and 130 mm, respectively. A 5% suspension was dispersed in 100 mL of distilled water then homogenized at 12,000 rpm at room temperature for 10 min.
Ultrasonication. Ultrasonication was performed with an ultrasonic cell crusher Ningbo Yinzhou SJIA Lab Equipment Co., Ltd. (Ningbo, China) using 400 W. One-hundred milliliters of the suspension were sonicated for 30 and 60 min at 60 • C.

Chemical Composition of Pineapple Leaf Fibers
The chemical composition of PLF before and after chemical treatments was observed. Cellulose and lignin content were measured by using Technical Association of the Pulp and Paper Industry (TAPPI) Standard T 9M-54 and TAPPI Standard T 13M-54, respectively. The holocellulose content, a combination of cellulose and hemicellulose content, was measured according to American Standard Testing Materials (ASTM) 1104-56.

X-ray Diffraction
All samples were dried in a drying oven at 60 • C before XRD was conducted to determine the index of crystallinity of PLF at each stage in the process (bleaching, acid hydrolysis, high-shear homogenizing and ultrasonication). XRD patterns of all samples were examined on a X'Pert PRO PANalytical instrument (Philips Analytical, Almelo, Netherlands), using CuKα radiation (λ = 0.154), 40 kV operating voltage and 30 mA. Intensity at 2θ was recorded from 10 to 30 • . The crystallinity index (CrI) was calculated by using the Segal method (Equation (1)).

Fourier Transform Infrared
FTIR characterization was performed by using a Perkin-Elmer FTIR spectrometer Frontier instrument (PerkinElmer, Inc., Waltham, MA, USA) with wave numbers of 600-4000 cm −1 . Before characterization, all samples were dried in an oven, drying at 50 • C until a constant weight was achieved.

Scanning Electron Microscopy
The surface morphology of the PLF fiber was investigated by using SEM. All samples were dried in dying oven until constant weight. The operation voltage during characterization was 10 kV.

Transmission Electron Microscopy
Five percent PLF cellulose was suspended in 100 mL distilled water. After that, it was ultrasonicated for 60 min to produce nanocellulose fibers. Its morphology was observed by using JEM-1400 Transmission Electron Microscopy (JEOL Ltd., Akishima, Tokyo, Japan) at 100 keV. The nanocellulose suspension was dropped onto a carbon film over a copper grid and then dried. The dried samples were directly observed via TEM at room temperature.

Thermal Gravimetric Analysis
Measurements of thermal stability of untreated and treated PLF were carried out by using DTG-60 SHIMADZU serial no. C30565000570 (Kyoto, Japan). The thermal analysis was done in a nitrogen atmosphere at a flow rate 50 mL/min. The heating rate was 20 • C/min.

Particle Size Analysis
The particle size distribution after 60 min ultrasonication was tested with a Malvern Zetasizer Nano ZS instrument (Malvern Instruments Limited, Worcestershire, UK). Samples of nanocellulose suspended in water were diluted and analyzed with a particle size analyzer using dynamic light scattering (DLS). Non Invasive Back Scattering technique was used with a 173 • detector angle using a HeNe 4 mW laser (633 nm). Measurements were repeated 3 times for each sample. Table 1 shows the main chemical composition of PLF at each stage of chemical treatment. Raw PLF has the largest percentage of hemicellulose, lignin, and extractive substances. The bleaching process reduced the amount of lignin and hemicellulose from 13.8% to 10.2%. This is due to the breaking of hemicellulose-lignin bonds and some lignin was hydrolyzed.

Main Composition of Pineapple Leaf Fibers before and after the Chemical Treatment
Hydrolyzed PLF consists of 81.3% cellulose, a similar figure to that contained in more intensively chemically treated (five cycles of treatment) wood, but lower than that of bamboo, wheat straw and flax fiber [8]. After acid hydrolysis, fiber diameter size decreased with the cellulose content was 20% higher than in the untreated PLF. This was due to removal of the amorphous content of the fiber which increased the crystalline cellulose fraction. The increasing proportion of crystalline cellulose leads to increases in thermal stability. This result is supported by XRD and TGA results and previous work [8,15,32].

Physical of Pineapple Leaf Fibers at Different Treatment
The physical structure of PLF before and after treatments is shown in Figure 1. Figure 1a shows the raw fiber. It is a brownish color due to the lignin, hemicelluloses, and extractive substances content as reported by previous studies [27]. After bleaching, PLF became white as the lignin and hemicellulose were removed during the process (Figure 1b). The removal of lignin and hemicellulose is also evident in the XRD and chemical composition results. Acid hydrolysis and high-shear homogenization results in a white suspension (Figure 1c,d) which indicates the pure cellulose content. Figure 1e shows the nanocellulose suspension after 60 min ultrasonication. The suspension was transparent due to the nano-sized fiber particles that are too small to reflect light. A similar phenomenon was observed in OPEFB nanofibers [16].  Figure 2 shows the XRD curve of all samples. Peaks appear at 2θ = 18 • and 22.6 • , suggesting the characteristic of amorphous and crystalline regions, respectively [31]. There is a significant difference in peak intensity between raw fiber and 60 min ultrasonicated fiber (Figure 2) indicating differences in crystallinity index [16]. The crystallinity index of raw fiber was 41.47% which was lower than any treated fiber (Figure 2 and Table 2). This is due to removed non-crystalline components and has been previously reported [2].  The highest index crystallinity was after acid hydrolysis (Table 2) as the amorphous content of the fiber was hydrolyzed and hemicellulose and lignin content were reduced so increased leaving a larger semicrystalline cellulose fraction [16,17]. After mechanical treatment, the crystallinity decreased. XRD measurements show that homogenization and ultrasonication result in fiber with a lower crystallinity index than acid hydrolysis. This is an indication that the intermolecular hydrogen bonds between the cellulose chains were broken destroying the crystalline structure of cellulose [8,29,30,32].

Fourier Transform Infrared
FTIR characterization was used to identify functional groups present in the chemical structure such as alkanes, esters, aromatic, and alcohols. Figure 3 and Table 3 present the FTIR spectra and functional groups identified in untreated and treated fiber. All samples show peaks at wavenumbers around 3328 cm −1 and 2900 cm −1 due to stretching vibration in hydroxyl (OH) and C-H groups, respectively [4].  The characteristic peak of lignin's aromatic ring vibration presents at wavenumber 1200-1300 cm −1 . This peak disappeared after chemical and mechanical treatment. This is due to the removal of lignin during the pulping and bleaching [8,17,32]. The OH group water absorption peak at around wavenumber 1640 cm −1 appears in all samples as expected from other studies [8,32]. Figure 4 shows SEM and TEM images of the morphological surface of PLF fiber before and after treatments. Figure 4a is an SEM image of raw PLF showing the fiber has a 50 µm diameter appears smooth and microfibrils are still bound to one another due to the presence of lignin and hemicellulose components [33].

Morphology of Different Treatment of Pineapple Leaf Fibers
After bleaching treatment ( Figure 4b) the bonding between lignin and hemicellulose has been broken due to the removal of the amorphous content resulting in microfibril bundles. This explanation is also supported by XRD crystallinity index data. The SEM image of PLF after acid hydrolysis treatment shows a reduction in fiber size (Figure 4c). The PLF cellulose consists of microfibril bundles with diameters of around 1-5 µm (Figure 4c). This is the result of depolymerization and has also been observed in previous research [17]. The SEM image in Figure 4d shows the morphological surface of PLF cellulose after homogenizing with reduced PLF diameter.

Morphological Properties of Nanocellulose
Figure 4e is a TEM image of PLF cellulose after high power intensity ultrasonication for 60 min. It shows nanocellulose appearing as individual 40-70 nm diameter thin particles with 88-1100 nm lengths. These results are similar to Zhao et al. (2013) who demonstrated that high-shear homogenization for 2 h was able to damage cellulose chains resulting in nanoparticle production.
Six-hour and ten-hour ultrasonication of cellulose chains has also been shown to result in 20-50 nm diameter nanoparticles [34,35]. The present study demonstrates nanocellulose production after a significantly shorter timeframe than has been reported previously.

Thermogravimetric Analysis and Derivative Thermogravimetry
Thermal stability of the PLF after each treatment was tested using thermogravimetric analysis from 35-550 • C. Thermogravimetric analysis (TGA) and the derivative thermogravimetry (DTG) curve are shown in Figure 5a,b, respectively. There are three regions of temperature degradation which occur in around 100 • C, 250-450 • C, and 450-550 • C, respectively.
The weight loss around 100 • C is likely attributed the evaporation water [22]. This is shown as a small trough on the left side of the DTG curve. All samples show a major weight loss in the 250-450 • C region as the hemicellulose and glycosidic linkages in cellulose were depolymerized and broken [14]. The onset degradation temperature of raw, bleached, acid hydrolysis fiber, high-shear homogenization, and ultrasonication were 215, 230, 300, 310, and 320 • C, respectively as shown in the sharp troughs in the DTG curve ( Figure 4b). Raw fiber has lower thermal stability than treated fiber.
From 450-550 • C (region 3) all samples were decomposed completely to ash [32]. However, raw fiber has more residue than treated fiber because of the hemicellulose content still left behind in fiber. This result is expected as similar trends have been observed in previous studies [32,36]. The most significant finding from TGA is that the higher degradation temperature of ultrasonicated fiber (320 • C) indicates superior thermal stability over untreated fiber (215 • C).

Particle Size Analysis
Fiber size after 60 min ultrasonication was determined using PSA. The nanofiber size distribution in a suspension (1 wt %) as measured by dynamic light scattering analysis is shown in Figure 6. The average diameter of these fibers was 68 nm and 91% were less than 100 nm which is the nanofiber range. The particle size distribution is relatively narrow. These results are further evidence of the successful production of nano sized cellulose fibers both in terms of length and width. Furthermore, the particle size distribution shows that the nanofibers obtained from PLF with mechanical treatment are relatively homogeneous. A similar range of fiber size was also reported for Helicteres isora nanofiber processed using steam explosion and homogenization [17].

Conclusions
PLF nanocellulose has been successfully fabricated using a new method. A combination of high-shear homogenization and ultrasonication reduced the need for extensive chemical pretreatment. Single stage pre-bleaching and acid hydrolysis were sufficient to produce microcellulose with a high degree of purity. This treatment is more environmentally friendly than previous methods using repeated bleaching [9]. One-hour sonication was sufficient to produce high purity nanocellulose fibers with an average of 68 nm diameters and length less than 88-1100 nm. Chemical analysis and FTIR testing of PLF indicates that most lignin, hemicelluloses, and extractive substances have been eliminated. The crystallinity of the nanocellulose fiber is 50% higher than that of the raw fiber and the thermal stability has increased to yield degradation temperatures around 320 • C. These properties suggest PLF nanofiber may be an effective reinforcing material for biocomposites.