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Chen, Y. W., Lee, H. V., and Abd Hamid, S. B. (2016). "Preparation of nanostructured cellulose via Cr(III)- and Mn(II)-transition metal salt catalyzed acid hydrolysis approach," BioRes. 11(3), 7224-7241.


Nanostructured cellulose was successfully prepared from native cellulose using a homogeneous catalytic H2SO4 hydrolysis pathway in the presence of Cr(III)- and Mn(II)-transition metal salts as the co-catalyst. The effect of transition metal salts with different valence states (Cr3+ and Mn2+) on the physicochemical properties (chemical characteristics, crystallinity index, nano-structure, thermal stability, and morphology) of prepared nanocellulose was investigated. Interestingly, TEM micrographs showed that the Cr(III)-treated and Mn(II)-treated nanocellulose exhibited a web-like nanostructured-surface with average diameters of 44.7 ± 13.2 nm and 58.4 ± 15.3 nm, respectively. XRD study revealed that the crystallinity of nanocellulose was increased because the catalytic degradation of the less crystalline regions of cellulose occurred at a faster rate than its crystalline phases. Cr(III)-treated nanocellulose was capable of rendering a higher crystallinity index (75.6 ± 0.1%) compared with Mn(II)-treated nanocellulose (72.3 ± 0.4%). Furthermore, a dynamic light scattering (DLS) study revealed that Cr(III)-treated nanocellulose showed a smaller distribution range (92% at 14 to 135 nm) compared with Mn(II)-treated nanocellulose (92% at 607 nm). A higher valence state for the Cr(III)-cation, with a trivalent state (+3), rendered a more effective hydrolysis reaction compared with the Mn(II)-cation, with a divalent state (+2), for preparing the nanocellulose.

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Preparation of Nanostructured Cellulose via Cr(III)- and Mn(II)-Transition Metal Salt Catalyzed Acid Hydrolysis Approach

You Wei Chen, Hwei Voon Lee,* and Sharifah Bee Abd Hamid

Nanostructured cellulose was successfully prepared from native cellulose using a homogeneous catalytic H2SO4 hydrolysis pathway in the presence of Cr(III)- and Mn(II)-transition metal salts as the co-catalyst. The effect of transition metal salts with different valence states (Cr3+ and Mn2+) on the physicochemical properties (chemical characteristics, crystallinity index, nano-structure, thermal stability, and morphology) of prepared nanocellulose was investigated. Interestingly, TEM micrographs showed that the Cr(III)-treated and Mn(II)-treated nanocellulose exhibited a web-like nanostructured-surface with average diameters of 44.7 ± 13.2 nm and 58.4 ± 15.3 nm, respectively. XRD study revealed that the crystallinity of nanocellulose was increased because the catalytic degradation of the less crystalline regions of cellulose occurred at a faster rate than its crystalline phases. Cr(III)-treated nanocellulose was capable of rendering a higher crystallinity index (75.6 ± 0.1%) compared with Mn(II)-treated nanocellulose (72.3 ± 0.4%). Furthermore, a dynamic light scattering (DLS) study revealed that Cr(III)-treated nanocellulose showed a smaller distribution range (92% at 14 to 135 nm) compared with Mn(II)-treated nanocellulose (92% at 607 nm). A higher valence state for the Cr(III)-cation, with a trivalent state (+3), rendered a more effective hydrolysis reaction compared with the Mn(II)-cation, with a divalent state (+2), for preparing the nanocellulose.

Keywords: Nanocellulose; Valence state; Crystallite structure; Selective catalytic hydrolysis; Morphology study

Contact information: Nanotechnology & Catalysis Research Centre (NANOCAT), Institute of Postgraduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia;

*Corresponding author:


Cellulose is a linear homo-polysaccharide composed of β-D-glucopyranose units linked with β-(1→4)-glucosidic linkages in the chair configuration. It is considered a renewable, environmentally friendly, and biocompatible raw material for the production of biodegradable products (Maepa et al. 2014). Nanocellulose can be prepared from natural cellulose via various hydrolysis treatments. Because of its nano-scale dimensions, nanocellulose exhibits various outstanding and excellent physicochemical properties compared with typical cellulose material, such as large specific surface area, high porosity, high aspect ratio, excellent tensile strength and modulus, and biodegradability (Cao et al. 2015). Also, the source material generally has a low cost. For these reasons, many studies have been carried out on the synthesis of nanocellulose fibers as reinforcement agents in polymer nanocomposites (Zimmermann et al. 2010). Environmentally friendly polymer composite materials based on cellulose have great advantages for emerging industrial applications such as applications in the electronic, biomaterial, water treatment, pharmaceutical, magnetic, and medical fields (Kalia et al. 2011; Chirayil et al. 2014). Chirayil et al. (2014) mentioned that the addition of nanocomposites into thermoplastic starch could enhance the tensile strength of the product significantly compared with pure isolated thermoplastic starch, and the addition of sugar beet chip nanofibrils isolated via high-pressure homogenization to a phenol formaldehyde and polyvinyl alcohol matrix demonstrated excellent reinforcement properties (Chirayil et al. 2014). Therefore, it is essential to isolate the nanocellulose from its micro-scale raw material with the purpose of maximizing the potential of the cellulose source to become the useful composite materials such as polyethylene oxide/cellulose nanocrystal (PEO/CNC) (Zhou et al. 2011) and poly(vinyl alcohol)/cellulose nanocrystal (PVA/CNC) (Huan et al. 2016).

Nanocellulose can be prepared from cellulosic materials via mechanical treatment, chemical treatment, or biological (enzymes) hydrolysis treatment (Fatah et al. 2014). Generally, a mechanical approach such as high-pressure homogenization, cryocrushing, ultrasonication, or grinding is the most direct method for the preparation of nanocellulose (Li et al. 2014b). However, the mechanical treatment pathway requires high energy consumption, repeated processing, or complex equipment (Lee et al. 2014). On the other hand, enzymatic hydrolysis is a costly treatment, the enzymes are difficult to recycle, and the treatment usually requires more time to complete the hydrolysis process (2 to 6 weeks) (Mora-Pale et al. 2011). In recent years, ionic liquids such as 1-butyl-3-methylimidazolium hydrogen sulfate (BmimHSO4) have been proposed as a potential hydrolyzing catalyst for cellulose depolymerization process, and the results showed that high crystallinity nanocellulose (95.8%) was isolated successfully from cotton linter model compound (84.4%) via ionic liquid catalyzed hydrolysis process by using BmimHSO4 at 90 oC for 1.5 h (Tan et al. 2015). In the same year, a new hydrolysis approach which comprised ethanol and peroxide solvothermal pretreatment followed by ultrasonic process has been developed by Li and his coworkers for isolation of nanocellulose (Li et al. 2016). They treated the wood flour with the proposed pretreatment under reaction conditions of 180 oC for 80 min, and the results revealed that the nanocellulose produced had higher crystallinity (79.37%) than raw material (70.72%) (Li et al. 2016). In spite of their prolific use, the capital cost for the nanocellulose production is one of the major concerns in the industrial applications. The utilization of ionic liquid is restricted for industrial scale applications due to most of the ionic liquids are expensive, incomplete chemical and toxicological data, and the process for recovery of the ionic liquid is a highly energy-consuming process (Wang et al. 2014). On the other hand, the application of ethanol and peroxide solvothermal hydrolysis approach for conversion of cellulose is always requires harsher conditions such as high temperature and longer pretreatment period (Li et al. 2016). Thus, from an economic viewpoint, this will eventually increase the capital cost of the production line and unfavorable for long term production in the industrial use.

For these reasons, strong acid hydrolysis by mineral acids such as sulfuric, phosphoric, and hydrochloric acid is a well-known method for the preparation of nanocellulose (Lee et al. 2014). The hydronium ions (H3O+) generated by mineral acid can effectively break down or hydrolyze the intramolecular and intermolecular bonding in cellulose fibers (Yahya et al. 2015). The depolymerization process of cellulose into its smaller dimensions normally happens faster on the defective crystalline parts in the long polymeric chains than the crystallinity phases as there are more active sites and defects regions for chemical reaction to be carried out (Usov et al. 2015). Although strong acid hydrolysis treatment is simple, some limitations still need to be considered, such as environmental pollution and difficulty in controlling the hydrolysis extent of cellulose, and the reaction is usually accompanied by uncontrollable over-degradation into undesired water-soluble monomers (e.g., glucose), as well as the corrosion of the reactor vessel equipment (Hamid et al. 2015). To overcome this, dilute or organic acids have been proposed to create a milder reaction, but such treatment normally requires a higher reaction temperature (>150 °C) or longer hydrolysis time (>1 h) (Alvira et al. 2010), and it was found to be less effective in reacting to the structure of lignocellulosic biomass (Yahya et al. 2015).

Recently, transition metal salt catalysts have received use as acid homogeneous catalysts for the cellulose hydrolysis process. Transition metal salt catalysts normally can be categorized based on their valence states, such as trivalent (FeCl3, Fe2(SO4)3, CrCl3, AlCl3) or divalent (FeCl2, FeSO4, CuCl2), and have high potential for acting as the hydrolysis catalyst for the degradation of the glycosidic linkage of cellulose during acid hydrolysis treatment (Liu et al. 2009; Yi et al. 2013). However, the valence state of the transition metal ion plays a significant role in hydrolysis efficiency. Li et al. (2015) reported that the crystallinity of nanocellulose was improved by approximately 19% from native cellulose after being treated by a trivalent Fe(III)-metal ion catalyst (Li et al. 2015). However, Mazlita’s study found that the divalent Ni(II)-inorganic salt with an optimum pH of 5.0 (1.0 M) was capable of increasing the crystallinity of the treated nanocellulose by approximately 7% compared with untreated cellulose (Yahya et al. 2015). The results of another study done by Li et al. (2013) to investigate the effect of Fe(III) and Cu(II) transition metal ions on the yield of nanocellulose indicated that nanocellulose treated with the ion with a higher oxidation state (Fe(III), which is trivalent) rendered an extra 7% higher crystallinity compared with a Cu(II)-catalyzed hydrolysis reaction (Li et al. 2013).

To achieve a technically feasible and high selectivity controllable hydrolysis pathway, Cr(NO3)3– and Mn(NO3)2 transition metal salts have been selected as a potential hydrolyzing agent for cellulose depolymerization process. Based on reported studies, Cr(III)- and Mn(II)-based transition metal catalysts have been found to have highly effective in breakage of extensive network of glycosidic linkage and hydrogen bonding within the cellulose matrix to separate cellulose fibrils. Shi et al. (2013) pointed out that, when the microcrystalline cellulose hydrolyzed by the [BMIM]Cl ionic liquid (9 mol%) in the presence of MnCl2 (3 mol%) as co-catalyst, a maximum HMF yield of 66.5% (selectivity 74.43%) was obtained under the reaction conditions of 120 oC and 1 h (Shi et al. 2013). On the other hand, Peng et al. (2010) reported that CrCl3transition metal salt was highly effective for conversion of cellulose into its simpler form (i.e.levulinic acid) under the optimized conditions of 200 oC for 180 min, with the maximum yield of 67 mol%. During the catalytic hydrolysis, the metal salt catalyst acts as a Lewis acid that possessed of high hydrolyzing ability to depolymerize the cellulose by disrupting its bonding system, and thus the enhanced the fermentable sugar yield as compared with dilute acid hydrolyzing alone (Nguyen and Tucker 2002). Therefore, by combining a Cr(NO3)3 metal ion catalyst with dilute H2SO4, one can potentially overcome the drawback of dilute acid hydrolysis since the combination of both pretreatment may initiate a synergistic effect, thus enhancing the hydrolysis efficiency of cellulose depolymerization into nanostructured cellulose under controllable reaction conditions. In fact, the advantage of using transition metal salt (e.g. Mn(II)- and Cr(III)-) instead of strong H2SO4 for acid hydrolysis is reducing acid consumption for depolymerization process, which prevent corrosive issues, increase the selectivity of nanocellulose product with lower severity conditions (mild temperature and shorter hydrolysis period).

Previous studies have reported on the utilization of different isolation methods for the production of cellulose nanoparticles. However, to the best of our knowledge, no studies considering on the isolation of solid intermediate crystallite nanocellulose via a Cr(III) and Mn(II)-catalyzed sulfuric acid hydrolysis process rather than water soluble reducing sugars. In addition, only limited studies have reported on the correlation effect between different valence states (+3, +2) transition metal salts and hydrolysis efficiency for producing nanocellulose with a higher crystallinity. In this context, the objectives of this study are as follows: (i) to explore the feasibility and the practicability of isolating nanocellulose from native cellulose via Cr(III)- and Mn(II)-catalyzed H2SO4 acid hydrolysis; (ii) to investigate the correlation between transition metal salts with divalent (Mn2+) and trivalent (Cr3+) catalysts on the efficiency of catalytic hydrolysis during cellulose depolymerization process; and (iii) to examine the changes in the physicochemical properties during conversion from macro- to nanocellulose in terms of crystallinity (X-ray diffraction; XRD), particle size distribution (dynamic light scattering; DLS), morphology (field emission scanning electron microscopy; FESEM, transmission electron microscopy; TEM, and atomic force microscopy; AFM), and thermal stability (thermogravimetric analysis; TGA). This study will explore the feasibility of using a new hydrolysis route to produce cellulose nanoparticles via a transition metal-catalyzed sulfuric acid hydrolysis approach that has the advantages of simplicity in operation and that no complicated equipment is required.



Alpha cellulose (white powder, analytical grade) was purchased from Sigma-Aldrich (USA). The chemical reagents used in this study, such as sulfuric acid (H2SO4, 95% to 97% purity), manganese (II) nitrate [Mn(NO3)2], and chromium (III) nitrate [Cr(NO3)3], were obtained from R&M Chemicals (Malaysia). All chemicals were used in their received form without further purification processing.


Preparation of nanocellulose

One gram of cellulose was treated with a mixture of 4 wt.% sulfuric acid and 25 mM of Mn(NO3)2 or Cr(NO3)3 transition metal salt catalyst. The suspension was heated to 80 °C for 45 min to complete the catalytic hydrolysis reaction. Subsequently, each sample was washed with cold deionized water and centrifuged repeatedly, followed by dialysis against distilled water until the pH of final suspension was around 4. The collected products were subjected to a freeze-drying process to obtain a fine white powder for further physicochemical characterization studies. The yield of nanocellulose was determined gravimetrically by drying the precipitate after the centrifugation (8000 rpm, 10 min) of 10 g aliquot at 40 oC under vacuum (Kos et al. 2014).

Physicochemical characterization

The Fourier transform infrared (FTIR) spectra of samples were analyzed with an FTIR spectrometer (Bruker IFS66/s, Germany) with a resolution of 4 cm−1 for 64 scans in the range of 4000 to 400 cm−1. The sample was mixed with KBr powder and compressed into a pellet with a ratio of 1:10 (w/w).

The crystalline structure of samples was analyzed using a wide-angle X-ray diffraction (WAXRD) powder diffractometer (PANalytical Empyrean, Netherlands) with a Cu-Kα radiation source. The X-ray diffractograms were recorded from 5° to 60° while operating at a power of 40 kV and a current of 40 mA. The crystallinity index (CrI) of the samples was calculated using Segal’s equation (Segal et al. 1959), as shown in Eq. 1,

CrI (%) = (I200Iam) / I200 × 100 (1)

where I200 and Iam refer to the intensity of the crystalline (2θ = 22.5°) and amorphous regions (2θ= 18.5°) of the samples, respectively.

The surface morphology of the samples was characterized using a field-emission scanning electron microscope (FESEM) (Hitachi SU8030, Japan). The analysis was performed at an accelerating voltage of 5 kV with the working distance of 4 mm and a secondary electron (SE) detector. The sample was coated with a layer of platinum using an auto-fine coater (JEOL JFC-1600, Japan) before analysis to prevent overcharging.

The particle dimensions and structure of nanocellulose were observed using a transmission electron microscope (TEM; Tecnai G2 F20 Series, FEI, USA), which was operated at an acceleration voltage of 200 kV. A diluted suspension (0.01 wt.%) of the sample was sonicated to disperse it and dropped on a copper grid. The average diameter of each nanocellulose fiber was estimated using Image-J software for 300 measurements.

Atomic force microscopy (AFM) imaging of the samples was visualized with a model Dimension 3100 scanning probe microscope (Brand Veeco, Russia). A drop of the sample suspension (0.001 wt.%) was placed on a silicon wafer surface and dried in a desiccator. When the excess liquid evaporated, the samples were observed in tapping mode at room temperature. The AFM measurements were determined with NanoScope Analysis software for 300 samples.

The thermal behavior of the sample was analyzed by a model Q500 brand TA (Australia) thermogravimetric analyzer (TGA). The sample was heated from room temperature to 600 °C at a constant heating rate of 10 °C/min under a nitrogen atmosphere.

The particle size distribution and zeta potential of the yielded nanocellulose was examined by dynamic light scattering (DLS) using a Zetasizer NanoZS Instrument (Malvern, model ZEN 3600, UK). The samples were diluted in water with a concentration of 0.1 wt.% and well-dispersed by sonication treatment before analysis was carried out. Triplicate tests were carried out and the average values were reported.


Chemical Structure Analysis (FTIR Study)

FTIR spectroscopic analysis of the native cellulose and chemically treated nanocellulose samples is shown in Fig. 1. The presence of the main peak at 3400 cm−1 is attributed to the O-H stretching band, caused by the hydrogen bonded hydroxyl group vibrations of cellulose I (Yahya et al. 2015). In addition, the peak at 2900 cm−1 corresponds to the aliphatic saturated symmetric C-H stretching vibration in cellulose (Hamid et al. 2015). The absorption peaks observed at 1640 and 1375 cm−1 are caused by the O-H bending mode of absorbed water and C-H bending within the polysaccharide aromatic rings of cellulose, respectively (Tan et al. 2015). FTIR peaks at 1430 cm−1 (CH2 scissoring motion in cellulose), 1161 cm−1 (C─C ring stretching band), 1110 cm−1(C─O─C pyranose ring stretching vibration), and 898 cm−1 (β─glycosidic linkages of cellulose glucose ring) are indicated as the typical bonds of cellulose I (Iβ) structure (Leung et al. 2011; Kumar et al. 2014).

As also illustrated in Fig. 1, similar FTIR patterns were found for Mn(II)- and Cr(III)-treated nanocellulose, which indicated that the chemical structures of nanocellulose were not altered after catalytic acid hydrolysis process, where the typical structure of the parent cellulose was being preserved (Chen et al. 2016a, b). As reported by Singha et al. (2011), when the metal ions absorbed on the yielded nanocellulose, the wavenumbers of the functional groups will be shifted (Singha et al. 2011). However, the wavenumber position of the isolated nanocellulose remained similar to native cellulose; this suggested that there is no any metal ion introduced to the surface of nanocellulose fiber. Furthermore, the absence of NO3‾ peak at the signal of 1384 cm-1indicated that the transition metal salt was completely removed during the centrifugation washing and dialysis process (Yahya et al. 2015).

Fig. 1. FTIR spectra of native cellulose, Mn(II)-, and Cr(III)-treated nanocellulose

Crystal Structure Analysis (XRD Study)

Figure 2 shows the XRD diffraction patterns obtained for cellulose and nanocellulose. The crystallinity of cellulose in the isolated nanocellulose is the main factor affecting its mechanical strength in the polymer matrix (Chirayil et al. 2014). Nanocellulose showed similar diffraction patterns to those of native cellulose, with diffraction peaks at 2θ = 16.5°, 22.5°, and 34.6°, which can be accredited to the typical cellulose I structure (Yahya et al. 2015). This suggests that the crystal integrity in highly ordered crystallite regions of Mn(II)- and Cr(III)-treated nanocellulose remained unchanged after catalytic acid hydrolysis treatment. This finding is in agreement with the FTIR results. On the other hand, the peak intensity of nanocellulose at 2θ = 22.5° gradually increased compared with cellulose, which indicated the successive removal of soft less crystalline parts in cellulose fibers during hydrolysis treatment.

Using Segal’s equation, the calculated crystallinity indices of cellulose, Mn(II)-treated, and Cr(III)-treated nanocellulose were found to be 43.7 ± 0.3%, 72.3 ± 0.4%, and 75.6 ± 0.1%, respectively. This showed a 58.9% and 70.7% increment for Cr(III)- and Mn(II)-treated nanocellulose compared with untreated cellulose, respectively. The results indicate that Cr(III)-metal ions rendered better hydrolytic efficiency and higher acid hydrolysis selectivity than Mn(II)-metal ions for breaking the glycosidic linkages of the cellulose defective crystalline regions while retaining crystalline regions in cellulose without over-hydrolysis to water-soluble monomer (e.g., glucose) by-products. From the results, it can be clearly seen that Cr(III)-treated nanocellulose rendered the highest crystallinity, which enabled better mechanical properties and tensile strength; this is beneficial for composite material synthesis applications (Chen et al. 2011c; Tan et al. 2015; Yahya et al. 2015).

As for industrial applications, crystallinity of nanocellulose is the most important factor to be considered as it has a greater influence on reinforcing capabilities than aspect ratio (Silvério et al.2013). In this study, the yielded nanocellulose prepared via transition metal ion catalyzed acid hydrolysis had higher crystallinity than that of nanomaterial derived from various biomasses via different hydrolysis treatments. Son and his coworker (Son and Seo 2015) conducted a study by preparing the nanocellulose from three major cellulose sources (wood, non-wood and marine pulp) in nature via strong sulfuric acid hydrolysis, and the results revealed that all the yielded nanocellulose (i.e. softwood, hardwood; cotton linters, cattail; and red algae) exhibited lower crystallinity (ranged from 61.5 to 71.7%) as compared with our current study (72.3 to 75.6%). This finding suggests that concentrated acid hydrolysis has high tendency to attack the highly crystalline regions and is accompanied by over-degradation, such that most of the cellulose polymeric chains tended to be solubilized into water soluble reducing sugar (glucose). This eventually lowers the quality of nanocellulose.

A similar study has been reported by Hu et al. (2014) in which the authors had proposed two different isolation methods for nanocellulose production derived from borer powder, namely ammonium persulfate oxidative depolymerization and H2SOhydrolysis approach. The crystallinity index of both yielded nanocellulose samples isolated via these two hydrolysis treatments was 62.75 and 69.84%, respectively (Hu et al. 2014). The low crystallinity might be due to the harsh conditions applied during the course of treatment.

Fig. 2. XRD patterns of native cellulose, Mn(II)-, and Cr(III)-treated nanocellulose

On the other hand, in a practical manufacturing process, the yield of nanocellulose obtained after the hydrolysis treatment should also be taken into account in order to minimize the cost of large-scale commercial production with maximize the nanocellulose production. Therefore, a compromise should be made between the nanocellulose yield and its crystallinity in the maximum range. The percentage of yield of nanocellulose produced in this study ranged from 83.5 to 85%. This result is similar to those reported for palm tree cellulose (84%) (Hamid et al. 2014) and microcrystalline cellulose (86%) (Karim et al. 2014) hydrolyzed by FeCl3 in HCl acidic medium but under higher temperature (91 to 98 oC) and longer hydrolysis duration (4 to 6 h) at optimum hydrolysis conditions. However, all the nanocellulose yields prepared via Cr(III)- and Mn(II)-catalyzed hydrolysis under mild reaction were much more higher than the value reported for the hydrolysis of cellulosic materials including rice straw cellulose (16.9%) (Jiang and Hsieh 2013), microcrystalline cellulose (28.6%) (Zhou 2012), and bamboo bleached fiber (30%) (Brito et al.2012) via H2SO4 (~65 wt.%) hydrolysis procedure. A recent study (Tang et al. 2015) conducted by Tang’s group found that the yield of nanocellulose produced via phosphoric acid hydrolysis followed by enzymatic hydrolysis was only able to achieve a yield of 23.98% with a crystallinity of 57.8%.

Table 1. Summary of Nanocellulose Characteristics Prepared using Transition Metal Salt Catalyzed Acid Hydrolysis Process

a Estimated from the dynamic light scattering (DLS) analysis

b Estimated from the transmission electron microscopy (TEM) analysis

c Crystallinity index estimated from Segal’s method

Reaction conditions: temperature, 80 oC; time, 45 min; concentration of H2SO4, 4 wt.%; concentration of metal salt catalyst, 25 mM

Surface Characterization Analysis (FE-SEM)

Scanning electron microscopy images of the cellulose and transition metal-treated nanocellulose are shown in Fig. 3. The cellulose material mostly consisted of aggregated long fibrils with an irregular shape (Fig. 3(a)). In addition, the compact structure of each cellulose fiber appeared to be assembled from hundreds to thousands of microfibrils, and each cellulose fiber was quite long and thus had a low aspect ratio (Tan et al. 2015). For Mn(II)- and Cr(III)-treated nanocellulose (Fig. 3(b) and (c)), it can be clearly observed that the original cellulose fibers were broken down and degraded to a great extent after hydrolysis treatment, which led to the long ribbon-like quality of the original fiber turning into short, rod-like structures.

The micro-sized cellulose fibrils tended to separate from the fibers’ bundles, which resulted in intermittent breakdown in its fibrillar structure with more individualized, smaller fragments. These findings are in excellent accordance with the reported literature (Qua et al. 2011). The yielded nanocellulose produced in this study showed a finer fibrillar structure and reduced fiber diameter compared with native cellulose (23.1 ± 11.5 μm). Furthermore, it was observed that the Cr(III)-treated nanocellulose (15.9 ± 6.2 μm) had a smaller diameter than Mn(II)-treated nanocellulose (19.0 ± 8.4 μm) because of the successive hydrolysis treatment catalyzed by Cr(III) metal ions in the degradation of defective crystalline regions due to the attacking of metal ion catalysts in this less crystalline regions and lead to the hydrolytic cleavage of the β-1,4-glycosidic linkages in cellulose polymeric chains.

In the present study, finer and shorter nano-dimensional fibrils were expected in the nanocellulose samples treated by different transition metal salt catalysts; however, this was not obvious from the FESEM micrographs, as shown in Fig. 3. This was accredited to the strong intermolecular hydrogen bonding within the cellulose chains. As a result, the cellulosic fibrils tended to be agglomerated during the lyophilizing process (Qua et al. 2011).

More insight into the particle size and morphology of the nanocellulose fibers in suspension was obtained through transmission electron microscopy (TEM) and atomic force microscopy (AFM) analyses.

Fig. 3. FE-SEM images of (a) native cellulose, (b) Mn(II)-, and (c) Cr(III)-treated sample

Particle Size Analysis (DLS) and Zeta Potential Analysis

Dynamic light scattering (DLS) is one of the most suitable techniques for a rapid evaluation and preliminary screening of yielded nanocellulose particle size distribution as compared with the more established electron microscopic study (Kos et al. 2014). The DLS technique is not able to accurately measure for rod-like materials; however, quantitative results regarding the hydrodynamic diameter of nanoparticles could be obtained quickly.

Because the DLS method offers the advantages of fast and simple preparation process, the initial and rough analysis for determining the nanocellulose particle size in suspension still has been conducted by this method in many published studies (Yu et al. 2013; Li et al. 2015). Figure 4 shows the comparative particle size distributions for nanocellulose prepared by Mn(II)- and Cr(III)-catalyzed hydrolysis processes, estimated by dynamic light scattering (DLS) analysis in a water suspension (Fig. 4). Based on the results, the particle size distribution of Mn(II)-treated nanocellulose showed two main groups: 91.6% were approximately 606.8 nm, and 8.4% were approximately 108.9 nm (Fig. 4(a)). Meanwhile, the hydrodynamic size distributions of the Cr(III)-treated nanocellulose, as shown in Fig. 4(b), were found to be 14.20, 135.8, and 458.5 nm with percentages of 2.2%, 89.7%, and 8.1%, respectively. Not surprisingly, the average hydrodynamic diameter of Cr(III)-treated nanocellulose (281.6 ± 55.3 nm) was found to be smaller than that of Mn(II)-treated nanocellulose (693.0 ± 34.8 nm), which indicated that Cr(III)-transition metal salt rendered better hydrolysis ability.

The colloidal performances of yielded nanocellulose were confirmed with zeta potential measurement. An absolute value lying within 0 to ± 15 mV reflects the onset flocculation and agglomeration of nanocellulose (Zhou 2012), whereas the values greater (or lower) than ± 25 mV were generally considered to be relatively stable for mutual repulsion to form a good stability colloidal suspension (Feng et al. 2015). In this study, the zeta potential for Mn(II)- and Cr(III)-treated nanocellulose was about ─42.5 and ─44.9 mV, respectively in the aqueous suspensions, thus indicative of the possibility to produce rather stable colloidal suspensions due to sufficient repulsive force. This is reasonably due to the presence of negatively charged sulfate groups introduced from H2SO4 into the nanocellulose surface during acid hydrolysis. This induced the increase in surface charges of the particles as well as led to the formation of an electrostatic layer covering the nanocellulose, thus minimizing it from aggregation and flocculation of particles (Hamid et al. 2015).

Fig. 4. Hydrodynamic size distributions of (a) Mn(II)- and (b) Cr(III)-treated nanocellulose

Morphological Analysis (TEM)

TEM images clearly illustrated that the nanocellulose isolated via both transition metal salt hydrolysis pathways exhibited entangled, complex spider-like network geometry with the presence of porous structures, as shown in Fig. 5. From the results, it was established that transition metal-catalyzed acid hydrolysis process was highly effective for hydrolyzing the micro-dimensional native cellulose fiber to its nanometer scale product. Based on the TEM micrographs, it was shown that nanocellulose treated with Mn(II)-metal ion catalyst had diameters mostly ranging between 40 and 80 nm, whereas Cr(III)-treated nanocellulose had a diameter range between 20 and 50 nm and were several microns long. The average fiber diameter showed that the former demonstrated a larger value (58.4 ± 15.3 nm) than the latter (44.7 ± 13.2 nm), with a fiber length of several micrometers. The smaller diameter of nanocellulose indicated that Cr(III) metal salt catalyst contributed to a highly efficient depolymerization process by attacking the defective crystalline parts at a faster rate than the crystallinity phases, as there are more active sites for chemical reactions to happen.

Additionally, some agglomeration of nanocellulose fibrils can be observed in certain places, while the rest are well separated. Similar phenomena were also reported from the preparation of cellulose nanofibrils from Helicters isora by acid hydrolysis (Chirayil et al. 2014). On the other hand, the entanglement structure among the yielded nanocellulose play an important role in force transferring from either fibrils to fibrils or matrix to fibrils to form a strong reinforcement when blended with other polymer materials (Tian et al. 2016).

In this study, the size distribution of all two types of nanocellulose (20 to 80 nm) was thus rather similar and relatively close to that reported for waste sackcloth (20 to 50 nm; Cao et al. 2015), borer powder (20 to 70 nm; Hu et al. 2014), rubberwood (10 to 90 nm; Jonoobi et al. 2011), sugarcane bagasse (20 to 60 nm; Kumar et al. 2014), flax (2 to 50 nm; Qua et al. 2011), and old corrugated container (15 to 80 nm; Zhang et al. 2015).

Fig. 5. TEM micrographs of (a) Mn(II)- and (b) Cr(III)-hydrolyzed nanocellulose

Morphological Study (AFM Analysis)

The surface morphology of yielded nanocellulose obtained using atomic force microscopy (AFM) analysis is displayed in Fig. 6. All the samples were observed in nano-dimensional rod-shape structure or needle-like nanoparticles. After the catalytic acid hydrolysis process, the original native cellulose long fibrils structure was successfully separated into smaller and finer fiber fragments (Lee et al. 2010). The AFM images show that the average diameters of Cr(III)-treated nanocellulose fibers (64.1 ± 31.7 nm) were smaller than those of Mn(II)-treated nanocellulose fibers (80.9 ± 30.3 nm). These findings correlate well with the TEM study.

The AFM results further verified that transition metal salt-catalyzed acid hydrolysis can easily break down the hydrogen bonding inside the cellulose matrix, mainly in defective less crystalline parts, and leads to gradual disintegration of the micro-scale cellulose fibers into nano-sized cellulose.

Fig. 6. AFM images and particle size distribution of (a) Mn(II)-, and (b) Cr(III)-treated nanocellulose

Thermal Stability Analysis (TGA Study)

Thermogravimetric (TG) curves and their derivatives (DTG) for cellulose and nanocellulose products are shown in Fig. 7. A minor weight loss that was found for all fiber samples below 100 °C can be attributed to the evaporation of absorbed moisture from the surroundings of the cellulose fiber. The native cellulose showed a typical decomposition behavior with an onset degradation temperature (Ton) of 250 °C and a maximum thermal degradation temperature (Tmax) of 353.5 °C. The decomposition of the native cellulose showed only the one-step pyrolysis process, as shown in the DTG curve.

In the case of the produced nanocellulose, the degradation process occurred within a narrower range with lower Tmax value (338 to 346 °C) compared with native cellulose, as summarized in Table 2. It was found that all the yielded cellulose nanoparticles possessed lower Tmax values because of their smaller particle size, high specific surface area, and active sulfate groups (Cheng et al. 2014; Tan et al. 2015). A similar phenomenon was previously reported for the production of cellulose nanofibrils from sugarcane bagasse via high-pressure homogenization because the produced nanomaterial consisted of a higher number of contact points to the heating source than crude cellulose (Saelee et al. 2016).

Fig. 7. (a) TG and (b) DTG curves of cellulose and yielded nanocellulose

In addition, the present finding was similar to several studies listed in Table 3, where the Tmaxvalue of yielded nanocellulose obtained from various biomass sources rendered lower values than that of crude cellulose material. The Tmax values of yielded nanocellulose derived from wheat straw (Tmax = 332.2 °C) (Chen et al. 2011), empty fruit bunch (Tmax = 339 °C) (Jonoobi et al. 2011), and mulberry bark (Tmax = 335 °C) (Li et al. 2009) are shown. Fortunately, all the cellulose nanoparticles were stable when the heating temperature was less than 280 °C. This is an important factor for the use of nanocellulose in various industrial applications, especially the thermoplastics field, as the processing temperature is normally higher than 200 °C (Cheng et al. 2014). Therefore, the thermal resistance of all the nanocellulose remained sufficient to ensure that nanocellulose can be processed at approximately 200 °C without any risk of degradation.

Table 2. TGA Profile of Nanocellulose Hydrolyzed by Cr(III)- and Mn(II)-Metal Salt

Table 3. Thermal Properties of Nanocellulose Isolated from Different Biomasses

Effect of Mn(II) and Cr(III) on Acid Hydrolysis Efficiency

Transition metal ions (Mn2+ or Cr3+) showed a similar acid hydrolysis mechanism as inorganic acid (H2SO4) during the depolymerization of cellulose. The metal ions (Cr3+ and Mn2+) were generated from transition metal salts capable of attaching to the high electronegativity of oxygen atoms in β-1,4-glycosidic bonds of the cellulose chain, which resulted in weakening of the bond strength between pyranose rings in the amorphous region and thus led to the easy rupture of the cellulose chain into smaller fiber fragments (Li et al. 2014a). Compared with single-charge protons (+1) from typical mineral acids (e.g., HCl or H2SO4), transition metal ions with higher valence states (e.g., +2, +3) can efficiently hydrolytically cleave the extensive network in the cellulose fibers. This indicates that more protons are available to pair with more oxygen atoms to render an extensive reduction in particle size by breaking the glycosidic linkages in the cellulose structure (Zhao et al. 2011). In nature, the depolymerization process occurred in a faster rate on the defects structure in cellulose matrix as there is randomly ordered and more active sites for chemical degradation process to happen.

Generally, the efficiency of cellulose hydrolysis depends on the acidity of the hydrolysis catalyst. The acidity of the Mn(II)- and Cr(III)-metal ions is attributed to the deprotonation of metal-ligand complex ions. When the water molecules (H2O) are bonded with the metal ions, they become more acidic than the metal ion alone (Li et al. 2013). This results in the generation of a higher concentration of H3O+ ion, as shown in Eqs. 2 and 3. Furthermore, a higher concentration of H3O+ ions was generated in the presence of Cr(III) ions as compared with Mn(II) ions because of its high valence state. Therefore, the Cr(III)-solution exhibits high acidity, which provides better acid hydrolysis efficiency for cellulose depolymerization (Zhao et al. 2007).

[Cr(H2O)6]3+ + 3H2O → [Cr(H2O)3(OH)3] + 3H3O+ (2)

[Mn(H2O)6]2+ + 2H2O → [Mn(H2O)4(OH)2] + 2H3O+ (3)


1. Nanocellulose was successfully prepared by Mn(II)- and Cr(III)-catalyzed acid hydrolysis under a moderate temperature of 80 °C with a short preparation time (45 min). The removal of amorphous regions from the cellulose fibers was confirmed by XRD, FESEM, TEM, DLS, AFM, and TGA techniques.

2. Based on the results, the trivalent Cr3+ metal ion showed better hydrolysis and produced nanocellulose with a higher crystallinity (75.6 ± 0.1%) with a smaller diameter (46.3 ± 15.5 nm) compared with the divalent Mn2+ ion, with a nanocellulose crystallinity of 72.3 ± 0.4% and a diameter of 62.3 ± 13.9 nm.

3. The morphology study revealed that Cr(III)- and Mn(II)-treated samples appeared in smaller fragments compared with long and aggregated cellulose fibers. Furthermore, the prepared nanocellulose exhibited a spider web-like geometry, which indicated that the amorphous region in the compact cellulose fibers was removed during the catalytic acid hydrolysis reaction.


The authors are grateful for the financial support of the Minister of Science, Technology and Innovation (MOSTI) e-Science Fund (SF002-2015) and the University of Malaya Postgraduate Research Grant Scheme PPP (PG063-2015A).


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Article submitted: March 22, 2016; Peer review completed: May 29, 2016; Revised version received and accepted: June 27, 2016; Published: July 13, 2016.

DOI: 10.15376/biores.11.3.7224-7241